First Isolation and Characterization of Lactococcus Garvieae From

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 Microlog, 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. Domeńech, Prieta, Fernańdez-Garayza´bal, Collins, Keywords: Biolog Microlog, Brazil, Lactococcus gar- Jones & Domıńguez (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 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, Domıńguez & both healthy (James, Hardman & Patterson 2000) Fernańdez-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, Berçot & 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 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 Microlog bacterial identification weight = 10 gm) were inoculated with 0.1 mL of Biolog Microlog bacterial identification (Hay- fivefold dilutions of this isolate yielding doses wood) was performed on all Brazilian fish isolates, ranging from 1 · 105 to 1 · 108 CFU/fish. Ten the bovine L. garvieae reference isolate (ATCC fish were used per dose and placed into individual 43921) and the fish E. seriolicida reference isolate tanks. For all trials, 10 control fish were inoculated (ATCC 49156) according to the manufacturerÕs with tryptic soy broth. Fish were monitored for instructions. Briefly, isolates were grown on Biolog 7 days and 20% of the morbid fish were removed BUG (Biolog, Inc.) agar and growth from each and necropsied for recovery of L. garvieae. plate removed with a sterile swab, re-suspended in 20 mL tubes containing Biolog dilution medium and adjusted to approximately 20% transmittance. Results Each inoculum was pipetted into a Biolog Gram- Conventional and Biolog characterization positive 96-well microplate and incubated at 35 C. The microplate was read at 4–6 h and again at All the Brazilian L. garvieae isolates studied here and 16–24 h using a microplate reader and the Micro- ATCC reference isolates were Gram-positive cocci, log Gram-positive database was used to yield a catalase and oxidase negative, and a-haemolytic on carbon utilization metabolic fingerprint and bacte- 5% SBA. Growth at 45 C, growth in broth rial identification. Identification confidence was containing 6.5% NaCl and bile-esculin tolerance based on % probability (PROB) and Similarity were positive for Brazilian isolates by conventional Index (SI) after 16–24 h incubation. methods. Likewise, the presence of pyrronidonyl arylamidase and leucine aminopeptidase enzymes, arginine deamination, and the Voges Proskauer Experimental infectivity trials (VP) reaction were positive for Brazilian isolates was Infectivity trials were initiated to determine the well as ATCC 49156 using conventional methods. ability of L. garvieae isolates to cause mortality in Sorbitol fermentation and hippurate and starch Nile tilapia and to fulfill KochÕs postulates. Tilapia hydrolysis were negative for all isolates. were determined to be free of L. garvieae by Of the six pintado and six tilapia isolates, three microbiological methods used in the initial isolation pintado (01-2408-1, 25-9590, 25-9590-1) and from whole frozen fish. Two L. garvieae isolates three tilapia (25-9938-2, 30-9630-6, 30-9630-11) (one from pintado, 01-2408-1 and one from tilapia, isolates were confirmed as L. garvieae by Biolog 30-9630-11) were used to inoculate two groups of (Table 1). These isolates gave a 100% PROB and a 10 tilapia (mean weight = 10 g). These isolates had SI range of 0.55–0.87. The Biolog database indi- been passed on SBA three times prior to their use in cated that there was 100% PROB that the ATCC this infectivitiy trial. Inoculum was prepared as reference strains were L. garvieae. Two tilapia previously described (Evans et al. 2006). Fish were isolates were identified as Enterococcus faecalis and injected intraperitoneally with 5 · 106 colony- another was not identified. One pintado isolate was forming units (CFU)/fish, placed into 57-L tanks identified as a Streptococcus sp. and two pintado and maintained at 28 C. The fish were monitored isolates were not identified. An SI of 0.5 is needed for disease signs and mortality for 7 days. Because to identify an isolate. Biolog positive substrate there were no disease signs or mortality, another utilization by L. garvieae and E. seriolicida reference Journal compilation trial using a different Brazilian tilapia isolate (25- isolates (ATCC 43921 and ATCC 49156), Biolog 2009 9938-2) and pintado isolate (02-2409-2) was database isolates, and Brazilian pintado and tilapia Blackwell Publishing Ltd No claim to original US government works 945 Journal of Fish Diseases 2009, 32, 943–951 J J Evans et al. Isolation of Lactococcus garvieae from tilapia and pintado

