共查询到20条相似文献,搜索用时 15 毫秒
1.
Gregorio Iraola Martín Hernández Lucía Calleros Fernando Paolicchi Silvia Silveyra Alejandra Velilla Luis Carretto Eliana Rodríguez Ruben Pérez 《Journal of veterinary science (Suw?n-si, Korea)》2012,13(4):371-376
Campylobacter (C.) fetus (epsilonproteobacteria) is an important veterinary pathogen. This species is currently divided into C. fetus subspecies (subsp.) fetus (Cff) and C. fetus subsp. venerealis (Cfv). Cfv is the causative agent of bovine genital Campylobacteriosis, an infectious disease that leads to severe reproductive problems in cattle worldwide. Cff is a more general pathogen that causes reproductive problems mainly in sheep although cattle can also be affected. Here we describe a multiplex PCR method to detect C. fetus and differentiate between subspecies in a single step. The assay was standardized using cultured strains and successfully used to analyze the abomasal liquid of aborted bovine fetuses without any pre-enrichment step. Results of our assay were completely consistent with those of traditional bacteriological diagnostic methods. Furthermore, the multiplex PCR technique we developed may be easily adopted by any molecular diagnostic laboratory as a complementary tool for detecting C. fetus subspecies and obtaining epidemiological information about abortion events in cattle. 相似文献
2.
3.
胎儿弯杆菌病TaqMan实时荧光定量PCR检测方法的建立 总被引:1,自引:0,他引:1
为建立胎儿弯杆菌(C.fetus)定量检测方法,本研究根据C.fetus毒力因子表面蛋白(SapA)基因序列设计引物和一条特异的TaqMan水解探针,建立了一种敏感、特异、重复性好的快速检测C.fetus的TaqMan荧光定量PCR方法.对该方法的特异性与敏感性研究,结果显示,该方法检测C.fetus结果均为阳性,而非C.fetus均为阴性;对带有SapA基因的阳性质粒的检测敏感性为10~8拷贝~10~2拷贝/μL范围内具有良好的线性关系,可敏感地检测到模板中13个拷贝的细菌DNA,其灵敏度是常规PCR方法的100倍.该方法具有简便、快速、特异性强、敏感性高等特点.该方法为C.fetus快速检测试剂盒的研制打下了良好的基础. 相似文献
4.
SA Mannering DM West SG Fenwick RM Marchant NR Perkins K O'Connell 《New Zealand veterinary journal》2013,61(6):358-363
AIMS: To genotype Campylobacter fetus subsp. fetus isolates cultured from sheep abortions submitted to diagnostic laboratories in New Zealand during the year 2000 breeding season. To compare the types found nationally with those found in the Hawke' Bay region in 1999, and strains held in the New Zealand Reference Culture Collection, Medical Section (NZRM) from a study published in 1987. METHODS: Campylobacter fetus subsp. fetus isolates cultured by veterinary diagnostic laboratories in the year 2000 breeding season, from sheep abortions from throughout New Zealand, were typed using pulsed-field gel electrophoresis (PFGE). In addition, seven freeze-dried C. fetus subsp. fetus isolates (strain numbers 2939–2945) from the NZRM, representing restriction types a–g found amongst sheep abortion isolates in a study published in 1987, were typed using PFGE. RESULTS: In total, 293 C. fetus subsp. fetus isolates from 200 farms were obtained from veterinary diagnostic laboratories. Twenty-two distinct PFGE profiles were identified amongst the isolates. PFGE type B1 was predominant in each region of New Zealand and was identified from 66% of farms overall. Of the C. fetus subsp. fetus restriction types a–g lodged with the NZRM, 3/7 had PFGE profiles indistinguishable from profiles found in the current study. The other four restriction types had PFGE profiles that were unique but similar to those found in the current study. CONCLUSIONS: PFGE type B1 was predominant amongst the C. fetus subsp. fetus isolates cultured from sheep abortions in each region of New Zealand in the year 2000, as was found in Hawke' Bay in 1999. The similarity between PFGE profiles of C. fetus subsp. fetus sheep abortion isolates from 1987 and 2000, and the relative prevalence of the PFGE groups, suggests that there has been no major genotypic shift in the population of C. fetus subsp. fetus implicated in sheep abortion in New Zealand during this time. 相似文献
5.
