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

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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.

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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.

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