DEVELOPING CHALLENGE MODELS AND VACCINE EFFICACY TESTS
AGAINST Streptococcus iniae FOR TWO ORNAMENTAL CYPRINID FISH: THE
RED-TAIL BLACK SHARK (Epalzeorhynchos bicolor) AND THE RAINBOW
SHARK (E. erythrurus)
DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
To my parents Francesca and Salvatore, my aunt Marilena, my uncle Ennio, and my wife
Juli-Anne. Thank you for your love and encouragement.
I want to thank my parents, Francesca and Salvatore; my aunt and uncle, Marilena
and Ennio; and my wife, Juli-Anne, for their help, support and love in these years. I want
also to thank Debra Anderson, Sonya Sampson, Marta Hartman, and Richard Miles for
their friendship, advice, and encouragement.
I thank the members of my supervisory committee (Drs. Roy Yanong, Jack Gaskin,
Richard Miles, Hugh Mitchell, John Tucker, Ruth Francis Floyd), Mr. Craig Watson
(Director of the Tropical Aquaculture Laboratory), and Dr.
Walter Parker (NOVARTIS-
Aqua Health LTD.) for their advice and help throughout my research. I thank Dr.
for teaching me about fish health and for the big revision of this dissertation, and Dr.
Gaskin for his great supervision and assistance during my stay in Gainesville.
I thank Dr. Debra Murie for helping me with the image analysis.
Special thanks go to Jason and Damon Diaz, Robin Sanderson, Scott Graves, and
Robert Leonard for assistance with fieldwork and in the laboratory. Thanks go to Carlos
Martinez, Sherry DeMayo, Tina Crosby, John Kao, and Bill Shields for their invaluable
assistance. I also want to thank all the University of Florida and University of South
Florida professors that have taught me so much in their courses.
This project was supported by a grant from USDA-CSREES.
TABLE OF CONTENT
. ... ..... ......... ..... .. ..... ... .. .......... .. .. .... ...... .. ......... ... ... ii
LIST OF TABLES .................................. .......................................... ..... .................. v
LIST OF FIGURES ........................... ................................................................................ x
A B ST R A C T .... ........... .. .. ........... .. ..... ....... .............. .... ......... ..... ...... .. .... .......... ...... ....... xiii
S *9..m**** C1(111)111 S1(1 C 111(1 111()1 CCC1111m****11 .45..mm$*.(11 *St m **C S m b e S S C e S* mS*SC 1m* *
RED-TAIL BLACK SHARK (Epalzeorhynchos bicolor) AND RAINBOW
SHARK (E. erythrurus) BIOLOGY
* CC *SC*lS Clm**SO CS *S C S C*S SC C C**S **Sl C.** CC S C... C S C*e me....C.C* C* SC
*.m ....... ....*em ....... m....S* mm *. *S.SC*...C S. CO *C1S ..m..S.C...* CCC1#.
STREPTOCOCCOSIS IN FISH
Host-Pathogen Interactions ...............
Antibiotics Treatments and Vaccines
C.. CCC S eeseee S C*S*Ce .. S C.*m S 55CCCS S* C SC C CC5
Fish Immune System......... .............. ..
Ontogenesis of the Immune System.
Immune Organs and T-Cell Ontogenesis:
Oral Vaccination ..........
Genomics and Proteomics App
Herd Imm unity ........................
. ... ... .. ...... ................... ........................ ................ 55
S... .... ..*..... ... ... S ..*............... ..................... ........ ........ ..5
............ .......................... ................ .. ................. ...61
.. ...................................................... ..... .... ......... .... 6 2
.... .... ... .... .... .... .... ... .... .... .... 62..
lied to Vaccine Design .............. .................. 66
..... ..... .. ..... .... ........ .. ... .. .. ..... .....................7 4
Immunostimulants and Experiment Design
......... ............... ... ... ... ........ .....76
.. ... ...... ........... ..... ...... .... ........... ........... ......................... ............ .. 8
...... ........ ................. .......... ...... ..... .. .......................... ............. 8
as ....... ............. ................ .. ........ .. ............ ... ........... ......................... 8
ans........................................... ... .. .... ...........*-**--................,....91
ans. ...... ... ... ... ....... .... ................. ....................... ............ ..... ...9 1
DESIGNING A BIOSECURITY LABORATORY AND RECIRCULATING
SYSTEMS FOR CHALLENGE AND VACCINE EXPERIMENTS WITH
........ ............ ............. ....... ...... ..... .. ............... .............. 9 4
.......................................................... .. .................9 6
Laboratory Design ...
Recirculating System s Design ...................................................................... 97
Sterilization and Start Up Protocols............................. ...................... ........ 97
Conclusion .................................. ......................... ..... ........................ ......... 98
DEVELOPING CHALLENGE MODELS AGAINST Streptococcus iniae IN
RED-TAIL BLACK SHARK (Epalzeorhynchos bicolor) AND RAINBOW
SHARK (E. erythrurus), FAM. Cyprinidae
......... ................ ...................... .............. .............. 9 2
<*1C.. ,*..*.. . C*.. *n C- .* *in** **C **C in....'*4 *C *Ci '.*C***i.**C.* C'**'*C-*C *"
EFFICACY OF DIFFERENT VACCINE FORMULATIONS AGAINST
Streptococcus iniae AND ADMINISTRATION ROUTES IN RED-TAIL
BLACK SHARK (Epalzeorhynchos bicolor), FAM. CYPRINIDAE
......... .......................................... ................. 1
.......................................................... ........ 1
... ...... ................... ... .. ... .................. ....* .. 1
.*................ . . 9 .. S ... e.. t.... ..................1
---------- -- ............................ 1-
IMPROVEMENT OF VACCINATION STRATEGIES AGAINST Streptococcus
iniae IN JUVENILE RED-TAIL BLACK SHARK (Epalzeorhynchos bicolor),
M methods .... .. .. ... .... .... .... ... ... ... .. ..... ... .... ..... .. ... .
. .. ....eee..t. .. . C.* 5**tt eC**t* *9S .. .t. .et. C. .t**. **' .. .*S t St* ** .
* C t t. t e ..... *. .. *** q tt tC e .** S .* St. ..** .** t .* *"t.*t .* C t Ce* tt*9. .* *
...... ........ 1
DIFFERENCES IN LEVELS OF PROTECTION PROVIDED BY DIFFERENT
VACCINE FORMULATIONS AGAINST Streptococcus iniae AMONG
JUVENILES AND ADULT FISH OF RAINBOW SHARK (Epalzeorhynchos
erythrurus) AND RED-TAIL BLACK SHARK (E. bicolor), FAM.
..t...... .... .e.t... ..t....e........................ ............................... 1
.*e.e SC** 9. t .. .. *. t. t t t*.ee... ...**. 9 *9..tt .e. ....t..tt.Ct.**.9**t"t*tt. I
DIFFERENCE IN RESISTANCE TO Streptococcus iniae INFECTIONS
BETWEEN THE WILD PHENOTYPIC RAINBOW SHARK (Epalzeorhynchos
erythrurus, FAM. CYPRINIDAE) AND AN ARTIFICIALLY SELECTED
PHENOTYPE (THE BLUE SHARK)
. e. .i.O t.. .. .... ... i. ...............e.. .. *** ** ** "'"
M methods .......... ....... ....................... ......................................t
.* t ^ "^ * .*' .. .. .. .. .. .. .. .. .. ... .. .. .. .. .. .. *. .. .* *-- -- '. .. *- '- *' '* '- -
14 ENHANCEMENT OF RESISTANCE TO Streptococcus iniae INFECTIONS IN
VACCINATED AND UNVACCINATED RED-TAIL BLACK SHARK
(Epalzeorhynchos bicolor, FAM. CYPRINIDAE) FED BETA-GLUCANS OR
M ethods . .. .... ....... .. ...... .. ............ .* ... .* .... 1
.....eth ds............ ................................................................... 1
. .. ..*.. ............ .. .... ... ... .. t. .. ....... .... ... .. .. .. ... ... 1
-- ..-. .... ............................I
EFFICACY OF VACCINATION OF ARTIFICIALLY AND STRIP-SPAWNED
BROODSTOCK RED-TAIL BLACK SHARK (Epalzeorhynchos bicolor, FAM.
CYPRINIDAE) AGAINST Streptococcus iniae
Introduction.............. .. ...... ... .................................. ......................... ............
M materials and M ethods ........ ...................... ................................................
Results ... .... ..... ....... ...S....t.. ..***..*. .*** *.***9. ........... ..... ...* ....... .....*. ...**
Discussion .................. ................... .................................... .. ......... .........
Conclusion ...................................................................................... .... .. ..........
AGGLUTINATION AND ELISA TESTS
Introduction. ............................ ............ ................... ......................................... 1
M materials and M methods .............................................................. .............. ................... .. 1
Results. ............... ........ .......... ................................. ............9.. 1
Discussion ..................................................... ................... .... ................... 1
Conclusion ................... ........... .................................................. .. ........... 1
17 CON CLUSION NI....................................................................................................197
LIST OF REFERENCES ......................................................... .....................................................200
BIOGRAPH ICA L SKETCH .......................................... ................... ..... ........... 236
LIST OF TABLES
First appearance of immune organs in different fish species. ..................................53
First appearance of lymphocytes in immune organs in different fish species .........53
Time of maturation of the immune organs .................... ...... ............. ......... .. ....54
Characteristic of S. iniae isolated from diseased rainbow shark................... .........11
Average cumulative mortality (%) 14 days after bacterial challenge for the
first experiment with RTB shark.
Average cumulative mortality (%) 14 days after bacterial challenge for the
two experiments with rainbow shark.
Average cumulative mortality (%) recorded in the first 12 hours after
injection of the vaccine formulations or of the bacterial challenge .......................124
Total average mortality (%) recorded for 12 days after challenge with S.
*.llll..........w. *. **g ...* m .*sla **# *S g. .S ,*. ** *S* *. q.* .
Average cumulative mortality (%) recorded in the first 12 hours after
injection of different volumes of the vaccine formulations
Total average mortality (%) in fish injected with different vaccines doses
recorded for 12
days after challenge with S. iniae .............
Total average mortality (%) recorded in RTB and rainbow sharks juveniles
and adults for 12 days after challenge with S. iniae ................... ............ .................. 153
Total average mortality (%) recorded in RTB,
12 days after challenge with S. iniae ...............
rainbow and blue sharks for
Total average mortality (%) recorded in RTB shark fed immunostimulants
and immunostimulants free-diets for 12 days after challenge with S. iniae. ......,..171
Total average mortality (%) recorded in RTB broodstock vaccinated 8
months before the experiment, for 12 days after challenge with S. iniae .............. 187
Range of koi and RTB shark serum dilutions tested in the indirect ELISA. .........196
Coating agents used in the indirect ELISA to detect koi or RTB or rainbow
shark Igs anti S. iniae, and coating agent dilutions that gave positive
* ** *S ** *S SS I*Se* ** ** SS ............ .................. .. .. .. .. ... .. .. .. ... ..1 9
LIST OF FIGURES
V iew of the biosecurity room .......................... .......................................... ...... 99
Close-up view of one of the recirculating systems ..............................................99
View of three of the four 400 L tanks used to store incoming water.....................99
View of one of the bead bioreactors ........... ........... ... ..... .. .................... 99
Average mortality rates (%) observed in Experiment 1 with RTB shark ............112
Average mortality rates (%) observed in Experiment
with RTB shark............l 12
Average mortality rates (%) observed in Experiment 3 with RTB shark............1 13
Average mortality rates (%) observed in Experiment 4 with rainbow shark....... 114
Average mortality rates (%) observed in Experiment
with rainbow shark.......1 14
Average mortality (%) recorded in the first 12 hours after injection of the
3 vaccine formulations or of the bacterial challenge ......... ....................... .........124
H&E staining of a posterior kidney section of a fish injected with the
H&E staining of a spleen section of a fish injected with the aluminum
Average mortality (%) recorded for 12 days after challenge with S. iniae.......... 126
Box plot of the mortality rate (%) in fish injected with different vaccines
doses recorded for 12 days after challenge with S. iniae ............ ......................... 136
H&E staining of a spleen section of a non-vaccinated fish ................................. 136
aluminum formulation ........................................
H&E staining of a posterior kidney section of a non-vaccinated fish .................137
H&E staining of a spleen section of a fish injected with the oil formulation......137
H&E staining of a posterior kidney section of a fish injected with the oil
H&E staining of a spleen section of a fish injected with the aluminum
H&E staining of a posterior kidney section of a fish injected with the
................... .. 138
*4S*S*5 *** S 55**. .139
Gram staining of a spleen section of a fish injected with the aluminum
H&E staining of a liver section of a fish injected with the aluminum
.......................... .................. 139
S......... S......... S *SS*SS*SSS *5955.1 0
Gram staining of a granuloma-like lesion in the spleen section of a fish
injected with the aluminum formulation...
Box plot of the mortality rate (%) recorded in fish vaccinated with the
aluminum formulation by bath or injection for 12 days after challenge
..................... 14 1
Rainbow and RTB juvenile sharks average mortality (%) recorded for 12
days after challenge with S.
Rainbow and RTB adult sharks average mortality (%) recorded for 12
days after challenge with S. iniae ..........
Box plot of the rainbow and RTB sharks average mortality (%) recorded
for 12 days after challenge with S.
.... ..... ............... ........ 1 5 3
Box plot of average mortality (%) recorded in the first experiment with
RTB, rainbow and blue sharks for 12 days after challenge with S. iniae............ 161
Box plot of average mortality (%) recorded in the second experiment with
RTB, rainbow and blue sharks for 12 days after challenge with S. iniae............ 162
aluminum formulation ........
in iae .........,,,.. ...........
with S. in iae ................... .
........._..,.., ..... 152
Box plot of average mortality (%) recorded in the first experiment with
RTB sharks fed immunostimulants and immunostimulants free-diets for
12 days after challenge with S. iniae ......... ... .... ..................... .... ...................172
Box plot of average mortality (%) recorded in the second experiment with
RTB shark fed immunostimulants and immunostimulants free-diets for 12
days after challenge with S. iniae.
Box plot of average mortality (%) recorded in RTB broodstock for 14 days
after challenge with S. in iae .......... ................... ......... ...... 18......6
Box plot of average mortality (%) recorded in RTB broodstock vaccinated
from 8 months, for 14 days after challenge with S. iniae................... ................. 18 7
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
DEVELOPING CHALLENGE MODELS AND VACCINE EFFICACY TESTS
AGAINST Streptococcus iniae FOR TWO ORNAMENTAL CYPRINID FISH: THE
RED-TAIL BLACK SHARK (Epalzeorhynchos bicolor) AND THE RAINBOW
SHARK (E. erythrurus)
Chair: Roy P. E.
Cochair: Jack Gaskin
Major Department: Fisheries and Aquatic Sciences
The purpose of our study was to test three autogenous vaccine formulations (a)
basic formula; b) basic formula with aluminum salt adjuvant; c) basic formula with oil
adjuvant) developed by NOVARTIS-Aqua Health LTD against Streptococcus iniae, for
two ornamental fishes: Epalzeorhynchos bicolor (red-tail black shark) (RTB shark) and
E. erythrurus (rainbow shark). These two species of fish were chosen because they are
considered economically important for the Florida ornamental industry, and they are very
susceptible to streptococcal diseases.
We first developed a challenge model against
iniae for the RTB shark, and then
tested it on rainbow sharks. The protection afforded by the three formulations and
The three vaccine formulations were then tested on rainbow sharks, and
differences in vaccine efficacy among juvenile and adult groups of both RTB and
rainbow shark were investigated. Results of these experiments showed that the vaccine
conferred a higher protection level to RTB than to rainbow sharks. Farmers have reported
that the blue shark (a color variant of the rainbow shark) is more resistant to S. iniae. Our
study confirmed this.
In another set of experiments, the immunostimulatory capability of beta-glucans
and nucleotides were tested. Both compounds decreased the mortality of RTB sharks
iniae compared to the controls.
The final step of our study was to
determine if the vaccines were effective in protecting RTB broodstock fish against S.
iniae infections resulting from the stress caused by artificial spawning. Results showed
that all three vaccines efficiently protected fish after artificial spawning.
Our study showed the efficacy of the two vaccine formulations with adjuvants
against S. iniae and the efficacy of two immunostimulant compounds, beta-glucans and
nucleotides for two species of ornamental fish. Furthermore, these challenge and vaccine
efficacy models can be used as templates in future studies with other species of
ornamental fish and other bacterial diseases.
The purpose of our study was to test three autogenous vaccine formulations (a
basic formula, and formulae with the addition of aluminum salt or oil as adjuvants)
against Streptococcus iniae, for two ornamental fish species, Epalzeorhynchos bicolor
(red-tail black shark) (RTB shark) and E. erythrurus (rainbow shark).
We chose these
two species of fish because they are considered economically important for the Florida
ornamental fish industry, and because they are very susceptible to streptococcal diseases.
We chose Streptococcus iniae infection because is an important disease in the
aquaculture industry, and has been reported worldwide causing up to 75% mortality.
