Presence of the Streptococcus suis suilysin gene and expression of MRP and EF correlates with high virulence in Streptococcus suis type 2 isolates

Nineteen Streptococcus suis type 2 isolates that had been analyzed previously for hemolysin production, ribotype, and virulence in pigs were examined for presence of the gene coding for suilysin by PCR amplification, and southern blot and hybridization techniques. Based on southern blot and hybridization analysis, all isolates tested contained at least a portion of the suilysin gene. PCR amplification of the entire gene resulted in gene fragments from five of the seven highly virulent isolates and none of the moderately virulent or avirulent isolates. Additional PCR analysis showed that mutation or deletions at the 5′ end of the suilysin gene in the less virulent isolates prevented amplification of the sly gene fragment from those isolates. The MRP+ (muramidase-released protein) EF+ (extracellular protein) phenotype was also expressed by the same five highly virulent/sly+ isolates.     

Serotype distribution and production of muramidase-released protein, extracellular factor and suilysin by field strains of Streptococcus suis isolated in the United States

Streptococcus suis is an important swine pathogen and a zoonotic agent. Differences in virulence have been noted among the 33 described serotypes, serotype 2 being considered the most virulent. In this study, we aimed at assessing the serotype distribution and the production of virulence-associated markers by strains recovered from diseased pigs in the United States (U.S.). Results showed that among the 100 strains evaluated, serotype 3 (20% of the isolates) and serotype 2 (17%) were the most prevalent. We then investigated the presence in these isolates of the genes sly, epf and mrp, encoding the virulence-associated markers suilysin (SLY), extracellular factor (EF) and muramidase-released (MRP) protein, respectively. The effective production of the markers by the strains was also verified. Results showed that the presence of the gene did not always correlate with actual expression of the respective protein. In the case of MRP, this was due, in most cases, to frameshift mutations at the 5′ end of the gene resulting in premature stop codons. The most prevalent phenotypes among U.S. strains were MRP⁺EF⁻SLY⁻ (40%) and MRP⁻EF⁻SLY⁺ (35%). Serotype distribution greatly differed from that reported in several European countries, as did the production of virulence markers, particularly for serotype 2. On the other hand, our results for the U.S. S. suis isolates are similar to those reported for Canadian strains, suggesting a common status in North America.     

Genetic organization and preferential distribution of putative pilus gene clusters in Streptococcus suis

Recent analyses of Streptococcus suis isolates using multilocus sequence typing (MLST) suggested the importance of sequence type (ST) 1 and ST27 complexes for animal hygiene and public health. In this study, to investigate whether pilus-associated genes in S. suis can be used as novel genetic markers for important clonal groups, we examined the correlation between STs and putative pilus-associated gene profiles in S. suis. Genomic searches using sequenced genomes and sequence data determined in several isolates revealed the presence of at least four distinct putative pilus gene clusters in S. suis (srtBCD, srtE, srtF, and srtG clusters). On the basis of the presence or absence of genes in the four clusters, 108 S. suis isolates from various origins were classified into 12 genotypes (genotypes A–L). Genotypes A and B, which possessed srtBCD plus srtF clusters and srtF plus srtG clusters, respectively, were the most common in isolates from diseased pigs and humans, and 29.9% and 59.8% of the isolates belonged to genotypes A and B, respectively. In contrast, only 4.8% and 28.6% of isolates from healthy carriers were genotypes A and B, respectively. MLST analysis showed the associations of genotypes A and B with ST1 and ST27 complexes, respectively. In addition, srtBCD and srtG clusters were preferentially distributed to ST1 and ST27 complex members, respectively. These results suggest that profiling of selected pilus-associated genes could be used as an easy screening method to monitor isolates important for S. suis infection.     

Identification of the tet(B) resistance gene in Streptococcus suis

The tetracycline resistance gene, tet(B), has been described previously in Gram negative bacteria. In this study tet(B) was detected in plasmid extracts from 17/111 (15%) Streptococcus suis isolates from diseased pigs, representing the first report of this resistance gene in Gram positive bacteria.     

ermB-mediated erythromycin resistance in Streptococcus uberis from bovine mastitis

The ermB gene was identified in 111 erythromycin resistant isolates of Streptococcus uberis from cases of bovine mastitis associated either with a constitutive (47/111) or an inducible (64/111) phenotype, as well as a phenotypic resistance to all macrolides tested. Resistance to lincosamides was identified in 14 other isolates of S. uberis from bovine mastitis cases and was mainly mediated by the linB gene; resistance conferred by a combination of two genes (linB–lnuD, ermB–linB) was also detected.     

