I had prepared this presentation with great care. You can rely on the data.

1. . Credit seminar
Major Advisor: Dr. K. S. Ramesh
Md. Abdullah-Al-Mamun
DFK- 1602
Department of Aquaculture
College of Fisheries, Mangaluru
Vaccination in Finfishes

2. 2025-2030
3.9%
29%
44%
50%
Introduction
 Aquaculture
Most promising
Steadily growing
(FAO, 2016)
93.4 mmt, 56%
73.8 mmt,

4. Transformed- extensive systems Semi-intensive &
Intensive systems
PROBLEMS :
1.Deterioration of water quality
2.Fall in health status of fishes
3.Prone to disease
4.Huge economic losses
Nearly 43 years ago, Snieszko (1974) proposed
Super intensive

5. Challenges in aquaculture

6. Fish diseases -Profile
McLoughlin, 2006

7. Measures to combat diseases
Chemotherapeutics
• Antibiotics
• Chemicals
Non Chemotherapeutics
• Probiotics
• Prebiotics
• Herbal components
Vaccines

8. What is Vaccine?
Intentional administration
of vaccines.
Vaccination?
Modern history of vaccination
Latin word Vacca, which means cow
Ellis (1988) defined vaccine as a ‘‘preparation of
antigens derived from pathogenic organisms, rendered
nonpathogenic by various means, which will stimulate
the immune system in such a way as to increase the
resistance to disease from subsequent infection by a
pathogen”
Vaccinology?
Is of more recent vintage. Salk
and Salk (1977) introduce the
term vaccinology.

9. In 1881 Pasteur suggested the word “vaccination” as
a tribute to the work of Jenner.
1796
Edward Jenner
Cowpox lesions
Smallpox lesions
Vaccine inoculation

10. Fish Vaccines- the pioneers
The first report of disease prevention using vaccines is
probably by Snieszko et al. (1938).
 The first report in English was written by Duff in 1942,
who showed protection against Aeromonas salmonicida in
trout.
 Fish vaccination in India, first attempt was made by
Karunasagar and his team in 1991 ( Karunasagar et al.,
1991)
 The first vaccine for aquaculture, a vaccine for prevention
of ERM/yersiniosis in salmonid fish, was licensed in USA in
1976.
 The first viral vaccine for fish was produced by a
Czechoslovakian company (Bioveta) in 1982.
A search on “fish vaccination”
in scientific data bases showed a
total of approx. 10,000 papers.
Only 100 at the end of the 1979.
(Guddin and Muiswinkel, 2013)

11. The Ideal Fish Vaccine?
• Sustained immunity and protection
• Safe
• mass application
• Cheap and cost effective
• Easily produced
• Stable
• Easily licensed
Vibriosis
Coldwater vibriosis
Furunculosis
ERM/Yersiniosis
Piscirickettsiosis
Columnaris
Enteric septicaemia of catfish
BKD
Pasteurellosis
Streptococciosis
SVC
IPN
ISA
IHN
VHS
CCVD
GCHD
-Major market in salmon
-Trout
-Expanding market in sea bass, sea
bream, tilapia, turbot, halibut, yellow
tail, cod etc.
TOTAL= 2

12. Current status of fish vaccines

13. Types of Vaccines Methods of administration
Inactivated vaccines
Live vaccines
Vaccines based on DNA-technology Injection
Oral
Immersion

14. 95% survival

15. Research Papers

16. PAPER 1
Global impact Factor: 3.148 (2016)
NAAS Score: 9.03 (2017)

17. Biofilms of
bacteria
Frustrated Phagocytosis

18. OBJECTIVES
In the present study, authors has conducted an experiment on MAB
based ELISA and protection upon challenge to evaluate biofilm oral
vaccine of A. hydrophila in carnivore C. striatus.

19. Brief overview of the paper
Isolation of bacteria and biofilm vaccine preparation
Collection of fry and acclimatization and grown up to 15 g
Oral vaccination for 20 days in FRP tank
Collection of serum for ELISA at different interval
Determination of LD50 for challenge test
Statistical analysis
Vaccines were shown significant positive results
Culture for 60 days with normal feed

21. Bacterial isolate and maintenance of pure culture
Isolation by
selective media (RS)
1 day old isolate
grow in 3% TSB
aliquots in 1.5 ml
micro centrifuge tub
Harvested pellet re-suspended in
TSB with 15% glycerol
centrifugation at 10,000 rpm
for 10 min
will be stored at – 200 c
for further use.
Confirmed by Koch’s Postulates

