investigating enteric coccidiosis in the endangered black

Investigating Enteric Coccidiosis in the Black-footed (Mustela nigripes) and

Domestic Ferret (Mustela putorius furo)

by

Adriana R Pastor

A Thesis

presented to

The University of Guelph

In partial fulfilment of requirements

for the degree of

Doctor of Veterinary Science

in

Zoological Medicine and Pathology

Guelph Ontario Canada

copy Adriana R Pastor November 2017

ABSTRACT INVESTIGATING ENTERIC COCCIDIOSIS IN THE BLACK-FOOTED (MUSTELA NIGRIPES) AND

DOMESTIC FERRET (MUSTELA PUTORIUS FURO)

Adriana R Pastor Advisors

University of Guelph 2017 Dr D A Smith

Dr J R Barta

Enteric coccidiosis is a major cause of death in both juvenile and adult black-footed ferrets (BFF

Mustela nigripes) in captive breeding programs that reduces the availability of animals for release to their

former North American range Coccidiosis is poorly understood in BFF but in vivo experimental infection

in this endangered host is untenable The goal of this research was to better characterize the etiologic

agents and natural history of enteric coccidiosis in BFF and to evaluate the domestic ferret (DF Mustela

putorius furo) as a model for experimental infection

Morphometric and molecular characterization of coccidia from BFF and DF was undertaken

Only Eimeria ictidea was identified in juvenile and adult BFF from 1999-2016 at the Toronto Zoo and

from BFF at the Louisville Zoo in 2016 Eimeria furonis and Isospora (=Cystoisospora) laidlawi were

identified in DF fecal and necropsy samples from Canadian and European diagnostic laboratories during

2008-2017 Molecular characterization of these parasites included generation of complete mitochondrial

genomes and nuclear 18S rDNA sequences for Eimeria ictidea and Eimeria furonis from BFF and DF

respectively Partial sequences were obtained from the same genetic targets from I (=C) laidlawi from

DF DNA isolation from formalin fixed paraffin embedded tissues and PCR amplicon sequencing

permitted identification of coccidia in BFF and DF tissues dating from 1999 to present

Retrospective and prospective analyses of medical and pathology records supplemented with

parasitological evaluation of repeated fecal samples was performed to determine the natural history of

coccidiosis in captive BFF Clinical signs and histopathologic changes associated with infection in BFF

were as described previously in the published literature Average yearly coccidia associated mortality

rates were 053 in adults and 195 in juveniles

Domestic ferrets were confirmed as experimental hosts of E ictidea isolated from BFF Seven of

10 juvenile DF inoculated with oocysts from a BFF developed patent infections and mild clinical disease

was observed in six of these seven Infection was confirmed via morphometric molecular and histologic

examination of samples from infected DF While much is still unknown about enteric coccidiosis in BFF

domestic ferrets provide a promising model for further investigation of this disease

iv

DEDICATION

For my mother Anna Pastorhellip

v

ACKNOWLEDGEMENTS

Itrsquos hard to believe that my residency and thesis have been completed and I have a lot of people

to thank for that

Dale and Graham the two people I wanted to be when I grew up thank you for your mentorship

for many years even before this program I know that you werenrsquot convinced that this project was DVSc

worthy when I first proposed it but Irsquom hoping that the results have changed your mind

I am sincerely grateful to all the members of my advisory committee Dale Smith John Barta and

Simon Hollamby for their insight support and interest in this project Dale you have been an exceptional

advisor I donrsquot know that I will ever get to your level but thank you for showing me that being a great

clinical zoo vet and pathologist are not mutually exclusive John thank you for spontaneously agreeing to

be my advisor when I came to you with this project proposal in my first semester for your energy and

enthusiasm and for supporting my widening interest in parasitology research

My heartfelt appreciation for the Toronto Zoo WHC veterinarians past and present Chris

Dutton Pauline Delnatte Simon Hollamby and Graham Crawshaw I have learned so much from all of

you that I will take forward into my future endeavours I appreciate the extra time you put in including

comps study sessions after-hours tecircte-agrave-tecirctes and the fact that your doors were open when I needed it

For the Toronto Zoo vet techs extraordinaire Michelle Lovering Cassia Devison Dawn

Mihailovic and Tasha Long ndash you have been indispensable during this program and there are not enough

words to express my gratitude

I would especially like to thank all the Wildlife Health Center staff (2013-2016) Mark Bongelli

Charles Guthrie Christine McKenzie Brian Telford Rick Vos Gerri Mintha Margaret Kolakowski

Andrew Lentini Rebecca Clark Lydia Attard Nigel Parr Paula Roberts Andrea Dada Mindy Waisglass

and Julie Digiandomenico for three very memorable years It is all of you that make the WHC such an

amazing place to be Irsquom not sure I have laughed so hard or so often as I did in that lunchroom and I hope

our paths will cross again

vi

I donrsquot think that I can truly express how thankful I am to Pathobiology laboratory technicians

Julie Cobean and Julia Whale Without your assistance patient teaching and friendship I would probably

still be screening fecal samples years from now and scratching my head as to how our lab protocols

actually work It is people like you who make sure graduate students become successful doctorates and I

canrsquot imagine Pathobio without you both in it

I would also like to thank my labmates in the Barta lab mdash Mian Hafeez Evelyn Rejman Rachel

Imai Perryn Kruth Ryan Snyder and Mosun Ogedengbe A special thank you goes to Alex Leveille

without whom my many adventures in parasitology research from coccidia to Babesia would not have

been as successful

To all the students who helped with ferret fecal sample processing data compilation and

necropsies Nathalie Ferriman Janessa Price Thisuri Eagalle Sarah Brisson thank you so much for your

hard work and excitement about my projecthellip even when it was very smelly

So many thanks to the amazing staff of Central Animal Facility - Linda Groocock Vicky Carson

Tony Cengija and Mary Fowler for the daily care and enrichment of my experimental ferrets Your

excitement about working with our ferrets and your assistance with all parts of the process helped made

this project a success

To Adriana Nielsen who was not only my better half but the other fifty percent of my brain for

several years It is your friendship fortitude and our endless phone conversations that got me through the

never-ending Toronto-Guelph commute and this program

To all the ldquoscope roomrdquo pathology co-residents past and present - thank you for being wonderful

friends and colleagues It is indeed rare to find so many amazing people in one place and I know this

program and my sanity would not have been the same without you

To the anatomic pathology faculty and senior graduate students - thank you for all the time

teaching and guidance you provided during my program While I canrsquot say that I have become an amazing

pathologist I can say that because of your mentorship I am a better diagnostician and the type of clinician

who asks better questions takes better samples and understands that you canrsquot ldquojust make a PCR for thatrdquo

vii

A special thank you to Tony van Dreumel who came out of retirement for a semester to try to teach the

Adrianas zoo pathology screening cases with you was always a pleasure

To all the lovely Histo Ladies PM room staff and the other AHL staff who helped me with

Toronto Zoo and HSC pathology cases along the way - I donrsquot think the anatomic path students could

survive without you Thank you for always smiling assisting and accommodating me even when I made

near-impossible processing requests during my weekly Guelph visits

I would also like to acknowledge and sincerely thank all the individuals who helped with resource

and sample acquisition for this project A special mention for those who went above and beyond because

of their interest in this project Don Duszynski who was instrumental in acquiring and then providing a

translator for many of the original mustelid Eimeria descriptions and Majda Globokar Nikola Pantchev

and Donald Martin who supplied my domestic ferret fecal samples and historical data

A shout-out to Julie Swenson Gary West and the Phoenix Zoo BFF team who fostered my love

of this endangered species and helped develop the idea for this project

As always I continue to go out into the world and pursue my dreams with the knowledge that I

have the support of my incredible family long-time friends and my partner Keith Morris I am so lucky

that my residency brought me home and that it afforded us all more time spent together For my aunt

Veronica Lacey who has never failed to believe in my potential and always pushed me to become an

academic ndash yoursquoll never get that PhD from me but I think this is pretty close Finally for my mother

Anna Pastor who never lived to see my greatest achievements but had absolute faith that I could reach

any goal I worked towardshellip this is for you

Finally none of this would have been possible without the generous support of the Toronto Zoo

Residency program and funding through the Barta Laboratory University of Guelph

Adriana Pastor

Toronto August 2017

viii

DECLARATION OF WORK PERFORMED

I declare that all the work reported in this thesis was performed by myself with the following

exceptions

Fecal samples were collected by personnel at the Toronto Zoo Louisville Zoo and participating

diagnostic laboratories

Fecal oocyst per gram counts (routine salt flotation and McMaster counts) were performed by

myself Julie Cobean Julia Whale Evelin Rejman Sarah Brisson Adriana Rodriguez and Perryn Kruth

Whole mitochondrial genome PCR and sequencing was performed by me in conjunction with

Julia Whale and Dr Mian Hafeez

Sequencing of PCR samples was performed at the University of Guelph Laboratory Services

(Guelph Ontario Canada) and results were obtained electronically

ix

TABLE OF CONTENTS

ABSTRACT ii

DEDICATION iv

ACKNOWLEDGEMENTS v

DECLARATION OF WORK PERFORMED viii

TABLE OF CONTENTS ix

LIST OF TABLES xiii

LIST OF FIGURES xiv

LIST OF APPENDICES xv

ABBREVIATIONS xvi

CHAPTER 1 LITERATURE REVIEW 1

11 INTRODUCTION 1

12 APICOMPLEXA 1

121 Brief introduction to apicomplexan pathogens 1

122 Life cycles of the Eimeria and Isospora species implicated in enteric coccidiosis 3

123 Methods of characterization 5

13 RECLASSIFICATION OF MAMMALIAN ISOSPORA 8

14 EIMERIID SPECIES CHARACTERIZED IN MUSTELIDS 8

141 The family Mustelidae 8

142 Eimeriid coccidia described from mustelids 9

143 Eimeriid coccidia described from domestic ferrets 16

144 Molecular characterization 19

145 Clinical signs of disease in domestic ferrets 21

146 Gross necropsy and histologic findings 21

15 INTRODUCTION TO ENTERIC COCCIDIOSIS IN THE BLACK-FOOTED FERRET

25

151 Natural history and conservation of the black-footed ferret in North America 25

152 Coccidia identified from black-footed ferrets 26

153 Morbidity mortality and clinical signs associated with enteric coccidiosis in black-footed

ferrets 28

16 TREATMENT PREVENTION AND CONTROL OF INFECTION BY EIMERIA SPP 29

161 Current recommendations for treatment of eimeriid coccidia in carnivores 29

x

162 Current recommendations for anticoccidial treatment and prophylaxis in domestic and

black-footed ferrets 30

17 VACCINES AGAINST COCCIDIA 32

171 Theory 32

172 Species successes in anticoccidial vaccination 34

18 RESEARCH GOALS AND OBJECTIVES 36

181 Objectives 36

182 Hypotheses 36

183 Applications 36

CHAPTER 2 MOLECULAR CHARACTERIZATION OF ENTERIC COCCIDIA FROM DOMESTIC

FERRETS (MUSTELA PUTORIUS FURO) 38

21 INTRODUCTION 39

22 MATERIALS amp METHODS 43

221 Fecal samples 43

222 Formalin fixed intestinal tissues 44


224 Phylogenetic analysis 46

23 RESULTS 47

231 Fresh fecal samples 47

232 Formalin fixed samples 48



24 DISCUSSION 50

CHAPTER 3 MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF ENTERIC

COCCIDIA ISOLATED FROM BLACK-FOOTED FERRETS (MUSTELA NIGRIPES) 60

31 INTRODUCTION 60

32 MATERIALS AND METHODS 64




33 RESULTS 66

331 Morphometric characterization 67


34 DISCUSSION 69

xi

CHAPTER 4 NATURAL HISTORY OF ENTERIC COCCIDIOSIS IN THE BLACK-FOOTED

FERRET (MUSTELA NIGRIPES) 78

41 INTRODUCTION 78


421 Toronto Zoo BFF breeding program 80

422 Fecal oocyst evaluation 81

423 Retrospective review of pathology records 82

424 Prospective modified necropsy protocol 82

425 Retrospective medical history review 83

43 RESULTS 83

431 Fecal oocyst evaluation and retrospective medical history review 83

432 Pathology 86

433 Morbidity and mortality 88

44 DISCUSSION 88

CHAPTER 5 EVALUATING THE DOMESTIC FERRET (MUSTELA PUTORIUS FURO) AS AN

EXPERIMENTAL MODEL FOR ENTERIC COCCIDIOSIS IN THE BLACK-FOOTED FERRET

(MUSTELA NIGRIPES) 104

51 INTRODUCTION 104


521 Animal care 106

522 Oocyst preparation 107

523 Experimental infections 108

524 Animal welfare 109

525 Hematology 110

526 Morphologic and molecular characterization 110

527 Necropsy protocol 111

53 RESULTS 111

531 Oocyst shedding 112


533 Clinical signs 113

534 Hematology 113

535 Necropsy 114

54 DISCUSSION 115

xii

CHAPTER 6 WHOLE MITOCHONDRIAL GENOME SEQUENCES OF TWO EIMERIA SPECIES

ISOLATED FROM DOMESTIC (MUSTELA PUTORIUS FURO) AND BLACK- FOOTED FERRETS


61 INTRODUCTION 129


621 Parasites 130

622 DNA isolation from coccidia in feces 131

623 Whole genome sequencing 131


63 RESULTS 133

64 DISCUSSION 134

CHAPTER 7 CONCLUSIONS AND FUTURE DIRECTIONS 145

REFERENCES 148

APPENDICES 157

xiii

LIST OF TABLES

Table 11 Morphometrics of Eimeria and Isospora (=Cystoisospora) species affecting mustelids 10

Table 21 Amplification primers for nuclear 18S rDNA and mitochondrial COI loci used in the

identification of enteric coccidia from domestic ferrets 55

Table 22 Summary of fecal samples from domestic ferrets submitted to two diagnostic laboratories

from 2008-2015 56

Table 23 Morphologic and molecular identification of coccidia from domestic ferrets 57

Table 31 Amplification primers for nuclear 18S rDNA mitochondrial COI and COIII loci used in the

identification of coccidia from black-footed ferrets 73

Table 32 Morphologic and molecular characterization of coccidia from fecal and FFPE necropsy

samples from black-footed ferrets 76

Table 33 Morphometric characterization of Eimeria ictidea oocysts from black-footed ferrets 77

Table 41 Eimeria ictidea shedding in black-footed ferret dam and kit family groups - 2014-2016 97

Table 42 Epidemiologic data for family groups of black-footed ferrets shedding Eimeria ictidea 98

Table 43 Shedding of Eimeria ictidea in adult black-footed ferrets - 2015-2016 99

Table 44 Epidemiologic data for adult black-footed ferrets shedding Eimeria ictidea 100

Table 45 Histologic findings from black-footed ferrets with enteric coccidiosis 101

Table 46 Incidence of coccidial infections in black-footed ferrets at the Cheyenne Mountain Zoo 102

Table 47 Yearly mortality associated with coccidiosis in black-footed ferrets at the Toronto Zoo 103

Table 51 Prepatent period and oocyst shedding of Eimeria ictidea in experimentally infected

domestic ferrets 126

Table 52 Results of oral inoculation of domestic ferrets with oocysts of Eimeria ictidea 127

Table 53 Distribution of coccidial life stages in intestinal tract of domestic ferrets orally

inoculated with oocysts of Eimeria ictidea 128

Table 61 PCR primers used to sequence the mitochondrial genome of Eimeria furonis 136

Table 62 PCR primers used to sequence the mitochondrial genome of Eimeria ictidea 137

Table 63 Coding regions in the mitochondrial genome of Eimeria furonis from a domestic ferret 138

Table 64 Coding regions in the mitochondrial genome of Eimeria ictidea from a black-footed ferret 139

Table 65 Pairwise comparison of coding regions in the mitochondrial genomes of Eimeria furonis

and Eimeria ictidea 140

xiv

LIST OF FIGURES

Figure 11 Phylogeny of the Apicomplexa 2

Figure 12 Classical life cycle of coccidian parasites 4

Figure 13 Morphologic characteristics used for identification of eimeriid oocysts 6

Figure 21 Life stages of Eimeria furonis within the small intestine of a domestic ferret 58

Figure 22 Phylogenetic relationships of coccidia (Eimeria ictidea Eimeria furonis and Isospora

(=Cystoisospora) laidlawi) from domestic or black-footed ferrets 59

Figure 31 Nuclear and mitochondrial genetic loci targeted by primers listed in Table 31 73

Figure 32 Morphometrics of Eimeria ictidea from a black-footed ferret (Mustela nigripes) 74

Figure 33 Nuclear 18S rDNA sequences of Eimeria ictidea to newly generated (see Chapter 2) and

published sequences of Eimeria furonis 75

Figure 34 Mitochondrial cytochrome c oxidase subunit I sequences of Eimeria ictidea to sequences

from other eimeriid parasites of carnivores 75

Figure 41 Oocyst per gram counts and shedding period of Eimeria ictidea from black-footed ferret

family groups from 2014-2016 95

Figure 42 Sexual life stages of Eimeria ictidea in the small intestine of a black-footed ferret 96

Figure 51 Exogenous life stages of Eimeria ictidea 123

Figure 52 Endogenous life stages of Eimeria ictidea within the small intestine of an experimentally

infected domestic ferret 124

Figure 53 Distribution of sexual and asexual life stages of Eimeria ictidea along the intestinal tract

of experimentally infected domestic ferrets 125

Figure 61 Map of the mitochondrial genome of Eimeria furonis 141

Figure 62 Map of the mitochondrial genome of Eimeria ictidea 142

Figure 63 Comparison of the mitochondrial genomes of Eimeria furonis and Eimeria ictidea 143

Figure 64 Phylogenetic relationships of coccidia from domestic and black-footed ferrets based on

complete mitochondrial genome sequences 144

xv

LIST OF APPENDICES

Appendix 1 Shedding of oocysts of Eimeria ictidea in black-footed ferret (Mustela nigripes) dam and

kit family groups from 2014-2016 158

Appendix 2a Hematology values for domestic ferrets (Mustela putorius furo) from 49-51 days of

age prior to experimental inoculation 161

Appendix 2b Serum biochemistry values for domestic ferrets (Mustela putorius furo) from

49-51 days of age prior to experimental inoculation 162

Appendix 3a Hematology values for domestic ferrets (Mustela putorius furo) inoculated orally

with Eimeria ictidea 163

Appendix 3b Serum biochemistry values for domestic ferrets (Mustela putorius furo) inoculated

orally with Eimeria ictidea 164

Appendix 4 Domestic ferret (Mustela putorius furo) weekly monitoring sheet 165

Appendix 5 Domestic ferret (Mustela putorius furo) 24 hour intensive monitoring sheet 167

Appendix 6 Domestic ferret (Mustela putorius furo) infection trial standard operating procedures 172

xvi

ABBREVIATIONS

ATP Adenosine triphosphate

BFF Black-footed ferret(s)

BI Bayesian inference

bp Base pair

CAPC Companion Animal Parasitology Council

CDS Coding DNA sequence

CITES Convention on International Trade in Endangered Species of Wild Fauna and Flora

COI Cytochrome c oxidase subunit 1

COIII Cytochrome c oxidase subunit 3

CytB Cytochrome b

DF Domestic ferret(s)

DNA Deoxyribonucleic acid

FFPE Formalin-fixed paraffin embedded tissue

IUCN International Union on the Conservation of Nature

L Length

LSU Large subunit

mt Mitochondrial

NaOH Sodium hydroxide

nu Nuclear

OPG Oocyst per gram count

PCR Polymerase chain reaction

rDNA Ribosomal DNA

SI Shape index

SND Single nucleotide difference

SOP Standard operating procedure

sp spp Species (singular plural)

SSP Species Survival Plan

SSU Small subunit

TMS Trimethoprim sulfadimethoxine

USFWS United States Fish and Wildlife Service

W Width

1

CHAPTER 1 LITERATURE REVIEW

11 INTRODUCTION

Black-footed ferrets (Mustela nigripes) are one of three wild ferret species worldwide Although

formerly distributed throughout the North American prairies black-footed ferrets (BFF) had been

extirpated from the majority of their range by the 1970s and were declared extinct in the wild in 1987

Since 1986 a multi-institutional effort has been breeding this species in captivity with reintroduction back

into the wild at select sites within Canada the USA and Mexico

Coccidial enteritis is a major cause of death in young captive black-footed ferrets (Bronson et al

2007) but coccidiosis can affect all age classes (personal observation) As a result fewer captive-bred

ferrets may be reared successfully for release to the wild The significance of coccidiosis in wild ferrets is

unknown Consequently the prevention and control of coccidial outbreaks is an important part of black-

footed ferret captive breeding programs and management This research is intended to improve the in situ

and ex situ health of the black-footed ferret through the provision of a better understanding of the

pathogenesis of enteric coccidiosis in this species and to pave the way for the investigation of novel

methods for disease treatment and control

12 APICOMPLEXA

121 Brief introduction to apicomplexan pathogens

The phylum Apicomplexa comprises a large number of eukaryotic intracellular parasitic

organisms many of which are of importance to human and veterinary medicine As indicated by their

name these parasites are characterized by the presence of an apical complex at the anterior aspect of the

infective stage of the life-cycle (Tenter et al 2002) The taxonomic classifications of members of the

Apicomplexa continue to be in a state of flux (reviewed by Adl et al 2005 Cavalier-Smith 2014 Tenter

et al 2002) For this reason a more simplified taxonomic structure has been used in this review (see

2

Figure 11) The subclass Coccidia is a speciose group within the Apicomplexa with most genera falling

into one of two major coccidian suborders within the Eucoccidiorida To date greater than 2000 species

of coccidia have been named (Duszynski Upton amp Couch nd Upton 2000) The adeleid coccidia

(suborder Adeleorina) include monoxenous (single host) and heteroxenous (multiple hosts) parasites in

genera such as Adelea Haemogregarina Hepatozoon and Karyolysus The eimeriorinid coccidia

(suborder Eimeriorina) include the typical intestinal coccidia such as Eimeria Isospora and Cyclospora

species belonging to the family Eimeriidae as well as tissue (cyst forming) coccidia such as

Cystoisospora Besnoitia Toxoplasma and Sarcocystis species that belong to the family Sarcocystidae

(Cox 1994)

Figure 11 Phylogeny of the Apicomplexa Numbers on branches and thickness indicate diversity

(ie named species) Taxonomic groupings demonstrated by the phylogenetic tree (1) subclass

Coccidia (2) suborder Adeleorina (3) suborder Eimeriorina (4) family Eimeriidae and (5) family

Sarcocystidae Adapted from Šlapeta J Morin-Adeline V (2011) Apicomplexa Levine 1970

Sporozoa Leucart 1879 httptolweborgApicomplexa2446 in The Tree of Life Web Project

httptolweborg

2

1

3

4

5

3

122 Life cycles of the Eimeria and Isospora species implicated in enteric coccidiosis

The life cycle of Eimeria species is considered the classical coccidian life cycle which is

typically completed in one host (monoxenous) with many Eimeria species parasitizing only a single host

species (stenoxenous) (Figure 12) The life cycle has two main phases of development one that takes

place within the host (endogenous) and the other that takes places outside of the host (exogenous)

Classically the endogenous stages of the Eimeria life cycle take place within the intestinal epithelium

however some Eimeria species undergo extraintestinal endogenous development such as Eimeria stiedae

in rabbits which replicates within the epithelium of the biliary tree During the exogenous phase of the

life cycle unsporulated oocysts that are shed in the feces of the host sporulate within the environment

resulting in the formation of four sporocysts within each oocyst (tetrasporocystic) Each sporocyst

contains two sporozoites (dizoic) Sporulation is affected by three main factors temperature moisture and

aerobic conditions (Fayer 1980)

Once ingested by the host the wall of the sporulated oocyst is broken to release sporocysts from

which the sporozoites (infective stage) excyst The freed sporozoites penetrate the intestinal epithelial

cells and undergo multiple mitotic divisions to form a single multinucleate meront The meront then

undergoes simultaneous cytokinesis to form first generation merozoites which leave the host cell to infect

new cells and undergo further asexual replications The undifferentiated uninucleate tissue stage of the

parasite within the intestinal epithelial cell is called a trophozoite The number of cycles of asexual

replication (merogony) is predetermined after which the last generation of merozoites penetrate host cells

and undergo sexual differentiation into male and female gamonts (gametogony) Each microgamont

(male) undergoes simultaneous fission to produce numerous motile microgametes each macrogamont

(female) develops into a single mature macrogamete Fertilization of a macrogamete by a motile

microgamete results in formation of a zygote that is rapidly enclosed in a thick wall to form an

unsporulated oocyst Oocysts are shed with the hostrsquos feces into the environment where they are

protected from desiccation and chemical disinfection by the oocyst wall Traditionally Eimeria species

4

have been differentiated based on the host species or host genus affected the site of endogenous life cycle

development and the microscopic cellular characteristics of the different life stages Interestingly

experimental cross infection of Eimeria species from their natural host to a novel host of a taxonomically

similar species has been successful in some cases (De Vos 1970 Levine and Ivens 1970 Haberkorn

1971) challenging the notion that Eimeria are truly stenoxenous parasites

Figure 12 Classical life cycle of coccidian parasites This apicomplexan life cycle includes both

sexual and asexual development The three processes in the life cycle are merogony (asexual

replication A-D) followed by gametogony (formation of gametes E-H) within the digestive tract

of the host with release of unsporulated oocysts (I) Exogenous sporogony (I-L) results in the

production of infective sporulated oocysts (L) Adapted from Barta 2001 with permission of the

author

The life cycle of Isospora spp is similar to that of species in the genus Eimeria (see Figure 12)

but the number of sporocysts and sporozoites differ sporulated oocysts contain two sporocysts (disporic)

5

each of which contains four sporozoites (tetrazoic) These characteristics are not unique to Isospora spp

because diasporic tetrazoic sporulated oocysts are also found in the genera Besnoitia Frenkelia

Hammondia Sarcocystis and Toxoplasma However the sporocysts in the latter parasites are

morphologically distinct in that they lack Stieda bodies

123 Methods of characterization

1231 Morphological features

Historically eimeriid coccidia have been classified based on the cellular morphology of the

different life stages (particularly the morphometrics of sporulated oocysts) where these stages occur in

the host and apparent host specificity (frequently assumed and not tested experimentally) The

morphological features and dimensions of oocysts and their components are important diagnostic features

because of the availability of these stages in clinical specimens these characteristics can include size

(length [L] width [W] shape index [SI=LW]) number of sporocysts wall morphology

presenceabsence of a micropyle micropyle cap residual body or polar granules for oocysts size number

of sporozoites wall morphology presenceabsence of Stieda body subStieda body paraStieda body or

residual body for sporocysts and presenceabsence of refractile bodies for sporozoites (see Figure 13)

Pertinent life cycle information includes type of life cycle (monoxenous versus heteroxenous) tissue

sites of merogony and gametogony (intestinal versus extraintestinal) and the presence or absence of

extraintestinal hypobiotic stages (eg dormozoites or hypnozoites) Further information used to

characterize coccidia that form tissue cysts generally includes details on life stages in the definitive and

intermediate hosts location and morphology of tissue cysts route(s) of transmission among host species

and morphologic descriptions of merozoites (eg tachyzoites or bradyzoites) in tissue culture

6

Figure 13 Morphologic characteristics used for identification of eimeriid oocysts 1) Oocyst in cross

section ol - oocyst length or - oocyst residual body ow - oocyst width pg - polar granule row -rough

outer wall 2) The top of a hypothetical oocyst mcd - depth of the micropyle cap mcw - width of the

micropyle cap mw - width of the micropyle sow - smooth outer wall 3) Sporocyst in cross section

psb - paraStieda body sb - Stieda body sl - sporocyst length sp - sporozoite sr - sporocyst residual

body srb - sporozoite refractile body ssb - subStieda body sw - sporocyst width From Duszynski D

Wilber PG (1997) A guideline for the preparation of species descriptions in the Eimeriidae Journal of

Parasitology 83(2)333-336 reproduced with permission of Allen Press Publishing Services

1232 Molecular characterization (genetic loci and methods)

More recently molecular techniques have been used to infer phylogenetic or evolutionary

relationships among coccidia and to reclassify taxonomic assignments to better reflect the evolutionary

history of these parasites Molecular data can be more informative than phenotypic data because recent

evolutionary divergence among coccidia is unlikely to be reflected in morphologic differences but may

be detectable using molecular data The principle behind the use of molecular sequencing to describe

evolutionary relationships is that nucleotide sequences like morphological features diverge over time

under selective pressure however nucleotide sequences evolve at a more regular rate than do

morphologic characteristics Phenotypic data is thus less likely to detect recent evolutionary divergence

Sequences that are more similar are inferred to be more closely related and to have diverged more

recently (Cox 1994) Molecular characterization can be performed using DNA RNA or protein

sequences Most of the early molecular phylogenetic analyses of coccidia performed used ribosomal RNA

sequences usually by PCR amplification of ribosomal DNA (rDNA) in the nuclear genome of the

7

parasites Ribosomes contain both small and large RNA subunits in eukaryotes the large ribosomal RNA

consists of two forms 5S and 28S while the small ribosomal RNA exists only as 18S Sequences from

several genetic loci have been used for characterization of parasites most commonly 18S rDNA 28S

rDNA and ribosomal internal transcribed spacer regions (ITS) from the nuclear genome and more

recently mitochondrial cytochrome c oxidase subunits I (COI) and III (COIII) however sequencing of

nuclear 18S rDNA (nu 18S rDNA) has been the most prevalent in the literature by far Early attempts to

use 5S RNA sequences formed unlikely phylogenies and too few 28S ribosomal DNA sequences have

been obtained to make this locus useful (Cox 1994 Tenter et al 2002) The disadvantage of nu 18S

rDNA is that it is comparatively poor at distinguishing among closely related eimeriid coccidial species

because of its conserved nature but for that reason the nu18S rDNA locus is useful for inferring

relationships among species with greater evolutionary divergence Although only exploited recently

because of the paucity of suitable PCR primers the mitochondrial COI locus appears to be more useful

for distinguishing closely related eimeriid coccidia (Ogedengbe Hanner amp Barta 2011) but COI

sequences are less useful for inferring more ancient relationships between highly divergent coccidial

species Consequently the combined use of nu 18S rDNA and mitochondrial COI sequencing has been

recommended for improved species description and phylogenetic analysis (El-Sherry et al 2013)

Molecular characterization has also been used for diagnostic purposes and is well-suited to the

identification of coccidia when information on host specificity parasite life cycle and life stages is not

available as the molecular (genetic) data is the same for a given parasite during each of its life cycle

stages This information can be particularly useful in identifying the relationship between different life

stages of heteroxenous parasites collected from different hosts (intermediate definitive) Furthermore for

previously unidentified coccidia or those for which limited information is available molecular

characterization could be used to predict likely definitive hosts or parasite life cycle traits based on

phylogenetic relationships to other known species

8

13 RECLASSIFICATION OF MAMMALIAN ISOSPORA

Recommendations have been made to reclassify the avian and mammalian Isospora into two

separate genera based on life cycle molecular phylogenetic studies and morphologic description of

sporulated oocysts (Frenkel 1977 Barta et al 2005) Due to their classical coccidian life cycle presence

of Stieda bodies within sporocysts and close phylogenetic association with Eimeria species the avian

Atoxoplasma and Isospora have been retained in the genus Isospora (see Barta et al 2005) Conversely

the presence of tissue life cycle stages lack of Stieda bodies within sporocysts and close phylogenetic

association with other genera within the family Sarcocystidae have required many mammalian Isospora

to be reclassified as members of the genus Cystoisospora Frenkel 1977 (Frenkel 1977 Barta et al 2005)

Consequently for the remainder of this thesis Isospora species from mustelids will be referred to as

Isospora (=Cystoisospora) to reflect their probable generic association

14 EIMERIID SPECIES CHARACTERIZED IN MUSTELIDS

141 The family Mustelidae

The family Mustelidae within the order Carnivora comprises a group of approximately 59

carnivorous mammalian species within 22 genera Native mustelids are found in terrestrial and aquatic

environments on almost every continent with the exception of Australia and Antarctica The Mustelidae

are classically divided into two subfamilies as defined by Wozencraft (2005) 1) Mustelinae (weasels

mink ferrets marten wolverine) the larger subfamily including the following genera Arctonyx Eira

Galictis Gulo Ictonyx Lyncodon Martes Meles Mellivora Melogale Mustela Neovison Poecilogale

Taxidea and Vormela and 2) Lutrinae (otters) including seven genera Aonyx Enhydra Hydrictis

Lontra Lutra Lutrogale and Pteronura More recently molecular data suggest the Mustelidae should be

separated into eight subfamilies although this is not universally accepted (Koepfli et al 2008 Lariviegravere

and Jennings 2009 Yu et al 2011)

9

142 Eimeriid coccidia described from mustelids

Ten named Eimeria species and twelve named Isospora (=Cystoisospora) species have been

described in the Mustelidae and are summarized in Table 11 This table includes information on host

range life cycle and detailed morphologic data used to identify and classify the individual parasites Two

coccidial parasites isolated from the Libyan striped weasel (Ictonyx libyca) and the European polecat

(Mustela putorius) initially ascribed to the genus Isospora Isospora zorillae and Isospora putori

respectively have since been reclassified as Sarcocystis spp (see footnote to Table 2 of Yi-Fan et al

2012)

10

Table 11 Morphologic characteristics of Eimeria and Isospora (=Cystoisospora) species affecting mustelids

Coccidial species Host genus and

species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Cytoisospora

eversmanni

Mustela

eversmanii

(Steppe polecat)

Mustela

putorius

(European

polecat)

Homoxenous L185 (16ndash20)

W 148 (16ndash12)

LW 13 (11ndash16)

M absent

PG absent

OR absent

L 115

(10ndash135)

W 98

(9ndash11)

LW 12

(11ndash15)

SB absent

SR present

SRB present Yi-Fan et al 2012

Svanbaev 1956

Nukerbaeva amp

Svanbaev 1973

1977

Cystoisospora

pavlovskyi

Mustela

eversmanii

Mustela

putorius

Homoxenous L 322 (29ndash36)

W 273 (265ndash285)

LW 12 (11ndash14)

M absent

PG absent

OR absent

L 195

(18ndash21)

W 144

(12ndash15)

LW 14

(12ndash15)

SB absent

SR present


Svanbaev 1956

Nukerbaeva amp

Svanbaev 1973

1977

Eimeria

baskanica^

Mustela

erminae

(ermine)

Homoxenous Oval with tapered

ends

L 112-126

W 84-98

M absent

PG absent

OR present

SR absent Bean shaped Nukerbaeva amp

Svanbaev 1977

Eimeria furonis Mustela

putorius

Mustela

putorius furo

(dom ferret)

Mustela

nigripes (BFF)

Mustela vison

(mink)

Homoxenous

Small intestine

rectum (H 1927)

Jejunumileum (BP

1993)

Spherical ndash

subspherical

L 11-14

W 10-13

OW 2 layers

M absent

PG absent

OR absent

Spindloid

L 8-9

W 4

SB present

SR present

Vermiform Blankenship-Paris

et al 1993

Hoare 1927 1935b

Jolley et al 1994

Nukerbaeva amp

Svanbaev

19731977

Williams et al 1988

1992 1996

Eimeria hiepei Mustela vison Homoxenous

Bile duct

Spherical

L 13-17

W 13-17

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

L 6

W 4

SB absent

SR absent

Banana shaped Davis et al 1953

Grafner et al 1967

11


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Eimeria ictidea Mustela

eversmanni

Mustela

nigripes

Mustela

putorius

Mustela

putorius furo

Homoxenous

Small intestine

Ovoid ndashellipsoid

L 13-27

W 13-21

OW 2 layers

M present

PG absent

OR absent

Ovoid

(irregular)

L 115

W65

SB present

SR present

- Hoare 1927 1935a

1935b

Jolley et al 1994

Litvenkova 1969

Svanbaev 1956

Tinar 1985

Williams et al 1988

1992

Eimeria irara Eira barbara

(tayra)

Homoxenous

Feces

Ovoid

L 21-25

W 18-20

OW outer

layer smooth

M absent

PG absent

OR absent

Ellipsoid

L 10-12

W 65

SB present

SR present

Elongate (one

end broader than

the other)

Carini amp da

Fonseca 1938

Eimeria melis Meles meles

(European

badger)

Homoxenous Ellipsoid

L 20plusmn018

W 157plusmn002

LW128plusmn0017

(112-15)

OW 2 layers

(outer

smooth)

M absent

PG present

OR present

Ovoid

L

119plusmn0018

W 65plusmn008

LW 183

(155-24)

SB present

L 90plusmn005

W 324plusmn0025

SRB present

Anwar et al 2000

Kotlan amp Pospesch

1933

Eimeria mustelae Mustela vison

Mustela nivalis

(snow weasel)

Homoxenous

Duodenumileum

Spherical or

Ellipsoid

L 18-26

W 14-24

OW 2 layers

M absent

PG present

OR absent

Ovoid

L 8

W 5

SB present

SR present

Broad at one

end and tapered

at other

L 7

W 3

Glebezdin 1978

Iwanoff-Gobzem

1934

Levine 1948

Musaev amp Veisov

1983

Tinar 1985

Eimeria sablii Martes zibellina

(sable)

Homoxenous

Gut

Spherical or

subspherical

L 112-126

W 112

OW 2 layers

M absent

OR absent

Ovoid

L 56

W 42

SR present

Elongate Nukerbaeva 1981

Eimeria sibirica Martes zibellina Homoxenous Ovoid

L avg 216

W avg 180

LW 1076

OW 2 layers

M absent

PG absent

OR absent

Ovoid

L 96-112

W 56-72

SR absent


Yakimoff amp

Gousseff 1934

Yakimoff amp

Terwinsky 1930

1931

12


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Eimeria vison

(Eimeria

mustelae)

Mustela

putorius

Mustela

putorius furo

Mustela vison

Homoxenous

Small intestine

+- large intestine

Ovoid

L 17-22

W 9-18

OW 2 layers

M absent

OR

sometimes

present

Ovoid or

Piriform

L 10

W 55

SB absent

SR present

Curved or Club

shaped

L 9

W 25

Foreyt amp Todd 1976

Foreyt et al 1977

Kingscote 1934

1935

Levine 1948

McTaggart 1960

Nukerbaeva amp

Svanbaev

19731977

Tinar 1985

Umurzakov amp

Nukerbaeva 1985

Wolter 1961

Zimmermann 1959

Isospora africana Ictonyx libyca

(Libyan striped

weasel)

Homoxenous

Feces

Spherical

L 25-27

W 25-27

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

Ovoid

L 15-17

W 10-12

SB absent

SR present

Elongate

L 135

W 3

Prasad 1961

Isospora altaica Mustela altaica

(mountain

weasel)

Homoxenous

Gut

Oval or spherical

L 280-336

W 252-280

LW 121 (111-

124)

OW 2 layers

M absent

PG absent

OR absent

Ovoid or

spherical

L 140-168

W 111-168

SR present

Svanbaev amp

Rachmatullina

1971

Isospora goussevi Mustela nivalis Homoxenous

Large intestine

Ovoid

L 224 (220-250)

W 174 (160-190)

LW 135 (133-

137)

OW 1 layer

PG present

OR present

Ovoid

L 120

(100-130)

W 70 (60-

80)

SB present

SR present

Elongate Musaev amp Veisov

1983

13


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Isospora

hoogstraali

Ictonyx libyca Homoxenous

Feces

Ellipsoid

L 37-41

W 32-34

OW 2 layers

(outer

smooth)

M absent

PG some

OR absent

Ovoid

L 19-21

W 13-15

SB absent

SR present

Club-shaped

L 18-19

W 4-6

Prasad 1961

Isospora laidlawi Mustela

putorius

Mustela

putorius furo

Mustela vison

Homoxenous

Feces

Intestinal contents

Ovoid L

320-368

W 272-304

OW 2 layers

M absent

PG absent

OR absent

Ellipsoid

L 208

W 144

SB absent

SR present

Sausage shaped Foreyt et al 1977

Hoare 1927

Levine 1948

McTaggart 1960

Nukerbaeva amp

Svanbaev 1973

1974 1977

Tinar 1985

Isospora lutrae Lutra lutra

(European

otter)

Lutra

canadensis

(North

American river

otter)

Homoxenous Spherical

L 312 (275-32)

W 296 (28-31)

LW 104

(10-112)

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

Ellipsoid

L 182 (17-

19)

W 144 (14-

16)

LW128

(12-14)

Sb absent

sSB absent

SR present

Spindle- shaped

L 124

W 25

SRB present

Torres et al 2000

Hoover et al 1985

Isospora

martessii

Martes zibellina Homoxenous

Gut

Ovoid short oval or

spherical

L 252 ndash 280 196

168

W 168 ndash 224 168

168

OW 2 layers

M absent

OR absent

Ovoid

L 112-168

W 84-112

SR present


Isospora melis Meles meles Homoxenous Ovoid

L 328plusmn034

W 269plusmn019

LW122 (110-

157)

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

Ellipsoid

L

215plusmn0166

W 14plusmn012

LW 155

(133-185)

SR absent

Round at one

end other end

tapered

L 142plusmn116

W 40plusmn017

SRB absent

Anwar et al 2000

Glebezdin 1978

Kotlan amp Pospesch

1933

Pelleacuterdy 1955

14


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Isospora

mustelae (nomen

nudum)

Martes martes Ovoid L

7 W

225

M present - - Galli-Valerio 1932

Isospora nivalis Mustela nivalis Homoxenous

Large intestine

Ovoid

L 206 (200-230)

W 184 (180-210)

LW 11 (109-111)

OW 1 layer

PG absent

OR absent

Ovoid

L 125

(120-130)

W 80 (70-

90)

SR present

Lemon or pear

shaped

Musaev amp Veisov

1983

Unnamed

ldquoCoccidiardquo^

Mustela

nigripes

Urinary bladder - - - - Jolley et al 1994

Unnamed

ldquoCoccidiardquo^

Mustela

nigripes

Trachea bronchus

bronchial glands

- - - - Jolley et al 1994

Unnamed

Eimeria sp^

Mustela

nigripes

Feces

intestinal contents

Ovoid

L 350-386

W 212-232

- - - Jolley et al 1994

Williams et al

1992

Unnamed

Eimeria sp^

Mustela

putorius furo

Small intestine - - - - Blankenship-Paris

et al 1993

Unnamed

Eimeria sp^

Mustela nivalis Homoxenous

Large intestine

Ovoid-ellipsoid L

2031 (1712-2162)

W 148 (1225-

1681)

LW 136 (121-16-

)

OW 1 layer

PG absent

OR absent

Ovoid or

pear-shaped

L 60-100

W 40-80

SR present

Elongate

L 50-90

W 30-70

Musaev amp Veisov

1983

Unnamed

Eimeria sp^

Martes martes

(marten)

Homoxenous Ovoid

L avg 216

W avg 180

LW 1076

OR absent 4 sporocysts

SR present

L 126

W 60

Yakimoff and

Gousseff 1934

Unnamed

Isospora sp^

Mustela

putorius furo

Feces - - - - Bell 1994

Unnamed

Isospora sp^

Mustela

putorius furo

Feces - - - - Bell 1994

Legend L = length W = width LW = length-width ratio avg = average OW = oocyst wall PG = polar granules M = micropyle SB = Stieda body sSB =

subStieda body OR = oocyst residuum SR = sporocyst residuum SRB = sporozoite refractile body ^ = species inquirendae - = no information provided by

author(s) = information obtained from secondary sources (primary reference could not be obtained) All measurements are in micrometers Bolded references

15

are those from which morphometric data were assembled Remaining references indicate other authors who have identified that parasite species in the same or

similar host

16

143 Eimeriid coccidia described from domestic ferrets

Three species of coccidia were originally described from 50 domestic ferrets (Mustela putorius

furo) Eimeria ictidea Eimeria furonis and Isospora (= Cystoisospora) laidlawi (Hoare 1927) All three

species were detected in feces from domestic ferrets at a research facility undergoing an outbreak of

canine distemper Sick ferrets appeared more frequently infected than healthy ones As per Hoare (1927)

none of the ferrets appeared to display clinical signs associated with protozoal infection For each

parasite the author described morphology of sporulated oocysts isolated from feces and sporulation time

(exogenous life stages) The pre-patent period (minimum duration of endogenous development) in an

inoculated naiumlve ferret was described only for E furonis and E ictidea due to insufficient sample size of

I (=C) laidlawi oocysts for an experimental infection trial Sporulation of oocysts occurred within 5-6

days for E furonis 3 days for E ictidea and 4 days for I (=C) laidlawi The sporulated oocysts of E

furonis were spherical with a double outer wall with a thin colourless outer layer and thick yellowish

inner layer no micropyle or residual body and measured on average 128 times 120 microm (length [L] 112-

144 width [W] 104-128 shape index [SI] 107) Unsporulated oocysts contained a zygote with a

diameter of 96 microm Sporocysts were spindle-shaped with one end constrictedblunted contained a

residual body and on average measured 8-88 times 4 microm Sporozoites were vermiform with one end

narrower than the other arranged head to tail and had a central nucleus a clear vacuole was identified in

some at the broad end The sporulated oocysts of E ictidea were oval or elliptical with a double outer

wall with a thin colourless outer layer and thick yellowish inner layer no micropyle or residual body

and measured on average 236 times 175 microm (L 184-272 W 128-208 shape index 135) The zygote in

unsporulated oocysts was elongate with a diameter of 15 times 12 microm when originally passed in feces but

became more spherical with time Sporocysts were irregularly oval with one end broad and the other

more constricted contained a residual body and on average measured 115 times 65 microm Sporozoites were

vermiform with one end narrower than the other arranged head to tail and had a central nucleus and a

clear vacuole at the broad end The sporulated oocysts of Isospora (=Cystoisospora) laidlawi were ovoid

with a double outer wall with a thin colourless outer layer and thick yellowish inner layer no micropyle

17

or residual body and measured on average 34 times 29 microm (L 320-368 W 272-304) Unsporulated

oocysts contained a spherical zygote with a diameter of 236 microm Two sporocysts were identified each

containing 4 sporozoites and no Stieda body sporocysts were elliptical contained a residual body and on

average measured on 208 times 144 microm Sporozoites were sausage shaped with one end slightly pointed

and had a central nucleus and a clear vacuole identified at the pointed end Sporozoites were arranged

with pointed ends all at the same pole of the sporocyst The pre-patent periods described for E furonis

and E ictidea were 6 days and 7 days respectively (Hoare 1927)

Since Hoarersquos initial description (Hoare 1927 Hoare 1935) multiple single case reports and

outbreaks of severe clinical disease associated with intestinal coccidiosis have been reported in domestic

ferrets Blankenship-Paris et al (1993) described a single case of a four-month-old domestic ferret that

presented depressed in thin body condition dehydrated and with pasty dark feces on the perineum This

ferret had been housed with its dam and another sibling neither dam nor sibling showed clinical signs of

enteric disease and both had negative fecal examination results on repeated evaluation Routine fecal

examination of the rest of the colony and necropsies on eight other ferrets in the colony revealed no

evidence of coccidial infection Enteric coccidiosis was determined to be the cause of disease in the four-

month-old ferret based on necropsy findings but the coccidia could not be speciated because diagnosis

was made on histologic findings only

Sledge et al ( 2011) described three separate outbreaks of severe enteric coccidiosis in domestic

ferrets from one ferret rescue centre (group 1) and two shelters (groups 2 and 3) all affected by the same

Eimeria sp The morphologic characteristics of sporulated oocysts were only described for group 1 no

coccidial oocysts were detected on direct smear or fecal flotation of diarrheic samples submitted from

groups 2 and 3 Oocysts were identified as spherical measuring 12-13 microm in diameter with four

sporocysts each containing two sporozoites Oocyst morphometrics histopathologic findings and nu 18S

rDNA partial sequences from all three groups were used collectively to confirm the coccidial species

identify in each outbreak as E furonis

18

Two cases of biliary coccidiosis with E furonis have been reported in domestic ferrets The first

was in a nine-week-old male ferret from a research facility (Williams Chimes amp Gardiner 1996) The

ferret presented with signs of hepatic disease and was negative for coccidia on fecal flotation and direct

smears Endogenous coccidial life stages were described from the gall bladder and liver on histologic

examination In tissue section the oocysts were oval to spherical and measured 125 times 120 microm Meronts

measured 108-130 times 89-93 microm and contained up to 16 merozoites The merozoites exhibited a double-

layered pellicle prominent conoid few rhoptries and many micronemes anterior to the nucleus Based on

the morphologic description of the life stages in this case the coccidia were identified by the authors as

an Eimeria species most likely E furonis Kaye et al (2015) described a second case of biliary

coccidiosis in an 18-month-old female pet domestic ferret with concurrent pure red cell aplasia In this

case all endogenous coccidial life stages were observed on histologic examination of the epithelium of

the extrahepatic biliary tree The oocysts were ovoid and measured 12 times 13 microm Meronts measured 12 times

15 microm and contained up to 16 merozoites each measuring 2 times 5 microm Based on the morphologic

description of the life stages in this case and nu 18S rDNA sequences the pathogen was also determined

to be E furonis Biliary coccidiosis has also been identified in mink (Mustela vison) with the etiologic

agent identified as Eimeria hiepei (Davis Chow amp Gorham 1953 Grafner Graubmann amp Dobbriner

1967)

Oocysts from Cystoisospora ohioensis have been reported from fecal samples collected from

healthy domestic ferret kits in a large American ferret breeding operation that were raised on the same

premise as juvenile domestic dogs (Patterson amp Fox 2007) The method of identification of this parasite

was not described by Patterson amp Fox A second similar institution reported the presence of a

Cystoisospora species also thought to be C ohioensis in routine fecal examination of their ferret colony

(Dr Bambi Jasmin personal communication) Coccidial identification in this case was performed by the

Animal Health Diagnostic Center at Cornell University The significance of these findings is unknown as

no clinical signs or histologic lesions have been described in domestic ferrets associated with shedding of

19

oocysts and the definitive host for C ohioensis is the domestic dog It is most likely that fecal

identification of C ohioensis represents a pseudoparasite in both of these cases or perhaps an

undescribed Cystoisospora sp that is morphologically indistinguishable from C ohioensis

It is difficult to estimate the prevalence of enteric coccidia within the North American domestic

ferret population Fecal samples submitted to university or large veterinary diagnostic laboratories from

domestic ferrets in Canada are uncommon and samples positive for coccidia appear infrequently (Dr

Donald Martin personal communication) Data from Idexx Vet Med Lab in Ludwigsburg Germany was

compiled to review the prevalence of coccidia and Giardia within fecal samples from domestic ferrets

(Pantchev et al 2011) The authors reported that of 284 fecal samples submitted from 2002-2004 18

(63) had detectable coccidial oocysts on fecal flotation Oocysts were identified based on morphologic

characteristics as E ictidea E furonis I (=C) laidlawi and another unidentified Isospora species

Comparative data from the same laboratory from 2009-2010 included sample submissions from 253

ferrets 21 (83) of which were positive for coccidial oocysts on fecal flotation Nine of the samples

were positive for E furonis three were positive with both E furonis and I(=C) laidlawi present eight

were positive only for I(=C) laidlawi and one sample contained both E furonis and E ictidea

identification in all cases was based on morphologic characteristics No statistically significant difference

in the occurrence of coccidial oocysts was detected when data from the two periods were compared

(Fisherrsquos exact test P=041) (Pantchev et al 2011)

144 Molecular characterization

Molecular characterization of Eimeria furonis was first performed by Abe et al (2008) using

oocysts purified from the feces of a single domestic ferret with clinical signs of coccidial enteritis Small

subunit ribosomal DNA (nu 18S rDNA) primers CYC1FE (5ʹ-TAC CCA ATG AAA ACA GTT T-3prime) and

CYC4RB (5prime-CGT CTT CAA ACC CCC TAC TG-3prime) were used to amplify a 347 base pair (bp) fragment

of nu 18S rDNA These primers were initially developed for molecular identification of Cyclospora

species but have since been shown to amplify nu 18S rDNA from several Eimeria species (Matsubayashi

20

et al 2005) The amplicon was sequenced (GenBank AB329724) and compared with previously

published partial nu 18S rDNA sequences from 40 Eimeria two Isospora and four Cyclospora species

The resulting phylogram grouped E furonis with E alabamensis (cattle) and E meleagrimitis (turkey) In

the same study the microscopic morphology of the oocysts was used to identify this coccidial species as