The species Campylobacter (C.) fetus is divided into the subspecies venerealis and fetus, which differ in epidemiology and clinical importance. The differences between these subspecies make an accurate distinction essential. Differentiation of C. fetus by traditional microbiological methods is only based on two reactions (tolerance to glycin, Na selenite reduction), in which C. fetus ssp. venerealis reacts negatively. However, the value of both reactions is limited. We used a specific PCR-based assay for identifying and differentiating the two C. fetus subspecies, which was recently developed by HUM et al. (1997). In this assay, a 764 bp amplicon is produced using primers MG3F and MG4R for both subspecies of C. fetus. In contrast to HUM et al. (1997), this amplicon was approximately 200 bp smaller. This discrepancy can't be explained. Afterwards, the primers VenSF and VenSR are used for differentiation. The identification of the sub-species venerealis is based on the presence of a 142 bp amplicon, which is not formed with subspecies fetus. The type strains of both C. fetus subspecies were used as positive controls. Non-specific reactions were not observed. In this PCR assay, 73 field strains were investigated (among them 24 C. fetus ssp. veneralis, 26 C. fetus ssp. fetus). In these investigations, the method has proved its diagnostic suitability. The results of the traditional microbiological differentiation of the C. fetus field strains could be confirmed by the PCR assay. In future, the traditional phenotypic characterization of C. fetus subspecies remains indispensable, but this PCR assay constitutes a valuable method for the confirmation of these results. 相似文献
6.
7.
Campylobacter fetus Bacteremia in a Healthy Patient Returning from a Trip to the Ecuadorian Amazonia 下载免费PDF全文
Campylobacter fetus is an opportunistic pathogen which causes bacteremia and other invasive infections in immunocompromised patients who have been exposed to livestock or ingested animal products (uncooked meat or unpasteurized milk). The present report describes a C. fetus infection in a healthy adult (immunocompetent) who returned from a visit to the Ecuadorian Amazonia and who did not report exposure to the typical sources of infection. 相似文献
8.
9.
10.
As a result of the high lability and slow growth of Campylobacter fetus subspecies, the laboratory diagnosis of bovine genital campylobacteriosis has always been difficult. This is especially true under South African conditions, where farms are far apart, laboratories are only present in major centres and there are high ambient temperatures. In order to overcome the shortcomings associated with traditional diagnostic methods, the implementation of a molecular assay was sought. This work describes how a previously published PCR assay (MG3F/ MG4R primers) was adapted, optimised and applied in the diagnostic laboratory to test preputial samples directly for the presence of Campylobacter fetus. Field evaluation of the assay revealed an analytical sensitivity and specificity of 85.7% and 99%, respectively. Subsequent genotyping and phenotyping of a diverse collection of South African field isolates revealed that South Africa has an unexpected and previously unreported high incidence of Campylobacter fetus subsp. venerealis biovar intermedius strains. These strains were not identified correctly by the subspecies-specific primer set evaluated. Until such time that cost- effective genotyping methods are available to diagnostic laboratories in South Africa, and other countries with these atypical Campylobacter fetus subsp. venerealis strains, the need for bacterial culture will persist. Identification to subspecies level of isolates at present remains dependent upon a single phenotypic criterion, namely tolerance to 1% glycine. 相似文献
11.
12.
Sensitivity of Direct Culture,Enrichment and PCR for Detection of Campylobacter jejuni and C. coli in Broiler Flocks at Slaughter 下载免费PDF全文
J. D. Rodgers E. Simpkin R. Lee F. A. Clifton‐Hadley A. B. Vidal 《Zoonoses and public health》2017,64(4):262-271
Broiler chicken flocks are a significant source of Campylobacter jejuni and Campylobacter coli that result in the major public health problem of campylobacteriosis. Accurate estimates of the prevalence of both C. coli and C. jejuni in flocks would enhance epidemiological understanding, risk assessment and control options. This study combined results from a panel of 10 detection tests (direct culture, enrichment and PCR) on caecal samples from flocks at slaughter. A parallel interpretation approach was used to determine the presence of Campylobacter spp. and for C. jejuni and C. coli individually. The sample was considered positive if at least one method detected the target and this interpretation was taken to represent a ‘proxy gold standard’ for detection in the absence of a gold standard reference test. The sensitivity of each individual method to detect Campylobacter spp., C. jejuni and C. coli was then estimated relative to the proxy gold standard. Enrichment in adapted Exeter broth (deficient in polymyxin B) with a resuscitation step was 100% sensitive, whilst direct culture on modified charcoal cefoperazone deoxycholate agar (mCCDA) was highly sensitive (97.9%). Enrichment methods using Preston broth and Bolton broth were significantly less sensitive. Enrichment in Exeter broth promoted the recovery of C. jejuni, whilst enrichment in Bolton broth favoured C. coli. A RT‐PCR detection test could identify 80% of flocks that were co‐colonised with both species. This study found that 76.3% (n = 127) of flocks were colonised with Campylobacter spp. The majority (95.9%) of Campylobacter‐positive flocks were colonised with C. jejuni; however, approximately one‐third of positive flocks were simultaneously colonised with both C. jejuni and C. coli. The findings highlight the impact of different detection methodologies on the accuracy of the estimated incidence of both C. jejuni and C. coli entering the abattoir within broiler flocks and the associated public health risks. 相似文献
13.
