Clonal relationships among avian Escherichia coli isolates determined by enterobacterial repetitive intergenic consensus (ERIC)-PCR

Forty-nine avian Escherichia coli isolates isolated from different outbreak cases of septicemia (24 isolates), swollen head syndrome (14 isolates) and omphalitis (11 isolates), and 30 commensal isolates isolated from poultry with no signs of illness were characterized by enterobacterial repetitive intergenic consensus (ERIC)-PCR technique and their serotypes were determined. The ERIC-PCR profile allowed the typing of the 79 isolates into 68 ERIC-types and grouped the isolates into four main clusters (A-D), with the omphalitis isolates being grouped with the commensals and separated from the septicaemia and swollen head syndrome. These results indicate that ERIC-PCR is a technique that could replace other molecular characterization techniques such as random amplification of polymorphic DNA (RAPD)-PCR and restriction fragment length polymorphism (RFLP), reinforce previous observations that omphalitis isolates are just opportunistic agents, and are consistent with many reports that specific genotypes are responsible for causing specific diseases. Most of the isolates were either nontypable or rough, supporting the need for alternative methods for typing these isolates.     

Host associated polymorphisms in the Corynebacterium pseudotuberculosis rpoB gene sequence

Corynebacterium pseudotuberculosis is a widespread facultative intracellular pathogen that causes caseous lymphadenitis disease in sheep and goats, and generates cutaneous abscesses and granulomas in horses and cattle. Although some genes have been studied for diagnostic and phylogenetic analysis within the genus Corynebacterium, at subspecies level the pathogen has been poorly analyzed. The aim of this study was to characterize C. pseudotuberculosis strains isolated from domestic animals, through the sequencing of a hypervariable rpoB gene segment. As result, there were identified host associated rpoB polymorphisms in strains infecting sheep, goats and horses from Chile. These differences suggest the existence of bacterial genotypes, in which the nucleotide similarity values were ranging from 98.8 to 99.8%. In conclusion, the analysis of polymorphisms in the partial rpoB sequence can be used as a diagnostic tool that differentiates C. pseudotuberculosis strains at subspecies level.     

Typing of Haemophilus paragallinarum strains by using enterobacterial repetitive intergenic consensus-based polymerase chain reaction

The enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) technique was used for fingerprinting of reference strains and Mexican isolates of Haemophilus paragallinarum. A total of nine ERIC patterns were given by the nine serovar reference strains of this bacteria. Two Modesto (C-2) reference strains from different sources showed the same ERIC pattern. Seventeen ERIC patterns were obtained among 29 Mexican isolates included in the study, belonging to serovars prevalent in Mexico (A-1, A-2, B-1, and C-2). Obtained results indicate that the ERIC-PCR technique could be used as a molecular laboratory tool for subtyping of H. paragallinarum.     

Strain differentiation of Indian isolates of Salmonella by ERIC-PCR

Enterobacterial repetitive intergenic consensus (ERIC) sequences are the repetitive elements present in the family enterobacteriacae. In the present study, ERIC-PCR (target ERIC sequence) was used for the molecular typing of 24 isolates of Salmonella serovars, namely abortusequi, choleraesuis, bareilly and dublin. In ERIC-PCR, seven molecular types were observed with ERIC-Cl primer, and nine molecular types with ERIC-C2 primer. When the results of both the ERIC-PCR were combined for molecular typing, 21 molecular types were observed, which indicated a high degree of discrimination. Both the ERIC primers are designed from the ERIC consensus sequence, yet they gave different profiles, indicating that they supplement each other. ERIC sequences were found to be useful targets for molecular typing. The different profiles observed appear to be due to differences in ERIC sequences and differences in inter-ERIC distance. The study indicates that ERIC-PCR is a very efficient tool for molecular typing of Salmonella species.     

Genotype analysis of Campylobacter jejuni isolated from broilers in Poland

Twenty-six Campylobacter jejuni strains isolated from poultry were analyzed by genotypic typing including ITS-profiling, REP- and ERIC-PCR. ITS-profiling revealed the presence of 8 different genotypes. Amplification of REP sequences by PCR gave similar results with 10 different genotypes. ERIC-PCR was found to be the most discriminatory for typing C. jejuni. As many as 13 different DNA patterns were obtained with this technique. Based on data obtained it was found that C. jejuni isolates recovered from broilers at the slaughterhouses in southwest Poland are characterized by a high degree of genetic heterogeneity.     