E furonis by comparison with published descriptions of E furonis E ictidea and E heipei by Hoare

(1927) Hoare (1935) and Grafner Graubmann amp Dobbriner (1967) respectively

Nuclear 18S rDNA was also used by Sledge et al (2011) for molecular identification of the

eimeriid coccidia implicated in the three distinct outbreaks of enteric disease in domestic ferrets As

described above initial identification and speciation of the coccidia was performed using morphologic

characteristics of the sporulated oocysts collected from feces in one of the three outbreaks being

investigated the oocysts were identified as E furonis Histologic sections of formalin fixed intestinal

segments from ferrets from each of the three outbreaks contained multiple coccidial life stages DNA was

then isolated from stored formalin-fixed tissues for further genetic analysis Using the partial nu 18S

rDNA gene sequence reported by Abe et al (2008) (GenBank AB329724) the following PCR primers

were created 5ʹ-ACA ATT GGA GGG CAA GTC TG-3ʹ and 5ʹ-GGCGAC AAG CCT GCT TGA AAC-

3ʹ PCR amplification produced a 247 bp amplicon from each of the three groups Analysis and

sequencing of amplicons from all three groups showed 100 homology to nucleic acid sequences

previously reported by Abe et al (2008) for the gene encoding E furonis nu 18S rDNA

Coccidia were identified within hepatobiliary lesions in a domestic ferret receiving

immunosuppressive therapy for red cell aplasia (Kaye et al 2015) DNA was extracted from frozen liver

and a 247 bp fragment of the nu 18S rDNA was amplified using the primers previously described by

Sledge et al (2011) and sequenced Kaye et al (2015) reported that the DNA sequence of the amplicon

was 100 homologous to the published nu 18S rDNA of E furonis and 95 homologous to the nu 18S

rDNA of E myoxi (rodent) E alabamensis (cattle) and I robini (avian)

21

145 Clinical signs of disease in domestic ferrets

Hoare (1927 1935b) in his initial descriptions of enteric coccidiosis in domestic ferrets

observed that clinical signs of intestinal disease were not evident The recent literature supports the

finding of subclinical disease but also describes signs ranging from mild transient diarrhea in young or

stressed animals to more severe disease with dehydration lethargy depression weight lossemaciation

inappetence and death (Blankenship-Paris et al 1993 Powers 2009 Sledge et al 2011 Hoefer et al

2012 Patterson et al 2014) Rectal prolapse has also been reported in ferrets with enteric coccidiosis

(Hillyer 1992 Hoefer et al 2012) In one study co-infection with coccidia and Lawsonia intracellularis

(Desulfovibrio sp) was diagnosed in 4 of 19 ferrets with proliferative bowel disease (Li et al 1996)

These ferrets presented with variable clinical signs including diarrhea lethargy anorexia weight loss

dehydration and emaciation

In the two reports of biliary coccidiosis clinical signs conformed to those expected with

hepatobiliary disease Williams et al (1996) described their case to have presented with emaciation poor

appetite abdominal distension and icterus Kaye et al (2015) described a one week history of lethargy

inappetence and icterus with serum biochemistry results consistent with cholestasis later clinical signs in

this case included melena anemia and cachexia

146 Gross necropsy and histologic findings

The pathology of enteric coccidiosis in domestic ferrets was described by Hoare (1927 1935b)

Two healthy domestic ferrets were experimentally inoculated one each with large numbers of mature

oocysts of either E furonis or E ictidea that were isolated during his initial work The inoculated ferrets

were killed humanely for histologic examination of intestinal sections at the time of first detection of fecal

oocyst shedding no clinical signs of coccidiosis were detected in these ferrets prior to death Infection

with E furonis resulted in invasion of the epithelium of the small intestine and rectum Within the small

intestine the parasites were concentrated in the tips of the villi but could be found to the level of the

22

opening of the crypts of Lieberkuumlhn In rectal sections life stages were limited to the epithelial ridges

between the openings of the glands of Lieberkuumlhn Organisms were located within the apical portion of

the epithelial cells and intensely infected regions exhibited multiple parasites within a single host cell

Both asexual and sexual life stages were present within the same sections Hoare (1927) described similar

histopathologic changes in naturally infected ferrets but the proportion of asexual versus sexual life

stages differed In natural infections sexual life stages were more numerous whereas in experimental

infections asexual life stages predominated these findings would be expected to correlate with the stage

of infection at which ferrets died or were humanely killed for tissue collection and would not be

reflective of differences between natural and experimental infection with this parasite Hoare also

described the morphology of the different endogenous stages including trophozoite (3-4 microm) merozoite

(stumpy sausage shaped L 3-4 microm W 2 microm) macrogamete (spherical 8 microm diameter with darkly

staining globular inclusions of reserve material) and microgamete (described as similar to those of other

Eimeria species) Two types of merogony are described from histologic sections the first with stumpy

merozoites as described above and the second with merozoites with elongated curved bodies and a

compact polar nucleus measuring 60 times 13 microm This second merogonic generation was observed almost

exclusively in the naturally infected ferrets and was associated with initiation of sexual differentiation and

reproduction

The pathology of experimental and non-experimental infection with E ictidea in domestic ferrets

was also described by Hoare (1927 1935b) Parasitic invasion of the epithelium was noted only in the

small intestine with patchy distribution of the parasite life stages throughout affected sections Within the

small intestinal villi the parasites were again concentrated in the tips of the villi with infected epithelial

cells never containing more than one parasite As each intracellular parasite grew it filled the entire host

cell displacing the nucleus to the base of the cell Predominantly sexual life stages were detected in tissue

sections with few asexual generations observed Interestingly the parasites were arranged into age

groups with forms of the same life stage grouped together within the affected epithelial sections this is in

23

contrast to E furonis where life stages of different maturities were found together in affected sections

Hoare described the morphology of the different endogenous stages of E ictidea including merozoites

(free within the lumen elongated vermiform with one pointed end and a nucleus located at the rounded

end 11 microm times 1 microm within the epithelium shortened and rounded 3-4 microm diameter) macrogametes

(elongated 20 times 7 microm occupying the entire host cell with darkly staining globular inclusions of reserve

material) and mature microgamonts (morphologically similar to those of other Eimeria species but larger

than those of E furonis) Of note a tissue reaction was observed specifically in association with more

developed life stages of E ictidea (eg mature meronts mature gamonts unsporulated oocysts) which

was not observed when cells contained earlier stages of development (eg trophozoites immature

gamonts) This tissue reaction was described by Hoare (1935a 1935b) as the development of an annular

constriction of the apical portion of the villus separating infected epithelial cells from unaffected cells

The constriction involved the epithelium but could also extend inwards into the core of the villus These

changes were associated with congestion of capillaries and extravasation of red blood cells within the

constricted segment and in some sections villar tip necrosis

In their case report of one domestic ferret Blankenship-Paris et al (1993) described the gross

pathologic lesions associated with intestinal coccidiosis in this case there was diffuse dilation and

reddening of the small intestine which was empty and the colon contained dark watery material

Histologic lesions were confined to the ileum and jejunum The jejunum exhibited thickening of the villi

with a crypt to villus ratio of 15 mild granulomatous inflammation in the lamina propria and large

numbers of coccidial meronts gamonts and oocysts within the enterocytes of the villar tips

The gross lesions described by Sledge et al (2011) from 20 domestic ferrets are as follows thin

body condition with moderate to marked dehydration perineal staining with diarrhea moderate dilation

of the small and large intestines and the presence of pasty tan to tarry black digesta within the distal small

intestine and colon Other findings in one to a small number of ferrets included enlarged pale tan livers

splenomegaly with dark red colouration and multiple superficial gastric or duodenal ulcers The

24

histologic lesions from 10 ferrets included moderate blunting and occasional fusion of jejunal and ileal

villi focal attenuation and erosion of the epithelium of the villar tips with exudation of fibrin neutrophils

and blood into the intestinal lumen in regions with severe erosion Intact epithelial cells at the villus tips

and rarely sloughed epithelial cells in the intestinal lumen contained numerous intracytoplasmic coccidia

representing a range of asexual and sexual life stages (meronts macrogamonts microgamonts and

oocysts) The subjacent lamina propria of the small intestine and of the large intestine exhibited moderate

lymphoplasmacytic infiltration with occasional neutrophils and congestion of blood vessels Marked

mucosal hemorrhage was identified in the most severely affected sections

Marked gross and histopathologic hepatobiliary lesions were described in a single ferret by

Williams et al (1996) On gross necropsy the liver was pale and enlarged with dilated firm bile ducts

and thickening of the gall bladder wall Similar gross necropsy findings were described by Kaye et al

(2015) marked dilation and mural thickening of the entire biliary tree (including gall bladder intrahepatic

and extrahepatic bile ducts) On histopathology Williams et al (1996) noted that the marked thickening

of the gallbladder wall was a result of cystic proliferation of mucosal glands which were separated by

tracts of fibrous connective tissue and marked granulomatous inflammation Liver sections exhibited

marked biliary hyperplasia marked periductular fibrosis and moderate periportal lymphoplasmacytic

cuffing There was multifocal papillary proliferation of bile duct epithelium and dilation of the bile ducts

and within the ductular lumens there were moderate numbers of lymphocytes and plasma cells small

numbers of degenerate neutrophils sloughed epithelial cells and debris All endogenous coccidial life

stages were present within the gall bladder and biliary epithelium with meronts visible in 20 of the

intact epithelial cells of the biliary tree and gallbladder and oocysts free within the lumen of the

intrahepatic bile ducts Similar lesions were present in the case described by Kaye et al (2015) and as

well as in juvenile and adult farmed mink (Mustela vison) with hepatobiliary coccidiosis (Davis Chow amp

Gorham 1953)

25


151 Natural history and conservation of the black-footed ferret in North America

Black-footed ferrets are one of only three wild ferret species worldwide the other species are the

European polecat (Mustela putorius) and the Siberian polecat or steppe polecat (Mustela eversmanii)

They are the only native North American ferret species and the most endangered North American

carnivore They are nocturnal carnivores whose diet and lifestyle are highly dependent on local prairie

dog (Cynomys sp) populations Prairie dogs comprise almost exclusively the diet for the BFF who also

use the complex burrow systems made by prairie dogs to escape their predators and raise their young

(Santymire et al 2014 USFWS BFF Recovery Program 2017)

While formerly distributed throughout the North America prairie ecosystem BFF were

considered extinct by the late 1950s In 1964 a single population was discovered in Mellette County

South Dakota Progressive decline of this population in subsequent years resulted in the decision by

United States Fish and Wildlife Service (USFWS) to initiate a captive breeding program for the species

From 1971-1973 four females and five males were captured for this purpose Despite successful breeding

no kits survived and the last adult ferret in this captive colony died in 1979 at that time BFF were again

presumed extinct in the wild based on annual surveys of the initial capture site In 1981 a dead BFF was

discovered by a ranch dog outside of Meeteetse Wyoming allowing wildlife biologists to identify

another colony of BFF This colony flourished until 1985 when an outbreak of canine distemper in the

BFF population and an outbreak of sylvatic plague in the local prairie dog population resulted in sharp

population declines From 1985 through 1987 all 24 of the remaining BFF were trapped and brought into

captivity to re-initiate the captive breeding program Six ferrets in this initial group died of canine

distemper while in captivity and of the remaining 18 survivors 7 bred successfully to create the founding

population of the current captive breeding population Today this captive breeding population consists of

approximately 300 BFF distributed among multiple institutions (Santymire et al 2014)

26

Since 1986 this multi-institutional effort has been breeding BFF in captivity with reintroductions

back into the wild in 28 selected locations in Canada the USA and Mexico Currently six facilities

participate in the BFF Species Survival Plan (SSP) the Toronto Zoo USFWS National Black-footed

Ferret Conservation Center National Zoorsquos Smithsonian Conservation Biology Institute Louisville

Zoological Garden Cheyenne Mountain Zoo and the Phoenix Zoo (Black-footed Ferret Recovery

Implementation Team 2011) As of 2011 over 8000 BFF kits had been produced in captive breeding

facilities (Black-footed Ferret Recovery Implementation Team 2011)

Multiple infectious diseases pose a significant risk to the captive breeding and post-release

survival of BFF including canine distemper and sylvatic plague Coccidiosis is recognized as a cause of

significant juvenile morbidity and mortality in captive breeding programs and can result in significant

population losses (Bronson et al 2007 Santymire et al 2014 USFWS BFF Recovery Program 2017)

152 Coccidia identified from black-footed ferrets

Eimeria ictidea and Eimeria furonis have been identified in black-footed ferrets based on

morphologic criteria (Jolley et al 1994) Jolley et al examined fecal samples from six captive BFF during

a distemper outbreak as well as samples from wild BFF They described one medium-sized ovoid

tetrasporic dizoic oocyst with a double wall presence of a polar body and lacking both an oocyst residual

body and micropyle The oocysts measured 232 microm (range 182-274) by 155microm (range 130-162) with

a SI of 150 The sporocysts were elongate with the presence of both sporocyst residuum and a Stieda

body Sporozoites contained prominent refractile bodies at the posterior end and were aligned anterior to

posterior within sporocysts These oocysts were shed by all six captive ferrets On histopathology of

intestinal sections merogony and gametogony were observed within the villar epithelium throughout the

small intestine but were concentrated in the jejunum Two morphologically distinct meronts were

detected in these sections one at the villar tips which was larger and lacking in undifferentiated mass

and the other at the base of the villi or rarely in the intestinal crypts Gametogony was predominantly

27

observed at the villar tips and was noted throughout the small intestine These organisms were considered

consistent with Eimeria ictidea based on descriptions by Hoare (1927) from domestic ferrets

A second small spherical to subspherical tetrasporic dizoic oocyst was documented that had a

pink double wall a granular residual body and lacked both oocyst polar body and micropyle This

smaller oocyst measured 126plusmn12 microm (108-152) by 119plusmn09 microm (101-129) with a SI of 106 The

sporocysts were elongate with the presence of a Stieda body and sporozoites contained refractile bodies

Similar to the larger Eimeria species described above merogony and gametogony were observed within

the villar epithelium throughout the small intestine with endogenous developmental stages most

numerous in the jejunum The meronts were small with 16 or fewer merozoites Micro- and

macrogamonts were observed clustered within the apical third of the villar epithelium as were meronts

Jolley et al (1994) determined these small spherical oocysts to be consistent with Eimeria furonis as

described by Hoare (1927) from domestic ferrets

Jolley et al (1994) described a third type of coccidial oocyst occasionally detected in small

numbers within the BFF fecal samples the authors did not state whether this third type of oocyst was

recovered from wild or captive BFF The oocysts measured 370plusmn13 microm (350-386) by 223plusmn23 microm

(212-232) with a SI of 106 Attempts to sporulate collected oocysts were largely unsuccessful and

corresponding endogenous stages were not identified on histopathologic examination of necropsied

ferrets precluding further morphologic identification of the parasite It should be noted that coccidial

oocysts with similar measurements had not been detected in wild or captive prey species available for

ingestion by BFF (Jolley et al 1994)

Previous to this report coccidial oocysts had been isolated from the feces of BFF in two captive

populations (Carpenter amp Hillman 1979 Williams et al 1988) The abstract by Carpenter amp Hillman

(1979) did not describe the oocysts whereas Williams et al (1988) stated that two Eimeria species (one

with larger oocysts and one with smaller oocysts) were identified within the fecal samples but they were

28

not identified further Interestingly Williams et al reported both Eimeria species to be shed in the feces

of all ferrets concurrently affected by distemper and by approximately 30 of the clinically healthy

ferrets at the time of investigation

Non-enteric coccidia have been reported from captive BFF in one facility by two authors (Jolley

et al 1994 Williams et al 1988) Both reports presumably describing the same case(s) noted the

presence of endogenous coccidial life stages in histologic sections of respiratory tissue and merozoites of

an unidentified coccidium in impression smears of the urinary bladder from BFF diagnosed with canine

distemper Meronts were observed within the epithelium of the trachea a large bronchus and associated

bronchial glands Jolley et al (1994) described the lesions as occurring in the same ferret whereas in the

earlier report by Williams et al (1988) they are described as occurring in two different ferrets There have

been no subsequent published reports of systemic coccidiosis in black-footed ferrets and no cases have

been identified within the pathology database of the Toronto Zoo captive BFF population or by the

current SSP pathologist (Dr Michael M Garner personal communication)

There is a significant information gap regarding the pre-patent periods and pathogenicity of both

identified Eimeria species in BFF and studies to further characterize the eimeriid coccidia of the BFF are

lacking

153 Morbidity mortality and clinical signs associated with enteric coccidiosis in black-footed ferrets

The clinical signs of enteric coccidiosis in black-footed ferrets include mucoid to hemorrhagic

diarrhea abdominal discomfort lethargy appetite loss vomiting and dehydration In some cases sudden

death precedes the development of diarrhea Both adult and juvenile BFF are affected by the disease

which causes significant morbidity and mortality in captive populations (Bronson et al 2007) One

retrospective study of the captive BFF population at the Smithsonian National Zoological Park

determined that the most common cause of death in juvenile BFF (aged 30 days ndash 11 months) was

gastrointestinal pathology (524 of juvenile deaths) with 636 of these cases caused by enteric

29

coccidiosis (Bronson et al 2007) Despite the significance of this disease to the captive population its

effect on morbidity and mortality in wild BFF populations is unknown To the authorrsquos knowledge no

routine surveys of fecal parasites have been conducted on wild-born or captive released BFF during

yearly spotlighting events at ferret release sites However samples may be collected opportunistically if

fecal material is identified within the traps used to catch wild BFF during yearly surveys at release sites

Where fecal samples have been analyzed a 13 prevalence of coccidiosis has been identified in wild

born BFF (Dr Rachel Santymire personal communication) Fecal samples have been collected from BFF

at four release sites within the USA Wind Cave National Park (South Dakota) Badlands (South Dakota)

Conata Basin (South Dakota) and Aubrey Valley (Arizona) and positive samples were identified only at

the first site (Dr Rachel Santymire personal communication) Although radio-telemetry has been used at

some release sites to determine sources of mortality and factors involved in survival its use is not

widespread Furthermore the nocturnal and fossorial lifestyle of the BFF is a significant impediment to

the surveillance and monitoring of disease in this species

16 TREATMENT PREVENTION AND CONTROL OF INFECTION BY EIMERIA SPP

161 Current recommendations for treatment of eimeriid coccidia in carnivores

Described anticoccidial therapies for carnivores come from research in domestic cats and dogs

infected by Cystoisospora species these tissue coccidia (family Sarcocystidae) are only distantly related

to the Eimeria species infecting the BFF and other ferrets Current therapeutic recommendations by the

Companion Animal Parasite Council (CAPC 2013) for treatment of described Cystoisospora species

isolated from cats and dogs include the following amprolium (300-400 mg daily for 5 days in dogs 110-

200 mg daily for 7-12 days in dogs 60-100 mgkg daily for 7 days in cats) amproliumsulfadimethoxine

(150 mgkg amprolium and 25 mgkg sulfadimethoxine daily for 14 days in dogs) diclazuril (25 mgkg

for one dose in cats) furazolidone (8-20 mgkg 1-2 times daily for 5 days in dogs and cats) ponazuril (20

mgkg daily for 1-3 days in dogs and cats) quinacrine (10 mgkg daily for 5 days in cats)

30

sulfadimethoxine (50-60 mgkg daily for 5-20 days in dogs and cats) sulfadimethoxineormetoprim (55

mgkg sulfadimethoxine and 11 mgkg ormetoprim daily for 7-23 days in dogs) sulfaguanidine (150 or

200 mgkg daily for 6 days or 100-200 mgkg every 8 hours for 5 days in dogs and cats) toltrazuril (10-

30 mgkg daily for 1-3 days in dogs) trimethoprimsulfonamide (30-60 mgkg trimethoprim daily for 6

days if gt4kg 15-30 mgkg trimethoprim daily for 6 days if lt4kg) (CAPC 2013) Notably the use of all

drugs listed by the CAPC is considered off-label with the exception of sulfadimethoxine

162 Current recommendations for anticoccidial treatment and prophylaxis in domestic and black-

footed ferrets

1621 Domestic ferrets

Recommended daily oral treatment regimens for enteric coccidiosis in domestic ferrets include

amprolium (19 mgkg once daily 05 mgkg) decoquinate (05 mgkg) sulfadimethoxine (300 mgkg in

drinking water) or sulfadiazine-trimethoprim (30 mgkg once daily) all administered for a minimum of

two weeks (Bell 1994 Patterson amp Fox 2007 Patterson et al 2014) Both the aforementioned

coccidiostats amprolium and decoquinate are sold in large formats and are ideal for use in larger

operations such as breeding facilities research facilities or rescue centers Other anticoccidial therapies

used in domestic ferrets include toltrazuril (20 mgkg) and ponazuril (30-50 mgkg) once daily It should

be noted that all anticoccidial therapy used in domestic ferrets is considered off-label drug use

Multiple follow up fecal examinations should be performed after the treatment regimen is

complete and large groups may need to be treated multiple times Routine cage cleaning is also important

to decrease the environmental oocyst burden and prevent re-infection and in the case of coccidial

outbreaks ferrets should be transferred to clean cages multiple times during the course of anticoccidial

therapy Disinfectants such as bleach or quaternary ammonium compounds or dry heat should be used

for effective environmental decontamination (Patterson et al 2014)

31

1622 Species Survival Plan recommendations for black-footed ferrets

Treatment and prophylaxis of enteric coccidiosis with oral sulfadimethoxine was previously

recommended by the BFF Species Survival Plan (SSP) However due to a suspicion of decreasing

efficacy of treatment ponazuril has been recommended recently for treatment Due to the perceived

exquisite sensitivity of BFF to enteric coccidia the current SSP recommendation for treatment is oral

ponazuril at 30 mgkg once if ferrets are to be transported anesthetized stressed or are otherwise

suffering from another illness or injury (even in the absence of clinical signs or fecal shedding) The same

single oral dose of 30 mgkg is also recommended for kits at weaning (30-35 days of age) post weaning

(40-45 days of age) and prior to anesthesia for initial examination and vaccines (50-60 days of age)

Large crowded or otherwise stressed litters should be administered 30 mgkg orally once every 7-10 days

during the period of stress For treatment of coccidial diarrhea diagnosed by fecal examination 30 mgkg

orally once every 7 days for two doses or 50 mgkg orally once daily for 3 days in food (repeated in 7

days) is recommended In BFF with clinical signs of dehydration administration of subcutaneous or

intravenous fluid therapy has been performed Additional therapy with other antibiotics is sometimes

provided in cases with severe clinical signs or where secondary or primary bacterial enteritis is suspected

There is no pharmacokinetic or pharmacodynamic information available for the use of

anticoccidial drugs in BFF or other Mustelidae and thus it is unknown whether the current dose or

frequency of administration is truly appropriate for treatment of coccidiosis In 2 to 3-month-old piglets

administered a single dose of ponazuril orally at 5 mgkg peak serum concentration occurred at 42 hours

(36-48 hr) and elimination half-life was ~56 days (Zou et al 2014) In llamas administered ponazuril as

a single dose of 20 mgkg orally peak serum concentration occurred at 84 hours and elimination half-life

was ~56 days (Prado et al 2011) In domestic cows administered ponazuril as a single 5 mgkg dose

orally peak serum concentration occurred at 48 hours and elimination half-life was 58 hours (Dirikolu et

al 2009) The relevance of serum drug concentrations for treating an intestinal infection that lacks

extraintestinal life stages is likely minimal because the highest drug dose will reach the site of concern

(intestines) and systemic distribution is not required

32

Furthermore no safety or efficacy studies have been performed in any ferret species to validate

the current uses of either sulfadimethoxine or ponazuril for treatment nor have the current recommended

treatment lengths been validated However anecdotal information based on current usage would indicate

that they are safe at the current dosages and frequencies of administration as no adverse effects have been

reported A recent efficacy study in shelter dogs and cats showed that oral ponazuril (50 mgkg)

administered once daily for 3 days was effective for treatment of infection with Cystoisospora as

determined by a reduction in or cessation of fecal oocyst shedding at 4 and 8 days post treatment

Treatment efficacy in this study was inversely correlated to fecal oocyst counts at the initiation of

treatment (Litster et al 2014) Interestingly efficacy of this dose compared to the other two treatment

groups (single 50 mgkg or 20 mgkg oral dose) did not seem to differ but no statistical analysis was

performed Given the ubiquitous use of ponazuril in captive breeding facilities and concerns regarding

resistance of coccidia species to sulfadimethoxine therapy information on minimum effective doses and

dose regimes would be necessary to inform appropriate future SSP treatment and management plans and

to minimize development of drug resistance

17 VACCINES AGAINST COCCIDIA

171 Theory

The development of resistance of protozoal parasites to chemotherapeutic agents has resulted in a

shift towards the development of vaccines for the protection of domestic livestock Immunity to enteric

coccidiosis in avian and mammalian species involves both humoral and cell mediated responses Eimeria

spp infection in sheep rats poultry and other species generally results in a protective immune response

against subsequent re-infections (Catchpole et al 1993 Shi et al 2000) Interestingly this is not the case

for some host parasite interactions for example a recent report indicated that primary infection with E

ninakohlyakimovae in goat kids did not provide protective immunity against subsequent challenge with

the same parasite (Ruiz et al 2013)

33

Vaccines can be divided into four general categories live vaccines inactivatedkilled vaccines

subunit vaccines and recombinant vaccines Live vaccines are orally administered using small numbers of

infectious oocysts or oocysts from strains with low pathogenicity and result in patent but ideally sub-

clinical infections in the host that will elicit a protective immune response Such live vaccines can be

produced using attenuated forms of the pathogen of interest for example in chickens using ldquoprecociousrdquo

strains of Eimeria spp These precocious strains undergo a reduced number of merogonic replications

within the host cells and thus fewer oocysts are shed in the feces of vaccinated animals This reduction in

endogenous merogonic cycles reduces the amount of damage to the intestinal epithelium as well as

reducing the number of oocysts contaminating the environment

Another strategy has been to use live parasites with truncated life cycles An example of this is

the Toxoplasma gondii vaccine developed to prevent abortion in sheep This parasite was passaged

multiple times through a mouse host resulting in an inability to produce tissue cysts (Meeusen et al

2007) This is desirable as the cyst stage of this parasite normally inhibited by the immune system can be

reactivated during periods of stress or immunocompromise The potential drawbacks of live vaccines

include 1) the ability to produce and isolate adequate numbers of coccidial oocysts to meet vaccine

production requirements 2) the potential development of clinical disease in the host as a result of

inoculation 3) the need for all susceptible individuals to receive the vaccine simultaneously to prevent

fecal-oral inoculation of unvaccinated animals with high doses of the infective agent likely to be present

in a shared environment through fecal shedding

Inactivated vaccines are produced when the microbe of interest is killed via application of heat

radiation or chemical treatment prior to inoculation into the host species While safer because they cannot

induce disease in the inoculated patient inactivated vaccines stimulate a reduced immune response

compared with live vaccines and are consequently less effective Subunit vaccines contain single or

multiple antigens of importance in initiating the host immune response rather than the entire pathogen of

concern Subunit vaccines cannot induce disease in the immunized host but are more difficult to produce

34

because they require a detailed understanding of host immune response to infection Recombinant

vaccines involve the genetic modification of a vector (virus or bacteria) one capable of infecting the host

of interest to contain DNA of the pathogen of interest These vectors induce an immune response in the

vaccinated host but as with subunit vaccines cannot induce disease However recombinant vaccines are

again difficult to produce because they require an in depth understanding of the life cycle stages genes

and antigens targeted by the host immune response to infection There are currently no recombinant

vaccines marketed in Canada for use in veterinary medicine against protozoal disease

Creation of effective vaccines against protozoal parasites is complicated by parasite antigenic

diversity during the different life cycle stages and among protozoal species and strains of the same species

(Meeusen et al 2007) Although most parasites induce some level of immunity in their host species the

immunological response to different parasite life stages and species has been poorly characterized for

most coccidia Furthermore many parasites have developed mechanisms to evade host immune responses

or to continue survive and replicate in and transmission by previously infected hosts Our limited

understanding of the immune responses against coccidial antigens has restricted commercial vaccine

production to live or attenuated vaccines (Meeusen et al 2007)

A notable disadvantage of anticoccidial vaccines is that they need to be developed for each

coccidial species of interest because of the species-specific nature of the immune responses this is a

considerable limitation compared with anticoccidial drugs that can have a much wider spectrum of action

(Vermeulen 2005) While the requirement for mass production of vaccine is a limiting factor for vaccines

developed for the agricultural industry this drawback would be less important for production of a vaccine

to be used in an endangered species

172 Species successes in anticoccidial vaccination

The first successful immunization against coccidiosis was reported in 1918 in dogs (Hall amp

Wigdor) In this report a dog that had previously recovered from coccidial infection with Diplospora

35

bigemina was fed three increasing doses of live non-attenuated coccidial culture (at 14 32 and 48 days

post recovery from primary infection) which resulted in no development of clinical signs and no oocyst

shedding for 11mdash18 days after each challenge Subsequently immunization of dogs and cats against

coccidia with protection lasting up to seven months was reported by Andrews (1926) Immunization of

albino rats to eimeriid infection after administration of three or more sublethal doses of Eimeria

nieschulzi via gastric intubation was reported by Morehouse (1938) further experiments showed that

sporozoites did not enter the host intestinal epithelium in immunized rats given a challenge dose

(Morehouse 1938) Similar findings were reported in chickens immunized against Eimeria tenella that

had 50 fewer intra-epithelial sporozoites following challenge compared to naiumlve birds (Augustine and

Danforth 1986) Conversely chickens previously inoculated with Eimeria acervulina exhibited more

intracellular sporozoites after challenge than naiumlve birds but sporozoites were not observed to develop in

previously immunized birds (Augustine and Danforth 1986) These findings provide further evidence that

the immune response to Eimeria spp may differ among host species

Vaccination against Eimeria species has been most successful and is most widely used in the

poultry industry particularly in breeder and layer flocks Almost all vaccines marketed for poultry are

live vaccines (attenuated and non-attenuated) Vaccination against other apicomplexan parasites in

domestic mammals has also been achieved but has been generally less effective for disease prevention

and is less widely available Marketed killed and inactivated (attenuated) vaccines include those

containing killed tachyzoites of Neospora caninum for cattle (Neoguard Merck Animal Health) and

chemically inactivated merozoites of Sarcocystis neurona for horses (EPM Vaccine Fort Dodge ndash no

longer in production) A subunit vaccine for Babesia canis in dogs uses cultured antigen (Pirodog

Merial) Available live vaccines include a vaccine against Toxoplasma gondii in sheep (Ovilis Toxovax

Intervet) that uses an attenuated temperature sensitive strain (S48)

36

18 RESEARCH GOALS AND OBJECTIVES

181 Objectives

a) To determine and characterize (morphologically and molecularly) the enteric coccidial species

currently affecting the black-footed ferret population

b) To describe the natural history of enteric coccidiosis in captive black-footed ferrets including

pre-patent period shedding frequency and burdens and morbidity and mortality rates

c) To compare molecular morphologic and life history characteristics of enteric coccidial species

identified in domestic ferrets to those in black-footed ferrets

d) To validate domestic ferrets as an experimental model for intestinal coccidiosis in the black-

footed ferret

182 Hypotheses

a) Multiple Eimeria species will be isolated from the black-footed ferret population

b) The Eimeria species identified from black-footed ferrets will be the same as those previously

described in domestic ferrets

c) A single pathogenic Eimeria species will be implicated in recorded outbreaks of clinical

coccidiosis during the period of study

d) Domestic ferrets can act as an experimental model of intestinal coccidiosis for black-footed

ferrets

183 Applications

The goal of this project is to better characterize the enteric coccidia of the endangered black-

footed ferret in order to set the stage for improved disease prevention and treatment To the authorrsquos

knowledge this project is the first attempt to isolate and perform molecular characterization of the

coccidial species endemic in the black-footed ferret population This information will be used to compare

these species to known coccidia from domestic ferrets and other related mammals As experimental work

37

cannot be carried out on enteric coccidiosis in the BFF due to its endangered status if the domestic ferret

can be validated as an experimental model studies of the patterns of anticoccidial resistance and

development of immunity against Eimeria spp can be undertaken in vivo The ultimate goal would be the

development of an autogenous vaccine used to improve survival of ferret kits and reduce morbidity and

mortality associated with coccidiosis in BFF captive breeding programs Based on clinical experience

stressful life events such a breeding weaning and transfer between institutions appear to increase the risk

of coccidial outbreaks in adult BFF As such vaccination could assist in reducing disease outbreaks in

BFF associated with various management activities There is no data on the significance of coccidiosis in

wild populations and limited means of disease surveillance following release vaccination during captive-

rearing or pre-release conditioning of BFF would be an ideal method of reducing the potential effects of

this disease in released and free-living BFF Increasing the numbers of ferrets being released to the wild

and releasing ferrets immune to the subsequent threat of coccidiosis would support the goals of the

conservation initiative for the black-footed ferret

38

CHAPTER 2 MOLECULAR CHARACTERIZATION OF ENTERIC COCCIDIA FROM

DOMESTIC FERRETS (MUSTELA PUTORIUS FURO)

This chapter has been submitted for publication as

Adriana R Pastor Dale A Smith and John R Barta (2017) Molecular Characterization of Enteric

Coccidia from Domestic Ferrets (Mustela putorius furo) Vet Parasitol Regional Studies and Reports (In

review)

ABSTRACT

Combined morphometric and molecular characterization of coccidia that infect domestic ferrets

(Mustela putorius furo) was completed to improve the diagnostic specificity of enteric lsquococcidiosisrsquo in

this host Coccidia positive fecal samples (n=11) and formalin fixed paraffin embedded intestinal tissues

(n=3) from domestic ferrets were collected from diagnostic laboratories in Canada and Europe An

average of 35 and 13 domestic ferret fecal samples per year were coccidia-positive when tested by

Canadian and European diagnostic laboratories respectively during the period 2008-2015 Oocyst

morphometrics and sequence genotyping at two loci (nuclear 18S rDNA [nu 18S rDNA] and

mitochondrial cytochrome c oxidase subunit I [mt COI]) were conducted on all samples The first nu 18S

rDNA and mt COI sequences for Isospora (=Cystoisospora) laidlawi and the first mt COI sequence for

Eimeria furonis were generated during this study Phylogenetic analysis of the mitochondrial COI

sequences demonstrated that E furonis was most closely related to E cf ictidea isolated from a black-

footed ferret (Mustela nigripes) and that I (=C) laidlawi was closely related to C canis and C felis The

identifications provided by diagnostic laboratories of the specific parasite species present in a sample

showed poor agreement with their identifications based on genotyping obtained in this study Molecular

techniques appear to be essential for accurate determination of coccidial species responsible for individual

and group outbreaks of coccidiosis and for further understanding of eimeriid host-parasite relationships

Key words coccidia Cystoisospora laidlawi domestic ferret Eimeria furonis Eimeria ictidea Mustela

putorius furo

39

21 INTRODUCTION

Coccidia are host-specific parasites of the phylum Apicomplexa with greater than 2000 species

named to date (Duszynski et al 2000 Upton 2000) The eimeriorinid coccidia (suborder Eimeriorina)

include typical intestinal coccidia such as Eimeria Isospora and Cyclospora species belonging to the

family Eimeriidae as well as tissue (cyst-forming) coccidia such as Cystoisospora Besnoitia Toxoplasma

and Sarcocystis species that belong to the family Sarcocystidae (see Cox 1994)

Enteric coccidia affect both domestic ferrets (Mustela putorius furo) and their wild counterparts

In his initial descriptions of enteric coccidiosis in domestic ferrets Hoare (1927 1935b) did not observe

clinical signs of intestinal disease associated with infection More recently it has been recognized that

enteric coccidiosis can result in clinical signs ranging from mild transient diarrhea to more severe disease

with dehydration lethargy depression weight lossemaciation inappetence and death (Blankenship-Paris

et al 1993 Powers 2009 Sledge et al 2011 Hoefer et al 2012 Patterson et al 2014) Rectal prolapse

has also been reported in ferrets with enteric coccidiosis (Hillyer 1992 Hoefer et al 2012) Disease

appears to be most common in young or stressed animals In one study co-infection with coccidia and

Lawsonia intracellularis (Desulfovibrio sp) was diagnosed in 4 of 19 ferrets with proliferative bowel

disease (Li et al 1996) these ferrets presented with clinical signs including diarrhea lethargy anorexia

weight loss dehydration and emaciation Two cases of biliary coccidiosis have also been reported in

domestic ferrets infection was associated with biliary epithelial hyperplasia cholecystitis and

cholangiohepatitis (Williams et al 1996 Kaye et al 2015)

Three species of coccidia affecting domestic ferrets were originally described and named by

Hoare (1927) Eimeria ictidea Eimeria furonis and Isospora (=Cystoisospora) laidlawi The three

species were detected in feces from 50 domestic ferrets involved in an outbreak of canine distemper at a

research facility For each parasite the author described the morphology of sporulated oocysts isolated

from feces as well as sporulation time (exogenous life stages) All subsequent reports of morphologic

diagnoses of these coccidia have been based on Hoarersquos original descriptions The pre-patent period

40

(minimum duration of endogenous development) in inoculated naiumlve ferrets was described for E furonis

and E ictidea as 6 and 7 days respectively (Hoare 1935b) The pre-patent period for I (=C) laidlawi

was not determined because the number of oocysts available was insufficient for an experimental

infection trial

Hoare (1927) described the sporulated oocysts of E furonis as follows spherical double outer

wall with a thin colourless outer layer and a thick yellowish inner layer no micropyle or residual body

and measuring on average 128 times 120 microm (length [L] 112-144 width [W] 104-128 shape index [SI]

107) Sporocysts were spindle-shaped with one end constrictedblunted contained a residual body and

on average measured 8-88 times 4 microm Sporozoites were vermiform with one end narrower than the other

arranged head to tail and each had a central nucleus a clear vacuole was identified in some sporozoites at

their broad posterior end

The sporulated oocysts of E ictidea were described as follows oval or elliptical with a double

outer wall with a thin colourless outer layer and a thick yellowish inner layer no micropyle or residual

body and measuring on average 236 times 175 microm (L 184-272 W 128-208 SI 135) Sporocysts were

irregularly oval with one end broad and the other more constricted contained a residual body and

measured 115 times 65 microm on average Sporozoites were vermiform with one end narrower than the other

arranged head to tail in the sporocysts and had a central nucleus and a clear vacuole at their broad

posterior end

The sporulated oocysts of I (=C) laidlawi were ovoid with a double outer wall with a thin

colourless outer layer and a thick yellowish inner layer had no micropyle or residual body and measured

on average 34 times 29 microm (L 320-368 W 272-304) A SI of 117 can be calculated from the original

mean dimensions Two sporocysts were identified each containing four sporozoites and no Stieda body

sporocysts were elliptical contained a residual body and measured 208 times 144 microm on average

Sporozoites were sausage shaped with one end slightly pointed and had a central nucleus and a clear

41

vacuole identified at the pointed end Sporozoites were arranged with pointed ends all at the same pole of

the sporocyst

Oocysts identified as Cystoisospora ohioensis have been reported from fecal samples collected

from healthy domestic ferret kits in a large American ferret breeding operation that also housed juvenile

domestic dogs (Patterson and Fox 2007) The method of identification of this parasite was not described

A second similar institution reported the presence of Cystoisospora (=Isospora) species also thought to

be C ohioensis in routine fecal examination of their colony (Dr Bambi Jasmin personal

communication) Identification in this case was performed by the Animal Health Diagnostic Center at

Cornell University and was based on morphometrics using light microscopy The significance of these

findings is unknown but no clinical signs or histologic lesions were described in the ferrets shedding

these oocysts The definitive hosts for C ohioensis are canids including the domestic dog

More recently molecular techniques have been used for the more precise identification of

coccidia Nucleotide sequences like morphological features diverge over time under selective pressure

however recent evolutionary divergence among coccidia is more likely to be reflected in molecular as

compared to morphologic differences Thus nucleotide sequences that are more similar are inferred to be

more closely related and to have diverged more recently (Cox 1994)

Molecular characterization of ferret coccidia has only been performed for one species Eimeria

furonis Abe et al (2008) extracted DNA from oocysts from the feces of a single domestic ferret with

clinical signs resulting from coccidial enteritis Using primers initially developed for molecular

identification of Cyclospora species (see Matsubayashi et al 2005) small subunit ribosomal DNA (nu

18S rDNA) primers CYC1FE (5ʹ-TAC CCA ATG AAA ACA GTT T-3prime) and CYC4RB (5prime-CGT CTT

CAA ACC CCC TAC TG-3prime) were used to amplify a 347 base pair fragment of nu 18S rDNA The

amplicon was sequenced (GenBank AB329724) and compared with previously published partial nu 18S

rDNA sequences from 40 Eimeria two Isospora and four Cyclospora species The resulting phylogram

42

grouped E furonis with E alabamensis (cattle) and E meleagrimitis (turkey) In the same study the

microscopic morphology of the oocysts was used to identify this coccidial species as Eimeria furonis by

comparison with Hoarersquos (1927 1935b) published descriptions of Eimeria furonis and Eimeria ictidea

Sledge et al (2011) also used nu 18S rDNA to identify Eimeria furonis as the cause of three

distinct outbreaks of enteric disease in domestic ferrets Initial identification was performed using

morphometrics of sporulated oocysts collected from feces in one of the three outbreaks being

investigated Formalin fixed paraffin embedded intestinal segments from ferrets from each of the

outbreaks contained multiple coccidial life stages when examined by light microscopy PCR amplification

of a 247 base pair (bp) amplicon of the nu 18S rDNA was generated from DNA isolated from stored

formalin-fixed tissues for further genetic analysis Analysis and sequencing of amplicons from all three

groups showed 100 identity to sequences previously reported by Abe et al (2008) for the gene encoding

E furonis nu 18S

In 2015 Kaye et al identified coccidia within hepatobiliary lesions in a domestic ferret receiving

immunosuppressive therapy for red cell aplasia DNA was extracted from frozen liver and a fragment of

the nu 18S rDNA was amplified using the primers previously described by Sledge et al(2011) The

authors reported that the DNA sequence of the amplicon had 100 identity to the published nu 18S

rDNA sequence of E furonis and 95 identity to the nu 18S rDNA of E myoxi (rodent) E alabamensis

(cattle) and Isospora robini (avian) (Kaye et al 2015)

It is difficult to estimate the current prevalence of enteric coccidia within the North American

domestic ferret population and no studies have been conducted to do so Fecal samples submitted to

veterinary diagnostic laboratories from domestic ferrets in Canada are uncommon and samples positive

for coccidia appear infrequently (Dr Donald Martin personal communication) Conversely in Europe

the prevalence of coccidia within the domestic ferret population appears to be higher based on

submissions to a large veterinary diagnostic laboratory in Germany Data from Idexx Vet Med Lab in

Ludwigsburg Germany was compiled to review the prevalence of coccidia and Giardia within fecal

samples from domestic ferrets (Pantchev et al 2011) The authors reported that of 284 fecal samples

43

submitted from 2002-2004 18 (63) had detectable coccidial oocysts on fecal flotation Based on

morphologic characteristics oocysts were identified as E ictidea E furonis I laidlawi (herein referred

to as I (=C) laidlawi as noted above) and another unidentified Isospora species Comparative data from

the same laboratory from 2009-2010 included sample submissions from 253 ferrets 21 (83) of which

were positive for coccidial oocysts on fecal flotation Nine of the samples were identified as containing E

furonis three contained both E furonis and I (=C) laidlawi eight contained only I (=C) laidlawi and

one sample contained both E furonis and E ictidea identification in all cases was again based on oocyst

morphometrics No statistically significant difference in the occurrence of coccidial oocysts was detected

when data from the two periods were compared (Fisherrsquos exact test P=041) (Pantchev et al 2011)

The purpose of the present study was to perform a more detailed molecular characterization of the

coccidial species isolated from domestic ferrets to estimate prevalence of the different coccidial species

within the Canadian domestic ferret population and to associate morphologic and molecular

characteristics of a greater range of enteric coccidial species in order to improve diagnostic accuracy

22 MATERIALS amp METHODS

221 Fecal samples

Multiple diagnostic laboratories within Ontario Canada1 and a major European diagnostic

laboratory 2 were solicited for fecal samples from domestic ferrets shedding coccidial oocysts Fecal

samples were diagnosed positive for coccidia based on fecal flotation and light microscopic identification

of Eimeria or Cystoisospora species Eleven samples were collected during the study period (from 2014-

2017) and preserved in potassium dichromate (25 wv) eight from Europe and three from Canada

Centrifugal flotation with saturated salt solution (Ryley et al 1976) was used to isolate oocysts from fecal

samples for genomic DNA extraction Genomic DNA extraction and purification were performed using a

1 Animal Health Laboratory Guelph ON Antech Diagnostics Canada Ltd Mississauga ON IDEXX Canada

Markham ON 2 Vet Med Labor GmbH Division of IDEXX Laboratories Ludwigsburg Germany

44

QIAamp DNA Mini Kit (Qiagen Hilden Germany) according to manufacturerrsquos instructions After

addition of DNAzol to the samples (Qiagen Hilden Germany) samples were vortexed using 05 mm

glass beads (Biospec Products Inc Bartlesville OK USA) prior to extraction in order to fracture the

oocyst walls and release the sporocysts Concentrations of the resultant DNA were estimating using a

Nanodrop 2000 spectrophotometer (NanoDrop Products Wilmington DE USA) and stored at 4 degC for

immediate use or minus20 degC for later use

For each laboratory the number of domestic ferret fecal sample submissions numbers diagnosed

positive for coccidial oocysts and number of each coccidial species identified in positive samples were

tabulated for each of the years 2008-2015

222 Formalin fixed intestinal tissues

Major diagnostic pathology services across Canada3 were contacted to identify cases of enteric

coccidiosis identified on necropsy of domestic ferrets Cases were considered positive based on the

presence of asexual or sexual life stages of the parasites in intestinal sections The histologic sections on

each positive case were reviewed re-described and organisms measured (AP DAS) Gross necropsy

reports for all cases were also reviewed to identify any clinical correlates associated with enteric

coccidiosis DNA was extracted from ten 5-6 microm scrolls of formalin fixed paraffin embedded tissue

(FFPE) using the QIAamp DNA FFPE Tissue Kit (Qiagen) as per manufacturer instructions


Regions from the nu 18S rDNA and mitochondrial cytochrome c oxidase subunit I (mt COI)

DNA were amplified by polymerase chain reaction (PCR) from each sample using the primers listed in

Table 21 PCR amplification was performed for all samples in a volume of 25 microl containing ~100 ng of

3 Animal Health Centre Abbotsford BC Animal Health Laboratory Guelph ON Faculteacute de meacutedecine veacuteteacuterinaire

Universiteacute de Montreacuteal Saint-Hyacinthe QC Histovet Surgical Pathology Guelph ON IDEXX Canada

Markham ON Prairie Diagnostic Services Inc Saskatoon SK

45

genomic DNA 1times PCR buffer 15 mM MgCl2 02 mM deoxyribonucleotide triphosphates (dNTPs) 400

nM of each primer and 1 U of Invitrogen Platinum Taq DNA Polymerase (Thermo Fisher Scientific

Toronto ON Canada) Reactions were performed on a Bio-Rad T100 PCR thermal cycler (Bio-Rad

Laboratories Singapore) Samples were denatured and Taq polymerase activated at 95 degC for 3 min then

subjected to 35 cycles of 94 degC for 30s anneal at 50-62 degC (see Table 21 for specific anneal conditions

for the various primer pairs) for 30s and extension at 72 degC for 30-75s (see Table 21) followed by a

final extension at 72 degC for 7 min Suitable DNA (ie genomic DNA from an Eimeria or Sarcocystis sp)

was included in the PCR reactions to act as a positive control for the reaction chemistry All amplification

products were subjected to electrophoretic separation using 15 submarine agarose gel stained with

ethidium bromide and visualized on an ultraviolet transilluminator (Spectronics Corporation New York

NY USA) DNA band size was determined by comparison with a 1 kb DNA ladder (GeneRuler 1kb Plus

DNA ladder Thermo Fisher Scientific Waltham MA USA) Bands were excised with a new sterile

scalpel blade and PCR products were purified from the gel using a QIAquick Gel Extraction Kit (Qiagen)

PCR products were cycle sequenced using an ABI Prism 7000 Sequence Detection System (Applied

Biosystems Inc Foster City CA USA) by the Molecular Biology Unit of the Laboratory Services

Division University of Guelph (Guelph ON Canada) using the amplification primers to obtain

sequences in both directions The resulting chromatograms were aligned and analyzed with Geneious Ver

818 or later (Biomatters Limited Auckland New Zealand) and high quality consensus sequences

generated The resulting consensus sequences were searched from within Geneious against publically

available sequences on the BLAST server (blastncbinlmnihgovBlastcgi) using the blastn search

algorithm against the nrnt database (GenBank+EMBL+DDBJ+RefSeq ndash AA or DNA) Resultant new

nucleotide sequences were submitted to GenBank

46

224 Phylogenetic analysis

To determine the phylogenetic affinities of the newly obtained sequences with sequences from

related apicomplexan taxa representative nu 18S rDNA and mt COI sequences were downloaded from

GenBank with special reference to sequences from parasites that infect members of the order Carnivora

Nuclear 18S and mt COI sequences were aligned independently using MAFTT v7017 (Katoh et

al 2002) executed from within Geneious and then concatenated into a combined nu18S rDNA mt COI

dataset Multiple sequences from a single parasite were used to generate consensus sequences for each

locus as described by Ogedengbe et al (2017) Aligned sequences were trimmed to the length of the

largest newly generated nu 18S sequence Phylogenetic trees were generated using Bayesian Inference

(BI) using MrBayes Ver 326 (Huelsenbeck and Ronquist 2001) executed from within Geneious the

combined nu 18S and mt COI alignment was partitioned to permit locus-appropriate substitution models

to be applied to each partition For the nu 18S sequence partition the general time reversible (GTR)

substitution model (nst=6) with gamma rate variation (ie a GTR+G+I model) was applied For the mt

COI sequence partition the codon (M1) substitution model (using translation table 4 [ie lsquometmtrsquo]) was

used instead of the GTR with the remaining parameters remaining the same

The resulting tree was rooted using a pair of adeleid coccidia (Hepatozoon spp) as the taxonomic

outgroup All BI analyses were run for a chain length of 1000000 with tree sampling every 1000

following a burn-in of 100000 with default settings of 4 heated chains and heated chain temp of 02

47

23 RESULTS

231 Fresh fecal samples

From 2008-2015 inclusive the Canadian diagnostic parasitology laboratory4 received an average

of 1206 (range 81-160) domestic ferret fecal samples yearly the European parasitology laboratory5

received a yearly average of 230 samples (range 213-270) The number of fecal samples diagnosed as

positive for coccidial oocysts per year on fecal flotation during this time averaged 35 (range 0-8) and

130 (range 6-20) for the Canadian and European laboratories respectively The diagnosing laboratories

used oocyst morphometrics to identify the species of coccidia present Almost all coccidia-positive

submissions to the Canadian laboratory were identified as containing an I (=C) species based on light

microscopy Coccidia in only three samples from the Canadian laboratory were identified as E furonis

one in each of 2010 2012 and 2014 E ictidea was not identified in any samples submitted to the

Canadian laboratory Approximately equal numbers of coccidia-positive samples from the European

laboratory were identified as E furonis and I (=C) laidlawi each year Only two samples from the

European laboratory contained oocysts that were identified as Eimeria ictidea using morphometrics one

from each of 2011 and 2013 Laboratory submissions to both laboratories are summarized in Table 22

Twelve fecal samples preserved in potassium dichromate were received for analysis by the

authors Eleven samples had previously been identified as containing a single coccidial species five

containing E furonis two containing E ictidea and four containing I (=C) laidlawi A final sample had

been identified as containing a mix of E furonis and Cystoisospora canis Results of microscopic and

molecular characterization of these samples are summarized in Table 23

4 IDEXX Canada Markham ON

5 Vet Med Labor GmbH Division of IDEXX Laboratories Ludwigsburg Germany

48

232 Formalin fixed samples

Only three cases of coccidiosis were identified in domestic ferrets within the databases of the five

diagnostic laboratories that participated in the retrospective study Histologic sections of intestine were

received from these three cases which originated in Ontario6 and Quebec7 The Quebec sample (P2010-I)

was collected in 2010 and the Ontario samples (93-40404 and 17-008571) in 1993 and 2017 respectively

