SOME CHARACTERISTICS OF AEROMONAS HYDROPHILA AND VIBRIO SPECIES ISOLATED FROM BACTERIAL DISEASE OUTBREAKS IN ORNAMENTAL FISH CULTURE IN SRI LANK4

: Bacterial fish disease outbreaksof freshwater ornamental fish hatcheries and farms, reported during the period from January,l993 to Febru-ary, 1994 in Sri Lanka were used in the study. Twelve fish species belonging to 11 wnera were involved. Gross external and internal lesions were recorded. Samples were plated on selective (Aeromonns agar, Pseudomonas agar and thiosulfate citrate bile sucrose anar -TCBS) media and non-selective (blood arrar and nutrient agar) media. ~ o t h &'I 20E and API GP test strips and conventi&al identification procedures were followed for characterization of .the isolates. Following identification, 11 selected antibiotics were tested for their effective- .ness against all the isolates using disk diffusion method. Aeromonas hydrophila was involved in 78.26% of the total (23) disease ofitbreaks while Vibrio sp. was invoked in the rest. Erodingfins, hemorrhagic lesionson the skin and atthe base of tail fin, sloughing scales, hemorrhagic intestinal wall and other visceral organs were among the lesions observed due to A. hydrophila infection. Lesions observed due to vibriosis were, erratic swimming, hemorrhagic patches an skin and on areas around anus,base of pelvic and tail fins, hemorrhagic peritoneum and visceral organs, fluid filled erythemic intestine and blindness among some survivors. All isolates ofA. hydrophila were resistant to the tested antibiotics of the families tetracycline and penicillin, and trimethoprim, trimethoprim+sulfamethoxazol and streptomycin. Antibiotics of the family quinolones (norfloxacin and flumequine),,nitrofurantoin and gentamicin proved to be the most efffcacious on A. hydrophila isolates. All Vibrio sp. isolates were sensitive to 11 antibiotics tested except colistin and streptomycin.


INTRODUCTION
As the global ornamental fish trade has been growing steadily over the years, the number ofhatcheries, farms and trade agencies has increased markedly over the last decade. In 1988, the global value of the aquarium industry was estimated at US $ 4 bi1lion.l Singapore, the world's largest exporter oftropical fish, earned US $38 million from this trade in 1988 while Sri Lanka which earned slightly over US $ 1 million in 1985 doubled its export in 1988 and has been projected to earn US $ 4 million in 1994 from this industry.lp2 Although the industry has been growing, almost all farmers experience setbacks in production during larval rearing, growout and transportation especially due to high mortality associated with diseases and related disorders which estimates roughly to about 40% ofthe total production.'

D.C. Hettiarachchi and C.H. Cheong
It is well established in m-namental fish'industry that bacterial infections are responsible for heavy losses from the farm level to the hobbyist tank. Gratzek et a13 and Shotts et ~1 .~ established that 15% ofthe 73 bags of pet fish originating from Southeast Asia had Aeromonas hydrophila.However, literature on bacterial diseases of ornamental fish culture in Sri Lanka is ~c a r c e .~ This work was therefore undertaken to investigate the bacterial disease outbreaks in ornamental fish culture in Sri Lanka, characterize the pathogens and to determine the sensitivity of the bacteria involved to commonly used antibiotics with a view to provide the farmers with the information on effective treatment.

METHODS AND MATERJALS
Bacterial diseases of freshwater ornamental fish that were reported to our laboratory from hatcheries and farms during the period January,1993 to February, 1994 were investigated. Physical examination: Fish were examined for the presence of external and internal parasites and macroscopic lesions were recorded. Samples for bacteriological culture were taken directly from lesions as well as from liver, kidney, peritonial cavity and intestinal contents aseptically. When fry and juveniles were involved, sampling was done according to Tanasomwang and Muroga6 and direct plating was carried out with the homogenate.
Media and culture techniques: Primary isolation of bacteria was carried out on Pseudomonas agar, Aeromonas agar,thiosulfate citrate bile sucrose agar,blood agar and nutrient agar (Oxoid, England). Plates were incubated a t room temperature (26OC ) for 24 to 48 h and predominant colonies, both from selective and non-selective media were subcultured and purified on nutrient agar. One such pure culture was taken from each outbreak of disease.

