This report presents the initial results of studies on the isolation and identification of the causative agent as well as possible immunization of the estuary groupers. It is hoped that by a vaccination programme, fish could be made immune to such disease. Results indicate: (1) Vibrio parahaemolyticus is pathogenic to estuary grouper (2) that the vaccines did not seem to protect the fish against the vibrio when challenged one week after vaccination, but that this was due to slow antibody production of the fish as salmonids challenged about 30-35 days after vaccination reported good protection against Vibrio anguillarum and other bacterial diseases. Experiments are now in progress to challenge the vaccinated groupers at a later stage

Chua, Thia-Eng

Ong, Bessie

Wong, See-Yong

English

Southeast Asian Fisheries Development Center, Aquaculture Department Institutional Repository (SEAFDEC/AQD Institutional Repository )

SEAFDEC/AQD Institutional Repository (SAIR)This document is downloaded at: 2013-07-02 05:37:48 CSTTitle Isolation, identification of causative agent of "red boil disease" in grouper(Epinephelus salmoides) and its possible control by vaccination.Author(s) Wong, See-Yong; Ong, Bessie; Chua, Thia-EngCitationWong, S.-Y., Ong, B., & Chua, T.-E. (1979). Isolation, identification ofcausative agent of "red boil disease" in grouper (Epinephelus salmoides)and its possible control by vaccination. In Proceedings of the InternationalWorkshop on Pen Cage Culture of Fish, 11-12 February 1979, Tigbauan,Iloilo, Philippines (pp. 81-87). Tigbauan, Iloilo, Philippines: AquacultureDepartment, Southeast Asian Fisheries Development Center; InternationalDevelopment Research Centre.Issue Date 1979URL http://hdl.handle.net/10862/1526http://repository.seafdec.org.phIsolation, identification of Causative Agent of 'Red Boil Disease' in Grouper (Epinephelus salmoides) and its Possible Control by VaccinationWong See-Yong, Bessie Ong and Chua Thia-Eng School of Biological Sciences University Sains MalaysiaINTRODUCTIONStudies of fish diseases have been sporadic and inadequate until recently when fish culture, sport fishing and aquarium fish industry have started to become more popular. Among the most studied diseases of fish are those caused by bacteria (Ander­son, 1974). Because of high ineffectivity and short generation time, bacterial pathogens produce severe effects on the host. Sinderman (1970) reported the most common bacteria pathogenic to marine fishes belong to species of Pseudomonas, Vibrio and Mycobacterium,The disease caused by vibrios is considered to be the most serious and affects both wild and cul­tured fishes. The causative agent of the 'red boil disease' is believed to be Vibrio anguillarum (Sinder­man, 1970; Smith, 1961; Akazawa, 1968).In Malaysia, Chua and Teng (1966) reported one serious disease in cultured groupers in floating fishpens in the Straits of Penang resembling the 'red boil disease' caused by Vibrio. The mortality of the fish has been reported to be as high as 90 per­cent. This report presents the initial results of stu­dies on the isolation and identification of the cau­sative agent as well as possible immunization of the estuary groupers. It is hoped that by a vaccination programme, fish could be made immune to such disease.MATERIALS AND METHODSIsolation of BacteriaA diseased grouper showing sluggish move­ment and external skin lesions was used. The fish was first anesthesized with 50 ppm tricanemethane- sulfonate (MS-222) and swabs from skin lesion, liver, kidney, and blood were then plated onto two non-selective media -- Trypticase Soy Agar (TSA) and seawater agar. Colonies from the two mediawere further plated onto three selective media: Thiosulfate Bile Salt Sucrose agar (TCBS). Rimler- Shotts agar (RS) and Cetrimide agar ((CA). All plates were incubated at room temperature (28°C).Presumptive Identification of PathogensThe colony and growth characteristics of the bacteria isolates on the three selective media were used in the presumptive identification of the patho­gens. TCBS agar selects for Vibrio species; Cetrimide selects for pseudomonads, and RS selects for aero- monads, some pseudomonads and coliforms.Green colony on TCBS agar indicates the pre­sence of Vibrio parahaemolyticus whereas yellow colonies indicate the presence of other vibrios (Saka- zaki, 1963; Kobayashi et  al . , 1963). Cetrimide