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Passive immunization of rainbow trout with chicken immunoglobins (IGY) Arasteh, Nikoo 2000

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PASSIVE IMMUNIZATION OF RAINBOW TROUT WITH CHICKEN IMMUNOGLOBULINS (IGY) by NIKOO ARASTEH B.Sc, Ferdowsi University of Mashhad, Iran, 1985 M.Sc, University of Tehran, 1992 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Food Science Program) We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH COLUMBIA August 2000 ©Nikoo Arasteh, 2000 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. 1 further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of poo^- S^-es^c*- j fco^c+U&t art- /^fiT' C**-£(M->>*JL SC^_ The University of British Columbia Vancouver, Canada DE-6 (2/88) A B S T R A C T Passive immunization as an alternative to vaccination or antibiotic therapy, involves use of a pathogen specific antibody raised in other animals to provide extended disease resistance to the host animals or humans. The egg yolks o f hens are a rich source of specific immunoglobulins (IgY) which can be easily extracted and incorporated into the human or animal diets. However, an effective method of IgY delivery into the host animal system is needed. In this study, enhancement o f rainbow trout resistance against Vibrio anguillarum infection has been used as a model to investigate passage of I g Y through the gut barrier into the bloodstream and the passive immunization that pathogen specific IgY may confer. High titers o f anti-K anguillarum I gY were raised in vaccinated hens, recovered from the water-soluble fraction (WSF) o f the egg-yolks and subsequently used in intraperitoneal (IP) injection, oral intubation or feeding of the trout. Western blotting of such IgY revealed a strong reactivity with V. anguillarum whole cell lysate and lipopolysaccharide, which was as strong as that o f the rabbit IgG and stronger than that of the fish I g M . Immunological properties of IgY as measured by E L I S A were not affected by freeze-drying, vacuum microwave drying, air-drying, or spray drying. IP injected anti-Vibrio I gY was transferred into the fish system in high enough levels to confer protection against Vibriosis in an experimental challenge. This protective effect which was retained at least 14 days post IgY injection, proved efficacy of pathogen-specific IgY in enhancement of disease resistance. To investigate absorption through trout digestive tract, I g Y was intubated both anally and orally at the levels of O.lmg and 1.4-2.7mg fish"1, respectively. Under the conditions of this study, anally intubated IgY (O.lmg) did not appear in the serum in a detectable level. Oral intubation of W S F led to absorption of IgY in an immunologically active form; however the levels were 800 to 2500 times lower than those resulting from UP injection. Among the detergents co-administered with IgY, deoxycholate, Mega9 and octyl-f3-glucoside mediated the highest enhancement of IgY absorption. Use of Mega9 raised serum IgY to levels only 12 to 18 times lower than the levels after TP injection o f a similar dose. Encapsulation of W S F in polylactide-co-glycolide did not improve IgY i i uptake. Oral administration of anti-Vibrio IgY in co-delivery with detergents resulted in different levels of protection of rainbow trout against Vibriosis following an immersion challenge, which in some cases was comparable to the protection offered by IP injection o f IgY. The efficacy o f continued feeding of specific IgY before and after exposure to the pathogenic bacteria has yet to be explored. T A B L E O F C O N T E N T S page A B S T R A C T i i T A B L E OF C O N T E N T S iv LIST OF T A B L E S ix LIST OF F I G U R E S x LIST OF A B B R E V I A T I O N S xiv A C K N O W L E D G E M E N T S xv i i D E D I C A T I O N S xv i i i 1. I N T R O D U C T I O N 1 1.1. B ackground of the study 2 1.2. Hypothesis 4 1.3. Objectives of the study 4 2. R E V I E W OF R E L A T E D L I T E R A T U R E 6 2.1. Vibriosis and other fish diseases 7 2.2. Antigenicity o f V. anguillarum and portals of entry 7 2.3. Vaccination 8 2.3.1. Intraperitoneal (IP) injection 8 2.3.2. Immersion vaccination 9 2.3.3. Oral vaccination 9 2.4. Antibodies as a replacement for antibiotics 10 2.5. Passive immunization 11 2.6. Use of chicken egg yolk antibodies (IgY) in passive immunization 12 2.7. Raising specific antibodies in chickens 14 2.8. Advantage of chicken IgY over other animal sources 14 2.9. Stability of IgY and differences with IgG 16 2.10. Methods of IgY preparation 18 2.11. Protein absorption in the digestive system 19 2.12. Time course and efficacy of macromolecule transfer into the blood circulation 20 iv 2.13. Protection from degradation in the GI tract 25 2.13.1. Encapsulation 26 2.13.2. Enhancement o f absorption using detergents 30 2.13.3. Immunostimulants 37 2.14. Dehydration techniques 38 2.14.1. Hot air-drying 38 2.14.2. Freeze-drying 39 2.14.3. Vacuum microwave-drying 40 3. M A T E R I A L S & M E T H O D S 42 3.1. Buffers 43 3.2. Animals 44 3.2.1. Chickens 44 3.2.2. Fish 44 3.3. Bacterial preparation 45 3.4. Vaccine preparation 48 3.5. Vaccination 49 3.5.1. Chickens 49 3.5.2. Fish 49 3.6. Preparation of IgY 51 3.7. Preparation of microcapsules 52 3.8. Feed preparation 54 3.9. Extraction o f I gY from the treated pellets 56 3.10. Blood collection from fish and serum preparation 56 3.11. Bacterial preparation for fish challenge studies 57 3.12. Lipopolysaccharide (LPS) and whole cell lysate ( W C L ) preparation 57 3.13. Anal intubation 58 3.14. Oral administration 59 3.14.1. Experiment 1. Using IgY in encapsulated form, in Mega9 and antacid, or in Na-pyrophosphate solution 59 v 3.14.2. Experiment 2. Tween detergents as absorption enhancing agents 60 3.14.3. Experiment 3. Comparative oral intubation 61 3.14.4. Experiment 4. Effect of various absorption enhancing agents 61 3.15. Challenge studies '. 63 3.15.1. Experiment 5. Preliminary feeding trial 64 3.15.2. Experiment 6 & 7. Challenge following oral administration of Mega9 and water soluble fraction of egg yolks (WSF) 66 3.15.3. Experiment 8. Challenge following oral intubation with absorption enhancing agents 68 3.15.4. Experiment 9. Challenge following feeding of absorption enhancing agents 69 3.15.5. Experiment 10. Challenge following LP injection of IgY 70 3.16. Analytical techniques 71 3.16.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- P A G E ) 71 3.16.2. Western blotting 72 3.16.3. Enzyme linked immunosorbent assay (ELISA) 73 3.16.3.1. Determination of total IgY content... 74 3.16.3.2. Determination of anti-Vibrio specific IgY titer 76 3.17. Drying of W S F 78 3.18. Dehydration of IgY-containing pellets 79 3.18.1. Initial dehydration & IgY incorporation 79 3.18.2. Freeze-drying.. 80 3.18.3. Vacuum microwave drying 81 3.18.4. Air-drying 81 v i 3.19. Statistical methods 81 3.20. Histological examination 82 4. R E S U L T S & D I S C U S S I O N 84 4.1. Cellular and surface antigens o f V. anguillarum interacting with Chicken IgY, rabbit IgG of fish I g M 85 4.2. Dehydration o f W S F 91 4.2.1. Dehydration rate 91 4.2.2. Stability o f I gY during dehydration when incorporated onto the pellets 93 4.2.3. Stability o f IgY during dehydration o f concentrated W S F 95 4.3. Post-vaccination IgY level in the yolk 99 4.4. Passive protection induced by EP injected specific anti-WAr/o I g Y to fish 104 4.5. Ana l administration of IgY 110 4.6. Absorption o f orally administered IgY into fish bloodstream... 112 4.6.1. Experiment 1. Using IgY in encapsulated form, in Mega9 and antacid, or in Na-pyrophosphate solution 112 4.6.2. Time course and levels of absorption after oral and anal administration of proteins 115 4.6.3. Efficacy of delivery into the blood using encapsulated proteins 118 4.6.4. Experiment 2. Tween detergents as absorption enhancing agents 120 4.6.5. Experiment 3. Comparative oral intubation 123 4.6.6. Experiment 4. Effects of various absorption enhancing agents 127 4.7. Challenge studies 131 4.7.1. Experiment 5. Preliminary feeding trial 131 v i i 4.7.2. Experiment 6, 7, 8 & 9. Challenge following oral administration of anti- Vibrio IgY 13 2 4.8. Enhancement of IgY uptake using detergents 141 4.9. Significance of IgY application in protection against diseases 144 4.10. Histological studies 149 5. C O N C L U S I O N 159 6. R E F E R E N C E S 164 7. A P P E N D I X 178 v i i i L I S T O F T A B L E S Page Table 4.1. Changes in IgY concentration due to dehydration treatments. 94 Table 4.2. Serum IgY levels & mortality rates in the fish IP injected with anti-K anguillarum specific IgY. 108 Table 4.3. Temporal absorption of IgY into the fish blood when encapsulated or co-administered with chemical compounds. 114 Table 4.4. Effect of Tween detergents in uptake of orally intubated IgY into the bloodstream. 121 Table 4.5. Effect o f chemical compounds in uptake of orally intubated IgY into the bloodstream. 124 Table 4.6. Lethality of various levels o f orally intubated detergents to rainbow trout. 128 Table 4.7. Effect of detergent in enhancement of I gY absorption. 129 Table 4.8. Serum IgY & mortality levels in preliminary feeding and challenge conducted at day 9. 133 Table 4.9. Serum IgY & mortality levels following oral intubation or feeding o f W S F and Mega9 (Experiment 6). 135 Table 4.10. Serum IgY & mortality levels after oral administration of W S F , Mega9 and antacid (Experiment 7). 137 Table 4.11. Serum IgY & mortality levels following oral administration of W S F , absorption enhancing agents and antacid (Experiment 8). 139 Table 4.12. Serum IgY & mortality levels following feeding of W S F , absorption enhancing agents and antacid (Experiment 9). 142 Table 4.13. Total I gY level in different pellet compositions (mg g"1 pellet). 148 ix LIST OF FIGURES Page Fig.2.1. Schematic mechanism of macromolecules intestinal uptake and transfer. 21 Fig.2.2. Passage of particulate materials from the intestinal lumen into the bloodstream. 22 Fig.2.3. The time course of appearance of bioactive proteins within the serum of orally and anally intubated fish. 24 Fig.2.4. Time-course of appearance o f human gamma globulin ( H G G ) in the serum o f Atlantic salmon following oral delivery o f free or P L G (50:50) encapsulated antigen. 29 Fig.2.5.(a). Chemical structure of Mega9 (nanonyle-n-glucamide) 32 Fig.2.5.(b). Chemical structure of deoxycholic acid (5-P-cholan-24-oic acid-3a, 12a-diol). 32 Fig.2.5.(c). Chemical structure of octyl P-glucoside (n-octyl-P-D-glucopiranoside). 33 Fig.2.5.(d). Chemical structure of L-cysteine ethylester. 33 Fig.2.5. (e, f). Chemical structures of polyoxyethylene ethers, (e) Triton X-100, (f) Triton X - l 14. 34 Fig.2.5.(g). Chemical .structure of C H A P S (3-3-cholamidopropyl dimethylammonio-1 -propanesulfonate) 3 5 Fig.2.5.(h). Chemical structure of C H A P S O (3-3-cholamidopropyl dimethylammonio-2-hydroxy-l-propane-sulfonate) 35 Fig.2.5.(i). Chemical structure of Tween-20 (polyoxyethylenesorbitan monolaurate). 36 Fig.2.5.(j). Chemical structure of Tween-80 (polyoxyethylenesorbitan monooleate). 36 Fig.3.1.(a &b). Design of a 75L fish holding tank. 46 Fig.3.2. Arrangement of fish holding facilities. 47 Fig.3.3. Schematic diagram of anti-Vibrio anguillarum vaccine preparation and vaccination of chickens. 50 Fig.3.4. Schematic diagram o f the IgY extraction from the hen eggs and application in the absorption and passive immunization studies in rainbow trout. 53 Fig.3.5. Schematic diagram of microcencapsulation of water-soluble fraction (WSF) of egg-yolks in polylactide-D-glucolide (PLG) 55 Fig.3.6. Schematic diagram o f fish challenge with V. anguillarum. 65 Fig.3.7. Procedure of sandwich E L I S A . 75 Fig.3.8. Procedure of antibody capture E L I S A . 77 Fig.4.1. (a) S D S - P A G E profile o f 15uL whole cell lysate ( W C L ) of V. anguillarum stained by coomassie blue, (b) S D S - P A G E profile o f 20uL proteinase K digested W C L of V. anguillarum stained by the L P S silver staining procedure. 88 Fig.4.2. Western blots o f V. anguillarum antigens (proteinase K digested whole cell lysate: lane marked as L P S ; whole cell lysate: lane marked as W C L ) . The primary antibody in immunoblotting was acquired from vaccinated (a) fish serum, (b) rabbit serum, (c) chicken egg yolk water-soluble fraction. 89 Fig.4.3. Western blots o f V. anguillarum antigens (proteinase K digested whole cell lysate: lane marked as L P S ; whole cell lysate: lane marked as W C L ) . The primary antibody in immunoblotting was acquired from non-vaccinated (a) fish serum, (b) rabbit serum, (c) chicken egg yolk water-soluble fraction. 90 Fig.4.4. Specific anti-K anguillarum I gY E L I S A values of a 10-fold diluted water-soluble fraction o f egg yolk before and after freeze-drying or vacuum microwave drying. 96 Fig.4.5. Specific anti-K anguillarum IgY E L I S A values of a 10-fold diluted water-soluble fraction o f egg yolk before and after spray-drying. 97 Fig.4.6. Fluctuation in total IgY concentration of a 10-fold diluted water-soluble fraction of egg-yolk. 101 Fig.4.7. Fluctuation in specific anii-V. anguillarum IgY titer of a 10-fold diluted water-soluble fraction o f egg yolk. 102 xi Fig.4.8. Levels o f specific mti-V. anguillarum IgY in the water-soluble fraction o f the egg yolks collected from immunized hens in different post-vaccination dates compared to pre-vaccination level (indicated as non-specific). 103 Fig.4.9. Cumulative 14-day post-challenge mortality rates in fish groups UP injected with P B S , non-specific or specific anti-K anguillarum IgY compared for each challenge date. 106 Fig.4.10. Cumulative 14-day mortality rates in groups of fish challenged at different post-injection dates compared within each treatment group (EP injected with P B S , non-specific or specific anti-K anguillarum IgY). 107 Fig.4.11. Serum IgY level in fish intubated with W S F alone, associated with Mega9 or sodium pyrophosphate and encapsulated in P L G 85:15 or P L G 50:50. EP injected W S F and intubated P B S served as positive & negative controls, respectively. 113 Fig.4.12. Microscopic photograph of P L G encapsulated water-soluble fraction (WSF) of egg yolks. 116 Fig.4.13. Effect of Tween-80 and Tween-20 at two concentration of 5% & 2.5% on the absorption of IgY into the rainbow trout bloodstream. 122 Fig.4.14. Effect o f Mega9, sodium pyrophosphate, sodium bicarbonate, 5% Tween-20 or some combinations of them on the absorption of IgY into the rainbow trout bloodstream. 125 Fig.4.15. Microscopic photographs of H & E stained cross section o f the intestinal tissues of a rainbow trout intubated with Mega9 and W S F (in experiment 6). 153 Fig.4.16. Microscopic photographs o f H & E stained cross section o f the intestinal tissues of an untreated rainbow trout (in experiment 6). 154 Fig.4.17. Microscopic photograph of H & E stained cross section of the intestinal tissues of a rainbow trout intubated with Mega9 and antacid. The dissected tissue was fixed in Bouin's solution. 155 Fig.4.18. Microscopic photograph of H & E stained cross section of the intestinal tissues of a rainbow trout intubated with deoxycholate and antacid. The dissected tissue was fixed in Bouin 's solution. 156 Fig.4.19. Microscopic photograph of H & E stained cross section of the intestinal tissues of a rainbow trout intubated with octyl-(3-glucoside and antacid. The dissected tissue was fixed in Bouin 's solution. 157 xn Fig.4.20. Microscopic photograph of H & E stained cross section o f the intestinal tissues of an untreated control rainbow trout. The dissected texture was fixed in Bouin 's solution. 158 Fig.7.1. Temporal trend of mortality rate in relation with total serum IgY level in the fish groups IP injected with anti-K anguillarum IgY. 179 Fig.7.2. Temporal trend of mortality rate in relation with anti-K anguillarum IgY titer in serum of fish following IP injection with anti-VibriolgY. 180 Fig.7.3. Temporal trend of anti-V. anguillarum IgY serum titer fluctuations in relation with total serum IgY level in LP injected fish with anti-Vibrio IgY. 181 Fig.7.4. Microscopic photograph of H & E stained cross section of the stomach tissues of a rainbow trout intubated with Mega9 and W S F (in experiment 6) sampled 7 days after intubation (at the day of challenge) and fixed in buffered formalin. 182 Fig.7.5. Microscopic photograph of H & E stained cross section of the pyloric caeca tissues o f a rainbow trout intubated with Mega9 and W S F (in experiment 6) sampled 7 days after intubation (at the day of challenge) and fixed in buffered formalin. 183 Fig.7.6. Microscopic photograph o f H & E stained cross section o f the intestinal tissues of a rainbow trout intubated with Mega9, antacid and W S F (in experiment 4) sampled 3 hours after intubation and fixed in buffered formalin. 184 Fig.7.7. Microscopic photograph of H & E stained cross section of the intestinal tissues of a rainbow trout intubated with deoxycholate, antacid and W S F (in experiment 4) sampled 3 hours after intubation and fixed in buffered formalin. 185 Fig.7.8. Microscopic photograph of H & E stained cross section of the intestinal tissues of a rainbow trout intubated with octyl-p-glucoside, antacid and W S F (in experiment 4) sampled 3 hours after intubation and fixed in buffered formalin. 186 Fig.7.9. Microscopic photograph of H & E stained cross section of the intestinal tissues of a rainbow trout intubated with P B S (in experiment 4) sampled 3 hours after intubation and fixed in buffered formalin. 187 x i i i LIST OF ABBREVIATIONS °c Degree(s) Celsius Kg Microgram(s) u L Microliter(s) abWSF Water-soluble fraction of egg yolks from vaccinated hens after absorption of antigen-specific IgY by formalin killed V. anguillarum A D A i r drying A N O V A Analysis of variance A P Alkaline-phosphatase B C I P 5-bromo-4-chloro-3-indolyl phosphate b G H Bovine growth hormone B S A Bovine serum albumin C Control treatment cc Cubic centimeter ccw Cheddar cheese whey cfu Colony forming units d Day db Dry base D C M Dichloromethane D E A Diethanolamine D X Sodium salt of deoxycholic acid E L I S A Enzyme-linked immunosorbent assay F D Freeze-drying Fig. Figure g Gram(s) GI tract Gastrointestinal tract h Hour(s) H & E Haemotoxylin and eosin h G G Human gamma globulin h G H Human growth hormone H R P Horseradish peroxidase IgG Immunoglobulin G I g M Immunoglobulin M IgY Egg yolk immunoglobulins M W Intermediate molecular weight I N Intubated IP Intraperitoneal kDa Kilodalton(s) kg Kilogram(s) L Liter(s) L M M L o w molecular weight protein standard marker xiv L P S Lipopolysaccharide L - W S F Lyophilized water-soluble fraction of egg yolks M Molar M 9 Mega9 mg Milligram(s) min Minute(s) m L Milliliter(s) m M Mil l imolar mmHg Millimeter of mercury M W Molecular weight N A Not available N B T Nitro blue tetrazolium N C Nitrocellulose ng Nanogram(s) nm Nanometer(s) N M W Nominal molecular weight N M W C Nominal molecular weight cut off nspWSF Non-specific water-soluble fraction of egg yolks from unvaccinated hens O B G Octyl-13-glucoside P + Top-dressed pellets P B S Phosphate buffered saline P K Proteinase-K P L G 50:50 Poly -lactide-co-glycolide with a lactide to glycolide ratio of 50:50 P L G 85:15 Poly -lactide-co-glycolide with a lactide to glycolide ratio of 85:15 P L G A Antigen containing P L G / 7 - N P P Alkaline p-nitrophenyl phosphate P V A Polyvinyl alcohol P Y Sodium pyrophosphate R I D Radial immuno-diffusion assay rpm Rotation per minute S B C Sodium bi-carbonate SDS Sodium-dodecyle sulfate S D S - P A G E Sodium-dodecyle sulfate-polyacrylamide gel electrophoresis S F U Simon Fraser University s G H Salmon growth hormone spWSF Specific water-soluble fraction of egg yolks from vaccinated hens T Test treatment T20 Tween-20 T80 Tween-80 T B S Tris buffered saline T N P - L P S Trinitrophenilated lipopolysaccharide T S A Tryptic Soy agar T S B Tryptic Soy broth T T B S T B S including Tween-20 U B C University of Brithish Columbia U F Ultrafiltration X V v/v Volume per volume V M Vacuum microwave V M D Vacuum microwave drying w/o Water in oi l w/o/w Water in oi l in water W C L Whole cell lysate W S F Water-soluble fraction of egg yolks ACKNOWLEDGMENTS I would like to offer my great appreciation to Dr. Timothy D . Durance, my thesis supervisor, for his invaluable guidance, encouragement and support throughout the course of study. I also wish to express my best thanks to other members of my advisory committee, Dr. Shuryo Nakai, Dr. Eunice Li -Chan and Dr. Lawrence Albright for their wise suggestions and kind and caring criticism which lighted my way through difficulties of research. I extend my thanks to Dr. A lex Yous i f for his technical consultation and guidance, to M r . Abdol-Hossein Amin i , Mrs. Angela Kummer, M r . Guillaume D a and Mrs. Parastoo Yaghmaee for their assistance, to Mr . Sherman Yee and M s . Valerie Skura for their continuous technical support, to Dr. Brent Skura, the graduate advisor for his consistent support and generous guidance in all occasions, Dr. Emanuel Aki ta for scientific advice, to M s . Joyce Tom and M s . Jeannette Law, the secretaries of the department, to M r . Michael Igallo and Ms . Julie Chow in the Histology Lab, Department of Pathology, to Dr. Helen Burt in the Department of Pharmaceutical Sciences and her lab manager, Dr. John Jackson for providing access to their laboratory facilities and offering me technical advice. M y special thanks go to all o f my friends and colleagues for their help and support. The financial support of University Graduate Fellowship (1996-1998) is also gratefully acknowledged. xv i i DEDICATIONS I dedicate this thesis to my dearest parents. I owe all and every success and happiness I have ever achieved to their unconditional love and sincere care and support o f all kinds, which has always lit up my life on ups and downs. And , to my brothers and their families whose love and emotional support have always brought me hope and strength. xv i i i CHAPTER ONE INTRODUCTION 1.1. Background of the study Control of fish diseases is of great concern in aquaculture because of the large population of fish and the high risk of disease transmission in an aqueous environment (Dunn et al., 1990). Vibrio anguillarum is one of the most important pathogens of marine and fresh water fish, causing up to 40% mortality in severe cases, with an annual loss of 11 mill ion British pounds due to the disease in Japan alone in the 1980's (Smith, 1988). Currently, antibiotics are widely used to treat infectious diseases in fish farming, although consumption o f their residues is not desirable for the final consumers, humans. Recently, environmental concerns, regulatory constraints, cost, and pathogen resistance have greatly diminished the appeal of antibiotic use and other chemotherapeutical methods in aquaculture (Palm Jr. et al., 1998). The alternative practice used to control disease outbreaks is vaccination, which in most common operations is administered by injection to each individual fish or by immersion in vaccine solution. Although commercial vaccination is effective and well established it involves considerable handling and is stressful to the fish (Smith, 1988). On the other hand, the availability of large amounts of relatively inexpensive immunoglobulins from egg yolk (IgY) offers the possibility o f using specific IgY for passive immunization by oral administration. Oral use of immunoglobulins is environmentally friendly and unlike antibiotics, elicits no side effects, disease resistance, possibility o f overdosing or occurrence of toxic residues, and there is no injection or handling involved. The efficacy o f IgY treatment to provide passive immunity against diseases has been investigated by several researchers. I gY has proven effective in protection against Escherichia coli infections in pigs (Yokoyama et 2 al, 1992; Marquardt, 1999) and in rabbits (O'Farrelly et al, 1992). In aquatic organisms, when anti-Edwardsiella tarda IgY and the disease causing bacteria were simultaneously administered orally to Japanese eels (Gutierrez et al, 1993; Hatta et al, 1993) a decrease in mortality and absence of the disease symptoms were observed in the challenge studies. Lee et al. (2000) reported a marginal reduction in mortality and intestinal infection caused by Yersinia ruckeri in rainbow trout when fed anti-7. ruckeri IgY either before or after immersion infection. However, intraperitoneal (LP) injection of the same IgY 4 hours before an immersion challenge presented a passive protection. Although uptake o f intact protein antigens through the skin o f bath-immunized rainbow trout has been confirmed (Ototake et al, 1996), passive bath-immunization does not seem either advantageous over passive oral immunization due to the stress to fish and need o f handling or more effective than bath-vaccination as indicated by a shorter immunization period conferred following administration. IgY is also more appealing for passive immunization than immunoglobulins from other animal sources with respect to reduced stress on the host animal, since no bleeding is required. Simple and industrially feasible methods o f IgY extraction from the egg yolk have already been developed. However, large-scale commercial use of IgY requires dehydration methods that confer minimal loss of activity. Vacuum microwave dehydration, a new drying technique, may provide a cost-effective method, to save antigenic properties of IgY. 3 1.2. Hypothesis 1 . Oral administration of specific chicken egg IgY raised against V. anguillarum w i l l decrease mortality level in rainbow trout challenged with this pathogenic bacteria, provided that IgY survives adverse effects of acidic conditions and digestive enzymes of the fish stomach. 2. Dehydration of IgY using freeze-drying, vacuum microwave drying or air-drying w i l l not destroy its immunogenicity. 3. Co-application of detergents w i l l enhance the uptake of orally administered IgY into the fish blood stream. 1.3. Objectives of the study The main objective of this study was to find an effective means of using chicken egg yolk immunoglobulin, IgY, specifically raised against certain disease causing organisms, to increase disease resistance of farm fish. The most practical approach seemed to be oral administration of IgY, with the immune enhancing materials added to the feed. Methods were sought to minimize destruction of the IgY molecule in the fish alimentary tract and maximize its absorption through the intestinal epithelia for uptake into the blood stream. Challenge studies using the causative organism was used to test the effectiveness of the delivery of the biologically active molecules, i.e. chicken IgY. In this study, Vibrio anguillarum served as model bacteria for causing diseases to fish. This approach, i f proven successful, could be modified and adapted for other fish diseases. 4 Other objectives of this research include: • To study antigen-antibody binding between V. anguillarum and chicken egg yolk IgY, in comparison with the binding of other immunoglobulins such as rabbit IgG and fish IgM. • To determine the best method of drying IgY for addition to fish feed. • To postulate a theory to explain how detergents may enhance absorption of protein molecules such as IgY through the fish gut into the circulating blood. 5 CHAPTER TWO Review of Related Literature 2.1. Vibriosis and other fish diseases Vibriosis is a bacterial disease of many salt-water fish including salmonids, the severity of which has increased with the expansion of fish farming. The most significant losses occur in cultured Pacific salmon, Atlantic salmon {Salmo salar) and rainbow trout (Oncorhynchus mykiss) grown in fresh water farms or in marine cage farms. Cultured non-salmonid fish including eel, yellow tail and red sea bream can also be affected (Ezura et al, 1980). In severe cases of vibriosis, mortality may be up to 40% of affected population. However, several vaccines have been developed and proven effective, especially when delivered by injection or immersion methods. The most commonly encountered fish pathogenic Vibrio species is V. anguillarum. It constitutes part of the normal microflora of the aquatic environment. The next most important Vibrio species is V. ordalii (Smith, 1988). Signs of vibriosis include hemorrhaging at the base of the fins, around the vent and gills and inside the mouth. Petechiae, necrotic lesions and diffuse hemorrhages can appear on the body surfaces. Internally, the intestine is often inflamed with some petechiae. It may be distended and filled with clear viscous fluid. V. anguillarum enters the fish by penetrating the descending intestine and rectum (Ransom et al, 1984). Vibrio bacteria can survive in the slime of uncleaned tanks, nets, build up of feces and unused feed. Such deposits can act as a reservoir for infection (Smith, 1988). 2.2. Antigenicity of V. anguillarum and portals of entry The major antigen o f V. anguillarum is a heat-stable (100-121°C), large molecular weight ( M W about 100 kDa) lipopolysaccharide (LPS) in the cell wall. 7 This L P S may not fully degrade in the acidic environment of the stomach (Kawai & Kusuda, 1983; Smith, 1988; Wong et al., 1992). Chart and Trust (1984) isolated two other minor heat labile proteins from the outer membrane having M W s of 49-51 kDa. Boesen et al. (1997) detected humoral antibody in fish injected with either of the extracellular products: cytoplasmic membrane proteins or outer membrane proteins with the latter eliciting the strongest reaction. They postulated the existence of undefined potent antigens among these proteins. Sites of entry of Vibr io include anal and oral routes, as well as gills and skin (Evelyn, 1984; Laurencin & Germon, 1987). However, kidneys, spleen, liver and lamina epithelia of the lower intestine may be infected as disease progresses (Nelson et ai, 1985). Although some reports emphasize the importance of posterior intestine to induce a sufficient immune response (Rombout & V a n den Berg, 1989), O'Donnell et al. (1994) have demonstrated uptake of L P S in the anterior part of the gut in brown trout and suggested that it can induce a systemic immune response. 