RESULTS
Aeromonas hydrophila was the most dominant isolate in-18 outbreaks out ofthe 23 cases investigated. The fish involved in these outbreaks were: Goldfish juveniles and adults, Kissing gourami fry, Angelfish juveniles, Giant gourami, Zebrafish; Guppies, Super serpae, Swordtail, Cuming's barb, and Cherry barb. Eroding fins, sloughing scales, hemorrhagic skin, intestine and other visceral organs, ulcerative necrosis a t the base of tail fin were among the lesions observed due to A. hydrophila infections.
The dominant isolate in the other five disease outbreaks was Vibrio sp. in which Swordtail, Black widow fish, Super serpae, Penguinfish, Angelfish, and Cherry barb were involved. Lesions observed due to vibriosis were, erratic swimming, hemorrhagic peritoneum and visceral organs, fluid filled erythemic intestine and blindness among some survivors. Many of the cultural and biochemical characteristics ofA.hydrophila isolated from the disease outbreaks were consistent while some varied among the isolates. Those inconsistent characters are shown in Table 1. Vibrio sp. isolated showed growth characteristics of 1-2 mm, low convex, circular colonies on blood agar (after 24 h a t 26 OC), less than 1 mm light green colonies onTCBS agar after 24 h which turned white (2mm) with a large yellow surrounding after'48 h. The 5 Vibrio sp. isolated showed almost the same characteristics except for reactions in Voges-Proskauer and salt requirement as shown in Tables 2 and 3. For comparison, the characteristics of the Vibrio sp. reporteds as the causative agent of vibriosis in the Kununa prawn are presented in these tables. All A. hydrophila isolates were sensitive to NX,AR,GM,and NI and resistant to AM and TC. Twenty seven percent were resistant to CL, 11.70 % were resistant 'to TM and TS. The Vibrio sp. were sensitive to the tested antibiotics except C0,NI and SM. The percentage sensitivity ofA. hydrophila and Vibrio sp. to the antibiotics are shown in Tables 4 and 5.

DISCUSSION
Aeromonas hydrophila was involved in 78.26% of the total (23) bacterial disease outbreaks investigated during the study period. This, a n opportunistic pathogen was reported to be the predominant pathogen found in lesions of Epizootic Ulcerative Syndrome (EUS) affected fishgJ0911. Costa and Wijeyaratne, 12 Subasinghe et aL9, Pathiratne et al.13 reported that over 21 species of brackish and freshwater fish in Sri L , d a were affected by EUS. EUSlesions(hemorrhages and ulcerative necrosis in the skin on various parts of the body) were observed in the present study on A. hydrophila affected fish'except larvae of Kissing Gourami and juveniles of Angelfish. Dixon et al.14 found that about 43 % of the Aeromonas isolates from a variety of tropical pet fish imported from Singapore were A. hydrophila which were causing monomicrobial infections in these fish. Shotts et aL4 also established A. hydrophila as a major disease problem in ornamental fish. These and the results of the'present study demonstrate the potential of A. hydrophila as a serious pathogen in ornamental fish industry.
A. hydrophila isolated in the present study were resistmt to TC. This finding is in agreement with the observation of Dixon et al.14,Supriyadi and Rukyani.16 In the present study, 27.77% of the Aeromonas isolates showed multiple resistant to TC and CL. This is comparable with the observations made by Aoki and Watanabel%ho isolated multiple drug resistant (to TC and CL) A. hydrophila from freshwater fish in Thailand. Of the antibiotics tested, GM,AR,and NXwere the most efficacious onA. hydrophilaisolates. Dixoneta1.14 also found the quinolone family antibiotics as the most effective antimicrobial drug on freshwater A. hydrophila from ornamental fish. Though the development of plasmid mediated resistant to quinolones is rare," Supryadi and

Diseases of O r n a~n t a l Fish 267
R u k y d l 6 noted. that 70% of the thirty A. hydrophila isolates from affected freshwater fish were resistant to AR in Indonesia.
The Vibrio sp. isolated from the five disease outbreaks were categorized as belonging taem species. Most of the characteristics of these isolates coincide with those of the Vibrio sp. reported by Pena et aL8 from diseased Kururna prawn, Pendeus japonicus. However, the true freshwater character of the present isolates was well supported by their ability to grow in salt level as low as 0.5%.
Internal and external lesions of the disease caused by this bacteria were similar to those of vibriosis described by Post.'* Vibriosis-of freshwater fish has not been recoided earlierin Sri Lanka. The list of susceptible freshwater fish species to vibriosis by Postl8 did not include any of the species involved in the present outbreaks of vibriosis.
All five Vibrio sp. isolated were sensitive to the eleven antibiotics tested except CO, NI i n d SM. In contrast, many authors reported resistant Vibrio sp. to commonly used antibiotics from marine environment^.'^.^^.^^ In conclusion, this investigation provides valuable information to the farmers on the two major bacterial killers of freshwater ornamental fish in Sri Lanka and a choice ofthe most effective antimicrobials against them. This also provides information for the scientists on the emergence of vibriosis of freshwater fish which has not been recorded earlier in this country.