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1.
The gram-negative bacterium Pasteurella multocida constitutes a heterogeneous species associated with wide range of disease in many animals. Isolates are classified into five groups based on capsular antigen (capA, B, D, E and F). Recently, a new valuable PCR-based method was introduced to determine the epidemiological correlation between P. multocida infection and existence of virulence genes including tbpA, pfhA, toxA and hgbB. However, this method is tedious and laborious. Thus, in the current study, we designed a reliable multiplex PCR method for rapid detection of virulence genes in P. multocida. Eighty seven strains of P. multocida isolated from various clinically healthy and infected hosts were examined by uniplex PCR method for each virulence associated genes. Based on our improved and simplified multiplex PCR method, rapid detection of four virulence genes was accomplished. It is proposed that its implementation may benefit the epidemiological investigations.  相似文献   

2.
Pasteurella multocida is a causative agent of many major diseases of which haemorrhagic septiciemia (HS) in cattle & a buffalo is responsible for significant losses to livestock sector in India and south Asia. The disease outcome is affected by various host- and pathogen-specific determinants. Several bacterial species-specific putative virulence factors including the capsular and virulence associated genes have been proposed to play a key role in this interaction. A total of 23 isolates of P. multocida were obtained from 335 cases of various clinically healthy and diseased cattle. These isolates were examined for capsule synthesis genes (capA, B, D, E and F) and eleven virulence associated genes (tbpA, pfhA, toxA, hgbB, hgbA, nanH, nanB, sodA, sodC, oma87 and ptfA) by PCR. A total of 19 P. multocida isolates belonging to capsular type B and 4 of capsular type A were isolated. All isolates of capsular type B harboured the virulence associated genes: tbpA, pfhA, hgbA, sodC and nanH, coding for transferrin binding protein, filamentous hemagglutinin, haemoglobin binding protein, superoxide dismutase and neuraminidases, respectively; while isolates belonging to capsular type A also carried tbpA, pfhA, hgbA and nanH genes. Only 50 % of capsular type A isolates contained sodC gene while 100 % of capsular type B isolates had sodC gene. The gene nanB and toxA were absent in all the 23 isolates. In capsular type A isolates, either sodA or sodC gene was present & these genes did not occur concurrently. The presence of virulence associated gene ptfA revealed a positive association with the disease outcome in cattle and could therefore be an important epidemiological marker gene for characterizing P. multocida isolates.  相似文献   

3.
AIMS: To examine pigs at slaughter in New Zealand for the presence of Pasteurella multocida, and to determine for isolates, their biochemical profiles, somatic and capsular types, and the presence or absence of the HSB and toxA genes, associated with haemorrhagic septicaemia (HS) and progressive atrophic rhinitis (PAR), respectively.

METHODS: Swabs from 173 lungs, 158 palatine tonsils and 82 nasal passages of pigs at two abattoirs in New Zealand were cultured for P. multocida using conventional techniques, and isolated colonies were subjected to biochemical tests for identification of biovars. Somatic serotyping was conducted using an agar gel immunodiffusion (AGID) test. Polymerase chain reaction (PCR) assays were used to confirm phenotypic identification of colonies using species-specific primers, capsule type using serogroup-specific primers and multiplex PCR, and to test for the presence of HSB and toxA genes.

RESULTS: Pasteurella multocida was isolated from 11/173 (6.4%) lung, 32/158 (20.2%) palatine tonsil and 5/82 (6.1 %) nasal swab samples, a total of 48 isolates from 413 samples (11.6%). Isolation rates per farm ranged from 1–53% of tissue samples collected from pigs 5–6 months of age. On phenotypic characterisation, isolates were allocated to seven main biovars, viz 1, 2, 3, 5, 9, 12, and a dulcitol-negative variant of Biovar 8, the majority (30/48) being Biovar 3. Of the 42 isolates for which somatic serotyping was conducted, 10% were Serovar 1, 79% were Serovar 3, 2% were Serovar 6,1, 2% were Serovar 12, and 7% could not be typed. All 48 isolates were confirned as P. multocida using a species-specific PCR. In the capsular multiplex PCR, 92% of isolates were Capsular (Cap) type A, 2% were Cap D, and 6% could not be typed. None of the samples were positive for the HSB or toxA genes.