On gross necropsy the small intestinal contents of case P2010-I were described as pasty mucoid

yellow-brown feces with some blood For case 93-40404 the small intestines were described as empty

but melena was present within the terminal portion of the large intestine Scant intestinal contents and

dark brown fecal material in the colon were described in case 17-008571

In all cases endogenous developmental stages of coccidia were visible in histological sections

(Figure 21 is exemplary of the findings from one case) Hematoxylin and eosin stained sections from

P2010-I contained two affected regions of small intestine The intestinal mucosa of the first region

contained numerous asexual life stages and moderate numbers of sexual life stages as well as a small

number of oocysts free within the lumen The second section contained tissues that were poorly

preserved nonetheless 0-4 oocysts per 400times field were identifiable within the intestinal lumen Two

regions of affected small intestine were identified from 93-40404 after screening of all submitted sections

both contained low numbers of sexual and asexual endogenous stages Within one region there were small

numbers of meronts within the intestinal mucosa and lamina propria The second region had small

numbers of oocysts within cells of the epithelium and lamina propria as well as free within the intestinal

lumen In case 17-008571 multiple sections of jejunum contained numerous coccidian meronts gamonts

6 Animal Health Laboratory Guelph ON

7 Faculteacute de meacutedecine veacuteteacuterinaire Universiteacute de Montreacuteal Saint-Hyacinthe QC

49

and oocysts within intestinal villi within the ileum scattered epithelial cells also contained these various

life stages

Average length and width of oocysts were measured from slide sections for all cases For P2010-

I oocyst average length and width were determined from seven oocysts to be 94 microm (range 85-105) and

75 microm (range 69-84) respectively with a SI of 125 (range112-140) Average length and width of

oocysts measured from 93-40404 were determined from 5 oocysts to be 2814 microm (range 229-341) and

233 microm (range 180-308) respectively with a SI of 123 (range 110-146) For the third case 17-

008571 only 2 oocysts were identified and average length and width of oocysts measured 982 microm (range

973-992) and 845 microm (range 821-870) respectively with a SI of 116 (range 114-118)


DNA was successfully extracted from all twelve fecal samples and two cases with formalin fixed

tissue samples Attempts at amplification of DNA extracted from sample 93-40404 using the primer pairs

listed in Table 21 were unsuccessful Molecular identification results and GenBank accession numbers

for the remaining samples are summarized in Table 23 Both the nu 18S rDNA and mt COI sequences

from I (=C) laidlawi were unique when compared with available sequences from other Cystoisospora

species within the public databases However sequences from I (=C) laidlawi were most similar to

sequences from C canis and C felis and somewhat more divergent from sequences from members of the

C ohioensis species complex Two apparent genotypes of E furonis were identified based on nu 18S and

mt COI sequencing results Genotype 1 represented by EU sample 9014 had 100 identity to previously

published sequences of the nu 18S locus from two isolates from Japan (GenBank AB239130 and

AB329724) Genotype 2 represented by EU sample 907 and Canadian sample 17-008571 had 994

identity at the nu 18S locus (3 single nucleotide differences [SNDs] over 561 base pair region [bp]) to the

three sequences above belonging to E furonis genotype 1 Pairwise alignment of mt COI sequences from

both genotypes identified only 2 SNDs (996 pairwise identity over 513 bp region) Partial mt COI

50

sequences of E furonis from both genotypes were only distantly related (941 pairwise identity 30

SNDs over 513 bp and 905 pairwise identity 49 SNDs over 513 bp respectively) to publicly available

sequences from Eimeria ictidea from the black-footed ferret (Mustela nigripes) (GenBank KT203399)

and Eimeria mephitidis from the striped skunk (Mephitis mephitis) (GenBank KT203398) the only other

Eimeria species infecting members of the Carnivora for which sequence was available


A phylogenetic reconstruction based on concatenated partial nu 18S rDNA and mt COI sequences

of E furonis I (=C) laidlawi and related coccidia is illustrated in Figure 22 The combined 18SCOI-

based tree demonstrates that the two Eimeria species from ferrets form a well-supported monophyletic

group that branches among a collection of other eimeriid coccidia that infect mammals The sarcocystid

parasite of the domestic ferret I (=C) laidlawi was found to group as the sister taxon to C canis that

together formed a monophyletic group with the closely related C felis all three of these closely related

Cystoisopora species possess comparatively large egg-shaped oocysts that are similar morphologically

24 DISCUSSION

The present work has generated the first nu 18S rDNA and mt COI sequences for Cystoisospora

laidlawi and the first mt COI sequence for Eimeria furonis both isolated from the domestic ferret In this

study histologic presence of organisms and microscopic identification of oocysts shed in feces have been

correlated with published and novel nu 18S and mt COI sequences

Eimeria ictidea was not identified in any Canadian sample and this coccidium was reported in

only 2 of 1840 fecal samples submitted from across the European Union (EU) to IDEXX Germany during

2008-2015 suggesting that E ictidea is not a frequent cause of enteric coccidiosis in domestic ferrets in

Canada or the EU

51

During the study period (2008-2015) almost twice as many domestic ferret fecal submissions

were made to the European as compared to the Canadian diagnostic laboratory however the prevalence

of coccidia-positive samples was similar The methodology used in this report cannot be used to

determine the actual prevalence of enteric coccidial infection (coccidiasis) or disease (coccidiosis) within

the domestic ferret population Fecal samples may be submitted to laboratories either as a result of

investigation into enteric disease or as part of a routine health examination Thus without historic

information accompanying each sample one can simply identify the proportion of positive samples and

compare the frequency of the finding of different coccidial species Prospective surveys of fecal samples

from healthy and sick domestic ferrets with greater sample size would be necessary to determine the true

prevalence of these parasites within the population and to infer their clinical significance

Comparatively few mitochondrial COI sequences have been generated for apicomplexan parasites

compared with other genetic loci the majority of published sequences obtained from Apicomplexa are

from nu 18S The disadvantage of using nu 18S rDNA sequences for parasite identification is that they

are poor at distinguishing among closely related eimeriid coccidia due to the highly conserved nature of

the nuclear ribosomal RNA locus In contrast mt COI sequences appear to be more useful for

distinguishing closely related coccidian species (Ogedengbe et al 2011) but are less useful than nu 18S

rDNA sequences for inferring more ancient relationships among more distantly related coccidia

Consequently the combined use of nu 18S rDNA and mt COI sequencing has been recommended for

improved species description and phylogenetic analysis (El-Sherry et al 2013) For these reasons both nu

18S and mt COI sequences were analysed in the present study

Despite adequate quantities of DNA extracted from the Ontario laboratory sample (93-40404)

successful amplification did not result with any primer pair (Table 21) Potential reasons for this include

degradation of formalin-fixed DNA into fragments too small for amplification with the desired primers

perhaps as a result of extended length of time in formalin prior to paraffin embedding or length of time

stored as FFPE tissue (23 years) or insufficient parasite DNA within the paraffin scrolls The primer pairs

52

used appear to be useful for most eimeriid coccidia (Ogedengbe 2015) and successfully amplified both

Eimeria species from DNA isolated from oocysts so it is unlikely that failure to amplify DNA from this

sample resulted from an inability of the primers used to recognize the parasite seen on section

Two genotypes of E furonis were identified in this study Genotype 1 was identified only from

samples originating from domestic ferrets in Europe but exhibited 100 identity based on nu 18S

sequencing with previously published sequences from both Japan and the USA Genotype 2 was

identified from samples originating from domestic ferrets in both Canada and Europe The small number

of single nucleotide differences between the two genotypes at two genetic loci in different genomes are

consistent with intraspecific variation (ie strain variation)

As might have been expected because of their morphological and host similarities nu 18S and mt

COI sequences of E furonis were determined to be most similar to an Eimeria species (E ictidea)

previously isolated from black-footed ferrets (Mustela nigripes) these eimeriid coccidia formed a

monophyletic group that was distinct from other eimeriid coccidia infecting mammals in the phylogenetic

analyses based on combined nu 18S rDNA and mt COI sequences Similarly the nu 18S rDNA and mt

COI sequences of I (=C) laidlawi are most similar to sequences from two other Cystoisospora species of

carnivores (C canis and C felis) that both have large egg-shaped oocysts comparable to those of I (=C)

laidlawi Both morphometrics and genotyping support the close relationships among these three

sarcocystid coccidia of carnivores These molecular data confirm that transfer of Isospora laidlawi to the

genus Cystoisospora by Barta et al (2005) is warranted

The previous light microscopic identifications of coccidial species in 3 of the 11 fecal samples

were not in agreement with the molecular findings These results were not surprising because light

microscopy has been shown to be an insensitive tool for distinguishing among apicomplexan parasites at

both the genus and species level Furthermore re-evaluation of these samples by the authors revealed that

many of the samples that were identified incorrectly based on morphometrics contained primarily

53

unsporulated oocysts making accurate identification based on microscopic appearance highly

challenging These findings further underscore the importance of molecular methods in accurate parasite

identification In the absence of molecular tools accurate measurement of oocyst size shape and

determination of SI can be useful for differentiating among species of Eimeria and Cystoisospora

however this can only be performed accurately on sporulated oocysts from feces Interestingly the size

and shape indices of oocysts of E furonis measured in histologic sections did not match those previously

described by Hoare (1927) for the same oocysts in feces despite molecular confirmation of identity

Thus measurements of oocysts in histologic sections are not recommended for use in coccidial

identification

Our observations highlight the utility of molecular methods for identifying enteric coccidia

infecting domestic ferrets and suggest that diagnoses based on morphological methods should perhaps be

limited to broad determinations of disease etiology (ie lsquococcidiosisrsquo or lsquococcidiasisrsquo) Using molecular

techniques we were able to differentiate morphologically similar coccidial species isolated from the feces

of domestic ferrets and specifically identify parasites seen in histological sections of ferret intestine

Molecular techniques thus appear to be essential for determining the coccidial species responsible for

individual and group outbreaks of coccidiosis and for further understanding of eimeriid host-parasite

relationships

ACKNOWLEDGEMENTS

Many thanks to Julia Whale and Alex Leveille for their assistance and encouragement during the course

of this project The authors would like to recognize the contributions of Dr Donald Martin (IDEXX

Canada) and Drs Nikola Pantchev and Majda Globokar (IDEXX Germany) for the contributions of data

and samples to this project The authors would also like to recognize the Laboratoire de Pathologie

(Service de diagnostic Faculteacute de meacutedecine veacuteteacuterinaire St Hyacinthe Quebec) and the Animal Health

Laboratory (Guelph Canada) for contributions of samples and data to this project Finally this project

was made possible through funding by the Toronto Zoo Residency Research Fund to DASAP and partial

54

funding from a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant

(400566) to JRB

55

Table 21 Amplification primers for nuclear 18S rDNA and mitochondrial COI loci anneal temperatures (Ta) extension times and expected PCR

product sizes used in the identification of enteric coccidia from domestic ferrets (Mustela putorious furo)

Gene Target Primer Pairs Primer Sequence (5ʹ-3ʹ) Size (bp) Ta (degC) Anneal (sec) Reference

nu 18S rDNA CYC1FE TACCCAATGAAAACAGTTT 560 52 45 Matsubayashi et al (2005)

CYC4RB CGTCTTCAAACCCCCTACTG Matsubayashi et al (2005)

Cocci_18S_595F CCGCGGTAATTCCAGCTCCAAT 216 62 30 Present study

Cocci_18S_847R GCTGMAGTATTCAGGGCGACAA Present study

Lank_18S_224F TCATAGTAACCGAACGGATC 1080 54 60 Ogedengbe (2015)

Api_SSU_2733R CGGAATTAACCAGACAAATC Mathew et al (2000)

mt COI COI_10F GGWDSWGGWRYWGGWTGGAC 500 52 30 Ogedengbe et al (2011)

COI_500R CATRTGRTGDGCCCAWAC Ogedengbe et al (2011)

COI 272F CAATTCTAYGATGCCGCWTT 222 52 30 Present study


Sdae-COI_260F GATCTTTATGTTYTTRATGCC 890 50 75 Ogedengbe (2015)

Sdae-COI_1147R CATTACCCATAACYACACC Ogedengbe (2015)

56

Table 22 Summary of fecal samples from domestic ferrets (Mustela putorius furo) submitted to two diagnostic laboratories from 2008-2015

No fecal samples positive for coccidia

No samples submitted

(percentage positive)

No samples positive for

Cystoisospora sp


Eimeria furonis


Eimeria ictidea

Year Canada Europe Canada Europe Canada Europe Canada Europe

2008 3140 (21) 6214 (28) 3 2 0 4 0 0

2009 2160 (12) 14214 (65) 2 9 0 5 0 0

2010 8127 (63) 20213 (94) 7 10 1 10 0 0

2011 0114 (0) 17215 (79) 0 9 0 7 0 1

2012 3108 (28) 10231 (43) 2 4 1 6 0 0

2013 281 (25) 16270 (59) 2 13 0 2 0 1

2014 6127 (47) 12234 (51) 5 6 1 6 0 0

2015 4108 (37) 9249 (36) 4 3 0 6 0 0

Total 28 (29) 104 (56) 25 56 3 46 0 2

Average

year 35 130 31 70 04 58 00 03

Legend Numbers in brackets refer to the percent of the total number of fecal samples submitted

57

Table 23 Morphologic and molecular identification of coccidia from domestic ferrets (Mustela putorius furo)

Sample ID Source

External Lab Morphologic

Diagnosis Morphologic Diagnosis (ARP) Molecular Diagnosis

mt COI GenBank

Accession

nu 18S rDNA

GenBank Accession

93-40404 FFPE enteric coccidia Histologic sample - - -

P2010-I FFPE enteric coccidia Histologic sample E furonis Identical to MF774036 Identical to MF774678

17-008571 FFPE NP Histologic sample E furonis Same as MF774036 Same as MF774678

17-008571 feces NP E furonis E furonis MF774036 MF774678

907 feces E furonis E furonis E furonis MF774035 MF774679

938 feces I (=C) laidlawi no oocysts visualized I (=C) laidlawi MF774037 MF774677

952-A feces E ictidea Cystoisospora sp I (=C) laidlawi Identical to MF774037 Identical to MF774677

9958 feces E furonis no oocysts visualized E furonis Identical to MF774034 Identical to MF774680

9011 feces E furonis E furonis E furonis Identical to MF774035 Identical to MF774679


9017 feces I (=C) laidlawi Cystoisospora sp I (=C) laidlawi Same as MF774037 Same as MF774677

9040 feces I (=C) laidlawi no oocysts visualized I (=C) laidlawi Same as MF774037 Same as MF774677

912-260 feces I (=C) laidlawi Cystoisospora sp I (=C) laidlawi Same as MF774037 Same as MF774677

CAN-2016-1 feces C canis + E furonis Cystoisospora sp I (=C) laidlawi MF774038 MF774676

Legend FFPE = formalin fixed paraffin embedded intestinal sections - = unsuccessful = morphologic diagnosis performed by JRB same as = 100 sequence

identity with listed GenBank entry over entire sequence length identical to = 100 sequence identity but shorter sequence than listed GenBank entry

58

Figure 21 Life stages of Eimeria furonis within the small intestinal epithelium of a domestic ferret

(Mustela putorius furo) Asexual life stages merozoites (black circle) Sexual life stages oocyst

(solid black arrow) macrogamonts (open arrows with labels) microgamont (dotted black arrow)

Hematoxylin and eosin staining scale bar = 25μm

25 microm

59


(=Cystoisospora) laidlawi) from domestic (Mustela putorius furo) or black-footed (Mustela

nigripes) ferrets based on partial nuclear 18S rDNA and mitochondrial COI sequences of these

parasites and related apicomplexan parasites A summary of the sources of the molecular data for

the remaining taxa included in this phylogenetic analysis are found in Supplementary Table 1 of

Ogedengbe et al (2017) Bayesian support is indicated for each node horizontal distance is

proportional to hypothesized evolutionary change (scale indicates sequence divergence of 10)

60

CHAPTER 3 MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF

ENTERIC COCCIDIA ISOLATED FROM BLACK-FOOTED FERRETS (MUSTELA

NIGRIPES)

ABSTRACT

Black-footed ferrets (BFF Mustela nigripes) are the only ferret species native to North America

and have been identified as endangered since 1967 Starting in 1986 a multi-institutional effort has been

breeding this species in captivity with successful reintroductions back into the wild Coccidiosis is

recognized as a cause of significant juvenile morbidity and mortality in captive breeding programs and

can result in significant population losses Little is known about the etiology of enteric coccidiosis in

BFF Coccidia positive fecal samples (n=12) and formalin fixed paraffin embedded intestinal tissues

(n=11) were obtained from BFF in the Toronto Zoo and Louisville Zoo Species Survival Plan (SSP)

populations Oocyst morphometrics and sequence genotyping at three loci (nuclear 18S rDNA

mitochondrial cytochrome c oxidase subunit I and mitochondrial cytochrome c oxidase subunit III) were

conducted Results suggest that the same Eimeria species E ictidea was the cause of enteric coccidiosis

in both SSP populations in both juvenile and adult age classes Wider research is indicated to determine

whether these findings are representative of the larger captive and wild BFF populations

31 INTRODUCTION

Black-footed ferrets (BFF) are one of only three wild ferret species worldwide the other two

being the European polecat (Mustela putorius) and the Siberian polecat or steppe polecat (Mustela

eversmanii) The BFF the only native North American ferret species was formerly distributed throughout

the North America prairie ecosystem but were considered extinct by the late 1950s In 1964 a single

population was discovered in Mellette County South Dakota Progressive decline of this population in

subsequent years resulted in the decision by United States Fish and Wildlife Service to initiate a captive

breeding program for the species From 1971-1973 four females and five males were captured for this

61

purpose Despite successful breeding no kits survived and the last adult ferret in this captive colony died

in 1979 BFF were again presumed extinct in the wild based on annual surveys of the initial capture site

In 1981 a dead BFF was discovered by a ranch dog outside of Meeteetse Wyoming allowing wildlife

biologists to identify another colony of BFF This colony flourished until 1985 when an outbreak of

canine distemper in this wild BFF population and an outbreak of sylvatic plague in the local prairie dog

population resulted in sharp population declines From 1985 through 1987 all 24 of the remaining BFF

were trapped and brought into captivity to re-initiate the captive breeding program Six ferrets in this

initial group died of canine distemper while in captivity and seven of the remaining eighteen survivors

are the founding population of the current captive breeding population Today this captive breeding

population consists of approximately 300 BFF distributed among multiple institutions (Santymire et al

2014)

Since 1986 a multi-institutional effort has been breeding BFF in captivity with reintroductions

back into the wild in selected locations in Canada the USA and Mexico Currently six facilities

participate in the BFF Species Survival Plan (SSP) the Toronto Zoo United States Fish and Wildlife

Services National Black-footed Ferret Conservation Center National Zoorsquos Smithsonian Conservation

Biology Institute Louisville Zoo Cheyenne Mountain Zoo and the Phoenix Zoo (Black-footed Ferret

Recovery Implementation Team 2011) In order to provide the best genetic matches BFF are transferred

among the six institutions for breeding Approximately 300-400 kits are produced annually between the

six SSP facilities with ~200 of these kits allocated for release to the wild yearly (Santymire et al 2014)

As of 2011 over 8000 BFF kits had been produced in captive breeding facilities (Black-footed Ferret

Recovery Implementation Team 2011)


survival of BFF including canine distemper and sylvatic plague (Santymire et al 2014 USFWS BFF

Recovery Program 2017) Coccidiosis is recognized as a cause of significant juvenile morbidity and

mortality in captive breeding programs and can result in significant population losses (Bronson et al

62

2007 Santymire et al 2014 USFWS BFF Recovery Program 2017) While the effects of the disease on

the wild population are not clear a prevalence of approximately 13 has been reported based on fecal

samples collected from wild BFF born at release sites (Dr R Santymire personal commication)

Coccidia are eukaryotic host-specific parasites of the phylum Apicomplexa affecting numerous

hosts within a wide taxonomic range Two species of coccidia Eimeria ictidea Hoare 1927 and Eimeria

furonis Hoare 1927 have been identified in black-footed ferrets based on morphometrics (Jolley et al

1994) Jolley et al examined fecal samples from six captive BFF during a distemper outbreak as well as

samples from wild BFF They described one medium-sized ovoid eimeriid oocyst with a double wall

presence of a polar body and lacking both an oocyst residual body and micropyle Oocysts of this Eimeria

species (sp) measured 232 times 155 microm (range 182-274 times 130-162) with a shape index (SI) of 150 The

sporocysts were elongate with the presence of both sporocyst residuum and a Stieda body Sporozoites

contained prominent refractile bodies at the posterior end and were aligned anterior to posterior within

sporocysts These oocysts shed by all six captive ferrets were considered consistent with Eimeria ictidea

based on descriptions by Hoare (1927) On histopathologic examination of intestinal sections parasites

undergoing merogony and gamogony were observed within the villar epithelium throughout the small

intestine but were concentrated in the jejunum (Hoare 1935b) parasite life stages were not described

from other tissuesorgans

A second small spherical to subspherical eimeriid oocyst was also documented in the captive

ferrets by Jolley et al (1994) this second species had a pink double oocyst wall a granular residual body

and lacked both oocyst polar body and micropyle This smaller species measured 126 times 119 microm (range

108-152 times 101-129) with a SI of 106 The sporocysts were elongate and possessed a Stieda body and

sporozoites contained refractile bodies Similar to the larger Eimeria sp described above merogonic and

gamogonic stages were observed within the villar epithelium throughout the small intestine but were

most numerous in the jejunum Jolley et al (1994) concluded these small spherical oocysts were

consistent with E furonis described by Hoare (1927) from domestic ferrets

63


numbers within BFF fecal samples however the authors did not state whether this third oocyst

morphotype was recovered from wild or captive animals The oocysts measured 370 times 223 microm (range

350-386 times 212-232) with a SI of 106 Attempts to sporulate collected oocysts were unsuccessful and




ingestion by BFF making it unlikely that this coccidial species would have been a pseudoparasite (Jolley

et al 1994)

Previous to this report by Jolley coccidial oocysts had been isolated from the feces of BFF in two

captive populations (Carpenter amp Hillman 1979 Williams et al 1988) The abstract by Carpenter amp

Hillman (1979) did not describe the oocysts whereas Williams et al (1988) stated that two Eimeria sp

(one with larger oocysts and one with smaller oocysts) were observed within the fecal samples but they

were not identified further Interestingly Williams et al reported both Eimeria sp to be shed in the feces



Non-enteric coccidia have also been reported by two authors from captive BFF at one facility

(Jolley et al 1994 Williams et al 1988) Both reports which presumably described the same case(s)

noted the presence of endogenous coccidial life stages in histologic sections of respiratory tissue and

merozoites of an unidentified coccidium in impression smears of the urinary bladder from BFF diagnosed

with canine distemper Meronts were observed within the epithelium of the trachea a large bronchus and

associated bronchial glands In the later report Jolley et al (1994) described the lesions as occurring in the

same ferret whereas in the earlier report by Williams et al (1988) they are described as occurring in two

different ferrets Paraffin blocks containing formalin fixed tissues from these cases have since been

discarded precluding further attempts at parasite identification with molecular methods Subsequent to

64

these reports further cases of systemic coccidiosis in BFF have neither been published nor identified

within the pathology database of the Toronto Zoo captive BFF population nor by the current SSP

pathologist (Dr Michael M Garner personal communication)

Previous characterization of coccidia from black-footed ferrets has been based on host species

affected tissues in the host and morphometric characterization of life stages in histologic sections and

oocyst characteristics using light microscopy It is known that morphologically similar Eimeria species

are not necessarily conspecific and may vary in host specificity and pathogenicity Molecular

characterization is thus required to accurately identify coccidia to the species level No molecular

characterization of coccidian parasites from black-footed ferrets has been performed to date

There is a significant information gap regarding which parasite species are implicated in

morbidity and mortality events associated with enteric coccidiosis in BFF and whether different coccidia

are associated with this disease in adult versus juvenile age classes or in different SSP institutions

Studies to further characterize the eimeriid coccidia of the BFF are warranted to improve the management

of this disease in the captive population The objectives of this research were to morphologically and

molecularly characterize coccidia associated with enteric disease in BFF at the Toronto Zoo and in other

SSP facilities

32 MATERIALS AND METHODS

321 Fecal samples

Twelve fecal samples were collected during the study period (from 2014-2016) and preserved in

potassium dichromate (25 wv aqueous) seven from the Toronto Zoo and five from the Louisville Zoo

Centrifugal flotation with saturated salt solution (Ryley et al 1976) was used to isolate and concentrate

oocysts from fecal samples for light microscopic examination and genomic DNA extraction

One to two drops of the supernatant from the centrifugal flotation were placed directly on a slide

and beneath a coverslip The morphology and dimensions of sporulated oocysts were documented using a

65

Provis AX70 photomicroscope (Olympus Canada Richmond Hill ON Canada) fitted with a digital

imaging device (Infinity3-1C Lumenera Corporation Ottawa ON Canada) controlled using iSolution

Lite image analysis software (Hoskin Scientific Burlington ON Canada) operated at a total

magnification of 1000times Morphologic features noted for each oocyst included oocyst wall morphology

number of sporocysts presence or absence of a micropyle micropyle cap residual body and polar

granules For sporocysts size number of sporozoites per sporocyst and presence or absence of Stieda

body and sporocyst residuum were noted Alignment of sporozoites within the sporocyst and

presenceabsence of refractile bodies within sporozoites were also described The sporulated oocyst

length and width measurements (in microm) were then used to calculate the SI for each measured oocyst

Morphologic and morphometric features were compared to previously published values for E furonis and

E ictidea from domestic and black-footed ferrets

Genomic DNA extraction and purification were performed using a QIAamp DNA Mini Kit

(Qiagen Hilden Germany) according to manufacturerrsquos instructions as described in Chapter 2 (Materials

amp Methods)


The pathology records of the Toronto Zoo were searched from 1993-2016 for cases of BFF

diagnosed with enteric coccidiosis on histopathology For each case slides of histologic sections from all

submitted intestinal segments were reviewed to confirm the presence of sexual andor asexual life stages

within the intestinal epithelium Scrolls (5-6 microm) were cut from the paraffin blocks containing affected

intestinal sections and DNA extracted from the formalin fixed paraffin-embedded tissue (FFPE) using

the QIAamp DNA FFPE Tissue Kit (Qiagen Toronto Ontario) as per manufacturerrsquos instructions

66


Molecular characterization of coccidial isolates was performed on oocysts purified from fresh

fecal samples (isolated as described above) that were collected from juvenile and adult ferrets from

2014-2016 and DNA extracted from FFPE samples of BFF intestine containing parasite life stages

Regions from the nuclear 18S (SSU) rDNA (nu 18S rDNA) mitochondrial cytochrome c oxidase

subunit I (mt COI) DNA and mitochondrial cytochrome c oxidase subunit III (mt COIII) DNA were

amplified by polymerase chain reaction (PCR) from each sample using the primers listed in Table 31 and

methodology described in the Materials amp Methods section of Chapter 2 Table 31 also contains the

specific anneal conditions used for the various primer pairs Genomic DNA from an Eimeria species of

poultry was included in the PCR reactions to act as a positive control for the reaction chemistry A

representative selection of the newly generated nucleotide sequences resulting from the above were

submitted to GenBank

DNA obtained from oocysts collected from fecal samples during the first year of the study (2014)

was used to generate a complete mitochondrial genome (see Chapter 6 for details) using primer pairs and

sequencing primers summarized in Table 31 All subsequent samples collected in 2015 and 2016 had

shorter mt COI and mt COIII sequences obtained to permit genotyping of all collected oocysts at these

two loci The location of each primer in the nu 18S mt COI and mt COIII genetic locus is illustrated in

Figure 31

33 RESULTS

From 2014-2016 coccidia-positive fecal samples were obtained from twelve BFF ferretsferret

groups from the Toronto Zoo and Louisville Zoo SSP populations (see Table 32) Nine samples were from

single housed adults between the ages of 1-5 years (63 MaleFemale) Two samples were from mixed

groups one pooled fecal sample from four adults (FERA-1 13 MF) and one fecal sample from a family

67

group consisting of a dam and five kits (23 MF) One fecal sample was collected from a juvenile male

ferret at the time of necropsy

Eleven BFF with enteric coccidiosis were identified in the Toronto Zoo necropsy reports from

1998-2016 and all were confirmed by histological re-evaluation (Table 32) Both juvenile (n=9 36 MF)

and adult ferrets (n=2 20 MF) were represented

331 Morphometric characterization

Twelve coccidia-positive fecal samples were identified from adult and juvenile BFF from 2014-

2016 by on site laboratories at either the Toronto Zoo or the Louisville Zoo Fecal flotation and light

microscopic re-examination of the samples identified coccidial oocysts in 10 of these 12 samples

Morphometric characterization was performed on six samples in which there was adequate

quantity and quality of sporulated oocysts for examination These included three samples from single-

housed adults one from a juvenile at the time of necropsy one of pooled feces from a group of adult

ferrets and one of pooled feces from a family group (dam and kits) Two of the three samples from

single-housed adults were from the same ferret on different dates in 2016 the dates of collection were

separated by a period in which shedding of oocysts was not identified on routine repeated fecal

examinations Oocysts were elliptical with a colourless double wall and contained four sporocysts each

with two sporozoites Sporocysts were ovoid and both Stieda body and residual body were present

Sporozoites exhibited an anterior to posterior alignment within the sporocysts and refractile bodies were

identified (Figure 32) Results for length width and shape index of sporulated oocysts including range

and average values are summarized in Table 33 and Figure 32 The average measurements based on the

results of all 148 oocysts measured were length 2398 microm (1859-3057) width 1855 microm (1373-2383)

and shape index 130 (101-160)

The same measurements were performed on 59 sporocysts from a single ferret (Noodle) and

results are as follows average length 1280 microm (898-1480) average width 738 microm (505-1028) and

average SI 176 (124-247) In one sample (Mohawk-2) sporozoites were visible free on the slide

68

Measurement of three of these provided an average length of 1068 microm (1044-1117) and an average

width of 341 microm (316-393)


Molecular characterization was successfully performed on oocysts from seven of 10 fecal

samples containing coccidial oocysts and FFPE tissue from nine of the 11 necropsy cases (see Table 32)

Attempts at amplification of DNA extracted from necropsy samples Z228-98 and Z137-14 using the

primer pairs listed in Table 31 were unsuccessful Similarly attempts at PCR and sequencing of DNA

extracted from fecal oocysts from two Toronto Zoo BFF Jenna and Ruckus were unsuccessful

Molecular identification results for the remaining samples are summarized in Table 32

Only one Eimeria species E ictidea was identified in all enteric coccidiosis cases diagnosed at

necropsy in both juvenile and adult BFF at the Toronto Zoo from 1998-2014 This same species was

identified in all Toronto and Louisville Zoo BFF fecal samples that were sequenced successfully (n=8)

with the exception of a single case from Louisville This Louisville ferret was identified as having a

rodent pseudoparasite (Eimeria species) in the submitted fecal sample the eimeriid pseudoparasite had

986 sequence identity at the mt COI locus to the murine coccidium Eimeria falciformis All sequences

generated for E ictidea exhibited 100 sequence identity at the mt COI and COIII loci

Novel nu 18S rDNA mt COI and mt COIII sequences were generated for E ictidea from both

geographic locations and deposited in GenBank (Accessions MF860826 MF860827 MF860823

MF860825 MF860822 MF860824) Sequences were compared to those previously published for related

eimeriid coccidia The nu 18S rDNA sequence from Eimeria ictidea isolated from the Toronto Zoo BFF

had 9736 identity (14 single nucleotide differences) to the previously published sequences from

isolates of E furonis from domestic ferrets (Mustela putorius furo) in Japan (GenBank AB239130 and

AB329724) and newly generated sequences from Canadian and European isolates (GenBank MF774678-

MF774680 see Chapter 2 and Figure 33) In contrast nu 18S rDNA sequence of E furonis from

domestic ferrets (see Chapter 2) showed 9953 to 100 identity (0 to 3 SND) to the Japanese

69

sequences Comparison of newly generated partial sequences of the mt COI region from E ictidea from

BFF to isolates of E furonis (GenBank MF774034-MF774036) from DF and E mephitidis (GenBank

KT203398) from the striped skunk (Mephitis mephitis) the only carnivore Eimeria sp for which a mt

COI sequence was previously available reveals only 9415 and 9084 sequence identity respectively

with these other Eimeria spp of carnivores (Figure 34)

34 DISCUSSION

This work presents the first nu 18S rDNA mt COI and mt COIII sequences (nu 18S rDNA -

MF860826 MF860827 mt COI - MF860823 MF860825 mt COIII - MF860822 MF860824) generated

from an intestinal eimeriid parasite of the BFF referred to here as E ictidea collected from multiple BFF

of different ages from two separate captive populations (Toronto Zoo Toronto Ontario Canada and

Louisville Zoo Louisville Kentucky USA)

The morphometric description of coccidial oocysts from BFF in this work are consistent with

previous descriptions of E ictidea from mustelids including BFF the Steppe polecat the European

polecat and domestic ferrets (Hoare 1927 Svanbaev 1956 Jolley et al 1994) Thus I propose the name

E ictidea for the enteric coccidium described from BFF reflecting the similarity in morphology host

species and location of infection in intestinal tissues yet recognizing the absence of species identification

by molecular techniques Molecular characterization of parasites that agree with the description of E

ictidea morphologically from various mustelid host species would allow not only for determination of

whether the parasites are conspecific but would also provide insight into the potential for cross-

transmission among related mustelid hosts

DNA extraction from FFPE samples allowed successful PCR and sequencing of small DNA

fragments (220 bp) in nine of the eleven cases in which the technique was attempted Age of the samples

did not appear to be the major factor associated with successful extraction of good quality DNA the two

samples for which it was unsuccessful were the most recent (2014) and oldest (1998) casesConsequently

it may be possible to use banked FFPE tissues from historic necropsy cases from other SSP institutions

70

and necropsies of wild-born or re-introduced ferrets to determine the identity of the coccidial species

underlying disease in these cases and to better characterize the disease in the greater BFF captive and wild

populations Williams et al (1988) were contacted regarding their historic FFPE samples but formalin

blocks were no longer available for these cases and thus comparisons could not be made Banked FFPE

samples were requested from other SSP institutions however the Convention on International Trade in

Endangered Species of Wild Fauna and Flora (CITES) restrictions on the international transport of DNA

from endangered species did not allow for sample acquisition during the period in which this research was

conducted

Evaluation of FFPE samples from the Toronto Zoo indicate that the same Eimeria species has

been implicated in deaths associated with enteric coccidiosis from 1999-2014 as well as episodes of

clinical disease in ferrets in the Toronto Zoo population from 2014-2016 Samples from coccidia-positive

BFF at the Louisville Zoo in 2016 also contained the same Eimeria species Finding the same parasite at

multiple SSP locations was expected because BFF are transferred among institutions on a yearly basis for

breeding and potential release Consequently these parasites have repeated opportunities to move

between institutions in infected hosts or on contaminated cage materials to become established at a new

location Furthermore the stress of transport and transfer to a new environment may precipitate shedding

of endemic coccidia and increase the risk of a coccidial outbreak this concern is reflected in the SSP

recommendations for prophylactic treatment of all BFF with anti-coccidial medication prior to shipment

(USFWS BFF Recovery Program 2017)

A single BFF from the Louisville SSP population not showing clinical signs consistent with

coccidiosis was identified as having a rodent Eimeria species in the submitted fecal sample

Morphometric characterization of oocysts in this sample was not performed due to the paucity of visible

oocysts however examination at 100times suggested that the oocysts in the sample were ovoid in shape and

of comparable size to oocysts identified in other BFF samples The finding of a rodent Eimeria in a BFF

fecal sample is not unexpected as whole rodents comprise a significant part of the captive BFF diet The

oocysts shed by the BFF were most likely acquired through ingestion of an infected prey item and thus

71

most likely represent pseudoparasitism Molecular characterization was however required to

differentiate this from a case of true enteric coccidiasis

Reports from the first captive BFF population derived from South Dakota indicate the presence

of an unidentified species of enteric coccidium (Carpenter and Hillman 1979) in this group before its

demise in 1979 No reports containing morphometric descriptions of the coccidia from this group were

found on literature review and all parasites of this group have been lost with their hosts All subsequent

reports on enteric coccidiosis in BFF are from ferrets derived from the second founder group from

Wyoming in the 1980s The frequent transfer of ferrets among SSP institutions within the captive

breeding program and to different release sites within North America would be expected to result in the

same Eimeria species being found in all populations The exception to this would be the potential for

cross-transfer of other eimeriid parasites to wild BFF from sympatric mustelid species such as the long-

tailed weasel (Mustela frenata) Jolley et al described two other species of enteric coccidia from this

second captive population in 1994 the first was similar to E furonis of domestic ferrets and the second a

large coccidian parasite of unknown genus Neither of these parasites was identified in the Toronto and

Louisville Zoo populations during the course of this study In order to determine whether these parasites

persist within the present-day BFF populations and their impact on this species more detailed

examination of coccidia-positive fecal samples from captive and wild BFF populations is recommended

Furthermore the molecular identification of enteric coccidia from historic and future necropsy samples of

wild and captive BFF could aid in determining the presence of and contribution to mortality events by

these additional coccidia species

ACKNOWLEDGEMENTS

The authors would like to recognize the Wildlife Health Centre staff at the Toronto Zoo for their

assistance with the collection of fecal samples from the BFF from 2014-2016 The authors would also like

to recognize the Louisville Zoo for their contribution of samples to this project Finally this project was

made possible through funding by the Toronto Zoo Residency Research Fund to DASAP and partial

72

funding from a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery

Grant (400566) to JRB

73

Table 31 Amplification primers used to sequence the nuclear 18S rDNA mitochondrial COI and COIII loci of Eimeria ictidea originating from

fecal and formalin-fixed paraffin embedded tissue samples from black-footed ferrets (Mustela nigripes) including anneal temeratures (Ta)

extension times and expected PCR product sizes

Figure 31 Nuclear and mitochondrial genetic loci targeted by primers listed in Table 31 and used to characterize Eimeria ictidea originating

from black-footed ferrets (Mustela nigripes)


18SrDNA Sarco_18S_123F TATCAGCTTTCGACGGTAGTGTATT 1480 60 30 Ogedengbe et al (2016)

ERIB10_REV CTTCCGCAGGTTCACCTACGG

mt COI T_Eim_COI_272F CAATTCTAYGATGCCGCWTT 220 52 30 Chapter 2 (Table 21)

COX1-500R CATRTGRTGDGCCCAWAC Ogedengbe et al (2011)

COI-400F GGDTCAGGTRTTGGTTGGAC 800 52 60 El-Sherry et al (2013)

COI-1202R CAAKRAYHGCACCAAGAGATA El-Sherry et al (2013)

mt COIII WG-MT_4140F AGAAAACCTAAAATCATCATGT 1000 52 60 Ogedengbe et al (2015)

Eimeriid_CO3_799R AAGTGAGTTCGCATGTTTAC Ogedengbe et al (2015)

74

Figure 32 A+B) Features and cellular contents of Eimeria ictidea from a black-footed ferret

(Mustela nigripes) Legend Oocyst dotted thin white arrow = polar granule Sporocyst dotted thin

black arrow = Stieda body thick white arrow= sporozoite refractile body thick black arrow=

residuum scale bars as indicated C) Shape index length and width measurements of sporulated

oocysts of Eimeria ictidea from black-footed ferrets (Mustela nigripes) Legend times indicates the

mean Dotted oval indicates one standard deviation around the mean

10 microm 5 microm

75

Figure 33 Comparison of nuclear 18S rDNA sequence alignment of Eimeria ictidea from two black-footed ferrets (Mustela nigripes) to

newly generated (see Chapter 2) and published sequences of Eimeria furonis from domestic ferrets (Mustela putorius furo)

Figure 34 Comparison of mitochondrial cytochrome c oxidase subunit I sequence alignment of Eimeria ictidea from two black-footed

ferrets (Mustela nigripes) to sequences from other eimeriid parasites of carnivores

Identity

Eimeria mephitidis ndash KT2033981

Eimeria ictidea ndash MF860823 (Guanella ndash LZ)

Eimeria ictidea ndash MF860825 (Mystery ndash MTZ)

Eimeria furonis ndash MF774034 (Type 1)


Identity

Eimeria ictidea ndash MF860827 (Guanella ndash LZ) Eimeria ictidea ndash MF860826 (Mystery ndash MTZ)

Eimeria furonis ndash MF774680 (Type 1) Eimeria furonis ndash AB3297241 (Type 1) Eimeria furonis ndash AB2391302 (Type 1)

Eimeria furonis ndash MF774678 (Type 2) Eimeria furonis ndash MF774679 (Type 2)

76

Table 32 Morphologic and molecular characterization of coccidia from fecal and FFPE necropsy samples from black-footed ferrets (Mustela

nigripes)

Sample ID Sample Source Year Age (years) Sex Source Microscopic Description^ Molecular Diagnosis

Z228-98 Toronto Zoo 1998 7 M FFPE rare asexual stages -

Z143-99 Toronto Zoo 1999 1 M FFPE sexual and asexual stages E ictidea

Z106-02 Toronto Zoo 2002 008 F FFPE rare asexual stages E ictidea

Z108-03 Toronto Zoo 2003 008 F FFPE sexual and asexual stages E ictidea







Z137-14 Toronto Zoo 2014 021 M FFPE

fresh feces

sexual and asexual stages

POS E cf ictidea

-

E ictidea

FERA_1 Toronto Zoo 2014 gt 1 MF fresh feces POS E cf ictidea E ictidea

Noodle Toronto Zoo 2015 1 M fresh feces POS E cf ictidea E ictidea

Ruckus Toronto Zoo 2016 3 F fresh feces POS -

Mystery Toronto Zoo 2016 1 M fresh feces POS E ictidea

Mohawk Toronto Zoo 2016 1 M fresh feces POS E cf ictidea E ictidea

Jenna Toronto Zoo 2016 1 F fresh feces POS -

Thrope Louisville Zoo 2016 3 M fresh feces NEG -

FloJean Louisville Zoo 2016 2 F fresh feces NEG -

Rigatoni Louisville Zoo 2016 5 M fresh feces POS rodent Eimeria

Guanella +kits Louisville Zoo 2016 2 015 F kits 2M3F fresh feces POS E cf ictidea E ictidea

Clive Louisville Zoo 2016 1 M fresh feces POS E ictidea

Legend FFPE= formalin fixed paraffin embedded Sex MF = samples from family groups containing both sexes - = PCR and sequencing unsuccessful ^ = life stages identified on

histologic section

= mitochondrial COI andor COIII sequencing results Presence (POS) or absence (NEG) of oocysts and morphological identification of oocysts based on measurements when

77

Table 33 Morphometric (length width shape index) characterization of Eimeria ictidea oocysts from fecal samples from black-footed ferrets

(Mustela nigripes)

Sample ID FERA - 1 ^ Z137-14 Noodle Mohawk -1 Mohawk - 2 Guanella Total

Number of oocysts 12 36 32 10 21 37 148

Length (microm) 2333 (2055-2583) 2456 (2111-2848) 2505 (2079-3008) 2779 (2590-3060) 2493 (2036-2822) 2139 (1859-2372) 2398 (1859-3057)

Width (microm) 1676 (1373-2180) 1835 (1643-2232) 1975 (1509-2360) 2253 (2092-2383) 1803 (1549-2017) 1751 (1610-1888) 1855 (1373-2383)

Shape index 135 (103-160) 134 (113-156) 127 (105-155) 124 (113-138) 139 (114-154) 122 (101-145) 130 (101-160)

Legend ^= mixed adult group = dam and kit group

78

CHAPTER 4 NATURAL HISTORY OF ENTERIC COCCIDIOSIS IN THE BLACK-

FOOTED FERRET (MUSTELA NIGRIPES)

ABSTRACT

Black-footed ferrets (BFF Mustela nigripes) the only native North American ferret species are

endangered throughout their former geographic range An intensive captive breeding program produces

animals to supplement re-established wild populations Coccidial enteritis is a major cause of morbidity in

young captive ferrets but the disease also affects adults Limited information is available on the

pathogenesis of intestinal coccidiosis in captive BFF and characterization of the natural history of the

disease for improved prevention and management is imperative The objectives of this research were to

determine morbidity and mortality rates in the Toronto Zoo captive BFF population as well as

characterizing the natural history of the disease in this species through evaluation of shedding patterns

body tissues affected pre-patent period and periods of enhanced host susceptibility to infection

Coccidia-associated mortality in BFF at the Toronto Zoo from 1997-2016 averaged 053 yearly in

adults (range 0-526) and 195 in juveniles (range 0-1667) Clinical signs and histologic lesions in

Toronto Zoo BFF were similar to those described in previous publications A seasonal influence on

oocyst shedding was identified in adult BFF and ferrets appeared to maintain persistent infection with E

ictidea shedding coccidia in multiple years A larger multi-institutional study is required to better

elucidate the natural history of enteric coccidiosis in this species

41 INTRODUCTION

Black-footed ferrets (BFF Mustela nigripes) are the only native North American ferret species

and are endangered throughout their former geographic range When the last remaining truly wild

population underwent serious decline as a result of disease the decision was made by the by United States

Fish and Wildlife Service to capture the remaining 24 animals and establish a captive breeding program

this occurred between 1985 and 1987 Only seven of the captured ferrets bred successfully and are the

79

founders of the current North American BFF population (USFWS BFF Recovery Program 2017) The

captive population which now numbers approximately 300 individuals is distributed among and managed

by six collaborating facilities these include the Toronto Zoo United States Fish and Wildlife Services

National Black-Footed Ferret Conservation Center National Zoorsquos Smithsonian Conservation Biology

Institute Louisville Zoo Cheyenne Mountain Zoo and the Phoenix Zoo (Black-footed Ferret Recovery

Implementation Team 2011 Santymire et al 2014) Since 1991 BFF have been released into

reintroduced into the wild at multiple sites within their former range and over 8000 BFF kits had been

produced in captive breeding facilities as of 2011(Black-footed Ferret Recovery Implementation Team

2011) Twenty-eight BFF reintroduction sites currently exist throughout North America however there

continues to be a need to support wild populations as only a four of the re-established groups are truly

self-sustaining

Enteric coccidiosis is recognized as a cause of significant morbidity and mortality in captive

breeding programs affecting both juvenile and adult animals (Bronson et al 2007 USFWS BFF

Recovery Program 2017) Two Eimeria species Eimeria ictidea and Eimeria furonis have been

identified from cases of entric coccidiosis in BFF (Jolley et al 1994) Jolley et al examined fecal samples

from both wild and captive BFF and provided detailed morphologic descriptions of the oocysts of both

Eimeria spp as well as descriptions of the intestinal pathology associated with infection Asexual and

sexual life stages of both of the aforementioned Eimeria spp were identified on histologic section within

the villar epithelium throughout the small intestine but were concentrated in the jejunum Intestinal

sections from BFF infected with E ictidea exhibited two morphologically distinct meronts one at the

villar tips which was larger and lacking in undifferentiated mass and the other at the base of the villi or

rarely in the intestinal crypts gamogony was predominantly observed at the villar tips and was noted

throughout the small intestine

80

Extraintestinal coccidia have also been reported from captive BFF at one facility (Jolley et al

1994 Williams et al 1988) The authors identified the presence of endogenous coccidial life stages in

histologic sections of respiratory tissue and in impression smears of the urinary bladder from BFF

diagnosed with canine distemper No subsequent reports of systemic coccidiosis in BFF have been

published or identified within the pathology database of the Toronto Zoo captive BFF population or by

the current SSP pathologist (Dr Michael M Garner personal communication)

Recent investigations into the etiologic agents of enteric coccidiosis in BFF at the Toronto Zoo

have identified a single Eimeria species associated with all cases of enteric coccidiosis and associated

mortality in juvenile and adult BFF from 1999-2016 Furthermore this pathogen was identified in fecal

samples based on morphologic and molecular characterization from adult and juvenile BFF in another

zoological collection (Louisville Zoo Kentucky USA) (see Chapter 3) This coccidium is

morphologically consistent with Hoarersquos original description of E ictidea (1927) and is referred to

henceforth as Eimeria ictidea

There is a significant information gap regarding the pathogenicity of E ictidea in BFF The

objectives of this research were to determine morbidity and mortality rates in the Toronto Zoo and

additional captive BFF SSP populations as well as characterizing the natural history of the disease in this

species through evaluation of shedding patterns body tissues affected pre-patent period and periods of

enhanced host susceptibility to infection


421 Toronto Zoo BFF breeding program

At the Toronto Zoo black-footed ferret breeding program all adult ferrets are housed

individually with the exception of dams and kits After the birth of the kits dams are housed with their

offspring from whelp date until removal at approximately 4-6 months of age Routine monthly fecal

81

examinations (direct examination and flotation) are performed in house for all ferrets in the breeding

program based on SSP recommendations to evaluate for the presence of coccidia

422 Fecal oocyst evaluation

Family groups

From 2014-2016 daily fecal examination for coccidial oocysts was initiated for all group-housed

dams and kits In 2014 fecal samples were collected daily from all dams and kits from weaning (30 days

after whelping) to 72 days post whelping Based on 2014 data in 2015 this surveillance was extended

from weaning (35 days post whelping) to 135 days of age Furthermore fecal samples were collected

from the dam for an additional 14 days after removal of kits In 2016 no fecal samples were collected

from dam and kit groups at the Toronto Zoo but samples were submitted from one group of dam and kits

from another SSP population at the Louisville Zoo (Kentucky USA)

Adults

From 2015-2016 daily fecal samples were also collected from all adult ferrets identified as

shedding coccidial oocysts on their monthly routine fecal examination and from clinically ill BFF

Samples were collected for 10-14 days after initial positive sample identification In 2016 fecal samples

were also submitted from four coccidia-positive adult ferrets from the Louisville Zoo population samples

were collected for 7 days post initial identification of shedding

Individual fecal samples were analyzed via flotation using the McMaster method followed by

routine flotation in saturated salt solution (Dryden et al 2005) to determine the presence or absence of

oocysts and oocyst burden (oocysts per gram of feces OPG) Temporal trends in oocyst shedding were

monitored Coccidia-positive ferrets were evaluated visually on a daily basis for presence of clinical signs

consistent with infection Infected juvenile ferrets and adult ferrets were treated with oral ponazuril or

toltrazuril regardless of the presence of clinical signs as per the black-footed ferret SSP

recommendations Based on these recommendations ponazuril is typically administered orally at 30-50

82

mgkg once daily for 3-7 days until clinical signs have resolved or oocyst shedding has been significantly

reduced (USFWS BFF Recovery Program 2017)

423 Retrospective review of pathology records


diagnosed with enteric coccidiosis on histopathology For each case gross necropsy reports were

reviewed and slides of histologic sections from all submitted intestinal segments re-examined to confirm

the presence of sexual andor asexual life stages within the intestinal epithelium and describe the

histologic lesions associated with presence of the parasite life stages

424 Prospective modified necropsy protocol

During the study period 2014-2016 necropsy protocols for all BFF were modified to improve

detection of coccidial life stages and better to determine which portions of the intestinal tract were

affected The entire length of the intestine from duodenum to anus was measured and intestinal contents

were flushed with 12 mL of sterile saline into a sterile container Intestinal contents were preserved in

25 potassium dichromate solution (mixed 11 with intestinal contents vv) for molecular diagnostics

Paired 2-cm long intestinal samples were collected from all sections of small and large bowel duodenum

(1) jejunum (6) ileum (1) and colon (2) The eight small intestinal samples were collected at equal

distances from the pyloric sphincter to the beginning of the colon and the distance from the pylorus noted

for each Colon samples were taken at 25 and 75 of the length of the colon One sample from each

pair was preserved in Serra solution (100 ethanol (60 vv) 37 formaldehyde (30 vv) glacial

acetic acid (10 vv) and the second sample was frozen Representative tissues from all internal organs

as well as additional intestinal samples skin muscle and brain were also collected and preserved in 10

buffered formalin Histopathologic examination was performed on all tissues collected

83

425 Retrospective medical history review

Medical histories of all BFF held by the Toronto Zoo since the initiation of the SSP program were

reviewed for data on frequency of occurrence of shedding of coccidial oocysts in adults and juveniles as

well as any association of shedding with clinical signs and administration of anticoccidial treatment Data

was tabulated yearly for adult and juvenile ferrets to determine annual morbidity and mortality rates

associated with enteric coccidiosis Medical records and pathology reports were solicited from the other