22. Heat inactivated at
1000 c for 50 min
Vaccine was incorporated
in the feed according to
Azad et al., (1997)
TSB (0.225 g)
Chitin flakes (0.3g)
Preparation of A. hydrophila biofilm vaccine
6 h shaking daily 4 days
120 strokes/min+ 100 ml D/W
(Azad et al., 1997)

23. Experimental Design for the Experiment
Control
1010 CFU/
fish/day
T1R1
T1R2
T1R3
T2R1
T2R2
T2R3
T3R1
T3R2
T3R3
1010 CFU/
fish/day
1010 CFU/
fish/day
1010 CFU/
fish/day
1010 CFU/
fish/day
PBS
PBS
PBS
BF FC
C. striatus (15±0.35 g)
Size of tank-250 lts
40 fingerlings / tank
Siphoned off every alternate d
Antigen dose was 1010 cfu/fish/day
Vaccinated feed was given at 5%
Duration of vaccination- 20 d
1010 CFU/
fish/day
Reared for 3 months
CIFA, Bhubaneswar (ICAR)
MBW 15 g

24. CFCBF
Sampling
0 dpv 10 dpv 20 dpv 30 dpv 40 dpv 50 dpv 60 dpv
Collection of serum

25. Development of ELISA
Coated with 0.1 µg/well of A. hydrophila
Serum samples added (dilution 1:640 in 0.2% BSA) and
incubated for 1hr at RT
Wash the plate with PBS-tween20 with PBS
C. Striatus IgM
A. hydrophila antigen

26. Add 0.1ml of Mab, incubated and washed
Mouse anti C striatus IgM (From
NBFGRI) (Primary Antibody)
Goat anti mouse IgG cojugated with HRP,
diluted 1:1200 (Secondary Antibody)
Chromogen and substrate were added
(TMB+H2O2)
O.D read at 450 nm

27. Determination of LD50 of A. hydrophila
104 105 106 107 108 109 1010
10 F10 F 10 F10 F10 F 10 F10 F10 F
0.1 ml PBS
Mortality was recorded at the end of 6, 12, 24, 48.
72, 96, 120, 144, 168, 192, 219 and 240 h post
injection.
According to Reed and Muench (1938)
IM

28. Challenge with A. hydrophila
25 F25 F 25 F
ControlBF FC
0.5 ml @ 109 cfu, IM
On the 60th day post vaccination (dpv).
Relative percent survival (RPS) was calculated according to Amend and Johanson (1981)
RPS = [1-(% mortality of vaccinated group/ % mortality in control group] × 100

29. Statistical analysis
The results obtained were analysed statistically using Chi square test.

30. Results and Discussion
Antibody response in C. striatus
Antibody response to biofilm of A.
hydrophila have been described in
herbivore carps and omnivore catfish by
employing antibody agglutination titre
method .
(Azad et. al., 1997, 1999; Nayak et. al., 2004)
Antibody response

31. LD50 of A. hydrophila in C. striatus
Lio-po et al., (1998) were reported severe lesions with experimental infection of C. Striatus
with A. hydrophila at a dosage 109 cfu within 3-4 days
0
1
2
3
4
5
6
7
8
9
10
1 2 3 4 5 6 7 8 9 10
Mortality Days
10 5 10 6 10 7 10 8 10 9 10 10107
105 106 108 109 1010

32. Vaccine efficacy upon lethal challenge
0
10
20
30
40
50
60
70
80
90
100
61 62 63 64 65 66 67 68 69 70 71 72 73 74 75
Biofilm Free Cell Control
Percentagecumulativemortality
Number of days
Treatment No of fish
challenged
No of fish
survived
Mortality % Survival RPS chi-square
test for
survivalit
y
Biofilm 75 69 6 92 88 Z >1.96
Free Cell 75 37 38 49.3 29.6
Control 75 21 54 28 ------
88
29.6
0
10
20
30
40
50
60
70
80
90
100
BF FC Control
Relativepercentagesurvival
Tretments
Percentage cumulative mortality
Relative percent survival (RPS)
Table: Protection C. striatus upon challenge with A.
hydrophila at 109 cfu
8%
50.66%
72%
Higher protection was comparable with findings of Azad et al., (1999) in rohu
with RPS of 68.29 with BF and 53.66 with FC vaccine. Similar findings also
reported by Nayak et al., (2004) where 100 and 33.30 with BF and FC
respectively.
88
29.6

33. The biofilm of A. hydrophila is ideal for oral vaccination of
carnivore fish such as C. striatus. Overall the biofilm oral vaccine
model of A. hydrophila is effective in herbivore, omnivore and
carnivore fishes in the aquaculture system.
Conclusion of the 1st paper

34. Laboratory of Aquatic Pathobiology, University of Copenhagen, Denmark.
PAPER 2
Global Impact Factor- 2.056 (2014)
NAAS Score- 8.05 (2017)

35. Bernasconi et al., 2002 has reported a classical
secondary immune response in human
immune system in SCIENCE journal.
Rijikers et al., 1980 describe
secondary immune response in carp
by repeated injection vaccination

36. Objectives
This study reports investigated to check the secondary
immune response in rainbow trout by repeated immersion
vaccination.

