Characterization of Lactococcus garvieae isolated from radish and broccoli sprouts that exhibited a KG+ phenotype, lack of virulence and absence of a capsule

AIMS: To identify Lactococcus garvieae isolates from radish and broccoli sprouts and compare them with virulent and less virulent mutant strains obtained from yellowtails with regard to KG phenotype, presence of a capsule and virulence towards yellowtails and mice. METHODS AND RESULTS: Comparative 16S rRNA gene sequence analysis of six isolates obtained from radish and broccoli sprouts indicated that they were L. garvieae (similarity >99%). They were compared with KG9502, Lg2 and ATCC49156 strains obtained from yellowtails. A less virulent mutant strain Lg2-S was obtained by Lg2 subculture. Biochemical characterization of the six strains resembled that of KG9502, Lg2, ATCC49156 and Lg2-S, except for saccharose and tagatose acidification and the presence of hippuricase. These six strains were nonpathogenic towards yellowtails and mice, nonsusceptible to bacteriophages and demonstrated heterogeneity on pulsed-field gel electrophoresis analysis. Using transmission electron microscopy, a capsule was observed in KG9502 and Lg2 but not in ATCC49156 and Lg2-S. CONCLUSIONS: We isolated L. garvieae strains that lacked pathogenicity towards yellowtails and mice from radish and broccoli sprouts; these were noncapsulated and exhibited KG(+) phenotype. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first documentation of L. garvieae isolated from terrestrial plants. These isolates exhibited genetic diversity; however, they were noncapsulated and nonpathogenic towards yellowtails and mice.     

Drug resistance and pulsed-field gel electrophoresis patterns of Lactococcus garvieae isolates from cultured Seriola (yellowtail, amberjack and kingfish) in Japan

AIMS: To investigate the existing antimicrobial susceptibility and genetic characteristics of Lactococcus garvieae isolates from cultured Seriola in Japan. METHODS AND RESULTS: Minimum inhibitory concentrations (MICs) of 14 antimicrobial agents for 170 isolates were determined using the agar dilution method. Seventy-five isolates (44.1%) were simultaneously resistant to erythromycin (EM) (MIC>or=2 microg ml-1), lincomycin (LCM) (MIC>or=128 microg ml-1) and oxytetracycline (OTC) (MIC>or=4 microg ml-1). Resistance to EM was grouped as intermediate- and high-level resistant by MIC values. All resistant isolates possessed ermB and tet(S) genes. The number of different bands between pulsed-field gel electrophoresis patterns of 25 isolates and two ATCC strains (isolated in 1974), determined using two enzymes (ApaI and SmaI), did not exceed 3. CONCLUSIONS: The present resistance pattern observed with ermB and tet(S) is similar to that observed in previous reports. Moreover, the genetic characteristics of L. garvieae isolates from a wide area in Japan in 2002 and ATCC strains were closely related. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that EM-, LCM- and OTC-resistant isolates have been present for 15 years and that L. garvieae strains with same origin have spread among Seriola spp. in Japan since 1974.     

Partial two-dimensional gel electrophoresis (2-DE) maps of Streptococcus iniae ATCC29178 and Lactococcus garvieae KG9408

To examine the proteomes of 2 important causative agents of fish streptococcosis, Streptococcus iniae ATCC29178 and Lactococcus garvieae KG9408, we used 2-dimensional gel electrophoresis (2-DE) followed by mass spectrometry to generate 2-DE maps of these type strains. Silver-stained 2-DE gels of S. iniae ATCC29178 and L. garvieae KG9408 revealed approximately 320 and 300 spots, respectively, and immobilized pH gradient strips (13 cm, pH 4 to 7) revealed that the majority of the detected spots were concentrated in the pH range of 4.5 to 5.5. The spots were randomly selected from the 2-DE profiles and identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry. The majority of the identified proteins were functionally related to energy and carbohydrate metabolism (e.g. enolase ATPase, glyceraldehyde-3-phosphate dehydrogenase) or translation and translocation (e.g. elongation factor G, elongation factor Tu, DNA-directed RNA polymerase alpha chain). These data, along with our partial 2-DE maps of S. iniae ATCC29178 and L. garvieae KG9408, may help suggest antigenic proteins for the development of effective diagnostic tools and vaccines against S. iniae and L. garvieae.     