新疆北部部分地区副猪嗜血杆菌的ERIC-PCR指纹聚类分析

为了解新疆北部部分规模化养猪场副猪嗜血杆菌(H.parasuis)分离株的基因型,本实验采用ERIC-PCR方法结合统计学分析软件,对来源不同的12株H.parasuis进行分子指纹图谱分析.结果表明12株分离株分别位于4个聚类中,各个菌株之间的遗传距离较近,并且含有长度为1 000 bp的相同条带,相同血清型的分离株在其分子指纹聚类上均位于同一个分支中.试验结果表明基于ERIC-PCR的分子指纹聚类分析结合传统的琼脂免疫扩散法可以准确地对不同分离株H.parasuis进行分型.试验结果显示H.parasuis在该地区广泛存在并具有多种不同的基因型,同时为该地区H.parasuis的免疫防治提供了重要的实验依据.     

Molecular genotyping of multinational ovine and caprine Corynebacterium pseudotuberculosis isolates using pulsed-field gel electrophoresis

Caseous lymphadenitis (CLA) is a chronic, suppurative disease, with a worldwide distribution, caused by Corynebacterium pseudotuberculosis. The clinical manifestation of CLA is known to vary between different countries, and has been postulated to be due to differences in the strains present in these countries. Forty-two sheep and goat isolates of C. pseudotuberculosis from Australia, Canada, Eire, The Netherlands and Northern Ireland were characterized by pulsed-field gel electrophoresis (PFGE), biotyping, antimicrobial susceptibility, and production of phospholipase D. The PFGE-determined genotypes of this multicentric collection were then compared with representative ovine and caprine isolates from a previously published panel of PFGE profiles of United Kingdom isolates. Digestion with SfiI generated 16-18 bands in the 48.5 and 290 kb range, and differentiated four distinct pulsotypes amongst the 36 ovine and 6 caprine strains which displayed remarkable homogeneity. Based on these results, it would appear that the genome of C. pseudotuberculosis is highly conserved, irrespective of the country of strain origin.     

Typing of Yersinia Enterocolitica Isolates by ITS profiling, REP- and ERIC-PCR

Thirty-five Yersinia enterocolitica strains isolated from humans, pigs and foxes were analysed by genotyping including intergenic transcribed sequence (ITS) profiling, REP- and ERIC-PCR. ERIC-PCR revealed the presence of seven different genotypes. Amplification of the 16S-23S rDNA spacer region by ITS profiling gave similar results with nine different genotypes. REP-PCR was found to be more discriminatory for typing of Y. enterocolitica than ERIC-PCR and ITS profiling. Fifteen different DNA patterns were obtained by this technique. Based on data obtained by three methods it was found that: (i) Y. enterocolitica strains belonging to the same serotype can represent different genotypes and vice versa; (ii) isolates recovered from humans, pigs and foxes exhibit limited heterogeneity and, independent of the origin, one or two prevailing genotypes were always observed; and (iii) many human Y. enterocolitica isolates shared common genotypes with porcine isolates.     

Molecular fingerprinting of strains of Yersinia ruckeri serovar O1 and Photobacterium damsela subsp. piscicida isolated in Italy

We studied the ribotypes, patterns of pulsed-field gel electrophoresis (PFGE) and interspersed repeated sequences (IRS)-PCR of 30 strains of Yersinia ruckeri O1 and 20 strains of Photobacterium damsela subsp. piscicida isolated from apparently unrelated epizootic outbreaks occurring on Italian fish farms between 1993 and 1999. All of the Y. ruckeri O1 strains had similar profiles, as demonstrated by all three typing methods, thus confirming the clonal structure of this species. The strains of P. damsela subsp. piscicida showed similar profiles when tested with ribotyping and PFGE; however, two slightly different profiles were distinguished by IRS-PCR using the primer ERIC2. The two profiles appeared to be related to the geographic origin of the strains, since each of them was specific to isolates from a certain area (i.e. northern or southern Italy). This result suggests that PCR fingerprinting may be a valuable tool in typing fish bacterial pathogens, which often present a great degree of genetic homogeneity.     

