PASSIVE IMMUNIZATION OF SALMONIDS AGAINST
FURUNCULOSIS AND VIBRIOSIS WITH CHICKEN EGG YOLE
IMMUNOGLOBULINS (IGY)
Abdolhossein Aminirissehei
Pham. D., Esfahan University of Medical Sciences, Iran, 1980
THESIS SUBMITIED IN PARTIAL. OF
THE REQUlREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
In the Department
of
Biological Sciences
O Abdolhossein Aminirissehei 2001
SIMON FRASER UNIVERSN
August 2001
AU rights reserved. This work may not be reproduced in whole or in part, by photocopy
or other rneans, witbout permission of the author.
The authot has granteci a non- e x c b licence allow9ig the National Li'brary of Canarin to reprduce, loan, distriiute or seU copies of this thesis in mimfo~in, paper or eiectronic formats.
The author retains ownership of the copyright in $ris thesis. Neither the thesis nor substantial extracts fiom it may be printed or othecwise reproduced without the author's pemiission.
L'auteur a accordé urie licence non exchisive permettant A la Bibiiothéque nationale du Canada de reproduire, @ter, distribaerou vendre des copies de cette dièse sous la forme de mirnfiche/film, de reproduction sur papier ou sur format électronique.
L'auteur conserve la propriété du droit d'auteur qui protège cette thbse. Ni la thèse ni des extrajits substantiels de d e c i ne doivent être imprimks ou autrement reproduits sans son autorisation,
Passive immunization of coho salmon (Oncorhynchus Risutch) and rainbow trout (O.
myKiss) was investigated using specific immunoglobuiins (IgYs), which were raised in
dornestic chickens (Gallus domesticus) and extracted fiom their egg yoik, against fish
pathogens of either Aeromonas salmonicida or Vibrio anguiIIarum. ûral treatments of
these fish with either anti-A. salmonicida or anti-V: anguillarum IgY did not provide
passive immune protection. When intraperitoneally (IP) injected, anti-V: anguiIIarum
IgY provided significant passive immune protection against V. anguillarum whereas
anti-A, salmonicida did not provide protection against A. salmonicida. In the latter case,
it appears the A. salmonicida was grown under conditions that restricted the formation of
ce11 surface antigens against which a protective antibody could be raised in chickens,
IgY h m unvaccinated chickens provided moderate passive immune protection against
K anguillulrum challenge as compared to 1gY fiom chickens vaccinated with this
bacterin that provided a strong passive immune protection. These results impiy that there
are c o m o n ce11 surface antigens of V , anguillarum on other bacteria to which the
chickens were normally exposed.
iP injection of anti-V: anguiIIarum IgY into rainbow trout provided signincant and
undimiriished immune protection against a lethal challenge by FI anguillarum for up to 2
weeks. Passive immune protection against V: anguiIImm in rainbow trout was provided
when the total IgY concentrations in rainbow trout were between the tested range 2.5 x
1W2 and 4.0 x lW3 mg/mL senun. If an& V: anguiIIarum immunogiobulins were assumai
to be 10 % of the total IgY, the equivalent concentrations of this specific IgY w d d be
between approximately 2.5 x 10'' and 4.0 x IO4 mglmL serum.
1 express my great appreciation to Dr. L. J. Albright, my thesis senior supervisor, for his invaluable support and encouragement through my course of study.
1 offer my best thanks to Dr. E.C.Y. Li-Chan of University of British Columbia for her guidance, and thanks to Dr. T. Borgford, of Simon Fraser University, two other my advisory committee members.
1 extend my best thanks to Dr. Alex Yousif for his technical consultation and guidance.
DEDICATIONS
In the Name of God, Most Compassionate, Most Mercifùl
1 dedicate this thesis to Mahzad, my lovely wife, for her support and patience especially for taking care of my lovely children Mohammad, Maryam, Marjan, and other home affairs, as well as providing me with cairn and peaceful conditions to concentrate on my studies.
To my great mother who always prays for me to be a successful peson in al1 waiks of my life.
And, to my late father who taught me how to be an active, resourceful, and patient person.
TABLE OF CONTENTS
Page
. . Examinmg Cornmittee Approval.. ..................... .. .......A
... A ~ ~ t . . . . . . ~ . . . . . . . . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ u ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ e ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~
Acknowledgements ...... .. ................... ..........................................a,... ...v
Micaîims.. ...... .............................................. ............................ ...ai .. .................................... .................... Table of Contents .................. ..VU
List of Tables ................. .. ....................a............. ,...... ..................... .Mx
List of Figures. ................................................................................. ..xi
Int~uct ion ............ ... ............................................................... ......l ................................................................................... Purpose. .3
Materiais and Methods ....................................................................... 5
........................................... Experimental animals (chickens and fish). 5
................................................................................. Antigens.. 5
................................... Chicken imrnunization and antibody production ..6
............................................... Preparation of column chromatograph.. 6
................................................. Extraction of injectable liquid IgY.. -7
................................................................................... ELISA ..8
........................................................ Preparation of egg yolk powder 9
..................................... Immunization of fish and challenge protocol.. IO
.................................................................. Chailenge procedure. 1 1
................................................................... Absorption of IgY.. 12
Immunization of fish and challenge protocol.. ...................................... 13
Duration of protection against K anguillamm foiiowing a single IP injection
... of specifi anti-Vibrio IgY; Immunization and challenge pmtocol.. 14
R ~ I W ~ S . ~ ~ ~ se,... W.ssm .......~se~~~~~......m.~.~...~..~m.~m..~....~I..a~..a.I.b.~~.~......~.~~.~~ 17
Extraction of IgY From egg yolk.. ................................................... f 7
EUSA ................................................................................... 17
Mortality and relative percent survival (RPS) of juvenile rainbow trout
treated with Anti-A. salmnicida IgY followed by exposure to
pathogenic A. salmonicida .................................................... 17
Mortality and relative percent survival (RPS) of juvenile coho dmon
treated with anti-K anguillanrm IgY followed by exposure to
pathogenic V. anguiilarum.. ............................................... -22
Mortality and relative percent suwivai (RPS) of juvenile coho salmon
IP injected with either (1) non-specific IgY (2) ami-V, anguillarum
IgY or (3) absorbed anti-V. anguillurzun IgY followed by exposure
to pathogenic K anguiii~~rum.. ......... .. ................................ -22
Mortaiity and relative percent suwivai (RPS) of juvenile rainbow trout
IP injected with either (1) non-specific IgY (2) anti-K anguillarum
IgY or (3) absorbai anti-K unguiilarum IgY followed by exposure
............................................ to pathogenk K anguilia m... .25
Duration of protection in rainbow trout against K anguillanun following
................................. an IP injection of anti-V. anguillanun IgY 28
Duration of total serum IgY in rainbow trout following an IP injection ......... 28
D i i i o n . . . . .... ...... ................ .., ........................................................... Titer in of IgY in egg yolk 37
Efficacy of antid. salmonicida IgY for passive immunization of juvenile
rainbow trout against A. salmonicida.., ................................... 37
Efficacy of anti-V. anguillarum IgY for passive immunization of juvenile
............................................... coho salmon against vibriosis 40
Efficacy of absotbed IgY for protecting rainbow bout against a lethal V.