SSP institutions to determine comparative morbidity and mortality rates associated with enteric

coccidiosis in BFF at other facilities Both morbidity and mortality rates were calculated as

incidenceattack rates with yearly adult population size or number of family groups (dam and kits) as the

denominator for morbidity rates and number of yearly deaths in each age class as the denominator for

mortality rates

43 RESULTS

431 Fecal oocyst evaluation and retrospective medical history review

Family groups

Fecal samples were collected from seven groups of dams and kits housed together at the Toronto

and the Louisville Zoos from 2014-2016 All data from first to last day of collection for all family groups

is listed in Appendix 1 selected pertinent data for each group is presented in Table 41 Five groups of

dams and kits were sampled in 2014 and one group in each of 2015 and 2016 Shedding occurred no

earlier than 55 days of kit-age in any of the groups and was identified from 55-81 days of age (Table 41

Table 42 Figure 41)

In 2014 fecal oocyst shedding was identified in three of the five surveyed groups In two of the

three groups (dams Poppy and Bumblefoot) changes to fecal colour and consistency were identified

concurrently with periods of oocyst shedding both groups shed higher numbers of oocysts than the other

dam and kit groups in 2014 and 2015 Both Poppy and Bumblefoot had had litters in the previous one and

84

two years prior to this study respectively based on medical record review these dams and their litters

were also diagnosed as shedding coccidial oocysts that were too numerous to count on direct exam and

fecal flotation Clinical signs in the previous years included dark tarry hemorrhagic or soft mucoid feces

and reduced appetite both groups received treatment with toltrazuril (Baycox Coccidiocide Solution

25 Bayer Inc Mississauga Canada) and trimethoprim sulfamethoxazole (Novo-Trimel Teva Canada

Ltd Scarborough Canada) (TMS) One of four kits from Poppyrsquos 2013 litter (Z113-13) died of enteric

coccidiosis three days after the group was diagnosed as shedding coccidial oocysts and the initiation of

treatment with TMS

In 2015 low grade fecal oocyst shedding (lt14 oocysts per gram of feces) without associated

clinical signs was noted in the Fiddlesticks group on three days during a seven day period from 63-69

days of kit-age and again for a single day at 128 days of kit-age The dam had been diagnosed and treated

for enteric coccidiosis in 2014 at which time she exhibited clinical signs of loose green feces to

hemorrhagic diarrhea lethargy and dehydration In 2016 she was diagnosed as shedding low numbers of

coccidia exhibited no clinical signs and did not receive treatment prior to resolution of shedding

In 2016 Guanella and kits shed oocysts over a nine day period and daily fecal oocyst shedding

ranged from 206 ndash 371714 OPG Combined treatment with ponazuril (first four days of shedding)

sulfadimethoxine injectable (first two days of shedding additional product information not available)

amoxicillin oral (first two days of shedding additional product information not available) penicillin

injectable (first two days of shedding additional product information not available) and subcutaneous

fluids (first two days of shedding additional product information not available) was administered to this

group Previous medical history was not available for this female for review

In 2014 and 2015 fecal oocyst shedding in all groups in the Toronto Zoo population started in the

three week period from the last week of July to mid-August In 2016 shedding was first identified in the

Louisville Zoo group in mid-July

85

Adults

Seven single-housed adult BFF (52 MF) were detected to have shed coccidia during the study

period (Table 43) Shedding periods lasted from 2-10 days and oocyst per gram counts ranged from 104

ndash 554274 (Table 44) Clinical signs were identified in four of the seven ferrets and consisted of

lossreduction of appetite (n=2) weight loss (n=1) lethargy (n=1) blood in feces (n=1) loose or runny

feces (n=3) soft mucoid feces (n=1) green colour of feces (n=2) Five of the seven adults received

treatment after detection of oocyst shedding two of which received treatment in the absence of clinical

signs Treatment consisted of oral toltrazuril in four cases toltrazuril in combination with trimethoprim

sulfamethoxazole in one case (Mohawk-A) and ponazuril and sulfadimethoxine (manufacturerrsquos

information not available) in one case (Clive) (Table 43)

Three of the adults in this study Mohawk Mystery and Jenna shed oocysts during multiple

different periods in 2016 Mohawk shed oocysts in May July and September of 2016 data from the first

two periods are reported in Tables 43 and 44 Mystery shed oocysts in June and July of 2016 Clinical

signs were observed only during the first shedding period and included poor appetite and hemorrhagic to

soft mucoid feces Jenna shed oocysts in July September and November of 2016 and again in February

and May of 2017 Although clinical signs were not detected in association with the initial period of

shedding in July 2016 (see Table 44) depressed mentation and hemorrhagic mucoid feces were identified

in the subsequent two shedding periods In both Mohawk and Jenna oocysts were not detected in feces on

multiple recheck and routine monthly fecal examination between shedding periods

Ruckus the fourth ferret shed low numbers of oocysts for two days in 2016 while housed alone

and had been reported to have shed oocysts during a 30 day period in 2014 while housed in a family

group with her kits Diarrhea to soft mucoid feces and loss of appetite were reported in 2014 Clinical

signs in 2016 consisted of small amounts of runny feces for a two day period two days after oocyst

shedding was no longer detected Treatment consisted of toltrazuril and TMS in 2014 and toltrazuril

again in 2016 Similarly Noodle shed low numbers of oocysts in July 2015 and had been identified as

shedding low numbers of oocysts during September of the previous year

86

The final ferret Rigatoni shed oocysts in feces in low numbers (54-732 OPG) over 5 days All

positive samples were pooled for molecular diagnostics and sequencing results showed 990 identity (7

single nucleotide differences SNDs) at the mt COI locus with a pair of rodent Eimeria species (ie

GenBank HM771682 JQ993704) (see Chapter 3) This ferret did not show clinical signs associated with

shedding and was not treated Previous medical history was not available for Clive the single adult ferret

from the Louisville Zoo

Based on sample collection dates and medical record review for the adult BFF for 2014-2016

shedding occurred during spring summer and fall with three ferrets shedding in May (Jenna Ruckus and

Mohawk) one ferret shedding in June (Mystery) three ferrets shedding in July (Mohawk Jenna Noodle)

one ferret shedding in August (Clive) two ferrets shedding in September (Noodle Jenna) and one ferret

shedding in November (Jenna)

432 Pathology


1993-2016 (Table 45) Cases were identified from 1998-2014 inclusive and all were confirmed by

histological re-evaluation (Chapter 3 Table 32) Both juvenile (n=9 36 MF) and adult ferrets (n=2 20

MF) were represented

Gross Pathology

Gross necropsy findings were similar across the 11 cases and included mucoid to fluid luminal

contents (n=7 636) beige to white pasty coating of the mucosal surface of the small intestine (n=6

545) and colon (n=4 364) gaseous dilation of intestinal segments (n=3 272) segmental enteritis

and hemorrhage (n=1 91) In one case Z228-98 no gross lesions were identified within the intestines

Impression smears of luminal contents or scrapings of intestinal mucosa were performed in four cases and

coccidia were identified in all four

Histopathology

87

Both sexual and asexual life stages were identified within the small intestinal segments in all

cases except Z228-98 and Z106-02 in which only rare asexual parasite life stages were identified (Table

45 Figure 42) Other histologic lesions seen in intestinal segments containing coccidia included

lymphoplasmacytic inflammation of the lamina propria (n=4) neutrophilic infiltration of the lamina

propria (n=2) villar necrosis (n=2) villar atrophy or blunting (n=3) and thrombi within the villar tips

(n=1)

Additional necropsy diagnoses included cholangiolar hyperplasia multiple hepatobiliary cysts

with suppuration renal adenocarcinoma apocrine gland adenocarcinoma (Z228-98) concurrent

clostridial enteritis (Z143-99) presumptive Salmonella sp septicemia (Z106-02) interstitial pneumonitis

(Z108-03) myocardial mineralization interstitial pneumonia and nephritis periportal hepatitis and

bacteremia (Z124-12) and suppurative esophagitis (Z137-14)

From 2014-2016 three black-footed ferrets were necropsied using the detailed protocol described

above Only one of the three cases Z137-14 was diagnosed with enteric coccidiosis based on

histopathology Two duodenal five jejunal two ileal and two colonic sections were collected at measured

lengths from the pylorus Parasite life stages were identified within the mucosal epithelium of all

intestinal segments extending from the distal duodenum (10-12 cm aboral to pylorus) through to the distal

colon (157-159 cm aboral from pylorus) The distal duodenal section contained asexual life stages only

with a single focus of epithelial cells containing meronts Sexual life stages (microgamonts

macrogamonts unsporulated oocysts) were identified within villar epithelial cells in all remaining

sections of the small intestine with numerous oocysts in the bowel lumen Mild lymphoplasmacytic

inflammation of the lamina propria was associated with the jejunal and ileal lesions and blunting of the

villi was identified within one jejunal segment The colonic sections contained small to moderate numbers

of sexual life stages identified within both superficial and deep crypt epithelium with occasional life

stages identified near the germinal cells Large numbers of oocysts and bacteria were identified within the

88

lumen of these colonic sections and both sections contained abscesses within the crypts The proximal

duodenum (0-2 cm) was the only section of the intestines not containing parasitic life stages

433 Morbidity and mortality

Annual morbidity rates for enteric coccidiosis at the Cheyenne Mountain Zoo and mortality rates

from enteric coccidiosis in Toronto Zoo BFF are summarized in Tables 46 and 47 During 2003-2016

yearly incidence of coccidiosis in adult BFF at the Cheyenne Mountain Zoo averaged 69 (range 0-

421) For family groups consisting of juvenile ferrets housed with their dams yearly incidence of

enteric coccidiosis averaged 115 (range 0-600)

From 1997-2016 coccidia-associated mortality in adult BFF at the Toronto Zoo averaged 053

yearly (range 0-526) with an average total mortality rate of 141 per year (range 0-526) For

juvenile ferrets (under 1 year of age) during the same period coccidia-associated mortality accounted for

an average of 133 of deaths yearly (range 0-100) with an overall average mortality rate of 170 per

year (range 0-3404) from all causes

Multiple additional SSP institutions provided partial medical and pathology data sets for use in

this study which were not sufficiently detailed to permit computation of morbidity and mortality rates for

those populations

44 DISCUSSION

The work described here supports previous clinical findings regarding the impact through both

morbidity and mortality associated with enteric coccidiosis in BFF No previous studies have been

undertaken to determine morbidity and mortality rates associated with enteric coccidiosis across BFF SSP

institutions

89

In a retrospective mortality study of captive BFF from 1984-2004 at the Smithsonianrsquos National

Zoological Park Bronson et al (2007) reported that gastrointestinal disease was the most common cause

of death in juvenile BFF (524) with 333 of juvenile mortality cases in the study caused by enteric

coccidiosis While the data is not directly comparable the findings reported here also reflect enteric

coccidiosis as a common cause of death in juveniles with increased mortality associated with the disease

compared with adult counterparts All Toronto Zoo mortalities in both juvenile and adult age classes for

which necropsy tissues were available have been attributed to infection with a single coccidia species

Eimeria ictidea (see Chapter 3)

Multiple SSP institutions provided partial medical and pathology data sets for use in this study

which were not sufficiently detailed to permit computation of morbidity and mortality rates for those

populations In future it would be useful to determine whether morbidity and mortality rates associated

with enteric coccidiosis vary among SSP institutions as this may allow for improved identification of

host parasite and environmental factors that increase risk

Clinical signs reported here are consistent with those described from both BFF and domestic

ferrets with enteric coccidiosis (Sledge et al 2011 Santymire et al 2014 USFWS BFF Recovery

Program 2017) Changes to fecal colour and consistency were the most common abnormalities identified

at the time of first detection of oocyst shedding While clinical signs in the cases described here do not

always correlate directly with the quantity of oocysts shed individuals shedding higher number of oocysts

showed clinical signs more frequently than those shedding lower numbers

In this study oocyst shedding from single-housed adult BFF ranged from 104 ndash 554274 oocysts

per gram Daily fecal samples produced from individual adult ferrets range in size from approximately 1-

16 grams In light of these findings during peak shedding from ~5times105 to 9times106 oocysts can be shed into

the environment in one day providing a massive infective dose The large numbers of oocysts shed

90

combined with confinement in a small enclosure space and hardiness of Eimeria oocysts in the

environment would be expected to markedly increase risk of infection in captive BFF

Oocyst shedding from family groups ranged from 0 ndash 371714 OPG The wide variability seen in

OPG counts between days as seen in Figure 41 and Table 41 may be accounted for by the staggered

initiation and resolution of shedding by different ferrets It is most likely that the source of infection in

these family groups is shedding by the dams some of which were identified to shed in multiple years

although environmental contamination cannot be excluded

Shedding in the adult ferrets was clustered during particular time periods specifically May July

and September If shedding is associated with stress and immunosuppression activities such as breeding

whelping electro-ejaculation of male ferrets and shipmenttransfer could act as stressors Whelping

which occurs primarily in May and June could also act as a stressor to other ferrets in the facility either

through social cues or as there would be associated changes in husbandry protocols The September

cluster could be associated with the transfer of ferrets between institutions kits are pulled during this time

and adult ferrets are moved among institutions thus changing the population dynamics of each SSP site A

large cluster of shedding was recorded in July With the exception of weaning of kits no other major

stressors are expected to occur during this time thus the increase in shedding by adults in this group

cannot be easily explained Interestingly shedding was not identified in ferrets from March through April

which is the typical breeding season and when ferrets are introduced for breeding a presumably stressful

time Shedding was not noted from December through April which could reflect reduced environmental

burdens due to low humidity levels as would be expected in a Nordic climate during the winter (which

would kill oocysts and thus block transmission) or may be consistent with reduced stress during this

period The results from single-housed adults are in contrast to the dam and juvenile ferret groups in

which oocyst shedding appeared to be correlated to a period of 55-81 days of kit age These results are

consistent with reports from other facilities of increased incidence of shedding by kits after 70 days of age


91

Retrospective and prospective review of histologic sections of intestines from affected BFF at the

Toronto Zoo showed the presence parasitic life stages in epithelial cells of both the small and large

intestines Neither Hoare (1935b) nor Jolley et al (1994) mentioned the presence of parasites in the large

intestine of experimentally infected domestic ferrets or naturally infected BFF respectively In the study

described here asexual and sexual life stages were identified within the epithelial cells of the small

intestinal villi from base to tip and were most numerous in jejunum This matches the description by

Jolley et al (1994) however Hoare (1935a b) found E ictidea to be present primarily in the villar tips

Jolley et al (1994) also described two morphologically distinct meronts of E ictidea within the small

intestinal sections one at the villar tip that was larger and lacking in undifferentiated mass and the other

at the base of the villi or in the intestinal crypts these findings were not echoed in this study as

merogonic stages were identified throughout the intestinal epithelium from villus to the base of the crypt

and no visual differences between meronts in any location were identified Hoare (1935a b) also

described a resulting annular constriction of the villus separating the affected and non-affected segments

this constriction was neither seen in the cases described here nor mentioned by Jolley et al (1994)

Whether these differences result from E ictidea from BFF and E ictidea from domestic ferrets being

different parasites or from differences in tissue tropism of a single parasite in two different hosts cannot

be ascertained from the available information

Histologic lesions such as necrosis hemorrhage villar atrophy and inflammation associated with

the presence of parasitic life stages were rare These changes are normally elicited by the host immune

system (inflammation) and the parasite (cellular rupture to release life stages resulting in hemorrhage and

necrosis) in response to infection In light of the fact that acute death occurred in a number of these ferrets

(Z113-13 Z117-13 Z118-13 Z119-13 Z137-14) in the absence of secondary disease processes and

with the intestinal epithelium intact but containing myriad parasitic life stages an alternative mechanism

for mortality associated with the infection must be proposed It is possible that these parasites elaborate

exotoxins during their life cycle and when at high density result in sudden death of the host with minimal

92

tissue changes The presence of parasitic life stages occupying the majority of both small and large

intestinal epithelial cells could also potentially impair fluid and protein movement in and out of the

mucosa however clinical signs associated with malabsorptive diarrhea were not identified in any of these

cases The presence of bacteria within the blood or other organ tissues was not identified in any cases and

consequently sepsis is unlikely to be the cause of death

Black-footed ferrets appear to maintain persistent infection with E ictidea Adult BFF in the

Toronto Zoo population shed coccidia in multiple years and in two adult ferrets multiple times in the

same year While the coccidia seen in all cases were not confirmed as E ictidea using molecular

techniques morphologic similarities and a lack of additional Eimeria spp identified on molecular work

undertaken suggest that only one species of parasite is and has been present in the collection Two dams

that had been identified as infected based on routine fecal screening in previous years presumably acted as

the source of infection to their litters of kits in multiple years While continued environmental

contamination cannot be ruled out these findings imply a failure of the immune response of the BFF to

clear infection with E ictidea or even to protect against sufficient replication of organisms to result in

clinical disease

Based on clinical experience and review of the literature BFF appear to be much more sensitive

to infection with E ictidea compared with their domestic counterparts In domestic ferrets subclinical

shedding of oocysts appears to be the most common with rare reports in the literature of overt disease

and that only in juveniles (Blankenship-Paris et al 1993 Abe et al 2008) However a single report exists

of three separate clinical outbreaks of Eimeria furonis infection in domestic ferrets under intensive

management with increased morbidity and mortality affecting all ages classes (Sledge et al 2011) The

role of genetics in the apparent increased susceptibility of BFF to enteric coccidiosis is unknown but the

current captive BFF population is derived from seven founders and inbreeding depression or familial

genetic susceptibility may play a role in their increased susceptibility to disease caused by E ictidea

93

Black-footed ferrets diagnosed with enteric coccidiosis during the course of the study were

treated with either ponazuril or toltrazuril sulfonamide drugs or often a combination of the two groups

of therapeutic agents Toltrazuril and ponazuril are triazine coccidiocides with proven efficacy against

both asexual and sexual life stages of mammalian and avian Eimeria spp (Mehlhorn and Aspock 2008)

The sulfonamides are antimicrobial drugs that exhibit coccidiostatic or coccidiocidal effects depending on

dose they act by blocking folate synthesis and have effects on first and second generation meronts

(asexual life stages) as well as potentially acting on sexual life stages (Mehlhorn and Aspock 2008)

Based on the limited data available from this study and the fact that treatment was initiated in almost all

adult BFF and family groups at the time of oocyst detection regardless of the presence of clinical disease

the effects of treatment on duration of clinical signs cannot be effectively evaluated It appears

subjectively that adult ferrets treated with toltrazuril and in one case a combination of toltrazuril and

TMS showed reduction in oocyst shedding after 3-5 days of oral anti-coccidial therapy (see Table 43)

The effects of treatment with either sulfonamides or triazines would be expected to reduce oocyst

shedding consequently the duration and amount of oocyst shedding reported in this study may not

accurately characterize the natural course of disease

Perceived resistance to sulfa drugs has been reported from multiple SSP facilities In light of this

and their potential negative effects on ferret reproduction (eg prevention of embryo implantation in the

uterus and impairment of sperm development) sulfonamides are no longer recommended by the SSP for

treatment of coccidia in this species (USFWS BFF Recovery Program 2017) The frequent and

widespread use of triazines in the management of enteric coccidiosis in BFF presents a risk for

development of resistance to this drug class in the future Neither pharmacokinetic (PK) nor

pharmacodynamic (PD) studies have been published to validate the dose and frequency of dosage in

either class of drugs in BFF and consequently it is unclear whether this perceived failure of some ferrets

to respond to treatment is based on true resistance versus inappropriate dosing The only work evaluating

ponazuril in BFF evaluated serum levels of ponazuril after a single oral dose of 50 mgkg and reported

94

therapeutic levels for 10 days after administration (USFWS BFF Recovery Program 2017) No

information was provided on number or age of ferrets that participated in the study or on how the

determination of what were therapeutic levels was made Furthermore as life cycles of the coccidia

affecting BFF are limited to the gastrointestinal tract and do not exhibit tissue stages the validity and

usefulness of assessing blood levels of ponazuril in determining appropriate dosage and dose schedules is

questionable Further work to determine the PK and PD of triazines in ferrets is warranted to provide safe

and efficacious treatment and to reduce the risk of development of resistance Furthermore the creation

and validation of a model for enteric coccidiosis in a related species would allow for in vivo studies of

drug resistance

95


(Mustela nigripes) family groups from 2014-2016

0

50000

100000

150000

200000

250000

300000

350000

400000

29

33

37

41

45

49

53

57

61

65

69

73

77

81

85

89

93

97

10

1

10

5

10

9

11

3

11

7

12

1

12

5

12

9

13

3

13

7

14

1

14

5

14

9

OP

G

Age of Kits

Poppy Bumblefoot Calico Aubrey Ruckus Fiddlesticks Guanella

96

Figure 42 Small intestinal epithelium of a black-footed ferret (Mustela nigripes) containing sexual

life stages of Eimeria ictidea Legend Solid black arrow = oocyst Hatched arrow = macrogamont

Outlined arrow = microgamont Hematoxylin and eosin staining scale bar = 25 microm

25 microm

97

Table 41 Shedding of oocysts of Eimeria ictidea in black-footed ferret (Mustela nigripes) dam and kit

family groups from 2014-2016

Collection Year 2014 2014 2014 2014 2014 2015 2016

Age of kits (days) Poppy Bumblefoot Calico Aubrey Ruckus Fiddlesticks Guanella^

29 - - - 0 - - -

30 - 0 - 0 - - -

- - - - -

34 0 0 - 0 0 - -

35 0 0 - 0 0 0 -

- -

46 0 0 - 0 0 0 -

47 0 0 - 0 0 0 -

48 0 lt14 - 0 0 0 -

- -

53 0 0 - 0 0 0 +

54 0 0 - 0 0 0 +

55 0 0 - 0 0 0 3717146

56 0 0 - 0 - 0 1084436

57 0 0 - 0 - 0 64133

58 0 324 - 0 0 0 20654

59 0 0 - 0 0 0 0

60 0 446688 - 0 0 0 0

61 0 934828 - 0 0 0 41111

62 0 530469 - 0 0 0

63 0 1617131 - 0 0 lt14

64 0 8396357 - 114 0 0

65 0 234876 - 5368 0 lt14

66 0 374625 - 9455 0 0

67 lt14 2311575 - 39579 0 -

68 0 603563 - 125051 0 0

69 - 4103036 - 27067 0 lt14

70 2470327 7759974 - 0 0 0

71 1073085 - 1784 0 0

72 3430966 - 0 0 0

73 4459536 - 7326 0 0

74 4842212 - 2 0 0

75 3627307 - 0 0 0

76 5801885 - 0 0 0

77 5994 0 0 0 0

78 589835 0 0 0

79 24815 0 0 0

80 144016 0 0 0

81 97862 0 0 0

82 0 0 0

127 0 0

128 0 2843

136 0 0

150 0

Numbers of Oocysts Shed (oocysts per gram of feces)

Dam Identity

Legend lt 14 = oocyst positive samples with less than 14 oocyst per gram of feces - = no sample recorded for this date underline = last

sampling date + = coccidia present but OPG count not performed = Toronto Zoo ferret ^ = Louisville Zoo ferret thick outer border =

days treatment was received = range of sequential dates between previous and subsequent number during which OPG counts were

performed and samples contained 0 oocysts

98

Table 42 Summary of epidemiologic data for family groups of black-footed ferrets (Mustela nigripes)

shedding oocysts of Eimeria ictidea

Dam Identity

Poppy

2014

Bumblefoot

2014

Aubrey

2014

Fiddlesticks

2015

Guanella

^

2016

Number of kits 7 5 3 4 5

Kit age (days) at time of

shedding 70-81 58-70 64-73 63-69 54-61

Shedding period (days) 15 13 11 7 9

OPG min 0 0 0 0 206

OPG max 48442 83963 2707 lt14 371714

Clinical signs YES YES NO NO NO

Treated - YES YES NO YES

Legend = Toronto Zoo ferret ^ = Louisville Zoo ferret OPG = oocysts per gram of feces

- = missing data

Table 43 Shedding of oocysts of Eimeria ictidea in single-housed adult black-footed ferrets (Mustela nigripes) 2015-2016


Collection

Year 2015 2016 2016 2016 2016 2016 2016

Ferret Identity Noodle Ruckus Mohawk-A Mohawk-B Mystery Jenna Clive^

Age (years) 1 3 1 1 1 1 1

1084058 0 + + + + +

+ 0 42307650 6286676 + 183150 554274

857808 16650 12805238 7777929 + 215710 377920

1604894 16650 309690 139860 + 0 25808

377042 0 599400 119880 + - 37294

554445 0 34688 385579 117920 0 5363

26640 0 16650 0 0 0 7500

0 10406 0 0 0 1090

0 20813 0 0 0

- 0 1761 0

0 0 0 0

- 0

0

0

Legend lt 1 = oocyst positive samples with less than 1 oocyst per gram of feces underline = last sampling date + = coccidia present but OPG

count not performed = Toronto Zoo ferret ^ = Louisville Zoo ferret thick outer border = days treatment was received

100

Table 44 Summary of epidemiologic data for single housed adult black-footed ferrets (Mustela nigripes) shedding oocysts of Eimeria ictidea

Noodle Ruckus Mohawk-A Mohawk-B Mystery Jenna Clive^

Sex M F M M M F M

Age (years) 1 3 1 1 1 1 1

Shedding period (days) 7 2 9 6 10 4 8

OPG min 266 166 104 1199 1761 1831 1090

OPG max 10840 166 423076 77779 - 2157 554274

Clinical signs YES YES NO NO YES NO YES

Treated NO YES YES YES YES YES YES

Legend = Toronto Zoo ferret ^ = Louisville Zoo ferret M = male F= female OPG = oocysts per gram of feces - = missing data

NOTE Mohawk-A and Mohawk-B refer to two separate episodes of oocyst shedding by the same ferret

101

Table 45 Histologic findings from necropsies of black-footed ferrets (Mustela nigripes) with enteric coccidiosis

Number of Sections Affected

Ferret

ID Year

Age

(years) Sex Coccidia in Intestinal Sections Small Intestinea Large Intestinea

Z228-98 1998 7 M rare asexual stages S - 01 A - 11 S - 01 A - 01

Z143-99 1999 1 M sexual and asexual stages S - 24 A - 24 S - 03 A - 03

Z106-02 2002 008 F rare sexual stages S - 14 A - 04 S - 01 A - 01

Z108-03 2003 008 F sexual and asexual stages S - 12 A - 12 S - 01 A - 01


Z124-12 2012 021 F sexual and asexual stages S - 24 A - 04 none






Legend a= xn where x is number of sections containing sexual or asexual lifestages n is the number of sections examined S = sexual life

stages A= asexual life stages

102

Table 46 Yearly incidence of coccidial infection in black-footed ferrets (Mustela nigripes) at the

Cheyenne Mountain Zoo


Year Adult Family

2003 116 (625) -

2004 819 (4211) -

2005 021 (000) 14 (2500)

2006 021 (000) 07 (000)

2007 023 (000) 08 (000)

2008 224 (833) 14 (2500)

2009 025 (000) 06 (000)

2010 326 (1154) 07 (000)

2011 125 (400) 09 (000)

2012 125 (400) 08 (000)

2013 028 (000) 05 (000)

2014 430 (1333) 09 (000)

2015 035 (000) 35 (6000)

2016 - 27 (2857)

Mean annual () 689 1155

Legend - = missing data xn= where x is the number of ferrets shedding coccidial oocysts and n is the

total number of adult ferrets or family groups in a given year () = incidence expressed as a percentage

103

Table 47 Yearly mortality rate and incidence of mortality associated with coccidial infection in

black-footed ferrets (Mustela nigripes) at the Toronto Zoo

Total Mortality

Coccidia Other Causes

Year Kit Adult Kit Adult

1997 015 (000) 023 (000) 315 (2000) 423 (1739) 1998 038 (000) 119 (526) 838 (2105) 919 (4734) 1999 047 (000) 119 (526) 1647 (3404) 119 (526) 2000 034 (000) 015 (000) 434 (1176) 315 (2000) 2001 032 (000) 016 (000) 532 (1563) 116 (625) 2002 150 (200) 020 (000) 450 (800) 220 (1000) 2003 227 (741) 018 (000) 327 (1111) 118 (555) 2004 020 (000) 016 (000) 620 (3000) 216 (1250) 2005 016 (000) 015 (000) 416 (2500) 215 (1333) 2006 030 (000) 016 (000) 230 (667) 016 (000) 2007 019 (000) 015 (000) 419 (2105) 215 (1333) 2008 034 (000) 016 (000) 1134 (3235) 316 (1875) 2009 017 (000) 016 (000) 017 (000) 116 (625) 2010 017 (000) 016 (000) 317 (1765) 316 (1875) 2011 011 (000) 016 (000) 111 (909) 216 (1250) 2012 111 (909) 017 (000) 111 (909) 317 (1765) 2013 424 (1667) 017 (000) 424 (1667) 317 (1765) 2014 126 (384) 017 (000) 326 (1154) 217 (1176) 2015 04 (000) 017 (000) 04 (000) 217 (1176) 2016 011 (000) 017 (000) 211 (1818) 117 (588)

Mean annual () 195 053 1594 1359

Legend xn= where x is the number of ferrets that died with coccidial infection or of other

causes and n is the total number of adult ferrets or kits in a given year () = incidence expressed

as a percentage

104

CHAPTER 5 EVALUATING THE DOMESTIC FERRET (MUSTELA PUTORIUS FURO) AS

AN EXPERIMENTAL MODEL FOR ENTERIC COCCIDIOSIS IN THE BLACK-FOOTED

FERRET (MUSTELA NIGRIPES)

ABSTRACT

The purpose of this study was to determine whether the domestic ferret (Mustela putorius furo) is

susceptible to an isolate of Eimeria ictidea originating from black-footed ferrets (BFF Mustela nigripes)

and thus could act as a suitable experimental model in which to investigate the pathogenesis and

management of this disease A pilot study was performed with 10 male intact juvenile domestic ferrets

Ferrets were administered an oral inoculum containing either a high dose (1 times 106 oocysts) moderate

dose (5 times 104 oocysts) or saline control and observed for shedding of oocysts and development of

clinical signs Seven of ten ferrets developed patent infection all of which had received the high dose

inoculum The prepatent period was 7-9 days and duration of shedding varied from 1-7 days Clinical

signs were identified in six of the seven infected ferrets and were consistent with those previously

described for enteric coccidiosis in domestic and BFF Parasite life stages were identified within the

intestines of four of the seven ferrets with patent infection and were limited to the distal jejunum and

ileum The demonstrated ability to produce patent infections in domestic ferrets following oral inoculation

of a high dose of E ictidea (1 times 106 oocysts) isolated from BFF provides an avenue for future

experimental investigations into the control and treatment of enteric coccidiosis in this endangered

species

51 INTRODUCTION

Black-footed ferrets (BFF Mustela nigripes) are one of only three ferret species worldwide

While formerly distributed throughout the North American prairies black-footed ferrets were declared

extinct in the wild in the 1980s Since 1986 a multi-institutional consortium has been breeding this

species in captivity with reintroductions back into the wild within their historic range in selected locations

in Canada the USA and Mexico Introduced colonies of BFF are present in Arizona Colorado Kansas

105

Montana New Mexico South Dakota Utah Wyoming and Chihuahua (Mexico) Reintroduction

attempts in Saskatchewan Canada have been unsuccessful to date


survival of BFF including distemper and sylvatic plague (Santymire et al 2014 USFWS BFF Recovery

Program 2017) Coccidiosis is a recognized cause of juvenile and adult morbidity and mortality in captive

breeding programs and can result in significant losses (Bronson et al 2007 Santymire et al 2014

USFWS BFF Recovery Program 2017) The effect of the disease on wild populations is unknown

Clinical signs of coccidiosis include mucoid to hemorrhagic diarrhea abdominal discomfort lethargy

appetite loss vomiting and dehydration Recent investigations into diseases affecting BFF at the Toronto

Zoo have identified a single Eimeria species E ictidea associated with all cases of enteric coccidiosis in

juvenile and adult BFF from 2014-2016 (see Chapter 3) This same Eimeria species was identified

retrospectively as the cause of juvenile and adult mortalities in previous years (1999 through 2014

inclusive) (Chapters 3 and 4) Furthermore this pathogen was identified in fecal samples based on

morphologic and molecular characterization from adult and juvenile BFF in another zoological collection

(Louisville Zoo Kentucky USA) (see Chapter 3)

Enteric coccidiosis also occurs in domestic ferrets (Mustela putorius furo) with three

morphologically distinct species of coccidia Eimeria ictidea Eimeria furonis and Isospora (=

Cystoisospora) laidlawi Both of the aforementioned Eimeria species have been identified in black-footed

ferrets based on morphologic criteria but molecular characterization was needed to confirm whether the

same species of parasite infects both ferret species (see Chapter 2 and 3) To this end nuclear and

mitochondrial sequences for E furonis and for I=(C) laidlawi were generated (Chapter 2) expanding the

existing limited sequence data from the nuclear 18S rRNA locus of Eimeria furonis Molecular

characterization of E ictidea from domestic ferrets was not possible because samples containing this

parasite were not available for study consequently it is unclear whether the same coccidium affects both

domestic and black-footed ferrets

106

There is no published information describing the pre-patent periods and pathogenicity of enteric

coccidia in BFF and given the conservation status of the BFF experimental work cannot be undertaken

in the natural host The purpose of this study was to determine whether the domestic ferret is susceptible

to E ictidea isolated from BFF if susceptible the domestic ferret could act as a suitable experimental

model in which to investigate the pathogenesis prevention and treatment of coccidiosis caused by E

ictidea


521 Animal care

Ten juvenile male intact ferrets of 48 (n=6) or 50 (n=4) days of age were obtained from a

commercial source (Marshall BioResources North Rose New York USA) and were housed in the

University of Guelph Central Animal Facility Isolation Facility Ferret weights on arrival ranged from

334-475 g (average= 3928 g) All ferrets were housed individually in wire bottom cages of 813 times 1117

times 457 cm size and were divided in equal numbers between two non-adjoining rooms They received ad

libitum access to Envigo Teklad Certified Global Ferret Diet (Madison Wisconsin USA) and water

changed daily Room temperature was maintained at 18-21 degC and a 16 hour light 8 hour dark

photoperiod was provided All personnel working with the ferrets were required to wear personal

protective equipment including disposable facemasks gloves gowns and bouffant caps Shoes were

provided for use in each room This study was carried out in accordance with the recommendations in the

Canadian Council on Animal Care guidelines The protocol was approved by the Animal Care Committee

of the University of Guelph (Animal Use Protocol 3289) and by both the Animal Welfare Committee

and Animal Care and Research Committee of the Toronto Zoo

An initial physical examination and blood collection were performed on each ferret by the

principal investigator (ARP) one day after arrival to assess health status prior to enrollment in the study

Ferrets were mask induced with isoflurane (Isoflurane USP Fresenius Kabi Richmond Hill Ontario) in

107

oxygen placed on a heat disc (SnuggleSafe Lenric C21 Ltd Littlehampton United Kingdom) weighed

examined and blood was collected from the jugular vein for routine CBC and biochemical profiles

All ferrets subsequently underwent an acclimation period of two weeks During this time fecal

samples were collected daily from each ferret and examined for the presence of coccidial oocysts using a

standard salt flotation technique (Dryden et al 2005) to ensure that all individuals were free of coccidia

prior to initiation of experimental work Any ferret positive for coccidia was to be removed from the

study

522 Oocyst preparation

Oocysts used for inoculation originated from fecal samples from two naturally infected BFF These

samples were stored in potassium dichromate for four weeks prior to oocyst purification and use in this

infection trial Stored fecal samples were mixed with distilled water and passed through a small sieve to

remove debris The strained contents were transferred to a 50 mL conical vial and topped up to 50 mL

with additional distilled water Samples were centrifuged (Sorvall ST40R Centrifuge Thermo Scientific)

at 2800 rpm (1315 timesG) for 10 minutes at 12 degC A drop of supernatant was evaluated microscopically at

100times for the presence of oocysts If oocysts were observed the supernatant was poured off into a second

50 mL conical tube and again topped up to 50 mL with distilled water and re-centrifuged under the same

conditions Otherwise the supernatant was discarded The pellets from both the first and second tubes

were combined with saturated salt solution at a 14 ratio by volume Oocysts were floated in the salt

solution by centrifugation at 1500 rpm (377 timesG) for 10 minutes at 12 degC The top 5 mL of supernatant

were collected and transferred to a clean 50 mL conical tube topped up to 40 mL with distilled water and

washed via centrifugation at 2800 rpm (1315 timesG) for 10 minutes After the wash step the supernatant

was again checked for presence of oocysts and discarded if no oocysts were observed The pellet was

collected and the presence of oocysts confirmed by examination of a drop placed on a clean glass slide at

100times Once verified the contents of the pellet of concentrated oocysts was placed in a 250 mL storage

container and mixed with approximately 200 mL of sterile saline (09 sodium chloride Hospira

108

Montreal Quebec) prior to storage for two to four weeks at 4 degC until inoculation Prior to inoculation a

McMaster count was performed to determine the number of oocysts per mL in order to determine

appropriate volume of inoculum

523 Experimental infections

Part 1

Five ferrets were randomly assigned to each of the control and infection groups After the

acclimation period on day 0 four ferrets in the infection group were inoculated orally with a high dose

oocyst suspension (1 times 106 oocysts in 025 mL of saline) mixed into 1 mL of FerreTone Skin amp Coat

Supplement (United Pet Group Inc Blacksburg Virginia USA) a fifth ferret was inoculated with a

moderate dose oocyst suspension (5 times 104 oocysts in 025 mL of saline) in the same volume of FerreTone

Ferrets in the control group were inoculated with a placebo (025 mL of saline) in 1 mL of FerreTone

Inoculation was performed by offering the oocyst suspension or placebo to the ferrets in a plastic

container

Fecal samples were collected daily from each inoculated ferret for 14 days post-inoculation

Samples were analyzed via fecal flotation using the McMaster method followed by routine flotation in

saturated salt solution (Dryden et al 2005) to determine the presence or absence of oocysts and oocyst

burden (oocysts per gram of feces [OPG])Temporal trends in oocyst shedding were monitored Ferrets

were evaluated visually twice daily for the presence of clinical signs of coccidial disease The first of

every two ferrets identified to shed oocysts was to be humanely killed at the time of peak oocyst shedding

(ie the first day that fecal oocyst counts remained static or declined) and necropsied to confirm the

presence of and describe parasitic replication in the intestinal mucosa Any remaining animals that shed

oocysts were to be monitored throughout the 14 day period following inoculation in order to determine

the duration and intensity of oocyst shedding for these individuals the total number oocysts shed during

patency was determined

109

Part 2

All ferrets from the infection group that did not shed oocysts during Part 1 (n=4) and all but one

ferret from the previous control group (n=4) were orally inoculated with the high dose oocyst suspension

(1 times 106 in 1 ml of saline) mixed with an equal volume of FerreTone Consequently between phases 1

and 2 all but one ferret were inoculated at least once with the BFF coccidia in order to increase

experimental animal numbers and determine if ferret age played a role in susceptibility to infection

One ferret from the previous control group was inoculated with a lower dose (2 times 105 oocysts in

075 mL of 09 saline mixed with 1 mL FerreTone) of oocysts that had been collected from the single

domestic ferret that shed in Part 1 oocysts were purified as described above for the initial inocula Fecal

collection and analysis were performed as previously As in Part 1 one in every two ferrets sequentially

identified to be shedding oocysts in feces was killed humanely at the time of peak shedding and a

complete necropsy examination performed The remainder of the ferrets observed to be shedding were

monitored for the full 14 days of the trial after which they were killed humanely and necropsied and total

number of oocysts shed during patency was determined All ferrets that did not shed coccidial oocysts

during the infection trial were rehomed at the end of the trial

524 Animal welfare

Ferrets were evaluated twice daily for development of clinical signs of coccidial disease and any

animal showing clinical disease was to be treated as determined by a veterinarian with supportive care

including fluid therapy A grading system for clinical signs including intervention points and removal

criteria was created for use during daily evaluation (see Appendices 3 and 4) Animals whose clinical

signs could not be ameliorated without the use of specific anticoccidial therapy were to be euthanized

Should the inoculation in Part 1 have resulted in clinical disease that required extensive treatment andor

necessitated euthanasia a lower number of oocysts would be used for subsequent inoculation in Part 2

Ferrets to be euthanized were anesthetized by mask induction with isoflurane in oxygen a 1 mL blood

110

sample was collected from the cranial vena cava and then an intracardiac dose of potassium chloride (2

mEqkg) was administered

525 Hematology

Blood was collected from all ferrets under isoflurane anesthesia at the time of pre-trial health

examination and again at the time of humane killing Blood was collected via jugular venipuncture

initially due to the small size of the ferrets at arrival and then by cranial vena cava venipuncture or

cardiocentesis prior to euthanasia Complete blood count and serum biochemistries were performed by the

Animal Health Laboratory of the University of Guelph Guelph Ontario

526 Morphologic and molecular characterization

Morphologic and molecular characterization of oocysts shed by the domestic ferrets during the

course of the infection trial was performed to ensure that the ferrets were shedding the same species of

Eimeria with which they were inoculated Oocysts were concentrated from positive fecal samples as

described above A drop of concentrated oocyst solution was viewed photographed and measured at

400times and 600times for comparison with previously determined morphometrics of Eimeria ictidea oocysts

(Chapter 3)

Regions from the mitochondrial cytochrome c oxidase subunit I and III (mt COI and mt COIII)

DNA were amplified by polymerase chain reaction (PCR) from each sample using primer pairs

400F1202R and -172F799R respectively For all PCR reactions samples were denatured at 95 degC for 5

min then subjected to 35 cycles of 94 degC for 30s anneal at 52degC for 30s and extension at 72 degC for 60s

followed by a final extension at 72 degC for 7 min PCR gel electrophoresis and sequencing methods used

were as described in the Materials amp Methods section of Chapter 2

The resulting consensus sequences were searched from within Geneious against previous

sequences for E ictidea produced by the authors and against publically available sequences on the

111

BLAST server (blastncbinlmnihgovBlastcgi) using the blastn search algorithm against the nrnt

database (GenBank+EMBL+DDBJ+RefSeq ndash AA or DNA)

527 Necropsy protocol

All humanely killed ferrets underwent a complete necropsy (Appendix 6) using the modified

protocol described in Materials amp Methods section 424 of Chapter 4

53 RESULTS

Initial physical examination was unremarkable with the exception of mild to moderate bilateral

ceruminous discharge within the external ear canal of all ferrets Complete blood count and serum

biochemistry results for all ferrets were within normal reference intervals for juvenile domestic ferrets

(Appendices 2a and b) (Fox 2014) Six days after arrival a single ferret (103) in the control group

developed mild upper respiratory signs consisting of sneezing and clear nasal and ocular discharge these

clinical signs were associated with mild dehydration and decreased food and water consumption The

ferret was treated with subcutaneous fluid therapy (10 mL Plasmalyte-A subcutaneous Baxter Alliston

Ontario) heat and supportive care and all clinical signs resolved within three days This ferret was

deemed healthy to participate in the remainder of the clinical trial A second ferret (105) in the control

group developed unilateral purulent ocular discharge 14 days after arrival one day prior to placebo

inoculation The ferret was treated topically twice daily for five days with Isathal ophthalmic gel (fusidic

acid 10 mgg Dechra Veterinary Products Inc Point-Claire Quebec) and the discharge resolved but

reoccurred within 2 days of treatment cessation Ocular examination showed mild conjunctivitis but no

evidence of corneal lesions and fluorescein staining did not indicate the presence of corneal ulceration

The ferret was treated for an additional six days with tobramycin ophthalmic solution (3 mgmL Sandoz

Tobramycin 03 Boucherville Quebec) after which clinical signs resolved completely No coccidial

oocysts were shed in feces from any of the ferrets during the two week acclimation period

112

531 Oocyst shedding

All ferrets readily ingested the inoculum with either placebo or concentrated oocysts In Part 1

one ferret (203) in the infection group which had received the high dose (1 times 106 oocysts) shed oocysts

on day 8 and day 9 after inoculation (Tables 51 52) This ferret was 71 days of age at the time shedding

was initially identified The ferret was killed humanely 11 days post inoculation later than had been

outlined in the protocol as processing of fecal samples had been delayed by two days resulting in late

detection of oocyst shedding in this individual Oocysts were not identified in the feces of the three

remaining ferrets that received the high inoculation dose the single ferret that received the lower dose (5

times 104 oocysts) or in the ferrets within the control group

In Part 2 six of eight ferrets inoculated with the high dose (1 times 106 oocysts) shed oocysts during

the 14 day observation period (Tables 51 52) Four of these ferrets were from the previous control

group One of the ferrets previously inoculated with the high dose inoculum in Phase 1 that had not shed

oocysts did shed oocysts after being inoculated a second time with the same dose during Phase 2 The

ferret that had previously received the low dose (5 times 104 oocysts) of oocysts in Phase 1 also shed after

inoculation with the high dose in Phase 2 Three ferrets did not shed oocysts after high dose inoculation in

Phase 2 one of these had been part of the previous control group the other two had received the high

dose inoculation previously in Phase 1

The pre-patent period ranged from 7-9 days (Table 51) with equivalent numbers of ferrets

commencing shedding on each of days 7 through 9 All six ferrets were between 91 and 93 days of age at

the time shedding was initially identified Oocyst per gram counts and shedding trends for all individuals

are shown in Table 51 Total oocyst shedding during patency was lt14 oocysts 8904 oocysts and

172291 oocysts for ferrets 201 104 and 105 respectively The two ferrets for which the prepatent period

was 9 days only shed oocysts for one day and in low numbers Oocysts were not identified in the feces of

the three remaining ferrets two of which received the high inoculation dose (1 times 106 oocysts) and the

third that received the lower inoculation dose (2 times 105 oocysts) (Table 52)

113


In all seven ferrets that shed oocysts the morphologic features and measurements (length width

shape index) of the shed oocysts were consistent with those of the E ictidea administered in the inoculum

(Figure 51) Molecular confirmation of the identity of the oocysts shed was successful in 3 out of the 7

ferrets (102 103 203) samples from the four remaining ferrets did not contain adequate quantity or

quality of DNA for confirmation

533 Clinical signs

Clinical signs associated with patent infection were identified in 6 of 7 ferrets (Table 52) These

signs included weight loss (n=5) diarrhea (n=1) mucoid soft feces (n=2) feces containing blood (n=2)

and malodorous feces (n=1) Appetite reduction was noted in two ferrets from the infection group in Part

1 between 6-8 days post infection however no oocyst shedding was detected from either ferret during

this time

534 Hematology

CBC and serum biochemistry values from ferrets collected during pre-trial health screening are

displayed in Appendices 1a and 1b Values obtained for ferrets euthanized during or after the

experimental trial are displayed in Appendices 3a and 3b Minor variances from reference range values

for CBC and serum biochemistry were identified in six of the seven ferrets with patent infection In all six

ferrets for which a complete serum biochemistry was obtained immediately prior to death creatinine

kinase (CK) values were elevated (see Appendix 3b) Ferret 103 exhibited a mild hypoalbuminemia (20

ref 24-40 gL) on ante-mortem serum biochemistry (see Appendix 3b)

114

535 Necropsy

No gross or histopathologic lesions were present and coccidia could not be identified in sections

of intestine from the single ferret (203) humanely killed in Part 1 For the ferrets humanely killed in Part

2 no evidence of diarrhea hematochezia or mucoid fecal material was identified grossly Ferret 105

killed at the termination of the experiment but still shedding low numbers of oocysts in its feces

exhibited a 7 cm region of congested mucosa within the distal jejunum

Coccidial life stages were identified in small intestinal sections from four of the seven ferrets that

were identified to shed oocysts at some point prior to necropsy (Figure 52 Tables 52 and 53) Affected

sections included jejunum in all four animals as well as ileum in one and were collected from 114 to 218

cm aboral from the pylorus (see Figure 53) Coccidia were not identified in sections of duodenum

proximal jejunum or large intestine however oocysts were identified within fecal material in the lumen

of the large intestine from one ferret (103) Of the 11-19 sections of intestine examined for each ferret the

number of sections containing parasites ranged from one to eight 1 section in ferret 102 2 sections in

ferret 201 4 sections in ferret 105 8 sections in ferret 103 A mix of sexual and asexual life stages was

observed within the enterocytes in small intestinal sections from ferret 103 the remainder of the ferrets

showed either asexual (102 201) or sexual (105) life stages in affected segments

Pathologic changes and additional histologic findings in small intestinal sections of ferrets with

enteric coccidia included rare regions of blunting of the villi and sloughing of the epithelium associated

with hemorrhage and inflammation The primary lesions identified were subjectively increased numbers

of eosinophils lymphocytes and plasma cells within the lamina propria of the small intestine and

similarly increased neutrophils lymphocytes and plasma cells within the lamina propria of the large

intestine Neutrophils were rarely present in intestinal crypts and glands

Other gross necropsy findings included two ferrets with renal cortico-medullary cysts and one

ferret with mild thickening of the esophageal mucosa midway along the esophageal length No histologic

changes were identified within a sample of esophagus taken from this region

115

54 DISCUSSION

The findings of this study show that domestic ferrets are susceptible to infection with the enteric

coccidium Eimeria ictidea isolated from black-footed ferrets Both morphometric and molecular

diagnostic methods were used to confirm that ferrets were shedding oocysts of the same species with

which they were inoculated Molecular characterization was successful in three of the seven ferrets that

developed patent infections and as no other coccidial species was identified during pre-trial observation

morphometry was considered to be confirmatory in the remaining four animals

We have referred to the eimeriid coccidium affecting BFF and used in this experimental trial as

E ictidea based on morphometric similarity of their oocysts with those of E ictidea as described from

domestic ferrets (see Chapter 3) There is limited published information on infection of domestic ferrets

with E ictidea outside of Hoarersquos original descriptions (1927 1935a b) which form the basis for all

subsequent identifications of E ictidea in domestic ferrets and in BFF Attempts to obtain exemplars of E

ictidea from domestic ferrets to characterize using molecular techniques were unsuccessful (Chapter 2)

Multiple diagnostic laboratories in Canada and Europe were solicited for coccidia-positive fecal samples

from domestic ferrets but no samples of E ictidea were received over a 4 year period (2014-2017)

Eimeria ictidea was identified based on microscopic examination in only two samples submitted to a

European diagnostic laboratory from 2008-2015 It is unproven whether the E ictidea described from

domestic ferrets and the E ictidea identified from black-footed ferrets and used in this experimental

work are the same or are simply morphologically indistinguishable Eimeria species However the

consistency in morphology host genus and location of infection within the intestinal tissues combined

with the successful cross-transmission of this parasite to domestic ferrets described in the present study

suggests they are likely conspecific

116

The pre-patent period (minimum duration of endogenous development) for infection with E

ictidea in the inoculated domestic ferrets ranged from 7-9 days (see Table 51) the pre-patent period for

this parasite in the BFF the natural host for this coccidium is unknown Hoare experimentally infected

naiumlve domestic ferrets with E ictidea derived from naturally occurring infection in this species (Hoare

1935b) The inoculated ferrets shed oocysts after a pre-patent period of 7 days consistent with the 7-9

days seen in the work described here with E ictidea

Shedding of oocysts was identified over a period of 1-7 days (see Table 51) and intensity ranged

from less than 14 up to 15624 OPG These results may be skewed with erroneously low duration of

shedding and number of oocysts shed as three of the seven ferrets were humanely killed at or prior to the

expected peak of oocyst shedding for tissue collection and histologic examination in order to increase the

probability of identifying parasite life stages within the intestinal sections Shedding periods were similar

to those identified in adult single-housed BFF which ranged from 2-9 days however oocyst per gram

counts from the domestic ferrets were consistently lower than OPG counts from BFF (104 - 554274

OPG) infected with the same parasite (see Chapter 4) Furthermore the total number of oocysts shed by

individual domestic ferrets (14 - 172291 oocysts) during patency was reduced compared to BFF despite

similar length of shedding period (see Chapter 4) The domestic ferrets in this study were naiumlve individual

juveniles whereas the BFF were adults 1-5 years of age some of which were showing clinical signs at

the time of oocyst shedding The relative influences of age species and previous exposure to the parasite

on our observations are unknown

Two different fecal flotation methods were used on all samples to increase the probability of

oocyst detection The McMaster method was used to provide accurate OPG counts for quantification of

oocyst shedding however this method had a minimum detection limit of ~13 oocysts per gram (1333