2.3. Vaccination Commercial Vibrio vaccines are inactivated cultures containing a mixture of whole cells and extracellular products of most commonly encountered species (Smith, 1988). Methods of vaccination include injection, oral administration and immersion. 2.3.1. Intraperitoneal (D?) injection The most effective method of immunizing fish is intraperitoneal (IP) injection. This technique allows the use of adjuvants, which enhance the magnitude of the 8 immune response. Injection ensures that each fish receives the exact dose of vaccine. However, it is very labor intensive and stressful to the fish since it requires anesthetization and handling. This method is not considered practical for fish with weights less than 15g (Ellis, 1988a; Smith, 1988). 2.3.2. Immersion vaccination Immersion vaccination consists of two methods, namely dip and bath vaccinations. Dip vaccination involves dilution of vaccine in a water container, removal of fish from the holding facility, immersion in vaccine solution for about 30 seconds and the return of the fish to the holding facility. The main portal of vaccine in this method is the gill tissue. In the bath method, vaccine is added directly into the holding tanks, thereby handling and stress to the fish is minimized. This technique is not stressful; however, it consumes more vaccine and since the vaccine solution is more dilute than in the former method, it requires a longer exposure time, approximately 1-2 hours, and requires oxygenation of the water. Immersion methods permit mass vaccination of the fish below 5g (Ellis, 1988a; Home & Ell is , 1988). 2.3.3. Oral vaccination Oral vaccination, which involves the incorporation of antigen into the feed over a suitable time course, is the only method economically suited to extensive aquaculture. This method offers a significant advantage for it reduces labor cost, is time saving, involves no handling and therefore limits stress to the fish, decreases the possibility for cross contamination with needles, does not require disposal of treatment 9 water and allows mass vaccination of fish of any size. However, oral vaccination requires larger doses of vaccine than the injection or immersion methods to achieve protection. Further disadvantages are poor stability o f the antigen in the digestive system and lack of control on the dosage for each fish, which is dependent on the feeding rate (McLean et al., 1999; Home & Ell is , 1988; Hart et al, 1988). Research is still continuing to optimize oral vaccination conditions and increase the potency of this method. 2.4. Antibodies as a replacement for antibiotics Antibiotics are commonly used in treatment of infectious diseases of humans and animals. However, with long-term use and/ or insufficient dosage of antibiotics, bacteria may mutate and exhibit resistance against the antibiotic. This may result in selection of resistant microbial strains. Antibiotic residues in the fish muscle may enter human food, another undesirable consequence of antibiotic use (Coleman, 1999; Siwicki et al., 1989). In total, environmental concerns, regulatory constraints, cost, and pathogen resistance have greatly diminished the appeal o f antibiotic use and other chemotherapeutical methods in aquaculture (Palm Jr. et al, 1998). Antibodies, on the other hand, inhibit pathogens by forming an antibody-antigen complex that inactivates the binding sites of bacteria to the host's cells. They produce no toxic metabolites and have few side effects. There is no possibility o f overdosing with the continuous use of antibodies (Coleman, 1999). The use of antibodies is not restricted to bacterial and fungal diseases but also has application to viral infection and neutralization of venom 10 (Hatta et al, 1997). A l l these factors suggest antibodies may have the potential to partially replace antibiotics in the future. 2.5. Passive immunizat ion Passive immunization involves use of an antibody raised in other animals to provide extended disease resistance to the target animals or humans. This approach has been investigated as an alternative to vaccination for prophylactic purposes, and to antibiotic therapy for therapeutic effects (Carlander et al, 1999; Bartz et al, 1980; Ebina et al, 1990; Marquardt, 1999; Hatta et al, 1993b; Yokoyama et al, 1992; Gutierrez et al, 1994). In fish, especially with respect to certain species, passive immunization may be considered as a potential alternative to vaccination (Hatta et al, 1997). For instance, salmonids do not develop a very effective level of immunity when vaccinated below 0.5-lg of weight and do not develop prolonged immune memory until approximately 4g (Ellis, 1988b). In another application, specific antibodies could be vertically transferred from injected female broodstock to salmonid eggs and embryos, although conferred protection is not maintained for long after the yolk sac is absorbed (Brown et al, 1997). Oral or systemic administration of specific immunoglobulins to certain antigens (bacteria, virus, venom, toxin, etc.) may be applied to neutralize biological activities o f the antigens. . However, administration of large amounts of antibody may be required in passive immunization (Hatta et al, 1997). Akhlaghi (1999) passively immunized rainbow trout using anti-K anguillarum antibodies raised in sheep, rabbits or rainbow trout via intraperitoneal (LP) or oral routes. IP injected trout with the immune serum o f all three species showed a 11 persistent and significant protective immunity against vibriosis in a challenge with the pathogenic bacteria up to a month post-injection. However, protection offered by trout anti-Vibrio serum was weaker than that of the others. The protection rate of passive immunization declined markedly after a month and was minimal after 3 months, while immersion vaccination with formalin killed V. anguillarum provided a high level o f protection even after 3 months. Oral passive immunization using purified sheep anti- V. anguillarum serum alone or when conjugated with E. coli heat labile toxin or when co-delivered with Q u i l - A saponin did not confer a significantly higher protection in treated trout than the untreated control group in challenge 15 and 30 days post immunization. The author concluded that BP passive immunization could offer an effective protection in trout against vibriosis, although the protection was not as prolonged as that provided by active vaccination. It was also indicated that the oral passive immunization might be a feasible approach but needs improvement in absorption of antibodies from the gut as no mix-Vibrio antibodies were detected in the sera of fish following oral administration. 2.6. Use of chicken egg yolk antibodies (IgY) in passive immunization The yolk of hens eggs is a valuable source of specific antibodies, which can be used in prophylactic or therapeutic treatments to confer passive immunity (Li-Chan, 1999). The effectiveness of oral administration of IgY in prevention of dental caries in rats (Hamada et al, 1991), rotaviral diarrhea in humans (Yolken et al., 1988) and rodents (Bartz et al, 1980; Ebina et al, 1990; Hatta et al., 1993b), and enterotoxigenic E. coli infection in piglets (Yokoyama et al., 1992) has been reported. Carlander et al. 12 (1999) successfully applied gargling of pathogen specific IgY to prevent chronic Pseudomonas aeroginosa colonization in the airways o f the patients with cystic fibriosis. IgY has also proven effective in promoting disease resistance in fish. Gutierrez et al. (1994) reported that mortality due to a natural infection by Edwardsiella tarda decreased when the eel feed was mixed with 1-3% whole egg powder obtained from chickens vaccinated against E. tarda. In another study, a mixture of anti- E. tarda IgY and the pathogen was cannulated into the stomach of Japanese eels after their intestinal mucosa was damaged using hydrogen peroxide solution (30%>). IgY treated eels survived while the control eels, which were infected with the same concentration of bacteria without adding IgY, died or showed the symptoms of the disease (Hatta, et al., 1994). Decreased intestinal necrosis due to Vibrio species in Japanese flounder larvae and a decrease in signs of vibriosis in Japanese shrimp when the animals diets contained anti-Vibrio IgY have also been reported (Gutierrez et al., 1994). Mine et al (1999) demonstrated that feeding encapsulated specific anti-Yersinia ruckeri IgY to rainbow trout either before or after an immersion infection could only produce a marginal reduction in mortality and intestinal infection. This same IgY passively protected rainbow trout against infection when it was administered by intraperitoneal (IP) injection four hours before an immersion challenge. 13 2.7. Raising specific antibodies in chickens High titers of specific antibodies can be obtained in egg yolk immunoglobulin (IgY) by immunization of laying hens against target antigens. Li-Chan (1999) reported that initial immunization by injection of bovine IgG (0.5-lmg mL"1 emulsion containing Freund's complete adjuvant) into the pectoralis muscle followed by two to four boosters of the antigen in incomplete adjuvant could maintain production of high titers of specific antibodies, which appear 5-6 weeks after the initial immunization and continue for up to a year. Hatta et al. (1997) vaccinated hens against E. tarda and boosted them weekly for four weeks with additional boosting at weeks 18, 30 and 34. The authors suggested that a shorter period of boosting would help maintain a high titer. Also, Poison et al. (1980a) vaccinated 20 weeks old hens with different protein antigens. Three to several weekly boosters, depending on the antigenicity of each specific antigen, followed the initial injection to elicit antibodies in adequate titer in the egg yolk. It was concluded that antigens with a molecular weight (MW) of equal or greater than human IgG (MW 150kDa) could elicit strong antigenic response in hens whereas lower M W antigens were considered poor antigens. 2.8. Advantage of chicken IgY over other animal sources Serum antibodies of hens are efficiently transferred and accumulated in the egg yolk. Yolk immunoglobulin (IgY) is therefore equivalent to the chicken's serum IgG, although the content is much higher in the yolk than in the hen's sera (Rose et al, 1974). Specific antibodies can also be prepared from sera or colostrum of immunized rabbits, goats, sheep and horses (Fichtali et al., 1993). However, chicken IgY has 14 advantages over other immunoglobulins in terms of yield, ease of collection and reduced stress on the host animal. Production of eggs with a high antibody titer is continuous throughout the year, whereas colostrum is only produced at parturition, and at other times, antibody content of milk is minimal. Specific antibody titer as high as l x l O 1 5 units per m L has been reported in immunized hens (Coleman, 1999). Approximately 3-4g of IgY could be obtained per laying hen each month, when immunized against bovine serum IgGi , IgG2 and IgG isolated from cheddar cheese whey. This corresponds to an annual yield of 40-50g IgY per hen (Akita & Li-Chan, 1998; Li-Chan, 1999), about 30 times the IgG produced in the serum of an immunized rabbit (Hatta et al, 1997), which must be sacrificed to collect the IgG. O f course IgY antibodies, like other immunoglobulins collected from other host animals, are polyclonal and contain antibodies specific to a variety of antigens to which the hen has been exposed. Levels o f antibody in IgY preparation have been reported to be 5% for anti-insulin antibody and 28% for anti-mouse IgG antibody (Hatta et al, 1997) or 10-15% of the total I gY when bovine IgG and lactoferrin were employed as antigens (Akita and Li-Chan, 1998; Li -Chan et al, 1998). There are other advantages that make IgY more favorable than mammalian IgG. Production of IgY does not involve bleeding and thus causes no harm to the animal and is compliant with animal welfare considerations (Larsson et al, 1993). It is cost effective and convenient (Poison et al, 1980b) and is easy to produce and maintain high levels o f antibody in eggs (Rose et al, 1974). 15 2.9. Stability of IgY and differences with IgG IgY is distinct from mammalian IgG in some respects. IgY has a slightly higher molecular weight of 180 kDa as compared with 150 kDa for IgG (Hatta et al, 1997). It also has a lower isoelectric point by one pH unit compared with the human IgG with a pi at pH 6.8 (Poison et al., 1980a) and confers some differences in structure (Shimizu et al., 1992). IgY neither binds rheumatoid factor in blood, which is a marker for inflammatory response (Larsson and Sjoquist, 1988) nor binds Staphylococcus protein A (Kronvall et al, 1974). However, antigen-binding capacity of IgY appears to be as strong as that of IgG (Warr et al., 1995). IgY is a reasonably heat-stable molecule, which retains most of its antibody activity after heating up to 70°C for 15 minutes (Shimizu etal, 1988). Denaturation endotherm (Tmax) of IgY has been reported to be 73.9°C and that of rabbit IgG as 77.0°C (Hatta et al, 1997). Shimizu et al (1988) showed that IgY was fairly stable at pH 4 or above, but lost the activity very rapidly below pH 4. The neutralization activities of IgY and Fab' fragments were partially retained even after 4 hours incubation at pH 2 and 37°C (Akita et al, 1998). Hatta et al (1993a) reported that the activity of anti-human rotavirus IgY determined by ELISA was completely lost at pH 2 after incubation for 4 hours at 37°C. At pH 3, loss of activity determined by ELISA was 80%, while it was only 30% at the same conditions when determined as neutralizing titer. Otani et al. (1991) found no residual IgY activity at pH 2 after incubation for 1 hour at 40°C, whereas both IgY and rabbit IgG were stable at pH range of 9.0 to 4.0. In general, IgY is reported to be more susceptible to acidic conditions (pH 2 and 3) than rabbit IgG (Otani et al, 1991; Hatta et al, 1993a). 16 Inactivation in low p H is attributed to conformational changes since no breakdown of polypeptide was observed on S D S - P A G E (Shimizu et al., 1988). However, these changes might not be severe enough to completely destroy the functional properties of the IgY molecule as an antibody (Li-Chan, 1999). IgY is fairly stable when exposed to trypsin or chymotrypsin but more sensitive than IgG to pepsin digestion at p H levels lower than 4. IgY activity was lost on incubation with pepsin at p H 2 after 15 minutes at 40°C but partially retained at p H 4, however it was quickly decreased until completely diminished after 120 minutes at p H 4 (Otani et al., 1991). IgY molecules incubated with pepsin at p H 2 were hydrolyzed into small peptides and no band corresponding to IgY was detected after one hour. On the contrary, at p H 4 heavy and light chains were clearly observed after 4 hours, although some new bands appeared between these two bands (Hatta et al., 1993b). Concentration, lyophilization and ultrafiltration have been reported to promote denaturation, aggregation or precipitation of antibodies, including IgY (Li-Chan, 1999; Aki ta and Li-Chan, 1998; Draber et al, 1995; M c C u e et al, 1988). Shimizu et al. (1988) reported that freezing and freeze-drying did not affect the activity of IgY unless it was repeated several times. In contrast, Chansarkar (1998) demonstrated that some loss of antibody activity might appear when IgY was frozen or freeze-dried at a concentration of 30mg mL^or especially l m g mL" 1 . Samples with a high protein concentration of 30mg mL" 1 underwent a drastic insolubilization, especially after freezing or freeze-drying. Insolubilization was reversible when the sample was diluted to lmg mL" 1 . However, proteins may suffer loss of activity during freeze-drying as a result of conformational changes, aggregation or adsorption. Careful 17 attention should be paid to process and formulation details which may lead to freezing and drying stresses (Li-Chan, 1999). 2.10. Methods of IgY preparation Several methods have been used to separate water-soluble proteins, including IgY, from the water-insoluble lipids and lipoproteins of the egg yolk. These methods, as reviewed by Akita & Nakai (1993) and Hatta et al. (1997), include extraction with organic solvents, precipitation with ethylene glycol, sodium dextran sulfate, polyacryl acid resins and alginate, carageenan or xanthan gum. Among all, the method reported by Ak i ta & Nakai (1992) and Fichtali et al. (1992) appears to be the simplest. This method involved 10-fold dilution of egg yolk with distilled water, acidified with 0.1 N HC1 to a final p H of 5.2. Centrifugation of this preparation following overnight sedimentation provided a water-soluble fraction (WSF) containing IgY at 1 5% purity (protein basis), which could be used as a semi-pure source of IgY or might be submitted to further purification steps. Lower levels of dilution, 4, 6, and 8 fold, were also examined. However, after overnight incubation, samples diluted 10 times or over resulted in a relatively clear supernatant with only slight lipid contamination. Extremes of pH, below 4.2 and above 9.0 prevented settling of egg yolk granules. The WSF was almost devoid of lipids in p H levels of 4.6 to 5.2 while the maximum recovery of IgY was obtained in the range of pH 5.0 to 5.2. Besides IgY, the other major proteins present in the WSF include a and (3-livetins and low-density lipoproteins (Akita & Nakai, 1992). 18 2.11. Protein absorption in the digestive system For a prolonged oral passive immunization o f fish, it is necessary that IgY reach the bloodstream via intestinal absorption. A variety of studies, both in fish and higher vertebrates, have demonstrated that the gut mucosal barrier does not completely prevent ingestion of micro particles and macromolecules and that the fish gastrointestinal (GI) tract, in particular, retains the capacity to absorb intact proteins and peptides (McLean et al, 1999). Scientific observations include the absorption of horseradish peroxidase (HRP) into the blood of rainbow trout intubated through oral and anal routes (McLean et al, 1999), transfer o f bovine growth hormone in a functional form through the GI tract of trout (Le Bai l et al, 1989), appearance of orally intubated bovine serum albumin in the serum of chum salmon (Fujino & Nagai, 1988) and transport of rabbit IgG into the circulation o f goldfish while preserving antigen binding activity (Nakamura et al, 1990). Sire and Vernier (1992) suggested that the posterior gut of teleost fish facilitates the absorption and digestion of protein arriving there. Macromolecules are reportedly absorbed from the gut lumen and transported into the bloodstream by transcellular (intracellular) mechanisms in which macromolecules enter the enterocytes via pinocytosis by the microvillus membrane. Intact molecules that remain after digestion reach the intercellular space or eventually the lamina propria by exocytosis. Other potential routes for macromolecule uptake include paracellular (intercellular) or transjunctional pathways. During this process, microparticles and associated solutes may be forced between entrocytes due to friction caused by peristalsis or rhythmic action of the circulation and this may help direct passage into 19 the lamina propria (O'Haggen, 1996; Walker, 1986; McLean & Ash, 1987a; McLean & Donaldson, 1990). In Fig.2.1 transcellular and paracellular modes of absorption are illustrated. Fig.2.2 demonstrates how particulate matters move from the gut into the bloodstream of mammals by way of the extrusion zones of intestinal v i l l i . This process may be accompanied by the passage o f bioactive proteins from the intestinal lumen. Although this phenomenon has not been demonstrated for the fish gut, it could represent a possible pathway for proteins (McLean & Donaldson, 1990). On the other hand, the cell sloughing process results in the formation of natural apertures through which lumenal contents may pass. Likewise, lesions o f the gut surface caused by dietary components such as shells, bones, scales, grits, etc. or as a result of parasitic infections, pollutants, toxins or due to malnutrition can form a portal for the absorption of macromolecules (McLean et al, 1999). 2.12. Time course and efficacy of macro molecule transfer into the blood circulation Various studies have been conducted to determine absorption efficacy and dose response of protein absorption from the GI tract of fish. In these studies, the time course and levels o f uptake have been explored. Oral and anal delivery o f human gamma globulin ( H G G ) resulted in the rapid transfer of antigen to the vicinity of capillaries and its subsequent passage to the systemic circulation and to the major body organs (Jenkins et al., 1991). Orally intubated human growth hormone (hGH) in carp reached maximum levels in the serum after 30 minutes and then gradually 20 Intracellular Fig.2.1. Schematic mechanism of macromolecules intestinal uptake and transfer. Intracellular (transcellular) pathway: Following adsorption and endocytosis of macromolecules by the microvilli of the intestinal epithelium, they are transported in phagosomes. When lysosomes merge, intracellular digestion occurs by hydrolysis process in the secondary lysosomes. The intact molecules that survive hydrolysis are transferred into the intercellular space by exocytosis. Intercellular (paracellular) pathway: Macromolecules may be forced into the intercellular space due to friction caused by peristalsis or rhythmic action of the circulatory system. (Adapted from Walker & Isselbacher, 1974, with modifications.) 21 Intestinal lumen Lamina propria Fig.2.2. Passage of particulate materials from the intestinal lumen into the bloodstream. Bioactive proteins may accompany them in the passage to the lamina propria through the intercellular spaces of intestinal vi l l i and eventually reach the bloodstream of mammals. (Adapted from Volkheimer, 1972, cited in McLean & Donaldson, 1990, with modification.) 22 decreased (Hertz et al, 1991). H R P appeared at a detectable level in serum 15 minutes after oral intubation in carp and peaked at 30 minutes (McLean & Ash, 1986). Direct administration of rabbit IgG into the gut of rainbow trout resulted in a significant level of absorption after 3 hours (Fujino et al, 1987). There are several other indications of transfer of proteins into the bloodstream of teleosts. Fig.2.3 (adapted from McLean & Donaldson, 1990) shows the time course of appearance of various bioactive proteins in the serum of orally and anally intubated fish. With respect to the efficacy of macromolecule uptake, Georgopoulou et al (1988) estimated up to 6% absorption of tracer protein in a substantially intact form into the blood circulation of rainbow trout. The same scientists reduced the estimate to 1% in a later work (Sire and Vernier, 1992). The latter value is closer to 0.07% net absorption of H R P which was detectable in an immunologically and enzymatically active form in rainbow trout (McLean et al, 1999). However, fish gut exhibits a great variation in macromolecule absorption between species and even between individuals of the same species (Smith, 1989; McLean et al, 1999). After reaching the blood circulation, proteins such as H R P are taken up by organs including kidneys, liver and spleen. H R P was found in a greater concentration in kidney, liver and plasma following anal intubation as compared to oral administration (McLean et al, 1999). However, Jenkins and colleagues (1994) demonstrated similar plasma absorption levels in both anal and oral administration of H G G to tilapia, when the protein was delivered without adjuvant or co-delivered with aluminum hydroxide. 23 O i I I I l i n — i — • 15 30 45 60 75 90 480 1440 Time (min) Fig.2.3. The time course of appearance of bioactive proteins within the serum of orally and anally intubated fish (mg or ng of protein in mL of serum). (1) Doggett et al. (unpublished data): horseraddish peroxidase (HRP) subsequent to oral administration to Mozambique tilapia (200ug g"1 body weight in O.lmL of 0.9% saline). (2) McLean & Ash (1986): HRP in common carp after oral introduction of 200ug g"1 in l m L of 0.9% saline. (3) Suzuki et al. (1988): Salmon gonadotropin in goldfish subjected to oral intubation of a crude piruitary homogenate containing 691ug gonadotropin ml' 1 . (4) Ash (1985) and McLean & Ash (unpublished data): HRP in rainbow trout after anal administration of lOOug g"1 in l m L of 0.9% saline. (5) McLean (1987): Human cationic trypsin (HCT) in common carp after oral delivery of 4ng g"1 in l m L of 0.9% saline. (6) McLean & Ash (unpublished data): HRP in goldfish after oral administration of 800ug g' 1 in 0.25mL of 0.9% saline. (7) Georgopoulou et a/.(1988): HRP in rainbow trout after oral administration of lOOug g"1 in l m L of isotonic saline. (8) McLean & Ash (1987b): HRP in rainbow trout after oral administration of lOOug g"1 in l m L of 0.9% saline. (9) McLean and colleagues (unpublished data): gonadotropin in young-of-the-year chinook salmon after oral administration of lOug g"1 body weight. (10) McLean and colleagues (unpublished data): recombinant bovine somatotropin in coho salmon parr after oral administration of 20ug g"1 in lmL of 0.9% saline. (From McLean & Donaldson, 1990) 24 There is evidence that uptake of intact proteins is a dose-dependent process. A relatively linear dose response was observed in the serum uptake of h G H when 0.1, 0.5 or 1 mg kg"1 body weight of it was orally delivered in 0 .05M deoxycholate solution into starved carp (Hertz et al, 1991). McLean and co-authors (1999) reported unpublished data from McLean & Ash demonstrating a relatively linear dose-dependence in plasma level of H R P following anal intubation in rainbow trout. This linear pattern was also observed between the administered dose and the accumulated H R P in liver, kidney and spleen following anal intubation as well as in the liver and spleen after oral intubation. In this study, a clear dose-dependence did not appear in the kidney or plasma levels following oral intubation. However, Georgopoulou and colleagues (1988) found a direct correlation between dose of intubated H R P at the pyloric curve o f the stomach and the quantities transferred into the plasma after 8 and 16 hours. These findings illustrate 1. potential of the fish gut to absorb proteins at a specific dose, 2. extent of natural variation between individual fish in absorption of macromolecules (McLean et al, 1999). 2.13. Protection from degradation in the GI tract Most gastrointestinally absorbed proteins and peptides are believed to be hydrolyzed at the brush border or by cytoplasmic enzymes (Sire and Vernier, 1992). However, various bioactive proteins and peptides retain physiological activity when administrated orally. Nakamura and co-authors (1990) suggested that permeability of the fish gut could be employed as a means of passively immunizing fish. In order to accomplish this objective, absorption of the bioactive protein macromolecules into the 25 fish bloodstream has to be improved. Several methods have been studied to improve oral uptake of proteins in fish, such as encapsulation in protective coatings, co-administration with antacids or proteolytic enzyme inhibitors and co-delivery with detergents. These methods have been more extensively reviewed by El l is (1995) and McLean et al. (1999). Use of encapsulation and co-administration of absorption enhancing agents are reviewed below. 