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Asian vaccine protects grouper juveniles against vibriosis

Asian vaccine protects grouper juveniles against vibriosis

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Published by aquaculturehealth
Vaccinating juvenile fish by injection has given a high level of protection against bacteria causing vibriosis, in a study involving cultured orange-spotted grouper in Thailand. The bivalent Vibrio vaccine was administered intraperitoneally to each fish. Although this method of administration is difficult to apply in routine farm management, the evidence from the study strongly suggests it should be a good prophylactic measure against mortalities due to Vibrio pathogens.
Vaccinating juvenile fish by injection has given a high level of protection against bacteria causing vibriosis, in a study involving cultured orange-spotted grouper in Thailand. The bivalent Vibrio vaccine was administered intraperitoneally to each fish. Although this method of administration is difficult to apply in routine farm management, the evidence from the study strongly suggests it should be a good prophylactic measure against mortalities due to Vibrio pathogens.

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Published by: aquaculturehealth on Jun 25, 2010
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Asian vaccine protects grouper juveniles against vibriosis
Published by
Disease control & management in the global aquaculture sector
Issue 1 - June 2010
 AquacultureHealth & DiseaseManagement
 
V
accinating juvenile fish byinjection has given a highlevel of protection againstbacteria causing vibriosis, ina study involving culturedorange-spotted grouper in Thailand. Thebivalent Vibrio vaccine was administeredintraperitoneally to each fish. Althoughthis method of administration is difficultto apply in routine farm management,the evidence from the study stronglysuggests it should be a good prophylacticmeasure against mortalities due to Vibriopathogens.The orange-spotted grouperEpinephelus coioides has formed part of a diversification of culture in Thailand overrecent years (de Silva and Phillips, 2007)that illustrates the expanding diversity of marine fish species being farmed in Asianaquaculture. Across the region, however,vibriosis has caused catastrophic eco-nomic losses in higher value species suchas grouper as well as the snapper andpompano.
A most common disease
Vibriosis is the most common diseaseaffecting the culture of marine finfish inthe Asian region and many epizootics havebeen attributedin particularto Vibriocholerae.Conventionaltreatmentsregionally useantibiotics.Unfortunately,these are oftenapplied incor-rectly. Therefore they fail to control thedisease and they can lead to the spread of bacterial resistance.Many other problems areassociated with the use of antibiotics. For example, theefficiency of oral delivery isquestionable because sick fishoften do not eat. Antibioticsthat are water soluble mayleach from feed. The efficacyof using some antibiotics suchas oxytetracycline is highlyreduced where they formcomplexes with calcium andmagnesium ions in seawateror hard water. Technically, of course, it should always be rememberedthat antibiotics cannot work unless theyare delivered to the correct target organor tissue at the appropriate concentration.Also, by their very nature, they act mainlyon bacterial pathogens; they have no directeffect on viruses and other pathogens.Vaccines, by contrast, can act againstboth bacteria and viruses and there iseven experimental evidence that they canbe active against parasitic infections. Butit is also important to remember anotherdifference in the fact that antibiotics curewhereas vaccines prevent. So while theapplication of an antibiotic is a curativemeasure or therapy against an existinginfection, a vaccine is applied preventivelyto prepare the immune system before aspecific challenge. What is more, the protec-tion secured by vaccinating normally lastslonger than that from applying antibiotics.
Vaccination reducesantibiotic useage
Data from Norway show that vaccina-tion of fish has greatly reduced the use of antibiotics in Norwegian salmon produc-tion. Over a period of two decades whilesalmonid production increased by a factorof 20 times, the weight of antibiotics usedeach year dropped from a high of over50 tonnes in 1984 to less than 2 tonnesin 2004. Additionally, the situation wherethe only fish vaccines available were forsalmonids is now changing. New vaccinesare being registered in Asia for Asian spe-cies, such as yellowtail in Japan, grass carp inChina and a recently launched commercialvaccine for use in Asian sea bass, tilapia andother species in some south-east Asiancountries (Komar et al., 2005).Various studies have been carried outinto the efficacy of various Vibrio vaccinetypes on the survival of different grouperspecies. In the present study, juvenilegrouper were injected with a bivalent Vibriovaccine prepared (NovaQsol Holdings Inc.,Philippines) from two virulent strains of V. harveyi and V. parahaemolyticus. Thesestrains had been isolated from diseased,moribund fish located in a farm near Phuket,Thailand.The vaccine’s safety was establishedby testing on a total of 240 juvenileorange-spotted grouper which had beenseparated into four groups of 60 fisheach. Sub-groups of 20 fish within eachgroup either were injected with 0.05 mlor 0.10 ml of vaccine, or given a 0.10 mlsaline injection as a positive control, orleft uninjected. Immunised and controlfish were then held for 21 days with dailymonitoring for mortality.