The first step in our study was to build a biosecure laboratory (Chapter 8). A
challenge model against S. iniae was developed for both fish species (Chapter 9).
tested the protection offered by the three formulations and different routes of applications
on juveniles and adults of both species (Chapters 10-12).
We then examined differences
in vaccine efficacy between rainbow and blue sharks (a selected phenotypic variant of
rainbow shark) (Chapter 13).
Immunostimulatory capabilities of beta-glucans and
nucleotides were tested on RTB shark (Chapter 14).
Then, we examined whether the
vaccines were effective in protecting RTB broodstock against S. iniae infections resulting
from the stress caused by artificial soawnin2 (Chanter 1
). Agglutination and ELITSA
RED TAIL BLACK (Epalzeorhynchos bicolor) AND THE RAINBOW SHARK (E.
erythrurus) BIOLOGY AND AQUACULTURE
Red-tail black shark (Epalzeorhynchos bicolor) and rainbow shark (E. erythrurus)
are important economic species for the ornamental fish industry. The name "shark" may
lead to the misunderstanding that these fish belong to the Elasmobranches, real sharks
and rays. In reality these fish belong to the class Actinopterygii,
family Cyprinidae. This
family includes the common carp (Cyprinus carpio) and danio (Danio spp.).
are called "shark" only for marketing purposes and because the dorsal fin is vaguely
reminiscent of the fin of a real shark.
Epalzeorhynchos bicolor has a black body with a red tail, while E. erythrurus has
a greenish-brown body with a large black spot at the base of the caudal fin; and the
dorsal, anal, and pelvic fins are red. Both species attain a maximum total length (TL) of
cm (Axelrod and Burgess, 1997). These fish are benthopelagic, tropical freshwater
fish. Their natural environment is water with a pH range of 6.
70C, and a hardness of 1
(>0.04 ppm) of NO2
, a temperature of
Sharks are very sensitive to high concentrations
-. The diet of the shark includes algae, periphyton, plant matter,
worms, crustaceans, insects, and zooplankton (Yang and Winterbottom, 1998).
S---------------------------------------------at. -- -~ 2 C ~..14 irfl.
-S Sn '-a a
These species originate from Southeast Asia, particularly the Chao Phraya
(Thailand) and Mekong basins. It is not clear if E. bicolor still exists in the wild (Baillie
et al., 2004). Presently, this species has been placed in the IUCN Red List in the category
of extinct in the wild.
The reasons for its decline in the wild are not clear.
fish trade has been accused of driving the species to extinction by overfishing, but no
evidence is documented (Kottelat and Whitten, 1996). Furthermore, most of the sharks
sold in the ornamental industry are farm raised and not caught in the wild. Habitat
modification may be a more likely cause of their population decline. In the 1970s many
dams were constructed in Thailand, and these probably severely impacted the natural
ecosystems of several large basins in the region. Furthermore, large swamps have been
drained, decreasing the natural habitat of this species (Dai and Yang, 2003).
Red-tail black and rainbow sharks can be raised and spawned commercially on
farms. Broodstock can be reared in ponds and in vats. The advantage of rearing
broodstock in vats is that they can be conditioned all year around. In ponds, fish are
conditioned naturally; and spawn in Florida only between May and September. The
indoor conditioning period lasts 6 weeks with fish kept at 280C and 16 h light.
Broodstock are then artificially spawned by induction with hormones (Rottmann et al.,
1991a,b). Each broodstock can be used for spawning up to 16 times in a period of 3 years
(personal communication by farmers).
In captivity, females become mature at 1.5 years
old, corresponding to a size of 10-13 cm. They can produce several thousand eggs.
In the third and fourth weeks, when fish are 4-8 mm long, they begin to be pigmented.
After 5-8 months, when the juveniles reach 2.5-5 cm, they are harvested and sold to the
pet market. Larger fish are also sold, but the market demand is less (personal
communication by farmers).
Streptococci are gram-positive, not motile, and catalase-negative bacteria. In
actively spreading lesions within host tissues, diplococcal and individual coccal forms are
common; in artificial media and purulent exudate, chains are more common. The length
of the chain tends to be inversely related to the adequacy of the culture medium. Factors
that tend to promote exaggerated chaining include conditions that impair growth (such as
unfavorable media, cold temperature, or antimicrobial agents); and also the presence of
antibody that reacts with cell wall antigens (Woo and Bruno, 1998). All streptococci are
lactic-acid bacteria, and derive their energy primarily from fermenting sugars, regardless
if they are grown aerobically or anaerobically. The accumulation of lactic acid in media
with high glucose content can limit bacterial growth if the pH is not buffered or
Several kinds of streptococci can be isolated from humans and other animals. The
first proposed classification of streptococci was based on the description of the type of
hemolytic reactions observed on blood agar plates. In this classification the bacteria are
divided into three groups: alpha, beta and gamma. A narrow zone of hemolysis surrounds
alpha-hemolytic streptococcal colonies, with unhemolyzed RBCs in an inner zone, and
complete hemolysis in an outer zone surrounding the colonies.
medium, and on the incubation time. Because of this feature, this streptococcal group is
also called the viridans group. Most of the streptococcal strains that cause human disease
belong to Group A.
Beta-hemolytic streptococci have an outer capsule, and produce a
wide clear zone of complete hemolysis in which no red cells are visible upon microscopic
examination. These bacteria can release two types of beta-hemolysin enzyme:
streptolysin O and streptolysin S.
Streptolysin O is destroyed by atmospheric oxygen,
and is therefore demonstrable only in deep colonies. Streptolysin S is stable in contact
with atmospheric oxygen, and is responsible for surface colony hemolysis. Beta
hemolytic streptococci are differentiated into a number of immunologic groups
designated by the letters A through O; in each group, different streptococcal types are
present. The groups are characterized by different external carbohydrates that act as
antigens, while specific antigen proteins characterize the types. Precipitin tests
(Lancefield) and aggregation reactions (Griffith) showed this difference in antigens.
Streptococcus iniae is included in the beta-hemolytic streptococcal group.
Gamma-streptococci produce no hemolysis either on the surface or within the agar.
classification based on the hemolysis pattern is not very satisfactory (Woo and Bruno,
1998) for the following reasons:
* Many species classified as beta-hemolytic are in fact not hemolytic (certain
members of the antigenic groups B, C, D, H, K, O and all group N).
* Most streptococci found in the gastrointestinal tract are nonhemolytic, but some
strains produce beta-hemolysis (group D).
STREPTOCOCCOSIS IN FISH
Streptococcal infections in fish result in septicemic diseases that have been
reported worldwide. They cause an estimated $150 million in annual losses of fish
production (Shoemaker and Klesius, 1997). Streptococcosis can cause up to
mortality in closed culture systems (Perera et al. 1994) and up to 50% in ponds (Eldar et
al. 1997). It is one of the most important diseases in South Africa, and Japan (Ferguson et
al. 1994), in yellowtail farming (Bang and Sung-Hee, 1993); and is among the most
important summer diseases in Italian and Israeli trout farming (Bercovier et al. 1997).
Streptococcosis can affect wild and farmed fish in both fresh and saltwater. Case reports
in saltwater fish include yellowtail, Seriola quinqueradiata (Aoki et al. 1990; Ikeda and
Minami, 1982; Park et al. 1987
Sako, 1998), turbot, Scophthalmus maximus (Romalde et
al. 2000), Japanese flounder, Paralichthys olivaceus (Nakatsugawa, 1983), mullet, Mugil
cephalus (Bunch and Bejerano, 1997), red drums, Sciaenops ocellatus (Eldar et al. 1999),
striped piggy, Pomadasys stridens, and variegated lizardfish, Synodus variegatus
(Colorni et al. 2002). Case reports in freshwater fish include tilapia, Oreochromis spp.
(Shoemaker et al. 2001), Mozambique tilapia, Tilapia mossambica (Ming-Chen et al.
1985), Nile tilapia, Tilapia nilotica (Kitao et al. 1981), hybrid tilapia, Tilapia aurea x T
al. 1995a; Kitao et al. 1981), eel, Anguillajaponica (Kusuda et al. 1978), ayu,
Plecoglossus altivelis (Kitao et al. 1981
Ugajin, 1981), snakeheads, Ophicephalus
striatus (Lio-Po et al. 1998), walking catfish, Clarias batrachus (Lio-Po et al. 1998),
zebra danio, Danio rerio, and pearl danio, D. albolineatus (Ferguson et al. 1994), clown
coaches, Botia macracanthus, rainbow shark, E. erythrurus, red-tailed black sharks, E.
bicolor, rosy barbs, Barbus conchonius (Russo data not published), tetras,
Hyphessobrycon sp., and African cichlids of the genera Nimbochromis and
Pelvicachromis (Yanong and Floyd, 2002). Cases of streptococcosis in wild fish are
reported in several publications (Baya et al. 1990; Colorni et al. 2002; Zlotkin et al.
The possibility of streptococcal outbreaks is increased if fish are stressed, as
happens with non-optimal water temperature, low dissolved oxygen, high nitrite levels,
and high culture densities (Bunch and Bejerano, 1997
Perera et al. 1997).
Several species of streptococci have been isolated from fish. Alpha-hemolytic
streptococci generally cause granulomatous inflammation; whereas beta and gamma-
hemolytic streptococci cause systemic infection localized in various organs (mainly
spleen, kidney, brain, liver, and eye), with the presence of granulomas in some
(Ferguson et al. 1994; Chang and Plumb, 1996). An alpha-hemolytic streptococcus was
isolated from hybrid tilapia (Al-Harbi, 1994) showing erratic and whirling swimming,
exophthalmia, hemorrhagic areas on the body surface, and the presence of fluid in the
abdominal cavity as the main clinical signs. Another alpha-hemolytic streptococcus, with
characteristics similar to S. faecalis and
faecium, was isolated from Siganus
affected the central nervous system. No external ulcerations and hemorrhages or other
signs were observed. Streptococcus difficile and S. shiloi were first isolated in Israel from
tilapia and trout with meningoencephalitis (Eldar, et al. 1994; 1995b). Two efficacious
vaccines against S. difficile, a formalin-killed and a bacterial extract based, have been
developed (Eldar et al. 1995b). Other streptococci isolated from fish are S. agalactiae
isolated from seabream and wild mullet (Evans et al. 2002), and S. parauberis isolated
from turbot (Domenech et al. 1996; Romalde et al. 2000).
Streptococcus iniae is the most commonly isolated streptococcus from fish
(Bromage et al. 1999; Eldar et al. 1999; Fiske and Johnson, 1998; Perera et al. 1994;
Sako, 1998). Streptococcus iniae causes a systemic disease characterized by
histological and external pathology and by characteristic fish behavioral changes. The
disease is associated with a strong inflammatory response by the host where acute
necrotizing reactions and systemic neutrophil and macrophage exudation can be observed
in several organs and tissues in conjunction with the presence of high numbers of
gram-positive cocci (Ferguson et al. 1994; Neely et al. 2002).
Externally, streptococcosis-induced lesions include pronounced congestion and
hemorrhages, especially around the base of the pectoral fins and over the heart.
Exophthalmia, corneal opacity, and intra-ocular and periorbital hemorrhages can also be
observed (Ferguson et al. 1994; Liu et al. 1990; Perera et al. 1998).
The main organs affected by S. iniae are the brain, spleen, and posterior kidney.
Pathological changes associated with the brain and nervous system are typical of
granulomas and massive cellular infiltration can be observed (Eldar et al. 1995a; Perera et
al. 1998). Spleen and posterior kidney are common targets because of their high
capability to trap bacteria. In the later stages of infection, there is an increase in size,
expressed as percent body weight, of the spleen and kidney (Ikeda and Minami, 1982).
Analysis of the spleen can reveal congestion and fibrinoid necrosis of the ellipsoidal
sheaths with loss of reticular macrophages. Bacteria can be widely scattered throughout
the parenchyma of the spleen, but can also be observed within macrophages (Ferguson et
al. 1994; Perera et al. 1998). Microscopic examination of the posterior kidney can reveal
severe congestion of the renal parenchyma, and pronounced degeneration and necrosis of
many tubules (Ferguson et al. 1994; Perera et al.
A large number of bacteria can be observed in the pericardial area, especially
within the hemorrhagic musculature associated with the pectoral girdle (Ferguson et al.
1994; Liu et al. 1990; Perera et al. 1998). Other organs that can be affected are the liver,
where sub-capsular petechial hemorrhages and granulomas have been observed; and the
intestinal tract, where bacteria have been observed in the lamina propria; but not in the
mucosa (Ferguson et al. 1994; Perera et al. 1998). An elevated number of bacteria can be
observed within blood vessels, especially in the capillary network of the gills, cranial
meninges, retina, and choroidal rete. The presence of bacteria in the capillaries caused
occasional thrombosis (Ferguson et al. 1994; Perera et al. 1998). Streptococcus iniae can
cause typical behavioral changes such as a swimming spinning pattern (Eldar et al.
1995a; Perera et al. 1998).
However, differently from the studies of Ferguson et al. (1994) and Perera et al. (1998),
during histological examination no granulomas were observed.
One concern with Streptococcus iniae is the possible transmission of the bacteria
from fish to humans. Few cases have been reported. Apparently the probability of such
transmission is low. It is a concern mainly with immunocompromised individuals (Goh et
al. 1998; Lau et al. 2003; Weinstein et al.
1996; 1997). However, recent reports in China
suggest a possible higher risk of S. iniae infections in Asian populations due to their habit
of cooking and eating fresh fish (Lau et al. 2003). Pulse-field gel electrophoresis (PFGE)
has showed that S. inae strains that cause disease in fish are genetically diverse from
commensal strains isolated from nondiseased fish (Fuller et al. 2001; Weinstein et al.
4.2. Host-Pathogen Interactions
Streptococcus iniae, like streptococci belonging to the Group B (GBS), shows a
tropism for the brain. This tropism is one of the main causes of death. The invasion rate
of S. iniae in the different organs has been experimentally observed in yellowtail (Kusuda
and Kawai, 1982
Kusuda and Kimura, 1978; Sako, 1998). In the first
challenge, there is an increase in the bacterial count of blood, spleen, and kidney;
followed by a decrease or a constant count value. In contrast,
days after challenge
there is a constant increase of bacterial count in the brain until the onset of mortality. The
exact mechanism used by S. iniae to cross the blood-brain barrier (BBB) is still not well
understood. It involves production of the cytolisin streptolysin
S (SLS) required for local
could elicit its own endocytosic uptake from the microvascular endothelial cells and so
pass the BBB (Nizet and Rubens, 2000). Otherwise, as observed with S. suis, S. iniae
could adhere and damage the microvascular endothelial cells through the action of SLS,
and then proceed from the circulation to the central nervous system (Charland et al.
The pathogenesis mechanisms of GBS have been studied in detail and resemble
those of systemic infection with S. iniae. For this reason, the GBS pathogenesis
mechanisms may be very useful in understanding the possible mechanisms used by S.
iniae (Miller and Neely, 2004). Invasion of the brain microvascular endothelial cells by
GBS causes an evident cellular injury and increased cell permeability because of the
cytotoxicity activity of the beta-hemolysin/cytolisin (b-h/c) that causes formation of pores
on the epithelial tissue (Nizet et al. 1997
Ring et al. 2002a). Streptococcal mutants with a
hyperhemolytic phenotype give evidence of the involvement of b-h/c on host tissue
damage. Histological examination of animals infected with these mutants shows higher
degree of necrotic tissue with these strains of bacteria (Ring et al. 2002a). The b-h/c is
also important for penetrating the BBB. In animals infected with mutants lacking b-h/c,
fewer bacteria penetrate the BBB, and the mortality rate and bacterial numbers in brain
increase with increased levels of b-h/c activity (Nizet et al. 1996). In addition, b-h/c has
proinflammatory, proapoptotic, and proinvasive properties that could contribute to
disease pathogenesis. Mice b-h/c production is associated with increased local and
systemic levels of proinflammatory cytokines, such as IL-6 and IL-1 released by injured
migration, and stimulate the respiratory burst, degranulation, and adherence of leukocytes
and neutrophils. The development of inflammatory exudates then promotes further
damage of the BBB and stimulation of apoptosis of neurons and other cells (Doran et al.
2003; Nizet et al. 1997). Leukocytic infiltration into the cerebrospinal fluid is a major
clinical diagnostic marker for acute bacterial meningitis.
Group B Streptococci may also induce epithelial damage indirectly by altering
host cell processes, such as the cell peroxidase-H202 system that can damage adjacent
cells by producing oxygen radicals. The observed hypotension in septic shock could also
be caused by an excess production of nitric oxide (NO) stimulated by the GBS (Ring et
al. 2000; 2002b). The GBS can also modify the arachinoic acid metabolism favoring the
production of prostaglandin E2 (Sbarra et al. 1987). Bacteria replication within CNS may
then provoke an overwhelming host inflammatory response that, in combination with the
disruption of the BBB, could be fatal (Doran et al. 2003). Streptococcus iniae, as with
GBS, could also secrete other factors that break down the extracellular matrix helping to
spread the bacteria, or that inactivate the inflammatory mediators, such as C5a, thus
decreasing recruitment, and the opsonophagocytic capacity of neutrophils (Cheng et al.
2002; Liu and Nizet, 2004).
