A Pseudomonas associated with disease in cultured rabbitfish Siganus rivulatus in the Red Sea

Experiments were carried out to study the cause of a disease outbreak among rabbitfish Sjganus rivulatus in the Saudi Arabian mariculture facility in the Red Sea and to investigate means of protecting fish against the disease. The causal agent of the disease proved to be a bacterium that was identified as Pseudomonas putrefaciens. When the bacterium was injected into healthy fish, it resulted in high mortality and produced the same clinical signs as those observed during the disease outbreak. A bacterin prepared by formalin killing was used to vaccinate fish. Fish vaccinated once by intraperitoneal Injection suffered 17 % less mortality than control fish when challenged with the hon~ologous bacterlum. Fish vaccinated twice by the same method suffered 40 % less mortality than control fish. Fish were also vaccinated by direct immersion in the bacterin but when these fish and their unvaccinated counterparts were challenged by dipping into a suspension of the homologous bacterium they did not show any clinical signs nor did any mortality occur The disease was thought to be stress-related. MATERIALS AND METHODS INTRODUCTION This study was undertaken to identify the responsible bacterium and to investigate the possibility of Rabbitfish Siganus rivulatus are indigenous to the immunizing fish against it. Indo-Pacific and now occur in the Red Sea and Eastern Mediterranean, where they are sought by fishermen and anglers because they are readily accepted by consumers. They are herbivorous but grow well on a low protein diet (Popper & Gunderman 1975) and tolerate Fish serving as the source of the pathogen were culture conditions (Thibaiti et al. 1984). Because of obtained as diseased specimens from the manculture these characteristics, the Saudi Arabian Ministry of facility. The chief clinical signs were discoloration, Agriculture and Water Resources has selected rabbithemorrhagic necrosis on the body and mouth, frayed fish for initiation of a maric.ulture program in the Red fins, and exophthalmia. All specimens were moribund Sea. and were extremely lethargic. During spring 1985 a disease outbreak occurred and Bacterial isolates were obtained from liver, ludney, resulted in a high mortality in the sea cages. Following and spleen on Brain Heart Infusion Agar (BHI agar) the initial epizootic, cases of the disease have frecontaining 3 % salt (NaC1). Subcultures of the prequently being diagnosed, the fish manifesting welldominating bacterium were made on the same medium defined clinical signs (mentioned below). Primary isoand were kept for further investigations. Maximum and lation from all diseased fish resulted in substantial minimum tolerance limits as well as optimum growth growth of bacteria whereas no parasites were evident conditions for salinity, temperature, and pH were on any fish. Bacterial infection was therefore thought to determined. Biochemical characteristics of the bacaccount for the disease. terium were determined at 35 'C using API 20 E according to the manufacturer's instructions (Analytical Profile Index 1983), as well as at 22 "C. All tests To whom correspondence should be directed were also supplemented by standard tube and other O Inter-ResearchIPnnted In F. R. Germany 178 Dis. aquat. Org. 3: 177-180, 1987 tests including Gram stain and growth on McConkey agar. To investigate the abillty of the bacterium to produce disease in healthy fish, a saline suspension of the organism, equivalent to McFarland no. 6 turbidity standard, was inoculated intraperitoneally into healthy fish (1 m1 fish-'). Control fish were inlected with 1 m1 of sterile saline. Average weight of fish used was 50 g and all fish were held in fiberglass tanks supplied with continuous flowing seawater and aeration. Dead fish were removed, necropsies were aseptically performed, and bacteria were isolated from liver, kidney, and spleen on plates of salt-supplemented BHI agar. Bacterial identification was carried out as previously described. To vaccinate fish, a bacterin was prepared by formalin-killing washed cells of the bacterium, harvested from BHI culture broth. The procedure of Saeed & Plumb (1986) was used. The bacterin was freeze-dried until used. Fish for the vaccine experiments were caught by fish traps in the Red Sea. Their average weight was 60 g. Fish were acclimated to 600 1 fiberglass tanks supplied with continuous flowing, aerated seawater. The water had salinity 38 %o, pH 8.2, and temperature between 29 and 31 "C. Fish were fed a commercial sinlung-type pelleted diet containing 30 % protein. Fish were vaccinated by intraperitoneal injection or by direct immersion in the bacterin. Fish were intraperitoneally injected with either a single dose of bacterin (0.2 mg dry weight in 1 m1 of 1 % saline) or with 2 doses of the bacterin (booster injection). For single injection, 3 groups of 30 fish each were used. Twenty of each group received the bacterin while the other 10 fish of each group were used as controls. Control fish were given an intraperitoneal injection of 1 m1 of sterile 1 % saline. For booster injection, 30 fish were used: 20 received the bacterin on Days 0 and 7 and 10 received saline on Days 0 and 7. Twelve d after the initial vaccination (5 d after the booster injection for fish vaccinated twice), samples of experimental and control fish were bled and their serum antibody titers were determined using formalinlulled cells of the bacterium. Sixty fish were used in an experiment to assess the efficacy of vaccination by immersion. Forty fish were immersed in the bacterin while 20 fish (controls) were left unvaccinated. Fish were vaccinated by immersion for 5 min in a vaccine that contained 0.5 mg bacterin (m1 seawater)-'. Vaccinated fish were then washed by dipping into clear seawater before returning them to the experimental tank. Unvaccinated controls were immersed for 5 min into straight seawater. Fish were bled after 12 d and their antibody titers were determ.ined as already described. Fish vaccinated by injection were challenged 22 d after the initial vaccine injection by intrapentoneal injection of 1 m1 of saline containing 2.3 X 10" cells of the bacterium. Mortality of vaccinated fish was compared to that of the controls. Fish vaccinated by immersion were challenged by bath exposure. This was done by immersing both vaccinated and control fish in a suspension of the bacterium in seawater for 10 min. The suspension contained 5.75 X 10" cells of the bacterium ml-'.