Journal of Fish Diseases 2009, 32, 943–951 doi:10.1111/j.1365-2761.2009.01075.x First isolation and characterization of Lactococcus garvieae from Brazilian Nile tilapia, Oreochromis niloticus (L.), and pintado, Pseudoplathystoma corruscans (Spix & Agassiz) J J Evans1, P H Klesius2 and C A Shoemaker2 1 USDA, ARS Aquatic Animal Health Laboratory, Chestertown, MD, USA 2 USDA, ARS Aquatic Animal Health Laboratory. Auburn, AL, USA Streptococcus genus, Streptococcus garvieae, these Abstract isolates originated from United Kingdom bovine Lactococcus garvieae infection in cultured Nile tilapia, mastitis cases (Collins, Farrow, Phillips & Kandler Oreochromis niloticus (L.), and pintado, Pseudo- 1983). Shortly after its characterization, enterococ- plathystoma corruscans (Spix & Agassiz), from Brazil is cal and lactic acid members of the Streptococcus reported. The commercial bacterial identification genus were transferred into two distinct genera, system, Biolog MicrologÒ, confirmed the identity of Enterococcus and Lactococcus (Schleifer & Kilpper- L. garvieae. Infectivity trials conducted in Nile Ba¨lz 1984; Schleifer, Kraus, Dvorak, Kilpper-Ba¨lz, tilapia using Brazilian Nile tilapia L. garvieae isolates Collins & Fischer 1985). Since this time, additional resulted in a median lethal dose-50 of 1.4 · 105 mammalian cases of L. garvieae have been reported colony-forming units (CFU)/fish. This is the first from humans (Elliott, Collins, Pigott & Facklam evidence of the presence of this pathogen from 1991). Concurrently, Kusuda, Kawai, Salati, Ban- Brazilian fish. In addition, this is the first report of ner & Fryer (1991) reported a novel fish pathogen, L. garvieae infection in either Nile tilapia or pintado. Enterococcus seriolicida, affecting yellowtail, Seriola Collectively, this evidence expands the geographical quinqueradiata (Temminck & Schlegel), and range of fish hosts, number of fish hosts harbour- amberjack, S. dumerili (Risso), in Japan. Recog- ing L. garvieae and carbon source utilization by nized as a pathogen of cultured fish in Japan L. garvieae fish isolates. Furthermore, the Biolog (Kusuda et al. 1991) and of trout throughout system may be an alternative technique to polymerase Mediterranean Europe (Eyngor, Zlotkin, Ghittino, chain reaction for the identification of L. garvieae and Prearo, Douet, Chilmonczyk & Eldar 2004) since discrimination between closely related bacterial 1991, debate over the correct taxonomy of this species. organism has generated substantial research. Dome´nech, Prieta, Ferna´ndez-Garayza´bal, Collins, Keywords: Biolog MicrologÒ, Brazil, Lactococcus gar- Jones & Domı´nguez (1993) provided phenotypic vieae, pintado, tilapia. and phylogenetic evidence of a close relationship between E. seriolicida and L. garvieae. Later research- Introduction ers, continents apart, engaged in phenotypic and genotypic studies of atypical subclinical isolates The taxonomy and characterization of Lactococcus from Brazilian water buffalos (Teixeira, Merquior, garvieae has had both a controversial and circuitous Vianni, Carvalho, Fracalanzza, Steigerwalt, Brenner history. First described as a member of the & Facklam 1996) and diseased rainbow trout, Correspondence J Evans, USDA, ARS Aquatic Animal Health Oncorhynchus mykiss (Walbaum), in Italy (Eldar, Laboratory, 118 B Lynchburg Street, Chestertown, MD 21620, Ghittino, Asanta, Bozzetta, Goria, Prearo & Journal compilation USA Bercovier 1996), regarded E. seriolicida as a synonym Ó 2009 (e-mail: [email protected]) Blackwell Publishing Ltd No claim to original US government works 943 Journal of Fish Diseases 2009, 32, 943–951 J J Evans et al. Isolation of Lactococcus garvieae from tilapia and pintado of L. garvieae, thereby re-linking Enterococcus with & Bercovier (1998) indicated these methods were of the Lactococcus genus. no value for routine identification and therefore As a result of the infrequent isolation of polymerase chain reaction (PCR) assays have L. garvieae from mammalian sources, it has been become a widely used tool for the identification regarded as either a contaminant of clinical cultures, of L. garvieae (Aoki, Park, Yamashita & Hirono or an opportunistic pathogen of low virulence, 2000; Goh, Facklam, Chang, Hill, Tyrrell, Burns, pathogenic only in immunocompromised hosts Chan, He, Rahim, Shaw & Hemmingsen 2000; (Ruoff, Whiley & Beighton 1999). Additional Pu, Dobos, Limsowtin & Powell 2002; Mata, clinical cases indicated it may be isolated from Gibello, Casamayor, Blanco, Domı´nguez & both healthy (James, Hardman & Patterson 2000) Ferna´ndez-Garayza´bal 2004). This report presents as well as immunosuppressed humans (Fefer, an alternative automated bacteriological identifica- Ratzan, Sharp & Saiz 1998; Mofredj, Baraka, tion system, Biolog, to facilitate