The polysaccharide capsule of Streptococcus protects the bacteria by decreasing
adhesion of immune cell receptors to the bacteria, (Tamura et al. 1994). Sialic acid
residues on the capsule can inhibit activation of the complement alternative pathway,
thereby interfering with the opsonophagocytic killing mechanism of the neutrophils, and
macrophages. The ability of S. iniae to survive in macrophages may be useful in
establishing a systemic bacteremia and for crossing the BBB within macrophages
migrating in the CNS. The ability to overcome the immune response of macrophages is
one characteristic that allows S. iniae to establish an infection. Another defense
mechanism used by S. iniae is inhibiting apoptosis and increasing necrosis of natural
cytotoxic cells in fish (NCC) (Doran et al. 2003; Fuller et al. 2001; Martin et al. 1992;
Taylor et al. 2001
Zlotkin et al. 2003).
Data collected in recent research shows that recognition of GBS by immune cells
and their later activation involves Toll-like receptors (TLRs). The same receptors may
also be involved in recognition of S. iniae (Henneke et al. 2001; 2002).
Both the innate and acquired parts of the immune system are involved during
Streptococcus infections. Nonspecific cytotoxic cells (NCC) are part of the nonspecific
immune system, and play an important role in fish during bacterial infections (Evans et
al. 1998; 2000a; Taylor et al. 2001; Weinstein et al. 1997). Nonspecific cytotoxic cells
have cytotoxic action. They release cytokines that increase phagocytosis by macrophages,
activate antigen-processing cells, and augment the inflammatory response. Nonspecific
cytotoxic cells become biochemically activated; this increases their cytotoxicity after the
first encounter with a stressor agent.
These responses are different from those occurring
during normal homeostatic regulation, where the encounter of NCC with a target cell
represented by a parasite or a tumor cell, causes apoptosis of the NCC. Prokaryotic DNA
can activate the cytotoxicity mechanism in NCC, and it is involved in the interactions of
cytotoxicity. Also treatment of bacterial DNA with DNAase or methylation of all
cytosine bases, abrogates the cytotoxic enhancement (Oumona et al. 2002).
observations indicate that DNA binding receptors on NCC and other immune cells have a
mechanism for preferring certain nucleotides. This mechanism should be similar to MHC
recognition and presentation of peptides, where certain amino acids serve to anchor the
peptide to the MHC antigen. Studies using synthetic oligonucleotides shown that
cytotoxic activation is enhanced by bacterial DNA fragments containing -GACGTT- and
-GTCGTT- sequences. These results also suggest that binding and internalization of
bacterial DNA is required for initiation signaling and cytokine release. Recognition of
bacterial DNA by NCC appears to be determined by a combination of sequence and
This mechanism may be similar to the recognition of antigenic
determinants by T and B-cell receptors. Apparently, different subsets of NCC could exist
with different specificities for bacterial DNA receptors, or individual NCC could contain
receptors with different specificities (Babiuk et al. 2000; Krieg, 2000; Scheule, 2000).
These results are important to better design and understand the mechanism of action of
Streptococcus iniae inhibits apoptosis and increases necrosis of NCC. The
advantage of a decreased number of apoptotic cells is that apoptotic cells are
phagocytosed and do not initiate an inflammatory response. In contrast, cells undergoing
necrosis are eventually lysed, and release several compounds that will activate the
inflammatory response. Another effect is increased antigen processing which increases
some stains of S. iniae where phosphatidylserine expression is inhibited in the NCC outer
membrane. Recognition of phosphatidylserine by macrophages is needed for initiating
the phagocytosis process (Taylor et al. 2001).
The possibility that protection against bacteria in fish mainly involves the cellular
immune system of the non-specific defense system could explain the results of several
vaccine studies (Eldar et al. 1995b; Evans et al. 2000b; Lillehaug et al. 1993; Taylor et al.
2001). They observed no correlation between antibody titers and protection. In other
studies the protection given by vaccination lasted only for a few months (Eldar et al.
1995b; Hortvitz et al. 1997
Iida et al. 1981).
Nevertheless, the humoral part of the immune system is important in the host
defense mechanism. In yellowtail (Kusuda and Tanaka, 1988) specific antibodies
increased the phagocytic activity and index of macrophages. This effect could not be
duplicated if complement alone was added to cell culture. However with both antibody
and complement, the phagocytic activity and index were significant higher. These results
suggest that during bacterial infections in fish, activation of the classical pathway of the
complement by antigen-antibody reaction is an important mechanism of defense.
Antibody production appears to be stimulated more by toxins released by
Streptococcus than by the bacteria itself (Kusuda and Takagi, 1983).
highlight the possibility that toxoid vaccines may work better than bacterial vaccines.
4.3. Antibiotics Treatments and Vaccines
The antibiotic used most to treat Streptococcus spp. infections is erythromycin
50S subunit of the bacterial ribosome eukaryotess do not have the 50S ribosomal subunit)
(Treves-Brown, 2000). Some erythromycin resistant strains of bacteria present mutations
in components of the 50S ribosomal subunit, and for this reason the antibiotic fails to
bind the ribosome. The non ionized from of the drug is considerably more permeable to
cells, and this probably explains the increased antimicrobial activity that is observed in
alkaline pH. The oral recommended dose for fish is 25-50 mg erythromycin/kg fish/day
for 4-7 days. The injectable dose is 10 to 20 mg/kg fish for 14 days.
Oxytetracycline was tested as a possible treatment for S. iniae, but efficacy varied
among experiments. Oxytetracycline inhibits the bacterial protein synthesis by blocking
the attachment of the complex tRNA-amino acid to the ribosome. Oxytetracycline can
less likely inhibits the protein synthesis in the host, because eukaryotic cells do not have a
tetracycline uptake mechanism (Treves-Brown, 2000). In blue tilapia infected with S.
iniae, administration for 14 days of medicated feed containing oxytetracycline at levels
between 50 and 100 mg OTC/kg fish/d fed at 2% body weight/day increased the survival
rate between 45 and 98% (Darwish et al. 2002). The minimum inhibitory concentration
(MIC) of oxytetracycline against multiple S. iniae isolates indicated a general sensitivity
at concentrations of 0.25-0.50 jig/mL. Completely different results were observed in
hybrid striped bass infected with S. iniae (Stoffregen et al. 1996). In this case, treatment
with Terramycin for Fish (Pfizer, Inc., Connecticut, USA) at doses of 82.7 mg drug/kg
fish per day for 10 days was unsuccessful. The different results could be explained partly
by the higher resistance of this bacterial strain to oxytetracycline with a MIC of
Amoxicillin has been used to treat infections from S. iniae with a range of dose of
30-120 mg/ kg fish body weight administered for 8 consecutive days (Darwish and
Ismaeil, 2003). Amoxicillin, as other penicillins, interferes with the bacteria cell wall
synthesis preventing the development of the peptidoglycan layer (Treves-Brown, 2000).
Another antibiotic tested as a possible treatment for streptococcosis in one study
is josamycin (Takemaru and Kusuda, 1988). Josamycin inhibits the protein synthesis by
binding the 23S rRNA or 50S bacterial ribosomal subunit (Treves-Brown, 2000). In the
study of Takemaru and Kusuda (1988), the MICs ofjosamycin, erythromycin, and
spiramycin were compared. Observed josamycin MIC values ranged between 0.10 and
0.39 pg/mL. They were higher than the values recorded for spiramycin, but lower than
the ones for erythromycin. The development of resistance of the bacteria to the antibiotics
after 9 successive subcultivations was 4-fold higher in MIC value for josamycin and 8-
fold higher for the other two antibiotics. These results support josamycin as a possible
treatment against streptococcosis.
Florfenicol is one of the newest antibiotics on the market, and it has been used
against several bacteria such as Aeromonas salmonicida, Yersinia ruckeri,
Flavobacterium psychrophilum, Vibrio salmonicida (Michel et al. 2003; Nordmo et al.
1998), and Edwardsiella ictaluri (Gaikowski et al. 2003). This antibiotic has also been
tested in ornamental fish (Yanong et al. 2002). Florfenicol binds reversibly the large
ribosomal subunit of bacteria and eukaryotes, and it also binds the peptidyl transferase
enzyme inhibiting the transfer of the growing polypeptide to the next amino acid
Streptococcus infections, as is reported by the label of Aquaflor (active ingredient
Florfenicol 500 mg/g, Schering Plough, Union, New Jersey) and from the results of some
investigations (Yanong et al. 2004).
Several vaccines have been developed against streptococci, mainly for tilapia and
rainbow trout. Protection levels given by the vaccines varied among experiments and
vaccine formulations (Akhlaghi et al. 1996; Bachrach et al. 2001; Eldar et al. 1995b;
Eldar et al. 1997
Klesius et al. 2000; Klesius et al. 2001; Lim and Klesius 2001a;
Palacios et al. 1993; Sakai et al. 1987
Shelby et al. 2002a). An effective combined
vaccine against Streptococcus sp. and Vibrio anguillarum has also been developed (Sakai
et al. 1995).
Fish Immune System
Fish are a heterogeneous group of animals that are physiologically and
anatomically very different. They are divided in the classes Agnatha, Chondrichthyes,
and Osteichthyes. Agnatha (Cyclostomes: hagfish and lamprey) lack a well-structured
mouth with bones such as maxillary and palatine, and has a cartilaginous skeleton.
Chondrichthyes (Elasmobranches: shark, rays) do not have a swim bladder and ossified
bones, but only a cartilaginous structure. Osteichthyes (salmon, tilapia) have ossified
bones. Teleost are included in the class Osteichthyes. Complexity of the immune system
differs substantially among Cyclostomes, Elasmobranches, and Osteichthyes. It is
possible to observe a gradual increase in complexity of the immune system (immune
organs, cells, soluble factors, and receptors) among these classes.
In mammals, the main lymphoid organs are thymus, spleen, bone marrow, lymph
nodes, and gut-associated tissues. Fish have lymph vessels, but lymph nodes have not
been observed, at least not with the structure present in mammals.
Bone marrow and
germinal centers also do not exist in fish, so other organs and tissues perform their
functions (Zapata et al. 1992
The main lymphoid organs in teleost fish are
thymus, kidney, spleen, and gut-associated lymphoid tissue. In Elasmobranches the main
(Culbreath et al. 1991; Rumfelt et al. 2002). Lampreys do not have true lymphoid organs,
but lymphohemopoietic tissues are present in various locations such as within the nephros
and intestinal lamina propria. Neither lampreys nor hagfish have a thymus, but lymphoid
accumulations that could be homologous to thymus, are present in the branchial area
(Mattison and Fange, 1977
Rowley et al. 1988). In fish, as in mammals, there is an
involution of the thymus with age; and in fish this process can also be related to season or
hormonal changes associated with sexual maturation (Honma and Tamura, 1984). In
some fish such as rays, flatfish and cod, the thymus continues to grow after sexual
maturation, whereas in eels it has already involuted before sexual maturity. In carp and
primitive sharks, the thymus does not appear to involute. In Zoarces viviparous (Bly,
1985) the thymus reaches is maximum size at 1 month preparturition when it occupies
0.83% of the total body volume. Thereafter, it decreased in size and by 3 months
postparturition occupies only 0.048% of the total body volume. Thymus involution is
characterized histologically by an increase in connective tissue, a decrease in lymphocyte
density, infiltration of adipocytes, the presence of reticular cells and collagenous fibers,
and the formation of epithelial cysts. However, the relationship of such changes to the
immune capabilities in older fish is not clear.
In mammals, the outer layer of the skin (stratum lucidum) is keratinized and
confers a physical barrier to the penetration of pathogens. In fish this layer is not
keratinized but is covered with mucus. The mucus protects the skin, decreasing the ability
of pathogens to attach to the skin, and it also contains several antibacterial compounds,
The fish immune system includes cells and soluble mediators that are involved in
the innate and acquired immunity. Fish present both B and T-cells that are involved in
acquired immunity. Although T-cells have not been formally identified in fish because
they lack.specific molecules such as CD4, CD8 or CD3, the genes encoding polypeptides
homologous to the TCR molecules have been reported in teleosts and elasmobranches
(Partula et al. 1995). Although in mammals the T-cells develop only in the thymus, it
appears that in fish an extrathymic population of T-cells is present. The results of some
studies have suggested the possibility that the lymphoid cell populations of the thymus
and kidney are acquired independently, as it has been observed in early thymectomy fish
that still have a normal lymphocyte population in the kidney (Grace and Manning, 1980).
B-cell lymphocytes can be recognized in fish by the presence of immunoglobulins on the
Macrophages, polymorphonuclear cells and non-specific cytotoxic cells are
involved in innate immunity in fish, as in mammals. Non-specific cytotoxic cells (NCC)
in fish are considered to be equivalent functionally to mammalian natural killer cells
(NK). NCC can lyse a wide spectrum of mammalian tumor cells and protozoan parasites
(Evans and Jaso-Friedmann, 1992) through the mediation of function-associated
molecules on the cell membrane (Harris et al. 1992). NCC cells are the smallest
leukocytes possessing a very pleomorphic, clefted nucleus and relatively little cytoplasm
with no cytoplasmic granules.
Villi are present on their surface, and they attach to their
target using long, membranous filaments, allowing many binding sites (Secombes, 1996).
and Jaso-Friedmann, 1992). In hagfish only a single population of granulocytes has been
reported (Mattison and Fange, 1977), but different subpopulations are distinguishable in
lampreys (Rowley et al. 1988). In Elasmobranches several subpopulations of
granulocytes have been identified, but there is an enormous heterogeneity among species
(Rowley et al. 1988). In teleost fish, different types of polymorphonuclear cells have been
detected, but there is an enormous variation among species. Furthermore, eosinophilic
and basophilic cells, when present, are not equivalent in function to those corresponding
to mammalians (Rowley et al. 1988; Zapata et al. 1996). In fish, as in mammals,
macrophages act as antigen presenting cells through MHC II, they activate lymphocytes
releasing IL-1, and they release reactive oxygen and nitrogen molecules for killing
pathogens (Ellsaesser and Clem, 1994). Fc receptors have also been identified on fish
macrophages (Haynes et al., 1988).
In Elasmobranches and Osteichthyes, as in mammals, antibodies represent the
humoral section of the adaptive branch of the immune system, but differences are
present. In Cyclostomes imunoglobulins have been observed in hagfish (Kronenberg et
al. 1994), but not in lamprey (Varner et al. 1991). IgM is present in fish, and some
species also produce IgG or unique Igs, but IgA, IgD, IgE have not been found. In
cartilaginous fish, at least sharks, skates and rays, three different types of imunoglobulins
have been found: IgM, IgNAR and IgW (Rumfelt et al. 2002; Voss and Sigel, 1972). Igl
exists in approximately equal amounts as a monomer (7S) and a pentamer (19S). In
teleost Igs appears to be a tetramer composed of four monomeric subunits, each
been observed, but this could be caused from a lack of detection (Lobb and Olson, 1988).
In fish the assembly of antibody molecules via disulfide crosslinking appears to be a
means by which structural diversity is generated.
This is distinctly different from what is
observed in mammalian species where intersubunit disulfide cross-linking appears to be
employed in a more uniform manner (Sanchez and Dominguez, 1991). In mammals,
somatic mutations cause a production of antibody with an increased affinity for the
antigen. Generally the process of somatic mutation has not been detected in fish, with the
exception of some species of elasmobranches (Hinds-Frey et al. 1993). In mammals, a
second exposure to an antigen stimulates a greater immune response with a faster and
greater production of imunoglobulins (mainly IgG). in contrast, in fish there is a lower
production of imunoglobulins after a second exposure to a pathogen compared to the
logarithmic increase in monomeric IgG observed in mammals (Arkoosh and Kaattari,
1991). Studies showed that during a secondary response, the increase of specific
antibody-producing cells is caused by a clonal proliferation of native and primed
lymphocytes, indicating that in fish immune memory is not well developed (Brooks and
Feldbush, 1981). Another important difference among the immune system of fish and
mammals is the absence of IgA in fish, even if a homologous function of IgA could be
present. Some studies have detecteded antigen specific antibody induced in the mucus of
the skin in orally vaccinated fish, while little serum antibody was produced (Rombout et
al. 1993). This is similar to what happens in humans, where the local exposure to a
pathogen on a mucosa stimulates IgA production in all the body mucosa.
identified in fish (Culbreath et al. 1991; Yano, 1996). In general, the complement system
in fish is effective against non-virulent gram-negative bacteria, but not against virulent
gram-negative and gram-positive bacteria. The alternative complement pathway can be
inhibited by the large amount of sialic acid present on virulent strains of gram-negative
bacteria (lida and Wakabayashi, 1993). In Cyclostomes, only components of the
alternative complement pathway have been detected, but the complement system lacks a
cytolytic activity (Ishiguro et al. 1992; Nonaka, 1994). Elasmobranches possess the
classical and alternative complement pathways that stimulate the formation of the
membrane attack complexes on target cells (Culbreath et al. 1991). The complement
system pathways in bony fish are very similar to those of mammals (Yano, 1996).