.................................................... anguillarum challenge.. .4 1
Duration of serum IgY versus challenge mortality in rainbow trout
. . following an iP injection ..................................................... 42
Duration of anti-V. anguillarum immune protection in rainbow trout
following IP injection of IgY ............................................... 43
A determination of the anti-V. anguillarum IgY concentrations required
to provide passive immune protection against a V, anguillarum
.................................................................. challenge.. .A4
.. COIICIUS~OIIS.*..~..~~...*...... .....o............ .........a n. . . .m*w~*wo**.s~nae***M
Refecences.. ................................................................................. ..A8
Table 1 Mortality and Relative Percent Survival (RPS) in Juvenile
rainbow trout after a bath-challenge with a lethai concentration of
live, virulent A. salmonicida, one day after IP injection and 7 days
after oral route administration of anti-A. salmonicida IgY ........... 23
Table 2 Mortaiity and Relative Percent Survival (RPS) in juvenile coho
saimon after a bathchallenge with a lethal concentration of live,
virulent V. anguillam, one day after IP injection and 7 days after
................ oral route administration of anti-V. anguillarum IgY .24
Table 3 Mortaiity and Relative Pexent Survival (RPS) in different treatment
groups of juvenile coho salmon up to 14 d pst-bath-chailenge with
............... a lethai concentration of live, virulent K anguillarum. 26
Table 4 Mortality and Relative Percent Survival (RPS) up to 14 d of post-
bath-challenge with a lethai concentration of live, virulent
K anguillarm, in juvenile rainbow mut previously IP injected
with non-absorbedand absorbed anti-Virio IgY ........................ 27
Table S Duration of protection in dierent treatment groups of juvenile
rainbow trout previously IP injected with specific and non-specific
IgY.. ...................... ... ........................................... -29
LIST OF FIGüRES
Page
Fig. 1 Elution profile of specific IgY (anti-A. salmonicida) via column
Fig. 2 Dilution endpoint determination by indirect ELISA of IgY (specific
for anti-Vibrio anguillarum), non-specific IgY, and absorbeci
specific IgY.. ................................................................ ..2W21
Fig. 3 Temporal trend of mortality rate in relation with total senim IgY levels
.......... in the fish groups IP injected with anti-V. anguillarum IgY.. W1
Fig. 4 Temporal trend of mortality rate in relation with anti-Vibrio IgY
titer in serurn of fish following IP injection of anti-Vibrio IgY ......... 33î34
Fig. 5 Temporal trend of anti-Vibrio IgY serum titer fluctuations in relation
.... with total senim IgY level in IP injected fish with anti-Vibrio IgY 35/36
INTRODUCTION
Cuitured fMsh, including salmonids, c m acquire a number of microbiai diseases. Many
of these diseases are caused by pathogenic bacteria. Examples of bacteriai-mediated
diseases of salmonids iaclude vibriosis, bacterial kidney disease (BKD), fiuunculosis,
enteric redrnouth, Yersiniosis, myxobacteriosis and Edwardsialosis. Some of the
microbial diseases are caused by vinises. Examples are inféctious hematopoietic
necrosis and infectious pancreatic necrosis (PN) of salmonids.
In general, vaccination of cultured fMsh, including salmonids, by the appropriate
vaccine at an early stage in their rearing cycle and the use of antibiotics in feed can
control most bacterial diseases of these animals. However, there are several bacterial
diseases for which an adequate vaccine has yet to be developed and which are not readily
controlled by antibiotic therapy. BKD as well as funinculosis in salrnonids are examples
of diseases for which these two treatment methods are unsatisfactory. In addition,
environmentai concems, regdatory restraints, cost and pathogen resistance have
diminished the appeai of antibiotic use in aquaculture. Viuses cannot be controlled with
antibiotics and there is currently no effective vaccine for most virus diseases, including
MN and IPN.
An alternative to actively immunking salmonids and administerhg antibiotics is passive
immunization of fbfïsh with immunoglobuiins b m other cultured animais, especiaüy
chickens. indeed, the potentiai availability of commercial quantities of relatively
inexpensive immunoglobulins (rgY) h m chicken egg yoks offers a good source of
spcific IgY for passive immunization against specific pathogens by oral administration
in feed.
IgY immunoglobulins are environmentally friendly. Unlike antibiotics, IgYs have
minimal side effects on the treated finfish. There is no disease resistance, or
development, possibility of overdosing or occurrence of toxic residues. If administered
orally in feed, there is no injection or handling of the finfish. in the case of virus
Uifections, passive immunization may be the only effective way of treating these
disease-causing microbes.
The use of IgY to passively immunize M s h against a number of bacterial diseases has
been investigated by several individuah. Gutierrez et al. (1993) showed that when
anti-Ehuardsiella tarda IgY and E. tarda were simultaneously administered orally to
Japanese eels, there was a decrease in mortality of the fish as well as a marked reduction
in disease signs as compared with the control challenged group where IgY was not used.
Lee et al. (2000) reported a reduction in moaality and intestinal infection due to the
pathogen, Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss) when anti-Y. ruckeri
IgY was included in the feed either before or following iafection by immersion. If the
IgY was administered 4 hours before the immersion challenge, the passive protection to
tbis disease organism was increased signiEicantly.
Ototake et al. (1996) demoustrateci that protein molecules of bovine semm albumin
would pass intact through the skia of rainbow trout. However, the amount that entered
the fish in this way was so small that it is uniikely that control of a microbial disease
would have been attained.
Oral administration of the IgY would appear to offer the best way of administering the
IgY. However, when administered in feed the amounts that pass the gut waii of rainbow
bout are low (Arasteh, 2000). This investigator did however demonstrate that when IgY
was CO-administered with any of the uptake enhancers, deoxycholate, Mega9 or
octyl-&glucoside, a significant enhancement of IgY absorption across the rainbow bout
gut occuned. Indeed, the use of the uptake enhancer, Mega9, raised s e m IgY Ievels
such that they were only 12 to 18 times lower than inûaperitoneal (IP) injection of a
similar dose in the rainbow mut.
Based upon these observations, it would appear that specific IgY may offer the potentiai
to be effectively used to passively immunize salmonids against certain microbial diseases
for which ttiere is currently no effective treatment (e.g. BKD, funincuiosis, MN and
IPN), since Arasteh (2000) has clearly shown that apparently therapeutic levels of IgY
can be attained in salmonids when orally treated with this immunoglobuiin.
The hypothesis of this study is as foliows:
Specfic domestic chicken (Gallus domesticus) egg yolk immunoglobulins can be used to
control vibriosis andfor furunculosis in rainbow trout (0. mykiss) and coho saIrnon (O.
Risurch) when administered in the feed of these animals,
The objectives of this siudy are as foiiows:
1. To determine the ability of oraiiy and interaperitoneaiiy (IP) administered specific and
non-specific IgY to pmtect rainbow trout and coho saimon agakt Aeromonas
salmonicida and Vibrio anguillmum, the causative pathogens of funuicuiosis and
vibriosis respectively.
2. To determine the duration of protection of specific IgY against a V , anguilhm
infection.
3. To estirnate the minimum dose of qxcific IgY required to provide protection
against V , anguillarum, when administeced in feed.
MATERIALS AND METHODS
Cbicken:
Six white bghotn chickens, G. domesticus, (appmximately one year old) h m the same
genetic stock, wete purchased from Coastiine Chicks, Abbotsford, British Columbia.
The chickens were bedded with wheat straw, nceived 14 hours of white light per day
and fed daily ad libitum. The temperature was kept between lS-%'C, which is optimal
for laying chickens (Longnecker, 1970).
Fish:
Naive f m e d raintiow trout (10-15 g), were purchased fiom Spring Valley Tmut
Hatchery, Langley, British Columbia. Wild coho salmon (10-15 g) were obtained from
the Capilano River Hatchery, Canada Dept. of Fisheries and Oceans. Al1 fish were
acclimated at f 17°C in Eresh water and a 12 hr photoperiod for two weeks prior to the
treatment.
Antigeas:
Virulent strains of the bacterial pathogens, A. salmonicida (drain # MT 26), and K
anguillarum (strain # MT 513), were provided by Microtek international Ltd. Saanichton,
British Columbia Vibrio angrcl'Ilmm was grown at room temperature for 24 hrs on
Tryptic Soy Agar (TSA) plates that had been supplemented with 1.5% sodium chioride.