Identification of a Streptococcus equi Strain Responsible for Four Outbreaks of Strangles in Colorado

The objective of this study was to use a new subtyping technique to determine the identity of five Streptococcus equi isolates from four outbreaks of strangles in Colorado during 2005−2006. All five of the isolates from the four strangles outbreaks in Colorado contained SeM allele 28. This SeM allele is typical of American isolates of S. equi and has previously been linked with strangles outbreaks in Minnesota in 1994 and Kentucky in 1995. Use of the new S. equi strain subtyping method allowed for characterization of recent S. equi isolates associated with outbreaks of strangles in Colorado. To our knowledge, this is the first report of typing of more recent isolates from North America. This approach, however, has been used in situations in the United Kingdom to differentiate the vaccine strain of S. equi from that of the wild type of the bacteria.     

Pheno- and genotyping of Rhodococcus equi isolated from faeces of healthy horses and cattle

The present study was designed to comparatively investigate 21 Rhodococcus equi isolates from the faeces of clinically healthy horses and cattle. The isolates were identified by cultural and biochemical properties and by PCR analysis. The latter, targeted to the gene coding for the 16S ribosomal RNA, revealed a species specific PCR product.The isolates were further characterised by serotyping with two typing systems, by haemagglutination tests and by plasmid and virulence protein profiling. Among the 21 cultures, four cultures contained plasmids, two of the four cultures expressed 15–17 kDa virulence proteins, no cultures contained 20 kDA virulence proteins. The 21 cultures were further analysed by DNA-finger-printing. This was performed by macrorestriction analysis of chromosomal DNA by pulsed-field gel electrophoresis (PFGE). The DNA-restriction patterns were different for most of the isolates indicating a clone heterogeneity among isolates from single farms.Serotyping, determination of virulence marker and PFGE analysis of R equi appeared to be useful for further characterisation of this species, possibly of importance for virulence estimation of single R equi isolates and for epidemiological studies.     

猪链球菌2型HA0609的分离及致病性研究

从江苏省某屠宰场猪的扁桃体中分离到1株细菌,通过培养特性、菌体形态、菌落形态、染色特性、生化试验以及荚膜多糖(cps)基因的PCR检测,确定为猪链球菌2型,命名为HA0609。本试验针对猪链球菌7种主要毒力因子——谷氨酸脱氢酶(gdh)、溶菌酶释放蛋白(mrp)、胞外因子(epf)、溶血素(sly)、纤连蛋白/血纤蛋白原结合蛋白(fpbs)、次黄嘌呤核苷酸脱氢酶(impdh)及毒力相关序列orf2,进行PCR检测。与已知强毒株比较,该菌株2种主要毒力因子sly和epf均为阴性。动物试验显示HA0609对猪、兔和Balb/c鼠均无致病性。     

Characterization of shiga toxin-producing Escherichia coli strains isolated from calves in Poland

Fecal samples from 67 3–5-months-old calves with diarrhea were screened for the presence of shiga toxin-producing Escherichia coli (STEC). Several accessory virulence factors genes were also tested. Among 192 E.coli isolates tested, 15 (7.6%) were found to harbour the shiga toxin 1 or 2 (stx1 or stx2) genes. The stx2-carrying samples were further subtyped by PCR for the stx2c, stx2d, and stx2e toxin variants. It was shown that stx2-positive bacteria mainly possessed the stx2c shiga toxin type gene. The enterohemolysin (hlyA) and intimin (eae) genes were found in seven (46.7%) STEC strains whereas the cytotoxic necrotizin factor 1 and 2 or the P fimbrial genes were detected in two isolates only. This study confirmed that calves are a reservoir of STEC strains (with all pathogenicity genes) that may be virulent for humans.     

Fast detection of Vibrio species potentially pathogenic for mollusc

Vibrio tasmaniensis, Vibrio splendidus and Vibrio neptunius species were distributed worldwide and associated with aquaculture and have been reported as the cause of diseases in aquatic organisms. Polyphasic analyses for bacterial identification are not feasible for routine diagnostic because of the time involved. The aim of this study is to design three PCR primer sets that can assist with fast detection of these species. They were designed from the 16S ribosomal RNA gene, and PCR conditions were found. Each PCR test successfully identified all the tested strains of each target species. The combined specificity of V. tasmaniensis and V. splendidus primer sets offered the best coverage (86%) in terms of separating target organisms from other related species. The primer set of V. tasmaniensis showed a lower sensitivity limit (500 fg of DNA) than the V. splendidus set (1 pg) and both sets gave positive amplification using homogenized tissues from inoculated clams, with 10² and 10⁴ cfu/g of clam, respectively. The primer set of V. neptunius was highly specific, showing only cross-reaction with V. parahaemolyticus species from 44 tested species. Its sensitivity limit was 100 pg of DNA. A small number of biochemical tests were proposed concurrently with the PCR to differentiate the cross-reacting bacteria. The time of detection of the three tested species was reduced and the further affected animals can be diagnosed in a rapid fraction of time. The detection of virulent strains of V. tasmaniensis pointed to the risk of mollusc culture outbreaks.     