Lysozyme is important for fighting all types of pathogens. Lysozyme is
distributed mainly in tissues rich in leucocytes, such as the head kidney, skin, gills, and
the alimentary tract. Fish lysozyme, in contrast to mammalian, has an antibacterial
activity against not only against gram-positive bacteria but also gram-negative bacteria in
the absence of complement (Grinde, 1989). Interferon (IFN) has been detected in bony
fish, but not in cyclostomes or cartilaginous fish (Yano, 1996). Transferrin and C-reactive
proteins have been found in fish with a similar activity as in mammals (Nakanishi et al.
Cytokines, TCR components, major histocompatibility complex I and II (MHC I
and MHC II) receptors have been identified in Elasmobranches and Osteichthyes, but the
genetic polymorphism of MHC genes is considerably lower than in mammals (Knight et
One of the major differences between the immune system of fish and mammals is
the effect that temperature has on the system. In fish, the rate of the development of the
immune system is influenced more by the external temperature and by the size of the fish
than by age (Tatner, 1996). The higher the temperature, the faster the fish grows and the
earlier the immune system develops. Rainbow trout start to produce antibodies when they
reach a weight of approximately 0.24 g without any dependence on their age (Tatner and
Manning, 1983; Tatner, 1986).
Seasonal changes in water temperature influence the functions of the fish immune
system. For example, the number of peripheral blood lymphocytes is minimum in winter
and maximum in the summer, although there are exceptions as in the brown trout where
the levels of lymphocytes are low in winter and summer and high in spring and autumn.
In winter, it is possible to observe regression in lymphoid tissues, particularly the thymus.
In spring and summer there are waves of thymocyte production, while degenerated
lymphoid cells can be found from the beginning of autumn and throughout winter. Higher
temperatures cause faster and higher antibody production (Rijkers et al. 1980).
Temperature has a different influence on the various parts and cells of the immune
system. For example, virgin T-cells rather than memory T-cells, B-cells or accessory
cells are particularly susceptible to the inhibitory influences of low temperature (Bly and
Clem, 1992; Miller and Clem, 1984). Low temperatures have a higher inhibitory effect on
T-cells than on B-cells. A possible explanation for this phenomenon is that B-cells have a
higher capability to modify the fluidity of their plasma membrane. B-cells can rapidly
phospholipids causing an increase of membrane viscosity. This may explain the
suppressed function at low temperatures. In fact, a lower membrane viscosity would
affect receptors and signal pathways that involve membrane-associated proteins (Bly et
al. 1990). Even if low temperatures have an inhibitory effect, the fish immune system is
able, at least in part, to counterbalance this effect. For example, in channel catfish serum
complement lytic activity is low in winter, while the antibody affinity increases. In this
way antibody can act as an opsin and activate the complement cascade (Clem and Small,
1970; Hayman et al. 1992). A similar mechanism involves C-reactive proteins in channel
catfish. CRP levels are low in winter, but at the same time their ligand avidity increases.
Temperature also influences the mammalian immune system, but in a minor way. This
difference is caused mainly by the ability of mammals to maintain a constant body
The immune system of sharks (Epalzeorhynchos sp.) has not been studied in any
research. However, because sharks belong to the family Cyprinidae, it is very plausible
that there are similarities between the shark, the common carp, Cyprinus carpio, and the
danio, Danio rerio, immune systems and ontogenesis of the immune systems. Because
sharks are warm water fish, as danio, it is plausible the timelines of the ontogenesis of the
immune system is more similar between these two species than between shark and carp.
5.2. Ontogenesis of the Immune System
The ontogenesis of the fish immune system has been studied only in a few species
(Ellis, 1988; Honma and Tamura, 1984; Tatner, 1996), such as nurse shark,
Zapata, 1998; Trede and Zon, 1998; Trede et al. 2001; Willett et al. 1997ab; Willett et al.
1999), common carp, Cyprinus carpio (Botham and Manning, 1981), rainbow trout,
Salmo gairdneri (Grace and Manning, 1980; Tatner and Manning, 1983; Tatner, 1986),
viviparous blenny, Zoarces viviparous (Bly, 1984; 1985), European flounder, Platichthys
flesus (Pulsford et al. 1994), channel catfish, Ictalurus punctatus (Petrie-Hanson and
Ainsworth, 2001), and angler fish, Lophius piscatorius (Fange and Pulsford, 1985).
Histological, immunological and biomolecular techniques have been used in these
studies. In histological analyses, tissues are first stained and then observed
microscopically. Immunological techniques utilize rabbit or mouse monoclonal
antibodies against the putative B and T-cells that can be detected by ELISA or
fluorescent staining techniques. Biomolecular techniques utilize the expression of several
genes involved in the ontogenesis process. One of the most delicate steps in this type of
analysis is the design of specific mRNA primers. Usually mRNA is isolated, amplified by
PCR and identified by Northern Blot analysis. In situ hybridization techniques can be
used for the detection of the time and of the organs where specific genes are expressed.
5.3. Immune Organs and T-Cell Ontogenesis: Histological Approaches
The thymus is the first immune organ to develop in fish and the first to become
lymphoid (Bly, 1985; Botham and Manning, 1981
Grace and Manning, 1980; Tatner and
Manning, 1983; Tatner, 1986; Trede et al. 2001; Willett et al. 1999). The second organ to
develop is the kidney followed by the spleen. The gut-associated lymphoid tissue
becomes lymphoid later than the kidney and the spleen. In general, the spleen remains
organs in rainbow trout from 1 to 15 months of age. Thymus, kidney and spleen grew
steadily over this period, but all attained their maximum relative weights expressed as a
percentage of body weight, at 2-3 months of age, indicating a time of intense activity and
importance in the maturation of the lymphoid system. The total number of leukocytes in
the organs increased with age, but when the size of the organ and the size of the fish were
taken into account, the actual leukocyte numbers decreased with age. The thymus showed
intense mitotic activity in the first few months, then slowed down. There were no signs of
massive cell death, and it was assumed that emigration of cells to the peripheral lymphoid
organs must have been occurring. Histological changes indicative of involution were
present in the thymus from 9 months onward. The spleen and the kidney showed an
increase in melanin deposition with age, with the kidney retaining a more varied range of
leukocyte types than the spleen. The number of circulating lymphocytes in the blood
remained remarkably constant throughout the first year of life.
The progressive development of the immune organs was also observed in
common carp (van Loon et al. 1982). The thymus contained the largest
pool, 70% or 3
lymphocyte cell pool, 5
x 105 cells at day 28, in the first 4 weeks. At two months of age the total
x 106 cells, was distributed for 38% in the mesonephros, 32% in
the thymus, 15% in the pronephros, 12% in the peripheral blood and 3% in the spleen.
In Zoarces viviparous, a viviparous marine fish, thymus has a paired structure
localized behind the last gill arch (Bly, 1985). The thymus lies in the angle between the
operculum and the body wall, and is covered by a single layer of epithelium. It is evident
densely stained, and is comprised of mainly lymphocytes and few reticular cells. The
other inner zone is lightly stained and contains fewer lymphocytes. However, there is no
distinct boundary between the two zones. The thymus is covered by a thin connective
tissue capsule and by collagen fibers. Macrophages can also be found. The head kidney is
seen at 3 months preparturition, and it contains mainly a few kidney tubules surrounded
by small erythrocytes. At 1 month preparturition the kidney also contains lymphoid
tissue, where granulocytes and macrophages can be found. The spleen is first observed at
two months preparturition. The organ is in close contact with the pancreas.
Differentiation in the spleen is evident at two months postparturition when two zones are
apparent: lightly stained lymphoid areas aggregated around the trabeculae, and dark
staining areas composed mainly of erythrocytes. These two zones may be analogous to
the white and red pulp of mammalian spleen. In the spleen, granulocytes and
macrophages are also present.
In rainbow trout (Grace and Manning, 1980; Razquin et al. 1990; Tatner and
Manning, 1983; Tatner, 1986), thymus is first observed at 4 days before hatching. At this
stage it consists of a thickened area of the pharyngeal epithelium containing a few
lymphoid cells interspersed among the epithelial cells, and of a thin connective tissue. At
4 days after hatching, thymic stellate cells, numerous thymocytes, and some macrophages
are present. The first connective tissue trabeculae in the thymic parenchyma are observed
about 14-21 days after hatching, and incipient subcapsular and outer thymic zones appear
at 1 month age. Thymic cysts develop from 1-2 months after hatching. Renal
intertubular loose connective tissue of the developing kidney. At hatching, those foci tend
to fuse and contain myelocytic cells interspersed among the cell processes of reticular
cells. Scattered large lymphoid cells can be clearly observed at 4 days after hatching, and
from this stage the amount of lympho-haemopoietic tissue increases in the kidney,
retaining a similar structure in both pro- and mesonephros, except for the absence of renal
tubules in the former. The spleen develops slowly, containing a poorly organized
haemopoietic red pulp at 12 days after hatching, formed by loose cell cords, mainly
occupied by erythroid cells and enlarged blood sinusoids. Some scattered lymphocyte,
macrophages and melanomacrophages occurred in the spleen about 14 days after
hatching. Small amounts of splenic white pulp can be observed in
month old trout,
when ellipsoids also develop.
The thymus of the channel catfish, Ictalurus punctatus (Petrie-Hanson and
Ainsworth, 2001), is first colonized by monocytes, suggesting that these cells may help
thymocyte proliferation and differentiation. The first immature lymphocytes can be seen
in the thymus after 5 days. Mature lymphocytes are detectable at 10 days post-hatch. The
intermediate stage of T lymphocytes is concentrated in the peripheral lighter staining area
(cortical zone); meanwhile mature lymphocytes are concentrated in the central darker
staining area (medulla region). Until approximately 3 months post hatch, the total number
of lymphocytes increases, then reaches a plateau.
In zebrafish, Danio rerio, a thymic primordium consisting of two layers of
ePithelial cells appears at 54 h after fertilization (Willett et al. 1997a). By 65 h
morphological mature. At 72 h post-fertilization kidney glomeruli can be seen. At 96 h
post-fertilization the pronephros begins to contain hematopoietic cells, mainly developing
erythrocytes and granulocytes. Fibroplastic reticular cells, that will constitute the
supporting apparatus for the developing hematopoietic cells, appear at 7 days
post-fertilization. Lymphocytes are detected after 3 weeks.
The first appearance of lymphocytes within an organ, rather than the first
appearance of the organ itself, is considered to be a better index of the maturation of the
lymphoid system (Tatner, 1996). The reproductive strategy among fish (such as the
length of egg incubation) and the environmental temperature are the main factors that
influence the time of maturation of the immune organs. The warmer the water
temperature, the faster development occurs. In general, the longer the hatching time, the
slower the development. For example trout eggs are incubated at 140C and hatch around
18 days; however, when they are incubated at lower temperatures, they develop slower.
Carp eggs are reared at 220C and hatch after 36 h. The gestation period in
4-5 months. Table 5-1 summarizes the first appearance of the immune system organs;
the first appearance of lymphocytes in those organs, and Table 5-3 when the
thymus can be considered immunologically mature.
Development of cell-mediated protection can be better analyzed using allograft
rejection responses. Allograft rejection in carp can occur as early as day 16 post-hatch
(Secombes and van Groningen, 1983), at day 26 post-hatch in rainbow trout, and at
months in Tilavia mossambica. These results suggest that cytotoxic T-cells mature earlier
It still is not well understood if during ontogenesis lymphocytes migrate from the
thymus to the kidney. The results of several studies have outlined the possibility that the
lymphoid cell populations of the thymus and kidney are acquired independently. In fact,
early thymectomized fish have a normal lymphocyte population in the kidney (Grace and
5.4. Immune Organs and T-Cell Ontogenesis: Biomolecular Approaches
Zebrafish have been used as a model in biomolecular studies for immune system
ontogenesis (Trede and Zon, 1998; Trede et al. 2001). One main reason is that the
genome of the zebrafish has been almost completely cloned. Moreover, because of the
small size (5 cm) of this fish, they are reared easily, they can be stocked at high density
(up to 70 adult per 19 L tank), their generation time is relatively short (3 months), and
females produce a large number of progeny (200-300 every week).
outside the mother's
The embryos develop
body and are easily accessible for physical manipulation, visual
inspection under dissecting microscope, whole mount in situ hybridization, and gene
transfer experiments. Thanks to the relatively short generation time, zebrafish lineages
may be created for specific mutant genes, and may be used for positional cloning of such
mutant genes. Zebrafish mutants with severe hematopoietic defects can still live beyond
the onset of thymopoiesis, this permits the analysis of lymphocyte development in
mutants that would be destined in the mouse to suffer early embryonic death from anemia
or other developmental defects (Trede et al. 2001). The juveniles of this fish are almost
transparent allowing a direct visualization of the temporal and spatial development of
The utilization of green fluorescent protein genes, the expression of which is
under the control of the promoter of the studied genes (such as Ick, for example), helps in
the direct visualization of the ontogenesis of the immune system. Additionally, proto-
oncogenes can be expressed under the control of the studied genes so as to cause tumors.
The animals with these malignant tumors can be utilized in several ways. For example,
monoclonal antibodies could be produced against zebrafish homologs of T-cell markers
such as CD3, CD4 and CD8. These transgenic experiments would also provide an animal
model of leukemia in lower vertebrates (Trede and Zon, 1998).
New techniques to study gene expression will apply retroviral insertions for the
generation of zebrafish mutants. A major advantage of this approach is the ease with
which genes responsible for the mutant phenotypes can be cloned (Trede et al. 2001).
These characteristics make the zebrafish an ideal model for studying the ontogenesis of
the vertebrate immune system.
As soon as the first lymphocytes are visible they are still not functionally active or
mature. For this reason some studies (Hansen, 1997
Hansen and Zapata, 1998; Trede and
Zon, 1998; Trede et al. 2001; Willett et al. 1997ab) have analyzed the gene expression of
the enzymes including RAG], RAG2, TdT,
Ick, GA TA, ikaros, or TdT, as markers of the
maturation of the immune system. These enzymes are involved in the maturation process
of the T and B-cells acting on the TCR and Igs
genes rearrangement. These studies have
confirmed the results obtained by histological investigations (Willett et al. 1997a). Brain
and thvmus express ikaros, indicating where early lymphoid development occurs.
Zon, 1998) expression of RAGI and RAG2 were first observed in the thymus at 92 h
post-fertilization marking, perhaps, the first rearrangement of TCR genes. At this time the
thymus is heavily populated with lymphocytes. The thymus enlarges for at least 1 month
continuing to express the RAG genes and changing shape from spherical to crescent. No
distinction of medulla and cortex is present at this stage. The expression of the gene Ick is
temporally and spatially parallel to the expression of the two RAG genes. Ikaros's
is found for the first time in the Intermediate Cell Mass (an intraembryonic region
between the somites and the yolk sac) at 24 h after fertilization. At day
is expressed in the dorsal aorta; and at day 3, it is expressed in the thymus. Pronephros
and kidney, which are respectively the larval and adult sites of hematopoiesis, do not
express RAG1 until day 21 indicating the later lymphoid function development of these
organs (Trede et al. 2001). The DNA polymerase Terminal Deoxynucleotidyl Transferase
(TdT) is one of the enzymes involved in the generation of functional diversity during
immunoglobulin and T-cell rearrangement. Northern blot analysis shows that TdT mRNA
is highly expressed within the thymus and to a lesser extent in the pronephros (Hansen,
1997). This research has also showed that RAG1 and TdT genes are expressed also in the
intestine, where few lymphocytes are present. This result suggests that the intestine of
fish, as in mammals, may serve as an alternative site for lymphocyte development. That
could indicate that the GALT is a location for gamma-delta T-cell homologues.
5.5. B-Cell Ontogenesis
The kidney seems to be the first lymphoid organ where IgM bearing cells can be
determine the age of maturation of B-cells. In common carp imunoglobulins were
detected on the surface of cells in thymus and pronephros from 14 days after fertilization
at 210C, and in mesonephros and spleen, from days 28 (Koumas-van Diepen et al. 1994).
From the same research, it was observed that the age of fish and the percentage of surface
immunoglobulin (sIg) cells in the lymphoid organs were positively correlated, with the
lowest percentage in 2
week old fish and the highest in the oldest fish. The percentage of
sig cells increased gradually from 3 to 16 months of age, with values being respectively
15.8-48.1% for blood, 9.7-21.6% for pronephros, 7.2-16.8% for mesonephros, 15.9-
21.6% for spleen and 1.5-3.7% for thymus. The percentages of cytoplasmic Ig cells were
very low or absent in spleen, thymus and blood. Plasma cells were not found in the
kidney until the fish were 1 month old (0.17%), and increased to 1% of the total cell
population by 8 months. This was supported by a steady increase in serum Igs from 3
weeks onward. In S. salar membrane IgM was detected at 41 days post-hatch, this
coincided with the onset of first feeding (Ellis, 1977). From this stage, the percentage of
positive lymphocytes increased steadily reaching 80% by day 48 post-hatch.