Molecular characterization of avian strains of Pasteurella multocida serogroup-A:1 based on amplification of repetitive regions by PCR

Repetitive extragenic palindromic (REP)-PCR (polymerase chain reaction), enterobacterial repetitive intergenic consensus (ERIC)-PCR, and single primer PCR assays were employed to characterize 66 strains of Pasteurella multocida serogroup A:1 isolated from avian species belonging to different regions of India. REP-PCR resulted in amplification of REP sequences from the genome which were in the range of approximately 200 to approximately 3000 bp and accounted for a total of 54 distinguishing profiles (D=0.99). ERIC-PCR analysis also generated amplified products in the range of approximately 200 to approximately 3200 bp categorizing strains into a total of 50 different profiles (D=0.98). Amplification of repetitive regions using a microsatellite primer (GTG)(5), resulted in clear distinctive bands ranging from approximately 200 to approximately 2400 bp. Strains were assigned to 43 profiles (D=0.96). No correlation could be drawn between genotypic profiles and avian hosts with their geographical area of origin. Avian strains of P. multocida serogroup A:1 were found to be highly heterogeneous with diverse profiles. REP-PCR was found to be highly discriminatory and simple method for differentiation of phenotypically similar strains. The present study also indicated that PCR based amplification of repetitive regions of P. multocida is a rapid technique with good discrimination and could be employed directly for routine typing of field isolates from fowl cholera outbreaks.     

Yersinia species isolated from sheep with enterocolitis

In 40 submissions to the Regional Veterinary Laboratory (RVL) Wagga Wagga from sheep in southern New South Wales from 1981 to 1989, 53 isolates of Yersinia sp were recovered from 45 sheep in 37 flocks. Of 53 isolates, 26 were identified as Y. pseudotuberculosis, 20 as Y. enterocolitica, 5 as Y. intermedia and 2 as Y. frederiksenii. Twelve isolates of Y. pseudotuberculosis tested in the slide agglutination test all belonged to serotype III. The 20 Y. enterocolitica isolates were categorised biochemically as biotype 5 strains and, of 6 isolates serotyped, all belonged to serogroup 2,3. Outbreaks of yersiniosis were most common in late winter and early spring and affected flocks often had experienced a change in husbandry. Infection with Yersinia sp was associated with diarrhoea, illthrift and mortality. At necropsy, congestion and occasionally thickening of the intestinal mucosa were observed in affected sheep. Gastrointestinal nematodiasis and coccidiosis often were concurrent findings. The characteristic histological lesion in sheep infected with Y. pseudotuberculosis was acute segmental suppurative erosive enterocolitis. There were no lesions consistently associated with Y. enterocolitica, Y. intermedia or Y. frederiksenii.     

Phenotypic and genetic characterization of Edwardsiella tarda isolated from pond sediments

The extent of genotypic and phenotypic diversity of Edwardsiella tarda isolated from pond sediment was assessed by SDS-PAGE, Plasmid Profiling and ERIC-PCR. SDS-PAGE of whole cell protein extracts reveals 20-23 discrete bands with molecular wt of 14-110 kDa. Several bands with molecular weight range of 38-83 kDa were present in all the isolates. Numerical analysis of protein electrophoregram delineated the isolates into four clusters. Two different types of plasmids having molecular mass of 23 kDa and 29 kDa were obtained by plasmid profiling. About 51% of the isolates carried both the plasmids. ERIC-PCR generates 3-7 bands with molecular mass of 14-1013 bp. Numerical analysis differentiated the ERIC pattern into 5 clusters at 60% similarly level. It was concluded that out of three methods ERIC-PCR was found to be more sensitive for intraspecific typing of E. tarda and can be used as a potential tool for epidemiological studies in the future.     

新疆牛源大肠杆菌O157∶H7的分离鉴定及ERIC-PCR基因分型研究

为了了解牛源大肠杆菌（E.coli）O157∶H7在新疆地区的污染状况以及遗传多样性,探究不同地区分离菌株的遗传关系,为控制牛源E.coli O157∶H7的传播提供试验依据。将采集的样品在EC肉汤中进行增菌（37 ℃、180 r/min）,接着将增菌液划线接种到SMAC平板上,37 ℃培养箱中过夜培养18 h左右。挑取SMAC平板上白色或无色单菌落接种MUG培养基,37 ℃培养18 h左右,将无荧光样品接种到SMAC平板上,37 ℃培养18 h左右,隔天挑取白色或无色单菌落进行PCR鉴定,具有rfbE和fliC基因条带的即为阳性菌株。将阳性菌株进行肠杆菌基因间重复共有序列扩增（ERIC-PCR）指纹图谱聚类分析,分析菌株之间的同源性关系。ERIC-PCR结果显示,相似性100%的菌株有3组。从伊犁地区分离到的菌株差异性最大,具有6种分型;其次是乌鲁木齐,具有4种分型。菌株来源多样性最多的在D簇,由此可见通过ERIC-PCR分型,可以进行溯源观察。ERIC-PCR能够区分特定采样点或物种的分离物,它能够证明从不同来源的菌株之间,存在着某些相似的ERIC特性,并聚集在同一个簇群中。该研究中筛选出的E.coli O157∶H7菌株具有广泛的遗传多样性,该方法对于检测不同物种间的细菌差异非常敏感。由此可见ERIC-PCR可以作为E.coli O157∶H7常规监测和鉴定的一个有效的工具。     