OPG calculated) because it is based on dilution of the initial sample with flotation media (saturated salt)

In samples with few oocysts oocysts may be missed or to be present in numbers below this detection

limit Routine salt flotation is in contrast performed using the entire sample allowing for detection of

117

small numbers of oocysts Consequently in cases where oocyst per gram counts were low shedding was

identified on routine salt flotation but not by the McMaster method and recorded as lsquopositiversquo but below

the detection limit of the enumeration method

Subclinical to clinical disease occurred in six of the seven ferrets that developed patent infection

with weight loss being the most frequent clinical sign Other clinical signs were typical of enteric

coccidiosis including diarrhea hematochezia and mucoid andor soft feces These clinical signs are

similar to those previously described for black-footed ferrets infected with this parasite (USFWS BFF

Recovery Program 2017 Chapter 4) and for domestic ferrets with enteric coccidiosis (Sledge et al

2011) Interestingly development of clinical disease was not described in the naiumlve domestic ferret

inoculated by Hoare (1935) with E ictidea derived from naturally occurring infection Based on review of

the literature severe clinical disease resulting from intestinal coccidiosis is rare in domestic ferrets

Black-footed ferrets however appear more susceptible to disease development and more frequently show

significant clinical signs No domestic ferret required treatment for clinical coccidiosis during the course

of this study It is possible that the more pronounced clinical signs associated with enteric coccidiosis in

BFF may result from the limited genetic diversity in a population derived from so few individuals and

increased susceptibility of BFF to other diseases such as sylvatic plague have been described in

comparison with their domestic counterparts (Williams et al 1994) Although unlikely recent acquisition

of E ictidea from a related host species such as the domestic ferret could have resulted in increased

pathogenicity and severity of clinical disease from infection with this parasite in BFF

Minor variances from reference range values for CBC and serum biochemistry were identified in

six of the seven ferrets with patent infection but only in one case (ferret 103) did this appear to be

correlated with infectiondisease In this animal a mild hypoalbuminemia (20 ref 24-40 gL) was noted

(Appendix 3b) On histopathology large regions of the small intestine contained parasite life stages

however inflammation lysis of epithelial cells and necrosis of affected areas that could be expected to

result in protein loss into the intestinal lumen were not identified

118

Creatine kinase values were elevated in all six ferrets for which a complete serum biochemistry

was obtained immediately prior to death these findings are consistent with release from CK rich tissues

such a skeletal muscle during venipuncture and manual restraint

The pathologic lesions identified within the intestinal sections of ferrets euthanized at the time of

oocyst shedding were similar to those identified in affected BFF but in most cases were less locally

extensive or widespread throughout the small intestine than those observed in necropsy cases of BFF (see

Chapter 4) Coccidia were seen in the histologic sections of four ferrets all three ferrets that were actively

shedding oocysts at the time of necropsy (102 103 105) and one of four in which necropsies were

performed after oocyst shedding had ceased (201)

Although the primary objective of the examination of histologic sections from affected ferrets

was to identify coccidial life stages attempts were made to describe the pathologic changes associated

with the presence of the parasite Despite necropsies being performed almost immediately after death and

the use of Serra solution fixative to improve parasite and tissue preservation the villi and villar

epithelium of the trimmed sections were frequently distorted or absent and consequently accurate

commentary on these areas was precluded and was made only on visible components of the lamina

propria and crypts or glands A deliberate decision was made not to kill and collect samples from the

saline inoculated control ferrets after Phase 1 and those not shedding oocysts during Phase 1 or 2 thus no

age-matched intestinal sections were available for comparison It is difficult to comment on the

significance of the inflammatory cells observed in the lamina propria of the small and large intestinal

segments or the proliferative rate of the crypt epithelium The only changes identified which may be

considered significant are the presence of neutrophils within the crypts and glands of small and large

intestinal sections respectively but these lesions were rare and not associated with the presence of

parasitic life stages

119

While no parasitic life stages were identified in intestinal sections from ferrets 104 203 and 205

one of the three ferrets ferret 104 exhibited lymphoplasmacytic inflammation and blunting of jejunal

villi in one section (36-38 cm aboral from pylorus) These findings might be expected as the most

extensive histologic lesions would occur associated with lysis of the intestinal epithelial cells as oocysts

were shed into the feces after which new intestinal epithelial cells would re-cover the denuded villar

surface Thus for those cases in which histologic examination was performed after shedding had ceased

presence of the protozoal life stages in the intestines would be expected to be significantly reduced or

absent

Hoare (1935a b) described a particular reaction to the presence of parasitic life stages of Eimeria

ictidea in the small intestine of domestic ferrets in which only the villar tips were affected and there was

resultant annular constriction of the villus separating the affected and non-affected segments These

particular changes were not identified in any of the ferrets in this study and had not been noted

retrospectively in naturally infected BFF (Chapter 4)

While patent infection and intestinal disease could be experimentally created in domestic ferrets

without equivalent experimental work in BFF it is difficult to fully compare the susceptibility to infection

and to the development of disease between the two species Eimeria species tend to be host specific thus

if E ictidea from BFF is not conspecific with E ictidea in domestic ferrets it might be expected that the

domestic ferret would be less susceptible to infection and the development of disease than is the BFF

Even if the two parasites are identical natural passaging through the BFF may alter affinity for the

domestic ferret

It appears that the infectious dose of oocysts of E ictidea derived from BFF required to initiate a

patent infection in domestic ferrets is high The ferrets that developed patent infections were administered

an inoculum containing 1 times 106 sporulated oocysts and even with this extremely high inoculating dose

only a proportion of inoculated ferrets became infected Neither the ferret that received the low dose

120

inoculum nor the ferret that received the passaged oocysts from Part 1 of the study shed oocysts during

the 14 day period post inoculation The latter finding was unexpected as fresh passaged oocysts would be

expected to contain larger proportions of viable oocysts and be comparatively more infective than oocysts

that had been stored for 2-4 weeks prior to inoculation

The only publication describing oral inoculation of Eimeria species in domestic ferrets (Hoare

1935b) did not quantify the number of oocysts administered A study performed in 16 farmed juvenile

mink (Mustela vison) administered 2000 sporulated oocysts of each of three coccidial species (I

laidlawi E vison and an unknown Eimeria species) resulting in patent infection with one of the three

species (Foreyt et al 1977) as determined by the presence of oocysts on fecal examination The authors

did not reveal which type of oocysts resulted in the infection

The number of oocysts required to result in infection in BFF is unknown Based on the

authorsrsquo observations of over 100000 oocysts per gram of feces being shed by black-footed ferrets into

cages of lt1m2 floor space (see Chapter 4) we estimated that under normal caged conditions animals

would likely ingest thousands of oocysts over a short period of time This was in part why a large number

of oocysts (up to 1000000 as available from our store of viable oocysts) was administered to each

domestic ferret in order to increase the probability that infection and shedding would result Furthermore

for the parasite to persist within the ferret population the total number of oocysts shed into the

environment would have to be several times higher than the infective dose required to generate a patent

infection otherwise the parasite would be expected to die out If E ictidea of domestic ferrets and E

ictidea of BFF are conspecific the difference in oocyst shedding between the host species during patent

infection could potentially explain the low prevalence of E ictidea reported from the domestic ferret

population (as seen in Chapter 2)

Both humoral and cell mediated immunity are involved in the immune response to coccidia The

role of maternal derived antibodies in combatting protozoal infection in mustelids has not been studied

121

but in carnivores maternal antibodies to viruses can last up to 16 weeks (Chappuis 1998) In poultry

maternal Eimeria-specific IgG is transferred via the egg yolk to offspring In one study breeding hens

were infected with a single species of Eimeria 28-39 days prior to lay Their hatched chicks were

challenged by inoculation with oocysts of the same and a related Eimeria species and showed reduced

oocyst shedding compared to age matched controls indicative of passive transfer of immunity (Smith et

al 1994) It is possible then that the presence or absence of maternal antibodies may be a factor in the

age at which ferrets are susceptible to coccidial infection and the development of disease The facility

from which the domestic ferrets were acquired has not previously detected Eimeria species on routine

fecal screening (Dr Bambi Jasmin personal communication) consequently it is unlikely that they would

have received maternal immunity to or been exposed to this parasite and thus can be considered to be

naive

Despite a theoretical lack of maternal immunity there did appear to be an effect of age on

susceptibility to infection In Part 1 when the ferrets were approximately 70 days of age only 1 of 4

ferrets inoculated with the high dose of oocysts developed patent infection In comparison in Part 2

when the ferrets were 91-93 days of age 3 of 4 ferrets that had been in the saline control group for Part 1

developed patent infections as did 2 of 4 ferrets that had been inoculated in Part 1 but had not shed

oocysts One of these previously inoculated ferrets was the individual that had received the lower dose of

oocysts Thus it appears that patent infection could be produced more easily in the older ferret kits

However the two ferrets that had previously been inoculated exhibited the shortest shedding periods (1

day) and lowest oocyst per gram counts and parasite life stages in these cases were rare (201) to absent

(205) on histologic examination of numerous sections of intestine It is thus possible that the primary

inoculation resulted in abbreviated infections or infections in which so few oocysts were shed that

infection was not detected Our observation of endogenous stages in the intestinal tissues of some of the

ferrets following cessation of oocyst shedding suggests that the pre-patent period and duration of patency

may vary considerably from animal to animal consequently it is possible that some of the kits would

122

have ultimately shed a few oocysts from the primary inoculum if followed beyond 14 days post-

inoculation Whether through an aborted or undetected infection previous exposure of these kits to the

parasite probably generated partial immunity against E ictidea and therefore the intensity of infection

upon challenge in previously exposed domestic ferret kits was reduced

The authors acknowledge the limitations of this initial pilot study however it was proven that

patent infection with E ictidea isolated from black-footed ferrets could be generated in a novel host the

domestic ferret Further studies will be required to investigate the effect of age on susceptibility to

infection as well as the possibility of development of immunity after exposure and its role in reducing

parasite replication and disease in subsequent infection With so few BFF in existence the use of BFF for

in vivo infection trials cannot be contemplated consequently refining the domestic ferret infection model

will be essential for carrying out research specifically intended to help manage coccidiosis in the

endangered black-footed ferret

Figure 51 Exogenous life stages of Eimeria ictidea shed from a domestic ferret (Mustela putorius

furo) experimentally inoculated with oocysts originating from black-footed ferrets (Mustela

nigripes) Unsporulated oocyst (solid black arrow) Sporulated oocyst (solid white arrow) Bright

field microscopy scale bar = 25 microm

25 microm

124

Figure 52 Life stages of Eimeria ictidea within the small intestinal epithelium of an experimentally

infected domestic ferret (Mustela putorius furo) A) Sexual life stages (micro- and macrogamonts

- white arrows) crowding the villar enterocytes all stages are found between the nucleus and

luminal surface of infected enterocytes Hematoxylin and eosin staining scale bar = 25microm B) At

higher magnification meronts (black arrows) and gamonts (open arrows) are crowded between the

enterocyte nuclei and brush border Hematoxylin and eosin staining scale bar = 25microm

25 microm

25 microm

125

Figure 53 Presence and location of sexual and asexual life stages of Eimeria ictidea within the

intestinal epithelium of domestic ferrets (Mustela putorius furo) (n=7) that developed patent

infection after experimental inoculation with oocysts originating from black-footed ferrets (Mustela

nigripes) Legend duod = duodenum jej= jejunum Sequential numbers for jejunal and colonic

sections represent the order aboral from the pylorus from which the samples were collected SI =

additional section(s) of small intestine whose aboral sequence was not recorded

0

1

2

3

duod jej 1 jej 2 jej 3 jej 4 jej 5 ileum colon 1 colon 2 SI

Nu

mb

er

of

Ferr

ets

Aff

ect

ed

Intestinal Section Containing Life Stages of Eimeria ictidea

Asexual life stages Sexual life stages

126

Table 51 Prepatent period and oocyst shedding patterns in domestic ferrets (Mustela putorius furo)

experimentally inoculated with 1 times 106 oocysts of Eimeria ictidea originating from black-footed ferret

(Mustela nigripes) that developed patent infections

Oocysts shed per gram of feces

Ferret Identity

Day post inoculation 102 103 104 105 201 203 205

1 0 0 0 0 0 0 0

2 0 0 0 0 0 0 0

3 0 0 0 0 0 0 0

4 0 0 0 0 0 0 0

5 0 0 0 0 0 0 0

6 0 0 0 0 0 0 0

7 lt 14 1807 0 0 0 0 0

8 11053 139 7091 0 156238 0

9 463 11733 lt 14 203 lt 14

10 578 7549 0 0 0

11 lt 14 0 0 0

12 0 lt 14 0

13 0 lt 14 0

14 0 lt 14 0

Legend ferret euthanized as of this date lt 14 = oocyst positive samples with less than 14 oocysts per

gram of feces

127

Table 52 Results of oral inoculation of domestic ferrets (Mustela putorius furo) with oocysts of Eimeria ictidea originating from black-footed

ferrets (Mustela nigripes)

Ferret

Identity

Results Part 1 Results Part 2

Inoculum Oocyst

Shedding

Presence

of clinical

disease

Coccidia

present in

sections

Inoculum Oocyst

Shedding

Presence

of clinical

disease

Coccidia

present in

sections

101 Saline N N - 2 times 105 oocystsa N N -

102 Saline N N - 1 times 106 oocysts Y N Y^

103 Saline N N - 1 times 106 oocysts Y Y Y^

104 Saline N N - 1 times 106 oocysts Y Y Nyen

105 Saline N N - 1 times 106 oocysts Y Y Yyen

201 1 times 106 oocysts N N - 1 times 106 oocysts Y Y Yyen

202 1 times 106 oocysts N N - 1 times 106 oocysts N N -

203 1 times 106 oocysts Y Y Nyen


205 5 times 104 oocysts N N - 1 times 106 oocysts Y Y N^

Legend includes any of the following weight loss diarrhea mucoid feces malodorous feces inappetence N = no Y= yes - = necropsy not

performed a = oocysts collected after passage through ferret 203 ^ = shedding oocysts at time of necropsy yen = not shedding oocysts at time of

necropsy

128

Table 53 Distribution of coccidial life stages in domestic ferrets (Mustela putorius furo) orally inoculated with oocysts of Eimeria ictidea

originating from black-footed ferrets (Mustela nigripes)

Ferret Identity

Intestinal level 102

103

104 105

201

203

205

Duodenum N N N N N N N

Jejunum 1 N N N N N N N


Jejunum 3 N S A N N N N N


Jejunum 5 N S A N S A N N

Ileum N S A N N N N N

Colon 1 N Na N N N N N


Unmeasured small intestinebc

S - 05

A - 15

S - 45

A - 25

S - 05

A - 05

S - 26

A - 06

S - 06

A - 06

S - 06

A - 06

S - 08

A - 08

Unmeasured large intestinebc

S - 01

A - 01

S - 01

A - 01

S - 01

A - 01

S - 02

A - 02

S - 01

A - 01 none

S - 01

A - 01

Legend N = no parasite life stages S = sexual life stages present A = asexual life stages present a= oocysts present in feces b=

additional sections of intestine for which the location measured from the pylorus was not obtained c= xn where x is number of

sections containing sexual or asexual lifestages n is the number of sections examined

129

CHAPTER 6 WHOLE MITOCHONDRIAL GENOME SEQUENCES OF TWO EIMERIA

SPECIES ISOLATED FROM DOMESTIC (MUSTELA PUTORIUS FURO) AND BLACK-

FOOTED FERRETS (MUSTELA NIGRIPES)

ABSTRACT

The complete mitochondrial (mt) genomes of Eimeria furonis and Eimeria ictidea (Eimeriidae

Coccidia Apicomplexa) originating from single fecal samples from a domestic (Mustela putorius furo)

and a black-footed ferret (Mustela nigripes) respectively were sequenced Both mt genomes were

circular-mapping with lengths of 6165 base pairs (Eimeria furonis - GenBank MF795598) and 6182

base pairs (Eimeria ictidea - GenBank KT203399) Genome organization and gene contents were

comparable with those of other publically available mt genomes from a variety of Eimeria species and

related coccidia there were three complete coding DNA sequence regions encoding cytochrome c

oxidase subunit I cytochrome c oxidase subunit III and cytochrome B and 33 regions encoding

fragmented rDNA Alignment of these mt genome sequences demonstrates a relatively high (945 340

single nucleotide differences [SNDs]) pairwise sequence identity between these Eimeria spp infecting

ferrets the majority of the SNDs resulted in synonymous codon changes with no changes to their protein

products Alignment of the newly sequenced mt genomes demonstrates and phylogenetic reconstructions

support the monophyly of these Eimeria spp of ferrets with another Eimeria sp of carnivores as the

sister taxon to this clade

61 INTRODUCTION

Coccidia are protozoal eukaryotic host-specific parasites of the phylum Apicomplexa and can

be divided into two major taxonomic suborders the eimerioirinid and adeleid coccidia The eimeriorinid

coccidia include both the typical intestinal coccidia (eg Eimeria Isospora Cyclospora) species

belonging to the family Eimeriidae as well as tissue or cyst-forming coccidia (eg Cystoisospora

130

Besnoitia Toxoplasma Sarcocystis) of the family Sarcocystidae (see Cox 1994) Ten species of

Eimeria and twelve species of Isospora (=Cystoisospora) have been described in the Mustelidae

(see Chapter 1 Table 11) Eimeria furonis has been reported in the European polecat (Mustela

putorius) domestic ferret (DF Mustela putorius furo) black-footed ferret (BFF Mustela nigripes) and

mink (Mustela vison) (Hoare 1927 Nukerbaeva and Svanbaev 1973 Jolley et al 1994) Eimeria ictidea

has been reported in the Steppe polecat (Mustela eversmanii) as well as the European polecat domestic

ferret and black-footed ferret (Hoare 1927 Svanbaev 1956 Jolley et al 1994) These reports are based on

the morphometric characteristics of oocysts identified in the feces of the aforementioned host species

without the use of molecular techniques to confirm specific parasite identities

Recently sequences of the mitochondrial cytochrome c oxidase subunit I gene (mt COI) and

nuclear small subunit ribosomal DNA (nu 18S rDNA) of E furonis originating from a domestic ferret (nu

18S rDNA GenBank MF774678-MF774680 mt COI GenBank MF774034-MF774036) and E ictidea

originating from a black-footed ferret (nu 18S rDNA GenBank MF860826-MF860827 mt COI

GenBank MF860823 MF860825) were generated (see Chapters 2 and 3) The parasite originating from

the black-footed ferret was identified as E ictidea based on morphologic similarity to the original

descriptions of E ictidea from domestic ferrets however sequence-based genotyping of E ictidea from

domestic ferrets has not been completed and consequently it has not been demonstrated unequivocally

that the two parasites are conspecific In the present work the complete mitochondrial genomes of E

furonis from the DF and E ictidea from the BFF are described and compared with the mitochondrial

genomes of related coccidia


621 Parasites

Two isolates of morphologically distinct Eimeria species were used in this study Isolate one

identified morphologically and by nu 18S rDNA and mt COI sequences as Eimeria furonis was obtained

131

from a fecal sample from a DF that was submitted for routine ova and parasite examination to a European

diagnostic laboratory8 Isolate two identified morphologically and by nu 18S rDNA and mt COI

sequences as Eimeria ictidea was obtained from a fecal sample from a BFF and was collected during

routine cage cleaning in a captive breeding facility (see Chapters 3 and 4) Fecal collection techniques for

the BFF were reviewed and approved by both the Animal Welfare Committee and the Animal Care and

Research Committee of the Toronto Zoo

622 DNA isolation from coccidia in feces

Genomic DNA was isolated from fecal derived coccidial oocysts as described section 221 of the

Chapter 2 Materials amp Methods Parasite DNA concentration was estimated using a Nanodrop 2000

spectrophotometer (NanoDrop Products Wilmington DE USA) and DNA was stored at 4 degC for


623 Whole genome sequencing

Mitochondrial whole genome amplification for both Eimeria species was initiated using sets of

mt-specific primers that generated overlapping polymerase chain reaction (PCR) fragments (Tables 61

and 62) PCR amplification was performed for all samples in a volume of 25 microl containing ~100 ng of



Toronto ON Canada) PCR reactions were performed on a Bio-Rad T100 PCR thermal cycler (Bio-Rad

Laboratories Singapore) using settings as described previously in the Materials amp Methods section of

Chapter 2 Table 61 details the specific anneal conditions used for the various primer pairs Genomic

DNA from either Eimeria maxima or Eimeria tenella acted as a positive control for the reaction

chemistry Gel electrophoresis purification and sequencing of the PCR amplification products were


132

performed as described in Chapter 2 The resulting chromatograms were aligned and analyzed with

Geneious Ver 818 or later (Biomatters Limited Auckland New Zealand) and high quality consensus

sequences generated The completed mt genome sequences were annotated by comparison with

previously annotated mt genomes from other Eimeria species (eg Eimeria innocua - KR1082961) and

the annotated mt genomes deposited in GenBank



related apicomplexan taxa representative whole mt genome sequences from eimeriid coccidia were

downloaded from GenBank A complete mt genome sequence from an unnamed Choleoeimeria sp was

used to root the ingroup taxa several small genomic rearrangements in the Choleoeimeria sp sequence

required some rearrangement of the genome sequence to unambiguously align homologous regions across

the complete mt genomes

Whole genome sequences were aligned using MAFTT v7017 (Katoh et al 2002) executed from

within Geneious the resulting alignment was examined by eye to adjust start and stop codon positions in

aligned coding DNA sequence [CDS] regions (ie mt COI mitochondrial cytochrome c oxidase subunit

III gene [mtCOIII] mitochondrial cytochrome b gene [CytB]) Phylogenetic trees were generated using

Bayesian Inference (BI) using MrBayes Ver 326 (Huelsenbeck and Ronquist 2001) executed from

within Geneious The aligned complete mt genomes were partitioned into coding (ie CDS) and non-

coding regions so that region-specific models of nucleotide substitution could be applied Characters in

the non-coding region were analysed with the general time reversible (GTR) model (Tavareacute 1986) with

the following parameters nucmodel=4by4 nst=6 rates=invgamma (ie GTR+I+G) Characters in the

coding regions were analysed using the codon nucleotide model (ie lset nucmodel=codon rates=gamma

ngammacat=4) using metazoan mitochondrial translation (ie code=metmt)

133

All BI analyses were run for a chain length of 1000000 with tree sampling every 1000


63 RESULTS

The whole mt genome sequences of the single isolates of E furonis and E ictidea were

respectively 6165 base pairs (bp) (Figure 61 GenBank MF795598) and 6182 bp (Figure 62 GenBank

KT203399) Content and organization of both mt genomes consisted of three protein-coding genes (mt

COI mt COIII and CytB) interspersed with large and small subunit ribosomal DNA (rDNA) fragments

Details of the various CDS and rDNA fragments are summarized in Table 63 (for E furonis) and Table

64 (for E ictidea)

Pairwise alignment of the mt genome sequences from E furonis and E ictidea demonstrated a

relatively high pairwise sequence identity (946 333 single nucleotide differences [SNDs]) between

these two parasites The bulk of the SNDs (676 225333) were clustered within the three CDS regions

that encode CytB mt COI and mt COIII (see Figure 63 and Table 65) However the majority of these

SNDs (826 186225) were synonymous codon changes that resulted in no changes to the protein

products Only 41 SNDs were involved in 34 amino acid changes distributed among the three CDS

The 33 rDNA fragments comprised 2108 and 2109 bp respectively of the mt genomes of

E furonis and E ictidea Pairwise comparison of these rDNA fragments demonstrated high (986 30

SNDs) sequence identity between the two parasites The remaining 778 and 794 bp respectively of the

mt genomes of E furonis and E ictidea were intergenic stretches between the various rDNA and CDS

regions these intergenic regions were more variable that other regions of the genomes with 78 SNDs

(almost 10 sequence divergence) Additionally all indels were restricted to these variable intergenic

regions

The BI phylogeny generated from aligned complete mt genomes (Figure 64) supported the close

relationship between E furonis and E ictidea within a clade of Eimeria species that include the only three

sequences available for Eimeria spp of carnivores Eimeria mephitidis from the striped skunk (Mephitis

134

mephitis Family Mephitidae) was the sister taxon to the two Eimeria species of ferrets (Family

Mustelidae)

64 DISCUSSION

This work generated the first complete mt genomes from coccidia that infect domestic and black-

footed ferrets (Carnivora Mustelidae) Eimeria mephitidis from the striped skunk Mephitis mephitis

(Carnivora Mephitidae) is the only other Eimeria species from a carnivore for which a complete mt

genome has been reported

Comparatively few eimeriid coccidia only 26 Eimeria species have been described from

carnivores there are 14 named species from the mustelids four from the procyonids four from the ursids

three from the herpestids and one from the viverrids (Duszynski et al 2000) The majority of coccidia

that infect the digestive tract of carnivores belong to the family Sarcocystidae including monoxenous or

facultatively heteroxenous Cystoisospora species or heteroxenous parasites in the genera Sarcocystis

Hammondia and Neospora So far as is known none of the parasites in the Sarcocystidae possess typical

apicomplexan mt genomes with 3 complete CDS and many rDNA fragments (Ogedengbe 2015)

The mt genomes from the two Eimeria sp of mustelid origin demonstrate the same structural

organization (ie the order and number of CDS and rDNA fragments) and circular mapping as the mt

genomes from other Eimeria spp and other closely related eimeriid coccidia such as Isospora

Cyclospora and Lankesterella species Despite the ability of the eimeriid sequences to be mapped

circularly the physical form of Eimeria spp mt genomes may be a linear concatemer of multiple genome

copies as demonstrated for Eimeria tenella (Hikosaka et al 2011) As in the mt genomes of other

eimeriid coccidia (Ogedengbe et al 2013 2014) the CDS for mt COIII demonstrated the highest

sequence divergence between E furonis and E ictidea the mt COI CDS was somewhat more conserved

and CytB CDS demonstrated the fewest SNDs

As expected based on limited sequence divergence between E furonis and E ictidea a BI

phylogenetic analysis using aligned complete mt genome sequences generated a tree that placed these

135

two Eimeria species that infect mustelids within a well-supported monophyletic group The sister taxon

for these ferret parasites was the only other Eimeria species from carnivores for which a complete mt

genome is available E mephitidis which infects hosts belonging to a different family of carnivores

Eimeriid parasites that infect closely-related definitive hosts are commonly found in a single or limited

number of clades based on mitochondrial and nuclear genetic loci (Ogedengbe et al in press)

Sequencing of the mt genomes and at least one nuclear genetic locus (ie nu 18S rDNA) from additional

Eimeria species infecting carnivores will be required to determine if all carnivore-specific Eimeria

species share a common ancestor

136

Table 61 PCR primer pairs and resulting fragments used for sequencing the mitochondrial genome sequence of an isolate of Eimeria furonis

originating from a fecal sample from a domestic ferret (Mustela putorius furo)

Fragment Primer names Primer sequences (5ʹ-3ʹ) Size (bp) Anneal Temp References

1 WG-MT_4140F AGAAAACCTAAAATCATCATGT 1000 52 Ogedengbe et al (2015)

Eim_CO3_799R AAGTGAGTTCGCATGTTTAC Ogedengbe et al (2015)

2 Eim_COI_19F ACTGCYGCAAACCATAARGAA 1700 60 Present study

Api_LSUG_UNI_R AGATAGGGAACAAACTGYCTCAA Present study

3 WG_MT_5416F GGTCCAGATAAGCGATCTCATG 3400 53 Ogedengbe et al (2013)

Eim_COI_1436R CACATTGTGTTCARATAAGTTA Present study

4 WG-MT_6219F GCATCCATCTACAGCTGCGG 500 55 Ogedengbe et al (2013)

WG-MT_344R GTAGGAATCTRAATTCCCAACC Ogedengbe et al (2013)

5 Api_LSUE UNI_F AGGTGCTCAGGGTCTTACCG 500 55 Present study

WG_MT_63R CTGGTATGGATGGATAACACT Ogedengbe et al (2015)

6 Lank_COB-30F CCAGGCCAACTGAACTCGTT 1300 55 Present study

q_Eim_COI_221R GGCATAACTACAAAGAARATCATA Present study

7 Cocci_MT_WG_F TACACCTAGCCAACACGAT 1600 55 Ogedengbe et al (2014)


137

Table 62 PCR primer pairs and resulting fragments used for sequencing the mitochondrial genome sequence of an isolate of Eimeria ictidea

originating from a fecal sample from a black-footed ferret (Mustela nigripes)


1 WG_MT_63R CTGGTATGGATGGATAACACT 2180 52 Ogedengbe et al (2015)

WG-MT_4140F AGAAAACCTAAAATCATCATGT Ogedengbe et al (2015)



3 WG-MT_3658F CTGGCGAGAAGGGAAGTGTG 1329 55 Ogedengbe et al (2013)



WG_MT_4072R GGTTGTTTCCATCTCGACTC Ogedengbe et al (2013)

138

Table 63 Coding regions within the mitochondrial genome of the eimeriid parasite Eimeria furonis from a domestic ferret (Mustela putorius faro)

139

Table 64 Coding regions of the mitochondrial genome of the eimeriid parasite Eimeria ictidea originating from a black-footed ferret (Mustela

nigripes)

Table 64 Features associated with the protein-coding regions of the mitochondrial genome of the eimeriid parasite Eimeria cf ictidea originating from a black-footed ferret (Mustela nigripes )

Protein coding regions (CDS) Sequence size (bp) Start position (bp) Stop position (bp) Direction Translation start codon Translation stop codon

Cytochrome c oxidase subunit I (COI) 1443 1343 2785 Forward ATG TAA

Cytochrome c oxidase subunit III (COIII) 756 4333 5088 Forward TTA TAA

Cytochrome b (CytB) 1080 226 1305 Forward ATG TAA

Ribosomal DNA fragments (rDNA) Product

SSUrRNA 46 17 62 forward RNA9 SSU8

SSUrRNA 77 104 180 forward SSUA SSU4 (partial)

SSUrRNA 35 181 215 forward RNA23t

LSUrRNA 20 2790 2809 forward RNA20 (partial) LSU

LSUrRNA 112 2835 2946 forward LSUF LSU11

LSUrRNA 106 2947 3052 forward LSUG LSU12

LSUrRNA 25 3087 3063 reverse LSU


LSUrRNA 16 3188 3173 reverse LSUC LSU4

SSUrRNA 33 3211 3243 forward SSU

SSUrRNA 61 3386 3326 reverse SSUF SSU12

LSUrRNA 74 3387 3460 forward RNA10 LSU13 (partial)

LSUrRNA 49 3476 3524 forward RNA11 LSU5

SSUrRNA 65 3533 3597 forward SSUD SSU10


SSUrRNA 30 3677 3706 forward RNA15 SSU



LSUrRNA 79 3899 3821 reverse LSUD LSU8

LSUrRNA 24 3931 3908 reverse RNA16 (partial)

SSUrRNA 92 4036 3945 reverse RNA8 SSU5


LSUrRNA 177 4316 4140 reverse LSUA LSU1



LSUrRNA 26 5342 5317 reverse LSUB LSU3

LSUrRNA 73 5447 5375 reverse RNA3 LSU7


SSUrRNA 119 5615 5497 reverse SSUB SSU6

LSUrRNA 80 5699 5620 reverse RNA7

LSUrRNA 188 5897 5710 reverse LSUE LSU9

SSUrRNA 33 6033 6001 reverse SSUE SSU11 (partial)

SSUrRNA 99 6156 6058 reverse RNA5SSU9

140

Table 65 Pairwise comparison of coding DNA and concatenated rDNA fragment sequences between the

mitochondrial genomes of Eimeria furonis originating from a domestic ferret (Mustela putorius furo) and

Eimeria ictidea originating from a black-footed ferret (Mustela nigripes)

Total length

(nucleotides)

Nucleotide

identity

Total amino

acids

Amino acid

identity

COI CDS 1443 934 (95) 480 975 (12)

COIII CDS 756 899 (76) 251 932 (17)

CytB CDS 1080 950 (54) 359 986 (5)

rDNA fragments 2109 985 (32) na na

Legend Numbers in brackets indicate the number of single nucleotide differences na = not

applicable

141

Figure 61 Circular mapping and organization of the mitochondrial genome content of Eimeria

furonis showing three protein-coding genes (COI COIII and CytB) interspersed with large and

small subunit rRNA fragments

142


ictidea showing three protein-coding genes (COI COIII and CytB) interspersed with large and


143

Figure 63 Pairwise comparison of coding DNA and concatenated rDNA fragment sequences of

the mitochondrial genomes of Eimeria furonis and Eimeria ictidea Legend Yellow bands

correspond to coding DNA fragments Red arrows correspond to rDNA fragments Arrows of both

colours indicate the forward or reverse direction of these segments

144

Figure 64 Phylogenetic relationships of coccidia (Eimeria furonis and Eimeria ictidea) from

domestic (Mustela putorius furo) and black-footed ferrets (Mustela nigripes) based on the complete

mitochondrial genome sequences these Eimeria species and a selection of related apicomplexan

parasites

145

CHAPTER 7 CONCLUSIONS AND FUTURE DIRECTIONS

The primary objective of this project was to better characterize the enteric coccidia of the

endangered black-footed ferret (BFF) in order to set the stage for improved disease prevention

management and treatment To the authorrsquos knowledge this work was the first attempt to isolate and

perform molecular characterization of the coccidial species endemic in the black-footed ferret population

and to characterize the natural history of the disease in this host

Two Eimeria species Eimeria ictidea and Eimeria furonis have been described from BFF and

domestic ferrets A single Eimeria species morphologically resembling E ictidea of the domestic ferret

and referred to in this work as E ictidea was identified from all historic and active cases of enteric

coccidiosis in the Toronto Zoo BFF population The same species was identified in both BFF family

groups and single-housed adults that shed coccidia during the study period (2014-2016) as well as from

historic necropsy samples of juvenile and adult Toronto Zoo BFF from 1999-2014 Furthermore this

same species was identified from both family groups and single-housed adults shedding coccidia at an

additional BFF Species Survival Plan institution the Louisville Zoo in 2016 These findings are contrary

to previous published reports that indicated multiple coccidia species were affecting captive and wild

BFF In order to determine whether these additional parasites continue to exist within the present-day

BFF population and their impact on this species further examination of coccidia-positive fecal samples

from captive and wild BFF populations is recommended Moreover determination of which parasite

species impact BFF morbidity and mortality would allow for development of targeted therapies for

disease management

Coccidia-positive fecal samples and necropsy samples from domestic ferrets were collected from

multiple diagnostic laboratories in Canada and Europe for comparison with results from BFF Eimeria

furonis and Isospora (=Cystoisospora) laidlawi were identified from fecal parasitology reports from

2008-2015 and in fecal samples obtained prospectively from 2014-2016 from domestic ferrets submitted

to Canadian and European laboratories E furonis was also identified in necropsy samples from 2010 and

146

2017 from two Canadian diagnostic laboratories No samples containing E ictidea were submitted to the

Canadian laboratories however samples containing E ictidea were identified twice by the European

diagnostic laboratory with one sample in 2011 and a second in 2013 Consequently it appears that E

ictidea is rarely identified from domestic ferrets Since no samples of E ictidea were acquired from

domestic ferrets during the study period it remains undetermined whether E ictidea from domestic

ferrets and E ictidea of BFF are conspecific Future molecular characterization of E ictidea isolated from

various mustelid host species would allow not only for determination of whether the parasites are

conspecific but would also provide insight into the potential for cross-transmission of parasites between

related mustelid hosts

The identifications provided by diagnostic laboratories of the specific parasite species present in

the domestic ferret fecal samples showed poor agreement with their identifications based on genotyping

obtained in this study In the authorrsquos opinion molecular techniques are essential tools for determining

the specific coccidial species responsible for individual and group outbreaks of coccidiosis and for

further understanding of the eimeriid host-parasite relationships To this end the nu 18S rDNA region and

whole mitochondrial (mt) genomes of E ictidea and E furonis were sequenced these mt whole genome

sequences are the first for the Eimeria species of mustelids to be entered into GenBank These sequences

may provide suitable targets for the development of highly discriminatory PCR-based methods of

identification that could be applied to fecal tissue or even formalin-fixed paraffin-embedded samples

(see Chapters 2 and 3) Methodological improvements such as less expensive next generation sequencing

methods that could be applied to diagnostics will depend on the availability of high quality reference

sequences such as generated herein Furthermore evaluation of the evolution of the eimeriid coccidia

using phylogenetic analysis based on additional whole mitochondrial genome sequences may allow for

more accurate determinations of relationships between parasite species and timing of their divergence

from common ancestors

One of the goals of this research was to investigate the possibility of using the domestic ferret as

an experimental model to study enteric coccidiosis in the BFF Experimental work cannot be carried out

147

in the BFF due to its endangered status We showed that the domestic ferret is susceptible to infection

with Eimeria ictidea originating from BFF Infected domestic ferrets showed similar clinical signs and

pathologic lesions to BFF strengthening the possibility of their serving as a suitable model Future

investigations would include studies of the domestic ferretrsquos immune response to enteric coccidial

infection in vivo drug trials including pharmacokinetic pharmacodynamic and efficacy studies of

anticoccidial medications investigations into patterns of drug resistance in coccidial species of ferrets

and oral vaccine development

Ultimately the goal of research on coccidiosis in BFF may be the development of an autogenous

vaccine to improve the survival of ferret kits and reduce coccidiosis-related morbidity and mortality in

BFF captive breeding programs It may be possible to use the domestic ferret to select for E ictidea that

are more highly adapted to this host perhaps with a commensurate loss of virulence to its original host If

this were to be the case domestic ferret-derived coccidia could be used in BFF as vaccine organisms

Alternately methods such as use of a bioshuttle (live vaccination with coccidia followed by anticoccidial

treatment to limit pathogenicity) might be useful for generating long-lasting immunological protection in

BFF against E ictidea Improving immunity through vaccination could assist in reducing coccidial

shedding and disease in BFF associated with stressful life events such a breeding weaning and transfer

between institutions There is minimal available data on the significance of coccidiosis in wild

populations of BFF and limited means of disease surveillance post release however it is logical to

assume that the stresses associated with release to the wild might result in clinical disease as happens with

stressed BFF in captivity Reducing morbidity and mortality associated with coccidiosis in BFF could

result in increased numbers of ferrets being released to the wild and higher survival of released ferrets

increasing the number of BFF in the wild supports the goals of the conservation initiative for the black-

footed ferret and will support the longer-term survival and recovery of this species

148

REFERENCES

Abe N Tanoue T Ohta G Iseki M (2008) First record of Eimeria furonis infection in a ferret Japan with

notes on the usefulness of partial small subunit ribosomal RNA gene sequencing analysis for

discriminating among Eimeria species Parasitol Res 103967ndash70 doi 101007s00436-008-1037-x

Adl SM Simpson AGB Farmer MA et al (2005) The new higher level classification of eukaryotes with

emphasis on the taxonomy of protists J Eukaryot Microbiol 52399ndash451 doi 101111j1550-

7408200500053x

Andrews JM (1926) Coccidiosis in mammals Am J Hyg 6784ndash798

Augustine PC Danforth HD (1986) A study of the dynamics of the invasion of immunized birds by

Eimeria sporozoites Avian Dis 30347ndash351

Barta JR Schrenzel MD Carreno R Rideout BA (2005) The genus Atoxoplasma (Garnham 1950) as a

junior objective synonym of the genus Isospora (Schneider 1881) species infecting birds and

resurrection of Cystoisospora (Frenkel 1977) as the correct genus for Isospora species infecting

mammals J Parasitol 91726ndash727 doi 101645GE-33411

Bell JA (1994) Parasites of Domesticated Pet Ferrets Compend Contin Educ Pract Vet 16617ndash620

Black-footed Ferret Recovery Implementation Team (2011) Captive Breeding

httpwwwblackfootedferretorgcaptive-breeding Accessed 8 Jan 2014

Blankenship-Paris TL Chang J Bagnell CR (1993) Enteric coccidiosis in a ferret Lab Anim Sci 43361ndash

363

Bronson E Bush M Viner T et al (2007) Mortality of captive black-footed ferrets (Mustela nigripes) at

Smithsonianrsquos National Zoological Park 1989 ndash 2004 J Zoo Wildl Med 38169ndash176

149

CAPC (2013) Current Advice on Parasite Control Intestinal Parasites - Coccidia

httpwwwcapcvetorgcapc-recommendationscoccidia Accessed 6 Jun 2015

Carpenter JW Hillman CN (1979) Husbandry reproduction and veterinary care of captive ferrets In

1978 Proceedings of the Annual Meeting of the American Association of Zoo Veterinarians

Washington DC pp 36ndash47

Catchpole J Norton CC Gregory MW (1993) Immunisation of lambs against coccidosis Vet Rec

13256ndash59

Cavalier-Smith T (2014) Gregarine site-heterogeneous 18S rDNA trees revision of gregarine higher

classification and the evolutionary diversification of Sporozoa Eur J Protistol 50472ndash495 doi

101016jejop201407002

Chappuis G (1998) Neonatal immunity and immunisation in early age  lessons from veterinary medicine

Vaccine 161468ndash1472

Cox FE (1994) The evolutionary expansion of the Sporozoa Int J Parasitol 241301ndash1316

Davis CL Chow TL Gorham JR (1953) Hepatic coccidiosis in mink Vet Med 48371ndash375

De Vos AJ (1970) Studies on the host range of ltigtEimeria chinchillaeltIgt de Vos and Van Der

Westhuizen 1968 Onderstepoort J Vet Res 3729ndash36

Dirikolu L Yohn R Garrett EF et al (2009) Detection quantifications and pharmacokinetics of

toltrazuril sulfone (Ponazuril) in cattle J Vet Pharmacol Ther 32280ndash288

Dryden MW Payne PA Ridley R Smith V (2005) Comparison of common fecal flotation techniques for

the recovery of parasite eggs and oocysts Vet Ther 615ndash28

Duszynski D Wilber PG (1997) A Guideline for the Preparation of Species Descriptions in the

Eimeriidae J Parasitol 83333ndash336

150

Duszynski DW Couch L Upton SJ (2000) The Coccidia of the World

httpbiologyunmeducoccidiacarniv2html Accessed 1 Sep 2017

El-Sherry S Ogedengbe ME Hafeez MA Barta JR (2013) Divergent nuclear 18S rDNA paralogs in a

turkey coccidium Eimeria meleagrimitis complicate molecular systematics and identification Int J

Parasitol 43679ndash685 doi 101016jijpara201303005

Evans HE An NQ (2014) Anatomy of the ferret In Fox JG Marini RP (eds) Biology and Diseases of the

Ferret 3rd edn Wiley Blackwell pp 23ndash67

Fayer R (1980) Epidemiology of Protozoan Infections The Coccidia Vet Parasitol 675ndash103

Foreyt WJ Todd AC Hartsough GR (1977) Anticoccidial Activity of Eight Compounds in Domestic

Mink AJVR 38391ndash394

Fox JG (2014) Normal Clinical and Biological Parameters In Fox JG Marini RP (eds) Biology and

Diseases of the Ferret 3rd edn John Wiley amp Sons Inc pp 157ndash185

Frenkel JK (1977) Besnoitia wallacei of Cats and Rodents  With a Reclassification of Other Cyst-

Forming Isosporoid Coccidia J Parasitol 63611ndash628

Grafner G Graubmann HD Dobbriner W (1967) Leberkokzidiose beim Nerz (Lutreola vison Schreb)

hervorgerufen durch eine neue Kokzidienart Eimeria hiepei n sp Monatshefte fur

Veterinearmedizin 22696ndash700

Haberkorn A (1971) Zur Wirtsspezifitat yon Eimeria contorta nsp (Sporozoa Eimeriidae) Z

Parasitenkd 37303ndash314

Hall MC Wigdor M (1918) Canine coccidiosis with a note regarding other protozoan parasites from the

dog J Am Vet Med Assn 5364ndash76

Hikosaka K Nakai Y Watanabe YI et al (2011) Concatenated mitochondrial DNA of the coccidian

151

parasite Eimeria tenella Mitochondrion 11273ndash278

Hillyer E V (1992) Gastrointestinal diseases of ferrets (Mustela putorius furo) J Small Anim Med 244ndash

45

Hoare CA (1927) On the coccidia of the ferret Ann Trop Med Parasitol 21313ndash320

Hoare CA (1935a) A histopathological reaction of a special type on the part of the intestinal villi in ferret

coccidiosis Trans R Soc Trop Med Hyg 292

Hoare CA (1935b) The endogenous development of the coccidia of the ferret and the histopathological

reaction of the infected intestinal villi Ann Trop Med Parasitol 29111ndash122

Hoefer HL Fox JG Bell JA (2012) Gastrointestinal Diseases In Quesenberry KE Carpenter JW (eds)

Ferrets Rabbits and Rodents Clinical Medicine and Surgery 3rd edn Elsevier Saunders St Louis

Missouri pp 27ndash45

Huelsenbeck JP Ronquist F (2001) MrBayes Bayesian inference of phylogenetic trees Bioinformatics

17754ndash755 doi 101093bioinformatics178754

Jolley WR Kingston N Williams ES Lynn C (1994) Coccidia Giardia sp and a Physalopteran

Nematode Parasite from Black-footed Ferrets (Mustela nigripes) in Wyoming J Helminthol Soc

Washingt 6189ndash94

Katoh K Misawa K Kuma K Miyata T (2002) MAFFT a novel method for rapid multiple sequence

alignment based on fast Fourier transform Nucleic Acids Res 303059ndash3066 doi

101093nargkf436

Kaye SW Ossiboff RJ Noonan B et al (2015) Biliary coccidiosis associated with immunosuppressive

treatment of pure red cell aplasia in an adult ferret (Mustela putoris furo) J Exot Pet Med doi

101053jjepm201504012

152

Koepfli KP Deere K Slater GJ et al (2008) Multigene phylogeny of the Mustelidae resolving

relationships tempo and biogeographic history of a mammalian adaptive radiation BMC Biol 610

Lariviegravere S Jennings AP (2009) Family Mustelidae (weasels and relatives) In Wilson DE Mittermeier

RA (eds) Handbook of the Mammals of the World Volume 1 Carnivores Lynx Edicions

Barcelona pp 564ndash656

Levine ND Ivens V (1970) The coccidian parasites (Protozoa Sporozoa) of ruminants

Li X Pang J Fox JG (1996) Coinfection with intracellular Desulfovibrio species and coccidia in ferrets

with proliferative bowel disease Lab Anim Sci 46569ndash571

Litster AL Nichols J Hall K et al (2014) Use of ponazuril paste to treat coccidiosis in shelter-housed

cats and dogs Vet Parasitol 202319ndash325 doi 101016jvetpar201403003

Matsubayashi M Takami K Abe N et al (2005) Molecular characterization of crane coccidia Eimeria

gruis and E reichenowi found in feces of migratory cranes Parasitol Res 9780ndash83

Meeusen ENT Walker J Peters A et al (2007) Current status of veterinary vaccines Clin Microbiol Rev

20489ndash510 doi 101128CMR00005-07

Mehlhorn H Aspock H (eds) (2008) Coccidial Drugs In Encyclopedia of Parasitology 3rd edn Springer

Berlin Heidelberg New York pp 269ndash286

Morehouse NF (1938) The Reaction of the Immune Intestinal Epithelium of the Rat to Reingection wiht

Eimeria nieschulzi J Parasitol 24311ndash317

Nukerbaeva KK Svanbaev SK (1973) Coccidia of fur bearing mammals in Kazakhstan Vestn Selrsquoskokh

Nauk Kazakh 1250ndash54

Ogedengbe JD Hanner RH Barta JR (2011) DNA barcoding identifies Eimeria species and contributes to

the phylogenetics of coccidian parasites (Eimeriorina Apicomplexa Alveolata) Int J Parasitol

153

41843ndash850

Ogedengbe ME (2015) DNA Barcoding of Apicomplexa Mitochondrial Evolution across the Phylum

University of Guelph

Ogedengbe ME El-Sherry S Ogedengbe JD et al Whatrsquos in a name Phylogenies based on combined

mitochondrial and nuclear sequences conflict with morphologically defined genera in the eimeriid

coccidia (Apicomplexa)

Ogedengbe ME El-Sherry S Whale J Barta JR (2014) Complete mitochondrial genome sequences from

five Eimeria species (Apicomplexa Coccidia Eimeriidae) infecting domestic turkeys Parasit

Vectors 7335 doi 1011861756-3305-7-335

Ogedengbe ME Hafeez MA Barta JR (2013) Sequencing the complete mitochondrial genome of Eimeria

mitis strain USDA 50 (Apicomplexa Eimeriidae) suggests conserved start positions for mtCOI- and

mtCOIII-coding regions Parasitol Res 1124129ndash4136 doi 101007s00436-013-3604-z

Pantchev N Gassmann D Globokar-Vrhovec M (2011) Increasing numbers of Giardia (but not

coccidian) infections in ferrets 2002 to 2010 Vet Rec 168519 doi 101136vrd2962

Patterson M Fox JG (2007) Parasites of Ferrets In Baker DG (ed) Flynnrsquos Parasites of Laboratory

Animals 2nd edn Wiley Blackwell pp 501ndash508

Patterson MM Fox JG Eberhard ML (2014) Parasitic Diseases In Fox JG Marini RP (eds) Biology and

Diseases of the Ferret 3rd edn Wiley Blackwell pp 553ndash572

Powers L V (2009) Bacterial and parasitic diseases of ferrets Vet Clin North Am - Exot Anim Pract

12531ndash561 doi 101016jcvex200906001

Prado ME Ryman JT Boileau MJ et al (2011) Pharmacokinetics of ponazuril after oral administration to

healthy llamas (Llama glama) Am J Vet Res 721386ndash9

154

Ruiz A Muntildeoz MC Molina JM et al (2013) Primary infection of goats with Eimeria ninakohlyakimovae

does not provide protective immunity against high challenge infections Small Rumin Res 113258ndash

266 doi 101016jsmallrumres201301006

Ryley J Meade R Hazelhurst J Robinson T (1976) Methods in coccidiosis research separation of

oocysts from faeces Parasitology 73311ndash326

Santymire R Branvold-Faber H Marinari PE (2014) Recovery of the Black-Footed Ferret In Fox JG

Marini RP (eds) Biology and Diseases of the Ferret 3rd edn Wiley Blackwell pp 219ndash231

Shi MQ Huther S Burkhardt E Zahner H (2000) Immunity in rats against Eimeria separata oocyst

excretion effects on endogenous stages and local tissue response after primary and challenge

infections Parasitol Res 86891ndash898

Sledge DG Bolin SR Lim A et al (2011) Outbreaks of severe enteric disease associated with Eimeria

furonis infection in ferrets (Mustela putorius furo) of 3 densely populated groups J Am Vet Med

Assoc 2391584ndash1588 doi 102460javma239121584

Smith NC Wallach M Petracca M et al (1994) Maternal transfer of antibodies induced by infection with

Eimeria maxima partially protects chickens against challenge with Eimeria tenella Parasitology

109551ndash557

Svanbaev SK (1956) Materials on the fauna of coccidia of wild mammals in western Kazakhstan Tr

Instituta Zool Akad Nauk Kazachskoi SSR 5180ndash191

Tavareacute S (1986) Some probabilistic and statistical problems in the analysis of DNA sequences Am Math

Soc Lect Math Life Sci 1757ndash86 doi citeulike-article-id4801403

Tenter AM Barta JR Beveridge I et al (2002) The conceptual basis for a new classification of the

coccidia Int J Parasitol 32595ndash616

155

Upton SJ (2000) Suborder Eimeriorina Leacuteger 1911 In Lee JJ Leedale GF Bradbury P (eds) An

Illustrated Guide to the Protozoa vol 1 2nd edn Allen Press Lawrence Kansas pp 318ndash339

USFWS BFF Recovery Program (2017) Black-footed Ferret Managed Care Operations Manual

(BFFMCOM)

Vermeulen AN (2005) Vaccination against coccidial parasites The method of choice In Proceeding of

the 9th International Coccidiosis Conference

Williams BH Chimes MJ Gardiner CH (1996) Biliary coccidiosis in a ferret (Mustela putorius furo) Vet