The ceils were harvested by scraping them off the aga surfaces and suspendhg them in
phosphate buffered saline (PBS) pH 7.1. The ceils were kilied with formalin, (0.3% vtv)
and le& ovemight at SOC. The bacteriai suspension was washed (x 3) with PBS pH 7.1
and stored at SOC until used.
The A. salmonicida suspension was prepared following the same procedure outlined for
K anguillamm except TSA was used without added sodium chloride.
Chicken immunization and antiboày production:
Chickens were allowed to acclimate to theu surroundings for two weeks prior to
treatment. Two groups, each consisting of two chickens, were injected intramuscularly
(breast muscle) with 1 mL of killed A. salmonicida or K anguillarum bacterin (at a
concentration of 1.1 x 107 chi (colony fonning units) m~.' suspended in 1mL Freund's
Adjuvant-Incomplete, Sigma). These A. salmonicida and V. anguillarum bacterins were
prepared as outlined above. Booster injections were repeated twice at 10 day intervals,
i.e. at day 10 and 20. Control eggs fiom non-injected chickens were collected one week
prior to the kt injection of the treated chickens and stored in 5' C until used.
Preparation of column chromatograph:
Twenty gram of carboxymethyl ceiiuiose powder (CM-32, Whatman, England), a cation
exchange medium, was mixed with 15 volumes of distilled water and placed at 5. C for
24 h prior to use. Na &de (0.02% wfv) was added as a microbiocide.
Foiiowing this 24 h time perîod, the gel was decanted and added to 0.5 N NaOH (gel :
NaOH : 1 : 15) for 30 min during which the suspension was shaken to evenly mix it.
The suspension was then fltered (Wbatman, England, No. 42) and the gel particles
washed with d i d e d HOH until the filtrate was pH4.0. The gel particles were then
added to 0.5 N HCI in a ratio of (gel : HCl : 1: 15) for 30 min, with occasional stirring,
tben the overlaying liquid was decanted and the gel particles washed with distilled HOH
until a pH of6.4 was observed.
This gel was added to 0.01 M phosphate buffer (PB) pH=5.4 at a gel : PB ratio of 1: 4.
This process was repeated twice with a 30 min incubation period between treatments.
This treated gel was then mixed with chicken IgY preparation (see immediately below),
100 mL gel was mked with 100 mL of prepared IgY solution. This mixture was shaken
occasionally and let to settle for 30 min. One-half of the overlaying liquid was removed
and the remainder packed in a chromatographic column (2.5 cm inside diameter, 50 cm
long).
Bound proteins were eluted fiom the column as described below.
Extraction of injectable iiquid IgY:
IgY was exüacted and purified as described by Fichtali et al. (1993). Briefly, two yolks
( h m eggs laid at 37 days f ier the fint injection) were separated h m egg white, diluted
@IO) with distilled water and the pH adjwted to 5.5 with 0.1 N HC1. The mixture was
left to sediment overnight at SOC and then filtered (Wbatman, No. 42). The filtrate was
then mixed with the prepared gel in a ratio of (1 : 1) and packed into the chromatographic
column.
Bound proteins were eluted fiom the column via step gradient (Mim Fractionator,
Gilson) elution. Coiiected fractions were monitored for protein using a
spectrophotometer (Bausch & Lomb Spectronic 21) at 280 m. Elution was iaitiated
with 0.01 M PB, pH 4.5 and continued until a zero absorbance was reached. At b t time
elution was continued using 0.2 M PB, pH 5. Most proteins eluted in this stage. EIution
continued with 0.2 M PB, pH 5 until again a zero absorbance was observed. The column
was then washed with 0.01 M PB containing NaCl, pH 4.8 followed by 0.01 M PB, at pH
5.4. The fractions with higher absorbance at 280 nm, Le. fractions No. 8 to 18 (Fig.1)
were pooled and used as an injection fluid for fish. This preparation was placed in 2 mL
centrifiige Eppendorf tubes and kept at -80" C until used.
ELISA:
Purified specific IgY (anti- V. anguillarum) h m immunized chickens or non-specific IgY
fiom unimmunized chickens were diluted and antibody titer was detemiined using
ELISA. Specific IgY was also absorbed with killed K anguilllruum (absorption of
specific IgY with killed K anguillarum is outlined in Materials and Methods), and
residual IgY activity was determined by ELISA.
Briefly, wells of ELISA plates (flat-bottom plate, Falcona No. 3915, USA) were coated
with antigen (kiiled bacterin was prepared as described above) and the plates were
incubated at 3 7 O ~ for one hour. The wels were then washed (x2) with PBS pH 7.1
containhg Tween-20 to which 250 pL of 0.5% milk (instant skim milk powder,
Carnation? in PBS was added per well as the blocking agent. Plates were then incubated
at 37'~ for 30 minutes. Mer incubation the plates were washed (xl) using PBS-Tween-20.
Diirent concenirations of ad-Vibrio IgY in PBS-Tween-20 were added to the weils
(100 pL / well) and incubated at 37°C for one hour. Plates were then washed with PBS-
Tween-20 (x5) to remove unbound proteins. Rabbit mti-chicken IgY Aikaiine Phosphate
Conjugate (Sigma) diluted 1:10,000 in PBS-Tween-20 was then added to the weUs (1 00
pL / weU) and incubated at 37" C for one hour. Plates were then washed with PBS-
Tween-20 (x3) and distilleci water (xl). pnitrophenol phosphate [(OS mghl), Sigma]
was added to the wells (100 pi., Iwell) as an enzyme substrate and d e r 10 minutes the
reaction was stopped by addition of 3N NaOH. Colour was memeci using a plate reader
(Bio-Khetics Reader; EL 3 12 E, USA) at a measurement wave length of 405 nm and a
reference wave length of 690 m. The results were plotted using Microsoft Excel.
Preparotion of egg y o k powder:
Three pre-cooled eggs (Sec) (previously collected Çom chickens vaccinated with either
A. sahonicida or V: anguilIarum bacûins) were cracked open and yolks were separated
from their whites, pooled and then rnixed with pre-cooled isopropyl aicohol (x 3 total
yok volume) with stining for 20 min. The solution was then filtered (Whatman, No. 42)
through a Buchner Fumel. The process of washing with pre-cooled isopropyl alcohol
was repeated 3 times.
To remove the remainder of the Lipids fiom the egg yolk the above washuig process was
repeated twice using pre-cooled acetone. The serni-purified egg proteins, fiom the last
washing process with acetone, were coliected, covered with a clean tissue paper, and Ieft
in the hood for 24 h to dry (Yousif, 1992). AU mixing was done on ice to niinimi.re
protein degradation. When dried, the extracted powder was ground, sieved (No. 120; 125
pm, mm, USA), and used to top-dress the fish feed (2% wfw). Herring oil was used to
seal the powder onto the feed pellets (10% w/w). Appmximately 3 g of cmde powder
was obtained h m each egg yolk.
The above process was repeated using control eggs (Le. eggs h m unvaccinated chickens).
Immunizatio~~ of îish and cbdienge pmtocol:
Naive juvenile rainbow trout or cobo salrnon (10-15 g) were wed. FoUowing an
accliiation period of 2 weeks at 17k1° C, 30 fish were randomly distributeci into eight
50 L circular tanks. The following design illustrates the arrangement and labeling of the
groups of fish in the tanks during the course of the expiment:
Control
Treated
I rep. 1 tep. II W. 1 rep. II
- -
Feeding
Tanks 1-4 represent the control group.
Tanks 5-8 represent the test group.