Typing of Yersinia Enterocolitica Isolates by ITS Profiling,REP‐ and ERIC‐PCR

Thirty‐five Yersinia enterocolitica strains isolated from humans, pigs and foxes were analysed by genotyping including intergenic transcribed sequence (ITS) profiling, REP‐ and ERIC‐PCR. ERIC‐PCR revealed the presence of seven different genotypes. Amplification of the 16S‐23S rDNA spacer region by ITS profiling gave similar results with nine different genotypes. REP‐PCR was found to be more discriminatory for typing of Y. enterocolitica than ERIC‐PCR and ITS profiling. Fifteen different DNA patterns were obtained by this technique. Based on data obtained by three methods it was found that: (i) Y. enterocolitica strains belonging to the same serotype can represent different genotypes and vice versa; (ii) isolates recovered from humans, pigs and foxes exhibit limited heterogeneity and, independent of the origin, one or two prevailing genotypes were always observed; and (iii) many human Y. enterocolitica isolates shared common genotypes with porcine isolates.     

Detection and Analysis of Shiga Toxin-producing Escherichia coli Isolates from Cattle and Sheep Sources in Some Regions of Xinjiang,China

Shiga toxin-producing Escherichia coli(STEC) is a new class of highly pathogenic food-borne pathogens carrying a prephage encoding one or two Shiga toxin genes. It has become an important public health issue that threatens human health. The present work aimed to characterize STEC strains isolated from cattle and sheep at various stages, in parts of Xinjiang, in terms of the presence of prevalence, genetic diversity, and antimicrobial susceptibility to 17 common antibiotics. Through amplification of four virulence genes (stx1, stx2, eae, hlyA)by PCR and ERIC-PCR genotyping to detection STEC isolates. In the present study, a total of 64 STEC strains were isolated from 431 samples from slaughterhouses, farms and markets. Of these, 31 (48.4%) of the isolates harbored stx1 + stx2, and only 29 (45.3%) of the isolates possessed stx1, only 4 (6.3%) of the isolates harbored stx2, and 1 isolates harbored all the 4 virulence genes. Drug sensitivity tests found that STEC strains displayed 7 antimicrobial resistance to midecamycin(61%), cephalothin(4.7%), cefoxitin(4.7%), ampicillin(3.1%), piperacillin(1.6%), tobramycin(1.6%), cefazolin(1.6%). The ERIC-PCR results showed a polymorphic distribution, which was divided into two clusters of A (36 strains) and B (28 strains). STEC strains isolated from cattle and sheep at various stages, in parts of Xinjiang, some of which might have the potential to cause food contamination and human diseases.     

新疆部分地区牛羊源产志贺毒素大肠埃希菌菌株检测与分析

产志贺毒素大肠埃希菌（Shiga toxin-producing Escherichia coli，STEC）是一类携带了前噬菌体编码的一种或两种志贺毒素基因的新发高致病性食源性病原菌，已成为威胁人类健康的重要公共卫生问题。为了解新疆部分地区牛、羊源各个环节产志贺毒素大肠埃希菌的感染情况及其遗传多样性，以及分离株对17种常见抗生素的敏感性，笔者采用PCR方法对STEC分离株进行了4种毒力基因（stx1、stx2、eae、hlyA）的检测和ERIC-PCR基因分型研究。结果表明：从屠宰场、养殖场和市场共431份样品中分离出产志贺毒素的大肠埃希菌64株，其中，编码stx1+stx2的STEC有31株（48.4%），只编码stx1的STEC有29株（45.3%），只编码stx2的STEC有4株（6.3%），4种毒力基因同时存在的有1株。药物敏感性检测发现STEC菌株对麦迪霉素（61%）、头孢噻吩（4.7%）、头孢西丁（4.7%）、氨苄西林（3.1%）、哌拉西林（1.6%）、妥布霉素（1.6%）、头孢唑啉（1.6%）等7种抗生素存在耐药。ERIC-PCR检测结果呈多态性分布，分为A（36株）和B（28株）两个簇。STEC菌株在新疆部分地区牛、羊源各个环节被检出，其中一些菌株可能会增加对食物的污染，从而引起人发病。     

