UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_2000-564967.pdf [ 22.66MB ]
Metadata
JSON: 831-1.0089669.json
JSON-LD: 831-1.0089669-ld.json
RDF/XML (Pretty): 831-1.0089669-rdf.xml
RDF/JSON: 831-1.0089669-rdf.json
Turtle: 831-1.0089669-turtle.txt
N-Triples: 831-1.0089669-rdf-ntriples.txt
Original Record: 831-1.0089669-source.json
Full Text
831-1.0089669-fulltext.txt
Citation
831-1.0089669.ris

Full Text

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  degree freely  at  this  the  thesis  in  partial  fulfilment  University  of  British  Columbia, I agree  available for  copying  of  department publication  this or of  reference  thesis by  this  for  his thesis  and study. scholarly  or for  her  of  requirements that the  1 further agree that  purposes  may  representatives.  financial  the  be  It  gain shall not  advanced  Library shall make  by  understood be  an  permission for  granted  is  for  allowed  the  extensive  head  that without  of  my  copying  or  my  permission.  Department  of  poo^- S^-es^c*-  The University of British C o l u m b i a Vancouver, Canada  DE-6  (2/88)  j  fco^c+U&t  art- /^fiT' C**-£(M->>*JL  it  SC^_  written  ABSTRACT Passive immunization as an alternative to vaccination or antibiotic therapy, involves use o f 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 o f I g Y 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 o f I g Y through the gut barrier into the bloodstream and the passive immunization that pathogen specific I g Y may confer. H i g h titers o f anti-K  anguillarum I g Y were raised i n vaccinated hens, recovered  from the water-soluble fraction ( W S F ) o f the egg-yolks and subsequently used i n intraperitoneal (IP) injection, oral intubation or feeding o f the trout. Western blotting o f such I g Y revealed a strong reactivity with  V. anguillarum whole cell lysate and  lipopolysaccharide, which was as strong as that o f the rabbit I g G and stronger than that o f the fish I g M . Immunological properties o f I g Y 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 g Y was transferred into the fish system i n high enough levels to confer protection against Vibriosis i n an experimental challenge. This protective effect which was retained at least 14 days post I g Y injection, proved efficacy o f pathogen-specific I g Y i n enhancement o f disease resistance. To investigate absorption through trout digestive tract, I g Y was intubated both anally and orally at the levels o f O.lmg and 1.4-2.7mg fish" , respectively. Under the 1  conditions o f this study, anally intubated I g Y (O.lmg) did not appear i n the serum i n a detectable level. Oral intubation o f W S F led to absorption o f I g Y i n 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 I g Y , deoxycholate, Mega9 and octyl-f3-glucoside mediated the highest enhancement o f I g Y absorption. Use o f Mega9 raised serum I g Y to levels only 12 to 18 times lower than the levels after TP injection o f a similar dose.  Encapsulation o f W S F in polylactide-co-glycolide did not improve I g Y ii  uptake. Oral administration o f anti-Vibrio I g Y in co-delivery w i t h detergents resulted in different levels o f protection o f 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 o f specific I g Y 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 ABSTRACT  ii  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  ACKNOWLEDGEMENTS  xvii  DEDICATIONS  xviii  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 o f the study  4  2. R E V I E W O F 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 o f chicken egg yolk antibodies (IgY) in passive immunization  12  2.7.  Raising specific antibodies in chickens  14  2.8.  Advantage of chicken I g Y over other animal sources  14  2.9.  Stability o f I g Y and differences with I g G  16  2.10.  Methods of I g Y preparation  18  2.11.  Protein absorption in the digestive system  19  2.12.  Time course and efficacy o f macromolecule transfer into the blood circulation  20  iv  2.13.  2.14.  Protection from degradation in the G I tract  25  2.13.1. Encapsulation  26  2.13.2. Enhancement o f absorption using detergents  30  2.13.3. Immunostimulants  37  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.  44  Chickens  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 o f I g Y  51  3.7.  Preparation o f microcapsules  52  3.8.  Feed preparation  54  3.9.  Extraction o f I g Y from the treated pellets  56  3.10.  B l o o d collection from fish and serum preparation  56  3.11.  Bacterial preparation for fish challenge studies  57  3.12.  Lipopolysaccharide ( L P S ) and whole cell lysate ( W C L ) preparation  57  3.13.  A n a l intubation  58  3.14.  Oral administration  59  3.14.1. Experiment 1. Using I g Y in encapsulated form, in Mega9 solution  and  antacid,  or  in  Na-pyrophosphate 59  v  3.14.2. Experiment  2. Tween detergents as  absorption  enhancing agents  60  3.14.3. Experiment 3. Comparative oral intubation 3.14.4. Experiment  4.  Effect  of  various  61  absorption  enhancing agents 3.15.  61  Challenge studies  '.  3.15.1. Experiment 5. Preliminary feeding trial 3.15.2. Experiment  6  &  7.  Challenge  63 64  following  oral  administration o f Mega9 and water soluble fraction of egg yolks ( W S F )  66  3.15.3. Experiment 8. Challenge following oral intubation with absorption enhancing agents 3.15.4. Experiment  9.  Challenge  following  68 feeding  of  absorption enhancing agents  69  3.15.5. Experiment 10. Challenge following LP injection o f IgY 3.16.  70  Analytical techniques  71  3.16.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( S D S - P A G E )  71  3.16.2. Western blotting  72  3.16.3. Enzyme linked immunosorbent assay ( E L I S A )  73  3.16.3.1.  Determination o f total I g Y content...  3.16.3.2.  Determination o f anti-Vibrio specific  I g Y titer  74  76  3.17.  Drying of W S F  78  3.18.  Dehydration o f IgY-containing pellets  79  3.18.1. Initial dehydration & I g Y incorporation  79  3.18.2. Freeze-drying..  80  3.18.3. Vacuum microwave drying  81  3.18.4. Air-drying  81  vi  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 4.1.  4.2.  84  Cellular and surface antigens o f V. anguillarum  interacting  with Chicken I g Y , rabbit I g G o f fish I g M  85  Dehydration o f W S F  91  4.2.1. Dehydration rate  91  4.2.2.  Stability  of  IgY  during  dehydration  when  incorporated onto the pellets 4.2.3.  93  Stability o f I g Y during dehydration o f concentrated WSF  95  4.3.  Post-vaccination I g Y level i n the yolk  4.4.  Passive protection induced by EP injected specific antiWAr/o I g Y to  99  fish  4.5.  A n a l administration o f I g Y  4.6.  Absorption  of  orally  104 110  administered  IgY  into  fish  bloodstream...  112  4.6.1. Experiment 1. Using I g Y in encapsulated form, in Mega9 and antacid, or i n Na-pyrophosphate solution 4.6.2.  112  Time course and levels o f absorption after oral and anal administration o f proteins  4.6.3. Efficacy  of  delivery  into  115 the  blood  using  encapsulated proteins 4.6.4.  Experiment  2. Tween  118 detergents as  absorption  enhancing agents 4.6.5. Experiment 3. Comparative oral intubation 4.6.6.  123  Experiment 4. Effects o f various absorption enhancing agents  4.7.  120  127  Challenge studies  131  4.7.1. Experiment 5. Preliminary feeding trial  131  vii  4.7.2.  Experiment 6, 7, 8 & 9. Challenge following oral administration o f anti- Vibrio I g Y  4.8.  Enhancement o f I g Y uptake using detergents  4.9.  Significance o f I g Y application in protection  4.10.  13 2 141 against  diseases  144  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  viii  LIST O F T A B L E S  Page Table 4.1. Changes in I g Y concentration due to dehydration treatments.  94  Table 4.2. Serum I g Y levels & mortality rates i n the fish IP injected with anti-K anguillarum specific I g Y .  108  Table 4.3. Temporal absorption o f I g Y into the fish blood when encapsulated or co-administered with chemical compounds.  114  Table 4.4. Effect o f Tween detergents i n uptake o f orally intubated I g Y into the bloodstream.  121  Table 4.5. Effect o f chemical compounds i n uptake o f orally intubated I g Y into the bloodstream.  124  Table 4.6. Lethality o f various levels o f orally intubated detergents to rainbow trout.  128  Table 4.7. Effect o f detergent in enhancement o f I g Y absorption.  129  Table 4.8. Serum I g Y & mortality levels i n preliminary feeding and challenge conducted at day 9.  133  Table 4.9. Serum I g Y & mortality levels following oral intubation or feeding o f W S F and Mega9 (Experiment 6).  135  Table 4.10. Serum I g Y & mortality levels after oral administration o f W S F , Mega9 and antacid (Experiment 7).  137  Table 4.11. Serum I g Y & mortality levels following oral administration o f W S F , absorption enhancing agents and antacid (Experiment 8).  139  Table 4.12. Serum I g Y & mortality levels following feeding o f W S F , absorption enhancing agents and antacid (Experiment 9).  142  Table 4.13. Total I g Y level i n different pellet compositions (mg g" pellet).  148  1  ix  LIST OF FIGURES Page Fig.2.1. Schematic mechanism o f 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 o f appearance o f bioactive proteins within the serum o f orally and anally intubated fish.  24  Fig.2.4. Time-course o f appearance o f human gamma globulin ( H G G ) i n 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 o f Mega9 (nanonyle-n-glucamide)  32  Fig.2.5.(b). Chemical structure o f deoxycholic acid (5-P-cholan-24-oic acid-3a, 12a-diol).  32  Fig.2.5.(c). Chemical glucopiranoside).  33  structure  o f octyl  P-glucoside (n-octyl-P-D-  Fig.2.5.(d). Chemical structure o f L-cysteine ethylester.  33  Fig.2.5. (e, f). Chemical structures o f polyoxyethylene ethers, (e) Triton X-100, (f) Triton X - l 14.  34  Fig.2.5.(g). Chemical .structure o f dimethylammonio-1 -propanesulfonate)  35  CHAPS  (3-3-cholamidopropyl  Fig.2.5.(h). Chemical structure o f C H A P S O dimethylammonio-2-hydroxy-l-propane-sulfonate)  (3-3-cholamidopropyl  Fig.2.5.(i). Chemical structure o f Tween-20 monolaurate).  (polyoxyethylenesorbitan  Fig.2.5.(j). Chemical structure o f Tween-80 monooleate).  (polyoxyethylenesorbitan  35  36  36  Fig.3.1.(a &b). Design o f a 7 5 L fish holding tank.  46  Fig.3.2. Arrangement o f fish holding facilities.  47  Fig.3.3. Schematic diagram o f anti-Vibrio anguillarum vaccine preparation and vaccination o f chickens.  50  Fig.3.4. Schematic diagram o f the I g Y extraction from the hen eggs and application i n the absorption and passive immunization studies in rainbow trout.  53  Fig.3.5. Schematic diagram o f microcencapsulation o f water-soluble fraction ( W S F ) o f egg-yolks i n polylactide-D-glucolide ( P L G )  55  Fig.3.6. Schematic diagram o f fish challenge with V. anguillarum.  65  Fig.3.7. Procedure o f sandwich E L I S A .  75  Fig.3.8. Procedure o f 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 ) o f V. anguillarum stained by coomassie blue, (b) S D S - P A G E profile o f 2 0 u L proteinase K digested W C L o f 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 i n immunoblotting was acquired from vaccinated (a) fish serum, (b) rabbit serum, (c) chicken egg yolk watersoluble 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 i n immunoblotting was acquired from nonvaccinated (a) fish serum, (b) rabbit serum, (c) chicken egg yolk watersoluble fraction. 90 Fig.4.4. Specific anti-K anguillarum I g Y E L I S A values o f 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 I g Y E L I S A values o f a 10-fold diluted water-soluble fraction o f egg yolk before and after spray-drying. 97 Fig.4.6. Fluctuation i n total I g Y concentration o f a 10-fold diluted watersoluble fraction o f egg-yolk.  101  Fig.4.7. Fluctuation i n specific anii-V. anguillarum I g Y titer o f a 10-fold diluted water-soluble fraction o f egg yolk. 102  xi  Fig.4.8. Levels o f specific mti-V. anguillarum I g Y in the water-soluble fraction o f the egg yolks collected from immunized hens in different postvaccination dates compared to pre-vaccination level (indicated as nonspecific).  103  Fig.4.9. Cumulative 14-day post-challenge mortality rates i n fish groups UP injected with P B S , non-specific or specific anti-K anguillarum I g Y compared for each challenge date.  106  Fig.4.10. Cumulative 14-day mortality rates i n groups o f 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 I g Y level i n fish intubated with W S F alone, associated with Mega9 or sodium pyrophosphate and encapsulated i n 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 o f P L G encapsulated water-soluble fraction ( W S F ) o f egg yolks.  116  Fig.4.13. Effect o f Tween-80 and Tween-20 at two concentration o f 5% & 2.5% on the absorption o f I g Y into the rainbow trout bloodstream.  122  Fig.4.14. Effect o f Mega9, sodium pyrophosphate, sodium bicarbonate, 5% Tween-20 or some combinations o f them on the absorption o f I g Y into the rainbow trout bloodstream.  125  Fig.4.15. Microscopic photographs o f H & E stained cross section o f the intestinal tissues o f 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 o f an untreated rainbow trout (in experiment 6).  154  Fig.4.17. Microscopic photograph o f H & E stained cross section o f the intestinal tissues o f a rainbow trout intubated with Mega9 and antacid. The dissected tissue was fixed in B o u i n ' s solution.  155  Fig.4.18. Microscopic photograph o f H & E stained cross section o f the intestinal tissues o f a rainbow trout intubated with deoxycholate and antacid. The dissected tissue was fixed in B o u i n ' s solution.  156  Fig.4.19. Microscopic photograph o f H & E stained cross section o f the intestinal tissues o f a rainbow trout intubated with octyl-(3-glucoside and antacid. The dissected tissue was fixed in B o u i n ' s solution.  157  xn  Fig.4.20. Microscopic photograph o f H & E stained cross section o f the intestinal tissues o f an untreated control rainbow trout. The dissected texture was fixed in B o u i n ' s solution.  158  Fig.7.1. Temporal trend o f mortality rate in relation with total serum I g Y level in the fish groups IP injected with anti-K anguillarum IgY.  179  Fig.7.2. Temporal trend o f mortality rate in relation with anti-K anguillarum I g Y titer i n serum o f fish following IP injection with antiVibriolgY.  180  Fig.7.3. Temporal trend o f anti-V. anguillarum I g Y serum titer fluctuations i n relation with total serum I g Y 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 o f challenge) and fixed in buffered formalin.  182  Fig.7.5. Microscopic photograph o f H & E stained cross section o f 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 o f challenge) and fixed in buffered formalin.  183  Fig.7.6. Microscopic photograph o f H & E stained cross section o f the intestinal tissues o f a rainbow trout intubated with Mega9, antacid and W S F (in experiment 4) sampled 3 hours after intubation and fixed i n buffered formalin.  184  Fig.7.7. Microscopic photograph o f H & E stained cross section o f the intestinal tissues o f 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 o f H & E stained cross section o f the intestinal tissues o f 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 o f H & E stained cross section o f the intestinal tissues o f a rainbow trout intubated with P B S (in experiment 4) sampled 3 hours after intubation and fixed in buffered formalin.  187  xiii  LIST OF ABBREVIATIONS  °c Kg  uL abWSF AD ANOVA AP BCIP bGH BSA C cc  ccw  cfu d db DCM DEA DX ELISA FD Fig. g G I tract h H&E hGG hGH HRP IgG IgM IgY M W IN IP kDa kg L LMM  Degree(s) Celsius Microgram(s) Microliter(s) Water-soluble fraction o f egg yolks from vaccinated hens after absorption of antigen-specific I g Y by formalin killed V. anguillarum A i r drying Analysis of variance Alkaline-phosphatase 5-bromo-4-chloro-3-indolyl phosphate Bovine growth hormone Bovine serum albumin Control treatment Cubic centimeter Cheddar cheese whey Colony forming units Day Dry base Dichloromethane Diethanolamine Sodium salt o f deoxycholic acid Enzyme-linked immunosorbent assay Freeze-drying Figure Gram(s) Gastrointestinal tract Hour(s) Haemotoxylin and eosin Human gamma globulin Human growth hormone Horseradish peroxidase Immunoglobulin G Immunoglobulin M E g g yolk immunoglobulins Intermediate molecular weight Intubated Intraperitoneal Kilodalton(s) Kilogram(s) Liter(s) L o w molecular weight protein standard marker  xiv  LPS L-WSF M M9 mg min mL mM mmHg MW NA NBT NC ng nm NMW NMWC nspWSF OBG P PBS PK P L G 50:50 P L G 85:15 PLGA +  /7-NPP  PVA PY RID rpm SBC SDS SDS-PAGE SFU sGH spWSF T T20 T80 TBS TNP-LPS TSA TSB TTBS UBC UF  Lipopolysaccharide Lyophilized water-soluble fraction o f egg yolks Molar Mega9 Milligram(s) Minute(s) Milliliter(s) Millimolar Millimeter o f mercury Molecular weight N o t available Nitro blue tetrazolium Nitrocellulose Nanogram(s) Nanometer(s) Nominal molecular weight Nominal molecular weight cut off Non-specific water-soluble fraction o f egg yolks from unvaccinated hens Octyl-13-glucoside Top-dressed pellets Phosphate buffered saline Proteinase-K Poly -lactide-co-glycolide with a lactide to glycolide ratio o f 50:50 Poly -lactide-co-glycolide with a lactide to glycolide ratio o f 85:15 Antigen containing P L G Alkaline p-nitrophenyl phosphate Polyvinyl alcohol Sodium pyrophosphate Radial immuno-diffusion assay Rotation per minute Sodium bi-carbonate Sodium-dodecyle sulfate Sodium-dodecyle sulfate-polyacrylamide gel electrophoresis Simon Fraser University Salmon growth hormone Specific water-soluble fraction o f egg yolks from vaccinated hens Test treatment Tween-20 Tween-80 Tris buffered saline Trinitrophenilated lipopolysaccharide Tryptic Soy agar Tryptic Soy broth T B S including Tween-20 University o f Brithish Columbia Ultrafiltration  XV  v/v VM VMD w/o w/o/w WCL WSF  Volume per volume V a c u u m microwave V a c u u m microwave drying Water in o i l Water in o i l in water Whole cell lysate Water-soluble fraction o f 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 o f study.  I also wish to express my best thanks to other members o f my advisory committee, Dr. Shuryo Nakai, Dr. Eunice L i - C h a n and Dr. Lawrence Albright for their wise suggestions and kind and caring criticism which lighted my way through difficulties o f research.  I extend my thanks to Dr. A l e x Y o u s i f for his technical consultation and guidance, to M r . Abdol-Hossein A m i n i , M r s . Angela Kummer, M r . Guillaume D a and Mrs. Parastoo Yaghmaee for their assistance, to M r . Sherman Y e e 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, D r . Emanuel A k i t a for scientific advice, to M s . Joyce T o m and M s . Jeannette L a w , the secretaries o f the department, to M r . Michael Igallo and M s . Julie C h o w in the Histology Lab, Department of Pathology, to Dr. Helen Burt in the Department o f 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 o f University Graduate Fellowship (1996-1998) is also gratefully acknowledged.  xvii  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. A n d , to my brothers and their families whose love and emotional support have always brought me hope and strength.  xviii  CHAPTER ONE INTRODUCTION  1.1. Background of the study Control o f fish diseases is o f great concern in aquaculture because o f the large population o f fish and the high risk of disease transmission in an aqueous environment (Dunn et al., 1990).  Vibrio anguillarum  is one o f the most important pathogens o f  marine and fresh water fish, causing up to 40% mortality in severe cases, with an annual loss o f 11 million British pounds due to the disease i n 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 o f antibiotic use and other chemotherapeutical methods in aquaculture (Palm Jr. et al.,  1998).  The  alternative practice used to control disease outbreaks is vaccination, which i n 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). O n the other hand, the availability o f large amounts o f relatively inexpensive immunoglobulins from egg yolk (IgY) offers the possibility o f using specific I g Y for passive immunization by oral administration.  Oral use o f immunoglobulins is  environmentally friendly and unlike antibiotics, elicits no side effects,  disease  resistance, possibility o f overdosing or occurrence o f toxic residues, and there is no injection or handling involved.  The efficacy o f I g Y treatment to provide passive  immunity against diseases has been investigated by several researchers.  I g Y 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 I g Y 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 o f 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 i n rainbow trout when fed anti-7. ruckeri I g Y either before or after immersion infection. However, intraperitoneal (LP) injection o f the same I g Y 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. I g Y 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 I g Y extraction from the egg yolk have already been developed. However, large-scale commercial use o f I g Y requires dehydration methods that confer minimal loss o f activity.  Vacuum  microwave dehydration, a new drying technique, may provide a cost-effective method, to save antigenic properties o f IgY.  3  1.2. Hypothesis 1.  Oral administration of specific chicken egg I g Y raised against V. anguillarum  will  decrease mortality level in rainbow trout challenged with this pathogenic bacteria, provided that I g Y survives adverse effects o f acidic conditions and digestive enzymes o f the fish stomach. 2.  Dehydration o f I g Y using freeze-drying, vacuum microwave drying or air-drying w i l l not destroy its immunogenicity.  3.  Co-application o f detergents w i l l enhance the uptake o f orally administered I g Y into the fish blood stream.  1.3. Objectives of the study The main objective o f this study was to find an effective means o f using chicken egg yolk immunoglobulin, I g Y , specifically raised against certain disease causing organisms, to increase disease resistance o f farm  fish.  The most practical  approach seemed to be oral administration o f I g Y , with the immune  enhancing  materials added to the feed. Methods were sought to minimize destruction o f the I g Y 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 o f the delivery o f 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 o f this research include: •  T o study antigen-antibody binding between V. anguillarum  and chicken egg yolk  IgY, in comparison with the binding o f other immunoglobulins such as rabbit I g G and fish I g M . •  T o determine the best method o f drying I g Y for addition to fish feed.  •  T o postulate a theory to explain how detergents may enhance absorption o f protein molecules such as I g Y through the fish gut into the circulating blood.  5  CHAPTER TWO Review of Related Literature  2.1. Vibriosis and otherfishdiseases Vibriosis is a bacterial disease o f many salt-water fish including salmonids, the severity o f which has increased with the expansion o f fish farming.  The most  significant losses occur in cultured Pacific salmon, Atlantic salmon {Salmo salar) and rainbow trout (Oncorhynchus farms.  mykiss) grown in fresh water farms or in marine cage  Cultured non-salmonid fish including eel, yellow tail and red sea bream can  also be affected (Ezura et al, 1980). In severe cases o f vibriosis, mortality may be up to 40% o f affected population. However, several vaccines have been developed and proven effective, especially when delivered by injection or immersion methods. The most anguillarum.  commonly encountered  fish  pathogenic  Vibrio  species is V.  It constitutes part o f the normal microflora o f the aquatic environment.  The next most important Vibrio species is V. ordalii (Smith, 1988). Signs o f vibriosis include hemorrhaging at the base o f 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.  distended and filled with clear viscous fluid. V. anguillarum  It may be  enters the fish by  penetrating the descending intestine and rectum (Ransom et al, 1984). Vibrio bacteria can survive in the slime o f uncleaned tanks, nets, build up o f 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 ( L P S ) in the cell wall.  7  This L P S may not fully degrade in the acidic environment o f the stomach (Kawai & Kusuda, 1983; Smith, 1988; W o n g et al., 1992). Chart and Trust (1984) isolated two other minor heat labile proteins from the outer membrane having M W s o f 49-51 kDa. Boesen et al. (1997) detected humoral antibody in fish injected with either o f the extracellular products: cytoplasmic membrane proteins or outer membrane proteins with the latter eliciting the strongest reaction.  