Studies on dietary supplements for the control of Aeromonas
hydrophila infection in rainbow trout (Oncorhynchus mykiss,
Walbaum)
Elijah J. Nya
A thesis submitted for the degree of
Doctor of Philosophy
School of Life Sciences, Heriot Watt University, Edinburgh, UK
August 2009
“The copyright in this thesis is owned by the author. Any quotation from the thesis or use of any
of the information contained in it must acknowledge this thesis as the source of the quotation or
information."
Abstract
Three compounds, garlic (Allium sativum), ginger (Zingiber officinale) and
lipopolysaccharide (LPS) were selected from an examination of ten dietary supplements
based on their performance in enhancing protection and immunity in rainbow trout
(Oncorhynchus mykiss, Walbaum) fingerlings, after challenge with Aeromonas
hydrophila. Thus, dietary garlic at doses of 0.5 g and 1.0 g/100 g of feed resulted in
mortalities of 4% (relative percent survival [RPS] = 91.75%), compared with 80%
mortalities in the controls. Use of dietary ginger and LPS led to 0% mortality (RPS =
100%), compared with 85% in the controls. Growth, feed conversion ratio (FCR) and
protein efficiency ratio (PER) were enhanced in all the experimental groups. The mode
of action involved stimulation of non-specific immunity by proliferation of immune
cells, enhancement of phagocytic, oxidative burst, lysozyme, antiprotease and
bacteriocidal activities, and modulation of digestive enzymes.
i
Dedication
To my daughter Edikan E. Nya of blessed memory
ii
Acknowledgements
I would like to thank God for his mercies upon my life and indeed, for sustaining me
throughout the period of this study. More importantly, my gratitude goes to my
supervisors, Prof. B. Austin and Dr. Alastair Lyndon, for their useful advice, support
and encouragement in editing and proof-reading this thesis. I appreciate greatly Dr.
Dawn Austin and Mrs Margaret Stobie for expertise and guidance during my laboratory
work and their motherly advice in time of need. Also members of our research group
Dr. Peter Morris, Dr. Susan, Dr Rahman, Sharif, John, Callum, Elham, Maite and Yan.
I acknowledge Dr M. Barker for her advice and help on DNA sequencing, and Sean
McMenamy for expertise on electrolyte analysis with flame emission photometry. Also,
I am grateful to Gina, Rob Young the computer technician, Elaine Legget and Mags
Munro of the School office and all others in the School of Life Sciences, who have in
no small measure, helped me in this course of study. Finally, I acknowledged the
patience of my wife Mrs Agnes Nya, for my absence from home all this time, and
acknowledge my sponsor Akwa Ibom State University of Technology Uyo, Nigeria, for
funding the entire program.
iii
ACADEMIC REGISTRY
Research Thesis Submission
Name:
Elijah J Nya
School/PGI:
Life Sciences
Version:
Final
(i.e. First,
Resubmission, Final)
Degree Sought
(Award and
Subject area)
Ph.D.
Declaration
In accordance with the appropriate regulations I hereby submit my thesis and I declare that:
1)
2)
3)
4)
5)
*
the thesis embodies the results of my own work and has been composed by myself
where appropriate, I have made acknowledgement of the work of others and have made
reference to work carried out in collaboration with other persons
the thesis is the correct version of the thesis for submission and is the same version as
any electronic versions submitted*.
my thesis for the award referred to, deposited in the Heriot-Watt University Library, should
be made available for loan or photocopying and be available via the Institutional
Repository, subject to such conditions as the Librarian may require
I understand that as a student of the University I am required to abide by the Regulations
of the University and to conform to its discipline.
Please note that it is the responsibility of the candidate to ensure that the correct version
of the thesis is submitted.
Signature of
Candidate:
Date:
Submission
Submitted By (name in capitals):
ELIJAH J NYA
Signature of Individual Submitting:
Date Submitted:
31.08.09
For Completion in Academic Registry
Received in the Academic
Registry by (name in capitals):
Method of Submission
(Handed in to Academic Registry; posted
through internal/external mail):
E-thesis Submitted (mandatory for
final theses from January 2009)
Signature:
Date:
iv
31.08.09
Declaration
I, Elijah J. Nya, hereby declare that I am the author of this thesis. All the work described
in this thesis is my own except where stated in the text. The work presented here has not
been accepted in any previous application for a higher degree. All the sources of
information have been consulted by myself and are acknowledged by means of
reference.
Elijah J. Nya
v
CONTENTS
Abstract………………………………………………………………………………….i
Dedication…………………………………………………………………………... ….ii
Acknowledgements…………………………………………………………………. ...iii
Submission form……………………………………………………………………….iv
Declaration………………………………………………………………………….......v
Contents………………………………………………………………………………...vi
List of Tables…………………………………………………………………………...xi
List of Figures………………………………………………………………………...xiii
Chapter 1 INTRODUCTION
1.1 Basis of aquaculture……………………………………………………………......1
1.2 Origin of aquaculture………………………………………………………………1
1.3 Definition of aquaculture………………………………………………………......2
1.4 Present state of aquaculture……………………………………………………….2
1.5 Bacterial diseases of farmed fish species………………………………………….5
1.6 Aeromonas hydrophila…………………………………………………………….10
1.6.1 Taxonomy of Aeromonas………………………………………………………...11
1.6.2 Diagnosis of Aeromonas hydrophila infections………………………………….11
1.6.3 Pathology…………………………………………………………………………12
1.6.4 Chemotherapy of Aeromonas hydrophila infections in fish………………….......13
1.6.5 Modern approaches to the control of fish diseases……………………………….14
1.7 Vaccines……………………………………………………………………………14
1.7.1 Inactivated vaccines………………………………………………………………16
1.7.2 Subunit vaccines………………………………………………………………….17
1.7.3 Live attenuated vaccines………………………………………………………….18
1.7.4 Recombinant vaccines……………………………………………………………19
1.7.5 DNA vaccines…………………………………………………………………….20
1.7.6 Adjuvants…………………………………………………………………………21
1.8 Probiotics………………………………………………………………………......21
vi
1.9 Fish immune system………………………………………………………………24
1.9.1 External defence mechanisms……………………………………………………24
1.9.2 Innate or non-specific immune mechanisms……………………………………..25
1.9.3 Acquired or specific immune mechanisms……………………………………….30
1.9.4 Exogenous factors…………………………………………………………….......32
1.9.5 Endogenous factors……………………………………………………………….34
1.10 The use of immunostimulants in aquaculture………………………………….34
1.10.1 Lipopolysaccharides ……………………………………………………………35
1.10.2 Chitin and chitosan………………………………………………………….......35
1.10.3 β 1, 3 Glucan…………………………………………………………………….36
1.10.4 Animal and plant extracts……………………………………………………….37
1.10.5 Dietary components…………………………………………………………......39
1.10.6 Other immunostimulants……………………………………………………......39
1.10.7 Timing of immunostimulant application…………………….………………….39
1.10.8 Administration methods for immunostimulants………………………………...40
1.10.9 Immunostimulant doses…………………………………………………………41
1.10.10 Growth activities of immunostimulants……………………………………….42
1.11 Aims and objectives……………………………………………………………...43
Chapter 2 MATERIALS and METHODS
2.1 Experimental fish…………………………………………………………….........44
2.2 Bacterial isolates………………………………………………………………......44
2.3 Characterisation of the bacterial isolates……………………………………......44
2.3.1 Micro-morphology………………………………………………………………..44
2.3.2 Motility…………………………………………………………………………...45
2.3.3 Catalase production………………………………………………………………45
2.3.4 Oxidase production……………………………………………………………….45
2.3.5 Haemolytic activity……………………………………………………………….45
2.3.6 Casein hydrolysis…………………………………………………………………46
2.3.7 Elastin hydrolysis………………………………………………………………...46
2.3.8 Gelatin hydrolysis…………………………………………………………….......46
2.3.9 Sensitivity to antibiotics………………………………………………………….46
2.4 API 20 E rapid identification system…………………………………………….46
2.5 Salt aggregation…………………………………………………………………...47
vii
2.6 16S rRNA gene sequencing……………………………………………………….47
2.6.1 DNA extraction……………………………………………………………….......47
2.6.2 Conventional PCR conditions……………………………………………………47
2.6.3 Primers used in the 16S rRNA sequencing………………………………………48
2.6.4 Agarose gel electrophoresis of DNA………………………………………….....48
2.6.5 Sequencing of purified PCR products……………………………………………49
2.7 Determination of LD 50 doses……………..………………………………….......50
2.8 Compounds evaluated for immunostimulatory activity……………………......51
2.8.1 Ginger, Zingiber officinale……………………………………………………….51
2.8.2 Garlic, Allium sativa……………………………………………………………...51
2.8.3 Rosemary, Rosmarinus officinalis……………………………………………......51
2.8.4 Clove, Eugenia aromaticum……………………………………………………...51
2.8.5 Other immunostimulants…………………………………………………………51
2.9 Preliminary screening of putative immunostimulants………………………….52
2.9.1 Determination of inhibitory activity……………………………………………...52
2.9.2 In vivo studies…………………………………………………………………….52
2.9.3 Experimental challenge and determination of growth performance…………......52
2.10 Mode of action……………………………………………………………………53
2.10.1 Induced cellular immune response……………………………………………...53
2.10.2 Population of blood cells……………………………………………………......53
2.10.3 Head kidney macrophages………………………………………………………54
2.10.4 Phagocytosis activity……………………………………………………………55
2.10.5 Respiratory burst activity………………………………………………………..55
2.10.6 Bacteriocidal activity……………………………………………………………56
2.11 Induced humoral immune response…………………………………………….56
2.11.1 Lysozyme activity……………………………………………………………….56
2.11.2 Serum anti-protease activity…………………………………………………….56
2.11.3 Natural haemolytic complement activity………………………………………..57
2.11.4 Serum biochemical parameters………………………………………………….58
2.11.5 Serum electrolyte indices………………………………………………………..58
2.12 Determination of effective dose of compounds for controlling
A. hydrophila infection…………………………………………………………..59
2.13 Digestive enzymes……………………………………………………………......59
2.13.1 Preparation of crude enzyme extracts……………………………………….......59
2.13.2 Pepsin activity……………………………………………………………….......60
viii
2.13.3 Total protease activity………………………………………………………......60
2.13.4 Alkaline phosphatase (AP) activity…………………………………………......61
2.13.5 Total proteolytic enzyme activity……………………………………………….61
2.13.6 Trypsin activity………………………………………………………………….61
2.14 Duration of protection……………………………………………………….......62
2.14.1 Determination of immunological parameters and protection…………………...62
2.14.2 Serum peroxide content…………………………………………………………62
2.15 Determination of active components of the beneficial compounds……….......63
2.15.1 Determination of inhibitory activity of allicin…………………………………..63
2.15.2 Determination of the minimum inhibitory concentration (MIC)………………..63
2.15.3 Determination of the mode of action of allicin… ……………………………....64
2.16 Statistical analysis………………………………………………………………..64
Chapter 3 RESULTS
3.1 Characteristics of the bacterial isolates………………………………………….65
3.2 Sensitivity to antimicrobial compounds…………………………………………66
3.3 Use of the API 20 E rapid identification system………………………………...67
3.4 16S rRNA gene sequencing……………………………………………………….68
3.5 Determination of LD50 of the bacterial isolates………………………….…........69
3.6 Preliminary screening of immunostimulants……………………………………69
3.6.1 Production of inhibitory activity by putative immunostimulants………………...71
3.7 Influence of dietary supplement on growth performance ………..……………72
3.8 Effect of dietary supplement on the survival of rainbow trout
after challenge with Aeromonas hydrophila……………………………………..74
3.9 Mode of action……………………………………………………………………..76
3.9.1 Haematological parameters………………………………………………………76
3.9.2 Population and partial differential blood cells………………………………........77
3.10 Immunological parameters……………………………………………………...80
3.10.1 Head kidney macrophages and phagocytosis activity………………………......80
3.10.2 Respiratory burst activity………………………………………………………..82
3.10.3 Bacteriocidal activity……………………………………………………………84
3.11 Induced humoral immune responses to dietary supplements………………...86
3.11.1 Lysozyme activity……………………………………………………………….86
ix
3.11.2 Serum anti-protease activity…………………………………………………….88
3.11.3 Natural haemolytic complement activity………………………………………..89
3.12 Serum biochemical parameters…………………………………………………90
3.12.1 Serum electrolyte indices………………………………………………………..92
3.13 Determination of the most effective dose of dietary supplements…………….93
3.14 Duration of protection after administration of dietary supplements………...94
3.14.1 Measurement of immunological parameters………………………………......103
3.15 Modulation of digestive enzymes by dietary supplements……………….......104
3.15.1 Pepsin and protease activities estimated from the stomach homogenates……..104
3.15.2 Alkaline phosphatase activity estimated from the small intestine and
brush border membrane……………………………………………………......106
3.15.3 Total proteolytic enzymes activity estimated from the small intestine
and hepatopancreas…………………………………………………………….107
3.15.4 Trypsin activity estimated from the small intestine
and the hepatopancreas……………………………………………………….108
3.16 The role of the garlic component allicin in controlling
Aeromonas hydrophila infection……………………………………………….109
3.16.1 Antimicrobial activity………………………………………………………….109
3.16.2 Protective effect of allicin………………………………………………….......109
3.16.3 Mode of action- haematological parameters………………………………......110
3.16.4 Biochemical parameters……………………………………………………….110
3.16.5 Immunological parameters…………………………………………………….111
3.16.5.1 Phagocytic activity………………………………………………………......111
3.16.5.2 Respiratory burst activity…………………………………………………….112
3.16.5.3 Lysozyme activity……………………………………………………………112
3.16.5.4 Bacteriocidal activity………………………………………………………...113
Chapter 4 DISCUSSION
Discussion and conclusions…………...……………………………………………..114
LIST OF REFERENCES……………………………………………………………138
x
LIST OF TABLES
Table 1 World aquaculture production in 1000 metric tonnes………………………......3
Table 2 Cultured Salmon: World production in 1000 metric tonnes……………………4
Table 3 Bacterial pathogens of fish (after Austin and Austin, 2007)……………………5
Table 4 Licensed live attenuated vaccines against Gram-negative bacteria
(after Klesius et al., 2005 with modifications)………………………………...19
Table 5 Licensed vaccines against bacteria (after Klesius et al., 2005
with modifications)…………............................................................................20
Table 6 Probiotics considered as biological control agents in aquaculture
(adapted from Balcazar et al., 2006)………………………………………......22
Table 7 Morphological and biochemical characteristics of the bacteria isolates………65
Table 8 Antimicrobial sensitivity profiles of bacterial isolates………………………...66
Table 9 Identification of bacterial isolates by API 20 E rapid identification system......67
Table 11 Closest relatives as determined by BLAST search and accession number......69
Table 12 Preliminary screening of ten putative dietary supplements…………………..70
Table 13 Production of inhibitory compounds against bacterial isolates………………72
Table 14 Growth performances of rainbow trout fed with supplemented
diet for 14 days………………………………………………………………73
Table 15 Haematological data of rainbow trout fed with supplemented
diets for 14 days…………………………………………………………........76
Table 16 Mean differential proportions of leucocytes in rainbow trout
fed dietary supplements for 14 days…………………………………………77
Table 17 Biochemical indices of rainbow trout after feeding with dietary
supplements for 14 days……………………………………………………..91
Table 18 Mean electrolyte indices of rainbow trout fed with dietary
supplements for 14 days……………………………………………………..92
Table 19 Comparison of different doses of dietary supplements administered
for 14 days before challenge with Aeromonas hydrophila…………………..93
Table 20 Effect of dietary supplements and their ability to lead to productive
immune response in rainbow trout after challenge with A. hydrophila……...94
Table 21 Physiological parameters in rainbow trout measured 14, 21 and 28 days
after stopping feeding dietary supplements………………………………….95
Table 22 Mean haematological parameters of rainbow trout fingerlings………………97
Table 23 Biochemical indices of rainbow trout fed immunostimulants for 14 days…..99
xi
Table 24 Mean electrolyte indices of rainbow trout fed dietary supplements…….......101
Table 25 Immunological indices after cessation of feeding with dietary
supplements………………………………………………………………...103
Table 26 Biometric indices of rainbow trout fed dietary supplements for 14 days.......105
Table 27 Haematological parameters of rainbow trout fed dietary
allicin for 14 days………………………………………………………......110
Table 28 Biochemical indices of rainbow trout fed allicin supplemented diet
for 14 days………………………………………………………………….111
Table 29 An overview of innate immunity components in fish and their mode
of action (after Abbas and Lichtman, 2006; Magnadottir, 2006)………….119
xii
LIST OF FIGURES
Figure 1 Rainbow trout farms, which consist of earthen ponds in Scotland…………….4
Figure 2.1-2.4 Adapted from leaflet of Fish Disease by Cipriano et al. (1984).............13
Figure 2.1 Scale protrusion on a carp Cyprinus carpio caused by A. hydrophila……...13
Figure 2.2 Abdominal distension and accumulation of ascitic fluid in goldfish
Carassius auratus caused by A. hydrophila………………………………...13
Figure 2.3 Exophthalmia…………………………………………………………….....13
Figure 2.4 Ulcerative syndrome………………………………………………………..13
Figure 3 16S rRNA, PCR of A. hydrophila and ORN2 DNA products…………..........68
Figure 4 Rainbow trout protected by garlic showing normal internal organs (spleen,
kidney and intestine), skin and fins after challenge with A. hydrophila…….71
Figure 5 Control fish exhibiting muscle necrosis after challenge with A. hydrophila…71
Figure 6.1 Percentage cumulative mortality of rainbow trout following
intraperitoneal injection with x106 cells / ml of A. hydrophila
after feeding garlic supplemented diet for 14 days…………………………74
Figure 6.2 Percentage cumulative mortality of rainbow trout following
intraperitoneal injection with x106 cells / ml of A. hydrophila
after feeding ginger supplemented diet for 14 days…………………….......75
Figure 6.3 Percentage cumulative mortality of rainbow trout following
intraperitoneal injection with x106 cells / ml of A. hydrophila
after feeding LPS supplemented diet for 14 days……………………...........75
Figure 7a- d Proliferations of immune cells in experimental groups of fish
a= Control, b= Ginger, c= Garlic and d= LPS treated blood…………….79
Figure 8.1 The effect of garlic supplemented diet on the phagocytic activity
(phagocytic ratio and index) of the head kidney macrophages of
rainbow trout, after 14 days feeding………………………………………..80
Figure 8.2 The effect of ginger supplemented diet on the phagocytic activity
(phagocytic ratio and index) of the head kidney macrophages of
rainbow trout, after 14 days feeding……………………………………......81
Figure 8.3 The effect of garlic supplemented diet on the phagocytic activity
(phagocytic ratio and index) of the head kidney macrophages of
rainbow trout, after 14 days feeding……………………………………......81
Figure 9.1 Effect of garlic supplemented diet on the superoxide anion
production by blood leucocytes of rainbow trout…………………………..82
xiii
Figure 9.2 Effect of ginger supplemented diet on the superoxide anion
production by blood leucocytes of rainbow trout………………………......83
Figure 9.3 Effect of LPS supplemented diet on the superoxide anion
production by blood leucocytes of rainbow trout………………………......83
Figure 10.1 Effect of garlic supplemented feed on the bacteriocidal activity of
rainbow trout serum………………………………………………….…….84
Figure 10.2 Effect of ginger supplemented feed on the bacteriocidal activity of
rainbow trout serum………………………………………………….…….85
Figure 10.3 Effect of LPS supplemented feed on the bacteriocidal activity of
rainbow trout serum………………………………………………….…….85
Figure 11.1 Effect of garlic supplemented diet on the lysozyme activity of
rainbow trout serum after 14 days feeding regimes……………………….86
Figure 11.2 Effect of feeding ginger supplemented diet for 14 days on
the lysozyme activity of rainbow trout………………………………........87
Figure 11.3 Effect of feeding LPS supplemented diet for 14 days on
the lysozyme activity of rainbow trout………………………………........87
Figure 12.1 Antiprotease activity of rainbow trout fed for 14 days
with diet supplemented with garlic…………………………………….......88
Figure 12.2 Antiprotease activity of rainbow trout fed with ginger for 14 days…….....88
Figure 12.3 Antiprotease activity of rainbow trout fed with LPS for 14 days…….…...89
Figure 13.1 Serum natural haemolytic complement activity of rainbow trout
fed with garlic supplemented diet for 14 days……………………………..89
Figure 13.2 Serum natural haemolytic complement activity of rainbow trout
fed with garlic supplemented diet for 14 days……………………………..90
Figure 13.3 Serum natural haemolytic complement activity of rainbow trout
fed with garlic supplemented diet for 14 days……………………………..90
Figure 14.1 Pepsin activity……………………………………………………………104
Figure 14.2 Protease activity……………………………………………………….....104
Figure 15 Alkaline phosphatase estimated from small intestine
and brush border membrane………………………………………………106
Figure 16 Total Proteolytic enzyme activities from small intestine
and hepatopancreas……………………………………………………......107
Figure 17 Trypsin activity from small intestine and hepatopancreas…………………108
Figure 18 Percentage cumulative mortality of rainbow trout following
intraperitoneal injection with x106 cells / ml of A. hydrophila
xiv
after feeding allicin supplemented diet for 14 days………………………..109
Figure 19 The effect of feeding allicin supplemented diet on
the phagocytic activity of rainbow trout, after 14 days feeding…………...111
Figure 20 Effect of feeding allicin supplemented diet on the superoxide
anion production by blood leucocytes of rainbow trout…………………...112
Figure 21 Effect of feeding allicin supplemented diet on the serum
lysozyme activity of rainbow trout…………………………………….......112
Figure 22 Effect of feeding allicin supplemented diet on the serum
bacteriocidal activity of rainbow trout……………………………………..113
xv
LIST OF ABBREVIATIONS and SYMBOLS
<
Less than
>
Greater than
~
approximately
Ab
Absorbance
BLAST
Basic local alignment search tools
bp
Base pair
BSA
Bovine serum albumin
CFU
Colony forming unit
CCV
Channel catfish virus
CRD
Completely randomized design
DNA
Deoxyribonucleic acid
e.g.
Example
et al.,
‘et alia’: and others
EDTA
Ethylenediaminetetra acetic acid
EGTA
Ethylene glycol-bis (2-aminoethoxy) tetra acetic acid
ELISA
Enzyme linked immunosorbent assay
FCA
Freund complete adjuvant
FCS
Foetal calf serum
FCR
Feed conversion ratio
g
Gram
GH
Growth hormone
h
Hour
HBSS
Hank’s balance salt solution
i.e.
‘idest’: that is
i.d.
Intra-dermal
i.m.
intramuscular
i.p.
intraperitoneal
i.v.
intra-venous
IHNV
infectious haematopoietic necrosis virus
IL-6
interleukin- 6
IL-10
interleukin- 10
Kg
kilogramme
Kb
kilobase
kDa
kilo Dalton
xvi
L
litre
LD50
lethal dose 50%
LPS
Lipopolysacharide
M
molar
min
minute
NBT
Nitroblue tetrazolium
NCBI
National Centre for Biotechnology Information
OD
Optical density
PBS
Phosphate buffered saline
PCR
Polymerase chain reaction
PFC
Plaque forming cells
PRR
Pattern recognition receptor
PER
Protein efficiency ratio
RNA
Ribonucleic acid
ROS
Reactive oxygen species
RPS
Relative percentage survival
sp.
Species
spp.
Species
SOD
Superoxide dismutase
SGR
Specific growth rate
SRBC
Sheep red blood cell
TAE
Tris Acetate-EDTA
TBE
Tris borate-EDTA
TLRs
Toll-like receptors
TNF-ά
Tumor necrosis factor -alpha
TSA
Tryptone soya agar
TSB
Tryptone soya broth
TCA
Trichloro acetic acid
UV
Ultra violet
µg
Microgram
µl
Micro litre
µg / ml
Microgram per millilitre
v/v
Volume by volume ratio
VHSV
Viral haemorrhagic septicaemia virus
w/v
Weight by volume ratio
xvii
w/w
Weight by weight ratio
xg
Multiples of gravity
xviii
PUBLICATIONS RESULTING FROM THIS WORK
Nya, E. J and Austin, B. (2009). Use of garlic (Allium sativum) to control Aeromonas
hydrophila infections in rainbow trout Oncorhynchus mykiss (Walbaum). Journal of
Fish Diseases JFD-77.R1. (In press)
Nya, E. J and Austin, B. (2009). Use of ginger Zingiber officinale Roscoe as
immunostimulant to control Aeromonas hydrophila infections in rainbow trout
Oncorhynchus mykiss (Walbaum). Journal of Fish Diseases JFD-2009 (In press).
Nya, E. J and Austin, B. (2009). Use of bacterial Lipopolysaccharide LPS as an
immunostimulant for the control of Aeromonas hydrophila in rainbow trout
Oncorhynchus mykiss, (Walbaum). Journal of Applied microbiology JAM2009 (In
press).
E. J. Nya; Dawood, Z and B. Austin (2009). Allicin prevents disease caused by
Aeromonas hydrophila in rainbow trout Oncorhynchus mykiss (Walbaum). Journal of
Fish Diseases (In press).
E.J. Nya and B. Austin. Development of immunity in rainbow trout (Oncorhynchus
mykiss, Walbaum) to Aeromonas hydrophila after the dietary application of garlic.
Fish and Shellfish Immunology. (To be submitted).
E. J. Nya and B. Austin. Dietary modulations of digestive enzymes by
immunostimulants in rainbow trout Oncorhynchus mykiss, Walbaum. Aquaculture.
(To be submitted).
xix
_____________________________________________________________________
Chapter 1 Introduction
1.1 Basis of aquaculture
Until the Neolithic era, human beings had depended on hunting and gathering of food
for subsistence. Fishing possibly might have developed as part of this fundamental need
for food. Since then fishing has witnessed a considerable transformation in methods of
capture and utilization. Interestingly, man adopted a form of cultivation that was
expected to stabilize production and bring it under control. Aquaculture developed from
this strategy to adopt more productive means to feed increasing human populations
(Pillay and Kutty, 2005).
1.2 Origin of aquaculture
Most writing about aquaculture refers to the long history of fish culture in Asia, ancient
Egypt and central Europe (Ling, 1977). The classic text of fish culture written around
500 BC by Fan Lei is believed to be proof that commercial fish culture existed in China
even before this period (Ling, 1977). Later writings suggested that fish culture existed
in Roman society (referred to as oyster culture) in the Middle Ages and in Egypt in
2500 BC. Fish culture was considered to have been introduced into several countries in
Asia and the Far East by Chinese immigrants, and to Europe during the Middle Ages in
monastic ponds. The history of aquaculture in Europe essentially started in the Middle
Ages with the introduction of common carp culture where it attained a social and
religious significance as the chosen food for special occasions. Carp culture continues
to flourish in Eastern European countries, this success led to its introduction into
modern Israel (Pillay and Kutty, 2005).
The propagation of trout has a fairly long history, originating in France in the 14 th
Century (Davis, 1956). Interest in trout culture centres on sports fishing and for
culinary purposes. Culturing occurs in almost all continents, having started in North
America during the 18th century (Davis, 1956). Later large scale commercial trout
culture developed in France, Denmark, Norway, Italy and Japan (Pillay and Kutty,
2005).
1
Chapter 1 introduction
_____________________________________________________________________
1.3 Definition of aquaculture
Aquaculture has been defined in many ways to denote all forms of culturing aquatic
animals and plants in fresh and brackish water and the marine environment (Pillay and
Kutty, 2005). The most current and internationally accepted definition has been given
by the Food and Agriculture Organization (FAO) of the United Nations (UN) as the
farming of aquatic organisms including fish, crustaceans, molluscs and aquatic plants
(FAO, 2001). The emphasis is on farming as a process of harnessing the forces of
nature to enhance production, such as control of disease and predators, regular stocking
and feeding. Most culture-based fisheries involving the practice in which species are
grown in specialized containers, ponds, cages, pens, raceways and various forms of nets
as in the case of molluscs are considered true forms of aquaculture (De Silva and
Anderson, 1994).
1.4 Present state of aquaculture
Aquaculture is now the fastest growing food-producing sector, contributing nearly 36%
to global fish supply in 2007 (FAO, Infofish, 2009). The rising global demand for fish
and seafood is largely supported by increasing supplies of aquaculture products,
particularly from developing countries. The world production of fish and fisheries
products in 2007 including aquatic plants increased by 2.7% to 156.37 million metric
tonnes (FAO, Infofish, 2009). The top six suppliers were China, India, Vietnam,
Indonesia, Thailand and Bangladesh. Freshwater carp, Cyprinus carpio, was the
dominant farmed fish species accounting to 38% of total production (Brander, 2007).
However, the species that showed good growth and gained consumer acceptance
worldwide were tilapia, catfish, salmonids and shrimp (Shelton and Rothbard, 2006;
Table 1).
2
Chapter 1 introduction
_____________________________________________________________________
Table 1. World aquaculture production in 1,000 metric tonnes.
Source
2003
2004
2005
2006
2007
Marine
25,379
27,362
28,661
29,946
31,327
Freshwater
21,834
23,832
25,341
27,085
29,102
3,003
3,301
3,729
4,287
4,760
50,216
54,495
57,731
61,318
65,189
Brackish water
Total including
Aquatic plants
Adapted from FAO, Infofish, 2009: Infofish.org
Rainbow trout (Oncorhynchus mykiss, Walbaum) is of North American origin, and is
one of the most widely introduced fish with virtually global distribution (Scott and
Crossman, 1969 and 1973). Rainbow trout belongs to the Salmonformes and is of the
Salmonidae family, but DNA studies showed they are genetically linked to
Oncorhynchus species, which comprise Pacific salmon in contrast to Atlantic salmon
(Salmo salar). Thus, the species was re-classified from Salmo to Oncorhynchus to
reflect the DNA status (Scott and Crossman, 1973).
Rainbow trout is an important species for aquaculture in temperate, tropical and
subtropical countries (Table 2), where it adapts well to the changing climate and water
temperature. Introduced to the UK and Europe from North America, it is now found
throughout the British Isles, where it is a popular species to farm due to its fast growth
rate and less stringent water quality demand. In the UK, trout production has been
maintained at ~16,000 tonnes for the last ten years, and in Scotland alone, 4,370 tonnes
of rainbow trout were produced from freshwater and 784 tonnes from seawater in 2000
(Jason, 2006; Fig. 1).
3
Chapter 1 introduction
_____________________________________________________________________
Table 2. Cultured salmon: World production in 1,000 metric tonnes.
Pacific
salmon
Japan
2003
2004
2005
2006
2007
2008
2009
9
10
12
10
10
10
10
Chile
97
103
115
115
120
113
70
Canada
17
21
21
10
8
7
7
New
Zealand
5
9
10
10
10
10
10
Total
128
143
158
145
148
140
97
Adapted from report prepared by Audun Lem, FAO Globefish, 2009.
Fig. 1. Rainbow trout farms, which consist of earthen ponds, in Scotland.
4
Chapter 1 introduction
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1.5 Bacterial diseases of farmed fish species
The increase in aquaculture is said to be paralleled with a corresponding increase in the
occurrence of infectious diseases, resulting often from high stocking densities and
stress conditions that favour the occurrence and spread of pathogens. Cultured fish
suffer a wide variety of bacterial, viral, parasitic and fungal diseases (see Austin and
Austin, 2007). Examples include motile aeromonad septicaemia, vibriosis, columnaris,
edwardsiellosis and furunculosis. Among these, the diseases caused by motile
aeromonads particularly Aeromonas hydrophila are widespread and affect a broad
range of mostly freshwater species. Motile aeromonads are implicated in a number of
disease conditions including haemorrhagic septicaemia, ulcerative conditions,
abdominal distensions, fin/tail rot and exophthalmia (Karunasagar et al., 1989; Austin
and Austin, 2007).
Table 3. Bacterial pathogens of fish (after Austin and Austin, 2007).
Pathogens
Disease
Host range
Geographical
distribution
Lactobacillosis
pseudo-kidney
disease
Salmonids
North America, UK
Clostridium botulinum
Botulism
Salmonids
Enterococcus
-
Rainbow
Denmark,
England
Italy
Anaerobes:
Gram-positive bacteria
The ‘lactic acid bacteria
Carnobacterium piscicola
USA
trout, Catfish
Lactobacillus spp.
Lactococcus garvieae
(Enterococcus seriolicida)
Lactobacillosis
pseudokidney
disease
Salmonids
Streptococcico
Many
spp.
sis/
Streptococcosis
5
North America, UK
fish Australia,
Europe,
Japan,
Israel,
Saudi Arabia, South
Chapter 1 introduction
_____________________________________________________________________
Africa,
USA
North America.
