keeping pathogens out of your zebrafish facility

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Keeping Pathogens out of Your Zebrafish Facility

Christopher M. Whipps & Carolyn ChangState University of New York,College of Environmental Science and Forestry (SUNY-ESF)Syracuse, NY

[email protected]

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Main Recommendations• Identify Pathogens• Prevent Introductions• Control Within Facility

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IntroductionMichael L. Kent and Zoltán Varga

Introduction: Use of Zebrafish in Research and Importance of Health and HusbandryHealth

Christopher M. Whipps, Christine Lieggi, and Robert Wagner Mycobacteriosis in Zebrafish Colonies

Justin L. Sanders, Virginia Watral, and Michael L. Kent Microsporidiosis in Zebrafish Research Facilities

Jan M. Spitsbergen, Donald R. Buhler, and Tracy S. Peterson Neoplasia and Neoplasm-Associated Lesions in Laboratory Colonies of Zebrafish Emphasizing Key Influences of Diet and Aquaculture System Design

Michael L. Kent, Claudia Harper, and Jeffrey C. Wolf Documented and Potential Research Impacts of Subclinical Diseases in Zebrafish

Marcus J. Crim and Lela K. Riley Viral Diseases in Zebrafish: What Is Known and Unknown

HusbandryStephen A. Watts, Mickie Powell, and Louis R. D'Abramo

Fundamental Approaches to the Study of Zebrafish NutritionAndrzej Nasiadka and Matthew D. Clark

Zebrafish Breeding in the Laboratory EnvironmentCarole Wilson

Aspects of Larval RearingChristian Lawrence and Timothy Mason

Zebrafish Housing Systems: A Review of Basic Operating Principles and Considerations for Design and FunctionalityMonte Matthews and Zoltán M. Varga

Anesthesia and Euthanasia in ZebrafishGeneral

George E. Sanders Zebrafish Housing, Husbandry, Health, and Care: IACUC Considerations

Stephen A. Smith Zebrafish Resources on the Internet

ILAR Journal Zebrafish Health and HusbandryVolume 53 Issue 2 June 2012

http://ilarjournal.oxfordjournals.org/content/53/2.toc

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Pathogens/Diseases of Concern

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0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

Pseudoloma neurophilia

Mycobacteriosis

Neoplasia

Hepatic megalocytosis

Myxozoans

Nephrocalcinosis

Gill epithelial hyperplasia and hypertrophy

Bacterial infection (non-acid fast)

Pseudocapillaria tomentosa

Other helminths

Pleistophora hyphessobryconis

Fungal infection

ZIRC Health Services Data (2006-2010)% facilities positive for a particular disease (5 yr. average)

ZIRC disease manual (https://zebrafish.org/health/diseaseManual)

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ZIRC Health Services Data (2006-2010)Just the infectious diseases

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%


Mycobacteriosis

Myxozoans

Bacterial infection (non-acid fast)


Other helminths

Pleistophora hyphessobryconis

Fungal infection

ZIRC disease manual (https://zebrafish.org/health/diseaseManual)

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Edwardsiella ictaluri

Images from Hawke et al. (2013)

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Edwardsiella ictaluri• Enteric Septicemia of Catfish• Hole in the Head Disease

http://www.ag.auburn.edu/fish/mediagallery/2013/08/13/bacterial-diseases-in-warmwater-fishes-37/

CM Whipps

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Edwardsiella ictaluri

Challenges

• Virulent

Advantages

• Acute infections – likely ID in quarantine

• Avoid pet store/ commercial fish

• Not environmentally stable

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http://zebrafish.org/health/Disease_images/CapillariaWetMounts4.jpg

• Nematode• Infects intestinal wall• Macroscopic (4-12 mm)

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Challenges

• Environmentally stable eggs

• Direct transmission

Advantages

• Screen water for eggs

• Can be treated

Capillaria egghttp://www.cdc.gov/dpdx/intestinalCapillariasis/gallery.html#eggs

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Myxozoanosis – Myxidium streisingeri

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• Associated with kidneys/urinary system• Nephritic ducts packed with plasmodia


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Image: Kent, M.L., and T.T. Poppe. 1998. Diseases of seawater netpen-reared salmonids. Pacific Biological Station Press, Nanaimo, British Columbia, Canada

• Typical myxozoan life cycle has 2 hosts

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Image: http://seedmagazine.com/place/place_labs-at-night.html#0


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Sump/Biofilter colonized by oligochaetes?