They postulated the existence o f  undefined potent antigens among these proteins. Sites o f entry o f Vibrio include anal and oral routes, as well as gills and skin (Evelyn, 1984; Laurencin & Germon, 1987). However, kidneys, spleen, liver and lamina epithelia o f the lower intestine may be infected as disease progresses (Nelson et ai, 1985). Although some reports emphasize the importance o f posterior intestine to induce a sufficient immune response (Rombout & V a n den Berg, 1989), O'Donnell et al. (1994) have demonstrated uptake o f L P S in the anterior part o f 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 o f whole cells and extracellular products o f most commonly encountered species (Smith, 1988). Methods o f vaccination include injection, oral administration and immersion.  2.3.1. Intraperitoneal (D?) injection The most effective method o f immunizing fish is intraperitoneal (IP) injection. This technique allows the use o f adjuvants, which enhance the magnitude o f the  8  immune response.  Injection ensures that each fish receives the exact dose o f 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 vaccinations.  o f two methods,  namely dip and bath  D i p vaccination involves dilution o f vaccine i n a water container,  removal o f fish from the holding facility, immersion in vaccine solution for about 30 seconds and the return o f the fish to the holding facility. The main portal o f 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 i n the former  method,  it requires  a longer exposure  time,  approximately 1-2 hours, and requires oxygenation o f the water. Immersion methods permit mass vaccination o f the fish below 5g (Ellis, 1988a; H o m e & Ellis, 1988).  2.3.3. Oral vaccination Oral vaccination, which involves the incorporation o f 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 o f treatment  9  water and allows mass vaccination o f fish o f any size.  However, oral vaccination  requires larger doses o f 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 o f control on the dosage for each fish, which is dependent on the feeding rate (McLean et al., 1999; H o m e & Ellis, 1988; Hart et al, 1988). Research is still continuing to optimize oral vaccination conditions and increase the potency o f this method.  2.4. Antibodies as a replacement for antibiotics Antibiotics are commonly used i n treatment o f infectious diseases o f humans and animals. However, with long-term use and/ or insufficient dosage o f antibiotics, bacteria may mutate and exhibit resistance against the antibiotic. This may result in selection o f resistant microbial strains.  Antibiotic residues i n the fish muscle may  enter human food, another undesirable consequence o f 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 o f 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 o f antibodies (Coleman, 1999).  The use o f antibodies is not restricted to bacterial and  fungal diseases but also has application to viral infection and neutralization o f 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 i m m u n i z a t i o n Passive immunization involves use o f 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, Ebina et al,  1990; Marquardt, 1999; Hatta et al,  Gutierrez et al,  1994).  1999; Bartz et al,  1980;  1993b; Yokoyama et al,  1992;  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 o f immunity  when vaccinated below 0 . 5 - l g o f 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 o f specific immunoglobulins to certain antigens (bacteria, virus, venom, toxin, etc.) may be applied to neutralize biological activities o f the antigens. . However, administration o f large amounts o f antibody may be required in passive immunization (Hatta et 1997). Akhlaghi (1999) passively immunized rainbow trout using anti-K  al,  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 i n a challenge with the pathogenic bacteria up to a month post-injection.  However, protection offered by  trout anti-Vibrio serum was weaker than that o f the others.  The protection rate o f  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 i n 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 i n absorption o f antibodies from the gut as no mix-Vibrio antibodies were detected in the sera o f fish following oral administration.  2.6. Use of chicken egg yolk antibodies (IgY) in passive immunization The yolk o f hens eggs is a valuable source o f specific antibodies, which can be used i n prophylactic or therapeutic treatments to confer passive immunity (Li-Chan, 1999). The effectiveness o f oral administration o f I g Y i n prevention o f dental caries in rats (Hamada et al, 1991), rotaviral diarrhea i n 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 o f pathogen specific I g Y to prevent chronic Pseudomonas aeroginosa colonization in the airways o f the patients with cystic fibriosis. I g Y has also proven effective in promoting disease Gutierrez et al.  resistance  in fish.  (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 o f anti- E. tarda I g Y and the pathogen was cannulated into the stomach o f Japanese eels after their intestinal mucosa was damaged using hydrogen peroxide solution (30%>). I g Y treated eels survived while the control eels, which were infected with the same concentration o f bacteria without adding I g Y , died or showed the symptoms o f the disease (Hatta, et al., 1994).  Decreased intestinal necrosis due to  Vibrio species in Japanese flounder larvae and a decrease i n signs o f vibriosis in Japanese shrimp when the animals diets contained anti-Vibrio I g Y have also been reported (Gutierrez et al., 1994).  M i n e et al (1999) demonstrated that feeding  encapsulated specific anti-Yersinia ruckeri I g Y to rainbow trout either before or after an immersion infection could only produce a marginal reduction i n mortality and intestinal infection. This same I g Y 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" emulsion 1  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 ( M W 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 o f yield, ease o f collection and reduced stress on the host animal. Production o f eggs with a high antibody titer is continuous throughout the year, whereas colostrum is only produced at parturition, and at other times, antibody content o f milk is minimal. Specific antibody titer as high as lxlO  1 5  units per m L has been reported in immunized hens (Coleman, 1999).  Approximately 3-4g o f I g Y could be obtained per laying hen each month, when immunized against bovine serum I g G i , IgG2 and I g G isolated from cheddar cheese whey. This corresponds to an annual yield o f 40-50g I g Y per hen (Akita & Li-Chan, 1998; L i - C h a n , 1999), about 30 times the I g G produced in the serum o f an immunized rabbit (Hatta et al, 1997), which must be sacrificed to collect the IgG. O f course I g Y antibodies, like other immunoglobulins collected from other host animals,  are  polyclonal and contain antibodies specific to a variety o f antigens to which the hen has been exposed. Levels o f antibody i n I g Y preparation have been reported to be 5% for anti-insulin antibody and 28% for anti-mouse I g G antibody (Hatta et al, 1997) or 1015% o f the total I g Y when bovine I g G and lactoferrin were employed as antigens (Akita and Li-Chan, 1998; L i - C h a n et al, 1998). There are other advantages that make I g Y more favorable than mammalian IgG.  Production o f I g Y 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 o f 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 o f the I g Y molecule as an antibody (Li-Chan, 1999). I g Y is fairly stable when exposed to trypsin or chymotrypsin but more sensitive than I g G to pepsin digestion at p H levels lower than 4. I g Y 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). I g Y molecules incubated with pepsin at p H 2 were hydrolyzed into small peptides and no band corresponding to I g Y was detected after one hour. O n 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 o f antibodies, including I g Y (Li-Chan, 1999; A k i t a 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 o f I g Y unless it was repeated several times. In contrast, Chansarkar (1998) demonstrated that some loss o f antibody activity might appear when I g Y was frozen or freeze-dried at a concentration o f 30mg m L ^ o r especially l m g mL" . 1  Samples with a high protein  concentration o f 30mg mL" underwent a drastic insolubilization, especially after 1  freezing or freeze-drying.  Insolubilization was reversible when the sample was  diluted to l m g mL" . However, proteins may suffer loss o f activity during freeze1  drying as a result o f 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 o f the egg yolk. These methods, as reviewed by A k i t a & 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. A m o n g all, the method reported by A k i t a & Nakai (1992) and Fichtali et al. (1992) appears to be the simplest.  This  method involved 10-fold dilution o f egg yolk with distilled water, acidified with 0.1 N HC1 to a final p H o f 5.2.  Centrifugation o f this preparation following overnight  sedimentation provided a water-soluble fraction ( W S F ) containing I g Y at 1 5 % purity (protein basis), which could be used as a semi-pure source o f I g Y or might be submitted to further purification steps. Lower levels o f 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 o f egg yolk granules. The W S F was almost devoid o f lipids in p H levels o f 4.6 to 5.2 while the maximum recovery o f I g Y was obtained i n the range o f p H 5.0 to 5.2. Besides IgY, the other major proteins present in the W S F 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 I g Y reach the bloodstream via intestinal absorption. A variety o f studies, both in fish and higher vertebrates, have demonstrated that the gut mucosal barrier does not completely prevent  ingestion  o f micro  particles  and macromolecules  and that the  fish  gastrointestinal (GI) tract, i n particular, retains the capacity to absorb intact proteins and peptides ( M c L e a n et al, 1999). Scientific observations include the absorption o f horseradish peroxidase ( H R P ) into the blood o f rainbow trout intubated through oral and anal routes ( M c L e a n et al,  1999), transfer o f bovine growth hormone i n a  functional form through the G I tract o f trout (Le B a i l et al,  1989), appearance o f  orally intubated bovine serum albumin i n the serum o f chum salmon (Fujino & Nagai, 1988) and transport o f rabbit I g G into the circulation o f goldfish while preserving antigen binding activity (Nakamura et al, 1990). Sire and Vernier (1992) suggested that the posterior gut o f teleost  fish  facilitates the absorption and digestion o f protein arriving there. Macromolecules are reportedly absorbed from the gut lumen and transported into the bloodstream by transcellular  (intracellular)  mechanisms  i n which  macromolecules  enterocytes v i a pinocytosis by the microvillus membrane.  enter  the  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 o f the circulation and this may help direct passage into  19  the lamina propria (O'Haggen, 1996; Walker, 1986; M c L e a n & A s h , 1987a; M c L e a n & Donaldson, 1990). In Fig.2.1 transcellular and paracellular modes o f absorption are illustrated. Fig.2.2 demonstrates how particulate matters move from the gut into the bloodstream o f mammals by way o f the extrusion zones o f 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). O n the other hand, the cell sloughing process results in the formation o f 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 o f parasitic infections, pollutants, toxins or due to malnutrition can form a portal for the absorption o f macromolecules ( M c L e a n 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 o f protein absorption from the G I tract o f 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 i n the rapid transfer o f antigen to the vicinity o f 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 villi 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 ( M c L e a n & A s h , 1986). Direct administration o f rabbit I g G into the gut o f rainbow trout resulted in a significant level o f absorption after 3 hours (Fujino et al,  1987). There are several  other indications o f transfer o f proteins into the bloodstream o f teleosts.  Fig.2.3  (adapted from M c L e a n & Donaldson, 1990) shows the time course o f appearance o f various bioactive proteins i n the serum o f orally and anally intubated fish. W i t h respect to the efficacy o f macromolecule uptake, Georgopoulou et al (1988) estimated up to 6% absorption o f tracer protein in a substantially intact form into the blood circulation o f 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 o f 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 i n macromolecule absorption between species and even between individuals o f the same species (Smith, 1989; M c L e a n 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  administration ( M c L e a n et al,  1999).  anal intubation as  compared  to oral  However, Jenkins and colleagues (1994)  demonstrated similar plasma absorption levels in both anal and oral administration o f H G G to tilapia, when the protein was delivered without adjuvant or co-delivered with aluminum hydroxide.  23  Oi  I  I  15  I  30  45  l  60 Time  i  n  75  — i — •  90 480  1440  (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 m L of serum). (1) Doggett et al. (unpublished data): horseraddish peroxidase (HRP) subsequent to oral administration to Mozambique tilapia (200ug g" body weight in O.lmL of 0.9% saline). (2) McLean & Ash (1986): H R P in common carp after oral introduction of 200ug g" 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' . (4) Ash (1985) and McLean & Ash (unpublished data): H R P in rainbow trout after anal administration of lOOug g" in l m L of 0.9% saline. (5) McLean (1987): Human cationic trypsin (HCT) in common carp after oral delivery of 4ng g" in l m L of 0.9% saline. (6) McLean & Ash (unpublished data): H R P in goldfish after oral administration of 800ug g' in 0.25mL of 0.9% saline. (7) Georgopoulou et a/.(1988): H R P in rainbow trout after oral administration of lOOug g" in l m L of isotonic saline. (8) McLean & Ash (1987b): H R P in rainbow trout after oral administration of lOOug g" 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" body weight. (10) McLean and colleagues (unpublished data): recombinant bovine somatotropin in coho salmon parr after oral administration of 20ug g" in l m L of 0.9% saline. (From McLean & Donaldson, 1990) 1  1  1  1  1  1  1  1  1  1  24  There is evidence that uptake o f intact proteins is a dose-dependent process. A relatively linear dose response was observed in the serum uptake o f h G H when 0.1, 0.5 or 1 mg kg" body weight o f it was orally delivered in 0 . 0 5 M deoxycholate 1  solution into starved carp (Hertz et al, 1991). M c L e a n and co-authors (1999) reported unpublished data from M c L e a n & A s h demonstrating  a relatively linear dose-  dependence in plasma level o f 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 o f 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 o f the fish gut to absorb proteins at a specific dose, 2. extent o f natural variation between individual fish in absorption o f macromolecules (McLean et al, 1999).  2.13. Protection from degradation in the GI tract M o s t 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 o f the fish gut could be employed as a means o f passively immunizing fish. In order to accomplish this objective, absorption o f the bioactive protein macromolecules into the  25  fish bloodstream has to be improved. Several methods have been studied to improve oral uptake o f proteins in fish, such as encapsulation in protective coatings, coadministration with antacids or proteolytic enzyme inhibitors and co-delivery with detergents. These methods have been more extensively reviewed by Ellis (1995) and M c L e a n et al. (1999).  Use o f encapsulation and co-administration o f absorption  enhancing agents are reviewed below.  2.13.1. Encapsulation Different methods o f encapsulation or coating have been employed to reduce digestive degradation o f oral vaccines and improve efficacy o f protein delivery into fish blood circulation.  Lillehaug (1989) incorporated lyophilized V. anguillarum  vaccine into a slow release pellet, "prill", or coated the vaccine with an acid-resistant film o f methylacrylic acid and acrylic acid ethyl ester. However, challenge with V. anguillarum following oral vaccination showed higher mortality levels i n the rainbow trout treated with protected vaccine than with an unprotected one. W o n g et al. (1992) sprayed  V. anguillarum vaccine onto dextrose beads followed by coating with  Eudragit L - 3 0 D . 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 - 3 0 D entericcoated microspheres for two different antigens, trinitrophenylated lipopolysaccharide ( T N P - L P S ) and a protein antigen, trinitrophenylated keyhole limpet haemocyanin (TNP-KLH).  T N P - L P S at the higher dose o f lOpg increased serum anti-TNP  26  antibody titer o f coho salmon. W i t h the protein antigen only the lowest dose o f 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. antigen.  However, there was no control group treated with unprotected  Oral vaccination o f 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 o f 10-30mg B S A was administered, encapsulation increased the absorption rate by 1.7 times.  However, when a low dose o f 5mg unprotected B S A  was applied, a level o f 7.1- 34.5u.g mL" B S A was detected in the serum while no B S A 1  was detected in the eels serum following administration o f protected B S A at the same dose (Nagai & Fujino, 1995).  Shimizu et al (1993) encapsulated chicken egg yolk  I g Y i n lecithin-cholesterol liposomes. N o efflux o f encapsulated I g Y was observed when incubated in P B S at 37°C up to 72 hours. Encapsulation reduced I g Y 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)  ( P L G ) is  another  type  of  biodegradable  microcapsule used in the study o f 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 o f the microparticles. However, about 60% o f the antigen was detectable in the posterior intestine and the bloodstream o f fish i n 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 i n the kidney for up to 7 days. In fish treated with P L G - H G G , the antigen was detectable i n 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 i n the kidney for up to 5 weeks. In vitro studies showed that when a polymer ratio o f 50:50 (lactide: glycolide) was used, 50%o o f the antigen was released in the first 2 weeks, whereas with the ratio o f 85:15 no release was detected over the entire 29 weeks o f study.  A k i t a & Nakai (1999)  prepared enteric-coated gelatin capsules o f I g Y with cellulose acetate phthalate. In vitro studies revealed a complete loss o f activity o f unprotected I g Y in p H 1.2, while the capsules were stable i n simulated acidic conditions o f stomach for 3 hours, but easily dissolved under alkaline conditions ( p H 8.0).  28  6  -a S3  s  6  J  &  f  -I  L  J  Ji JS U3  T i m e (post-oral 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 ); • P L G - H G G (2.5mg fish" ). (From O'Donnell et al., 1996) 1  1  29  2.13.2. Enhancement of absorption using detergents Use o f a variety o f non-ionic detergents and bile salts has been investigated as a means o f enhancement o f protein absorption from the fish gut. Oral intubation o f 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 o f h G H . The levels o f h G H i n deoxycholate solution which appeared i n the circulation of starved and fed carp were estimated to be at least 2 % and 0.5% o f the intubated dose, respectively. It was postulated that deoxycholate might increase the availability of protein molecule for absorption through formation o f complexes i n which the hydrophilic end o f deoxycholate is attached to the protein molecule and the lipophilic end to the gut lipid membrane (Hertz et al, 1991). Administration o f Q u i l - A saponin caused a considerable increase in the uptake of orally and anally intubated human gamma globulin, H G G , i n tilapia, a gastric fish. In this study, Q u i l - A saponin was observed to have a number o f physical effects on the intestinal enterocytes o f tilapia, such as loosening intercellular junctions, increasing the pinocytosis o f 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. Q u i l - A also caused the microvilli o f the enterocytes to become shortened and damaged. Other possible modes o f action was postulated to be neutralization o f gastric and intestinal proteolytic enzymes, hence an increased level o f antigen reaching port o f absorption i n the intestine (Jenkins et al, 1991). In contrast, Akhlaghi (1999), who co-administered sheep antibodies with Q u 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 o f non-ionic detergents, N-D-gluco-Nmethylalkanamide, properties.  as exhibiting  high  solubilization power and  non-denaturing  These detergents attained all o f the desirable properties o f commercially  used non-denaturing detergents o f the time, such as Triton X - 1 0 0 and octyl-p-Dglucoside for membrane studies, while at the same time they were produced in a higher yield and lower cost. from this family.  Mega-9, nonanoyl-Af-methylglucamide, is a detergent  Oral co-administration o f Mega-9 enhanced gastrointestinal uptake  of H R P by rainbow trout. Tissue accumulation o f H R P was further enhanced when a combination o f Mega-9 and soybean trypsin inhibitor was used (McLean and A s h , 1990).  These authors reported gelatinization o f mucus lining o f the G I tract and  formation o f mucus clumps due to Mega9.  In another study, simultaneous use o f  Mega-9 and sodium bicarbonate enhanced the growth rate conferred by oral intubation of recombinant bovine somatotropin in coho salmon, O. kisutch ( M c L e a n et al., 1990). M a n y different detergents have been tested for solubilization effect on lipid bilayer membranes. sulfonate)  and  C H A P S (3[(3-Cholamidopropyl)dimethylammonio]-l-propane-  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 w h i c h 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 o f some o f these detergents are shown in Fig.2.5 (a-j).  31  0 11 11  CHc^CHL^CHo' C c - NCHo 1  CH H  9  i  - C1- O H HO - C - H H 1 -C-OH H I -C-OH i  CH OH 2  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  OCH (CH ) Chi  1111  2  HO  2  6  OH  Fig.2.5.(c). Chemical structure of octyl p-glucoside (n-octyl-p-D-glucopyranoside).  HN 2  H  9  HSCH C — C - O C H C H 2  2  3  HCI  Fig.2.5.(d). Chemical structure of L-cysteine ethylester.  33  CH3 H  3  C —  CH3  ,  .  C-CH2-C—/ C H  C H  3  V-O(CH CH 0>-H 2  2  3  N = approx. 9 . 5  (e)  C1 H  CH  3  H C—(j-CH -(j 3  2  r/~1 T T  '_/ V 3  K  )—  f~*  f*  v  CH  3  CH  7  0(CH CH 0)-H  \\  2  2  N  3  N = 7 to 8  (0 Fig.2.5. (e, f). Chemical structures of polyoxyethylene ethers, (e) Triton X-100, (f) Triton X114.  34  Fig.2.5.(h). Chemical structure o f C H A P S O (3-3-cholamidopropyl dimethylammonio2-hydroxy-1 -propane-sulfonate).  35  HO(Cr^CH 0) 2  w  xS  ,(OCH2CH ) OH 2  |  X  H(OCH CH ) OH 2  2  Q  Y  CH20(CrtCH20) -iCH2CH 0-C-CH2(CH2)9CrH3 Z  2  Sum of w + x + y + z = 20  Fig.2.5.(i). Chemical structure o f Tween-20 (polyoxyethylenesorbitan monolaurate).  HO(CriCH 0) 2  ^(CCHpH^OH  w  D  YKOCHjCr^JyOH  O  CH 0(C|-bCH 0) . Cr^CH CwC^H (CH ) Cr^CHsCHCH ^ 2  2  z  1  2  2  2  7  2  S u m o f w + x + y + z = 20  Fig.2.5.(j). Chemical structure o f Tween-80 (polyoxyethylenesorbitan monooleate).  36  2.13.3. Immunostimulants Various chemical compounds, called "immunostimulants", are known to increase resistance o f 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 o f muramyl- and lipo-peptides (Raa, Yano et al.,  1991).  1996;  Galeotti,  1998).  Similar  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 o f cytotoxic cells (McLean et al., 1 9 9 9 ; Anderson,  1992).  N i k l et al.  challenge after a single  UP  (1992)  reported an improved survival in bacterial  injection o f P-glucan. De Baulny et al.  (1996)  found no  reduction i n 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 o f rainbow trout following administration o f glucan has also been reported (Anderson & Jenny,  1993).  Siwicki et al.  (1994)  observed elevations in oxidative radical release,  phagocytic activity, killing potential o f neutrophils, as w e l l as total immunoglobulin and total plasma protein levels after feeding fish with a variety o f immunostimulant compounds. A challenge with Aeromonas salmonicida revealed a greater resistance in rainbow trout fed immunostimulants in the diet.  37  2.14. Dehydration techniques The main objective o f dehydration as a preservation method is to convert perishable products to stable materials by reducing their water activity. Depending on the water content o f the fresh product, weight and perhaps volume o f the dehydrated material may be reduced. Consequently, it can be more easily stored for an extended time or transported to the sites o f demand.  A variety o f techniques are commonly  used to dehydrate food products at the industrial level.  In all these techniques an  energy supply is needed for concurrent transfer o f heat into and the water mass out o f the material.  Dehydration involves the manipulation o f temperature to evaporate  water followed by removal o f water vapor after separation from the dehydrated material (Jayaraman & Das Gupta, 1992). There are some advantages and limitations associated with each o f these techniques, based on which the method o f 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 o f the dried product (Yang & Atallah, 1985; L i n et al, 1998). Hot air drying is usually carried out at temperatures o f 60°C to 90°C or even greater in order to achieve efficient drying.  L o n g drying  38  times at this temperature combined with available liquid water in the material can cause  undesirable  changes  to the  properties o f the dried product.  structural, physico-chemical and  functional  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 products o f high quality.  food  Although all the above mentioned restrictions are  disadvantageous to the application o f 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 o f high quality commodities.  In this method, frozen products are transferred to the drier  chamber i n w h i c h the pressure is reduced to a level at which water sublimates from ice crystals to vapor i n a low temperature (Somogyi & L u h , 1975). Freeze-drying usually results i n the least structural and chemical damage to dried products when compared with other dehydration methods.  The low processing temperatures, the absence o f  liquid water and l o w concentration o f O2 in the dehydration environment combine to minimize degradative effects such as protein denaturation, enzymatic and oxidative reactions.  Sublimation o f water from the rigid frozen structure prevents collapse o f  the solid matrix o f the dried product and results in a porous, non-shrunken structure. However, slow drying rates and use o f 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 o f water from the frozen phase are energy intensive (Durance, 2000). The high expense o f the process is considered the major limitation o f this technique. Since the cost o f freeze-drying depends on the amount o f 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 d r y i n g Vacuum microwave ( V M ) drying is an emerging technique, which often offers improved quality for dehydrated products.  In this method, drying takes place i n a  microwave oven chamber in which pressure is reduced.  Applying microwave  electromagnetic energy under vacuum provides the advantages o f 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 lowtemperature 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 o f the dried product. The quality o f this dried food is i n some cases comparable to that o f freeze-dried foods while the operating costs are estimated to be closer to those o f forced convention air-drying technology (Durance, 2000). Vacuum microwave drying has been used in dehydration o f plant and animal materials such as sweet basil ( Y o u s i f et al., 1999), carrot slices ( L i n et al., 1998), potato chips (Durance & L i u , 1996), cranberry (Yongsawatdiguul & Gunasekaran, 1996) and shrimp ( L i n et  40  al., 1999). In all cases, vacuum microwave dried products showed better retention o f key constituents and sensory properties than air-dried materials.  However, a major  disadvantage o f this method is the high cost o f energy required to generate microwave power. The economics o f 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 o f moisture by lower cost heat treatments (Drouzas & Schubert, 1996).  When a major portion o f  water content is removed by lower cost dehydration methods, microwave drying can be used for a quick and efficient removal o f the remaining moisture from the interior parts o f the product without overheating the pre-dehydrated product (Attiyate, 1979). Durance & W a n g (1999) who used a combination o f air-drying and vacuum microwave drying to dry tomatoes, calculate the costs o f the process.  The authors  concluded that in spite o f the higher cost o f electricity energy used in vacuum microwave drying than that o f 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 i n the final portion o f 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 C 0 ; 2.93g N a H C 0 ; 0.2g N a N ; I L H 0), 2  3  3  3  Phosphate-buffered  2  saline  (PBS) at p H 7.4 (8g N a C l ; 0.2g K H P 0 ; 1.15g N a H P 0 ; 0.2g K C I ; 0.2g N a N ; I L 2  4  2  4  3  H 0), Phosphate-buffered saline-Tween (PBS-Tween) at p H 7.4 (0.5mL Tween 20; 2  I L H 0), 2  blocking buffer (0.5% skim milk [Carnation, Nestle, D o n M i l l s , O N ,  Canada] in P B S ) , 10% diethanolamine ( D E A ) buffer (97.0mL diethanolamine; O.lg M g C l , 6 H 0 ; 0.2g N a N ; HC1 to p H 9.8; H 0 to I L ) . 2  2  3  2  P B S at p H 7.1 was used to suspend bacteria or dissolve W S F or chemicals (8g NaCl; 1.21gK HP0 ; 0.34gKH PO ; 1LH 0). 2  4  2  4  2  S D S - P A G E buffers included sample buffer (12.5mL o f 0 . 5 M Tris, HC1, p H 6.8; 20mL o f 10% S D S solution; l O m L glycerol; 5mL 2-mercaptoethanol; 2 m L 0.1% bromophenol blue; 50.5rnL H 0 ; total volume lOOmL), electrode buffer at p H 8.3 2  (3.03g Tris base; 14.41g glycine; 20mL 10% S D S solution; Ff 0 to I L ) . 2  Western blot transfer buffer ( 2 5 m M Tris; 192mM glycine; 20%> v/v methanol) stored i n a dark glass bottle at 4°C. Immun-blotting buffers include Tris buffered saline ( T B S ) at p H 7.5 ( 2 0 m M Tris; 5 0 0 m M NaCl), Tween-20-TBS wash solution ( T T B S ) at p H 7.5 (0.05% Tween20 i n T B S ) , antibody buffer (0.1% bovine serum albumin i n T T B S ) , alkalinephosphatase (AP) substrate stock solution I (50mg mL" 5-bromo-4-chloro-3-indolyl 1  phosphate (BCEP), Bio-Rad  170-653), stock solution II (50mg mL" nitro blue 1  tetrazolium ( N B T ) , Bio-Rad 170-6532), AP-buffer (lOOmM Tris, l O O m M N a C l , 5 m M M g C l , p H 9.5). 2  43  Fixative buffer for histological tissue samples included 10% buffered formalin solution (4g N a H P 0 , 6.5g N a H P 0 , lOOmL o f 37% pure formaldehyde, H 0 to 1L) 2  4  2  4  2  and Bouin's solution (1500mL o f a 21g L " picric acid aqueous solution, 500 m L o f 1  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 o f 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 A n i m a l Care Facility o f 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 o f this study, were obtained from local fish farms. They were acclimated i n an indoor 500L holding tank for a minimum o f 15 days before being split into 7 5 L 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 o f the fish holding tanks and the mentioned tank equipment. W a r m 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% o f their body weight, which were obtained from MooreClark 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 o f 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 i n 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 L t d . , Sannichton, B C , Canada) was rehydrated according to the manufacturer's instructions. It was then grown in lOOmL sterile  Tryptic  Soy  Broth  ( T S B ) (Difco  Laboratories,  Detroit,  MI, USA),  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 o f 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 7 5 L fish holding tank. (1). Water inlet, (2). Running water distributor, (3). Air inlet, (4). Standpipe drain.  46  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.  47  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 o f antigenicity and binding characteristics o f I g M to Vibrio antigens. prepare the vaccine, the frozen V. anguillarum  To  suspension maintained at -78°C  (described i n section 3.3) was thawed, vortexed and subsequently used to inoculate Tryptic Soy Agar plates ( T S A ) (Difco Laboratories) with 2 5 p L 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 o f the culture, the inoculum was streaked on T S A plates. ambient temperature.  Plates were then incubated for 24 hours at  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 o f 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 o f Damon, U S A ) in 5 0 m L 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 o f killed Vibrio in suspension to prepare the vaccine was adjusted to give an absorbance o f 5 at 540nm (As^nm 5 ) . =  Fig.3.3 shows the  process o f vaccine preparation and vaccination o f chickens in brief. A surface culture o f the bacteria was prepared on T S A plates before being  48  formalin-killed.  The concentration o f the bacterial cells i n the final suspension for  vaccine preparation was calculated to be 5x10 cfu m l / based on plate enumeration. 7  1  A n equal volume o f Freund's complete adjuvant (Sigma, F 5881) was subsequently mixed with the bacterial suspension for the first injection.  Boosters were also  prepared i n 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 o f age, the  first  intramuscular  injections  o f l m L anti-F! anguillarum  vaccine  were  administered to the chickens. One m L boosters were alternated between right and left sides o f 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 i n 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 i n antigenicity and binding characteristic studies. Twenty naive juvenile rainbow trout (average weight 35g) were acclimated for 15 days to a water temperature o f 12-13°C. A volume o f 0. l m L o f vaccine was intraperitoneally  49  Harvest after 24h @ 25°C  m  ST  Formaldehyde (0.3% in P B S )  t-rt  o  n <—  Freund's adjuvant  Washed, 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 . l m L booster preparations 14 days after the initial injections.  Fish were anesthetized with 0.5mL L " o f 2-phenoxy ethanol 1  (Anachemia A C - 7 1 9 0 P) prior to injection. B l o o d collection from the caudal vein was performed after 14 days to prepare serum as a source o f specific anti-P! anguillarum I g M . 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 I g Y potential to reach the fish bloodstream and confer a passive protection against diseases.  I g Y was also used to study its binding characteristics to V. anguillarum.  The I g Y preparation method was according to A k i t a & Nakai (1992), with some modifications. To prepare I g Y , egg yolk was carefully separated from the egg white and the yolk sac using paper towel to remove the final layer o f 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 o f 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 o f 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 5 B plus, Sorvall Instruments, Dupont, N e w T o w n , C T , U S A ) .  The supernatant was filtered through  glass w o o l and filter paper (Whatman #4).  Glass w o o l was used to exclude fat  residues. The filtrate, containing the water-soluble fraction o f egg yolk (WSF), was used as a source o f semi-pure I g Y 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 o f 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 - W S F ) was prepared  using a freeze-dryer (VirTis Research Equipment, Gardiner, N Y , U S A ) with a shelf temperature o f 20°C, a chamber pressure o f lOOpmHg and a condenser temperature o f -55°C. Fig.3.4 summarizes the process o f I g Y 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 I g Y preparation. Each batch contained eggs o f 2 or in some cases 4 consecutive weeks. Eggs used i n the drying studies were white eggs obtained from a local supermarket.  A larger ultrafiltration apparatus ( U F Spiral System, K o c h Membrane  Systems Inc., Wilmington Massachusetts, U S A ) equipped with a spiral membrane o f 30kDa N M W cut off was used i n this part o f the study to concentrate the W S F as one whole batch.  3.7. Preparation of microcapsules T o prevent I g Y degradation in the gastrointestinal tract o f fish, IgY-containing microcapsules were produced. 150mg o f the lyophilized W S F was dissolved in 1.5mL of d - H 0 , vortexed and centrifuged to remove the insoluble matter. The supernatant 2  52  Yolk  | Dilute x 10 in HC1 & d - H 0 final pH=5.2 2  Over night (5>4°C  Ultrafiltration Lyophilization (L-WSF)  Incorporation into the pellets  hollow fiber cartridge 10000NMWC  Centrifuged @16000xg @4°C  Supernatant: Water-Soluble Fraction ( W S F )  Orally intubated  Fed  I g Y absorption studies  Challenge studies  Fig.3.4. Schematic diagram o f the I g Y extraction from the hen eggs and application i n the absorption and passive immunization studies in rainbow trout. I g Y was recovered in the water-soluble fraction ( W S F ) o f the egg yolks, concentrated by ultrafiltration and lyophilized. The lyophilized W S F ( L - W S F ) was administered to fish v i a the oral route or LP injection to study the absorption o f I g Y and the protection I g Y may confer.  53  was then added to a solution o f 400mg poly(DL-lactide-co-glycolide) with a lactide:glycolide ratio o f 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 4 0 u L o f 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 G m b H , Switzerland) to produce a water in oil (w/o) emulsion. The mix was gradually added to a 2.5% solution o f polyvinyl alcohol ( P V A ) (98% hydrolyzed, 13,000-23,000 Daltons, Aldrich Chemical Company, Inc.) while stirring @ 900 rpm with a D y n a - M i x 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 0 followed 2  by centrifugation (Beckman G P R , U S A ) @ 3,000 rpm for 10 minutes and then dried at ambient temperature.  Size o f microspheres was determined by Particle Size  Analyzer (Coulter LS130, Coulter Electronics o f N e w England Inc., Amherst, Mass., U S A ) . Microscopic photographs o f the microparticles were produced using a camera attached  to an Olympus B H - 2 light microscope (Olympus G m b H , Hamburg,  Germany). Fig.3.5 shows the process o f preparation o f microcapsules.  3.8. Feed preparation T o examine I g Y uptake into the fish blood from feed, and to test the potential of this immunoglobulin in conferring passive immunity against diseases, I g Y 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)  1.5 ml d-H 0  400 mg PLG 85:15/50:50  2  10 ml DCM  <—  40 nl Span 80  centrifuge Supernatant  homogenize w/o emulsion  PLGA (w/o/w)  2.5h-  Air dried @ room temp. Microcapsules C30 um)  600 r^  Fig.3.5. Schematic diagram o f microencapsulation o f water-soluble fraction ( W S F ) o f egg-yolks i n polylactide-D-glucolide ( P L G ) with two different ratios o f lactide to glycolide, 85:15 or 50:50. Water i n o i l in water (w/o/w) emulsion o f W S F i n P L G i n dichloromethane ( D C M ) was prepared by the aid o f 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 o f 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 T o determine the I g Y level i n 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 o f each diet was soaked in l O m L P B S and blended (Ultra Turrax T25 DC A Labortechnik, I K A Works, Inc., Willmington, NC, USA).  Following overnight incubation at 4 ° C , the samples were vortexed and  centrifuged ( I E C Centra-7R Refrigerated Centrifuge) at 4000 rpm and 10°C for 20 minutes. The supernatant was collected after filtration through filter paper Whatman #4 i n order to measure total I g Y as well as anti-K anguillarum I g Y content. A l l samples were prepared in triplicate.  3.10. Blood collection from fish and serum preparation T o determine I g Y 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 o f 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 G m b H , 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 o f viable frozen bacterial suspension (described in section 3.3) was thawed, vortexed and pipetted into a 4 L flask containing 800mL o f 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, M i l l i p o r e Corporation, Bedford, M A , U S A ) . reached  The apparatus was stopped when the turbidity o f the culture  A54o m = 2.6. n  Approximately l O m L o f the bacterial suspension  was  transferred into a sterile 50mL capped Falcon tube and temporarily stored on ice until applied to the buckets o f salted water provided for fish challenge.  3.12. Lipopolysaccharide (LPS) & whole cell lysate (WCL) preparation Lipopolysaccharide ( L P S ) & whole cell lysate ( W C L ) were used to study the antigenic characteristics o f V. anguillarum in chickens (IgY), rabbits (IgG) and fish (IgM).  To prepare whole cell lysate ( W C L ) o f 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 . incubation at ambient temperature,  After 24 hours  a full loop o f V. anguillarum colonies was  transferred into a 1.5mL capped microtube and l m L o f P B S ( p H 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 o f 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. supernatant was transferred into a fresh tube.  The  This W C L preparation was used in  S D S - P A G E electrophoresis. V. anguillarum lipopolysaccharide ( L P S ) was prepared by protein digestion o f W C L using a solution o f proteinase K (PK), (0.0025g P K [ M E R K , 24568] i n l m L o f sample buffer, where l O p L o f this solution contained 25pg P K ) . To 5 0 p L o f the heated W C L , l O u L o f P K preparation was added and incubated at 60°C for 60 minutes. This was according to the procedure o f Hitchcock & B r o w n (1983). L P S prepared using this method was used for S D S - P A G E electrophoresis.  3.13. Anal intubation T o investigate crossing o f the I g Y molecules through the gut barrier, 0. l m L o f a concentration o f l m g mL" I g Y (Sigma 1-4881) was anally intubated into each fish 1  with an average weight o f 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. T w o fish were sampled at each time. Sandwich E L I S A was performed to determine I g Y levels in the serum o f 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 o f I g Y in the G I tract o f trout and I g Y 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 - W S F ) o f the egg-yolk from immunized chickens, which contained 12.4% total I g Y , was used as a source o f I g Y in all treatments. In the test treatments (T), rainbow trout (29g average weight) were orally intubated (IN) with 1. L - W S F solution i n sterile P B S , p H 7.1, 2. L - W S F i n 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 o f sodium pyrophosphate ( P Y ) (0.044g N J L ^ O ? , 10 H 0 in 2  lOmL o f 0.44M N a C l ) , p H 9.4, 4. L - W S F microencapsulated in P L G 85:15, 5. L - W S F microencapsulated i n P L G 50:50. Microcapsules had a theoretical I g Y loading o f 3.4%) (w/w) and an average particle size o f 29.39±5.75uxn for P L G 50:50 and 30.32±5.48u,m for P L G 85:15. microcapsules.  Fig.4.12 shows a microscopic photograph o f these  T w o control treatments (C) were also applied.  A positive control  59  group was intraperitoneally (LP) injected with L - W S F i n sterile P B S and a negative control group was orally intubated with sterile P B S . In treatments 1 to 3 fish were intubated with 2 0 0 u L o f lOOmg mL" L - W S F solution which provided 2.68mg total 1  IgY per fish or 92.4mg I g Y kg" average body weight.  In treatments 4 and 5, the  1  intubation material consisted o f 2 0 0 u L o f a 200mg mL" L - W S F containing P L G 1  suspension in P B S .  In the latter treatments, fish were theoretically receiving 46.9mg  I g Y kg" average body weight, which was equal to 1.36mg I g Y per fish. Positive 1  control fish were LP injected with 200uL o f a lOOmg mL" L - W S F solution in P B S . In 1  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 till week 5 for treatments 4 and 5. Sample size consisted o f 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 g Y levels i n 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 o f I g Y through the fish gut. In all cases, the source o f I g Y was L - W S F containing 12.4% total IgY. Each rainbow trout (16g average weight) was orally intubated with 2 0 0 u L of lOOmg mL" L - W S F solution, which provided 2.68mg total I g Y per fish or 167.5mg 1  kg" average body weight. The treatments were 1. L - W S F i n P B S , 2. L - W S F i n a 1% 1  60  solution o f 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 i n a solution o f 1% sodium bicarbonate containing either o f 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 T o confirm the results o f 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 i n P B S containing 5% Mega9, 4. L - W S F i n sodium pyrophosphate solution containing 5% Mega9, 5. L - W S F in a solution o f 1% sodium bicarbonate, 6. L - W S F i n a solution o f 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 2 0 0 p L o f lOOmg mL" L - W S F solution. This provided 2.68mg total I g Y per fish 1  or 100.75mg kg" average body weight. 1  B l o o d 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 o f concentrations o f various chemicals known for their absorption enhancing effect was tested to determine the highest concentration o f each substance w h i c h could safely be fed to juvenile rainbow trout (average weight 25g).  61  The highest safe level was defined as the highest concentration o f 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 o f these  substances are illustrated in Fig.2.5.(a-j). Saponin (Sigma S-4521) ( l m g , 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 - 1 0 0 (Sigma X-100) and Triton X-114 (Sigma X 114); C H A P S (3-3-cholamidopropyl dimethylammonio-l-propanesulfonate) C-3020); C H A P S O (3-3-cholamidopropyl  (Sigma  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" ). The concentration used, the mortality rate caused by 1  each concentration, and the highest safe concentrations o f these chemicals are reported in Table 4.6. To test the efficacy o f these detergents in enhancement o f I g Y absorption from the gut, juvenile rainbow trout (25g average weight, 4 fish per group) were orally intubated with lOOpL o f 1% N a H C 0 3 solution including 40mg o f non-specific L - W S F (containing 150mg g" o f I g Y ) to which the selected chemicals at the highest safe 1  concentration, were added.  A control group was intubated with W S F suspended in  P B S . B l o o d was collected 4 hours after intubation to assess I g Y uptake level into the circulation in each treatment.  62  3.15. Challenge studies To investigate the efficacy o f I g Y in passive immunization o f trout, fish were exposed to the pathogenic bacteria following administration o f anti-K anguillarum IgY. A bacterial preparation (described in section 3.11) was used in an appropriate amount as determined i n a pre-challenge test. Prior to each challenge, a pre-challenge test was performed using three groups o f 15 juvenile rainbow trout to determine the suitable concentration o f V. anguillarum preparation to be used i n the target challenge study. Three different bacterial concentrations o f the same preparation were used for these three groups. To conduct the pre-challenge test, fish were transferred from the holding tanks into the 2 5 L buckets containing 10L o f 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 o f 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 i n the untreated fish in the prechallenge, was used later i n the target challenge experiment. In the challenge experiments, the proper concentration o f V. anguillarum preparation was added to a 200L tank o f salt water and mixed w e l l to make an even concentration for all treatments o f the same experiment.  Fish o f each holding tank  were transferred into a separate 2 5 L bucket containing 10L o f 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 o f I g Y into the fish to examine its efficacy in protection against  Vibriosis in experimental  challenge.  3.15.1. E x p e r i m e n t 5. Preliminary feeding trial A preliminary feeding experiment was performed to study the absorption o f I g Y 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" average body weight o f total I g Y day" ). 