37. Brief overview of the paper
6 treatment group having one Control group (Total 7)
Commercial immersion vaccines,
Serum collection before and after challenge for ELISA
Spleen tissue collection after post challenge for IHC
Statistical analysis
RBT does not raise a classical secondary response
Rearing 3 months, having single dose for 30 s/ monthly
Collection of egg and rearing for 6 months and grown up to 5 g

39. Fish collection , experimental groups and vaccination
Control (pure water) 3 X vaccinated (Nov. D. J.) 2 X vaccinated (Nov. Dec.)
1 X vaccinated (November)
Antigen- Y. ruckeri biotype 1 & 2
Vaccine dose- 5 x 109 cfu/ml
Vaccine types- Formalin inactivation
64 fish
1 X vaccinated (December)
1 X vaccinated (January) 2 X vaccinated (Dec. & Jan.)
Vaccination period- 30 seconds
Vaccine frequency- Once per month
7 Gr, 2 X
Rearing for 6 months
Fed once/day, 1% BW
MBW 5 g, Total 900 fish usedCollected in
April, 2015

40. Fish infection facility (exposure to live bacteria)
group
64 fish
24 fish
64 fish
24 fish
700 l
100 l
Challenge with Danish strain of Y. ruckeri O1 biotype 2
Caudal fin was punctured (10 perforation)
Dose of live bacteria- 7.7 x 108 CFU
Challenged fish
After 3 months
Sampling
After 3 months, before challenge
20 days (3 weeks) after the challenge
27 days after the challenge for IHC
Duration of challenge- 90 s
Experimental fish were monitored and
Moribund fish were removed and re-isolate the bacteria
(Dalsgaard and Madsen, 2000)
Stored at -80 0C

41. Development of ELISA
Coated with 100 µl/well of Y. ruckeri in bicarbonate
coating buffer
Overnight incubation with 40C
Serum samples added (100 µl, dilution 1:50, 1:100 in 2%
BSA) and incubated for 12 hr at 40C
Wash the plate with washing buffer, 3 x
RBT IgM
Y. ruckeri antigen
Washed 3 x with PBS

42. Incubated for 1 h on shaker (50 rpm and washed 3x
Mouse anti-salmonid Ig (from Germany) (Primary Antibody)
Rabbit anti mouse IgG (from Germany) conjugated with HRP,
diluted 1:1200 (Secondary Antibody)
Chromogen (TMB) plus substrate
Absorbance (O.D) read at 450 nm
Stopped the reaction adding 1 N HCL

43. Sections (4 µm)

44. Statistical analysis
One way Anova
Tukey’s post hoc
test

45. Results and Diss.
Group no. Treatment Group
ID
% Mortality
1 Control (No vaccination at all) (---) 16%
2 3 x vaccinated (Nov. Dec. Jan.) (+++) NM
3 2 x vaccinated (Nov. Dec.) (++-) NM
4 1 x vaccinated (December) (-+-) 8%
5 1 x vaccinated (November) (+--) 4%
6 1 x vaccinated (January) (--+) NM
7 2 x vaccinated (Dec. January) (-++) 6%

46. Antibody responses determined by ELISA

47. Immunohistochemistry
(4 months after first vaccination)
Negative control Vaccinated
IgM+ lymphocytes
Melano-
macrophages cell

48. Densities of cells/mm2)
Rainbow trout immunized by repeated immersion vaccination, which increases prolong protection
(Chettri et al., 2013, 2015)
However, no gradual elevation of spleen lymphocyte counts was found in fish receiving none, one or two
immersion vaccinations. This investigation therefore could not describe a secondary trout immune response
comparable to a classical mammalian secondary response which is differ from Bernasconi et al., 2002 and
Rijikers et al., 1980).

49. This might be due to the short immersion time (30 s) and dilution of the vaccine
(1:10) may limit the uptake of antigen when compared to injection vaccinated fish.

50. Repeated immersion immunization or exposure to live bacteria did
not show a classical secondary immune response as described in
mammals, only IgM+ lymphocytes in spleen was significantly
increased after three times immunizations.
Conclusion of the 2nd paper

51. Final conclusion
Disease prevention by vaccination is one of the milestones of
modern medicine and vaccine is essential to the continued
development of aquaculture around the world.