Image c/o Mike Kent

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Many Remaining Questions


Challenges

• Unknown life cycle

• Impact on host??? (kidney function studies?)

Advantages

• Likely requires oligochaete host

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Signs of Disease

Skinny fishBent spine


(Matthews et al. 2001 J. Eukaryot. Microbiol., 48(2), 227–233)

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Histology

Observation of xenomas and spores in tissue sections


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Figure from:Sanders et al., 2013 - Verification of Intraovum Transmission of a Microsporidium of Vertebrates: Pseudoloma neurophilia Infecting the Zebrafish, Danio rerio. PLoS One

Microsporidian spore

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Challenges

• Environmentally stable/resistant spores

• Fish may not have clinical signs (chronic infection)

• Spores intra-ovum

• Spores resistant to disinfectants (more to come…)

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Mycobacteriosis

• Swollen belly

• Emaciation

• Skin ulcers

• General malaise

• Exopthalmia

Signs of mycobacteriosis

None at all?

• Granuloma

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Mycobacteriosis

Caused by Mycobacterium spp.

• Over 100 species in the genus

• Infect wide range of animals

• Acute to chronic infections

• Notoriously difficult to treat

• Fastidious

• Environmentally derived

• Form biofilms

http://www.futura-sciences.com/magazines/sante/infos/actu/d/vie-afrique-situation-urgence-tuberculose-6992/

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Mycobacteriosis

Mycobacterium spp. of zebrafish

Zebrafish• M. chelonae

• M. fortuitum

• M. marinum

• M. haemophilum

• M. peregrinum

• M. abscessus

• M. gordonae

• M. fortuitum-like

• M. marinum-like

• Mycobacterium spp....

Culture and PCR of infected tissues

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Mycobacteriosis

Many species of Mycobacterium

•Some common, others less so

•M. marinum and M. haemophilum more serious pathogens

Variation within hosts

•TU fish more susceptible

•Other lines of fish with some genetic resistance?

Different bugs + Different fish = Different Impacts

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Mycobacteriosis

Direct cause of mortality

Contributing cause of mortality

Morbidity

Source of non-protocol variation

Safety of employees

Mycobacterium marinum zoonotic

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Transmission

• Sharing of fish between facilities

• Vertical transmission?

http://www.epa.gov/region5/superfund/ecology/images/fishcartoon.gif

• Feeding on infected tissues•Important to remove “sick” fish

• ‘Ubiquitous’ in aquatic systems• Role of Biofilms in tsm

http://unsolvedmysteries.oregonstate.edu/microarray_02

Mycobacteriosis

http://www.epa.gov/region5/superfund/ecology/

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Zambrano (2005) Cell, 123(5): 762

http://paulorwin.blogspot.com/

Biofilms harbor and protect mycobacteria

Biofilms

Tank swabs

Water sump

Food (-ve)

Sampling at a Zebrafish facility

Mycobacteriosis

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Biofilm transmission... (?)-When tanks are cleaned, does the suspension of biofilm in water

increase transmission of mycobacteria??

Scrubbed+ debris

siphoned laterEntire tank replaced

Low risk? Higher risk?

Scrubbed+ increased

flow to flush debris

Low risk?