1  1  Mega9 content o f the diet in  treatment 1 ( T I ) was 30mg per fish or 325mg kg" average body weight per day for the 1  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" average body weight o f Mega9 per 1  day for 7 days. There were 8 fish (89.7g average weight) i n each group, which were starved for 4 days pre-treatment.  Three fish o f each treatment were sacrificed to  collect blood on the second day, 6 hours after feeding. B l o o d was collected from the remaining 5 fish o f 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 o f 1.2x10 cfu mL" was used to challenge fish by immersion. 6  1  64  Fig.3.6. Schematic diagram of fish challenge with V. anguillarum. Overnight growth in TSB at room temperature was stopped at A 5 4 0 m =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. n  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 o f specific anti-V. anguillarum I g Y to enhance fish resistance against vibriosis when administered alone or i n conjunction with Mega9. This experiment consisted o f nine different feeding or intubation treatments carried out for 7 days. The W S F o f the egg yolks obtained from immunized hens (spWSF) was used as the source o f anti-P  anguillarum I g Y and W S F  o f egg yolks from non-immunized hens (nspWSF) as a source o f 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 s p W S F 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 , n s p W S F and Mega9 ( d l ) , +  regular pellets (d2-7); C 2 . P , nspWSF and Mega9 ( d l ) , P and nspWSF (d2-7); C 3 . +  +  oral intubation (IN) o f spWSF and Mega9 i n 1% N a H C 0  solution ( d l ) , P and +  3  s p W S F (d2-7); C4. oral intubation o f absorbed (ab) spWSF and Mega9 i n 1% NaHC0  solution ( d l ) , P and ab-spWSF (d2-7); C 5 . P and Mega9 ( d l ) , pellets (d2+  3  +  7); C 6 . pellets (1-7); C 7 . P and spWSF (dl-7). T o provide absorbed spWSF for C 4 , a +  suspension o f formalin-killed  V. anguillarum was prepared  (A540nm=5). lOmL o f W S F  from the eggs o f immunized hens was exposed to l m L o f the bacterial suspension for 1 hour with occasional mixing. It was then centrifuged ( I E C 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. C 4 was designed to test whether any component o f the spWSF  66  other than I g Y has an effect on resistance against vibriosis. Mega9 content o f the diet was 22mg fish" or approximately 396mg kg" body weight day" when fed or 5% when 1  intubated.  1  1  In all cases Mega9 was accompanied by 4.4mg fish" or approximately 1  80mg kg" body weight (or in case o f intubation 1%) o f sodium bicarbonate ( S B C ) . 1  The spWSF or nspWSF was added in a concentration that provided 5.5mg total I g Y fish" or approximately lOOmg total I g Y kg" body weight day" . These amounts were 1  1  1  very similar to those used in experiment 1. In case o f intubation, 2 0 0 u L o f intubation solution was applied to each  fish.  There were either 2 or 4 tanks containing 16  rainbow trout (55.23g average weight) per treatment. B l o o d was collected from four fish o f each treatment to test I g Y concentration in the serum at the day o f challenge. Fish were exposed to a concentration between 2.48 x 10 and 2.64 x 10 cfu mL" o f V. 5  anguillarum  for 30 minutes on the 8  th  5  1  day, 20 hours after the last feeding.  Mortality  rate was monitored for 14 days post challenge. A l l the details i n challenge conditions were identical to experiment 5. Experiment 7 was designed to confirm results from experiment 6. treatment or control group consisted o f 3 tanks o f 16 fish (average 30g).  Each  Feeding  regimens were as follows: T I . P with Mega9 and spWSF (dl-2), P and spWSF (d3+  +  7); C l . P , Mega9 and n s p W S F (d3-7); C 2 (positive control), intubated Mega9 and +  specific W S F ( d l ) , P , Mega9 and spWSF (d2), P and spWSF (d3-7); C 3 . pellets ( d l +  +  7); C4. P and spWSF (dl-7). Where applicable, pellets fed at a rate o f 2% average +  body weight per day, Mega9 as lOmg fish" or approximately 333mg kg" body weight 1  1  or 5% in solution when intubated, N a H C 0 3 as 2mg fish' or approximately 66mg kg" 1  1  body weight or 1% in solution when intubated, spWSF or nspWSF (fed or intubated)  67  in a concentration that provided 6.5mg total I g Y fish" or approximately 217mg total 1  I g Y kg" body weight day" . Volume o f intubation solution was 200pL. Challenges 1  1  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 cfu mL" . One fish out o f each tank was used for 4  1  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 I g Y oral uptake would be improved by use o f absorption enhancing agents and whether this approach would lead to an enhanced protection o f fish against Vibriosis. Based on the results o f experiment 4, the three most effective chemicals, Mega9 (M9), octyl-P-glucoside ( O p G ) and Na-deoxycholate ( D X ) , were selected as I g Y absorption enhancing agents. In T l , T 2 and T 3 , M 9 , O p G or D X , as well as spWSF were dissolved i n 1% S B C solution (200pL fish" ) orally intubated (IN) into the fish (40g average weight) at the 1  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 i n 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 I g Y fish" or approximately 153mg kg" body weight 1  day" .  1  M 9 and O p G were added as 5% (lOmg fish" or approximately 250mg kg"  1  body weight day" ) into the I N suspension or 15mg fish" (approximately 375mg kg"  1  1  1  1  1  68  body weight day" ) when top-dressed onto the pellets. The amounts o f D X were 1% 1  (2mg fish" or approximately 50mg kg" body weight day" ) when intubated or 3mg 1  1  1  fish" (approximately 75mg kg" body weight day" ) when added onto the pellets. Each 1  1  1  treatment consisted o f 3 tanks o f 16 fish.  B l o o d collection and challenge practices  were performed as in the previous experiment.  Bacterial concentration in the  challenge water was 2.5 x 10 cm mL" . 4  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 o f oral intubation and feeding o f spWSF and absorption enhancing agents was used. Since intubation is not feasible in commercial aquaculture practice, experiment 9 was conducted to test the efficacy o f pathogen specific I g Y in protection against diseases, when it was incorporated into feed.  In this experiment, fish (45g  average weight) i n 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 T 4 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 o f T I i n experiment 8. A negative control (C2) was fed regular pellets day 1-7. The concentrations o f the detergents in feed were increased to 20mg fish" day" or approximately 440mg kg" body weight day" for M 9 and O 0 G 1  and 4 m g fish"  1  1  1  1  or approximately 88mg kg" body weight day" for D X . 1  1  The  concentration o f intubated M 9 was 5% volume (lOmg fish" or approximately 222mg 1  69  kg" body weight). 1  Enough spWSF was used to provide 8.8mg I g Y fish" day" or 1  approximately 196mg kg" body weight day" . 1  1  Fish o f different treatments were  exposed to 1.7 to 1.9 x 10 cfu mL" o f V. anguillarum 4  1  for 30 minutes in an immersion  1  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 I g Y was capable o f conferring disease protection when delivered efficiently into the blood circulation o f rainbow trout. Fish (68g average weight) were challenged at days 1, 3, 7 or 14 following injection with lOOuL o f a preparation o f anti-K anguillarum I g Y (spWSF) into the intraperitoneal (IP) cavity o f rainbow trout i n order to study duration o f I g Y effectiveness in enhancement o f disease resistance. at the base o f the pelvic fins.  The injection was applied  T w o control groups were LP injected with the same  volume o f either P B S or nspWSF. Concentrations o f total I g Y in spWSF and nspWSF preparations were 1.33mg mL" and 0.15mg mL" , respectively. 1  Each treatment  1  consisted o f 3 tanks o f 16 fish for each date o f challenge. B l o o d was collected at the day o f challenge from one fish from each tank.  Bacterial concentration used in  immersion challenge was 8.2 X 10 , 3.2 X 10 , 3.3 x 10 and 2.0 x 10 cfu mL" at days 4  1, 3, 7 and 14, respectively.  5  5  5  1  Conditions o f 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 o f 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 o f the gel, 15pL o f W C L , 2 0 p L o f L P S , 5 p L o f low molecular weight protein marker ( L M M ) or 5 p L o f prestained L M M (Bio-Rad S D S - P A G E Standard 161-0304 and 161-0305, respectively) were loaded.  Discontinuous S D S -  P A G E o f the samples was run at a constant current o f 2 5 m A at 400v (Electrophoresis Power Supply E P S 500/400, Pharmacia Fine Chemicals, Uppsala, Sweden), using a Mini-PROTEAN® II Slab C e l l (Bio-Rad Laboratories, Hercules, C A , U S A ) vertical gel electrophoresis system.  M o r e details on casting gels and operating the slab cell  can be found i n the guidebook o f B i o - R a d Laboratories. Each 10 well 0.7mm gel was loaded w i t h 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 w i t h 0.025%) coomassie brilliant blue R-250. Stained gels were then shrunk i n 50% methanol and dried using a vacuum gel drying system ( G e l Slab Drier G S D - 4 , Pharmacia Fine Chemicals, Sweden).  The gel with the prestained marker was used in Western  blotting. Silver staining o f 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 o f specific I g Y and I g M , respectively. Pre-immunization samples were also collected to be used as negative controls as a measure o f non-specific reactions between the immunoglobulins with W C L or L P S o f V. anguillarum. Specific anti-K anguillarum rabbit I g G was obtained from Microtek International L t d . (Sanichton, B . C . , Canada, # AS006) and non-specific rabbit I g G from Pierce (Rockford, LL, U S A , #31207). Polypeptides  obtained  electrophoretically transferred  from  SDS-PAGE  electrophoresis  were  from the gel onto a 0.45uxn nitrocellulose ( N C )  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 B i o - R a d recommended procedure. Immunoblotting o f the N C membrane was performed according to B i o - R a d immunblot instruction manual at ambient temperature while shaking gently, with some modification.  The constituents o f 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 ) i n 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 ( A P )  substrate and the reaction was stopped by addition o f 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 o f rabbit IgG, chicken I g Y 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 o f 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 I g G (Jackson ImmunoResearch Laboratories Inc., West Grove, P A , U S A , # 111-055-003) at a concentration o f 0.12ug m L " , AP-rabbit anti-chicken I g G (Sigma, C-1161) at a 1  concentration o f 0.23ixg mL" , and AP-labeled goat anti-trout immunoglobulin ( K P L , 1  Gaithersberg, M D , U S A . # 05-29-05) at a concentration o f 0.2ug mL" . 1  solutions as well as the blocking buffer were freshly prepared.  The antibody  T o prepare A P -  substrate, 6 6 p L o f N B T and 33 u L o f B C D were added t o l O m L o f AP-buffer for each 3  N C membrane.  3.16.3. Enzyme linked immunosorbent assay (ELISA) E L I S A was performed to determine the total I g Y , as well as specific anti-K anguillarum I g Y content o f the W S F , its retentate and the lyophilized powder in each  73  batch o f egg yolks, as well as  in fish serum and the extract o f 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 I g Y content o f 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 o f l p g mL"  1  rabbit anti-chicken I g G in carbonate coating buffer and incubated for 60 minutes at 37°C.  After washing the wells three times with PBS-Tween, 2 5 0 p L o f 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 o f IgY-containing sample solutions or a standard solution o f I g Y (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" I g Y and samples were diluted to fit within the range. 1  Plates were  washed four times and wells were applied with lOOpL o f the conjugate (anti-chicken I g G 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 o f lOOpL  substrate solution into each well. Substrate solution contained 0.5mg alkaline pnitrophenyl phosphate (p-NPP, Sigma 104-105) per m L D E A buffer. Development o f 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. Rinse the unbound antibody. Block the rest of the surface with a neutral protein and then rinse the unbound protein.  1 y y y v  Add the sample to the solid phase antibody. The solid phase antibody specifically captures any target antigen within a complex sample. Rinse the unbound sample material.  y  A f 1 v v Y  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  Enzyme-labelled antibody  Capture antibody  Target antigen  •  Colored product ol enzyme substrate  Fig.3.7. Procedure o f sandwich E L I S A (adapted from Rittenburg, 1990, with modification).  Standard and samples were assayed in triplicate. Readings o f the pure I g Y range o f 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 I g Y 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 I g Y titer o f the samples.  Formalin- killed V. anguillarum suspension was prepared  following the procedure described i n vaccine preparation except that the final concentration was adjusted to As40nm 1 and stored at -20°C after being distributed i n =  1.5mL  sterile capped tubes.  It was then thawed and diluted ten times i n carbonate  coating buffer to coat the wells o f E L I S A plates (lOOuL per well).  Blocking,  conjugate and the substrate were identical to those in determination o f total I g Y . A series o f doubling dilutions o f each sample in P B S - T w e e n was added into the wells in triplicates or duplicates (lOOuL per well).  A panel o f 8 dilutions o f the negative  control samples o f the same nature formed the basis for determination o f anti-Vibrio I g Y titer in the test samples.  M e a n 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 o f each triplicate or duplicate wells o f the test sample above this value was considered as antigen positive and the corresponding dilution was accepted as the specific anti-K anguillarum I g Y 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 enzymelabeled 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 Target antigen  Primary antibody  Q  Colored product of enzyme substrate  Fig.3.8. Procedure o f antibody capture E L I S A (adapted from Rittenburg, 1990, modification).  3.17. Drying of W S F To facilitate commercial use o f IgY as a feed ingredient to fortify fish health, feasible preservation methods had to be sought. Dehydration o f W S F as a source of IgY was investigated since a dried W S F powder does not need a large storage space and is easy to preserve. Ultrafiltration (UF) retentate of W S F was dried using three different methods. 1. Freeze drying for 48 hours at shelf temperature o f 20°C, pressure of 0. l m m H g and a condenser temperature o f -55°C (using a freeze-drier manufactured by V i r T i s Research Equipment, Gardiner, N Y , U S A ) .  2. Vacuum-microwave drying,  using a household microwave oven (Matsushita Electric Ind. Co. Ltd., Japan) with a maximum power o f 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 o f the oven and the desiccator lid. A volume of 20mL of W S F 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 o f 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 0 W 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 4 2 0 W for 9 minutes. The temperature of the dried W S F at the end of process was 31°C. 3. Spray drying o f the W S F after the addition of lactose (analytical grade, B D H Chemicals, Toronto, Canada) to a final concentration of 2 5 % , providing a higher level o f 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 Spraydrier (SD-04 L a b Plant Manufacturing L t d . , Leeds, England).  A l l o f the dried  products were screened using B i o R a d protein assay and the result was used to prepare solutions o f 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  I g Y titer i n the  samples.  3.18. Dehydration of IgY-containing pellets To investigate the stability o f I g Y 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 I g Y  concentrations i n the pellets before and after dehydration were evaluated using E L I S A . Details on incorporation o f I g Y into the pellets and dehydration o f them are described below.  3.18.1. Initial dehydration & IgY incorporation Three kilograms o f commercial fish pellets having a moisture content o f 8.76% (dry base, db) were dehydrated using a 1.5kW vacuum microwave ( V M ) dehydrator, operating at 2 4 5 0 M H z microwave frequency (EnWave Corporation, Port Coquitlam, B C , Canada). Five cheesecloth bags containing 500g pellets each, were placed i n the drying cavity o f the V M drier (a rotating cylinder o f approximately 0.27m radius and 0.3m length w h i c h tumbled the sample during drying) i n two batches o f 2 or 3 bags.  79  Microwave power o f OkW for 1 minute followed by 1.5kW for 5 minutes and again OkW for 3 minutes was employed at a constant absolute pressure o f 50mmHg to dry the pellets.  A l l 5 bags o f pellets were mixed in a sealed plastic box and incubated  overnight at ambient temperature to equilibrate the moisture among all.  Final  moisture content o f the mixed pellets as determined by drying i n 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 predehydration o f pellets was performed to increase their liquid absorption capacity. The ultrafiltration retentate o f W S F prepared from the yolk o f commercial white eggs was sprayed onto the dehydrated pellets. The resultant moisture content o f 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 I g Y content o f the samples was determined using the E L I S A technique. Temperature measurement o f the pellets was performed using an Infra-red Thermometer (Model 39650-04, Cole-Parmer Instrument, Chicago, I L , U S A ) .  3.18.2. Freeze-drying 250g o f 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 o f 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 o f OkW for 1.5 minutes followed by 1.5kW for 5 minutes and OkW for 3 minutes at a constant absolute pressure o f 50mmHg was used to dehydrate 500g o f 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 , W a l - D o r Industried Ltd., N e w Hamburg, Ontario, Canada) was used at 90°C for 8 minutes and 45 seconds to dry 300g o f the wet pellets. Temperature o f the pellets at the end o f the drying process was 60°C.  3.19. Statistical methods M e a n values o f immunologically active I g Y in the serum o f the test and control fish following different treatments, as well as I g Y concentration in the pellets were calculated. The observations were analyzed using one way analysis o f variance (ANOVA).  Fisher's Least Significant Difference ( L S D ) multiple range test was  employed for pair-wise comparison o f the means o f different treatments i n each set o f data. The data collected from mortality rates i n fish following bacterial infection was processed in the same manner.  To satisfy statistical considerations concerning  normality and homogeneity o f variances, data were transformed when necessary. Lilliefors (Conover, 1980) method was used to test data for normality o f distribution and Levene's (Snedecor & Cochran, 1980) method was employed to test for the homogeneity o f variances.  A l l o f the statistical evaluations were performed at the  81  significance level o f a=0.05.  Microsoft Excel version 97 was used to calculate  mortality values and to determine I g Y 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 G I tract due to oral intake o f 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. A l s o in experiment 4, a similar sampling was performed 3 hours after intubation o f W S F in a 1% antacid solution, coadministered 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 o f W S F i n 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 o f 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 o f intestinal microvilli. 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 o f chicken I g Y was considered as a means of passive immunization against vibriosis. A s a preliminary step, the potential o f I g Y of the immunized hens to bind membrane proteins and lipopolysaccharide ( L P S ) o f V. anguillarum was examined. This was also compared to the binding characteristics o f rabbit I g G and rainbow trout I g M . 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 o f prominent bands around a M W o f 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 i n 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 o f V. anguillarum, obtained from silver stained S D S - P A G E gel.  This profile consists o f a typical ladder-like  pattern o f bands, which was described by Tsai & Frasch (1982).  The micro-  heterogeneity o f the intermediate molecular weight ( I M W ) fraction o f V. anguillarum L P S is apparent with four distinct bands being clearly resolved. The four bands i n the approximate M W regions o f 22, 30, 45 and greater than 97.4kDa represent L P S molecules that differ from one another largely in the number o f 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 o f whole cell components o f 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 I g Y reacted strongly with L P S species in the M W zones o f 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 i n 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 o f L P S and no staining was observed with components o f the O-polysaccharide.  Hastings & Ellis (1988) found that although rabbit antibodies  recognized 14 components in the extracellular products o f Aeromonas salmonicida, rainbow trout antibodies reacted with only four. Fish are known to produce a much smaller repertoire o f 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 o f 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 o f L P S is a distinct possibility. However, none o f the latter is a strong assumption since electrophoresis and Western blotting has been employed in similar conditions for  86  chicken I g Y and rabbit I g G 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 I g Y recognized a cellular protein o f approximately 14kDa M W . Fig.4.3 shows the reactivity pattern o f antibodies o f non-vaccinated animals with V. anguillarum  antigens in Western blots. Noteworthy was the strong reaction o f  antibodies o f non- vaccinated chicken and fish with a cellular protein i n the vicinity o f 32kDa M W .  N o reactivity with L P S or W C L was observed w i t h the serum o f non-  vaccinated rabbit. The antigen-antibody reaction shown in Fig.4.2 explains why I g Y used in passive immunization, especially when obtained from the egg yolk o f vaccinated hens, could confer protection in fish against a subsequent experimental infection with V. anguillarum. anguillarum  According to the reaction observed between V.  antigens and I g Y from non- vaccinated chickens (Fig.4.3), a partial  protection would be understandable i f conferred by passive immunization o f fish using I g Y o f non- vaccinated chickens.  87  WCL  M  (a)  M  LPS  (b)  Fig.4.1. (a) S D S - P A G E profile o f 15pL whole cell lysate ( W C L ) o f V. anguillarum stained by coomassie blue, (b) S D S - P A G E profile o f 20pX proteinase K digested W C L o f V. anguillarum ( L P S ) 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  (a)  WCL  WCL  M  (b)  LPS  WCL  LPS  (<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  M  WCL  LPS  WCL  LPS  106.0 80.0 49.5 32.5 27.5 18.5  (a)  (b)  (c)  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 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 i n the column " M " in kilodaltons.  90  4.2. Dehydration of WSF In order to use I g Y as a feed ingredient to enhance fish resistance against diseases in commercial scale, dehydration o f W S F was investigated as a preservation method. A dried W S F powder is a source of I g Y that does not need a large storage space and is easy to store and transport. A s a preliminary method, as recommended in the literature, ultrafiltration (UF) was used to remove most o f the water from W S F prior to dehydration using more advanced dehydration methods.  U F concentrate o f  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 o f both studies are detailed in the following sections.  4.2.1. Dehydration rate To suggest a feasible method o f producing IgY-containing fish feed pellets, various dehydration methods were compared.  