Pathol 33437ndash439 doi 101177030098589603300412

Williams ES Mills K Kwiatkowski DR et al (1994) Plague in a Black-footed (Mustela nigripes)

presence J Wildl Dis 30581ndash585

Williams ES Thome ET Appel MJG Belitsky DW (1988) Canine Distemper in Black-Footed (Mustela

nigripes) from Wyoming J Wildl Dis 24385ndash398

Wozencraft WC (2005) Order Carnivora In Wilson DE Reeder DM (eds) Mammal Species of the

World A Taxonomic and Geographic Reference 3rd edn Johnrsquos Hopkins University Press

Baltimore Maryland p 2142

Yi-Fan C Le Y Yin D et al (2012) Emendation of 2 Isospora Species (Apicomplexa Eimeriidae)

Infecting the Steppe Polecat Mustela eversmanii Lesson 1827 in China to the Genus

Cystoisospora (Apicomplexa Sarcocystidae) Comp Parasitol 79147ndash152 doi 10165445311

Yu L Peng D Liu J et al (2011) On the phylogeny of Mustelidae subfamilies analysis of seventeen

nuclear non-coding loci and mitochondrial complete genomes BMC Evol Biol 1192 doi

1011861471-2148-11-92

Zou M Guo G Zhao Y Zhang Q (2014) Detection quantifications and pharmacokinetics of ponazuril in

156

healthy swine J Vet Pharmacol Ther 37598ndash602 doi 101111jvp12126

157

APPENDICES

158

Appendix 1 Shedding of oocysts of Eimeria ictidea in black-footed ferret (Mustela nigripes) dam and kit


Collection Year 2014 2014 2014 2014 2014 2015 2016


29 - - - 0 - - -

30 - 0 - 0 - - -

31 - 0 - 0 - - -

32 - 0 - 0 - - -

33 - 0 - 0 - - -

34 0 0 - 0 0 - -

35 0 0 - 0 0 0 -

36 0 0 - 0 0 0 -

37 - 0 - 0 - 0 -

38 - 0 - 0 - 0 -

39 - 0 - 0 - 0 -

40 0 0 - 0 0 0 -

41 - 0 - 0 - 0 -

42 0 0 - 0 0 - -

43 0 0 - 0 0 0 -

44 0 0 - - 0 0 -

45 0 0 - 0 0 - -

46 0 0 - 0 0 0 -

47 0 0 - 0 0 0 -

48 0 lt14 - 0 0 0 -

49 0 0 - 0 0 0 -

50 0 0 - - 0 0 -

51 0 0 - 0 0 0 -

52 0 0 - 0 0 0 -

53 0 0 - 0 0 0 +

54 0 0 - 0 0 0 +

55 0 0 - 0 0 0 3717146

56 0 0 - 0 - 0 1084436

57 0 0 - 0 - 0 64133

58 0 324 - 0 0 0 20654

59 0 0 - 0 0 0 0

60 0 446688 - 0 0 0 0

61 0 934828 - 0 0 0 41111

62 0 530469 - 0 0 0

63 0 1617131 - 0 0 lt14

64 0 8396357 - 114 0 0

65 0 234876 - 5368 0 lt14

66 0 374625 - 9455 0 0

67 lt14 2311575 - 39579 0 -

68 0 603563 - 125051 0 0

69 - 4103036 - 27067 0 lt14

70 2470327 7759974 - 0 0 0

71 1073085 - 1784 0 0

72 3430966 - 0 0 0

73 4459536 - 7326 0 0

74 4842212 - 2 0 0

75 3627307 - 0 0 0

76 5801885 - 0 0 0

77 5994 0 0 0 0

78 589835 0 0 0


Dam Identity

159

Appendix 1 continued Shedding of oocysts of Eimeria ictidea in black-footed ferret (Mustela nigripes)

dam and kit family groups from 2014-2016

Collection Year 2014 2014 2014 2014 2014 2015 2016


79 24815 0 0 0

80 144016 0 0 0

81 97862 0 0 0

82 0 0 0

83 0 0 0

84 0 0 0

85 0 0 0

86 0 0 0

87 0 0 0

88 0 0 0

89 0 0 0

90 0 0 0

91 0 0 0

92 0 - -

93 0 - 0

94 0 - 0

95 0 0 -

96 0 - 0

97 0 0 -

98 0 0 0

99 0 0 0

100 0 0 0

101 0 0 0

102 0 0 0

103 0 0 0

104 0 0 0

105 0 0 -

106 0 0 0

107 0 0 0

108 - 0 0

109 0 0 -

110 0 0 0

111 0 0 -

112 0 0 0

113 0 0 -

114 0 0 -

115 0 0 -

116 0 0 -

117 0 -

118 0 -

119 0 0

120 0 0

121 0 0

122 0 -


Dam Identity

160



Collection Year 2014 2014 2014 2014 2014 2015 2016


123 0 0

124 0 -

125 0 -

126 0 0

127 0 0

128 0 2843

129 0 0

130 0 -

131 0 0

132 0 0

133 0 0

134 0 0

135 0 0

136 0 0

137 0

138 0

139 0

140 0

141 0

142 0

143 0

144 0

145 0

146 -

147 0

148 0

149 0

150 0

Legend lt 14 = oocyst positive samples with less than 14 oocyst per gram of feces - = no sample recorded for this date

underline = last sampling date + = coccidia present but OPG count not performed = Toronto Zoo ferret ^ = Louisville Zoo

ferret thick outer border = days treatment was received


Dam Identity

161

Appendix 2a Hematology values for domestic ferrets (Mustela putorius furo) from 49-51 days of age

prior to experimental inoculation

Test Reference

Intervala

Reference

Intervalb

101 102 103 104 105 201 202 203 204 205

Age (days) adult 70 50 50 50 50 50 48 48 48 50 48

WBC (x 109L) 27-112 53-120 86 64 77 97 76 48 8 72 104 76

RBC (x 1012

L) 50-108 55ndash74 66 58 45 5 48 47 67 5 5 42

Hb (gL) 87-177 104ndash136 121 106 90 58 94 91 122 96 98 80

HCT (LL) 04 - 051 029ndash037 037 033 027 030 029 027 038 030 030 024

MCV (fL) 44-52 478ndash548 55 56 60 60 60 58 57 59 61 58

MCH (pg) 15-18 175ndash191 18 18 20 12 20 20 18 19 20 18

MCHC (gL) 325-362 347ndash370 331 327 328 196 325 337 321 327 324 331

RDW () 12-16 - 134 127 139 139 133 131 122 136 131 127

Platelets (x 109L) 54-695 629ndash775 777 329 529 480 640 402 534 336 527 363

MPV (fL) 5-10 - 78 78 96 75 66 74 76 81 74 82

TS Protein (gL) 49-76 - 54 51 - - - - - - - -

Seg Neuts (x 109L) 1-8 15ndash48 292 141 216 281 205 187 152 151 354 251

Lymphocytes (x 109L) 1-63 28ndash63 525 416 516 64 509 254 608 468 645 456

Monocytes (x 109L) 0-09 01ndash05 026 07 031 039 038 034 024 094 031 038

Eosinophils (x 109L) 0-13 01ndash06 009 013 008 01 008 005 016 007 010 015

Basophils (x 109L) 0-02 0 009 0 0 0 0 0 0 0 0 0

Polychromasia 2-5 - 5-10 2-5 10-15 10-15 10-15 10-15 2-5 10-15 10-15 5-10

Anisocytosis Occ 1+ 1+ 1+ 1+ 1+ 1+ 1+

HJ bodies rare rare rare rare rare

crenation Occ

poikilocytosis Occ Occ Occ

shift platelets Occ Occ

hemolysis Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg

lipemia mild mild mild mild mild Neg Neg Neg mild mild

Ferret Identity

Legend - = reference value unavailable bolded = outside reference range a = adult ferret reference ranges from Animal Health Laboratory

(University of Guelph Guelph Canada) b

= reference ranges for 10-week old ferrets (Fox JG 2014) Neg = negative Occ = occasional

162

Appendix 2b Serum biochemistry values for domestic ferrets (Mustela putorius furo) from 49-51 days of

age prior to experimental inoculation

Test Reference

Intervala

Reference

Intervalb

101 102 103 104 105 201 202 203 204 205

Age (days) adult 70 50 50 50 50 50 48 48 48 50 48

Calcium (mmolL) 185-242 253-302 239 233 241 244 24 221 242 234 253 242

Phosphorus (mmolL) 112-256 278-475 239 267 29 264 31 218 286 284 315 303

Magnesium (mmolL) 08-139 - 08 08 07 08 08 06 08 07 08 08

Sodium (mmolL) 147-159 146-154 149 149 149 149 148 144 153 148 152 152

Potassium (mmolL) 37-57 47-83 44 46 42 46 48 45 47 44 47 46

Chloride (mmolL) 111-129 115-121 110 112 115 113 113 110 119 117 117 120

Carbon dioxide (mmolL) 17-29 13-27 18 18 18 17 17 16 15 15 19 17

Anion gap (mmolL) 6 - 23 - 25 24 20 24 23 23 24 20 21 20

NaK ratio - - 34 32 35 32 31 32 33 34 32 33

Total protein (gL) 51-75 44-56 49 46 44 47 44 41 52 45 49 43

Albumin (gL) 24-40 26-32 29 26 28 28 28 25 28 27 29 25

Globulin (gL) 19-41 17-24 20 20 16 19 16 16 24 18 20 18

AG ratio 053-167 13ndash12 145 130 175 147 175 156 117 15 145 139

Blood urea nitrogen (mmolL) 45-153 71-139 83 89 107 11 136 73 119 105 118 135

Creatinine (umolL) 8-67 53-124 50 58 68 59 50 7 68 41 62 92

Glucose (mmolL) 32-91 64-138 47 54 54 42 55 53 57 56 52 59

Cholesterol (mmolL) 294-894 619-860 413 386 375 393 344 349 227 357 373 346

Total bilirubin (umolL) 2 to 7 - 1 L 0 L 0 0 0 1 0 0 0 0

Conjugated biilrubin (umolL) - 0-10 1 0 0 1 1 1 0 0 1 0

Free bilirubin (umolL) 0-2 0-15 0 0 0 0 0 0 0 0 0 0

ALKP (UL) 13-237 117ndash277 180 169 172 215 175 168 241 184 177 179

GGT (UL) 0-40 2ndash20 1 1 6 10 4 0 1 1 5 9

AST (UL) - 63ndash152 61 58 48 61 64 59 93 61 58 69

ALT (UL) 39-196 95ndash544 95 105 89 105 106 82 234 137 115 156

CK (UL) 74-294 - 513 330 496 560 530 492 793 539 479 724

Amylase (UL) - - 23 28 35 35 29 28 29 24 36 23

Lipase (UL) - - 65 63 60 64 62 56 67 69 60 68

Calculated osmo (mmolL) - - 298 300 301 301 303 289 311 300 308 311

Ferret Identity


(University of Guelph Guelph Canada) b = reference ranges for 10-week old ferrets (Fox JG 2014)

163

Appendix 3a Hematology values for domestic ferrets (Mustela putorius furo) inoculated orally with

Eimeria ictidea

Test Reference

Intervala

Reference

Intervalb

102 103 104 105 201 203 205

Age (days) adult 98-112 92 91 99 99 97 74 92

Days post inocculation 8 7 15 15 15 11 10

WBC (x 109L) 27-112 52ndash150 94 124 81 85 88 85 142

RBC (x 1012

L) 50-108 62ndash92 71 62 73 60 65 63 65

Hb (gL) 87-177 127ndash159 122 110 122 98 103 114 102

HCT (LL) 04-051 030ndash043 037 033 037 031 032 035 032

MCV (fL) 44-52 50ndash54 53 53 52 51 50 55 49

MCH (pg) 15-18 16-21 17 18 17 16 16 18 16

MCHC (gL) 325-362 351ndash426 328 332 325 319 320 326 317

RDW () 12-16 - 122 126 129 129 145 127 145

Platelets (x 109L) 54-695 376ndash610 524 413 445 117 303 42 429

MPV (fL) 5-10 - 8 78 72 66 67 14 8

TS Protein (gL) 49-76 - 66 63 58 66 55 59 59

Seg Neuts (x 109L) 1-8 21ndash62 160 446 203 170 211 170 554

Lymphocytes (x 109L) 1-63 16ndash79 761 657 551 646 59 646 682

Monocytes (x 109L) 0-09 01ndash02 019 087 024 026 044 026 085

Eosinophils (x 109L) 0-13 03ndash09 0 037 032 009 026 009 099

Basophils (x 109L) 0-02 0 0 012 0 0 009 0 0

Polychromasia 2-5 - 1-3 0-2 0-2 1-3 1-3 2-5 5-10

Anisocytosis Occ Occ Occ

HJ bodies rare

crenation

rouleaux

poikilocytosis


hemolysis Neg

Ferret Identity

Legend - = reference value unavailable bolded = outside reference range a = adult ferret reference ranges from Animal Health

Laboratory (University of Guelph Guelph Canada) b

= reference ranges for 10-week old ferrets (Fox JG 2014) Neg = negative

Occ = occasional = many platelet clumps

164

Appendix 3b Serum biochemistry values for domestic ferrets (Mustela putorius furo) inoculated orally

with Eimeria ictidea

Test Reference

Intervala

Reference

Intervalb

102 103 104 105 201 203 205

Age (days) adult 98-112 92 91 99 99 97 74 92


Calcium (mmolL) 185-242 245-268 243 222 238 233 232 236 240

Phosphorus (mmolL) 112-256 200-323 292 223 248 298 268 288 306

Magnesium (mmolL) 08-139 - 06 08 08 08 09 08

Sodium (mmolL) 147-159 148-155 147 150 150 148 148 150

Potassium (mmolL) 37-57 45-55 42 41 51 41 50 44

Chloride (mmolL) 111-129 114-124 114 113 118 112 111 115

Carbon dioxide (mmolL) 17-29 16-24 16 21 19 20 12 18

Anion gap (mmolL) 6 - 23 - 21 20 18 20 30 21

NaK ratio - - 35 37 29 36 30 34

Total protein (gL) 51-75 49-64 52 55 55 49 56 57

Albumin (gL) 24-40 30-36 28 20 27 27 26 29 27

Globulin (gL) 19-41 19-30 32 28 28 23 27 30

AG ratio 053-167 11-17 063 096 096 113 107 090

Blood urea nitrogen (mmolL) 45-153 50-150 92 87 119 92 110 98 149

Creatinine (umolL) 8-67 706-1414 56 26 49 40 50 46 79

Glucose (mmolL) 32-91 688-943 52 59 64 71 58 17 66

Cholesterol (mmolL) 294-894 440-640 453 429 262 475 370 285

Total bilirubin (umolL) 2 to 7 - 0 0 0 0 1 1

Conjugated biilrubin (umolL)- - 1 0 0 0 0 0

Free bilirubin (umolL) 0-2 - 0 0 0 0 1 1

ALKP (UL) 13-237 41-181 124 120 213 120 146 170 196

GGT (UL) 0-40 1-2 2 1 3 0 0 2

AST (UL) - 47-128 48 95 104 66 100 100

ALT (UL) 39-196 78-279 133 110 140 203 158 183 281

CK (UL) 74-294 - 382 765 1190 578 680 930

Amylase (UL) - - 24 37 31 28 33 35

Lipase (UL) - - 60 65 72 72 86 79

Calculated osmo (mmolL) - - 296 305 305 300 296 309

Ferret Identity



= reference ranges for 10-week old ferrets (Fox JG 2014)

165

Appendix 4 Domestic ferret (Mustela putorius furo) weekly monitoring sheet

Mon

8 AM

Mon

4 PM

Tues

8 AM

Tues

4 PM

Wed

8 AM

Wed

4 PM

Thurs

8 AM

Thurs

8 PM

Fri

8 AM

Fri

4 PM

Sat

8 AM

Sat

4 PM

Sun

8AM

Sun

4 PM

Mentation

Weight (g)

Respiratory

Rate

Vomit

(+ ++ +++)

Diarrhea

(+ ++ +++)

Urination

(+ ++ +++)

Defecation

(+ ++ +++)

Food

offered

Food

remaining

Water remaining

(ml)

Treatments

Other

observations

Initials of

observer

166

Animal ID ________________________________________ Week ______________

Monitoring Criteria

Mentation BAR (bright alert responsive) QAR (quiet alert responsive) depressed lethargic quiet

Weight measured in grams to be performed once weekly (pre-inoculation) and once daily (post-inoculation)

Respiratory rate measured in breaths per minute

Vomit + (small amount) ++ (moderate amount) +++ (large amount)

Diarrhea + (small amount) ++ (moderate amount) +++ (large amount)

Urination + (small amount) ++ (normal amount) +++ (large amount)

Defecation + (one pile) ++ (two piles) +++ (3+ piles)

Food offered Y (yes) N (no)

Food remaining A (all) P (partial) N (none)

Please note any additional observations in the appropriate section

Monitoring Times

Pre-inoculation ndash ONCE daily at 8am during cage cleaningfeeding

Post-inoculation ndash TWICE daily at 8am and 4pm

Critical patients (as determined by veterinary examination) ndash 3-6 times daily (based on veterinarian recommendation) ndash switch to 24

hour care sheet

Intervention Points

If any ferret develops vomiting diarrhea goes off food or exhibits gt 3 weight loss both the PI and graduate student listed below are

to be notified and the animal will be assessed to determine further treatment plans

Removal Criteria

If an individual ferret develops severe gastrointestinal or systemic disease during the course of the study as characterized by abnormal

mentation marked dehydration 10 weight loss within 24-48 hours profuse diarrhea unresponsive to treatment anorexia of 2 or

more days or frequent vomiting unresponsive to treatment the individual will be removed from the study and humanely killed

167

Appendix 5 Domestic ferret (Mustela putorius furo) 24 hour intensive monitoring sheet

Animal ID ___________________________________________ Date___________________

0700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800

Mentation

Weight (g)

Heart Rate

Respiratory Rate

Temperature (C)

Water offered

(mL)

Water consumed

(mL)

Food offered

(type and

amount in

grams)

Food consumed

(type and

amount in

grams)

Presence of urine

Presence of feces

168

Character of

feces

Presence of

vomit

Character of

vomit

Medications to

be administered

Other

observations

169

1900 2000 2100 2200 2300 2400 100 200 300 400 500 600

Mentation

Weight (g)

Heart Rate

Respiratory Rate

Temperature (C)

Water offered

(mL)

Water consumed

(mL)

Food offered

(type and amount

in grams)

Food consumed

(type and amount

in grams)

Presence of urine

Presence of feces

Character of

feces

Presence of vomit

Character of

vomit

170

Medications to be

administered

Other

observations

Monitoring Criteria


Weight measured in grams to be performed once weekly (pre-inocculation) and once daily (post-inocculation)






171




Monitoring Times


hour care sheet

Intervention Points



Removal Criteria




172

Appendix 6 Domestic ferret (Mustela putorius furo) infection trial standard operating procedures

Daily Fecal Collection

1 Collection of all feces passed in a 24 hour period will be performed once daily for all ferrets from

July 13 to July 24 inclusive

2 CAF Isolation staff will collect all fecal material present at the time of daily cage cleaning and

place in individual pre-labelled plastic bags (one per cage)

3 The amount of non-fecal matter (bedding etc) collected should be as minimal as possible

4 CAF isolation staff will record fecal character observations daily for each sample by ticking the

appropriate box on the baggie label

5 Isolation staff will place samples in the necropsy cooler (4 degC) while awaiting collection by

summer student

6 Monday to Friday fecal samples will be collected by A Rodriguez and brought for processing to

the Barta Lab in Pathobiology Samples collected Saturday and Sunday will be put on hold in a

refrigerator (4degC) until pick up on Monday

Example Bag label

Ferret ID Date

Weight of Feces

Fecal Character Normal

Soft

Liquid

Bloody

Abnormal odour

Physical Examination and Health Assessments

1 Manual restraint by hand or in towel by CAF Isolation staff and project personnel If required

examination +- blood collection may be performed under general anesthesia with isoflurane

2 Physical examination body weight HR RR temperature to be performed by PIs (Adriana Pastor

and Dale Smith)

3 Blood collection (~1mL per ferret) is to be performed from the jugular vein if under manual

restraint or the cranial vena cava under anesthesia by PIs using a 25g needle and 1cc syringe

Blood will be collected into small heparinized tubes and submitted to AHL for

CBCBiochemistry

4 All physical examination findings will be recorded on the Exam Sheet

5 Any minor wounds will be treated as appropriate (to be determined by PIs)

Inoculation of Ferrets with Coccidia

1 All ferrets to be inoculated will have been confirmed negative on daily fecals for two weeks

173

2 Brief physical examination by PIs to confirm that the ferrets are healthy to continue in study

3 The concentrated oocyst solution in sterile saline (up to a volume of 1mL) will be combined with

up to 1 mL of FerreTone (or another highly palatable substance if more preferred by the ferrets)

and ferrets will be allowed to consume the mixture ad lib while being monitored

4 Control ferrets will be administered saline only (equal mL to oocyst solution) with 1 mL of

FerreTone (or other substance as used for experimental group)

5 If ferrets refuse to consume the mixture oral inoculation via syringe of concentrated oocyst

solution will be performed by PIs under manual restraint

6 If ferrets are resistant to manual restraint for oral inoculation then inoculation will be performed

by PIs under general anesthesia via gastric tube (8 Fr red rubber)

Euthanasia Protocol

1 Ferrets to be euthanized will be masked down with isoflurane under manual restraint (or in an

anesthetic chamber as judged most appropriate by the PI)

2 Once anesthetized the ferret will be weighed and any blood fecal or other samples will be

collected as required (as determined by PIs)

3 Once an adequate plane of anesthesia is obtained (as determined by PIs) potassium chloride at a

dose of 2 mEq K+kg will be administered either via vena caval puncture or cardiac puncture to

induce cardiac arrest

4 Presenceabsence of respirations heartbeat corneal reflex will be used to assess death

Necropsy Protocol

1 Measure the ferret from nose to tail base (body length)

2 Perform standard necropsy but start with gastrointestinal tract first then thoracic and abdominal

viscera

3 Gently flush the entire contents of the intestine with 12 mL saline into a sterile urine cup

Potassium dichromate should be added in a 11 volumevolume ratio to the same container and

mixed with the combined intestinal contents and saline Label the container with the ferretrsquos ID

ferret group ID date and place on appropriate shelf in the refrigerator (4 degC) in the Barta lab

4 Measure the length of the gut from duodenum to anus

5 Collect paired sections of intestine for histological sectioning and frozen These sections should

be collected along the entire length of the gut from duodenum to rectum (see below for GI length

calculations)

6 For histological sections cut a 2 cm long section of bowel open completely on one side place on

a pre-cut section of box-board and staple both ends to the board to create a flat section Place all

gastrointestinal sections (attached to the board) in Serra fixative solution (100 ethanol (60

VV) 37 formaldehyde (30 VV) glacial acetic acid (10 VV)) for fixation and trim in

cross (transverse) sections

7 Adjacent to each sample removed for histopathology remove another 2 cm section intact and

place in a labelled Whirl-pack for freezing

8 Box-board and pre-labelled Whirl-packs should be labelled in pencil and sharpie respectively

with the following information Animal ID Zoo pm number section of gut (information should

include the region of the gut - jejunum colon etc and the length from the pylorus to the section)

174

Ferret gastrointestinal length calculations (from Evans amp An 2014)

Adult ferret body length 36-41 cm

NB In domestic ferrets there is ~51 ratio of small intestine to body length

Adult domestic ferret GI lengths

Small intestine ndash 182-198 cm

Large intestine - ~10 cm long 06 cm diameter (colon ndash 7 cm rectum ndash 2 cm anus ndash 1 cm)

Based on the above information

Small intestine Six sections of the small intestine from duodenum to ileum will be collected each ~ 25-40

cm apart depending on the size of the ferret

Large intestine Collect two sections of colon (4 cm each) at 25 and 75 of the length of the colon from

the junction of the small and large intestine to the rectum Smaller ferrets may allow only on section

based on colon length

Laboratory SOPs

General notes on processing fecal samples

1 Samples will be collected from the isolation facility necropsy room cooler daily from Monday-

Friday for the entire 6 weeks of the project

2 Upon transfer to the lab each baggie will be weighed after filling to determine the amount of

feces (grams) in each bag and that number entered onto the label on the bag

3 All information regarding fecal processing will be entered onto the spreadsheet for the individual

ferret including ID weight of feces feces character date of sample collection date of sample

processing fecal flotation method presence or absence of oocysts oocyst quantification

Fecal Processing - Weeks 1+2 (acclimation period)

1 After weighing transfer the contents of one baggie to a small sieve on top of a paper cup

2 Fill a single glass container (for fecal flotation) with saturated salt solution

3 Slowly pour small amounts of the solution over the feces mix and strain liquid contents in the paper

cup using a tongue depressor

4 Dispose of the remaining fecal matter in the sieve

5 Pour the contents of the paper cup back into the glass container and cover slip for 5-7 minutes

6 Place coverslip on a clean glass slide and viewscan under microscope at 10x power for presence of

oocysts

7 Record all findings on the provided spreadsheet

8 Notify A Pastor if any oocysts are detected If detected contents of the slide should be flushed back

into the glass container with distilled water and contents of glass container should be transferred to a

50 mL plastic conical tube Clean to remove salt as per Barta lab SOP and mix with potassium

dichromate equal parts by volume to amount of oocysts in diH2O Place a checkmark on the lid of the

50 mL conical vial containing the remainder of the feces and store in the lab fridge at 4 degC

175

Fecal Processing - Weeks 3-7 (infection trial)

1 After weighing transfer the entire contents of one baggie to a small sieve on top of a paper cup

2 Slowly pour small amounts of distilled water over the feces (enough to wet) mix and strain liquid

contents in the paper cup using a tongue depressor until feces appear almost dry


4 Pour the contents of the paper cup into a 50 mL conical tube If samples are not going to be counted

on the same day then mix potassium dichromate (25 wv) 11 by volume with sieved fluid from

the paper cup and store at 4 degC (refrigerator) until ready to perform OPG counts

If proceeding with the count the same day

5 Pipette and place one drop of the sieved fecal fluid on a slide to determine approximate oocyst

concentration If oocysts rare to none ndash dilute 12 during step 6 if moderate numbers ndash dilute 19 if

too numerous to count ndash dilute 199 (or perform serial dilutions of 10x from initial 19 dilution)

6 Transfer x mL of the mixture from the 50 mL conical tube into a clean 15 mL conical tube and mix

with appropriate amount of saturated salt solution for desired dilution

7 Fill both sides of the McMaster counter chamber and count the number of oocysts per side (total for

one side= total number of oocysts from all 6 sections of the chamber )

8 Average the total counts from both sides

9 Use the following calculation to determine the oocyst per gram count

OocystsmL = oocysts counted times 666 times dilution (ie 3 if dilution 12)

OPG= oocystmL times total volume recorded at end of step 4


11 Notify A Pastor if any oocysts are detected If detected place a checkmark on the lid of the 50 mL

conical vial containing the remainder of the feces and store in the lab fridge at 4 degC

12 For all samples for which no oocysts are detected during steps 6-8 follow up with routine salt

flotation of the remainder of the fecal sample from step 6 (use instructions for fecal processing from

weeks 1-2)

ABSTRACT INVESTIGATING ENTERIC COCCIDIOSIS IN THE BLACK-FOOTED (MUSTELA NIGRIPES) AND

DOMESTIC FERRET (MUSTELA PUTORIUS FURO)

Adriana R Pastor Advisors

University of Guelph 2017 Dr D A Smith

Dr J R Barta

Enteric coccidiosis is a major cause of death in both juvenile and adult black-footed ferrets (BFF

Mustela nigripes) in captive breeding programs that reduces the availability of animals for release to their

former North American range Coccidiosis is poorly understood in BFF but in vivo experimental infection

in this endangered host is untenable The goal of this research was to better characterize the etiologic

agents and natural history of enteric coccidiosis in BFF and to evaluate the domestic ferret (DF Mustela

putorius furo) as a model for experimental infection

Morphometric and molecular characterization of coccidia from BFF and DF was undertaken

Only Eimeria ictidea was identified in juvenile and adult BFF from 1999-2016 at the Toronto Zoo and

from BFF at the Louisville Zoo in 2016 Eimeria furonis and Isospora (=Cystoisospora) laidlawi were

identified in DF fecal and necropsy samples from Canadian and European diagnostic laboratories during

2008-2017 Molecular characterization of these parasites included generation of complete mitochondrial

genomes and nuclear 18S rDNA sequences for Eimeria ictidea and Eimeria furonis from BFF and DF

respectively Partial sequences were obtained from the same genetic targets from I (=C) laidlawi from

DF DNA isolation from formalin fixed paraffin embedded tissues and PCR amplicon sequencing

permitted identification of coccidia in BFF and DF tissues dating from 1999 to present

Retrospective and prospective analyses of medical and pathology records supplemented with

parasitological evaluation of repeated fecal samples was performed to determine the natural history of









iv

DEDICATION


v

ACKNOWLEDGEMENTS


to thank for that























vi









been as successful


















vii























Adriana Pastor

Toronto August 2017

viii



exceptions









ix

TABLE OF CONTENTS

ABSTRACT ii

DEDICATION iv

ACKNOWLEDGEMENTS v



LIST OF TABLES xiii

LIST OF FIGURES xiv


ABBREVIATIONS xvi


11 INTRODUCTION 1

12 APICOMPLEXA 1













25




ferrets 28



x




171 Theory 32



181 Objectives 36

182 Hypotheses 36

183 Applications 36



21 INTRODUCTION 39






23 RESULTS 47





24 DISCUSSION 50



31 INTRODUCTION 60





33 RESULTS 66



34 DISCUSSION 69

xi



41 INTRODUCTION 78







43 RESULTS 83


432 Pathology 86


44 DISCUSSION 88




51 INTRODUCTION 104


521 Animal care 106




525 Hematology 110



53 RESULTS 111




534 Hematology 113

535 Necropsy 114

54 DISCUSSION 115

xii




61 INTRODUCTION 129


621 Parasites 130




63 RESULTS 133

64 DISCUSSION 134


REFERENCES 148

APPENDICES 157

xiii

LIST OF TABLES





from 2008-2015 56

























xiv

LIST OF FIGURES


























xv

LIST OF APPENDICES














xvi

ABBREVIATIONS




bp Base pair






CytB Cytochrome b





L Length

LSU Large subunit

mt Mitochondrial


nu Nuclear



rDNA Ribosomal DNA

SI Shape index





SSU Small subunit



W Width

1


11 INTRODUCTION














12 APICOMPLEXA








2









(Cox 1994)






httptolweborg

2

1

3

4

5

3


























4











author



5






















6






















7

























8
























9








2012)

10



species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Cytoisospora

eversmanni

Mustela

eversmanii

(Steppe polecat)

Mustela

putorius

(European

polecat)


W 148 (16ndash12)

LW 13 (11ndash16)

M absent

PG absent

OR absent

L 115

(10ndash135)

W 98

(9ndash11)

LW 12

(11ndash15)

SB absent

SR present


Svanbaev 1956

Nukerbaeva amp

Svanbaev 1973

1977

Cystoisospora

pavlovskyi

Mustela

eversmanii

Mustela

putorius


W 273 (265ndash285)

LW 12 (11ndash14)

M absent

PG absent

OR absent

L 195

(18ndash21)

W 144

(12ndash15)

LW 14

(12ndash15)

SB absent

SR present


Svanbaev 1956

Nukerbaeva amp

Svanbaev 1973

1977

Eimeria

baskanica^

Mustela

erminae

(ermine)


ends

L 112-126

W 84-98

M absent

PG absent

OR present


Svanbaev 1977


putorius

Mustela

putorius furo

(dom ferret)

Mustela

nigripes (BFF)

Mustela vison

(mink)

Homoxenous

Small intestine

rectum (H 1927)

Jejunumileum (BP

1993)

Spherical ndash

subspherical

L 11-14

W 10-13

OW 2 layers

M absent

PG absent

OR absent

Spindloid

L 8-9

W 4

SB present

SR present


et al 1993

Hoare 1927 1935b

Jolley et al 1994

Nukerbaeva amp

Svanbaev

19731977

Williams et al 1988

1992 1996


Bile duct

Spherical

L 13-17

W 13-17

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

L 6

W 4

SB absent

SR absent


Grafner et al 1967

11


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References


eversmanni

Mustela

nigripes

Mustela

putorius

Mustela

putorius furo

Homoxenous

Small intestine


L 13-27

W 13-21

OW 2 layers

M present

PG absent

OR absent

Ovoid

(irregular)

L 115

W65

SB present

SR present

- Hoare 1927 1935a

1935b

Jolley et al 1994

Litvenkova 1969

Svanbaev 1956

Tinar 1985

Williams et al 1988

1992


(tayra)

Homoxenous

Feces

Ovoid

L 21-25

W 18-20

OW outer

layer smooth

M absent

PG absent

OR absent

Ellipsoid

L 10-12

W 65

SB present

SR present

Elongate (one

end broader than

the other)

Carini amp da

Fonseca 1938


(European

badger)


L 20plusmn018

W 157plusmn002

LW128plusmn0017

(112-15)

OW 2 layers

(outer

smooth)

M absent

PG present

OR present

Ovoid

L

119plusmn0018

W 65plusmn008

LW 183

(155-24)

SB present

L 90plusmn005

W 324plusmn0025

SRB present

Anwar et al 2000

Kotlan amp Pospesch

1933


Mustela nivalis

(snow weasel)

Homoxenous

Duodenumileum

Spherical or

Ellipsoid

L 18-26

W 14-24

OW 2 layers

M absent

PG present

OR absent

Ovoid

L 8

W 5

SB present

SR present

Broad at one

end and tapered

at other

L 7

W 3

Glebezdin 1978

Iwanoff-Gobzem

1934

Levine 1948

Musaev amp Veisov

1983

Tinar 1985


(sable)

Homoxenous

Gut

Spherical or

subspherical

L 112-126

W 112

OW 2 layers

M absent

OR absent

Ovoid

L 56

W 42

SR present



L avg 216

W avg 180

LW 1076

OW 2 layers

M absent

PG absent

OR absent

Ovoid

L 96-112

W 56-72

SR absent


Yakimoff amp

Gousseff 1934

Yakimoff amp

Terwinsky 1930

1931

12


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Eimeria vison

(Eimeria

mustelae)

Mustela

putorius

Mustela

putorius furo

Mustela vison

Homoxenous

Small intestine

+- large intestine

Ovoid

L 17-22

W 9-18

OW 2 layers

M absent

OR

sometimes

present

Ovoid or

Piriform

L 10

W 55

SB absent

SR present

Curved or Club

shaped

L 9

W 25


Foreyt et al 1977

Kingscote 1934

1935

Levine 1948

McTaggart 1960

Nukerbaeva amp

Svanbaev

19731977

Tinar 1985

Umurzakov amp

Nukerbaeva 1985

Wolter 1961

Zimmermann 1959


(Libyan striped

weasel)

Homoxenous

Feces

Spherical

L 25-27

W 25-27

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

Ovoid

L 15-17

W 10-12

SB absent

SR present

Elongate

L 135

W 3

Prasad 1961


(mountain

weasel)

Homoxenous

Gut

Oval or spherical

L 280-336

W 252-280

LW 121 (111-

124)

OW 2 layers

M absent

PG absent

OR absent

Ovoid or

spherical

L 140-168

W 111-168

SR present

Svanbaev amp

Rachmatullina

1971


Large intestine

Ovoid

L 224 (220-250)

W 174 (160-190)

LW 135 (133-

137)

OW 1 layer

PG present

OR present

Ovoid

L 120

(100-130)

W 70 (60-

80)

SB present

SR present


1983

13


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Isospora

hoogstraali


Feces

Ellipsoid

L 37-41

W 32-34

OW 2 layers

(outer

smooth)

M absent

PG some

OR absent

Ovoid

L 19-21

W 13-15

SB absent

SR present

Club-shaped

L 18-19

W 4-6

Prasad 1961


putorius

Mustela

putorius furo

Mustela vison

Homoxenous

Feces

Intestinal contents

Ovoid L

320-368

W 272-304

OW 2 layers

M absent

PG absent

OR absent

Ellipsoid

L 208

W 144

SB absent

SR present


Hoare 1927

Levine 1948

McTaggart 1960

Nukerbaeva amp

Svanbaev 1973

1974 1977

Tinar 1985


(European

otter)

Lutra

canadensis

(North

American river

otter)


L 312 (275-32)

W 296 (28-31)

LW 104

(10-112)

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

Ellipsoid

L 182 (17-

19)

W 144 (14-

16)

LW128

(12-14)

Sb absent

sSB absent

SR present

Spindle- shaped

L 124

W 25

SRB present

Torres et al 2000

Hoover et al 1985

Isospora

martessii


Gut

Ovoid short oval or

spherical

L 252 ndash 280 196

168

W 168 ndash 224 168

168

OW 2 layers

M absent

OR absent

Ovoid

L 112-168

W 84-112

SR present



L 328plusmn034

W 269plusmn019

LW122 (110-

157)

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

Ellipsoid

L

215plusmn0166

W 14plusmn012

LW 155

(133-185)

SR absent

Round at one

end other end

tapered

L 142plusmn116

W 40plusmn017

SRB absent

Anwar et al 2000

Glebezdin 1978

Kotlan amp Pospesch

1933

Pelleacuterdy 1955

14


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Isospora

mustelae (nomen

nudum)


7 W

225



Large intestine

Ovoid

L 206 (200-230)

W 184 (180-210)

LW 11 (109-111)

OW 1 layer

PG absent

OR absent

Ovoid

L 125

(120-130)

W 80 (70-

90)

SR present

Lemon or pear

shaped

Musaev amp Veisov

1983

Unnamed

ldquoCoccidiardquo^

Mustela

nigripes


Unnamed

ldquoCoccidiardquo^

Mustela

nigripes

Trachea bronchus

bronchial glands


Unnamed

Eimeria sp^

Mustela

nigripes

Feces

intestinal contents

Ovoid

L 350-386

W 212-232


Williams et al

1992

Unnamed

Eimeria sp^

Mustela

putorius furo


et al 1993

Unnamed

Eimeria sp^


Large intestine

Ovoid-ellipsoid L

2031 (1712-2162)

W 148 (1225-

1681)

LW 136 (121-16-

)

OW 1 layer

PG absent

OR absent

Ovoid or

pear-shaped

L 60-100

W 40-80

SR present

Elongate

L 50-90

W 30-70

Musaev amp Veisov

1983

Unnamed

Eimeria sp^

Martes martes

(marten)

Homoxenous Ovoid

L avg 216

W avg 180

LW 1076


SR present

L 126

W 60

Yakimoff and

Gousseff 1934

Unnamed

Isospora sp^

Mustela

putorius furo

Feces - - - - Bell 1994

Unnamed

Isospora sp^

Mustela

putorius furo

Feces - - - - Bell 1994




15


similar host

16



























17


























18

















1967)









19


























20

























21

























22

















reproduction









23


























24
























Gorham 1953)

25

























26

























27

























28
















lacking









29

























30








footed ferrets
















31



























32
















171 Theory









33


























34

























35
























36


181 Objectives








footed ferret

182 Hypotheses







ferrets

183 Applications






37














38






review)

ABSTRACT


















putorius furo

39

21 INTRODUCTION

























40




infection trial














posterior end







41


the sporocyst























42












E furonis nu 18S















43















221 Fecal samples










44

























45























46



















47

23 RESULTS






















48























49


life stages























50














24 DISCUSSION








Canada or the EU

51


























52

























53









identification








relationships

ACKNOWLEDGEMENTS








54


(400566) to JRB

55
















56






Cystoisospora sp


Eimeria furonis


Eimeria ictidea


2008 3140 (21) 6214 (28) 3 2 0 4 0 0

2009 2160 (12) 14214 (65) 2 9 0 5 0 0

2010 8127 (63) 20213 (94) 7 10 1 10 0 0

2011 0114 (0) 17215 (79) 0 9 0 7 0 1

2012 3108 (28) 10231 (43) 2 4 1 6 0 0

2013 281 (25) 16270 (59) 2 13 0 2 0 1

2014 6127 (47) 12234 (51) 5 6 1 6 0 0

2015 4108 (37) 9249 (36) 4 3 0 6 0 0

Total 28 (29) 104 (56) 25 56 3 46 0 2

Average

year 35 130 31 70 04 58 00 03


57


Sample ID Source



mt COI GenBank

Accession

nu 18S rDNA

GenBank Accession

















58





25 microm

59








60



NIGRIPES)

ABSTRACT













31 INTRODUCTION








61











2014)















62


























63









et al 1994)

















64
















SSP facilities


321 Fecal samples







65














amp Methods)








66


















Figure 31

33 RESULTS





67


























68


























69






34 DISCUSSION




















70








conducted



















71





















ACKNOWLEDGEMENTS





72



73















74







10 microm 5 microm

75





Identity






Identity




76


nigripes)













fresh feces


POS E cf ictidea

-

E ictidea













histologic section


77


(Mustela nigripes)



Length (microm) 2333 (2055-2583) 2456 (2111-2848) 2505 (2079-3008) 2779 (2590-3060) 2493 (2036-2822) 2139 (1859-2372) 2398 (1859-3057)

Width (microm) 1676 (1373-2180) 1835 (1643-2232) 1975 (1509-2360) 2253 (2092-2383) 1803 (1549-2017) 1751 (1610-1888) 1855 (1373-2383)

Shape index 135 (103-160) 134 (113-156) 127 (105-155) 124 (113-138) 139 (114-154) 122 (101-145) 130 (101-160)


78



ABSTRACT
















41 INTRODUCTION






79











self-sustaining













80
























81




Family groups








Adults













82























83











mortality rates

43 RESULTS


Family groups






Table 42 Figure 41)





84








treatment with TMS

















85

Adults


























86












432 Pathology





Gross Pathology







Histopathology

87






(n=1)



















88
















those populations

44 DISCUSSION




institutions

89
























90


























91


























92















clinical disease










93


























94









drug resistance

95



0

50000

100000

150000

200000

250000

300000

350000

400000

29

33

37

41

45

49

53

57

61

65

69

73

77

81

85

89

93

97

10

1

10

5

10

9

11

3

11

7

12

1

12

5

12

9

13

3

13

7

14

1

14

5

14

9

OP

G

Age of Kits


96




25 microm

97



Collection Year 2014 2014 2014 2014 2014 2015 2016


29 - - - 0 - - -

30 - 0 - 0 - - -

- - - - -

34 0 0 - 0 0 - -

35 0 0 - 0 0 0 -

- -

46 0 0 - 0 0 0 -

47 0 0 - 0 0 0 -

48 0 lt14 - 0 0 0 -

- -

53 0 0 - 0 0 0 +

54 0 0 - 0 0 0 +

55 0 0 - 0 0 0 3717146

56 0 0 - 0 - 0 1084436

57 0 0 - 0 - 0 64133

58 0 324 - 0 0 0 20654

59 0 0 - 0 0 0 0

60 0 446688 - 0 0 0 0

61 0 934828 - 0 0 0 41111

62 0 530469 - 0 0 0

63 0 1617131 - 0 0 lt14

64 0 8396357 - 114 0 0

65 0 234876 - 5368 0 lt14

66 0 374625 - 9455 0 0

67 lt14 2311575 - 39579 0 -

68 0 603563 - 125051 0 0

69 - 4103036 - 27067 0 lt14

70 2470327 7759974 - 0 0 0

71 1073085 - 1784 0 0

72 3430966 - 0 0 0

73 4459536 - 7326 0 0

74 4842212 - 2 0 0

75 3627307 - 0 0 0

76 5801885 - 0 0 0

77 5994 0 0 0 0

78 589835 0 0 0

79 24815 0 0 0

80 144016 0 0 0

81 97862 0 0 0

82 0 0 0

127 0 0

128 0 2843

136 0 0

150 0


Dam Identity





98



Dam Identity

Poppy

2014

Bumblefoot

2014

Aubrey

2014

Fiddlesticks

2015

Guanella

^

2016



shedding 70-81 58-70 64-73 63-69 54-61


OPG min 0 0 0 0 206

OPG max 48442 83963 2707 lt14 371714




- = missing data



Collection

Year 2015 2016 2016 2016 2016 2016 2016


Age (years) 1 3 1 1 1 1 1

1084058 0 + + + + +

+ 0 42307650 6286676 + 183150 554274

857808 16650 12805238 7777929 + 215710 377920

1604894 16650 309690 139860 + 0 25808

377042 0 599400 119880 + - 37294

554445 0 34688 385579 117920 0 5363

26640 0 16650 0 0 0 7500

0 10406 0 0 0 1090

0 20813 0 0 0

- 0 1761 0

0 0 0 0

- 0

0

0



100



Sex M F M M M F M

Age (years) 1 3 1 1 1 1 1


OPG min 266 166 104 1199 1761 1831 1090

OPG max 10840 166 423076 77779 - 2157 554274





101



Ferret

ID Year

Age















102




Year Adult Family

2003 116 (625) -

2004 819 (4211) -

2005 021 (000) 14 (2500)

2006 021 (000) 07 (000)

2007 023 (000) 08 (000)

2008 224 (833) 14 (2500)

2009 025 (000) 06 (000)

2010 326 (1154) 07 (000)

2011 125 (400) 09 (000)

2012 125 (400) 08 (000)

2013 028 (000) 05 (000)

2014 430 (1333) 09 (000)

2015 035 (000) 35 (6000)

2016 - 27 (2857)




103



Total Mortality



1997 015 (000) 023 (000) 315 (2000) 423 (1739) 1998 038 (000) 119 (526) 838 (2105) 919 (4734) 1999 047 (000) 119 (526) 1647 (3404) 119 (526) 2000 034 (000) 015 (000) 434 (1176) 315 (2000) 2001 032 (000) 016 (000) 532 (1563) 116 (625) 2002 150 (200) 020 (000) 450 (800) 220 (1000) 2003 227 (741) 018 (000) 327 (1111) 118 (555) 2004 020 (000) 016 (000) 620 (3000) 216 (1250) 2005 016 (000) 015 (000) 416 (2500) 215 (1333) 2006 030 (000) 016 (000) 230 (667) 016 (000) 2007 019 (000) 015 (000) 419 (2105) 215 (1333) 2008 034 (000) 016 (000) 1134 (3235) 316 (1875) 2009 017 (000) 016 (000) 017 (000) 116 (625) 2010 017 (000) 016 (000) 317 (1765) 316 (1875) 2011 011 (000) 016 (000) 111 (909) 216 (1250) 2012 111 (909) 017 (000) 111 (909) 317 (1765) 2013 424 (1667) 017 (000) 424 (1667) 317 (1765) 2014 126 (384) 017 (000) 326 (1154) 217 (1176) 2015 04 (000) 017 (000) 04 (000) 217 (1176) 2016 011 (000) 017 (000) 211 (1818) 117 (588)

Mean annual () 195 053 1594 1359



as a percentage

104




ABSTRACT















species

51 INTRODUCTION






105


























106






ictidea


521 Animal care

















107







study



















108





Part 1








container












109

Part 2















524 Animal welfare









110



525 Hematology












(Chapter 3)









111






53 RESULTS


















112

531 Oocyst shedding

























113







533 Clinical signs





this time

534 Hematology








114

535 Necropsy

























115

54 DISCUSSION






















116


























117


























118
























119








absent












domestic ferret





120

























121











naive















122

















25 microm

124







25 microm

25 microm

125







0

1

2

3


Nu

mb

er

of

Ferr

ets

Aff

ect

ed



126





Ferret Identity


1 0 0 0 0 0 0 0

2 0 0 0 0 0 0 0

3 0 0 0 0 0 0 0

4 0 0 0 0 0 0 0

5 0 0 0 0 0 0 0

6 0 0 0 0 0 0 0

7 lt 14 1807 0 0 0 0 0

8 11053 139 7091 0 156238 0

9 463 11733 lt 14 203 lt 14

10 578 7549 0 0 0

11 lt 14 0 0 0

12 0 lt 14 0

13 0 lt 14 0

14 0 lt 14 0


gram of feces

127



Ferret

Identity


Inoculum Oocyst

Shedding

Presence

of clinical

disease

Coccidia

present in

sections

Inoculum Oocyst

Shedding

Presence

of clinical

disease

Coccidia

present in

sections













necropsy

128



Ferret Identity


103

104 105

201

203

205











S - 05

A - 15

S - 45

A - 25

S - 05

A - 05

S - 26

A - 06

S - 06

A - 06

S - 06

A - 06

S - 08

A - 08


S - 01

A - 01

S - 01

A - 01

S - 01

A - 01

S - 02

A - 02

S - 01

A - 01 none

S - 01

A - 01




129




ABSTRACT















61 INTRODUCTION





130






















621 Parasites



131
























132
























133



63 RESULTS






64 (for E ictidea)













regions




134


Mustelidae)

64 DISCUSSION























135









136


















137












138


139


nigripes)








































140




Total length

(nucleotides)

Nucleotide

identity

Total amino

acids

Amino acid

identity

COI CDS 1443 934 (95) 480 975 (12)

COIII CDS 756 899 (76) 251 932 (17)

CytB CDS 1080 950 (54) 359 986 (5)



applicable

141




142




143





144




parasites

145




















disease management






146



























147
























148

REFERENCES






7408200500053x












363



149







13256ndash59



101016jejop201407002













150























151



45
















101093nargkf436



101053jjepm201504012

152














20489ndash510 doi 101128CMR00005-07









153

41843ndash850








Vectors 7335 doi 1011861756-3305-7-335











12531ndash561 doi 101016jcvex200906001



154
















109551ndash557







155




(BFFMCOM)