2.13.1. Encapsulation Different methods of encapsulation or coating have been employed to reduce digestive degradation of oral vaccines and improve efficacy of protein delivery into fish blood circulation. Lillehaug (1989) incorporated lyophilized V. anguillarum vaccine into a slow release pellet, "pr i l l " , or coated the vaccine with an acid-resistant film of methylacrylic acid and acrylic acid ethyl ester. However, challenge with V. anguillarum following oral vaccination showed higher mortality levels in the rainbow trout treated with protected vaccine than with an unprotected one. Wong et al. (1992) sprayed V. anguillarum vaccine onto dextrose beads followed by coating with Eudragit L-30D. Serum and mucus antibody levels were significantly higher in coho salmon orally vaccinated with the protected vaccine than the unprotected one, although challenge with live bacteria did not reveal any advantage for protected vaccine. Piganelli et al. (1994) also used dextrose beads and Eudragit L-30D enteric-coated microspheres for two different antigens, trinitrophenylated lipopolysaccharide (TNP-LPS) and a protein antigen, trinitrophenylated keyhole limpet haemocyanin ( T N P - K L H ) . T N P - L P S at the higher dose of lOpg increased serum anti-TNP 26 antibody titer of coho salmon. With the protein antigen only the lowest dose of 0.5ug induced a serum antibody response after oral vaccination. The authors suggested that the higher doses might not be optimal in eliciting an immune response. In the same study, oral vaccination was found to be as effective as injection and better than the immersion method. However, there was no control group treated with unprotected antigen. Oral vaccination of rainbow trout and carp with microencapsulated anti-K anguillarum vaccine using alginate microparticles resulted in a better uptake and a better memory formation (Joosten et al, 1997). In another study, bovine serum albumin ( B S A ) was encapsulated by cellulose acetate phthalate and orally intubated into eel. When a dose of 10-30mg B S A was administered, encapsulation increased the absorption rate by 1.7 times. However, when a low dose of 5mg unprotected B S A was applied, a level of 7.1- 34.5u.g mL" 1 B S A was detected in the serum while no B S A was detected in the eels serum following administration of protected B S A at the same dose (Nagai & Fujino, 1995). Shimizu et al (1993) encapsulated chicken egg yolk IgY in lecithin-cholesterol liposomes. No efflux of encapsulated IgY was observed when incubated in P B S at 37°C up to 72 hours. Encapsulation reduced IgY activity loss under acidic conditions and markedly increased its resistance to pepsin hydrolysis. However, no in vivo study was conducted. Poly(DL-lactide-co-glycolide) (PLG) is another type of biodegradable microcapsule used in the study of protein delivery into the fish gastrointestinal tract. Jeffery et al. (1993) studied the optimum conditions for P L G microparticle preparation using ovalbumin as a model antigen. Lavelle et al. (1997) encapsulated H G G in P L G and administrated it orally into rainbow trout. They found an increased retention time 27 in the stomach and delayed entry into the intestine when H G G was encapsulated. Detection o f antigen fragments in gut contents was attributed to proteolysis o f H G G present at the surface of the microparticles. However, about 60% of the antigen was detectable in the posterior intestine and the bloodstream of fish in an intact form, suggesting that antigen was only partially protected. Oral immunization with P L G -H G G did not result in a greater serum antibody level than in the case of unprotected H G G until after boosting with unprotected H G G . O'Donnell et al. (1996) orally intubated Atlantic salmon with P L G - H G G or free H G G . Free H G G was detected in the serum from 15 minutes to 3 days post intubation, in the posterior intestinal epithelium for up to 2 days and in the kidney for up to 7 days. In fish treated with P L G - H G G , the antigen was detectable in the serum from 15 minutes to two days with two higher peaks after 6 days and 5 weeks (Fig.2.4). H G G was observed in the posterior intestinal epithelium for up to 3 days and in the kidney for up to 5 weeks. In vitro studies showed that when a polymer ratio of 50:50 (lactide: glycolide) was used, 50%o of the antigen was released in the first 2 weeks, whereas with the ratio of 85:15 no release was detected over the entire 29 weeks of study. Aki ta & Nakai (1999) prepared enteric-coated gelatin capsules of IgY with cellulose acetate phthalate. In vitro studies revealed a complete loss of activity of unprotected IgY in p H 1.2, while the capsules were stable in simulated acidic conditions o f stomach for 3 hours, but easily dissolved under alkaline conditions (pH 8.0). 28 6 -a S3 s 6 & f J L - I J Ji JS U3 T i m e ( p o s t - o r a l i m m u n i s a t i o n ) Fig.2.4. Time-course of appearance of human gamma globulin (HGG) in the serum of Atlantic salmon following oral delivery of free or P L G (50:50) encapsulated antigen. Values are means ± standard deviations based on JV=3 for each individual time point. S Soluble H G G ; • P L G -H G G (5mg fish 1); • P L G - H G G (2.5mg fish"1). (From O'Donnell et al., 1996) 29 2.13.2. Enhancement of absorption using detergents Use of a variety of non-ionic detergents and bile salts has been investigated as a means of enhancement of protein absorption from the fish gut. Oral intubation of h G H in a solution of .0.05M deoxycholate and 0.4% N a H C 0 3 resulted in up to 1000-fold increase in serum level as compared to fish intubated with an aqueous solution of h G H . The levels of h G H in deoxycholate solution which appeared in the circulation of starved and fed carp were estimated to be at least 2% and 0.5% of the intubated dose, respectively. It was postulated that deoxycholate might increase the availability of protein molecule for absorption through formation o f complexes in which the hydrophilic end of deoxycholate is attached to the protein molecule and the lipophilic end to the gut lipid membrane (Hertz et al, 1991). Administration of Q u i l - A saponin caused a considerable increase in the uptake of orally and anally intubated human gamma globulin, H G G , in tilapia, a gastric fish. In this study, Q u i l - A saponin was observed to have a number of physical effects on the intestinal enterocytes of tilapia, such as loosening intercellular junctions, increasing the pinocytosis of luminal contents and fusion with the plasma membrane as well as direct effect on the microvilli . A l l o f these functions serve to increase the permeability of intestine to macromolecules. Qu i l -A also caused the microvill i o f the enterocytes to become shortened and damaged. Other possible modes of action was postulated to be neutralization of gastric and intestinal proteolytic enzymes, hence an increased level o f antigen reaching port of absorption in the intestine (Jenkins et al, 1991). In contrast, Akhlaghi (1999), who co-administered sheep antibodies with Qu i l -A to rainbow trout via oral route, did not detect any increase in absorption from the gut. 30 Hildreth (1982) introduced a new class of non-ionic detergents, N-D-gluco-N-methylalkanamide, as exhibiting high solubilization power and non-denaturing properties. These detergents attained all o f the desirable properties of commercially used non-denaturing detergents of the time, such as Triton X-100 and octyl-p-D-glucoside for membrane studies, while at the same time they were produced in a higher yield and lower cost. Mega-9, nonanoyl-Af-methylglucamide, is a detergent from this family. Oral co-administration of Mega-9 enhanced gastrointestinal uptake of H R P by rainbow trout. Tissue accumulation of H R P was further enhanced when a combination of Mega-9 and soybean trypsin inhibitor was used (McLean and Ash, 1990). These authors reported gelatinization of mucus lining of the GI tract and formation of mucus clumps due to Mega9. In another study, simultaneous use of Mega-9 and sodium bicarbonate enhanced the growth rate conferred by oral intubation of recombinant bovine somatotropin in coho salmon, O. kisutch (McLean et al., 1990). Many different detergents have been tested for solubilization effect on lipid bilayer membranes. C H A P S (3[(3-Cholamidopropyl)dimethylammonio]-l-propane-sulfonate) and C H A P S O (3[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-l-propanesulfonate), the nondenaturing zwitterionic detergents, and octylglucoside, the nonionic detergent, were among a few which elicited the best results (Womack et al., 1983). Helenius et al. (1979) considered P-D-octylglucoside and bile salts, such as deoxycholate and cholate, as excellent membrane solubilizers. Chemical structures of some of these detergents are shown in Fig.2.5 (a-j). 31 CHc^CHL^CHo' C 0 11 11 c - NCHo 1 CH 9 H i - C - O H HO 1 - C - H H 1 - C - O H H I - C - O H i CH 2OH Fig.2.5.(a). Chemical structure of Mega9 (nonanoyl-n-methylglucamide). Fig.2.5.(b). Chemical structure of deoxycholic acid (5-p-cholan-24-oic acid-3a, 12a-diol). 32 HOCK O HO 1111 OCH 2(CH 2) 6Chi HO OH Fig.2.5.(c). Chemical structure of octyl p-glucoside (n-octyl-p-D-glucopyranoside). H 2N H 9 HSCH 2C — C - O C H 2 C H 3 HCI Fig.2.5.(d). Chemical structure of L-cysteine ethylester. 33 CH3 C H 3 , . H 3 C — C - C H 2 - C — / V - O ( C H 2 C H 2 0 > - H C H 3 C H 3 N = approx. 9.5 (e) C H 3 C H 3 H 3 C — ( j - C H 2 - ( j K )— 0 ( C H 2 C H 2 0 ) - H 1 ' _ / V r/~1 T T f~* f * \\ v 7 N C H 3 C H 3 N = 7 to 8 (0 Fig.2.5. (e, f). Chemical structures of polyoxyethylene ethers, (e) Triton X-100, (f) Triton X-114. 34 Fig.2.5.(h). Chemical structure of C H A P S O (3-3-cholamidopropyl dimethylammonio-2-hydroxy-1 -propane-sulfonate). 35 H O ( C r ^ C H 20) w xS,(OCH2CH2)XOH | H ( O C H 2 C H 2 ) Y O H Q CH20(CrtCH20) Z-iCH2CH 20-C-CH2(CH2)9CrH3 Sum of w + x + y + z = 20 Fig.2.5.(i). Chemical structure of Tween-20 (polyoxyethylenesorbitan monolaurate). HO(Cr iCH 2 0) w ^ ( C C H p H ^ O H D YKOCHjCr^JyOH O C H 2 0 ( C | - b C H 2 0 ) z . 1 C r ^ C H 2 C w C ^ H 2 ( C H 2 ) 7 C r ^ C H s C H C H 2 ^ Sum o fw + x + y + z = 20 Fig.2.5.(j). Chemical structure of Tween-80 (polyoxyethylenesorbitan monooleate). 36 2.13.3. Immunostimulants Various chemical compounds, called "immunostimulants", are known to increase resistance of livestock and humans, as well as fish and shellfish, to infections. These include bacteria, microbial products, complex carbohydrates, animal extracts, plant extracts, cytokines and lectins, synthetic compounds such as dipeptide bestatin, and a number of muramyl- and lipo-peptides (Raa, 1 9 9 6 ; Galeotti, 1 9 9 8 ) . Similar Yano et al., 1 9 9 1 ) . Immunostimulants have been shown to induce non-specific and humoral defense mechanisms such as oxidative activity o f neutrophiles, engulfment potential o f phagocytic cells and activities of cytotoxic cells (McLean et al., 1 9 9 9 ; Anderson, 1 9 9 2 ) . N i k l et al. ( 1 9 9 2 ) reported an improved survival in bacterial challenge after a single UP injection of P-glucan. De Baulny et al. ( 1 9 9 6 ) found no reduction in mortality in a challenge study after oral administration o f P-glucan; however, an increase in white blood cell count was observed. Increase in total antibody level and non-specific protection capabilities in bacterial challenge of rainbow trout following administration of glucan has also been reported (Anderson & Jenny, 1 9 9 3 ) . Siwicki et al. ( 1 9 9 4 ) observed elevations in oxidative radical release, phagocytic activity, ki l l ing potential of neutrophils, as wel l as total immunoglobulin and total plasma protein levels after feeding fish with a variety of immunostimulant compounds. A challenge with Aeromonas salmonicida revealed a greater resistance in rainbow trout fed immunostimulants in the diet. 3 7 2.14. Dehydration techniques The main objective of dehydration as a preservation method is to convert perishable products to stable materials by reducing their water activity. Depending on the water content of the fresh product, weight and perhaps volume of the dehydrated material may be reduced. Consequently, it can be more easily stored for an extended time or transported to the sites of demand. A variety of techniques are commonly used to dehydrate food products at the industrial level. In all these techniques an energy supply is needed for concurrent transfer of heat into and the water mass out of the material. Dehydration involves the manipulation of temperature to evaporate water followed by removal of water vapor after separation from the dehydrated material (Jayaraman & Das Gupta, 1992). There are some advantages and limitations associated with each of these techniques, based on which the method of choice would be selected for each specific application. Three dehydration techniques that have been used in this dissertation are discussed below. 2.14.1. Hot air-drying In this dehydration process the product is exposed to a hot air current and the moisture is removed from it by evaporation. Generally, thermal damage incurred during drying is proportional to the temperature and time involved. The high temperature and long drying times associated with conventional hot-air drying often causes heat damage and adversely affects the quality of the dried product (Yang & Atallah, 1985; L i n et al, 1998). Hot air drying is usually carried out at temperatures of 60°C to 90°C or even greater in order to achieve efficient drying. Long drying 38 times at this temperature combined with available liquid water in the material can cause undesirable changes to the structural, physico-chemical and functional properties of the dried product. These conditions may also affect solubility and promote enzymatic and oxidative reactions (Durance, 2000). Therefore alternative energy efficient dehydration techniques have been sought to manufacture food products of high quality. Although all the above mentioned restrictions are disadvantageous to the application of air-drying, it is the least expensive dehydration method after sun drying, while some other dehydration methods such as freeze-drying are only economically feasible for high value products (Brown, 1973). 2.14.2. Freeze-drying In recent decades, freeze-drying has become widely used in production of high quality commodities. In this method, frozen products are transferred to the drier chamber in which the pressure is reduced to a level at which water sublimates from ice crystals to vapor in a low temperature (Somogyi & Luh, 1975). Freeze-drying usually results in the least structural and chemical damage to dried products when compared with other dehydration methods. The low processing temperatures, the absence of liquid water and low concentration of O2 in the dehydration environment combine to minimize degradative effects such as protein denaturation, enzymatic and oxidative reactions. Sublimation of water from the rigid frozen structure prevents collapse of the solid matrix of the dried product and results in a porous, non-shrunken structure. However, slow drying rates and use of vacuum makes freeze-drying expensive (Liapis, 1987). Furthermore, since the equipment is large and expensive, capital costs 39 of industrial scale installations are very high. Energy cost is also higher than in other drying methods because both freezing and evaporation of water from the frozen phase are energy intensive (Durance, 2000). The high expense of the process is considered the major limitation of this technique. Since the cost of freeze-drying depends on the amount of water removed, pre-treatment using methods such as osmotic drying can reduce the processing expenses (Bolin et al., 1983). 2 .14 .3. V a c u u m microwave drying Vacuum microwave ( V M ) drying is an emerging technique, which often offers improved quality for dehydrated products. In this method, drying takes place in a microwave oven chamber in which pressure is reduced. Applying microwave electromagnetic energy under vacuum provides the advantages of both vacuum drying and microwave drying (Yongsawatdiguul & Gunasekaran, 1996). The low temperature and quick mass transfer conferred by vacuum (Huxsoll & Morgan, 1968), combined with a fast energy transfer by microwave heating provide rapid low-temperature drying. Inhibition o f oxidation due to reduced air pressure in the drying chamber helps preserve physico-chemical properties such as tissue structure, color and sensory qualities of the dried product. The quality of this dried food is in some cases comparable to that of freeze-dried foods while the operating costs are estimated to be closer to those of forced convention air-drying technology (Durance, 2000). Vacuum microwave drying has been used in dehydration of plant and animal materials such as sweet basil (Yousif et al., 1999), carrot slices (Lin et al., 1998), potato chips (Durance & L i u , 1996), cranberry (Yongsawatdiguul & Gunasekaran, 1996) and shrimp (Lin et 40 al., 1999). In all cases, vacuum microwave dried products showed better retention of key constituents and sensory properties than air-dried materials. However, a major disadvantage of this method is the high cost of energy required to generate microwave power. The economics of the process restricts its application to the cases where high quality standards are required in the dried product or to a complementary method employed for quality improvement after initial removal o f a portion of moisture by lower cost heat treatments (Drouzas & Schubert, 1996). When a major portion of water content is removed by lower cost dehydration methods, microwave drying can be used for a quick and efficient removal of the remaining moisture from the interior parts of the product without overheating the pre-dehydrated product (Attiyate, 1979). Durance & Wang (1999) who used a combination of air-drying and vacuum microwave drying to dry tomatoes, calculate the costs of the process. The authors concluded that in spite of the higher cost of electricity energy used in vacuum microwave drying than that of natural gas used in conventional drying, the combination method resulted in a lower overall cost which was less expensive than either air drying or vacuum microwave alone. This lower cost was attributed to the dehydration efficiency in microwave processing, especially in the final portion of the drying process. 41 CHAPTER THREE MATERIALS & METHODS 42 3.1. Buffers Buffers used in the E L I S A assay included carbonate coating buffer at p H 9.6 (1.59g N a 2 C 0 3 ; 2.93g N a H C 0 3 ; 0.2g N a N 3 ; I L H 20), Phosphate-buffered saline (PBS) at p H 7.4 (8g N a C l ; 0.2g K H 2 P 0 4 ; 1.15g N a 2 H P 0 4 ; 0.2g K C I ; 0.2g N a N 3 ; I L H 20), Phosphate-buffered saline-Tween (PBS-Tween) at p H 7.4 (0.5mL Tween 20; I L H 20), blocking buffer (0.5% skim milk [Carnation, Nestle, Don Mi l l s , O N , Canada] in PBS) , 10% diethanolamine ( D E A ) buffer (97.0mL diethanolamine; O.lg M g C l 2 , 6H 2 0; 0.2g N a N 3 ; HC1 to p H 9.8; H 2 0 to IL) . P B S at p H 7.1 was used to suspend bacteria or dissolve W S F or chemicals (8g N a C l ; 1 . 2 1 g K 2 H P 0 4 ; 0 . 3 4 g K H 2 P O 4 ; 1 L H 2 0 ) . S D S - P A G E buffers included sample buffer (12.5mL of 0 .5M Tris, HC1, p H 6.8; 20mL of 10% SDS solution; lOmL glycerol; 5mL 2-mercaptoethanol; 2mL 0.1% bromophenol blue; 50.5rnL H 2 0 ; total volume lOOmL), electrode buffer at p H 8.3 (3.03g Tris base; 14.41g glycine; 20mL 10% SDS solution; Ff 20 to IL) . Western blot transfer buffer (25mM Tris; 192mM glycine; 20%> v/v methanol) stored in a dark glass bottle at 4°C. Immun-blotting buffers include Tris buffered saline (TBS) at p H 7.5 (20mM Tris; 500mM NaCl) , Tween-20-TBS wash solution (TTBS) at p H 7.5 (0.05% Tween-20 in TBS) , antibody buffer (0.1% bovine serum albumin in T T B S ) , alkaline-phosphatase (AP) substrate stock solution I (50mg mL" 1 5-bromo-4-chloro-3-indolyl phosphate (BCEP), Bio-Rad 170-653), stock solution II (50mg mL" 1 nitro blue tetrazolium (NBT) , Bio-Rad 170-6532), AP-buffer ( lOOmM Tris, l O O m M N a C l , 5 m M M g C l 2 , p H 9.5). 43 Fixative buffer for histological tissue samples included 10% buffered formalin solution (4g N a H 2 P 0 4 , 6.5g N a 2 H P 0 4 , lOOmL of 37% pure formaldehyde, H 2 0 to 1L) and Bouin's solution (1500mL of a 21g L" 1 picric acid aqueous solution, 500 mL of 37% pure formaldehyde, lOOmL glacial acetic acid). 3.2. Animals Chickens were used to produce immunoglobulins, which were extracted from the egg yolks for the purposes of this study. Fish served as the animal model for disease protection studies. 3.2.1. Chickens Four 19 week old brown leghorn (Gallus domesticus) laying hens were obtained from Coast Line Farm, British Columbia (B.C.) , Canada and maintained at Animal Care Facility of Simon Fraser University (SFU), Burnaby, B . C . , Canada. 3.2.2. Fish Naive juvenile rainbow trout {Oncorhynchus mykiss), which ranged from 16 to 90g for various experiments of this study, were obtained from local fish farms. They were acclimated in an indoor 500L holding tank for a minimum o f 15 days before being split into 75L tanks. Tanks were equipped with adjustable running water distributors and air inlet tubes as well as water drainage. Fig.3.1a and Fig.3.1b show the interior of the fish holding tanks and the mentioned tank equipment. Warm water was supplied through a central heating system. Fig.3.2 shows the arrangement o f the 44 fish holding tanks as well as water and air supply piping. Fish were fed with 2mm food pellets weighing 1-2% of their body weight, which were obtained from Moore-Clark in Vancouver, B .C . , Canada unless otherwise mentioned. Starvation periods before each experiment were 2-3 days unless otherwise stated. The photoperiod was adjusted to 12-hour intervals of light and dark throughout the experiment. Water temperature was maintained between 12 and 13°C. 3.3. Bacterial preparation The stock bacterial preparation was used to provide vaccines for the fish and hens, coating for indirect E L I S A to detect pathogen specific immunoglobulins, and to supply pathogenic bacterial preparation in challenge studies. It was also used in the binding characterization o f pathogen specific immunoglobulins from various animal sources. To provide the bacterial preparation, lyophilized Vibrio anguillarum strain M T 513 (obtained from Microtek International Ltd. , Sannichton, B C , Canada) was rehydrated according to the manufacturer's instructions. It was then grown in lOOmL sterile Tryptic Soy Broth (TSB) (Difco Laboratories, Detroit, M I , U S A ) , supplemented with 1.5% N a C l in 250mL Erlenmeyer flasks. The broth medium was gently swirled at an ambient incubation temperature (22±2 °C) for 24 hours and then l m L aliquots were dispensed into 1.5mL capped Eppendorf tubes containing 150pL of 50% (v/v) sterile glycerol. The suspension was mixed and stored at -78°C until used. 45 (b) Fig.3.1.(a & b). Design of a 75L fish holding tank. (1). Water inlet, (2). Running water distributor, (3). Air inlet, (4). Standpipe drain. 4 6 Fig.371 Arrangement of fish holding facilities. (1). Fish holding tank, (2). Warm water supply piping. (3). Adjustable water inlet valve, (4). Waste water drainage, (5). Air inlet tubing. 4 7 3.4. Vaccine preparation Anti-P". anguillarum vaccine was prepared to vaccinate hens in order to produce pathogen specific chicken egg-yolk immunoglobulins (IgY). The vaccine was also used to immunize fish in order to provide fish immunoglobulins (IgM) for the study of antigenicity and binding characteristics of I g M to Vibrio antigens. To prepare the vaccine, the frozen V. anguillarum suspension maintained at -78°C (described in section 3.3) was thawed, vortexed and subsequently used to inoculate Tryptic Soy Agar plates (TSA) (Difco Laboratories) with 25pL droplets using the drop-plate method. T S A was supplemented with 1.5% sodium chloride, ensuring conditions specific to Vibrio growth. To test the purity and sterility of the culture, the inoculum was streaked on T S A plates. Plates were then incubated for 24 hours at ambient temperature. The bacteria were harvested and subsequently suspended in sterile phosphate buffered saline (PBS), p H 7.1. The suspension was fixed by addition of formalin to a final concentration of 0.3% (v/v) while incubating at 5°C for 3 hours with occasional agitation. The suspension was then centrifuged (EEC Centra-7R Refrigerated Centrifuge, International Equipment Company, a division of Damon, U S A ) in 50mL capped Falcon tubes at 4,000 rpm for 20 minutes at 5°C. The supernatant was discarded and fresh P B S was added to remove formalin residue, followed by vortexing and centrifuging the suspension. This step was repeated three times. The final concentration of killed Vibrio in suspension to prepare the vaccine was adjusted to give an absorbance of 5 at 540nm (As^ nm = 5 ) . Fig.3.3 shows the process of vaccine preparation and vaccination of chickens in brief. A surface culture of the bacteria was prepared on T S A plates before being 48 formalin-killed. The concentration of the bacterial cells in the final suspension for vaccine preparation was calculated to be 5x10 7 cfu m l / 1 based on plate enumeration. A n equal volume of Freund's complete adjuvant (Sigma, F 5881) was subsequently mixed with the bacterial suspension for the first injection. Boosters were also prepared in the same manner with Freund's incomplete adjuvant (Sigma, F 5506) and injected into hens or fish. 3.5. Vaccination 3.5.1. Chickens Chickens were vaccinated to produce anti-Vibrio antibodies, which could be recovered from their egg yolks. These were needed in antigenicity studies as well as the immunoglobulin uptake and disease protection experiments. A t 31 weeks of age, the first intramuscular injections of l m L anti-F! anguillarum vaccine were administered to the chickens. One m L boosters were alternated between right and left sides of the breast at 1, 2, 3, 4, 7, 9, 11, 15, 20, 25, 31, 47, 60 weeks after the initial injection to maintain a stable antibody level in the egg yolks until egg collection was terminated. 3.5.2. Fish Fish were vaccinated against V. anguillarum to produce antigen specific antibodies (IgM) to be used in antigenicity and binding characteristic studies. Twenty naive juvenile rainbow trout (average weight 35g) were acclimated for 15 days to a water temperature of 12-13°C. A volume of 0. l m L of vaccine was intraperitoneally 49 Harvest after 24h @ 25°C m Formaldehyde (0.3% in PBS) ST t-rt o n Freund's Washed, <— adjuvant Adjusted @ A540 nm = 5 Fig.3.3. Schematic diagram o f anti-Vibrio anguillarum vaccine preparation and vaccination o f chickens. 50 administered into each fish followed by O. lmL booster preparations 14 days after the initial injections. Fish were anesthetized with 0.5mL L" 1 o f 2-phenoxy ethanol (Anachemia AC-7190 P) prior to injection. Blood collection from the caudal vein was performed after 14 days to prepare serum as a source of specific anti-P! anguillarum IgM. Ice tuberculin syringes (27 G V2) were used for injection and blood collection. 3.6. Preparation of IgY Egg-yolk immunoglobulins (IgY) were needed throughout the project to study IgY potential to reach the fish bloodstream and confer a passive protection against diseases. IgY was also used to study its binding characteristics to V. anguillarum. The IgY preparation method was according to Aki ta & Nakai (1992), with some modifications. To prepare IgY, egg yolk was carefully separated from the egg white and the yolk sac using paper towel to remove the final layer of white adhering to the yolk sac. A needle was used to break the yolk sac and release the yolk content into a measuring cylinder. The yolk, with a p H of approximately 6.5, was diluted 10 times in distilled de-ionized water and acidified with 0.1 N H Q to a final p H of 5.2. The suspension was stirred and subsequently stored at 5°C over night. The following day, it was centrifuged @ 16,000 x g for 30 minutes at 4°C (Sorvall® R C 5B plus, Sorvall Instruments, Dupont, NewTown, CT, U S A ) . The supernatant was filtered through glass wool and filter paper (Whatman #4). Glass wool was used to exclude fat residues. The filtrate, containing the water-soluble fraction o f egg yolk (WSF), was used as a source of semi-pure IgY throughout the study. W S F was concentrated by ultrafiltration using a hollow fiber cartridge with a 10,000 molecular weight cut-off 51 ( A / G Technology Corporation, Needham, M A , U S A ) to 7-15 times the concentration of the supernatant. Due to limited capacity of the ultrafiltration system, the supernatant was sometimes stored at -20°C until required. The concentrate (retentate) was again stored at -20°C until needed. A l l frozen batches o f the retentate were thawed, pooled and re-frozen until used. Lyophilized W S F (L-WSF) was prepared using a freeze-dryer (VirTis Research Equipment, Gardiner, N Y , U S A ) with a shelf temperature of 20°C, a chamber pressure of lOOpmHg and a condenser temperature of -55°C. Fig.3.4 summarizes the process of IgY preparation until administered to fish. Non-specific IgY-containing W S F was extracted from the eggs collected before vaccination. Starting 6 weeks after the first vaccination, the eggs were used to provide specific anti-K anguillarum IgY-containing W S F . Eggs were stored at 5°C between 1 to 6 months prior to IgY preparation. Each batch contained eggs of 2 or in some cases 4 consecutive weeks. Eggs used in the drying studies were white eggs obtained from a local supermarket. A larger ultrafiltration apparatus (UF Spiral System, K o c h Membrane Systems Inc., Wilmington Massachusetts, U S A ) equipped with a spiral membrane of 30kDa N M W cut off was used in this part of the study to concentrate the W S F as one whole batch. 3.7. Preparation of microcapsules To prevent IgY degradation in the gastrointestinal tract of fish, IgY-containing microcapsules were produced. 150mg of the lyophilized W S F was dissolved in 1.5mL of d - H 2 0 , vortexed and centrifuged to remove the insoluble matter. The supernatant 52 Y o l k | Dilute x 10 in HC1 & d - H 2 0 final pH=5.2 Over night (5>4°C Centrifuged @ 1 6 0 0 0 x g @ 4 ° C Lyophilization (L-WSF) Incorporation into the pellets Ultrafiltration hollow fiber cartridge 10000NMWC Supernatant: Water-Soluble Fraction ( W S F ) Orally intubated Fed IgY absorption studies Challenge studies Fig.3.4. Schematic diagram o f the IgY extraction from the hen eggs and application in the absorption and passive immunization studies in rainbow trout. IgY was recovered in the water-soluble fraction (WSF) o f the egg yolks, concentrated by ultrafiltration and lyophilized. The lyophilized W S F ( L - W S F ) was administered to fish via the oral route or LP injection to study the absorption o f I g Y and the protection IgY may confer. 