Abstract: Vibrio anguillarum and Lactococcus garvieae which causes mixed infections were isolated for the first time from rainbow trout farms in the Southwest of Turkey. The outbreak occurred at 15°C water temperature in June 2009. The diseased fish showed erratic swimming, dark discoloration, bilateral exopthalmia, haemorhages on the fins, operculum, the liver, spleen and lower intestines with mortality ranging from 30-50% in the pools. The identities of the isolates were confirmed as V. anguillarum O1 and L. garvieae by biochemical and serological tests using API 20 Strep, API 20 E and API Strept system test strips and also V. anguillarum (ATTC 43305) used as antigens for agglutination. The epizootic disease was effectively controlled by oral administration of enrofloxacin.

INTRODUCTION

Lactococcus garvieae (L. garvieae) is the species of clinical significance in humans and animals. The original description of L. garvieae dates back to 1984 when it was first isolated from a mastitic udder of a cow in the United Kingdom (Vela et al., 2000). On the other hand, regardless of its limited importance as a mastitogenic agent, L. garvieae turned out to be the most important risk factor in the Mediterranean sea because it causes specific losses approximately 50% in the total production of in European trout industry (Ghittino and Prearo, 1992).

Prieta et al. (1993) reported L. garvieae as a fish pathogen in Europe. Bacteriologic and molecular studies confirmed L. garvieae as the etiological agent of a hemorrhagic septicemia in cultured trout characterized by bilateral exophthalmia, darkening of the skin, congestion of the intestine, liver, kidney, spleen and brain and by characteristic hemorrhagic enteritis (Domenech et al., 1993). In 1991, a new enterococcal species Enterococcus seriolicida was described as a new pathogen responsible for an infection of eels and yellowtail with symptoms identical to those caused by L. garvieae in trout (Rodkhum et al., 2005).

In Japan, fish diseases due to gram positive cocci were described as Enterococcus seriolicida during the 1970. Indeed, 16S rRNA sequencing and DNA hybridization studies confirmed that L. garvieae and E. seriolicida are the same species (Domenech et al., 1993). Vibrio anguillarum is one of the most important pathogens in fish of several countries and in many of fish species. Primary identification of V. anguillarum can be performed by using biochemical criteria such as those described by Baumann and Schubert (1994). The V. anguillarum usually attaches to the segments of intestine in rainbow trout and turbot (Olsson et al., 1996; Spanggaard et al., 2000).

The gut has also been reported to be the initial site of the infection in turbot larvae (Grisez et al., 1996). Vibriosis affects some fish species such as turbot (Scophthalmus maximus), pacific salmon (Oncorhynchus kisutch), atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), sea bass (Dicentrarchus labrax), striped bass (Morone saxatilis), cod (Gadus morhua), Japanese and European eel (Anguilla japonica and Anguilla anguilla), yellowtail (Seriola quinqueradiata) and ayu (Plecoglosus altivelis) (Rodkhum et al., 2005). The aim of the present study was to examine the mixed infections accounted for by Lactococcus garvieae and Vibrio anguillarum O1 in rainbow trout.

MATERIALS AND METHODS

Sampling: The outbreak was first observed in a rainbow trout fish farm in the Southwest of Turkey in June 2009. During the infection period, behaviors of the sick fish and mortalities were recorded.

A total of 20 moribund fish of 80-200 g were collected from the farm and transported to the laboratory of fish diseases in Faculty of Fisheries, Ege University. Infected fish samples were described by their gross external and internal symptoms and examined by bacteriological investigations of some organs.

Isolation and identification of bacteria: Samples of spleen, kidney and liver were subjected to microbiological examination by inoculation on trypticase soy agar (Difco) supplemented with 5% defibrinated sheep blood (BTSA) and incubated at 21°C for 48 h. Pure cultures of the different colony types were obtained on BTSA.

A presumptive identification of the strains was performed by Gram staining, catalase, oxidase activity, motility and resistance to O/129 vibriostatic agent. API 20 Strep test and API 50 CH tests (BioMerieux S.A., France) were used to determine biochemical characteristics of L. garvieae according to the manufacturer’s instruction at incubation temperature of 24±0.5°C. For the biochemical tests of V. anguillarum, API 20E system was carried out at 21°C for 48 h. (BioMerieux S.A., France). Slide agglutination test was performed on all isolated strains using raised rabbit serum against V. anguillarum O1 (ATTC 43305) as in that of Toranzo et al. (1987).

Antimicrobial sensitivity test: All isolates were tested for their sensitivity to 9 antibiotics on Mueller-Hilton agar using the Kirkby-Bauer disc diffusion technique (Stokes et al., 1993). A loopful of a 24 h culture was placed in the center of the petri plate with media and spread with a dry swab.

Antibiotic sensitivity-impregnated discs were then placed on the plates and following incubation at 21°C for 24 h the zones of inhibition were measured.

RESULTS AND DISCUSSION

During the infection period, water temperature was measured as 15±0.5°C. Clinical signs of the disease were erratic swimming, bilateral exophthalmia and hemorrhage in the eyes, skin discoloration, yellowish and reddish necrotic lesions and ulcers in the abdominal muscle, hemorrhage and erosion in pectoral, abdominal and anal fins and also hemorrhage in mouth of some fish. Mortality rates varied from 30-50% in different pools of the farm.

Necropsy showed typical findings such as enlarged spleen, pale gills, liver and kidney as well as a petechial hemorrhage in liver, pyloric caeca, peritoneal and subperitoneal membranes and perivisceral fat in lower intestines. As a result of bacteriological culture from the diseased fish, L. garvieae and V. anguillarum from the same fish were grown whereas from the some fishes in the farm either L. garvieae or V. anguillarum were also isolated.