Lactococcus piscium
Lactobacillosis
pseudokidney
disease
Rainbow trout North America
Oncorhynchus
mykiss
Streptococcus difficilis
(Str. agalactiae)
Meningoencephalitis
Carp
Israel
Cyprinus
carpio,
tilapia Oreochromis spp.
(Streptococcus faecalis
subsp. liquefaciens
Streptococcus iniae
(Str. shiloi).
Meningoencephalitis,
Streptococcico
sis/Streptococcosis
various fresh- Australia, Europe,
water fish spp
Israel, Japan, Saudi
Lactobacillosis
pseudokidney
disease,
peritonitis
septicaemia
Atlantic
Australia, France,
salmon
North America
brown trout,
rainbow trout
Bacillus spp.
Septicaemia
freshwater sp.
Nigeria
Coryneform bacteria
Corynebacterio
sis
micrococcosis
salmonids
England
rainbow trout
England
Mycobacterium spp
Nocardia spp. (Noc
asteroides, Noc
seriolae)
Mycobacteriosis
most fish spp
Worldwide
Planococcus sp.
-
Most fish spp.
Worldwide
Renibacterium
Bacterial
salmonids
England
Vagococcus salmoninarum
Arabia, S. Africa,
USA
Aerobic Gram-positive rod
& cocci
Micrococcus luteus
(fish tuberculo
sis)
Nocardiosis
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Chapter 1 introduction
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salmoninarum
kidney
salmonids
disease (BKD;
Dee disease
Europe, Japan
N&S America
Rhodococcus sp.
Ocular oedema
Chinook
salmon
O.
tshawytscha
Canada
Streptomyces salmonis
(Streptoverticillum
salmonis)
Streptomycosis
Salmonids
USA
Aeromonas bestiarum
A. caviae
septicaemia
Atlantic
salmon
USA
Turkey
A. hydrophila, Aer.
liquefaciens, Aer.
punctata
haemorrhagic
Many fresh
septicaemia,
water fish
motile
species
aeromonas
septicaemia,
redsore disease
fin rot
Worldwide
-
Europe, India,
USA
Aeromonadaceae
representatives
Enterobacteriaceae
Representatives
Citrobacter freundii
salmonids,
sunfish Mola
mola,
Carp
Cyprinus
carpio
Edwardsiella tarda
(Paracolobactrum
anguillimortiferum,
Edw. anguillimortifera)
redpest,
various
edwardsiellosis freshwater
emphysematou fish species
s putrefactive
disease of catfish
Japan, USA
Escherichia vulneris
septicaemia
various
freshwater
fish species
Turkey
Hafnia alvei
haemorrhagic
septicaemia
cherry salmon Bulgaria,
O.
masou, England
7
Japan,
Chapter 1 introduction
_____________________________________________________________________
rainbow trout
Klebsiella pneumoniae
fin and
disease
tail rainbow trout
Scotland
Serratia liquefaciens
septicaemia
Atlantic
salmon,
turbot
France
Scotland
Serratia plymuthica
-
Rainbow trout Scotland,
Australia
Yersinia intermedia
-
Atlantic
salmon
Australia
Yersinia ruckeri
enteric
redmouth
(ERM)
salmonid
blood spot
salmonids
Australia,
N/America
Europe
‘Cytophaga rosea’
Flavobacterium
branchiophilum
gill disease
gill disease
salmonids
salmonids
Europe, USA
Europe, Korea
Japan, USA
Flavobacterium columnare
(Flexibacter/cytophaga
columnaris)
columnaris,
saddleback
disease
many
freshwater
fish species
Worldwide
Flavobacterium hydatis
(Cytophaga aquatilis)
gill disease
salmonids
Europe, USA
Flavobacterium
psychrophilum
(Cytophaga psychrophila)
bacterial gill
disease, coldwater disease,
rainbow trout
fry syndrome
Salmonids
Australia,
Europe, USA
Flexibacter maritimus
gill disease,
black patch
necrosis
many freshwater fish spp
Europe, Japan
Sporocytophaga sp.
Saltwater
columnaris
salmonids
Scotland, USA
Spain
Cytophaga-Flavobacterium
Flexibacter group
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Chapter 1 introduction
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Pseudomonads
Pseudomonas
anguilliseptica
red spot
(Sekiten-byo)
rainbow trout,
Finland, Japan
marine fish sp
particularly
eels Anguilla
anguilla,
A. japonica
France, Spain,
Scotland
Pseudomonas
chlororaphis
-
Amago trout
O. rhodurus
Japan
Pseudomonas
fluorescens
generalised
septicaemia
Most fish spp.
Worldwide
Pseudomonas
pseudoalcaligenes
skin ulceration
rainbow trout
Scotland
skin lesions
Atlantic
salmon
Iceland
-
rainbow trout, Germany, Spain
sturgeon
Portugal
Acipenser
sturio
Vibrio anguillarum
(Listonella
anguillarum)
vibriosis
Most marine Worldwide
fish spp.
V. logei
skin lesions
Atlantic
salmon
V. ordalii
vibriosis
most marine Worldwide
fish spp
V. salmonicida
coldwater
vibriosis, Hitra
disease
Atlantic
salmon
Canada,
Scotland
V. viscosus
winter
ulcer Atlantic
disease
salmon
syndrome
Iceland,
Norway
Vibrios
Moritella marina
(V. marinus)
Plesiomonas
shigelloides
9
Iceland
Norway
Chapter 1 introduction
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Moraxellaceae
Representatives
Acinetobacter sp
Acinetobacter
disease
Atlantic
salmon
Channel
catfish
Norway, USA
Small Morphologically
Simple bacteria
Piscirickettsia
salmonis
coho salmon Salmon
syndrome
salmonid
rickettsial
septicaemia
Canada,
Chile
Norway
Miscellaneous
Pathogens
Janthinobacterium
lividum Streptobacillus
anaemia
rainbow trout Scotland, Iceland
Atlantic
salmon
1.6 Aeromonas hydrophila
A. hydrophila is a heterotrophic, free-living, Gram-negative bacterium, commonly
found in freshwater and occasionally in marine waters (Larsen and Jensen, 1977). This
organism may also be found in areas where the climate is warm, in saltwater, estuarine,
chlorinated and unchlorinated waters, and aerobic and anaerobic environments (Hayes,
2006). It is considered to have a worldwide distribution (Austin and Austin, 2007). A.
hydrophila comprises part of the normal microbial floral of freshwater fish, but is an
opportunist pathogen, being converted from a commensal to a pathogenic state under
stress conditions (Davis et al., 1978). Stress is known to weaken the immune
mechanism in fish (Fast et al., 2008). Stressed fish, as a result of overcrowding or other
stressors, witness many physiological changes which can lead to metabolic imbalances,
increases in protein hydrolysis, increase released of cortisol from adrenal tissue with
attendant biochemical exhaustion and immune suppression. Thus, the stressed fish
become susceptible to opportunist bacterium, such as A. hydrophila (Hazen et al.,
1978). In particular, A. hydrophila is known to cause diseases in fish, frogs, lizards and
humans (Eddy, 1960; Graevenitz and Merisch, 1968).
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Chapter 1 introduction
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1.6.1 Taxonomy of Aeromonas
The genus Aeromonas belongs in the Family Aeromonadaceae, of the Order
Aeromonadales. Two distinct forms exist, the more heterogeneous motile form and the
homogenous non-motile form (Joseph and Carnahan, 2000). At present, the species
recognised include: A. allosaccharophila, A. bestiarum, A. caviae, A. encheleia, A.
eucrenophila, A. hydrophila, A. jandaei, A. popoffii, A. schubertii, A. sobria, A. trota
and A. veronii from the motile group, and A. salmonicida and possibly A. media from
the non-motile group (Smith, 1963; USFDA, 2006). A. hydrophila was first legitimately
published as Bacillus hydrophila by Chester (1901) and as Aeromonas punctata by
Zimmerman (1890) in the family Vibrionaceae (Popoff and Veron, 1976). On the basis
of molecular genetic evidence, the group was proposed to constitute a new family, i.e.
Aeromonadaceae (Colwell et al., 1986). MacInnes et al. (1979) concluded that the
overall classification of motile aeromonads is far from being resolved.
1.6.2 Diagnosis of Aeromonas hydrophila infections
Diagnosis is based on isolation and identification of cultures from infected fish (Austin
and Austin, 2007). Isolation is readily done from surface lesions and kidney swabs by
the use of standard, non-selective bacteriological media, such as nutrient agar or
tryptone soya agar, and selective media such as peptone beef extract glycogen agar
(PBG; McCoy and Filcher, 1974). With incubation at 20-25oC for 24–48 h, typically on
non-selective media, cream, round, raised, shiny entire colonies of 2–3 mm. diameter
develop (Austin and Austin, 2007). Diagnosis is effectively done upon examination of
key phenotypic traits of pure cultures (Austin and Austin, 2007). Boulanger et al.
(1977) highlighted the value of the fermentative metabolism, Gram-staining reaction,
oxidase and catalase, and production of arginine dihydrolase. Serological methods, such
as slide agglutination, latex agglutination and fluorescent antibody techniques, are
considered effective in confirming the presence of the bacterial pathogen (Eurell et al.,
1978).
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Chapter 1 introduction
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1.6.3 Pathology
Traditionally, A. hydrophila has been recognised as the causative agent of
haemorrhagic septicaemia/ motile aeromonas septicaemia, skin ulceration, fin/tail rot
and red sore disease (Fig. 2; Haley et al., 1967). Often, healthy fish suddenly develop
swimming abnormalities, pale gills, bloat (= abdominal distension) and dermal/ ocular
ulcerations. The skin ulcers may occur at any site and are surrounded by a bright red
rim of tissue (Randy, 1991). Austin and Austin (2007) described haemorrhagic
septicaemia as characterised by the presence of surface lesions which lead to the
sloughing off of scales. Internally, there may be ascitic fluid accumulation, anaemia and
necrosis in the organs notably kidney and liver resulting in a high mortality rate
(Huizinga et al., 1979). The pathogen has the ability to adhere to selected host T-cells
via the action of ‘adhesins’ (Trust et al., 1980). These adhesins appear extremely
selective, recognizing D mannose and L fructose side chains on the surface of
eukaryotic cells. With attachment, the host becomes at the mercy of the pathogen
(Austin and Austin, 2007). The mechanism of cell and tissue damage involves of both
endo and exotoxins.
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Chapter 1 introduction
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Fig. 2.1 Scale protrusion on a carp
Cyprinus carpio caused by A. hydrophila.
2.2) abdominal distension & accumulation
of ascitic fluid in goldfish Carassius
auratus caused by A. hydrophila
2.3) Exophthalmia
2.4) Ulcerative syndrome
Fig. 2.1 -2.4. Adapted from leaflet of Fish Disease by Cipriano et al. (1984).
1.6.4 Chemotherapy of A. hydrophila infections in fish
The most effective chemotherapeutic agents in the treatment of haemorrhagic
septicaemia include oxytetracycline, chloramphenicol, chlortetracycline and penicillin:
streptomycin mixture added to water at the rate of 10-15 mg/l. Oxytetracycline (=
Terramycin) is a drug of choice for treating any disease caused by A. hydrophila in fish.
The compound is approved by both USFDA and European medicines agency (EMEA)
for use in fish ponds with channel catfish and salmonids, and is administered orally at
0.050- 0.075g/kg of fish daily for ten days. Treatments are withheld for 21 days before
any fish stock are consumed. Effective results are noted within 2-3 days treatments
(Meyer, 1964; Meyer and Collar, 1964). Chloramphenicol (= chloromycetin) is
13
Chapter 1 introduction
_____________________________________________________________________
effective at treating fish orally. However, its use in food fish has been discouraged,
since it is a drug of choice for human diseases such as typhoid fever. Wright and Snow
(1975) reported that acriflavine used at the rate of 0.5-0.7g/l for 15 min or iodine (=
Betadine) 0.1-0.15 g/l for 15 min were successful in disinfecting eggs of largemouth
bass, Micropterus salmoides, preventing contamination of aquarium facilities and
stocks. Piromidic acid administered orally has been found effective in reducing A.
hydrophila infections (Katae et al., 1979). Furanace is also effective against A.
hydrophila infections when administered by immersion 5 10 min in water containing 12 mg/l of furanace or by holding fish for 1 week in 0.1 mg/l of furanace in water.
However, furanace can be toxic to fish if used carelessly (Mitchell and Plumb, 1980).
1.6.5 Modern approaches to the control of fish disease
Until recently, disease management strategies were based mainly on chemotherapy
(Aoki, 1992). However, the emergence of drug resistance in pathogens, problems
associated with drug residues in cultured fish, and awareness towards environmental
pollution problems emanating from the use of chemotherapeutants have led to greater
focus on alternative methods of disease management (Tonguthai and Chanratchakool,
1992). In recent years, disease prevention by means of optimal husbandry and use of
vaccines, immunostimulants and probiotics has been increasingly recognised.
1.7 Vaccines
Vaccination is recognised as having great potential in aquaculture, and some vaccines
are commercially available (Adam et al., 1995). Song and Kou (1981) injected eels
with various A. hydrophila preparations and obtained serum antibody levels which were
highest following use of live attenuated cells, formalised or heated-killed cells and
sonicated cell extracts. They also found significant differences of protection between
eels vaccinated with attenuated live cells and controls upon challenge with virulent
strains. A natural epizootic proved the effectiveness of immunization of catfish with a
combined A. hydrophila and Flexibacter columnaris heat inactivated whole cell vaccine
when administered by injection or immersion but not by feeding (Schachte, 1978).
14
Chapter 1 introduction
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However, circulating and secretory antibody titres were recorded for all three
treatments with the injected fish having the highest serum titre followed by the
immersed fish. The immersed fish showed the highest level of gut mucus antibody,
followed by the orally treated group.
Immunostimulants have been used as adjuvants in vaccine formulations, especially ß-13 and ß-1-6 glucans, and led to good antibody responses specifically when used to
replace oil-based adjuvants (Kawakami et al., 1998). Davis and Hayasaka (1984)
investigated the effect of the immunostimulant ‘Ette’ on eels immunized and
challenged with A. hydrophila. This substance increased the survival rate of naïve fish
whereas it enhanced antibody production and phagocytic activity in fish injected
intravenously.
Lamers and De Haas (1985) used A. hydrophila as a model antigen in carp to examine
the effect of routes of exposure and antigen preparation on antigen processing in
lymphoid tissue and on production of antibody. Injected heat inactivated vaccines gave
slightly higher serum antibody titres than formalized cells. Interestingly, the antibody
induced by formalized cells was mainly directed to lipopolysaccharide (LPS). The
humoral response after a second injection seemed to be priming dependent, thus
injection of low numbers of cells evoked poor memory while optimal effects were
obtained with matching priming and second doses. Single immersion in heat inactivated
cells did not induce humoral response but did so upon secondary challenge. Immersion
evoked a measurable agglutinating antibody response at 1, 3 or 8 months after priming
(Lamers and De Haas, 1985).
Early reports on fish vaccines relates to vibriosis and furunculosis (Duff, 1942). Since
then, many studies have been conducted on the factors influencing fish immune
responses. Vaccines development is based on the principle of acquired immunity
(Galindo-Villegas and Hosokawa, 2004). On the contrary, fish depend more heavily on
non-specific (innate) defence mechanisms than do mammals (Anderson, 1992).
15
Chapter 1 introduction
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Example of commercially available vaccines are AquaVac® ERM- for use in healthy
rainbow trout to prevent Enteric Redmouth Disease caused by Yersinia ruckeri;
AquaVac® Furovac 5 Vibrio; AquaVac® Vibrio; AquaVac® Vibrio oral - for the
reduction in mortality due to furunculosis and vibriosis disease caused by Aeromonas
salmonicida and Vibrio anguillarum (biotype I and II ) in healthy salmonids;
AquaVac® Ergosan - a completely natural feed ingredient which aids the non-specific
immune response to a range of pathogens.
Moreover, vaccine development and the process of vaccination are relatively slow and
costly and, so far, have mainly been applied to fish species and diseases of importance
in the West. Therefore, immunostimulants have a significant role to play in health
management strategies in aquaculture.
1.7.1 Inactivated vaccines
Inactivated vaccines are killed preparations of micro-organisms delivered by injection
with adjuvant or administered by water-borne exposure or by immersion. Inactivation
can be by heat treatment (70oC for 10 min) and chemicals, including formaldehyde
(0.1- 0.3%), beta-propiolactone or binary ethylenamine (Klesius et al., 2005; Habib et
al., 2006 and Hossain et al., 2009). This traditional strategy form the basis for most
whole cell vaccines that have been developed commercially (Evelyn, 1997). An
inactivated vaccine has a high degree of safety and is easy to develop, and include
autogenous products which are preparations of killed pathogens isolated from a specific
site and restricted for use in the same farm. To be effective, they must be used in the
present of adjuvant, and offer relatively short duration of protection (Ada, 1990).
Example of inactivated commercial vaccines are Norvax® Strep Si - a water-based
inactivated vaccine against Streptococcus iniae infections in fish, AquaVac® ERM inactivated Yersinia ruckeri vaccine (Hagerman strain) for immersion or injection,
AquaVac® ERM oral and AquaVac® Furavac 5 oral - inactivated Aeromonas
salmonicida vaccine for the prevention of furunculosis.
16
Chapter 1 introduction
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1.7.2 Subunit vaccines
Subunit vaccines consist of whole or portion of protein extracted from disease agents,
typically protein units, segment or regions that bear the protective antigens. They are
the immunogenic protein unit that stimulate immunity (Leong, 1993). However, they
may be prepared by purifying native antigen directly from pathogenic cultures, from
recombinant DNA involving insertion of all or part of the gene coding for particular
antigen into bacterial vectors, yeast or viruses which then produce large number of the
protein in vitro. The development of synthetic peptide vaccines is a form of subunit
vaccine. The approach requires a detailed knowledge of the epitope structure of the
productive antigen (Brown et al., 1993), but has a high level of safety since no virulent
genes are involved. Most bacterial expression system or vectors used are E. coli and
baculovirus (Leong et al., 1997; Lorenzen and Olesen, 1997). Gilmore et al. (1988)
reported the first subunit vaccine of recombinant DNA origin. They cloned infectious
haematopoietic necrosis virus (IHNV) glycoprotein gene into an E. coli expression
vector to produce trp E fusion protein that was successful in immunizing rainbow trout
and chinook salmon, O. tshawytscha by immersion. Xu et al. (1991) produced plasmid
coding for sequential portions of the IHNV glycoprotein, achieving the best protection
in rainbow trout fry. Manning and Leong (1990) used several clones of Sp strains of
infectious pancreatic necrosis virus (IPNV) segment A to construct a series of trp E
expression vectors that were used to synthesize large amounts of A segment encoded
protein in E. coli which was used as an experimental subunit vaccine, with good
protection against IPNV administered by immersion (Cumulative mortality was 44 % in
the control and 4 % among vaccinated fish). Example of subunit commercial vaccines
are Intervet’s Compact® IPN used against infectious pancreatic necrosis virus in
Salmon and AquaVac FNM
PLUS
IPN – containing protein antigens cloned from VP2
and VP3 of the IPN Virus produced in a yeast expression system, use against
furunculosis and IPN disease caused by Aeromonas salmonicida and IPN virus in
healthy Atlantic salmon.
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Chapter 1 introduction
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1.7.3 Live attenuated vaccines
This category of vaccine comprise live modified preparations of pathogenic organisms
created by deletion of specific genes known for virulence or by serial culturing by using
natural occurring mutants with cross-reacting antigens. These vaccines generally offer
good protection but there are concerns about reversion to virulence and the possible
effect on other aquatic organisms, transfer of resistant plasmid and cost effectiveness.
However, they form the basis of most human and animal vaccines (Leong and Fryer,
1993). In their preparation, two or more genes may be knocked out or deleted, or
inactivated for the product to remain stable without reversion to pathogenic organism
(Uzzau et al., 2005). Another way is to use infectious clones of disease pathogen or
ones created through isolation of the entire genome of the disease causing agent and
then modifying them. Genetic markers or DNA probes are use to distinguished the
strain used in the vaccine from the natural isolates. The vector-based systems used are
those of bacteria, viruses or plants carrying a gene from another disease agent. Roberti
et al. (1992) developed an attenuated strain of IHNV by growing wild-type virus in
neutralizing monoclonal antibodies until a mutant that was resistant to neutralization
was developed; some had altered growth properties and were attenuated in virulence.
The products provided sufficient protection to rainbow trout against IHNV challenge.
Vaughan et al. (1993) created an aromatic dependent mutant of A. salmonicida using a
kanamycin resistance cassette to inactivate the arc-A-gene of the bacterium. The
deletion of the gene created a mutant that was unable to grow in the fish, highly stable
resulting in significant attenuation of virulence for trout (Vaughan et al., 1993).
Example of live attenuated commercial vaccine is Intervet’s AquaVac ESC® - use
against Edwardsiella ictaluri in Channel catfish Ictalurus punctatus.
18
Chapter 1 introduction
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Table 4. Licensed live attenuated vaccines against Gram-negative bacteria
Klesius et al., 2005).
Pathogen
Disease
Species
E. ictaluri
Enteric
septicaemia.
Catfish
F. columnaris
Columnaris
Catfish,
salmonids
Name of
company
Schering
Plough
-do-
(after
Delivery
Immersion
Immersion
1.7.4 Recombinant vaccines
Recombinant vaccines are subunit products containing only part of the whole organism
or synthetic peptides representing the basic portion of a protein that elicited the immune
response (Winton, 1998). Several systems or vectors are used to express recombinant
protein. Whole cell expression system or vectors are prokaryotic cells e.g. E. coli and
eukaryotic e.g. mammalian cells, avian cells, insecT-cells or yeasT-cells. Developments
of recombinant vaccines involve the use of recombinant DNA technology to insert
copies of the genes from protective antigens into a virus or bacterium that can infect the
host and possibly replicate within it but without causing the disease. During
replications, high level of recombinant antigens are produced that will stimulate the
host immune systems. This approach is also used to create multivalent vaccines that
stimulate protection against several pathogens. For example, Zang and Hanson (1996)
developed a recombinant channel catfish virus expressing a foreign gene. They inserted
the lac Z gene from E. coli within the thymidine kinase (TK) gene of channel catfish
virus (CCV) so that the new construct CCV lac Z was TK, but able to deliver the
reporter genes into cells where it was expressed sufficiently. Noonan et al. (1995)
developed a recombinant vector using a strain of A. salmonicida that had been
attenuated by deletion of a 1410 base-pair (bp) segment of the vapA gene encoding for
Para crystalline surface protein array (= A-layer). Fragment of the glycoprotein gene of
VHSV and IHNV were cloned into a bacterial vector in the present of the plac promoter.
Immunoblotting revealed the inducible expression of E. coli. The plasmid was
mobilized into the A440 strain of A. salmonicida by conjugation. Vaccinated fish
infected with IHNV or VHSV by cohabitation showed good survival compared to
controls.
19
Chapter 1 introduction
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1.7.5 DNA vaccines
Plasmids, which contain genes from a disease agent and a promoter, had been used to
initiate protein expression from the gene in the host animal (Rodriguez and Whitton,
2000). The recombinant plasmids containing foreign genes are purified from bacteria
and the ‘naked DNA’ is injected directly into the animal intramuscularly (i.m.) or
intradermally (i.d.). An immune response is elicited to the protein expressed from the
foreign gene. Anderson et al. (1996) constructed plasmid-vector encoding IHNV
nucleoprotein and glycoprotein genes in the presence of cytomegalovirus as promoter.
The DNA plasmid was used to injected rainbow trout fry and held 6weeks before
challenge by immersion in wild-type virus. Non-immunized fish and PBS injected
controls had 65% and 67% mortalities, respectively, compared to immunized fish
containing glycoprotein alone and in combination with plasmid-nucleoprotein genes
which experienced mortalities of 17% and 15%, respectively. However, genetic
vaccines involving injection of plasmid DNA coding for protective antigens (Tang et
al., 1992) are considered to be effective against viral, bacterial and parasitic diseases
(Robinson et al., 1997).
Table 5. Licensed vaccines against bacteria (after Klesius et al., 2005 with
modification).
Pathogen
Disease
Species
Name/company Delivery
Piscicrickettsia
salmonis
Piscicrickettosis
Salmonids
Pharmaq AS
Oral,
injection
V. anguillarum
Vibriosis
Schering
Plough ltd
Oral,
injection
V. anguillarumV. ordalii
Vibriosis
V.
parahaemolyticus
Vibriosis
Ayu,
Salmonids
Yellowtails.
Salmonids,
Cod
Halibut, Sea
bass
Sea bream,
Amberjack,
Yellowtails.
Shrimps.
V. salmonicida
Coldwater
Salmonids.
20
-do-do-
Oral,
injection
-do-
Immersion
-do-
Injection
Chapter 1 introduction
_____________________________________________________________________
vibriosis.
Yersinia ruckeri
Enteric
mouth
red Salmonids.
-do-
Oral,
injection
Gram-positive
bacteria
Lactococcus
garvieae
Lactococcosis
Renibacterium
salmoninarum
Bacterial kidney
disease
Streptococcus
iniae
Streptococcosis
Rainbow
trout
Amberjack,
Yellowtails.
Salmonids
Tilapia,
Rainbow
trout
Intervet
Immersion,
Injection.
Schering
Plough
Injection
-do-
Immersion,
Injection
1.7.6 Adjuvants
Adjuvants have been shown to exert a powerful influence on the immune response by
acting as antigenic depots and thus rendering the antigen more immunogenic.
Examples include immunostimulants (Vadstein, 1997; McCumber et al., 1981),
Freund’s complete adjuvant (FCA) (Avtalion et al., 1980) and dimethyl sulphoxide
(DMSO) (Anderson et al., 1984).
1.8 Probiotics
Probiotics may be considered as an alternative to antimicrobials in disease control
strategies of cultured fish. Probiotics have been defined by different workers but the
definition by Fuller (1989) has attracted much attention. He defined probiotics as live
microbial feed supplements which beneficially affect the host animal by improving its
intestinal balance (Fuller, 1989). Furthermore, FAO/WHO (2001) referred to probiotics
as live micro-organisms which confer health benefits on the host when administered in
adequate amounts. Probiotics enhance the performance of the intestinal microbial flora
by colonizing the gut and depriving pathogens of adhesion sites and nutrients
(Gatesoupe, 1999). Research on Probiotic in aquaculture focused initially on fish
juveniles, but subsequently concern was shifted to fish larvae and shellfish (Verschuere
et al., 2000). To date, most probiotics used in aquaculture belong to Lactobacillus,
Carnobacterium, Aeromonas, Bacillus, Pseudomonas and Vibrio genera.
21
Chapter 1 introduction
_____________________________________________________________________
Table 6. Probiotics considered as biological control agents in aquaculture (adapted
from Balcazar et al., 2006).
Probiotics
Source
Used on
Delivery
References
Streptococcus
lactis
Lactobacillus
bulgaricus
?
Turbot larvae
Scophthalmus
maximus
Enrichment of
live food.
Garcia de la
Banda et al.
(1992)
Lactobacillus
sp.
Carnobacterium
sp.
Vibrio
alginolyticus
Rotifers
(Brachionus
plicatilis)
Turbot larvae
Enrichment of
rotifers
Gatesoupe
(1994)
Commercial
Shrimp
hatchery
Atlantic
salmon
(Salmo salar)
Bathing in
bacterial
suspension
Austin et al.
(1995)
V. pelagius
Turbot larvae
Turbot
Addition to
culture water
Ringo and
Vadstein
(1998)
Carnobacterium Intestines of
divergens
Atlantic
salmon
Atlantic cod
fry (Gadus
morhua)
Addition to
diets
Gildberg and
Mikkelsen
(1998)
Carnobacterium intestine of
sp.
Atlantic
salmon
Atlantic
salmon
Addition to
diets
Robertson et
al. (2000)
Bacillus
megaterium
B. subtilis,
B. polymyxa
B. licheniformis
Commercial
product
(Biostart)
Channel
catfish
(Ictalurus
punctatus)
Addition to
diets
Queiroz and
Boyd (1998)
G-probiotics
Commercial
product
Tilapia
(Oreochromis
niloticus)
Addition to
diets
Naik et al.
(1999)
Pseudomonas
fluorescens
Pseudomonas
Iced freshwater fish
(Lates
niloticus)
Rainbow trout Addition to
(Oncorhynchus culture water
mykiss)
22
Gram et al.
(1999)
Chapter 1 introduction
_____________________________________________________________________
Lactobacillus
rhamnosus
ATCC 53103
Cultured
collection
Rainbow trout
Addition to
diets
Nikoskelainen
et al. (2001)
L. rhamnosus
JCM 1136
Cultured
collection
Rainbow trout
Addition to
diets
Panigrahi et al.
(2004)
Aeromonas
Digestive tract
hydrophila, V.
Of rainbow
fluvialis,
trout
Carnobacterium
sp.,Micrococcus
Luteus
Rainbow trout
-do-
Irianto and
Austin (2002)
Enterococcus
faecium SF-68
Commercial
product
(Cernivet)
Turbot larvae,
Tetraselmis
Copepod-fed
larvae
Eel (Anguilla
anguilla)
Addition to
diets
Chang and Liu
(2002)
Turbot larvae
Addition to
cultured water
Hjelm et al.
(2004)
Intestines of
Rohu (Labeo
rohita), carp
L. rohita
Addition to
diet.
Ghosh et al.
(2004)
Rosebacter sp.
strain 27-4
Bacillus
circulans
23
Chapter 1 introduction
_____________________________________________________________________
1.9 Fish immune system
The immune system of fish has witnessed a surge in interest over the past two decades,
occasioned by the demand of the fish farming industry for the control of infectious
diseases. Immunology had been defined as the study of the organs, cells and molecules
responsible for recognition and disposal of foreign or non-self materials that enters the
body usually in the form of life threatening infectious microbes (Playfair and Chain,
2005). Although fish are poikilothermic, aquatic vertebrates, they possess a system of
defence mechanism displaying many similarities with those of their mammalian
counterparts. Moreover, it is now indisputable that fish are closer to mammals than to
any invertebrate taxon (Ellis, 1982; Manning, 1984). The first line of defence
mechanism present in fish is the innate or non-specific mechanism (Fletcher, 1982).
The specific immune mechanisms conferring acquired resistance to disease require
adaptive processes within the immune system and this forms the second line of defence.
These two categories of immunity often act in concerted effort with each other as they
are in many ways not mutually exclusive, being inter-dependent (Ellis, 1982).
1.9.1
External defence mechanisms
The presence of fish scales, mucus surfaces of the intact skin and mucus membranes
lining the viscera hollow of the gastro intestinal tract, the respiratory tract, urinary tract,
and the gills are significant barriers to the entry of potential pathogens. The mucus, a
viscous fluid composed mainly of glycosylated proteins called mucins suspended in an
electrolytes solution, is secreted constantly over these surfaces by the goblet-cells.
Mucus serves many functions including protection from pathogens by preventing their
attachment (Austin and McIntosh, 1988), by continuously sloughing off and is
produced at an increasing rate following stress, injury or disease. In addition, there are
numerous antimicrobial secretions on these surfaces, which contribute to keeping
infectious agents at bay (Austin and McIntosh, 1988). These include lysozyme (also
found in egg white), lactoferrin, and peroxidase (Playfair and Chain, 2005), lectins and
proteolytic enzymes e.g. trypsin-like or cathepsin proteases (Ellis, 1981; Alexander and
Ingram, 1992). More recently, mucus lining the gills has been found to contain nitric
oxide (Campos-Perez et al., 2000) and the skin mucus, anti-microbial peptides and
24
Chapter 1 introduction
_____________________________________________________________________
protein (Ebran et al., 1999; Fernandes and Smith, 2002). More specialised defence
mechanisms include the extreme acidity of the stomach (about pH 2.0), the phagocytic
cells in the epithelium of the bronchus and the gills (Mittal et al, 1980).
1.9.2
Innate or non-specific immune mechanisms
The innate or non-specific immune system consists of humoral (free in the serum or
body fluids) and cellular immunity.
The humoral element of the non-specific immune mechanism indicates the presence of
antibody in the blood or body fluid elicited in response to the stimulus of a foreign
protein or other antigen (Cruickshank, 1965). The ranges of beneficial macro-molecules
include:
Complement - a thermolabile biochemical cascade that helps clear pathogens from an
organism and produces widespread inflammatory effects as well as lysis of bacteria.
The complement system consists of a number of small proteins found in the circulating
blood as inactive zymogens (= enzyme precursors). When activated by one of several
stimulants (either directly by microbes or antibody-antigen complex), proteases in the
system cleave specific proteins to release cytokines and initiate spiralling cascade
which further causes cleavages, leading to massive response and activation of the cellkilling and membrane attack complex. More than 20 proteins and protein fragments
make up the complement system, including serum proteins and cell membrane
receptors. These proteins are synthesized mainly in the liver, and they account for about
5% of the globulin fraction of blood serum (Janeway et al., 2001).
