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First Isolation of Vagococcus Salmoninarum from Cultured Rainbow Trout (Oncorhynchus Mykiss, Walbaum) Broodstocks in Turkey

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First Isolation of Vagococcus Salmoninarum from Cultured Rainbow Trout (Oncorhynchus Mykiss, Walbaum) Broodstocks in Turkey Bull. Eur. Ass. Fish Pathol., 31(6) 2011, 235 First isolation of Vagococcus salmoninarum from cultured rainbow trout (Oncorhynchus mykiss, Walbaum) broodstocks in Turkey B. I. Didinen1*, A. Kubilay1, O. Diler1, S. Ekici1, E. E. Onuk2 and A. Findik3 1Suleyman Demirel University, Egirdir Fisheries Faculty, Egirdir, Isparta, Turkey; 2Ondokuz Mayıs University, Faculty of Veterinary Medicine, Department of Fish Diseases; 3Ondokuz Mayıs University, Faculty of Veterinary Medicine, Department of Microbiology, Samsun, Turkey Abstract In this study, we isolated Vagococcus salmoninarum for the first time from rainbow trout in Turkey. The disease outbreak occurred in post-spawning broodstocks at a water temperature of 12.6°C during March-April 2011. The cumulative mortality rate was approximately 55% within 2 months. Infected trout exhibited lethargy, a marked loss of equilibrium, anorexia, dark coloration, mono and bilateral exophthalmos, eyeball disruption, furuncles and erosive lesions on both sides of the body, hemorrhage in the abdomen, jaw, mouth, and anus, and prolapse of the anus. Necropsy findings included hyperemia, visceral hemorrhage, and fibrinous deposits in the heart (ventricle and atrium), liver, and spleen. Samples for bacteriological examinations were collected from the kidney, liver, spleen, and ovary by using sterile swabs; these samples were streaked onto trypticase soy agar plates, and incubated at 22°C for 48 h. Four bacterial isolates were obtained from sick fish. The Gram-positive V. salmoninarum isolates were identified by their morphological, physiological, and biochemical features. The identification was confirmed by PCR with the amplification of highly specific 16S rRNA gene for V. salmoninarum. Although the isolates were sensitive to erythromycin and doxycycline in vitro; antibiotic treatments were ineffective. Introduction Coldwater Gram-positive coccal infections eased adult rainbow trout in Oregon, USA; this are less widespread and include vagococcosis isolate has become the type strain for the newly caused by V. salmoninarum (Wallbanks et al., proposed taxon V. salmoninarum (Wallbanks et 1990), carnobacteriosis caused by Carnobacterium al., 1990; Daly, 1999). maltaromaticum (formerly Lactobacillus piscicola; Collins et al., 1987) , and Lactococcus piscium V. salmoninarum has been reported in rainbow infection (Williams et al., 1990). trout in Australia (Tasmania), France, Italy, and Spain (Schmidtke and Carson, 1994; Michel et In 1968, a Lactobacillus was isolated from dis- al., 1997; Ghiino et al., 2004; Ruiz-Zarzuela et * Corresponding author’s e-mail: [email protected] Bull. Eur. Ass. Fish Pathol., 31(6) 2011, 236 al., 2005; Salogni et al., 2007) and in Atlantic examination were performed on diseased fish salmon (Salmo salar) and brown trout (Salmo on site. There was no relationship between the trua) in Norway (Schmidtke and Carson, regular losses of fish and temperature of water 1994). because water temperature was constant. Doxy- cycline and erythromycin were administered Vagococcosis is an emerging disease in the orally to control disease outbreak. The doses of European trout industry, affecting subadult doxycycline and erythromycin were 20 mg/kg or adult rainbow trout, with mortality rates bw for 7 days and 100 mg/kg bw for 14 days, of 20–50%. Outbreaks occur at water tempera- respectively. The affected fish did not respond tures of 10–12°C, and are associated with post- to treatment. Therefore all of the remaining fish spawning stress in broodstocks (Michel et al., in the pond were humanely destroyed in order 1997; Ruiz-Zarzuela et al., 2005; Austin and to prevent the further spread of disease. Austin, 2007). Isolation and identification of bacteria V. salmoninarum is included in the list of Gram- For bacterial isolation, samples were obtained positive bacteria responsible for chronic infec- from the kidney, liver, spleen, and ovary of each tions that do not respond to control procedures fish, streaked on trypticase-soy agar plates (TSA, and pose a serious threat to broodstocks and Merck), and incubated at 22°C for 48 h. Single large fish (Michel et al., 1997). colonies were restreaked on the same media to obtain pure isolates. Four isolates obtained from The aim of this study was the characterization one sick fish (liver, ovary, spleen, and kidney) of V. salmoninarum isolated from rainbow trout were identified by physiological, biochemical, broodstocks in a farm located in the Mediter- and enzymatic characterization. Routine tests ranean region of Turkey. were carried out for determination of biochemi- cal characteristics of the bacteria, as described Materials and Methods previously (Cowan and Steel, 1970; Collins and Fish Sampling Lyne, 1976; Holt, 1994). A total of 12 rainbow trout (body weight 1800– 2200 g) suspected of showing the clinical signs A presumptive identification of the isolates of the disease were