53 was then added to a solution of 400mg poly(DL-lactide-co-glycolide) with a lactide:glycolide ratio of 50:50 ( P L G 50:50) (Aldrich Chemical Company, Inc., U S A ) or P L G 85:15 (Lactel, Birmingham Polymers Inc., Birmingham, A L , U S A ) in lOmL dichloromethane ( D C M ) ( B D H Chemicals, Toronto, Canada) containing 40uL of a surfactant agent, Span-80, and blended with a P O L Y T R O N blender ( K I N E M A T I C A GmbH, Switzerland) to produce a water in oil (w/o) emulsion. The mix was gradually added to a 2.5% solution of polyvinyl alcohol ( P V A ) (98% hydrolyzed, 13,000-23,000 Daltons, Aldr ich Chemical Company, Inc.) while stirring @ 900 rpm with a Dyna-Mix overhead stirrer (Model 143, Fisher Scientific, Fairlawn, N J , U S A ) . After 10 minutes, the speed was reduced to 600 rpm and continued for 2.5 hours to obtain a w/o/w emulsion. Antigen-containing P L G ( P L G A ) was washed 4 times with d - H 2 0 followed by centrifugation (Beckman GPR, U S A ) @ 3,000 rpm for 10 minutes and then dried at ambient temperature. Size of microspheres was determined by Particle Size Analyzer (Coulter LS130, Coulter Electronics of New England Inc., Amherst, Mass., U S A ) . Microscopic photographs of the microparticles were produced using a camera attached to an Olympus B H - 2 light microscope (Olympus GmbH, Hamburg, Germany). Fig.3.5 shows the process of preparation of microcapsules. 3.8. Feed preparation To examine IgY uptake into the fish blood from feed, and to test the potential of this immunoglobulin in conferring passive immunity against diseases, IgY-containing pellets were produced. When a fabricated diet was used, all necessary additives including W S F , antacid or absorption enhancing agents were dissolved in 54 L-WSF (12.4% IgY) centrifuge 1.5 ml d-H20 Supernatant PLGA (w/o/w) Air dried @ room temp. Microcapsules C30 um) 400 mg PLG 85:15/50:50 10 ml 40 nl DCM <— Span 80 homogenize 2.5h-w/o emulsion 600 r^ Fig.3.5. Schematic diagram o f microencapsulation of water-soluble fraction (WSF) of egg-yolks in polylactide-D-glucolide ( P L G ) with two different ratios o f lactide to glycolide, 85:15 or 50:50. Water in oi l in water (w/o/w) emulsion of W S F in P L G in dichloromethane ( D C M ) was prepared by the aid of a sufactant (Span-80) and stirring for 2.5 hours. The antigen containing microcapules ( P L G A ) were then recovered by 4 times centrifugation and washing with d-H20 and were air dried at room temperature. 55 P B S , p H 7.1 and top-dressed onto commercial pellets by multiple spray applications. Pellets were then dried at ambient temperature using a fast air draft under the fume hood. Marine oil (3% weight of pellets) was subsequently sprayed onto the pellets to seal the top dressing and reduce additive loss in the water tanks before the uptake by fish. 3.9. Extraction of IgY from the treated pellets To determine the IgY level in fabricated fish feed pellets, an extract o f such pellets had to be prepared. A l l top-dressed pellets produced as feed diets in different feeding studies, as well as the pellets prepared for dehydration studies, were sampled and stored at -20°C until used. A l g sample of each diet was soaked in lOmL P B S and blended (Ultra Turrax T25 DC A Labortechnik, I K A Works, Inc., Willmington, N C , U S A ) . Following overnight incubation at 4°C, the samples were vortexed and centrifuged (IEC Centra-7R Refrigerated Centrifuge) at 4000 rpm and 10°C for 20 minutes. The supernatant was collected after filtration through filter paper Whatman #4 in order to measure total I gY as well as anti-K anguillarum I gY content. A l l samples were prepared in triplicate. 3.10. Blood collection from fish and serum preparation To determine IgY levels in the fish serum following different treatments, and to study antigenicity and binding characteristics o f fish I g M in reaction with V. anguillarum, fish blood had to be collected. Blood was collected from the caudal vein using a sterile syringe, following anesthesia of fish with 2-phenoxy ethanol. The 56 collected blood was then transferred into sterile 1.5mL capped Eppendorf micro test tubes to store for 2 hours in ambient temperature and overnight at 4°C before being centrifuged (Eppendorf Centrifuge 5415 C, Eppendorf GmbH, Hamburg, Germany) at 12,000 x g for 4 minutes to separate the serum. The serum was subsequently transferred into new tubes in sterile conditions and stored at -20°C until required. 3.11. Bacterial preparation for fish challenge studies A V. anguillarum preparation was needed to challenge fish with the pathogen in order to determine their resistance against the disease-causing bacteria. A 1.5mL tube of viable frozen bacterial suspension (described in section 3.3) was thawed, vortexed and pipetted into a 4 L flask containing 800mL of sterile T S B supplemented with 1.5% N a C l . It was then incubated overnight at ambient temperature while shaking. During this time, the culture was aerated with sterilized air passing through a 0.22(j.m sterile syringe filter membrane ( M i l l e x - G V Filter Unit, Mil l ipore Corporation, Bedford, M A , U S A ) . The apparatus was stopped when the turbidity of the culture reached A54onm = 2.6. Approximately lOmL of the bacterial suspension was transferred into a sterile 50mL capped Falcon tube and temporarily stored on ice until applied to the buckets of salted water provided for fish challenge. 3.12. Lipopolysaccharide (LPS) & whole cell lysate (WCL) preparation Lipopolysaccharide (LPS) & whole cell lysate ( W C L ) were used to study the antigenic characteristics of V. anguillarum in chickens (IgY), rabbits (IgG) and fish (IgM). To prepare whole cell lysate ( W C L ) of V. anguillarum, frozen bacterial 57 suspension (described in section 3.3) was thawed, vortexed and subsequently used for surface culture on T S A plates supplemented with 1.5 % N a C l . After 24 hours incubation at ambient temperature, a full loop of V. anguillarum colonies was transferred into a 1.5mL capped microtube and l m L o f P B S (pH 7.1) was added in sterile conditions, vortexed and centrifuged (Eppendorf Centrifuge 5415C) for 3 minutes at 5,000 rpm. The supernatant was aspirated off and 0. l m L o f sample buffer (described in section 3.1) was added for every l m g wet weight of cells and vortexed 30 seconds to completely dissolve the cells. It was then boiled for 5 minutes and centrifuged for 3 minutes to eliminate any possible non-dissolved cells. The supernatant was transferred into a fresh tube. This W C L preparation was used in S D S - P A G E electrophoresis. V. anguillarum lipopolysaccharide (LPS) was prepared by protein digestion of W C L using a solution of proteinase K (PK), (0.0025g P K [ M E R K , 24568] in l m L of sample buffer, where lOpL of this solution contained 25pg P K ) . To 50pL of the heated W C L , lOuL of P K preparation was added and incubated at 60°C for 60 minutes. This was according to the procedure of Hitchcock & Brown (1983). L P S prepared using this method was used for S D S - P A G E electrophoresis. 3.13. Anal intubation To investigate crossing of the IgY molecules through the gut barrier, 0. l m L o f a concentration of l m g mL" 1 IgY (Sigma 1-4881) was anally intubated into each fish with an average weight of 21g which had been starved for 24 hours. Blood was collected from caudal vein every 2 hours post intubation for 24 hours and was 58 continued at 27, 30, 48 and 72 hours. Two fish were sampled at each time. Sandwich E L I S A was performed to determine IgY levels in the serum of the test fish as well as the control fish, which were anally intubated with P B S . 3.14. Oral administration To study the survival of IgY in the GI tract of trout and IgY uptake into the bloodstream, oral intubation experiments using various delivery approaches were conducted. The oral administration studies are numbered from 1 to 9 as follows: 3.14.1. Experiment 1. Using IgY in encapsulated form, in Mega9 and antacid, or in Na-pyrophosphate solution Lyophilized W S F (L-WSF) of the egg-yolk from immunized chickens, which contained 12.4% total IgY, was used as a source of I gY in all treatments. In the test treatments (T), rainbow trout (29g average weight) were orally intubated (IN) with 1. L - W S F solution in sterile P B S , p H 7.1, 2. L - W S F in a 1% w/v sodium bicarbonate solution (SBC) , as antacid, containing 5% Mega9 (M9) (Nonanoyl-n-methylglucamide, I C N Biomedical Inc., Ohio, U S A ) , a non-ionic detergent, p H 8.26, 3. L - W S F in a solution of sodium pyrophosphate (PY) (0.044g N J L ^ O ? , 10 H 2 0 in lOmL of 0.44M NaCl) , p H 9.4, 4. L - W S F microencapsulated in P L G 85:15, 5. L - W S F microencapsulated in P L G 50:50. Microcapsules had a theoretical IgY loading of 3.4%) (w/w) and an average particle size of 29.39±5.75uxn for P L G 50:50 and 30.32±5.48u,m for P L G 85:15. Fig.4.12 shows a microscopic photograph of these microcapsules. Two control treatments (C) were also applied. A positive control 59 group was intraperitoneally (LP) injected with L - W S F in sterile P B S and a negative control group was orally intubated with sterile PBS . In treatments 1 to 3 fish were intubated with 200uL of lOOmg mL" 1 L - W S F solution which provided 2.68mg total IgY per fish or 92.4mg IgY kg"1 average body weight. In treatments 4 and 5, the intubation material consisted of 200uL of a 200mg mL" 1 L - W S F containing P L G suspension in P B S . In the latter treatments, fish were theoretically receiving 46.9mg IgY kg" 1 average body weight, which was equal to 1.36mg IgY per fish. Positive control fish were LP injected with 200uL of a lOOmg mL" 1 L - W S F solution in P B S . In all cases fish blood was collected from the caudal vein using a l m L syringe at 30 minutes, 1,3, and 24 hours. For all except the controls, sampling was carried out on days 2, 3, 5, 7, and 14. Blood collection continued at weekly intervals t i l l week 5 for treatments 4 and 5. Sample size consisted of 3 fish per sampling event. Serum was prepared as described previously and an E L I S A assay was employed to determine total I gY levels in fish serum. 3.14.2. Experiment 2. Tween detergents as absorption enhancing agents This experiment was performed to study the effect o f Tween detergents (Tween-20, polyoxyethylenesorbitan monolaurate and Tween-80, polyoxyethylenesorbitan monooleate), as well as an antacid on the absorption of IgY through the fish gut. In all cases, the source of IgY was L - W S F containing 12.4% total IgY. Each rainbow trout (16g average weight) was orally intubated with 200uL of lOOmg mL" 1 L - W S F solution, which provided 2.68mg total IgY per fish or 167.5mg kg"1 average body weight. The treatments were 1. L - W S F in P B S , 2. L - W S F in a 1% 60 solution of sodium bicarbonate, 3. L - W S F in P B S containing 5% Tween-80 (T80), 4. L - W S F in P B S containing 2.5% Tween-80. In all other treatments L - W S F was employed in a solution of 1% sodium bicarbonate containing either of the following detergents: 5. Tween-80 (5%), 6. Tween-80 (2.5%), 7. Tween-20 (T20) (5%), 8. Tween-20 (2.5%). Blood samples (3 fish per treatment) were collected at 1 and 4 hours post intubation. 3.14.3. Experiment3. Comparative oral intubation To confirm the results of the two previous experiments, a similar experiment was performed with 7 treatments: 1. L - W S F in P B S , 2. L - W S F in sodium pyrophosphate solution (identical to experiment 1), 3. L - W S F in P B S containing 5% Mega9, 4. L - W S F in sodium pyrophosphate solution containing 5% Mega9, 5. L - W S F in a solution of 1% sodium bicarbonate, 6. L - W S F in a solution of 1% sodium bicarbonate containing 5% Mega9, 7. L - W S F in P B S containing 5% Tween-20. After 40 hours starvation each rainbow trout (26.6g average weight) was orally intubated with 200pL o f lOOmg mL" 1 L - W S F solution. This provided 2.68mg total IgY per fish or 100.75mg kg" 1 average body weight. Blood samples (3 fish per treatment) were collected at 1 and 3 hours post intubation. 3.14.4. Experiment 4. Effect of various absorption enhancing agents In a pre-test, a range of concentrations of various chemicals known for their absorption enhancing effect was tested to determine the highest concentration of each substance which could safely be fed to juvenile rainbow trout (average weight 25g). 61 The highest safe level was defined as the highest concentration of each chemical tested that did not cause any lethal effect within 10 days on any o f the 4 treated fish. These substances, as listed here with the tested concentrations indicated in brackets, were orally intubated into fish in an aqueous solution. The chemical structures of these substances are illustrated in Fig.2.5.(a-j). Saponin (Sigma S-4521) ( lmg, 100, 50, 10 pg fish"1); Mega9 (nonanoyl-n-methylglucamide) ( ICN 150056) (5%); Na-deoxycholate (deoxycholic acid, sodium salt: 5-p-cholan-24-oic acid-3a, 12a-diol) (Sigma D-6750); octyl P-glucoside (n-octyl-P-D-glucopiranoside) (Sigma O-8001); polyoxyethylene ethers, Triton X-100 (Sigma X-100) and Triton X-114 (Sigma X -114); C H A P S (3-3-cholamidopropyl dimethylammonio-l-propanesulfonate) (Sigma C-3020); C H A P S O (3-3-cholamidopropyl dimethylammonio-2-hydroxy-l-propane-sulfonate) (Sigma C-3649), all at (5%, 3%, 1%, 0.5%); L-cysteine ethylester (Sigma C-2757) (0.9, 0.3, O.lmg fish"1). The concentration used, the mortality rate caused by each concentration, and the highest safe concentrations o f these chemicals are reported in Table 4.6. To test the efficacy of these detergents in enhancement of IgY absorption from the gut, juvenile rainbow trout (25g average weight, 4 fish per group) were orally intubated with lOOpL of 1% N a H C 0 3 solution including 40mg of non-specific L - W S F (containing 150mg g"1 of IgY) to which the selected chemicals at the highest safe concentration, were added. A control group was intubated with W S F suspended in P B S . Blood was collected 4 hours after intubation to assess IgY uptake level into the circulation in each treatment. 62 3.15. Challenge studies To investigate the efficacy of IgY in passive immunization of trout, fish were exposed to the pathogenic bacteria following administration of anti-K anguillarum IgY. A bacterial preparation (described in section 3.11) was used in an appropriate amount as determined in a pre-challenge test. Prior to each challenge, a pre-challenge test was performed using three groups of 15 juvenile rainbow trout to determine the suitable concentration of V. anguillarum preparation to be used in the target challenge study. Three different bacterial concentrations of the same preparation were used for these three groups. To conduct the pre-challenge test, fish were transferred from the holding tanks into the 25L buckets containing 10L of salted water, to which the pathogenic bacteria were added. Tapwater was supplemented with 0.9% iodine-free table salt to make the conditions favorable for Vibrio. The buckets were aerated during the 30 minutes course of exposure to bacteria. Contaminated fish were then transferred to their holding tanks equipped with aeration and fresh running water. The bacterial concentration, which caused 70% mortality in the untreated fish in the pre-challenge, was used later in the target challenge experiment. In the challenge experiments, the proper concentration of V. anguillarum preparation was added to a 200L tank of salt water and mixed wel l to make an even concentration for all treatments of the same experiment. Fish of each holding tank were transferred into a separate 25L bucket containing 10L of such bacterial preparation for immersion challenge. The challenge was conducted similar to pre-challenge as discussed previously. Mortality rates were always monitored for 14 days post-challenge. Fig.3.6 provides a summary o f the challenge process. 63 Various approaches as described below were applied in delivery of IgY into the fish to examine its efficacy in protection against Vibriosis in experimental challenge. 3.15.1. Experiment 5. Preliminary feeding trial A preliminary feeding experiment was performed to study the absorption of IgY into the fish blood circulation when Mega9 was included in the diet. Feed pellets (1.3% average body weight) were top-dressed with Mega9 and W S F (8mg per fish or 87mg kg" 1 average body weight of total IgY day"1). Mega9 content of the diet in treatment 1 (TI) was 30mg per fish or 325mg kg" 1 average body weight per day for the first 2 days. For the next 5 days, Mega9 was eliminated from the diet. In treatment 2 (T2), the diet contained 6mg per fish or 65mg kg" 1 average body weight of Mega9 per day for 7 days. There were 8 fish (89.7g average weight) in each group, which were starved for 4 days pre-treatment. Three fish of each treatment were sacrificed to collect blood on the second day, 6 hours after feeding. Blood was collected from the remaining 5 fish of each treatment on the seventh day, 6 hours after feeding. Four fish from each treatment were saved after blood collection to be used in a preliminary challenge study with V. anguillarum, which was performed at day 9. A bacterial concentration of 1.2x106 cfu mL" 1 was used to challenge fish by immersion. 64 Fig.3.6. Schematic diagram of fish challenge with V. anguillarum. Overnight growth in TSB at room temperature was stopped at A540 nm =2.6. This bacterial preparation was used to inoculate salted water for the fish challenge. Fish were transferred into fresh water tanks after 30 minutes exposure to the pathogenic bacteria. The mortality was monitored 14 days post challenge. 65 3.15.2. Experiments 6 & 7. Challenge following oral administration of Mega9 and water-soluble fraction of egg-yolks ( W S F ) Experiment 6 was designed to examine the efficacy of specific anti-V. anguillarum IgY to enhance fish resistance against vibriosis when administered alone or in conjunction with Mega9. This experiment consisted of nine different feeding or intubation treatments carried out for 7 days. The W S F of the egg yolks obtained from immunized hens (spWSF) was used as the source of anti-P anguillarum IgY and W S F of egg yolks from non-immunized hens (nspWSF) as a source of non-specific total IgY. Diets for the treatment (T) and control (C) groups were as follows: T l . pellets (1.2% average body weight) top-dressed (P + ) with spWSF and Mega9 on the first day (d l ) followed by feeding regular pellets on days 2 to 7 (d2-7); T2. P + , spWSF and Mega9 (d l ) , Mega9 was eliminated on days 2-7; C l . P + , nspWSF and Mega9 (d l ) , regular pellets (d2-7); C2. P + , nspWSF and Mega9 (dl) , P + and nspWSF (d2-7); C3. oral intubation (IN) of spWSF and Mega9 in 1% N a H C 0 3 solution (dl) , P + and spWSF (d2-7); C4. oral intubation o f absorbed (ab) spWSF and Mega9 in 1% N a H C 0 3 solution (d l ) , P + and ab-spWSF (d2-7); C 5 . P + and Mega9 (dl) , pellets (d2-7); C6. pellets (1-7); C7. P + and spWSF (dl-7). To provide absorbed spWSF for C4, a suspension o f formalin-killed V. anguillarum was prepared (A540nm=5). lOmL of W S F from the eggs of immunized hens was exposed to l m L o f the bacterial suspension for 1 hour with occasional mixing. It was then centrifuged ( IEC Centra-7R) at 4000rpm and the absorption was repeated for the supernatant. The second time, the supernatant was filtered through a 0.22pm sterile syringe filter membrane to eliminate any bacterial residues. C4 was designed to test whether any component of the spWSF 66 other than IgY has an effect on resistance against vibriosis. Mega9 content of the diet was 22mg fish"1 or approximately 396mg kg"1 body weight day"1 when fed or 5% when intubated. In all cases Mega9 was accompanied by 4.4mg fish"1 or approximately 80mg kg" 1 body weight (or in case of intubation 1%) of sodium bicarbonate (SBC). The spWSF or nspWSF was added in a concentration that provided 5.5mg total IgY fish"1 or approximately lOOmg total IgY kg"1 body weight day"1. These amounts were very similar to those used in experiment 1. In case of intubation, 200uL of intubation solution was applied to each fish. There were either 2 or 4 tanks containing 16 rainbow trout (55.23g average weight) per treatment. Blood was collected from four fish of each treatment to test IgY concentration in the serum at the day of challenge. Fish were exposed to a concentration between 2.48 x 10 5 and 2.64 x 10 5 cfu mL" 1 o f V. anguillarum for 30 minutes on the 8 t h day, 20 hours after the last feeding. Mortality rate was monitored for 14 days post challenge. A l l the details in challenge conditions were identical to experiment 5. Experiment 7 was designed to confirm results from experiment 6. Each treatment or control group consisted of 3 tanks of 16 fish (average 30g). Feeding regimens were as follows: T I . P + with Mega9 and spWSF (dl-2), P + and spWSF (d3-7); C l . P + , Mega9 and nspWSF (d3-7); C2 (positive control), intubated Mega9 and specific W S F (dl) , P + , Mega9 and spWSF (d2), P + and spWSF (d3-7); C3. pellets ( d l -7); C4. P + and spWSF (dl-7). Where applicable, pellets fed at a rate of 2% average body weight per day, Mega9 as lOmg fish"1 or approximately 333mg kg" 1 body weight or 5% in solution when intubated, N a H C 0 3 as 2mg fish'1 or approximately 66mg kg"1 body weight or 1% in solution when intubated, spWSF or nspWSF (fed or intubated) 67 in a concentration that provided 6.5mg total IgY fish"1 or approximately 217mg total IgY kg"1 body weight day"1. Volume of intubation solution was 200pL. Challenges were carried out under the same conditions as in experiment 5 except that the bacterial concentration was 5.1 or 5.4 x l O 4 cfu mL" 1 . One fish out of each tank was used for blood collection. 3.15.3. Experiment 8. Challenge following oral intubation with absorption enhancing agents The objective o f this experiment was to test whether IgY oral uptake would be improved by use of absorption enhancing agents and whether this approach would lead to an enhanced protection of fish against Vibriosis. Based on the results o f experiment 4, the three most effective chemicals, Mega9 (M9), octyl-P-glucoside (OpG) and Na-deoxycholate (DX) , were selected as IgY absorption enhancing agents. In T l , T2 and T3, M 9 , O p G or D X , as well as spWSF were dissolved in 1% S B C solution (200pL fish"1) orally intubated (IN) into the fish (40g average weight) at the first day or added to pellets (P + ) (1.2% average body weight) to be fed on day 2. The absorption-enhancing agent was eliminated from the feed on days 3-7. In T4, fish were intubated with spWSF in S B C on dayl and fed pellets containing spWSF on days 2-7. S B C was added at 1% pellet weight to treatments 1-3. A negative control group ( C l ) was intubated with P B S on day 1 and fed commercial pellets on days 2-7. spWSF provided 6.12mg total IgY fish"1 or approximately 153mg kg" 1 body weight day"1. M 9 and O p G were added as 5% (lOmg fish"1 or approximately 250mg kg"1 body weight day"1) into the I N suspension or 15mg fish"1 (approximately 375mg kg" 1 68 body weight day"1) when top-dressed onto the pellets. The amounts of D X were 1% (2mg fish"1 or approximately 50mg kg"1 body weight day"1) when intubated or 3mg fish"1 (approximately 75mg kg" 1 body weight day"1) when added onto the pellets. Each treatment consisted of 3 tanks of 16 fish. Blood collection and challenge practices were performed as in the previous experiment. Bacterial concentration in the challenge water was 2.5 x 10 4 cm mL" 1 . 3.15.4. Experiment 9. Challenge following feeding of absorption enhancing agents This experiment was conducted to complete the results o f experiment 8 where a combination of oral intubation and feeding of spWSF and absorption enhancing agents was used. Since intubation is not feasible in commercial aquaculture practice, experiment 9 was conducted to test the efficacy of pathogen specific IgY in protection against diseases, when it was incorporated into feed. In this experiment, fish (45g average weight) in the test treatments were fed pellets (1.5% average body weight) containing absorption enhancing agents and antacid (1% pellet weight). Experimental design for T I to T4 was similar to the previous experiment except that there was no intubation applied on the first day and instead, fish were fed the same diet on both the first and the second days. A positive control group ( C l ) received oral intubation on the first day identical to that of T I in experiment 8. A negative control (C2) was fed regular pellets day 1-7. The concentrations of the detergents in feed were increased to 20mg fish"1 day"1 or approximately 440mg kg" 1 body weight day"1 for M 9 and O 0 G and 4mg fish"1 or approximately 88mg kg" 1 body weight day"1 for D X . The concentration of intubated M 9 was 5% volume (lOmg fish"1 or approximately 222mg 69 kg"1 body weight). Enough spWSF was used to provide 8.8mg IgY fish"1 day"1 or approximately 196mg kg" 1 body weight day"1. Fish of different treatments were exposed to 1.7 to 1.9 x 10 4 cfu mL" 1 o f V. anguillarum for 30 minutes in an immersion challenge. A l l other details were similar to experiment 8. 3.15.5. Challenge following IP injection of IgY This experiment was performed to test whether pathogen specific IgY was capable of conferring disease protection when delivered efficiently into the blood circulation of rainbow trout. Fish (68g average weight) were challenged at days 1, 3, 7 or 14 following injection with lOOuL of a preparation of anti-K anguillarum IgY (spWSF) into the intraperitoneal (IP) cavity of rainbow trout in order to study duration of IgY effectiveness in enhancement of disease resistance. The injection was applied at the base of the pelvic fins. Two control groups were LP injected with the same volume of either P B S or nspWSF. Concentrations of total IgY in spWSF and nspWSF preparations were 1.33mg mL" 1 and 0.15mg mL" 1 , respectively. Each treatment consisted of 3 tanks of 16 fish for each date of challenge. Blood was collected at the day of challenge from one fish from each tank. Bacterial concentration used in immersion challenge was 8.2 X 10 4, 3.2 X 10 5, 3.3 x 10 5 and 2.0 x 10 5 cfu mL" 1 at days 1, 3, 7 and 14, respectively. Conditions of challenge were identical to previous experiments. 70 3.16. Analytical techniques 3.16.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Gel electrophoresis was used to characterize W C L and L P S of V. anguillarum and resolve different components associated with them. S D S - P A G E sample buffer under reducing conditions (with 2-mercapoethanol) and electrode buffer as well as 4% stacking and 12% separating acrylamide gels were prepared according to Laemmli (1970). In each well of the gel, 15pL of W C L , 20pL of L P S , 5pL of low molecular weight protein marker ( L M M ) or 5pL of prestained L M M (Bio-Rad S D S - P A G E Standard 161-0304 and 161-0305, respectively) were loaded. Discontinuous SDS-P A G E of the samples was run at a constant current of 25mA at 400v (Electrophoresis Power Supply E P S 500/400, Pharmacia Fine Chemicals, Uppsala, Sweden), using a Mini-PROTEAN® II Slab Cel l (Bio-Rad Laboratories, Hercules, C A , U S A ) vertical gel electrophoresis system. More details on casting gels and operating the slab cell can be found in the guidebook of Bio-Rad Laboratories. Each 10 well 0.7mm gel was loaded with identical samples in the right and the left sides. The gel with L M M was carefully cut in half for silver staining and coomassie blue staining with 0.025%) coomassie brilliant blue R-250. Stained gels were then shrunk in 50% methanol and dried using a vacuum gel drying system (Gel Slab Drier GSD-4 , Pharmacia Fine Chemicals, Sweden). The gel with the prestained marker was used in Western blotting. Silver staining of proteins ( W C L and L P S ) was performed according to Tsai &Frasch(1982) . 71 3.16.2. Western blotting Western blotting was used to study antigenicity o f V. anguillarum W C L and L P S (section 3.12) in chickens (IgY), rabbits (IgG) and fish (IgM). For this purpose, W S F o f chicken egg yolk and fish serum were prepared from immunized animals against V. anguillarum to be used as sources of specific IgY and IgM, respectively. Pre-immunization samples were also collected to be used as negative controls as a measure of non-specific reactions between the immunoglobulins with W C L or L P S of V. anguillarum. Specific anti-K anguillarum rabbit IgG was obtained from Microtek International Ltd . (Sanichton, B . C . , Canada, # AS006) and non-specific rabbit IgG from Pierce (Rockford, LL, U S A , #31207). Polypeptides obtained from S D S - P A G E electrophoresis were electrophoretically transferred from the gel onto a 0.45uxn nitrocellulose (NC) membrane (Bio-Rad), using a tank blotting system, M i n i Trans-Blot Cell (Bio-Rad) for 1 hour at 100V and 250mA at 4°C according to Bio-Rad recommended procedure. Immunoblotting of the N C membrane was performed according to Bio-Rad immun-blot instruction manual at ambient temperature while shaking gently, with some modification. The constituents of all buffers are described previously in "Buffers". The N C membrane was briefly washed in T B S and blocked in 1% bovine serum albumin ( B S A ) in T B S overnight. It was then rinsed with T T B S and washed in this buffer twice for 10 minutes. The N C membrane was divided into two parts. Halves were transferred into the first antibody solution containing either specific anti-K anguillarum or non-specific antibodies and remained there for 2 hours. After rinsing and washing twice with T T B S , the N C membranes were incubated in the secondary 72 antibody (conjugate) for 1 hour followed by rinsing and washing twice with T T B S and once with T B S to remove Tween-20 in order to avoid interference with color development. The N C membranes were exposed to alkaline-phosphatase (AP) substrate and the reaction was stopped by addition of distilled water after color development. Developed blots were kept in the dark until scanned using Adobe Photoshop version 5.0. The specific anti-K anguillarum titer of rabbit IgG, chicken IgY or fish I g M was determined by E L I S A assay as being 128,000, 6,400 or 1,280, respectively. Based on these results, working dilutions were selected as 1:16000, 1:800, 1:160, respectively, to bring the concentration of the different antibodies to the same level. The same dilutions were employed for non-specific antibodies with respect to the same species. The conjugates used were AP-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, P A , U S A , # 111-055-003) at a concentration of 0.12ug mL" 1 , AP-rabbit anti-chicken IgG (Sigma, C-1161) at a concentration o f 0.23ixg mL" 1 , and AP-labeled goat anti-trout immunoglobulin ( K P L , Gaithersberg, M D , U S A . # 05-29-05) at a concentration of 0.2ug mL" 1 . The antibody solutions as well as the blocking buffer were freshly prepared. To prepare A P -substrate, 66pL of N B T and 33 u L of B C D 3 were added tolOmL o f AP-buffer for each N C membrane. 