Complement activation can be through 3 pathways: the classical complement pathway
CCP, the alternative complement pathway ACP and the mannose-binding lectin
pathway LCP. All these pathways have been identified in fish with exception of jawless
fish (Galindo-Villegas and Hosokawa, 2004). The CCP is known to be triggered by the
binding of antibody to cell surface. Whereas the ACP is activated directly by bacteria
pathogens, viruses, fungi and is independent of antibody. The LCP is initiated by
binding of a protein complex consisting of mannose- binding lectin MBL and serine
proteases to mannose residues on the pathogen surface; this is also initiated
independent of antibody.
25
Chapter 1 introduction
_____________________________________________________________________
Lysozyme in the serum. In fish, lysozyme plays important role in the innate defense
mechanisms against invasion by pathogens (Murray and Fletcher, 1976). Lysozyme,
also known as muramidase or N-acetylmuramide glycanhydrolase, comprises a family
of enzymes which function by attacking peptidoglycans in the cell walls of bacteria,
especially Gram-positive bacteria and hydrolyzing the glycosidic bond that connects Nacetylmuramic acid with the fourth carbon atom of N-acetylglucosamine. It does this by
binding to the peptidoglycan molecule in their binding site, causing distortion to the 4th
sugar molecules in the hexasaccharide (the D ring). In this stressed condition, the
glycosidic bond is easily broken leading to damage to the bacterial cell walls by
hydrolysing the 1, 4-beta-linkages between N-acetylmuramic acid and N-acetyl-Dglucosamine residues in the peptidoglycan. Lysozyme is found in a number of
secretions, such as tears, saliva, and mucus lining the skin, gills, gastrointestinal wall
and urino-genital tract. Lysozyme is also present in cytoplasmic granules of the
neutrophils and monocyte (Murray and Fletcher, 1976). These immune cells probably
accounted for the serum lysozyme activity.
Inhibitors. These are molecules that bind and interfere with the metabolic activity of
invading microbes, either by depriving them of available nutrients or by disrupting their
metabolic pathways and blocking their enzyme's activity (Galindo-Villegas and
Hosokawa, 2004). Inhibitors include:
Transferrin- an iron-binding glycoprotein which plays an active role in iron transport
and delivery (Putnam, 1975). The main role of transferrin is to deliver iron from the
absorption site in the digestive tract, red blood cell and macrophages to all tissues. To
achieve this, a transferrin receptor on the surface of the cell binds to the iron molecule,
and as a consequence is transported in a vesicle to the cell where it is required. The
receptor helps maintain iron homeostasis in the cells by controlling iron concentrations
(Macedo and de Sousa, 2008). Transferrin is associated with the innate immune system,
in the sense that it is found in mucosa and binds iron, thus creating an environment low
in free iron, where few bacterial pathogens are able to survive. The level of transferrin
in the blood is therefore an important indicator of host – pathogen susceptibility (Yano,
1996).
26
Chapter 1 introduction
_____________________________________________________________________
Antiproteases – these are enzyme protease inhibitors within the serum, which function
to regulate the hydrolysis of protein. There is speculation that they may play an
important role in the innate defense mechanism against bacterial infections (Ellis,
2001).
Antimicrobial peptides or antibacterial peptides – are also known as host defense
peptides. Antimicrobial peptides comprises of diverse group of molecules divided into
subgroups on the basis of their amino acid composition and structure (Yeaman and
Yount, 2003). Antimicrobial peptides include two or more positively charged residues
provided by arginine, lysine and histidine (Papagianni, 2003). An example is Misgurin
found in Loach Misgurnus anguillicaudatus, This is a 21-amino-acid peptide with
antimicrobial activity against a broad spectrum of micro organisms but without
significant haemolytic activity (Park et al., 1997). The modes of action include bacterial
membrane disruption, interference with metabolism, and targeting cytoplasmic
components. Antibacterial peptides have been identified from the mucus secretions of
most fish species. An example is Histone H2A obtained from skin secretions of rainbow
trout (Smith et al., 2000); Pleurocidin identified from natural flounder Pleuronectes
americanus- this compound protects coho salmon from V. anguillarum infections (Jia
et al., 2000); and Pardaxin a shark repellent peptide idenified from Moses sole fish
(Pardachirus marmoratus; Oren and Shai, 1996). Hepcidin is another antimicrobial
peptides isolated from the gill of hybrid striped bass, white bass (Morone
chrysops) × striped bass (M. saxatilis). This compound has a strong antimicrobial
activity against Escherichia coli (Shike et al., 2002).
Lectins – these are sugar-binding proteins, highly specific for their sugar moieties.
They play a role in biological recognition phenomena involving cells and proteins. For
example, lectin receptor recognizes hydrolytic enzymes containing mannose-6phosphate, and subsequently targets these proteins for delivery to the lysosomes for
killing. Lectins also recognize carbohydrates that are found exclusively on pathogens.
Examples are the lectin complement activation pathway LCP and Mannose binding
lectin MBL. Some bacteria use lectins to bind themselves to the cells of the host
organism during infection. Lectins can block this attachment and subsequent invasion
(Tort et al., 2003). However, lectins are known for binding to sugar moieties on the
27
Chapter 1 introduction
_____________________________________________________________________
surface of bacterial pathogens, thus resulting in agglutination (Galindo-Villegas and
Hosokawa, 2004). They may bind to soluble carbohydrates or to carbohydrate moieties
which are part of a glycoprotein or glycolipid present in animal cells thereby causing
agglutination or precipitation in the cells. They are also involved in the induction and
activation of complement activity (Arason, 1996).
Lysins- these are protein molecules that are responsible for cell lysis. They comprise of
lytic enzymes (lysins), such as hydrolases, chitinases and lysozymes, which are
produced during infection with the ability to lyse bacterial cells or the cascade of
several enzymes as observed in the complement system (Galindo-Villegas and
Hosokawa, 2004).
Cellular immunity results from an enhanced capacity of a particular cell or tissue to
counteract infection (Cruickshank, 1965). The phagocytes together with the natural
killer cells (NK), granulocytes, macrophages, cytotoxic cells and the dendritic cells
form the cellular arm of non-specific defences, the two subsets (the humoral and
cellular) do not function in isolation; all parts of the immune system work together like
a network. They co-operate in many instances in the discharge of their functions, for
example, the opsonisation through complement/C-reactive protein/phagocytosis (Ellis,
1986). Cells in the innate immune system do not recognise specific antigenic patterns,
as in the case of acquired immune responses. Instead, they recognise pathogens by
means of highly conserved structure and complement (Frazer et al., 1998). The
phagocytic cells include monocytes, macrophages, granulocytes and dendritic cells
(Steinman, 1991). These cells are specialized for the pursuit, capture, ingestion and
intracellular destruction of invading microbes, i.e. by phagocytosis.
Phagocytosis is the cellular process of phagocytic engulfment of foreign cells,
including pathogens, with subsequent killing and digestion of the foreign material. The
occurence in fish is described as the most primitive defense mechanism with the initial
step of movement of immune cells to the site of infection. This movement is
nondirectional (= chemokinesis) as well as directional (= chemotaxis; Galindo-Villegas
and Hosokawa, 2004). With movement, comes attachment via lectins enhanced by
opsonization (Ainsworth et al., 1994) and finally the engulfment and killing.
28
Chapter 1 introduction
_____________________________________________________________________
Granulocytes- are also known as polymorphonuclear leukocytes PML because of the
varying shapes of the nucleus, which is usually lobed and segmented. In fish, the most
abundant of the 3 types of granulocytes are neutrophils: followed by the eosinophils
and basophils. However the presence of the last two types of granulocytes in fish is
disputed ((Ellis, 1997). Granulocytes are mobile, phagocytic and responsible for the
formation of reactive oxygen species ROS involved in intracellular killing (GalindoVillegas and Hosokawa, 2004).
Non specific cytotoxic cells NCC mediate the acute innate cytotoxic responses of fish
and may be the precursor of natural killer (NK) cells (Evans and Jaso-Friedmann,
1992). They form defense against viral and parasitic infections, and possibly provide
defence against tumour development. Fish NCC have been assayed by their capacity to
lyse different pathogenic organisms.
NK cells are cytotoxic lymphocytes that make up a major component of the innate
immune system. NK cells play a major role in cell apoptosis and necrosis, and rejection
of tumors. NK cell activity is highly regulated, and is activated in response to
interferons or macrophage -derived cytokines.
Dendritic cells (DCs) are innate immune cells. Their main function is in antigen
presentation i.e. they process antigenic materials and present it on the surface to other
cells of the immune system, thus functioning as antigen- presenting cells.
DCs are present in tissues that are in contact with the external environment, such as the
skin, where there are called langerhans cells (Langerhans, 1868), and the inner lining of
the respiratory tract; gastrointestinal tracts and the gills (Lovy et al., 2006). Once
activated, they migrate to the lymphoid tissues where they interact with T-cells and Bcells to initiate the immune response.
Inflammation - is a complex type of molecular and cellular event that is designed to
protect the host from microbial invasion, clear damaged tissue and facilitate the repair
process (Bols et al., 2001). Not much detail is known about Inflammation in fish, but it
has been stated that it is similar to the mammalian counterpart (Secombes, 1996). With
the activation of inflammation comes increased vascular permeability and
vasodilatation, activation of blood clotting system, and infiltration of phagocytic cells
29
Chapter 1 introduction
_____________________________________________________________________
into the injured tissue. Inflammation is initiated when tissues are injured, or there is the
presence of foreign cell, and when phagocytosis could not prevent infection.
Inflammatory reactions occur in fish but relatively little is known about the actual
mechanisms (Ellis, 1986).
Lymphocytes are derived from the lymphoid stem cell of the central thymus (GalindoVillegas and Hosokawa, 2004). These cells are capable of differentiating into T or B
lymphocytes, which occur in the peripheral organs (spleen and kidney) and in the
circulatory and other tissues policing the body for non self materials or pathogens
(Trede and Zon, 1998). The ability to recognise antigens through surface receptors
make lymphocytes ideal for adaptive responses in fish (Playfair and Chain, 2005).
T-cells are involved in cell- mediated immunity whereas B-cells are primarily
responsible for humoral immunity, i.e. related to antibody production. In response to
pathogenic invasion, some T-cells i.e. T helper cells (Th cells) produce cytokines that
signal the immune response, while other T-cells, namely the cytotoxic T-cells, produce
toxic molecules that induce apoptosis of the infected cells. Following activation, B and
T-cells leave a lasting memory of the antigens they have encountered in the form of
memory cells. Throughout the lifetime of the fish, these memory cells will remember
each specific pathogen encountered, and in response mount a strong immune response,
in the event that the pathogen is detected again.
1.9.3
Acquired or specific immune mechanisms
Acquired immunity is specific in the sense that it protects against one particular
pathogen or its toxic products (Cruickshank, 1965). In fish, antibody forming
mechanisms varies according to the type of antigens and B-cells stimulated (Singleton,
2004). The basic structure of the immunoglobulin consists of four polypeptide chains,
i.e. two similar heavy chains and two identical light chains. The four chains being
linked to form the Y-shaped molecules by disulphide (S-S) and other bonds (Singleton,
2004). In the plasma, immunoglobulins occur primarily in the monomeric form with a
molecular weight of ~160,000, but in extra vascular tissues such as respiratory and
gastro intestinal secretions, bile and cutaneous mucus they are in a dimeric form (Lobb
and Clem, 1981).
30
Chapter 1 introduction
_____________________________________________________________________
Antibody diversity appears to be limited in fish, but there is increased functional
affinity (Secombes and Resink, 1984). Antibodies have been known to play an
important role in defence mechanisms (Vilain et al., 2005) by antigen induction and
isolation to active germinal centres (Boes, 2000), neutralisation of bacterial pathogens
and probably their toxins and adhesins (Ellis, 1998), complement activation leading to
lysis of pathogenic cells (Yano et al., 1985), antibody dependent-cell-mediated
cytotoxicity through macrophage activation (Whyte et al., 1990), opsonisation of
foreign cells such as bacteria directly or indirectly through complement activation
leading to enhanced phagocytosis (Griffin, 1983; Sakai, 1984) and mediation of
hypersensitivity responses (Groven et al., 1980).
The development of an antibody response is a complex sequence of events following
antigenic stimulation. There is both functional and genetic evidence that fish, like
mammals, have a network of signalling molecules, cytokines and chemokines that
control and co-ordinates the innate and acquired immune responses (Secombes et al.,
1999; Secombes, 2002). Two categories of molecular patterns are believed to induce
antibody response. Foreign or pathogen associated molecular patterns (antigens) and
molecular patterns exposed through inflammations caused by damage of the host’s own
tissues, due to injury, necrotic changes and natural cell death, signalling danger to the
immune system (Elward and Gasque, 2003). Tissue damage often results in local
inflammatory responses associated with increased permeability of the endothelium, upregulation of leucocytes, adhesion molecules on the endothelial cells and extravasations
or movement of the recruited leucocytes into the site of injury (Gallucci and Matzinger,
2001). The dendritic cells of the skin, formerly described as langerhans cells
(Langerhans, 1868) and characterised as the most important antigen presenting cells
(Steinman, 1991), migrate to the site of inflammation and take up pathogens or cell
debris, process the materials and transport it to the lymphoid tissues for antigen
presentation. This leads to the adaptive immune system activation and induction of
pattern recognition receptors (PRR). These are soluble components expressed as
receptors on phagocytes and other immune cells (Elward and Gasque, 2003).
There is evidence for ß-1,3 glucan receptors in salmon macrophages and on catfish
neutrophils (Engstat and Robertsen, 1994). Receptors for LPS binding activity have
31
Chapter 1 introduction
_____________________________________________________________________
also been described in rainbow trout and sea bream macrophages (Mulero et al., 2001).
The elimination of the pathogenic organisms often leaves the host with specific
memory enabling it to respond more effectively to reinfection with the same microorganism, a condition termed acquired resistance (Akbar et al., 1988; Playfair and
Chain, 2005). The mechanism of memory resides in the activation of B lymphocytes.
These cells retain the ability to produce antigen-specific antibodies rapidly if the body
is subsequently challenged with the same antigen (Singleton, 2004). Memory has been
demonstrated in fish for both humoral and cellular immune responses (Rijkers, 1982).
However, tolerance (negative memory) has been shown to take place especially in
immature fish (Manning et al., 1982). Cell mediated immunity attributed to antigenspecific T lymphocytes (= T-cells) has been observed in fish in association with
allograft rejection (Botham et al., 1980), delayed type hypersensitivity (Bartos and
Somer, 1981), macrophage migration inhibition and immuno-suppression (Smith et al.,
1980). The immune response of fish is modulated by both exogenous and endogenous
factors:
1.9.4
Exogenous factors
The exogenous factors that can influence the immune-competence of fish include
environmental temperature (Alcorn et al., 2002). Temperature is known to effect
antibody production, onset and magnitude of primary response (Avtalion et al., 1973),
memory cells formation and magnitude of the secondary response (Rijkers et al., 1980).
Avtalion et al. (1980) observed that low temperature favours the development of T-cell
suppressor activity. It was also shown that T-cells are more susceptible to low
temperature than B-cells, possibly due to differential membrane homoviscosity i.e.
differences in membrane resistant to flow of forces (permeability) acting between the
sticky molecules
(Abruzzini et al., 1982). The sensitivity of T- cells to extreme
temperature has also been reported in catfish (Bly and Clem, 1994). The immune
response of fish is relatively temperature independent. An example of this is the wide
temperature range of codfish phagocytic activity which appears to be as active at 0 oC,
as at 12oC (Alcorn et al., 2002).
Some authors assert that the innate immune response is active at low temperatures
whereas acquired immune parameters such as lymphocyte activity and antibody
production are more effective at higher temperature (Miller and Clem, 1984; Bly and
32
Chapter 1 introduction
_____________________________________________________________________
Clem, 1992; Alcorn et al., 2002). In codfish, the spontaneous haemolytic and
antiprotease activity was more active at 1oC and 7oC than at 14oC, whereas the opposite
was the case for the serum-IgM concentration (Magnadottir et al., 1999). There are,
however, instances of low temperature having adverse effects on innate parameters, for
example rainbow trout acclimatized at different temperatures showed lower phagocytic
and complement activity at 5oC than at 20oC (Nikoskelainen et al., 2004). Similarly, a
drop in water temperature from 22oC to 10oC had adverse effects on the protective role
of channel catfish mucus (Quiniou et al., 1998). On the whole, the optimal temperature
for expression of the immune response is known to be associated with the
environmental temperatures experienced by a particular species in its natural habitat.
Seasonality in the induction of the immune response appears to be linked to the effect
of temperature, either directly or indirectly. For example, Horne et al. (1982) found
lower protection in fish vaccinated during the winter. Moreover, Yamaguchi et al.
(1980) observed seasonal differences in the titres of agglutination and cytolytic
antibodies. Immunosuppressive effects of pollution and stress resulting in higher
disease susceptibility are well known (Kollner et al., 2002). Stress brings about cortisol
mediated immunosuppression as indicated by low antibody production and accessory
cells functions. Food additives such as vitamins, lipids or high carbohydrate content
may or may not enhance immune parameters but can still be of general benefit as
regards to growth and survival (Lygren et al., 1999).
The dose of antigen administered may affect the latent period and height of the primary
response (Lamers et al., 1985). It may also affect the level of the secondary response
and the affinity of the produced antibodies (Ambrosius et al., 1982; Fiebig et al., 1983).
Moreover, the dosage is important in inducing tolerance (Avtalion et al., 1973). The
route of antigen administration has a great effect on the outcome of any immunization.
i.m. injection is superior to i.p. for primary responses (Harris, 1973; Ingram and
Alexander, 1976) whereas it is superior to i.v. injection for the secondary response
(Rijker et al., 1980). Secondary responses occur only if the antigen is given through the
same route as primary immunization (Lamers et al., 1985). Oral administration may
induce systemic tolerance as does intracardiac injection (Udey and Fryer, 1978). The
nature of the antigen can determine the quantity and quality of the immune response.
Soluble antigens differ in the response they elicit, and their chemical modification may
33
Chapter 1 introduction
_____________________________________________________________________
result in differences as regards the expression of the immune response (Avtalion et al.,
1980). The fish species also has an effect on the immune response to certain antigens
(Stolen et al., 1982). The extent to which an antigen will be regarded as foreign or
pathogenic by the recipient fish can also determine the magnitude of the response
(Rodgers and Austin, 1985).
1.9.5.
Endogenous factors
Genetic traits influences immune competence and disease susceptibility of fish (Salte et
al., 1993). However, attempts to apply selective breeding for important parameters have
been hampered by the specificity of different innate characters. Ontogenic development
can lead to tolerance, particularly with T- dependent antigens (Manning and Mughal,
1985). The onset of specific immune parameters varies greatly between fish species,
even between closely related species like the salmonids (Schroda et al., 1998).
In general, the acquired immune system develops late in marine fish species which
depend on innate defence mechanism for the first 2 to 3 months after hatching (Schroda
et al., 1998). In zebra fish, phagocytic activity was detected in the embryo prior to
hatching, and at 2 days post fertilization in the carp embryo (Romano et al., 1998).
Lysozyme has been detected in fertilized eggs and the larval stages of several fish
species namely sea bass, tilapia and salmonids (Cecchini et al., 2000). Studies have
shown that the presence of lysozyme in eggs and embryo can prevent the transfer of
bacterial fish pathogens from mother to progeny (= vertical transfer) (Yousif et al.,
1994). With the application of genetic and proteomic methods major progress can be
expected in this field of fish immunology in the future.
1.10 The use of immunostimulants in aquaculture
The application of immunostimulants in aquaculture is described as an innovative
approach to enhancing the non-specific defence mechanism of fish to diseases
(Robertsen et al., 1994). This approach has the multiple benefit of being effective
against a wide range of bacteria and viruses, suitable for many species of fish and easy
to apply as it can be administered orally in feeds. Immunostimulants are inevitably
34
Chapter 1 introduction
_____________________________________________________________________
naturally occurring compounds that modulate the non-specific immune mechanisms by
enhancing the host resistance against diseases (Bricknell and Dalmo, 2005).
Immunostimulants are considered to be safer and more environmental friendly than
chemotherapeutics, and their range of efficacy is often wider than that of vaccination
(Sakai, 1999). According to Anderson (1992), immunostimulants are often grouped by
either their functions, origin (= sources) and consist of heterogeneous groups, biological
substances, bacterial, algae-derived, animal derived, nutritional factors, herbal
/medicinal plants, synthetic products and hormones.
In aquaculture, there are >20 different compounds which have potential for use as
immunostimulants (Anderson, 1992) among which are chitin, (Sakai et al., 1992),
dimerized lysozymes (Siwicki et al., 1998), nisin (Villamil et al., 2003), glucan from
yeast (Anderson, 1997), and chitosan from arthropods and shellfish (Siwicki and
Anderson, 1994). Potentially, they can make cost effective dietary supplements due to
the relative low cost of their source ingredients. The use of immunostimulants given as
dietary supplement can improve the innate defense of aquatic animals principally fish
and thus providing resistance to pathogens during period of stress, such as grading, sea
transfer and vaccination (Robertsen, 1999).
1.10.1
Lipopolysaccharide (LPS)
LPS is a cell wall component of Gram negative bacteria such as A. hydrophila,
Salmonella Shigella, Neisseria, Pseudomonas and E. coli (Kenneth, 2002). Bacterial
LPSs are B-cell mitogens which stimulate and induce proliferation of B-cells and not Tcells (Manning and Nakanishi, 1996). LPS injected into red sea bream have been shown
to enhance macrophage phagocytic activity (Salati et al,. 1987). MacArthur et al.
(1985) reported that plaice (Pleuronectes platessa) injected with LPS showed increased
macrophage migratory activity. In vitro, LPS stimulates phagocytosis and the
production of superoxide anions in Atlantic salmon (Solem et al., 1995). Similarly, LPS
stimulates the production of macrophage activating factor in goldfish lymphocytes
(Neumann et al., 1995).
1.10.2 Chitin and chitosan
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Chapter 1 introduction
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Chitin is a polysaccharide and forms one of the main component of insect and
crustacean exoskeletons and the cell walls of many fungi (Sakai, 1999). Sakai et al.
(1992) reported that rainbow trout injected with chitin showed stimulated macrophage
activity and an increased resistance to V. anguillarum infection. Also, yellowtail
injected with chitin alone showed increased protection against Photobacterium
damselae subsp. piscicida (= P. piscicida), which continued until 45 days after
treatment (Kawakami et al., 1998). Chitin did not show an adjuvant effect in yellowtail
(Nishimura et al., 1985; Kawakami et al., 1998). Chitosan is obtained by Ndeacetylation from chitin. It is produced industrially from crab shell waste, but research
has been carried out on the use of alternative sources for chitosan mainly from fungi,
which contain chitin and chitosan in vivo in their cell wall (White et al., 1979).
Chitosan stimulates the immune system and has antibacterial activity (Suzuki and
Watanabe, 1992). Brook trout (Salvelinus fontinalis) injected or immersed in chitosan
solutions showed increased protection against A. salmonicida infection (Anderson and
Siwicki, 1994). Similar results were obtained with rainbow trout administered chitosan
orally (Siwicki et al., 1994). Rainbow trout treated with chitosan by injection or
immersion showed increases in immunological parameters in the blood, such as lytic
activity, and myeloperoxidase and total Ig concentration (Anderson et al., 1995).
1.10.3 ß-1, 3 Glucan
Glucans are macro-molecules of glucose with ß-1,3 and ß-1,6 chains. The immune
stimulatory effect of glucan and peptidoglucan has been well studied in fish (Sakai,
1999). Intraperitoneal injection of yeast glucan (ß-1,3 and 1,6 linked glucan) into
Atlantic salmon resulted in increased resistance to V. anguillarum, V. salmonicida and
Y. ruckeri (Robertsen et al., 1990). Chen and Ainsworth (1992) reported that catfish
injected with yeast glucan showed increased resistance to Edwardsiella ictaluri.
However, Thompson et al. (1995) reported that rainbow trout injected with yeast
glucan did not show enhanced protection against V. anguillarum infection. Raa et al.
(1992) reported that oral administration of yeast glucan to Atlantic salmon increased
protection against V. anguillarum and V. salmonicida. Tiger shrimp immersed in yeast
glucan solution (0.5 and 1.0 mg/ml) gave enhanced protection against V. vulnificus
36
Chapter 1 introduction
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infection (Sung et al., 1994). Moreover, yeast glucan enhances the lysozyme activity in
Atlantic salmon, rainbow trout and turbot (Thompson et al., 1995; Baulny et al., 1996.);
and complement activity, bacterial killing and superoxide production of macrophages in
rainbow trout, Atlantic salmon and catfish (Chen and Ainsworth, 1992; Yoshida et al.,
1995).
Peptidoglucan prepared from Brevibacterium lactofermentum increased phagocytosis in
yellowtail and resistance to Enterococcus seriola infection (Itami et al., 1996). The
efficacy of peptidoglucan was also demonstrated against vibriosis in rainbow trout
(Matsuo and Miyazano, 1993) and yellow head Baculovirus infection in black tiger
shrimp (Boonyaratpalin et al., 1995).
1.10.4 Animal and plant extracts
The extracts from some invertebrates have immunostimulatory effects (Sakai, 1999).
An extract from the marine tunicate, Ecteinascida turbinate (Ete), and a glucoprotein
fraction of the water extract (Hde) from abalone, Haliotis discus hannai, enhanced the
killing of tumour cells in vitro and inhibited tumour growth in vivo (Sigel et al., 1970;
Uchida et al., 1987). Eel injected with Ete showed enhanced phagocytosis and
increased survival following A. hydrophila challenge (Davis and Hayasaka, 1984).
Also, the heat extract from firefly squid, Watasenia scintillans, stimulated the immune
system of rainbow trout such as the production of superoxide anion, potential killing
activity of the macrophages and lymphocytic transformation in vitro (Siwicki et al.,
1994).
The long history of the medicinal use of garlic is well known. The intrinsic medicinal
properties of garlic, garlic extracts and some garlic constituents in vivo have been
widely documented (Augusti and Sheela, 1996). Several antioxidant compounds,
mainly polyphenols such as flavenoids and sulphur-containing compounds, have been
described in garlic (Banerjee et al., 2002). Garlic extracts increase glutathione peroxide,
superoxide dismutase (SOD), and catalase activities in vascular cells in culture (Wei
and Lau, 1998). Much information on the antibacterial, antifungal and antiprotozoal
properties of garlic are well known (Soffar and Mokhtar, 1991). Garlic extracts provide
a suitable basis for new therapies because they possess well established antimicrobial
37
Chapter 1 introduction
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actions (O’Gara et al., 2000). Angelo et al. (1998) reported inhibition of
Mycobacterium marinum growth in sea bass when treated with garlic extract.
Immunostimulatory effects of the dietary intake of ginger, Zingiber officinale, as a
medicinal plant on rainbow trout had been reported to enhance extracellular respiratory
burst and phagocytic activities of blood leucocytes (Suheyla et al., 2003). Ginger
extracts is said to have a broad range of biological responses (activities including antisecretory, antibacterial, antifungal, gastric, anticonvulsant, analgesic, antiulcer,
antitumor and other activities in humans (Newall et al., 1996; Ohara et al., 1996;
Miller, 1998). Other studies showed that gingerol helps counter liver toxicity by
increasing bile secretion (gingerol is one of the primary constituents of ginger
responsible for the pungent smell in ginger; Langmean and Rampton, 2001). Ginger is
highly valued all over the world as a spice, culinary herb and condiment, medicinal
agent and home remedy. It is hopeful that new research will undoubtedly reveal new
values for this ancient herb.
The use of rosemary, Rosmarinus officinalis as dietary treatment against Streptococcus
iniae in tilapia, Oreochromis nileticus, had been reported to be effective in inhibiting
bacterial growth. Rosemary is considered to have a bacteriostatic effect and known to
be a rich source of active metabolites used in traditional medicine to help relax muscles
including the smooth muscles of the uterus and digestive tract (Karamanolic et al.,
2000). Agriculturally, it is grown as a companion plant with cabbage, beans, carrots and
sage. The dried leaves are used in sachets to scent clothes and linen and to deter moths.
The history of clove is similar to that of nutmeg and mace as spices and medicinal
plants. Clove contains eugenol which is an effective local anaesthetic and has been used
in dentistry (Soto and Burhanuddin, 1995). Clove is the most stimulating of all
aromatics, used either in powders or infusions, and has analgesic and antiseptic
properties. Other constituents of clove, such as vanillin and iso-eugenol, have been
reported to have antimicrobial activity (Alqareer et al., 2006).
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Chapter 1 introduction
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1.10.5 Dietary components
Vitamin C is essential for normal growth and for several physiological functions in fish
(Halver, 1989). High levels of dietary vitamin C are reported to increase resistance to
Edwardsiella tarda and E. ictaluri infections in channel catfish (Durve and Lovell,
1982; Li and Lovell, 1985). Hardie et al. (1991) reported that treatment with high doses
of vitamin C increased complement activities in catfish and Atlantic salmon. The
activation of macrophages was reported in Atlantic salmon (Thompson et al., 1993) and
turbot (Robert et al., 1995). High doses of vitamin C, i.e. >1000 mg/kg) stimulated
macrophage activating factors followed by lymphocyte proliferation (Lygren et al.,
1999).
1.10.6 Other immunostimulants
Other immunostimulants, such as vitamin A, E, soybean protein, trace elements of zinc,
iron, copper and selenium, have all been tested in fish (Galinda-Villegas and
Hosokawa, 2004). Rainbow trout treated orally with soybean protein showed increased
leucocyte activities including phagocytosis, bacterial killing and the production of
superoxide (Rumsey et al., 1994). Furthermore, vitamin E enhanced both humoral and
cellular defences in mammals. Blazer and Wolke (1984) reported that specific and cellmediated immunity and macrophage phagocytosis were all compromised in rainbow
trout fed with vitamin E depleted diets. Moreover, Hardie et al. (1990) reported that
Atlantic salmon fed vitamin E depleted diets had significantly increased mortality rate
following A. salmonicida infection compared to fish receiving commercial diet
enriched with vitamin E. Wise et al. (1993) showed that catfish fed with high doses of
vitamin E had increased phagocytic indices and superoxide anion production by
leucocytes. It should be emphasised that the importance of diet in fish immune
responses has been reviewed thoroughly by Landolt (1989) and Blazer (1992).
1.10.7 Timing of immunostimulant application
Anderson (1992) proposed that immunostimulants should be administered to stock
before the outbreak of disease to reduce losses. There is evidence that
immunostimulants can be used to reversed autoimmune problems like immune-
39
Chapter 1 introduction
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suppression and tolerance caused by stress and salinity (Kitao and Yoshida, 1986).
These workers reported that rainbow trout injected with hydrocortisone showed
suppressed phagocytic activity of peritoneal and kidney leucocytes, and this
suppression of phagocytosis were reversed by injection of the immunostimulant FK565.
1.10.8 Administration methods for immunostimulants
Different farm conditions arising from scale of production and environmental changes
have given rise to different methods of application of immunostimulants. The three
basic methods reported by many authors are injection, immersion and oral uptake:
Injection: This method has been reported to enhance the function of leucocytes and to
enable protection against pathogens (Galindo-Villegas and Hosokawa, 2004). However,
this method is suitable only for intensive aquaculture and requires the fish be handled
or confined to a small space during the operation. Furthermore, it is labour intensive,
relatively time consuming and becomes impracticable when fish weigh <15 g (Sakai,
1999).
Immersion: This method is also suitable for intensive aquaculture and requires the fish
to be confined in a small space for a few hours or days during the operation. Baba et al.
(1993) reported that carp immersed in levamisole solution (10 µg/ml for 24 h) showed
activated phagocytic activities, chemotactic ability and the production of active oxygen
in head kidney phagocytes and enhanced protection against A. hydrophila. This effect
lasted for > 2 weeks. Anderson et al. (1995) demonstrated that rainbow trout immersed
in glucan or chitosan showed increased protection against A. salmonicida after
treatment for 3 days, however, this effect was transient and was not present after 14
days. Although the benefits of immersion have been reported by many authors, the
dilution, the level of efficacy and the exposure time need further investigation.
Oral uptake: oral administration is the only method economically suited for extensive
aquaculture. It is not stressful and allows mass administration regardless of fish size
and of course can be administered only in artificial diets (Galindo-Villegas and
40
Chapter 1 introduction
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Hosokawa, 2004). Oral administration of immunostimulants has already been reported
for glucans, EF 203, lactoferrin, levamisole and chitosan. Their applications resulted in
enhancement of leucocyte function and protection against infectious diseases such as
furunculosis, vibriosis and streptococcosis (Sakai, 1999). Nevertheless, the oral method
is not without disadvantages as it required sufficient quantity of source ingredient in
order to achieve the desired protection. There is the problem of precision in amount
taken up by the fish and may only be suitable for used with dietary supplementation
(Galindo-Villegas and Hosokawa, 2004).