华北地区牛源无乳链球菌的分离鉴定及生物学特性

【目的】了解华北地区牛源无乳链球菌的生物学特性。【方法】在2012到2015年间从内蒙古自治区、河北、北京等地隐性乳房炎557份奶牛乳样中分离、收集无乳链球菌。采用纸片扩散法和PCR的方法对这些菌株分别进行耐药谱测定、荚膜分子分型、表面蛋白基因及毒力因子的检测。【结果】无乳链球菌的分离率为5.03%,其药物敏感性与其他地区无明显差别。分离到的28株无乳链球菌均属于荚膜Ia型,且毒力基因基本相同并且其表面蛋白均属于未定型。【结论】华北不同地区的无乳链球菌有相似的药物敏感性和毒力基因。为奶牛乳房炎无乳链球菌疫苗的研制及药物防治提供理论依据。     

Diversity of lactic acid bacteria associated with fish and the fish farm environment, established by amplified rRNA gene restriction analysis

Lactic acid bacteria have become a major source of concern for aquaculture in recent decades. In addition to true pathogenic species of worldwide significance, such as Streptococcus iniae and Lactococcus garvieae, several species have been reported to produce occasional fish mortalities in limited geographic areas, and many unidentifiable or ill-defined isolates are regularly isolated from fish or fish products. To clarify the nature and prevalence of different fish-associated bacteria belonging to the lactic acid bacterium group, a collection of 57 isolates of different origins was studied and compared with a set of 22 type strains, using amplified rRNA gene restriction analysis (ARDRA). Twelve distinct clusters were delineated on the basis of ARDRA profiles and were confirmed by sequencing of sodA and 16S rRNA genes. These clusters included the following: Lactococcus raffinolactis, L. garvieae, Lactococcus l., S. iniae, S. dysgalactiae, S. parauberis, S. agalactiae, Carnobacterium spp., the Enterococcus "faecium" group, a heterogeneous Enterococcus-like cluster comprising indiscernible representatives of Vagococcus fluvialis or the recently recognized V. carniphilus, V. salmoninarum, and Aerococcus spp. Interestingly, the L. lactis and L. raffinolactis clusters appeared to include many commensals of fish, so opportunistic infections caused by these species cannot be disregarded. The significance for fish populations and fish food processing of three or four genetic clusters of uncertain or complex definition, namely, Aerococcus and Enterococcus clusters, should be established more accurately.     

Differentiation of Shewanella putrefaciens and Shewanella alga on the basis of whole-cell protein profiles, ribotyping, phenotypic characterization, and 16S rRNA gene sequence analysis.

Seventy-six presumed Shewanella putrefaciens isolates from fish, oil drillings, and clinical specimens, the type strain of Shewanella putrefaciens (ATCC 8071), the type strain of Shewanella alga (IAM 14159), and the type strain of Shewanella hanedai (ATCC 33224) were compared by several typing methods. Numerical analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell protein and ribotyping patterns showed that the strains were separated into two distinct clusters with 56% +/- 10% and 40% +/- 14% similarity for whole-cell protein profiling and ribotyping, respectively. One cluster consisted of 26 isolates with 52 to 55 mol% G + C and included 15 human isolates, mostly clinical specimens, 8 isolates from marine waters, and the type strain of S. alga. This homogeneous cluster of mesophilic, halotolerant strains was by all analyses identical to the recently defined species S. alga (U. Simidu et al., Int. J. Syst. Bacteriol, 40:331-336, 1990). Fifty-two typically psychrotolerant strains formed the other, more heterogeneous major cluster, with 43 to 47 mol% G + C. The type strain of S. putrefaciens was included in this group. The two groups were confirmed by 16S rRNA gene sequence analysis. It is concluded that the isolates must be considered two different species, S. alga and S. putrefaciens, and that most mesophilic isolates formerly identified as S. putrefaciens belong to S. alga. The ecological role and potential pathogenicity of S. alga can be evaluated only if the organism is correctly identified.     