3.16.3. Enzyme linked immunosorbent assay (ELISA) E L I S A was performed to determine the total IgY, as well as specific anti-K anguillarum I gY content of the W S F , its retentate and the lyophilized powder in each 73 batch of egg yolks, as well as in fish serum and the extract of the fabricated fish pellets. A l l the necessary buffers were prepared according to Kummer et al. (1992) as previously described in "buffers". 3.16.3.1. Determination of total IgY content A sandwich E L I S A (Fig.3.7) was conducted to determine total IgY content of the samples. Flat-bottomed 96-well polystyrene microtiter plates, Immulon 2 (Dynex Technologies, Inc., Chantilly, V A , U S A . #3455), were coated with lOOpL of l p g mL" 1 rabbit anti-chicken IgG in carbonate coating buffer and incubated for 60 minutes at 37°C. After washing the wells three times with PBS-Tween, 250pL of blocking buffer was added to the wells and incubated for 30 minutes at 37°C. The plates were washed three more times and then lOOpL of IgY-containing sample solutions or a standard solution of IgY (Sigma 1-4881) was dispensed into the wells and incubated for 60 minutes at 37°C. Standard solutions were diluted in PBS-Tween to contain 0.78- 50ng mL" 1 IgY and samples were diluted to fit within the range. Plates were washed four times and wells were applied with lOOpL o f the conjugate (anti-chicken IgG alkaline phosphatase, developed in rabbit, Sigma A-7191) diluted in PBS-Tween. After 60 minutes incubation at 37°C, plates were washed four times with PBS-Tween followed by a final rinse with distilled water. The last step was addition of lOOpL substrate solution into each well. Substrate solution contained 0.5mg alkaline p-nitrophenyl phosphate (p-NPP, Sigma 104-105) per mL D E A buffer. Development of yellow color caused by enzyme-substrate reaction was measured at 405nm, using E L I S A plate reader (Labsystems i E M S Reader M F , Labsystems O Y , Finland). 74 Adsorb the antibody to the surface of the flat-bottomed microtiter plate wells. 1 y y y v y Rinse the unbound antibody. Block the rest of the surface with a neutral protein and then rinse the unbound protein. Add the sample to the solid phase A antibody. The solid phase antibody f specifically captures any target 1 v v Y antigen within a complex sample. Rinse the unbound sample material. Add the enzyme-conjugated antibody, specific for the target antigen, to the solid phase. This labeled antibody attaches to any antigen captured by the solid phase antibody. Rinse the unbound enzyme-labeled antibody. Add the enzyme substrate to the solid phase. The amount of the colored product that develops is proportional to the amount of antigen in the sample. KEY: Solid phase Capture antibody Target antigen Enzyme-labelled antibody • Colored product ol enzyme substrate Fig.3.7. Procedure of sandwich E L I S A (adapted from Rittenburg, 1990, with modification). Standard and samples were assayed in triplicate. Readings of the pure IgY range of sample were used to establish a standard curve. Microsoft Excel version 97 was used to transform absorbance values from E L I S A plates to IgY concentrations using a linear regression equation. 3.16.3.2. Determination of anti-Vibrio specific IgY titer E L I S A technique (Fig.3.8) was employed to determine specific anti- V. anguillarum IgY titer o f the samples. Formalin- kil led V. anguillarum suspension was prepared following the procedure described in vaccine preparation except that the final concentration was adjusted to As40nm = 1 and stored at -20°C after being distributed in 1.5mL sterile capped tubes. It was then thawed and diluted ten times in carbonate coating buffer to coat the wells of E L I S A plates (lOOuL per well). Blocking, conjugate and the substrate were identical to those in determination of total IgY. A series of doubling dilutions of each sample in PBS-Tween was added into the wells in triplicates or duplicates (lOOuL per well). A panel of 8 dilutions of the negative control samples of the same nature formed the basis for determination of anti-Vibrio IgY titer in the test samples. Mean o f the absorbance readings o f triplicate or duplicate negative control wells was used to calculate the mean o f the panel plus two times the standard deviation (mean + 2 SD). The smallest mean o f absorbance readings of each triplicate or duplicate wells of the test sample above this value was considered as antigen positive and the corresponding dilution was accepted as the specific anti-K anguillarum IgY titer (Catty & Raykundalia, 1988, p. 127). 76 Adsorb the antigen to the surface of the flat-bottomed microtiter plate wells. Rinse the unbound antibody. Block the rest of the surface with a neutral protein and then rinse the unbound protein. Add the sample to the solid phase antigen. The solid-phase antigen specifically captures any target antibody within a complex sample. Rinse the unbound sample material. Add the enzyme-conjugated antibody, specific for the target globulin, to the solid phase. This labeled antibody attaches to any primary antibody captured by the solid phase antigen. Rinse the unbound enzyme-labeled antibody. Add the enzyme substrate to the solid phase. The amount of the colored product that develops is proportional to the amount of antigen in the sample. K E Y : | | Solid phase Target antigen Primary antibody Q Colored product of enzyme substrate Fig.3.8. Procedure of antibody capture E L I S A (adapted from Rittenburg, 1990, modification). 3.17. Drying of WSF To facilitate commercial use of IgY as a feed ingredient to fortify fish health, feasible preservation methods had to be sought. Dehydration of WSF as a source of IgY was investigated since a dried WSF powder does not need a large storage space and is easy to preserve. Ultrafiltration (UF) retentate of WSF was dried using three different methods. 1. Freeze drying for 48 hours at shelf temperature of 20°C, pressure of 0. lmmHg and a condenser temperature of -55°C (using a freeze-drier manufactured by VirTis Research Equipment, Gardiner, NY , USA). 2. Vacuum-microwave drying, using a household microwave oven (Matsushita Electric Ind. Co. Ltd., Japan) with a maximum power of 700W, which was modified to function as a vacuum microwave drier. For this purpose, a glass desiccator, placed inside the oven, was connected to a vacuum pump via tubing through the holes made on the top wall of the oven and the desiccator lid. A volume of 20mL of WSF U F retentate in a 250mL beaker was placed inside the desiccator. Another 250mL beaker containing 150mL water was placed in the oven outside the desiccator to absorb some microwave power in order to prevent burning of the W S F solid contents, especially when there was not enough free water left in the sample due to evaporation during the drying process. Vacuum to absolute pressure of 20mmHg was applied and the microwave power was run at 0W for 1 minute to let enough vacuum build up in the desiccator before heating, followed by 70W for 1 minute, 210W for 3 minutes, 350W for 3 minutes and 420W for 9 minutes. The temperature of the dried WSF at the end of process was 31°C. 3. Spray drying of the WSF after the addition of lactose (analytical grade, B D H Chemicals, Toronto, Canada) to a final concentration of 25%, providing a higher level of solids 78 content for a more efficient spray drying. Spray drying was conducted at inlet and outlet temperatures o f 150°C and 92-102°C, respectively, using a Lab-Plant Spray-drier (SD-04 Lab Plant Manufacturing Ltd. , Leeds, England). A l l o f the dried products were screened using BioRad protein assay and the result was used to prepare solutions of the dried samples at a similar protein concentration level. These solutions were used in E L I S A to determine the specific anti-K anguillarum IgY titer in the samples. 3.18. Dehydration of IgY-containing pellets To investigate the stability o f IgY during dehydration when incorporated into the fish feed pellets, IgY-containing pellets were produced and dehydrated using three methods; freeze-drying, vacuum-microwave drying, and air-drying. The IgY concentrations in the pellets before and after dehydration were evaluated using E L I S A . Details on incorporation of IgY into the pellets and dehydration of them are described below. 3.18.1. Initial dehydration & IgY incorporation Three kilograms of commercial fish pellets having a moisture content of 8.76% (dry base, db) were dehydrated using a 1.5kW vacuum microwave ( V M ) dehydrator, operating at 2450MHz microwave frequency (EnWave Corporation, Port Coquitlam, B C , Canada). Five cheesecloth bags containing 500g pellets each, were placed in the drying cavity o f the V M drier (a rotating cylinder o f approximately 0.27m radius and 0.3m length which tumbled the sample during drying) in two batches of 2 or 3 bags. 79 Microwave power of OkW for 1 minute followed by 1.5kW for 5 minutes and again OkW for 3 minutes was employed at a constant absolute pressure of 50mmHg to dry the pellets. A l l 5 bags of pellets were mixed in a sealed plastic box and incubated overnight at ambient temperature to equilibrate the moisture among all. Final moisture content of the mixed pellets as determined by drying in a hot air oven (Blue M Electric Co. , Blue Island, EL, U S A ) at 100°C for 36 hours was 5.3% (db). This pre-dehydration of pellets was performed to increase their liquid absorption capacity. The ultrafiltration retentate of W S F prepared from the yolk of commercial white eggs was sprayed onto the dehydrated pellets. The resultant moisture content of the wet pellets was 27% (db). Three different drying techniques, freeze-drying, vacuum microwave drying and hot air dying were employed to dry the W S F -containing pellets. Triplicate samples were collected from untreated pellets as well as pellets sprayed with W S F or dried using all three different methods. Subsequently, total IgY content of the samples was determined using the E L I S A technique. Temperature measurement of the pellets was performed using an Infra-red Thermometer (Model 39650-04, Cole-Parmer Instrument, Chicago, IL, U S A ) . 3.18.2. Freeze-drying 250g of the wet IgY-containing pellets were spread on aluminum trays and freeze-dried (Labcono Corp., Kansas City, M O , U S A ) at a shelf temperature of 20°C, pressure o f 0. l m m H g and a condenser temperature o f -55°C. 80 3.18.3. Vacuum-microwave drying A microwave power of OkW for 1.5 minutes followed by 1.5kW for 5 minutes and OkW for 3 minutes at a constant absolute pressure of 50mmHg was used to dehydrate 500g of the pellets sprayed with W S F . 3.18.4. Air-drying A belt counter current air-dryer ( V E R S - A - B E L T , Wal-Dor Industried Ltd., New Hamburg, Ontario, Canada) was used at 90°C for 8 minutes and 45 seconds to dry 300g of the wet pellets. Temperature of the pellets at the end of the drying process was 60°C. 3.19. Statistical methods Mean values of immunologically active IgY in the serum of the test and control fish following different treatments, as well as IgY concentration in the pellets were calculated. The observations were analyzed using one way analysis of variance ( A N O V A ) . Fisher's Least Significant Difference (LSD) multiple range test was employed for pair-wise comparison o f the means of different treatments in each set o f data. The data collected from mortality rates in fish following bacterial infection was processed in the same manner. To satisfy statistical considerations concerning normality and homogeneity of variances, data were transformed when necessary. Lilliefors (Conover, 1980) method was used to test data for normality of distribution and Levene's (Snedecor & Cochran, 1980) method was employed to test for the homogeneity of variances. A l l o f the statistical evaluations were performed at the 81 significance level of a=0.05. Microsoft Excel version 97 was used to calculate mortality values and to determine IgY concentrations employing a standard linear regression equation. Statistical analysis was performed using S Y S T A T version 8.0 (SPSS Inc., Chicago, EL, U S A ) . 3.20. Histological examinations To study possible changes in the fish GI tract due to oral intake of detergents, histological examinations were conducted. In experiments 7, 8, and 9, three fish o f each treatment were killed by a blow to the head at the day o f challenge and immediately dissected to expose the viscera. Also in experiment 4, a similar sampling was performed 3 hours after intubation of W S F in a 1% antacid solution, co-administered with a detergent into the fish stomach as detailed in section 3.16.4. Samples were also prepared from the control fish intubated with P B S or a solution of W S F in P B S . In all cases, the entire intestine (after the pyloric caeca to the anal opening) was removed and immediately preserved in the fixative solution (10% buffered formalin as described in section 3.1). Histological cross sections of the specimens were prepared and subsequently stained with haemotoxylin and eosin ( H & E ) and mounted onto glass slides for microscopic examination to study possible changes occurred in the brush border and columnar epithelia of intestinal microvill i . Microscopic photographs from the intestinal tissues were taken using a Carl-Zeiss microscope (Axioskop M C I 0 0 , West Germany) equipped with a camera. In the experiment 6, three untreated fish as well as three fish from the treatment intubated with spWSF and Mega9 were sampled for histological studies 3 hours after intubation. 82 Intestine, stomach and pyloric caeca were removed, fixed and examined similarly. In this experiment, microscopic photographs from the stomach and pyloric caeca tissues were also taken. To make sure that formalin had completely and immediately reached all parts of the intestine for a proper fixation, an untreated fish was sacrificed and the entire intestine, after being removed, was flushed with buffered formalin a few times and subsequently fixed in the same fixative solution. In another trial, 3 hours after intubation with 300pL of a 5% concentration of Mega9 or octyl-p-glucoside, or 1% Na-deoxycholate all dissolved in 1% N a H C 0 3 solution, the intestine of rainbow trout (80g average weight) was removed in a similar manner. A lateral cut was made along the entire length of the intestine and immediately fixed in Bouin's solution. Three fish from each treatment, as well as untreated fish and fish intubated with sterile PBS were sampled for similar histological studies using H&E stained cross sections of the entire intestine. 83 CHAPTER FOUR RESULTS & DISCUSSION 84 4.1. Cellular and surface antigens of V. anguillarum interacting with chicken IgY, rabbit IgG or fish IgM In the present study, administration of chicken IgY was considered as a means of passive immunization against vibriosis. As a preliminary step, the potential of IgY of the immunized hens to bind membrane proteins and lipopolysaccharide (LPS) of V. anguillarum was examined. This was also compared to the binding characteristics of rabbit IgG and rainbow trout IgM. The whole cell lysate ( W C L ) preparation contains cell envelope and cytoplasmic membrane proteins, L P S fractions and other cellular proteins. F i g A l . a shows the coomassie blue stained S D S - P A G E profile o f the W C L . Presence of prominent bands around a M W of 40kDa corresponds with the major outer membrane protein ( M O M P ) observed in this region by Chart & Trust (1984). Appearance o f these bands was also reported by Knappskog et al. (1993) for V. anguillarum isolated from diseased cod. Clusters o f closely positioned bands in the regions o f 14-20kDa and 44-68kDa M W were also observed. F i g A l . b illustrates the electrophoretic L P S profile of V. anguillarum, obtained from silver stained S D S - P A G E gel. This profile consists of a typical ladder-like pattern of bands, which was described by Tsai & Frasch (1982). The micro-heterogeneity of the intermediate molecular weight ( IMW) fraction of V. anguillarum L P S is apparent with four distinct bands being clearly resolved. The four bands in the approximate M W regions of 22, 30, 45 and greater than 97.4kDa represent L P S molecules that differ from one another largely in the number of the O-polysaccharide repeat units and thus exhibit variable chain length. The fast migrating phospholipid and lipid A was appeared below M W o f 14.4kD. This fraction occurred at the same 85 M W region for all virulent V. anguillarum serotypes examined by Knappskog et al. (1993). The antigenicity of whole cell components of V. anguillarum in rabbit, chicken and fish was assessed by the Western blot technique. A s demonstrated in Fig.4.2, rabbit polyclonal antibodies, IgG, as well as chicken IgY reacted strongly with L P S species in the M W zones of approximately 27.5, 49.5, 75 and 95kDa. The core phospholipid and oligosaccharide-lipid A fraction proved non-antigenic as judged from lack o f staining in the L P S regions lower than 14.4kDa upon reaction with antibodies in the Western blot. Rainbow trout antibodies, however, reacted only with L M W (27.5kDa) species of L P S and no staining was observed with components of the O-polysaccharide. Hastings & El l is (1988) found that although rabbit antibodies recognized 14 components in the extracellular products of Aeromonas salmonicida, rainbow trout antibodies reacted with only four. Fish are known to produce a much smaller repertoire of antibodies than mammals (Du Pasquier, 1982). Thus, the reduced LPS-antibody reactivity seen with rainbow trout antibodies may simply represent inability o f fish to react with more than a small proportion o f the L P S epitopes o f a given bacterium. Other causes for the low reactivity o f the fish antibodies with L P S may be attributed to conformational changes or alterations in accessibility of the L P S epitope resulting from electrophoretic and N C transfer treatments. Steric hindrance due to long O- polysaccharide chains which may prevent rainbow trout I g M from binding to specific epitopes on the higher M W species of L P S is a distinct possibility. However, none of the latter is a strong assumption since electrophoresis and Western blotting has been employed in similar conditions for 86 chicken IgY and rabbit IgG that resulted in higher antigen-antibody reactions. Antibodies o f all three animals reacted with those cellular proteins in W C L o f V. anguillarum in the L M W and high M W regions. However, fish antibodies did not react with the 95kDa M W antigens and only chicken IgY recognized a cellular protein of approximately 14kDa M W . Fig.4.3 shows the reactivity pattern of antibodies of non-vaccinated animals with V. anguillarum antigens in Western blots. Noteworthy was the strong reaction of antibodies of non- vaccinated chicken and fish with a cellular protein in the vicinity of 32kDa M W . N o reactivity with L P S or W C L was observed with the serum of non-vaccinated rabbit. The antigen-antibody reaction shown in Fig.4.2 explains why IgY used in passive immunization, especially when obtained from the egg yolk of vaccinated hens, could confer protection in fish against a subsequent experimental infection with V. anguillarum. According to the reaction observed between V. anguillarum antigens and IgY from non- vaccinated chickens (Fig.4.3), a partial protection would be understandable i f conferred by passive immunization o f fish using IgY o f non- vaccinated chickens. 87 WCL M LPS M (a) (b) Fig.4.1. (a) S D S - P A G E profile of 15pL whole cell lysate ( W C L ) of V. anguillarum stained by coomassie blue, (b) S D S - P A G E profile o f 20pX proteinase K digested W C L of V. anguillarum (LPS) stained by the L P S silver staining procedure. Core oligosaccharaide-lipid A fraction is indicated by an arrow. Molecular weight standards are presented in the columns " M " in kilodaltons. 88 M LPS WCL WCL M LPS WCL LPS (a) (b) (<0 Fig.4.2. Western blots of V. anguillarum antigens (proteinase K digested whole cell lysate: lane marked as LPS; whole cell lysate: lane marked as WCL). The primary antibody used in immunoblotting was acquired from vaccinated (a) fish serum, (b) rabbit serum, (c) chicken egg yolk water-soluble fraction. Molecular weight standards are presented in the columns "M" in kilodaltons. 89 WCL LPS 106.0 80.0 49.5 M WCL LPS W C L LPS 32.5 27.5 18.5 (a) (b) ( c ) Fig.4.3. Western blots of V. anguillarum antigens (proteinase K digested whole cell lysate: lane marked as L P S ; whole cell lysate: lane marked as W C L ) . The primary antibody used in immunoblotting was acquired from non-vaccinated (a) fish serum, (b) rabbit serum, (c) chicken egg yolk water-soluble fraction. Molecular weight standards are presented in the column " M " in kilodaltons. 90 4.2. Dehydration of WSF In order to use IgY as a feed ingredient to enhance fish resistance against diseases in commercial scale, dehydration of W S F was investigated as a preservation method. A dried W S F powder is a source of IgY that does not need a large storage space and is easy to store and transport. As a preliminary method, as recommended in the literature, ultrafiltration (UF) was used to remove most of the water from W S F prior to dehydration using more advanced dehydration methods. U F concentrate of W S F was dehydrated using freeze-drying, spray drying and vacuum microwave drying. Furthermore, to produce IgY-containing feed pellets W S F concentrate was sprayed onto the commercial pellets followed by dehydrating the wet pellets using hot air drying, freeze-drying and vacuum microwave drying. The results of both studies are detailed in the following sections. 4.2.1. Dehydration rate To suggest a feasible method of producing IgY-containing fish feed pellets, various dehydration methods were compared. One of the considerations in this comparison was the efficiency of dehydration. As described in materials & methods, fish feed pellets were sprayed with W S F of egg yolks and subsequently dehydrated. Dehydration of fish feed pellets using three methods: vacuum-microwave drying ( V M D ) , freeze-drying (FD) and air drying (AD) occurred at different dehydration rates. In the conditions of this study, the moisture in 500g of pellets was reduced from 27% (db) to 6.7% in only 5 minutes and the temperature at the end of the process was 45°C, when V M D was used. The corresponding final moisture content and pellet 91 temperature for 300g of pellets which had undergone A D for 8 minutes, 45 seconds at an air temperature of 90°C were 8.76% (db) and 60°C. The final moisture content and pellet temperature for 250g pellet samples dehydrated using F D for 72 hours were 1.74%> and 20°C. Therefore, dehydration rates were calculated as being equal to 2.44, 1.25 and 0.004 kg water kg"1 dry matter hour"1 for V M D , A D and F D , respectively. In this way, V M D was 610 and 1.95 times faster than F D and A D , respectively. L i n et al. (1998, 1999) observed a distinctively faster dehydration rate for V M D as compared to A D and F D when dehydrating shrimps or carrot slices. They reported a dehydration rate of 6 to 16 times faster for V M D when different microwave power and vacuum levels were used than for A D . The dehydration rate was 123 to 288 times faster for V M D than for F D in the case o f shrimps, and 1470 times faster in the experiment with carrot slices. These results are concurrent with the results of the present study in which dehydration occurred in a much faster rate in V M D than in A D and especially than F D . However, due to the different vacuum and microwave power levels used in their studies and the varying nature of the test materials, the dehydration rates acquired were different from this study. The rapid mass transfer in V M D resulted from high internal pressure generated by microwave energy combined with the low chamber pressure provided by vacuum. The large vapor pressure differential between the center and the surface of the product helps the quick removal of moisture. Although vacuum level affected the speed of dehydration, the more significant factor proved to be the microwave power (Lin et al., 1999). Although air-drying in the present study was only 1.95 times slower than V M D , it is known to induce a higher level of damage to the quality o f the dried 92 product (Yang & Atallah, 1985). For instance, oxidative degradative reactions are more likely in A D due to the application of high temperature (Durance, 2000). Therefore, V M D provides a higher quality product in a shorter time than A D . Although V M D proved a favorable dehydration method, since this method is not yet completely developed for dehydration of liquids, in the preparation of stock W S F powder throughout this study F D was used. 4.2.2. Stability of IgY during dehydration when incorporated onto the pellets Spraying of the W S F onto the fish feed pellets and dehydration o f these IgY-containing pellets was used in this research to provide a feed with a potential of fortifying fish resistance against Vibriosis. In order for this diet to serve the purpose, IgY has to remain stable during dehydration since preservation of IgY functional properties counts as an important measure in its efficacy in passive immunization. To investigate stability of IgY during dehydration of pellets, IgY-containing pellets were examined before and after dehydration. The IgY concentrations in the extract of the wet and dehydrated pellets, determined by E L I S A , are summarized in Table 4.1. Dehydration of pellets by A D , V M D and F D methods reduced the amount of functional I gY less than 1% that was not a significant decrease (p < 0.05). One-way analysis of variance ( A N O V A ) was performed for reduction of IgY due to dehydration (IgY concentration in dried pellets subtracted from that of the wet pellets, db). The results indicated that there was no significant difference between the three techniques with regards to the damage incurred to IgY (p=0.991). One-way A N O V A also showed that there was no significant difference between the total IgY 93 Table 4.1. Changes in IgY concentration due to dehydration treatments IgY concentration in pellets (mg g"1) IgY reduction (%) V M D F D A D V M D F D A D wet 1.689+0.096 a 1.658+0.159 a 1.696+0.129 a dried 1.686±0.106 a 1.658±0.196 a 1.679±0.171 a 0.21 0.01 0.99 Concentration values are mean ± standard deviation of 3 samples. V M D : Vacuum microwave drying; FD: Freeze-drying; A D : Air-drying. Common letters within each column indicate no significant difference (p<0.05). 94 concentration in pellets before and after drying, with the p values calculated as 0.899, 0.968 and 0.999 for A D , V M D and F D , respectively. Therefore all three dehydration methods used in this study could be considered as safe and non-destructive for IgY under the described conditions. 4.2.3. Stability of IgY during dehydration of concentrated WSF In the previous section, effect of dehydration on total I gY activity when incorporated into the pellets was studied. However, anti-P. anguillarum IgY activity and its preservation during dehydration was o f a special interest in the present study. Specific anti-K anguillarum IgY relative titer in W S F of the eggs collected from the vaccinated hens was determined by the E L I S A technique, using doubling dilution as described in the materials & methods. This titer was also measured for the W S F after dehydration with three different methods: V M D , F D and spray drying (SD). Specific anti- V. anguillarum IgY titer was 3200 for all samples. The effect of the non-specific reaction of IgY present in the W S F from non-vaccinated hens was eliminated by comparing the E L I S A readings o f the specific IgY-containing samples to the non-specific W S F sample in calculating the titer, as mentioned in materials & methods. Fig.4.4 and Fig.4.5 display the anti-P! anguillarum IgY relative concentration in W S F before and after dehydration with different techniques. Due to the lack of space in a single microtiter plate for all the samples, the same W S F sample before drying was used in both E L I S A plates as a measure of comparison. As shown in these figures, the concentrations o f the specific I gY are similar in all four samples. This confirms that IgY was stable under all dehydration treatments employed in this study. 95 1.2 j E c 1 - -u> o "* @) 0 . 8 - -4) O 0 . 6 - -c re .Q 0 . 4 - -O in .Q 0 . 