Pathol 33437ndash439 doi 101177030098589603300412













1011861471-2148-11-92


156


157

APPENDICES

158



Collection Year 2014 2014 2014 2014 2014 2015 2016


29 - - - 0 - - -

30 - 0 - 0 - - -

31 - 0 - 0 - - -

32 - 0 - 0 - - -

33 - 0 - 0 - - -

34 0 0 - 0 0 - -

35 0 0 - 0 0 0 -

36 0 0 - 0 0 0 -

37 - 0 - 0 - 0 -

38 - 0 - 0 - 0 -

39 - 0 - 0 - 0 -

40 0 0 - 0 0 0 -

41 - 0 - 0 - 0 -

42 0 0 - 0 0 - -

43 0 0 - 0 0 0 -

44 0 0 - - 0 0 -

45 0 0 - 0 0 - -

46 0 0 - 0 0 0 -

47 0 0 - 0 0 0 -

48 0 lt14 - 0 0 0 -

49 0 0 - 0 0 0 -

50 0 0 - - 0 0 -

51 0 0 - 0 0 0 -

52 0 0 - 0 0 0 -

53 0 0 - 0 0 0 +

54 0 0 - 0 0 0 +

55 0 0 - 0 0 0 3717146

56 0 0 - 0 - 0 1084436

57 0 0 - 0 - 0 64133

58 0 324 - 0 0 0 20654

59 0 0 - 0 0 0 0

60 0 446688 - 0 0 0 0

61 0 934828 - 0 0 0 41111

62 0 530469 - 0 0 0

63 0 1617131 - 0 0 lt14

64 0 8396357 - 114 0 0

65 0 234876 - 5368 0 lt14

66 0 374625 - 9455 0 0

67 lt14 2311575 - 39579 0 -

68 0 603563 - 125051 0 0

69 - 4103036 - 27067 0 lt14

70 2470327 7759974 - 0 0 0

71 1073085 - 1784 0 0

72 3430966 - 0 0 0

73 4459536 - 7326 0 0

74 4842212 - 2 0 0

75 3627307 - 0 0 0

76 5801885 - 0 0 0

77 5994 0 0 0 0

78 589835 0 0 0


Dam Identity

159



Collection Year 2014 2014 2014 2014 2014 2015 2016


79 24815 0 0 0

80 144016 0 0 0

81 97862 0 0 0

82 0 0 0

83 0 0 0

84 0 0 0

85 0 0 0

86 0 0 0

87 0 0 0

88 0 0 0

89 0 0 0

90 0 0 0

91 0 0 0

92 0 - -

93 0 - 0

94 0 - 0

95 0 0 -

96 0 - 0

97 0 0 -

98 0 0 0

99 0 0 0

100 0 0 0

101 0 0 0

102 0 0 0

103 0 0 0

104 0 0 0

105 0 0 -

106 0 0 0

107 0 0 0

108 - 0 0

109 0 0 -

110 0 0 0

111 0 0 -

112 0 0 0

113 0 0 -

114 0 0 -

115 0 0 -

116 0 0 -

117 0 -

118 0 -

119 0 0

120 0 0

121 0 0

122 0 -


Dam Identity

160



Collection Year 2014 2014 2014 2014 2014 2015 2016


123 0 0

124 0 -

125 0 -

126 0 0

127 0 0

128 0 2843

129 0 0

130 0 -

131 0 0

132 0 0

133 0 0

134 0 0

135 0 0

136 0 0

137 0

138 0

139 0

140 0

141 0

142 0

143 0

144 0

145 0

146 -

147 0

148 0

149 0

150 0





Dam Identity

161



Test Reference

Intervala

Reference

Intervalb

101 102 103 104 105 201 202 203 204 205

Age (days) adult 70 50 50 50 50 50 48 48 48 50 48

WBC (x 109L) 27-112 53-120 86 64 77 97 76 48 8 72 104 76

RBC (x 1012

L) 50-108 55ndash74 66 58 45 5 48 47 67 5 5 42

Hb (gL) 87-177 104ndash136 121 106 90 58 94 91 122 96 98 80

HCT (LL) 04 - 051 029ndash037 037 033 027 030 029 027 038 030 030 024

MCV (fL) 44-52 478ndash548 55 56 60 60 60 58 57 59 61 58

MCH (pg) 15-18 175ndash191 18 18 20 12 20 20 18 19 20 18

MCHC (gL) 325-362 347ndash370 331 327 328 196 325 337 321 327 324 331

RDW () 12-16 - 134 127 139 139 133 131 122 136 131 127


MPV (fL) 5-10 - 78 78 96 75 66 74 76 81 74 82

TS Protein (gL) 49-76 - 54 51 - - - - - - - -





Basophils (x 109L) 0-02 0 009 0 0 0 0 0 0 0 0 0

Polychromasia 2-5 - 5-10 2-5 10-15 10-15 10-15 10-15 2-5 10-15 10-15 5-10



crenation Occ





Ferret Identity




162



Test Reference

Intervala

Reference

Intervalb

101 102 103 104 105 201 202 203 204 205

Age (days) adult 70 50 50 50 50 50 48 48 48 50 48

Calcium (mmolL) 185-242 253-302 239 233 241 244 24 221 242 234 253 242


Magnesium (mmolL) 08-139 - 08 08 07 08 08 06 08 07 08 08

Sodium (mmolL) 147-159 146-154 149 149 149 149 148 144 153 148 152 152

Potassium (mmolL) 37-57 47-83 44 46 42 46 48 45 47 44 47 46

Chloride (mmolL) 111-129 115-121 110 112 115 113 113 110 119 117 117 120


Anion gap (mmolL) 6 - 23 - 25 24 20 24 23 23 24 20 21 20

NaK ratio - - 34 32 35 32 31 32 33 34 32 33

Total protein (gL) 51-75 44-56 49 46 44 47 44 41 52 45 49 43

Albumin (gL) 24-40 26-32 29 26 28 28 28 25 28 27 29 25

Globulin (gL) 19-41 17-24 20 20 16 19 16 16 24 18 20 18

AG ratio 053-167 13ndash12 145 130 175 147 175 156 117 15 145 139



Glucose (mmolL) 32-91 64-138 47 54 54 42 55 53 57 56 52 59





ALKP (UL) 13-237 117ndash277 180 169 172 215 175 168 241 184 177 179

GGT (UL) 0-40 2ndash20 1 1 6 10 4 0 1 1 5 9

AST (UL) - 63ndash152 61 58 48 61 64 59 93 61 58 69

ALT (UL) 39-196 95ndash544 95 105 89 105 106 82 234 137 115 156

CK (UL) 74-294 - 513 330 496 560 530 492 793 539 479 724

Amylase (UL) - - 23 28 35 35 29 28 29 24 36 23

Lipase (UL) - - 65 63 60 64 62 56 67 69 60 68


Ferret Identity



163


Eimeria ictidea

Test Reference

Intervala

Reference

Intervalb

102 103 104 105 201 203 205

Age (days) adult 98-112 92 91 99 99 97 74 92


WBC (x 109L) 27-112 52ndash150 94 124 81 85 88 85 142

RBC (x 1012

L) 50-108 62ndash92 71 62 73 60 65 63 65

Hb (gL) 87-177 127ndash159 122 110 122 98 103 114 102

HCT (LL) 04-051 030ndash043 037 033 037 031 032 035 032

MCV (fL) 44-52 50ndash54 53 53 52 51 50 55 49

MCH (pg) 15-18 16-21 17 18 17 16 16 18 16

MCHC (gL) 325-362 351ndash426 328 332 325 319 320 326 317

RDW () 12-16 - 122 126 129 129 145 127 145


MPV (fL) 5-10 - 8 78 72 66 67 14 8

TS Protein (gL) 49-76 - 66 63 58 66 55 59 59





Basophils (x 109L) 0-02 0 0 012 0 0 009 0 0

Polychromasia 2-5 - 1-3 0-2 0-2 1-3 1-3 2-5 5-10


HJ bodies rare

crenation

rouleaux

poikilocytosis


hemolysis Neg

Ferret Identity





164



Test Reference

Intervala

Reference

Intervalb

102 103 104 105 201 203 205

Age (days) adult 98-112 92 91 99 99 97 74 92


Calcium (mmolL) 185-242 245-268 243 222 238 233 232 236 240


Magnesium (mmolL) 08-139 - 06 08 08 08 09 08

Sodium (mmolL) 147-159 148-155 147 150 150 148 148 150

Potassium (mmolL) 37-57 45-55 42 41 51 41 50 44

Chloride (mmolL) 111-129 114-124 114 113 118 112 111 115


Anion gap (mmolL) 6 - 23 - 21 20 18 20 30 21

NaK ratio - - 35 37 29 36 30 34

Total protein (gL) 51-75 49-64 52 55 55 49 56 57

Albumin (gL) 24-40 30-36 28 20 27 27 26 29 27

Globulin (gL) 19-41 19-30 32 28 28 23 27 30

AG ratio 053-167 11-17 063 096 096 113 107 090



Glucose (mmolL) 32-91 688-943 52 59 64 71 58 17 66





ALKP (UL) 13-237 41-181 124 120 213 120 146 170 196

GGT (UL) 0-40 1-2 2 1 3 0 0 2

AST (UL) - 47-128 48 95 104 66 100 100

ALT (UL) 39-196 78-279 133 110 140 203 158 183 281

CK (UL) 74-294 - 382 765 1190 578 680 930

Amylase (UL) - - 24 37 31 28 33 35

Lipase (UL) - - 60 65 72 72 86 79


Ferret Identity




165


Mon

8 AM

Mon

4 PM

Tues

8 AM

Tues

4 PM

Wed

8 AM

Wed

4 PM

Thurs

8 AM

Thurs

8 PM

Fri

8 AM

Fri

4 PM

Sat

8 AM

Sat

4 PM

Sun

8AM

Sun

4 PM

Mentation

Weight (g)

Respiratory

Rate

Vomit

(+ ++ +++)

Diarrhea

(+ ++ +++)

Urination

(+ ++ +++)

Defecation

(+ ++ +++)

Food

offered

Food

remaining

Water remaining

(ml)

Treatments

Other

observations

Initials of

observer

166

Animal ID ________________________________________ Week ______________

Monitoring Criteria











Monitoring Times




hour care sheet

Intervention Points



Removal Criteria




167


Animal ID ___________________________________________ Date___________________

0700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800

Mentation

Weight (g)

Heart Rate

Respiratory Rate

Temperature (C)

Water offered

(mL)

Water consumed

(mL)

Food offered

(type and

amount in

grams)

Food consumed

(type and

amount in

grams)

Presence of urine

Presence of feces

168

Character of

feces

Presence of

vomit

Character of

vomit

Medications to

be administered

Other

observations

169

1900 2000 2100 2200 2300 2400 100 200 300 400 500 600

Mentation

Weight (g)

Heart Rate

Respiratory Rate

Temperature (C)

Water offered

(mL)

Water consumed

(mL)

Food offered

(type and amount

in grams)

Food consumed

(type and amount

in grams)

Presence of urine

Presence of feces

Character of

feces

Presence of vomit

Character of

vomit

170

Medications to be

administered

Other

observations

Monitoring Criteria








171




Monitoring Times


hour care sheet

Intervention Points



Removal Criteria




172











summer student




Example Bag label

Ferret ID Date

Weight of Feces


Soft

Liquid

Bloody

Abnormal odour





and Dale Smith)




CBCBiochemistry





173











Euthanasia Protocol









Necropsy Protocol



viscera








calculations)











174













Laboratory SOPs

















oocysts







175


























weeks 1-2)









iv

DEDICATION


v

ACKNOWLEDGEMENTS


to thank for that























vi









been as successful


















vii























Adriana Pastor

Toronto August 2017

viii



exceptions









ix

TABLE OF CONTENTS

ABSTRACT ii

DEDICATION iv

ACKNOWLEDGEMENTS v



LIST OF TABLES xiii

LIST OF FIGURES xiv


ABBREVIATIONS xvi


11 INTRODUCTION 1

12 APICOMPLEXA 1













25




ferrets 28



x




171 Theory 32



181 Objectives 36

182 Hypotheses 36

183 Applications 36



21 INTRODUCTION 39






23 RESULTS 47





24 DISCUSSION 50



31 INTRODUCTION 60





33 RESULTS 66



34 DISCUSSION 69

xi



41 INTRODUCTION 78







43 RESULTS 83


432 Pathology 86


44 DISCUSSION 88




51 INTRODUCTION 104


521 Animal care 106




525 Hematology 110



53 RESULTS 111




534 Hematology 113

535 Necropsy 114

54 DISCUSSION 115

xii




61 INTRODUCTION 129


621 Parasites 130




63 RESULTS 133

64 DISCUSSION 134


REFERENCES 148

APPENDICES 157

xiii

LIST OF TABLES





from 2008-2015 56

























xiv

LIST OF FIGURES


























xv

LIST OF APPENDICES














xvi

ABBREVIATIONS




bp Base pair






CytB Cytochrome b





L Length

LSU Large subunit

mt Mitochondrial


nu Nuclear



rDNA Ribosomal DNA

SI Shape index





SSU Small subunit



W Width

1


11 INTRODUCTION














12 APICOMPLEXA








2









(Cox 1994)






httptolweborg

2

1

3

4

5

3


























4











author



5






















6






















7

























8
























9








2012)

10



species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Cytoisospora

eversmanni

Mustela

eversmanii

(Steppe polecat)

Mustela

putorius

(European

polecat)


W 148 (16ndash12)

LW 13 (11ndash16)

M absent

PG absent

OR absent

L 115

(10ndash135)

W 98

(9ndash11)

LW 12

(11ndash15)

SB absent

SR present


Svanbaev 1956

Nukerbaeva amp

Svanbaev 1973

1977

Cystoisospora

pavlovskyi

Mustela

eversmanii

Mustela

putorius


W 273 (265ndash285)

LW 12 (11ndash14)

M absent

PG absent

OR absent

L 195

(18ndash21)

W 144

(12ndash15)

LW 14

(12ndash15)

SB absent

SR present


Svanbaev 1956

Nukerbaeva amp

Svanbaev 1973

1977

Eimeria

baskanica^

Mustela

erminae

(ermine)


ends

L 112-126

W 84-98

M absent

PG absent

OR present


Svanbaev 1977


putorius

Mustela

putorius furo

(dom ferret)

Mustela

nigripes (BFF)

Mustela vison

(mink)

Homoxenous

Small intestine

rectum (H 1927)

Jejunumileum (BP

1993)

Spherical ndash

subspherical

L 11-14

W 10-13

OW 2 layers

M absent

PG absent

OR absent

Spindloid

L 8-9

W 4

SB present

SR present


et al 1993

Hoare 1927 1935b

Jolley et al 1994

Nukerbaeva amp

Svanbaev

19731977

Williams et al 1988

1992 1996


Bile duct

Spherical

L 13-17

W 13-17

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

L 6

W 4

SB absent

SR absent


Grafner et al 1967

11


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References


eversmanni

Mustela

nigripes

Mustela

putorius

Mustela

putorius furo

Homoxenous

Small intestine


L 13-27

W 13-21

OW 2 layers

M present

PG absent

OR absent

Ovoid

(irregular)

L 115

W65

SB present

SR present

- Hoare 1927 1935a

1935b

Jolley et al 1994

Litvenkova 1969

Svanbaev 1956

Tinar 1985

Williams et al 1988

1992


(tayra)

Homoxenous

Feces

Ovoid

L 21-25

W 18-20

OW outer

layer smooth

M absent

PG absent

OR absent

Ellipsoid

L 10-12

W 65

SB present

SR present

Elongate (one

end broader than

the other)

Carini amp da

Fonseca 1938


(European

badger)


L 20plusmn018

W 157plusmn002

LW128plusmn0017

(112-15)

OW 2 layers

(outer

smooth)

M absent

PG present

OR present

Ovoid

L

119plusmn0018

W 65plusmn008

LW 183

(155-24)

SB present

L 90plusmn005

W 324plusmn0025

SRB present

Anwar et al 2000

Kotlan amp Pospesch

1933


Mustela nivalis

(snow weasel)

Homoxenous

Duodenumileum

Spherical or

Ellipsoid

L 18-26

W 14-24

OW 2 layers

M absent

PG present

OR absent

Ovoid

L 8

W 5

SB present

SR present

Broad at one

end and tapered

at other

L 7

W 3

Glebezdin 1978

Iwanoff-Gobzem

1934

Levine 1948

Musaev amp Veisov

1983

Tinar 1985


(sable)

Homoxenous

Gut

Spherical or

subspherical

L 112-126

W 112

OW 2 layers

M absent

OR absent

Ovoid

L 56

W 42

SR present



L avg 216

W avg 180

LW 1076

OW 2 layers

M absent

PG absent

OR absent

Ovoid

L 96-112

W 56-72

SR absent


Yakimoff amp

Gousseff 1934

Yakimoff amp

Terwinsky 1930

1931

12


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Eimeria vison

(Eimeria

mustelae)

Mustela

putorius

Mustela

putorius furo

Mustela vison

Homoxenous

Small intestine

+- large intestine

Ovoid

L 17-22

W 9-18

OW 2 layers

M absent

OR

sometimes

present

Ovoid or

Piriform

L 10

W 55

SB absent

SR present

Curved or Club

shaped

L 9

W 25


Foreyt et al 1977

Kingscote 1934

1935

Levine 1948

McTaggart 1960

Nukerbaeva amp

Svanbaev

19731977

Tinar 1985

Umurzakov amp

Nukerbaeva 1985

Wolter 1961

Zimmermann 1959


(Libyan striped

weasel)

Homoxenous

Feces

Spherical

L 25-27

W 25-27

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

Ovoid

L 15-17

W 10-12

SB absent

SR present

Elongate

L 135

W 3

Prasad 1961


(mountain

weasel)

Homoxenous

Gut

Oval or spherical

L 280-336

W 252-280

LW 121 (111-

124)

OW 2 layers

M absent

PG absent

OR absent

Ovoid or

spherical

L 140-168

W 111-168

SR present

Svanbaev amp

Rachmatullina

1971


Large intestine

Ovoid

L 224 (220-250)

W 174 (160-190)

LW 135 (133-

137)

OW 1 layer

PG present

OR present

Ovoid

L 120

(100-130)

W 70 (60-

80)

SB present

SR present


1983

13


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Isospora

hoogstraali


Feces

Ellipsoid

L 37-41

W 32-34

OW 2 layers

(outer

smooth)

M absent

PG some

OR absent

Ovoid

L 19-21

W 13-15

SB absent

SR present

Club-shaped

L 18-19

W 4-6

Prasad 1961


putorius

Mustela

putorius furo

Mustela vison

Homoxenous

Feces

Intestinal contents

Ovoid L

320-368

W 272-304

OW 2 layers

M absent

PG absent

OR absent

Ellipsoid

L 208

W 144

SB absent

SR present


Hoare 1927

Levine 1948

McTaggart 1960

Nukerbaeva amp

Svanbaev 1973

1974 1977

Tinar 1985


(European

otter)

Lutra

canadensis

(North

American river

otter)


L 312 (275-32)

W 296 (28-31)

LW 104

(10-112)

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

Ellipsoid

L 182 (17-

19)

W 144 (14-

16)

LW128

(12-14)

Sb absent

sSB absent

SR present

Spindle- shaped

L 124

W 25

SRB present

Torres et al 2000

Hoover et al 1985

Isospora

martessii


Gut

Ovoid short oval or

spherical

L 252 ndash 280 196

168

W 168 ndash 224 168

168

OW 2 layers

M absent

OR absent

Ovoid

L 112-168

W 84-112

SR present



L 328plusmn034

W 269plusmn019

LW122 (110-

157)

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

Ellipsoid

L

215plusmn0166

W 14plusmn012

LW 155

(133-185)

SR absent

Round at one

end other end

tapered

L 142plusmn116

W 40plusmn017

SRB absent

Anwar et al 2000

Glebezdin 1978

Kotlan amp Pospesch

1933

Pelleacuterdy 1955

14


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Isospora

mustelae (nomen

nudum)


7 W

225



Large intestine

Ovoid

L 206 (200-230)

W 184 (180-210)

LW 11 (109-111)

OW 1 layer

PG absent

OR absent

Ovoid

L 125

(120-130)

W 80 (70-

90)

SR present

Lemon or pear

shaped

Musaev amp Veisov

1983

Unnamed

ldquoCoccidiardquo^

Mustela

nigripes


Unnamed

ldquoCoccidiardquo^

Mustela

nigripes

Trachea bronchus

bronchial glands


Unnamed

Eimeria sp^

Mustela

nigripes

Feces

intestinal contents

Ovoid

L 350-386

W 212-232


Williams et al

1992

Unnamed

Eimeria sp^

Mustela

putorius furo


et al 1993

Unnamed

Eimeria sp^


Large intestine

Ovoid-ellipsoid L

2031 (1712-2162)

W 148 (1225-

1681)

LW 136 (121-16-

)

OW 1 layer

PG absent

OR absent

Ovoid or

pear-shaped

L 60-100

W 40-80

SR present

Elongate

L 50-90

W 30-70

Musaev amp Veisov

1983

Unnamed

Eimeria sp^

Martes martes

(marten)

Homoxenous Ovoid

L avg 216

W avg 180

LW 1076


SR present

L 126

W 60

Yakimoff and

Gousseff 1934

Unnamed

Isospora sp^

Mustela

putorius furo

Feces - - - - Bell 1994

Unnamed

Isospora sp^

Mustela

putorius furo

Feces - - - - Bell 1994




15


similar host

16



























17


























18

















1967)









19


























20

























21

























22

















reproduction









23


























24
























Gorham 1953)

25

























26

























27

























28
















lacking









29

























30








footed ferrets
















31



























32
















171 Theory









33


























34

























35
























36


181 Objectives








footed ferret

182 Hypotheses







ferrets

183 Applications






37














38






review)

ABSTRACT


















putorius furo

39

21 INTRODUCTION

























40




infection trial














posterior end







41


the sporocyst























42












E furonis nu 18S















43















221 Fecal samples










44

























45























46



















47

23 RESULTS






















48























49


life stages























50














24 DISCUSSION








Canada or the EU

51


























52

























53









identification








relationships

ACKNOWLEDGEMENTS








54


(400566) to JRB

55
















56






Cystoisospora sp


Eimeria furonis


Eimeria ictidea


2008 3140 (21) 6214 (28) 3 2 0 4 0 0

2009 2160 (12) 14214 (65) 2 9 0 5 0 0

2010 8127 (63) 20213 (94) 7 10 1 10 0 0

2011 0114 (0) 17215 (79) 0 9 0 7 0 1

2012 3108 (28) 10231 (43) 2 4 1 6 0 0

2013 281 (25) 16270 (59) 2 13 0 2 0 1

2014 6127 (47) 12234 (51) 5 6 1 6 0 0

2015 4108 (37) 9249 (36) 4 3 0 6 0 0

Total 28 (29) 104 (56) 25 56 3 46 0 2

Average

year 35 130 31 70 04 58 00 03


57


Sample ID Source



mt COI GenBank

Accession

nu 18S rDNA

GenBank Accession

















58





25 microm

59








60



NIGRIPES)

ABSTRACT













31 INTRODUCTION








61











2014)















62


























63









et al 1994)

















64
















SSP facilities


321 Fecal samples







65














amp Methods)








66


















Figure 31

33 RESULTS





67


























68


























69






34 DISCUSSION




















70








conducted



















71





















ACKNOWLEDGEMENTS





72



73















74







10 microm 5 microm

75





Identity






Identity




76


nigripes)













fresh feces


POS E cf ictidea

-

E ictidea













histologic section


77


(Mustela nigripes)



Length (microm) 2333 (2055-2583) 2456 (2111-2848) 2505 (2079-3008) 2779 (2590-3060) 2493 (2036-2822) 2139 (1859-2372) 2398 (1859-3057)

Width (microm) 1676 (1373-2180) 1835 (1643-2232) 1975 (1509-2360) 2253 (2092-2383) 1803 (1549-2017) 1751 (1610-1888) 1855 (1373-2383)

Shape index 135 (103-160) 134 (113-156) 127 (105-155) 124 (113-138) 139 (114-154) 122 (101-145) 130 (101-160)


78



ABSTRACT
















41 INTRODUCTION






79











self-sustaining













80
























81




Family groups








Adults













82























83











mortality rates

43 RESULTS


Family groups






Table 42 Figure 41)





84








treatment with TMS

















85

Adults


























86












432 Pathology





Gross Pathology







Histopathology

87






(n=1)



















88
















those populations

44 DISCUSSION




institutions

89
























90


























91


























92















clinical disease










93


























94









drug resistance

95



0

50000

100000

150000

200000

250000

300000

350000

400000

29

33

37

41

45

49

53

57

61

65

69

73

77

81

85

89

93

97

10

1

10

5

10

9

11

3

11

7

12

1

12

5

12

9

13

3

13

7

14

1

14

5

14

9

OP

G

Age of Kits


96




25 microm

97



Collection Year 2014 2014 2014 2014 2014 2015 2016


29 - - - 0 - - -

30 - 0 - 0 - - -

- - - - -

34 0 0 - 0 0 - -

35 0 0 - 0 0 0 -

- -

46 0 0 - 0 0 0 -

47 0 0 - 0 0 0 -

48 0 lt14 - 0 0 0 -

- -

53 0 0 - 0 0 0 +

54 0 0 - 0 0 0 +

55 0 0 - 0 0 0 3717146

56 0 0 - 0 - 0 1084436

57 0 0 - 0 - 0 64133

58 0 324 - 0 0 0 20654

59 0 0 - 0 0 0 0

60 0 446688 - 0 0 0 0

61 0 934828 - 0 0 0 41111

62 0 530469 - 0 0 0

63 0 1617131 - 0 0 lt14

64 0 8396357 - 114 0 0

65 0 234876 - 5368 0 lt14

66 0 374625 - 9455 0 0

67 lt14 2311575 - 39579 0 -

68 0 603563 - 125051 0 0

69 - 4103036 - 27067 0 lt14

70 2470327 7759974 - 0 0 0

71 1073085 - 1784 0 0

72 3430966 - 0 0 0

73 4459536 - 7326 0 0

74 4842212 - 2 0 0

75 3627307 - 0 0 0

76 5801885 - 0 0 0

77 5994 0 0 0 0

78 589835 0 0 0

79 24815 0 0 0

80 144016 0 0 0

81 97862 0 0 0

82 0 0 0

127 0 0

128 0 2843

136 0 0

150 0


Dam Identity





98



Dam Identity

Poppy

2014

Bumblefoot

2014

Aubrey

2014

Fiddlesticks

2015

Guanella

^

2016



shedding 70-81 58-70 64-73 63-69 54-61


OPG min 0 0 0 0 206

OPG max 48442 83963 2707 lt14 371714




- = missing data



Collection

Year 2015 2016 2016 2016 2016 2016 2016


Age (years) 1 3 1 1 1 1 1

1084058 0 + + + + +

+ 0 42307650 6286676 + 183150 554274

857808 16650 12805238 7777929 + 215710 377920

1604894 16650 309690 139860 + 0 25808

377042 0 599400 119880 + - 37294

554445 0 34688 385579 117920 0 5363

26640 0 16650 0 0 0 7500

0 10406 0 0 0 1090

0 20813 0 0 0

- 0 1761 0

0 0 0 0

- 0

0

0



100



Sex M F M M M F M

Age (years) 1 3 1 1 1 1 1


OPG min 266 166 104 1199 1761 1831 1090

OPG max 10840 166 423076 77779 - 2157 554274





101



Ferret

ID Year

Age















102




Year Adult Family

2003 116 (625) -

2004 819 (4211) -

2005 021 (000) 14 (2500)

2006 021 (000) 07 (000)

2007 023 (000) 08 (000)

2008 224 (833) 14 (2500)

2009 025 (000) 06 (000)

2010 326 (1154) 07 (000)

2011 125 (400) 09 (000)

2012 125 (400) 08 (000)

2013 028 (000) 05 (000)

2014 430 (1333) 09 (000)

2015 035 (000) 35 (6000)

2016 - 27 (2857)




103



Total Mortality



1997 015 (000) 023 (000) 315 (2000) 423 (1739) 1998 038 (000) 119 (526) 838 (2105) 919 (4734) 1999 047 (000) 119 (526) 1647 (3404) 119 (526) 2000 034 (000) 015 (000) 434 (1176) 315 (2000) 2001 032 (000) 016 (000) 532 (1563) 116 (625) 2002 150 (200) 020 (000) 450 (800) 220 (1000) 2003 227 (741) 018 (000) 327 (1111) 118 (555) 2004 020 (000) 016 (000) 620 (3000) 216 (1250) 2005 016 (000) 015 (000) 416 (2500) 215 (1333) 2006 030 (000) 016 (000) 230 (667) 016 (000) 2007 019 (000) 015 (000) 419 (2105) 215 (1333) 2008 034 (000) 016 (000) 1134 (3235) 316 (1875) 2009 017 (000) 016 (000) 017 (000) 116 (625) 2010 017 (000) 016 (000) 317 (1765) 316 (1875) 2011 011 (000) 016 (000) 111 (909) 216 (1250) 2012 111 (909) 017 (000) 111 (909) 317 (1765) 2013 424 (1667) 017 (000) 424 (1667) 317 (1765) 2014 126 (384) 017 (000) 326 (1154) 217 (1176) 2015 04 (000) 017 (000) 04 (000) 217 (1176) 2016 011 (000) 017 (000) 211 (1818) 117 (588)

Mean annual () 195 053 1594 1359



as a percentage

104




ABSTRACT















species

51 INTRODUCTION






105


























106






ictidea


521 Animal care

















107







study



















108





Part 1








container












109

Part 2















524 Animal welfare









110



525 Hematology












(Chapter 3)









111






53 RESULTS


















112

531 Oocyst shedding

























113







533 Clinical signs





this time

534 Hematology








114

535 Necropsy

























115

54 DISCUSSION






















116


























117


























118
























119








absent












domestic ferret





120

























121











naive















122

















25 microm

124







25 microm

25 microm

125







0

1

2

3


Nu

mb

er

of

Ferr

ets

Aff

ect

ed



126





Ferret Identity


1 0 0 0 0 0 0 0

2 0 0 0 0 0 0 0

3 0 0 0 0 0 0 0

4 0 0 0 0 0 0 0

5 0 0 0 0 0 0 0

6 0 0 0 0 0 0 0

7 lt 14 1807 0 0 0 0 0

8 11053 139 7091 0 156238 0

9 463 11733 lt 14 203 lt 14

10 578 7549 0 0 0

11 lt 14 0 0 0

12 0 lt 14 0

13 0 lt 14 0

14 0 lt 14 0


gram of feces

127



Ferret

Identity


Inoculum Oocyst

Shedding

Presence

of clinical

disease

Coccidia

present in

sections

Inoculum Oocyst

Shedding

Presence

of clinical

disease

Coccidia

present in

sections













necropsy

128



Ferret Identity


103

104 105

201

203

205











S - 05

A - 15

S - 45

A - 25

S - 05

A - 05

S - 26

A - 06

S - 06

A - 06

S - 06

A - 06

S - 08

A - 08


S - 01

A - 01

S - 01

A - 01

S - 01

A - 01

S - 02

A - 02

S - 01

A - 01 none

S - 01

A - 01




129




ABSTRACT















61 INTRODUCTION





130






















621 Parasites



131
























132
























133



63 RESULTS






64 (for E ictidea)













regions




134


Mustelidae)

64 DISCUSSION























135









136


















137












138


139


nigripes)








































140




Total length

(nucleotides)

Nucleotide

identity

Total amino

acids

Amino acid

identity

COI CDS 1443 934 (95) 480 975 (12)

COIII CDS 756 899 (76) 251 932 (17)

CytB CDS 1080 950 (54) 359 986 (5)



applicable

141




142




143





144




parasites

145




















disease management






146



























147
























148

REFERENCES






7408200500053x












363



149







13256ndash59



101016jejop201407002













150























151



45
















101093nargkf436



101053jjepm201504012

152














20489ndash510 doi 101128CMR00005-07









153

41843ndash850








Vectors 7335 doi 1011861756-3305-7-335











12531ndash561 doi 101016jcvex200906001



154
















109551ndash557







155




(BFFMCOM)




Pathol 33437ndash439 doi 101177030098589603300412













1011861471-2148-11-92


156


157

APPENDICES

158



Collection Year 2014 2014 2014 2014 2014 2015 2016


29 - - - 0 - - -

30 - 0 - 0 - - -

31 - 0 - 0 - - -

32 - 0 - 0 - - -

33 - 0 - 0 - - -

34 0 0 - 0 0 - -

35 0 0 - 0 0 0 -

36 0 0 - 0 0 0 -

37 - 0 - 0 - 0 -

38 - 0 - 0 - 0 -

39 - 0 - 0 - 0 -

40 0 0 - 0 0 0 -

41 - 0 - 0 - 0 -

42 0 0 - 0 0 - -

43 0 0 - 0 0 0 -

44 0 0 - - 0 0 -

45 0 0 - 0 0 - -

46 0 0 - 0 0 0 -

47 0 0 - 0 0 0 -

48 0 lt14 - 0 0 0 -

49 0 0 - 0 0 0 -

50 0 0 - - 0 0 -

51 0 0 - 0 0 0 -

52 0 0 - 0 0 0 -

53 0 0 - 0 0 0 +

54 0 0 - 0 0 0 +

55 0 0 - 0 0 0 3717146

56 0 0 - 0 - 0 1084436

57 0 0 - 0 - 0 64133

58 0 324 - 0 0 0 20654

59 0 0 - 0 0 0 0

60 0 446688 - 0 0 0 0

61 0 934828 - 0 0 0 41111

62 0 530469 - 0 0 0

63 0 1617131 - 0 0 lt14

64 0 8396357 - 114 0 0

65 0 234876 - 5368 0 lt14

66 0 374625 - 9455 0 0

67 lt14 2311575 - 39579 0 -

68 0 603563 - 125051 0 0

69 - 4103036 - 27067 0 lt14

70 2470327 7759974 - 0 0 0

71 1073085 - 1784 0 0

72 3430966 - 0 0 0

73 4459536 - 7326 0 0

74 4842212 - 2 0 0

75 3627307 - 0 0 0

76 5801885 - 0 0 0

77 5994 0 0 0 0

78 589835 0 0 0


Dam Identity

159



Collection Year 2014 2014 2014 2014 2014 2015 2016


79 24815 0 0 0

80 144016 0 0 0

81 97862 0 0 0

82 0 0 0

83 0 0 0

84 0 0 0

85 0 0 0

86 0 0 0

87 0 0 0

88 0 0 0

89 0 0 0

90 0 0 0

91 0 0 0

92 0 - -

93 0 - 0

94 0 - 0

95 0 0 -

96 0 - 0

97 0 0 -

98 0 0 0

99 0 0 0

100 0 0 0

101 0 0 0

102 0 0 0

103 0 0 0

104 0 0 0

105 0 0 -

106 0 0 0

107 0 0 0

108 - 0 0

109 0 0 -

110 0 0 0

111 0 0 -

112 0 0 0

113 0 0 -

114 0 0 -

115 0 0 -

116 0 0 -

117 0 -

118 0 -

119 0 0

120 0 0

121 0 0

122 0 -


Dam Identity

160



Collection Year 2014 2014 2014 2014 2014 2015 2016


123 0 0

124 0 -

125 0 -

126 0 0

127 0 0

128 0 2843

129 0 0

130 0 -

131 0 0

132 0 0

133 0 0

134 0 0

135 0 0

136 0 0

137 0

138 0

139 0

140 0

141 0

142 0

143 0

144 0

145 0

146 -

147 0

148 0

149 0

150 0





Dam Identity

161



Test Reference

Intervala

Reference

Intervalb

101 102 103 104 105 201 202 203 204 205

Age (days) adult 70 50 50 50 50 50 48 48 48 50 48

WBC (x 109L) 27-112 53-120 86 64 77 97 76 48 8 72 104 76

RBC (x 1012

L) 50-108 55ndash74 66 58 45 5 48 47 67 5 5 42

Hb (gL) 87-177 104ndash136 121 106 90 58 94 91 122 96 98 80

HCT (LL) 04 - 051 029ndash037 037 033 027 030 029 027 038 030 030 024

MCV (fL) 44-52 478ndash548 55 56 60 60 60 58 57 59 61 58

MCH (pg) 15-18 175ndash191 18 18 20 12 20 20 18 19 20 18

MCHC (gL) 325-362 347ndash370 331 327 328 196 325 337 321 327 324 331

RDW () 12-16 - 134 127 139 139 133 131 122 136 131 127


MPV (fL) 5-10 - 78 78 96 75 66 74 76 81 74 82

TS Protein (gL) 49-76 - 54 51 - - - - - - - -





Basophils (x 109L) 0-02 0 009 0 0 0 0 0 0 0 0 0

Polychromasia 2-5 - 5-10 2-5 10-15 10-15 10-15 10-15 2-5 10-15 10-15 5-10



crenation Occ





Ferret Identity




162



Test Reference

Intervala

Reference

Intervalb

101 102 103 104 105 201 202 203 204 205

Age (days) adult 70 50 50 50 50 50 48 48 48 50 48

Calcium (mmolL) 185-242 253-302 239 233 241 244 24 221 242 234 253 242


Magnesium (mmolL) 08-139 - 08 08 07 08 08 06 08 07 08 08

Sodium (mmolL) 147-159 146-154 149 149 149 149 148 144 153 148 152 152

Potassium (mmolL) 37-57 47-83 44 46 42 46 48 45 47 44 47 46

Chloride (mmolL) 111-129 115-121 110 112 115 113 113 110 119 117 117 120


Anion gap (mmolL) 6 - 23 - 25 24 20 24 23 23 24 20 21 20

NaK ratio - - 34 32 35 32 31 32 33 34 32 33

Total protein (gL) 51-75 44-56 49 46 44 47 44 41 52 45 49 43

Albumin (gL) 24-40 26-32 29 26 28 28 28 25 28 27 29 25

Globulin (gL) 19-41 17-24 20 20 16 19 16 16 24 18 20 18

AG ratio 053-167 13ndash12 145 130 175 147 175 156 117 15 145 139



Glucose (mmolL) 32-91 64-138 47 54 54 42 55 53 57 56 52 59





ALKP (UL) 13-237 117ndash277 180 169 172 215 175 168 241 184 177 179

GGT (UL) 0-40 2ndash20 1 1 6 10 4 0 1 1 5 9

AST (UL) - 63ndash152 61 58 48 61 64 59 93 61 58 69

ALT (UL) 39-196 95ndash544 95 105 89 105 106 82 234 137 115 156

CK (UL) 74-294 - 513 330 496 560 530 492 793 539 479 724

Amylase (UL) - - 23 28 35 35 29 28 29 24 36 23

Lipase (UL) - - 65 63 60 64 62 56 67 69 60 68


Ferret Identity



163


Eimeria ictidea

Test Reference

Intervala

Reference

Intervalb

102 103 104 105 201 203 205

Age (days) adult 98-112 92 91 99 99 97 74 92


WBC (x 109L) 27-112 52ndash150 94 124 81 85 88 85 142

RBC (x 1012

L) 50-108 62ndash92 71 62 73 60 65 63 65

Hb (gL) 87-177 127ndash159 122 110 122 98 103 114 102

HCT (LL) 04-051 030ndash043 037 033 037 031 032 035 032

MCV (fL) 44-52 50ndash54 53 53 52 51 50 55 49

MCH (pg) 15-18 16-21 17 18 17 16 16 18 16

MCHC (gL) 325-362 351ndash426 328 332 325 319 320 326 317

RDW () 12-16 - 122 126 129 129 145 127 145


MPV (fL) 5-10 - 8 78 72 66 67 14 8

TS Protein (gL) 49-76 - 66 63 58 66 55 59 59





Basophils (x 109L) 0-02 0 0 012 0 0 009 0 0

Polychromasia 2-5 - 1-3 0-2 0-2 1-3 1-3 2-5 5-10


HJ bodies rare

crenation

rouleaux

poikilocytosis


hemolysis Neg

Ferret Identity





164



Test Reference

Intervala

Reference

Intervalb

102 103 104 105 201 203 205

Age (days) adult 98-112 92 91 99 99 97 74 92


Calcium (mmolL) 185-242 245-268 243 222 238 233 232 236 240


Magnesium (mmolL) 08-139 - 06 08 08 08 09 08

Sodium (mmolL) 147-159 148-155 147 150 150 148 148 150

Potassium (mmolL) 37-57 45-55 42 41 51 41 50 44

Chloride (mmolL) 111-129 114-124 114 113 118 112 111 115


Anion gap (mmolL) 6 - 23 - 21 20 18 20 30 21

NaK ratio - - 35 37 29 36 30 34

Total protein (gL) 51-75 49-64 52 55 55 49 56 57

Albumin (gL) 24-40 30-36 28 20 27 27 26 29 27

Globulin (gL) 19-41 19-30 32 28 28 23 27 30

AG ratio 053-167 11-17 063 096 096 113 107 090



Glucose (mmolL) 32-91 688-943 52 59 64 71 58 17 66





ALKP (UL) 13-237 41-181 124 120 213 120 146 170 196

GGT (UL) 0-40 1-2 2 1 3 0 0 2

AST (UL) - 47-128 48 95 104 66 100 100

ALT (UL) 39-196 78-279 133 110 140 203 158 183 281

CK (UL) 74-294 - 382 765 1190 578 680 930

Amylase (UL) - - 24 37 31 28 33 35

Lipase (UL) - - 60 65 72 72 86 79


Ferret Identity




165


Mon

8 AM

Mon

4 PM

Tues

8 AM

Tues

4 PM

Wed

8 AM

Wed

4 PM

Thurs

8 AM

Thurs

8 PM

Fri

8 AM

Fri

4 PM

Sat

8 AM

Sat

4 PM

Sun

8AM

Sun

4 PM

Mentation

Weight (g)

Respiratory

Rate

Vomit

(+ ++ +++)

Diarrhea

(+ ++ +++)

Urination

(+ ++ +++)

Defecation

(+ ++ +++)

Food

offered

Food

remaining

Water remaining

(ml)

Treatments

Other

observations

Initials of

observer

166

Animal ID ________________________________________ Week ______________

Monitoring Criteria











Monitoring Times




hour care sheet

Intervention Points



Removal Criteria




167


Animal ID ___________________________________________ Date___________________

0700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800

Mentation

Weight (g)

Heart Rate

Respiratory Rate

Temperature (C)

Water offered

(mL)

Water consumed

(mL)

Food offered

(type and

amount in

grams)

Food consumed

(type and

amount in

grams)

Presence of urine

Presence of feces

168

Character of

feces

Presence of

vomit

Character of

vomit

Medications to

be administered

Other

observations

169

1900 2000 2100 2200 2300 2400 100 200 300 400 500 600

Mentation

Weight (g)

Heart Rate

Respiratory Rate

Temperature (C)

Water offered

(mL)

Water consumed

(mL)

Food offered

(type and amount

in grams)

Food consumed

(type and amount

in grams)

Presence of urine

Presence of feces

Character of

feces

Presence of vomit

Character of

vomit

170

Medications to be

administered

Other

observations

Monitoring Criteria








171




Monitoring Times


hour care sheet

Intervention Points



Removal Criteria




172











summer student




Example Bag label

Ferret ID Date

Weight of Feces


Soft

Liquid

Bloody

Abnormal odour





and Dale Smith)




CBCBiochemistry





173











Euthanasia Protocol









Necropsy Protocol



viscera








calculations)











174













Laboratory SOPs

















oocysts







175


























weeks 1-2)

v

ACKNOWLEDGEMENTS


to thank for that























vi









been as successful


















vii























Adriana Pastor

Toronto August 2017

viii



exceptions









ix

TABLE OF CONTENTS

ABSTRACT ii

DEDICATION iv

ACKNOWLEDGEMENTS v



LIST OF TABLES xiii

LIST OF FIGURES xiv


ABBREVIATIONS xvi


11 INTRODUCTION 1

12 APICOMPLEXA 1













25




ferrets 28



x




171 Theory 32



181 Objectives 36

182 Hypotheses 36

183 Applications 36



21 INTRODUCTION 39






23 RESULTS 47





24 DISCUSSION 50



31 INTRODUCTION 60





33 RESULTS 66



34 DISCUSSION 69

xi



41 INTRODUCTION 78







43 RESULTS 83


432 Pathology 86


44 DISCUSSION 88




51 INTRODUCTION 104


521 Animal care 106




525 Hematology 110



53 RESULTS 111




534 Hematology 113

535 Necropsy 114

54 DISCUSSION 115

xii




61 INTRODUCTION 129


621 Parasites 130




63 RESULTS 133

64 DISCUSSION 134


REFERENCES 148

APPENDICES 157

xiii

LIST OF TABLES





from 2008-2015 56

























xiv

LIST OF FIGURES


























xv

LIST OF APPENDICES














xvi

ABBREVIATIONS




bp Base pair






CytB Cytochrome b





L Length

LSU Large subunit

mt Mitochondrial


nu Nuclear



rDNA Ribosomal DNA

SI Shape index





SSU Small subunit



W Width

1


11 INTRODUCTION














12 APICOMPLEXA








2









(Cox 1994)






httptolweborg

2

1

3

4

5

3


























4











author



5






















6






















7

























8
























9








2012)

10



species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Cytoisospora

eversmanni

Mustela

eversmanii

(Steppe polecat)

Mustela

putorius

(European

polecat)


W 148 (16ndash12)

LW 13 (11ndash16)

M absent

PG absent

OR absent

L 115

(10ndash135)

W 98

(9ndash11)

LW 12

(11ndash15)

SB absent

SR present


Svanbaev 1956

Nukerbaeva amp

Svanbaev 1973

1977

Cystoisospora

pavlovskyi

Mustela

eversmanii

Mustela

putorius


W 273 (265ndash285)

LW 12 (11ndash14)

M absent

PG absent

OR absent

L 195

(18ndash21)

W 144

(12ndash15)

LW 14

(12ndash15)

SB absent

SR present


Svanbaev 1956

Nukerbaeva amp

Svanbaev 1973

1977

Eimeria

baskanica^

Mustela

erminae

(ermine)


ends

L 112-126

W 84-98

M absent

PG absent

OR present


Svanbaev 1977


putorius

Mustela

putorius furo

(dom ferret)

Mustela

nigripes (BFF)

Mustela vison

(mink)

Homoxenous

Small intestine

rectum (H 1927)

Jejunumileum (BP

1993)

Spherical ndash

subspherical

L 11-14

W 10-13

OW 2 layers

M absent

PG absent

OR absent

Spindloid

L 8-9

W 4

SB present

SR present


et al 1993

Hoare 1927 1935b

Jolley et al 1994

Nukerbaeva amp

Svanbaev

19731977

Williams et al 1988

1992 1996


Bile duct

Spherical

L 13-17

W 13-17

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

L 6

W 4

SB absent

SR absent


Grafner et al 1967

11


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References


eversmanni

Mustela

nigripes

Mustela

putorius

Mustela

putorius furo

Homoxenous

Small intestine


L 13-27

W 13-21

OW 2 layers

M present

PG absent

OR absent

Ovoid

(irregular)

L 115

W65

SB present

SR present

- Hoare 1927 1935a

1935b

Jolley et al 1994

Litvenkova 1969

Svanbaev 1956

Tinar 1985

Williams et al 1988

1992


(tayra)

Homoxenous

Feces

Ovoid

L 21-25

W 18-20

OW outer

layer smooth

M absent

PG absent

OR absent

Ellipsoid

L 10-12

W 65

SB present

SR present

Elongate (one

end broader than

the other)

Carini amp da

Fonseca 1938


(European

badger)


L 20plusmn018

W 157plusmn002

LW128plusmn0017

(112-15)

OW 2 layers

(outer

smooth)

M absent

PG present

OR present

Ovoid

L

119plusmn0018

W 65plusmn008

LW 183

(155-24)

SB present

L 90plusmn005

W 324plusmn0025

SRB present

Anwar et al 2000

Kotlan amp Pospesch

1933


Mustela nivalis

(snow weasel)

Homoxenous

Duodenumileum

Spherical or

Ellipsoid

L 18-26

W 14-24

OW 2 layers

M absent

PG present

OR absent

Ovoid

L 8

W 5

SB present

SR present

Broad at one

end and tapered

at other

L 7

W 3

Glebezdin 1978

Iwanoff-Gobzem

1934

Levine 1948

Musaev amp Veisov

1983

Tinar 1985


(sable)

Homoxenous

Gut

Spherical or

subspherical

L 112-126

W 112

OW 2 layers

M absent

OR absent

Ovoid

L 56

W 42

SR present



L avg 216

W avg 180

LW 1076

OW 2 layers

M absent

PG absent

OR absent

Ovoid

L 96-112

W 56-72

SR absent


Yakimoff amp

Gousseff 1934

Yakimoff amp

Terwinsky 1930

1931

12


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Eimeria vison

(Eimeria

mustelae)

Mustela

putorius

Mustela

putorius furo

Mustela vison

Homoxenous

Small intestine

+- large intestine

Ovoid

L 17-22

W 9-18

OW 2 layers

M absent

OR

sometimes

present

Ovoid or

Piriform

L 10

W 55

SB absent

SR present

Curved or Club

shaped

L 9

W 25


Foreyt et al 1977

Kingscote 1934

1935

Levine 1948

McTaggart 1960

Nukerbaeva amp

Svanbaev

19731977

Tinar 1985

Umurzakov amp

Nukerbaeva 1985

Wolter 1961

Zimmermann 1959


(Libyan striped

weasel)

Homoxenous

Feces

Spherical

L 25-27

W 25-27

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

Ovoid

L 15-17

W 10-12

SB absent

SR present

Elongate

L 135

W 3

Prasad 1961


(mountain

weasel)

Homoxenous

Gut

Oval or spherical

L 280-336

W 252-280

LW 121 (111-

124)

OW 2 layers

M absent

PG absent

OR absent

Ovoid or

spherical

L 140-168

W 111-168

SR present

Svanbaev amp

Rachmatullina

1971


Large intestine

Ovoid

L 224 (220-250)

W 174 (160-190)

LW 135 (133-

137)

OW 1 layer

PG present

OR present

Ovoid

L 120

(100-130)

W 70 (60-

80)

SB present

SR present


1983

13


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Isospora

hoogstraali


Feces

Ellipsoid

L 37-41

W 32-34

OW 2 layers

(outer

smooth)

M absent

PG some

OR absent

Ovoid

L 19-21

W 13-15

SB absent

SR present

Club-shaped

L 18-19

W 4-6

Prasad 1961


putorius

Mustela

putorius furo

Mustela vison

Homoxenous

Feces

Intestinal contents

Ovoid L

320-368

W 272-304

OW 2 layers

M absent

PG absent

OR absent

Ellipsoid

L 208

W 144

SB absent

SR present


Hoare 1927

Levine 1948

McTaggart 1960

Nukerbaeva amp

Svanbaev 1973

1974 1977

Tinar 1985


(European

otter)

Lutra

canadensis

(North

American river

otter)


L 312 (275-32)

W 296 (28-31)

LW 104

(10-112)

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

Ellipsoid

L 182 (17-

19)

W 144 (14-

16)

LW128

(12-14)

Sb absent

sSB absent

SR present

Spindle- shaped

L 124

W 25

SRB present

Torres et al 2000

Hoover et al 1985

Isospora

martessii


Gut

Ovoid short oval or

spherical

L 252 ndash 280 196

168

W 168 ndash 224 168

168

OW 2 layers

M absent

OR absent

Ovoid

L 112-168

W 84-112

SR present



L 328plusmn034

W 269plusmn019

LW122 (110-

157)

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

Ellipsoid

L

215plusmn0166

W 14plusmn012

LW 155

(133-185)

SR absent

Round at one

end other end

tapered

L 142plusmn116

W 40plusmn017

SRB absent

Anwar et al 2000

Glebezdin 1978

Kotlan amp Pospesch

1933

Pelleacuterdy 1955

14


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Isospora

mustelae (nomen

nudum)


7 W

225



Large intestine

Ovoid

L 206 (200-230)

W 184 (180-210)

LW 11 (109-111)

OW 1 layer

PG absent

OR absent

Ovoid

L 125

(120-130)

W 80 (70-

90)

SR present

Lemon or pear

shaped

Musaev amp Veisov

1983

Unnamed

ldquoCoccidiardquo^

Mustela

nigripes


Unnamed

ldquoCoccidiardquo^

Mustela

nigripes

Trachea bronchus

bronchial glands


Unnamed

Eimeria sp^

Mustela

nigripes

Feces

intestinal contents

Ovoid

L 350-386

W 212-232


Williams et al

1992

Unnamed

Eimeria sp^

Mustela

putorius furo


et al 1993

Unnamed

Eimeria sp^


Large intestine

Ovoid-ellipsoid L

2031 (1712-2162)

W 148 (1225-

1681)

LW 136 (121-16-

)

OW 1 layer

PG absent

OR absent

Ovoid or

pear-shaped

L 60-100

W 40-80

SR present

Elongate

L 50-90

W 30-70

Musaev amp Veisov

1983

Unnamed

Eimeria sp^

Martes martes

(marten)

Homoxenous Ovoid

L avg 216

W avg 180

LW 1076


SR present

L 126

W 60

Yakimoff and

Gousseff 1934

Unnamed

Isospora sp^

Mustela

putorius furo

Feces - - - - Bell 1994

Unnamed

Isospora sp^

Mustela

putorius furo

Feces - - - - Bell 1994




15


similar host

16



























17


























18

















1967)









19


























20

























21

























22

















reproduction









23


























24
























Gorham 1953)

25

























26

























27

























28
















lacking









29

























30








footed ferrets
















31



























32
















171 Theory









33


























34

























35
























36


181 Objectives








footed ferret

182 Hypotheses







ferrets

183 Applications






37














38






review)

ABSTRACT


















putorius furo

39

21 INTRODUCTION

























40




infection trial














posterior end







41


the sporocyst























42












E furonis nu 18S















43















221 Fecal samples










44

























45























46



















47

23 RESULTS






















48























49


life stages























50














24 DISCUSSION








Canada or the EU

51


























52

























53









identification








relationships

ACKNOWLEDGEMENTS








54


(400566) to JRB

55
















56






Cystoisospora sp


Eimeria furonis


Eimeria ictidea


2008 3140 (21) 6214 (28) 3 2 0 4 0 0

2009 2160 (12) 14214 (65) 2 9 0 5 0 0

2010 8127 (63) 20213 (94) 7 10 1 10 0 0

2011 0114 (0) 17215 (79) 0 9 0 7 0 1

2012 3108 (28) 10231 (43) 2 4 1 6 0 0

2013 281 (25) 16270 (59) 2 13 0 2 0 1

2014 6127 (47) 12234 (51) 5 6 1 6 0 0

2015 4108 (37) 9249 (36) 4 3 0 6 0 0

Total 28 (29) 104 (56) 25 56 3 46 0 2

Average

year 35 130 31 70 04 58 00 03


57


Sample ID Source



mt COI GenBank

Accession

nu 18S rDNA

GenBank Accession

















58





25 microm

59








60



NIGRIPES)

ABSTRACT













31 INTRODUCTION








61











2014)















62


























63









et al 1994)

















64
















SSP facilities


321 Fecal samples







65














amp Methods)