CONCLUSION: Serovars or capsular types of P. multocida associated with HS or PAR in pigs were not detected. Establishment of species-specific, capsular and toxin PCR assays allowed the rapid screening of isolates of P. multocida, while serotyping provided an additional tool for epidemiological and tracing purposes.  相似文献   

4.
A multiplex PCR method for rapid and sensitive diagnosis, differentiating three pathogenic Yersinia groups such as the highly pathogenic Y. enterocolitica, including serotype O8, low pathogenic Y. enterocolitica, and Y. pseudotuberculosis, was developed. Four primer pairs were chosen to detect the genes fyuA, ail, inv, and virF, responsible for the virulence in pathogenic Yersinia species. Under the multiplex PCR conditions, the unique band patterns for the highly pathogenic Y. enterocolitica, low pathogenic Y. enterocolitica, and Y. pseudotuberculosis were generated from Yersinia strains. The detection limit of this method was 101–103 CFU per reaction tube. This multiplex PCR method could detect highly pathogenic Y. enterocolitica O8 from the wild rodent fecal samples that were culture-positive. Therefore, the new multiplex PCR method developed in this study is a useful tool for rapid and sensitive diagnosis, distinguishing three pathogenic Yersinia groups.  相似文献   

5.
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6.
本研究以致泻性大肠杆菌(DEC)的4种毒力因子EAST1、LT、STa和STb为模板设计特异性引物并优化各项反应条件,以建立仔猪DEC的多重PCR检测方法.该方法具有较好的特异性,只对DEC产生特异性条带,对非DEC及其他病菌则呈阴性.利用所建立的多重PCR方法对吉林省各猪场采集的样本进行检测,结果表明,在测定的猪场中...  相似文献   

7.
The purpose of this study was to develop a multiplex PCR that can detect porcine endogenous retrovirus (PERV) proviral genes (pol, envA, envB, envC) and porcine mitochondrial DNA, using a dual priming oligonucleotide (DPO) system. The primer specifically detected the PERV proviral genes pol, envA, envB, envC, and porcine mitochondrial DNA only in samples of pig origin. The sensitivity of the primer was demonstrated by simultaneous amplification of all 5 target genes in as little as 10 pg of pig DNA containing PERV proviral genes and mitochondrial DNA. The multiplex PCR, when applied to field samples, simultaneously and successfully amplified PERV proviral genes from liver, blood and hair root samples. Thus, the multiplex PCR developed in the current study using DPO-based primers is a rapid, sensitive and specific assay for the detection and subtyping of PERV proviral genes.  相似文献   

8.
Pasteurella multocida is the causative agent of many diseases of economic importance in veterinary medicine and is characterized by high zoonotic potential. Pet animals can be infected and play a major role as carriers. This study aimed to characterize the genetic diversity of P. multocida isolated from dogs, cats and rabbits, and to evaluate their antimicrobial susceptibility profiles. A total of 620 animals were studied; 51 were positive for P. multocida and 92 strains were isolated. 60.9% of the strains belonged to the capsular type A, while the remaining were classified as non-typeable. The hgbA, ptfA, sodC, tadD and hsf2 genes were more frequent among the rabbit strains. Sulfonamides and cotrimoxazole presented the highest resistance rate, followed by erythromycin. PFGE clustered strains according to host species. Our results indicate that P. multocida from companion animals carry several virulence factors and are resistant to antimicrobials commonly used in human and veterinary medicine.  相似文献   