Monitoring water quality
As with all 840 fish involved in thestudy, these grouperhad been purchasedfrom a private farmin Thailand when theywere approximately5.75g in weight andapproximately 4.31 cmin length. There werekept in plastic aquariafor an acclimatizationperiod of 14 days andthe sacrifice of twofish to verify that theywere free of Vibrio.Throughout the study, half of the water ineach tank was exchanged every alternateday. A commercial test kit (AQUA-VBC,Thailand) was used to monitor water qual-ity parameters of pH, hardness, ammonia,nitrite and alkalinity. Salinity was measuredeach week with a refractometer.The experimental bacterial challengefor the vaccine assessment consisted of Vibrio cholerae that had been isolatedfrom moribund sea bass and cultured at theVeterinary Medical Aquatic Animal ResearchCentre of the Faculty of Veterinary Scienceat Chulalongkorn University, Thailand.Bacteria were identified using API® 20E(BioMerieux, Madrid, Spain) and the APIprofiles compared with the API database(Apilab Plus, version 3.3.3.; BioMerieux).To establish virulence, doses rangingfrom 103 to 1010 CFU of bacteria per fishwere injected intra-peritoneally into 540orange-spotted juveniles. Each dose had3 replicates with 20 fish in each replicate.Control groups were challenged with sterile
This report from Thailand describes a trial in which an injectable vaccine successfully protected orange-spotted grouper from an experimental challenge with Vibrio pathogens
Asian vaccine protects grouper juveniles against vibriosis
by Jhn S Clark, Jirasak Tangtrngpirs and Nantarika Chans
Figure 1. Determination of LD50 and LD100 for V. chol-erae in juvenile spotted grouper.Septicaemia of hepatic tissue
Table 1:
Means of water quality parameters.ParametersMean level and S.D.ParametersMean level and S.D.Temperature27.5±0.5°CAlkalinity60±19.27 ppmpH7.66±0.23Ammonia0.88±0.77 ppmSalinity34pptNitrite1.56±1.55 ppmHardness>1000 ppm23.15±18.61bc77.59
Vibriosis |
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Health & Disease Management | Vibriosis
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fry, quarantine measures, optimized feedingand good husbandry techniques. Monitoringdiseases by surveillance and reporting isessential, as is good sanitation and anemphasis on being responsible in the useof chemicals and antibiotics when diseasesoccur.There will certainly be a role for vac-cination as the overall emphasis changesto preventing diseases rather than treatingor curing them. Vaccination on its own isnot sufficient on its own to guarantee highsurvival and profitability (see box), but it isone of the tools to employ in good healthmanagement strategies.production, development and sustainableexpansion of marine fish aquaculture inthe Asian region. Most Asian aquacultureis operated on a small scale. Added to theother factors mentioned and the diversityof organisms in tropical waters, this contrib-utes to a truly challenging disease situationin which pathogens have various opportuni-ties for entry points. Only now are movesbeing made to correct the failure to collect,report and analyse epidemiological data inthe event of a disease outbreak.On a more positive note, more attentionhas been given in past few years to the iden-tification of aetiological agents involved infish disease epidemics. The following studiesgive a solid indication of the diversity of major pathogens affecting the fish farmingindustry in Asia (Bondad-Reantaso et al.,2005; Komar et al., 2005; Labrie et al., 2005;Leong et al., 2005, 2006 and Tan et al., 2003).Meanwhile, significant progress has beenmade in the field of vaccine research anddevelopment (Grisez and Tan, 2005).But as Asian aquaculture continues togrow, inevitably its problems with diseasewill worsen unless key steps are taken. Newdisease control concepts must be imple-mented. This includes the use of healthywere significantly lower than for all infectedgroups. Although not significantly differentfrom the 0.10 ml vaccinated fish for per-centage mortality, the 0.05 ml vaccine groupregistered a lower RPS of 45.76%.
Higher survival andlower mortality
Clearly, therefore, injecting 0.10 ml of the bivalent vaccine gave a higher rela-tive survival and the lowest mortality rateof any group in the test. The test alsodemonstrated that the European model of using vaccination as a primary prophylactictool could be applied successfully in Asia toreduce losses in spotted grouper juvenilesfrom a Vibrio infection.This points to a new direction in Asiangrouper culture for health management. Sofar, the development of effective strategies inAsia for disease prevention and control hasbeen hampered by a lack of data on the widevariety of disease-causing agents present andthe immune systems of Asian fish species.Technical support in such matters as diseasediagnosis is often lacking at farm level.There is also a stark contrast betweenthe Asian situation and modern salmonidaquaculture in countries such as Norway.Intensified salmon production has broughta separation between the hatchery sitesproducing fry and those for grow-out,together with optimized feed strategies andvirtually disease-free fingerlings. However,the segregation in year classes that isobligatory for salmon in Europe does notyet exist in Asia.Asian farms also commonly producedifferent species of fish at the same site,they make widespread use of trash fish asfeed and their fry are often wild-caught orderived from wild-caught brood stock. Withfarm licensing and zoning policies virtuallynon-existent in most Asian countries, thespread of diseases is promoted by havingtoo many fish and too many farms in aconcentrated area.It explains why the salmon farms of Norway or Chile consider asurvival rate of lower than95% as a sign of a diseaseoutbreak, whereas a survivalrate of 50% at fish farms inAsia is often considered tobe acceptable.