One o f the considerations in this  comparison was the efficiency o f dehydration. A s described i n materials & methods, fish feed pellets were sprayed with W S F o f egg yolks and subsequently dehydrated. Dehydration o f fish feed pellets using three methods: vacuum-microwave drying ( V M D ) , freeze-drying (FD) and air drying ( A D ) occurred at different dehydration rates. In the conditions o f this study, the moisture i n 500g o f pellets was reduced from 27% (db) to 6.7% i n only 5 minutes and the temperature at the end o f the process was 4 5 ° C , when V M D was used.  The corresponding final moisture content and pellet  91  temperature for 300g o f pellets which had undergone A D for 8 minutes, 45 seconds at an air temperature o f 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 k g water kg" dry matter hour" for V M D , A D and F D , respectively. In 1  1  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 o f 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 o f the present study i n which dehydration occurred i n a much faster rate i n V M D than i n A D and especially than F D . However, due to the different vacuum and microwave power levels used i n their studies and the varying nature o f 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 l o w chamber pressure provided by vacuum. The large vapor pressure differential between the center and the surface o f the product helps the quick removal o f moisture. Although vacuum level affected the speed o f dehydration, the more significant factor proved to be the microwave power ( L i n 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 o f 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 o f 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 o f liquids, in the preparation o f 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 o f the W S F onto the fish feed pellets and dehydration o f these I g Y containing pellets was used in this research to provide a feed with a potential o f fortifying fish resistance against Vibriosis. In order for this diet to serve the purpose, IgY has to remain stable during dehydration since preservation o f I g Y functional properties counts as an important measure in its efficacy in passive immunization. To investigate stability o f I g Y during dehydration o f pellets, IgY-containing pellets were examined before and after dehydration. The I g Y concentrations i n the extract o f the wet and dehydrated pellets, determined by E L I S A , are summarized in Table 4.1. Dehydration o f pellets by A D , V M D and F D methods reduced the amount o f functional I g Y less than 1% that was not a significant decrease (p < 0.05). One-way analysis o f variance ( A N O V A ) was performed for reduction o f I g Y due to dehydration (IgY concentration in dried pellets subtracted from that o f the wet pellets, db). The results indicated that there was no significant difference between the three techniques with regards to the damage incurred to I g Y (p=0.991).  One-way A N O V A  also showed that there was no significant difference between the total I g Y  93  Table 4.1. Changes in IgY concentration due to dehydration treatments I g Y reduction (%) I g Y concentration in pellets (mg g" ) FD V MD AD FD VMD 1.689+0.096 a 1.658+0.159 a 1.696+0.129 a 0.01 1.686±0.106 a 1.658±0.196 a 1.679±0.171 a 0.21 1  wet  dried 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).  AD 0.99  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 I g Y under the described conditions.  4.2.3. Stability of IgY during dehydration of concentrated WSF In the previous section, effect o f dehydration on total I g Y activity when incorporated into the pellets was studied. However, anti-P. anguillarum  I g Y activity  and its preservation during dehydration was o f a special interest i n the present study. Specific anti-K anguillarum  I g Y relative titer in W S F o f the eggs collected from the  vaccinated hens was determined by the E L I S A technique, using doubling dilution as described i n 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 I g Y titer was 3200 for all samples. The effect o f the non-specific reaction o f I g Y 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 nonspecific 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 I g Y relative concentration in W S F before and after dehydration with different techniques.  Due to the lack o f  space i n 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 o f comparison. A s shown in these figures, the concentrations o f the specific I g Y are similar i n all four samples.  This  confirms that I g Y was stable under all dehydration treatments employed in this study.  95  Specific Anti-V. anguillarum  y = 0.0035X - 0.0495X + 0.176 2  E c u> o  "*  1.2  IgY Absorbance •  j  0.9939  Non-specific W S F  e  Specific W S F  A  Freeze dried  X  V M dried  y = 0.0285X - 0.3995x + 1.4139 2  1 --  0.9973 y = 0.0256X - 0.3802x + 1.433 2  0.8 --  @) 4) 0 . 6 - O c 0.4 - .Q O in 0 . 2 - .Q  0.9991 0.0282X - 0.4031X + 1.4616 2  0.999  re  . . . Poly. (Non-specific WSF) Poly. (Specific W S F )  <  • Poly. (Freeze dried) 1  2  3  4  Dilution factor (100x2  5 n-1  6  7  -Poly. (VM dried)  , n= 1 to 8)  Fig.4.4. Specific anti-K anguillarum I g Y E L I S A values o f a 10-fold diluted watersoluble fraction o f egg yolk before and after freeze-drying or vacuum microwave drying.  96  Specific Anti-V. anguillarum 1.2  j  E 1 c o •<* 0.8 IO  <§)  0.6 u c 0.4 0)  IgY Absorbance  y = 0.0037x • 0.0521X +0.1812 R =0.9963 2  y=0.0255X • 2  0.3602X  +1.2881  R =0.9974 2  y=0.0248X • 0.3494X +1.2431 R = 0.9971  specific W S F spray dried  2  2  Poly, (non-specific WSF)  nCS o 0.2 It)  <  non-specific W S F  2  Poly, (specific W S F )  0 -  •Poly, (spray dried)  Dilution factor (100 x 2 n-1 , n= 1 to 8)  Fig.4.5. Specific anti-K anguillarum I g Y E L I S A values of a 10-fold diluted watersoluble fraction o f egg yolk before and after spray-drying.  97  These findings support the apparent stability o f I g Y top-dressed onto fish feed pellets observed in the previous dehydration experiment.  Similarly, Shimizu et al.  (1992) observed no change in the anti-mouse I g G activity o f chicken I g Y or rabbit IgG after heating for 30 minutes at 62.5°C.  I g Y activity decreased drastically after  heating for 15 minutes at 70°C or higher, and that o f rabbit I g G decreased at 75-80°C. In another study, Shimizu et al. (1988) found that anti-El coli L P S activity o f I g Y 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 I g G 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 I g Y when top-dressed onto the pellets or as dehydrated solely when compared with the I g Y level in the wet pellets or i n 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 o f dehydration studies.  The results o f the present study are in accordance with those obtained by  Shimizu et al. (1988) who observed no loss in I g Y activity due to freezing or freezedrying unless repeated several times.  However, Chansarkar (1998) reported a 40%  decrease in the absorbance value at 405nm due to freezing followed by when I g Y activity was determined by E L I S A technique.  freeze-drying  The samples i n that study  were 90% pure I g Y which were frozen at -80°C in a concentration o f 30mg mL" or 1  98  l m g mL" in a high salt ( 1 . 5 M N a C l ) or low salt (0.14M N a C l ) solution prior to 1  freeze-drying.  4.3. Post-vaccination IgY level in the yolk Efficiency o f I g Y production following vaccination o f 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 ( W S F ) o f the egg yolks from immunized and non-immunized hens was extracted in batches o f biweekly or i n some cases monthly collections o f eggs.  In each batch, concentration o f total I g Y was  measured using the E L I S A technique.  The average concentration o f total I g Y was  5.93 ± 1.26mg mL" yolk or 91.4mg egg" over the whole period o f egg collection. 1  1  This is approximately half the I g Y (12.53mg mL" yolk) collected by A k i t a & Nakai 1  (1992) at the same p H and using the same recovery method but from the egg yolk o f white leghorn hens. However, they estimated I g Y 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" month" , 2.38g I g Y is 1  1  obtainable per hen i n a month or 28.56g hen" year" . 1  1  Fig.4.6 illustrates the fluctuation i n the concentration o f total I g Y in the W S F (10-fold dilution o f egg yolk) over the period o f egg collection. The post-vaccination total I g Y concentration in the W S F obtained after completion o f initial vaccination followed by 5 weekly boosting varied i n the range o f 0.44-0.94mg mL" W S F , which 1  was slightly higher than pre-vaccination values.  This increased level o f I g Y was  maintained over the period o f screening except for a few sampling points. The eggs o f vaccinated hens were not used for 6 weeks after the first vaccination due to reports o f  99  a low titer o f specific I g Y in the yolk during this period (Li-Chan, 1999). no data was collected on I g Y concentration during this period. anguillarum  Therefore,  The specific anti-P".  I g Y titer o f 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  the  o f the W S F samples  discontinuous variable.  for E L I S A  data points  appeared as a  N o consistent trend was observed between boosting and  increase in the titer. However, the boosters obviously helped maintain a high titer o f specific I g Y in the W S F . Since the W S F o f 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 o f non-specific reaction between the V. anguillarum  coating o f the E L I S A  plates and the W S F o f non-immunized hen eggs was considered as the base line, the specific titer o f 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 I g Y in eggs from immunized hens as compared to the non-specific control W S F extracted from the eggs o f non-immunized hens. Other researchers have used different  protocols with respect to vaccination o f  chickens. L i - C h a n (1999) immunized chickens against bovine serum I g G 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 anti-CCW I g Y levels remained at lower levels between  100  T o t a l IgY L e v e l in the  WSF  T o t a l IgY  Date of e g g c o l l e c t i o n  Fig.4.6. Fluctuation i n total I g Y concentration o f a 10-fold diluted water-soluble fraction o f egg-yolk. Arrows indicate the initial and booster injections.  101  A n t i - V . anguillarum  IgY Titer in the  >-  WSF  • sp-lgY titer  cn o a>  Q.  (0  Date of e g g c o l l e c t i o n Fig.4.7. Fluctuation in specific anti-K anguillarum I g Y titer o f a 10-fold diluted water-soluble fraction o f egg yolk. Arrows indicate the boosting dates. Data is not available for the first 6 weeks after initial vaccination.  i  102  S p e c i f i c A n t i - V . anguillarum 0.3  E c u> o  ® 0) o  c  ra  i  ^ n o n - s pecific E3 24-Apr  0.25 -  A 22-May  0.2 -  X29-May  0.15 -  X5-Jun  0.1 -  w  0.05 -  <  0 -  X)  e 12-Jun  i  X!  o  IgY A b s o r b a n c e  + 19-Jun -26-Jun  1  2  3 3  4  Dilution factor (800x2  5  6  n= 0 to 5)  — 3-Jul • 17-Jul  Fig.4.8. Levels o f specific anti-F! anguillarum I g Y in the water-soluble fraction o f the egg yolks collected from immunized hens i n different post-vaccination dates compared to pre-vaccination level (indicated as non-specific).  103  weeks 10 and 24.  In both cases, the I g Y level was considerably higher than pre-  immunization levels at all times.  Boosting at week 25 increased both types o f  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 o f boosters with longer intervals produced a prolonged high titer i n the case of anti-IgG while more frequent boosting might be helpful to sustain a higher titer o f anti-CCW I g Y . Poison et al. (1980a) vaccinated 20 weeks old hens with different protein antigens.  Three to several weekly boosters, depending on the antigenicity o f each  specific antigen, followed the initial injection to elicit antibodies in adequate titer i n the egg yolk. The authors suggested that molecular weight ( M W ) o f antigen was an important factor i n eliciting antibodies i n 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 o f specific I g Y in the eggs throughout the 16 weeks o f 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 g Y i n 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 I g Y and duration o f such protection was studied. This experiment was conducted jointly with Abdul-Hossein Aminirissehei at the Department o f  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 o f fish receiving specific I g Y than those groups IP injected either with non-specific I g Y or with P B S in all challenge experiments performed after different periods o f time (p < 0.05).  N o significant  difference was observed between mortality levels o f the P B S and non-specific I g Y treated groups on any challenge date.  Fig.4.9 demonstrates these results.  As  illustrated i n 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 o f the total serum I g Y level i n all treatments and specific anti-P". anguillarum I g Y titer in the fish groups IP injected with specific I g Y , as well as cumulative mortality rates are summarized i n Table 4.2. In a similar challenge study, Aminirissehei (1999) found a greater disease resistance i n the fish D? injected with anti-K anguillarum I g Y than those which received the same dose o f non-specific I g Y .  Lee et al. (2000) found no Yersinia  ruckeri in the intestine or kidney o f rainbow trout 7 days after an immersion challenge with the pathogenic bacteria when fish were EP injected with specific anti-F. ruckeri I g Y 4 hours before challenge.  However, the bacterium was detected i n both kidney  and intestinal tissues o f the control fish groups injected non-specific I g Y 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 o f rainbow trout using anti-K anguillarum antibodies raised i n sheep and rabbits via  105  Temporal Post-Challenge Mortality Rate 120 -,  Day 1  Day3  Day 7  Day14  Post-injection challenge date  Fig.4.9. Cumulative 14-day post-challenge mortality rates in fish groups EP injected with P B S , non-specific or specific anti-K anguillarum I g Y 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  o ra  80 -  >«  60 -  rtal  100 -  40 -  +J  o  a  a  L T f  20 0 -  specific IgY  non-specific IgY  PBS  IP injection treatment Fig.4.10. Cumulative 14-day mortality rates in groups o f fish challenged at different post-injection dates compared within each treatment group (IP injected with P B S , nonspecific or specific anti-P! anguillarum IgY). Values are the mean o f 3 tanks ± standard deviation. Similar letters indicate no significant difference within each treatment (Specific I g Y : 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 Day 14  Day 7  Day 3  Challenge date (days post-injection)  Day 1  Vibrio count in challenge (cfu mL" )  8.2xl0  Treatment 1  Mortality (%)  11.1 ± 3 . 8 a *  31.1 ± 7 . 7 a  24.4 ± 13.9 a  17.8 ± 13.9 a  Anti-K  2.56E+04 ± 5.20E+02 256  1.47E+04 ± 3.17E+03 64  1.15E+04± 4.55E+03 32  3.80E+03 ± 3.26E+03 32  IgY (IP)  Total serum IgY * (ng mL" ) Anti-Vibrio IgY titer  Control 1  Mortality (%)  75.6 ± 10.2 b  75.5 ± 3 . 8  62.2 ± 16.8 b  57.8 ± 26.9 b  PBS (IP)  Total serum IgY (ng mL" )  4.84E-01 ± 4.42E-01  6.26E-01 ± 3.33E-01  2.95E-01 ± 2.54E-01  1.02E-01 ± 1.71E-02  Control 2  Mortality (%)  71.1 ± 10.2 b  71.1 ± 27.8  57.8 ± 7 . 7 b  82.2 ± 7.7  Non-specific* IgY (IP)  Total serum IgY (ng mL" )  1.02E+02± 1.29E+02  1.69E+04± 6.94E+03  5.22E+03 ± 4.73E+03  3.74E+03 ± 1.41E+03  1  £  8  1  anguillarum  1  1  4  3.2 xlO  3.3 xlO  3  b  b  3  2.0 xlO  3  b  (p<0.05). Total IgY level i n anti-Vibrio IgY injection preparation^ .33mg mL" . Total IgY level in non-specific IgY injection preparation^. 15mg mL" . Mortality value is mean cumulative 14 days post-challenge mortality rate of 3 tanks ± standard deviation. 1  ¥  1  §  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 o f the same study showed that non-immune sheep sera conferred no protection. The results o f the present study and the reviewed studies, prove that specific antibodies raised in other animals, including chicken I g Y , are capable o f enhancing resistance to disease-causing bacteria when it reaches the fish system in sufficient amounts. It also indicates that protection arises from specific I g Y activity against the causative organism, not from any other source that might exist in the water-soluble fraction o f the egg-yolk, nor from a non-specific reaction between I g Y and the bacterial pathogen. A s illustrated i n Fig.7.1 (in the appendix), total serum I g Y level in the groups o f fish EP injected with anti- Vibrio I g Y showed a general significant decreasing trend during the course o f the study, although the levels at days 3 and 7 were not significantly different.  The highest level o f serum I g Y at day 1 corresponded to the  lowest mortality rate among four different challenge dates. However, temporal trends o f the significant levels for total I g Y were not similar to that o f the mortality rates, which did not change significantly over time.  There was no significant linear  correlation (p = 0.365) between total serum I g Y levels o f the sampled fish and the mortality rate (%)  following  an immersion infection (r  relationship was studied for the  fish  2  = 0.403) when this  groups EP injected with specific anti-K  anguillarum I g Y on different challenge dates. Serum I g Y and mortality rate values used for this calculation represent the average o f three samples collected at each challenge date (one fish from each experimental tank). A l s o as shown in Fig.7.2 (in the appendix), the highest anti-K anguillarum I g Y titer in the fish serum corresponded to the lowest mortality rate.  A linear regression analysis indicated no significant  109  correlation between anti-Vibrio 0.309).  serum titer and the mortality rate (r =0.477, p = 2  This provides a similar conclusion conceived from the relationship o f total  serum I g Y level and mortality rate discussed above. Corresponding total I g Y levels and specific anti-P anguillarum  I g Y 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 I g Y levels had descending trends over time. However, it was not possible to calculate the ratio o f specific to total serum I g Y level due to the use o f different measurement techniques and units for these two purposes.  A k i t a & L i - C h a n (1998)  found that approximately 10% o f the total I g Y obtained from egg yolk preparation o f the hyperimmunized hens was specific to bovine IgG. In their study, they measured the amount o f affinity purified specific anti-bovine I g Y using a P J D method where the results could be translated to weight per volume amounts o f IgY. In the present study, it was not feasible to measure the amount o f purified specific anti-K anguillarum I g Y in the fish serum due to the small volumes o f 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 I g Y was considered to eliminate the stomach barrier from the absorption system.  ELISA  results showed no uptake in the serum following anal intubation o f lOOpg I g Y 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 o f the triplicate blood samples collected at 24, 27, 30, 48 and 72 hours showed transfer o f I g Y into the blood circulation o f fish. In contrast, other researchers have reported transport o f proteins into the circulatory system o f fish following anal intubation. M c L e a n & A s h (data published in M c L e a n et al, 1999) detected up to 70ng mL" horseradish peroxidase ( H R P ) in the 1  plasma o f rainbow trout after anal delivery o f a dose o f lOOug g" body weight. 1  Jenkins et al. (1994) also measured a maximum o f 60ug mL" o f human gamma 1  globulin ( H G G ) i n the plasma o f tilapia after an anal dose o f 2mg fish" with a weight 1  of 30-50g. A maximum level o f 2ug mL" o f salmon growth hormone (sGH) was 1  observed i n 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 o f 200 or 500ng mL" o f bovine growth hormone (bGH), using two different 1  assays, after anal intubation o f a l m g dose into 30-45g rainbow trout. It is noteworthy that in the above studies the dose o f administered protein was 5 to 21 times higher than that applied i n the present study, except for s G H , which was lower than that used here.  Since intestinal absorption o f proteins following anal  administration is reported to be dose-dependent ( M c L e a n et al., 1999; Hertz et al, 1991; Georgopoulou et al,  1988), the lack o f I g Y 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 o f I g Y and variation in starvation periods before intubation.  Effect o f starvation was reported by Hertz et al. (1991),  who found significantly higher absorption o f 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 I g Y in the G I tract o f trout and I g Y uptake into the bloodstream, oral administration of W S F as a crude source o f I g Y was applied through intubation as well as incorporation into the feed.  T o enhance intestinal absorption,  various detergents were co-administered with the W S F . Microencapsulation using polylactide-co-glycolide ( P L G ) was also used to protect I g Y 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 o f a non-ionic detergent, Mega9, in antacid solution, as well as pyrophosphate  and microencapsulation in P L G was examined  enhancement o f I g Y absorption i n an oral intubation trial.  for  IP injected W S F and  intubated P B S served as positive and negative controls, respectively.  A s shown in  Fig.4.11 and Table 4.3, absorption o f I g Y 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. I g Y uptake i n this approach was 14 to 158 fold higher at different sampling times when compared with L - W S F dissolved i n P B S . Concentration o f total  112  I D  C O  C D O  in  BS  ho  in  LG LG  g>  /rop  C O  -a  O L O  CO C D  Q _  • •0  s  i  i  i  s ea u n C D  •g 2? a "CD  C D  >  +-» -*-> cd o  -C  CD  OH OH CO  CO  OH o f  2 O ft a S 8 .3 o> o  £ a CD  CD  S i CO O  o.  "I CO +J  03  C D  T3 CD  3 o  co CO cd  C/3  «f  £  d P-  1  O "O  13  .g "a | i  oo  * J-a d  ccj  >  1  -o  CD  *H  O CD  2 d^ m  .aft CD  O  & r>.  —  B  o  in  CO  td ro  C+H  o  OH  CD C/3  °  m  .  00  d 5  CD  0 CD  o o o  GO  CD  (|Ui/6u) uoijejjuaouoo  fc  'PH  >  113  oo  £  eight.  oo  LI  S  .§«•:  SP os o  53 »  •—  1  >  cj <+H o X5  —  S3  1 o  bO <=>  o ts  T3  u  ts  -* J  S3  'uo o cn  II J=i  o  1  c3  o  &  T1/31  O  Pi  T3 (30  SP t»"  '53 I  * la £ SJ  O  o  S  If  |gS ° 3  o  .1  >> 'bO  t-l  >H  <=> Of) PH  «  GO ON  « «  & II «  0 4  si  ^ O O  ^ ON II  M Xj  S  oo o  '53  ° -5 & <u U  00  D.  *  g 2& 00 op-r  serum I g Y 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 I g Y level in the serum, the difference was not significant at any sampling time. However, serum I g Y level after oral intubation o f L - W S F dissolved in P B S or pyrophosphate solution was significantly higher than the negative control (intubated P B S ) . I g Y uptake into the blood was always significantly higher in the fish EP injected with L - W S F in P B S than I g Y uptake in any other treatment.  Encapsulation o f L - W S F in P L G 50:50 or P L G  85:15 (with an average particle size o f 2 9 . 3 9 ± 5 . 7 5 u m for P L G 50:50 & 3 0 . 3 2 ± 5 . 4 8 u m for P L G 85:15, Fig.4.12) did not result i n satisfactory I g Y serum levels under the conditions o f this experiment.  The I g Y 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 o f I g Y in the intubated  material was theoretically 1.36mg fish" i n P L G treatments, lower than the 2.68mg 1  fish" used in the other treatments. However, this fact does not justify the observation 1  that serum I g Y 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 I g Y 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" I g Y in serum following oral intubation o f unprotected W S F was 1  reached in 1 hour, then gradually decreased. pyrophosphate was added.  The same trend was observed when  Co-delivery with Mega9 and antacid resulted in a high  absorption level o f 1740ng mL" at 30 minutes, a level that was maintained for 24 1  hours with a slight increase at 3 hour (2500ng mL" ). 1  Serum I g Y subsequently  decreased to some extent but was still clearly detectable even after 2 weeks at a level of 29.3ng mL" . A similar pattern was reported by Hertz et al. (1991) who obtained 1  maximum plasma levels 30 minutes after oral intubation o f h G H , when delivered alone or in conjunction with deoxycholate.  