52. References
AMEND D.F. and JOHNSON, K.A., 1981. Current status and future needs of Vibrio anguillarum bacterin. Dev Biol Stand., 49: 403-17.
AZAD, I. S., SHANKAR, K. M. and MOHAN, C. V., 1997. Evaluation of an Aeromonas hydrophila biofilm oral vaccination of carps.
IM; Diseases in asian aquaculture III (T.W. Flegal and I.H.Macrae, eds.), Fish health section, AFS, Manila, Pp.181-186.
AZAD, I.S., SHANKAR, K.M. and MOHAN, C.V., 1999. Biofilm vaccine of Aeromonas hydrophila – standardization of dose and
duration for oral vaccination of carps. Fish and shellfish Immunol., 9:519-528.
BERNASCONI, N. L., TRAGGIAI, E., & LANZAVECCHIA, A. 2002. Maintenance of serological memory by polyclonal activation of
human memory B cells. Science, 298, 2199–2202.
BRUDESETH, B.E., WIULSRØD, R., FREDRIKSEN, B.N., LINDMO, K., LØKLING, K.E., BORDEVIK, M., STEINE, N., KLEVAN,
A. and
CHETTRI, J. K., DESHMUKH, S., HOLTEN-ANDERSEN, L., JAAFAR, R. M., DALSGAARD, I. and BUCHMANN, K. 2013.
Comparative evaluation of administration methods for a vaccine protecting rainbow trout against Yersinia ruckeri O1 biotype 2 infections.
Vet. Immunol. Immunopathol., 154: 42–47.
CHETTRI, J. K., JAAFAR, R. M., SKOV, J., KANIA, P. W., DALSGAARD, I. and BUCHMANN, K., 2015. Booster immersion
vaccination using diluted Yersinia ruckeri bacterin confers protection against ERM in rainbow trout. Aquaculture, 440: 1–5.
DALSGAARD, I. and MADSEN, L., 2000. Bacterial pathogens in rainbow trout, Oncorhynchus mykiss (Walbaum), reared at Danish
freshwater farms. Journal of Fish Diseases, 23: 199–209.

53. DUFF, D. C. B., 1942. The oral immunization of trout against Bacterium salmonicida. J Immunol., 44:87-94.
ELLIS, A.E., 1988. Ontogeny of the immune system in teleost fish. In: Ellis AE, editor. Fish Vaccination. London, UK: Academic Press; pp. 1–50.
FAO, 2016. The State of World Fisheries and Aquaculture. Food and Agriculture Organization of the United Nations, Rome, ISBN 92-5-105177-1.
KARUNASAGAR I, ROSALIND G. and KARUNASAGAR, I., 1991. Immunological response of the Indian major carps to Aeromonas hydrophila
vaccine. J Fish Diseases 14:413–417
LIO-PO G.D., ALBRIGHT, L. J., MICHEL, C. and LEANO, E.M., 1998. Experimental induction of lesions in snakeheads (Ophicephalus striatus)
and catfish (Clarias batrachus) with Aeromonas hydrophila, Aquaspirillum sp., Pseudomonas sp. and Streptococcus sp. J Applied Ichthyol., 14: 75-
79.
NAYAK, D.K., ASHA, A., SHANKAR, K.M. and MOHAN, C.V., 2004. Evaluation of biofilm of Aeromonas hydrophila for oral vaccination of
Clarias batrachus – a carnivore model. Fish Shellfish Immunol., 16: 613–19.
REED, L. J. and MUENCH, H., 1938. A simple method of estimating 50% end points. Am. J. Hyg. 27: 493-497.
SALK, J. and SALK, D., 1977. Control of influenza and poliomyelitis with killed virus vaccines. Science. 834-47
SNIESZKO, S.F., 1970. Immunization of fishes: a review. J Wildl Dis, 6:24-30.
SOOD, N., CHAUDHARY D. K., RATHORE G., SINGH A. and LAKRA, W.S., 2011. Monoclonal antibodies to snakehead, Channa striata
immunoglobulins: Detection and quantification of immunoglobulin-positive cells in blood and lymphoid organs. Fish Shellfish Immunol., 30(2): 569-
575.
YIN, Z., LAM, T. J. and SIN, Y. M., 1997. Cytokine-mediated antimicrobial immune response of catfish, Clarias gariepinus, as a defence against
Aeromonas hydrophila. Fish Shellfish Immunol., 7(2): 93–104.