Fish in tanks 3,4,7 and 8 were fed the basic diet (at 1% body weightlday) throughout the
course of the experiment (i.e. reguiar fi& feed # 3 purchased fiom Mwre Clarke
Vancouver, BC.). Fish in tanks 5 and 6, however, received the basic diet that had been
top-dressed with a known concentration of egg yoik powder (Le. 2% w/w of either ad-A.
salmonicida or V. anguillarum IgY in yolk powder). The second control group (tanks 1
and 2) received the basic diet top-dressed witb the control yolk powder aione (i.e. without
anti-A. salmonicida or K anguillarum IgY).
Following 7 d of feeding (on the day 8) fish in tanks 7 and 8 were IP injected with 100 pi,
of the purified antid. salmonicida or Y. anguillarum IgY (at the concentration of 1 mg
protein fish-'). Control fish (tanks 3 and 4) were IP injected with purified IgY (at a
concentration of 1 mg protein fis&') h m a control egg yolk.
On day 9 fish in al1 groups were immersion challenged (see below) with an LD50 (-5.0~
106 c h m ~ " ) of a live, vident strain of either A. salmonicida or Y. anguillarum.
Mortality was monitored for 2 weeks foliowing the challenge, during whicb time the fish
were fed as stated above.
ChaUenge procedure:
Wendemyer et ai. (1990) reviewed suitabIe protocols for bacteriai challenges in fish.
Vibrio anguiIlarum was grown as previously described. When the broth culture reached
0D6,,=2.2, the culture was placed on ice and diiuted with h s h sterile culture medium to
a nnal 0D6,,=0.22.
Five milliliters of the culture was mixed with 100 L of aerated water supplemented with
0.9% NaCl in a circular tank. This challenge water then was divided equally into eight
containers (20 L each) and amted. The bath challenge was carried out by placing 20 fish
in each container of water for 30 min. Fish were then returned to their 50 L circular
tanks. A water sample was collected fiom the challenge water and the colony forming
units (ch) detennined using the drop plate method on TSA + 1.5% NaCl.
Mortalities due to vibriosis were monitored and tallied for 14 days post-challenge. The
gut contents of randomly selected fish were examined and cultured to detennine if the
cause of death had been due to vibriosis. In al1 cases Vibrio sp. was isolated h m the
cultures.
Absorption of IgY:
Killed K anguillmm cells (prepared as described above), were diluted with sterile PBS
(pH 7.1) to give a bacterid preparation with a concentration of OD,,, = 0.29. The
extracted IgY was diluted with PB 0.01 M, pH 5 to a concentration of 0.0 1 mg protein
mL1. into 2 mL Eppendorftubes were added 1 mL of bacterial preparation and 1 mL of
prepared IgY, the mixture was vortexed and stored at 5" C overnight.
The Eppendorftubes were then centrifiiged (5000 rpm) for 10 min and the supemtant of
each tube was mixed with 1 mL of diluted bacterial preparation (prepared earlier),
vortexed and stored ovdght at 5" C. The samples were then centrifugai again, the
supematants pooled, filtered and used as absorbed anti-vibrio IgY. The same procedure
was used to prepare absorbed non-specific IgY.
Immunizrition of 6sh and challenge protocol:
Foiiowing an acclimation p e r d of 2 weeks at 1 Si 1 C, fish were randomly disûibuted
into 12 circuiar 50 L tanks (test group 16 and control group 10 fish per tank).
The following design illustrates the arrangement of various groups of fish in tanks during
the course of the experiment:
control cc0 Treated
Absorbed
Tanks 1-6 represent the control groups.
Tanks 7-1 2 represent the test groups.
Fish in tanks 1,2,3 and 7,8,9 each were iP injecteci with 100 pL non-specific IgY and
specific IgY, respectively (at a concentration of 2.75 mg protein fish").
Fish in tanks 4,5,6 and 20,11,12 were IP injected but withlOO @ absorbed non-
specific IgY and absorbed specific IgY, respectively (each at a concentration of 2.75 mg
protein fish").
Ail fish were bath challenged 24 h d e r the injection by exposure to an LD,, (- 5.0 x 106
cft mL-') of a live, virulent sîrain of K anguillmum. MortaIity was monitored for two
weeks following the challenge.
Duration of proteetion against V. onguillarum foliowiug a single iP injection of
specific anti-vibrio IgY; Immunizatioa and challenge protocol:
Rainbow trout (mean weight of 68g) were used. Following an acclimation period for 2
weeks at 14il0 C, the fish were randomly disûibuted into 36 circular 50 L tanks, each
with 16 fish.
The following design illustrates the arrangement of various groups of fish in tanks durhg
the course of the experiment:
Control 1 Control II Treated
PBS
Day 3 (J(J(J - --
Day 7 QQQ
Fish in the treated group were iP injected with 100 pL specific IgY (at a concentration
of 2.75 mg fish-') whereas fish in control II fish were IP injected with 100 pi, non-
specific IgY (at a concentration oE2.75 mg fish-'). The fish in control 1 gmup were IP
injected with 100 pL stecile PBS pH 7.1
The fish in al1 groups were f d ihe basic, complete diet throughout the course of
expriment (Moore Clarke, Vancouver, BC) at a rate of 1 % of their body m w day".
Fish in tanks 1 to 9 were bath-challenged 24 h after injection by exposure to an LDH,
(average - 2.4xld chi of a live, virulent srniin of V. anguillunun
The fish in tanks 10 to 18, 19 to 27 and 28 to 36 wete bath challenged in the same manner
at 1,3,7 and 14 d after injection. Before each challenge, blood was collected from one
fish per tank for an ELlSA experiment to detemine the semm concentration of IgY in the
fish in each stage. N. Arasteh, University of British Columbia determined the
conceniration of IgY using ELISA. Mortality was monitored for 2 weeks following each
challenge.
Relative percent survival (RPS) was calculateci via the following equation:
(8 mortality in test group) RPS= [1 - ] XI00
(% mortality in conml group)
Data were statistically anaIyzed by using Student's T test (hstat).
RESULTS
Extraction of IgY from egg yolk:
The elution profile of IgY h m the column chromatograph showed a significant iacrease
in absorbance of eluted IgY when the column was eluted with 0.2M PB pH 5.4 using step
gradient method (Fig. 1). Since fraction nwnbers 7 to 18 contained most of the 280 nm
absorbing materials, these were collected and pooled. This pooled material was used as a
source of IgY. The concentration of IgY in pooled material was 5.23 + 1.75 mg mL-'.
ELISA
Men analyzed by ELISA (Fig. 2), the non-specific and absorbed IgY showed a constant
low absorbance with dilution, whereas, the specific IgY showed decreasing absorbance
with ùicreased dilution. At the 1:1280 dilution the titer of the specific IgY was at the
base line of the non-specific IgY and the absorbed IgY (Fig. 2).
Mortality and relative percent survival (RPS) of juvenile rainbow trout treated with
a n W salnaonicida IgY followed by exposure to pathogenic A. salmonicida:
There was no si@cant difference in survival between those juvenile rainbow trout
orally treated with ad-A. salmonicida IgY 24 h prior to an exposure to a letbal
concentration of A. salmonicida, and the control rainbow bout that were orally treated
with non-specific IgY (Table 1).
Fig. 1 Elution profile of IgY (anti-A. salmonicida) via column chromatograph,
CM-32 ceilulose ( 14.5 x 2.3 cm, Whatman, UK) equilibrated in 0.01 M PB pH 5.4
bufYer. Elution was done with O.2M PB buffer pH 5.0.
Fraction Number
Fig. 2 Dilution endpoint determination by indirect ELISA of IgY (specific for anti-V.
anguillarum), non-specüic IgY and absorbed specific IgY. Each point is a mean of four
readings.
+Spcific IgY +Non-speciîic IgY +Absaibsd IgY
r
IgY dilutions
There was aiso no significant diiemce in survivd between those juvenile rainbow mut
IP injected with anti-A. suhonicida IgY 24 h prior to an exposure to a lethal
concentration of A. salmonicida and the control rainbow trout which were IP injected
with non-specific IgY (Table 1).