A l s o , protein delivery to the fish  bloodstream v i a the oral route reached its peak after 15-30 minutes in several other studies (reviewed by M c L e a n & 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 o f 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 i n oral and anal intubation o f H G G when delivered alone or in conjunction with aluminum hydroxide or cholera toxin P-subunit. M c L e a n & A s h (reported in M c L e a n et al, 1999) observed a higher plasma level o f H R P after anal delivery than after oral delivery. These reports are i n conflict with the results o f the present study, in which anal intubation o f I g Y did not lead to absorption into the blood.  Possible explanations were discussed previously, under the heading " A n a l  administration o f I g Y ' .  117  4.6.3. Efficacy of delivery into the blood using encapsulated proteins Encapsulation o f W S F in P L G 50:50 or P L G 85:15 was used with the hope o f increased protection o f I g Y 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 o f I g Y into the bloodstream o f rainbow trout. However, use o f these polymers with different levels o f success in protection o f 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 ( H G G ) . They detected little H G G in the plasma o f the fish intubated with unprotected antigen and none in high molecular weight fragments or intact form, while i n the fish receiving P L G - H G G the level o f intact protein antigen reaching the bloodstream was increased.  These researchers concluded that encapsulation partially protected the  antigen from proteolysis i n the digestive tract.  They also suggested that the antigen  was not sufficiently released from the microparticles.  O ' D o n n e l 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 o f 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 i n the serum o f orally intubated Atlantic salmon was rapid at 15 minutes, peaked at 1 hour and cleared in 4 days. The uptake o f 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 i n higher levels and greater persistence o f 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 o f release as  118  being: (1) an initial release o f surface bound and poorly encapsulated antigen, (2) diffusion, and (3) degradation o f the polymer matrix. The present study, in contrast to the study o f O'Donnell et al. (1996) with H G G , did not demonstrate a peak in the serum I g Y level following intubation with P L G encapsulated W S F in the entire three week period o f the study (data at week 3, available only for encapsulated I g Y , is not shown). The amount o f in vivo release o f W S F as judged by the levels o f I g Y 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 o f I g Y 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 o f protein that could be encapsulated per gram o f P L G was judged inadequate for the specific purpose o f the present study. Jeffery et al. (1993) found that when the amount o f encapsulated protein exceeded a ratio o f 1:5, the surface o f particles appeared pitted and some collapsed.  Use o f W S F , which  contains low purity I g Y , makes P L G encapsulation even less effective in delivering sufficient amounts o f pure I g Y into the fish bloodstream under the conditions o f the present study. The costs o f encapsulating materials and the volumes o f encapsulated I g Y that fish would need to be fed were considered to be prohibitive. It was estimated that, assuming a 100% efficacy o f entrapment and release, 1.5g o f P L G costing $20  119  would be needed to deliver a 520mg daily dose o f 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 I g Y intestinal absorption as possible substitutes for Mega9. This class o f detergents has been considered safe for use i n human foods i n a limited concentration as noted in Food & Drug A c t & Regulations o f Canada, 1994 (pages 6712A to 67-14). In "Experiment 1", the highest I g Y level appeared after 1-3 hours post oral intubation.  Therefore, the study o f I g Y absorption i n co-delivery with Tween  detergents was limited to the first 4 hours after intubation. A s shown i n Table 4.4 and Fig.4.13, serum I g Y level did not improve significantly after 1 hour when Tween-20 or Tween-80 was added to the L - W S F solution i n P B S or antacid.  However, at 4 hour sampling time, a 2.5% solution o f  Tween-80 i n P B S improved the I g Y uptake as compared to the solution o f L - W S F i n P B S . This level was not significantly higher than that o f the treatment with L - W S F solution i n sodium bicarbonate.  N o significant increase was observed when antacid  replaced P B S i n any o f the applied concentrations o f the detergents.  Another  disadvantage associated with Tween-80 was poor solubility i n P B S or 1% sodium bicarbonate ( S B C ) solution.  120  Table 4.4. Effect of Tween detergents in uptake of orally intubated IgY into the bloodstream. . I g Y ( n g m L ' ) 1 hour I g Y ( n g m L ' ) * 4 hours Oral intubation treatment 206 ± 4 5 . 0 a* 176+101 a* 2.5% Tween80-PBS 167 ± 6 3 . 5 ab a 131±121 2.5% T w e e n 8 0 - N a H C O (1%) ab 165 ± 1 1 7 95.7 ± 8 5 . a 5% Tween80-PBS ab 147 ± 3 2 . 1 223 +75.7 a 2.5% T w e e n 2 0 - N a H C O (1%) 146 ± 6 7 . 7 ab 189±151 a 5%.Tween20-NaHCO (1%) 122.00 ± 6 9 . 0 ab 88.7 ± 4 9 . 7 a 5% T w e e n 8 0 - N a H C O (1%) ab 112 ± 58.8 91.0 ± 2 5 . 9 a W S F - N a H C 0 (1%) 77.3 ± 3 9 . 3 b a WSF-PBS 110 ± 10.0 * Common letters within each column indicate no significant difference between the means (p<0.05). Values are mean of 3 fish ± standard deviation. Oral intubation: 200uL of lOOmg mL' fish' L-WSF containing 12.4% total IgY (2.68mg IgY fish" =167.5mg IgY kg' average body weight), including additives when indicated. Average fish weight = 16g Blood was collected at 1 & 4 hours post intubation. 1  §  1  1  3  3  3  3  3  s  §  1  1  1  1  121  Effect of T w e e n Detergents on IgY Uptake 400 350 ^  300  E O) 250  £  >  E 3  ab  200  01 h ab  04 h  150 \  CD  (0  100 50  WSFantacid WSF-PBS  Oral intubation treatment Fig.4.13. Effect o f Tween-80 and Tween-20 at two concentration o f 5% & 2.5% on the absorption o f I g Y into the rainbow trout bloodstream. Detergents were prepared in P B S or 1% sodium bicarbonate solution. Values are mean o f serum o f 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 o f the previous oral intubation trials, a combination experiment was performed.  Table 4.5 and Fig.4.14 compare absorption enhancing  effects o f Tween-20 to that o f Mega9 and sodium pyrophosphate.  Although co-  administration o f Tween-80 provided a better absorption o f I g Y in the previous experiment, due to its poor solubility, a 5% solution o f Tween-20 in P B S was used in this experiment. In all cases except for L - W S F solution i n P B S , serum I g Y level was higher at 3 hours than at 1 hour sampling time. Four significantly different levels o f I g Y uptake into the blood were observed. The highest level coincided with the use o f L - W S F i n 1% sodium bicarbonate solution containing 5% Mega9. The second and the third highest levels conferred via co-administration o f 5% solution o f Mega9 i n P B S and 5% Mega9 in pyrophosphate solution, respectively. None o f the other solutions, i.e. pyrophosphate, 1% sodium bicarbonate, or 5% Tween-20 increased the absorption o f I g Y when compared to the levels obtained from W S F dissolved i n P B S . The results suggest that the strongest effect in enhancement o f I g Y uptake was conferred by use o f Mega9, which acted synergistically when concurrently applied w i t h antacid. The absorption o f intact proteins into the bloodstream o f rainbow trout following oral administration is reported to be dose dependent (Georgopoulou et al., 1988). M c L e a n 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 o f horseradish peroxidase ( H R P ) 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 WSF-Mega9 ^-NaHCOs WSF-Mega9 WSF-Mega9-pyrophosphate WSF-pyrophosphate §  1  ¥  I g Y C n g m L ' V 1 hour 181±147 NA** NA  I g Y C n g m L - ) 3 hours 1  3  292 ± 52.2 188 ± 0 . 7 1 106 ± 3 0 . 5 23.8 ± 16.2 21.9 ± 8 . 8 0 13.6 ± 10.7 8.68 ± 6 . 5 9 significant difference between the  a* b c d d d d means  7.20 ± 1.11 8.85 ± 4 . 5 7 WSF- N a H C 0 30.5 ±41.6 WSF-PBS 6.39 ± 1.50 WSF-Tween 20 * Common letters within each column indicate no (p<0.05). Values are mean of 3 fish ± standard deviation. Oral intubation: 200pL of lOOmg mL" fish L-WSF containing 12.4% total IgY (2.68mg IgY fish' =100.75mg IgY kg" average body weight), including additives when indicated. Mega9 or Tween-20: 5% in indicated solution. Pyrophosphate: 0.044g Na4P 0 ,10 H 0 in lOmL of 0.44M NaCl, p H 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. 3  £  s  §  1  1  1  1  £  ¥  2  7  2  124  Effect of Chemical compounds on IgY Uptake 400 350 300  -I  • 1 h  H3h  250  >  200  E 150 3  I-  in 100  H jj  50  d  0 WSF-Megantacid  WSF-Meg  WSF-Meg-  WSF-pyro  WSFantacid  pyro  WSF-PBS  WSF-T20 5%  Oral intubation treatment Fig.4.14. Effect o f Mega9, sodium pyrophosphate, sodium bicarbonate, 5% Tween-20 or some combinations o f them on the absorption o f I g Y into the rainbow trout bloodstream. Values are mean o f 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 o f protein reaching the sites o f intestinal uptake may help enhance absorption. Use o f sodium bicarbonate in the present study probably elevated the p H o f the stomach and consequently contributed to the increase in the amount o f I g Y available for intestinal absorption. The observed absorption enhancing effect o f antacid co-delivered with Mega9 was consistent with the result reported by M c L e a n et al. (1990) who detected an increased growth stimulating effect o f 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.  A l s o i n accordance with our results, M c L e a n & 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 i n the present study to enhance I g Y absorption from fish G I tract was incorporation o f pyrophosphate which is authorized for human use (Food & Drug A c 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, Velji & 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 o f this compound did not lead to a  significantly elevated I g Y 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 o f administration for each o f these non-ionic detergents, was further orally intubated into the fish concomitantly with W S F to examine its enhancing effect on I g Y uptake. Table 4.6 shows the concentration levels o f each chemical used i n this experiment along with their relevant mortality rates incurred in the intubated fish. The highest safe level for each detergent is also shown i n this table. Table 4.7 illustrates the levels o f various detergents used in oral intubation o f fish with IgY. The serum I g Y levels obtained 4 hours after co-administration o f 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 I g Y absorption when compared with W S F dissolved in a P B S solution.  Serum I g Y level in these treatments appeared to be  significantly higher than the levels achieved following co-administration o f all other tested detergents and also higher than that o f P B S treated fish. CFfAPS, C F I A P S O , Triton X - l 14 and saponin at the levels used in the present study did not contribute to an increase in I g Y uptake and resulted i n serum I g Y levels not significantly different from that observed when W S F was intubated in a solution o f 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 o f I g Y .  These levels were  significantly lower than that offered by W S F solutions i n P B S .  127  Table 4.6. Lethality of various levels of orally intubated detergents to rainbow trout.  Oral intubation treatment  8  Mortality rate  Levels of detergent  Highest safe level*  (%)  ¥  5% 0, 0, 0, 0 5%, 3%, 1%, 0.5% Octyl-B-glucoside 1% 100, 75, 0, 0 5%, 3%, 1%, 0.5% Na-deoxycholate 5 % 0 5% Mega9 1% 25, 25, 0, 0 5%, 3%, 1%, 0.5% CHAPS 1% 25, 25,' 0, 0 5%, 3%, 1%, 0.5% CHAPSO 1% 50, 25, 0, 0 5%, 3%, 1%, 0.5% Triton X-114 1 mg fish' 1,0.1,0.05, 0.01 mg fish' 0, 0, 0,0 Saponin 50, 0, 0, 0 3% 5%, 3%, 1%, 0.5% Triton X-100 0, 0,0 0.9 mg fish" 0.9,0.3, O.lmg fish' L-cysteine * Highest safe level of the detergent is the highest level tested which did not cause any mortality. Oral intubation: 200pL of detergent solution fish" . Cumulative mortality rate among 4 fish 10 days after oral intubation with detergent solution. Average fish weight = 25g. 1  1  1  §  1  1  ¥  128  Table 4.7. Effect of detergent in enhancement of IgY absorption. Concentration of detergent Serum IgY * (ng mL" ) Oral intubation treatment 776 + 361 a* 5% Octyl-B-glucoside + W S F 1% 715+455 a Na-deoxycholate + W S F 5 % 312 ±77.1 a Mega9 + WSF 46.3 ±45.8 b PBS + W S F 44.9 ±23.9 b 1% CHAPS + WSF 28.4 ± 17.3 b 1% CHAPSO + WSF 24.7 ± 12.2 b 1% Triton X - l 1 4 + W S F 5 mg mL" 21.7 ± 11.1 b Saponin + W S F 3% 9.50 + 13.7 c Triton X-100 + WSF 4.74 ±5.35 c 4.5 mg mL" L-cysteine + W S F 4.57+4.90 PBS (negative control) * 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' of IgY-containing detergent solution. Blood sampling at 4 hour post intubation of WSF. Average fish weight = 25g. 1  8  1  1  c  $  §  1  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 o f h G H when deoxycholate in antacid solution was concurrently orally intubated.  Mega9  absorption enhancing effect is also in agreement with M c L e a n et al. (1990) and M c L e a n & A s h (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 o f the most effective detergents in releasing proteins from membrane bounded compartments without denaturing them. They also found a similar protein releasing power i n deoxycholate and Triton X - 1 0 0 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 I g Y 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 o f these detergents and their ability in releasing proteins from cell membrane cannot be concluded. Co-delivery o f saponin ( l m g fish" ) did not elicit an absorption enhancing 1  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 anguillarum  following intubation o f 5mg  fish"  1  anti-F!  sheep I g G co-administered with Q u 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" dose o f saponin into 1  tilapia. The contrary results observed in different  fish  studies might be due to  divergence between the species, size o f the studied fish, nutrition, molecular weight and structure o f protein molecule, conditions o f experiment such as temperature, photoperiod and intensity o f light, as well as the detection systems employed. A s the present study and the related literature have shown, considerable individual variations exist within the same species.  This fact along with the small numbers o f fish (3-6)  sampled in various studies (Akhlaghi, 1999; Jenkins et al., 1991; Hertz et ai, Lee et al., 2000; M o r i y a m a et al,  1991;  1990; M c L e a n & A s h , 1990) contributes to large  experimental variability, which could explain some o f the discrepancy observed by different researchers.  4.7. Challenge studies T o test the efficacy o f oral passive immunization o f trout, fish were exposed to the pathogenic bacteria in experimental challenge trials following oral administration o f anti-K anguillarum  chicken egg I g Y . Various approaches as described i n materials  & method were considered in oral delivery o f IgY. In the following, the result o f 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 o f I g Y absorption when incorporated into the fish  131  feed. T w o 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 I g Y level o f 6.8 and 13.Ong mL" at day 2 and as 11.7 and 13.6ng mL" at day 7 for the fish 1  1  receiving 2 days o f high dose or 7 days o f 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 I g Y concentrations (p=O.S). In an immersion challenge with V. anguillarum,  mortality rates were 100% for  untreated fish and the group receiving 7 days o f low content Mega9 diet. Surprisingly, only 50%) mortality was recorded for the group receiving a diet high in Mega9 for 2 days and devoid o f it for the rest o f the time.  There was only 2 days resting time  between blood collection and challenge o f 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 g Y in protection o f 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 7day treatment period when challenge was performed, total serum I g Y appeared in  132  Table 4.8. Serum IgY & mortality levels in preliminary feeding and challenge*" conducted at day 9. , _ , Serum I g Y (ng m L ) * day 2 16.8 + 10.4 a* 13.0 + 6.04 a - 1  Treatment  Semm I g Y (ng m L " ) *  Mortality rate  day 7 11.7 + 3.47 13.6 ± 8 . 4 1  (%)  1  50 a Treatment 1 100 a Treatment 2 100 Untreated * 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 day' =87mg IgY kg' 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. Values are mean ± standard deviation of 4 fish. V. anguillarum concentration for challenge = 1.2 X 10 cfu mL' . Average fish weight = 89 g.  §  1  1  1  ¥  £ s  11  6  1  133  significantly higher levels in C3 & C 4 than most o f 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 o f C 7 and T2 in which fish received pellets containing W S F from immunized hens ( P - s p W S F ) for the +  whole 7-day period o f this study. I g Y 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 C 7 , occurred in C 3 .  This group (C3) which also  showed the highest I g Y concentration in the serum, received I N s p W S F - M 9 at the first day and P - s p W S F - M 9 for the rest o f study period. +  There was no significant  difference between the mortality rate o f any other treatment groups. The fact that the mortality rate i n C 4 did not come close to that o f C3 indicates that the protective effect is due to the specificity o f I g Y , since diet o f C 4 was identical to C3 except that the specific anti-K anguillarum I g Y was absorbed by the antigen. The high mortality rate in C 5 , which received M 9 without any I g Y , 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 o f 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). Serum IgY (ng mL" )* Mortality (%) Treatment 96.7±3.87 a 31.0126.5 be* T l - d 1: P -M9-spWSF; d 2-7: pellets 91.7±6.36 a 58.3 ±27.3 ab T2- d 1: P -M9-spWSF; d 2-7: P -spWSF 93.3 ± 0 . 0 0 a c 21.3 ±8.35 C l - d 1: P -M9-nspWSF; d 2-7: pellets 90.0 ± 4.67 a be 40.9 ± 10.2 C2- d 1: P -M9-nspWSF; d 2-7: P -nspWSF 65.7±18.7 b 120 ±54.4 a C3- d 1: IN M9-spWSF; d 2-7: P -spWSF 86.7 ± 9.40 a a C4- d 1: IN M9+abspWSF; d 2-7: P -abspWSF 104 ±35.2 90.0 ± 8.60 a d 2.04 ± 1.57 C5- d 1: P - M 9 (no WSF); d 2-7: pellets 93.4 ± 9.40 a 0.76 ±0.86 e C6 (Untreated)- d 1-7: pellets 80.0 ± 9.48 ab 46.0 ± 17.6 ab C7- d 1-7: P -spWSF Abbreviations as described in the list of abbreviations are: T: test treatment, C: control, d: day(s), P : treated pellets, M 9 : Mega9, sp: specific, nsp: non-specific, W S F : 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. Oral intubation volume: 200uL. Specific or non-specific IgY: 5.5mg IgY fish' day" =100mg IgY kg" average body weight. Mega9 & N a H C 0 : 396 & 80mg kg" 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 10 cfu mL' ): 20 hours after the last feeding for 30 minutes. Blood collection @ the day of challenge. Average fish weight = 55g. 1  +  £  ¥  +  £  +  §  +  +  §  +  11  £  +  §  +  £  +  §  §  +  +  $  £  §  11  1  1  1  1  3  5  1  135  Results o f experiment 7 are illustrated in Table 4.10. In this trial, group C 2 had a significantly higher level o f total serum I g Y than all other treatments at the end o f the7-day study period. C 2 received I N spWSF, M 9 and sodium bicarbonate ( S B C ) on day 1, followed by feeding the same ingredients incorporated into the pellets on day 2 and P - s p W S F on days 3-7. +  The untreated control group (C3) showed a  significantly lower level o f total serum I g Y than all other groups, which were not significantly different from each other in this respect.  C 2 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 o f C 2 , although 20% lower, was not significantly  different from T l and C 4 , which for the whole course o f study were fed P - s p W S F +  with or without M 9 & S B C , respectively.  Mortality rate in C l was significantly  higher than T l and C 4 . Therefore, i n the spectrum o f this trial, feeding o f specific anti-P! anguillarum I g Y could enhance resistance o f the trout to vibriosis but feeding o f non-specific I g Y could not offer any protection. In fact, the mortality rate o f C l , receiving nspWSF, was at the same level with C 3 , the untreated control. This result is consistent with the results obtained from the IP injection o f IgY, in which mortality rate in the group receiving non-specific I g Y was not significantly different from that o f 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 i n all treatment groups ( T l , T 2 ,  136  Table 4.10. Serum IgY & mortality levels after oral administration of W S F , Mega9 and  Serum IgY (ng mL") 151 ±24.2 T I - dl-2: P -M9-SBC-spWSF; d3-7: P -spWSF * 144 ± 16.8 C l - dl-2: P -M9-SBC-nspWSF; d3-7: P -nspWSF C2- dl: IN M9-SBC-spWSF; d2: P -M9-SBC-spWSF; 201 ±36.4 d3-7: P -spWSF 1.84 ±2.72 C3 (untreated)- dl-7: pellets 120 ± 6.24 C4- dl-7: P -spWSF  Treatment  1  +  +  +  +  1  +  £  s  Mortality (%) b* 70.5 ± 16.7 bc b 93.3 ±6.67 a a 58.8+20.0 c  +  c 93.33± 0.00 ab b 71.1 + 16.8 bc  +  A U U 1 U V JdLlVJllD  CIO U ^ V i i L / w u  i i i  UIAW  iijv  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). Values are mean ± standard deviation of 3 fish. Values are mean ± standard deviation of 3 tanks. ' Oral intubation volume: 200uL fish" . Specific or non-specific IgY: 6.5mg IgYfish" day" =217mg IgY kg" average body weight. Mega9: lOmg fish" =333mg kg' average body weight; NaHC0 : 2mgfish" =66mg kg" average body weight. Pellets: 2% average body weight, coated with marine oil (3% pellet weight). Exposure to V. anguillarum (5.1-5.4 X 10 cfu mL"): 20 hours after the last feeding for 30 minutes. Blood collection @ the day of challenge. Averagefishweight = 30g. +  s  £  1  1  1  1  1  1  1  1  3  4  1  137  T3 & T4) received I N spWSF on the first day followed by P - s p W S F for the rest o f +  the period o f the study.  The diets o f 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.  A s shown i n Table 4.11, receiving I N s p W S F and non-ionic  detergents on the first day ( T I , T 2 & T3) did not result i n a lower mortality i n such treated groups than the fish receiving I g Y without a detergent (T4). The mortality rate in C l , although approximately 1.6 times higher than T I , T 2 and T 3 , was not significantly different from any o f the treatments.  Absorption o f I g Y into the  bloodstream appeared at the highest levels i n T2 and T 3 , 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 T 4 (devoid o f detergent i n the diet) showed serum I g Y levels significantly higher than the negative control and lower than T 2 and T 3 . This result suggests that Mega9 was not as effective as the other two detergents i n the enhancement o f I g Y uptake. The same trend o f 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 i n experiment 8, the difference between the mortality levels o f the three detergents was not significant.  Although a linear correlation with a r = 0.68 was 2  found between total serum I g Y 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 I g Y used always contained specific anti-V. anguillarum activity.  138  Table 4.11. Serum IgY & mortality levels following oral administration of WSF, Serum IgY (ng mL" ) 183 ± 3 5 . b*  Mortality  512+ 142 a  47.9 ±15.5 a  373 ± 1 7  a  46.7 ± 2 3 . 1 a  151 ±24.2 b 8.36 ±3.45 c  59.1 ±20.1 a 77.1 ±20.6 a  $  Treatment  1  T l - d l : IN 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 C l (negative control)- d l : IN PBS ; d2-7: pellets 11  +  £  (%) 50.0 ±14.3 a  +  +  +  +  +  +  ADUreVlcltlUIlS  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, M 9 : Mega9, OpG: octyle-P-glucoside, D X : deoxycholate, sp: specific, W S F : 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' . IgY: 6.12mg fish' day" =153mg kg" average body weight day" . Mega9 & octyl-P-glucoside (intubated): 5% volume=10mg fish" =250mg kg' average body weight. Mega9 & octyl-P-glucoside (in pellets): 1.5 X intubation=15mg fish' =375mg kg" average body weight. Na-deoxycholate (intubated): 1% volume=2mg fish" 50mg kg" average body weight. Na-deoxycholate (in pellets): 1.5 X intubation =3mg fish" 75mg kg" 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 (2.5X10 cfu mL" ): 21 hours after the last feeding for 30 minutes. Blood collection @ the day of challenge. Average fish weight = 40g. +  $  £  11  1  1  1  1  1  1  1  1  1=  1  1=  4  1  1  1  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 T I 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 = 0.26 was found between serum IgY 2  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 (r = 0.512,/? < 0.001) appeared to depend upon the method of delivery. 