1.10.9
Immunostimulant doses
The results from some experiments in which several compounds were tested as
immunostimulants suggested dose and exposure times have importance. Kajita et al.
(1990) showed that the chemiluminescent effects of phagocytic cells in rainbow trout
were increased by injection of levamisole at 0.1 and 0.5 mg/kg. However, these
workers also reported that the injection of 5 mg/kg of levamisole did not produce this
immunostimulatory effect. Robertsen et al. (1994) observed that the increase in
respiratory burst activity of glucan-treated macrophages was maximal at concentrations
of 0.1 – 1.0 µg/ml, whereas at 10 µg/ml no effect was seen, and at 50 µg/ml glucan was
inhibitory. Comparable effects in function were also observed in lymphocytes. Kitao et
al. (1987) determined that high doses of FK 565 (10 µg/ml) did not increase the
numbers of plaque forming cells (PFC) against Yersinia ruckeri, although the optimum
dose (5 µg/ml) increased PFC. The effects of immunostimulants are therefore not
directly dose dependent, and high doses may not enhance and may even inhibit the
immune response.
The effects of long-term exposure of fish to immunostimulants either orally or
immersion are still unclear (Sakai, 1999). Matsuo and Miyazano (1993) reported that
rainbow trout treated with peptidoglucan orally for 56 days did not show protection
after challenge with V. anguillarum, although fish treated for 28 days showed increased
protection. Yoshida et al. (1995) reported that the number of nitroblue tetrazolium
(NBT) positive cells in African catfish increased following oral administration of
glucan or oligosaccharide over 30 days, but not over 45 days.
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Chapter 1 introduction
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1.10.10
Growth activities of immunostimulants
Several workers have reported growth promoting activities associated with the use of
immunostimulants in aquaculture. Thus, Boonyaratpalin et al. (1995) reported that
black tiger shrimp fed with peptidoglycan supplemented diets showed better feed
conversion rates and growth than the control fed a normal diet. This effect was
observed with 0.01% peptidoglycan supplementation, but not with the highest level, i.e.
0.1%. Sung et al. (1994) demonstrated that black tiger shrimp grew faster with glucan
immersed at the 0.5, 1.0, 2.0 mg/ml than the controls. Sakai et al. (1996) observed that
growth hormones (GH) function as immunostimulants and enhance macrophages
activities of fish. Thus, rainbow trout injected with GH exhibited increased resistance
against V. anguillarum. Certainly, there may be close correlations between growth and
immunostimulation.
However, the deleterious side effects of immunostimulants have not been investigated.
Overdoses and long-term administration of immunostimulants reduce their efficacy (Ian
and Roy, 2005). Research has not yet been carried out on the influence of
immunostimulants on the maturation and spawning of fish, a developmental stage that
is very important in aquaculture.
It is realised that when fish spawn the immune system become suppressed by sex
hormones (Wang and Belosevic, 1995). The use of immunostimulants may lead to the
recovery of the suppressed immune system. The possibility of their causing
disturbances in the sexual maturation and other essential functions associated with
spawning are yet to be confirmed (Sakai, 1999).
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Chapter 1 introduction
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1.11 Aims and Objectives
The aim of this research was to
1. Examine the effect of dietary supplements in enhancing the immune defence
mechanisms of rainbow trout Oncorhynchus mykiss against A. hydrophila
infections.
2. Specifically, to establish the dose(s) of dietary supplements most effective at
preventing infection by A. hydrophila.
3. Determine the possible mode of action (s) of the dietary supplements in
immunomodulation.
4. Assess the dietary effect of the immunostimulants on growth performance of
rainbow trout.
5. Determine the survival rate of rainbow trout fed with dietary supplements,
following challenge with A. hydrophila.
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Chapter 2 Materials and Methods
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Chapter 2 Materials and Methods
2.1 Experimental fish
Rainbow trout Oncorhynchus mykiss of average wet weight 15 ± 0.2 g were obtained
from commercial fish farms in Scotland, and kept in aerated free-flowing freshwater at
12oC, (precise age/sex, were not given by the producer).The health status was examined
upon the arrival for evidence of diseases after Austin and Austin (1989), by random
sampling and aseptically streaking out their spleen and kidney on a plate of tryptone
soya agar (Oxoid) and incubated at 28oC for 48 h. If dense virtually pure culture growth
was obtained, the isolate was identified after Austin and Austin (2007).
2.2 Bacterial isolates
Two isolates of A. hydrophila AH1 and ORN2 were obtained from Barramundi and Koi
carp, respectively, in England, and were provided from the culture collection of the
School of Life Sciences, Heriot-Watt University, Edinburgh, UK. Stock cultures were
kept as suspensions in 0.9 % (w/v) saline supplemented with 10 % (v/v) glycerol at 70oC. Cultures were routinely grown on TSA or tryptone soya broth (TSB, Oxoid) at
25oC.
2.3 Characterisation of the bacterial isolates
2.3.1 Micro-morphology
Cultures were grown for 24 h on TSA at 25oC, with visual inspection of the colony
forming units, colour, size, shape and texture. Smears were stained by Hucker’s
modification of Gram-stain (Hucker and Conn, 1923), and examined at a magnification
of x1000 on a Kyowa light Microscope. The staining reactions and micromorphology of
the cells were noted.
2.3.2. Motility
The presence of motility was determined from wet preparations at x1000 on a Kyowa
light microscope.
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Chapter 2 Materials and Methods
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2.3.3 Catalase production
The bacterial culture was spotted into a drop of 5 % (v/v) hydrogen peroxide (SigmaAldrich) on a glass slide. A positive reaction was indicated by effervescence within 5
min.
2.3.4. Oxidase production
The method of Kovacs (1956) was used. Thus, a piece of Whatman No.1 filter paper
was
soaked
with
1%
(w/v)
oxidase
reagent
(1%
N,N,N,N-tetramethyl-p-
phenylenediamine dihydrochloride; Sigma) solution onto which was streaked bacterial
growth (grown overnight) from TSA plates. A positive reaction was recorded by the
development of a purple/blue colour within 30 s.
2.3.5 Haemolytic activity
Columbia agar (Oxoid) was supplemented with 5% (v/v) sheep blood (Oxoid).
Inoculated plates were incubated at 250C and examined daily for up to 7 days. haemolysis was recorded by the presence of an opaque greenish zone around the area of
growth. ß-haemolysis was recorded by the appearance of a clear zone around the areas
of growth.
2.3.6 Casein hydrolysis
Casein hydrolysis was observed after incubation at 28oC for up to 7 days from plates of
nutrient agar (Oxoid) supplemented with 10% (v/v) of sterile (115oC for 20 min)
skimmed milk (Oxoid). A positive reaction was recorded by the presence of a clear zone
around the bacterial growth. This was confirmed by adding 2 M HCl to the plates
(Harrigan, 1998), whereby the continued presence of the clear zone indicated casein
degradation. HCl is added to the medium to confirm changes that occur during this
reaction and secondly HCl act as electron acceptor, becoming reduced by the bacteria
enzymes, which results in a clear zone (white) in the medium, thus confirming casein
hydrolysis.
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Chapter 2 Materials and Methods
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2.3.7 Elastin hydrolysis
Elastin hydrolysis was examined by using nutrient agar supplemented with 0.3% (w/v)
elastin (Sigma-Aldrich) followed by incubation at 28oC for 7 days whereupon a positive
reaction was indicated by a zone of clearing.
2.3.8 Gelatin hydrolysis
Gelatin hydrolysis was examined according to the method of Loghothetis and Austin
(1996). Briefly, a plate of nutrient agar supplemented with 0.4 % (w/v) gelatin (Oxoid)
was seeded and incubated at 28oC for 48 h after which the plates were held at 4oC for 30
min before addition of a saturated ammonium sulphate solution. The presence of clear
zones around the growth area was indicative of a positive reaction.
2.3.9 Sensitivity to antibiotics
Antibiograms were performed on TSA plates seeded with a heavy suspension in saline
of the bacterial culture. Antibiotic sensitivity discs (Mastring) were aseptically placed
onto the freshly prepared lawns with incubation at 28oC for 48 h. Sensitivity was
indicated as the presence of a clear zone (of ≥ 3 mm) around the discs.
2.4. API 20E rapid identification system
Analytical profile index for Enterobacteriaceae API 20E kits (BioMerieux) were used
according to the manufacturer’s instructions except that incubation was at 25oC for 25
h. A comparison of the results was made to the BioMerieux database.
This kit provides an easy way to identify members of the Enterobacteriaceae and
associated organisms. The kit comprises of plastic strip holding 20 mini-test tubes. The
strip is normally inoculated with a saline suspension of a pure bacterial culture (as per
manufacturer's instructions). This process also rehydrates the desiccated medium in
each tube. A few tubes are completely filled (CIT, VP and GEL) and some tubes are
overlaid with mineral oil such that anaerobic reactions can be carried out (ADH, H2S,
LDC, ODC and URE). Incubation is in a humidity chamber for 24 hours at 37°C. After
the incubation, the colour reactions are noted (some with the aid of added reagents).
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Chapter 2 Materials and Methods
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The reactions and the oxidase reaction done separately, and the data are converted to a
seven-digit code, which is entered into the manufacturer's computerised database
(BioMerieux, Inc; Hazelwood, MO), identification is usually given to genus and
species.
2.5. Salt aggregation
This test was carried out to ascertain the surface characteristic of the bacterial isolates.
A dense suspension of the bacterial culture was prepared from overnight culture in
phosphate buffered saline, pH 7.4 (PBS; Oxoid). One drop was mixed with an equal
amount of 0.2 M ammonium sulphate in PBS on a glass microscope slide. The slide was
gently rocked for 2 min and observed against a dark background for aggregation of
cells.
2.6
16S rRNA gene sequencing
2.6.1 DNA extraction
The bacterial cultures were grown in TBS for 24 h at 28oC, and harvested by
centrifugation at 3000 x g for 10 min at 4oC, washed twice and resuspended in 500 µl
(0.5 ml) PBS, the bacterial cells were lysed with Tris- EDTA, and the DNA extracted
using a DNA extraction Kit (a DNeasy Tissue Kit; Qiagen) following the
manufacturer’s protocol for extraction of genomic DNA.
2.6.2 Conventional PCR conditions
In a sterile 0.2 ml amplification tube (Greiner), several components of PCR reaction
mixtures were seeded to perform the PCR in the following order: 25 µl of biomix buffer
(Bioline) solution containing 1.0 unit Taq DNA polymerase (Amersham Bioscience);
1.0 µl of forward and reverse primers (MWG- Biotech); 1.0 µl of template DNA; 2.0
mM stock solution of four d NTPs, 1.5 mM MgCl2 , 32 mM (NH4)2SO4, 125 mM TrisHCl and 0.02 % (v/v) Tween 20, and 15.0 µl sterile MilliQ (Millipore) water to achieve
a final volume of 50 µl.
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Chapter 2 Materials and Methods
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The nucleic acids were amplified using an icycler (Bio-Rad) and the denaturation,
annealing and polymerization (extension) times were as follows: initial denaturation at
96oC for 4 min, followed by 30 cycles of denaturation at 95oC for 30 s; annealing at
55oC for 30 s; polymerization (extension) at 72oC for 1 min, and a final extension at
72oC for 7 min. Controls, without target DNA, were included in the amplification
process alongside with test samples. The test reaction mixtures were stored at -20oC
until needed. DNA molecular markers used in the study were Gene Ruler TM 1 kb DNAl
ladders (MBI Fermentas) and provided 14 discrete fragments in base pairs (bp): 10000,
8000, 6000, 5000, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 750, 500 and 250 bps.
2.6.3 Primers used in the 16S rRNA sequencing
The DNA sequencing assay was carried out following the methodology of Brunt and
Austin (2005), with slight modifications. Briefly, the bacterial cells were lysed and the
DNA extracted using a DNeasy tissue Kit (Qiagen), following the protocol for isolation
of total DNA from animal tissues. DNA templates were amplified by the polymerase
chain reaction (PCR) on a Perkin Elmer Gene APP 2400, using the universal primers.
27 F (5 ‘ AGAGTTTGATCMTGGCTCAG-3’
685 r
3
(5 ‘ TCTRCGCATTYCACCGCTAC-3’) Lane (1991) obtained from MWG
Biotech.
2.6.4 Agarose gel electrophoresis of DNA
For the assessment of DNA extracted from bacterial isolates, (A. hydrophila AH1 and
ORN2), agarose gel electrophoresis was carried out using the Horizon 58 Gel
Electrophoresis Apparatus (Gibco, BRL). One g of Agarose (Integra Bioscience) was
dissolved in 100 ml of Tris- EDTA (TBE) buffer by heating in a microwave oven. After
the dissolution, the solution was allowed to cool at room temperature, before 1.0 µl of
ethidium bromide (Sigma-Aldrich) was added. The gel solution was thoroughly mixed
and poured into a taped gel casting tray with 14 wells integrated comb, and left to cool
and solidified in a cool room for 4 h. After this, the solidified mass of agarose gel was
transferred into a PCR chamber and electrophoresis buffer–Tris-acetate EDTA (TAE)
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Chapter 2 Materials and Methods
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was poured into the PCR chamber to cover the agarose gel to the top, followed by
careful removal of the comb to reveal the 14 sample wells.
5 µl of a gel loading buffer was prepared to include:
0.25 % (w/v) bromophenol blue
0.0125 ml
0.25 % (w/v) ethylene cyanol
0.0125 ml
25 % (v/v) glycerol
1.25 ml
1 % (w/v) sodium dodecyl sulphate (SDS)
0.05 ml
150 mM EDTA at pH 8
1.5 ml of 0.5 M EDTA
Sterile water
2.25 µl to achieve a final volume of 5 µl.
10-20 µl of sample DNA was mixed with 2 µl of gel loading buffer before loading the
wells with the sample DNA solution. A marker (10000 bps) was loaded on one of the
wells for a comparative molecular weight determination of the DNA. A blank (without
target DNA sample) was pipetted into one other well for use alongside the test sample.
Electrophoresis was then carried out by connecting the electrophoresis chamber to the
source of power and allowed to run for 2 h at 93 V or for an unspecified number of
times until the tracking dye had diffused to the bottom of the agarose gel. Following
electrophoresis, the DNA banding patterns on the agarose gel was immediately
observed with a ultra violet (UV) Tran illuminator (UV Products). Photographic records
were taken using a gel documentation unit (Amersham Bioscience).
2.6.5 Sequencing of purified PCR products
The purified PCR products were sequenced using 20 µl reaction mixtures containing 4
µl of big dye deoxy Terminator Cycle Sequencing Kit (Applied Biosystems), 4 µl of x 5
sequencing buffer, 1 µl of either forward or reverse primer (5p mol/µl), 2 µl of purified
PCR products and 9 µl of sterile MilliQ (Millipore) water. Cycle sequencing reactions
were accomplished by initial denaturation at 98oC for 5 min, followed by 25 cycles of
denaturation at 96oC for 10 s, annealing at 50oC for 5 s and extension at 60oC for 4 min.
Cycle sequenced templates were purified by the addition of 40 µl of 75% (v/v)
isopropanol (Sigma-Aldrich), then incubated at room temperature for 10 min to allow
for precipitation of the DNA. Extractions were centrifuged at 10,000 x g for 20 min to
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Chapter 2 Materials and Methods
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pellet the DNA. The overlying supernatants were removed, and then the resultant pellets
were washed thrice with 125 µl of 70 % (v/v) isopropanol to remove intemperate dyes
and repeated thrice. The resultant samples were dehydrated at 90oC for 1 min.
Purified templates (dehydrated samples) were resuspended in 25 µl of template
suppression reagent (TSR, Applied Biosystems) heated at 95oC for 2 min, and were
analysed using an ABI Prism 310 Genetic Analyser (Applied Biosystems). The
resulting chromatograms were examined using Chromas Pro Version 1.21 software
(Technelysium), and forward and reverse sequences were compared and corrected using
GENETYX version 7.0.3 packed programs (Software Development Co.). The resulting
16S rRNA gene sequences were compared in a BLAST search with those in the
National Library of Medicine database.
2.7 Determination of LD50 doses
The lethal dose 50% (LD50) of the pathogens was determined using rainbow trout of
average weight 12 g. The fish were divided into 5 groups using random sampling
method. Each group containing 12 animals and 6 of which were injected
intraperitoneally (i.p.) and intramuscularly (i.m.) with 0.1 ml volumes of 10 fold
dilutions of freshly prepared AHI bacterial suspensions in saline ranging from 102, 103,
104, 105, and 106 viable cells/fish, and observed for 7 days. The same were repeated for
ORN2 bacterial suspensions. Dead fish were subjected to standard microbiological and
pathological examinations. The LD50 value was calculated after the method of Reed and
Muench (1938).
Thus:
Critical dilutions between which 50 % mortality lies =
[Mortality above 50 % - 50 / mortality above 50 % - mortality below 50 %].
Therefore LD50 %= dilution above 50 % mortality + critical dilution between which
50 % mortality lies.
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2.8 Compounds evaluated for immunostimulatory activity
2.8.1 Ginger, Zingiber officinale
Fresh rhizomes of ginger were obtained from a supermarket in Edinburgh, Scotland.
The ginger was washed, peeled, shredded and dried for 24 h at room temperature. The
dried ginger was then ground using a pestle and mortar and sieved using a household
sifter (1/1cm wire mesh, reinforced mesh with metal body), and mixed directly with
commercial fish feed (Biomar) to achieve 0 g (control), 0.05 g, 0.1 g, 0.5 g and 1.0 g
ginger/100 g of feed. The modified feed was stored in screw cap bottles at room
temperature until needed.
2.8.2 Garlic, Allium sativa
Oven-dried garlic bulbs were obtained from a local supermarket in Edinburgh and
crushed using a household garlic press, and mixed with commercial fish feed (Biomar,
Denmark) to achieve 0 g, 0.05 g 0.1 g, 0.5 g and 1.0 g/100 g of feed. The modified feed
was stored in screw cap bottles at room temperature.
2.8.3. Rosemary, Rosmarinus officinalis
Rosemary was obtained in packets form of shredded dry leaf from a supermarket in
Edinburgh. This was grinded into a fine powder and mixed with the basal diet (Biomar)
at the rate of 0 g, 0.05 g 0.1 g, 0.5 g and 1.0 g/100 g of feed and stored in an airtight
container at room temperature until needed.
2.8.4 Clove, Eugenia aromaticum
Clove was obtained in packets as powder, from a supermarket in Edinburgh. This was
mixed with the basal diet (Biomar) at the rate of 0 g, 0.05 g 0.1 g, 0.5 g and 1.0 g/100 g
of feed and stored in an airtight container at room temperature until needed.
2.8.5 Other compounds
A range of other compounds was evaluated, and included ß-1, 3 glucan, chitin, chitosan,
vitamin C, and lipopolysaccharide (LPS) which was obtained from Sigma and were
mixed with the commercial feeds at the rate of 0 g, 0.05 g 0.1 g, 0.5 g and 1.0 g/100 g of
feed, except LPS which was used at the rate of 0, 1. 87, 3.75, 7.5, 15 mg/100g, in view
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of their toxicity at high dose (Gultvik et al., 2002; Nayak et al., 2008). These feeds were
stored at 4oC until needed.
2.9 Preliminary screening of putative immunostimulants
The ability of the compounds to confer health benefits to rainbow trout against A.
hydrophila infections was examined initially to produce a shortlist of useful products
for further detailed study. The preliminary screening methods focuses on the
determination of inhibitory activity of the compounds and in vivo study to determined
the protective effect of these compounds.
2.9.1 Determination of inhibitory activity
Antimicrobial activity was assessed Austin et al. (1992) and Patrizia et al. (2003).
Briefly, bacterial suspension were streaked on plates of TSA and incubated for 24 h at
28 C. A drop (0.25 µl) or grains of the compounds were spotted with a micro-dilutor
handle onto the inoculated TSA plates with further incubation for 24 h at 28oC.
Antimicrobial activity was indicated by a zone of inhibition on the lawn.
2.9.2 In vivo studies
The effect of compounds was assessed in experiments with rainbow trout average wet
weight 15 ± 0.2 g. Thus, 125 fish were randomly distributed in five experimental
groups following a completely randomized design (CRD) and maintained in aerated,
dechlorinated water at 12oC. The fish were fed with diets containing 0 g, 0.05 g 0.1 g,
0.5 g and 1.0 g/100 g of each compound, except LPS which was used at 0, 1.87, 3.75,
7.5 and 15 mg/100g-of feed. Feeding was twice daily to satiation for 14 days before
challenge with A. hydrophila.
2.9.3 Experimental challenge and determination of growth performance
The challenge was by i.p. injection with 0.1 ml suspensions of A. hydrophila AH1
containing 106 cells/ml. Previous work had determined the LD 50% to be x 104 cells/
ml. This was adjusted to a higher dose of x106 cells/ml for the challenge to achieve
approximately 80% mortalities depending on the growth of cultured isolates and to
allow for minor differences in age and size of fish with their attendant differences in
resistance. Different batches of fish stock from the aquaria, which comprises of
unspecified sex (genetic status = polyploidy) were used.
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Mortalities were recorded over 14 days, and any dead or moribund fish examined
bacteriologically to confirm the presence of A. hydrophila (after Austin and Austin,
2007).
The relative percentage survival (RPS) was calculated according to Amend (1981).
Sub-groups of 10 fish were used to determine growth performance in which the
percentage weight gain, specific growth rate (SGR), feed conversion ratio (FCR) and
protein efficiency ratio (PER) were determined according to Choudhury et al. (2005):
Wt. gain % = Final wt. – Initial wt / Initial wt. X 100.
SGR
= Loge of Final wt. – Loge of Initial wt. / No. of days
FCR
= Feed given (dry wt.) / Body wt. gain (wet wt.)
PER
= Net wt. gain (wet wt.) / protein fed.
RPS
= [1- (mortality in treatment group/mortality in control group) x 100]
2.10 Mode of action
2.10.1 Induced cellular immune response
Separate groups of 125 rainbow trout were fed with the experimental compounds for 14
days and used to determine the possible modes of action and effect on immune
parameters. Thus, blood was collected by venepuncture from groups of 10 freshly killed
fish, and transferred into vacuette tubes containing heparin as anticoagulant (Greiner) to
prevent clotting. This blood was used for determination of haematocrit (Hct),
haemoglobin (Hb) content, and total erythrocyte and leucocyte counts. For this, the
blood was diluted to 102 and 103 in PBS at pH 7.2, and the number of leucocytes and
erythrocytes counted after Sarder et al. (2001). Duplicate blood samples were also
collected and allowed to clot in at room temperature for 2 h, and stored overnight at 4oC
before the clotted blood was centrifuged at 3000 rpm for 10 min at 4oC, and the serum
collected and stored at -70oC until use.
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2.10.2 Population of blood cells
To obtain the differential leucocyte counts, smears from whole blood were prepared on
slides with fixation for 5 min in 96 % (v/v) methanol. After 10 min air- drying at a room
temperature, the slides were stained with May-Grunwald/Giemsa solutions for light
microscopy according to Sarder et al. (2001). Three areas were randomly chosen in a
slide and examined at a magnification of x400 for differential cell count according to
Pavlidis et al. (2007). The packed cell volume (PCV) was determined according to a
method described by Shoemaker et al. (2003) by using standard plain heparin coated
microhaematocrit tubes, 75 mm long with 1.1–1.2 mm internal diameter. Blood samples
were touched with the heparinized capillary tube and the blood drawn three-quarters of
the way up the tube by capillary action. The tubes were sealed at one end with sealant,
i.e. Critoseal. The samples were then centrifuged in a MSA microhaematocrit centrifuge
for 5 min at 7000 rpm. The length of the column containing pack red blood cells and
buffy supernatant was measured in a microhaematocrit reader.
Haemoglobin concentration was assessed following the cyano-methahaemoglobin
method (Van Kampen and Zijlstra, 1965), using a commercially available kit
(Quantichrom haemoglobin assay kit from BioAssay systems) and recording the
absorbance at OD 400 nm in a Tecon micro plate reader.
2.10.3 Head kidney macrophages
The isolation of head kidney macrophages was done for the evaluation of phagocytic,
respiratory burst and bacteriocidal activities. Using aseptic technique, the head kidneys
were removed from fish, crushed in a Jencons tissue grinder and diluted 1:10 in RPMI
1640 (Gibco-Invitrogen), filtered (0.22 µm Millipore Millex porosity filters) in RPMI
1640 containing 1 µl/100 m of penicillin and streptomycin (Sigma-Aldrich), 0.2 mg/100
ml heparin (Sigma-Aldrich) and 0.1% (v/v) foetal calf serum (FCS, Sigma-Aldrich)
(Sakai et al., 1995). Macrophages were obtained by forcing the suspension through 100
µL nylon mesh (Simon) before layering onto a 34/51 % (v/v) percoll gradient (SigmaAldrich) in Hank’s Balanced Salt Solution (HBSS) (Sigma-Aldrich) and centrifuging at
2,500 rpm for 25 min at 4oC. The cells were collected from the interface and washed
twice with RPMI 1640 and adjusted to 105 cells/ml using a haemocytometer slide at a
magnification of x 400 on a Kyowa microscope. Cell viability was determined by using
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trypan blue exclusion in which cell suspensions were stained with 0.4 % (w/v) trypan
blue (Sigma-Aldrich) and the percentage of viable (unstained) and dead (stained) cells
was scored after Sakai et al. (1995).
2.10.4 Phagocytosis activity
The phagocytic activity of the head kidney macrophages was examined by a method
according to Sakai et al. (1995). Volumes of 1 ml (106 cell/ml) of cell suspension was
spread over triplicate glass microscope slides and incubated for 60 min at 18oC to allow
for attachment of the cells. The slides were washed in fresh RPMI 1640 medium to
remove unattached cells before adding 1.0 ml of 0.8 µm diameter latex bead suspension
(Sigma–Aldrich) and adjusted to contain 109 latex beads/ml. This was incubated at 18oC
for 60 min. Thereafter the slides were washed 3 times in fresh medium and fixed for 35 min at room temperature with 96% (v/v) methanol, air- dried and stained by Giemsa
method (Sakai et al., 1995). The slides were examined at x400 magnification and
triplicate groups of 100-200 cells were counted to determine the proportion of cells with
ingested latex beads. The phagocytic activity (PA) was calculated as:
PA = Number of phagocytosing cells/Total number of cells x 100.
The phagocytic index (PI) was determined by the number of latex beads phagocytosed
per cell.
2.10.5 Respiratory burst activity
The respiratory burst activity of the macrophages was determined from the reduction of
nitroblue tetrazolium (NBT; Sigma-Aldrich) to formazan as a measure of super oxide
anion (O2-) production, after Secombes (1990) as modified by Stasiack and Bauman
(1996). Briefly, samples (50 µl) was pipetted into the wells of U-bottom microlitre
plates (Nunc) and incubated at 37oC for 1 h to facilitate cell adhesion. The supernatant
was removed and the cells washed 3 times in PBS. Thereafter, 50 µl of 0.2% (w/v) NBT
was added and incubated for further 1 h. The cells were then fixed with 100% (v/v)
methanol for 2-3 min. and washed again, 3 times with 30% (v/v) methanol. The plates
were air-dried at room temperature before 60 µl of 2 N potassium hydroxide (KOH)
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(Sigma–Aldrich) were added to each well to dissolve the resulting formazan blue
crystals. The OD was read in a Tecan ELISA reader) at OD 540 nm.
Parallel experiments using whole blood cells was also carried out to evaluate the
respiratory burst activity of the neutrophils in experimental groups of fish and the
controls according to Kumar et al. (2005).
2.10.6 Bacteriocidal activity
The bacteriocidal assay was performed according to Selvaraj et al. (2005). Briefly,
bacterial cells of the pathogen were grown in TSB for 24 h and adjusted to x 10 7
CFU/ml in 0.9% (w/v) saline. Thereafter, 0.1 ml volumes of the bacterial suspensions
was removed and mixed with 0.1 ml of macrophage suspensions (adjusted to x 106
cells/ml). Subsequently, 0.04 ml of fresh rainbow trout serum collected from 10 fish
was added and incubated for 2 h with shaking every 15 min. in a water bath at 27oC.
Finally, 0.1 ml of the bacteria-macrophage mixture was diluted with 9.9 ml of sterile
distilled water to release living bacteria from phagocytosis. This was serially diluted to
101-106 and plated on TSA plates with incubation overnight at 37oC. The number of
colonies was counted. To confirm that macrophages were actually killing the bacteria, a
control assay was carried out in the absence of macrophage to give 100% survival of all
bacterial dilutions.
2.11 Induced humoral immune response
2.11.1 Lysozyme activity
The lysozyme activity of serum was measured by using a method based on the ability of
lysozyme to lyse cells of Micrococcus lysodeikticus (Lange et al., 2001). Briefly, 100 µl
of sera in four-twofold serial dilution (1/5 - 1/40) in sodium phosphate buffer (SPB;
0.05 M, pH 6.2) was pipetted into the wells of a 96-well micro titre plate (Nunc) and
mixed with 100 µl of 0.4 mg/ml suspension of M. lysodeikticus in SPB (Sigma–
Aldrich). The micro titre plate was incubated at 22oC and the OD was read on a Tecan
ELISA reader at 590
nm,
after 0, 15, 30 and 60 min. For a positive control, serum was
replaced by a serial dilution of hen egg white lysozyme (ICN) starting at 1.6 µg/ml. For
a negative control, buffer replaced serum. A unit of lysozyme activity was defined as
the amount of serum causing a decrease in the OD reading of 0.001/min. (Brunt and
Austin, 2005).
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2.11.2 Serum antiprotease activity
The serum anti-protease activity was measured according to Magnadottir et al. (1999).
Thus, 20 µl of serum was incubated with 20 µl of standard trypsin solution (SigmaAldrich, T-7409), 1000- 2000 BAEE 5 mg/ml) for 10 min at 22oC. Thereafter, 200 µl of
0.1 M PBS (pH 7.2) and 250 µl of 2% (w/v) of azocasein solution (20 mg/ml PBS) were
added and incubated for 1 h at 22oC. The reaction was then terminated with the
addition of 500 µl of 10 % (v/v) trichloro acetic acid (TCA) and incubated for 30 min at
22oC. The mixture was centrifuged at 600 x g for 5 min and 100 µl of the supernatant
was transferred to a 96-microwell flat bottom plate containing 100 µl of 1 N NaOH per
well. The OD was read at 430
nm.
An enzyme activity of 100% was obtained by not
adding serum to the reaction mixture. The control for each test sample was obtained by
buffer which replaced both serum and trypsin. The percentage inhibition of trypsin
activity was calculated by comparing the average of the absorbance reading with the
value of the 100% enzyme activity thus allowing the calculation of residual activity of
trypsin based upon the amount (µl) of serum used in term of percentage trypsin
inhibition as described by Zuo and Woo (1997).
% Trypsin inhibition = % Trypsin OD – Test sample OD / % Trypsin OD x 100.
2.11.3 Natural haemolytic complement activity
The alternative complement pathway activity was assayed according to a method
described by Selvaraj et al. (2005) using sheep red blood cells (SRBC) in Elsevier’s
solution (Oxoid) as a target. The SRBC were washed in phenol red-free HBSS
containing Mg2+ and ethylene glycol-bis (2- aminoethoxy) - tetra acetic acid (EGTA)
and resuspended at 3% (v/v) in HBSS containing Mg2+ and EGTA. Aliquots of 100 µl
of test serum as complement source were serially diluted in HBSS to give a final serum
concentration ranging from 10% to 0.078% and mixed with an equal volume (100 µl) of
SRBC in duplicate wells of 96-well plates. After incubation for 90 min at 22oC, the
samples were centrifuged at 400 x g for 5 min at 4oC to remove unlysed SRBC. The
values of maximum (100%) and minimum haemolysis were obtained by adding 100 µl
of distilled water to 100 µl samples of SRBC with and without serum, respectively
(Diaz-Rosales et al., 2006). Lysis curves were obtained by plotting percentage
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haemolysis against the volume of test serum added on a log10- log10 scaled paper. The
volume yielding 50% haemolysis (ACH 50, unit/ml) was determined for each treatment.
2.11.4 Serum biochemical parameters
Serum biochemical parameters were analysed using a kit (Quantichrom) from Bio
Assay Systems. Serum total protein was estimated by a method based on an improved
Bradford assay (Bradford, 1976). Thus, standard and serum samples were diluted in
distilled water according to the manufacturer’s instructions, and 10 µl of both diluted
standard and samples were transferred into duplicate wells of clear bottom 96-well
plates. Then, 200 µl of working reagent was added to each well and mixed gently. The
OD of standard and test samples was measured against a blank in a Tecan microplate
reader at 595nm.