In rainbow trout cytoplasmic IgM was first observed in embryos at 12 days before
hatch with eggs reared at 140C (Castillo et al. 1993; Razquin et al. 1990). At this stage no
cells with sIgM were present. Lymphocytes with sIgM were first observed at 8 days
before hatch. Unfertilized eggs contained detectable amounts of IgM indicating that
transfer of IgM from mother to embryo is possible in salmonids. Cells with sIgM first
annear in the kidney 4-5 days after hatching. By 1 month after hatching, IgM positive
activities in the non-lymphoid cells indicated that a certain degree of maturation of the
cellular stroma of the developing lymphoid organs of trout was reached before or at the
time when IgM-expressing cells could be observed. This could suggest the possibility
that both macrophages and some stromal cells are involved in the maturation of B-cells.
However, reticular cells with alkaline phosphatase are not observed in the spleen red pulp
of trout. This could explain the difference in haemopoietic and lymphoid functions
between kidney and spleen. In splenic parenchyma, reticular cells with acid phosphatase
activity are present before the appearance of the first IgM positive cells. That cell type
appears to form the extravascular microenvironment of the red pulp, which is an
important erythropoietic site (Zapata, 1983). In contrast, the development of alkaline
phosphatase reticular cells in the white pulp of the spleen is not related to the occurrence
of the first IgM positive cells in this organ. The development of IgM positive cells in the
thymus might be related to the development of the connective tissue trabeculae, rather
than with the differentiation of alkaline phosphatase and acid phosphatase-positive cells.
In fact, cells with IgM are not present in the thymus until the development of thymic
trabeculae, and IgM positive cells are concentrated at the border between the thymus
inner and outer zones where trabeculae ramify into several branches.
In mammals, macrophages play an important role in the B-cell maturation
process. It appears that in fish these cells have the same function. Melanomacrophage
centers may be functionally analogous to the germinal centers of higher vertebrates where
the localization of antigen on the surface of dendric cells has been implicated in the
al. 1990). Furthermore, kidney and spleen of trout fry are functional phagocytic organs
prior the acquisition of lymphoid cells (Tatner and Manning, 1983). Development of
splenic ellipsoids, which are considered involved in antigen trapping in fish, appear to be
related in time to the differentiation of the white pulp rather than with the early
differentiation of splenic IgM positive cells (Lamers and De Hass, 1985).
Similar results have been observed in channel catfish (Petrie-Hanson and
Ainsworth, 2001). In renal interstitial tissue, macrophages are present before B-cells
suggesting a possible role in B-cell differentiation. In channel catfish larvae mature
B-cells appear in the anterior kidney before their detection in any other organ, confirming
the role of the kidney in early lymphopoiesis. B-cells are detected in the kidney at 7 days
post-hatch, in the thymus at 10 days, and in the spleen at 14 days after hatching.
Interestingly, in research conducted by Petrie-Hanson and Ainsworth (2001) Igs positive
cells were absent in the thymus from day 14 to 19. On day 21 putative B-cells were
observed again in the thymus. This suggests a wave-like, B-lymphocyte maturation
process in the thymus. At hatch, the spleen of catfish contains only erythrocytes and blast
cells. Monocytes are present by day 3. By day 5 neutrophil and immature B-lymphocytes
are present. Mature B-cells are observed from day 14 after hatching. At this time there is
still not any recognizable organization of the splenic lymphoid compartment. T-cells can
be found by day 28. There is an increase in the T-cell population until
hatch and at this stage T-cells appear to be more numerous than B-cells. T and
B-lymphocyte populations are localized adjacent to the arterioles and along some of the
The response to thymus-dependent and thymus-independent antigens in fish
involves different immune cells, and these responses develop at different ages. B-cells
respond to thymus-independent antigens, while helper T-cells and accessory cells
respond to thymus-dependent antigens. Antibody production has been observed in 23
days old rainbow trout, however, juveniles respond earlier to thymus-independent than to
thymus-dependent antigens (Paterson and Fryer, 1974). Carp fry, 4- weeks old, were
unable to mount a plaque-forming cell response to sheep red blood cells, that are
thymus-dependent antigens (van Loon et al. 1982). In contrast, 8-week-old carp fry were
able to mount a primary humoral response against human gamma globulin, a
thymusdependent antigen, and against formalin-killed Aeromonas salmonicida, which is
a thymus-independent antigen. Rainbow trout also mount a response to thymus-
independent antigens earlier (2 months old age) than to thymus-dependent ones
(3 months old age) (Tatner, 1986). If juveniles are exposed too early to thymus-
dependent antigen, they can develop a tolerance state. These results are important for
vaccination strategies. Fry can be successfully vaccinated only when they can respond to
antigen, and vaccines based on thymus-independent antigens can be used earlier.
In Elasmobranches, Leydig's
organ, the intestinal spiral valve, spleen and the
epigonal organs are important in B-cell maturation (Rumfelt et al. 2002). In
Elasmobranchs the epigonal and the Leydig organs are homologous to the mammalian
bone marrow. The Leydig organ is attached to the esophagus and consists of a reticulum
packed with large number of leukocytes with few arteries and capillaries present. The
development. Putative lymphocytes of various sizes are abundant in this organ and they
form loose follicle-like aggregates with scattered plasma cells. The epigonal organ is
similar in structure and organization, and it is physically attached to the gonads. It is a
primary lymphoid tissue for the entire life of the animal. At least one of these organs is
present in all cartilaginous fish; for example in nurse shark only the epigonal organ is
present. As in other vertebrates, the spleen is highly differentiated, has a vascular
structure, and is the major site for antigen presentation for antibody production.
In the cartilaginous sharks, skates, and rays, three different types of
imunoglobulins have been found: IgM, IgNAR and IgW (Rumfelt et al. 2002).
in approximately equal amounts as a monomer (7S) and a pentamer (19S). In neonatal
nurse sharks there are very low levels of IgM; at 1 month of age, the level of IgM is
similar to the adult one. However, some IgM may be passed from the mother by the egg
yolk. In neonatal sharks, high levels of a different IgM are present in the blood. Cells of
the spleen and epigonal organs secrete other forms of IgM, and a germline-joined
non-diverse VDJ gene encodes it. This type of IgM is secreted throughout the adult life
only in the epigonal organ. IgM is first expressed in the liver, than in the kidney, spleen,
and Leydig/epigonal organ when the juveniles are close to hatch. IgNAR is expressed
mainly in adults, while IgW has been identified only in the supernatant of in vitro
cultures of splenocytes. It is also interesting to note that in adult sharks MHC class II
mRNA is highly expressed in the spleen, thymus, gill, and intestine (Ohta and Flajnik,
unpublished results). In the spleen, MHC II is expressed on slgM+
cells (B-cells) and on
the surface, (2) large white pulp areas presenting cells without IgM and with few
scattered IgM and IgNAR secretary B-cells, (3) red pulp with IgM putative secretary
cells localized principally close to venous sinuses. The Igs" and MHC II1
second zone are associated with MHC II'
cells in the
cell (dendric cells) and they resemble
lymphocytes in size and appearance. For these reasons they are considered putative T-
cells. In newborn sharks, the spleen is populated mainly by sIgM
cells (B-cells), and
these cells have low expression of MHC II. They are also present in lower numbers than
in adults. T-cell zones are not yet present. In newborn shark the epigonal organ contains
developing granulocytes, blast cells, lymphocytes and plasma cells. The genes RAG 1 and
TdT are expressed in these organs during the entire life of the animal, and the
transmembrane form of IgM mRNA is predominantly found. These data and the fact that
the present sIgM cells are not secretary, suggest that in reality these cells are more likely
developing naive B-cells. The expression of RAG1 gene is 10 times higher in the thymus
than in the epigonal organ, and this gene is not expressed in spleen or muscle. This
confirms that the thymus is the site for T-cell maturation in shark (Rumfelt et al. 2002).
Data collected in several studies (Irwin and Kaatari, 1986; Kaatari and Irwin,
1985; Sanchez et al. 1995) have shown that there is an organ dependent heterogeneity in
B-cell populations. These differences are also related with the specific function of the
immune organs. The anterior kidney of coho salmon possesses lymphocytes with a more
restricted antigen recognition profile of antibody specificities than do lymphocytes from
the posterior kidney or spleen. The heterogeneity profiles of the posterior kidney
by variation in antibody affinity or to the actual absence of select antigenic specificities in
different B-cell populations. Differences in the response to mitogens can be also observed
among B-cell populations of different organs. Anterior kidney cells can respond only to
LPS, while splenic cells can respond to LPS and PPD. These results must be related to
the function of these organs: the anterior kidney is more a hemapoietic center and can be
considered the homologue of mammalian bone marrow.
Differences in organ function can be also observed in the antigen trapping
process. In the spleen antigens are probably held as immune complexes, and are located
extracellularly, associated with the metalophil reticular fibers present mainly in the
ellipsoid walls, scattered throughout the peri-ellipsoid areas, and the melanomacrophage
centers. In the kidney, tissue antigens are present mainly intracellularly within the
reticulo-endothelial system and free macrophages. The thymic and the
reticulo-endothelial system of the heart atrium play no part in the uptake of antigen. The
antigen trapping process could be involved in the induction of B memory cells and in the
negative feedback control of antibody production (Ellis, 1988).
5.6. Nonspecific Immunity
Non-specific immunity involving both cellular and soluble factors plays an
important role in the defense mechanisms of fish both in the adult and during earlier
stages of development. Fish are most likely entirely dependent on non-specific immunity
during the period of free-living existence prior to immune system maturation. Injection of
colloidal carbon particles has been used to study the phagocytic activity in different
present in connective tissues, under the skin, in the gut, and in the gills. Some
macrophages are also observed in the kidney. By day 18 the kidney has become efficient
at trapping carbon. At 8 months the main sites of localization of phagocytosed material
are.the spleen, kidney, heart, and macrophages in the blood. In 2 week old carp, the
phagocytic pattern is similar to that in the adult, and the kidney and spleen trap the largest
amount of carbon.
In addition to the nonspecific lectins and hemagglutinins found in fish eggs and
newly hatched fry, fry possess an efficient phagocytic system (Tatner and Manning,
1983; Tatner and Home, 1984). Rainbow trout fry 4 days post-hatch have an efficient
phagocytic system, and administered carbon is engulfed by free-wandering macrophages
that accumulate under the skin and in the connective tissue, gut, and gills. By day 14
post-hatch the pattern of antigen-trapping was similar to that found in adults, with the
fixed macrophages of the spleen and kidney sequestering the carbon particles from the
Passive immunity from mother to fry has been demonstrated in a few species, and
C-reactive protein and lectin-like agglutins, that should be maternally derived, have been
found in ova of several fish (Tatner, 1996). In rainbow trout the levels of IgM in the
whole fish increased gradually to reach a peak, in terms of milligrams per gram tissue, at
time of hatch, and then slowly declined to initial values by 2 months post hatch (Castillo
et al. 1993). Differences in the feto-maternal and neonate-maternal relationships were
observed among oviparous plaice, Pleuronectes platessa, the viviparous blenny with
into plaice eggs, the young blenny, and swordtail. The earlier lymphoid development in
the blenny compared to the oviparous species could result in the embryo being
immunologically competent while still within the ovary, and capable of mounting an
immune response against both maternal and sibling antigens. The finding that
immunoglobulin can be detected in the eggs and larvae of fish raised the possibility that
hen (mother) fish could be vaccinated to provide their offspring with protection against
various pathogens. This would be particularly beneficial in the case of pathogens that are
transmitted vertically and to which the young are particularly susceptible, such as IPN
5.7. Interactions between the Immune and Neuroendocrine Systems
Neuroimmunoendocrinology is a new interdisciplinary field of research that has
recently emerged, and it involves the study of the bidirectional communication between
the immune and the neuroendocrine systems. These systems are closely and finely
interconnected through innervations and a bidirectional communication that uses the
same set of signal molecules, such as hormones and lymphokines. Most of the studies in
the neuroimmunoendocrinology field have been conducted on mammals, but fishes have
also been target of study, as several recent reviews have shown (Harris and Bird, 2000;
Weyts et al. 1999; Yada and Nakanishi, 2002). There are several lines of evidence that
the sympathetic nervous system (SNS) plays a role in immune regulation: (1) primary
and secondary lymphoid organs are innervated by sympathetic noradrenergic nerves;
(2) synapse-like contacts among sympathetic nerve terminals and T and B-cells in the
Quinn et al. 1997); (4) norepinephrine (NE) is released in lymphoid organs following
immunization (Kohm et al. 2000); (5) NE can alter lymphoid cell functions (Moynihan et
al. 2004); (6) the nervous system acts as an endocrine system when activated neurones
produce an electric stimulation of the terminal part of axon, thus releasing
neuromediators, such as norepinephrine, that will influence the functions of the target
cells. In addition, the histological structure of some endocrine glands resembles the
structure of some parts of the nervous system, as can be observed in the neurohypophysis,
composed mainly of axons originating in the hypothalamus. Also in fish, the sympathetic
nerves have a regulatory function on the immune system (Flory, 1989).
External and internal stimuli are registered by the nervous system and passed to
the hypothalamus. The hypothalamus, through nerve axons or hormones, inhibits or
stimulates the pituitary to release a second group of hormones that will target specific
organs, tissues, and glands regulating their physiology. During these processes several
negative feedback mechanisms are present to finely control the release of hormones and
their effects. For example, under specific stimuli, the nervous system stimulates the
hypothalamus to release the thyrotropin-releasing hormone (TRH) that will stimulate the
hypophysis to secrete thyroid-stimulating hormone (TSH). TSH will stimulate the thyroid
to synthesize and release the thyroid hormones triiodothyronine (T3) and thyroxine (T4).
Both these hormones will target specific tissues and organs to stimulate precise
physiological processes, but they will also produce a negative feedback on both
hvyothalamus and hypophysis inhibiting the release of TRH and TSH. In this way an
As mentioned previously, hormones can influence and be released by the immune
system. It is well known that hormones released under stress conditions can affect the
immune system (Blalock, 1989; Sanders et al. 1997
Schreck et al. 2001), but they are not
the only ones. For example, TRH stimulates both the adenohypophysis and T-cells to
release TSH (Kruder and Blalock, 1986). TSH, produced by the hypophysis or T-cells,
stimulates the thyroid to synthesize and release the thyroid hormones triiodothyronine
(T3) and thyroxine (T4). Chorionic gonadrotopin (CG) can be also produced by T-cells
under gonadotropin releasing hormone (GnRH) stimulation by the hypothalamus.
Somatostatin can block CG production both from the adenohypophysis and T-cells. The
adrenocorticotropic hormone (ACTH) can suppress the immune response to both
T-dependent and T-independent antigens, lymphokines and IFN-gamma production by
T-cells, and Igs synthesis and macrophage activation by IFN-gamma (Blalock, 1989).
The effect of opioids on the immune system is very variable dependently on the type of
opioid peptide and immune cell. For example, alpha-endorphins are potent suppressors of
antibody production, but not beta or gamma-endorphins (Johnson et al. 1982).
Alpha-endorphins have multiple effects on the immune system dependently on the cell
type: they block T helper cells from producing and/or secreting interleukins needed for
antibody production, they inhibit B-cells from producing antibody and transforming
plasma cells. Beta-endorphins enhance T-cell proliferation and differentiation to
cytotoxic T-cells, stimulate cytotoxicity in NK cells, and they are chemotactic factors for
monocytes and neutrophils. Neuropeptide Y (NPY) can have a stimulatory or inhibitory
radicals in peritoneal monocytes, and macrophage phagocytic activity; increases
monocytes mobilization, leukocytes adherence capability, and immune cell redistribution.
Neuropeptide Y can produce a shift in Th2 response by suppressing Thl cells release of
IFN-gamma and stimulating the production of IL-4 by Th2 cells, thus causing an increase
in serum antibodies. Neuropeptide Y can also influence the immunomodulatory effects of
other neurotransmitters, thereby acting as a neuroimmune co-transmitter (Ahmed et al.
2001; Bedoui et al. 2003).
Gastrointestinal hormones can also modulate the immune system. Substance P
increases T-cell proliferation, degranulation of basophils and mast cells, enhances
macrophage phagocytosis, stimulates 02, H202 and tromboxane B2 production (Goetzi et
al. 1985; Payan and Goetzi, 1985). Substance P and somatostatin can also modulate cells
involved in delayed-type hypersensitivity reactions and cell-mediated immunity.
Somatostatin stimulates release of histamine from mast cells, but suppresses histamine
and leukotriene D4 release from basophils. It also suppresses T-cell proliferation. These
two hormones can have similar effects, such as stimulating macrophage phagocytosis, but
they can also have opposite effects: substance P can increase thymidine and leucine
uptake from T-cells in the presence or absence of mitogens, while somatostatin inhibits
this process. The effects of these hormones can be different even on the same cell type
located in different tissues. For example, tissue mast cells and basophils in the circulation
do not release mediators in response to neuropeptides. Instead, nanomolar to
subpicomolar concentrations of somatostatin can suppress Ig-E mediated histamine and
Vasoactive intestinal peptide can influence the immune system
stimulating cytotoxic T-cell, but not B-cell proliferation. This effect can be explained by
the presence of VIP receptors on T, but not in B-cells.
The immunoregulatory function of hormones has also been demonstrated in fish.
Sex steroids, especially androgens, can suppress the immune system (Hou et al. 1999;
Yamaguchi et al. 2001). Melatonin also appears to modulate the immune system (Yada
and Nakanishi, 2002).