Classification and characterization of lactic acid bacteria isolated from the intestines of common carp and freshwater prawns

Fifty-four isolates of lactic acid bacteria were obtained from the intestines of the common carp (Cyprinus carpio) and freshwater prawns (Macrobrachium rosenbergii) in Nakorn-Pathom Province, Thailand. All isolates were Gram-positive and catalase-negative cocci that did not produce gas from glucose and formed dl or L(+) lactic acid only. Most isolates were able to grow in broth at pH 9.6, in 6.5% NaCl (w/v) and 40% (w/v) bile. These isolates were divided into six groups (A-F) by sugar fermentation patterns. Strains in the groups A, B, C, and D showed intergroup DNA homology values of above 73.8%, indicating that these groups were composed of a single species. Following phylogenetic analysis, strains E 1, E 7, and E 26 from groups A, E, and F were placed in the clusters of the genera Lactococcus, Pediococcus, and Enterococcus, respectively. The type strains of Lactococcus garvieae, Pediococcus acidilactici, and Enterococcus faecium were the most closely related species with E 1, E 7, and E 26 in the phylogenetic tree, respectively. The DNA-DNA hybridization results indicated that strains in groups A (including groups B, C, and D), E, and F could be identified as belonging to the species Lactococcus garvieae, Pediococcus acidilactici, and Enterococcus faecium, respectively. Lactococcus garvieae was the dominant member of the population, accounting for 90.7% of the isolates.     

Lactococcus garvieae infection in the giant freshwater prawn Macrobranchium rosenbergii confirmed by polymerase chain reaction and 16S rDNA sequencing.

An epizootic bacterial infection in the giant freshwater prawn Macrobranchium rosenbergii occurred in Taiwan from May to June 1999. The cumulative mortality was approximately 30 to 75%. The diseased prawns showed opaque and whitish muscles and were approximately 2 mo old with total lengths from 5 to 6 cm. Histopathologically, they showed marked edema and necrotic lesions with inflammation in the muscles and hepatopancreas. Bacteria isolated using brain heart infusion medium or tryptic soy agar were Gram-positive and ovoid. Three isolates from diseased prawns at different farms were tested using the API 20 Strepsystem and conventional tests and identified as Lactococcus garvieae. Experimental infections with these isolates gave gross signs and histopathological changes similar to those seen in the naturally infected prawns. The LD50 value of isolate MR1 was 6.6 x 10(5) colony forming units/prawn. Identification of MR1 was confirmed by a PCR assay for L. garvieae that gave the expected amplicon of 1100 bp. In addition, its 16S rDNA sequence (GenBank accession number AF283499) gave 99% sequence identity to Enterococcus seriolicida (synonym L. garvieae; GenBank accession number AF061005). This is the first report of confirmed L. garvieae infection in prawn aquaculture.     