表观健康猪群携带猪链球菌2型毒力因子的分布特征

为了研究钦州市猪链球菌2型(SS2)的毒力因子分布特征,采用多重PCR方法对从我市健康猪扁桃体分离的11株SS2相关毒力基因——荚膜多糖(Cps2J),溶菌酶释放蛋白(Mrp),胞外蛋白因子(EF),溶血素(Sly),进行毒力基因检测。检测结果显示:毒力基因型为Mrp+epf+Sly+共有8株,毒力基因型为Mrp+epf+Sly-共有1株,这两种毒力基因型可归为强毒力基因型,占此次健康猪源毒株的81.8%;另外Mrp-epf-Sly-共有2株,属于弱毒和无毒基因型,占健康猪源毒株的18.2%。通过结果分析,预示我市目前SS2的主要流行菌株是同时具有4种毒力因子的高致病性菌株。     

The intramacrophagic environment of Brucella suis and bacterial response

Phagocytes have developed various antimicrobial defense mechanisms to eliminate pathogens. They comprise the oxidative burst, acidification of phagosomes, or fusion of phagosomes with lysosomes. Facultative intracellular bacteria, in return, have developed strategies counteracting the host cell defense, resulting in intramacrophagic survival.Until lately, only very little was known about the phagosomal compartment containing Brucella spp., the environmental conditions the bacteria encounter, and the pathogen’s stress response. Recently, we have determined that the phagosomes acidify rapidly to a pH of 4.0–4.5 following infection, but this early acidification is crucial for intracellular replication as neutralization results in bacterial elimination. A vacuolar proton-ATPase is responsible for this phenomenon that is not linked to phagosome–lysosome fusion. On the contrary, in vitro reconstitution assays revealed association only between phagosomes containing killed B. suis and lysosomes, describing the absence of phagolysosome fusion due to specific recognition inhibition for live bacteria. Further evidence for the necessity of an intact, acidic phagosome as a predominant niche of brucellae in macrophages was obtained with a strain of B. suis secreting listeriolysin. It partially disrupts the phagosomal membranes and fails to multiply intracellularly.How does B. suis adapt to this environment? We have identified and studied a series of genes that are involved in this process of adaptation. The bacterial heat shock protein and chaperone DnaK is induced in phagocytes and it is essential for intracellular multiplication. A low-level, constitutive expression of dnaK following promoter exchange does not restore intramacrophagic survival. Another chaperone and heat shock protein, ClpB, belonging to the family of ClpATPases, is important for the resistance of B. suis to several in vitro stresses, but does not contribute to intramacrophagic survival of the pathogen. Additional bacterial genes specifically induced within the phagocyte were identified by an intramacrophagic screen of random promoter fusions to the reporter gene gfp. A large majority of these genes are encoding proteins involved in transport of nutrients (sugars, amino acids), or cofactors, such as nickel. Analysis of the intracellular gene activation reveals that low oxygen tension is encountered by B. suis.Altogether, these results suggest three major stress conditions encountered by brucellae in the phagosome: acid stress, starvation and low oxygen tension.     

猪囊尾蚴西昌分离株线粒体cox1和nad1基因的序列测定与种系发育分析

应用PCR技术对3个猪囊尾蚴西昌分离株的线粒体细胞色素C氧化酶第Ⅰ亚基(cox1)基因全序列和烟酰胺腺嘌呤二核苷酸还原酶第一亚基(nad1)基因部分序列进行扩增,分析其遗传变异,并用Mega 5.0程序NJ法绘制种系发育树,探讨不同地区来源的猪囊尾蚴种系发育关系。测序结果显示,猪囊尾蚴的cox1基因全序列长度为1 620bp,nad1基因部分序列长度为796bp。cox1和nad1基因序列均存在一定的遗传变异,且cox1基因序列的变异高于nad1序列。种系发育分析结果显示,所有猪囊尾蚴分离株构成一个分支,3个西昌分离株均属于猪带绦虫亚洲基因型。结果表明,cox1和nad1基因均可用于猪带绦虫的分子分类,为猪带绦虫病/猪囊尾蚴病的分子诊断奠定了基础。     

Identification and differentiation of avirulent and virulent Rhodococcus equi using selective media and colony blotting DNA hybridization to determine their concentrations in the environment

Selective agar media have been used for many years to facilitate the isolation of Rhodococcus equi from environmental and clinical samples. However, characterisation of R. equi still requires the use of immunochemical or polymerase chain reaction (PCR) analysis to differentiate between virulent and avirulent isolates. Here, we describe a novel method to detect and differentiate between R. equi isolates using colony blotting and DNA hybridization. Radiolabelled PCR product derived from the R. equi rrnA gene and specific hybridization conditions enabled differentiation of colonies of R. equi from environmental species, whilst radiolabelled PCR product derived from the R. equi vapA gene, under specific hybridization conditions, allowed differentiation between avirulent and virulent R. equi. This technique has the potential to be used for quantitative screening of large environmental and clinical samples for both avirulent and virulent R. equi. Its use in ecological and epidemiological studies of R. equi has the potential to improve understanding of the relationship between the environment, the foal and the disease.     