identification of Cadranel, LeMaitre, Kloeti & Dumont 2000; L. garvieae, to discriminate between L. garvieae Fihman, Raskine, Barrou, Kiffel, Riahi, Berc¸ot & and L. lactis and to produce metabolic fingerprinting Sanson-Le Pors 2006) and those with gastro- of L. garvieae isolates for epidemiological analysis. intestinal disorders (Wang, Shie, Chen, Huang, Hsieh, Wen, Lin & Wu 2007), rendering this pathogen potentially zoonotic. Furthermore, its Materials and methods presence in Brazilian water buffalos with subclinical Conventional bacteriological tests mastitis (Teixeira et al. 1996), agricultural food products (Barakat, Griffiths & Harris 2000; Villani, Acaqua Imagen Service, Brazil collected three Aponte, Blaiotta, Mauriello, Pepe & Moschetti morbid Nile tilapia, Oreochromis niloticus (L.), 2001; Rantsiou, Urso, Iacumin, Cantoni, Cattaneo, and three morbid pintado, Pseudoplathystoma Comi & Cocolin 2005; Zamora 2005), aquacul- corruscans (Spix & Agassiz), from aquaculture tural products (Wang et al. 2007) and domestic facilities with daily fish mortalities and sent whole animals (Pot, Devriese, Ursi, Vandamme, frozen fish in individual plastic bags to the United Haesebrouck & Kersters 1996), has posed sugges- States Department of Agriculture (USDA), Agri- tions that L. garvieae be regarded as a potential cultural Research Service (ARS), Aquatic Animal zoonotic agent (Vendrell, Balca´zar, Ruiz-Zarzuela, Health Research Laboratory, Auburn, AL,USA, for de Blas, Girone´s&Mu´zquiz 2006). Additionally, isolation and identification of the bacterial patho- L. garvieae has been reported from an aquatic gens. Microbiological samples were obtained from mammalian host (Evans, Pasnik, Klesius & thawed fish brains and streaked onto de-fibrinated Al-Ablani 2006). 5% sheep blood agar plates (SBA; Remel). The Despite the isolation of L. garvieae from aquatic plates were incubated at 28 °C for 18–20 h and the species, food products, cattle and humans, and its predominant alpha haemolytic colonies were significance in clinical veterinary and medical selected for further phenotypic analyses. The microbiology, identification of L. garvieae has not phenotypic characterization was based on presence been possible solely by a semi-automated bacterio- of haemolysis, Gram staining, colony differentia- logical system such as API Rapid ID 32 Strep tion and standard biochemical tests. Oxidase (bioMe´rieux). This system cannot discriminate (Becton Dickinson) negative, Gram-positive cocci between L. garvieae and L. lactis subsp. lactis, were inoculated onto tryptic soy agar slants (Difco another human pathogen (Elliott et al. 1991). The Laboratories), starch agar plates (Remel) for starch supplementation of phenotypic characterization hydrolysis, and in brain–heart infusion broth (Becton with other characterization techniques; determina- Dickinson) for growth determination at 45 °Cand tion of clindamycin susceptibility, sodium dodecyl in appropriate media for other biochemical tests. sulphate–polyacrylamide gel electrophoresis (Elliott These tests included catalase (Sigma-Aldrich), et al. 1991), DNA, RNA and restriction fragment- haemolysis on SBA, 6.5% salt tolerance (Becton length polymorphism (Eldar, Goria, Ghittino, Dickinson), fermentation of sorbitol, arginine deam- Zlotkin & Bercovier 1999; Eyngor et al. 2004) has ination, bile-esculin tolerance, methyl red Voges culminated in a better identification and/or dis- Proskauer (Sigma-Aldrich), hippurate hydrolysis, Journal compilation crimination between L. garvieae, L. lactis and related pyrronidonyl arylamidase (Remel) and leucine Ó 2009 bacterial species. However, Zlotkin, Eldar, Ghittino aminopeptidase (Sigma-Aldrich) (Shoemaker & Blackwell Publishing Ltd No claim to original US government works 944 Journal of Fish Diseases 2009, 32, 943–951 J J Evans et al. Isolation of Lactococcus garvieae from tilapia and pintado Klesius 1997; MacFaddin 2000). For comparison conducted as above. No disease signs or mortality purposes, two American Type Culture Collection were observed in the group of tilapia inoculated (ATCC) reference isolates; ATCC 43921 type isolate with the pintado isolate. Fifty percent mortality of L. garvieae originating from a clinical case of occurred in the tilapia inoculated with the Brazilian bovine mastitis (Collins et al. 1983; Schleifer et al. tilapia isolate. The Brazilian tilapia isolate was 1985) and ATCC 49156 Enterococcus seriolicida from re-isolated from the brain of one of the dead- a yellowtail (Kusuda et al. 1991) were also charac- challenged fish. This virulent tilapia isolate was terized as above. subsequently used for median lethal dose (LD50) estimation according to the procedures of Reed & Muench (1938). Six doses were used. Tilapia (mean Biolog MicrologÒ bacterial