2 - -< Specific Anti-V. anguillarum IgY Absorbance y = 0.0035X2 - 0.0495X + 0.176 0.9939 y = 0.0285X2 - 0.3995x + 1.4139 0.9973 y = 0.0256X2 - 0.3802x + 1.433 0.9991 0.0282X2 - 0.4031X + 1.4616 1 2 3 4 Dilution factor (100x2 5 n-1 0.999 6 7 , n= 1 to 8) • Non-specific W S F e Specific W S F A Freeze dried X VM dried . . . Poly. (Non-specific W S F ) Poly. (Specific WSF) • Poly. (Freeze dried) -Poly. (VM dried) Fig.4.4. Specific anti-K anguillarum IgY E L I S A values of a 10-fold diluted water-soluble fraction of egg yolk before and after freeze-drying or vacuum microwave drying. 9 6 Specific Anti-V. anguillarum IgY Absorbance 1.2 j E 1 -c IO o •<* 0.8 -<§) 0) 0.6 -u c CS 0.4 -n o It) 0.2 -< 0 -y = 0.0037x2 • 0.0521X +0.1812 R2=0.9963 y=0.0255X2 • 0.3602X +1.2881 R 2=0.9974 y=0.0248X2 • 0.3494X +1.2431 R 2 = 0.9971 n-1 Dilution factor (100 x 2 , n= 1 to 8) non-specific W S F specif ic W S F spray dried Poly, (non-specific WSF) Poly, (specific WSF) •Poly, (spray dried) Fig.4.5. Specific anti-K anguillarum IgY E L I S A values of a 10-fold diluted water-soluble fraction of egg yolk before and after spray-drying. 9 7 These findings support the apparent stability o f IgY top-dressed onto fish feed pellets observed in the previous dehydration experiment. Similarly, Shimizu et al. (1992) observed no change in the anti-mouse IgG activity of chicken IgY or rabbit IgG after heating for 30 minutes at 62.5°C. IgY activity decreased drastically after heating for 15 minutes at 70°C or higher, and that of rabbit IgG decreased at 75-80°C. In another study, Shimizu et al. (1988) found that anti-El coli L P S activity of IgY obtained from vaccinated chickens was almost intact after 15 minutes at 62.5°C and only slightly (< 5%) reduced after 15 minutes at 65°C. A similar heat stability has been reported for bovine serum IgG, cow's milk IgG and human milk IgG, which retained full activity after 30 minutes at 62.7, 60 or 62.5°C. However, all o f these immunoglobulins were affected when heated above 70°C (Li-Chan et al., 1995; Goldsmith et al, 1983). In our study, freeze-drying did not affect the activity o f IgY when top-dressed onto the pellets or as dehydrated solely when compared with the IgY level in the wet pellets or in the W S F before freeze-drying. It was noteworthy that all o f the W S F samples were frozen at least once before being used in any kind of dehydration studies. The results of the present study are in accordance with those obtained by Shimizu et al. (1988) who observed no loss in IgY activity due to freezing or freeze-drying unless repeated several times. However, Chansarkar (1998) reported a 40% decrease in the absorbance value at 405nm due to freezing followed by freeze-drying when IgY activity was determined by E L I S A technique. The samples in that study were 90% pure IgY which were frozen at -80°C in a concentration o f 30mg mL" 1 or 98 l m g mL" 1 in a high salt (1 .5M NaCl) or low salt (0.14M NaCl) solution prior to freeze-drying. 4.3. Post-vaccination IgY level in the yolk Efficiency o f IgY production following vaccination of hens was studied. Approximately 26 eggs were collected from each hen per month and 15.4mL yolk per egg was obtained on average. The water-soluble fraction (WSF) of the egg yolks from immunized and non-immunized hens was extracted in batches o f biweekly or in some cases monthly collections o f eggs. In each batch, concentration of total IgY was measured using the E L I S A technique. The average concentration of total IgY was 5.93 ± 1.26mg mL" 1 yolk or 91.4mg egg"1 over the whole period of egg collection. This is approximately half the IgY (12.53mg mL" 1 yolk) collected by Aki ta & Nakai (1992) at the same p H and using the same recovery method but from the egg yolk of white leghorn hens. However, they estimated IgY concentration in the W S F using a radial immuno-diffusion assay (RED) which is different from the method used in the present study. Based on the present results and 26 eggs hen"1 month"1, 2.38g IgY is obtainable per hen in a month or 28.56g hen"1 year"1. Fig.4.6 illustrates the fluctuation in the concentration of total I gY in the W S F (10-fold dilution of egg yolk) over the period of egg collection. The post-vaccination total IgY concentration in the W S F obtained after completion of initial vaccination followed by 5 weekly boosting varied in the range of 0.44-0.94mg mL" 1 W S F , which was slightly higher than pre-vaccination values. This increased level o f IgY was maintained over the period of screening except for a few sampling points. The eggs of vaccinated hens were not used for 6 weeks after the first vaccination due to reports of 99 a low titer of specific IgY in the yolk during this period (Li-Chan, 1999). Therefore, no data was collected on IgY concentration during this period. The specific anti-P". anguillarum IgY titer of the W S F determined by E L I S A varied between the two subsequent titers o f 12800 and 25600 (Fig.4.7). Due to doubling dilution in the preparation of the W S F samples for E L I S A the data points appeared as a discontinuous variable. N o consistent trend was observed between boosting and increase in the titer. However, the boosters obviously helped maintain a high titer of specific IgY in the W S F . Since the W S F of the pre-vaccination eggs was used as a base to determine the specific titer (see materials & methods for details), it was not possible to determine a titer for pre-vaccination eggs using this method. However, as the degree of non-specific reaction between the V. anguillarum coating o f the E L I S A plates and the W S F of non-immunized hen eggs was considered as the base line, the specific titer of such eggs could be assumed equal to zero. Fig.4.8 presents an example o f the magnitude o f the differences between the levels o f detected specific IgY in eggs from immunized hens as compared to the non-specific control W S F extracted from the eggs of non-immunized hens. Other researchers have used different protocols with respect to vaccination of chickens. L i -Chan (1999) immunized chickens against bovine serum IgG and cheddar cheese whey ( C C W ) with boosting at 3, 5, 7 and 25 weeks after the initial immunization. A n t i - C C W and anti-IgG antibodies peaked at weeks 5 and 6 in the egg yolk and began decreasing after 9 and 12 weeks, respectively. However, anti-IgG antibody levels remained high with a slight fluctuation, until week 24 when it reached its lowest level, whereas ant i -CCW IgY levels remained at lower levels between 100 Total IgY Level in the W S F Total IgY Date of egg col lect ion Fig.4.6. Fluctuation in total IgY concentration of a 10 - fold diluted water-soluble fraction o f egg-yolk. Arrows indicate the initial and booster injections. 101 >-cn o a> Q . (0 Anti-V. anguillarum IgY Titer in the W S F • sp-lgY titer Date of egg col lect ion Fig.4.7. Fluctuation in specific anti-K anguillarum I gY titer of a 10-fold diluted water-soluble fraction of egg yolk. Arrows indicate the boosting dates. Data is not available for the first 6 weeks after initial vaccination. i 102 Specific Anti-V. anguillarum IgY A b s o r b a n c e 0.3 i E c 0.25 -u> o 0.2 -® 0) 0.15 -o c ra X ! 0.1 -o 0.05 -w X) < 0 -3 i 1 2  4 5 6 Dilution factor (800x2 n= 0 to 5) ^ n o n - s pecif ic E3 24-Apr A 22-May X 2 9 - M a y X 5 - J u n e 12-Jun + 19-Jun - 2 6 - J u n — 3-Jul • 17-Jul Fig.4.8. Levels o f specific anti-F! anguillarum IgY in the water-soluble fraction of the egg yolks collected from immunized hens in different post-vaccination dates compared to pre-vaccination level (indicated as non-specific). 103 weeks 10 and 24. In both cases, the IgY level was considerably higher than pre-immunization levels at all times. Boosting at week 25 increased both types of antibodies close to their maximum levels. This fact shows that immunization against different antigens may need different protocols to obtain the optimum output as fewer numbers of boosters with longer intervals produced a prolonged high titer in the case of anti-IgG while more frequent boosting might be helpful to sustain a higher titer of ant i -CCW IgY. Poison et al. (1980a) vaccinated 20 weeks old hens with different protein antigens. Three to several weekly boosters, depending on the antigenicity of each specific antigen, followed the initial injection to elicit antibodies in adequate titer in the egg yolk. The authors suggested that molecular weight ( M W ) of antigen was an important factor in eliciting antibodies in hens. Only antigens with a M W equal or greater than 150kDa appeared to produce good responses in hens. Lee et al. (2000), who immunized white leghorn hens against Y. ruckeri, boosted the hens only at 2, 3, 4, 5 and 10 weeks after the initial immunization. They achieved a high titer of specific IgY in the eggs throughout the 16 weeks of study, except for a drop in titer after 8 weeks. Considering all other studies, it could be possible to achieve a satisfactory high level o f specific I gY in the egg yolk using fewer boosters. 4.4. Passive protection induced by D? injected anti-Vibrio IgY to fish Inducing passive protection in rainbow trout after a single EP injected dose o f anti-K anguillarum IgY and duration of such protection was studied. This experiment was conducted jointly with Abdul-Hossein Aminirissehei at the Department of 104 Biological Sciences, Simon Fraser University, at Burnaby, B . C . , Canada. Immersion challenge, as described in materials & methods, after 1, 3, 7 and 14 days resulted in significantly lower mortality levels in the groups of fish receiving specific IgY than those groups IP injected either with non-specific IgY or with P B S in all challenge experiments performed after different periods of time (p < 0.05). N o significant difference was observed between mortality levels o f the P B S and non-specific IgY treated groups on any challenge date. Fig.4.9 demonstrates these results. As illustrated in Fig.4.10, the cumulative 14 days post-challenge mortality rates within each treatment group did not change significantly for the fish groups challenged after different time intervals from the initial EP injection. The collective results of the total serum IgY level in all treatments and specific anti-P". anguillarum IgY titer in the fish groups IP injected with specific IgY, as well as cumulative mortality rates are summarized in Table 4.2. In a similar challenge study, Aminirissehei (1999) found a greater disease resistance in the fish D? injected with anti-K anguillarum IgY than those which received the same dose o f non-specific IgY. Lee et al. (2000) found no Yersinia ruckeri in the intestine or kidney of rainbow trout 7 days after an immersion challenge with the pathogenic bacteria when fish were EP injected with specific anti-F. ruckeri IgY 4 hours before challenge. However, the bacterium was detected in both kidney and intestinal tissues of the control fish groups injected non-specific IgY or saline. Akhlaghi (1999) obtained a significant protection against vibriosis in a challenge with the pathogenic bacteria up to a month following the passive immunization of rainbow trout using anti-K anguillarum antibodies raised in sheep and rabbits via 105 Temporal Post-Challenge Mortality Rate 120 -, Day 1 D a y 3 Day 7 D a y 1 4 Post-injection challenge date Fig.4.9. Cumulative 14-day post-challenge mortality rates in fish groups EP injected with PBS , non-specific or specific anti-K anguillarum IgY compared for each challenge date. Values are the mean o f 3 tanks ± standard deviation. Similar letters indicate no significant difference within each challenge date (Day 1: lower case, Day 3: double lower case, Day7: upper case, Day 14: double upper case letters) (p < 0.05). 106 Post-Challenge Mortality Rate 120 i 100 -o 80 -ra >« 60 -+ J rtal 40 -o 20 -0 -a a L T f specific IgY non-specific IgY PBS IP injection treatment Fig.4.10. Cumulative 14-day mortality rates in groups of fish challenged at different post-injection dates compared within each treatment group (IP injected with P B S , non-specific or specific anti-P! anguillarum IgY). Values are the mean of 3 tanks ± standard deviation. Similar letters indicate no significant difference within each treatment (Specific IgY: lower case, Non-specific IgY: double lower case, P B S : upper case letters)(p> < 0.05). 107 Table 4.2. Serum IgY levels & mortality rates in the fish D? injected with anti-K Challenge date (days post-injection) Day 1 Day 3 Day 7 Day 14 Vibrio count in challenge (cfu mL"1) 8.2xl04 3.2 xlO 3 3.3 xlO3 2.0 xlO 3 Treatment 1 Anti-K £ anguillarum IgY (IP) Mortality (%) 8 11.1 ± 3 . 8 a * 2.56E+04 ± 5.20E+02 31.1 ± 7 . 7 a 1.47E+04 ± 3.17E+03 24.4 ± 13.9 a 1.15E+04± 4.55E+03 17.8 ± 13.9 a 3.80E+03 ± 3.26E+03 Total serum IgY * (ng mL"1) Anti-Vibrio IgY titer 256 64 32 32 Control 1 PBS (IP) Mortality (%) 75.6 ± 10.2 b 4.84E-01 ± 4.42E-01 75.5 ± 3 . 8 b 6.26E-01 ± 3.33E-01 62.2 ± 16.8 b 2.95E-01 ± 2.54E-01 57.8 ± 26.9 b 1.02E-01 ± 1.71E-02 Total serum IgY (ng mL"1) Control 2 Non-specific* IgY (IP) Mortality (%) 71.1 ± 10.2 b 1.02E+02± 1.29E+02 71.1 ± 27.8 b 1.69E+04± 6.94E+03 57.8 ± 7 . 7 b 5.22E+03 ± 4.73E+03 82.2 ± 7.7 b 3.74E+03 ± 1.41E+03 Total serum IgY (ng mL"1) (p<0.05). Total IgY level in anti-Vibrio IgY injection preparation^ .33mg mL"1. ¥ Total IgY level in non-specific IgY injection preparation^. 15mg mL"1. § Mortality value is mean cumulative 14 days post-challenge mortality rate of 3 tanks ± standard deviation. $ Serum IgY value is mean of 3 fish ± standard deviation. Sample size: 3 tanks of 16 fish per treatment. Average fish weight = 68g. Volume of injection: lOOuLfish"1. 108 intraperitoneal (EP) injection. Other results of the same study showed that non-immune sheep sera conferred no protection. The results of the present study and the reviewed studies, prove that specific antibodies raised in other animals, including chicken IgY, are capable of enhancing resistance to disease-causing bacteria when it reaches the fish system in sufficient amounts. It also indicates that protection arises from specific IgY activity against the causative organism, not from any other source that might exist in the water-soluble fraction of the egg-yolk, nor from a non-specific reaction between IgY and the bacterial pathogen. A s illustrated in Fig.7.1 (in the appendix), total serum IgY level in the groups of fish EP injected with anti- Vibrio IgY showed a general significant decreasing trend during the course of the study, although the levels at days 3 and 7 were not significantly different. The highest level o f serum IgY at day 1 corresponded to the lowest mortality rate among four different challenge dates. However, temporal trends of the significant levels for total I gY were not similar to that of the mortality rates, which did not change significantly over time. There was no significant linear correlation (p = 0.365) between total serum IgY levels of the sampled fish and the mortality rate (%) following an immersion infection (r 2 = 0.403) when this relationship was studied for the fish groups EP injected with specific anti-K anguillarum I gY on different challenge dates. Serum IgY and mortality rate values used for this calculation represent the average of three samples collected at each challenge date (one fish from each experimental tank). Also as shown in Fig.7.2 (in the appendix), the highest anti-K anguillarum IgY titer in the fish serum corresponded to the lowest mortality rate. A linear regression analysis indicated no significant 109 correlation between anti-Vibrio serum titer and the mortality rate (r2=0.477, p = 0.309). This provides a similar conclusion conceived from the relationship of total serum IgY level and mortality rate discussed above. Corresponding total IgY levels and specific anti-P anguillarum IgY titers during the course o f the study are plotted in Fig.7.3 (in the appendix) for the fish groups EP injected with specific IgY. A s demonstrated in this figure, both specific and total serum IgY levels had descending trends over time. However, it was not possible to calculate the ratio of specific to total serum IgY level due to the use of different measurement techniques and units for these two purposes. Aki ta & Li -Chan (1998) found that approximately 10% of the total IgY obtained from egg yolk preparation of the hyperimmunized hens was specific to bovine IgG. In their study, they measured the amount of affinity purified specific anti-bovine IgY using a P J D method where the results could be translated to weight per volume amounts of IgY. In the present study, it was not feasible to measure the amount o f purified specific anti-K anguillarum IgY in the fish serum due to the small volumes of the serum samples. 4.5. Anal administration of IgY Since the stomach conditions might elicit destructive effects on protein molecules before reaching the absorption sites o f intestine, anal intubation o f IgY was considered to eliminate the stomach barrier from the absorption system. E L I S A results showed no uptake in the serum following anal intubation of lOOpg IgY fish"1 (21g average weight) for the first 22 hours post intubation. Only one o f the three fish sampled at 24 hours conferred a positive result. When this experiment was repeated 110 with different sample collection time intervals, none of the triplicate blood samples collected at 24, 27, 30, 48 and 72 hours showed transfer of IgY into the blood circulation of fish. In contrast, other researchers have reported transport of proteins into the circulatory system of fish following anal intubation. McLean & Ash (data published in McLean et al, 1999) detected up to 70ng mL" 1 horseradish peroxidase (HRP) in the plasma of rainbow trout after anal delivery of a dose of lOOug g"1 body weight. Jenkins et al. (1994) also measured a maximum of 60ug mL" 1 o f human gamma globulin ( H G G ) in the plasma of tilapia after an anal dose o f 2mg fish"1 with a weight of 30-50g. A maximum level o f 2ug mL" 1 o f salmon growth hormone (sGH) was observed in the plasma o f rainbow trout following anal administration o f 200ug per 103g fish (Moriyama et al., 1990). L e B a i l et al. (1989) detected a maximum plasma level of 200 or 500ng mL" 1 of bovine growth hormone (bGH), using two different assays, after anal intubation of a l m g dose into 30-45g rainbow trout. It is noteworthy that in the above studies the dose of administered protein was 5 to 21 times higher than that applied in the present study, except for s G H , which was lower than that used here. Since intestinal absorption of proteins following anal administration is reported to be dose-dependent (McLean et al., 1999; Hertz et al, 1991; Georgopoulou et al, 1988), the lack of IgY in trout plasma following anal administration in the present study might be attributed to the low dose applied. Other possible factors could be the larger molecular weight of IgY and variation in starvation periods before intubation. Effect of starvation was reported by Hertz et al. (1991), who found significantly higher absorption of orally intubated h G H into the blood after 111 12 days starvation than 7 or one day and no h G H in fish intubated 6 hours or less, post feeding. 4.6. Absorption of orally administered IgY into the fish bloodstream To study survival o f IgY in the GI tract of trout and IgY uptake into the bloodstream, oral administration of W S F as a crude source of IgY was applied through intubation as well as incorporation into the feed. To enhance intestinal absorption, various detergents were co-administered with the W S F . Microencapsulation using polylactide-co-glycolide (PLG) was also used to protect IgY from the stomach degradation. The results are discussed for each experiment in the following sections. 4.6.1. Experiment 1. Using IgY in encapsulated form, in Mega9 and antacid, or in Na-pyrophosphate solution In this experiment, effect of a non-ionic detergent, Mega9, in antacid solution, as well as pyrophosphate and microencapsulation in P L G was examined for enhancement of I gY absorption in an oral intubation trial. IP injected W S F and intubated P B S served as positive and negative controls, respectively. As shown in Fig.4.11 and Table 4.3, absorption of IgY into the fish blood peaked at 1 or 3 hours after administration, in all treatments. The most effective orally intubated preparation was L - W S F administered in conjunction with 5% Mega9 in a 1% sodium bicarbonate (antacid) solution. IgY uptake in this approach was 14 to 158 fold higher at different sampling times when compared with L - W S F dissolved in P B S . Concentration o f total 112 I D O C O L O C D ho in C O O in C O g> /rop LG LG B S Q _ • • 0 s i i i o o o (|Ui/6u) uoijejjuaouoo -a C D s ea u n C D •g 2 ? a "CD C D > +-» -*-> cd o - C CD OH OH CO CO OH of 2 O ft a S 8 .3 o> o £ a CD CD S i + J "I C D 3 o co CO cd C/3 « f £ d P-1 O "O 1 3 | i .g oo "a * J > 1 m . a f t CO O o. CO 03 T3 CD -a d ccj -o CD *H O CD 2 d^  CO td CD O & r>. — o i n ro C+H o d 5 CD 0 CD GO CD fc 'PH > B OH CD ° C/3 m . 0 0 113 oo £ oo eight. eight. LI S SP .§«•: s oo •—1 53 » > — cj <+H o X 5 S3 1 bO <=> o o ts S3 -* J o II J=i 1 o 1/1 Pi T 3 O (30 '53 * la £ SJ3 | g >> 'bO t-l > H <=> Of) ^ O ON O II M X j SP t»" I o I f S ° PH « s i ^ S ° -5 U D . 00 T3 u ts u 'o o cn c3 & T3 O S o .1 GO « ON « 0 4 & II « oo o '53 & <u g * 00 op- r 2 &serum IgY in this treatment was significantly higher than all other oral intubation treatments at all sampling times. Although substituting sodium pyrophosphate solution for P B S to dissolve L - W S F increased the mean detectable IgY level in the serum, the difference was not significant at any sampling time. However, serum IgY level after oral intubation of L - W S F dissolved in P B S or pyrophosphate solution was significantly higher than the negative control (intubated PBS) . I gY uptake into the blood was always significantly higher in the fish EP injected with L - W S F in P B S than IgY uptake in any other treatment. Encapsulation of L - W S F in P L G 50:50 or P L G 85:15 (with an average particle size o f 29.39±5.75um for P L G 50:50 & 30.32±5.48um for P L G 85:15, Fig.4.12) did not result in satisfactory IgY serum levels under the conditions of this experiment. The IgY levels associated with encapsulation treatments never significantly exceeded that o f the negative control. These were both significantly smaller than the levels achieved when L - W S F was intubated in an unprotected form. It is noteworthy that the concentration of I gY in the intubated material was theoretically 1.36mg fish"1 in P L G treatments, lower than the 2.68mg fish"1 used in the other treatments. However, this fact does not justify the observation that serum IgY levels in P L G treatments were close to P B S treated fish and much smaller than the fish intubated with unprotected L - W S F . 4.6.2. Time course and levels of absorption after oral or anal administration of proteins The levels o f IgY uptake into the blood and its fluctuations over time when delivered alone or with a detergent were studied. In the present study, the peak level 115 Fig. 4 .12 . Microscopic photograph of P L G encapsulated water-soluble fraction (WSF) of egg yolks. Magnification, as demonstrated by the scale bar, is 150 fold. 116 of 128ng mL" 1 IgY in serum following oral intubation o f unprotected W S F was reached in 1 hour, then gradually decreased. The same trend was observed when pyrophosphate was added. Co-delivery with Mega9 and antacid resulted in a high absorption level o f 1740ng mL" 1 at 30 minutes, a level that was maintained for 24 hours with a slight increase at 3 hour (2500ng mL" 1). Serum IgY subsequently decreased to some extent but was still clearly detectable even after 2 weeks at a level of 29.3ng mL" 1 . A similar pattern was reported by Hertz et al. (1991) who obtained maximum plasma levels 30 minutes after oral intubation of h G H , when delivered alone or in conjunction with deoxycholate. Also , protein delivery to the fish bloodstream via the oral route reached its peak after 15-30 minutes in several other studies (reviewed by McLean & Donaldson, 1990, as shown in Fig.2.3). In contrast, Georgopoulou et al. (1988) observed the peak o f H R P delivery to blood o f trout 12-16 hours after oral intubation, with the first appearance after 7-8 hours. Jenkins et al. (1994) reported that maximum plasma levels were reached 6 hours after oral delivery of H G G to tilapia, whether administered with or without adjuvant, although the first peak appeared after 15 minutes. These authors achieved a similar level o f absorption in oral and anal intubation o f H G G when delivered alone or in conjunction with aluminum hydroxide or cholera toxin P-subunit. McLean & A s h (reported in McLean et al, 1999) observed a higher plasma level of H R P after anal delivery than after oral delivery. These reports are in conflict with the results of the present study, in which anal intubation of IgY did not lead to absorption into the blood. Possible explanations were discussed previously, under the heading " A n a l administration of I g Y ' . 117 4.6.3. Efficacy of delivery into the blood using encapsulated proteins Encapsulation of W S F in P L G 50:50 or P L G 85:15 was used with the hope of increased protection of IgY against the adverse gastric conditions and a higher absorption into the serum. In the present study, this approach did not lead to a higher absorption rate of IgY into the bloodstream of rainbow trout. However, use of these polymers with different levels of success in protection of proteins against degradation and enhanced serum uptake has been reported by other scientists. Lavelle et al. (1997) orally intubated rainbow trout with P L G 50:50 encapsulated human gamma globulin (HGG) . They detected little H G G in the plasma of the fish intubated with unprotected antigen and none in high molecular weight fragments or intact form, while in the fish receiving P L G - H G G the level of intact protein antigen reaching the bloodstream was increased. These researchers concluded that encapsulation partially protected the antigen from proteolysis in the digestive tract. They also suggested that the antigen was not sufficiently released from the microparticles. O'Donnel l et al. (1996) detected no H G G release from P L G 85:15 microparticles into P B S over a 29 week period of in vitro studies while P L G 50:50 was associated with a considerable release o f the antigen. The appearance o f unprotected H G G in the serum o f orally intubated Atlantic salmon was rapid at 15 minutes, peaked at 1 hour and cleared in 4 days. The uptake of P L G - H G G was also rapid and dose related with the first appearance at 15 minutes and a high peak at 3 hours. Encapsulated H G G resulted in higher levels and greater persistence of antigen than the free H G G and presented 2 more peaks at 6 days and 5 weeks (Fig.2.4). Hora et al. (1990) described the three phases of release as 118 being: (1) an initial release of surface bound and poorly encapsulated antigen, (2) diffusion, and (3) degradation of the polymer matrix. The present study, in contrast to the study of O'Donnell et al. (1996) with H G G , did not demonstrate a peak in the serum IgY level following intubation with P L G encapsulated W S F in the entire three week period of the study (data at week 3, available only for encapsulated IgY, is not shown). The amount of in vivo release of W S F as judged by the levels of IgY present in the serum, was only significantly higher for P L G 50:50 than for P L G 85:15 at 3 hours and 2 week sampling times. Both P L G treatments offered unsatisfactory delivery of IgY into the serum. This result could be due to a poor antigen entrapment in the microparticle production process or to inadequate release o f W S F . Although P L G is a favored co-polymer for oral delivery o f proteins due to its biodegradability and non-toxic composition (Lavelle et al., 1997), this approach was not pursued further because the amount of protein that could be encapsulated per gram of P L G was judged inadequate for the specific purpose o f the present study. Jeffery et al. (1993) found that when the amount of encapsulated protein exceeded a ratio of 1:5, the surface of particles appeared pitted and some collapsed. Use of W S F , which contains low purity IgY, makes P L G encapsulation even less effective in delivering sufficient amounts o f pure IgY into the fish bloodstream under the conditions o f the present study. The costs of encapsulating materials and the volumes of encapsulated IgY that fish would need to be fed were considered to be prohibitive. It was estimated that, assuming a 100% efficacy of entrapment and release, 1.5g of P L G costing $20 119 would be needed to deliver a 520mg daily dose of L - W S F per fish to elicit protective effects. 4.6.4. Experiment 2. Tween detergents as absorption enhancing agents Non-ionic detergents from the Tween family (polyoxyethylenesorbitans) were tested for their impact on IgY intestinal absorption as possible substitutes for Mega9. This class of detergents has been considered safe for use in human foods in a limited concentration as noted in Food & Drug Act & Regulations o f Canada, 1994 (pages 67-12A to 67-14). In "Experiment 1", the highest I gY level appeared after 1-3 hours post oral intubation. Therefore, the study of IgY absorption in co-delivery with Tween detergents was limited to the first 4 hours after intubation. A s shown in Table 4.4 and Fig.4.13, serum IgY level did not improve significantly after 1 hour when Tween-20 or Tween-80 was added to the L - W S F solution in P B S or antacid. However, at 4 hour sampling time, a 2.5% solution of Tween-80 in P B S improved the IgY uptake as compared to the solution o f L - W S F in P B S . This level was not significantly higher than that of the treatment with L - W S F solution in sodium bicarbonate. N o significant increase was observed when antacid replaced P B S in any o f the applied concentrations of the detergents. Another disadvantage associated with Tween-80 was poor solubility in P B S or 1% sodium bicarbonate (SBC) solution. 