Table 1:	The API 50CH profile for L. garvieae isolated from farm
+ : Positive; - : Negative

Isolated streptococci were described as non-motile Gram (+) cocci in short chains and oxidase and catalase reactions were negative. Growth occurred at 24°C. The API 20 Strept rapid identification system resulted in a profile 5143110. According to the API 20 Strept test and API 50 CH test results, the biochemical characteristics of isolated L. garvieae are shown in Table 1 and 2, respectively. API 20 E profile for V. anguillarum was 3207726. The morphologic and penotypic characteristics of V. anguillarum isolated from the farm are shown in Table 3.

In a slide agglutination tests with anti V. anguillarum O1 (ATCC 43305) serum showed that all strains were positive. Antibiyogram tests results for L. garvieae and V. anguillarum are shown in Table 4. There were parallelism in both L. garvieae and V. anguillarum in that they revealed identical antimicrobial susceptibility patterns, susceptibility to amoxicillin, ampicillin, enrofloxacin, flumequin, norfloxacin and to oxolonic acid and resistance to erythromycin, oxytetracycline and trimethoprim-sulphamethoxazole (1.25+23.75). Lactococcosis is a kind of disease caused by L. garvieae which has been particularly devastating freshwater culture of salmonid fish. The first outbreak of Lactococcosis was in the rainbow trout fish farm in Spain in 1988 (Ghittino and Prearo, 1992). Since then lactococcosis has caused heavy mortality in rainbow trout in Italy, Australia, South Africa, England, France and Israel (Ghittino and Prearo, 1992; Prieta et al., 1993).

Table 2:	The API Strept profile for L. garvieae isolated from farm
+ : Pozitive ; - : Negative

Table 3:	The morphologic and biochemical properties for V. anguillarum isolated from farm
+ : Positive; - : Negative

The agent of this disease was first isolated as Enterococcus seriolicida from the trout farms in Turkey, 1997 (Cagirgan and Tanrikul, 1995) causing hemorrhagic septicaemia, ascites, bilateral exophthalmus and hemorrhage of the intestine, liver, kidney, spleen and brain. L. garvieae led to 80% of mortality in fish of 200±g at 18°C (Cagirgan and Tanrikul, 1997).

Table 4:	Antibiotic susceptibility profile of the isolates of V. anguillarum and L. garvieae
*Trimethoprim-sulphadiazine (1/5); S : Sensitive; R : Resistant

Streptococcosis was also diagnosed from Onchorhynchus mykiss in Iran (Soltani et al., 2005). The affected fish in Iran have been showed to have the same symptoms such as erratic swimming, dark discoloration, haemorrhages on the base of fins and exophthalmia with cataract with fish mortality varying from 20-40%.

Internally, there were haemorrhagic livers, congestion of spleen and kidney and occasionally ascites in the peritoneal cavity. It was reported in Iran that they have the same symptoms as those found by us. However, the isolated pathogen was more similar to the Streptococcus iniae than L. garvieae (Cagirgan and Tanrikul, 1995; Soltani et al., 2005). There is no available information on isolation of Streptococcus iniae in Turkey but L. garvieae has been isolated rutinely (Domenech et al., 1993; Altun et al., 2004).

L. garvieae has been causing differents level of mortality in various countries but it has led to mortality of approximately 50% in total production accross Mediterranean of trout industry (Ghittino and Prearo, 1992). Eldar et al. (1999) reported the three biotypes of L. garvieae. Vela et al. (2000) also identified the thirteen biotypes. API profiles of Spanish strains were the similar to Turkish strains (Prieta et al., 1993). The present results show that the strains concerned were parallel with those found by Cagirgan (2004).

V. anguillarum is widespread across the world. V. anguillarum was isolated from salmon (Winton et al., 1983; Muroga, 1975) and from different marine species (Toranzo and Barja, 1990). Tanrikul et al. (2005) and Tanrikul (2007) reported that API 20 E strips were used for identification of V. anguillarum O1 which was isolated from both cultured sea bass (Dicentrarchus labrax) and rainbow trout (Onchorynchus mykiss).

According to the API 20 E test results; this isolate was identified as V. anguillarum O1 which were similar strains to those by Tanrikul et al. (2005) and Tanrikul (2007). So far, natural mixed infection has not been isolated in Turkey. Both L. garvieae and V. anguillarum O1 were isolated from infected fish.

Clinical signs are the same as those in other reported lactococcosis (Domenech et al., 1993; Cagirgan and Tanrikul, 1997, 1995; Salati et al., 1996) and vibriosis (Muroga, 1975; Toranzo and Barja, 1990). Antimicrobial susceptibility tests were conducted using from isolates of mix infection. L. garvieae and V. anguillarum were susceptible to amoxicillin, ampicillin, enrofloxacin, flumequin, norfloxacin and oxolonic acid. Endo et al. (1975) and Takahashi et al. (1976) proposed oxolonic acid and sulphonamids for vibriosis treatment, respectively. According to the Cagirgan and Tanrikul (1995) erythromycin and oxytetracycline were effective chemotherapeutic agents for streptococcosis.

Generally in Turkey, erythromycin is used against streptococcosis while oxytetracycline is also used against the rainbow trout fry syndrome. However, trimethoprim-sulphamethoxazole is employed against enteric red mouth. For these reasons, effects of some drugs are decreased for these bacteria. In this case, treatment with enrofloxacin was made against these two bacteria strains.

CONCLUSION

In this study, infected fish were treated with enrofloxacin (50 mg/kg/day for 7 days orally) and mortalities were completely controlled at the end of treatment period.