Lactococcus lactis subspecies lactis also causes white muscle disease in farmed giant freshwater prawns Macrobrachium rosenbergii

From May to August 2001 in Taiwan, 27 farms for the giant freshwater prawn Macrobrachium rosenbergii experienced white tail disease outbreaks in animals approximately 3 to 5 mo old, with total lengths from 6 to 8 cm. Examination of the infected prawns revealed not only previously reported Lactococcus garvieae (16 farms) but also the novel L. lactis subsp. lactis (10 farms). One farm had shrimp infected with both bacteria. In the farms with L. lactis infections, the cumulative mortality was approximately 25 to 60%. Gross signs of disease were opaque and whitish muscles, while histopathology included marked edema and necrotic lesions, with inflammation in the muscles and hepatopancreas. Bacteria isolated using brain/heart infusion medium or tryptic soy agar were Gram-positive and ovoid. Eleven isolates from different farms were identified as L. lactis subsp. lactis using API 20 Strep and Rapid ID32 Strep tests and using PCR assays specific for the L. lactis subsp. lactis 16S rDNA gene (650 bp amplicon) and for the 16S to 23S rDNA interspacer region (380 bp amplicon). In addition, sequencing of the full 16S rDNA genes of 2 of the isolates (MR17 and MR26; GenBank Accession Numbers AF493058 and AF493057, respectively) revealed 99.9% identity between the isolates and 98.7% identity to several complete 16S rRNA sequences of L. lactis subsp. lactis at GenBank. Experimental infections with our isolates gave gross signs and histopathological changes similar to those seen in naturally infected prawns. The mean lethal dose of 4 isolates and the reference strain L. lactis subsp. lactis BCRC 10791 ranged from 4.2 x 10(6) to 2.5 x 10(7) colony-forming units prawn(-1), indicating virulence similar to that previously reported for L. garvieae. This is the first report confirming L. lactis subsp. lactis as a pathogen in juvenile and adult prawns from aquaculture.     

牛源无乳链球菌长尾噬菌体的特性

【目的】分离鉴定噬菌体,对其生物学特性进行研究,并筛选候选毒株为防控牛源无乳链球菌的感染提供依据。【方法】分别采用从牛奶或环境中分离、溶原菌诱导两种方法分离鉴定无乳链球菌噬菌体,利用双层琼脂平板法纯化。将新分离鉴定毒株与前期已分离鉴定的源自乳腺炎牛奶的无乳链球菌噬菌体JX01进行分析和比较,包括噬菌体透射电镜形态观察、对55株无乳链球菌和其他细菌的宿主谱鉴定、噬菌体基因Eco R I、Sal I、Xba I或Pst I的酶切图谱、最适MOI、吸附曲线和一步生长曲线、不同保存条件下的稳定性等。【结果】分离鉴定的3株噬菌体LYGO9、HZ04和p A11(诱导自牛源菌株HAJL2011070601)与JX01比对分析,结果显示,4株噬菌体均为长尾噬菌体;Eco R I、Sal I、Xba I、Pst I的酶切图谱分获4、3、3或2种带型,显示4株噬菌体为不同毒株;均特异性裂解牛源无乳链球菌,对42株牛源无乳链球菌的裂解率如下:LYGO9为28.6%(12/42)、p A11为31%(13/42)、HZ04为47.6%(20/42)、JX01为54.8%(23/42);同时,LYGO9与p A11、HZ04和JX01分别有共同宿主11、12和11株;HZ04与JX01有共同宿主18株,提示它们具有同源性。LYGO9感染宿主的潜伏期短,仅5 min,平均裂解量为30。分离株在SM液中4°C至少可保存1个月。【结论】分离鉴定的3株牛源无乳链球菌噬菌体均为长尾噬菌体,其中LYGO9潜伏期短、裂解量较大。     

Occurrence of tetracycline resistance genes tet(M) and tet(S) in bacteria from marine aquaculture sites