agar inhibits most bacteria except pseudomonads (Shotts, 1975). Yellow colonies on RS agar indicate the presence of pathogens Aeromonas hydrophila (Shotts and Rimler, 1973).Identification and Confirmation of Vibrio spp.The growth of organism on TCBS agar was presumptively identified as Vibrio. The green colo­nies on TCBS are also presumptively identified as Vibrio parahaemolyticus and yellow colonies as other vibrios.PathogenicityPathogenicity was investigated using Kochs' hypothesis. Experiment was done on the estuary grouper, Epinephelus salmoides and two other spe­cies of fishes, Tilapia mossambica and gold fish (Carassius auratus). Two separate isolates from TCBS agar were used for the test.The test fish were starved for 24 hours, anes- thesized with 50 ppm MS-222 and given intraperi- toneal injection of active 24-hour bacteria cultureTable 1. Growth of isolates on three selective mediaGR30 GR32 GR34 GR35 GR36 GR37TCBS green yellow green green green yellowRS — — — — — —Cetrimide — — — — — —suspended in 0.1 ml nutrient broth. The control were injected with 0.1 ml nutrient broth only.The treated fish were then observed daily over a period of one week for mortality and development of symptoms. Re-isolation of bacteria from perito­neal fluid, lesions (if present), kidney and blood of the test fish were made on TCBS agar. Re-isolated bacteria were further identified accordingly.Immunization of fishBacteria vaccine was prepared from an isolate according to methods described by Fryer et al., (1976) and Campbell et al., (1970) with some modi­fications. Suspension of 18-24 hours culture of V. parahaemolyticus in sterile saline was either subjec­ted to 90°C in a water bath for 1 hour or treated with 1 percent formalin-saline solution for 1 hour at room temperature. The heat-killed vaccine or for- malinized vaccine were washed twice with sterile saline at 23,000 g for 20 minutes at 4°C. The vac­cines were stored at 4°C at a concentration of 1-2 x 109 cells/ml until used. The sterility of the vaccines were tested on TCBS plates and nutrient broth tubes. Absence of growth after 48 hours incubation was taken as an indication of sterility of the vaccines.Groupers of approximately the same size were used for the experiments. The fish were anesthesized with 50 ppm MS-222 and injected with 0.1 ml vac­cine (equivalent to 1 x 109 cells/ml) intraperitoneal-ly or intramuscularly. Heat-killed vaccine, formalin- killed vaccine, heat-killed vaccine with complete Freund's adjuvant were injected i.p. to 3 different groups of fish. One heat-killed vaccine was also in­jected intramuscularly. Booster dose was done by injecting i.p. with 0.5 x 109 cells/ml administered in 2 injections over a one week period. Controls were uninjected.One week after immunization the fish were challenged with virulent cells from the isolate. The fish was observed for mortality over a one week period.RESULTS AND DISCUSSION Isolation of bacteria from various tissuesFour yellow isolates were obtained from TSA agar plates which were designated as GR30, GR31, GR32 and GR33. GR30 was isolated from liver; GR31 from kidney; GR32 from blood from the heart and GR33 from the lesion, blood, liver and kidney respectively. Two cultures (GR31 and GR33) were subsequently lost during the transfer. The results indicated that the tissues of the test fish were infected.Presumptive identification of the isolatesThe six isolates were placed on three selective media, namely TCBS, RS and Cetrimide agar. The results are shown in Table 1.Gold Fish Tilapia GrouperGR35 (V. parahaemolyticus) 4/4* 4/4 3/4GR32 (V. alginolyticus) 0/4 0/4 0/4Control (NB only) 0/4 0/4 0/4The results indicate that ail the isolates were vibrios. The green colonies were tentatively identi­fied as Vibrio parahaemolyticus due to their large, green, non-sucrose fermenting colonies on the TCBS agar. The yellow colonies were tentatively identified as other vibrios due to their yellow sucrose fermen­ting colonies on the TCBS agar. The yellow sucrose fermenting colonies may be V. cholerae, V. alginoly- ticus or others.. Since all the cultures did not grow on the RS and Cetrimide agars, it was assumed that the isolates were not pseudomonads, aeromonads, or other coliform bacteria.Physiological and biochemical reactions of vibriosFurther identification of the vibrios was done on two isolates were identified and confirmed