Mortality and relative percent suMvai of juvenile coho salmon treated with anti-Y.
anguillarunr IgY foUowed by exposure Co pathogenic Y. anguillarum:
There was no significant difference in survivai between those juvenile coho salmon
oraliy treated with anti-Y, anguillarum IgY 24 h prior to an exposure to a lethal
concentration of K anguiIIarum and the control rainbow trout that were oraily treated
with non-specific IgY (Table 2).
There was however, a significant difference in survivai between those juvenile coho
salmon IP injected with anti-K anguillarum IgY 24 h prior to an exposure to a lethal
concentration of V. anguillarum and the control coho salmon that were il? injected with
non-specific IgY (Table 2).
Mortality and relative percent survival of juvenile coho salmon IP injected with
either (1) non-speeüïc IgY (2) anti-K anguiilarum IgY or (3) absorbed aati-Y.
anguillarum IgY foiiowed by exposure ta pathogenic V. anguillarum:
There was a significant clifference in survivaI between those juvenile coho saitnon IP
injected with anti-V. anguillarum aud the control coho salmon that were iP injected with
Table 1. Mortality and Relative Percent Suntival (RPS) in juveniie rainbow bout &er a
bath- challenge with a lethal concentration of live, vident A. salmonicida, one
day d e r IP injection and 7 days a h oral route administration of anti-A.
suhonicida IgY.
CONTROL (Non-spccific tgY)
TREATMENT ORAL IP ORAL IP
TREATED (Ami-AsaImmicidn IgY) (14/60) ( 9 w
indicac*r iht ratio a l monality io thc cotai numbcr oParpcrimcnraI f i h in each group. samc teucrs indieatc no signiticant difiraicc bcowtcn the means (% 0.05).
Fish in the test group were fed a diet containimg crude 2% wlw anti-A. saIrnonici& IgY
for 7 d or IP injected once with purïfied &-A. salmonicida IgY (at the concentration of 1
mg proiein fi&'). Control fish were treated similady but with non-specific IgY. Al1 fish
were then challenged (24 h aftet injection) with a virulent strain of A. saIrnonici&
Mortalities were monitored and tallied for 2 weeks.
Table 2. Mortality and Relative Percent Survival (RPS) in juvenile coho salmon &er a
bath-challenge with a lethal concentration of live, virulent K anguillarum, one
day &er IF injection and 7 days after oral route adaiinistration of anti- Vibrio
anguiIIarum IgY.
I I RELATIVE % SURVIVAL
TREATMENT
* indicaics thc ratio of monality IO thc lMal number of utpaimrntai [ish in each gmup. samc Icmn indicatc no signifiant dif inncc khvccn the mcans (P<O.05).
CONTROL
Fish in the test group were fed a diet containing c ~ d e 2% w/w anti-Y. anguillarum IgY for 7 d
or iP injected once with purified anti-V. m g u i l l m IgY (at the concentraion of 1 mg protein
fish" ). Control fish were treated similarly but wiîh non-specific IgY. Al1 fish were then
challenged (24 h &er injection) with a live, virulent strain of Y. mguiiIarum Mortalities were
monitored and tallied for 2 weeks.
ORAL IP
56.6 a* 50.9 b
ORAL Il'
non-specific IgY (TabIe 3). The former fish had a percent mortaiity of 1 3% whereas the
latter fish had a percent mortaIity of9.8%.
M e n the anti-Y: anguillarum IgY was absorbed with killed K anguiIlarum cells for a
period of 24 h, its abiiity to protect naive juvenile coho salmon against a chailenge of this
pathogenic bacteriwn was significantly degraded, Le., approximately 32% of the fish
that had been IP injected with absorbai IgY 24 hours prior to a lethal V: anguillarum
challenge died (Table 3).
Mortality and relative sumival of juvenile rair~bow trout IP injected with either (1)
non-specific IgY (2) anti-V. anguilkrrum IgY or (3) absorbed anti-Y. anguiliurum
IgY foliowed by exposure to pathogenic V. anguillarum:
Thete was a sigaificant difference in sunival between those juvenile rainbow trout IP
injected with either (1) anti-K anguillarum IgY or (2) absorbed anti-K anguillarum IgY,
24 h prior to an exposure to a letha! concentration of f? anguillarum (Table 4). The
percent mortality in the latter case was approximately doubled as compared to the
former.
There was aiso a sigaincant difference in survival between those juvenile rainbow bout
IP injected with either (1) non-specific IgY or (2) absorbed non-specific IgY, 24 h prior
to an exposure to a lethal concentration of K anguiIIarum (Table 4). The percent
mortality in the latter case was approximately doubled as compared to the former.
Table 3. Mortality and Relative Percent Swvival (RPS) in different treatment groups of
juvenile coho salmon up to 14 d pst-bath-challenge with a lethal concentration
iive, Wulent K anguiliantm.
TREATMENT I TOTAL RFILATLVE%SURMVAL (te injection) MORTALITY
--- -
CONTROL 9.8 a'
indicstcs thc mtio o f mortality to the total number ofcxprimcnlal fmh in cach gmup. diffcrcnt lcttcm indicatc signiticant diffcnncc bctwccn the mcans (PO.05).
Fish in treated 1 group IP injected (at the concentration of I mg protein fisk') once with purified
anti-K anguillarum IgY. Fish in treated II group IP injecteci once wiih purified but absorbed
anti-Y. anguillarum IgY. Fish in control group were treated similarly but with non-specific
IgY. Al1 fish were then challenged (24 h afler injection) with a Iive, virulent strain of Y.
anguiilarum. Monalities were monitored and mllied for 2 weeks.
TREATED 1 (Anti-Yibrio IgY)
TREATED II (Absorbcd anti-Yibrio IgY)
1.8 b (Ulm
32 c (3611 12)
82
Table 4. Mortality and Relative Percent SurYival (RPS) up to 14 d of post-bath-
challenge with a lethal concentration of live, vident anguillarum, in juvenile
rainbow trout previously IP injected with non-absorbed and absorbed anti-Virio
IgY.
TREATMENT (IP injection)
CONTROL 1 40 a* 83 a 1 - -
I Absorbed
TREATED (Anti-Vibrlo IgY)
TOTAL % MORTALlTY
Non-absorbed Absorbed
* indicatcs numbcr o f mortality in totd numbcr o f f ~ h in each group. samc lettes indiatc significant diEcrencc khvrcn the mans (PXl.05).
Fish in lhc mtcd group wcrr dividcd inîo two gmups. F i h in group one wcrc IP injcctcd
once with puritied anti-Y. anguillarum IgY. Fisb in group two were [P injected once with
purified but absorbed anti-Y. anguiIlarum [gY, Controt fish were treated similarly but wiîh
non-specific IgY. All fish were then challenged (24 h aAer injection) with a live, virulent snah of
Y. anguillarum. Mortalities were monitored and tallied Cor 2 weeks.
RELATIVE % SURVIVAL
There was no significant diffetence between the percent mortaiity of the fish treated with
either non-specific IgY and anti-V. angui1Imrn IgY or between absorbed non-specific
IgY and absorbed anti-K a n g u i l h m IgY (Table 4).
Duration of protection in rainbow m u t against VI unguillarum foUowing an IP
injection of anti-K anguillurum IgY;
The data of Table 5 show that for 14 d following an iP injection of anti-K anguillurum
IgY, the cumulative 14 d post-challenge mortality rates within a treatrnent group did not
change significantly for the groups chdlenged at different time intervals following the
initiai IP injection. Within each group there was no significant ciifferences in mortaiity
rates.
The rainbow trout IP injected with anci- V. unguiIImm IgY expressed significantly more
resistance to a lethal challenge to pathogenic V , anguiIIarum as compared to both the PBS
injection and non-specific IgY injected trout.