Occurrence of tetracycline resistance genes encoding ribosomal protection proteins was examined in 151 tetracycline-resistant bacterial isolates from fish and seawater at coastal aquaculture sites in Japan and Korea. The tet(M) gene was detected in 34 Japanese and Korean isolates, which included Vibrio sp., Lactococcus garvieae, Photobacterium damsela subsp. piscicida, and unidentified Gram-positive bacteria. The majority of these bacterial isolates displayed high-level resistance with a minimum inhibitory concentrations (MICs) equal to or greater than 250 microg/ml of oxytetracycline and only four isolates had MICs less than 31.3 microg/ml. 16S rDNA RFLP typing of tet(M)-positive Vibrio isolates suggests that these are clonal populations of the same phylotype specific to a particular location. One Vibrio clone (phylotype III), however, is widely disseminated, being detected during different sampling years, at different locations, and in different fish species in both Japan and Korea. The tet(S) gene was detected in L. garvieae from yellowtail in Japan and in Vibrio sp. from seawater in Korea. This is the first report of tet(S) occurrence in Gram-negative facultative anaerobes. These results suggest that tet(M) and tet(S) genes are present in fish intestinal and seawater bacteria at aquaculture sites and could be an important reservoir of tetracycline resistance genes in the marine environment.     

Primary bacterial pathogens in bottlenose dolphins Tursiops truncatus: needles in haystacks of commensal and environmental microbes

Bacterial cultures of marine mammal samples often yield multiple genera and species, and it can be difficult to determine if a cultured bacterium is a primary pathogen or an incidental finding. To determine the relative risk of bacterial isolates among Atlantic bottlenose dolphins Tursiops truncatus at the United States Navy Marine Mammal Program (MMP), retrospective data on isolates cultured during June 1987 through June 2007 were organized into a novel, 5-tier risk categorization system limited to sole bacteria cultured from internal organ or fluid samples. Of 2586 bacterial isolates cultured, only 34 (1.3%) and 25 (1.0%) were sole isolates attributed to morbidity and mortality, respectively, and only 19 (0.7%) isolates were associated with mortalities without evidence of fungal or viral co-infections. Highest risk bacterial isolates were most likely to be identified in pleural fluid (33.3% of pleural fluid samples with bacterial isolates had only one genus), followed by renal (23.1%) and splenic (11.1%) tissue. Sole Staphylococcus aureus isolates were identified as the highest risk bacterial pathogens in the MMP dolphin population, accounting for 0.4 % of total bacterial isolates over a 20 yr period. In summary, isolation of sole bacterial isolates definitively associated with morbidity and mortality in marine mammals was uncommon in the MMP population. Our proposed risk categorization system may be useful in determining high risk pathogens among other marine mammal populations.     

Complete genome sequence and comparative analysis of the fish pathogen Lactococcus garvieae

Lactococcus garvieae causes fatal haemorrhagic septicaemia in fish such as yellowtail. The comparative analysis of genomes of a virulent strain Lg2 and a non-virulent strain ATCC 49156 of L. garvieae revealed that the two strains shared a high degree of sequence identity, but Lg2 had a 16.5-kb capsule gene cluster that is absent in ATCC 49156. The capsule gene cluster was composed of 15 genes, of which eight genes are highly conserved with those in exopolysaccharide biosynthesis gene cluster often found in Lactococcus lactis strains. Sequence analysis of the capsule gene cluster in the less virulent strain L. garvieae Lg2-S, Lg2-derived strain, showed that two conserved genes were disrupted by a single base pair deletion, respectively. These results strongly suggest that the capsule is crucial for virulence of Lg2. The capsule gene cluster of Lg2 may be a genomic island from several features such as the presence of insertion sequences flanked on both ends, different GC content from the chromosomal average, integration into the locus syntenic to other lactococcal genome sequences, and distribution in human gut microbiomes. The analysis also predicted other potential virulence factors such as haemolysin. The present study provides new insights into understanding of the virulence mechanisms of L. garvieae in fish.     