as V. parahaemolyticus (GR35) and V. alginolyticus (GR32) using the criteria described by Sakazaki(1973).Pathogenicity of bacteriaTable 2 shows the results of the pathogenicity test of V. parahaemolyticus (GR35) and V. alginoly­ticus (GR32) on three species o f fish.All fish injected appeared to succumb to iso­late GR35 but not to isolate GR32. Diseased fish were darker in colour and might develop hemorrha­gic abscess if  they survived long enough. In most cases, disease course was so rapid that external symptoms were not present. From this experi­ment, it may be concluded that the V. parahae­molyticus is pathogenic to actuary grouper and V. alginolyticus is relatively harmless.The re-isolation of the pathogens on TCBS agar from diseased fish indicated the presence of both green and yellow colonies characteristic of GR32 and green colony designated GR35 were identical with GR32 and GR35, respectively as indicated in Table 2. This suggests that both types of vibrios were the normal inhabitants of the fish. The outbreak of the disease was probably due to invasion of the V. parahaemolyticus once the body defence declined.The presence of V. parahaemolyticus and V. alginolyticus was also confirmed by analysis of the water samples taken from the culture station.The confinement of grouper in the cage culture condition could cause serious stress to the fish (Chua and Egen, 1977) and this probably reduces fish resistance to the vibrio infection. It is not sur­prising to find V. parahemolyticus and other vibrios in the water samples since the station is polluted with human sewage and vibrios are known to survive very well in coastal water where organic matters are abundant (Kaneko and Colwell, 1973).Effectiveness of vaccinesTable 3 shows the protective nature of the various vaccines in grouper. From the result, all the vaccines did not seem to protect the fish against the vibrio when challenged one week after intraperito- neal or intramuscular administration o f the vaccines. Both heat-killed or formalin-killed vaccines did not seem to protect the fish from the challenge.Table 2. Mortality in experimental fishes*Total dead injected after 7 days.Table 3. Type of vaccines against mortalityType of vaccine Route of vaccination MortalityHeat-killed i.p. 6/10Heat-killed i.m. 7/10Heat-killed booster i.p. 6/10Heat-killed, Freund's adjuvant i.p. 7/10Formalin-killed i.p. 7/10Control Uninjected 6/10All the groupers whether immunized or not showed about 60-70 percent mortality when chal­lenged with the virulent bacteria. The result appears to be somewhat misleading and might indicate the lack of protection of various vaccines. The lack of protection is probably due to the early challenge of the virulent bacteria before the development of im­munity in the fish has occurred. The fish are cold­blooded animals and the time for antibody produc­tion may need a longer period to develop.The prediction appears to be somewhat true when the antibody titer of the fish was examined. Figures 1 and 2 indicate the level of antibody titers in the various groups of fish immunized with heat- killed or formalin-killed vaccines. The antibody titers were very low (i.e. around 2) when examined one week after vaccination. However, the antibody titers had increased to three times two weeks after immunization. Further work has to be done to cla­rify this aspect. The challenge of the fish indeed has been done in salmonids about 30-50 days after ad­ministration of vaccines and are reported to be very effective in the protection of the fish against Vibrio anguillarum and other bacterial diseases (Antipa and Amend, 1976; Fryer et al ., 1976). Experiment is now in progress to  challenge the fish at a later stage and also to follow the antibody titer for a longer period. From the preliminary result as indicated inthe figures, booster injection and injection of vac­cine with Freund's adjuvant produced higher titer of antibody within a shorter period of time.The data obtained show a relatively high V. parahaemolyticus count on the water samples ob­tained from the cultured station. The number of V. parahaemolyticus counts was comparable to that at Tokyo Bay. This high count was not surprising as there were direct discharge of raw sewage into the water around the grouper farm. There are evidences to prove that rearing of estuary grouper in relatively clean water on the other