Prolactin and growth hormone enhance both the cellular and
humoral immune responses, and are important for the proliferation of B-cells (Sakai et al.
1996a, b, c; Yada et al. 1999). The decrease in immune responses of hypophysectomized
fish demonstrated the importance of hormonal modulation of the immune system (Yada
et al. 1999; Yada and Azuma, 2002).
As in mammals, gastrointestinal hormones, such as
substance P, somatostatin, and calcitonin gene-related peptide, may regulate the immune
functions of fish (McGillis and Figueiredo, 1996; Ndoye et al. 1991).
Lymphokines are usually associated with the immune system, but they can also
influence the neuroendocrine system.
When IL-1 enters the circulation, it can act as a
hormonal signal. IL-1 may pass through the fenestrated endothelium in the brain (median
eminence) and stimulate corticotrophin-releasing hormone (CRH) secretion from the
CRH nerve-terminals that will influence the physiological activity of other parts of the
body. In addition, IL-1 may activate the brain endothelial cells to produce IL-1, IL-6, and
prostaglandins, and to secrete other mediators into the brain (Tilders et al. 1994). As for
hormones, lymphokines can also be produced by the neuroendocrine system, for example
axis in response to infection and inflammation (Cohen and Kinney, 2001; Tumbull and
Rivier, 1995; Yada and Nakanishi, 2002).
Hormone and lymphokine receptors have been identified in cells of both immune
and neuroendocrine systems. IL-1 has two distinct receptors, Type I and II. Type I is
present on T-cells, fibroblasts and endothelial cells; Type II on B-cells and macrophages.
IL-1 receptors are present also in the choroid plexus, pituitary gland, and hippocampus.
Even if some hormones and lymphokines are produced and interact with both
immune and neuroendocrine systems, some differences are present. For example, ACTH
is produced by the hypothalamus, but lymphocytes and macrophages can also synthesize
it. The hypothalamus can synthesize two distinct forms of ACTH that differ in their
amino acid sequence (1-39; 1-24). Interestingly,
both forms of ACTH have the same
steroidogenic activity, but only the ACTH 1-39 can regulate the immune system (Johnson
et al. 1982). At least in this context, cells of the immune system, but not cells of the
neuroendocrine systems, can distinguish among closely related or truncated peptides
The interaction between the nervous and immune system is also evident from the
cell types common to both systems: astrocytes-macrophages. Even if astrocytes are
considered a type of macrophage, there are differences between these two cell groups.
During inflammation, astrocytes are involved in interacting with invading T-cells.
Activated T-cells are capable of passing the blood-brain barrier and thus infiltrate the
brain parenchyma. In the CNS, T-cells can recognize peptides presented by astrocytes via
than Thi (Aloisi et al. 1998). Astrocytes, even if they belong to the macrophage cell
group, play an important role in nerve regeneration and function. Astrocytes are the
major source of nerve growth factor (NGF). NGF constitutes one of the members of the
neurotrophin family, and it influences growth, differentiation, survival, and maintenance
of peripheral and central neurons. NGF can also act as a protective agent in
neuroinflammatory diseases (Oren et al. 2004). In addition to astrocytes, NGF can also be
released by oligodendrocytes and microglia, and to a certain extent by Thl cells
(Santambrogio et al. 1994). Cells from both the nervous and immune system express the
functional NGF receptor TrkA, suggesting that NGF participates in the interactions
between these two systems. Astrocytes restimulate preferentially Th2 cells because these
cells releasing IL-4 and IL-10 induce NGF secretion by astrocytes; while IL-2 and
IFN-gamma released by Th2 do not stimulate NGF secretion (Oren et al. 2004).
The interaction between immune and neuroendocrine systems is more complex
than expected because there may be a bidirectional relation among immune cells and
behaviors. Mast cells can reside in the brain or they can migrate within the brain after
inflammation. In mammals it has been demonstrated that the migration of mast cells in
the brain can be triggered not only by inflammatory processes, but also by specific
behaviors (Silver et al. 1996). For example in sexually active doves, after a brief period
of courtship, there is a marked increase in mast cells in the medial habenula (brain)
compared to control birds that have a relative absence of mast cells. During courtship
there is an increase of gonadal steroids that could influence mast cell migration. In fact,
elevated also during the 4-5 week period when parents take care of the eggs and young.
There is a reason why mast cells migrate specifically into the medial habenula: this
structure in the brain is a major integrative link between septal nuclei and the midbrain.
Another example where mast cell migration and activity is influenced by behavior
is seen in rats where stress was induced by social isolation (Silver et al. 1996). In stressed
rats the mean number of mast cells in the thalamus was reduced by 90%. Sex hormones
can directly and indirectly influence the synthesis and release of the granular content of
mast cells. However, under these physiological conditions the secretion of granular
content occurs without inflammation or tissue damage. Also, the interactions between
mast cells and elements of the peripheral nervous system have been documented for
peripheral ganglia, connective tissue, and gut mucosa. In these tissues and in the brain,
mast cells could mediate alterations in blood flow, neurotransmission, and local immune
responses such as switching of IgE in B-cells. Mast cells can produce up to 290
mediators, including histamine, interleukins 1-6, granulocyte-macrophage colony
stimulating factor, a variety of proteoglycans, neuropeptides, nitric oxide, and
chemoattractants for eosinophils and monocytes (Silver et al. 1996). A more clear effect
of mast cells on behavioral changes is given in an experiment of induction of an anxiety
status in rat (Ikarashi and Yuzurihara, 2002). In this experiment, several drugs that
stimulate histamine release from mast cells and neurons, or that block histamine receptors
with a histamine release inhibition effect, were used in combination and injected
intracerebroventricularly. After 60 minutes from injection, the time spent in light areas
a dark-light boundary. The results of the experiment show that the release of histamine
due to drug administration causes an increase in the anxious state of rats.
The effect of
the different drugs shows also that histamine can be released by mast cells or neurons.
Table 5-1. First appearance of immune organs of different fish species.
Organ first appearance
Species TempC Thymus Kidney Spleen Reference
Salmo gairdneri 14 5 dprh Grace and Manning, 1980;
Tatner and Manning, 1983;
Cyprinus carpio 22 2 dph 2 dph 5 dph Botham and Manning, 1981
Danio rerio 28 54 hpf 72 hpf Trede et al. 2001;
Willett et al. 1999
Zoarces viviparous 3 months 3 months 2 months Bly, 1985
Abbreviations: dprh= days pre-hatch, dph= days post-hatch, hpf= hours
First appearance of lymphocytes in immune organs of different fish
First appearance of lymphocytes
Species TempC Thymus Kidney Spleen GALT Reference
Salmo salar 4-7 22 dprh 14 dprh 42 dph Ellis, 1977
Salmo gairdneri 14 3 dph 5 dph 6 dph 13 dph Grace and Manning, 1980;
Tatner and Manning, 1983
Ictalurus punctatus 5 dph 7 dph 14 dph Petrie-Hanson and
Cyprinus carpio 22 3 dph 6 dph 8 dph Botham and Manning, 1981
Barbus conchonius 23 4 dph 4 dph 7 dph Grace, 1981
Danio rerio 28 65 hpf 3 weeks Trede et al. 2001;
Willett et al. 1999
Zoarces viviparous 2 mprep I mprep After birth Bly, 1985
Abbreviations: GALT= Gut Associated Lymphoid Tissue, dprh= days
pre-hatch, dph= days post-hatch, hpf= hours post-fertilization, mprep= months
Time of maturation of the immune organs.
Development to mature lymphocytic organ
Species TempoC Thymus Kidney Spleen Reference.
Salmo gairdneri 14 5 dph Grace and Manning, 1980;
Tatner and Manning. 1983;
Ictalurus punctatus 10 dph Petrie-Hanson and
Cyprinus carpio 22 5 dph Botham and Manning, 1981
Danio rerio 28 1 week Trede et al. 2001;
Willett et al. 1999
Abbreviations: dph= days
The purpose of vaccination is to provide an individual with resistance to a
particular pathogen without having to undergo a potentially risky infection.
confers a protection against disease, not necessarily infection. Thus, vaccines protect
against outbreaks of disease, but not necessarily against persistent infection in an
asymptomatic carrier state.
Vaccination works by inducing a protective immune response
that,, through memory cells persists for a relatively long period of time, though the
precise longevity varies. The term "immunity"
refers to this memory state. This memory
state varies among different pathogens, and also varies among fish species against the
same pathogen. Natural exposure to infection acts as a booster to the immunity produced
by vaccination, provided the memory cells are still present. In the absence of natural
exposure, booster vaccination is necessary to maintain the level of immunity. Specificity
and memory are two of the key elements exploited by vaccination since the adaptive
immune response is stronger after the second encounter with antigen. The vaccine may
stimulate non-specific defense mechanisms that provide a degree of protection over a
short time, but these mechanisms lack a memory component.
Vaccines must be safe, have no side effects, and should induce a high level of
most cases, some degree of antigen purification or enrichment is necessary. In this last
case it is important to identify the antigens that stimulate a protective immune response.
Vaccines can be classified as dead, live, and those that are genetically engineered.
Dead vaccines are composed of inactivated pathogens or extracts. Live vaccines are
composed of attenuated pathogens with low or no virulence. Live vaccines are developed
by serial passage of a pathogen in culture, or by using naturally occurring mutants and
cross-reacting strains, or using recombinant DNA technology deleting specific genes
coding for virulence factors. Compared to dead vaccines, live vaccines produce a higher
stimulation especially of the cellular branch of the immune system, and for this reason in
general, they produce a greater immune response (Marseden et al. 1996). However, a
problem associated with live vaccines is the possibility that the pathogen can revert to a
virulent state or that it can still possesses a residual virulence. In these cases, infection
and mortality will be the result of fish exposed to the pathogen. Genetically-engineered
vaccines are generally composed of a purified highly immunogenic antigen that has been
expressed in a microorganism through insertion of DNA coding for that specific antigen.
Generally, these vaccines provide superior protection because they are able to stimulate a
more complete immune response resulting in a broader and more long-lasting immunity
(Winton, 1998). Another significant advantage of genetically-engineered vaccines is the
ability to construct multivalent preparations that can protect fish against several
pathogens or different strains of the same pathogen.
All microorganisms and their products are composed of many different antigens,
those associated with virulence, could be caused by an adaptation of the pathogen to
avoid destruction by the host's immune response. Antigens may induce antibody
production, but, unless these antibodies can neutralize the components of pathogenicity,
they are valueless in giving protection. For this reason, assays of blood antibody levels
following vaccination may give no indication of the degree of protection unless the
antibodies are directed against pathogenic antigens. For example, when salmonids have
been vaccinated with specific Aeromonas salmonicida bacterins, antibodies against the
pathogen can be detected in the blood, but the fish are not protected (Ellis, 1985).
The form in which the antigen is administered may be very important. Generally,
soluble antigens are administered in adjuvant to elicit antibody production. Oil-based
vaccines have proven to give high levels of protection (Midtlyng et al. 1996a; Nordmo
and Ramstad, 1997). In oil-based vaccines, it is important that the dispersed phase is
homogenous to guarantee a reproducibility of injection. In the case of large oil droplets
the dispersion tends to phase separate, with the possible sedimentation of droplets, and
formation of a high-density lower phase or flotation of oil droplets.
This will result in
non-uniform dosing during the vaccination period. Phase separation can be avoided or
minimized by producing vaccines with a small droplet size. Small droplet size is also
preferable in vivo because fine dispersion will lead to an even distribution of the vaccine
intracoelomically (Muller and Harnisch, 1998). There is also an optimum size for
macrophage uptake and lymphatic absorption, the parameters determining vaccine
efficacy and antigenic response. In in vitro studies, highest particle uptake from
because the particles approach the size of the macrophage itself. Studies on lymphatic
absorption of particulates demonstrate that optimum size is below 1 pm (Davis et al.
Ideally, the vaccine dispersion should be stable after shaking the vaccine solution,
at least for one day. In vaccine production, it is also important to reduce the viscosity of
the formula. A low viscosity spreads more easily in the coelom and requires less injection
force, thereby reducing the chances of possible lesions in the coelom. However, in some
case, a lower viscosity may cause a too fast release of antigen, and a wider diffusion of
the vaccine causing a too generalized inflammation.
The route of antigen administration is also important. Generally, intramuscular
injection of antigen elicits higher antibody production than bath exposure (Fukuda and
Kusuda, 1981; Midtlyng et al. 1996b). Another consideration is that the immunogenicity
of antigens may vary among fish species.
In bacteria, serotype differences are frequently associated with variation in the
structure of the lipopolysaccharides, LPS. If antigens which represent different
pathogenic groups or strains are involved in eliciting a protective immune response, then
the different serotypes must be incorporated into the vaccine to protect against the
different serotypes of the pathogen. Such vaccines are called multivalent.
Adjuvants are substances that enhance the immune response. They comprise a
variety of compounds whose mechanisms of action are variable and in many cases not
well understood. Administering antigen with adjuvant can considerably enhance the
adjuvant, and it may be significant that in both cases the protective antigens are
lipopolysaccharide, considered to be a T-independent antigen. Protection against other
diseases has been achieved only when antigen was given in adjuvant (Aeromonas
vaccine). In these cases the adjuvant may either improve the immunonogenicity of the
antigens or have a non-specific stimulatory effect on the non-specific defense
mechanisms, or both (Ellis, 1988).
One of the problems in using adjuvants, is that they can cause side effects or
severe local tissue responses (Lillehaug; Midtlyng, 1996; Midtlyng et al. 1996b).
The most effective adjuvant and one of the most widely used by experimental
immunologists is Freund'
Complete Adjuvant, FCA, which is a mixture of killed
Mycobacterium tuberculosis in a mineral oil in which the antigen in saline solution is
emulsified. The emulsion can only be administered by injection. FCA can cause
undesirable side effects, such as local granuloma formation, autoimmune diseases, and
tuberculin sensitization. By intramuscular injection, it induces sterile abscesses with
extensive local muscle necrosis. Intracoelomic injection is less traumatic, but visceral
adhesions and granuloma formation may result. Slight reduction in fish growth rate can
Freund's Incomplete Adjuvant, FIA, lacks the Mycobacterium. It is used in the
same way as FCA and has only slightly less traumatic side effects, but it is not as
effective an immunopotentiator as FCA.
Other injectable adjuvants are aluminum hydroxide gel mixed with saponin,
6.3. Injection Vaccination
The most effective way to immunize fish is by intracoelomic injection. This
method permits the use of adjuvant that enhances the magnitude of the immune response.
This method has some disadvantages, principally by requiring anesthetization and
handling, both of which can be quite stressful for the fish. It is also very labor intensive
and can be difficult to apply to small fish. Nevertheless, the overall smaller amount of
vaccine required for this method, makes it more economical for fish larger than 40 g
Intramuscular injection on the dorsal surface of the fish is rarely used mainly
because of the possibility of creating unsightly scarring, and because of potential leakage
of the inoculums before absorption. However, salmon broodstock are usually injected
intramuscularly in the "dorsal sinus", a potential space between musculature in front of
the dorsal fin (Mitchell, personal communication).
The swim bladder has also been used as a site for vaccine administration (Endo et
al. 1995). In some species of fish, the swim bladder is surrounded by well-vascularized
thin collagenous membranes that facilitate the absorption of the vaccine in to the blood
stream without causing as much damage to the body as intracoelomic or intramuscular
injections. Moreover, the swim bladder has a large spaced lumen and the surrounding
membrane can prevent the backflow and spread of the injected agents. Absorption of
injected agents can take up to 7 days. This slow ratio of absorption can be useful in some
treatments against diseases, but it can also cause pathological damages to the organ wall.
6.4. Immersion Vaccination
The first methods developed for immersion vaccination required a hyperosmotic
immersion step. The concept behind this technique was that a hyperosmotic media would
have induced a greater absorption of antigen through the gills and skin of the fish. It was
quickly realized that the hyperosmotic step was unnecessary and often very stressful. For
this reason, direct immersion methods were developed. There are two main immersion
methods, the dip and the bath. The substantial differences among the methods are the
time of exposure to the antigen and the concentration of antigen in the water, the higher
the concentration, the shorter the time. In dip vaccination fish are kept in the water with a
vaccine dilution of 1:3 or 1:10 or 1:100 for 5-30 seconds. In the bath method fish are kept
for an hour in the water with a vaccine dilution of 1:500-1:5000. A further variation of
these methods is the spray method in which fish are sprayed with the vaccine after being
removed from the water (Fukuda and Kusuda, 1981).
The uptake of some bacterial antigens, such as Aeromonas sp., by fish following
immersion has been demonstrated to be quite effective.
The mechanism of uptake is not
precisely understood, but the gills appear to be the main route of entry, with the skin and
lateral line system possibly playing a role. The failure to immunize fish against many
diseases by the immersion method is probably not mainly associated with inadequate
uptake of the antigens, but with the nature of the ensuing immune response. There is also
the probability that mucosal immunity, and not systemic immunity, is produced. This
hypothesis seems to be proven by the fact that while in injection vaccination there are
immersion vaccinations stimulate integumentary immunity, which is quite sufficient to
protect against natural exposure, but not to challenge by injection.