Table 1 Identification of Lactococcus garvieae and other isolates isolates together and the other two tilapia isolates fromNiletilapia,Oreochromisniloticus,pintado,Pseudoplathystoma (30-9630-6, 30-9630-11) together with those corruscans, and American Type Culture Collection (ATCC) isolates either not identified (02-2409-2, 25- reference isolates using the BIOLOG identification systems 9590, 25-9590-1, 25-9590-2) or identified as Biolog Enterococcus faecalis (11-9446-6, 30-9630-7) by

Source Isolate ID PROB SIa Biolog. The L. garvieae ATCC 43921 reference strain utilized 37 of the Biolog compounds and Pintado 01-2408-1 Lactococcus 100 0.72 differed from the Biolog database isolates only in garvieae 01-2408-2 Streptococcus sp. – 0.19 its ability to utilize sucrose. The E. serolicida 02-2409-2 No ID – 0.35 ATCC 49156 reference strain utilized 31 carbon 25-9590 L. garvieae 100 0.60 sources but unlike the ATCC 43921 and Biolog 25-9590-1 L. garvieae 100 0.87 25-9590-2 No ID – 0.49 database isolates this strain did not utilize mannan, Tilapia 11-9446-6 Enterococcus 100 0.61 a-methyl-d-glucoside, b-methyl-d-glucoside, pal- faecalis atinose or turanose. The utilization pattern by 25-9938-2 L. garvieae 100 0.62 26-9939-4 No ID – 0.48 ATCC 49156 was almost identical to the pintado 30-9630-6 L. garvieae 100 0.55 and tilapia (25-9938-2) isolates with the exception 30-9630-7 E. faecalis 99 0.53 of utilization of arbutin and d-gluconic acid by 30-9630-11 L. garvieae 100 0.73 Yellowtailb ATCC 49156 L. garvieae 100 0.97 ATCC 49156 and not the Brazilian isolates, and Bovinec ATCC 43921 L. garvieae 100 0.87 utilization of palatinose and a-hydroxybutyric acid

