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.     

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.     

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.     

Polymerase chain reaction amplification of repetitive intergenic consensus and repetitive extragenic palindromic sequences for molecular typing of Pseudomonas anguilliseptica and Aeromonas salmonicida

The aim of this study was to evaluate the use of two molecular techniques, repetitive extragenic palindromic polymerase chain reaction (REP-PCR) and repetitive intergenic consensus PCR (ERIC-PCR), as epidemiological tools with which to discriminate among genetically distinct strains within two bacterial fish pathogens, Pseudomonas anguilliseptica and Aeromonas salmonicida. A total of 30 A. salmonicida and 52 P. anguilliseptica were analyzed. For P. anguilliseptica, three different major fingerprints were obtained with both techniques, which defined three genomic groups: one was composed of strains isolated from eels Anguilla spp., the second of strains from turbot Scophthalmus maximus and blackspot seabream (also known as red seabream) Pagellus bogaraveo, and the third of strains from other fish species, such as gilthead seabream (also known as gilthead bream) Sparus auratus, sea bass Dicentrarchus labrax (also known as European bass Morone labrax), and salmonids. In the case ofA. salmonicida, promising results were obtained with both techniques for subspecies differentiation. Thus, two genomic profiles were obtained by ERIC-PCR. The first profile consisted of A. salmonicida subsp. salmonicida strains isolated from the different hosts. The second profile was composed of two A. salmonicida subsp. masoucida and one A. salmonicida subsp. achromogenes. Using REP-PCR, three genotypes were obtained within this pathogen that were related to the diverse subspecies analyzed. In summary, both methodologies are useful for typing distinct strains associated with different host species and therefore are helpful in epidemiological studies of P. anguilliseptica. In contrast, in the case of A. salmonicida, more studies are needed to determine their utility in discriminating the subspecies salmonicida from the other two subspecies.     

Molecular fingerprinting of Riemerella anatipestifer by repetitive sequence PCR.

Riemerella anatipestifer is a gram-negative rod-shaped bacterium associated with epizootic infections in poultry. A total of 35 R. anatipestifer isolates including the type strain ATCC11845T, reference and field strains for 18 different serotypes were characterized by repetitive sequence based-PCR (rep-PCR) with outwardly-directed primers based on the repetitive extragenic palindromic (REP) consensus sequence. This technique was applied by using either extracted genomic DNA or preparation of whole bacterial cells harvested directly from plate cultures. Rep-PCR discriminated the R. anatipestifer isolates into 19 electrophoretic types. DNA fingerprints obtained from rep-PCR of extracted genomic DNA or from preparations of whole cells yielded comparable patterns. Substantial variation was seen among the rep-PCR fingerprints of different serotypes. Moreover, different polymorphisms of the rep-PCR fingerprints were evident among epidemiologically unrelated isolates of the same serotype. These results suggest the presence of repetitive extragenic palindromic-like elements within the genome of R. anatipestifer that can be used in some isolates to discriminate between different strains belonging to the same serotype. Rep-PCR may serve as a useful molecular tool for subtyping R. anatipestifer isolates for epidemiologic investigations. The whole cell procedure offers the advantage of ease of performance requiring only small quantities of cells.     

Emerging avian pathogenic Escherichia coli strains belonging to clonal groups O111:H4-D-ST2085 and O111:H4-D-ST117 with high virulence-gene content and zoonotic potential

The present study characterizes, for the first time, two emerging avian pathogenic Escherichia coli (APEC) clonal groups of serogroup O111: O111:H4-D-ST117 and O111:H4-D-ST2085. The clonal group O111:H4-D-ST117 was already present in APEC strains isolated between 1991 and 2000, and was still present in strains isolated between 2004 and 2009, showing long time evolution according to the virulence-gene differences and macrorestriction profiles. Among ST117 strains, two virulence profiles could be distinguished: papG II-positive tsh-negative strains which satisfied criteria for extraintestinal pathogenic E. coli (ExPEC), and papG II-negative tsh-positive strains without ExPEC status. Interestingly, we have detected a human septicemic O111:H4-D-ST117 ExPEC strain isolated from a hemocultive in 2000 whose macrorestriction profile showed >85% similarity with four APEC strains of the study. The clonal group O111:H4-D-ST2085 was exclusively detected in 17 APEC strains isolated in 2008 and 2009, and showed short time evolution based on its homogeneity since all were nalidixic acid-resistant, all had ExPEC status, and most carried papG II and tsh genes. From the clinical point of view, O111:H4-D-ST2085 seems a successful clonal group that could be the result of the epidemiological evolution of O111:H4-D-ST117. Due to the increasing prevalence of both clonal groups among clinical APEC isolates, their high virulence-gene content, and zoonotic potential, we suggest them as possible candidates for the development of a future vaccine against avian colibacillosis.     