Comparison of ITS profiling,REP- and ERIC-PCR of Salmonella Enteritidis isolates from Poland

Thirty-one Salmonella Enteritidis strains isolated from chickens, broilers and hens were analysed by genotypic typing including REP-PCR. ERIC-PCR and ITS profiling (PCR-ribotyping). Analysis of DNA banding patterns generated by REP-PCR revealed the presence of 22 different genotypes, which were grouped by dendrogram analysis into three distinct lineages (maximum similarity approx. 50%). Each isolate of S. Enteritidis analysed by ERIC-PCR generated an individual DNA pattern. Again, these isolates could be divided into three distinct genomic groups (maximum similarity approx. 60%) by their ERIC-PCR fingerprints. REP- and ERIC-PCR were found to be more discriminatory for typing of S. Enteritidis than ITS profiling. Amplification of the 16S-23S rDNA spacer region gave nine different profiles of DNA, subdivided into two closely related groups by dendrogram analysis. In summary, data obtained by genotyping methods for S. Enteritidis isolates from regions located in the south-west and the central parts of Poland revealed an enormous heterogeneity among analysed samples, and proved that REP- and ERIC-PCR are highly discriminatory techniques, which can be used, in addition to conventional methods, in epidemiological studies of S. Enteritidis infections.     

Genetic comparison of Campylobacter jejuni isolated from different cattle farms

To compare the genotypes of Campylobacter jejuni, isolates of cattle origin were collected from 9 Polish farms and genotyped by ERIC-PCR. We identified 28 genotypes among the 43 C. jejuni isolates, and demonstrated high genomic diversity. The highest level of diversity was observed in strains isolated from stanchion-barn animals in opposition to those from the loose-housing system.     

Visceral caseous lymphadenitis in thin ewe syndrome: isolation of Corynebacterium, Staphylococcus, and Moraxella spp from internal abscesses in emaciated ewes

The relationship between the visceral form of caseous lymphadenitis and a chronic debilitating condition of mature sheep designated as the thin ewe syndrome was investigated. Internal abscesses were found during necropsy in 81% of animals with thin ewe syndrome and Corynebacterium pseudotuberculosis (C ovis) was recovered from 86% of the animals with internal abscesses. Other pyogenic bacteria, including C pyogenes, C equi, Staphylococcus epidermis, S aureus, and Pseudomonas aeruginosa were often recovered in association with C pseudotuberculosis. Moraxella sp was recovered in 41% of the animals with internal abscesses. In some abscesses, Moraxella sp was the dominant microorganism isolated and in others, they were outnumbered only by C pseudotuberculosis. Species isolated included M bovis, M osloensis, and M nonliquefaciens. The potential importance of Moraxella sp to the cause and pathogenesis of the thin ewe syndrome is not known. The results of the present study indicate that visceral caseous lymphadenitis is either an important contributing factor to the development of thin ewe syndrome or that the presence of thin ewe syndrome may predispose affected sheep to the development of visceral caseous lymphadenitis. A skin test reagent prepared by sonicating C pseudotuberculosis was of limited value in detecting animals with visceral caseous lymphadenitis. Only 56% of the animals with abscesses caused by C pseudotuberculosis gave positive delayed-type hypersensitivity skin test responses.     

Immunisation against ovine caseous lymphadenitis: comparison of Corynebacterium pseudotuberculosis vaccines with and without bacterial cells

Sheep were immunised with Corynebacterium pseudotuberculosis vaccines prepared from cell-free toxoid or from toxoid with formalin-killed cells of C pseudotuberculosis added. Resistance of sheep to infection was tested 6 months after immunisation by inoculation with caseous lymphadenitis pus. The outcome was assessed 3 months later by slaughter and inspection of the sheep for lesions of caseous lymphadenitis. immunised sheep were adequately protected against infection as shown by a significant reduction in the number of sheep exhibiting lesions compared with control sheep, and by fewer abscesses in affected vaccinated sheep than in affected control sheep. The protective potency of the vaccines was not improved by the inclusion of cells of C pseudotuberculosis.