9.
Pasteurella multocida is a pathogenic, Gram-negative bacterium that is commonly found as normal flora in nasopharynx of variety of wild and domestic animals. Numerous virulence factors have been described for P. multocida isolates which include adherence and colonization factors, iron-regulated and acquisition proteins, extracellular enzymes such as neuraminidase, lipopolysaccharide (LPS), capsule and a variety of outer membrane proteins (Omp). OmpA has a significant role in stabilizing the cell envelope structure by providing physical linkage between the outer membrane & peptidoglycan. It has been shown to mediate P. multocida -host cells interaction via heparin and/or fibronectin binding and therefore act as an important invasive molecule which could determine the final outcome of initial infection. Comparative nucleotide sequence analysis of ompA gene of P. multocida has revealed that despite extensive genetic diversity in ompA of P. multocida, most sequences could be classified into two major allele classes namely ompA allele (I) and allele (II). The P. multocida recovered from nasal cavity of bovine and belonging to two ompA classes were tested for their differential virulence. In vitro pathogenicity studies on Madin Darby Bovine Kidney (MDBK) cell line employing adhesion and invasion assays indicated that P. multocida strain with ompA (I) is more invasive than P. multocida strain with ompA (II). In vivo studies in mice further reiterated that the isolates harbouring ompA(I) were comparatively more virulent to isolates harbouring ompA (II).  相似文献   

10.
为了建立猪源大肠杆菌对氟苯尼考、β-内酰胺类与粘杆菌素耐药基因的多重PCR检测方法,本研究以氟苯尼考耐药基因floR、β-内酰胺耐药基因CTX-M、粘杆菌素耐药基因mcr-1作为目的基因,设计3对特异性引物,通过对多重PCR反应体系及条件的优化,成功建立了多重PCR检测方法。该方法的灵敏度为1.46×10^5CFU/m L,具有高度特异性、敏感性和可重复性。本方法的建立为大肠杆菌中常见耐药基因的快速检测及分子流行病学调查提供了新的手段。  相似文献   

11.
Streptococcus equi subspecies equi, S equi subspecies zooepidemicus, and S dysgalactiae subspecies equisimilis are β-hemolytic Streptococci, often isolated from horses with respiratory or genital diseases. The aim of this study was (i) defining and validating a multiplex polymerase chain reaction (PCR) protocol for identifying these Streptococci in bacterial cultures and for detecting them directly in equine clinical specimens, and (ii) defining and validating a cheap DNA extraction protocol for clinical specimens. When respiratory and genital samples from symptomatic and asymptomatic horses were tested by bacterial culture and by multiplex PCR, all the 150 samples culture-positive for S equi, S zooepidemicus, or S equisimilis were also positive by PCR. Of 150 culture-negative samples, 143 were negative by PCR. Seven samples were positive by PCR but negative by bacteriology. The multiplex PCR protocol described in this study is proven suitable for a sensitive, specific, and rapid detection and identification of S equi, S zooepidemicus, and S equisimilis in cultured bacterial colonies, as well as in clinical specimens from symptomatic or asymptomatic horses. The inclusion of internal control primers in the PCR protocol excludes false-negative results. A cheap DNA extraction method has been also validated for swabs, tracheal aspirates, bronchoalveolar lavage, and guttural pouches lavage samples.  相似文献   

12.
Isolates of Pasteurella multocida recovered from infected humans (n = 15) were characterized by traditional and molecular microbiological methods and were compared with cat-derived strains (n = 5). The most prevalent subspecies among strains from human infections was P. multocida subsp. septica (80%), and nearly all isolates showed a similar combination of virulence-associated genes. MLST analysis classified the 20 P. multocida strains into 16 different sequence types, and we assigned 11 new sequence types (ST), however, only one of those (ST 334) was shared by two human and one cat isolates. P. multocida subsp. septica strains formed a distinct phylogenetic group within the species. The strains showed resistance to erythromycin, clindamycin and sulfamethoxazole, and with two exceptions, resistance to tilmicosin was also detected. Each strain was susceptible to ampicillin, streptomycin, gentamycin, tetracycline, doxycycline, cefazolin, cefpodoxime, chloramphenicol, florfenicol and enrofloxacin. Common characteristics (virulence profile and antibiotic sensitivity pattern) shared by strains isolated from humans and cats support the view that domestic cats may serve as a potential reservoir for P. multocida.  相似文献   