Identification of patterns of transmission of Salmonella within swine production systems using pulsed field gel electrophoresis (PFGE) and repetitive sequence polymerase chain reaction (REP-PCR): a quantitative analysis

Pulsed field gel electrophoresis (PFGE) using 3 enzymes (Spe I, Xba I, Avr II) and repetitive sequence polymerase chain reaction (REP-PCR) with 3 primers (BOX, ERIC, REP) were compared with respect to their validity as a method for identifying transmission of Salmonella on swine farms. Sixty-eight isolates of Salmonella were obtained from feces of swine, cats, mice, and birds, insect body parts, water and floor samples, and boot scrapings collected on 9 swine farms in Illinois USA. Genetic distances between isolates were calculated using the Dice matching coefficient. Cluster analysis of distance matrices was conducted using the UPG-MA algorithm. There was no significant difference between PFGE and REP-PCR in the genetic diversity detected; however, REP-PCR differentiated between 14 pairs of isolates which PFGE identified as identical. There were no significant differences between PFGE and REP-PCR in identifying all or most close genetic links as isolates from the same farm, the same building, and from the same sampling visit, suggesting ecological validity for both methods. Thus, REP-PCR should be considered as an acceptable and perhaps preferable alternative to PFGE as a genotyping method for studies of Salmonella transmission.     

Cloning and sequencing of the iss gene from a virulent avian Escherichia coli

Control of colibacillosis is important to the poultry industry. We have found that the presence of a gene for increased serum survival, iss, is strongly correlated with Escherichia coli isolated from birds with colibacillosis. Therefore, the iss gene and its protein product, Iss, are potential targets for detection and control of avian colibacillosis. The iss gene was amplified from a virulent avian E. coli isolate and sequenced. The sequences of the gene and the predicted protein product were compared with those of iss from a human E. coli isolate and lambda bor. The iss gene from the avian E. coli isolate has 96.8% identity with the iss gene from the human E. coli isolate and 89.4% identity with lambda bor. The Iss protein from the avian isolate has 87% identity with Iss from the human isolate and 90% identity with Bor. The low identity between the two Iss proteins is because of a frame-shift in their respective coding sequences. In sum, iss from this avian E. coli isolate is very similar to iss from a human E. coli isolate, but because of a frameshift mutation in the coding sequence of iss from the human E. coli isolate, Iss proteins from avian and human E. coli isolates have only 87% identity. The strong association of iss with E. coli isolated from birds with colibacillosis, suggests that this sequence be studied for its value as a marker or target to be used in colibacillosis control.     

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.     

氟苯尼考长效制剂对鸡大肠杆菌病的疗效试验

用试管二倍稀释法测定了氟苯尼考与丁胺卡那霉素对大肠杆菌质控菌株ATCC25922,菌株K88、HD1047和临床分离的36株大肠杆菌的体外抗菌活性,进行了鸡大肠杆菌病的临床疗效试验.结果质控菌株和K88、HD1047株对氟苯尼考均敏感,36株临床分离菌株对氟苯尼考的敏感率为96.2%.在临床疗效试验中,氟苯尼考长效口服液和注射剂组的死亡率低于感染对照组和丁胺卡那霉素对照组(P《0.01),治愈率及有效率高于感染对照组和丁胺卡那霉素对照组(P《0.01),表明氟苯尼考长效制剂对鸡大肠杆菌病的治疗有很好的疗效并有较好的应用前景.     

6株副猪嗜血杆菌基因组DNA的PCR指纹图谱研究

根据肠道菌基因闻重复一致序列，设计了一对特异性引物，采用ERIC-PCR和RAPD技术，研究了副猪嗜血杆菌6个分离菌株的指纹图谱和DNA多态性。结果表明，6个分离株的PCR指纹图谱与15个标准血清型指纹图谱相比较可分辨出4种血清型；6个分离株的RAPD研究结果均表现出多态性。有意义的是，6个菌株的多态性DNA片段也能明显将其分为4种类型的副猪嗜血杆菌，与特异性引物PCR结果相一致。该研究可作为流行病学调查和该菌的分子分型快速诊断方法的基础。     

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.     

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.     

鸭致病性大肠杆菌的分离鉴定及其生物学特性分析

鸭致病性大肠杆菌病是危害养鸭业的最为重要的细菌性传染病之一。本研究分离鉴定了65株鸭致病性大肠杆菌,并对其O血清型、耐药谱以及毒力相关基因进行了检测。结果表明：大肠杆菌O78,O2和O1为主要流行血清型,各占分离株的75％、11％和5％。对15种常规抗菌素或药物的药敏试验结果表明：100％的分离株对利福平和红霉素耐药;94％以上的分离株对头孢噻吩等10种抗菌素或药物耐受;70％以上的分离株对氯霉素、卡那霉素、庆大霉素、壮观霉素和头孢噻肟敏感。对14种可能的大肠杆菌毒力相关基因的检测结果表明：ompA、pfs和luxS三种基因高度保守,在分离菌株中的检出率为100％;iss、iuCD、tsh、fimC、cvaC和iroC基因的检出率达到72％以上,为重要的鸭致病性大肠杆菌毒力相关基因。     