To produce the standard curve, the OD value of a blank was deducted from the standard
OD value and plotted against the standard concentration. Then, the OD values of the
serum samples were plotted onto the standard curve to obtain the sample protein
concentration. The albumin content was estimated by the bromocresol green binding
method (Kamphuis et al., 2001) using a commercial kit from BioAssay Systems. Thus,
standards were diluted in distilled water according to the manufacturer’s instruction and
serum samples were serially two-fold diluted in distilled water. Then, 20 µl of diluted
standard and samples were transferred into wells of clear-bottom 96-well plates before
200 µl of working reagent was added to each well and mixed gently with incubation for
5 min at a room temperature. The OD was taken against a blank at 620nm in a Tecan
microplate reader. The OD values of the samples were plotted onto the standard curve
to obtain the sample albumin concentration. The globulin content was calculated by
subtracting albumin values from serum total protein. Albumin/globulin ratio was
estimated by dividing albumin values by those of globulin (Jha et al., 2007).
2.11.5 Serum electrolyte indices
Electrolytes, i.e. calcium (Ca++), magnesium (Mg++), sodium (Na+), potassium (K+) and
ferrous (Fe+) ppm/ml were determine by flame emission photometry according to
Rehulka (2000) using an automated system – Atomic Absorption Spectrometer (Perkin
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Elmer precisely Analyst 200) with appropriate standard. Thus, the standard was diluted
in distilled water according to the manufacturer’s instructions by carrying out a 10-fold
dilution. After which, 5 ml of this dilution was added to 5 ml of distilled water to obtain
5 ppm. This was further diluted to obtain 2.5 ppm, 1.25 ppm and 0.625 ppm. With the 3
lowest dilutions, i.e. 0.625, 1.25 and 2.5 ppm, calibrations were done. After calibration
of the instrument, following the machine protocols, blank (distilled water) was analysed
first, follow by standard and then the serum samples, all in triplicate.
2.12 Determination of effective dose of compounds for controlling A. hydrophila
infection
The effective doses were determined in experiments using compounds dosed at 0 g (=
controls), 0.05 g, 0.1 g, 0.5 g and 1.0 g/100 g of feed, except for LPS which was used at
0, 1.87, 3.75, 7.5 and 15 mg/100g of feed. The fish were fed for 14 days and challenged
as before. The cumulative mortalities were recorded daily for 14 days. Dead and
moribund fish were removed and examined after Austin and Austin (2007). For this,
swabs were taken from the internal organs of dead fish, particularly from the kidney and
spleen, and spread onto TSA plates with incubation at 25oC for 48 h to determine the
presence or absence of A. hydrophila. Survivors were sacrificed at the end of the
experiments, and examined to confirm that they were free from infections. The
cumulative mortality and RPS of different treatment groups were calculated according
to Burrells et al. (2001).
2.13 Digestive enzymes
2.13.1 Preparation of crude enzyme extracts
After the 14 days feeding periods, the fish were starved for 24 h before sacrifice by
overdose in anaesthetic (MS-222 Tricaine Methane Sulphonate) and removal of the
intact digestive tracts. In all, groups of 10 fish were used to determine enzymatic
activity. The procedures were as described by Borlongan (1990). For this, the fish were
weighed individually and then incised ventrally. The digestive tract of each fish was
dissected and ligated at points to isolate specific regions, i.e. the stomach, pylori caeca,
liver/ pancreas, small intestine and posterior intestine (for brush border membrane).
Tissues from each region were washed gently with distilled water and weighed. Pooled
tissues were homogenized using a mechanical dispenser (Tissue grinder homogenizer
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glass vessel of 30 ml capacity, Jencons) and then subjected to an ultrasonic cell
disintegrator (Sonicator- MSE). The homogenates were centrifuged at 15000 rpm for 30
min at 4oC. A clear supernatant were obtained and used as the crude extract in
subsequent assays. The pH of the clear supernatant was determined with a digital pH
meter. Total protein content of each tissue (stomach, liver/pancreas, pylori caeca and the
intestine) were determined by a method based on an improved Bradford assay
(Bradford, 1976).
2.13.2 Pepsin activity
Pepsin activity was determined by the method described Sarath et al. (1989). The acid
pepsin activity from the stomach crude enzyme extract was measured at pH 3.0 and 6.5
at 20oC, with haemoglobin as substrate. The incubation mixture consisted of 2.5 ml of
2% bovine haemoglobin dissolved in 32% urea in 25 mM phosphate buffer at pH 2.0
and 0.4 ml of homogenate enzyme sample with incubation for 10 min at 20oC. The
reaction ended with the addition of 5.0 ml of TCA (5.0%). For a blank, TCA was added
before the enzymatic extracts. The mixtures were allowed to stand for 30 min at room
temperature, and then filtered through Whatman No. 3 filter paper. The Absorbance
(Ab) of the filtrates was taken at 700 nm with reference to the blank:
Specific activity (U) was determined according to Natalia et al. (2004) and expressed
as:
[Ab (filtrate) – Ab (blank) / 10 min x mg protein] x 100.
2.13.3 Total protease activity
The total protease activity of stomach crude extract was estimated at pH 3.0 and 9.0 at
25 oC with the use of the azocasein digestion assay described by Garcia-Carreno (1992).
For this, 10 µl of enzyme homogenate was incubated with 500 µl of 0.5% azocasein in
Tris-HCl buffer (50 mM; pH 7.5) for 10 min. The reaction was stopped with 500 µl of
20% trichloroacetic acid (TCA). Samples were centrifuged at 12,000 x g for 5 min. and
the absorbance was recorded at 405 nm for 3 min. For a control, TCA was added first to
the extract before the addition of the substrate.
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Specific activity of the total protease activities (U) was calculated as:
[Ab (supernatant) – Ab (blank) / 10 min x mg protein] x 100.
2.13.4. Alkaline phosphatase (AP) activity
The AP activity of the intestinal brush border membrane was determined by measuring
the hydrolysis of paranitro-phenol by the mucosal enzymes thus yielding paranitrophenol and inorganic phosphate, which was then measured spectrophotometrically at
410 nm (Harpaz and Uni, 1999) using a Tecan ELISA reader. Homogenized enzyme
extract (20 µl) was added to 100 µl of alkaline buffer solution (Sigma-Aldrich) and
incubated at 37oC for 5 min. The addition of 2.0 ml of 0.05 N NaOH stopped the
reaction. The absorbance was then recorded at 410 nm. A unit of alkaline phosphatase
activity was calculated as the amount of enzyme activity liberating 1.0 mM of pnitrophenol/min. The extinction coefficient of p-ntrophenol used for the calculation was
18.45 mMM/cm (Lemieux et al., 1999).
2.13.5 Total proteolytic enzyme activity
The total proteolytic enzyme activity was estimated from the intestinal and hepatopancreas crude enzyme homogenates at pH 6.50 and 6.42 respectively, with incubation
at 25oC by the use of azocasein as substrate according to Garcia-Carreno and Haard
(1993). Briefly, 10 µl of enzyme homogenate was incubated with 0.5 ml of 0.5%
azocasein in 50 mM Tris-HCl buffer, pH 7.5 for 10 min. the reaction was stopped with
the addition of 0.5 ml of 20% TC). Samples were centrifuged at 14,000 rpm for 5 min
and the absorbance recorded at 405 nm for 3 min. For azocasein, one unit of proteolytic
activity corresponds to the amount of enzymes releasing 1.0 µg azocasein/min. under
the assay condition. The extinction coefficient of azocasein (3648) was used for
calculating proteolytic activity.
2.13.6. Trypsin activity
Trypsin activity was assayed from the intestinal and hepato-pancreas crude enzyme
extract at pH 6.50 and 6.42, respectively, with incubation at 25oC using benzoylarginine-p-nitroanilide (BAPNA) as substrate according to Erlanger et al. (1961).
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Enzyme homogenate (200 µl) was mixed with Tris-HCl buffer [(0.1 M, pH 8.3
containing NaCl (1M) and CaCl2 (10 mM)] before 200 µl BAPNA (1.0 mM) was
added. After 1 h incubation at 25oC, the reaction was ended by adding 200 µl of 20%
acetic acid. Absorbance was recorded at 415 nm for 3 min. Trypsin activity was
calculated according to the formula of Erlanger et al. (1961).
Ab (test/ min) x 1000 x volume of reaction mixture/8800 x mg protein in the reaction
mixture.
Here, 8800 is the molar extinction coefficient of BAPNA (Erlanger et al., 1961).
2.14 Duration of protection
Separate groups of 125 rainbow trout were fed with compounds, as before, and used to
determine the effect on immune parameters and duration of protection. For this, rainbow
trout fingerlings were fed with the experimental compounds for 14 days. Physiological
factors, biochemical, immunological, haematological parameters and electrolyte indices
were evaluated after a further 14, 21 and 28 days before challenge with A. hydrophila
AH1 isolate.
2.14.1 Determination of immunological parameters and protection
As before, mortalities after challenge were monitored over 14 days, and any dead or
moribund fish examined bacteriologically to confirm the presence of A. hydrophila
(after Austin and Austin, 2007). The RPS was calculated after Amend (1981).
2.14.2 Serum peroxidase content
The total peroxidase content of the serum was measured according to Diaz-Rosales et
al. (2006). Here, 15 µl of serum was diluted with 35 µl of Ca
2+
and mg2+ free HBSS
(Sigma-Aldrich) in a flat-bottom 96-well plate. Then, 50 µl of 20 mM 3,3, 5,5tetramethylbenzidine hydrochloride (TMB, Sigma-Aldrich) and 5 mM H2O2 (SigmaAldrich), both substrates of peroxidase, were added. Serum volume of 150 µl was
transferred from each well to a new 96-well plate and incubated for 2 min. The colour
change reaction was stopped by adding 50 µl of 2 M sulphuric acid and the OD was
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read at 540
nm
in a Tecan ELISA reader. Standard samples without serum were also
analysed.
Other immunological parameters such as total erythrocyte and leucocyte counts,
phagocytic activity and index, bacteriocidal activity, lysozyme activity, respiratory burst
activities and serum anti-protease activity were as described previously (Nya and
Austin, 2009).
2.15 Determination of active components of the beneficial compounds
In order to gain insight into the mode of action(s) of garlic components, one
commercially prepared allicin product (Allimed® liquid) at 0, 0.5 and 1.0%
concentrations was studied, using a range of in vitro and in vivo methods. Thus, 150 fish
were randomly distributed into 3 experimental groups with 2 replicates, following a
CRD and fed with 0, 0.5 and 1.0 ml Allimed® liquid/100g of basal feed for 14 days
before challenge with A. hydrophila AH1 isolate.
2.15.1 Determination of inhibitory activity of allicin
The antimicrobial property of allicin against A. hydrophila was determined from zones
of clearing on freshly seeded lawns after incubation for 24 h at 37oC (Austin et al.,
1992; Patrizia et al., 2003). Briefly, Suspension of the bacterial cultures (0.1ml) was
transferred aseptically to Petri dish of standard trypsin soya agar (TSA; Oxoid) and
streaked out with sterile swab to spread the bacteria evenly and then, incubated for 24 h
at 37oC for a standard lawn formation. A drop (0.25 ml) of liquid Allicin were spotted
on the lawn plates and further incubated for 24 h at 37oC. Antimicrobial or inhibitory
activity of Allicin commercial products was indicated by a clear zone of inhibition on
the lawn area around the spot of the producer. The inhibitory effect of the products was
quantified by measuring the clearing zones in millimetres.
2.15.2 Determination of the minimum inhibitory concentration (MIC)
The MIC was estimated according to Cai et al., (2007). For this, bacterial cultures were
prepared in TSB with overnight incubation at 37OC.
Then, the cultures were
centrifuged at 3000 x g for 10 min at 4oC before the cells were washed twice in PBS,
pH 7.4, and the pellets resuspended in fresh buffer. The concentration was adjusted to
5.6 x 107 cells/ml as determined by means of a haemocytometer slide (Improved
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Neubauer Type, Merck) at a magnification of x400 on a Kyowa light microscope.
Suspensions of bacterial cultures (0.1 ml) were streaked out with a sterile swab to
spread the bacteria evenly TSA plates to facilitate standard lawn formation.
A two–fold dilution of allicin was made and tested on the plates of the bacterial
cultures. The results were recorded after plates were incubated at 37oC for 24 h. MIC4580
was estimated as the lowest concentration of allicin that inhibited 45-80 % of the
bacterial growth.
2.15.3 Determination of mode of action of allicin
The determination of haemoglobin (Hb) content, total erythrocyte and leucocyte counts,
phagocytic activity and index, bacteriocidal activity, lysozyme, respiratory burst and
serum anti-protease activities were performed as previously described (Nya and Austin,
2009).
2.16 Statistical analysis
Values for each parameter measured were expressed as the arithmetic mean ± standard
error (SE). Effects of dietary compounds on growth performance, haematological,
biochemical and immunological parameters were tested using one-way ANOVA and a
comparison of the mean values was done by using Duncan’s multiple range tests
(Duncan, 1955), at the 5 % level of significance. The software programme SPSS
(Version 14.0) (SPSS) for Windows was used.
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Chapter 3 Results
3.1 Characteristics of the bacterial isolates
The two cultures of A. hydrophila AH1 and ORN2 showed only minor differences in
their characteristics as shown in Table 7. Essentially, the characteristics are consistent
with the description of A. hydrophila (Austin and Austin, 2007).
Table 7 Morphological and biochemical characteristics of the bacterial isolates
Characteristics
AH1
ORN2
Colour
Creamy /yellow
Creamy / yellow
Size
3-4 mm diameter
3-4 mm diameter
Shape
Conical
Conical
Texture
Viscous
Less viscous
Gram staining reaction
–
–
Rods
+
+
Motility
+
+
Cocci
–
–
Capsular material
–
–
Endospores
–
–
Oxidase
+
+
Catalase
+
+
nitrate
+
+
Reduction of nitrite
–
–
 haemolysis
–
–
 haemolysis
+
+
Casein hydrolysis
+
+
Elastin hydrolysis
+
+
Salt aggregation
+/–
+/–
Colony morphology:
Biochemical characteristic
Reduction of nitrite to
+ = Positive reaction, – = Negative reaction, +/– = Weak reaction
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3.2 Sensitivity to antimicrobial compounds
The antimicrobial sensitivity profiles of the isolates are presented in Table 8. Thus, the
cultures were sensitive to chloramphenicol, colistin sulphate, cotrimoxazole, gentamycin,
nalidixic
acid,
nitrofurantoin,
streptomycin,
sulphamethizole,
sulphatriad
and
tetracycline, but resistant to ampicillin, cephalothin, carbenicillin and penicillin G (Table
8).
Table 8 Antimicrobial sensitivity profiles of bacterial isolates.
Antibiotics
Bacterial isolates
A. hydrophila AH1
ORN2
a. Ampicillin (10 µg)
R
R
b. Chloramphenicol (25µg)
S
S
c. Penicillin G (1 International Unit)
R
R
d. Streptomycin (10µg)
S
S
e. Sulphatriad (200µg)
S
S
f. Tetracycline (25µg)
S
MR
g. Ampicillin (25µg)
R
MR
h. Gentamycin (10µg)
S
S
i. Carbenicillin (100µg)
R
R
j. Nalidixic acid (30µg)
S
S
k. Nitrofurantoin (50µg)
S
S
l. Sulphamethizole (200µg)
S
S
m. Tetracycline (100µg)
S
S
n. Cotrimoxazole (25µg)
S
S
o. Cephalothin (5µg)
R
R
p. Colistin sulphate (25µg)
S
S
S = Sensitive, R = Resistance, MR = Moderate resistance.
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3.3 Use of the API 20E rapid identification system
The API 20E rapid identification system enabled identification of both isolates (Table 9).
Thus, AH1 and ORN2 were identified as A. hydrophila with 100 % and 99 % confidence,
respectively, according to the manufacturer’s database.
Table 9 Identification of bacterial isolates by the API 20E rapid identification system.
Test
Bacterial isolate
AH1
ORN2
a. ß- galactosidase
+
+
b. Arginine dihydrolase
+
+
c. Lysine decarboxylase
+
–
d. Ornithine decarboxylase
–
–
e. Citrate utilization
–
–
f. Hydrogen sulphide production
–
–
g. Urease production
–
–
h. Tryptophan deaminase production
–
–
i. Indole production
+
+
j. Voges-Proskauer reaction
+
+
k. Gelatinase production
+
+
m. Glucose
+
+
n. Mannose
+
+
o. Inositol
–
–
p. Sorbitol
–
–
q. Rhamnose
–
–
l. Production of acid from:
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r. Saccharose
+
+
s. Melezitose
–
–
t. Amygdalin
+
+
u. Arabinose
–
–
v. Oxidase production
+
+
+ = positive, – = negative reaction
3.4 16S rRNA gene sequencing
The two isolates were identified using partial sequencing of the16S ribosomal RNA gene
which revealed homologies of > 99 % to A. hydrophila according to the NCBI BLAST
database. In particular, the forward and reverse sequences of AH1 produced 100 %
alignment with A. hydrophila AN-1, AE57, AE55 and AE53, i.e. Accession No. AY
987735.1, AY987734.1, AY987733.1 and 987732.1 respectively. The analysis of the
ORN2 sequence produced 99 % alignment with A. hydrophila ATCC 7966 and CCM
7232 with Accession No. CP 00046.1 and D0 207728.2 respectively (Fig. 3; Table 11).
The size of nucleotide (amplicon) fragments in base pairs (bp) were 1400 - 1500 bps.
500bp
250bp
C A O
Fig. 3. 16S rRNA, PCR of A. hydrophila AH1and ORN2 DNA products.
(C= Control, A=AH1, O= ORN2).
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Table 11 Closest relative as determined by BLAST search and accession number.
Bacterial isolate
Aeromonas
hydrophila AH1
Aeromonas
hydrophila
ORN2
Closest relative obtained from Blast
search
Aeromonas hydrophila strain AE57 16
rRNA gene
Identity
(%)
100
Accession
number
AY987734.1
Aeromonas hydrophila strain AN-1 16S
rRNA gene
100
AY987735.1
Aeromonas hydrophila subsp. AE55
16S rRNA gene
100
AY987733.1
Aeromonas sp. AE53 16S rRNA gene,
complete sequence
100
AY987732.1
Aeromonas punctata strain MPT4 16S
rRNA gene
100
DQ979324.1
Aeromonas sp. GPTSA100-19 16S
rRNA gene, partial sequence
100
DQ859921.1
Aeromonas hydrophila strain
CCM7232 partial 16S rRNA gene
99
DO207728.2
Aeromonas hydrophila strain ATCC
7966 16S rRNA gene
99
CP00046.1
3.5 Determination of LD50 of the bacterial isolates
The LD50 of the two strains was determined to be 1.7 x 103 and 1.7 x 104 CFU/fish for A.
hydrophila ORN2 and AH1 respectively, calculated from i.p. injection only, which was
also used throughout the study. Infection by i.m injection could not be estimated due to
some problems with the infected fish, and consequently were not used in the study.
3.6 Preliminary screening of immunostimulants
Results of the preliminary screening of ten novel candidate immunostimulants are
summarized in Table 12 and 13. Three compounds, i.e. garlic (Allium sativum; Fig. 4),
ginger (Zingiber officinale) and LPS led to enhanced protection of 95, 71 and 100 %
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relative percentage survival (RPS) respectively, compared to the controls (Fig. 5).
Consequently, these compounds were examined further.
Table 12 Preliminary screening of ten putative dietary supplements used at 1 % dose,
except LPS (15 mg per 100 g of feed).
Compound
No. of
mortalities
1±0.0
No. of
survivals
24±0.0
RPS (%)
Garlic
No. of fish
challenged
25
Clove
25
9±1.0
16±1.0
57
β1,3-glucan
25
9±3.0
16±3.0
57
Ginger
25
6±1.0
19±1.0
71*
Chitosan
25
11±2.0
14±2.0
48
Chitin
25
14±1.0
11±1.0
33
Vitamin C
25
14±2.0
11±2.0
33
LPS
25
0±0.0
25±0.0
100*
Rosemary
25
11±1.0
14±1.0
48
Tea (Camelia
sinensis)
25
8±1.0
17±1.0
62
Control
25
21±1.0
4±1.0
-
95*
*= selected for in-depth studies. Two replicates were used in the trials. Data are
presented as Mean±SE (n = 2).
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Fig. 4. Rainbow trout protected by garlic showing normal internal organs (spleen, kidney
and intestines), skin and fins after challenge with A. hydrophila.
Fig.5. Control fish exhibiting muscle necrosis after challenge with A. hydrophila
3.6.1
Production of inhibitory activity by putative immunostimulants
The production of inhibitory properties against AH1 and ORN2 is summarised in Table
13. Essentially, the data revealed that rosemary, clove, garlic and vitamin C but not
chitin, chitosan, ginger, LPS, β1,3-glucan or tea were inhibitory to AH1 and ORN2.
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Table 13 Production of inhibitory compounds against bacterial isolates
Compounds tested
Bacterial isolate
Ginger
A. hydrophila AH1
MS
ORN2
R
Rosemary
S
S
Clove
S
S
Garlic
S
S
Vitamin C
S
S
LPS
MS
R
β1,3-Glucan
R
R
Chitin
R
R
Chitosan
R
R
Tea (Camelia sinensis)
MS
R
S = sensitive, i.e. with a zone of clearing of ≤ 6mm, MS = moderate sensitive with
narrow zone of clearing; R = resistance, i.e. the absence of any zone of clearing.
3.7 Influence of dietary supplements on growth performance of rainbow trout
The feeding of supplemented diets to rainbow trout generally stimulated appetite. In
particular, during the first 4 days of use, the fish showed a better feeding response
compared with the controls. The specific growth rate (SGR) of the fish was 1.2±0.4 in the
control group and 1.9±0.2 and 2.0±0.9 in 0.5 g and 1.0 g garlic 100 g-1 of feed
respectively. The feed conversion ratio (FCR) and protein efficiency ratio (PER) was also
enhanced in supplemented diets treated group compared with the control. The overall
data are summarized in Table 14.
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Chapter 3 Results
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Table 14. Growth performance of rainbow trout fed with supplemented diet for 14 days.
Treatment
Group
% Weight Gain
SGR
FCR
PER
(g/100 g of feed)
Control
49.2±6.3
1.2± 0.4
2.0±0.5
0.9± 0.1
Garlic
0.05
50.2±3.4
1.4± 0.1
1.8±0.9
1.0± 0.1
0.1
55.8±3.1
1.6± 0.1
1.5±0.9
1.2± 0.1
0.5
57.7±3.5
1.9± 0.2
1.2±0.8
1.5± 0.1
1.0
60.7±3.1
2.0± 0.9
1.1±0.9
1.7± 0.1
Control
18.4±1.5
0.2±0.02
0.5±0.04
0.2± 0.04
0.05
19.2±0.7
0.5±0.02
0.2±0.1
0.3± 0.03
0.1
23.8±0.6
0.7± 0.02
0.2±0.1
0.4± 0.03
0.5
26.2±0.4
0.7± 0.003
0.1±0.1
0.5± 0.02
1.0
31.4±0.4
0.8± 0.002
0.1±0.1
0.6± 0.02
Control
11.1±1.6
0.3± 0.02
0.4±0.02
0.2± 0.10
1.875
11.7±1.8
0.3± 0.03
0.3± 0.1
0.2± 0.04
3.75
20.6±1.7
0.6± 0.04
0.2±0.1
0.4± 0.03
7.5
18.1±2.2
0.5± 0.04
0.2±0.1
0.3± 0.01
15
14.3±1.4
0.4± 0.03
0.3±0.1
0.3± 0.03
Ginger
(mg LPS/100 g
of feed).
Data expressed as Mean ± SE, P < 0.05, n = 10. SGR, Specific growth rate, FCR, Feed
conversion Ratio, PER, Protein efficiency ratio.
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3.8 Effect of dietary supplements on the survival of rainbow trout after challenge with
A. hydrophila (AH1)
The use of feed additives for 14 days led to a marked reduction in mortalities after
challenge with A. hydrophila (Fig. 6.1, .2 and .3). Thus, in fish fed with garlic, the total
cumulative percentage mortality was 88 % in the control group and 12 %, (RPS = 86 %),
8 % (RPS = 91%), 4 % (RPS = 96 %) and 4 % (RPS = 96 %), in the groups which
received 0.05, 0.1, 0.5 and 1.0 g garlic/100 g of feed, respectively (Fig. 6.1) Similar data
were recorded for use of ginger which led to 64 % mortality in the controls compared to
16 % (RPS = 75 %), 4 % (RPS = 94 %) and 0 % (RPS = 100 %) mortalities in the groups
which received 1.0 g, 0.05 g and 0.5 g ginger/100 g of feed, respectively (Fig. 6.2). Also,
the fish fed with LPS led to 45 % mortalities in the controls within 7 days post challenge,
whereas among the experimental groups the were 5 % mortalities (RPS= 89 %) in those
which received 1.875 mg and 3.75mg/100 g of feed (Fig. 6.3), and 10 % (RPS= 78 %)
and 15 % (RPS=67 %) mortalities for those dosed with 7.5 mg and 15 mg LPS/100 g of
feed, respectively. In all, the survivors of the treated groups did not show any sign of
cumulative mortality (%)
disease at the end of experiments.
100
80
control
60
0.05g %
0.1g %
40
0.5g %
20
1g %
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
days after challenge with A. hydrophila
Fig. 6.1. Percentage cumulative mortality of rainbow trout following intraperitoneal injection
with x106 cells /fish of A. hydrophila after feeding garlic supplemented diet for 14 days.
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Chapter 3 Results
Cumulative mortality (%)
_____________________________________________________________________
70
60
control
50
0.05g %
40
0.1g %
30
0.5g %
20
1g %
10
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14
Days after challenge with A. hydrophila
Fig. 6.2. Percentage cumulative mortality of rainbow trout following intraperitoneal
injection with x106 cells /fish of A. hydrophila after feeding ginger supplemented diet for
14 days.
100
90
cumulative mortality (%)
80
70
control
60
1.875mg
50
3.75mg
7.5mg
40
15mg
30
20
10
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
day after challenge
Fig. 6.3. Percentage cumulative mortality of rainbow trout following intraperitoneal
injection with x106 cells/fish of A. hydrophila after feeding LPS supplemented diet for 14
days.
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Chapter 3 Results
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3.9 Mode of action
3.9.1 Haematological parameters
The results of the haematological parameters of rainbow trout after use of supplemented
diets are included in Table 15. Overall, the dietary compounds induced significant
changes (P < 0.05) in erythrocyte and leucocyte counts, compared with the control.
However, there was an increase in the erythrocyte count in fish which received 0.5 g and
1.0 g garlic/100 g of feed; the same trend is seen in ginger and LPS (Table 15). The
percentage haematocrit was significant (P < 0.05) when compared to the control group
(Table 15). However, there were not any significant differences in the haemoglobin
contents of the control and the treatment groups. In fact, the haemoglobin content
oscillated from the control values of 0.59±0.05 % to 0.71±0.0 % in 0.5 g garlic/100 g of
feed. The same trend is seen in all other treatments (Table 15).
Table 15 Haematological data of rainbow trout fed with supplemented diets for 14 days.
Treatment
(g/100 g of feed)
RBC
(x106/ml)
Control 6.4±1.7b
Garlic
0.05
6.0±3.1b
104.7±1.9c
31.6±1.6a
0.5±0.3
0.1
7.1±2.1b
228.5±1.6b
27.5±1.9b
0.5±0.3
0.5
12.9±0.7a
289.6±0.9a
27.8±0.5b
0.7±0.0
1.0
14.1±2.7a
247.7±2.1b
26.0±3.2b
0.8±0.4
Control 2.4±1.7b
164.1±1.5c
24.6±1.8b
0.8±0.0
0.05
2.9±3.3b
112.3±2.3c
29.2±1.1a
0.6±0.0
0.1
6.6±1.6b
187.6±1.1b
28.2±1.7a
0.6±0.2
0.5
9.1±3.1a
243.7±1.0a
27.0±0.7a
0.7±0.1
1.0
12.9±9.2a
233.1±1.9b
28.3±4.9a
0.7±0.4
Control 6.8±0.7b
84.1±1.9c
23.6±1.6b
0.8±0.0
7.6±0.5b
87.5±1.2c
33.8±1.0a
0.7±0.0
Ginger
(mg LPS/100 g
of feed).
Group
1.875
WBC
(x 103/ml)
164.1±1.5c
Hct. (%)
Hb. (g %)
24.6±1.8c
0.6±0.2
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Chapter 3 Results
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3.75
8.4±1.1b
248.3±1.8a
35.6±2.0a
0.6±0.0
7.5
12.6±1.9a
299.9±0.9a
34.4±0.7a
0.8±0.2
15
17.5±0.9a
173.1±0.9b
25.0±2.6b
0.7±0.4
Data expressed as M±SE. significant (P < 0.05; n=10). Hct = Haematocrit, Hb =
Haemoglobin. Mean values with the same superscripts in the same treatment column are
not significantly different (P < 0.05, n =10).
3.9.2 Population and partial differential blood cells
The differential leucocyte counts were also affected by dietary supplements. In particular,
the lymphocyte level showed a significant difference (P < 0.05) in groups receiving 1.0 g
garlic/100 g of feed, increasing from the control value of 21.94±2.2 % to 33.87±12.24 %
in the group fed with garlic. In fish receiving ginger, the proportion of lymphocytes
increased from 32.4±0.7 in the controls to 35.0±1.1 % in the experimental group, which
received 1.0 g ginger/100 g of feed (Table 16). The lymphocyte level showed significant
differences (P < 0.05) in groups receiving 15 mg and 3.75 mg LPS/100 g of feed,
increasing from the control value of 20.46 % to 43.56 % and 43.30 %, respectively.
Higher numbers of neutrophils and monocytes were observed in all experimental groups.
Lower numbers of thrombocytes were found in all experimental groups. Eosinophils and
basophils were absent in all the samples (Fig. 7 a-d).
Table 16 Mean differential proportions of leucocyte in rainbow trout fed dietary
supplements for 14 days.
Treatment
Group
Control
Lymphocyte Monocyte
s (%)
s (%)
21.9±2.2c
11.7±1.4
Neutrophils
(%)
59.2±1.5
Thrombocytes
(%)
7.1±0.7
(g/100g of feed)
Garlic
0.05
24.1±1.2b
13.0±0.7
53.7±0.7
9.2±0.7
0.1
21.5±1.0c
13.0±1.2
55.3±4.0
10.3±1.1
0.5
24.0±0.8b
14.6±3.5
57.1±9.7
4.3±0.5
1.0
33.9±1.2a
16.2±2.5
45.5±6.1
4.5±0.8
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Ginger
(mg LPS /100g
of feed).
Control
32.4±0.7
16.2±0.9b
35.2±0.6
16.2±0.9
0.05
29.6±1.4
25.9±1.2a
38.9±1.0
5.6±0.8
0.1
30.2±1.7
28.6±1.4a
35.7±1.0
5.5±0.7
0.5
30.5±3.5
26.3±3.5a
38.40±1.6
4.8±0.3
1.0
35.0±1.0
18.0±1.5b
40.3±9.4
6.7±0.5
Control
20.5±1.0b
13.6±1.0c
54.6±1.0a
11.3±1.0
1.875
40.69±1.0a
20.0±1.0b
30.5±1.7b
8.4±0.7
3.75
43.3±1.7a
24.6±1.2b
28.1±1.0b
4.2±1.0
7.5
41.6±1.0a
28.5±1.5a
25.6±7.4c
4.4±2.0
15
43.6±1.9a
17.0±1.5c
36.2±4.6b
3.3±1.2
Data expressed as M±SE. significant (P < 0.05; n=10). Mean values with the same
superscripts in the same treatment column are not significantly different.
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a
b
c
d
Fig.7 a- d. Proliferation of cells in experimental groups of fish; a= control, b= ginger,
c= garlic and d= LPS treated blood.
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Chapter 3 Results
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3.10 Immunological parameters
3.10.1 Head kidney macrophages and phagocytosis activity
The head kidney leucocytes comprised macrophages (mature monocytes), granulocytes,
lymphocytes and thrombocytes. The phagocytic activity of the isolated head kidney
macrophages are shown in Fig. 8.1, .2 and .3. It was recorded that the phagocytic activity
of the head kidney macrophages was greatly affected by the dietary supplements. The
phagocytic ratio was significantly (P < 0.05) higher in fish fed 1.0 g garlic/100 g of feed,
i.e. 70.33 % followed by 0.5 g garlic/100 g of feed namely 40.80 % and compared with
the control group with 19.70 % (Fig. 8.1). In ginger, the phagocytic ratio was higher, i.e.