120 Table 4.4. Effect of Tween detergents in uptake of orally intubated IgY into the bloodstream. . Oral intubation treatment § IgY ( n g m L ' 1 ) 1 1 hour IgY ( n g m L ' 1 ) * 4 hours 2.5% Tween80-PBS 176+101 a * 206 ± 4 5 . 0 a * 2.5% Tween80-NaHCO 3 (1%) 1 3 1 ± 1 2 1 a 167 ± 6 3 . 5 ab 5% Tween80-PBS 95.7 ± 8 5 . a 165 ± 1 1 7 ab 2.5% Tween20-NaHCO 3 (1%) 223 +75.7 a 147 ± 3 2 . 1 ab 5%.Tween20-NaHCO 3 (1%) 1 8 9 ± 1 5 1 a 146 ± 6 7 . 7 ab 5% Tween80-NaHCO 3 (1%) 88.7 ± 4 9 . 7 a 122.00 ± 6 9 . 0 ab W S F - N a H C 0 3 (1%) 91.0 ± 2 5 . 9 a 112 ± 58.8 ab W S F - P B S 110 ± 10.0 a 77.3 ± 3 9 . 3 b * Common letters within each column indicate no significant difference between the means (p<0.05). s Values are mean of 3 fish ± standard deviation. § Oral intubation: 200uL of lOOmg mL' 1 fish'1 L-WSF containing 12.4% total IgY (2.68mg IgY fish"1 =167.5mg IgY kg' 1 average body weight), including additives when indicated. Average fish weight = 16g Blood was collected at 1 & 4 hours post intubation. 121 400 350 ^ 300 E O) 250 £ > 200 E 3 CD (0 100 50 Effect of Tween Detergents on IgY Uptake 150 \ ab ab 0 1 h 0 4 h WSFantacid WSF-PBS Oral intubation treatment Fig.4.13. Effect of Tween-80 and Tween-20 at two concentration of 5% & 2.5% on the absorption o f IgY into the rainbow trout bloodstream. Detergents were prepared in P B S or 1% sodium bicarbonate solution. Values are mean of serum of 3 fish. Similar letters within each time indicate no significant difference (1 hour: upper case, 3 hours: lower case) (p < 0.05). 122 4.6.5. Experiment 3. Comparative oral intubation To confirm the findings of the previous oral intubation trials, a combination experiment was performed. Table 4.5 and Fig.4.14 compare absorption enhancing effects of Tween-20 to that of Mega9 and sodium pyrophosphate. Although co-administration of Tween-80 provided a better absorption of IgY in the previous experiment, due to its poor solubility, a 5% solution of Tween-20 in P B S was used in this experiment. In all cases except for L - W S F solution in P B S , serum IgY level was higher at 3 hours than at 1 hour sampling time. Four significantly different levels of IgY uptake into the blood were observed. The highest level coincided with the use of L - W S F in 1% sodium bicarbonate solution containing 5% Mega9. The second and the third highest levels conferred via co-administration of 5% solution of Mega9 in P B S and 5% Mega9 in pyrophosphate solution, respectively. None of the other solutions, i.e. pyrophosphate, 1% sodium bicarbonate, or 5% Tween-20 increased the absorption of IgY when compared to the levels obtained from W S F dissolved in P B S . The results suggest that the strongest effect in enhancement of IgY uptake was conferred by use of Mega9, which acted synergistically when concurrently applied with antacid. The absorption of intact proteins into the bloodstream of rainbow trout following oral administration is reported to be dose dependent (Georgopoulou et al., 1988). McLean et al. (1999) also reported unpublished data from M c L e a n & A s h who observed a direct relationship between dose and uptake of horseradish peroxidase (HRP) when anally intubated into rainbow trout. However, they did not find a clear dose-dependent pattern in orally intubated H R P . Hertz et al. (1991) also reported a linear dose-response relationship for orally intubated human growth hormone (hGH) 123 Table 4.5. Effect of chemical compounds in uptake of orally intubated IgY into the bloodstream. , ___ Oral intubation treatment § I g Y C n g m L ' V 1 hour IgYCngmL-1 ) 3 3 hours WSF-Mega9 ^-NaHCOs 1 1 8 1 ± 1 4 7 292 ± 52.2 a* WSF-Mega9 N A * * 188 ± 0 . 7 1 b WSF-Mega9-pyrophosphate ¥ N A 106 ± 3 0 . 5 c WSF-pyrophosphate 7.20 ± 1.11 23.8 ± 16.2 d W S F - N a H C 0 3 8.85 ± 4 . 5 7 21.9 ± 8 . 8 0 d W S F - P B S 30.5 ± 4 1 . 6 13.6 ± 10.7 d WSF-Tween 20 £ 6.39 ± 1.50 8.68 ± 6 . 5 9 d * Common letters within each column indicate no significant difference between the means (p<0.05). s Values are mean of 3 fish ± standard deviation. § Oral intubation: 200pL of lOOmg mL"1 fish 1 L-WSF containing 12.4% total IgY (2.68mg IgY fish'1 =100.75mg IgY kg"1 average body weight), including additives when indicated. £ Mega9 or Tween-20: 5% in indicated solution. ¥ Pyrophosphate: 0.044g Na4P207,10 H 2 0 in lOmL of 0.44M NaCl, pH 9.4. ^NaHCOB: 1% solution. Blood was collected at 1 & 3 hours after intubation. Average fish weight = 27 g. ** N A : Data is not available. 124 Effect of Chemical compounds on IgY Uptake > E 3 I -in 400 350 300 -I 250 200 150 100 50 0 H jj • 1 h H3h d WSF-Meg- WSF-Meg WSF-Meg- WSF-pyro WSFantacid WSF-PBS WSF-T20 antacid pyro Oral intubation treatment 5% Fig.4.14. Effect of Mega9, sodium pyrophosphate, sodium bicarbonate, 5% Tween-20 or some combinations of them on the absorption of IgY into the rainbow trout bloodstream. Values are mean of the serum o f 3 fish. Similar letters indicate no significant difference (p < 0.05). 125 in starved fish. If the protein absorption is truly dose-dependent, any attempt that increases the amount of protein reaching the sites of intestinal uptake may help enhance absorption. Use of sodium bicarbonate in the present study probably elevated the p H of the stomach and consequently contributed to the increase in the amount of IgY available for intestinal absorption. The observed absorption enhancing effect of antacid co-delivered with Mega9 was consistent with the result reported by McLean et al. (1990) who detected an increased growth stimulating effect of orally intubated somatotropin in rainbow trout when administered simultaneously with Mega9 and sodium bicarbonate. They speculated that the observed result was due to an increased serum level o f the hormone. Also in accordance with our results, McLean & A s h (1990) obtained an 86% increase in plasma levels o f H R P 45 minutes after it was orally co-delivered with 5% Mega9 into rainbow trout. The other possibility investigated in the present study to enhance IgY absorption from fish GI tract was incorporation o f pyrophosphate which is authorized for human use (Food & Drug Ac t & Regulations, 1994, pages 67-16A, 67-3 7 A and 67-53). Although this compound has not been used for absorption enhancing effects before, Vel j i & Albright (1985) successfully used it as a deflocculent to disperse bacterial cells and the hope here was to observe a mild destructive effect on the intestinal mucus layer. However, the use of this compound did not lead to a significantly elevated IgY uptake. 126 4.6.6. Experiment 4. Effects of various absorption enhancing agents Various chemicals known for their absorption enhancing effect were tested in different concentrations (as described in materials & methods) to determine the highest concentration that might be fed to juvenile rainbow trout without causing any mortality among the 4 treated fish. Such concentration, considered as the highest safe level of administration for each of these non-ionic detergents, was further orally intubated into the fish concomitantly with W S F to examine its enhancing effect on IgY uptake. Table 4.6 shows the concentration levels o f each chemical used in this experiment along with their relevant mortality rates incurred in the intubated fish. The highest safe level for each detergent is also shown in this table. Table 4.7 illustrates the levels of various detergents used in oral intubation of fish with IgY. The serum I g Y levels obtained 4 hours after co-administration of the detergent and W S F are also shown. The application o f octyl P-glucoside, Na-deoxycholate and Mega9 was associated with a significantly higher IgY absorption when compared with W S F dissolved in a P B S solution. Serum IgY level in these treatments appeared to be significantly higher than the levels achieved following co-administration of all other tested detergents and also higher than that of P B S treated fish. CFfAPS, CFIAPSO, Triton X - l 14 and saponin at the levels used in the present study did not contribute to an increase in IgY uptake and resulted in serum I g Y levels not significantly different from that observed when W S F was intubated in a solution of P B S . Triton X - l 0 0 and L-cysteine conferred levels not significantly different from the negative control, when fish were intubated with P B S lacking any source of IgY. These levels were significantly lower than that offered by W S F solutions in P B S . 127 Table 4.6. Lethality of various levels of orally intubated detergents to rainbow trout. Oral intubation treatment8 Levels of detergent Mortality rate (%)¥ Highest safe level* Octyl-B-glucoside Na-deoxycholate Mega9 CHAPS CHAPSO Triton X-114 Saponin Triton X-100 L-cysteine 5%, 3%, 1%, 0.5% 5%, 3%, 1%, 0.5% 5% 5%, 3%, 1%, 0.5% 5%, 3%, 1%, 0.5% 5%, 3%, 1%, 0.5% 1,0.1,0.05, 0.01 mg fish'1 5%, 3%, 1%, 0.5% 0.9,0.3, O.lmg fish' 1 0, 0, 0, 0 100, 75, 0, 0 0 25, 25, 0, 0 25, 25,' 0, 0 50, 25, 0, 0 0, 0, 0,0 50, 0, 0, 0 0, 0,0 5% 1% 5% 1% 1% 1% 1 mg fish' 1 3% 0.9 mg fish"1 * Highest safe level of the detergent is the highest level tested which did not cause any mortality. § Oral intubation: 200pL of detergent solution fish"1. ¥ Cumulative mortality rate among 4 fish 10 days after oral intubation with detergent solution. Average fish weight = 25g. 128 Table 4.7. Effect of detergent in enhancement of IgY absorption. Oral intubation treatment8 Concentration of detergent Serum IgY * (ng mL"1) Octyl-B-glucoside + WSF 5% 776 + 361 a* Na-deoxycholate + WSF 1% 715+455 a Mega9 + WSF 5% 312 ±77.1 a PBS + WSF 46.3 ±45.8 b CHAPS + WSF 1% 44.9 ±23.9 b CHAPSO + WSF 1% 28.4 ± 17.3 b Triton X - l 14+WSF 1% 24.7 ± 12.2 b Saponin + WSF 5 mg mL"1 21.7 ± 11.1 b Triton X-100 + WSF 3% 9.50 + 13.7 c L-cysteine + WSF 4.5 mg mL" 1 4.74 ±5.35 c PBS (negative control) 4.57+4.90 c * Common letters indicate no significant difference between the means (p<0.05). $ Values are the mean ± standard deviation of 3 fish. § Oral intubation with 200uLfish' 1 of IgY-containing detergent solution. Blood sampling at 4 hour post intubation of WSF. Average fish weight = 25g. 129 The satisfactory results that were obtained with deoxycholate are consistent with Hertz et al. (1991) who observed a 1000-fold increase in plasma level of h G H when deoxycholate in antacid solution was concurrently orally intubated. Mega9 absorption enhancing effect is also in agreement with McLean et al. (1990) and McLean & Ash (1990) who, respectively, detected an increased growth stimulation by somatotropin when co-administered with Mega9 and antacid or an elevated H R P plasma level after oral co-delivery with 5% Mega9 into rainbow trout. Womack et al. (1983) recognized octylglucoside as one of the most effective detergents in releasing proteins from membrane bounded compartments without denaturing them. They also found a similar protein releasing power in deoxycholate and Triton X-100 which were all less effective than C H A P S and C H A P S O . Hildreth (1982) reported a similar effect on cell membranes exposed to Mega9. In the present study, a considerably lower IgY uptake was mediated by C H A P S or C H A P S O than by octylglucoside, deoxycholate, or Mega9. Based on all these observations, a strong relationship between the absorption enhancing effect of these detergents and their ability in releasing proteins from cell membrane cannot be concluded. Co-delivery o f saponin ( l m g fish"1) did not elicit an absorption enhancing effect when compared with the W S F delivered in P B S solution. This result is consistent with that reported by Akhlaghi (1999) who observed no uptake into the serum and no protection conferred following intubation of 5mg fish"1 anti-F! anguillarum sheep IgG co-administered with Qu i l -A saponin in micellar form into trout stomach. In contrast, Jenkins et al. (1991) reported an enhanced enteric uptake 130 of human gamma globulin when co-delivered with a 20ug fish"1 dose of saponin into tilapia. The contrary results observed in different fish studies might be due to divergence between the species, size of the studied fish, nutrition, molecular weight and structure of protein molecule, conditions of experiment such as temperature, photoperiod and intensity of light, as well as the detection systems employed. As the present study and the related literature have shown, considerable individual variations exist within the same species. This fact along with the small numbers of fish (3-6) sampled in various studies (Akhlaghi, 1999; Jenkins et al., 1991; Hertz et ai, 1991; Lee et al., 2000; Moriyama et al, 1990; McLean & Ash, 1990) contributes to large experimental variability, which could explain some of the discrepancy observed by different researchers. 4.7. Challenge studies To test the efficacy of oral passive immunization of trout, fish were exposed to the pathogenic bacteria in experimental challenge trials following oral administration of anti-K anguillarum chicken egg IgY. Various approaches as described in materials & method were considered in oral delivery o f IgY. In the following, the result of each challenge experiment is discussed separately. 4.7.1. Experiment 5. Preliminary feeding trial A preliminary experiment was conducted to test the potential o f a non-ionic detergent, Mega9, in enhancement of IgY absorption when incorporated into the fish 131 feed. Two different feeding protocols, as described in the materials & methods, were used. E L I S A results, as shown in Table 4.8, indicated an average serum IgY level o f 6.8 and 13.Ong mL" 1 at day 2 and as 11.7 and 13.6ng mL" 1 at day 7 for the fish receiving 2 days o f high dose or 7 days of low dose Mega9 diets, respectively, while the W S F was provided throughout the entire 7-day course o f feeding. There was no significant difference between any o f these IgY concentrations (p=O.S). In an immersion challenge with V. anguillarum, mortality rates were 100% for untreated fish and the group receiving 7 days of low content Mega9 diet. Surprisingly, only 50%) mortality was recorded for the group receiving a diet high in Mega9 for 2 days and devoid of it for the rest of the time. There was only 2 days resting time between blood collection and challenge of the same individual fish and they were not fed during this period. Considering these facts as well as the stress the fish underwent in handling and blood collection, 50% survival rate was a strong indication o f increased resistance with treatment 1 diet. However, since only 4 fish were challenged in each group, individual differences could greatly contribute to the error effect. 4.7.2. Experiments 6, 7, 8 & 9. Bacterial challenge following oral administration of anti-Vibrio IgY The objective o f Experiment 6 as detailed in the materials & methods, was to study efficacy o f specific anti-K anguillarum I gY in protection of fish against vibriosis when the W S F was orally administered to trout with or without the absorption-enhancing agent, Mega9 (M9). A s Table 4.9 illustrates, at the end o f a 7-day treatment period when challenge was performed, total serum IgY appeared in 132 Table 4.8. Serum IgY & mortality levels in preliminary feeding and challenge*" conducted at day 9. , _ , Treatment Serum IgY (ng m L - 1 ) * Semm IgY (ng mL"1 )* Mortality rate day 2 day 7 (%) Treatment 1 § 16.8 + 10.4 a* 11.7 + 3.47 a 50 Treatment 2 ¥ 13.0 + 6.04 a 13.6 ± 8 . 4 1 a 100 Untreated £ 100 * Common letters within each column indicate no significant difference between the means § Treatment 1. Days 1-2: pellets (1.3% average body weight) containing spWSF & 2.5% Mega9; days 3-7: pellets and spWSF only (8mg IgY fish 1 day'1 =87mg IgY kg' 1 average body weight). ¥ Treatment 2. Day 1-7: pellets (1.3% average body weight) containing spWSF & 0.5% Mega9. £ Untreated fish were fed commercial pellets. s Values are mean ± standard deviation of 4 fish. 11 V. anguillarum concentration for challenge = 1.2 X 106 cfu mL' 1 . Average fish weight = 89 g. 133 significantly higher levels in C3 & C4 than most of the other treatments. In these two control treatments fish were orally intubated (IN) with W S F and M 9 on the first day. However, these levels were not significantly different from those of C7 and T2 in which fish received pellets containing W S F from immunized hens (P + -spWSF) for the whole 7-day period of this study. IgY levels in the untreated control group (C6) were significantly lower than all other treatments. The lowest mortality rate, which was significantly lower than all but C7, occurred in C3 . This group (C3) which also showed the highest I gY concentration in the serum, received I N spWSF-M9 at the first day and P + - s p W S F - M 9 for the rest of study period. There was no significant difference between the mortality rate of any other treatment groups. The fact that the mortality rate in C4 did not come close to that o f C3 indicates that the protective effect is due to the specificity of IgY, since diet of C4 was identical to C3 except that the specific anti-K anguillarum IgY was absorbed by the antigen. The high mortality rate in C5, which received M 9 without any IgY, verifies that Mega9 by itself does not trigger an enhanced resistance against the disease, so, it can not be considered as an immunostimulant agent. It could also be concluded that oral intubation can offer higher I g Y uptake and better protection against the disease than incorporation into the pellets. The mortality rates in all groups were high enough to speculate that the virulence of the bacterial pathogen at the dose used for this challenge was so high that it undermined the treatment effect. Accordingly, experiment 7 was performed to retest the same effects. 134 Table 4.9. Serum IgY & mortality levels following oral intubation or feeding of WSF and Mega9 (Experiment 6). Treatment T l - d 1: P +-M9-spWSF; d 2-7: pellets ¥ T2- d 1: P +-M9-spWSF; d 2-7: P +-spWSF C l - d 1: P +-M9-nspWSF; d 2-7: pellets C2- d 1: P +-M9-nspWSF; d 2-7: P+-nspWSF C3- d 1: IN 1 1 M9-spWSF; d 2-7: P +-spWSF C4- d 1: IN M9+abspWSF; d 2-7: P+-abspWSF C5- d 1: P + -M9 (no WSF); d 2-7: pellets C6 (Untreated)- d 1-7: pellets C7- d 1-7: P +-spWSF Serum IgY (ng mL" 1)* Mortality (%) 31.0126.5 be* 9 6 . 7 ± 3 . 8 7 £ a 58.3 ±27.3 ab 9 1 . 7 ± 6 . 3 6 £ a 21.3 ±8.35 c 93.3 ± 0 . 0 0 § a 40.9 ± 10.2 be 90.0 ± 4.67 § a 120 ±54.4 a 6 5 . 7 ± 1 8 . 7 £ b 104 ±35.2 a 86.7 ± 9.40 § a 2.04 ± 1.57 d 90.0 ± 8.60 £ a 0.76 ±0.86 e 93.4 ± 9.40 § a 46.0 ± 17.6 ab 80.0 ± 9.48 § ab Abbreviations as described in the list of abbreviations are: T: test treatment, C: control, d: day(s), P + : treated pellets, M9: Mega9, sp: specific, nsp: non-specific, WSF: water-soluble fraction of egg-yolks, IN: intubated, ab: absorbed. * Common letters within each column indicate no significant difference between the means (p<0.05). $ Values are mean ± standard deviation of 3 or 4 fish. £ Values are mean ± standard deviation of 4 tanks. § Values are mean ± standard deviation of 2 tanks. 11 Oral intubation volume: 200uL. Specific or non-specific IgY: 5.5mg IgY fish'1 day"1 =100mg IgY kg"1 average body weight. Mega9 & N a H C 0 3 : 396 & 80mg kg"1 average body weight, respectively. Pellets: 1.2% average body weight, coated with marine oil (3% pellets weight). Exposure to V. anguillarum (2.48-2.64 X 105 cfu mL' 1 ): 20 hours after the last feeding for 30 minutes. Blood collection @ the day of challenge. Average fish weight = 55g. 135 Results of experiment 7 are illustrated in Table 4.10. In this trial, group C2 had a significantly higher level of total serum IgY than all other treatments at the end o f the7-day study period. C2 received I N spWSF, M 9 and sodium bicarbonate (SBC) on day 1, followed by feeding the same ingredients incorporated into the pellets on day 2 and P + - spWSF on days 3-7. The untreated control group (C3) showed a significantly lower level of total serum IgY than all other groups, which were not significantly different from each other in this respect. C2 was the only treatment, which exhibited a significantly lower mortality rate than untreated control (C3). This was also lower than C l , which received P + - n s p W S F - M 9 - S B C on days 1-2 and P + -nspWSF on days 3-7. Mortality of C2, although 20% lower, was not significantly different from T l and C4, which for the whole course of study were fed P + - spWSF with or without M 9 & S B C , respectively. Mortality rate in C l was significantly higher than T l and C4. Therefore, in the spectrum o f this trial, feeding o f specific anti-P! anguillarum IgY could enhance resistance of the trout to vibriosis but feeding o f non-specific I gY could not offer any protection. In fact, the mortality rate of C l , receiving nspWSF, was at the same level with C3, the untreated control. This result is consistent with the results obtained from the IP injection of IgY, in which mortality rate in the group receiving non-specific IgY was not significantly different from that of the P B S injected control group. In experiment 8, two other absorption-enhancing agents, deoxycholate ( D X ) and octyl-p-glucoside (opg), which proved most effective in experiment 4, were added to the experimental plan. In experiment 8, fish in all treatment groups ( T l , T2, 136 Table 4.10. Serum IgY & mortality levels after oral administration of WSF, Mega9 and Treatment Serum IgY s (ng mL"1) £ Mortality (%) T I - dl-2: P+-M9-SBC-spWSF; d3-7: P+-spWSF * Cl - dl-2: P+-M9-SBC-nspWSF; d3-7: P+-nspWSF C2- dl: IN1 M9-SBC-spWSF; d2: P+-M9-SBC-spWSF; d3-7: P+-spWSF C3 (untreated)- dl-7: pellets C4- dl-7: P+-spWSF 151 ±24.2 b* 144 ± 16.8 b 201 ±36.4 a 1.84 ±2.72 c 120 ± 6.24 b 70.5 ± 16.7 bc 93.3 ±6.67 a 58.8+20.0 c 93.33± 0.00 ab 71.1 + 16.8 bc A U U 1 U V J d L l V J l l D CIO U ^ V i i L / w u i i i UIAW i i j v v / i V T <.MMV^W —. . „ „ 7 _ day(s), P+: treated pellets, M9: Mega9, sp: specific, nsp: non-specific, WSF: water-soluble fraction of egg-yolks, IN: intubated, SBC: sodium bicarbonate. * Common letters within each column indicate no significant difference between the means i><0.05). s Values are mean ± standard deviation of 3 fish. £ Values are mean ± standard deviation of 3 tanks. ' Oral intubation volume: 200uL fish"1. Specific or non-specific IgY: 6.5mg IgY fish"1 day"1 =217mg IgY kg"1 average body weight. Mega9: lOmg fish"1 =333mg kg'1 average body weight; NaHC03: 2mg fish"1 =66mg kg"1 average body weight. Pellets: 2% average body weight, coated with marine oil (3% pellet weight). Exposure to V. anguillarum (5.1-5.4 X 104cfu mL"1): 20 hours after the last feeding for 30 minutes. Blood collection @ the day of challenge. Average fish weight = 30g. 137 T3 & T4) received I N spWSF on the first day followed by P + - spWSF for the rest of the period of the study. The diets of T I , T2 or T3 contained M 9 , D X or 0(3G accompanied by S B C on the days 1 & 2, respectively. Fish in the negative control group ( C l ) were intubated with P B S the fist day and fed commercial pellets for the rest o f the time. As shown in Table 4.11, receiving I N spWSF and non-ionic detergents on the first day (TI , T2 & T3) did not result in a lower mortality in such treated groups than the fish receiving IgY without a detergent (T4). The mortality rate in C l , although approximately 1.6 times higher than T I , T2 and T3, was not significantly different from any of the treatments. Absorption of IgY into the bloodstream appeared at the highest levels in T2 and T3, which received 0(3G and D X on the first 2 days, respectively. T I (receiving M 9 on the first 2 days), as well as T4 (devoid of detergent in the diet) showed serum IgY levels significantly higher than the negative control and lower than T2 and T3. This result suggests that Mega9 was not as effective as the other two detergents in the enhancement of IgY uptake. The same trend of absorption was observed in the study where different absorption enhancing agents were used, with O p G and D X eliciting the highest levels followed by M 9 . However in experiment 8, the difference between the mortality levels of the three detergents was not significant. Although a linear correlation with a r 2 = 0.68 was found between total serum IgY level and the mortality rate o f different treatments, linear regression analysis showed this correlation was not significant (p = 0.068). In both experiments 8 & 9, detergents were accompanied by 1% antacid and the IgY used always contained specific anti-V. anguillarum activity. 138 Table 4.11. Serum IgY & mortality levels following oral administration of WSF, Treatment Serum IgY $ (ng mL"1) Mortality £ (%) T l - d l : IN 1 1 M9-SBC-spWSF; d2: P +-M9-SBC-spWSF; d3-7: P +-spWSF T2- d l : IN OpG-SBC-spWSF; d2: P +-OpG-SBC-spWSF; d3-7: P +-spWSF T3- d l : IN DX-SBC-spWSF; d2: P + -DX-SBC-spWSF; d3-7: P +-spWSF T4- d l : IN spWSF; d2-7: P +-spWSF Cl (negative control)- d l : IN PBS ; d2-7: pellets 183 ± 3 5 . b* 512+ 142 a 373 ± 1 7 a 151 ±24.2 b 8.36 ±3.45 c 50.0 ±14.3 a 47.9 ±15.5 a 46.7 ±23.1 a 59.1 ±20.1 a 77.1 ±20.6 a A D U r e V l c l t l U I l S cU5 U ^ U I U & U ill U L ^ II.IL U I auuiuTiuuv/iu w.*w. • . „ v u f c ... V ) , day(s), P + : treated pellets, M9: Mega9, OpG: octyle-P-glucoside, D X : deoxycholate, sp: specific, WSF: water-soluble fraction of egg-yolks, IN: intubated, SBC: sodium bicarbonate. * Common letters within each column indicate no significant difference between the means (p<0.05). $ Values are mean ± standard deviation of 3 fish. £ Values are mean ± standard deviation of 3 tanks. 11 Oral intubation volume: 200uL fish'1. IgY: 6.12mg fish'1 day"1 =153mg kg"1 average body weight day"1. Mega9 & octyl-P-glucoside (intubated): 5% volume=10mg fish"1=250mg kg' 1 average body weight. Mega9 & octyl-P-glucoside (in pellets): 1.5 X intubation=15mg fish'1=375mg kg"1 average body weight. Na-deoxycholate (intubated): 1% volume=2mg fish"1=50mg kg"1 average body weight. Na-deoxycholate (in pellets): 1.5 X intubation =3mg fish"1=75mg kg"1 average body weight. NaHCOj: 1% intubated volume or pellet weight. Pellets: 1.2% average body weight, coated with marine oil (3% pellet weight). Exposure to V. anguillarum (2.5X104cfu mL"1): 21 hours after the last feeding for 30 minutes. Blood collection @ the day of challenge. Average fish weight = 40g. 139 Intubation does not seem feasible in a commercial aquaculture practice. Therefore in experiment 9, the same ingredients used in experiment 8 were incorporated into the feed for the complete term of the study, except for a positive control group (Cl), which received an intubated dose on the first day. Results as illustrated in Table 4.12, showed that serum IgY levels in all different treatments where fish received P +-spWSF-SBC and a detergent on days 1 & 2 were not significantly different from each other or from the fish which received P+-spWSF without a detergent or SBC. However, IgY uptake in all these treatments was significantly higher than in the untreated control. The lowest mortality occurred in C1 and T3 in which fish received IN spWSF-M9-SBC at day one or P +-spWSF-DX-SBC on days 1 & 2, respectively. These groups were the only ones with a significantly lower mortality rate than the untreated control. However, these morality rates were not significantly lower than those of the groups that received P +-spWSF-OPG-SBC on days 1 & 2 followed by P+-spWSF on days 3-7 (T2) or P+-spWSF for the entire seven days (T4). The low mortality rate observed in C l and T3 was significantly different from that of TI receiving P +-spWSF-M9-SBC on the first two days followed by P + -spWSF. The mortality rate in none of the other groups was lower than the untreated control. Although a linear correlation with an r 2 = 0.26 was found between serum IgY level and the mortality rate of different treatments, linear regression analysis showed that the correlation was not significant (p = 0.307). The relationship between protection and serum IgY concentration while significant (r2 = 0.512,/? < 0.001) appeared to depend upon the method of delivery. When intubated with absorbance enhancing agents, serum IgY levels may be 140 substantially elevated compared to feeding trials. However protection, although improved, was not enhanced in proportion to the serum IgY level. Thus IP injection resulted in serum IgY concentration in the order of 10 4 ng ml" 1, intubation 3 x 10 2 ng ml" 1 and feeding 1 x 10 2 ng ml" 1. However, in some cases, protection conferred by feeding was comparable to protection with IP injection. A possible explanation is that IgY in other locations in the body contributed to protection. For instance, IgY in the gut has been shown to provide some protection (Hatta et al., 1994). IgY transport through the serum to other organs such as kidneys, spleen and liver may also reduce mortality. These other tissues were not examined in this study. 