Identification (ID) confidence was based on % probability (PROB) and by the Brazilian isolates and not by ATCC 49156. Similarity Index (SI) after 16–24 h incubation. Biolog compounds not utilized by ATCC, Biolog, aA Similarity Index of 0.5 is needed to identify an isolate in Biolog. or Brazilian fish Lactococcus garvieae isolates are bAmerican Type Culture Collection (ATCC) 49156 reference strain for Enterococcus serolicida from Kusuda et al. (1991). provided in Table 3. cATCC 43921 reference strain for L. garvieae from Collins et al. (1983). Experimental infectivity trials isolates previously identified as L. garvieae by Biolog are presented in Table 2. The Brazilian pintado The pintado isolate was not virulent when tested for isolate (01-2408-1) and three tilapia isolates tilapia under the experimental conditions. The (25-9938-2, 30-9630-6, 30-9630-11) collectively passaged tilapia isolate (25-9938-2) produced utilized 43 of the 95 (45%) carbon sources. Sixty- 70–100% mortality at 1.5 · 105 CFU/fish and at seven percent of the carbon sources utilized were doses ranging from 1 · 106 to 1 · 108 CFU/fish sugars and sugar derivatives followed by nucleosides within 7 days (Fig. 1). Forty percent mortality was and nucleotides (12%), carboxylic acids and methyl noted at a dose of 5 · 105 CFU/fish at 7 days. Fifty esters, amino acids, and alcohols (12%) and poly- percent mortality was achieved within 3 days for mers (9%). Twenty-nine carbon sources were all doses tested except 5 · 105 CFU/fish. The 5 utilized by all four fish isolates, ATCC reference estimated median LD50 was 1.41 · 10 CFU/fish. isolates and Biolog database isolates. The pintado The Brazilian tilapia isolate was re-isolated from the isolate and tilapia 25-9938-2 isolate exhibited iden- brain of all of the experimental tilapia sampled. tical substrate utilization, utilizing 31 compounds. The other two tilapia isolates (30-9630-6, 30-9630- Discussion 11) were more versatile in substrate utilization, utilizing 38 compounds and differed from each Lactococcus garvieae is a fish pathogen affecting other only in utilization of mannan, d-melibiose, numerous fish species globally (Vendrell et al. d-raffinose and stachyose. Eight compounds used by 2006). Until now, no evidence of L. garvieae has these tilapia isolates; a-cyclodextrin, b-cyclodextrin, been reported from Brazilian fish. Increasing arbutin, b-methyl-d-glucoside, a-d-lactose, sucrose, Brazilian tilapia culture (Kubitza 2004) may have d-gluconic acid, 2,3-butanediol were not utilized by spurred the L. garvieae outbreaks as disease the pintado or tilapia 25-9938-2 isolates. Con- epizootics are often reported from burgeoning versely, a-hydroxybutyric acid utilized by the pin- aquaculture production. The evidence of L. garvieae tado isolate was not used by these two tilapia infection in Brazilian Nile tilapia and pintado Journal compilation isolates. A Biolog dendrogram of all of the fish expands the geographical range of fish hosts and 2009 isolates grouped the pintado and tilapia (25-9938-2) number of fish hosts harbouring L. garvieae. Blackwell Publishing Ltd No claim to original US government works 946 Journal of Fish Diseases 2009, 32, 943–951 J J Evans et al. Isolation of Lactococcus garvieae from tilapia and pintado

Table 2 Biolog substrate utilization by Biolog database Lactococcus garvieae isolates (BI), American Type Culture Collection (ATCC) reference isolates, and Brazilian tilapia (T) and pintado (P) L. garvieae isolates

Substrates ATCC 43921 ATCC 49156 BI 01-2408-1 P 25-9938-2 T 30-9630-6 T 30-9630-11 T

Polymers a-Cyclodextrin xx b-Cyclodextrin xx Dextrin xxxx xx Mannan x x x Sugars and sugar derivatives Amygdalin xxxx xx Arbutin x x x x x N-acetyl-D-glucosamine xxxx xx N-acetyl-D-mannosamine xxxx xx D-cellobiose xxxx xx D-fructose xxxx xx D-galactose xxxx xx Gentiobiose xxxx xx D-gluconic acid x x x x x A-D-glucose xxxx xx A-D-lactose xx Maltose xxxx xx Maltotriose xxxx xx D-mannose xxxx xx D-mannitol xxxx xx D-melibiose x 3-Methyl-glucose xxxx xx a-Methyl-D-glucoside x x b-Methyl-D-glucoside x x x x Palatinose x x x x x D-psicose xxxx xx D-rafﬁnose x D-ribose xxxx xx Salicin xxxx xx D-sorbitol xxxx xx Stachyose x Sucrose xxx D-trehalose xxxx xx Turanose x x Carboxylic acids and methyl esters a-Hydroxybutyric acid x Methyl pyruvate xxxx xx Pyruvic acid xxxx xx Amino acids, peptides and related chemicals Alaninamide xxxx xx L-alanyl-glycine xxxx xx Alcohols 2,3-Butanediol xx Glycerol xxxx xx Nucleosides and nucleotides Adenosine xxxx xx 2-Deoxyadenosine xxxx xx Inosine xxxx xx Thymidine xxxx xx Uridine xxxx xx Totals 37 31 36 31 42 38

Bold substrates and shading indicate all isolates utilized that carbon source. Italicized substrates are common compounds in Biolog and API rapid ID 32 Strep. A blank space indicates that the carbon source was not used in Biolog, while an ÔxÕ indicates that the carbon source was utilized.