A disadvantage of vaccination by immersion compared to injection is that much
more vaccine is required. In addition, immersion vaccinations done at high concentration
can be toxic.
6.5. Oral Vaccination
Oral vaccination is a preferred method since it is suitable for mass administration
to fish of all sizes, imposes no handling stress on the fish, and does not interfere with
routine husbandry practices. Oral vaccination is the only method suitable for extensive
pond rearing of fish where catching the fish prior to harvesting for injection or immersion
vaccination is impractical. However, oral vaccination has some intrinsic limitations. In
the case of killed vaccines large amounts are required, thus increasing the cost, and there
is the uncertainly of individual dosage concentration since each fish consumes a different
amount of food. Unfortunately, the most important limitation of oral vaccines is their
poor potency, and many trials with many different vaccines have resulted in low or
inconsistent levels of protection (Ellis, 1988). A major problem seems to be the
destruction of antigens in the stomach and foregut before they reach the immune sensitive
areas of the lower gut (Rombout et al. 1985). To overcome this problem, enteric-coated
spheres can be used. Microparticles are used as carrier systems and they are capable of
partially protecting and entrapping antigens against enzymatic degradation. Experiments
with rats (O'Hagan et al. 1989) and with coho salmon (Piganelli et al. 1994) have shown
Another reason for a low protection state following oral immunization is that oral
vaccine may induce a mucosal, but not a systemic response. A semi-autonomous mucosal
immune system exists in fish. Stimulation of any mucosa can lead to antibody production
in.all mucosal compartments (gills, skin, guts),
without necessarily stimulating a systemic
response and inducing a memory state. Induction of mucosal immune responses via the
oral route often requires multiple oral administrations (Joosten et al. 1997
Lin et al.
2000; O'Hagan, 1992).
6.6. Biomolecular Vaccines
The application of biomolecular techniques to vaccine design can help produce
vaccines that confer higher protection and are safer than vaccines produced by
conventional techniques. In fish, research into recombinant DNA technology has focused
mainly on viral vaccines. Currently, there are only a few vaccines available, including
one vaccine against Infectious Pancreatic Necrosis Virus (IPNV) (Leong, 1993; Leong
and Fryer, 1993
Leong et al. 1997
Lorenzen, 1999; Midtlyng, 1997). Expression-based
vaccines can be divided into several different groups: 1) those agents that replicate within
the host, such as attenuated strains; 2) recombinant viral and bacterial vectors; and
3) nonreplicating agents that serve only as vehicles to deliver nucleic acid sequences,
such as retroviral sequences and genetic immunization.
The first step in the production of a recombinant DNA vaccine is to identify the
proteins/antigens of the pathogen that stimulate a protective immune response in the host.
More than one antigen may be protective, and some antigens may stimulate some parts of
cultured so the protective antigens can be produced on a large scale. Because no
infectious agent is present, these types of vaccines have a high level of safety, and low
production costs after initial research and development (Winton, 1998).
Bacterial expression systems based upon E. coli may lack the ability to
glycosylate and correctly fold some types of proteins, resulting in less effective
protection. In these cases another expression system that is able to produce a protein in its
correct native conformation, such as baculovirus vector growth in insect cells, must be
chosen. However, these vectors can make the vaccines too expensive. These alternative
expression systems were used to produce a vaccine for rainbow trout against the viral
haemorrhagic septicemia virus (VHSV) (Lecocq-Xhonneux et al. 1994). The cloned gene
for one virus glycoprotein was inserted into a baculovirus expression system and the
recombinant protein was produced in insect cells. The recombinant protein had a slighter
lower molecular weight than the native viral protein and displayed a different pattern of
glycosylation. The recombinant protein was transported to the plasma membrane and
induced cell fusion in a manner similar to the authentic protein. The vaccine based on this
recombinant protein conferred a protection to rainbow trout similar to that conferred by
killed and attenuated vaccines, and neutralizing antibodies recognized it.
With recombinant DNA technology, besides inserting sections of DNA into the
genome of a cell, it is also possible to delete sections. Specific deletion of a gene that
codes for a virulence factor in a pathogen, while preserving other protective antigens,
allows the production of attenuated forms where reversion to virulent wild type is
In addition to traditional genetic immunization and recombinant DNA technology,
there are several other biomolecular techniques that have been applied to vaccine
Genetic immunization using naked DNA is the most recent approach in vaccine
design. This technology is based on the observation that skeletal muscle cells injected
with purified plasmid DNA express plasmid-encoded proteins. In mammals the specific
immune response after DNA vaccination is caused by antibodies, T-helper cells and
Protein engineering. The aim of this technique is to improve immunogenicity of
an antigen or to mass produce it cheaply. Protective antigens may be made more
immunogenic by cleavage of epitopes that stimulate the
T-cell suppressor. The protective
epitopes of an antigen may be composed of a short sequence of amino acids that can be
artificially synthesized quite cheaply for use as vaccines in combination with appropriate
Anti-idiotype vaccines. These are antibodies, the antigen-binding site of which
mimics the structure of a protective antigen. They are produced by first raising antibodies
to the appropriate antigen in a suitable animal. The specific antibodies produced
(idiotypes) are then used to raise further antibodies that react with the antigen-binding
site of the first antibody (anti-idiotypic antibodies). The antigen-binding site of the anti-
idiotype can be used as a vaccine since it resembles stoichiometrically the original
antigen and will induce formation of antibodies which cross reacts with it. Such vaccines
Genomics and Proteomics Applied to Vaccine Design
New approaches to vaccine development are important because there are several
infectious diseases for humans and animals for which the traditional approaches have
failed to produce effective vaccines. Moreover, especially for humans, regulatory
authorities often require that vaccines meet high standards of safety. For these reasons,
there is increased interest in the application of the new technologies known as genomics,
proteomics, and transcriptomics to human and animal vaccine design.
Genomics, proteomics and transcriptomics have become important tools for
increased understanding of the biology of pathogens (viral, bacterial and parasitic) on a
large scale, giving information on which genes and proteins are expressed and under
which conditions. This information can help assign functions to unknown genes and
proteins, decipher the mechanism of pathogenicity of organisms, reveal new possible
targets for drugs, and assist in the design of new vaccine candidates for humans and
animals (Barret et al. 2000; Betts, 2002; Cordwell et al. 2001; Grandi, 2001; Klade, 2002;
Moxon and Rappuoli, 2002; Nilsson, 2002; Potter and Babiuk, 2001; Tarleton and
Kissinger, 2001; Teixeira et al. 2002; Wallace and Wallace, 2002). For example, antigens
present only in virulent bacteria strains may make good vaccine candidates.
DNA microarrays are one of the most useful tools for the study of the
transcriptome, the complete set of transcripts produced by an organism. In this technique
DNA elements are arranged as spots in a grid pattern on a solid support. These arrayed
targets are hybridized with complex probes prepared from an RNA population for a
Gene expression profiling is performed by two-color hybridization. DNA labeled with
different fluorochromes is produced from RNA isolated from two experimental
conditions. The two populations are combined in equal quantities and hybridized to the
array. Scanning the array gives the quantitative fluorescence intensities for each fluor in
each spot, from which ratios are calculated to give relative expression levels for each
gene. This analysis can reveal even shifts of hundreds of genes expressed. Such
techniques have been used to study the whole genome of Streptococcus pneumoniae to
identify suitable antigens for vaccine production (Wizemann et al. 2001).
Through exploring the effects of drug action using DNA microarrays, it is
possible to identify genes within pathways that may have not been previously realized,
and may provide alternative drug targets. Moreover, the generation of signature profiles
of drug action may allow prediction of the mode of action of novel compounds.
Improvements in the sensitivity of this technique may be achieved through amplifying
mRNA, thus facilitating analysis of small amounts of RNA recovered from tissues or
Another technique for studying genome expression is In Vivo Expression
Technology (IVET). Plasmid libraries are created with random fragments of bacterial
chromosomes fused to promoterless genes that encode an antibiotic resistance gene. A
single crossover between the plasmid and the corresponding homologous region on the
chromosome produces mutant bacteria. The bacteria are then injected in to antibiotic
treated animals. At this point only the mutant bacteria that have the antibiotic resistance
Salmonella typhimurium, Pseudomonas aeruginosa, Yersinia enterocolitica, Vibrio
cholerae and Staphylococcus aureus (Grandi, 2001).
In Signature Tagged Mutagenesis (STM), bacterial mutants are created by random
insertion of a set of transposomes differing by a short synthetic nucleotide sequence
(TAG), so that each mutant can then be recognized by its TAG. The bacteria mutants are
then injected in to animals and the bacterial clones with a mutation in a gene needed for
pathogenesis will not survive. The missing mutant clones can be detected by recovering
bacteria and doing a TAG-specific DNA hybridization, in this way all the genes required
for infection can be identified. For example, in a study on Group B Streptococcus,
out of 1600 transposome mutants were defective for survival in the animal host, and only
50% of these mutants have insertions in known genes (Jones 2000).
Proteomics, in addition to describing the protein complement to the genome, can
provide important information on proteins expressed at defined points in time or under
defined conditions, their location within the cell, and the existence of post-translational
modifications. Protein analysis can be more useful than mRNA analysis for several
reasons. The final products of genes are proteins; mRNAs can be present in a cell but not
translated; and from a single mRNA molecule, several proteins can be produced, as in the
case of some hormones such as calcitonin and calcitonin-related gene. Both hormones are
translated by the same mRNA, but by different splicing occurring in the brain or in the
thyroid. The simple analysis of mRNA would not detect all these different proteins
produced by the same mRNA. Stability, half-life, post-transcriptional, co-translation and
environmental stimuli may not completely represent the expression of a corresponding
protein in vivo.
Protein analysis, however, can present some difficulties that are not encountered
in genome analysis, such as target abundance and molecular recognition. Another
difference is that target DNA or RNA can be amplified by PCR. There are no similar
techniques that easily allow amplification of target proteins. In genome studies,
sequences can be recognized by oligonucleotide probes, but for protein analysis,
recognition is mediated by antibodies, other proteins or ligands, and each of them is very
specific for only one or a few proteins.
In proteomic analysis, several techniques are utilized, with the most common
being two-dimensional gel electrophoresis (2DE). Two-dimensional gel electrophoresis is
a technique that can be applied to a complex protein mixture, such as that extracted from
whole cells and tissues. The proteins migrating in a pH gradient gel are first separated
based on their charge (isoelectric point).
Then, the proteins migrate according to their
molecular mass, in a direction perpendicular to the first migration axis. Size fractionation
is achieved by first equilibrating the isoelectric focusing gel in a 2% solution of the
detergent sodium dodecylsulphate (SDS) which binds non-specifically to all proteins and
confers a uniform negative charge. After fractionation, the protein gel is stained using, for
example, silver staining or Coomassie brilliant blue or non-covalent fluorescent dyes.
The outcome is a unique pattern of dots, each dot representing a protein. This pattern can
be used as a protein fingerprint of the cell. Typically more than 1,000 distinct proteins
separation of up to 10,000 protein spots.
Western blotting of the gel and detection of
antigens with patient sera can help distinguish immunorelevant proteins.
This technique can be utilized to easily compare protein expression of a particular
celH in different situations by just comparing the 2DE gels and looking for a spot absent
or present in one of the gels. However, a problem for this type of analysis is that the
conditions in which the gels are prepared and run should be the same in each experiment,
and this can be difficult to accomplish.
To enhance separation of proteins, instead of 2DE, reverse phase microcapillary
liquid chromatography can be utilized. Multidimensional chromatography can also be
applied. This technique consists of serial chromatographic separation according to
Identification of proteins on the 2DE gel can be achieved. First, proteins inside
the gel are digested with trypsin. The resultant peptide mixtures can be analyzed with
mass spectrometry, measuring the mass of each peptide obtained from the digestion of
the same protein. Each protein produces a characteristic peptide mixture that can be used
as a fingerprint. The measure of the mass of the peptide can be correlated to the amino
acid composition of each peptide, and then the amino acid sequence of the protein can be
determined. This fingerprint can be compared with theoretical digests of proteins in
databases through a bioinformatics tool in order to identify the protein. Otherwise,
peptides can be separated by size-exclusion chromatography (SEC) and reversed-phase
(RP) chromatography, then directly analyzed by mass spectrometry (MS).
resolving power of 2DE in conjunction with narrow-range immobilized pH gradients and
subcellular prefractionation has been estimated to reveal up to 75% of protein genome,
while the remaining 20-30% cannot be resolved due to technical limitations associated
with this technology. Moderate or low abundant proteins could not be observed in the
2DE, especially if a nearby spot representing a high abundant protein obscures their
signal. Scarce proteins may be completely undetected. Fractionating the total protein
sample and running a gel specific for particular types of proteins, such as membrane
proteins or proteins with a particular isoelectric pH, can overcome this problem.
Prefractionating complex mixtures into specific cellular compartments, such as
membrane or extracellular or intracellular proteins, is important because for pathogenic
organisms, proteins of interest are most likely confined to the membrane or extracellular
fractions. However, due to their intrinsically hydrophobic nature, membrane proteins can
be difficult to solubilise in gel. Recently, several new solubilising agents have been
developed and have allowed the visualization of significantly more hydrophobic
membrane-associated proteins (Cordwell et al. 2001). This type of analysis was
performed on Streptococcus agalactiae to identify the major outer surface proteins that
can be used for vaccine production (Hughes et al. 2002).
Bacterial proteins secreted during growth may be important for several pathogenic
processes, including toxicity or cell-signaling, but the proteomic analysis of such proteins
can be difficult due to the presence of contaminating proteins from undefined sources or
from supplement animal sera. In some bacteria, the analysis of such proteins could be
important for vaccine production were identified analyzing the secreted proteins of group
A Streptococcus (Lei et al. 2000).
Another technology used to study the proteome of a target cell or tissue is protein
arrays. Protein arrays are in theory very similar to DNA arrays being miniature devices
that have proteins or molecules that recognize other proteins. However, protein
microarrays have more limitations than DNA microarrays. First there is no procedure that
amplifies the sample proteins as there is for DNA. Second, while all DNA sequences are
constituted by the same four nucleotides that are similar in their chemical properties,
proteins are comprised of
amino acids with different chemical properties. The
principles of molecular recognition are very different among amino acids and also vary
among different proteins. For this reason the same reaction conditions can never be
applied for all proteins. Third, in a DNA array, labels are incorporated evenly and do not
interfere with hybridization. Fourth, solid substrate, such as nylon or glass, does not
interfere with hybridization. In contrast, for proteins, labeling is much more variable and
both labeling and attachment to a substrate may interfere with protein binding by
affecting protein folding or by blocking the binding site. In these arrays, antibodies are
attached to the array or chip surface.
Antibody arrays can detect specific proteins. Because antibodies are highly
discriminatory, they are suitable for detailed analysis of protein profiles and expression
levels. There are three different types of antibody arrays. The first is a standard
immunoassay in which the antibodies are immobilized and are used to capture labeled
allows exposure of two different protein samples at the same time, each labeled with a
different fluorophore. The second technique consists of a miniature sandwich assay in
which unlabelled proteins are captured from the solution by antibodies on the array after
which the proteins are detected by a secondary antibody. This technique requires that the
two antibodies recognize different parts of the proteins. The advantage of this technique
is that it is not necessary to label the proteins, a process that is variable and sometimes
inefficient. The third technique involves a tertiary detection system, and therefore it is
more sensitive. The principle of this technique is that the immobilized antibody captures
a protein. After, a secondary antibody linked to a circular oligonucleotide will recognize
the protein. In the presence of a circular DNA template and dNTPs, DNA polymerase
will amplify the circular DNA template producing a long DNA concameter containing
hundreds of copies of the template. Then a fluorescent-labeled oligonucleotide probe can
be added to detect the DNA template.
More recent techniques in proteomics use a microfluid device instead of a gel.
These devices should have a higher sensitivity because of the faster kinetics of binding in
solution than in a solid phase, and they should have a higher throughput because the
arrays are constructed in three dimensions.
Another technique to identify a new possible targets for vaccine production is in
silico analysis. This technique employs algorithms involving known gene sequences to
predict genes encoding secreted, surface-associated antigens and virulence factors. This
technique analyzes the genomic sequence of pathogens to detect proteins with
virulence factors can be detected by searching for unknown genes that are co-regulated
with known virulence genes. The selected genes are expressed in E. coli and the
expressed proteins are purified and tested for their effect on the immune system. This
technique is very fast because it uses robotic stations. Five scientists can produce and
purify 100 proteins each month. However, a limitation of this technique is that it can not
be used to detect new families of virulence factors. In a study on Chlamydia pneumoniae,
147 membrane-associated proteins were identified, 91% of them were successfully
expressed in E. coli, and 58 proteins induced Igs that bind C. pneumoniae (Grandi, 2001).
Collectively, these technologies will allow the identification of most of the
antigens targeted by the immune system under certain pathological situations. Once the
target antigens are identified, their genes must be cloned and expressed, then the
synthesized proteins must be purified and tested in vitro and in vivo for identification of
candidates for vaccine production. Moreover, the comparison of multiple immunomes
may allow the discovery of immunogenic structural features shared and conserved among
different pathogens, which could form the basis of broadly protective vaccines.