Comparative genome analysis of Lactococcus garvieae using a suppression subtractive hybridization library: discovery of novel DNA signatures

Lactococcus garvieae, the pathogenic species in the genus Lactococcus, is recognized as an emerging pathogen in fish, animals, and humans. Despite the widespread distribution and emerging clinical significance of L. garvieae, little is known about the genomic content of this microorganism. Suppression subtractive hybridization was performed to identify the genomic differences between L. garvieae and Lactococcus lactis ssp. lactis, its closest phylogenetic neighbor, and the type species of the genus Lactococcus. Twenty-seven clones were specific to L. garvieae and were highly different from Lactococcus lactis in their nucleotide and protein sequences. Lactococcus garvieae primer sets were subsequently designed for two of these clones corresponding to a pyrH gene and a novel DNA signature for application in the specific detection of L. garvieae. The primer specificities were evaluated relative to three previously described 16S rRNA gene-targeted methods using 32 Lactococcus and closely related strains. Both newly designed primer sets were highly specific to L. garvieae and performed better than did the existing primers. Our findings may be useful for developing more stable and accurate tools for the discrimination of L. garvieae from other closely related species.     

Occurrence and drug-resistance of beta-hemolytic streptococci

The aim of this study was the analysis of drug-resistance and frequency appearance of beta-hemolytic streptococci strains which were isolated in 2003-2005 in the University Hospital at the L. Rydygier Collegium Medicum in Bydgoszcz University of Nicolaus Copernicus in Toruń. Among investigeted beta-hemolytic streptococci the most frequency isolated species was S. agalactiae. All isolates examined in our study were susceptible to penicillin, the higest rate of resistance was found for tetracycline. The rates of resistence to macrolide-lincosamide-streptogramin B (phenotyp MLS(B)) were as follows: S. agalactiae (18.7%), S. pyogenes (10.1%), group G streptococci (10.6%) and group C streptococci (8.0%). In our study we presented also a special case patient from which in investigeted period S. agalactiae was isolated twenty eight times. For ten chromosomal DNA isolated from this patient three different PFGE profiles were obtained.     

Shewanella and Photobacterium spp. in oysters and seawater from the Delaware Bay

Shewanella algae, S. putrefaciens, and Photobacterium damselae subsp. damselae are indigenous marine bacteria and human pathogens causing cellulitis, necrotizing fasciitis, abscesses, septicemia, and death. Infections are rare and are most often associated with the immunocompromised host. A study was performed on the microbiological flora of oysters and seawater from commercial oyster harvesting sites in the Delaware Bay, New Jersey. From 276 water and shellfish samples tested, 1,421 bacterial isolates were picked for biochemical identification and 170 (12.0%) of the isolates were presumptively identified as S. putrefaciens, 26 (1.8%) were presumptively identified as P. damselae subsp. damselae, and 665 (46.8%) could not be identified using the API 20E identification database. Sequencing of the 16S rRNA genes of 22 S. putrefaciens-like isolates identified them as S. abalonesis, S. algae, S. baltica, S. hafniensis, S. marisflavi, S. putrefaciens, Listonella anguillarum, and P. damselae. Beta-hemolysis was produced by some S. algae and P. damselae isolates, while isolates of S. baltica and L. anguillarum, species perceived as nonpathogenic, also exhibited beta-hemolysis and growth at 37 degrees C. To our knowledge, this is the first time these beta-hemolytic strains were reported from shellfish or seawater from the Delaware Bay. Pathogenic Shewanella and Photobacterium species could pose a health threat through the ingestion of contaminated seafood, by cuts or abrasions acquired in the marine environment, or by swimming and other recreational activities.     