Duration of total serum IgY in rainbow trout following an IP injection:
The data of Fig. 3 show that during the 14 d of this triai the total serum IgY injected into
the rainbow trout significady decreased, although the concentrations at d 3 and 7 were
not signif?cantIy different h m each other.
The lowest mortality of the injected bout was at day 1 pst-injection. However, the
mortalities on days 3 and 7 were not significady different fiom each other, while that of
day 14 was greater than day 1, but less than either of days 3 or 7.
Table 5. Duration of protection in different treatment groups of juvenile rainbow
trout previously IP injecteci with specific and non-specific IgY
TREATMENT 1 TOTAL % MORTALITY 1 RELATIVE KSURWAL
I I
BACTERIAL 1
DAY POST INJECTION
CONTROL II 1 71.1 a 71.1 a 57.7 a 82.2 a 1 6 6 8 42
1 3 7 14
CONTROL 1 ( P M injection)
TREATED (Anti- fibrio IgY)
1 3 7 14
75.5 a' 75.5 a 62.2 a 57.7 a (34145)' (34145) (28145) (26145)
indicaîing numbcr of mortelity in totd nunibcr of fsh in cach p u p same I e w n indicatc no signifiant diffcrena ktwm itK mcans (P4.05).
Fish in the treated group were iP injected once with purifieci anti-Y. anguillarum IgY. Fish in
control II and control 1 groups were also ïP injected once with non-specific IgY and PBS
respectively, All fish were then challenged with a live, virulent main of Y. anguiIlarum on days
1,3,7, and 14 post injection. Mortalities were moaitored and tallied for 2 weeks. Bacteria1
counts refer to water-borne challenge doses,
Fig. 3 Temporal trend of mortality rate in relation with total serum IgY levels in the fish
groups IP injected with anti-V. anguiIZamm IgY. Values are the mean of 3 replicates
k standard deviation. The % mortality vaiues are h m the Wated" data of table 5.
Mortality rab & total m m IgY leval following IP injection with anti-Vibria IgY
Day 1 Day 3 Day 7 Day t4
Poat-injecüon thalkngr date
There was no signincaat hear correlation (P= 0.365) between total serum IgY
concentrations of the fish and the percent mortalities following an immersion challenge.
The values noted here represent the mean of tbree fish coiiected fiom each experimental
tank on each challenge date.
When the relative concentrations of anti-K anguillumm IgY titer (128) in the sera was
greatest, the mortality of the trout was the least (Fig. 4). At lesser titers of 64 and 32
the mortalities were greater (Fig. 4). But, linear regression analysis showed no signincant
correlation between the mortalities and the anti-Vibrio IgY titer at these lesser titers (i =
0.477, P = O. 309).
A comparison of total IgY concentrations and specific anti-V. anguiilarum IgY titers
shows that both decreased with time in the rainbow trout, following the IP injection (Fig. 5).
However, a direct comparison cannot be made since the assays for each were different
therefore, the concentration units were different.
Fig. 4 Temporal trend of morîality rate in relation with anti-Vibrio IgY titer in serum of
fish following IP injection with anti-Vibrio IgY. Values are the mean of 3
replicates (* standard deviation for mortality rate). Similar letters indicate no
significant daerence.
(Pc 0.05). The % morîality numbers are fiom the "treated" data of Table 5.
Mortality rate & anti-K anguillamm IgY titer following IP injection wlth an& Wbrio IgY
Day 1 Day 3 Day 7 Day 14
Polt-injection chilknga data
Fig, 5 Temporal trend of anti-Vibrio IgY senun titer fluctuations in dations with total
semm IgY level in IP injected fish with anti-l'?brio IgY. Values are the mean of 3
replicates (* standard deviation for total IgY).
Anti-Y. anguiIIarum IgY titer 6 total senim IgY Ievd in IP injecteci fkh
Day 1 Day 3 Day 7 Day 14
Polt-injection challenge date
DISCUSSION
Titre of lgY in egg y o k
Akita and Nakaai (1992) reported a concentration of total IgY in the eggs they analyzed
as 12.53 mg IgY ml" of yolk whereas Arasteh (2000) reported an IgY concentration of
5.93 i 1.26 mg mL" in the egg yoks that she aaalyzed.
The results of the present study, which show a concentration of 5.23 + 1.75 mg IgY rnL-'
of yolk, are similar to those of Arasteh (2000) and approximately haif of the values
detennined by Akita and Nakai (1992). This difference is aîûibuted to the fact that the
latter investigators used a radio immuno-difiion (RID) assay whereas the ELISA assay
technique used in the present investigation was very simila to the ELISA assay used by
Arasteh (2000).
Efficacy of anti-A. salmonicidu IgY for passive immunization of juveniie rainbow
tmut against A. sul~nicida:
Previous resestrçh has shown that salmonids, induding tainbow trout, can be both
actively and passively immunized aga& a number of bacterial-mediated diseases,
including funincdosis (causative agent =A. salmonicida) and vibriosis (causative agent
= V. mguiIlmum).
Spence and Fryer (1965) intra-abdominally injected 0.5 mL of a pooled senun sample
(which agglutinated A. sahonicida ceils) fiom 20 rainbow trout (previously
intra-abdominally vaccinated with an A. salmonicidz bacterin) into coho &on and
determined their susceptibility to a letbal exposure to this pathogen. Those salmon
passively irnmunized in this way with the an& A. salmonicida antiserum showed fewer
mortaiities than the fish injected with either non-immune serum or not treated. The
authors attributed this protection to the presence of anti-A. salmonicida immunoglobulins
in the injected serum.
McCarthy et al. (1983) M e r demoostrated that antisenun prepared fiom female New
Zealand white rabbits injected with the rough strain (with the A-layer proteins) of A.
salmonicida could passively protect naive sockeye salmon against a lethal challenge of
this pathogen. However, poor (or no) protection was observed if smooth ceUs (without
the A-layer protein) were used to vaccinate the rabbits.
Cipriano (1983) noted that the serum of rainbow trout that displayed an innate resistance
to funincuiosis wouid passively protect naive brook trout (Salvelinwfontinalis) against a
Iethal furuncdosis challenge whereas the serum fkom Atlantic salmon (Salmo salar) that
showed no innate resistance to fiininculosis could not passively immunize brook trout
against this disease.
These trials unequivocaüy demonsaated that anti-A. salmonicida immunoglobulins, can
in certain circumstances, protect at least some salmonids against a lethal exposure to A.
salmonicida.
Based on these previous observations, the trials carried out in the present snidy shodd
have shown protection for the rainbow trout against a lethal challenge by A. salmonicida
when semi-purified chicken IgY was injected (Table l), since an autoagglutinating strain
was used (This is indicative of a pathogenic A. salmonicida). That no protection was
observai (Table 1) may perhaps be explaineci by the observations of Bricknell et al.
(1997, 1999). Brickneii et ai. (1999) noted that a bacterin made fiom formalized whole
ceils of A. salmonicida that had been grown under uon-restricted conditions was
effective in pmtecting Atlantic salmon against a lethal challenge to this pathogen. The
authors attributed this to the presence of hn-regulated outer membrane proteins
(ROMPS) that were induced whea the cells were grown under iron-resûicted conditions.
The ROMPS induced specsc and protective antibodies in target saimonids. In a
previous investigation, Bricknell et al. (1997) noted that significant immune protection
was not achieved with polysaccharide extracted fiom the ce11 walls of A. salmonicida
whereas the hot phenol-extracted polysaccharide fiom the broth-culture supernatant of
this bacterium led to protection against an experimental challenge by Wulent AI
salmonicida. The authors suggested that the antiserum to this material was capable of
passively protecting the salmon against the lethal toxicity of phenyl methyl sulphonyl-
treated extracellular products.