13.
Salmonella (S.) Typhimurium and S. Enteritidis are the major causative agents of food-borne illnesses worldwide. Currently, a rapid detection system using multiplex real-time polymerase chain reaction (PCR) has been applied for other food-borne pathogens such as Escherichia coli, Staphylococcus aureus and Streptococcus spp. A multiplex real-time PCR was developed for the simultaneous detection of Salmonella spp., especially S. Typhimurium and S. Enteritidis, in beef and pork. For the specific and sensitive multiplex real-time PCR, three representative primers and probes were designed based on sequence data from Genbank. Among the three DNA extraction methods (boiling, alkaline lysis, and QIAamp DNA Mini Kit), the QIAamp DNA Mini Kit was the most sensitive in this study. The optimized multiplex real-time PCR was applied to artificially inoculated beef or pork. The detection sensitivity of the multiplex real-time PCR was increased. The specificity of the multiplex real-time PCR assay, using 128 pure-cultured bacteria including 110 Salmonella isolates and 18 non-Salmonella isolates, was 100%, 100% and 99.1% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The sensitivity was 100%, 100% and 91.7% for Salmonella spp., S. Typhimurium and S. Enteritidis, respectively. The multiplex real-time PCR assay developed in this study could detect up to 0.54 ± 0.09 and 0.65 ± 0.07 log10 CFU/ml for S. Typhimurium and S. Enteritidis for beef, 1.45 ± 0.21 and 1.65 ± 0.07 log10 CFU/ml for S. Typhimurium and S. Enteritidis for pork, respectively, with all conditions optimized. Our results indicated that the multiplex real-time PCR assay developed in this study could sensitively detect Salmonella spp. and specifically differentiate S. Typhimurium from S. Enteritidis in meats.  相似文献   

14.
猪链球菌2型及其毒力因子检测多重PCR的建立与应用   总被引：8，自引：3，他引：8  
根据相关文献设计并合成引物，建立了能同时检测猪链球菌2型（cps）及其重要毒力因子溶菌酶释放蛋白（mrp）和细胞外因子（epf）的多重PCR方法。目的片段的大小分别为885bp（mrp）、675bp（cps）和443bp（epf）。对参考菌株、人工攻毒病料和临床收集病料的检测结果显示，该多重PCR特异性强、敏感性高，可直接从临床病料中检测出猪链球菌2型，并能鉴定其毒力因子表型。  相似文献   

15.
Neurologic diseases are common in domestic cats, and infectious agents are suspected to be the primary cause in 30–45% of cases. Among infectious etiologies, those of bacterial origin are only sporadically characterized in the literature, with few of these reports correlating gross and histologic findings with confirmatory bacteriologic identification. Here, we describe bacterial meningitis and meningoencephalomyelitis associated with Pasteurella multocida in 3 domestic cats. Purulent exudate expanding the cerebral meninges was grossly evident in 2 of the cases. In all 3 cases, histologic changes included multifocal suppurative-to-necrosuppurative meningitis and/or meningoencephalomyelitis of variable severity. Intralesional colonies of gram-negative, short rod-shaped to coccobacillary bacteria were evident histologically in only 1 case. P. multocida was confirmed by routine bacteriologic culture in all cases. Based on our cases, we hypothesize that the upper respiratory system serves as the main portal of entry for P. multocida, leading to invasion of the central nervous system and possible systemic hematogenous dissemination. A case series of meningoencephalomyelitis associated with P. multocida infection in cats has not been reported previously, to our knowledge. We also review briefly other causes of meningoencephalomyelitis in cats.  相似文献   