2  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 I g Y level. Thus IP injection resulted in serum I g Y concentration in the order o f 10 ng ml" , intubation 3 x 10 ng 4  1  2  ml" and feeding 1 x 10 ng ml" . However, in some cases, protection conferred by 1  2  1  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, I g Y in the gut has been shown to provide some protection (Hatta et al., 1994).  I g Y 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 o f the intubation and feeding studies, shows that oral intubation o f octyl-P-glucoside, deoxycholate and Mega9 can induce a more efficient uptake o f I g Y into the bloodstream o f rainbow trout when compared to the intubated I g Y devoid o f detergents. Although intubated Mega9 followed by feeding IgY-containing pellets i n experiment 8 did not significantly increase the serum I g Y level,  contrasting  observations  o f the  previous  intubation  studies,  especially  experiment 1, proves the potential o f Mega9 in enhancement o f I g Y uptake. different  observation i n experiment  8 might be attributed  to large  The  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 I g Y uptake  after  intubation, did not elicit a significant increase in I g Y 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" ) 110+17.4 156 + 51.3 144 + 42.8 158 + 66.5 165+70.8  a* a a a a  Mortality (%) 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  0.56 + 0.16  b  68.6 + 30.5  £  1  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 T 4 - d l - 7 : P -spWSF C l (positive control)- d l : I N M9-SBC-spWSF; d2: P -M9-SBC-spWSF; d3-7: P -spWSF C2 (negative control)- dl-7: pellets +  +  +  +  +  +  +  1  +  +  J~VU U l ^ V I C I U I W I I O  CIO  UWOWAISWI*  '11  L . . v.  n u .  v  &  ~  _ ,  a  _ .  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" . IgY: 8.8mg fish" day" =196mg kg" average body weight day" . Mega9 (intubated): 5% volume=10mg fish" =222mg kg" average body weight. Mega9 & octyl-P-glucoside (in pellets): 20mg fish" 440mg kg" average body weight. Na-deoxycholate (in pellets): 4mg fish" 88mg kg" average body weight. N a H C 0 : 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 10 cfu mL" ): 20 hours after the last feeding for 30 minutes. Blood collection @ the day of challenge. Average fish weight = 45g. +  £  1  1  1  1  1  1  1  1=  1=  1  1  3  4  1  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 o f 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 o f 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 G I tract, and therefore the food left i n the gut could have interfered with the absorption o f IgY. O n later days, when I g Y and the additives were carried by pellets, I g Y could have been bound to the food ingredients even before introduction into the fish gut. O n the other hand, the presence o f feed could extend the digestion process in the stomach when compared with the intubation o f a solution o f I g Y associated with detergents and antacid. A longer exposure o f I g Y to the digestive enzymes and acidic conditions o f the stomach would make it more susceptible to destruction. Lee et al. (2000) reported that the activity o f anti-F. ruckeri I g Y 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 i n 30 minutes after feeding w h i c h increased again after 3 hours. Shimizu et al. (1988) found that the  143  activity o f anti-E.coli I g Y 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 I g Y was completely lost in exposure to pepsin at p H 2 while it was 6 3 % retained at p H 4.  These studies show that sensitivity o f I g Y to gastric enzymes is p H dependent.  This fact may explain the positive results obtained from combined application o f antacid and detergents i n the present study.  Antacid would have increased stomach  p H and consequently helped retain I g Y activity to a greater extent, especially when feed was absent in intubation treatments.  Intubation could thus accelerate passage o f  material through the gastric section o f the G I tract. Hofer (1982) suggested that following periods o f extended fasting, protease enzymatic systems might undergo adaptation allowing higher levels o f ingested protein to escape degradation.  If this is true, a higher I g Y 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 o f Mega9 with anti-K". anguillarum I g Y 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 o f I g Y incorporated into the pellets also increased the disease resistance in some cases. However, i n most of the cases the extent o f offered protection was not significantly different from the control.  Intubation followed by feeding o f I g Y co-delivered with Mega9 decreased  144  the mean mortality rate to a lower level than in the group treated with anti-Vibrio I g Y devoid o f detergents by a factor o f 1.6 in experiment 9.  However the level o f  difference  the  was  not  statistically  significant  in  any  of  challenge  Administration o f deoxycholate led to a significant decrease in mortality.  trials. Use o f  octyl-P-glucoside, although resulted in the highest I g Y uptake in both experiments, did not offer a better protection against the disease. The overall result o f the challenge studies following oral administration o f I g Y is that use o f specific anti- Vibrio I g Y , in one form or the other, induced an enhanced protection against vibriosis in all trials.  The only exception was experiment 8, i n  which the protection due to use o f I g Y was higher than the control group but the difference was not statistically significant. Considering results o f experiment 8 along with the other trials, it can be concluded that oral administration o f anti-F. anguillarum  I g Y 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 o f I g Y was used to prepare the fabricated pellets, the dose reaching each fish was uncertain due to competition between the individuals and possible leaching o f I g Y into the water before being consumed by the fish. A s illustrated in Table 4.13, the amount o f total I g Y incorporated into the pellets through spWSF in experiments 7, 8 and 9 was not significantly different i n various treatments o f the same experiment. The commercial pellets or pellets top-dressed with Mega9 (without W S F ) did not show any I g Y content.  145  Detergents, although capable o f enhancing I g Y 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  I g Y but no  detergent. Intraperitoneal (D?) injection o f specific I g Y led to a significantly higher protection o f rainbow trout against V. anguillarum in an immersion challenge when compared to the groups EP injected with non-specific I g Y or P B S . This protective effect was persistent when fish were challenged 1, 3, 7 or 14 days post EP injection o f specific I g Y , which shows that specific I g Y 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 i n experiment 9 and the antiVibrio I g Y treated and P B S treated control groups in the EP experiment at days 3 and 7.  In both o f these experiments, there was a significant difference between the  mortality o f the negative control (62%-75%) and that o f the anti-Vibrio treated groups with mortality levels o f 24%-31% i n EP injected trials and 35.6% in experiment 9, when I g Y was fed i n conjunction with deoxycholate and antacid. This result suggests that feeding o f I g Y 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 I g Y confers protection. The result o f 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 o f specific I g Y 4 hours before challenge. These researchers obtained only a marginal reduction i n mortality and intestinal infection after feeding the same  146  specific I g Y 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.  L o w e r contents o f the egg-yolk powder in the feed did not elicit  protective effect.  Hatta et al. (1994) cannulated a mixture o f anti- E. tarda I g Y and  the pathogen into the stomach o f Japanese eels after their intestinal mucosa was damaged. I g Y treated eels survived while the control eels died or showed the symptoms o f the disease.  Although they did not detect any immunologically active  I g Y i n the serum o f eel, a high level o f anti-£. tarda I g Y was maintained in the G I tract for 7 days. These results suggest that simultaneous introduction o f specific I g Y and pathogenic bacteria to the digestive system o f fish can reduce mortality.  This  effect could be attributed to direct local neutralizing action o f specific I g Y at the site of bacterial infection i n the digestive tract before being absorbed. Therefore, a better protection could be possibly obtained in the present study i f the diet containing antiVibrio I g Y was continued after the challenge, too. A l s o an increase i n diet I g Y could be helpful to elevate the serum I g Y levels. In all o f the challenge experiments o f the present study, there was approximately 20 hours between the last feeding and the challenge. The fish were fed regular commercial diets i n the post-challenge period.  147  Experiment 9 Experiment 8 Additives to pellets Experiment 7 8.7 ± 0 . 9 a NA 13.4 ± 1 . 5 a* spWSF 11.4 ± 1.9 a nspWSF 11.9 ± 0 . 5 a M9-nspWSF NA 9.7 ± 1.7 a M9-spWSF 14.2 ± 2 . 2 a 8.4 ± 0 . 5 a a 9.4 ± 1 . 7 OpG-spWSF 11.6 ± 1 . 2 a 8.8 ± 0 . 5 a DX-spWSF 0.001 ± 0 . 0 b 0.001 ± 0 . 0 b 0.001 ± 0 . 0 b no additive Mega9 Abbreviations as described in the list of abbreviations are: M9: Mega9, glucoside, D X : deoxycholate, sp: specific, nsp: non-specific, W S F : 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. Data is not available. ¥  11  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)  lysophosphotidylcholine increased  reported ileal  that  in rats,  non-ionic  permeability to intact  detergent,  proteins  due  to  enterocyte sloughing and cell rupture caused by osmotic shock. Jenkins et al. (1991) observed an appreciable increase in the uptake o f orally and anally delivered human gamma globulin when co-administered with Q u i l - A saponin. They detected a number o f 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 o f luminal contents, fusion with the plasma membrane and causing the cellular microvilli o f the enterocytes to become shortened and damaged. A l l o f these effects generally serve to increase the permeability o f the intestine to macromulecules. Although the effects o f Mega9 on the intestinal tissues o f fish have not been investigated i n such detail, M c L e a n & 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 o f the G I tract to gelatinize, forming clumps o f 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 o f 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 I g Y , dissects o f stomach, pyloric caeca and anterior intestine were fixed in buffered formalin as detailed i n materials & methods. Microscopic examination o f the H & E stained G I tissues demonstrated the removal o f the striated border and partial disruption o f the columnar epithelia o f intestinal microvilli.  The stomach and pyloric caeca textures were not affected (Fig.7.4 &  Fig.7.5 in the appendix).  The intestine o f the untreated control fish seemed intact.  Fig.4.15 and Fig.4.16 display the histological cross-sections o f the intestines o f 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, i n all o f these samples, including the untreated and  P B S treated fish the same degree o f 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 i n the appendix illustrates microscopic photographs o f some o f these samples. T o investigate the cause o f this unexpected observation, the same fixative i.e. buffered formalin was injected through digestive lumen o f an untreated fish to ensure a thorough fixation. histological slides still indicated the same extent  o f degradation,  experiments B o u i n ' s solution was used to fix the specimens.  Since the in all later  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 o f  the cross sections o f the entire intestine revealed no severe tissue disruption in any o f the samples. This result suggests that the tissue damage primarily observed was due to the inefficiency o f 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 o f I g Y molecule into the blood circulation.  Fig.4.17 to Fig.4.20  illustrate microscopic photographs o f H & E stained cross section o f the intestinal tissue o f the Mega9, Na-deoxycholate, octyl-P-glucoside or P B S intubated, and untreated fish fixed i n B o u i n ' 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  Georgopoulolou etal,  studies (Jenkins et al,  1985; Georgopoulolou etal,  1991; L e B a i l  et al,  1988; Rombout etal,  1989;  1985).  The histological examinations o f the present study led to rejection o f the hypothesis that Mega9, Na-deoxycholate or octyl-P-glucoside might have enhanced protein absorption through physical damage to the enterocyte lining o f 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 o f the untreated control and no disruption was noticed in the epithelial villi.  However the other hypothesis o f M c L e a n & A s h  151  (1990) that the gelling action o f 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 o f 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, i n 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. T o examine changes in more detail at the intercellular and microvilli level, electromicroscopic studies are required. In the absence o f such studies the possibility of loosening o f intercellular junctions, increase in pinocytosis o f luminal content, fusion with the plasma membrane and damage to microvilli could be considered as the subjects o f 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  •::M  Aid  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 o f specific immunoglobulin production in hens in which immunoglobulins can be recovered from the egg yolk, and compatibility o f this procedure with animal rights concerns, make chicken egg yolk immunoglobulins (IgY) a favorable source o f 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. amount  o f work has  been reported  on oral  delivery  Although a considerable o f proteins,  including  immunoglobulins to aquacultured species, more research is needed to develop effective and industrially-feasible methods o f 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 o f pathogen specific I g Y w i l l decrease the mortality o f rainbow trout following bacterial infection.  2. Dehydration o f I g Y w i l l  not affect  its immunogenicity. 3. C o -  administration o f detergents w i l l enhance the oral uptake o f I g Y into the fish blood. The results revealed the followings: 1. A strong antigen-antibody reaction occurred between V. anguillarum antigens and I g Y obtained from the egg yolk o f vaccinated hens. 2.  H i g h titers o f specific anti- V. anguillarum I g Y were effectively raised in vaccinated laying hens.  The water-soluble fraction ( W S F ) o f the egg yolks o f  160  vaccinated hens was a good source o f semi-pure IgY. The I g Y o f W S F could be concentrated using ultrafiltration, an established technology in the food industry. 3.  Immunological properties o f I g Y were retained when W S F was dehydrated using the techniques o f 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 o f I g Y in a reactive form seems feasible. 4. A n a l intubation o f pure I g Y did not prove effective i n uptake o f I g Y into the blood under the conditions o f the present study.  This might be due to the use o f  insufficient doses o f I g Y or to insufficient starvation o f fish prior to treatment. 5. Orally administered I g Y was absorbed into the bloodstream o f 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. A m o n g a wide range o f detergents tested herein, use o f the non-ionic detergents, Mega9 and octyl-p-glucoside as w e l l as the bile salt, deoxycholate improved I g Y uptake significantly, following simultaneous oral intubation with IgY-containing W S F . The I g Y levels following EP injection were only 12 to 18 times higher than the levels after oral co-administration with Mega9. 7. Lyophilized W S F was microencapsulated as a source o f I g Y in polylactide-coglycolide ( P L G ) with a lactide: glycolide ratio o f 50:50 or 85:15.  Oral intubation  161  of microencapsulated I g Y did not lead to a higher serum I g Y level than the unprotected I g Y over a 2-week period o f study.  Limitation o f encapsulation  capacity, poor entrapment, and incomplete release may have contributed to the poor uptake o f IgY. L o w purity o f I g Y in W S F could have added to the problem, too. 8. EP injection o f W S F from the egg yolk o f immunized hens led to a high serum I g Y level and a significantly decreased  mortality rate following  challenge with the pathogenic bacteria.  an immersion  This proved the efficacy o f I g Y in  enhancement o f disease resistance in rainbow trout. However, EP injection might not be considered as a practical method o f passive immunization in aquaculture. 9. Oral administration o f anti-K anguillarum I g Y on feed pellets alone or in codelivery w i t h detergents, offered different levels o f protection o f rainbow trout against Vibriosis following an immersion challenge, which in some cases was as effective as EP injection o f IgY, but this result was not consistent throughout the study and needs further research.  The efficacy o f continued feeding o f higher  doses o f specific I g Y before and after exposure to the pathogen has to be investigated. Areas yet to be explored i n this field include in vivo stability and functionality o f I g Y , storage life o f W S F and I g Y , development o f alternative IgY-containing products  such as coated  I g Y concentrate for oral application and legislative  requirements. Further research is also needed to improve oral uptake o f immunoglobulins when incorporated into the feed.  Different aspects, such as feeding intervals, diet  162  composition and dose o f fed I g Y should be studied. Since dose dependence o f protein uptake has been documented, use o f higher I g Y 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 I g Y 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 i n the literature.  Although better informed  speculation about the mechanism o f absorption enhancement is now possible this phenomenon is not yet fully understood.  163  CHAPTER SIX REFERENCES  Akhlaghi, M , 1999. Passive immunization o f fish against vibriosis, comparison o f intraperitoneal, oral and immersion routes. Aquaculture 180, 191-205. Akita, E . M . & Li-Chan, E . C . Y . , 1998. Isolation o f bovine immunoglobulin G subclasses from milk, colostrum and whey using immobilized egg yolk antibodies. Journal ofDairy Science 81, 54-63. Akita, E . M . , Li-Chan, E . C . Y . , Nakai, S., 1998. Neutralization o f enterotoxigenic Escherichia coli heat labile toxin by chicken egg yolk immunoglobulin (IgY) and its antigen binding fragments. Food and Agricultural Immunology 10, 161-172. Akita, E . M . & Nakai, S., 1999. Preparation o f enteric-coated gelatin capsules o f I g Y with cellulose acetate phthalate. In Egg Nutrition and Biotechnology (Sim, J.S., Nakai, S., and Guenter, W . , eds.) pp. 301-310, C A B I Publishing, C A B International, Wallingford, U K . Akita, E . M . & Nakai, S., 1993. Comparison o f four purification methods for the production o f immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E. coli strain. Journal ofImmunological Methods160, 207-214. Akita, E . M . & Nakai, S., 1992. Immunoglobulins from egg yolk: isolation and purification. Journal of Food Science 57, 629-634. Aminirissehei A . - H , 1999. Personal communication. Department Sciences, Simon Fraser University, Burnaby. B . C . , Canada.  o f Biological  Anderson, D . P . , 1992. Immunostimulants, adjuvants, and vaccine carriers in fish: Applications to aquaculture. Annual Review of Fish Diseases 2, 281-307. Anderson, D . P . & Jeney, G . , 1993. Immunostimulation and protection from diseases in rainbow trout by glucans given by injection or bath. European Aquaculture Society. Special Publications 19. Available as: From Discovery to Commercialization. Oostende (Belgium): European Aquaculture Society, 199. Ash, R., 1985. Protein digestion and absorption. In Nutrition and Feeding in Fish (Cowey, C . B . , Mackie, A . M . and B e l l G . R . , eds) pp 69-93. Academic Press, Orlando, Florida, U S A .  165  Attiyate,Y., 1979. Microwave vacuum drying- First industrial application. Food Engineering International 4(1), 30-31. Bartz, C . R . , Conklin, R . H . , Tunstall, C . B . , Steele, J . H . , 1980. Prevention o f murine rotavirus infection with chicken egg immunoglobulin. Journal of Infectious Diseases 152, 439-441. Boesen, H . T . , Pedersen, K . , K o c h , C , Larsen, J.L., 1997. Immune response o f rainbow trout ( Oncorhyncus mykiss) to antigenic preparations from Vibrio anguillarum serogroup 01. Fish & Shellfish Immunology 7, 543-553. B o l i n , H . R . , Huxsoll, C C , Jackson R., N g , K . C . , 1983. Effect o f osmotic agents and concentration on fruit quality. Journal of Food Science 48, 202-205. Brown, A . H . , 1973. Drying rates and estimation o f drier capacity. In F o o d Dehydration (Arsdel, W . B . , Copley, M . J . , and Morgan, A L , eds.) 2 ed., V o l . 1, pp.347-350. A V I Publishing C o . Inc., Westport, Connecticut, U S A . n d  Brown, L . L . , Evelyn, T.P.T., Iwama, G . K . , 1997. Specific protective activity demonstrated in eggs o f broodstock salmon injected with rabbit antibodies raised against a fish pathogen. Diseases of Aquatic Organisms 31, 95-101. Carlander, D . , Kollberg, H . , Wejaker, P . E . , Larson, A . , 1999. Prevention o f chronic Pseudomonas aeroginosa colonization by gargling with specific antibodies: a preliminary report. In Egg Nutrition and Biotechnology (Sim, J.S., Nakai, S., and Guenter, W . , eds.) pp. 371-374. C A B I Publishing, C A B International, Wallingford, UK. Catty, D . & Raykundalia, C , 1988. E L I S A and related enzyme immunoassays. In Anibodies, a Practical Approach (Catty, D . , ed.) V o l . 2 , pp. 97-154, TRL Press at Oxford University Press, Oxford, England. Chansarkar, N . L . , 1998. Studies on structural stability o f hens egg yolk immunoglobulin (IgY). M S c Thesis, The University o f British Columbia, Vancouver, British Columbia, Canada. Chart, H . & Trust, T.J., 1984. Characterization o f the surface antigens o f the marine fish pathogens, Vibrio anguillarum and Vibrio ordalii. Canadian Journal of Microbiology 30, 703-710. Coleman, M . , 1999. Using egg antibodies to treat diseases. In Egg Nutrition and Biotechnology (Sim, J.S., Nakai, S., and Guenter, W . , eds.) pp. 351-370. C A B I Publishing, C A B International, Wallingford, U K . Conover, W . J . , 1980. Practical nonparametric statistics, 2  n d  edition, pp.357-362. John  W i l e y & Sons, Inc., N Y , U S A .  166  Draber, P., Drabevora, E . , Novakova, M . , 1995. Stability o f monoclonal M antibodies Freeze-dried in the presence o f trehalose. Journal of Immunological Methods 181, 3743. de Baulny, M . O . , Quentel, C , Fournier, V . , Lamour, F., L e Gouvello, R., 1996. Effect of long-term oral administration o f beta-glucan as an immunostimulant or an adjuvant on some non-specific parameters of the immune response of turbot Scophthalmus maximus. Diseases of Aquatic Organisms 26, 139-147. Drouzas, A . E . , Schubert, H . , 1996. Microwave application in vacuum drying o f fruits. Journal of Food Engineering 28, 203-209. Dunn, E . J . , Polk, A . , Scarrett, D.J., Olivier, G . , Lall, S., Goosen, M . F . A . , 1990. Vaccines in aquaculture: The search for an efficient delivery system. Aquaculture Engineering 9, 23-32. D u Pasquire, L . , 1982. Antibody diversity i n lower vertebrates-why is it so restricted? Nature 296, 311-313. Durance, T . D . , 2000. Vacuum microwave dehydration of food. Monograph.21, 1-8. Tezukyama College, F o o d Sciences, Nara City, Japan. Durance, T . D . & L i u , F., 1996. Production of potato chips. U . S . Patent 5,676,989. Durance, T.D., Wang, J . H . , 1999. Energy utilization and quality in vacuum microwave and convective air dehydration o f tomatoes. In Proceedings of Canadian Institute of Food Science & Technology, 6-9 June 1999, Okanagan, British Columbia, Canada. Oral presentation abstract N o . 