The cells of A. salmonicida used in this investigation were grown in a medium that was
not restricted with regard to its iron content. Hence, it would appear very Lilcely that the
Ùon resfncted outer membrane components of the A. salmonicida cells wete not
expressed and the cells did not therefore express surface antigens against wbich
protective immunoglobulins could be raised in the chicken,
Efficacy of antl-V. anguillarum IgY for passive immunization of juvenile coho
sahon against vibriosis:
A number of investigators (see below) have show that several finfish, including
salmonids, may also be protected against V. anguillarum infections by passive
immunization. Kawai and Kusuda (1983) employed a V. anguillarum bacterin to oraUy
vaccinate ayu and reported a ûansfer of passive immunity to naive ayu. Hm11 et al.
(1975) demonstrated that rainbow trout s e m antibodies would protect naive rainbow
bout against a K anguillarum challenge. Akhlaghi (1999) found that anti-V. anguillaruni
whole sera could be readily raised in sheep, rabbits and rainbow trout and be effective in
controlliig vibriosis when injected into naive rainbow trout prior to a bath challenge with
a lethal concentration of A. salmonicida. Velji (1992), in an exhaustive study,
detennined that naive coho salmon that received plasma fiom coho salmon that were
IP-vaccinated with a V. anguillarum extraceliular lipopolysaccharide (LPS), displayed 83
% survival. When a similar group of naive coho salmon were injected with s e m fiom a
group of coho saimon that had been previously vaccinated orally with extracellular-LPS,
the enhauced SUTVival was 33 %.
Based upon the observations reported here and those reported by other investigators (see
above), it appeats that passive irnmunization c m be an effective means of protecting
salmonids against at least several bacterial diseases, including vibriosis, regardless of
whether the antibody source is chicken ova or sera of rabbits, sheep or rainbow trout.
The resuits reported here (refer to Fig. 4) clearly show that i? anguiIIarum acts as an
excellent and consistent source of antigens for production of protective antibodies in
sahonids and other animals, whereas A. salmonicida does not consistentiy produce
protective immunoglobulins. The main protective ceii surface antigen determinant
of V. anguillarum is LPS, which is consistently produced (Velji, 1992), regardless
of the culture medium whereas the main pmtective ceIl d a c e antigens of A,
salmnicida are growth medium dependent. Accordingly, the remainder of the
research focused on the use of K anguillarum (which provided a source of
consistent antigen) for studying the use of domestic chicken IgY for protecting
rainbow trout against lethal K anguillarum challenges. Research focused on
factors that influenced this protection process.
Efficacy of absorbed IgY for protecting rainbow trout against a letbal Y.
anguillrirum challenge:
As expected, absorption of anti- V: anguillarum IgY with whole cells of VI
anguillmm markedly degraded the ability of the anti-i? anguillarum IgY to
protect naive rainbow trout against a lethal challenge with V. anguillarum (Table
3) since the percent mortality of the anti-VI anguillarum IgY treated fish was 1.8
% whiie that of the fish treated with absorbed anti-VI anguillarum IgY was 32 %.
This decrease is attributed to removai of most, if not dl, specific IgY that binds to
the SUffaçe antigens of this pathogen.
The apparent protection given to the trout by the control IgY (9.8%) is of interest
since this is significantiy ciiffirent h m the absorbed anti-vibrio IgY (32%). It would
appear that the control IgY contains protective immunoglobulins against K anguillaium.
These observations strongly indicate that unabsorbeci non-specific IgY contains
immunogiobuli tbat bind to whole ceUs of K anguiIIarum in spite of the fsact that the
chickens were nut exposai tù this bacterium (V. anguillarum is a marine bacteriun that is
not a resident of temsûial animais and their fieshwater and soi1 environments).
That the conml non-specific IgY and the anti-Y: anguiiCarrmi IgY have presumed
common immunoglobuiins is confirmeci by the data of Table 4. In each case, absorption
of the IgY with V: anguiilarum ceUs greatiy compromised the ability of the IgY to
provide immune protection. These results strongly imply that the control and the F!
anguiIIarum treated chickens were exposed to common antigens.
Duration of serum IgY versus challenge mortaüty in rainbow trout following an ïP
injection:
The results reported here show that the significant protection against a lethal chalenge
by V. anguiilmm occurred throughout the 14 days of the trial (Table 5). There was no
significant diffecence between the mortalities observed when the fish were challenged at
days 1,3,7 and 14, pst-treatment with specific a d - K anguillurum IgY.
The difference in % mortality noted in the control groups (non-specific IgY) (Tables 4
and Table 5) is due to the use of digerent set of chickens in the Iatter experiment.
These data cornoborate those reported by Akhlaghi (1 9W) who observed good protection,
for 1 month pst-treatment, of rauibow mut passiveiy immunized with sheep, rabbit or
rainbow trout anti-Y: mguiZlarum sera. The protection, due to passive immunization
declined, markedly d e r one month although it was stiIl ptesent at 2 months. Akhlaghi
(2996) noted a similar temporal protection when sheep, rabbit and trout
anti-S~reptococcus spp. immune sera were used to passiveiy immuaize rainbow mut
against this pathogen. One wouid have expected these imrnunogiobulins to be quickly
cleared fiom the blood ofthis animal.
The data reported here (Table 5) and those of Akhlaghi (1997, 1999) raise significant
questions regardhg the persistence of foteign proteins such as chicken IgY and sheep and
rabbit irnrnunuglobulins in fish such as a rainbow trout. E.g. how long do therapeutic
quaatities of immunoglobulins adrninistered for passive protection of the rainbow mut
rernain in the animal?, and wi a determination of the minimum serum chicken IgY
concentration that provides protection be determinai?
To investigate this, an expriment was jointly conducted with N. Arasteh of the
Faculty of Graduate Studies (Food Science Program), University of British Columbia.
The ~ s u i t s of this expriment are discussed below.
Duration of anti-Y. anguiiIatum immune protection in minbow trout foiiowing an
IP injection of IgY:
Akhiaghi (1996,1993) has shown that when immunogbbulins of sheep and rabbits are P
administered to rainbow trout, these proteins reached their highest concentrations
between 6 and 24 h following each administration. Because of these observations, it was
therefore decided to take one day post IP injection as the thne at which the administered
non-specific IgY and mti-V. anguiKarum IgY were at their miuamal concentrations and
meastue the subsequent concentrations for a period of 14 days t h e d e r . The data (Fig.
3) show that tbere was a deçrease with time in the senun total IgY. There was a
significant difference in concentrations betweea day 1, day 3-7 and day 14. Titre of
anti-V. anguillurum IgY in the senun showed a significant decrease between day 1 and
day 3, through day 14. However, there was no significant difference between days 3
and 14 (Fig. 4).
Akhlaghi (1996, 1999) noted a similar pattern of decrease in sheep and rabbit anti-F!
anguillarum immunoglobulins in the sera rainbow trout that had been IP injected with
these proteins. He found that the maximal concentrations occurred within 24 h of IP
injections of the immunoglobulins. And, in a subsequent three-month period of time, the
immunoglobulins decreased to undetectable levels.
Akhlaghi (1 996, 1999) found that good protection against a lethal V . anguillarum
challenge occurred for 1 rnonth following IP injections of either the sheep or rabbit
immunoglobulins. However, the protection then decreased significantly with time such
that Little remained by 3 months p s t IP injection.
These results, show that the protection pmvided by passive immwiization of chicken ova
IgY as administered by IP occurs for at least for 14 days, and, perhaps longer if one
considers the data of Akhlagh. (1996,1999) with regard to sheep and rabbit
immunoglobulins.
A detemination of the anti-V. anguiIIarum IgY concentrations requireà to provide
passive immune protection agaiast a K mguiitarum challenge:
Knowledge of the minimal amounts of the total and specifïc IgY required in nnfish sera
to control microbial üifections, such as that caused by V. anguillarum, would be usefiil if
this technique of passive immunization is to be used for therapeutic purposes.