Molecular subtyping of Staphylococcus aureus isolates from cases of bovine mastitis in Brazil.

Sixty-six isolates of Staphylococcus aureus obtained from milk samples of dairy cows suffering from subclinical mastitis in southern Brazil were analysed by five different molecular typing methods. These included the analysis of plasmid profiles, the analysis of coagulase (coa) gene polymorphisms by PCR amplification of the 3' terminal region of the coa gene, the PCR-based detection of polymorphisms in the X region of the protein A gene (spa), the PCR-directed analysis of variations in the spacer region between 16S and 23S rRNA, and the comparison of pulsed-field gel electrophoretically separated genomic SmaI fragment patterns. The molecular typing methods were supplemented with the biochemical characterization of the isolates and the determination of their in-vitro susceptibility to 14 different antibiotics. All genotypic and phenotypic typing methods were analyzed for their ability to discriminate between the isolates. Macrorestriction analysis proved to be the most discriminatory single method (D = 0.96) followed by rRNA spacer typing (D = 0.85), coa PCR (D = 0.82), and spa PCR (D = 0.80).     

Complete nucleotide sequence analysis of the oncogene “Meq” from serotype 1 Marek’s disease virus isolates from India

1. A study was undertaken to characterise the oncogene Meq at the molecular level for three serotype 1 Marek’s disease virus (MDV) field isolates from vaccinated poultry flocks which had encountered a Marek’s disease outbreak in the southern part of India. The isolates were named Ind/TN/11/01, Ind/KA/12/02 and Ind/TN/12/03. The oncogene Meq was amplified by PCR and sequenced.

2. The isolates were shown to have a homology for the Meq gene of 99.1–99.8% with various isolates from China and 98.5–99.2% with isolates from Europe and the USA. Alignment analysis of the nucleotide sequences showed that nucleotide mutations at 5 different positions in the Meq gene displayed perfect regularity in MDVs circulating in the southern part of India, which could be considered as features of field MDVs recently prevalent in this area.

3. In addition, the mutation in the Meq gene at positions 251, 260 and 437 was unique and coincides with very virulent strains from China GX0101, GXY2 and a Hungarian strain ATE. The mutation at positions 283 and 300 was unique and coincides with the very virulent strain ATE of Hungary. There were also single nucleotide mutations at positions 155 (A–T), 369 (A–C), 462 (C–A) and 548 (C–T) observed in the isolate Ind/TN/12/03.

4. Phylogenetic analysis of Meq sequences revealed that field MDVs in this area evolved independently but have similarities with very virulent strains from China, and that Meq has more similarities with the very virulent Hungarian strain.     

Virulence genes and plasmid profiles in Rhodococcus equi isolates from domestic pigs and wild boars (Sus scrofa) in Brazil

The virulence genes and plasmid profiles of 23 Rhodococcus equi isolates from 258 lymph nodes from domestic pigs (129 nodes with lesions and 129 without lesions) and 120 lymph nodes from slaughtered wild boars (60 nodes with lesions and 60 without) were characterized. R. equi was obtained from 19 lymph nodes of domestic pigs, 17 with, and two without lesions, and from four lymph nodes with lesions, from wild boars. The 23 isolates were tested for the presence of vapA and vapB genes, responsible for the 15–17 and 20 kDa virulence-associated proteins, respectively, by PCR in order to characterize as virulent (VapA), intermediately virulent (VapB) and avirulent. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases to estimate size and compare their polymorphisms. Of the 19 domestic pigs strains, seven (36.8%) were avirulent and 12 (63.2%) were intermediately virulent, with the intermediately virulent isolates being plasmid types 8 (8 isolates), 10 (2 isolates), 1 (1 isolate) and 29 (1 isolate). The plasmid type of four strains isolated from wild boars was also intermediately virulent type 8. None of the domestic pigs and wild boar isolates showed the vapA gene. These findings demonstrate a high occurrence of plasmid type 8 in isolates from pigs and wild boars, and the similarity of plasmid types in the domestic pigs, wild boars and human isolates in Brazil.