Murray et al. (2011 Comp Med)

Fish density also important (crowding stress)

Mycobacteriosis

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Biofilms

Fish

‘DNA fingerprinting’Whipps et al. (2008)

DAO 82, 45-54

Several other biofilm mycobacteria

are not found in fish

(not shown)

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Mycobacteriosis

Challenges

• Several species

• Acute to chronic infections

• Form surface biofilms (no need for fish)

• Waxy hydrophobic cell wall (tough to kill)

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Main Recommendations• Identify Pathogens• Prevent Introductions• Control Within Facility

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Prevent Introductions

• Source History

• Quarantine

• Disinfected Eggs Only

• Specific Pathogen-Free Fish

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Prevent Introductions – SPF fishSpecific Pathogen Free fish

•Artificial Diet•Controlled personnel access/procedures•Monitoring/screening population and morts•‘Retiring’ fish at 1yr •Sentinel screening

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Prevent Introductions – SPF fish

Pseudoloma-free fish availableMycobacteria difficult to eliminate

Table from Kent et al. (2011)

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Prevent Introductions - Quarantine

• Separate System

• 2 weeks – 3 months (depending on pathogen of concern or history of fish)

• Screening recommended for:• Morts• subsample

• Regular cleaning between housing incoming fish

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Prevent Introductions - Quarantine

• Weeks • Likely good for acute infections• i.e., Edwardsiella ictaluri

• 6 wks - months• Mycobacterium haemophilum• Mycobacterium marinum

• May or may not see signs of:• Other Mycobacterium spp.• Pseudocapillaria tomentosa• Pseudoloma neurophilia• Myxidium streisingeri

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Prevent Introductions – Eggs Only

Quarantine

Surface disinfect eggs /embryos

Main Facility

Shipment from lab X

Nursery

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Focus testing on mycobacteria•Very common•Same methods likely effective on other bacteria

Notes on other pathogens•Pseudocapillaria

•Eggs very resistant•Screening in quarantine should be effective

•Myxidium•Not known to be associated with eggs•Eggs-only should avoid

•Pseudoloma•Spores very resistant•Intra-ovum a possibility•Screening parents important

Sanders et al., 2013 -PLoS One

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What do you do when mycobacteria detected?

Depopulate

Disinfect system1000ppm bleach >1hr

Re-establish via ‘eggs only’ policy

Disinfection - Mycobacteria

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Follow up

Mycobacteria re-establish in biofilms within weeks after bleaching (and fish added)

A few cases of infected fish since (M. chelonae)

No further cases of M. haemophilum (as of Feb 2015)


ILAR Journal 2012, 53(2) 95-105

Described outbreak of M. haemophilumDepopulation, re-establishment

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Some treatments:•Bleach (25-50ppm, 5-10min)•Bleach (150ppm, 5-10min)•PVP-Iodine (100ppm, 2min) + bleach (30ppm, 10min)

Rinse or inactivate bleach with water/sodium thio

Surface disinfect

General Method of Surface Disinfection

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Chlorine Bleach Hydrogen Peroxide Povidone Iodine

Current

Usage

• Zebrafish

• 5-10 min @ 25-150

ppm

• Catfish

• 450-900 ppm in a bath

or flow-through

• Salmonids &

Zebrafish

• 2-5 min @ 100 ppm

Our

Study

• Clorox®

• Unbuffered &

Buffered

• CDFB

• 50, 100 & 150 ppm

• 5 and 10 minutes

• Drugstore brand

• Unbuffered

• 15 000 & 30 000 ppm

• 5 minutes

• Drugstore brand

• Unbuffered

• 12.5-100 ppm

• 5 minutes

Mycobacteria - Disinfectant Choice


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Not all disinfectants are equalPercent survival differs significantly (p<0.05) between treatments.

Mycobacterium chelonae

• Less than 1% survival for:

• Buffered Bleach

• PVP-I

Bleach (CDFB)

Bleach (MILQ)

Hydrogen Peroxide

PVP Iodine


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Not all species are equally susceptiblePercent survival differs significantly (p<0.05) between species.

Mycobacterium chelonaeMycobacterium abscessus

Mycobacterium peregrinumMycobacterium gordonae


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One week post plating differences in colony growth are observed in representative

(A) M. abscessus, (B) M. chelonae, (C) M. gordonae, and (D) M. peregrinum

control plates and (A’, B’, C’, D’) 25 ppm iodine treatment plates.