66


















Figure 31

33 RESULTS





67


























68


























69






34 DISCUSSION




















70








conducted



















71





















ACKNOWLEDGEMENTS





72



73















74







10 microm 5 microm

75





Identity






Identity




76


nigripes)













fresh feces


POS E cf ictidea

-

E ictidea













histologic section


77


(Mustela nigripes)



Length (microm) 2333 (2055-2583) 2456 (2111-2848) 2505 (2079-3008) 2779 (2590-3060) 2493 (2036-2822) 2139 (1859-2372) 2398 (1859-3057)

Width (microm) 1676 (1373-2180) 1835 (1643-2232) 1975 (1509-2360) 2253 (2092-2383) 1803 (1549-2017) 1751 (1610-1888) 1855 (1373-2383)

Shape index 135 (103-160) 134 (113-156) 127 (105-155) 124 (113-138) 139 (114-154) 122 (101-145) 130 (101-160)


78



ABSTRACT
















41 INTRODUCTION






79











self-sustaining













80
























81




Family groups








Adults













82























83











mortality rates

43 RESULTS


Family groups






Table 42 Figure 41)





84








treatment with TMS

















85

Adults


























86












432 Pathology





Gross Pathology







Histopathology

87






(n=1)



















88
















those populations

44 DISCUSSION




institutions

89
























90


























91


























92















clinical disease










93


























94









drug resistance

95



0

50000

100000

150000

200000

250000

300000

350000

400000

29

33

37

41

45

49

53

57

61

65

69

73

77

81

85

89

93

97

10

1

10

5

10

9

11

3

11

7

12

1

12

5

12

9

13

3

13

7

14

1

14

5

14

9

OP

G

Age of Kits


96




25 microm

97



Collection Year 2014 2014 2014 2014 2014 2015 2016


29 - - - 0 - - -

30 - 0 - 0 - - -

- - - - -

34 0 0 - 0 0 - -

35 0 0 - 0 0 0 -

- -

46 0 0 - 0 0 0 -

47 0 0 - 0 0 0 -

48 0 lt14 - 0 0 0 -

- -

53 0 0 - 0 0 0 +

54 0 0 - 0 0 0 +

55 0 0 - 0 0 0 3717146

56 0 0 - 0 - 0 1084436

57 0 0 - 0 - 0 64133

58 0 324 - 0 0 0 20654

59 0 0 - 0 0 0 0

60 0 446688 - 0 0 0 0

61 0 934828 - 0 0 0 41111

62 0 530469 - 0 0 0

63 0 1617131 - 0 0 lt14

64 0 8396357 - 114 0 0

65 0 234876 - 5368 0 lt14

66 0 374625 - 9455 0 0

67 lt14 2311575 - 39579 0 -

68 0 603563 - 125051 0 0

69 - 4103036 - 27067 0 lt14

70 2470327 7759974 - 0 0 0

71 1073085 - 1784 0 0

72 3430966 - 0 0 0

73 4459536 - 7326 0 0

74 4842212 - 2 0 0

75 3627307 - 0 0 0

76 5801885 - 0 0 0

77 5994 0 0 0 0

78 589835 0 0 0

79 24815 0 0 0

80 144016 0 0 0

81 97862 0 0 0

82 0 0 0

127 0 0

128 0 2843

136 0 0

150 0


Dam Identity





98



Dam Identity

Poppy

2014

Bumblefoot

2014

Aubrey

2014

Fiddlesticks

2015

Guanella

^

2016



shedding 70-81 58-70 64-73 63-69 54-61


OPG min 0 0 0 0 206

OPG max 48442 83963 2707 lt14 371714




- = missing data



Collection

Year 2015 2016 2016 2016 2016 2016 2016


Age (years) 1 3 1 1 1 1 1

1084058 0 + + + + +

+ 0 42307650 6286676 + 183150 554274

857808 16650 12805238 7777929 + 215710 377920

1604894 16650 309690 139860 + 0 25808

377042 0 599400 119880 + - 37294

554445 0 34688 385579 117920 0 5363

26640 0 16650 0 0 0 7500

0 10406 0 0 0 1090

0 20813 0 0 0

- 0 1761 0

0 0 0 0

- 0

0

0



100



Sex M F M M M F M

Age (years) 1 3 1 1 1 1 1


OPG min 266 166 104 1199 1761 1831 1090

OPG max 10840 166 423076 77779 - 2157 554274





101



Ferret

ID Year

Age















102




Year Adult Family

2003 116 (625) -

2004 819 (4211) -

2005 021 (000) 14 (2500)

2006 021 (000) 07 (000)

2007 023 (000) 08 (000)

2008 224 (833) 14 (2500)

2009 025 (000) 06 (000)

2010 326 (1154) 07 (000)

2011 125 (400) 09 (000)

2012 125 (400) 08 (000)

2013 028 (000) 05 (000)

2014 430 (1333) 09 (000)

2015 035 (000) 35 (6000)

2016 - 27 (2857)




103



Total Mortality



1997 015 (000) 023 (000) 315 (2000) 423 (1739) 1998 038 (000) 119 (526) 838 (2105) 919 (4734) 1999 047 (000) 119 (526) 1647 (3404) 119 (526) 2000 034 (000) 015 (000) 434 (1176) 315 (2000) 2001 032 (000) 016 (000) 532 (1563) 116 (625) 2002 150 (200) 020 (000) 450 (800) 220 (1000) 2003 227 (741) 018 (000) 327 (1111) 118 (555) 2004 020 (000) 016 (000) 620 (3000) 216 (1250) 2005 016 (000) 015 (000) 416 (2500) 215 (1333) 2006 030 (000) 016 (000) 230 (667) 016 (000) 2007 019 (000) 015 (000) 419 (2105) 215 (1333) 2008 034 (000) 016 (000) 1134 (3235) 316 (1875) 2009 017 (000) 016 (000) 017 (000) 116 (625) 2010 017 (000) 016 (000) 317 (1765) 316 (1875) 2011 011 (000) 016 (000) 111 (909) 216 (1250) 2012 111 (909) 017 (000) 111 (909) 317 (1765) 2013 424 (1667) 017 (000) 424 (1667) 317 (1765) 2014 126 (384) 017 (000) 326 (1154) 217 (1176) 2015 04 (000) 017 (000) 04 (000) 217 (1176) 2016 011 (000) 017 (000) 211 (1818) 117 (588)

Mean annual () 195 053 1594 1359



as a percentage

104




ABSTRACT















species

51 INTRODUCTION






105


























106






ictidea


521 Animal care

















107







study



















108





Part 1








container












109

Part 2















524 Animal welfare









110



525 Hematology












(Chapter 3)









111






53 RESULTS


















112

531 Oocyst shedding

























113







533 Clinical signs





this time

534 Hematology








114

535 Necropsy

























115

54 DISCUSSION






















116


























117


























118
























119








absent












domestic ferret





120

























121











naive















122

















25 microm

124







25 microm

25 microm

125







0

1

2

3


Nu

mb

er

of

Ferr

ets

Aff

ect

ed



126





Ferret Identity


1 0 0 0 0 0 0 0

2 0 0 0 0 0 0 0

3 0 0 0 0 0 0 0

4 0 0 0 0 0 0 0

5 0 0 0 0 0 0 0

6 0 0 0 0 0 0 0

7 lt 14 1807 0 0 0 0 0

8 11053 139 7091 0 156238 0

9 463 11733 lt 14 203 lt 14

10 578 7549 0 0 0

11 lt 14 0 0 0

12 0 lt 14 0

13 0 lt 14 0

14 0 lt 14 0


gram of feces

127



Ferret

Identity


Inoculum Oocyst

Shedding

Presence

of clinical

disease

Coccidia

present in

sections

Inoculum Oocyst

Shedding

Presence

of clinical

disease

Coccidia

present in

sections













necropsy

128



Ferret Identity


103

104 105

201

203

205











S - 05

A - 15

S - 45

A - 25

S - 05

A - 05

S - 26

A - 06

S - 06

A - 06

S - 06

A - 06

S - 08

A - 08


S - 01

A - 01

S - 01

A - 01

S - 01

A - 01

S - 02

A - 02

S - 01

A - 01 none

S - 01

A - 01




129




ABSTRACT















61 INTRODUCTION





130






















621 Parasites



131
























132
























133



63 RESULTS






64 (for E ictidea)













regions




134


Mustelidae)

64 DISCUSSION























135









136


















137












138


139


nigripes)








































140




Total length

(nucleotides)

Nucleotide

identity

Total amino

acids

Amino acid

identity

COI CDS 1443 934 (95) 480 975 (12)

COIII CDS 756 899 (76) 251 932 (17)

CytB CDS 1080 950 (54) 359 986 (5)



applicable

141




142




143





144




parasites

145




















disease management






146



























147
























148

REFERENCES






7408200500053x












363



149







13256ndash59



101016jejop201407002













150























151



45
















101093nargkf436



101053jjepm201504012

152














20489ndash510 doi 101128CMR00005-07









153

41843ndash850








Vectors 7335 doi 1011861756-3305-7-335











12531ndash561 doi 101016jcvex200906001



154
















109551ndash557







155




(BFFMCOM)




Pathol 33437ndash439 doi 101177030098589603300412













1011861471-2148-11-92


156


157

APPENDICES

158



Collection Year 2014 2014 2014 2014 2014 2015 2016


29 - - - 0 - - -

30 - 0 - 0 - - -

31 - 0 - 0 - - -

32 - 0 - 0 - - -

33 - 0 - 0 - - -

34 0 0 - 0 0 - -

35 0 0 - 0 0 0 -

36 0 0 - 0 0 0 -

37 - 0 - 0 - 0 -

38 - 0 - 0 - 0 -

39 - 0 - 0 - 0 -

40 0 0 - 0 0 0 -

41 - 0 - 0 - 0 -

42 0 0 - 0 0 - -

43 0 0 - 0 0 0 -

44 0 0 - - 0 0 -

45 0 0 - 0 0 - -

46 0 0 - 0 0 0 -

47 0 0 - 0 0 0 -

48 0 lt14 - 0 0 0 -

49 0 0 - 0 0 0 -

50 0 0 - - 0 0 -

51 0 0 - 0 0 0 -

52 0 0 - 0 0 0 -

53 0 0 - 0 0 0 +

54 0 0 - 0 0 0 +

55 0 0 - 0 0 0 3717146

56 0 0 - 0 - 0 1084436

57 0 0 - 0 - 0 64133

58 0 324 - 0 0 0 20654

59 0 0 - 0 0 0 0

60 0 446688 - 0 0 0 0

61 0 934828 - 0 0 0 41111

62 0 530469 - 0 0 0

63 0 1617131 - 0 0 lt14

64 0 8396357 - 114 0 0

65 0 234876 - 5368 0 lt14

66 0 374625 - 9455 0 0

67 lt14 2311575 - 39579 0 -

68 0 603563 - 125051 0 0

69 - 4103036 - 27067 0 lt14

70 2470327 7759974 - 0 0 0

71 1073085 - 1784 0 0

72 3430966 - 0 0 0

73 4459536 - 7326 0 0

74 4842212 - 2 0 0

75 3627307 - 0 0 0

76 5801885 - 0 0 0

77 5994 0 0 0 0

78 589835 0 0 0


Dam Identity

159



Collection Year 2014 2014 2014 2014 2014 2015 2016


79 24815 0 0 0

80 144016 0 0 0

81 97862 0 0 0

82 0 0 0

83 0 0 0

84 0 0 0

85 0 0 0

86 0 0 0

87 0 0 0

88 0 0 0

89 0 0 0

90 0 0 0

91 0 0 0

92 0 - -

93 0 - 0

94 0 - 0

95 0 0 -

96 0 - 0

97 0 0 -

98 0 0 0

99 0 0 0

100 0 0 0

101 0 0 0

102 0 0 0

103 0 0 0

104 0 0 0

105 0 0 -

106 0 0 0

107 0 0 0

108 - 0 0

109 0 0 -

110 0 0 0

111 0 0 -

112 0 0 0

113 0 0 -

114 0 0 -

115 0 0 -

116 0 0 -

117 0 -

118 0 -

119 0 0

120 0 0

121 0 0

122 0 -


Dam Identity

160



Collection Year 2014 2014 2014 2014 2014 2015 2016


123 0 0

124 0 -

125 0 -

126 0 0

127 0 0

128 0 2843

129 0 0

130 0 -

131 0 0

132 0 0

133 0 0

134 0 0

135 0 0

136 0 0

137 0

138 0

139 0

140 0

141 0

142 0

143 0

144 0

145 0

146 -

147 0

148 0

149 0

150 0





Dam Identity

161



Test Reference

Intervala

Reference

Intervalb

101 102 103 104 105 201 202 203 204 205

Age (days) adult 70 50 50 50 50 50 48 48 48 50 48

WBC (x 109L) 27-112 53-120 86 64 77 97 76 48 8 72 104 76

RBC (x 1012

L) 50-108 55ndash74 66 58 45 5 48 47 67 5 5 42

Hb (gL) 87-177 104ndash136 121 106 90 58 94 91 122 96 98 80

HCT (LL) 04 - 051 029ndash037 037 033 027 030 029 027 038 030 030 024

MCV (fL) 44-52 478ndash548 55 56 60 60 60 58 57 59 61 58

MCH (pg) 15-18 175ndash191 18 18 20 12 20 20 18 19 20 18

MCHC (gL) 325-362 347ndash370 331 327 328 196 325 337 321 327 324 331

RDW () 12-16 - 134 127 139 139 133 131 122 136 131 127


MPV (fL) 5-10 - 78 78 96 75 66 74 76 81 74 82

TS Protein (gL) 49-76 - 54 51 - - - - - - - -





Basophils (x 109L) 0-02 0 009 0 0 0 0 0 0 0 0 0

Polychromasia 2-5 - 5-10 2-5 10-15 10-15 10-15 10-15 2-5 10-15 10-15 5-10



crenation Occ





Ferret Identity




162



Test Reference

Intervala

Reference

Intervalb

101 102 103 104 105 201 202 203 204 205

Age (days) adult 70 50 50 50 50 50 48 48 48 50 48

Calcium (mmolL) 185-242 253-302 239 233 241 244 24 221 242 234 253 242


Magnesium (mmolL) 08-139 - 08 08 07 08 08 06 08 07 08 08

Sodium (mmolL) 147-159 146-154 149 149 149 149 148 144 153 148 152 152

Potassium (mmolL) 37-57 47-83 44 46 42 46 48 45 47 44 47 46

Chloride (mmolL) 111-129 115-121 110 112 115 113 113 110 119 117 117 120


Anion gap (mmolL) 6 - 23 - 25 24 20 24 23 23 24 20 21 20

NaK ratio - - 34 32 35 32 31 32 33 34 32 33

Total protein (gL) 51-75 44-56 49 46 44 47 44 41 52 45 49 43

Albumin (gL) 24-40 26-32 29 26 28 28 28 25 28 27 29 25

Globulin (gL) 19-41 17-24 20 20 16 19 16 16 24 18 20 18

AG ratio 053-167 13ndash12 145 130 175 147 175 156 117 15 145 139



Glucose (mmolL) 32-91 64-138 47 54 54 42 55 53 57 56 52 59





ALKP (UL) 13-237 117ndash277 180 169 172 215 175 168 241 184 177 179

GGT (UL) 0-40 2ndash20 1 1 6 10 4 0 1 1 5 9

AST (UL) - 63ndash152 61 58 48 61 64 59 93 61 58 69

ALT (UL) 39-196 95ndash544 95 105 89 105 106 82 234 137 115 156

CK (UL) 74-294 - 513 330 496 560 530 492 793 539 479 724

Amylase (UL) - - 23 28 35 35 29 28 29 24 36 23

Lipase (UL) - - 65 63 60 64 62 56 67 69 60 68


Ferret Identity



163


Eimeria ictidea

Test Reference

Intervala

Reference

Intervalb

102 103 104 105 201 203 205

Age (days) adult 98-112 92 91 99 99 97 74 92


WBC (x 109L) 27-112 52ndash150 94 124 81 85 88 85 142

RBC (x 1012

L) 50-108 62ndash92 71 62 73 60 65 63 65

Hb (gL) 87-177 127ndash159 122 110 122 98 103 114 102

HCT (LL) 04-051 030ndash043 037 033 037 031 032 035 032

MCV (fL) 44-52 50ndash54 53 53 52 51 50 55 49

MCH (pg) 15-18 16-21 17 18 17 16 16 18 16

MCHC (gL) 325-362 351ndash426 328 332 325 319 320 326 317

RDW () 12-16 - 122 126 129 129 145 127 145


MPV (fL) 5-10 - 8 78 72 66 67 14 8

TS Protein (gL) 49-76 - 66 63 58 66 55 59 59





Basophils (x 109L) 0-02 0 0 012 0 0 009 0 0

Polychromasia 2-5 - 1-3 0-2 0-2 1-3 1-3 2-5 5-10


HJ bodies rare

crenation

rouleaux

poikilocytosis


hemolysis Neg

Ferret Identity





164



Test Reference

Intervala

Reference

Intervalb

102 103 104 105 201 203 205

Age (days) adult 98-112 92 91 99 99 97 74 92


Calcium (mmolL) 185-242 245-268 243 222 238 233 232 236 240


Magnesium (mmolL) 08-139 - 06 08 08 08 09 08

Sodium (mmolL) 147-159 148-155 147 150 150 148 148 150

Potassium (mmolL) 37-57 45-55 42 41 51 41 50 44

Chloride (mmolL) 111-129 114-124 114 113 118 112 111 115


Anion gap (mmolL) 6 - 23 - 21 20 18 20 30 21

NaK ratio - - 35 37 29 36 30 34

Total protein (gL) 51-75 49-64 52 55 55 49 56 57

Albumin (gL) 24-40 30-36 28 20 27 27 26 29 27

Globulin (gL) 19-41 19-30 32 28 28 23 27 30

AG ratio 053-167 11-17 063 096 096 113 107 090



Glucose (mmolL) 32-91 688-943 52 59 64 71 58 17 66





ALKP (UL) 13-237 41-181 124 120 213 120 146 170 196

GGT (UL) 0-40 1-2 2 1 3 0 0 2

AST (UL) - 47-128 48 95 104 66 100 100

ALT (UL) 39-196 78-279 133 110 140 203 158 183 281

CK (UL) 74-294 - 382 765 1190 578 680 930

Amylase (UL) - - 24 37 31 28 33 35

Lipase (UL) - - 60 65 72 72 86 79


Ferret Identity




165


Mon

8 AM

Mon

4 PM

Tues

8 AM

Tues

4 PM

Wed

8 AM

Wed

4 PM

Thurs

8 AM

Thurs

8 PM

Fri

8 AM

Fri

4 PM

Sat

8 AM

Sat

4 PM

Sun

8AM

Sun

4 PM

Mentation

Weight (g)

Respiratory

Rate

Vomit

(+ ++ +++)

Diarrhea

(+ ++ +++)

Urination

(+ ++ +++)

Defecation

(+ ++ +++)

Food

offered

Food

remaining

Water remaining

(ml)

Treatments

Other

observations

Initials of

observer

166

Animal ID ________________________________________ Week ______________

Monitoring Criteria











Monitoring Times




hour care sheet

Intervention Points



Removal Criteria




167


Animal ID ___________________________________________ Date___________________

0700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800

Mentation

Weight (g)

Heart Rate

Respiratory Rate

Temperature (C)

Water offered

(mL)

Water consumed

(mL)

Food offered

(type and

amount in

grams)

Food consumed

(type and

amount in

grams)

Presence of urine

Presence of feces

168

Character of

feces

Presence of

vomit

Character of

vomit

Medications to

be administered

Other

observations

169

1900 2000 2100 2200 2300 2400 100 200 300 400 500 600

Mentation

Weight (g)

Heart Rate

Respiratory Rate

Temperature (C)

Water offered

(mL)

Water consumed

(mL)

Food offered

(type and amount

in grams)

Food consumed

(type and amount

in grams)

Presence of urine

Presence of feces

Character of

feces

Presence of vomit

Character of

vomit

170

Medications to be

administered

Other

observations

Monitoring Criteria








171




Monitoring Times


hour care sheet

Intervention Points



Removal Criteria




172











summer student




Example Bag label

Ferret ID Date

Weight of Feces


Soft

Liquid

Bloody

Abnormal odour





and Dale Smith)




CBCBiochemistry





173











Euthanasia Protocol









Necropsy Protocol



viscera








calculations)











174













Laboratory SOPs

















oocysts







175


























weeks 1-2)

vi









been as successful


















vii























Adriana Pastor

Toronto August 2017

viii



exceptions









ix

TABLE OF CONTENTS

ABSTRACT ii

DEDICATION iv

ACKNOWLEDGEMENTS v



LIST OF TABLES xiii

LIST OF FIGURES xiv


ABBREVIATIONS xvi


11 INTRODUCTION 1

12 APICOMPLEXA 1













25




ferrets 28



x




171 Theory 32



181 Objectives 36

182 Hypotheses 36

183 Applications 36



21 INTRODUCTION 39






23 RESULTS 47





24 DISCUSSION 50



31 INTRODUCTION 60





33 RESULTS 66



34 DISCUSSION 69

xi



41 INTRODUCTION 78







43 RESULTS 83


432 Pathology 86


44 DISCUSSION 88




51 INTRODUCTION 104


521 Animal care 106




525 Hematology 110



53 RESULTS 111




534 Hematology 113

535 Necropsy 114

54 DISCUSSION 115

xii




61 INTRODUCTION 129


621 Parasites 130




63 RESULTS 133

64 DISCUSSION 134


REFERENCES 148

APPENDICES 157

xiii

LIST OF TABLES





from 2008-2015 56

























xiv

LIST OF FIGURES


























xv

LIST OF APPENDICES














xvi

ABBREVIATIONS




bp Base pair






CytB Cytochrome b





L Length

LSU Large subunit

mt Mitochondrial


nu Nuclear



rDNA Ribosomal DNA

SI Shape index





SSU Small subunit



W Width

1


11 INTRODUCTION














12 APICOMPLEXA








2









(Cox 1994)






httptolweborg

2

1

3

4

5

3


























4











author



5






















6






















7

























8
























9








2012)

10



species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Cytoisospora

eversmanni

Mustela

eversmanii

(Steppe polecat)

Mustela

putorius

(European

polecat)


W 148 (16ndash12)

LW 13 (11ndash16)

M absent

PG absent

OR absent

L 115

(10ndash135)

W 98

(9ndash11)

LW 12

(11ndash15)

SB absent

SR present


Svanbaev 1956

Nukerbaeva amp

Svanbaev 1973

1977

Cystoisospora

pavlovskyi

Mustela

eversmanii

Mustela

putorius


W 273 (265ndash285)

LW 12 (11ndash14)

M absent

PG absent

OR absent

L 195

(18ndash21)

W 144

(12ndash15)

LW 14

(12ndash15)

SB absent

SR present


Svanbaev 1956

Nukerbaeva amp

Svanbaev 1973

1977

Eimeria

baskanica^

Mustela

erminae

(ermine)


ends

L 112-126

W 84-98

M absent

PG absent

OR present


Svanbaev 1977


putorius

Mustela

putorius furo

(dom ferret)

Mustela

nigripes (BFF)

Mustela vison

(mink)

Homoxenous

Small intestine

rectum (H 1927)

Jejunumileum (BP

1993)

Spherical ndash

subspherical

L 11-14

W 10-13

OW 2 layers

M absent

PG absent

OR absent

Spindloid

L 8-9

W 4

SB present

SR present


et al 1993

Hoare 1927 1935b

Jolley et al 1994

Nukerbaeva amp

Svanbaev

19731977

Williams et al 1988

1992 1996


Bile duct

Spherical

L 13-17

W 13-17

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

L 6

W 4

SB absent

SR absent


Grafner et al 1967

11


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References


eversmanni

Mustela

nigripes

Mustela

putorius

Mustela

putorius furo

Homoxenous

Small intestine


L 13-27

W 13-21

OW 2 layers

M present

PG absent

OR absent

Ovoid

(irregular)

L 115

W65

SB present

SR present

- Hoare 1927 1935a

1935b

Jolley et al 1994

Litvenkova 1969

Svanbaev 1956

Tinar 1985

Williams et al 1988

1992


(tayra)

Homoxenous

Feces

Ovoid

L 21-25

W 18-20

OW outer

layer smooth

M absent

PG absent

OR absent

Ellipsoid

L 10-12

W 65

SB present

SR present

Elongate (one

end broader than

the other)

Carini amp da

Fonseca 1938


(European

badger)


L 20plusmn018

W 157plusmn002

LW128plusmn0017

(112-15)

OW 2 layers

(outer

smooth)

M absent

PG present

OR present

Ovoid

L

119plusmn0018

W 65plusmn008

LW 183

(155-24)

SB present

L 90plusmn005

W 324plusmn0025

SRB present

Anwar et al 2000

Kotlan amp Pospesch

1933


Mustela nivalis

(snow weasel)

Homoxenous

Duodenumileum

Spherical or

Ellipsoid

L 18-26

W 14-24

OW 2 layers

M absent

PG present

OR absent

Ovoid

L 8

W 5

SB present

SR present

Broad at one

end and tapered

at other

L 7

W 3

Glebezdin 1978

Iwanoff-Gobzem

1934

Levine 1948

Musaev amp Veisov

1983

Tinar 1985


(sable)

Homoxenous

Gut

Spherical or

subspherical

L 112-126

W 112

OW 2 layers

M absent

OR absent

Ovoid

L 56

W 42

SR present



L avg 216

W avg 180

LW 1076

OW 2 layers

M absent

PG absent

OR absent

Ovoid

L 96-112

W 56-72

SR absent


Yakimoff amp

Gousseff 1934

Yakimoff amp

Terwinsky 1930

1931

12


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Eimeria vison

(Eimeria

mustelae)

Mustela

putorius

Mustela

putorius furo

Mustela vison

Homoxenous

Small intestine

+- large intestine

Ovoid

L 17-22

W 9-18

OW 2 layers

M absent

OR

sometimes

present

Ovoid or

Piriform

L 10

W 55

SB absent

SR present

Curved or Club

shaped

L 9

W 25


Foreyt et al 1977

Kingscote 1934

1935

Levine 1948

McTaggart 1960

Nukerbaeva amp

Svanbaev

19731977

Tinar 1985

Umurzakov amp

Nukerbaeva 1985

Wolter 1961

Zimmermann 1959


(Libyan striped

weasel)

Homoxenous

Feces

Spherical

L 25-27

W 25-27

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

Ovoid

L 15-17

W 10-12

SB absent

SR present

Elongate

L 135

W 3

Prasad 1961


(mountain

weasel)

Homoxenous

Gut

Oval or spherical

L 280-336

W 252-280

LW 121 (111-

124)

OW 2 layers

M absent

PG absent

OR absent

Ovoid or

spherical

L 140-168

W 111-168

SR present

Svanbaev amp

Rachmatullina

1971


Large intestine

Ovoid

L 224 (220-250)

W 174 (160-190)

LW 135 (133-

137)

OW 1 layer

PG present

OR present

Ovoid

L 120

(100-130)

W 70 (60-

80)

SB present

SR present


1983

13


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Isospora

hoogstraali


Feces

Ellipsoid

L 37-41

W 32-34

OW 2 layers

(outer

smooth)

M absent

PG some

OR absent

Ovoid

L 19-21

W 13-15

SB absent

SR present

Club-shaped

L 18-19

W 4-6

Prasad 1961


putorius

Mustela

putorius furo

Mustela vison

Homoxenous

Feces

Intestinal contents

Ovoid L

320-368

W 272-304

OW 2 layers

M absent

PG absent

OR absent

Ellipsoid

L 208

W 144

SB absent

SR present


Hoare 1927

Levine 1948

McTaggart 1960

Nukerbaeva amp

Svanbaev 1973

1974 1977

Tinar 1985


(European

otter)

Lutra

canadensis

(North

American river

otter)


L 312 (275-32)

W 296 (28-31)

LW 104

(10-112)

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

Ellipsoid

L 182 (17-

19)

W 144 (14-

16)

LW128

(12-14)

Sb absent

sSB absent

SR present

Spindle- shaped

L 124

W 25

SRB present

Torres et al 2000

Hoover et al 1985

Isospora

martessii


Gut

Ovoid short oval or

spherical

L 252 ndash 280 196

168

W 168 ndash 224 168

168

OW 2 layers

M absent

OR absent

Ovoid

L 112-168

W 84-112

SR present



L 328plusmn034

W 269plusmn019

LW122 (110-

157)

OW 2 layers

(outer

smooth)

M absent

PG absent

OR absent

Ellipsoid

L

215plusmn0166

W 14plusmn012

LW 155

(133-185)

SR absent

Round at one

end other end

tapered

L 142plusmn116

W 40plusmn017

SRB absent

Anwar et al 2000

Glebezdin 1978

Kotlan amp Pospesch

1933

Pelleacuterdy 1955

14


species

Life cycle

Location

Oocyst shape and

size

Oocyst

description

Sporocyst

description

Sporozoite

description

References

Isospora

mustelae (nomen

nudum)


7 W

225



Large intestine

Ovoid

L 206 (200-230)

W 184 (180-210)

LW 11 (109-111)

OW 1 layer

PG absent

OR absent

Ovoid

L 125

(120-130)

W 80 (70-

90)

SR present

Lemon or pear

shaped

Musaev amp Veisov

1983

Unnamed

ldquoCoccidiardquo^

Mustela

nigripes


Unnamed

ldquoCoccidiardquo^

Mustela

nigripes

Trachea bronchus

bronchial glands


Unnamed

Eimeria sp^

Mustela

nigripes

Feces

intestinal contents

Ovoid

L 350-386

W 212-232


Williams et al

1992

Unnamed

Eimeria sp^

Mustela

putorius furo


et al 1993

Unnamed

Eimeria sp^


Large intestine

Ovoid-ellipsoid L

2031 (1712-2162)

W 148 (1225-

1681)

LW 136 (121-16-

)

OW 1 layer

PG absent

OR absent

Ovoid or

pear-shaped

L 60-100

W 40-80

SR present

Elongate

L 50-90

W 30-70

Musaev amp Veisov

1983

Unnamed

Eimeria sp^

Martes martes

(marten)

Homoxenous Ovoid

L avg 216

W avg 180

LW 1076


SR present

L 126

W 60

Yakimoff and

Gousseff 1934

Unnamed

Isospora sp^

Mustela

putorius furo

Feces - - - - Bell 1994

Unnamed

Isospora sp^

Mustela

putorius furo

Feces - - - - Bell 1994




15


similar host

16



























17


























18

















1967)









19


























20

























21

























22

















reproduction









23


























24
























Gorham 1953)

25

























26

























27

























28
















lacking









29

























30








footed ferrets
















31



























32
















171 Theory









33


























34

























35
























36


181 Objectives








footed ferret

182 Hypotheses







ferrets

183 Applications






37














38






review)

ABSTRACT


















putorius furo

39

21 INTRODUCTION

























40




infection trial














posterior end







41


the sporocyst























42












E furonis nu 18S















43















221 Fecal samples










44

























45























46



















47

23 RESULTS






















48























49


life stages























50














24 DISCUSSION








Canada or the EU

51


























52

























53









identification








relationships

ACKNOWLEDGEMENTS








54


(400566) to JRB

55
















56






Cystoisospora sp


Eimeria furonis


Eimeria ictidea


2008 3140 (21) 6214 (28) 3 2 0 4 0 0

2009 2160 (12) 14214 (65) 2 9 0 5 0 0

2010 8127 (63) 20213 (94) 7 10 1 10 0 0

2011 0114 (0) 17215 (79) 0 9 0 7 0 1

2012 3108 (28) 10231 (43) 2 4 1 6 0 0

2013 281 (25) 16270 (59) 2 13 0 2 0 1

2014 6127 (47) 12234 (51) 5 6 1 6 0 0

2015 4108 (37) 9249 (36) 4 3 0 6 0 0

Total 28 (29) 104 (56) 25 56 3 46 0 2

Average

year 35 130 31 70 04 58 00 03


57


Sample ID Source



mt COI GenBank

Accession

nu 18S rDNA

GenBank Accession

















58





25 microm

59








60



NIGRIPES)

ABSTRACT













31 INTRODUCTION








61











2014)















62


























63









et al 1994)

















64
















SSP facilities


321 Fecal samples







65














amp Methods)








66


















Figure 31

33 RESULTS





67


























68


























69






34 DISCUSSION




















70








conducted



















71





















ACKNOWLEDGEMENTS





72



73















74







10 microm 5 microm

75





Identity






Identity




76


nigripes)













fresh feces


POS E cf ictidea

-

E ictidea













histologic section


77


(Mustela nigripes)



Length (microm) 2333 (2055-2583) 2456 (2111-2848) 2505 (2079-3008) 2779 (2590-3060) 2493 (2036-2822) 2139 (1859-2372) 2398 (1859-3057)

Width (microm) 1676 (1373-2180) 1835 (1643-2232) 1975 (1509-2360) 2253 (2092-2383) 1803 (1549-2017) 1751 (1610-1888) 1855 (1373-2383)

Shape index 135 (103-160) 134 (113-156) 127 (105-155) 124 (113-138) 139 (114-154) 122 (101-145) 130 (101-160)


78



ABSTRACT
















41 INTRODUCTION






79











self-sustaining













80
























81




Family groups








Adults













82























83











mortality rates

43 RESULTS


Family groups






Table 42 Figure 41)





84








treatment with TMS

















85

Adults


























86












432 Pathology





Gross Pathology







Histopathology

87






(n=1)



















88
















those populations

44 DISCUSSION




institutions

89
























90


























91


























92















clinical disease










93


























94









drug resistance

95



0

50000

100000

150000

200000

250000

300000

350000

400000

29

33

37

41

45

49

53

57

61

65

69

73

77

81

85

89

93

97

10

1

10

5

10

9

11

3

11

7

12

1

12

5

12

9

13

3

13

7

14

1

14

5

14

9

OP

G

Age of Kits


96




25 microm

97



Collection Year 2014 2014 2014 2014 2014 2015 2016


29 - - - 0 - - -

30 - 0 - 0 - - -

- - - - -

34 0 0 - 0 0 - -

35 0 0 - 0 0 0 -

- -

46 0 0 - 0 0 0 -

47 0 0 - 0 0 0 -

48 0 lt14 - 0 0 0 -

- -

53 0 0 - 0 0 0 +

54 0 0 - 0 0 0 +

55 0 0 - 0 0 0 3717146

56 0 0 - 0 - 0 1084436

57 0 0 - 0 - 0 64133

58 0 324 - 0 0 0 20654

59 0 0 - 0 0 0 0

60 0 446688 - 0 0 0 0

61 0 934828 - 0 0 0 41111

62 0 530469 - 0 0 0

63 0 1617131 - 0 0 lt14

64 0 8396357 - 114 0 0

65 0 234876 - 5368 0 lt14

66 0 374625 - 9455 0 0

67 lt14 2311575 - 39579 0 -

68 0 603563 - 125051 0 0

69 - 4103036 - 27067 0 lt14

70 2470327 7759974 - 0 0 0

71 1073085 - 1784 0 0

72 3430966 - 0 0 0

73 4459536 - 7326 0 0

74 4842212 - 2 0 0

75 3627307 - 0 0 0

76 5801885 - 0 0 0

77 5994 0 0 0 0

78 589835 0 0 0

79 24815 0 0 0

80 144016 0 0 0

81 97862 0 0 0

82 0 0 0

127 0 0

128 0 2843

136 0 0

150 0


Dam Identity





98



Dam Identity

Poppy

2014

Bumblefoot

2014

Aubrey

2014

Fiddlesticks

2015

Guanella

^

2016



shedding 70-81 58-70 64-73 63-69 54-61


OPG min 0 0 0 0 206

OPG max 48442 83963 2707 lt14 371714




- = missing data



Collection

Year 2015 2016 2016 2016 2016 2016 2016


Age (years) 1 3 1 1 1 1 1

1084058 0 + + + + +

+ 0 42307650 6286676 + 183150 554274

857808 16650 12805238 7777929 + 215710 377920

1604894 16650 309690 139860 + 0 25808

377042 0 599400 119880 + - 37294

554445 0 34688 385579 117920 0 5363

26640 0 16650 0 0 0 7500

0 10406 0 0 0 1090

0 20813 0 0 0

- 0 1761 0

0 0 0 0

- 0

0

0



100



Sex M F M M M F M

Age (years) 1 3 1 1 1 1 1


OPG min 266 166 104 1199 1761 1831 1090

OPG max 10840 166 423076 77779 - 2157 554274





101



Ferret

ID Year

Age















102




Year Adult Family

2003 116 (625) -

2004 819 (4211) -

2005 021 (000) 14 (2500)

2006 021 (000) 07 (000)

2007 023 (000) 08 (000)

2008 224 (833) 14 (2500)

2009 025 (000) 06 (000)

2010 326 (1154) 07 (000)

2011 125 (400) 09 (000)

2012 125 (400) 08 (000)

2013 028 (000) 05 (000)

2014 430 (1333) 09 (000)

2015 035 (000) 35 (6000)

2016 - 27 (2857)




103



Total Mortality



1997 015 (000) 023 (000) 315 (2000) 423 (1739) 1998 038 (000) 119 (526) 838 (2105) 919 (4734) 1999 047 (000) 119 (526) 1647 (3404) 119 (526) 2000 034 (000) 015 (000) 434 (1176) 315 (2000) 2001 032 (000) 016 (000) 532 (1563) 116 (625) 2002 150 (200) 020 (000) 450 (800) 220 (1000) 2003 227 (741) 018 (000) 327 (1111) 118 (555) 2004 020 (000) 016 (000) 620 (3000) 216 (1250) 2005 016 (000) 015 (000) 416 (2500) 215 (1333) 2006 030 (000) 016 (000) 230 (667) 016 (000) 2007 019 (000) 015 (000) 419 (2105) 215 (1333) 2008 034 (000) 016 (000) 1134 (3235) 316 (1875) 2009 017 (000) 016 (000) 017 (000) 116 (625) 2010 017 (000) 016 (000) 317 (1765) 316 (1875) 2011 011 (000) 016 (000) 111 (909) 216 (1250) 2012 111 (909) 017 (000) 111 (909) 317 (1765) 2013 424 (1667) 017 (000) 424 (1667) 317 (1765) 2014 126 (384) 017 (000) 326 (1154) 217 (1176) 2015 04 (000) 017 (000) 04 (000) 217 (1176) 2016 011 (000) 017 (000) 211 (1818) 117 (588)

Mean annual () 195 053 1594 1359



as a percentage

104




ABSTRACT















species

51 INTRODUCTION






105


























106






ictidea


521 Animal care

















107







study



















108





Part 1








container












109

Part 2















524 Animal welfare









110



525 Hematology












(Chapter 3)









111






53 RESULTS


















112

531 Oocyst shedding

























113







533 Clinical signs





this time

534 Hematology








114

535 Necropsy

























115

54 DISCUSSION






















116


























117


























118
























119








absent












domestic ferret





120

























121











naive















122

















25 microm

124







25 microm

25 microm

125







0

1

2

3


Nu

mb

er

of

Ferr

ets

Aff

ect

ed



126





Ferret Identity


1 0 0 0 0 0 0 0

2 0 0 0 0 0 0 0

3 0 0 0 0 0 0 0

4 0 0 0 0 0 0 0

5 0 0 0 0 0 0 0

6 0 0 0 0 0 0 0

7 lt 14 1807 0 0 0 0 0

8 11053 139 7091 0 156238 0

9 463 11733 lt 14 203 lt 14

10 578 7549 0 0 0

11 lt 14 0 0 0

12 0 lt 14 0

13 0 lt 14 0

14 0 lt 14 0


gram of feces

127



Ferret

Identity


Inoculum Oocyst

Shedding

Presence

of clinical

disease

Coccidia

present in

sections

Inoculum Oocyst

Shedding

Presence

of clinical

disease

Coccidia

present in

sections













necropsy

128



Ferret Identity


103

104 105

201

203

205











S - 05

A - 15

S - 45

A - 25

S - 05

A - 05

S - 26

A - 06

S - 06

A - 06

S - 06

A - 06

S - 08

A - 08


S - 01

A - 01

S - 01

A - 01

S - 01

A - 01

S - 02

A - 02

S - 01

A - 01 none

S - 01

A - 01




129




ABSTRACT















61 INTRODUCTION





130






















621 Parasites



131
























132
























133



63 RESULTS






64 (for E ictidea)













regions




134


Mustelidae)

64 DISCUSSION























135









136


















137












138


139


nigripes)








































140




Total length

(nucleotides)

Nucleotide

identity

Total amino

acids

Amino acid

identity

COI CDS 1443 934 (95) 480 975 (12)

COIII CDS 756 899 (76) 251 932 (17)

CytB CDS 1080 950 (54) 359 986 (5)



applicable

141




142




143





144




parasites

145




















disease management






146



























147
























148

REFERENCES






7408200500053x












363



149







13256ndash59



101016jejop201407002













150























151



45
















101093nargkf436



101053jjepm201504012

152














20489ndash510 doi 101128CMR00005-07









153

41843ndash850








Vectors 7335 doi 1011861756-3305-7-335











12531ndash561 doi 101016jcvex200906001



154
















109551ndash557







155




(BFFMCOM)




Pathol 33437ndash439 doi 101177030098589603300412













1011861471-2148-11-92


156


157

APPENDICES

158



Collection Year 2014 2014 2014 2014 2014 2015 2016


29 - - - 0 - - -

30 - 0 - 0 - - -

31 - 0 - 0 - - -

32 - 0 - 0 - - -

33 - 0 - 0 - - -

34 0 0 - 0 0 - -

35 0 0 - 0 0 0 -

36 0 0 - 0 0 0 -

37 - 0 - 0 - 0 -

38 - 0 - 0 - 0 -

39 - 0 - 0 - 0 -

40 0 0 - 0 0 0 -

41 - 0 - 0 - 0 -

42 0 0 - 0 0 - -

43 0 0 - 0 0 0 -

44 0 0 - - 0 0 -

45 0 0 - 0 0 - -

46 0 0 - 0 0 0 -

47 0 0 - 0 0 0 -

48 0 lt14 - 0 0 0 -

49 0 0 - 0 0 0 -

50 0 0 - - 0 0 -

51 0 0 - 0 0 0 -

52 0 0 - 0 0 0 -

53 0 0 - 0 0 0 +

54 0 0 - 0 0 0 +

55 0 0 - 0 0 0 3717146

56 0 0 - 0 - 0 1084436

57 0 0 - 0 - 0 64133

58 0 324 - 0 0 0 20654

59 0 0 - 0 0 0 0

60 0 446688 - 0 0 0 0

61 0 934828 - 0 0 0 41111

62 0 530469 - 0 0 0

63 0 1617131 - 0 0 lt14

64 0 8396357 - 114 0 0

65 0 234876 - 5368 0 lt14

66 0 374625 - 9455 0 0

67 lt14 2311575 - 39579 0 -

68 0 603563 - 125051 0 0

69 - 4103036 - 27067 0 lt14

70 2470327 7759974 - 0 0 0

71 1073085 - 1784 0 0

72 3430966 - 0 0 0

73 4459536 - 7326 0 0

74 4842212 - 2 0 0

75 3627307 - 0 0 0

76 5801885 - 0 0 0

77 5994 0 0 0 0

78 589835 0 0 0


Dam Identity

159



Collection Year 2014 2014 2014 2014 2014 2015 2016


79 24815 0 0 0

80 144016 0 0 0

81 97862 0 0 0

82 0 0 0

83 0 0 0

84 0 0 0

85 0 0 0

86 0 0 0

87 0 0 0

88 0 0 0

89 0 0 0

90 0 0 0

91 0 0 0

92 0 - -

93 0 - 0

94 0 - 0

95 0 0 -

96 0 - 0

97 0 0 -

98 0 0 0

99 0 0 0

100 0 0 0

101 0 0 0

102 0 0 0

103 0 0 0

104 0 0 0

105 0 0 -

106 0 0 0

107 0 0 0

108 - 0 0

109 0 0 -

110 0 0 0

111 0 0 -

112 0 0 0

113 0 0 -

114 0 0 -

115 0 0 -

116 0 0 -

117 0 -

118 0 -

119 0 0

120 0 0

121 0 0

122 0 -


Dam Identity

160



Collection Year 2014 2014 2014 2014 2014 2015 2016


123 0 0

124 0 -

125 0 -

126 0 0

127 0 0

128 0 2843

129 0 0

130 0 -

131 0 0

132 0 0

133 0 0

134 0 0

135 0 0

136 0 0

137 0

138 0

139 0

140 0

141 0

142 0

143 0

144 0

145 0

146 -

147 0

148 0

149 0

150 0





Dam Identity

161



Test Reference

Intervala

Reference

Intervalb

101 102 103 104 105 201 202 203 204 205

Age (days) adult 70 50 50 50 50 50 48 48 48 50 48

WBC (x 109L) 27-112 53-120 86 64 77 97 76 48 8 72 104 76

RBC (x 1012

L) 50-108 55ndash74 66 58 45 5 48 47 67 5 5 42

Hb (gL) 87-177 104ndash136 121 106 90 58 94 91 122 96 98 80

HCT (LL) 04 - 051 029ndash037 037 033 027 030 029 027 038 030 030 024

MCV (fL) 44-52 478ndash548 55 56 60 60 60 58 57 59 61 58

MCH (pg) 15-18 175ndash191 18 18 20 12 20 20 18 19 20 18

MCHC (gL) 325-362 347ndash370 331 327 328 196 325 337 321 327 324 331

RDW () 12-16 - 134 127 139 139 133 131 122 136 131 127


MPV (fL) 5-10 - 78 78 96 75 66 74 76 81 74 82

TS Protein (gL) 49-76 - 54 51 - - - - - - - -





Basophils (x 109L) 0-02 0 009 0 0 0 0 0 0 0 0 0

Polychromasia 2-5 - 5-10 2-5 10-15 10-15 10-15 10-15 2-5 10-15 10-15 5-10



crenation Occ





Ferret Identity




162



Test Reference

Intervala

Reference

Intervalb

101 102 103 104 105 201 202 203 204 205

Age (days) adult 70 50 50 50 50 50 48 48 48 50 48

Calcium (mmolL) 185-242 253-302 239 233 241 244 24 221 242 234 253 242


Magnesium (mmolL) 08-139 - 08 08 07 08 08 06 08 07 08 08

Sodium (mmolL) 147-159 146-154 149 149 149 149 148 144 153 148 152 152

Potassium (mmolL) 37-57 47-83 44 46 42 46 48 45 47 44 47 46

Chloride (mmolL) 111-129 115-121 110 112 115 113 113 110 119 117 117 120


Anion gap (mmolL) 6 - 23 - 25 24 20 24 23 23 24 20 21 20

NaK ratio - - 34 32 35 32 31 32 33 34 32 33

Total protein (gL) 51-75 44-56 49 46 44 47 44 41 52 45 49 43

Albumin (gL) 24-40 26-32 29 26 28 28 28 25 28 27 29 25

Globulin (gL) 19-41 17-24 20 20 16 19 16 16 24 18 20 18

AG ratio 053-167 13ndash12 145 130 175 147 175 156 117 15 145 139



Glucose (mmolL) 32-91 64-138 47 54 54 42 55 53 57 56 52 59





ALKP (UL) 13-237 117ndash277 180 169 172 215 175 168 241 184 177 179

GGT (UL) 0-40 2ndash20 1 1 6 10 4 0 1 1 5 9

AST (UL) - 63ndash152 61 58 48 61 64 59 93 61 58 69

ALT (UL) 39-196 95ndash544 95 105 89 105 106 82 234 137 115 156

CK (UL) 74-294 - 513 330 496 560 530 492 793 539 479 724

Amylase (UL) - - 23 28 35 35 29 28 29 24 36 23

Lipase (UL) - - 65 63 60 64 62 56 67 69 60 68


Ferret Identity



163


Eimeria ictidea

Test Reference

Intervala

Reference

Intervalb

102 103 104 105 201 203 205

Age (days) adult 98-112 92 91 99 99 97 74 92


WBC (x 109L) 27-112 52ndash150 94 124 81 85 88 85 142

RBC (x 1012

L) 50-108 62ndash92 71 62 73 60 65 63 65

Hb (gL) 87-177 127ndash159 122 110 122 98 103 114 102

HCT (LL) 04-051 030ndash043 037 033 037 031 032 035 032

MCV (fL) 44-52 50ndash54 53 53 52 51 50 55 49

MCH (pg) 15-18 16-21 17 18 17 16 16 18 16

MCHC (gL) 325-362 351ndash426 328 332 325 319 320 326 317

RDW () 12-16 - 122 126 129 129 145 127 145


MPV (fL) 5-10 - 8 78 72 66 67 14 8

TS Protein (gL) 49-76 - 66 63 58 66 55 59 59





Basophils (x 109L) 0-02 0 0 012 0 0 009 0 0

Polychromasia 2-5 - 1-3 0-2 0-2 1-3 1-3 2-5 5-10


HJ bodies rare

crenation

rouleaux

poikilocytosis


hemolysis Neg

Ferret Identity





164



Test Reference

Intervala

Reference

Intervalb

102 103 104 105 201 203 205

Age (days) adult 98-112 92 91 99 99 97 74 92


Calcium (mmolL) 185-242 245-268 243 222 238 233 232 236 240


Magnesium (mmolL) 08-139 - 06 08 08 08 09 08

Sodium (mmolL) 147-159 148-155 147 150 150 148 148 150

Potassium (mmolL) 37-57 45-55 42 41 51 41 50 44

Chloride (mmolL) 111-129 114-124 114 113 118 112 111 115


Anion gap (mmolL) 6 - 23 - 21 20 18 20 30 21

NaK ratio - - 35 37 29 36 30 34

Total protein (gL) 51-75 49-64 52 55 55 49 56 57

Albumin (gL) 24-40 30-36 28 20 27 27 26 29 27

Globulin (gL) 19-41 19-30 32 28 28 23 27 30

AG ratio 053-167 11-17 063 096 096 113 107 090



Glucose (mmolL) 32-91 688-943 52 59 64 71 58 17 66





ALKP (UL) 13-237 41-181 124 120 213 120 146 170 196

GGT (UL) 0-40 1-2 2 1 3 0 0 2

AST (UL) - 47-128 48 95 104 66 100 100

ALT (UL) 39-196 78-279 133 110 140 203 158 183 281

CK (UL) 74-294 - 382 765 1190 578 680 930

Amylase (UL) - - 24 37 31 28 33 35

Lipase (UL) - - 60 65 72 72 86 79


Ferret Identity




165


Mon

8 AM

Mon

4 PM

Tues

8 AM

Tues

4 PM

Wed

8 AM

Wed

4 PM

Thurs

8 AM

Thurs

8 PM

Fri

8 AM

Fri

4 PM

Sat

8 AM

Sat

4 PM

Sun

8AM

Sun

4 PM

Mentation

Weight (g)

Respiratory

Rate

Vomit

(+ ++ +++)

Diarrhea

(+ ++ +++)

Urination

(+ ++ +++)

Defecation

(+ ++ +++)

Food

offered

Food

remaining

Water remaining

(ml)

Treatments

Other

observations

Initials of

observer

166

Animal ID ________________________________________ Week ______________

Monitoring Criteria











Monitoring Times




hour care sheet

Intervention Points



Removal Criteria




167


Animal ID ___________________________________________ Date___________________

0700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800

Mentation

Weight (g)

Heart Rate

Respiratory Rate

Temperature (C)

Water offered

(mL)

Water consumed

(mL)

Food offered

(type and

amount in

grams)

Food consumed

(type and

amount in

grams)

Presence of urine

Presence of feces

168

Character of

feces

Presence of

vomit

Character of

vomit

Medications to

be administered

Other

observations

169

1900 2000 2100 2200 2300 2400 100 200 300 400 500 600

Mentation

Weight (g)

Heart Rate

Respiratory Rate

Temperature (C)

Water offered

(mL)

Water consumed

(mL)

Food offered

(type and amount

in grams)

Food consumed

(type and amount

in grams)

Presence of urine

Presence of feces

Character of

feces

Presence of vomit

Character of

vomit

170

Medications to be

administered

Other

observations

Monitoring Criteria








171




Monitoring Times


hour care sheet

Intervention Points



Removal Criteria




172











summer student




Example Bag label

Ferret ID Date

Weight of Feces


Soft

Liquid

Bloody

Abnormal odour





and Dale Smith)




CBCBiochemistry





173











Euthanasia Protocol









Necropsy Protocol



viscera








calculations)











174













Laboratory SOPs

















oocysts







175


























weeks 1-2)

investigating enteric coccidiosis in the endangered black

Documents