4.8. Enhancement of IgY uptake using detergents A n overall view to the results of the intubation and feeding studies, shows that oral intubation of octyl-P-glucoside, deoxycholate and Mega9 can induce a more efficient uptake of IgY into the bloodstream of rainbow trout when compared to the intubated IgY devoid of detergents. Although intubated Mega9 followed by feeding IgY-containing pellets in experiment 8 did not significantly increase the serum I g Y level, contrasting observations of the previous intubation studies, especially experiment 1, proves the potential of Mega9 in enhancement of IgY uptake. The different observation in experiment 8 might be attributed to large individual differences in fish and the small number o f samples. In some cases, the same dose o f mti-Vibrio IgY, antacid and detergents which increased the IgY uptake after intubation, did not elicit a significant increase in IgY absorption when it was incorporated into the feed. 141 Table 4.12. Serum IgY & mortality levels following feeding of WSF, absorption Treatment Serum IgY * (ng mL"1) Mortality £ (%) T I - dl-2: P +-M9*-SBC-spWSF; d3-7: P +-spWSF T2- dl-2: P +-OpG-spWSF; d3-7: P +-spWSF T3- dl-2: P + -DX-SBC-spWSF; d3-7: P +-spWSF T4-dl-7: P +-spWSF C l (positive control)- d l : I N 1 M9-SBC-spWSF; d2: P +-M9-SBC-spWSF; d3-7: P +-spWSF C2 (negative control)- dl-7: pellets 110+17.4 a* 156 + 51.3 a 144 + 42.8 a 158 + 66.5 a 165+70.8 a 0.56 + 0.16 b 73.3 + 11.5 a 66.5 +22.8 ab 35.6+10.2 b 57.8 + 15.4 ab 35.4 + 21.9 b 68.6 + 30.5 a J ~ V U U l ^ V I C I U I W I I O CIO U W O W A I S W I * ' 1 1 L . . v. n u . v & ~ _ , _ . day(s), P + : treated pellets, M9: Mega9, OpG: octyle-P-glucoside, D X : deoxycholate, sp: specific, WSF: water-soluble fraction of egg-yolks, IN: intubated, SBC: sodium bicarbonate. * Common letters within each column indicate no significant difference between the means (p<0.05). Values are mean ± standard deviation of 3 fish. £ Values are mean ± standard deviation of 3 tanks. ' Oral intubation volume: 200uL fish"1. IgY: 8.8mg fish"1 day"1 =196mg kg"1 average body weight day"1. Mega9 (intubated): 5% volume=10mg fish"1=222mg kg"1 average body weight. Mega9 & octyl-P-glucoside (in pellets): 20mg fish"1=440mg kg"1 average body weight. Na-deoxycholate (in pellets): 4mg fish"1=88mg kg"1 average body weight. N a H C 0 3 : 1% intubated volume or pellet weight. Pellets: 1.5% average body weight, coated with marine oil (3% pellet weight). Exposure to V. anguillarum (1.7-1.9 X 104 cfu mL"1): 20 hours after the last feeding for 30 minutes. Blood collection @ the day of challenge. Average fish weight = 45g. 142 Hertz et al. (1991), who applied deoxycholate to enhance intestinal absorption o f human growth hormone (hGH) in carp, demonstrated that level o f serum h G H was dependent upon length o f starvation period prior to intubation of h G H . The absorption level was higher after 12 days starvation than 7 or one day. N o h G H could be detected in fish intubated 6 hours or less, post feeding. The authors postulated that the time interval from the last feeding might reflect the amounts of indigestible and digestible food left in the fish gut which may compete for absorption with the target protein or inhibit its absorption by binding with it. The same assumption could be applied to the present study, where fish were fed pellets from day 2 post-intubation and blood sampling was performed after 7 days. The pre-intubation starvation period was usually 2 or 3 days, which might not have been long enough to clear the GI tract, and therefore the food left in the gut could have interfered with the absorption o f IgY. On later days, when IgY and the additives were carried by pellets, IgY could have been bound to the food ingredients even before introduction into the fish gut. On the other hand, the presence of feed could extend the digestion process in the stomach when compared with the intubation of a solution of IgY associated with detergents and antacid. A longer exposure of IgY to the digestive enzymes and acidic conditions of the stomach would make it more susceptible to destruction. Lee et al. (2000) reported that the activity of anti-F. ruckeri IgY decreased rapidly after 2 hours residence in the stomach and was completely lost 5 hours after oral administration. This coincided with a rapid decrease o f stomach p H in 30 minutes after feeding which increased again after 3 hours. Shimizu et al. (1988) found that the 143 activity o f anti-E.coli IgY was sensitive to pepsin, especially at p H levels lower than 4. However, in another study (Hatta et al., 1993b) the activity o f anti-human rotavirus IgY was completely lost in exposure to pepsin at p H 2 while it was 63% retained at p H 4. These studies show that sensitivity of IgY to gastric enzymes is p H dependent. This fact may explain the positive results obtained from combined application of antacid and detergents in the present study. Antacid would have increased stomach p H and consequently helped retain IgY activity to a greater extent, especially when feed was absent in intubation treatments. Intubation could thus accelerate passage of material through the gastric section of the GI tract. Hofer (1982) suggested that following periods of extended fasting, protease enzymatic systems might undergo adaptation allowing higher levels of ingested protein to escape degradation. If this is true, a higher IgY uptake level might have been obtained by applying a longer starvation period prior to the feeding experiments. However, this is perhaps not feasible in the real aquaculture practice where exposure to the pathogenic bacteria is not controlled. 4 .9. Significance of IgY application in protection against diseases Co-administration of Mega9 with anti-K". anguillarum IgY via oral intubation followed by feeding o f I g Y led to a decreased mortality, when compared with the untreated control in all trials except experiment 8. Oral feeding of IgY incorporated into the pellets also increased the disease resistance in some cases. However, in most of the cases the extent of offered protection was not significantly different from the control. Intubation followed by feeding of IgY co-delivered with Mega9 decreased 144 the mean mortality rate to a lower level than in the group treated with anti-Vibrio IgY devoid of detergents by a factor of 1.6 in experiment 9. However the level of difference was not statistically significant in any o f the challenge trials. Administration of deoxycholate led to a significant decrease in mortality. Use of octyl-P-glucoside, although resulted in the highest IgY uptake in both experiments, did not offer a better protection against the disease. The overall result of the challenge studies following oral administration o f IgY is that use of specific anti- Vibrio IgY, in one form or the other, induced an enhanced protection against vibriosis in all trials. The only exception was experiment 8, in which the protection due to use of IgY was higher than the control group but the difference was not statistically significant. Considering results of experiment 8 along with the other trials, it can be concluded that oral administration o f anti-F. anguillarum IgY offers an improvement in resistance against an immersion challenge with the causative organism. One o f the factors that could have caused the inconsistent results in feeding experiments or intubation treatments followed by feeding, is the possibility o f uneven feeding. Although the same preparation and concentration of IgY was used to prepare the fabricated pellets, the dose reaching each fish was uncertain due to competition between the individuals and possible leaching of IgY into the water before being consumed by the fish. A s illustrated in Table 4.13, the amount of total IgY incorporated into the pellets through spWSF in experiments 7, 8 and 9 was not significantly different in various treatments of the same experiment. The commercial pellets or pellets top-dressed with Mega9 (without W S F ) did not show any IgY content. 145 Detergents, although capable of enhancing IgY uptake, affected the mortality in various experiments to different degrees and did not always reduce the mortality to lower levels when compared to the treatments containing anti-Vibrio IgY but no detergent. Intraperitoneal (D?) injection of specific IgY led to a significantly higher protection of rainbow trout against V. anguillarum in an immersion challenge when compared to the groups EP injected with non-specific IgY or P B S . This protective effect was persistent when fish were challenged 1, 3, 7 or 14 days post EP injection of specific IgY, which shows that specific I gY can offer an enduring passive immunity against vibriosis i f absorbed in an effective level. The interesting finding was similarity between the mortality levels in deoxycholate fed and control groups in experiment 9 and the anti-Vibrio IgY treated and P B S treated control groups in the EP experiment at days 3 and 7. In both of these experiments, there was a significant difference between the mortality of the negative control (62%-75%) and that of the anti-Vibrio treated groups with mortality levels of 24%-31% in EP injected trials and 35.6% in experiment 9, when I g Y was fed in conjunction with deoxycholate and antacid. This result suggests that feeding of IgY could be considered as an effective approach for disease protection. However, further research is needed to overcome the practical problems and better understand the mechanisms by which IgY confers protection. The result of EP injection experiment is consistent with the results reported by Lee et al. (2000) who passively protected rainbow trout against an immersion challenge with Y. ruckeri by EP injection of specific IgY 4 hours before challenge. These researchers obtained only a marginal reduction in mortality and intestinal infection after feeding the same 146 specific IgY either before or after the challenge. However, Gutierrez et al. (1994) reported that mortality due to a natural infection by Edwardsiella tarda decreased when the eel feed contained at least 3% egg-yolk powder obtained from the vaccinated hens for 2 weeks. Lower contents of the egg-yolk powder in the feed did not elicit protective effect. Hatta et al. (1994) cannulated a mixture of anti- E. tarda IgY and the pathogen into the stomach of Japanese eels after their intestinal mucosa was damaged. I gY treated eels survived while the control eels died or showed the symptoms of the disease. Although they did not detect any immunologically active IgY in the serum o f eel, a high level o f anti-£. tarda IgY was maintained in the GI tract for 7 days. These results suggest that simultaneous introduction o f specific I gY and pathogenic bacteria to the digestive system of fish can reduce mortality. This effect could be attributed to direct local neutralizing action o f specific I gY at the site of bacterial infection in the digestive tract before being absorbed. Therefore, a better protection could be possibly obtained in the present study i f the diet containing anti-Vibrio IgY was continued after the challenge, too. Also an increase in diet I gY could be helpful to elevate the serum IgY levels. In all of the challenge experiments of the present study, there was approximately 20 hours between the last feeding and the challenge. The fish were fed regular commercial diets in the post-challenge period. 147 Additives to pellets Experiment 7 Experiment 8 Experiment 9 spWSF 13.4 ±1 .5 a* N A 8.7 ±0 .9 a nspWSF 11.4 ± 1.9 a M9-nspWSF 11.9 ±0.5 a N A M9-spWSF 14.2 ±2 .2 a 9.7 ± 1.7 a OpG-spWSF 9.4 ±1 .7 a 8.4 ±0 .5 a DX-spWSF 11.6 ±1 .2 a 8.8 ±0.5 a no additive 0.001 ±0 .0 b 0.001 ±0.0 b 0.001 ±0.0 b Mega9 ¥ Abbreviations as described in the list of abbreviations are: M9: Mega9, glucoside, D X : deoxycholate, sp: specific, nsp: non-specific, WSF: water-soluble fraction of egg-yolks. * Common letters within each column indicate no significant difference between the means (p<0.05). Values are mean ± standard deviation of 4 samples. 11 Data is not available. 148 4.10. Histological studies There are a few reports in the literature indicating that detergents may enhance macromolecule absorption through induced damage to the intestinal epithelium. Tagesson and co-authors (1985) reported that in rats, non-ionic detergent, lysophosphotidylcholine increased ileal permeability to intact proteins due to enterocyte sloughing and cell rupture caused by osmotic shock. Jenkins et al. (1991) observed an appreciable increase in the uptake of orally and anally delivered human gamma globulin when co-administered with Q u i l - A saponin. They detected a number of physical effects induced by saponin on the intestinal enterocytes o f tilapia in immuno-microscopic examinations. These effects include loosening intercellular junctions, increasing the pinocytosis of luminal contents, fusion with the plasma membrane and causing the cellular microvill i o f the enterocytes to become shortened and damaged. A l l o f these effects generally serve to increase the permeability of the intestine to macromulecules. Although the effects of Mega9 on the intestinal tissues of fish have not been investigated in such detail, McLean & A s h (1990) reported that coincidental delivery o f this non-ionic detergent enhanced the uptake o f horseradish peroxidase into the trout blood circulation. It was observed that Mega9 caused the mucus lining of the GI tract to gelatinize, forming clumps of mucus that upon gentle squeezing o f fish abdomen readily exuded from the vent. The authors postulated that the gelling action might, by partial removal of the physical barrier presented by the mucus, have provided increased protein-enterocyte interactions leading to the enhanced antigen uptake. They also suggested that Mega9 might have caused physical 149 damage to the enterocyte lining o f the gut. However, the tissues o f fish gut were not histologically studied. To investigate the mechanism through which non-ionic detergents, specifically Mega9 may enhance protein absorption, histological examinations were performed. In the first challenge trial (experiment 6), where Mega9 was orally intubated into fish as an absorption enhancing agent along with IgY, dissects of stomach, pyloric caeca and anterior intestine were fixed in buffered formalin as detailed in materials & methods. Microscopic examination of the H & E stained G I tissues demonstrated the removal o f the striated border and partial disruption of the columnar epithelia o f intestinal microvill i . The stomach and pyloric caeca textures were not affected (Fig.7.4 & Fig.7.5 in the appendix). The intestine of the untreated control fish seemed intact. Fig.4.15 and Fig.4.16 display the histological cross-sections of the intestines of the Mega9 treated and control fish, respectively. In all later challenge experiments, as well as the experiment with the absorption enhancing chemical substances (experiment 4), similar histological studies were performed. Unfortunately, in all o f these samples, including the untreated and P B S treated fish the same degree of disintegration and tissue damage to the intestinal brush border and the v i l l i was observed. Fig.7.6 to Fig.7.9 in the appendix illustrates microscopic photographs o f some of these samples. To investigate the cause o f this unexpected observation, the same fixative i.e. buffered formalin was injected through digestive lumen of an untreated fish to ensure a thorough fixation. Since the histological slides still indicated the same extent of degradation, in all later experiments Bouin 's solution was used to fix the specimens. It is noteworthy that 150 buffered formalin and Bouin's solution were both recommended as fixatives o f choice for histological studies o f fish tissues (Yasutake & Wales, 1983). In the next trial, rainbow trout intestine from the untreated or intubated groups with P B S , Mega9, Na-deoxycholate, or octyl-P-glucoside was removed and fixed in Bouin's solution as described in materials & methods. Microscopic observations of the cross sections of the entire intestine revealed no severe tissue disruption in any of the samples. This result suggests that the tissue damage primarily observed was due to the inefficiency of formalin fixation and that none o f the three chemicals induced such a remarkable tissue rupture in the fish gut, which could be assumed to mediate an enhanced uptake of IgY molecule into the blood circulation. Fig.4.17 to Fig.4.20 illustrate microscopic photographs of H & E stained cross section of the intestinal tissue o f the Mega9, Na-deoxycholate, octyl-P-glucoside or P B S intubated, and untreated fish fixed in Bouin 's solution, respectively. Microscopic photographs o f some other specimens are presented in the appendix for a more thorough comparison. Glutaraldehyde and paraformaldehyde are the other two most commonly used fixatives in other fish studies (Jenkins et al, 1991; L e Ba i l et al, 1989; Georgopoulolou etal, 1985; Georgopoulolou etal, 1988; Rombout etal, 1985). The histological examinations of the present study led to rejection of the hypothesis that Mega9, Na-deoxycholate or octyl-P-glucoside might have enhanced protein absorption through physical damage to the enterocyte lining of the gut, since no drastic damage to the epithelial cell lining was observed. The brush border in the detergent treated fish was as intact as that of the untreated control and no disruption was noticed in the epithelial v i l l i . However the other hypothesis of McLean & Ash 151 (1990) that the gelling action of Mega9 might, by partial removal o f the mucus barrier, have provided increased protein-enterocyte interactions leading to the enhanced antigen uptake, does not contradict the observations made in the present study and could be considered as a possible mechanism. The effect of Na-deoxycholate detergent on the absorption process is unclear. However, Hertz et al. (1991) speculated that protein absorption might be mediated through the formation o f complexes, in which the hydrophilic part o f deoxycholate is attached to the protein molecule while the lipophilic part penetrates through the gut lipid membrane, thus increasing the availability o f the protein molecule for absorption. To examine changes in more detail at the intercellular and microvill i level, electromicroscopic studies are required. In the absence of such studies the possibility of loosening of intercellular junctions, increase in pinocytosis of luminal content, fusion with the plasma membrane and damage to microvill i could be considered as the subjects of further studies. 152 Fig. 4.15! Microscopic photographs of H&E stained cross section of the intatfinal tissues of a rainbow trout intubated with Mega9 and WSF (in experiment 6) sampled 7 days after intubation (at the day of challenge) and fixed in buffered formalin. Magnification is 100 fold. 153 Aid •::M mimM IfPiiiii Illi HJ 1 IlllltlfitM Fig. 4.16. Microscopic photographs of H&E stained cross section of the intestinal tissues of an untreated rainbow trout (in experiment 6) fixed in buffered formalin. Magnification is 100 fold. 154 Fig. 4.17. Microscopic photograph of H & E stained cross section of the intestinal tissues of a rainbow trout intubated with Mega9 and antacid. The dissected tissue was sampled 3 hours after intubation and fixed in Bouin's solution. The entire intestine was rolled oyer after a lateral cut was made along the length of the intestine. Therefore, the cross section depicts the entire intestine. Magnification: 100 fold. 155 Fig. 4.18. Microscopic photograph of H & E stained cross section of the intestinal tissues of a rainbow trout intubated with deoxycholate and antacid. The dissected tissue was sampled 3 hours after intubation and fixed in Bouin's solution. The entire intestine was rolled over after a lateral cut was made along the length of the intestine. Therefore, the cross section depicts the entire intestine. Magnification: 100 fold. 156 Fig.4.19. Microscopic photograph of H & E stained cross section of the mtestinal tissues of a rainbow trout intubated with octyl-p-glucoside and antacid. The dissected tissue was sampled 3 hours after intubation and fixed in Bouin's solution. The entire intestine was rolled over after a lateral cut was made along the length of the intestine. Therefore, the cross section depicts the entire intestine. Magnification: 100 fold. 157 Fig. 4.20. Microscopic photograph of H & E stained cross section of the intestinal tissues of an untreated control rainbow trout. The dissected texture was fixed in Bouin's solution. The entire intestine was rolled over after a lateral cut was made along the length of the intestine. Therefore, the cross section depicts the entire intestine. Magnification: 100 fold. 158 CHAPTER FIVE CONCLUSIONS Passive immunization using pathogen specific antibodies raised in other animals has been investigated in animals and humans by several researchers for prophylactic and therapeutic purposes with some promising results. Ease and efficiency of specific immunoglobulin production in hens in which immunoglobulins can be recovered from the egg yolk, and compatibility of this procedure with animal rights concerns, make chicken egg yolk immunoglobulins (IgY) a favorable source of specific antibodies for passive immunization against disease. The oral delivery route offers technical and economic advantages in disease prevention or resistance enhancement strategies in teleosts. Although a considerable amount o f work has been reported on oral delivery o f proteins, including immunoglobulins to aquacultured species, more research is needed to develop effective and industrially-feasible methods of delivery. Special attention should be paid to absorption enhancement o f target proteins with biologically active structures. These proteins may need to be protected from gastric degradation in teleosts. In the present study, three hypotheses were tested. 1. Oral administration of pathogen specific IgY w i l l decrease the mortality of rainbow trout following bacterial infection. 2. Dehydration of I gY w i l l not affect its immunogenicity. 3. Co-administration of detergents w i l l enhance the oral uptake of I gY into the fish blood. The results revealed the followings: 1. A strong antigen-antibody reaction occurred between V. anguillarum antigens and IgY obtained from the egg yolk of vaccinated hens. 2. H igh titers of specific anti- V. anguillarum IgY were effectively raised in vaccinated laying hens. The water-soluble fraction (WSF) of the egg yolks of 160 vaccinated hens was a good source of semi-pure IgY. The IgY of W S F could be concentrated using ultrafiltration, an established technology in the food industry. 3. Immunological properties of IgY were retained when W S F was dehydrated using the techniques of freeze-drying, vacuum microwave drying or spray drying or when W S F was incorporated into the commercial fish pellets and subsequently dehydrated using freeze-drying, vacuum microwave drying or hot air drying. Based on this pilot scale result, large-scale production of IgY in a reactive form seems feasible. 4. Ana l intubation o f pure IgY did not prove effective in uptake o f IgY into the blood under the conditions of the present study. This might be due to the use of insufficient doses of IgY or to insufficient starvation of fish prior to treatment. 5. Orally administered IgY was absorbed into the bloodstream of rainbow trout in an immunologically active form. However, the serum levels of IgY were significantly lower after oral delivery than intraperitoneal (EP) injection. The I g Y levels following EP injection were 800 to 2500 times higher than the levels after oral administration. 6. Among a wide range of detergents tested herein, use of the non-ionic detergents, Mega9 and octyl-p-glucoside as wel l as the bile salt, deoxycholate improved IgY uptake significantly, following simultaneous oral intubation with IgY-containing W S F . The IgY levels following EP injection were only 12 to 18 times higher than the levels after oral co-administration with Mega9. 7. Lyophil ized W S F was microencapsulated as a source of IgY in polylactide-co-glycolide (PLG) with a lactide: glycolide ratio of 50:50 or 85:15. Oral intubation 161 of microencapsulated IgY did not lead to a higher serum IgY level than the unprotected IgY over a 2-week period of study. Limitation of encapsulation capacity, poor entrapment, and incomplete release may have contributed to the poor uptake of IgY. L o w purity of IgY in W S F could have added to the problem, too. 8. EP injection of W S F from the egg yolk of immunized hens led to a high serum IgY level and a significantly decreased mortality rate following an immersion challenge with the pathogenic bacteria. This proved the efficacy o f IgY in enhancement of disease resistance in rainbow trout. However, EP injection might not be considered as a practical method of passive immunization in aquaculture. 9. Oral administration of anti-K anguillarum IgY on feed pellets alone or in co-delivery with detergents, offered different levels of protection of rainbow trout against Vibriosis following an immersion challenge, which in some cases was as effective as EP injection of IgY, but this result was not consistent throughout the study and needs further research. The efficacy o f continued feeding of higher doses of specific IgY before and after exposure to the pathogen has to be investigated. Areas yet to be explored in this field include in vivo stability and functionality of IgY, storage life o f W S F and IgY, development of alternative IgY-containing products such as coated IgY concentrate for oral application and legislative requirements. Further research is also needed to improve oral uptake of immunoglobulins when incorporated into the feed. Different aspects, such as feeding intervals, diet 162 composition and dose of fed IgY should be studied. Since dose dependence of protein uptake has been documented, use of higher IgY oral doses may lead to a more effective uptake and possibly further protection against disease. There is also a potential for research on the mechanism o f passive protection that specific IgY can confer against the target pathogen. Use o f some additives such as enzyme inhibitors to protect protein molecules against gastric digestion and detergents to enhance intestinal absorption in fish has been suggested in the literature. 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Journal of Food Processing & Preservation 20, 145-156. Yousif, A . N . , Seaman, C . H . , Durance, T.D. , Girard, B . , 1999. Flavor volatiles and physical properties of vacuum microwave and air-dried sweet basil (Ocimum basilicum L. ) . Journal of Agricultural & Food Chemistry 47, 4777-4781. 177 CHAPTER SEVEN APPENDIX Mortality Rate & Total Serum IgY Level Fol lowing IP Injection with Ant\-Vibrio IgY 50 40 i 30 >rtal 20 10 0 i a a % M o rta lity - *—lgY(mg/m l ) D a y 1 D a y 3 D a y 7 D a y 14 Post-injection chal lenge date Fig .7 . 1 . Temporal trend o f mortality rate in relation with total serum IgY level in the fish groups EP injected with anti-K anguillarum IgY. Values are the mean of 3 replicates ± standard deviation. Similar letters indicate no significant difference (p < 0.05). 179 Mortality Rate & Anti-V. anguillarum IgY Titer Following IP Injection with Anti-Vibrio IgY lity D a y 1 D a y 3 D a y 7 D a y 14 Post - injec t ion c h a l l e n g e date Fig.7.2. Temporal trend of mortality rate in relation with anti-K anguillarum IgY titer in serum o f fish following EP injection with anti- Vibrio IgY. Values are the mean of 3 replicates (± standard deviation for mortality rate). Similar letters indicate no significant difference (p < 0.05). 180 >• O) o cu Q. (0 Ant i -V . anguillarum IgY Titer & Tota l Serum IgY Leve l In IP Injected Fish >-3 o E E D a y 1 D a y 3 D a y 7 D a y 14 Post - in jec t ion c h a l l e n g e date - • — T o t a l IgY Fig.7.3. Temporal trend of anti-K anguillarum IgY serum titer fluctuations in relation with total serum IgY level in IP injected fish with aa&-Vibrio IgY. Values are the mean of 3 replicates (± standard deviation for total IgY). 181 Fig . 7 .4. Microscopic photograph of H & E stained cross section of the stomach tissues of a rainbow trout intubated with Mega9 and W S F (in experiment 6) sampled 7 days after intubation (at the day of challenge) and fixed in buffered formalin. Magnification: 280 fold. 1 8 2 Fig. 7.5. Microscopic photograph of H&E stained cross section of the pyloric caeca tissues of a rainbow trout intubated with Mega9 and WSF (in experiment 6) sampled 7 days after intubation (at the day of challenge) and fixed in buffered formalin. Magnification: 280 fold. 183 F i g . 7.6. Microscopic photograph, of H & E stained cross Section of the intestinal tissues of a rainbow trout intubated with Mega9, antacid and W S F (in experiment 4) sampled 3 hours after intubation and fixed in buffered formalin. Magnification: 100 fold. 184 Fig. 7.7. Microscopic photograph of H & E stained cross section of the mtestinal tissues of a rainbow trout intubated with deoxycholate, antacid and WSF (in experiment 4) sampled 3 hours after intubation and fixed in buffered formalin. Magnification: 100 fold. 185 Fig. 7.8. Microscopic photograph of H&E stained cross section of the intestinal tissues of a rainbow trout intubated with octyl-JJ-glucoside, antacid and WSF (in experiment 4) sampled 3 hours after intubation and fixed in buffered formalin. Magnification: 100 fold. 186 Fig. 7.9. Microscopic photograph of H & E stained cross section of the mtestinal tissues of a rainbow trout intubated with PBS (in experiment 4) sampled 3 hours after intubation and fixed in buffered formalin. Magnification: 100 fold. 187 

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