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Chlorine bleach•Variable by species, 100ppm+ for 10 min largely effective•Buffered (to pH7) chlorine very effective at 50ppm+

Peroxide•Moderately effective at 3% •This level likely lethal to fish

Iodine•25ppm+ effective

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Impacts of Germicides on Fish - Chlorine

•Impacts of chlorine (0, 6.25, 12.5, 25, 50, 100 ppm)•5 vs. 10 minutes•Fish at 6 and 24 hours post fertilization•Buffered and unbuffered chlorine

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Kent et al. (2014) DAO 107; 235-240

Mortality (buffered chlorine)

Mortality (unbufferedchlorine)

Malform(buffered chlorine)

Malform(unbufferedchlorine)

6 hpf – 5min exposure 6 hpf – 10min exposure

24 hpf – 5min exposure 24 hpf – 10min exposure


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Kent et al. (2014) recommend 100ppm chlorine (unbufferred):•10min for fish 6 hpf•5 min for fish 24 hpf

Mycobacterium peregrinumMycobacterium gordonae

Our data on Mycobacterium spp.

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Viability of embryo disinfection with povidone-iodine tested:

2 Developmental Stages• 6 and 24 hours post-fertilization (hpf)

3 Treatment Concentrations• 12.5, 25, and 50 ppm

2 Treatment Times• 2 and 5 min

2 Brands of PVP-I• Drugstore and Ovadine (Western Chemical)

Impacts of Germicides on Fish - Iodine

Modeled on chlorine protocol of Kent et al. (2014)

Chang, Amack, Whipps (In Prep)

Incubate in 96 well plate at 28.5°C

until 5 dpf

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RO H2O

RO H2O

RO H2O

RO H2O

PVPISol’n

E2 Medium

6 hpf

24 hpf

Embryos at 2 Developmental Stages


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Concentration and duration of treatment solutions has an effect on zebrafish health at 6 hpf exposures.

Percent mortality differs significantly (p<0.05) for 50 ppm 5 min treatments.

NS mortality between buffersfor 25 ppm at 6 hpf

Drugstore Brand (Unbuffered) Ovadine (Buffered)

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Impacts of Germicides on Fish - IodineConcentration and duration of treatment solutions has an effect on

zebrafish health at 24 hpf exposures.Percent mortality differs significantly (p<0.05) for 50 ppm 5 min treatments.

Ovadine (Buffered)

NS mortality between buffersfor 12.5 & 25 ppm at 24 hpf

NS between treatments

Drugstore Brand (Unbuffered)

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Rinsing alone is not enough to remove mycobacteria

RO H2O

RO H2O

RO H2O

RO H2O

PVPISol’n

E2 Medium

RO H2ORO H2O RO

H2ORO H2O

25 ppm 5 min PVPI

E2 Medium

105 cfu/ml

Incubate:Overnight

28.5°C50 rpm

Average518 571 cfu/mL

Average56 666 cfu/mL

No Growth

Average2611 cfu/mL

No Growth No Growth

Average2383 cfu/mL

Control


Rinse only

Iodine & rinse

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Impacts of Germicides on Fish - IodineFresh Preparation of Iodine is criticalAvoid light exposure

Test solutions after 24 hrs

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• Treatment with PVPI not harmful to embryos except 50 ppmfor 5 min

• 6 hpf embryos were generally more resilient to treatments compared to 24 hpf

• No difference in embryo mortality between unbuffered and buffered iodine at 12.5 and 25 ppm

• Rinsing embryos is not enough to eradicate mycobacteria

Recommendations for Egg Disinfection- Iodine

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• PVPI may be a better alternative to chlorine bleach for preventing mycobacterial spread between facilities

• Facilities should incorporate a embryo disinfection protocol to suit their needs– Take into account:

• Source

• Strain

• Age

• Generally recommend disinfecting embryos at 25 ppm PVPI for 5 min– Modified as necessary

Recommendations for Egg Disinfection- Iodine

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Grant R24OD010998

• Mike Kent

• Virginia Watral

• Christine Lieggi

• Jeff Amack

• Trisha Lynn Honors

• Kristen Doerr

• Elle Palmer

• Erica Colicino

• Elizabeth DiPaolo

• Team Chen

• Hadi Jabbar Al-Hasnawi

• Whipps Fish and Wildlife

Disease Lab

• Many others...

Acknowledgements

keeping pathogens out of your zebrafish facility

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