55.3 %, 45.7 % and 26.7 %, when administered 1.0 g, 0.5 g and 0.1 g ginger/100 g of
feed, compared to 18.75 % of the controls (Fig. 8.2).
The phagocytic ratio was
significantly (P < 0.05) higher in fish fed 7.5 mg LPS/100 g of feed, i.e. 50.20 %,
followed by 15 mg LPS/100 g of feed (35.0 %) and 3.75 mg LPS/100 g (29.16 %)
% phagocytic activity
compared to the controls at 18.75 % (Fig. 8.3).
90
80
70
60
50
40
30
20
10
0
a
b
c
0
c
c
0.05
0.1
phag. Ratio
phag. Index
0.5
1
g garlic / 100 g of feed
Fig. 8.1: The effect of garlic supplemented diet on the phagocytic activity (phagocytic
ratio and index) of the head kidney macrophages of rainbow trout, after 14 days feeding.
Data are expressed as mean ± SE, n = 10. Bars with different superscripts are
significantly different (P < 0.05, n =10).
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Chapter 3 Results
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% phagocytic activity
70
a
60
b
50
40
30
c
c
phag. ratio
phag. index
c
20
10
0
0
0.05
0.1
0.5
1
g ginger / 100 g of feed
Fig. 8.2. The effect of feeding ginger supplemented diet on the phagocytic activity
(phagocytic ratio and index) of the head kidney macrophages of rainbow trout, after 14
days feeding. Data are expressed as mean ± SE, n = 10. Bars with different superscripts
are significantly different (P < 0.05, n =10).
% phagocytic activity
70
a
60
50
b
c
40
30
c
phag. ratio
phag. index
c
20
10
0
0
1.875
3.75
7.5
15
mg LPS / 100 g of feed
Fig. 8.3. The effect of feeding LPS supplemented diet on the phagocytic activity
(phagocytic ratio and index) of the head kidney macrophages of rainbow trout, after 14
days feeding. Data are expressed as mean ± SE, n = 10. Bars with different superscripts
are significantly different (P < 0.05, n =10).
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3.10.2 Respiratory burst activity
The production of superoxide anion, as examined by Nitroblue tetrazolium (NBT)
reduction was significantly (P < 0.05) influenced by diet supplementation (Fig. 9.1, .2,
.3). A significant (P < 0.05) increase in the respiratory burst activity was observed in fish
treated with 0.1 g and 0.5 g garlic/100 g of feed, with 0.558 ± 0.37 and 0.242± 0,032 OD
nm
respectively, compared with the control group with 0.188± 0.035 (Fig.9.1). Also, a
significant increase in the respiratory burst activity, i.e. 0.675± 0.35 and 0.266± 0.01, was
observed in fish treated with 0.1 g and 0.5 g ginger/100 g of feed, respectively, compared
with the 0.142± 0.25 of the controls (Fig. 9.2). Also, parallel increases in respiratory burst
activity was observed in fish treated with 3.75 mg and 7.5 mg LPS/100 g of feed with
levels of 0.477± 0.2 and 0.254±0.04 respectively, as compared with the control group
with 0.142± 0.25 (Fig. 9.3).
Optical density at (540 nm)
0.7
a
0.6
0.5
0.4
b
0.3
0.2
b
b
b
0.1
0
0
0.05
0.1
0.5
1
g garlic / 100 g of feed.
Fig. 9.1: Effect of garlic supplemented diet on the respiratory burst activity by blood
leucocytes of rainbow trout. Values are expressed as mean± SE. Bars with the same
superscripts are not significantly different (P < 0.05, n =10).
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Optical density at (540 nm)
0.9
a
0.8
0.7
0.6
0.5
b
0.4
0.3
c
c
c
0.2
0.1
0
0
0.05
0.1
0.5
1
g ginger / 100 g of feed.
Fig. 9.2. Effect of feeding ginger supplemented diet on the respiratory burst activity by
blood leucocytes of rainbow trout. Values are expressed as mean± SE. Bars with the
same superscripts are not significantly different (P < 0.05, n =10).
0.7
a
optical density at (540 nm)
0.6
0.5
0.4
0.3
b
c
c
0
1.875
c
0.2
0.1
0
3.75
7.5
15
mg LPS / 100 g of feed
Fig. 9.3. Effect of feeding LPS supplemented diet on the respiratory burst activity by
blood leucocytes of rainbow trout. Values are expressed as mean± SE. Bars with the
same superscripts are not significantly different (P < 0.05, n =10).
83
Chapter 3 Results
_____________________________________________________________________
3.10.3 Bacteriocidal activity
The effect of dietary supplements on the macrophage bacteriocidal activity is shown in
Fig. 10.1, .2 and .3. Bacteriocidal activity was significantly (P < 0.05) higher in groups
fed with 1.0 g, 0.5 g and 0.1 g garlic/100 g of feed and in fish fed with 0.1 g, 0.5 g and
1.0 g ginger/100 g of feed when compared with the controls. LPS was most effective at
7.5 and 15 mg LPS/100 g of feed (Fig. 10.1, .2 and .3). The viable bacterial colony
2000
1800
1600
1400
1200
1000
800
600
400
200
0
c
1
Garlic
without serum
b
0.
5
0.
1
0.
05
a
1
wi
th
ou
ts
er
um
a
a
0
No. of bacterial colonies
counts were significantly (P < 0.05) lower in these groups compared with controls.
g garlic / 100 g of feed
Fig. 10.1: Effect of garlic supplemented feed on the bacteriocidal activity of rainbow
trout. Values expressed as M±SE. Bars with different superscripts are significantly
different (P < 0.05, n =10).
84
Chapter 3 Results
2000
1800
1600
1400
1200
1000
800
600
400
200
0
c
c
ginger
without serum
b
w
ith
ou
ts
1
er
um
a
0.
5
0.
1
0.
05
b
0
No. of bacterial colonies
_____________________________________________________________________
g ginger / 100 g of feed
Fig. 10.2. Effect of feeding ginger supplemented diet for 14 days on bacteriocidal activity
of rainbow trout. Values are expressed as mean± SE, n=10.
Bars with different
superscripts are significantly different. (P < 0.05).
450
Number of bacterial colonies
400
350
b
300
b
250
LPS
b
200
without serum
150
a
100
a
50
w
it h
ou
t
se
ru
m
15
7.
5
3.
75
1.
87
5
0
0
mg LPS / 100 g of feed
Fig. 10.3. Effect of feeding LPS supplemented diet on the bacteriocidal activity of
rainbow trout. Values expressed as M±SE. Bars with different superscripts are
significantly different (P < 0.05, n =10).
85
Chapter 3 Results
_____________________________________________________________________
3.11 Induced humoral immune responses to dietary supplements
3.11.1 Lysozyme activity
The impact of dietary supplements on serum lysozyme activity is shown in Fig. 11.1, .2
and .3. The results indicated that serum lysozyme activity was significantly (P < 0.05)
higher in groups fed with 0.1 g and 1.0 g garlic/100 g of feed at 15 min, 30 min and 60
min reading time, when compared with the controls. With 0.1 g ginger/100 g feed, the
serum lysozyme activity was also increased significantly (P < 0.05) at 15, 30 and 60 min
(Fig. 11.2). Conversely in the groups receiving 3.75 mg, 7.5 mg and 1.875 mg LPS/100
g, serum lysozyme activity was significantly (P < 0.05) higher at 15 min, but at 30 min
and 60 min, only 3.75 mg LPS/100 g exhibited high lysozyme activity when compared
lysozyme activity (unit/ml)
with the controls (Fig. 11.3).
2500
a
2000
a
1500
1000
c
b
c
b
b
b
b
b
c
b
a
a
b
500
0
15 30 60 15 30 60 15 30 60 15 30 60 15 30 60
min. min. min. min. min. min. min. min. min. min. min. min. min. min. min.
0
0.05
0.1
0.5
1
g garlic / 100 g of feed
Fig. 11.1: Effect of garlic supplemented diet on lysozyme activity of rainbow trout after
14 day feeding regimes. Values are expressed as mean± SE, P < 0.05, n=10. Bars with
different superscripts in the same sampling time are significantly different (P< 0.05, n
=10).
86
Chapter 3 Results
lysozyme activity (unit/ml)
_____________________________________________________________________
3000
2500
2000
1500
1000
500
0
a
a
b
c
b
b
b
a
b
c
b
b
c
d
c
15 30 60 15 30 60 15 30 60 15 30 60 15 30 60
min. min. min. min. min. min. min min. min. min. min. min. min. min. min.
0
0.05
0.1
0.5
1
g ginger /100 g of feed
Fig. 11.2. Effect of feeding ginger supplemented diet for 14 days on lysozyme activity of
rainbow trout. Values are expressed as mean± SE, n=10. Bars with different superscripts
lysozyme activity (unit/ml)
in the same sampling time are significantly different. (P < 0.05).
3000
2500
2000
1500
1000
500
0
a
a
c
b
b
b
b
a
b
b
b
b
d
c
c
15
30
60
15
30
60
15
30
60
15
30
60
15
30
60
min. min. min. min. min. min. min. min. min. min. min. min. min. min. min.
0
1.875
3.75
7.5
15
mg LPS /100 g of feed
Fig. 11.3. Effect of feeding LPS supplemented diet for 14 days on lysozyme activity of
rainbow trout. Values are expressed as mean± SE, n=10. Bars with different superscripts
in the same sampling time are significantly different. (P < 0.05).
87
Chapter 3 Results
_____________________________________________________________________
3.11.2 Serum anti-protease activity
The dietary supplements had significant (P < 0.05) effect on the serum anti-protease
activity compared with the controls (Fig. 12.1, .2 and .3). However, 0.05 g and 0.1 g
garlic/100 g of feed did not lead to any significant (P < 0.05) increase in the anti-protease
activity. Conversely, feeding fish with ginger at 0.5 g and 1.0 g/100 g of feed has a
significant (P < 0.05) effect on serum anti-protease activity, i.e. 68.85 % and 65.87 %
respectively, compared with the 34.6 % of the controls (Fig. 12.2). Moreover, 15 mg
LPS/100 g of feed showed high anti-protease activities with 61.56 % compared to the
control with 41.42 %.
90
% trypsin inhibition
80
70
b
60
b
50
a
a
0.5
1
b
40
b
30
20
10
0
0
0.05
0.1
g garlic / 100 g of feed
Fig. 12.1 Anti-protease activity of rainbow trout fed for 14 days with diet supplemented
with garlic. Data are expressed as M± SE. Bars with the same superscripts are not
significantly different (P < 0.05, n =10).
90
% trypsin inhibition
a
a
80
70
60
50
b
c
b
40
30
20
10
0
0
0.05
0.1
0.5
1
g ginger / 100 g of feed
Fig. 12.2. Anti-protease activity of rainbow trout fed with ginger for 14 days. Data are
expressed as M± SE. Bars with the same superscripts are not significantly different (P <
0.05, n =10).
88
Chapter 3 Results
_____________________________________________________________________
80
a
% trypsin inhibition
70
60
b
50
b
b
c
40
30
20
10
0
0
1.875
3.75
7.5
15
mg LPS / 100 g of feed
Fig. 12.3. Anti-protease activity of rainbow trout fed with LPS for 14 days. Data are
expressed as M± SE. Bars with the same superscripts are not significantly different (P <
0.05, n =10).
3.11.3 Natural haemolytic complement activity
The results of serum alternative complement pathway activity, measured by volume of
test serum (/ml) yielding 50% haemolysis (ACH50 Unit/ml), is shown in Fig, 13.1, .2 and
.3. The highest activity was observed in 0.1 g garlic and 7.5 mg LPS/100 g of feed with
32.2±0.3 and 19.80±1.0 Units/ml, respectively, compared with (50.0±1.4 Units/ml) in the
controls. In ginger supplementation, the highest activity was observed in 0.5 g ginger/100
g of feed, followed by 1.0 g and 0.1 g ginger/100 g of feed, with 39.8±1.07, 40.0±0.67
and 41.36±0.96 Units/ml respectively, compared with (190.0±1.35 Units/ml) in the
controls.
70
ACH 50 % (units / ml)
60
c
c
50
b
b
0.5
1
a
40
30
20
10
0
0
0.05
0.1
g garlic / 100 g of feed
Fig. 13.1. Serum natural haemolytic complement activity of rainbow trout fed with garlic
supplemented diet for 14 days. Data are represented as Mean±SE (n=10). Bars with
different superscripts are significantly different (P < 0.05).
89
Chapter 3 Results
ACH 50 % (units / ml)
_____________________________________________________________________
300
c
250
200
b
150
100
a
a
a
0.1
0.5
1
50
0
0
0.05
g ginger / 100 g of feed
Fig. 13.2. Serum natural haemolytic complement activity of rainbow trout fed with
ginger supplemented diet for 14 days. Data are represented as Mean±SE (n=10). Bars
ACH 50 % (units / ml)
with different superscripts are significantly different (P < 0.05).
70
60
50
c
c
c
40
a
30
a
20
10
0
0
1.875
3.75
7.5
15
m g LPS / 100 g of feed
Fig. 13.3. Serum natural haemolytic complement activity of rainbow trout fed with LPS
for 14 days. Data are represented as Mean±SE (n=10). Bars with different superscripts
are significantly different (P < 0.05).
3.12 Serum biochemical parameters
The effect of dietary supplements on the serum biochemical parameters of rainbow trout
fingerlings is shown in Table 17. The serum total protein content was significantly (P <
0.05) higher in groups fed with 15 mg, 3.75 mg and 7.5 mg LPS/100 g of feed, i.e.
2.56±0.18 mg/ml, 1.74±0.15 mg/ml and 1.51±0.13 mg/ml respectively, compared with
1.09±0.18 mg/ml in the control group. The comparative values for the groups, which
received 0.1 g and 0.5 g ginger/100 g of feed, were 1.77±0.16 mg/ml and 1.68±0.15
mg/ml, respectively. The same trend was seen in garlic supplemented diets. The albumin
90
Chapter 3 Results
_____________________________________________________________________
content and albumin/globulin ratio (A/G) did not vary significantly (P < 0.05) in any of
the treated groups compared with the controls.
Table 17 Biochemical indices of rainbow trout after feeding with dietary supplements for 14
days.
Treatment Group
Garlic
Ginger
(mg LPS/
100 g of
feed).
Total protein Albumin
Globulin
Albumin: globulin
g/100 g feed
(mg/ml)
(mg/ml)
(mg/ml)
ratio.
0
1.47±0.1c
0.26±0.0
1.21±0.1c
0.2±0.0
0.05
1.18±0.1c
0.25±0.0
0.93±0.0c
0.3±0.0
0.1
1.94±0.1b
0.22±0.0
1.72±0.1b
0.1±0.1
0.5
1.38±0.1c
0.23±0.0
1.15±0.1c
0.2±0.0
1.0
2.51±0.2a
0.31±0.1
2.21±0.1a
0.1±0.2
0
1.47±0.1c
0.26±0.0
1.2±0.1b
0.2±0.0
0.05
1.05±0.1c
0.23±0.0
0.9±0.0c
0.3±0.0
0.1
1.77±0.1b
0.30±0.0
1.7±0.1b
0.1±0.1
0.5
1.68±0.1b
0.27±0.0
1.1±0.1b
0.2±0.0
1.0
2.34±0.2a
0.23±0.1
2.2±0.1a
0.1±0.0
0
1.09±0.2c
0.24±0.1
0.85±0.1c
0.3±0.1
1.875
1.15±0.2c
0.24±0.0
0.91±0.2c
0.3±0.0
3.75
1.74±0.2b
0.26±0.1
1.48±0.1b
0.2±0.1
7.5
1.51±0.1b
0.96±0.1
0.55±0.1c
0.8±0.0
15
2.56±0.2a
0.46±0.1
2.10±0.1a
0.2±0.1
Data expressed as M±SE. Data with different superscripts in the same treatment group
and columns are significantly different (P < 0.05, n = 8).
91
Chapter 3 Results
_____________________________________________________________________
3.12.1 Serum electrolyte indices
The result of the electrolyte indices i.e. ion metabolism, which is a biomarker of acidbase regulations, is presented in Table 18. The values of calcium were observed to be
higher relative to the controls in groups fed with ginger and LPS. Magnesium and
potassium was significantly (P < 0.05) higher in groups treated with 0.5 g of garlic and
ginger; and 7.5 mg of LPS, as compared with the controls.
Table 18 Mean electrolyte indices of rainbow trout fed with dietary supplements for 14 days
Treatment
Calcium
Magnesium
Iron
Sodium
Potassium
(mg/l)
(mg/l)
(mg/l)
(mg/l)
(mg/l)
Control
2.48±0.00a
0.71±0.01b
0.73±0.01
2.39±0.02a
1.67±0.02a
0.1 g garlic
1.42±0.01b
0.56±0.01b
0.34±0.01
1.43±0.03b
0.30±0.00b
0.5 g garlic
2.24±0.01a
1.26±0.01a
0.40±0.02
2.31±0.13a
1.83±0.01a
1.0 g garlic
2.14±0.01a
0.59±0.01b
0.57±0.00
2.40±0.09a
1.71±0.04a
Control
2.18±0.01
1.17±0.01a
0.23±0.01
2.30±0.0a
1.46±0.02a
0.1 g ginger
2.31±0.02
0.69±0.01b
0.28±0.00
1.48±0.07b
0.38±0.00b
0.5 g ginger
2.14±0.02
1.36±0.01a
0.40±0.01
2.33±0.03a
1.84±0.01a
1.0 g ginger
2.31±0.12
0.72±0.00b
0.66±0.01
2.41±0.08a
1.88±0.02a
Control
2.40±0.00
0.73±0.01b
0.34±0.00
1.50±0.06b
0.24±0.00b
3.75 mg LPS 2.07±0.01
0.68±0.01b
0.34±0.01
1.50±0.07b
0.35±0.00b
7.5 mg LPS
2.23±0.04
1.23±0.00a
0.41±0.01
2.27±0.07a
2.10±0.02a
15 mg LPS
2.54±0.02
0.78±0.01b
0.66±0.00
2.42±0.11a
1.94±0.01a
92
Chapter 3 Results
_____________________________________________________________________
Data are presented as Mean±SE (n = 3). Data with different superscripts in the same
treatment group and column are significantly different (P < 0.05).
3.13 Determination of the most effective dose of the dietary supplements
The effective dose was determined after oral administration for 14 days. Taking into
considerations, the effects of different doses (concentrations) of dietary compounds on
the enhancement of immune mechanisms to overcome pathogenic infections by A.
hydrophila and growth parameters, two doses from each dietary compound were chosen.
(See Table 19). The effectiveness of these doses was also reflected on the mode of
actions of the respective compounds.
Table 19 Comparison of different doses of dietary supplements administered for 14 days
after challenge with A. hydrophila (AH1)
Treatment
No. of fish
No. of mortality
% mortality
Control
25
22±1.0
88±1.0
Relative %
survival (RPS)
-
0.05 g garlic
25
9±1.0
36±1.0
59
0.1 g garlic
25
6±2.0
24±2.0
73
0.5 g garlic
25
1±0.0
4±0.0
96*
1.0 g garlic
25
1±0.0
4±0.0
96*
Control
25
19±2.0
76±2.0
-
0.05 g ginger
25
8±1.0
32±1.0
58
0.1 g ginger
25
5±1.0
20±1.0
74
0.5 g ginger
25
1±0.0
4±0.0
95*
1.0 g ginger
25
0±0.0
0±0.0
100*
Control
25
21±1.0
84±1.0
-
1.875 mg LPS
25
6±1.0
24±1.0
71
93
Chapter 3 Results
_____________________________________________________________________
3.75 mg LPS
25
3±1.0
12±1.0
86*
7.5 mg LPS
25
2±1.0
8±1.0
91*
15 mg LPS
25
11±2.0
44±2.0
47.6
*= most effective doses of dietary supplements. All treatments were replicated twice.
Data are presented as Mean±SE (n = 2).
3.14 Duration of protection after administration of dietary supplements
The result of duration of protection of rainbow trout fed dietary supplements for 14 days
and experimental challenges at 14, 21 and 28 days after withdrawal of dietary
supplements is summarized in Table 20. The result indicated a steady reduction in the
level of protection in rainbow trout after 14 days following challenge with A. hydrophila.
Thus, 14 days after ending the administration of garlic dosed at 0.5 g/100 g, the RPS was
85%, decreasing to 71 % and 69 % after 21 and 28 days, respectively. Similarly after 14
days, of feeding 1.0 g garlic/100 g of feed and 14 days withdrawals of supplement, the
RPS was 80 %, it reduces to 50 % after 21 days, and 28 % at 28 days. The same trend
was observed in ginger and LPS fed fish (Table 20). After challenge, diseased fish
displayed abdominal distension, necrosis, ascitic fluid and exophthalmia.
Table 20. Effect of dietary supplements and their ability to lead to protective immune
response in rainbow trout after challenge with A. hydrophila.
Treatment
Control
Mortality (%)
---------------------------------------14 days
21 days
28 days
80±1.0
84±2.0
92±0.5
RPS (%)
-----------------------------------------14 days
21 days
28 days
-
0.5g garlic
12±1.0
24±1.0
28±0.5
86
71
69
1.0g garlic
16±1.0
40±1.0
64±1.0
80
50
28
0.5g ginger
12±0.0
12±1.0
32±1.0
85
86
65
1.0g ginger
12±1.0
20±1.0
44±0.5
85
72
57
94
Chapter 3 Results
_____________________________________________________________________
3.75mgLPS 8±1.4
12±0.0
25±0.3
91
86
73
7.5 mg LPS 8±2.8
24±1.0
44±0.5
86
70
52
Data are presented as Mean±SE, (n = 2).
Table 21. Physiological parameters in rainbow trout measured 14, 21 and 28 days after
stopping feeding dietary supplements.
body wt.
(%)
length (cm3) gutted wt.
(g)
SGR (%)
Condition
factor (%)
Control
18.7±1.0
38.6±1.1
2.3±0.2
1.2±0.0
5.8±0.0
0.5 g garlic
18.9±1.3
30.4±2.6
1.9±0.2
0.9±0.0
6.0±0.1
1.0 g garlic
23.4±1.0
39.4±1.1
2.3±0.1
1.2±0.0
6.3±0.2
0.5 g ginger
19.1±0.9
32.6±2.0
2.1±0.2
1.1±0.0
5.6±0.1
1.0 g ginger
23.0±1.1
41.9±1.6
3.1±0.2
1.2±0.0
6.2±0.2
3.75mg LPS 22.6±0.8
43.7±1.4
3.0±0.2
1.1±0.1
6.1±0.1
7.5 mg LPS
19.3±1.2
37.5±1.8
2.1±0.2
1.1±0.1
5.6±0.0
Control
13.4±0.2
27.7±1.0
1.2±0.2
0.8±0.0
5.5±0.1
0.5 g garlic
13.1±0.8
29.4±1.6
1.6±0.2
0.9±0.0
5.4±0.1
1.0 g garlic
12.4±0.8
28.8±1.6
0.9±0.2
0.9±0.0
5.3±0.0
0.5 g ginger
13.3±0.9
29.5±1.7
1.6±0.2
0.8±0.0
5.9±0.1
1.0 g ginger
14.3±0.3
30.3±1.0
1.8±0.1
0.9±0.0
6.0±0.1
3.75mg LPS 13.3±0.7
28.3±1.6
1.5±0.1
0.9±0.0
5.5 ±0.0
7.5 mg LPS
29.0±1.3
1.7±0.1
0.8±0.0
5.8±0.1
Treatment
A
B
12.9±0.7
95
Chapter 3 Results
_____________________________________________________________________
C
Control
13.4±1.5
30.0±1.8
1.1±0.2
0.8±0.0
3.6±0.1
0.5 g garlic
13.7±0.9
31.02±1.5
1.1±0.3
0.9±0.1
3.8±0.0
1.0 g garlic
13.6±1.4
30.0±1.9
1.1±0.2
0.9±0.0
3.7±0.0
0.5 g ginger
12.0±0.9
29.4±1.7
0.9±0.1
0.8±0.0
3.1±0.1
1.0 g ginger
12.5±0.3
28.9±1.0
1.0±0.1
0.8±0.0
3.6±0.1
3.75mg LPS 11.1±0.7
28.2±1.3
1.1±0.2
0.7±0.0
3.7±0.1
7.5 mg LPS
28.7±1.6
0.9±0.1
0.8±0.0
3.6±0.1
12.5±0.8
A = 14 days after stopping feeding with dietary supplements. B = 21 days after stopping
feeding with dietary supplements. C = 28 days after stopping feeding with dietary
supplements. Data are expressed as M±SE, n = 10.
96
Chapter 3 Results
_____________________________________________________________________
Table 22. Mean haematological parameters of rainbow trout fingerlings
Treatment
RBC
WBC
Monocytes
Lymphocytes
Neutrophils
Thrombocytes
(x 106)
(x 103)
(%)
(%)
(%)
(%)
Control
6.2±2.4b
336.0±0.8b
14.3±0.6b
33.3±2.1
37.0±1.5
14.8±0.6
0.5 g garlic
4.2±2.0b
292.0±1.6b
18.5±0.5a
32.3±1.2
36.9±0.6
12.3±0.3
1.0 g garlic
9.1±3.0a
460.0±1.2a
17.5±0.9a
32.5±1.5
34.2±1.7
15.8±1.5
0.5g ginger
3.8±1.4b
258.0±1.0b
18.2±0.9
27.3±0.6
40.0±0.9
14.5±0.3
1.0g ginger
8.3±2.4a
380.0±0.8b
21.6±0.9
27.6±1.2
37.9±1.9
12.9±0.6
3.75mgLPS
4.0±2.4b
230.0±1.2c
22.5±1.5
36.0±2.4
28.1±0.9
13.6±0.6
7.5 mg LPS
8.8±2.0a
420.0±1.0a
13.6±0.9
36.0±0.6
38.4±1.5
12.0±0.6
Control
5.9±0.2b
330.0±0.2
24.7±0.7a
24.0±0.6c
37.1±0.9a
14.2±0.3a
0.5 g garlic
10.8±0.8a
220.0±1.0
19.2±0.9b
26.3±0.6c
38.0±0.9a
16.5±0.4a
1.0 g garlic
14.0±1.4a
330.0±0.4
23.6±1.5b
34.9±2.0b
29.1±0.9b
12.6±0.6b
A
B
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0.5 g ginger
8.3±1.3
500.0±0.7
28.0±1.2a
34.6±1.5b
28.0±1.2b
9.3±0.9c
1.0 g ginger
6.3±0.8
426.0±0.4
26.1±1.2a
32.9±1.5b
30.7±0.6b
10.2±0.6b
3.75mg LPS 7.5±1.1
464.0±0.5
22.6±1.2b
38.7±1.2a
29.0±1.2b
9.7±0.5c
7.5 mg LPS
6.1±0.7
418.0±0.6
26.1±0.6a
37.0±1.2a
29.4±0.6b
7.6±0.9c
Control
5.8±0.6
321.0±0.4b
22.0±1.0c
34.3±0.7
35.9±0.2a
11.8±0.3
0.5 g garlic
6.2±0.8
240.0±0.6c
27.1±0.6a
36.0±0.2
28.2±0.6c
8.8±0.9
1.0 g garlic
6.7±0.8
430.0±0.5a
24.3±0.8b
32.7±1.1
30.4±0.6b
12.6±0.9
0.5 g ginger
5.8±1.4
256.0±1.0c
19.2±0.8c
26.3±0.5
40.1±0.8a
14.5±0.3
1.0 g ginger
6.2±0.5
326.0±0.3b
26.1±1.2a
31.4±1.4
32.0±0.6b
10.3±0.5
3.75mg LPS 6.1±2.4
230.0±1.2c
21.4±1.4c
35.9±2.3
28.1±0.8c
13.6±0.5
7.5 mg LPS
414.0±0.5a
27.0±0.5a
35.5±1.2
29.3±0.6c
7.7±0.8
C
6.3±0.7
A = 14 days after stopping feeding with dietary supplements. B = 21 days after stopping feeding with dietary supplements. C = 28 days after
stopping feeding with dietary supplements. Data are expressed as M±SE, n = 10. Values with different superscripts in the same day/group and
column are significantly different (P < 0.05, n =10).
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Table 23. Biochemical indices of rainbow trout fed immunostimulants for 14 days
Treatment
Total protein
Albumin
Globulin
Albumin: globulin
(mg/ml)
(mg/ml)
(mg/ml)
Control
1.3±0.1
0.2±0.1
1.0±0.1
0.2±0.0
0.5 g garlic
1.4±0.2
0.2±0.2
1.2±0.1
0.2±2.0
1.0 g garlic
1.1±0.1
0.3±0.1
1.0±0.0
0.3±0.1
0.5 g ginger
1.4±0.1
0.2± 0.1
1.2±0.1
0.2±1.0
1.0 g ginger
1.2±0.1
0.2±0.0
1.0±0.1
0.2±0.0
3.75 mg LPS
1.9±0.1
0.1± 0.0
1.8±0.1
0.1±0.0
7.5 mg LPS
1.2±0.1
0.2±0.0
1.0±0.1
0.2±0.0
Control
1.4±0.1
0.4±0.2
1.0±0.0
0.4±0.2
0.5 g garlic
1.5±0.1
0.2±0.2
1.3±0.1
0.1±2.0
1.0 g garlic
1.3±0.1
0.5±0.2
1.0±0.1
0.5±2.0
A
B
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0.5 g ginger
1.4±0.1
0.2± 0.1
1.2±0.1
0.2±1.0
1.0 g ginger
1.8±0.2
0.3± 0.1
1.5±0.0
0.2±0.1
3.75 mg LPS
1.7±0.2
0.3± 0.2
1.4±0.0
0.2±0.2
7.5 mg LPS
1.3±0.1
0.4± 0.2
0.9±0.1
0.4±2.0
Control
1.2±0.2
0.4±0.3
0.8±0.0
0.5±0.3
0.5 g garlic
1.2±0.2
0.2±0.3
1.0±0.2
0.2±1.5
1.0g garlic
1.3±0.3
0.3±0.2
1.0±0.0
0.3±0.2
0.5 g ginger
1.3±0.1
0.3± 0.1
1.1±0.1
0.3±1.0
1.0 g ginger
1.2±0.12
0.4± 0.1
0.8±0.0
0.5±0.1
3.75 mg LPS
1.3±0.2
0.3± 0.1
1.0±0.2
0.3±0.5
7.5 mg LPS
1.3±0.1
0.4 ± 0.1
0.9±0.0
0.4±0.1
C
A = 14 days after stopping feeding with dietary supplements. B = 21 days after stopping feeding with dietary supplements. C = 28 days after
stopping feeding with dietary supplements. Data are expressed as M±SE, n = 10.
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Table 24. Mean electrolyte indices of rainbow trout fed dietary supplements
Treatment
Calcium
(mg/l)
Magnesium
(mg/l)
Iron
(mg/l)
Sodium)
(mg/l)
Potassium
(mg/l)
Control
1.1±0.0c
0.2±0.0
0.2±0.0
2.3±0.1
0.7±0.0
0.5 g garlic
1.1±0.0c
0.6±0.0
0.4±0.0
2.3±0.1
0.9±0.0
1.0 g garlic
1.8±0.0b
0.7±0.0
0.5±0.0
2.4±0.0
1.0±0.0
0.5 g ginger
1.7±0.0b
0.5±0.0
0.4±0.0
2.3±0.0
1.1±0.0
1.0 g ginger
2.4±0.1a
0.8±0.0
0.5±0.0
2.6±0.1
1.3±0.0
3.75 mg LPS
2.0±0.0a
0.7±0.0
0.4±0.0
2.2±0.1
1.2±0.1
7.5 mg LPS
1.7±0.0b
0.7±0.0
0.3±0.0
2.2±0.1
1.2±0.0
Control
1.4±0.0b
0.4±0.0
0.2±0.0
1.5±0.0
1.2±0.0
0.5 g garlic
1.9±0.0a
0.8±0.0
0.4±0.0
1.7±0.0
1.7±0.0
1.0 g garlic
1.8±0.1a
0.7±0.0
0.6±0.0
1.6±0.0
1.4±0.0
0.5 g ginger
1.1±0.0b
0.6±0.0
0.4±0.0
2.3±0.1
0.9±0.0
A
B
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1.0 g ginger
1.2±0.0b
0.8±0.0
0.3±0.0
1.6±0.1
1.9±0.0
3.75 mg LPS
1.1±0.0b
0.6±0.0
0.4±0.0
2.3±0.1
1.1±0.0
7.5 mg LPS
1.3±0.0b
0.7±0.0
0.4±0.0
1.6±0.1
1.9±0.0
Control
2.0±0.0
0.5±0.0
0.4±0.0
1.7±0.0
1.0±0.0
0.5 g garlic
2.0±0.0
0.7±0.0
0.8±0.0
1.7±0.0
1.4±0.0
1.0 g garlic
1.8±0.0
0.7±0.0
0.7±0.0
1.6±0.0
1.5±0.0
0.5 g ginger
1.4±0.0
0.4±0.0
0.2±0.0
1.6±0.0
1.1±0.0
1.0 g ginger
1.7±0.1
0.8±0.0
0.6±0.0
1.6±0.1
1.4±0.0
3.75 mg LPS
1.5±0.0
0.5±0.0
0.2±0.0
2.3±0.1
0.7±0.0
7.5 mg LPS
1.2±0.0
0.6±0.0
0.4±0.0
2.3±0.1
0.9±0.0
C
A = 14 days after stopping feeding with dietary supplements. B = 21 days after stopping feeding with dietary supplements. C = 28 days after
stopping feeding with dietary supplements. Data are expressed as M±SE. Superscripts indicate significantly different results (P < 0.05, n =3)
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3.14.1 Measurement of immunological parameters
The production of respiratory burst was significantly influenced (P < 0.05) by dietary
garlic (Table 25). Furthermore, a significant (P < 0.05) increase in respiratory burst
activity, i.e. 0.3±0.4 OD, was recorded in fish which received 0.5 g garlic /100 g feed,
compared to 0.2±0.0 of the controls. Although respiratory burst production 28 days
after feeding with garlic was lower, the data were nevertheless higher than the controls.