In studies of isolates from water buffalos with sucrose and mannitol in conventional tests. They subclinical mastitis, Teixeira et al. (1996) reported proposed to broaden the original description of atypical L. garvieae phenotypic traits including L. garvieae (ATCC 43921; Collins et al. 1983) to Journal compilation growth at 45 C, growth in broth containing include variants that grow at 45 C. The Brazilian 2009 6.5% NaCl, and failure to produce acid from ﬁsh isolates from this study and ﬁsh reference isolate Blackwell Publishing Ltd No claim to original US government works 947 Journal of Fish Diseases 2009, 32, 943–951 J J Evans et al. Isolation of Lactococcus garvieae from tilapia and pintado

Table 3 Compounds not utilized by ATCC, Biolog, or ﬁsh 110 Lactococcus garvieae isolates 100

Polymers 90 Inulin 80 Tween 40 Tween 80 70 Sugars and sugar derivatives 60 L-arabinose 50 D-arabitol L-fucose 40 D-galacturonic acid 30 m-Inositol 20 Lactulose Mean % cumulative mortality D-melezitose 10 a-Methyl-D-galactoside 0 a-Methyl-D-mannoside 0 1 2 3 4 5 6 7 8 9 10 L-rhamnose Days after challenge Sedoheptulosan D-tagatose Figure 1 Cumulative percent mortality among six groups of 10 Xylitol Nile tilapia each injected intraperitoneally with either 1.5 · 105 D-xylose (hourglass), 5.0 · 105 (circle), 1.0 · 106 (triangle), 5.0 · 106 Sugar phosphates 7 8 Fructose-6-phosphate (inverted triangle), 1.0 · 10 (hatched square) and 1.0 · 10 Glucose-1-phosphate (square) colony-forming units L. garvieae/fish. No mortality was Glucose-6-phosphate seen in fish injected with tryptic soy broth. D-l-a-glycerol phosphate Alcohols 2,3-Butanediol and ATCC 43921 and ATCC 49156 reference Nucleosides and nucleotides isolates also utilized mannitol in the Biolog system. Adenosine-5¢-monophosphate Thymidine-5¢-monophosphate Although positive mannitol fermentation has con- Uridine-5¢-monophosphate sistently been reported for ATCC 43921 and Amino acids, peptides, and related chemicals ATCC 49156 reference isolates and fish isolates N-acetyl-L-glutamic acid D-alanine regardless of methodology employed (Kusuda et al. L-asparagine 1991; Teixeira et al. 1996; Eldar et al. 1999; Vela, L-glutamic acid Va´zquez, Gibello, Blanco, Moreno, Lie´bana, l Glycyl- -glutamic acid ´ ´ ´ ´ L-pyroglutamic acid Albendea, Alcala,Mendez, Domınguez & Fernan- L-serine dez-Garayza´bal 2000; Ravelo, Magarinõs, Romalde Putrescine & Toranzo 2001), reports of sucrose, lactose, Carboxylic acids and methyl esters Acetic acid tagatose and cyclodextrin fermentation for ATCC b-Hydroxybutyric acid 43921 and ATCC 49156 reference isolates and fish c-Hydroxybutyric acid isolates have varied between L. garvieae studies. The p-Hydroxyphenyl acetic acid a-Ketoglutaric acid results of our conventional tests for the Brazilian a-Ketovaleric acid fish isolates as compared with conventional tests for c-Hydroxybutyric acid buffalo isolates as reported by Teixeira et al. (1996) p-Hydroxyphenyl acetic acid a-Ketoglutaric acid were identical with the exception of mannitol a-Ketovaleric acid (negative for buffalos and positive for fish) and Lactamide lactose (positive for buffalos and tilapia; negative for D-lactic acid methyl ester D-malic acid pintado and ATCC 49156). Despite the disparity L-malic acid in carbohydrate utilization derived from multi-test Mono-methyl succinate and conventional systems, the phenotypic hetero- Propionic acid Succinamic acid geneity of L. garvieae originating from different Succinic acid hosts and geographical regions has been proposed based on acidification of selected carbohydrates, d-tagatose, sucrose (saccharose), ribose, mannitol (ATCC 49156; Kusuda et al. 1991), like other fish and cyclodextrin and/or the presence of the isolates (Eldar et al. 1999), also grew at 45 C and enzymes pyroglutamic acid arylamidase and Journal compilation 6.5% NaCl indicating the likelihood that this is not N-acetyl-b-glucosaminidase (Eldar et al. 1999; Vela 2009 atypical for L. garvieae isolates. Brazilian isolates et al. 2000). Eldar et al. (1999) proposed three Blackwell Publishing Ltd No claim to original US government works 948 Journal of Fish Diseases 2009, 32, 943–951 J J Evans et al. Isolation of Lactococcus garvieae from tilapia and pintado