6.8. Herd Immunity
Concomitant with research on vaccine development, there is a need for an
improved understanding of how best to use vaccines to protect the community as well as
the individual. Epidemiological and economic issues are at least as important as
technological ones (Anderson and May, 1985; Waltner-Toews, 1989).
Herd immunity is the ability of groups of animals to resist becoming infected or to
The persistence of an infectious disease within a population requires that the
density of susceptible individuals exceed a critical value such that, on average, each
primary case of infection generates at least one secondary case. It is therefore not
necessary to vaccinate everyone within a community to eliminate infection, but it is
sufficient to vaccinate a percentage of the population so as to reduce the susceptible
fraction below the critical point. The central problem for the epidemiologist then is to
calculate the proportion of the population that should be vaccinated. This calculation is
not easy because many factors must be considered, such as desired level of vaccine
protection, primary infection rate, transmission rate, incubation time, duration of acquired
immunity, age-related changes in infection susceptibility, best age for immunization,
genetic factors, population density, and behavioral factors. For example a more dense
population requires higher rates of vaccination (Anderson and May, 1985; Waltner-
When the concept of herd immunity is kept in mind, it becomes clear why the use
of vaccines that confer low or moderate protection is still beneficial. In fact, these
vaccines may slow down or stop the rate of disease transmission. A reduction in the rate
of transmission may be helpful especially when the production periods are not too long,
as in aquaculture where the production period is generally of one or two years.
Immunostimulants and Experiment Design
The term immunostimulants is applied to several compounds of diverse chemical
structure that have been shown to stimulate the immune system (Anderson, 1992; Raa,
The exact mechanisms of the immune system stimulation are still not
well understood, and there are still doubts on the real efficacy of some of these
compounds. For many immunostimulants, such as vitamin C,
have shown diverse and even conflicting efficacy results. However, comparing the results
of different studies is not an easy task due to differences in experimental protocols
including variations in doses, methods, and length of administration.
During experimental analyses, the body's
response to immunostimulant
administration over time is often not considered. This class of compounds can stimulate
the immune system, but prolonged dosing will produce a homeostatic response with
consequent decreases in stimulation. In addition, different immune parameters can be
stimulated at different doses and times. One example of these issues is evident in research
examining vitamin C administered to gilthead seabream (Spaurus aurata) (Ortuno et al.
1999). In this study, fish were fed diets containing 0.5
and 3 g/kg ascorbic acid for
8 and 10 weeks. Some of the non-specific immune parameters were enhanced by dietary
not affected. After 8-10 weeks, the serum complementary activity was almost the same in
all the trials. Even when high levels of ascorbic acid were maintained in the serum, the
immune responses did not improve, but returned to their normal levels. These data
suggests that after a transient enhancement of the non-specific immune response as a
result of vitamin C supplementation, an adjustment of the immune response to the new
conditions of higher ascorbic acid availability occur. If this hypothesis is correct, it might
explain the absence of effect described in some previous studies with this vitamin when
long administration periods were tested.
Body storage capability for different immunostimulants is another consideration
when experimental results are analyzed. In studies where different doses of an
immunostimulant that can be stored in the body, are tested, lack or a non-significant
difference in results among diets may be due primarily to body storage of the compound.
For this reason before the experiment it is important to feed diets without
Additional factors include storage capabilities and retention period. These may
differ, for a particular compound, among organs. For example, in rainbow trout after the
fish were switched for two weeks to the control diets, plasma and liver ascorbic acid
levels were strongly reduced meanwhile the level was still high in the head kidney. The
longer retention of vitamin C in the head kidney is related to its lymphoid function:
leukocytes are able to store elevated amounts of ascorbic acid in their cytosol and,
therefore, require a longer period of time to become depleted (Verlhac et al. 1996).
development, reproduction, wound healing, metal ion metabolism, response to stressors,
immune response, and perhaps lipid metabolism through the action of carnitine synthesis.
Most teleost fish do not synthesize vitamin C because they lack the enzyme
L-gulonolactone oxidase. This vitamin is synthesized in other phylogenetic groups of
fish, such as sea lamprey, Petromyzon marinus, Atlantic dogfish, Squalus acanthias,
sterlet sturgeon, Acipenser ruthenus, white sturgeon, Acipenser transmontanus,
paddlefish, Polyodon spathula, and in some teleost such as Atlantic halibut, Hippoglossus
hippoglossus, and Atlantic salmon, Salmo salar, (Dabrowski, 1994; Dabrowski and
Clereszko 2001; Maeland et al. 1999; Maeland and Waagboe, 2000; Moreau, 1999).
Signs of vitamin C deficiency in fish include decreased growth, scoliosis,
lordosis, internal and fin hemorrhages, distorted gill filaments, fin erosions, anorexia, and
increased mortality (Halver et al. 1969; MacConnell, and Barrows, 1993).
Organ and tissue concentrations of ascorbic acid are correlated to dietary intake
levels (Hardie et al. 1991; Verlhac et al. 1996). Brain, thymus, head kidney, and
leukocytes concentrate more vitamin C and maintain its level for longer periods than
other organs, such as liver, in cases of dietary deficiencies. Liver and head kidney are the
primary storage organs for vitamin C in fish. The high level of vitamin C found in
thymus, brain, head kidney, and leukocytes may be related to the requirement for
antioxidant substances to maintain cellular membrane integrity to preserve vital organs
from oxidative processes, and to allow the optimal functioning of immune cells. The high
level of this compound in the head kidney could also be related to the lymphopoietic
Trunk kidney and spleen are also able to store large amounts of vitamin C.
Vitamin C is involved in the synthesis of cortisol and catecholamine hormones
involved in the stress response. In a stressed state, plasma cortisol levels increase and the
immune system efficiency decreases. Cortisol causes changes in numbers and proportions
of circulating leukocytes, generally resulting in lymphocytopenia and neutrophilia.
Cortisol also has an effect on the non-specific immune system causing inhibition of
respiratory burst activity, phagocytic activity, and leukocyte migration. Cortisol also
decreases the mitogen response and antibody production (Barton and Iwama 1991).
Vitamin C can improve the immune system by acting as a brake on steroidogenesis
through peroxidation of unsatured fatty lipids and so preventing their conversion into
cholesters that are important components of the cortisol. For this reason, during stressful
situations, the requirement of vitamin C increases.
Vitamin C plays a significant role in the immune response and resistance to
infectious diseases primarily by stimulating components of the non-specific immune
system, including the complement pathway, serum lysozyme, macrophages, and
neutrophils phagocytic activity (Hardie et al. 1991; Li and Lovell, 1985; Ortuno et al.
1999; Roberts et al. 1995; Verlhac et al. 1996). During phagocytosis, immune system
cells produce reactive oxygen radicals. These compounds have potent microbicidal
activity, but they are also auto toxic to fish macrophages (Secombes et al. 1988).
Administration of ascorbic acid has been shown to increase survival of fish challenged
with pathogens. Increased survival was observed in salmon challenged with Aeromonas
Labeo rohita, challenged with E. tarda (Sahoo and Mukherjee, 2002); in rainbow trout
challenged with the ciliate parasite Ichthyophthirius multifilis (Wahli et al. 1986).
However, in several other studies, supplementation of vitamin C did not improve
the immune system or the resistance of fish to bacteria or parasites (Bell et al. 1984; Lim
et al. 2000; Lygren et al. 1999; Sealey and Gatlin, 2002). In channel catfish,
administration of vitamin C increased macrophage migration, but did not increase
survival against E. ictaluri (Lim et al. 2000).
Ascorbic acid is also involved in mineral metabolism, influencing absorption and
decreasing metal toxicity.
Vitamin C transforms metals, such as cadmium, nickel, lead,
into their reduced forms, which are less efficiently absorbed and more rapidly excreted.
7.3. Vitamin E
Vitamin E, acting as an antioxidant, protects biological membranes and assists in
optimal functioning of the immune system. Phospholipids of mitochondria, endoplasmic
reticulum, and plasma membranes possess special affinities for alpha-tocopherol. Being a
lipid soluble antioxidant, vitamin E protects the highly unsaturated fatty acids in cells
against oxidative degeneration by terminating free-radical-initiated chain reactions and
decreasing the production of lipid peroxides and reactive oxygen species. Because these
chemicals are autotoxic and can destroy neutrophils and macrophages, they can adversely
affect the immune system (Lall and Olivier, 1991).
Alpha-tocopherol also stimulates the
phagocytic response, and total superoxide dismutase activity in liver and muscle (Lygren
et al. 1999).
Vitamin E significantly increases immunity and decreases mortality in Indian
Vitamin E deficiency results in decreased T and B lymphocyte function and
phagocytic index in rainbow trout challenged with Yersinia ruckeri (Blazer et al. 1984),
coelomic macrophage function in rainbow trout (Blazer, 1982), as well as serum
complement function and serum capability to opsonize bacteria in Atlantic salmon
(Hardie et al. 1990; Montero et al. 1999).
In some studies no positive response on the immune system was observed with
administration of vitamin E (Li et al. 1993; 1998) nor was there resistance to bacterial
challenge (Lall et al. 1988; Sealey and Gatlin, 2002).
Vitamin A and other carotenoids can influence the innate and specific immune
responses. These compounds have high affinity for toxic oxygen radicals, which result
mainly from lipid peroxidation, but which are also generated during the respiratory burst
of white blood cells in the initial response to an infection (Benedich, 1989).
reason the carotenoids can enhance the ability of macrophages, killer T-cells and
cytotoxic T-cells to kill tumor cells and bacteria. This capability is demonstrated in
homeothermic animals where p-carotene and canthaxanthin can enhance B and T-cell
proliferation in vivo (Benedich and Shapiro, 1986). However the effect of these
compounds on the immune system of fish has not been well investigated.
In a study on rainbow trout, vitamin A and astaxanthin were shown to have only
limited potential as immunomodulatory agents (Thompson et al. 2001). Fish fed diets
deficient in vitamin A and astaxanthin presented lower serum antiprotease and classical
phagocyte respiratory burst activity were unaffected by astaxanthin or vitamin A
deficient diets. The results of this research showed that deficiencies of vitamin A and
astaxanthin can cause a decrease of serum antiprotease activity and this could affect the
resistance of fish to external pathogens. In fish, the antiproteases play an important role in
neutralizing the extracellular proteases of bacterial pathogens, and a2-macroglobulin
level in particular has been reported to be an important correlate of protection against
infection by Aeromonas salmonicida (Ellis, 1985).
7.5. Folic Acid
The effect of folic acid on the immune system was investigated in channel catfish
(Duncan and Lovell, 1994).
Deficiency of folic acid caused binucleated erythrocytes,
spectacle cells (abnormally dividing erythrocytes with two nuclei joined by a strand of
nuclear material), hemocytoblasts (immature blood cells), cytoplasmic clearing,
poikilocytosis (variation in shape of erythrocytes), pyknosis (condensation of the nucleus
of erythrocytes), smudge cells (disintegrating blood cells), macrocytes, erythrocytes with
irregular nuclear borders, and segmented or spreading neutrophils. In the same research it
was observed that fish challenged with E. ictaluri showed maximum survival and
antibody production when folic acid was fed with a high level of vitamin C, but only high
supplementation of folic acid improved survival when the diet contained a marginal level
of vitamin C. In contrast, in Nile tilapia, Tilapia nilotica, administration of folic acid did
not increase antibody levels nor did it improve survival of fish challenged with
Streptococcus iniae (Lim and Klesius 2001).
Iron is an essential element for the functioning of organs and tissues of higher
animals, including fish, because of its important role in oxygen transport and cellular
respiration. Iron is also one of the most important micronutrients for its effect on immune
system functions and host defense against infection. Chemotactic response of coelomic
macrophages to Edwardsiella ictaluri exoantigen was depressed in iron-deficient channel
catfish (Barros et al. 2002; Lim and Klesius, 2001; Sealey et al. 1997), but this
abnormality was remedied by feeding an iron repleted diet for 4 weeks (Lim and Klesius,
2001). However, a high level of supplemental iron may be harmful to fish due to the
delicate balance between the requirement of iron for host defense mechanisms and the
requirement of iron to sustain microbial growth (Sealey et al. 1997).
Fish can absorb soluble iron from the water across the gill membrane and
intestinal mucosa, but feed is considered the major source of iron for fish due to low
concentrations of soluble iron in natural waters (Lim et al. 2000). The total dietary iron
requirement for optimum growth, feed efficiency, hematological values, and immune
response of juvenile channel catfish has been determined to be about 30 mg/kg diet
(Sealey et al. 1997).
Several studies have examined the combined effects of vitamin C and iron
supplementation on fish immune system function because ascorbic acid is known to be
involved in the metabolism of iron in animals.
Vitamin C enhances the absorption of iron
from the intestine by reducing ferric iron to ferrous state, a more soluble form that is
with iron-binding proteins, apoferritin and transferritin, into tissue ferritin. High levels of
dietary ascorbic acid in the absence of dietary iron may accelerate iron deficiency and
increase the severity of microcytic anemia in channel catfish (Lim et al. 2000).
Significant interactions between dietary levels of ascorbic acid and iron were
observed in channel catfish with regard to hematological values and survival of fish
challenged with E. ictaluri (Lim et al. 2000). Mean macrophage migration in the absence
or presence of E. ictaluri exoantigen was significantly higher for fish fed the iron
supplemented diets, but neither dietary level of iron nor ascorbic acid nor their interaction
influenced the survival level of juvenile channel catfish against E. ictaluri challenge.
However, the onset of mortality was earlier for fish fed the iron-deficient diet.
Selenium is an essential trace element for all animals. It is a component of
enzymes such as glutathione peroxidase (GSH-Px). This enzyme catalyzes reactions
necessary for the conversion of hydrogen peroxide and fatty acid hydroperoxides into
water and fatty acid alcohols by using reduced glutathione, thereby protecting cell
membranes against oxidative damage. GSH-Px acts along with vitamin E as a biological
antioxidant to protect polyunsaturated phospholipids in cellular and subcellular
membranes from peroxidative damage. Tissues or cellular components that are inherently
low in glutathione peroxidase will not be affected by selenium, but will be still protected
by vitamin E, which acts as an antioxidant by a mechanism not involving GSH-Px. Wang
(1996) found that selenium deficiency in the presence of dietary vitamin E seriously
immune response is protecting cell membranes against peroxide damage during
phagocytosis. Production of hydrogen peroxide in phagocytes is necessary in the process
of killing microbes. However, the overproduction or leakage of peroxides into cytoplasm
can damage the cell membranes of macrophages when GSH-Px activity is low.
Selenium also has a protective effect against the toxicity of heavy metals such as
cadmium and mercury. In addition, selenium is important for normal humoral and
cellular immune response. Selenium plays a role in antibody production through the
proliferation and protection effects of GSH-Px on B-lymphocytes. Suppressed antibody
production appears to be a common result of selenium deficiency among animals.
Chinook salmon fed a selenium deficient diet showed greater mortality from
Renibacterium salmonarium infection (Thorarinsson et al. 1994).
The concentration and source of selenium can influence the immune response to
bacterial challenge (Wang,
1996; Wang et al. 1997). In channel catfish dietary selenium
concentrations for maximum survival from E. ictaluri challenge was 0.20 mg/kg for fish
fed Se-Methionine, and 0.40 mg/kg for fish fed Se-Yeast (Se-Y) or Na2SeO3. Antibody
production generally increased as dietary concentration of selenium increased. Antibody
titer was highest for fish fed Se-Y, intermediate for fish fed Se-M and lowest for fish fed
Na2SeO3. Macrophage chemotactic response was higher for fish fed Se-M and Se-Y than
for the control and Na2SeO3. A possible explanation for these results is that amino acid
bound trace minerals have higher biological values than inorganic forms because the
elements remain chelated after intestinal absorption, are transported intact to target
Supplementation of vitamin E or selenium to channel catfish at higher doses than
the recommended levels of one or both nutrients may enhance macrophage functions
(Wise et al. 1993). However, selenium and vitamin E do not complement each other,and
neither compensates for a deficiency of the other. Megadoses of selenium used for
disease therapy may involve risks because of the high toxicity of this mineral to animals.
Fish can absorb selenium present in the water across the gills and digestive tract
(Lim et al. 2001). The uptake of selenium as selenite across the gills is very efficient even
at low waterbone concentrations. At least in rainbow trout, the level of selenium in the
water can affect the dietary selenium requirement (Hodson and Hilton, 1983).
Selenium has been found to be required in the diet of Atlantic salmon, rainbow
trout and channel catfish (Lim et al. 2001), with the actual requirement dependent upon
the source ingested, polyunsaturated fatty acids, vitamin E content in the diet, and the
concentration of selenium in the water.
Zinc is required for normal growth, development and function of all animal
species. The primary functions of zinc are based on its role as a cofactor in several
enzyme systems and as a component of a large number of metalloenzymes. Zinc plays an
important role in immune defense mechanisms. Zinc is fundamental to T-cell function
and a deficiency leads to thymus atrophy and decrease thymic hormone and lymphokine
production in mice (Fraker et al. 1977). Zinc may also affect resistance to parasites and
bacteria. For example, mice fed a zinc deficient diet for 8 days were significantly more