The same trend is seen with the use of dietary ginger and LPS (see Table 25). Use of
garlic at 0.5 g and 1.0 g/100 g of feed had no significant (P < 0.05) effect on the serum
peroxidase activity, as levels declined over the 28 day withdrawal period (Table 25)
Table 25. Immunological indices after cessation of feeding with dietary supplements
Treatment
Oxidative burst activity
14 days
peroxidase activity
21 days
28 days
14 days
21 days
28 days
Control
0.20±0.0b
0.15±0.0b
0.12±0.0b
2.5±0.0
2.4±0.0
1.5±0.0
0.5g garlic
0.30±0.0a
0.24±0.0a
0.18±0.0a
2.9±0.0
2.4±0.0
1.7±0.0
1.0g garlic
0.22±0.0b
0.21±0.0a
0.17±0.0a
3.2±0.0
1.9±0.0
1.8±0.0
0.5g ginger
0.21±0.0b
0.16±0.0b
0.15±0.0b
2.3±0.0
2.1±0.0
1.6±0.0
1.0g ginger
0.25±0.0a
0.19±0.0a
0.18±0.0b
3.1±0.0
2.0±0.0
1.5±0.0
3.75mgLPS
0.20±0.0b
0.14±0.0b
0.12±0.0b
2.2±0.0
2.1±0.0
1.5±0.0
7.5 mg LPS
0.22±0.0b
0.18±0.0b
0.14±0.0b
3.5±0.0
2.4±0.0
1.9±0.0
Data are presented as Mean±SE (n=4). Data with different superscripts in the same
sampling group and column are significantly different (P < 0.05).
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3.15 Modulation of digestive enzymes by dietary supplements
The pH for stomach (ST) was lowest in the dietary garlic treated group with pH 5.79,
followed by LPS with pH 6.16, compared to control with pH 6.32 (Table 26). The small
intestine (SI) and pyloric intestine (PI) pH were lower in the treatments groups, than in
the controls, except in hepato-pancreas (HP) where the pH of the control was the same
with dietary ginger group.
3.15.1 Pepsin and protease activities estimated from the stomach homogenates
Dietary garlic induced the highest pepsin activity and thus highest digestive capacity
(Fig. 14.1). This was followed by LPS. However, the activity of gastric protease was
highest in LPS supplemented diet, followed by garlic, but was lowest in ginger (Fig.
14.2).
a
140
120
25
100
pepsin
activity (u/
m g/ m in)
pH 3
60
pH 6.5
c
15
pH 3
pH 9
10
b
40
a
a
b
20
p r o t ease
act ivit y (
U / mg / min)
80
20
a
30
5
c
b
d
c
c
0
control
0
control
Garlic
Ginger
Garlic
Ginger
LPS
g / 10 0 g o f f eed
LPS
g / 100 g of feed
Fig. 14.1 pepsin activity.
Fig. 14.2 protease activity.
Bars with different superscripts are significantly different (P < 0.05, n=5)
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Table 26. Biometric indices of rainbow trout fed dietary supplements for 14 days.
Treatments body wt. wt. gain
length
G. I wt.
S. T wt.
(g)
(cm3)
(g)
(g)
(%)
P. I wt.
S. I wt.
(g)
(g)
H. P wt. C. F HSI DSI
(g)
(%) (%) (%)
S.T
P. I
S. I
H. P
(pH) (pH) (pH) (pH)
Control
31.83±3.9 52.58±0.2 48.65±4.5bc 2.59±0.2 0.69±0.1 0.90±0.1b 0.49±0.1 0.60±0.1 5.3 1.9 8.2b
6.3
6.5
6.5
6.4
Garlic
36.66±2.8 76.67±0.3 55.56±3.4ab 2.86±0.3 0.94±0.1 1.46±0.2a 0.50±0.1 0.58±0.1 5.2 1.6 7.8c
5.7
6.2
6.5
6.3
Ginger
32.51±2.4 55.56± 0.2 44.35±3.1c 2.39±0.4 1.07±0.2 0.81±0.1b 0.44±0.1 0.46±0.0 5.4 1.4 7.4c
6.2
6.4
6.5
6.4
LPS
31.08±1.5 48.99± 0.1 52.14±1.4bc 2.93±0.2 0.81±0.2 1.12±0.1b 0.52±0.0 0.53±0.1 5.6 1.7 9.4a
6.1
6.3
6.3
6.3
Data are expressed as M±SE. Values with different superscripts in the same column are significantly different (P < 0.05, n =5).
G.I =gastrointestinal tract, ST = stomach, P.I = pyloric intestine, S.I = small intestine, H.P = hepato-pancreas, C.F = condition factor,
HSI = hepato-somatic index and DSI = digestive somatic index.
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3.15.2 Alkaline phosphatase activity estimated from the small intestine and brush
border membrane
The same pattern of enzymatic activities in pepsin extracts was found for alkaline
phosphatase, with the highest level of activity obtained with brush border membrane
from LPS supplemented diet, followed by garlic (Fig. 15). Ginger and the controls
were the same. However, the alkaline phosphatase activity in the small intestine was
significantly high with dietary garlic, followed by ginger.
3
a
a
Alkaline phosphatase (u / ml)
2.5
b
2
b c
c
c
c
Small Intestine
1.5
Brush border
1
0.5
0
control
Garlic
Ginger
LPS
g / 100 g of feed
Fig. 15. Alkaline phosphatase from small intestine and brush border membrane.
Data are expressed as mean ± SE, n = 5. Bars with different superscripts are
significantly different (P < 0.05).
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3.15.3 Total proteolytic enzymes activity estimated from the small intestine and
hepatopancreas
Enzymes with proteolytic activity in the liver-pancreas of rainbow trout did not show
any effect with the use of dietary supplements (Fig. 16). However, their activity in the
small intestine was significantly high with dietary garlic treatment. Dietary LPS
induced activity was the same with the control but different from the dietary ginger
(Fig. 16).
total proteolytic enzymes activities (u / ml / min)
2
1.8
1.6
1.4
1.2
Small Intestine
1
Hepato-pancreas
0.8
0.6
0.4
0.2
0
control
Garlic
Ginger
LPS
g / 100 g of feed
Fig. 16. Total proteolytic enzyme activity from small intestine and hepatopancreas.
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3.15.4 Trypsin activity estimated from the small intestine and the hepatopancreas
The result of the trypsin activity indicated that the lowest trypsin activity was found
in the intestine of groups administered with dietary garlic and ginger, compared to the
control which showed high trypsin activity (Fig. 17). However, trypsin activity in the
hepatopancreas was also significantly lower in these treatment groups except with
LPS which was the same as the control. The overall high trypsin activity was
obtained from the control group.
0.3
trypsin activity (u / ml / min)
0.25
b
a
b
c
a
a
0.2
b
0.15
Small intestine
b
Hepato-pancreas
0.1
0.05
0
control
Garlic
Ginger
LPS
g / 100 g of feed
Fig. 17. Trypsin activity from small intestine and hepatopancreas. Data are expressed
as mean ± SE, n = 10. Bars with different superscripts are significantly different
(P < 0.05).
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3.16 The role of the garlic component, allicin in controlling A. hydrophila
infection
3.16.1 Antimicrobial activity
Allicin was strongly inhibitory against A. hydrophila producing large, i.e. > 10 mm
diameter zones on lawns of A. hydrophila. The MIC 50 was calculated as > 400 µl/ml.
3.16.2 Protective effect of allicin
The result of experimental challenges after 14 days feeding of allicin supplemented
diet is summarized in Fig. 18. The result indicated a marked reduction in mortality
after challenge with A. hydrophila. Thus, 80 % mortalities were recorded for the
controls compared to 8 % (RPS = 90 %) and 0 % (RPS = 100 %) mortalities in the
groups which were fed with 0.5 ml allicin/100 g of feed and higher dose of 1.0 ml
allicin/100 g of feed, respectively (Fig. 18).
90
80
Cumulative mortality (%)
70
60
Control
50
0.5 ml allicin/100g of
f eed
40
1.0 ml allicin/100g of
f eed
30
20
10
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14
day afte r challe nge
Fig. 18. Percentage cumulative mortality of rainbow trout following intraperitoneal
injection with 106 cells/fish of A. hydrophila after feeding allicin supplemented diet
for 14 days.
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3.16.3 Mode of action - haematological parameters
The result indicated that the number of erythrocytes was significantly (P < 0.05)
higher in experimental groups compared with the controls (Table 27). Overall, a
higher erythrocyte count was found in the group fed with the higher dose of allicin
(13.60±0.3), compared with the controls (12.45±0.2). Conversely, the leucocyte
numbers decreased in these groups relative to the controls (Table 27).
Table 27. Haematological parameters of rainbow trout fed dietary allicin for 14 days.
Treatment RBC
(x 106)
WBC
(x 103)
Mono.
(%)
Lymph.
(%)
Neutro.
(%)
Thromb. Hb
(%)
(%)
Control
12.5±0.2b 175.0±0.1a 18.7±0.5 36.2±1.7 37.1±1.6a 7.9±0.5
0.5 ml
12.7±0.2b 132.0±0.4b 22.5±0.8 33.8±0.5 32.5±1.4b 11.3±0.5 0.8±0.2
1.0 ml
13.6±0.3a 122.0±0.3b 19.1±0.6 34.5±0.8 36.9±0.8a 9.5±0.5
0.8±0.2
0.8±0.2
Data are expressed as M±SE. Values with different superscripts in the same column
are significantly different (P < 0.05, n= 10).
3.16.4 Biochemical parameters
Serum total protein contents was significantly (P < 0.05) higher in the group
receiving the higher dose of allicin, with 2.3±0.1 mg protein/ml compared with
1.6±0.0 mg protein/ml in the controls (Table 28). The albumin content and
albumin/globulin ratio (A/G) did not vary significantly (P < 0.05) in any of the
experimental groups compared with the controls. However, the globulin fractions
were significantly (P < 0.05) higher in the group which received the higher dose, i.e.
2.1±0.1 mg globulin/ml, compared with 1.3±0.1 mg globulin/ml in the controls
(Table 28).
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Chapter 3 Results
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Table 28. Biochemical indices of rainbow trout fed allicin supplemented diets for 14
days.
Treatment
Total protein
(mg/ml)
Albumin
(mg/ml)
Globulin
(mg/ml)
Albumin/Globulin
ratio.
Control
1.6±0.0b
0.3±0.1
1.3±0.0b
0.2±0.1
0.5 ml
1.9±0.1b
0.4±0.1
1.5±0.0b
0.3±0.1
1.0 ml
2.3±0.1a
0.3±0.0
2.1±0.1a
0.1±0.0
Data are expressed as M±SE. Values with different superscripts in the same column
are significantly different (P < 0.05, n= 10).
3.16.5 Immunological parameters
3.16.5.1 Phagocytic activity
The phagocytic activity was greatly affected by dietary allicin (Fig. 19). Thus, the
group fed with the higher dose of allicin had a value of 39.2 % compared to the 13.6
% of the controls. Moreover, a higher phagocytic index than the controls was also
recorded (Fig. 19).
50
45
a
% phagocytic activity
40
a
35
30
phag. ratio
25
20
phag.index
b
15
10
5
0
0
0.5 ml
1.0 ml
m L allicin/ 100 g of feed
Fig. 19. The effect of feeding allicin supplemented diet on the phagocytic activity
(phagocytic ratio and index) of rainbow trout, after 14 days feeding. Data are
expressed as mean ± SE, n = 10. Bars with different superscripts are significantly
different (P < 0.05, n =10).
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3.16.5.2 Respiratory burst activity
The production of super oxide anion was influenced by allicin (Fig. 20). In particular,
the respiratory burst was 0.2±0.0 % in the group which received the lower dose, as
OD 540 nm
compared with the controls at 0.1± 0.0 %.
0.16
0.155
0.15
0.145
0.14
0.135
0.13
0.125
0.12
0.115
a
b
b
0
0.5
1
m L allicin/ 100 g of feed
Fig. 20. Effect of feeding allicin supplemented diet on the superoxide anion
production by blood leucocytes of rainbow trout. Values are expressed as mean± SE.
Bars with different superscripts are significantly different (P < 0.05, n =10).
3.16.5.3 Lysozyme activity
There were significant (p < 0.05) differences in serum lysozyme activity in the
lysozyme activity (unit/ mL)
experimental groups at 15 and 30 min when compared with the controls (Fig. 21).
2500
a
2000
1500
b
a
a
b
1000
b
c
c
c
500
0
15 min 30 min 60 min 15 min 30 min 60 min 15 min 30 min 60 min
0
0.5
1
mL allicin/ 100g of feed
Fig. 21. Effect of feeding allicin supplemented diet on the serum lysozyme activity of
rainbow trout. Values are expressed as mean± SE, n=10. Bars with different
superscripts in the same sampling time are significantly different. (P < 0.05).
112
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3.16.5.4 Bacteriocidal activity
Serum bacteriocidal activity was significantly (P < 0.05) higher in the experimental
groups, with 165.0 x 103 and 344.0 x 103 CFU/ml respectively for the lower and
higher doses compared with the control, i.e. 582.0 x 103 CFU/ ml (Fig. 22).
number of bacterial
colonies
12000
10000
8000
c
b
6000
a
4000
2000
0
0
0.5
1
w ithout serum
m L allicin/ 100 g of feed
Fig. 22. Effect of feeding allicin supplemented diet on the serum bacteriocidal
activity of rainbow trout. Values expressed as M±SE. Bars with different superscripts
are significantly different (P < 0.05, n=10).
113
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Chapter 4 Discussion
With increasing investment and expansion in aquaculture production, there is a
corresponding increase in disease outbreaks caused by infectious agents. Indeed, disease
outbreaks are considered to be the main constraint to aquaculture production,
worldwide, and are undoubtedly responsible for the economic risk faced by the sector
(Riquelme et al., 1997). The problems of disease have attracted the attention of many
investigators (see Austin and Austin, 2007). Indeed, disease outbreaks are known to be
the result of the interaction between the host, the disease causing agent (= pathogen) and
the environment (= stressor) (Austin and Austin, 2007). An imbalance in these three
factors leads to the likelihood of an outbreak of disease (Pillay and Kutty, 2005).
Aeromonas hydrophila is a well recognised pathogen particularly of freshwater fish
with an almost worldwide occurrence (Austin and Austin, 2007). However, the
organism is not only associated with fish diseases, but has been implicated as possible
cause of human gastroenteritis leading to diarrhoea. In immunocompromised
individuals, the organism may cause wound infections leading to septicaemia
(Subashkumar et al., 2007).
To prevent diseases caused by this organism, various measures have been adopted, of
which the use of antibiotics is commonplace. To a lesser extent, vaccines have been
considered, although the process of immunization and vaccine development is costly
and relatively slow (Saitanu et al., 1994; Austin and Austin, 2007). Moreover, disease
prevention in animals used for food is slowly moving away from the dominant use of
antimicrobial compounds as concerns about tissues residues and the development and
spread of antibiotic resistance have been increasingly voiced. Therefore, the adoption of
immunoprophylactic agents in disease prevention and control has gained widespread
acceptance especially as new and effective products enter the marketplace (Petrunov et
al., 2007; Rajendiran et al., 2008). In addition, there is widespread interest in probiotics
and immunostimulants, both of which may enhance the immune state of the recipient
animal (Leishman and Brundick, 2004). However, the use of live organisms as
probiotics has raised concerns about the possible acquisition of virulence factors, such
as on plasmids, and thus the development of a pathogenic state although there is not any
evidence that this situation has ever arisen in practice (Murray and Peeler, 2005; Gibson
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et al., 1998). Justifiably, the use of immunostimulants remains an attractive option for
use in disease control strategies in aquaculture. Certainly, their application is regarded
as innovative in enhancing the innate defence mechanism of fish (Robertsen et al.,
1994). As such, immunostimulants have been defined by Bricknell and Dalmo (2005) as
“naturally occurring compounds that modulate the non-specific immune system by
increasing the host resistance against diseases which in most circumstances are caused
by pathogens”.
Studies on the effectiveness of immunostimulants for the control of bacterial fish
pathogens have been conducted with fish models involving feeding regimes followed by
challenge with virulent cultures (McCarthy, 1983; Amend, 1981). For example,
Kawakami et al. (1998) injected yellowtail with chitin and reported increased protection
for up to 45 days after administration when challenged with Photobacterium damselae
subsp. piscicida. In another example, Robertsen et al. (1990) injected yeast glucan (ß1,3 and ß-1,6- glucan), which was prepared from cell walls of Saccharomyces
cerevisiae, intraperitoneally into Atlantic salmon. The result was resistance to challenge
with V. anguillarum, V. salmonicida and Y. ruckeri. Furthermore, Chen and Ainsworth
(1992) reported that catfish, which were injected with yeast glucan, became resistant to
challenge with Edwardsiella ictaluri. Indeed in the present study, garlic, ginger and LPS
were demonstrated to be beneficial to rainbow trout, leading to enhancement of immune
parameters and resistance to challenge by A. hydrophila.
In addition to immune
stimulation, there was evidence of in vitro inhibition of the target pathogen, which is
common with other previous reports (Tsao et al., 2007; Goswami and Prasad, 2000;
Shadkchan et al., 2004). However, it should be emphasised that inhibitory activity in
vitro does not necessarily reflect the mode of action in vivo (Gram et al., 2001; Balcázar
et al., 2006).
The initial screening exercise in this study led to the identification of three compounds
with immunostimulatory properties. Of these, garlic has a long history of dietary and
medicinal applications as an anti-infective agent (Lawson, 1998; Reuter et al., 1996).
Interest in garlic as an immunostimulant for aquaculture follows its use in human
medicine and agriculture as a formidable prophylactic and therapeutic agent (Amagase
et al., 2001). The mechanisms believed to be involved in the health benefit of garlic
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relate to the present of S-allyl cysteine SAC, which is a water soluble organo-sulphur
compound (Banerjee et al., 2003), allicin (diallyl thiosulphinate; Amagase et al., 2001)
and lectin (believed to be the most abundant protein in garlic; Fenwich and Hanley,
1985). Garlic is known to modulate the immune response by promoting lymphocyte
synthesis (Zhang et al., 1997), phagocytosis, respiratory burst, cytokine and natural
killer cells activities (Sumiyoshi 1997, Imai et al., 1994, Numagami et al., 1996; Kyo et
al., 2001), and inhibition of tumour metabolism (Lamm and Riggs, 2001). Therefore, it
is not surprising that garlic led to a high level of protection against challenge with A.
hydrophila.
The second compound highlighted in this study was the rhizome of ginger, Zingiber
officinale. Ginger belongs to the family Zingiberaceae, which has been recognised for
centuries as a source of spice. This was used for treatments of many ailments,
worldwide (Langner et al., 1998). Ginger has broad-spectrum prophylactic and
therapeutic functions (Ernst and Pittler, 2000). Studies indicates that ginger exerts
biological effects as an antimicrobial agent, with activity against many bacteria, fungi
and parasites (Agarwal, 2001; Martin et al., 2001; Endo et al., 1990), and demonstrates
anti-inflammatory, anti-oxidative (Chrubasik et al., 2005; Kim et al., 2007; Grzanna et
al., 2005) and immuno-modulatory actions (Benny et al., 2004; Ali et al., 2007). There
has been a growing awareness of the potential benefits of medicinal plants, including
ginger, as dietary supplements/immunostimulants for use in aquaculture. This follows
their use (medicinal plants) with mice, chicken and human cell lines for combating
gastrointestinal tract infections, ulcerative conditions by activating bile secretion,
enhancing pancreatic lipase activity and stimulation of the immune system (Benny et
al., 2004; Langner et al., 1998; Chrubasik et al., 2005; Ardo et al., 2008; Cao and Lin,
2003; Lin and Zhang, 2004; Shan et al., 1999). Ahmed et al. (2000) highlighted the
protective action in rats fed with 1% ginger leading to significantly attenuated
malathion induced oxidative stress. Subsequently, Jagetia et al. (2003) pretreated (by
injection) mice with ginger rhizome extracts, and recorded protection against severity of
symptoms and mortalities attributed to gastrointestinal related deaths. Moreover, rats
fed with ginger oil supplemented diets for 26 days were protected from experimental
infection with Mycobacterium (Sharma et al., 1994).
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LPS was the third compound identified in this study. LPS is an endotoxin, being a major
component of the outer cell wall membrane of Gram-negative bacteria, and consists of
polysaccharides extending from the outer cell surface and a lipid portion embedded
inside the membrane. This lipid portion is known to be responsible for eliciting
immunostimulatory responses in animal models (Akira and Hemmi, 2003). While its
molecules are highly deleterious and trigger septic shock in mammals, fish have been
recognised to be resistant to (endo-) toxic shock (Mackenzie et al., 2006). LPS is
considered to induce beneficial biological functions in fish and other animal species by
eliciting non-specific immune responses, such as activation of complement alternative
pathway, phagocytic activity of the macrophages, and proliferation of B-cells
(Uchiyama, 1982, Salati et al., 1987; Salati et al., 1989; Velji et al., 1990). In particular,
LPS stimulation of macrophages leads to the induction of cytokines, specifically TNFά, IL-6 and IL-10, with enhanced protection against disease (Akira and Hemmi, 2003).
Clearly, there are benefits to the administration of comparatively low quantities of LPS
to fish, although most work has reflected the use of injection techniques and have
inevitably been related to vaccine studies, i.e. by explaining the mode of action of
vaccines for the protection of infection by Gram-negative bacteria. For example, Baba
et al. (1988) reported the protection of carp (Cyprinus carpio) to challenge by A.
hydrophila following the administration of crude LPS. Also, Al-Harbi and Austin
(1992) reported increase survival of turbot (Scophthalmus maximus) against Cytophagalike bacteria following i.p. injection of crude LPS. In another example, Selvaraj et al.
(2004) reported that i.p. injection of LPS to A. hydrophila infected carp and led to the
enhanced survival after challenge.
Seven compounds were eliminated from detailed study, although β-1,3 glucan, vitamin
C, chitin and chitosan have been used to elevate non-specific immune responses in fish
previously (Siwicki and Anderson 1994; Suzuki et al., 1992; Siwicki et al., 1994;
Anderson 1997; Thompson et al., 1995; Kawakami et al., 1998; Li and Lovell, 1985;
Lygren et al., 1999; Sakai et al., 1999). In particular, injection of Atlantic salmon with
β-glucan enhanced resistance to various bacterial fish pathogens (Robertsen et al.,
1990). Also of relevance to this study, injection of β-glucan in combination with LPS
led to protection of carp following challenge with A. hydrophila (Selvaraj et al., 2006).
It is interesting to note that the source and method of extraction for some
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immunostimulants has been considered to affect their potency (Wang and Wang, 1997;
Engstad et al., 1992). Perhaps, this may explain why the 7 compounds were not found
to be particularly useful in this study, despite a literature pointing to their success.
The question is how does feeding garlic, ginger or LPS to rainbow trout lead to
protection against infection by A. hydrophila? Is there a threshold effect by which
feeding with these compounds corrects possible deficiencies in diet leading to a
pronounced effect? Certainly with garlic, ginger and LPS, there was a statistically
significant effect on immune parameters, which are assumed to be responsible for the
beneficial effect, i.e. the compounds acted as immunostimulants.
The result was
activation of immune cells such as B- and T-lymphocytes, macrophages, neutrophils,
monocytes and dendritic cells (Lo et al., 1999). Interestingly, these immune cells act by
the recognition of pathogen associated molecular patterns (PAMPs), which are highly
conserved motif regions on the surface of most pathogens (Werling and Jungi, 2003;
Elward and Gasque, 2003). Consequently, invasion of the host by pathogens, means that
these highly conserved motifs are readily recognised by the pattern recognition
receptors (PRRs) expressed on the surface of immune cells (Akira et al., 2006). As cell
receptors and agonists, they bind to the cells and trigger a response such as cytokine
production, phagocytic stimulation, complement activity and activation of antigen
presenting cells (APC). This activation of immune cells includes the proliferation of
leucocytes and endothelial cells, which in turn leads to increased expression of adhesion
molecules and the subsequent migration of the cells to the site of infection (Table 29;
Luster, 2002). This process leads ultimately to the elimination of the pathogens and thus
protection of the host animal. Purcell et al. (2006) in their phylogenetic study, identified
several receptor molecules in fish including the toll-like receptors (TLR; Kawai and
Akira, 2005; Meijer et al., 2003; Jault et al., 2004).
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Table 29. An overview of innate immunity components in fish and their mode of
action (after Abbas and Lichtman, 2006; Magnadottir, 2006).
Component
of
Reference
innate Mode of action
immunity
Cellular components
Neutrophils
Induce phagocytosis and Magnadottir,
Galindo-Villegas
activates
phagocyte
Hosokawa, 2004
secretions.
Monocyte/ macrophages
Causes phagocytosis and Abbas and Lichtman, 2006
cytokine
2006;
and
production,
secrete growth factors and
enzymes to repair injures
tissues and stimulate Tlymphocytes
Natural killer (NK) cells
Induction of apoptosis of Hamerman et al., 2005
infected cells. Synthesis
and secretion of IFN-γ
Humoral components
Complement system
Enhance
binding
promote
phagocytic Ellis, 2001
of
microbes,
inflammatory
activity at the site of
infection, causes osmotic
lysis and apoptotic death.
Interferons
Inhibition
of
119
virus Alexander and
1992; Ellis, 2001
Ingram,
Chapter 4 Discussion
_____________________________________________________________________
replications
Transferrin
Iron
binding
activity, Bayne and Gerwick, 2001
growth
inhibitors
of
bacteria,
activation
of
macrophages
Lytic enzymes
Opsonization of microbes Galindo-Villegas
Hosokawa, 2004
to enhance phagocytosis
Antiproteases
Restrictions of bacterial Ellis, 2001
and
growth and invasions in
vivo.
Lectins
Causes agglutinations and Galindo-Villegas
complement activation
C-reactive proteins (CRP)
Opsonized
microbes
and
Hosokawa, 2004
to Baldo and Fletcher, 1973
facilitate phagocytosis by and Nakanishi et al., 1991
the macrophages induce
cytokines production and
complement activation
Antibacterial peptides
Bacterial membrane dis- Smith et al., 2000; Yeaman
ruption, interfering with
and Yount, 2003)
metabolism and targeting
cytoplasmic components.
The approach in this study involving the application of potentially useful compounds to
the host followed challenge has been widely used by others (e.g. Gildberg and
Mikkelsen, 1998; Gram et al., 1999; Verschuere et al., 2000). Certainly, the results of
this study reinforce the commonly held view that the use of dietary compounds as
immunostimulants is an innovative approach to disease prevention and control in
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Chapter 4 Discussion
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aquaculture (Robertseen et al., 1994; Petrunov et al., 2007; Rajendiran et al., 2008).
Clearly, the data support previous studies which also highlighted the effects of dietary
garlic on disease resistance in a wide range of farmed fish species. For example, Sahu et
al. (2007) reported enhanced protection following 60 days feeding of dietary garlic to
rohu (Labeo rohita) fingerlings (average weight = 10 ±2 g). Similarly, Rao et al. (2006)
reported a reduction in mortality also in L. rohita after feeding with 0.5% Achyranthes
aspera followed by challenge with A. hydrophila. Moreover, Logambal et al. (2000)
emphasised that intraperitoneal injection of O. sanctum leaf extracts into O.
mossambicus led to enhance disease resistance to A. hydrophila. Indeed, the protective
effects of various immunostimulatory compounds led to protection of fish against a
range of pathogens (Robertsen et al., 1990, Matsuyama et al., 1992; Yano et al., 1989).
So, it is not surprising that garlic was identified as an extremely useful dietary
supplement in this study. Consideration of the precise nature of the beneficial molecules
in garlic will be detailed later.
Good nutrition is critical in promoting good health of all species, including those raised
in aquaculture (Pillay and Kutty, 2005). A healthy condition is needed if the animals are
to survive and grow rapidly to table size, which after all is the most important goal in
aquaculture. Artificial diets prepared from different feedstuffs (including cereals and
fish meal, depending on the species to be fed) in contrast to natural diets, such as those
comprising zoo- and phyto-plankton, and filamentous algae, are the primary sources of
commercial feeds in modern aquaculture (Delbert and Gatlin, 2002).
The artificial diets should provide essential nutrients needed for proper growth, and may
also serve for the oral delivery of specialist compounds, including immunostimulants
and nutraceuticals, to fish (Delbert and Gatlin, 2002). It is obvious that the precise
composition of feed will have a major impact on the health of fish. This begs the
question as to whether or not commercial diets are adequate when supplementation with
comparatively small quantities of compounds, such as garlic, ginger and LPS, have a
dramatic effect on health. It is well documented that fish are varied in their ability to
metabolize dietary carbohydrates, proteins and lipids, and to distribute and store these
food components in body tissue (Sheridan and Kittilson, 2004). It was reported by NRC
(1993) that the capacity for nutrient utilization also influenced growth of fish especially
when receiving dietary supplements. A recommendation was made that there should be
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a proper balance of energy-yielding nutrients for sustaining growth and achieving feed
utilization in cultured fish species.
The realisation that dietary supplements and oral immunostimulants enhance disease
resistance, improve overall health, and impact on growth and body composition matches
the observations from this study (Delbert and Gatlin, 2002).
Specifically and in
comparison to this study, it has been reported previously that feeding dietary garlic to
tilapia led to high growth performance (Diab et al., 2002). Similarly, Shalaby et al.
(2006) obtained significantly increased weight gain in Nile tilapia after feeding with
garlic. Yet, the specific weight gain and feed conversion will reflect feeding rates and
fish size (Hardy and Barrow, 2002). Overall, the results of this study are in agreement
with those of Shalaby et al. (2006) and Khattab et al. (2004), who found out that dietary
garlic enhanced feed intake, SGR, FCR and PER in Nile tilapia. Similar results were
obtained in experiments with rainbow trout (Gomes et al., 1993).
The benefits of dietary ginger on growth rate of rainbow trout were in agreement with
the results of Dugenci et al. (2003), who obtained optimum SGR and condition factors
(CF) in rainbow trout after feeding diet containing 0.1% and 1% ginger for 3 weeks.
Certainly other compounds have been found to influence growth rate. For example,
Gopalakannan and Arul (2006) fed common carp with dietary chitosan and levamisole,
and reported enhanced growth rate. Also, Chen et al. (2005) obtained increased body
weight in black grunt and bannes pompano of 23.7% and 46.0%, respectively, after
feeding recombinant yeast containing 1% Japanese sea bass growth hormone (GH).
Indeed, positive effects of LPS as determined in this study certainly matched previous
work, including the study of Gultvik et al. (2002), who obtained significant increase in
mean weight of Atlantic salmon fry after feeding for 64 days with 0.03% and 0.01%
LPS.
The ability of any dietary supplement to modulate the immune system is considered an
important mechanism in assessing its mode of action (Engwerda et al., 2001). The
immune system of teleosts like their mammalian counterparts comprise highly
specialised cells which work together to ensure protection from the invasion of potential
pathogens (Iwama and Nakanishi, 1996; Abbas and Lichtmann, 2006).
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Chapter 4 Discussion
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In fish, the adaptive and innate immune systems may be readily distinguished from each
other. The former is not active initially but has to be stimulated in the form of exposure
to antigens leading to the activation of [immune] memory. This response is specific
(Agrawal et al., 1998). In contrast, the innate immune system occurs throughout the life
of the animal, and comprises physical barriers, and cellular and humoral components
(Table 29). Innate immunity is arguably the first line of defense against invasion of
pathogens, and involves with a non-specific, inducible form of response. To date, most
immunostimulants, e.g. β-glucans (Yano et al., 1989, Robertsen et al., 1990, Jeney et
al., 1997; Siwicki et al., 1994) and levamisole (Mulero et al., 1998; Ispir and Yonar,
2007), examined in fish have been determined to induce a non-specific immune
response (Anderson, 1992).