11. Ebina, T., Tsukuda, K . , Umezu, K . , Nose, M . , Tsuda, K . , Hatta, H . , K i m , M . Yamamoto, T., 1990. Gastroenteritis i n suckling mice caused by human rotavirus can be prevented with egg yolk immunoglobulin (IgY) and treated with a protein bond polysaccharide preparation (PSK). Microbiology and Immunology 34, 617-629. Ellis, A . E . , 1995. Recent development Pathology 30, 293-300.  in oral vaccine delivery systems. Fish  Ellis, A . E . , 1988a. General principles o f fish vaccination. In Fish Vaccination (Ellis, A . E . , ed.) pp. 1-31 London, Academic Press. Ellis, A . E . , 1988b. Optimization factors for fish vaccination. In Fish Vaccination (Ellis, A . E . , ed.) pp. 32-46. London, Academic Press. Evelyn, T.P.T., 1984. Immunization against pathogenic Vibrios. In Symposium on Fish Vaccination: Theoretical Background and Practical Results on Immunization  167  against Infectious Diseases, (de Kinkelin., P., ed.) pp. 121-150. Office Internationl des Epizooties, Paris, France. Ezura, Y . , Tajima, K . , Yoshimizu, M . , Kimura, T., 1980. Studies on the taxonomy and serology o f causative organisms o f fish vibriosis. Fish Pathology 14, 167-179. Fichtali, I , Charter, E . A . , L o , K . V . , Nakai, S., 1992. Separation o f egg yolk immunoglobulins using an automated liquid chromatography system. Biotechnology and Bioengineering 40, 1388-1394. Fichtali, J., Charter, E . A . , L o , K . V . , Nakai, S., 1993. Purification o f antibodies from industrially separated egg yolk. Journal of Food Science 58, 1282-1285. Food & D r u g A c t & Regulations with amendments to December Department o f National Health & Welfare o f Canada.  15,  1994.  Fujino, Y . & Nagai, A . , 1988. The ingestion o f bovine serum albumin into the serum through the intestine o f chum salmon oncorhynchus keta Walbaum. Journal of Faculty of Marine Science and Technology of Tokai Unniversity 26, 155-166. Fujino, Y . , Ono, S., Nagai, A . , 1987. Studies on uptake o f rabbit immunoglobulin into columnar epithelium cell in the gut o f rainbow trout, Salmo gairdneri. Bulletin of the Japanese Society of Scientific Fisheries 53, 367-370. Galeotti, M . , 1998. Some aspects o f the application o f immunostimulants and a critical review o f methods for their evaluation. Journal of Applied Ichthyology/Z. Angew. Ichthyol. 14, 189-199. Georgopoulou, U . , Dabrowski, K . , Sire, M . F . , Vernier, J . M . , 1988. Absorption o f intact proteins by intestinal epithelium o f trout. Demonstration by luminescence enzyme immunoassay and cytochemistry. Cell & Tissue Research 251, 145-152. Georgopoulou, U . , Sire, M . F . , Vernier, J . M . , 1985. Macromolecular absorption o f proteins by epithelial cells o f the posterior intestinal segment and heir intracellular digestion in the rainbow trout. Ultrastructural and biochemical study. Biology of Cell 53,269-282. Goldsmith, S.J., Dickson, J.S., Barnhart, H . M . , Toledo, R , Eitenmiller, R . R . , 1983. I g A Ig, I g M and Iactoferrin contents o f human milk during lactation and the effect o f processing and storage. Journal of Food Protection 46, 4-7. Gutierrez, M . A . , M i y a z a k i , T., Hatta, H . K i m , M . , 1993. Protective properties o f egg yolk I g Y containing anti-Edwardsiella tarda antibody against paracolo disease in Japanese eel, Anguilla japonica Temminck & Schlegel. Journal of Fish Diseases 16, 113-122.  168  Gutierrez, M . A . , M i y a z a k i , T., Mabe, K . , Hatta, H . , K i m , M . , Akashi, S., 1994. Field tests o f passive immunization with anti-Edwardsiella tarda egg yolk antibody in Japanese eel. In: 3 Asian Fisheries Forum, Asian Fish Society, Manilla, PI, pp. 317319. rd  Hamada, S., Torikoshi, T., M i n a m i , T., Kawabata, S., Hiraoka, J., Fujiwara, T., Ooshima, T., 1991. Oral passive immunization against dental caries in rats by use o f hen egg yolk antibodies specific for cell-associated glucosyltransferase of Streptococcus mutans. Infection & Immunity 5, 4161 -4167. Hart, S., Wrathmell, A . B . , Harris, J.E., Grayson, T . H . , 1988. Gut immunology in fish: a review. Development in Comparative Immunology 12, 453-480. Hastings, T S . & Ellis, A . E . , 1988. The humoral immune response o f rainbow trout, Salmo gairdneri Richardson, and rabbits to Aeromonas salmonicida extracellular products. Journal of Fish Diseases 11, 147-160. Hatta, H , Mabe, K . , K i m , M . , Yamamoto, T., Gutierrez, M . A , Miyazaki, T., 1994. Prevention o f fish disease using egg yolk antibody. In Egg Uses and Processing Technologies: New Developments (Sim, J.S. and Nakai S. eds.) pp.241-249 C A B International Wallingford, U K . Hatta, H , Ozeki, M . , Tsuda, K . , 1997. E g g yolk antibody and its application. In Hen Eggs: Their Basic and Applied Science (Yamamoto, T. Juneja, L . R . , Hatta, H , K i m , M . eds.) pp. 151-178 C R C Press Inc., Boca Raton, Florida. Hatta, H , Tsuda, K . , Akashi, S., K i m , M . , Yamamoto, T., Ebina, T., 1993b. Oral passive immunization effect o f antihuman rotavirus I g Y and its behavior against proteolytic enzymes. Bioscience, Biotechnology and Biochemistry 57, 1077-1081. Hatta, H . , Tsuda, K . , Yamamoto, T., K i m , M . , Akashi, S., 1993a. Productivity and some properties o f egg yolk antibody (IgY) against human rotavirus compared with rabbit IgG. Bioscience, Biotechnology and Biochemistry 57, 450-454. Helenius, A , M c C a s l i n , D . R , Fries, E . , Tanford, C , 1979. Properties o f detergents. Methods in Enzymology 56, 734-749. Hertz, Y . , Tchelet, A . , Madar, Z . , Gerter, A , 1991. Absorption o f bioactive human growth hormon after oral administration in common carp (Cyprinus carpid) and its enhancement by deoxycholate. Journal of Comparative Physiology B 161, 159-163. Hildreth, J . E . K . , 1982. A^-D-Gluco-A'-methylalkanamide compounds, a new class o f non-ionic detergents for membrane biochemistry. Biochemical Journal 207, 363-366.  169  Hitchcock, P.J. & B r o w n T . M . , 1983. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes i n silver-stained polyacrylamide gels. Journal of Bacteriollogy 154, 269-277. Hofer, R . , 1982. Protein digestion and proteolytic activity in the digestive tract o f an omnivorous cyprinid. Comparative Biochemistry andBiophysiology A 72, 55-63. Hora, M . S . , Rana, R . K . , Nunberg, J . H . , Tice, T.R., Gilley, R . M . , Hudson, M . E . , 1990. Release o f human serum albumin from poly(lactide-co-glycolide) microspheres. Pharmaceutical  Research 7, 1190-1194.  Home, M . T . & Ellis, A . E . , 1988. Strategies o f fish vaccination. In Fish ( A . E . Ellis, ed.) pp. 55-66. London: Academic Press.  Vaccination  Huxsoll, C C . & Morgan, Jr. A . L , 1968. Microwave dehydration o f potatoes and apples. Journal of Food Science 22, 47-52. Jayaraman, K . S . , Das Gupta, D . K . , 1992. Dehydration o f fruits and vegetables- recent development in principals and techniques. Drying Technology 10, 1-50. Jeffery, H . , Davis, S.S., O'Hagan, D . T . , 1993. The preparation and characterization o f poly(lactide-co-glycolide) microparticles. II. The entrapment o f a model protein using a (water-in-oil)-in-water emulsion solvent evaporation technique. Pharmaceutical Research 10, 362-368. Jenkins, P . G . , Harris, J.E., Pulsford, A . L . , 1991. Enhanced enteric uptake o f human gamma globulin by Q u i l - A saponin i n Oreochromis mossambicus. Fish & Shellfish Immunology 1, 279-295. Jenkins, P . G . , Wrathmell, A . B . , Harris, J.E., Pulsford, A . L . , 1994. The effects o f different adjuvants o n intestinal antigen absorption and subsequent immune responses of the tilapia Oreochromis mossambicus. Fish & Shellfish Immunology 4, 167-177. Joosten, P . H . M . , Tiemersma, E . , Threels, A . , Caumartin-Dhieux, C , Rombout, J . H . W . M . , 1997. Oral vaccination o f fish against Vibrio anguillarum using alginate microparticles. Fish & Shellfish Immunology 7, 471-485. Kawai, K . & Kusuda, R , 1983. Efficacy o f the lipopolysaccharide vaccine against vibriosis i n cultured ayu. Canadian Translations of Fisheries & Aquatic Sciences 5027, 14 pp. Knappskog, D . H . , Rodseth, O . M . , Slinde. E . , Endersen, C , 1993. Immunochemical analysis o f Vibrio anguillarum strains isolated from cod, Gadus morhua L . , suffering from vibriosis. Journal of Fish diseases 16, 327-338.  170  Kronvall, G . , Seal, U . S . , Svensson, S., Williams, R . C . Jr., 1974. Phylogenetic aspects of streptococcal protein A-reactive serum globulins in birds and mammals. Acta Pathalogica et Microbiologica Scandinavica, Section B, Microbiology & Immunology 82, 12-18. Kummer, A . , Kitts, D . D . , Li-Chan, E . , Losso, J . N . , Skura, B . J . , Nakai, S., 1992. Quantification o f bovine I g G in milk using enzyme-linked immunosorbent assay. Food & Agricultural Immunology 4, 93-102. Laemmli, U . K . , 1970. Cleavage o f structural proteins during the assembly o f the head of bacteriophage T4. Nature 227, 680-685. Larsson, A . , Balow, R., Lindahl, T . L . , Forsberg, P., 1993. Chicken antibodies: taking advantage o f evolution, a review. Poultry Science 72, 1807-1812. Larsson, A . & Sjoquist, J., 1988. Chicken antibodies: a tool to avoid false positive results in latex fixation tests. Journal of Immunological Methods 108, 205- 208. Laurencin, F . B . & Germon, E . , 1987. Experimental infection o f rainbow trout Salmo gairdneri R , by dipping i n suspension o f Vibrio anguillarum: ways o f bacterial penetration; influence o f temperature and salinity. Aquaculture 67, 303-206. Lavelle, E . C . , Jenkins, P . G , Harris, J.E., 1997. Oral immunization o f rainbow trout with antigen microencapsulated in poly (DL-lactide-co-glycolide) microparticles. Vaccine 15, 1070-1079. L e B a i l , P . Y . , Sire, M . F . , Vernier, J . M . , 1989. Intestinal transfer o f growth hormone into the circulatory system o f rainbow trout Salmo gairdneri. Interference by granule cells. Journal of Experimental Zoology 251,101-107. Lee, S.B., Mine, Y . , Stevenson, R . M . W . , 2000. Effects o f hen egg yolk immunoglobulin i n passive protection o f rainbow trout against Yersinia ruckeri. Journal of Agricultural & Food Chemistry 48, 110-115. Liapis, A . I . , 1987. Freeze Drying. In Handbook of Industrial Drying (Mujumdar, A S . , ed.) pp. 295-326. L i - C h a n , E . C . Y . , 1999. Applications o f egg immunoglobulins i n immunoaffmity chromatography. In Egg Nutrition and Biotechnology (Sim, J.S., Nakai, S., and Guenter, W . , eds.) pp. 323-339, C A B I Publishing, C A B International, Wallingford, UK. L i - C h a n , E . C . Y . , Ler, S.S., Kummer, A . , Akita, E . M . , 1988. Isolation o f lactoferrin by immunoaffmity chromatography using egg y o l k ' antibodies. Journal of Food Biochemistry 22, 179-195.  171  L i - C h a n , E . C . Y . , Kummer, A . , Losso, J . N . , Kitts, D . D . , Nakai, S., 1995. Stability o f bovine immunoglobulins to thermal treatment and processing. Food Research International 28, 9-16. Lillehaug, A . , 1989. Oral immunization o f rainbow trout, Salmo gairdneri Richardson, against vibriosis with vaccines protected against digestive degradation. Journal of Fish diseases 12, 579-584. L i n , T . M . , Durance, T . D . , Seaman, C . H . , 1998. Characterization o f vacuum microwave, air and freeze-dried carrot slices. Food Research International 31, 111117. L i n , T . M . , Durance, T . D . , Seaman, C . H . , 1999. Physical and sensory properties o f vacuum microwave dehydrated shrimp. Journal of Aquatic Food Product Technology 8(4)41-53. Marquardt, R . R . , 1999, Control o f intestinal diseases i n pigs by feeding specific chicken egg antibodies. In Egg Nutrition and Biotechnology (Sim, J.S., Nakai, S., and Guenter, W . , eds.) pp. 289-299, C A B I Publishing, C A B International, Wallingford, UK. M c C u e , J.P., Sasagava, P . K . , H e i n R . H . , 1988. Changes induced in antibodies by isolation methods. Biotechnology and Applied Biochemistry 10, 63-71. M c L e a n , E . , 1987. Intact  protein absorption i n teleosts.  Doctoral dissertation.  University o f Bradford, Bradford, U . K . M c L e a n , E . & A s h , R , 1990. Modified uptake o f protein antigen horseradish peroxidase ( H R P ) , following oral delivery to rainbow rout, Oncorhynchus mykiss. Aquaculture 87, 373-379. M c L e a n , E . & A s h , R , 1986. The time-course o f appearance and net absorption o f horseradish peroxidase ( H R P ) presented orally to juvenile carp Cyprinus carpio (L). Comparative Biochemistry and Physiology A Comparative Physiology 84, 687-690. M c L e a n , E . & A s h , R , 1987a. Intact protein (antigen) absorption i n fishes: mechanism and physiological significance. Journal of Fish Biology 31, 219-223. M c L e a n , E . & A s h , R , 1987b. The time-course o f appearance and net absorption o f horseradish peroxidase ( H R P ) presented orally to rainbow Salmo gairdneri (Richardson). Comparative Biochemistry and Physiology A Comparative Physiology 88,507-510. M c L e a n , E . & Donaldson, E . M . , 1990. The absorption o f bioactive proteins by the fish gastrointestinal tract: a review. Journal of Aquatic Animal Health 2, 1-11.  172  M c L e a n , E , Donaldson, E . M . , Dye, H . M . , Souza, L . M . , 1990. Growth acceleration o f coho salmon (Oncorhynchus kisutch) following oral administration o f recombinant bovine somatotropin. Aquaculture 91, 197-203. M c L e a n , E . , Ronsholdt, B . , Sten, C , Najamuddin, 1999. Gastrointestinal delivery o f peptide and protein drugs to aquacultured teleosts. Aquaculture 177, 231-247. Mine, Y . , Lee, S.B., Stevenson, R . M . W . , 1999. Applications o f egg immunoglobulins in immunoaffinity chromatography. In Egg Nutrition and Biotechnology (Sim, J.S., Nakai, S., and Guenter, W . , eds.) pp.341-350. C A B I Publishing, C A B International, Wallingford, U K . Moriyama, S., Takahashi, A . , Hirano, T., Kawauchi, H . , 1990. Salmon growth hormone is transported into the circulation o f rainbow trout Oncorhynchus mykiss, after intestinal administration. Comparative Physiology 160, 251-257. Nagai, A . & Fujino, Y . , 1995. Application o f enteric-coated microcapsule in oral administration o f bovine serum albumin into eel. Fisheries Science 61, 796-799. Nakamura, O., Kobayashi, M . , Suzuki, Y . , Aida, K . , Hanyu, I., 1990. Transport o f orally administered rabbit I g G into blood circulation o f goldfish. Nippon Suisan Gakkaishi 56, 1749-1753. Nelson, J.S., Rohovec, S., Fryer, J.L., 1985. Location o f Vibrio anguillarum in tissues o f infected rainbow trout (Salmo gairdneri) using the fluorescent antibody technique. Fish Pathology 20, 229-23 5. N i k l L . , Albright L J., Evelyn T P T . , 1991. Influence o f seven immunostimulants on the immune response o f coho salmon to aeromonas-salmonicida. Diseases of Aquatic Organisms 12, 7-12. N i k l , L . , Albright, L . J . , Evelyn, T.P.T., 1992. Immunostimulants hold promise in furunculosis prevention. Bulletin of the Aquaculture Association of Canada 92, 49-52. O'Donnell, G . B . , Reilly, P., Davidson, G . A . , Ellis, A . E . , 1996. The uptake o f human gamma globulin incorporated into poly (DL-lactide-co-glycolide) miroparticles following oral intubation i n Atlantic salmon, Salmo salar L . Fish & Shellfish Immunology 6, 507-520. O'Donnell, G . B . , Smith P.R., Kilmartin, J.J., Morgan, A . P . , 1994. Uptake and fate o f orally administered bacterial lipopolysaccharide in brown trout (Salmo trulla). Fish & Shellfish Immunology 4, 285-299. O'Farrelly, C , Branton, D . , Wanke, C . A . , 1992. Oral ingestion o f egg yolk immunoglobulin from hens immunized with an enterotoxigenic Escherichia coli strain  173  prevents diarrhea in rabbits challenged with the same strain. Infection & Immunity 60, 2593-2597. O'Hagen, D . T . , 1996. The intestinal uptake o f particles and the implications for drug and antigen delivery. Journal of Anatomy 189, 477-482. Otani, H . , Matsumoto, K . , Saeki, A . , Hosono, A , 1991. Comparative studies on hen egg yolk I g Y and rabbit serum I g G antibodies. Lebenesmittel Wissenschaft und Technologie 24, 152-158. Ototake, M . , Iwama, G . K . , Nakanishi, T., 1996. The uptake o f bovine serum albumin by the skin o f bath immunized rainbow trout Oncorhynchus mykiss. Fish & Shellfish Immunity 6,321-333. Palm Jr., R . C . , Landolt, M . L . , Busch, R . A . , 1998. Route o f vaccine administration: effects on the specific humoral response i n rainbow trout Oncorhynchus mykiss. Diseases of Aquatic Organisms 33,157-166. Piganelli, J.D., Zhang, J.A., Christensen, J . M . , Kaattari, S.L., 1994. Enteric coated microspheres as an oral method for antigen delivery to salmonids. Fish & Shellfish Immunology 4, 179-188. Poison, A , V o n Wechmer, M . B . , Fazakerley, G . , 1980a. Antibodies to proteins from yolk o f immunized hens. Immunological Communications 9, 495-514. Poison, A . , V o n Wechmer, M . B . , V a n Regenmortel, M . H . V . , 1980b. Isolation o f viral I g Y antibodies from egg yolks o f immunized hens. Immunological Communications 9, 475-493. Raa, J., 1996. The use o f immunostimulants in fish and shellfish farming. Reviews in Fisheries Science 4, 229-288. Ransom, D . P . , Lannan, C . N . , Rohovec, J.S., Fryer, J . L . , 1984. Comparison o f histopathology caused by Vibrio anguillarum and Vibrio ordalii and three species o f Pacific salmon. Journal of Fish Diseases 7, 107-115. Rittenburge, J . H . 1990.Fundamentals o f immunoassay. In Development and Application of Immunoassay for Food Analysis (Rittenburge, J . H . , ed.) pp. 29-57, Elsevier Science Publishers, Ltd., U K . Rombout, J . H . W . M . , Lamers, C . H . J . , Helfrich, M . H . , Dekker, A , Taverne-Thiele, J.J., 1985. Uptake and transport o f intact macromolecules in the intestinal epithelium o f carp (Cyprinus carpio L . ) and the possible immunological implications. Cell Tissue Research 293,519-530.  174  Rombout, J.H. W . M . & van den Berg, 1989. Immunological importance o f the second gut segment o f carp. I. Uptake and processing o f antigens by epithelial cells and macrophages. Journal of Fish Biology 35, 179-186. Rose, M . E . , Orlans, E . , Butteress, N . , 1974. Immunoglobulin classes in the hen egg: their segregation in yolk and white. European Journal of Immunology 4, 521-523. *  Shimizu, M . , Fitzsimmons, R . C . , Nakai, S., 1988. Anti-£. coli immunoglobulin Y isolated from egg yolk o f immunized chickens as a potential food ingredient. Journal of Food Science 53,1360-1366. Shimizu, M , M i w a , Y . , Hashimoto, K . , Goto, A . , 1993. Encapsulation o f chicken egg yolk immunoglobulin G (IgY) by liposomes. Bioscience, Biotechnology & Biochemistry 57, 1445-1449. Shimizu, M . , Nagashima, H , Sano, K . , Hashimoto, K . , Ozeki, M . , Tsuda, K . , Hatta, H , 1992. Molecular stability o f chicken and rabbit immunoglobulin G . Bioscience, Biotechnology and Biochemistry 56, 270-274. Sire, M . F . & Vernier, J . - M . , 1992. Intestinal absorption o f protein in teleost Comparative Biochemistry & Physiology A 103, 771-781.  fish.  Siwicki, A . K . , Anderson, D . P . , Dixon, O . W . , 1989. Comparisons o f non-specific and specific immunodulation by oxolinic acid, oxytetracycline and levamisole in salmonids. Veterinary Immunology and Immunopathology 23, 195-200. Siwicki, A . K . , Anderson, D . P . , Rumsey, G . L . , 1994. Dietary intake o f immunostimulants by rainbow trout affect non-specific immunity and protection against fiirunculosis. Veterinary Immunology & Immunopathology 41, 125-139. Smith, L . S . , 1989. Digestive functions in teleost fish. In Fish Nutrition ed.) pp. 331-421. Academic Press, San Diego.  (Halver, J.R.  Smith, P . D . , 1988. Vaccination against vibriosis. In Fish Vaccination ( A E . Ellis, ed.) pp. 67-84. London: Academic Press. Snedecor, G . W . & Cochran, W . G . , 1980. Statistical Methods. 7 The Iowa State University Press, Ames, Iowa, U S A .  th  edition, pp 253-254.  Somogyi, L . P . , L u h , B.S., 1975. Dehydration o f fruits. In Commercial Fruit Processing (Woodroof, J . G . and L u h , B . S . , eds.) pp. 374-429. A V I Publishing C o . Inc., Westport, Coonecticut, U S A . Suzuki, Y . M , Kobayashi, M . , Aida, K . , Hunyu, I. 1988. Transport o f physiologically active gonadotropin into the circulation in goldfish, following oral administration o f  175  salmon pituitary extract Journal of Comparative Physiology B, Biochemical, Systemic & Environmental Physiology 157, 753-758. Tagesson, C , Franzen, L . , Dahl, G . , Westrom, B . , 1985. Lysophosphotidylcholine increases rat ileal permeability to macro molecules. Gut 26, 369-377. Tsai, C - M . & Frasch, C . E . , 1982. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Analytical Biochemistry 119, 115-119. Velji, M . I . & Albright, L . J . , 1986. Microscopic enumeration o f attached marine bacteria o f seawater, marine sediment, fecal matter, and kelp blade samples following pyrophosphate and ultrasound treatments. Canadian Journal of Microbiology 32, 121126. Volkheimer, G . , 1972. Persorption. Georg Thieme Verlag, Stuttgart, Germany. Walker, W . A . , 1986. Antigen handling Gastroenterology 15, 1-20.  by  the  small  intestine.  Clinics in  Walker, W . A . , Isselbacher, K . J . , 1974. Uptake and transpot o f macromolecules by the intestine: possible role i n clinical disorders. Gastroenterology 67, 531-550. Warr, G . W . , Magor, K . E . , Higgins, D . A . , 1995. IgY: clues to the origin o f modern antibodies. Immunology Today 16, 392-398. Womack, M . D . , Kendall, D . A . , MacDonald, R . C . , 1983. Detergent effects on enzyme activity and solubilization o f lipid bilayer membrane. Biochemica Biophysica Acta 733, 210-215. Wong, G . , Kaattari, S.L., Christensen, J . M . , 1992. Effectiveness o f an oral enteric coated vibrio vaccine for use in salmonid fish. Immunological Investigations 21, 353364. Yang, C.S.T. & Atallah, W . A . , 1985. Effect o f four drying methods on the quality o f intermidiate moisture lowbush blueberries. Journal of Food Science 50, 1233-1237. Yano T., Matsuyama, H . , Mangindaan, R.E.P., 1991. Polysaccharide-induced protection o f carp Cyprinus carpio L . , against bacterial infection. Journal of Fish Diseases 14, 577-582. Yasutake, W . T . & Wales, J . H . , 1983. Microscopic Anatomy o f Salmonids: A n Atlas, p. 181. United States Department o f the Interior, Fish and Wildlife Service, Resource Publication 150, Washington D C , U S A . Yokoyama, H . , Peralta, R . C . , Diaz, R , Sendo, S , Ikemori, Y . , Kodama, Y . , 1992. Passive immunization effect o f chicken egg immunoglobulins against experimental  176  enterotoxigenic Escherichia 60, 998-1007.  coli infection in neonatal piglets. Infection & Immunity  Yolken, R . H . , Leister, F., Wee, S B . , Miskuff, R., Vonderfetcht, S., 1988. Antibodies to rotavirus in chickens eggs: a potential source o f antiviral immunoglobulins suitable for human consumption. Pediaterics 81, 291-295. Yongsawatdiguul, J. & Gunasekaran, S., 1996. Microwave vacuum drying o f cranberries: Part II: quality evaluation. 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 o f vacuum microwave and air-dried sweet basil (Ocimum basilicum L . ) . Journal of Agricultural & Food Chemistry 47, 4777-4781.  177  CHAPTER SEVEN APPENDIX  Mortality Rate & T o t a l S e r u m IgY L e v e l F o l l o w i n g IP Injection with Ant\-Vibrio IgY 50  i  30  >rtal  40  20  i a a % M o rta lity -*—lgY(mg/ml)  10 0 Day  1  Day  3  Day  7  D a y 14  P o s t - i n j e c t i o n c h a l l e n g e date  F i g . 7 . 1 . Temporal trend o f mortality rate in relation with total serum I g Y level i n the fish groups EP injected with anti-K anguillarum IgY. Values are the mean o f 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  Day 1  Day3  Day7  Post-injection challenge  D a y 14  date  Fig.7.2. Temporal trend o f mortality rate i n relation with anti-K anguillarum I g Y titer i n serum o f fish following EP injection with anti- Vibrio I g Y . Values are the mean o f 3 replicates (± standard deviation for mortality rate). Similar letters indicate no significant difference (p < 0.05).  180  A n t i - V . anguillarum IgY T i t e r & T o t a l S e r u m IgY L e v e l In IP Injected F i s h  >• O)  >-  o cu  o  3  E E  - • — T o t a l IgY  Q. (0  Day 1  Day 3  Day 7  D a y 14  P o s t - i n j e c t i o n c h a l l e n g e date  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.  182  Fig. 7.5. Microscopic photograph of H & E stained cross section of the pyloric caeca 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.  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 W S F (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  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0089669/manifest

Comment

Related Items