L. garvieae biotypes for fish and bovine strains of the Biolog system compared with API systems originating from Europe, Asia and Australia. In an include standardized media employed for isolation expanded study of trout, yellowtail, bovine, water (BUG) and inoculation (inoculation fluid) of buffalo and human isolates from various countries, bacterial inoculum into 96-well plates, the presence Vela et al. (2000) later proposed 13 intraspecies of 95 compounds from various compound classes, biotypes with seven of these biotypes containing fish internal L. garvieae reference database isolate(s) for isolates based on a combination of carbohydrate comparison to sample isolates, dendrogram capa- acidification and enzyme presence. In this classifi- bilities, and the ability to create a ÔUserÕ database for cation scheme, ATCC 43921 and ATCC 49156 individual organisms, atypical variants, or group of reference isolates belonged to different biotypes. In organisms for comparison to database isolate(s). Of addition, two Brazilian water buffalo isolates each the 95 compounds present in the Biolog system, 33 singularly constituted a different biotype and are present in the API Rapid ID 32 Strep and CH contained none of the isolates from any of the 50 systems and 62 compounds are not. Further- other 11 biotypes, suggesting Brazilian buffalo more, Biolog has 341 Gram-positive organisms of isolates may be genetically distinct from isolates veterinary and medical importance including fish examined from the eastern hemisphere. Interest- pathogens in their GP database from which to ingly, human isolates from both Brazil and the compare and identify isolates. The Biolog isolate(s) United States and the yellowtail reference strain and the bovine ATCC 43921 isolate had almost (ATCC 49156) comprised one biotype. Unfortu- identical profiles differing only in sucrose utiliza- nately, no biotype agreement could be established tion. It is likely this ATCC isolate serves as the between the studies of Eldar et al. (1999) and Vela standard to which unknown isolates are compared, et al. (2000). hence the high PROB of being identified and high These classification schemes have been met with SI in the Biolog system. The Brazilian capsulated some controversy. In later studies, Ravelo et al. pintado isolate and tilapia 25-9938-2 isolate exhib- (2001), employing 23 geographically diverse fish ited identical substrate utilization, utilizing 31 isolates, indicated a high level of biochemical compounds in the Biolog system. The Ômetabolic homogeneity among these strains regardless of their fingerprintÕ of these isolates corresponded most origin of isolation. These researchers suggested that closely with the fish ATCC 49156 isolate. variations in acid production from sucrose, lactose, Despite having the same metabolic profiles as tagatose and cyclodextrin using API Rapid ID 32 each other and the fish ATCC 49156 isolate, the Strep system vs. conventional methods were the pintado isolate, unlike the tilapia isolate, was not result of different culture media, inoculum density experimentally infective to tilapia. In experimental and incubation temperature and times employed. LD50 studies by Kawanishi, Yoshida, Yagashiro, Furthermore, they refuted the use of previously Kijima, Yagyu, Nakai, Murakami, Morita & Suzuki reported biotyping schemes employing carbo- (2006), a capsulated Japanese yellowtail isolate was hydrate utilization for differentiation of L. garvieae significantly more pathogenic to yellowtail than an biotypes. Thus, caution should be exercised when uncapsulated Spanish trout isolate indicating that comparing L. garvieae isolates to previous charac- the host source and capsule presence may play a role terizations which used API systems. The ability of in virulence. Although not tested for pathogenicity, Rapid ID 32 Strep and other API systems to two of the Brazilian tilapia isolates were more identify L. garvieae, to discriminate between the versatile in carbon source utilization than the similar species L. lactis subsp. lactis and L. garvieae yellowtail ATCC 49156 reference isolate and and biotype isolates appears to be variable and the pintado isolate and better corresponded to the unreliable (Collins et al. 1983; Elliott et al. 1991; bovine ATCC 43921 reference isolate and Biolog Domeńech et al. 1993; Facklam & Elliott 1995). database isolate(s). Biolog results indicate that The Biolog system contains L. lactis lactis but did Brazilian fish isolates are similar to both piscine not identify Brazilian isolates as such. In the absence and mammalian ATCC reference isolates. The of PCR assay technology known to discriminate biodiversity of these Brazilian fish isolates compared between L. lactis subsp. lactis and L. garvieae, the with strains from different hosts and geographical identification of L. garvieae by the Biolog system areas may be answered by employing molecular Journal compilation poses an alternative to API Rapid ID 32 Strep and epidemiological techniques. Molecular techniques 2009 CH 50 systems and supplemental tests. Advantages have highlighted genetic clonality and diversity and Blackwell Publishing Ltd No claim to original US government works 949 Journal of Fish Diseases 2009, 32, 943–951 J J Evans et al. Isolation of Lactococcus garvieae from tilapia and pintado