It has been established that activation of a non-specific immune response is usually
through processes such as leucocyte proliferation including macrophages, neutrophils,
lymphocyte and non specific cytotoxic cells (Secombes, 1996; Duncan and Klesius,
1996; Sahu, 2004; Kaisho, 2005; Zhang et al., 1997; Ahmad-Nejad et al., 2002),
phagocyte stimulation, cytokine production, activation of cell receptors e.g. lectin
receptors, stimulation of both B- and T- cells, complements cascade and antigen
presenting cells (APC; Lo et al., 1999; Jiang et al., 1995). Here, fish macrophages and
neutrophils have phagocytic, chemotactic, bacteriocidal and respiratory burst activity
and their assessment is an invaluable indicator of the health status of fish population
(Lamas and Ellis, 1994, Rodriguez et al., 2003; Palic et al., 2005). Stimulation of innate
immunity is considered to be the most likely explanation for the beneficial effect of
garlic, ginger and LPS observed in this study. The overall results parallel those of
Martins et al. (2002), who investigated the effect of dietary garlic on the erythrocyte,
leucocyte and haematocrit content. Furthermore, Sahu et al. (2007) reported increased
number of erythrocytes and leucocytes following 60 days of feeding of garlic to rohu
fingerlings. I concur with the opinion of Kyo et al. (2001) that garlic is indeed a
promising immunostimulant and a biological immune modifier capable of maintaining
the homeostasis of immune functions and stimulating necessary immune mechanisms. It
should be emphasised that eosinophils and basophils were not observed in the blood of
rainbow trout, although their presence in fish is disputed (Ellis, 1977; Hendrick et al.,
1986).
The data for use of LPS followed the pattern for garlic and ginger in which
significant changes were documented in the erythrocyte and leucocyte count.
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Chapter 4 Discussion
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This mirrors previous in vivo and in vitro studies (Clem et al., 1985). Interestingly, i.p.
injection of LPS leads to localisation in the head kidney and spleen (Dalmo and
Bogwald 1996), with stimulation of macrophage and cytokine production, such as
TNFα, IL-1, IL-6 and IL-10 (Akira and Hemmi, 2003).
The kidney is an important haematopoietic organ in fish (Galindo-Villegas and
Hosokawa, 2004). Yet, fish lack bone marrow and lymphoid nodes, which are present in
their mammalian counterparts (Press and Evensen, 1999). Therefore, the kidney in
addition to the spleen, the thymus and the liver serves as the site of formation of blood
cells (Galindo-Villegas and Hosokawa, 2004). The posterior part of the kidney is
concerned with blood filtration and excretory functions, whereas the anterior kidney, i.e.
the head kidney, is the main site of blood cell differentiation involving the synthesis of
immune cells (mostly monocytes) considered as immature macrophages (Takahashi,
2001). However, it is argued that monocytes represent the circulating macrophage
population, and should be considered fully functional cell types (Stafford et al., 2001).
The head kidney is composed of melanomacrophages, and these are aggregates of
macrophages, lymphocytes and plasma cells (Agius and Robert, 2003).
Melanomacrophages have been suggested to act in concerted efforts with the
endothelial cells and spleen in trapping antigens from the blood stream, and may play a
role in immunogenic memory (Brathgjerd and Evensen, 1996; Secombes et al., 1982).
These all important immunocompetent organs in teleost fish have been shown to be
influenced by dietary immunostimulants (Sakai, 1999; Dalmo et al., 2003; GalindoVillegas and Hosokawa, 2004). In this study, the head kidney macrophages were
involved in phagocytic activity, respiratory burst and bacteriocidal activity. Phagocytic
activity of the head kidney macrophage and other immune cells is an important defence
mechanism against pathogenic organisms (Dalmo et al., 2003). The phagocytic activity
in garlic, as observed in this study, matches previous work with dietary yeast in juvenile
rohu (Choudhury et al., 2005). On a similar theme, Torrecillas et al. (2007).
demonstrated high phagocytic activity in fish fed 0.4% dietary mannan oligosaccharides
(MOS). Also, comparable results were obtained using sea bass (Montero et al., 2005).
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Chapter 4 Discussion
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Undoubtedly, it is possible that the mode of action of garlic and other dietary
supplements on the immune system could involve stimulation of mannose binding lectin
(MBL; Amagase et al., 2001). In particular, lectin is regarded as the most abundant
protein in garlic (Fenwich and Hanley, 1985). Lectins occurred in nature and are
considered to bind to bacterial cells and trigger complement cascade and subsequently
phagocytosis (Janeway, 1993; Zhu et al., 2006; Magnadottir, 2006). Conversely, it is
recognized that mannose constitutes an important surface component of cells including
A. hydrophila (Merino et al., 1996). Here, the mannose-specific lectin is used as a mean
of attachment of the bacterial cells to the gut epithelium of the host, thus serving as
adhesins mediating the binding of bacterial cells with phagocytic cells (Wright et al.,
1989).
The situation regarding the mode of action of ginger may reflect the immunostimulating
effect through its bioactive constituent, i.e. gingerol, which induces the activity of
interleukin-6 (IL-6) a potent B-lymphocyte stimulant (Benny et al., 2004). Moreover,
since B-lymphocyte activation requires the activation of T helper cells as well, it is
therefore expected to entail phagocytic activities.
It is well established that LPS affects the phagocytic cell activities of the head kidney
macrophages in vivo. Thus, Wright et al. (1990) and Ulevitch (1993) demonstrated that
macrophage cells express a LPS receptor in complex with LPS-binding protein present
in serum. Binding of Gram-negative bacterial endotoxins to the receptors is a function
of LPS-induced macrophage activation, thus initiating host defences against bacterial
pathogens (Tobias and Ulevitch, 1993). LPS stimulated macrophage and monocytic
cells rapidly release mediators, especially cytokines, interleukins IL-1, IL-6 and tumour
necrosis factor TNFά that in turn activate other cells implicated in host defence
mechanisms (Gallay et al., 1993). Glauser et al. (1991) suggested that feeding large
amounts of LPS might induce overproduction of cytokines by immune cells, which
could lead to multiple organ failure and death. Indeed, in this study, the groups fed with
the highest amounts of LPS showed elevated mortality rates compared to other groups
(including the controls). Although head kidney macrophages were stimulated in this
study, Salati et al. (1987) reported an enhanced phagocytic activity of blood leucocytes
when eels received E. tarda derived LPS. Similarly, Stafford et al. (2003) showed that
cultured goldfish macrophages responded to LPS by the increased expression of
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Chapter 4 Discussion
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cytokines and IL-1 receptors (IL-1 R), which exert a major role in the induction of
immune responses.
Respiratory burst activity involves the rapid release of ROS such as superoxide anions
(O2-), hydrogen peroxide (H2O2) and hydroxyl radicals (OH) by immune cells mainly
neutrophils during phagocytosis, and is viewed as an important indicator of innate
immunity in fish (Miyazaki, 1998; Jeney and Anderson, 1993). ROS are recognised to
be toxic to pathogenic bacteria (Itou et al., 1996; Hardie et al., 1996). Conversely, many
cellular lipids and polypeptides are vulnerable to attack by ROS resulting in cellular
damage. Immunostimulants modulate non-specific defence mechanisms in fish by upregulating the release of ROS by immune cells (Jeney and Anderson, 1993; Miyazaki,
1998). The overall results obtained in this study are in broad agreement with those of
Choudhury et al. (2005), who observed high activity in rohu fed with 0.4% yeast RNA.
Indeed, comparable results were also obtained by Sakai et al. (2001) using carp, which
were fed with nucleotide obtained from yeast RNA. Of relevance to the current study,
Sahu et al. (2007) reported increased activity in rohu juveniles fed with 0.1%, 0.5% and
1.0% garlic. Furthermore, it has been reported that most of these phytochemical
compounds act synergistically or additively, and exert their antioxidant property by
scavenging ROS (Borex, 2001; Kim et al., 2001), thus enhancing the neutrophilic
activity of the cellular enzymes in the hosT-cells.
Ginger has been shown to be endowed with potent antioxidant properties and is an
effective scavenger of superoxide radicals, which has been proposed as one of the
possible mechanisms of its protective action against autotoxicity and lethality (Jagetia et
al., 2003, Kim et al., 2007, Hirahara, 1974; Krishnakantha and Lokesh, 1993, Borex,
2001; Kim et al., 2001).
In agreement with the results of this study, Solem et al. (1995) demonstrated that LPS
increases the respiratory burst activity of rainbow trout. In particular, the use of dietary
LPS led to increases in the production of superoxide radicals as reported previously
(Nayak et al., 2008; Gultvik et al., 2002).
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Serum bacteriocidal activity is a mechanism noted for killing and clearing of pathogenic
organisms in fish (Ellis, 1999; 2001). In this study and in agreement with previous
workers (e.g. Rao et al., 2006; Misra et al., 2006), serum bacteriocidal activity was
significantly elevated, indicating that various humoral immune factors involved in both
specific and non-specific immunities are found in the serum. In comparison, Misra et
al., (2006) observed high serum bacteriocidal activity after multiple injections of rohu
fingerlings with β-glucan. Similarly, Sahu et al., (2007) reported an enhanced serum
bacteriocidal activity in rohu fed with garlic. This finding seems to be common with
other immunostimulants (e.g. Jorgensen et al., 1993).
Lysozyme is a major humoral immune defense mechanism, and is prodigally present in
the serum, mucus and eggs of fish (Yano, 1996; Ellis, 1999). The compound is enzymic,
being produced by the immune cells especially the monocytes, macrophages and
granulocytes, and exerts an important role in innate immune responses (Magnadottir et
al., 2005). Serum lysozyme activity presents a first line of defence mechanism together
with lytic factors by preventing adhesion, colonization and attacking the peptidoglycan
in the cell wall of bacterial pathogens. This results in the prevention of disease (Misra et
al., 2004; 2006). Modulation of lysozyme concentration in the serum has been shown to
be achieved by immunostimulants and infection with pathogenic micro-organisms
(Siwicki and Studnicka, 1987; Panigrahi et al., 2004). In another report, Lapatra et al.,
(1998) noted a significant increase in lysozyme activity in the serum of rainbow trout
fed with β-glucan for 28 days. Also, Christybapita et al., (2007) observed significantly
enhanced lysozyme activity after 1, 2 or 3 weeks of feeding tilapia with leaf extracts of
the medicinal plant, Eclipta alba. Furthermore, Paulsen et al., (2003) noted elevated
lysozyme activity in Atlantic salmon following administration of immunostimulants.
Although ginger has not been previously associated with enhancing lysozyme activity,
the same may not be stated of LPS (Paulsen et al., 2003).
Trypsin is a major protease found in the living cells that causes hydrolysis of protein.
Fish blood is recognised to possess a number of antiprotease or protease inhibitors, such
as 1-antiprotease, 2-antiplasmin and 2- macroglobulin (Ellis, 2001). Inhibition of
these proteases can regulate the hydrolysis of protein in vivo (inside the cell) and thus
upregulate the mechanism of immune defence against pathogens. Furthermore, the
presence of this inhibitor in the serum of treated fish can activate the complex
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biochemical system in the blood that may potentiate immune defence mechanisms
against pathogenic organisms (Tremacoldi and Pascholati, 2002). In this study, serum
antiprotease activities were elevated, which is in agreement with the previous work of
Vasudeva and Chakrabarti (2004), who obtained an enhanced anti-protease activity in
rohu after administering herbal ingredients orally. Similarly, Vasudeva and Chakrabarti
(2005) reported increased antiprotease activity in Indian carp treated with the medicinal
herb Achyranthes aspera.
The alternative complement pathway is a thermolabile cascade known for bacterial
killing and clearing from the host (Ellis, 2001).
Complement activation by
immunostimulants may occur via the classical complement pathway (CCP) or the
alternative complement pathway (ACP; Merino et al., 1991). The CCP is activated by a
complex between antibody with bacterial surface antigens and sometimes directly by
lipid A moiety of LPS (Morrison and Kline, 1977). The ACP is initiated independent of
antibody (Quinn et al., 1977; Boshra et al., 2006). In the present study, inhibition of
CCP by serum treatment with Mg2+ EGTA did not affect the bacteriocidal activity of the
complement system. Thus, ACP may probably be involved in the haemolytic
complement activity against the bacterial pathogen.
This study revealed that dietary uptake of garlic and ginger resulted in increased
alternative complement activity, which is in agreement with the work of Mulero et al.,
(1998), who noticed a significant serum complement activity after 10 weeks
administration of 500 mg levamisole/kg to gilthead sea bream Sparus aurata. Similarly,
the use of LPS as a dietary supplement led to increased alternative complement activity
although a previous study did not report any marked haemolytic activity in sea bass,
Dicentrarchus labrax, and Atlantic halibut, Hippoglossus hippoglossus, sera (Lange and
Magnadottir, 2003).
Increases in the serum total protein, albumin and globulin contents of fish populations
are considered to reflect a stronger innate immunity (Wiegertjes et al., 1996). Albumin
and globulin fractions are certainly important for maintaining a healthy immune system
(Jha et al., 2007). In particular, it has been asserted that the gamma globulin fractions is
the source of all immunological protein of the blood, whereas albumin is essential for
maintaining the osmotic pressure needed for normal distribution of body fluids, and acts
128
Chapter 4 Discussion
_____________________________________________________________________
as plasma carrier and non-specific ligand with many binding domains (Shenkin et al.,
1996). A lower albumin/globulin ratio in the blood serum is an indication that there is a
greater globulin concentration in the total protein compared to the albumin content,
which was the situation in the current study. However, since the gamma globulin
fraction constitutes the largest part of the overall globulin content (Looney, 2005), it
could be inferred that dietary garlic and ginger have stimulatory effects on the
biochemical indices of rainbow trout and hence the immune response. This inference is
supported by the increased survival of rainbow trout fed with these compounds
following experimental infection with A. hydrophila. Furthermore, the data for use of
LPS reinforced this view although Selvaraj et al., (2004) reported a reduction in serum
total protein after i.p. injection in carp. It is interesting to note that Ingram and
Alexander (1980) attributed such decreases in protein level to handling and injection
stress.
It is pertinent to reflect on the dose(s) of dietary supplements that were most effective at
controlling A. hydrophila infection in rainbow trout. Certainly, it has been asserted that
dosage or level of inclusion of the compounds is critical in determining their success or
indeed failure. Invariably, proper dosage is the key to obtaining optimal enhancement of
the host response (Sakai, 1999). High dosages may not be economical and may lead to
harmful effects such as immunosuppression, and considerable variation in effect may
occur (Delbert and Gatlin, 2002). Indeed, Sakai (1999) noted that:
‘the use of high dose of immunostimulant may or may not lead to higher disease
protection as the effect of immunostimulant is not directly dose dependent’.
Overall, the results of this study supported the previous findings of Sahu et al., (2007),
who noted the effectiveness of 5.0 g and 10.0 g garlic/kg in rohu when challenged with
A. hydrophila.
Electrolytes have been recognised to be associated with various biological functions in
fish. For example, calcium, one of the most abundant cations in fish, beside its role in
the maintenance of acid- base equilibrium and cell membrane formation, serves for
blood clotting, nerve transmissions and activation of enzyme activity (Lall, 2002). In
the cell membrane, calcium is closely bound to phospholipids and in this state regulates
the permeability of the membrane for uptake of substances by the cell. Conversely,
magnesium is an important co-factor in many enzymatic reactions and is known to exert
129
Chapter 4 Discussion
_____________________________________________________________________
an important role in osmoregulation and respiratory mechanism in fish (Houston, 1985).
Magnesium support activation of the immune system enhances the activity of
macrophages and natural killer cells (NK; Jamroz, 2005). Iron, which exists as
haemoglobin in erythrocytes, transferrin in plasma and as ferritin in serum (Bernat,
1983), supports the killing activity of the neutrophils and respiratory tract mucosa.
Within the immune system iron is thought to form lactoferrin, which is a
multifunctional protein with antimicrobial activity (Jamroz, 2005). Many reports have
shown that deficiency of these electrolytes in fish result in a weakness of the immune
system, reduced growth and high rates of mortality (Jamroz, 2005; Cowey et al., 1977;
Knox et al., 1983). It was clear in this study that the dietary supplements did not have
any significant effect on the electrolytes, with the exception of magnesium which was
upregulated by garlic and LPS. Indeed, a similar study by Liao et al., (2007) using
chitosan did not reveal any effect on the status of the electrolytes. This was supported
by Rehulka (2000), who reported lower levels of electrolytes in rainbow trout
administered with astaxanthin for 84 days.
Digestion involves the breakdown of the major macronutrients, namely carbohydrates,
proteins and lipids, into components that may pass through the cell walls of the
alimentary tract and be absorbed into the bloodstream (Pillay and Kutty, 2005). These
breakdowns are made possible through the activity of enzymes (Pfeffer et al., 1991). It
is relevant to note that Pfeffer et al., (1991) asserted that digestion of food components
is a clear indication of their bioavailability, and the capacity is species specific and
varies with food source. It has been argued that in the case of fish, growth may be
affected by many environmental and physiological factors (Blier et al., 1997; Weatherly
and Gill, 1995). Also, the activity of digestive enzymes and the absorption of nutrients
derived from digested food are among the important metabolic processes that guarantee
a high sustainable energy budget that leads to increased growth rate (Blier, 1997).
Furthermore, the capacity of fish for growth may be constrained by several factors at the
level of digestion, and include indigestion, poor assimilation of nutrients, feed
conversion and efficiency which relates to feed quality and its ability to sustain growth,
which in turn depends on the capacity of the digestive enzymes (Jobling, 1995;
Weatherly and Bill, 1987).
130
Chapter 4 Discussion
_____________________________________________________________________
Like the situation with mammals, there are several enzymes involved in digestive
activities in fish. Pepsin, which is a major protease secreted by the gastric glands of the
stomach, initiates the digestion (of proteins) by hydrolysing and denaturing the protein
(Lauff and Hofer, 1984). Trypsin and chymotrypsin are secreted by the pancreas, which
in most fish is diffuse, and therefore not easily isolated (Hidalgo et al., 1999). The
secretions are expressed in the intestinal lumen, and regulate the dietary protein level of
the host (Cahu et al., 2004). Interestingly, trypsin secretion is known to occur at a high
level in fish larvae fed diet supplemented with native proteins. Conversely, secretion is
reduced when diets have a high level of hydrolysate (Cahu et al., 2004).
Among the pancreatic enzymes of fish, trypsin has received more interest in recent
times. Moreover, intestinal enzymes are considered to be responsible for terminal
digestion of protein peptides (Torrisen et al., 1994; Zambonino and Cahu, 2007). These
enzymes are found in the cytosol and brush border membrane of the enterocyte
(Zambonino and Cahu, 2007). Cytosolic enzymes include principally dipeptidase and
tripeptidase (Shils et al., 2005; Zambonino and Cahu, 2007), which complete the
hydrolysis of protein by reducing peptides to free amino acids (Zambonino and Cahu,
2007).
The incorporation of some food substances or additives in diets is known to stimulate
the activity of cytosolic enzymes and consequently facilitates the assimilation of amino
acids (Cahu and Infante, 1995). However, with the maturation of enterocyte cytosol, the
cytosolic enzymes decrease giving way to the development of brush border membrane
enzymes, of which alkaline phosphatase and leucine aminopeptidase are the most
important (Ma et al., 2005). Alkaline phosphatase is associated with the absorption and
transport of lipid and carbohydrate across the intestinal cell wall (Fraisse et al., 1981).
Meanwhile, leucine aminopeptidase degrades protein into smaller peptides and free
amino acids for adsorption (Sabapathy and Teo, 1993; Natalia et al., 2004). Dietary
supplements are also recognised to influence the maturation processes of intestinal
enzymes. For example, amines and polyamines of low- molecular weights are
commonly found in feed supplemented with fish meal (Bardocz et al., 1993). In
rainbow trout fry, replacement of fish meal supplemented diet with full fat soybean
meal was determined to result in reduced growth (Dabrowski et al., 1989).
131
Chapter 4 Discussion
_____________________________________________________________________
The high rate of secretion and release of digestive enzymes means the availability of
nutrients in fish for growth and metabolic processes. Thus, their availability may be
limited by the enzymatic activity present (Krogdahl et al., 1994; Cohen et al., 1981).
Against this background, it should be explained that the aim of this study was to
examine whether the increased growth rate and heightened immune response recorded
in rainbow trout after administration of dietary immunostimulants reflected the
modulation of digestive enzymes. Interestingly, it has been reported that the fish
digestive systems have the ability to respond to different feed supplementations. For
example, Garcia-Carreno et al., (2002) reported that by varying the quality and quantity
of protein in feed, fish responded appropriately with a corresponding production of
alkaline phosphatase activity. These workers noted that fish digestive systems could be
modulated by external factors involving feed treatment.
In line with this consideration, it was observed in this study that variations in most
enzymatic activities occurred among the various treatments. This could also be further
explained by the specificity of enzyme activity, as most of the enzymes had high
activity at acid and near alkaline conditions. Here, the pH of the enzyme extracts was
slightly different from that of the controls, thus offering optimal condition for high
enzymatic activity. Also, this may have contributed to the high digestibility of diets
resulting in relatively better growth of the fish.
In animal models, it is established that feed intake is related to body weight (Koong et
al., 1985; Sainz and Bentley, 1997; McLead and Baldwin, 2000). The biometric indices
of rainbow trout fed with dietary supplements showed indications of enhanced feed
intake, which is reflected in the growth of some gastrointestinal organs and the body
weight, as compared with the controls.
This could only be in response to feed
treatments. The enzymes responsible for proteolysis in the intestine are the serine
protease, mainly trypsin and chymotrypsin (Krogdahl et al., 1994). The amount of
amino acid in fish is limited by the rate of nutrient produced from these digestive
enzymes (Dabrowski, 1983, Krogdahl et al., 1994; Torrisen et al., 1994). Dabrowski et
al. (1989) asserted that in rainbow trout fry replacement of fish meal with full-fat
soybean meal resulted in reduced growth. Furthermore, Kakade et al., (1973) confirmed
that ~30 - 50% of the inhibitory effect of soybean meal on growth is from the soybean
trypsin inhibitors. In contrast in this study, trypsin inhibition did not seem to impose any
132
Chapter 4 Discussion
_____________________________________________________________________
limitation to growth in rainbow trout fingerlings when administered with the dietary
supplements. Certainly, one of the outcomes of the research was the realisation that
dietary garlic and ginger possessed powerful anti-trypsin activity against A. hydrophila
infections. This was reflected by the low trypsin activity induced by these dietary
supplements. That their anti-trypsin activity did not manifest in growth reduction in
rainbow trout could be explained by the gastric absorption of protein biproducts by acid
protease mainly pepsin, which was observed to be significantly higher than in the
controls. Gastric absorption of food nutrients especially protein biproducts, i.e. amino
acid, in fish had been previously reported (Austreng, 1978; Dabrowski and Dabrowska,
1981).
Furthermore, the high activity of alkaline phosphatase in the intestinal brush border
membrane enterocytes observed in this study could also compensate for this anti-trypsin
activity. It is apparent that alkaline phosphatase is expressed by the matured brush
border membrane of the enterocytes, and is therefore an indicator of enterocytes
functionality (Traber et al., 1992; Uni et al., 1998). Their presence at high level in the
intestinal epithelium indicated a potentially high absorption capacity, which was
observed here, and is in line with the previous work of Reshkin et al., (1989). These
workers studied the effect of dietary growth hormones (GH) in tilapia, and concluded
that variable growth patterns could be attributed to changes in the intestinal brush
border membrane transport of nutrients. In view of the present findings, it is concluded
that dietary garlic, ginger and LPS modulated enzymatic activities of the fish, and thus
contributed to growth performance.
The use of immunostimulants as dietary supplements is recognized to improve the nonspecific defence mechanism in fish, thus providing resistance to infection (Jeney and
Jeney, 2002; Petrunov et al., 2007). Interestingly, it has been argued that the fish innate
immune system lacks memory, and as such the duration of protection will inevitably be
shorter than the specific or adaptive immune response (Anderson, 1992). Also, it has
been considered that long-term exposure to immunostimulants lead to
immune
suppression and tolerance insofar as the immune system becomes de-sensitized thereby
losing its sensitivity (Bricknell and Dalmo, 2005; Bagni et al., 2000). However, the use
of dietary immunostimulants has led to protection in fish against a range of bacterial
fish pathogens (Sahu et al., 2007; Shalaby et al., 2005; Delaha and Garagusi, 1985).
133
Chapter 4 Discussion
_____________________________________________________________________
For example, Shalaby et al., (2005) recorded high level of protection in tilapia after
receiving dietary garlic followed by challenge with A. hydrophila. Furthermore,
Cavallito and Bailey (1944) suggested that dietary garlic provides a suitable basis for
new therapies because of their antimicrobial and immunological properties.
Of relevance to the present study, a previous investigation using brook trout, Salvelinus
fontinalis, which were administered with chitosan by 30-min immersion led to reduced
protection 14 days later (Anderson, 1994). At 21 days after administration of chitosan,
there were not any significant differences in protection levels noted. This is in line with
the view that long-term application of immunostimulants leads to immunosuppression
and loss of effectiveness of the compounds (Siwicki et al., 1990; Bricknell and Dalmo,
2005). It is speculative whether or not a similar effect could have happened in this
study.
Modulation of non-specific defence mechanisms in the fish used in this work may have
been chiefly by activation of the released of ROS by immune cells. This might explain
the significant increased respiratory burst activity, measured by the reduction of NBT to
formazan as indicator of superoxide anion (02- ) production. This reactive oxygen
species include superoxide radicals and hydrogen peroxide, which are known to be toxic
to pathogenic bacteria (Itou et al., 1996; Hardie et al., 1996). Of relevance, Sahu et al.,
(2007) reported and increase in NBT activity over control diet in rohu juveniles fed
0.1%, 0.5% and 1.0% doses of garlic. Moreover, this significant difference between the
treatment groups and the controls agreed with the finding of Choudhury et al., (2005),
who observed a high NBT activity in rohu which received 0.4% yeast RNA.
Comparable results were also obtained by Sakai et al., (2001) using carp, which were
fed with nucleotide derived from yeast RNA. Certainly, the proliferation rate and
number of lymphocytes produced is important for the magnitude and duration of
protection (Eggset et al., 1997). This supports the views that the persistence of an
immune activator may be a critical factor in maintaining long-term protection. With
garlic, various bioactive compounds have been found to exhibit immunological
properties and are detectable in blood after oral uptake (Rose et al., 2005, Amagase,
2006; Steiner and Li, 2001).
Also in comparison to this study, the condition factor
(CF) was reduced in rainbow trout as reported by White and Fletcher (1985).
134
Chapter 4 Discussion
_____________________________________________________________________
It is noteworthy that CF has been regarded as a useful bio indicator of stress (Anderson
et al., 2003), and is reflected in changes in energy budgets (Smolders et al., 2003). In
the present study, it is possible that the deterioration in CF may be a consequence of
disrupted metabolic processes, resulting from the withdrawal of dietary supplements
from the diet.
Furthermore, the changes in levels of blood electrolyte ions may be explained by
reduced energy metabolism as considered previously (Lall, 2002). It is interesting to
note that similar results were documented by Bradbury et al., (1991) in rainbow trout
treated with central nervous seizure agents. Notwithstanding, this study has affirmed
that the protective property of dietary garlic, ginger and LPS extends beyond the 14 day
period of application.
Although there is a clear benefit to the use of garlic and ginger, a question remains
about the nature of the active ingredient(s). In this connection, it has been established
that intact garlic bulbs contain high amounts of γ-glutamyl cysteine as storage peptides,
which become hydrolysed naturally during processing and storage to alliin and S-allyl
cysteine (SAC; Arnualt and Auger, 2006; Annu et al., 2005; Arnault et al., 2005). Alliin
is converted by alliinase to allicin diallyl thiosulphinate, which is an extremely unstable
compound that is responsible for the pungent smell of garlic (Block, 1985), and
becomes quickly decomposed into different organo-sulphur compounds, i.e. diallyl
sulphide (DAS), diallyl disulphides (DADS), polysulphides and ajoenes (Amagase et
al., 2001; Shadkchan et al., 2004). Unlike allicin, S-allyl cysteine is a more stable,
odourless; bio available and water-soluble compounds, and may well contribute to the
pharmacological and immunological activities of garlic (Corzo-Martinez et al., 2007).
Allicin diallyl thiosulphinate is an active but volatile compound of garlic, and has been
implicated in antibacterial activity against a wide range of Gram-positive and Gramnegative bacteria, and anti-viral, anti-fungal and anti-protozoal activity (Pai and Platti,
1995; Ankri and Mirelman, 1999; Gupta and Potter, 2001; Weber et al., 1992; Sofar and
Mokhtar, 1991; Pszczola, 2002; Rose et al., 2005).
From this study, the value of allicin for the control of A. hydrophila infection in rainbow
was clearly demonstrated. This is not so surprising as allicin has been reported to be
inhibitory against bacterial pathogens of clinical significance including Escherichia coli
135
Chapter 4 Discussion
_____________________________________________________________________
and Staphylococcus aureus (Tsao and Yin, 2001; Bjarnsholt et al., 2005). The MIC
values recorded in this study were in line with the work of Cai et al., (2007) who
reported a value against Staphylococcus of >512 µg/ml. The mode of action of allicin
may well include inhibition of cysteine protease, scavenging and trapping of free
radicals (hydroxyl, superoxide anions and hydrogen peroxides) and initiation of the
inhibition of thiol containing protein in the cells of the pathogens (Coppi et al., 2006;
Rabinkov et al., 1998; Ankri et al., 1997). This might well explain the increased values
of superoxide anion production in the study. It has been suggested that some of the
protein-based sulphur compounds in garlic, such as S-allyl cysteine SAC, S-ethyl
cysteine, N-acetyl cysteine, lectin and pectin, which are stable, odourless and
bioavailable, may be responsible for priming pharmacological and immunological
protections (Corzo-Martinez et al., 2007).
The involvement of allicin in stimulating immune parameters would certainly suggest
that this compound may well explain in part the beneficial effect of garlic. It is relevant
to note the work of Kuttan (2000), who studied the immunomodulatory effect of
naturally occurring sulphur compounds, namely S-ethyl cysteine, N-acetyl cysteine and
allicin derivatives, i.e. diallyl sulphide DAS and diallyl disulphide DADS and diallyl
trisulphide DATS, in mice, and concluded that the compounds potentiated stem-cell
proliferation and differentiation. However, allicin has been considered to be a transient
compound, being rapidly decomposed or hydrolysed into the various sulphur containing
compounds indicated above. It has certainly been argued that the breakdown products of
allicin exhibit the antibacterial and antifungal effects of garlic (Tansey and Appleton,
1975). Notwithstanding, allicin has a definite role in disease control in rainbow trout.
It is clear that the laboratory based work has highlighted the value of dietary
supplements. Now, field trials are needed to determine the potential value of these
products in the fish farm environment.
136
Chapter 4 Discussion
_____________________________________________________________________
Conclusions
Ten dietary supplements were evaluated and garlic, ginger and LPS examined in detail.
The key findings were:
1.
A 14 day feeding regime with dietary garlic, ginger and LPS was effective for up
to 21 days after withdrawal in terms of protecting rainbow trout against challenge
by A. hydrophila.
2.
The most effective dose(s) at enhancing the non-specific immune response for
optimal protection and growth performance were 0.5 g and 1.0 g /100 g of feed
for garlic and ginger, and 1.87 mg and 3.75 mg LPS/100 g of feed for LPS.
3.
The modes of action involved the stimulation of innate immune response in terms
of phagocytosis, respiratory burst, lysozyme, bacteriocidal and antiprotease
activities, and proliferation of immune cells, production of inhibitory substances
and digestive enzymes.
4.
The oral administration of garlic, ginger and LPS was effective in up-regulating
the non-specific immune response mechanisms of rainbow trout against A.
hydrophila infections.
5.
The dietary supplements stimulated the digestive enzymes in the stomach, small
intestine and brush border membrane, thereby contributing to the growth
performance of rainbow trout.
6.
The garlic component allicin diallyl thiosulphinate and its derivatives have
antibacterial activity. The protein-based sulphur compound S- allyl cysteine and
its derivatives have immunological functions.
7.
Garlic and ginger dietary supplements stimulated gastric absorption of protein byproduct by up-regulating pepsin activity.
8.
Garlic and ginger have been shown to possess trypsin inhibitor, by down- regulating intestinal trypsin activities in the digestive system.
137
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