Strain variation in Mycobacterium marinum fish isolates

A molecular characterization of two Mycobacterium marinum genes, 16S rRNA and hsp65, was carried out with a total of 21 isolates from various species of fish from both marine and freshwater environments of Israel, Europe, and the Far East. The nucleotide sequences of both genes revealed that all M. marinum isolates from fish in Israel belonged to two different strains, one infecting marine (cultured and wild) fish and the other infecting freshwater (cultured) fish. A restriction enzyme map based on the nucleotide sequences of both genes confirmed the divergence of the Israeli marine isolates from the freshwater isolates and differentiated the Israeli isolates from the foreign isolates, with the exception of one of three Greek isolates from marine fish which was identical to the Israeli marine isolates. The second isolate from Greece exhibited a single base alteration in the 16S rRNA sequence, whereas the third isolate was most likely a new Mycobacterium species. Isolates from Denmark and Thailand shared high sequence homology to complete identity with reference strain ATCC 927. Combined analysis of the two gene sequences increased the detection of intraspecific variations and was thus of importance in studying the taxonomy and epidemiology of this aquatic pathogen. Whether the Israeli M. marinum strain infecting marine fish is endemic to the Red Sea and found extremely susceptible hosts in the exotic species imported for aquaculture or rather was accidentally introduced with occasional imports of fingerlings from the Mediterranean Sea could not be determined.     

Isolation of St. Louis encephalitis virus from a killer whale

We report the isolation of St. Louis Encephalitis (SLE) virus from a mature male killer whale (Orcinus orca). This represents the first isolation of SLE virus from a marine mammal. The animal presented with reduced appetite, rapidly became lethargic and subsequently died. Virus-induced CPE was observed in a dolphin cell line, SP-1K (ATCC CCL 78), inoculated with brain, kidney, and lung tissues obtained at necropsy. Electron microscopy of infected SP-1K cells revealed the presence of virions having morphology and size resembling members of the Flaviviridae. Final identification as SLE virus was made by neutralization and immunofluorescence staining tests.     

Isolation and genetic characterization of Toxoplasma gondii from striped dolphin (Stenella coeruleoalba) from Costa Rica

Toxoplasma gondii infection in marine mammals is of interest because of mortality and mode of transmission. It has been suggested that marine mammals become infected with T. gondii oocysts washed from land to the sea. We report the isolation and genetic characterization of viable T. gondii from a striped dolphin (Stenella coeruleoalba), the first time from this host. An adult female dolphin was found stranded on the Pacific Coast of Costa Rica, and the animal died the next day. The dolphin had a high (1:6400) antibody titer to T. gondii in the modified agglutination test. Severe nonsuppurative meningoencephalomyelitis was found in its brain and spinal cord, but T. gondii was not found in histological sections of the dolphin. Portions of its brain and the heart were bioassayed in mice for the isolation of T. gondii. Viable T. gondii was isolated from the brain, but not from the heart, of the dolphin. A cat fed mice infected with the dolphin isolate (designated TgSdCol) shed oocysts. Genomic DNA from tachyzoites of this isolate was used for genotyping at 10 genetic loci, including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico, and this TgSdCo1 isolate was found to be Type II.     

Comparison of Lactococcus garvieae strains isolated in northern Italy from dairy products and fishes through molecular typing

Aims:  To investigate the genetic relatedness between Lactococcus garvieae strains isolated from fish and dairy samples collected in northern Italy, using random-amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR), Sau -PCR and amplified fragment length polymorphism (AFLP).
Methods and Results:  Eighty-one isolates from bovine and caprine dairy products ( n  = 53) and from diseased rainbow trouts and other fishes ( n  = 28) were examined. All methods showed a typeability of 100%, repeatability ranging from 84·4% to 97·5% and discriminatory powers from 0·798 to 0·986. Dairy and fish strains revealed a low genetic relatedness as they are often grouped into distinct clusters. RAPD analysis discriminated 52 genotypes when primer M13 was used, whereas with primer P5 only 27 genotypes were identified. When Sau -PCR was performed, 13 genotypes were detected while AFLP analysis allowed the differentiation of 32 genotypes.
Conclusion:  L. garvieae strains isolated from dairy samples are generally not related to those collected from fish lactococcosis outbreaks.
Significance and Impact of the Study:  L. garvieae strains exhibit a genetic diversity related to the specific animal host they colonize. RAPD M13 fingerprinting proved to be a molecular tool for comparing isolates, whereas Sau -PCR and AFLP analyses were useful techniques to investigate the distribution of L. garvieae populations in the environment.