The data of Fig. 5 show that total chicken IgY concentration was approximately 2.5 x
10'' mg/mL of senun of the rainbow mut. This concentration decreased to
approximately 4 x 10" mg/mL of trout senun by 14 days post IP injection. And, at this
lower concentration, passive immune protection was pmvided by the chicken IgY.
However, it was not possible to determine the concentration of specific anti-V
anguillarum IgY in the s e m since the assay for titer of this material was on a relative
basis with the use of ELISA.
Akita and Li-Chan (1998) approached this problem by measuring the amount of affinity
purifieci specific anti-bovine IgG in a total IgY preparation by the use of a RID method.
They determhed that approximately 10 % of the total IgY obtained fiom their egg yolk
preparation was specific to the bovine IgG antigen.
If one assumes that approximately 10 % of the total IgY used in the experiment described
in (Fig. 5) is anti-V. anguiIIarum IgY, this would imply that the anti-V, anguilIanim IgY
concentrations varied h m approximately 2.5 x 1om3 to 4 x 104 mg/mL of senim of the
h b o w trout during the 14 days course of the experiment.
The present study evaluated the ability of non-specifïc and specific immunoglobulins
(IgYs) of chicken egg yolks to passively imunize two species of salmonids (rainbow
mut and coho salrnon) against two common bacteriai diseases (furw?culosis and
vibriosis).
Oral treatments of these salmonids with anti-A. salmonicida or anti-V: anguillarum IgYs
did not provide passive immunization against lethai challenges of A. salmonicida and V:
anguillarum, respectively.
Intraperitoneai injection of either non-specific IgY or anti-V: anguillarum IgY
provided variable or significant passive immune protection, respectively, against a lethal
K anguillamnt challenge.
M e n non-specific IgY and anti-K anguillmm IgY were each absorbed with K
anguiIlmm celb prior to king used to passively unaiunize naive rainbow mut, each
IgY preparation lost its ability to passively immunize juvenile rainbow trout agaiast a
lethal V: anguillarum challenge. These results strongly imply that the unvaccinated
control and the V. anguillarum-vaccinated ûeated chickens were exposed to cornmon
antigens, These may be bacteria with common sutface antigens.
Serum concentrations between the tested range of 2.5 xlO'L to approximately 4.0 x 10'~
mg anti-V. anguiIlunun IgY mL" were effective in protecting juvenile rainbow trout
against a lethal challenge of V. anguiIIarum. This Iower concentration occurred 2 weeks
after the IP injection of anti-K anguillarum IgY into the trout. Un the assumption that
the anti-V: anguiIIarum immu~ïoglobulins were appmximately 10 % of the anti-K
anguillamm IgY, îhis wouid imply that the proteciive anti-K an8i(ilimm
immunoglobulin concentrations in tbe serum of the treated fish varied h m
appmximatel y 2.5 x 1 O'3 to 4 x 104 mg CUL".
The results of the present study and others (e.g. AMaghi, 1996,1999) indicate that good
passive immune protection against vibriosis can occur for up to one month pst-IP-
treatment with specific anti-K anguilIurum IgY.
Akhlaghi, M. (1996). Comparison of passive and active immunization of fish against streptoccosis (enterococcis). J . Fish Dis. 19: 25 1-258.
Akhlaghi, M. (1999). Passive immunization of fish against vibriosis, cornparison of intraperitoneal, oral and immersion routes. Aquacult. 180: 19 1-205.
Akita, E. M., E.C.Y. Li-Chan (1998). Isolation of bovine immunoglobulin G subclasses from milk, colostrum and whey using immobilized egg yolk antibodies. J . Dairy Science 8 1 : 54-63.
Akita, E.M., S. Nakaai (1992). ImmunogIobulins frorn egg yoik: isolation and purification. J , Food Science 57: 629-634.
Arasteh, N. (2000). Passive immunization of rainbow trout with chicken immunoglobulins (IgY). Ph.D. thesis, University of British Columbia, Canada.
Bricknell, I.R., T.J. Bowden, J. Lomax, A.E. EHis (1997). Antibody response and protection of Atlantic salmon (Salmo salar) immunized with an extraceilular polysaccharide of Aeromonus salrnonicida Fish & Shelysh Immunol. 7: 13-16.
Bricknell, I.R., J.A. King, T.J. Bowden, AE. Ellis (1999). Duration of protective antibodies, and the correlation with protection in Atlantic salrnon (Salmo salar L.), following vaccination with an Aeromonar salmonicida vaccine containing iron-regulated outer membrane proteins and sectetory potysaccharide. Fish di Shelljish Immunol. 9: 139-15 1
Cipriano, R.C. (1 983). Resistance of salmonids to Aeromonas salmonicida: Relations between agglutinins and neutralizing activities. Trans. Amer. Pish. Soc. 112: 95-99.
Fichtali, J., E.A. Charter, K.V. Lo, S. Nakai (1993). Purification of antibadies h m industrially separated egg yolk. J. Food Science 58: 1282-1285.
Gutierrez, MA., T. Miyazaki, H. Hatta, M. Kim (1993). Pmtective properties of egg yolk IgY containing anti-Edwardsiella tarda antibody against paracolo disease in Japanese eel, Anguilla japonica Temminck 8i Schlegel. J . Fish Diseases 16: 113-122.
Harrell, L.W., H.M- Edinger, H.O. Hodgins (1975). Humoral factors important in resistance of salmonid fish to bacterial disease: L S e m antiôody pmtection of rainbow trout (Salmo gairhrf) against vibriosis. Aquacuk 6: 21 1-219.
Kawai, K., R. Kusuda (1983). Efficacy of the lipopolysaccharide vaccine against vibriosis in cultured ayu. Can. Trum. Fish. Aquat. Sci 5027: 14 pp.
Lee, S.B., Y. Mine, R.M.W. Stevenson (2000). Effects of hen egg yolk immunoglobulin in passive protection of rainbow trout against Yersinia ruckeri. J. Agriculhral & Food Chemistry 48: 110-1 15.
Longnecker, B. M. (1 970). Measurement of Graft vs. Host Relations on the Choriailantoic Membrane of the Chicken in Transplantation. 9: 6 Williams and WiIkens, Co. USA.
McCarthy, D.H., D.F. Amend, K.A. Johnson, J .V. Bloom ( 1 983). Aeromonas salmonicida: determination o f an aatigen associated with protective immunity and evaiuation of an expetùnental bacterin. J. Fish Dis, 6: 155-1 74.
Otatake, M., G.K., Iwama, T. Nakaaishi (1996). The uptake of bovine senim albumin by the skin of bath irnmwiized rauibow trout Oncorhynchus mykiss. Fish & ShellJish Immunol. 6: 321-333.
Spence, K.D., J.L. Fryer, K.S. Pilcher (1965). Active and passive immunization of certain Salmonid fishes against Aeromonus salmonicida. Can. J. Micriobiol. 1 1 : 397-405.
Velji, M.I. (1992). Studies on the immune response of coho salmon (Oncorhynchus kisutch) to lipopolysaccharide antigens produced by Vibrio ordalii, a causative agent of vibriosis in saimon. Ph.D. thesis, Simon Fraser University, Buniaby, Canada 123 pp.
Wendemyer, G.A., Barton, D.J. McLeay (1990). Stress and acclimation. Pages 451-489. In: Methods for fish biology. C.B. Schreck and P.B. Moyle (editors).
Amer. Fish. Soc., Bethesda, MaryIaad.
Yousif, A. N. (1992). Studies on antimicrobid factors in salmonid eggs with special reference to coho salmon Oncor~chus Aisutch (walbaum). PhD. thesis, Simon Fraser University, Burnaby, Canada 65 pp.