Analyses of Dutch Haemophilus parasuis isolates by serotyping, genotyping by ERIC-PCR and Hsp60 sequences and the presence of the virulence associated trimeric autotransporters marker

In this study, 117 isolates of Haemophilus parasuis from organs and tissues from pigs showing clinical signs, were characterised and compared with 10 H. parasuis reference strains. The isolates were subjected to the 16S rRNA gene PCR and subsequently serotyped, genotyped by 60-kDa heat shock protein (Hsp60) gene sequences, the enterobacterial repetitive intergenic consensus (ERIC) PCR and a multiplex PCR for the detection of the vtaA virulence associated trimeric autotransporter genes. Serotyping revealed the presence of 13 H. parasuis serovars. Serovars 3 and 10 were not detected, and 16 of the 117 H. parasuis isolates could not be typed by specific antisera. All isolates were positive in the 16S rRNA gene specific H. parasuis PCR. ERIC-PCR revealed a very heterogeneous pattern with 61 clusters; based on a 90% agreement. In total, 46 different Hsp60 sequence types were detected. Using 98% sequence similarity, as threshold for separation, 22 separate Hsp60 sequence clusters were distinguished. There was no correlation between H. parasuis serovars and ERIC-PCR clusters or Hsp60 sequence types, but both the ERIC-PCR and the Hsp60 sequence typing are suited as markers for H. parasuis molecular-epidemiology studies. In total, 102 H. parasuis swine isolates corresponded to the virulence associated group 1 vtaA type. The group 1 vtaA was detected in 12 different serovars. Only four of the 46 Hsp60 sequence types were not associated with the group 1 vtaA. This study shows that Dutch H. parasuis isolates from pigs with clinical signs have both a high serovar and genotypic lineage diversity. A majority of the known serovars contain the group 1 vtaA.     

宁夏部分地区鸡大肠埃希菌的分离与血清型鉴定

为调查宁夏地区鸡大肠杆菌病的流行情况,采集宁夏4个地区的103个规模化鸡场疑似病料151份,通过病原菌分离鉴定及生化试验,鉴定出鸡大肠埃希菌109株,平均分离率为72.19%(109/151);动物试验证实,59株分离菌有致病性,占分离菌54.13%(59/109);对致病性菌株进行血清型鉴定,确定血清型种类13种38株,占致病性菌株64.41%(38/59);优势血清型分别为O15、O18、O78、O88,占定型菌株63.16%(24/38)。     

Virulence factors and genetic variability of uropathogenic Escherichia coli isolated from dogs and cats in Italy

In this study, the association between virulence genotypes and phylogenetic groups among Escherichia (E.) coli isolates obtained from pet dogs and cats with cystitis was detected, and fingerprinting methods were used to explore the relationship among strains. Forty uropathogenic E. coli (UPEC) isolated from dogs (n = 30) and cats (n = 10) in Italy were analysed by polymerase chain reaction (PCR) for the presence of virulence factors and their classification into phylogenetic groups. The same strains were characterized by repetitive extragenic palindromic (REP)- and enterobacterial repetitive intergenic consensus (ERIC)-PCR techniques. We found a high number of virulence factors such as fimbriae A, S fimbriae (sfa) and cytotoxic necrotizing factor 1 (cnf1) significantly associated with phylogenetic group B2. We demonstrated a high correlation between α-hemolysin A and pyelonephritis C, sfa, and cnf1 operons, confirming the presence of pathogenicity islands in these strains. In addition, UPEC belonging to group B2 harboured a greater number of virulence factors than strains from phylogenetic groups A, B1, and D. REP- and ERIC-PCR grouped the UPEC isolates into two major clusters, the former grouping E. coli strains belonging to phylogenetic group B2 and D, the latter grouping those belonging to groups A and B1. Given the significant genetic variability among the UPEC strains found in our study, it can be hypothesized that no specific genotype is responsible for cystitis in cats or dogs.     

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.     

山东省部分地区禽源致病性大肠杆菌的血清流行病学调查与耐药性检测

为调查山东省禽源致病性大肠杆菌流行的血清型及耐药性,从山东部分地区的45家养禽场分离到致病性大肠杆菌96株,应用微量平板凝集试验进行了血清型鉴定,共鉴定出18种血清型,其中优势血清型6种,分别为O78、O2、O15、O18、O143、O88,占定型致病性菌株的64％。抗菌药物敏感性试验发现,96株大肠杆菌对20种药物有不同程度的耐药性。75％以上的菌株对氨苄青霉素、阿莫西林、土霉素等5种抗菌药耐药,50％以上的菌株对卡那霉素、多西环素、环丙沙星等7种抗菌药表现为耐药;所有分离株存在多重耐药现象,75％的受检菌对9种或9种以上的被测药物耐药。结果表明,O78、O2、O15、O18、O143、O886种血清型是山东省部分地区近年来禽源致病性大肠杆菌的优势血清型,且禽源致病性大肠杆菌的耐药现象严重,有必要加强耐药性检测,以指导兽医临床合理使用抗菌药物。     

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.