part of the island has cut down tremendously the incidence of this disease (Chua and Teng, 1978).Although the Straits of Penang is heavily pol­luted with raw sewage, it is an ideal culture site as it  is protected from strong currents and tidal waves. If the fish can be immuned against vibriosis, then the large water area in the straits can be utilized for fish production. Although antibiotics and sulfa drugs have been used to combat vibriosis, they also give rise to  many undesirable side effects (Evelyn, 1977). Vaccination appears to be a more effective and eco­nomical method. The present observation of the antibody formation after vaccination is encouraging as there have been evidence of success of immuniza­tion on other marine fish against vibriosis caused by V. anguillarum.Fig. 1. Antibody Response of Groupers 1 week after Vaccination.Fig. 2. Antibody REsponse of Groupers 2 Weeks after Vaccination. Note the Overall Increase in Agglutinating Antibiotics from that of 1 Week after Vaccination.REFERENCESAkazawa, H. (1960). Bacterial disease of marine fishes. In Symposium of bacterial fish disease. Bulletin of Jap. Sec. of Sc. Fish. 34(3): 271 - 272.Anderson, D.P. (1974). Fish immunology. Ed. Snieszko S.F. and H.R. Axelrod. TFH Publi­cation Inc. Ltd., New Jersey.Anderson, J.I.W. and D.A. Conray (1970). Vibrio disease in marine fishes and shellfishes. Am. Fish. Sec. Spec. Publ. 5. Washington, D.C.Antipa, R. and D.F. Amend (1976). immunization of Pacific salmon: comparison o f intraperito- neal injection and hyperesmotic infiltration of Vibrio anguillarum and Aeromonas salmoni- cide bacteria. J. Fish. Res. Board Can: 203- 208.Campbell, D.H., J.S. Garvey, N.E. Cremer and D.H. Suesdorfo (1970). Methods in Immunology. W.A. Benjamin Inc. N.Y. p. 129-130.Chua, T.E. and S.K. Teng (1977). Floating fishpens for rearing fishes in coastal waters, reservoirs and mining pools in Malaysia. Fish. Bull. No. 20. 36 pp. Ministry of Agriculture, Malaysia.Chua, T.E. and S.K. Teng (1978). Economic produc­tion of estuary grouper, Epinephelus salmoi­des Maxwell, reared in floating net-cages. Paper presented to the International Founda­tion for Science Regional Aquaculture Work­shop, Penang.Cisar, J.O. and J.L. Fryer (1969). An epizcotic of vibriosis in chinook salmon. Bull. Wildl. Dis. Assoc. 5:73-76.Evelyn, T.P.T. (1971). First records of vibriosis in pacific salmon culture in Canada and Taxo­nomic status of the responsible bacterium, Vibrio anguillarum. J. Fish. Res. Board Can. 28:517-525.Evelyn, T.P.T. (1977). Immunization of salmonids. In International Symposium on diseases of cul­tured salmonids.Fryer, J.L., A.J.S. Nelson and R.S. Garrison (1972). Vibriosis in fish. Prog. Fish Food Sci. 5:129- 133.Fryer, J.L., J.D. Rohovec, G.L. Tebbit, J.S. Mc- Michael and K.S. Pilcher (1976). Vaccina­tion for control of infectious diseases in pacific salmon. Fish pathology 10:155-164.Horie, S., K. Saheki and M. Okuzumi (1967). Quan­titative enumeration of Vibrio parahaemoly­ticus in ocean and estuarine waters. Bull. Jap. Sec. Sci. Fish. 33:126-130 ( In Japanese).Hugh, R. and R. Sukazaki (1972). Minimal number of characters for the identification of Vibrio parahaemolyticus. Pub. 30, 133.Kaneko, T. and R.R. Colwell (1973). Ecology of Vibrio parahaemolyticus in Chesapeake Bay. J. of Bact. 113:24-32.Kobayashi, T., S. Enormoto, R. Sakazaki and S. Kuwahara (1963). A new selective medium of Pathogenic vibrios: T.C.B.S. Agar (Modified Narkanishi's Agar) Jap. J. Bacteriol. 18, 10- 11, 387-391.Krantz, G.E., R.R. Colwell and E. Lovelace (1969). Vibrio parahaemolyticus from the blue crab (Callinectes sapidus in Chesapeake Bay. Science 164, 1286-1287.Kusuda, R. (1966). Study of the ulcer disease of marine fishes. Proc. First U.S. - Japan Joint Conf. Microbiol. 1966, 13 pp.Kashiro, T. (1964). The distribution of Vibrio para­haemolyticus in the natural environment. p. 263-277. In T. F jun io  and H. Fukumi (ed.), Vibrio parahaemolyticus. Nayashoten, Tokyo, Japan (In Japanese).Rucker, R.R., J. Earp and E.J. Ordal (1953). Infec­tious disease of pacific salmon. Trans. Am. Fish. Soc. 83:297-312.Rucker, R.R. (1959). Vibrio infections among marine and fresh water fish. Prog. Fish Cult, 21:22-25.Sakazaki, R. (1963). Vibrio parahaemolyticus Tokyo, Osseido.Sakazaki, R. (1973). 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A new selective medium of Pathogenic vibrios:

An epizcotic of vibriosis in chinook salmon.

Bacterial disease of marine fishes. In Symposium of bacterial fish disease.

Bacterial diseases of fishes: diagnostic procedures for gram-negative pathogens.

Cont rol of contamination with Vibrio parahaemolyticus in seafoods and iso lation and identification of the Vibrio in the microbiological society of food,

Diseases of Pacific salmon, their prevention and treatment.

Ecology of Vibrio parahaemolyticus in Chesapeake Bay.

Economic produc tion of estuary grouper, Epinephelus salmoi des Maxwell, reared in floating net-cages. Paper presented to the International Founda tion for Science Regional Aquaculture Work

First records of vibriosis in pacific salmon culture in Canada and Taxo nomic status of the responsible bacterium, Vibrio anguillarum.

Floating fishpens for rearing fishes in coastal waters, reservoirs and mining pools in

immunization of Pacific salmon: comparison of intraperitoneal injection and hyperesmotic infiltration of Vibrio anguillarum and Aeromonas salmonicide bacteria.

Immunization of salmonids.

Infec tious disease of pacific salmon.

Medium for the isolation of Aeromonas hydrophila. Ap plied Microbiology.

Minimal number of characters for the identification of Vibrio parahaemolyticus.

Principal diseases of marine fish and shellfish.

Quan titative enumeration of Vibrio parahaemoly ticus in ocean and estuarine waters.

Study of the ulcer disease of marine fishes.

The distribution of Vibrio para haemolyticus in the natural environment.

Vaccina tion for control of infectious diseases in pacific salmon.

Vibrio disease in marine fishes and shellfishes.

Vibrio infections among marine and fresh water fish.

Vibrio parahaemolyticus from the blue crab (Callinectes sapidus in Chesapeake Bay.

Isolation, identification of causative agent of "red boil disease" in grouper (Epinephelus salmoides) and its possible control by vaccination

Isolation, identification of causative agent of "red boil disease" in grouper (Epinephelus salmoides) and its possible control by vaccination

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