16.
牛产肠毒素大肠杆菌毒力因子多重PCR检测方法的建立   总被引：6，自引：1，他引：6  
通过多重PCR扩增产肠毒素大肠杆菌（enterotoxigentic E.coli，ETEC）的毒力因子F41菌毛、K99菌毛和STa肠毒素的编码基因来检测和鉴定ETEC。试验中对影响PCR扩增的dNTP、Mg^2＋、引物浓度以及退火温度等因素进行优化，在优化条件的基础上，确定多重PCR的特异性和灵敏性，以此建立同时检测ETEC多个毒力因子的多重PCR方法。用该方法对分离于犊牛腹泻和犊牛肠毒血症的7株大肠杆菌进行检测，结果2株为F41、K99和STa阳性，4株为F41、STa阳性，1株为K99STa阳性。这与玻片凝集试验检测菌毛的结果一致。试验表明，该方法特异性强、敏感性高、简便、快速，适用于临床鉴定和检测牛ETEC菌株。  相似文献   

17.
Three multiplex real-time TaqMan PCR assays were developed for the detection of Escherichia coli virulence factor genes in veterinary samples. Target virulence factors chosen were the fimbriae K88 (F4), K99 (F5), F41, F17, F18 and 987p (F6) and the toxins LT, STa and CDT IV. Detection of genes coding GAD were included in each assay as an internal control. These assays allow rapid identification of virulence factor genes using identical cycling conditions on an Mx3000Ptrade mark real-time PCR machine with the capacity to test up to 20 strains for 9 virulence genes in 1h.  相似文献   

18.
本研究旨在建立一种快速鉴定致猪水肿病大肠埃希菌的多重PCR检测方法.分别针对大肠埃希菌16S rDNA、志贺毒素Stx2e A亚基和菌毛F18ab A亚基保守序列设计合成3对特异性引物,优化多重PCR反应条件,并进行特异性和敏感性检测.结果显示,阳性对照菌株扩增产物大小分别为1 062、733和313 bp.特异性和灵敏性检测结果表明,与肠炎沙门菌、多杀性巴氏杆菌、胸膜肺炎放线杆菌、副猪嗜血杆菌、支气管败血波氏杆菌和猪链球菌等猪常见致病菌均无交叉反应;菌体直接扩增法最低检出量为1 875 CFU.利用建立的多重PCR检测方法对分离收集的128株大肠埃希菌进行鉴定,得到36株致猪水肿病大肠埃希菌,其中30株既有菌毛F18ab又产志贺毒素Stx2e,另外6株仅产志贺毒素Stx2e.结果表明,本试验所建立的多重PCR检测方法对致猪水肿病大肠埃希菌的快速诊断和流行病学调查具有一定的应用价值.  相似文献   

19.
An exhaustive biochemical characterisation of 60 porcine Pasteurella multocida clinical isolates recovered from lesions indicative of pneumonia, previously confirmed by PCR and all belonging to the capsular serogroup A, was performed by means of four commercial systems. The API 20NE correctly identified almost all isolates (95%), but only 60% could be ascribed to this species by the API 20E method. The high diversity exhibited by the API 50CHB/E system, with six different patterns, does not advise its use as additional system for a definitive identification at the species level, but this method could be a potential tool for characterising P. multocida isolates below this level. The more uniform reactions yielded by the API ZYM test make this system helpful in the confirmatory identification of this organism. The high variability (20 profiles) obtained when the four systems are taken together also suggests their usefulness for epidemiological purposes in order to sub-type P. multocida isolates.  相似文献   

20.
A set of four specific primers for six regions of kmt1 gene from a species specific region was designed for developing the loop-mediated isothermal amplification diagnostic method of swine Pasteurella multocida (Pm-LAMP). After the Pm-LAMP was carried out at 63°C for 1 h, the LAMP products could be visually confirmed using fluorescent dyes as detection reagent under UV-illumination. In sensitivity, the detection limit of the Pm-LAMP was 10 cfu/mL, and was 1 log less than that of the PCR method. In specificity, the Pm-LAMP did not amplify genomic DNA of swine common respiratory pathogens. Furthermore, based on results for clinical swab samples (n = 31) using PCR detection as golden standard, relative sensitivity of the Pm-LAMP was 100%, relative specificity of the Pm-LAMP was 90.9%, and percentage of observation agreement was 93.5% (Kappa = 0.85). The Pm-LAMP method should be a useful diagnostic tool for rapid and visible detection of swine Pasteurella multocida.  相似文献   

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