contributed to a better understanding of the and meningoencephalitis in fish. Current Microbiology 32, epidemiology of L. garvieae from fish and other 85–88. infection sources (Eldar et al. 1999; Vela et al. Eldar A., Goria M., Ghittino C., Zlotkin A. & Bercovier H. 2000; Ravelo, Magarinõs, Lopez-Romalde, Toranzo (1999) Biodiversity of Lactococcus garvieae strains isolated from fish in Europe, Asia, and Australia. Applied and Environmental & Romalde 2003; Schmidtke & Carson 2003; Microbiology 65, 1005–1008. Eyngor et al. 2004; Kawanishi et al. 2006). Vela Elliott J.A., Collins M.D., Pigott E. & Facklam R.R. (1991) et al. (2000) demonstrated genetic relatedness Differentiation of Lactococcus lactis and Lactococcus garvieae between bovine ATCC 43921 and water buffalo from humans by comparison of whole-cell protein patterns. isolates, although Kawanishi et al. (2006) demon- Journal of Clinical Microbiology 29, 2731–2734. strated homogeneity between Japanese yellowtail Evans J.J., Pasnik D.J., Klesius P.H. & Al-Ablani S. (2006) First isolates but not between isolates obtained from report of Streptococcus agalactiae and Lactococcus garvieae from terrestrial animals. Eyngor et al. (2004) contend a wild bottlenose dolphin (Tursiops truncatus). Journal of Wildlife Diseases 42, 561–569. that endemic L. garvieae isolates possess a clonal structure while major genetic heterogeneity is Eyngor M., Zlotkin A., Ghittino C., Prearo M., Douet D.G., Chilmonczyk S. & Eldar A. (2004) Clonality and diversity of displayed where L. garvieae is sporadic. Previously the fish pathogen Lactococcus garvieae in Mediterranean recognized from Brazilian water buffalos (Teixeira countries. Applied and Environmental Microbiology 70, et al. 1996), L. garvieae occurrence in Brazilian fish 5132–5137. poses the question whether these strains are Facklam R.R. & Elliott J.A. 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