collected from a farm in was performed using the following tests: the Mediterranean region of Turkey during Gram staining and cell morphology, motility, March-April 2011. The land-based farm has a oxidase and catalase activities, oxidative and hatchery unit and concrete ponds with a total fermentative degradation of glucose with O/F production capacity of 10 tons per year. The basal medium (Merck) supplemented with 1% farm is not included in a health-improvement glucose, H2S production on triple-sugar iron program. The water supply for the farm is medium (TSI, Merck), indole production, starch ground water and is not filtered or pre-treated. hydrolysis, hemolysin production, growth at Mortalities in the brood stock were observed different temperatures (10°C, 20°C, 37°C, and in fiVeen-day intervals during the disease 42°C) and pH 9.6, growth on trypticase-soy outbreak. Bacteriological and parasitological broth containing 6.5% NaCl (Merck) and Mac- Bull. Eur. Ass. Fish Pathol., 31(6) 2011, 237 Conkey agar. Additional tests were performed Antimicrobial sensitivity using API 20 NE, API 20 E, API 20 Strep, and Antimicrobial tests were performed on the API 50 CH systems (bioMérieux) (Schmidtke kidney isolate using disc diffusion assay on and Carson, 1994; Ghiino et al., 2004; Ruiz- Mueller–Hinton agar (Oxoid). NCCLS stand- Zarzuela et al., 2005; Austin and Austin, 2007). ards were used for the evaluation of the results In all the tests, V. salmoninarum NCIMB 13133 (NCCLS, 2001). was used as a reference. Results and discussion Genotypic confirmation of identification This is the first report of V. salmoninarum isolation Total DNA used for PCR applications were from broodstocks of rainbow trout in Turkey. extracted from cultures(positive, negative The disease outbreak occurred in post-spawning control strains and the kidney isolate) using fish in a farm during photoperiod studies at a DNeasy tissue kit (Qiagen) according to the water temperature of 12.6°C during March- manufacturer’s instruction. Lactococcus garviae April 2011. Cumulative mortality aributed ATCC 43921 and V. salmoninarum NCIMB 13133 to this pathogen was as high as 55% within 2 were used as negative and positive controls, re- months. This mortality rate was higher than the spectively. Confirmation of identification of the rates previously reported by other researchers kidney isolate at the species level was performed (Michel et al., 1997; Ruiz-Zarzuela et al., 2005). by a PCR as described by Ruiz-Zarzuela et al. Although earlier reports indicated that the out- (2005).The highly specific 16S rRNA gene was breaks occurred during post spawning period, selected for the identification. The sequences in our case other factors may have aributed to of the primers used for the amplification of the this outbreak, i.e. the fish were under stressful gene were as follows: pSal-I: 5’- GTT TTA GCC conditions, including overcrowding, poor water GCA TGG CTG AGA TAT-3’; pSal-2:5’- AGG quality, and photoperiod application. TGG GAA CAG TTA CTC TCC CA-3’.The am- plification of the target gene was performed in Diseased trout showed listlessness, a marked a 50-μl mixture containing 10 microliters of the loss of equilibrium, swimming difficulty, mono extracted DNA, 1X PCR buffer (50 mmol KCl, and bilateral exophthalmos, eyeball disrup- 20 mmol Tris HCl), 5 μl of 25 mmol MgCl2, tion, paleness of the gills, prolapsed anus, and 100 μmol l-1 of each dNTP, 100 pmol of each melanosis. External hemorrhage was observed primer, and 1 U of Taq polymerase. The am- in the jaw, eye, mouth, abdomen, ventral fins, plification was carried out with the following and anus. Furuncles were observed on the thermocycling conditions: initial denaturation back of the opercular and caudal regions, and at 94°C for 3 min, 35 cycles of denaturation at erosive lesions were observed on the sides of 94°C for 1 min, annealing at 55°C , for 1 min , the body. Internal symptoms included bloody extension at 72°C for 1 min and a final extension ascitic fluid in the body cavity; transparent fluid step at 72°C for 10 min. The PCR products were accumulation and hemorrhage in the stomach; loaded onto 2% agarose gel containing 1 mg/ fibrinous deposits on the heart (ventricle and ml ethidium bromide,separated and visualized atrium), liver and spleen; paleness of the liver; under ultraviolet light. hyperemia and hemorrhage in the ovary and Bull. Eur. Ass. Fish Pathol., 31(6) 2011, 238 intestine; yellow and bloody gelatinous exudate AVer cultivation of samples from internal in the intestine; congestion in the blood vessels organs, white small, smooth colonies of 0.5 of the air bladder and muscles; and petechial mm diameter were recovered from all fish on hemorrhages in fat tissue, liver, and muscle TSA. Compared to other isolates, the growth wall (Figure 1). The most typical symptoms of kidney isolate took longer and the colonies were fibrinous deposits on the heart (ventricle were less intense. Ghiino et al. (2004) have and atrium), spleen, and liver and hemorrhage reported similar results. In their study, isolates in the viscera. Clinical findings were similar of V. salmoninarum were obtained from 100% to those previously described by Michel et al.
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