The biggestconstraint toprocution
Disease undoubtedlyrepresents one of the big-gest constraints to thelated by the Reed and Muench procedure asthe basis for deciding the optimal challengedose to be used during testing of vaccineefficacy.Figure 1 illustrates the clear doseresponse curve found from the virulenceassays. The LD100 dose (killing 100% of fish within three days) for this particularisolate of Vibrio cholerae was 1x1010 CFU/ml, with the LD50 established as 1x108CFU/ml.At 21 days after immunization, the vacci-nated and control fish were challenged withthe LD100 as a 0.10 ml injection adminis-tered intraperitoneally while the positivecontrols received 0.10 ml of sterile saline.Monitored for three days post-challenge,there was a 100% mortality rate for the fishin the positive control group. All dead fish inthe challenge experiment were confirmedby re-culture and re-identification using API20E as positive for Vibrio cholerae infection.Results of the efficacy test in Table 1confirmed that giving 0.10 ml of the vaccineprovided the highest rate of protectionagainst Vibrio cholerae. This vaccination gaverelative percentage survival (RPS) values of up to 77.59% compared with the relevantcontrols. Its percentage mortality rates0.9 % (v/v) saline. The aim was to findpathogen doses that would kill 50% and100% of injected fish within three days, sothat the mean lethal dose could be calcu-
Authors
Dr John S Clark
obtained his Ph.D.in micro-encapsulation at Heriot-WattUniversity in Edinburgh, Scotland in 1983.He currently works on formulation of anti-parasitics, vaccines, nutritional/health products for a variety of clients.Email: aquavax2003@yahoo.co.uk 
Dr Nantarika Chansue
is theAssociate Professor and Directorof the Veterinary Medicine AquaticAnimal Research Centre (VMAARC) atChulalongkorn University in Bangkok.She specializes in disease identifica-tion and control methods for shrimp,marine and freshwater fish, turtles andaquatic mammals. Aquatic immunoto-xicology has been her major researchinterest for some 20 years.
Professor Jirasak Tangtrongpiros
 is the head of the Veterinary MedicineAquatic Animal Research Centre(VMAARC) at ChulalongkornUniversity in Bangkok, Thailand. Hespecialises in aquatic animal diseaseand his primary interest is in methodsfor disease prevention and control.
Acknowledgements
The authors would like to convey their thanks to all the staff of VMAARC inChulalongkorn University, BangkokThailand, and to the supporting staff of the Research Institute at Rayong, Rayong Province, Thailand, and finally to Drs. Gee,Na, Lin and Jim for their endless patience.References are available on request.
Measures to sustain asuccessful aquaculture
(Tan et al., 2006)
Recommended
1.
Use healthy (not necessarilycheap) fry
2.
Quarantine incoming animals
3.
Use formulated pelleted feed
4.
Grade fish periodically
5.
Monitor water quality
6.
Record diseases and feeding
7.
Observe withdrawal period of drugs
8.
Remove dead fish at least once aday
9.
Clean and disinfect equipment
10.
Vaccination if available
NOT recommended
1.
Place your farm too close toothers
2.
Have several species in one farm
3.
Use fingerlings from unknownsources
4.
Overstock (to overcome lowsurvival)
5.
Use trash fish
6.
Overfeed
7.
Use drugs without diagnosis
8.
Leave or throw dead fish in thewater
9.
Restock fish without cleaning thecages
10.
Ignore diseases until heavy mor-tality occurs
Table 2:
Percentage mortality of juvenile orange-spotted grouper,non-vaccinated and vaccinated by two intraperitoneally injecteddoses of a Vibrio vaccine and relative percentage survival (RPS)after challenge with Vibrio cholerae.GroupMortality*RPSNCControl, no infection2.63±3.72c97.44PCControl, normal saline IP100.00±0.00a0.00T10.05 ml. vaccine IP54.04±31.73b45.76T20.10 ml. vaccine IP23.15±18.61bc77.59
Data was subjected to two way analysis of variance using an SPSSprogram. Significant differences were determined at P<0.05.
Acute peritonitis
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