14.
Giardia intestinalis is a pathogenic protozoan which infects humans and a wide range of animal hosts, including cats and dogs(1). However, the status of animals in New Zealand with respect to Giardia infection has not received much attention, so we undertook a preliminary study of cats and dogs in Palmerston North and Hamilton to determine the prevalence of infection, as indicated by the presence of cysts in faeces. 相似文献
15.
Bonnie Chaban Shirley Chu Steven Hendrick Cheryl Waldner Janet E. Hill 《Canadian journal of veterinary research》2012,76(3):166-173
The detection and subspeciation of Campylobacter fetus subsp. venerealis (CFV) from veterinary samples is important for both clinical and economic reasons. Campylobacter fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis, a venereal disease that can lead to serious reproductive problems in cattle, and strict international regulations require animals and animal products to be CFV-free for trade. This study evaluated methods reported in the literature for CFV detection and reports the translation of an extensively tested CFV-specific polymerase chain reaction (PCR) primer set; including the VenSF/VenSR primers and a real-time, quantitative PCR (qPCR) platform using SYBR Green chemistry. Three methods of preputial sample preparation for direct qPCR were evaluated and a heat lysis DNA extraction method was shown to allow for CFV detection at the level of approximately one cell equivalent per reaction (or 1.0 × 103 CFU/mL) from prepuce. The optimized sample preparation and qPCR protocols were then used to evaluate 3 western Canadian bull cohorts, which included 377 bulls, for CFV. The qPCR assay detected 11 positive bulls for the CFV-specific parA gene target. DNA sequence data confirmed the identity of the amplified product and revealed that positive samples were comprised of 2 sequence types; one identical to previously reported CFV parA gene sequences and one with a 9% sequence divergence. These results add valuable information towards our understanding of an important CFV subspeciation target and offer a significantly improved format for an internationally recognized PCR test. 相似文献
16.
为建立一种能鉴别鸡毒支原体(MG)强、弱毒株的快速检测方法,本研究根据GenBank中MG强毒株和弱毒株的基因组序列,选取特异性保守区序列设计了2对引物和2奈探针,分别用于强弱毒株和弱毒株的检测,优化反应条件,建立了能区分MG强、弱毒株的荧光定量PCR检测方法.该法特异性强,对鸡常见呼吸道病原体的反应均为阴性;灵敏度高,可检测到100拷贝/μL的模板;稳定性好,批内和批间试验Ct值的变异系数小.本研究建立的MG强、弱毒鉴别检测方法简便、快捷,为该病的防控与净化提供新方法、新思路. 相似文献
17.
Campylobacteriosis is the most commonly notified illness in New Zealand. Whilst the importance of commercial poultry in campylobacteriosis is well established, little is known about the possible role of chickens kept at home as a direct animal/faecal contact or consumption exposure pathway. The aim of this study was to determine the prevalence and genetic diversity of Campylobacter spp. in domestic backyard chicken flocks in the Canterbury region of New Zealand. Poultry faecal samples were collected from 35 domestic 'backyard' poultry flocks from urban and rural properties around the Canterbury Region of New Zealand. A total of 291 samples were collected and tested for the presence of thermotolerant Campylobacter spp. and positive isolates were analysed using pulsed-field gel electrophoresis (PFGE) using both SmaI and KpnI enzymes. There was a high prevalence of Campylobacter spp. with 86% of flocks testing positive. Campylobacter jejuni alone, Campylobacter coli alone and both C. jejuni and C. coli were detected in 20 (57%), 2 (6%) and 8 (23%) of the flocks respectively. SmaI/KpnI PFGE analysis identified 50 different genotypes across the 35 flocks. Genotype diversity richness was highest on the lifestyle block and farm properties with 43 different genotypes isolated, whilst urban properties displayed the least richness with 12 genotypes isolated. Rural flocks tended to have more different genotypes in a given flock than urban flocks. Comparison of the genotypes with the PulseNet Aotearoa Campylobacter database showed that 28 of the genotypes had previously been isolated from human cases of campylobacteriosis. Many of these were also indistinguishable from Campylobacter spp. previously isolated from retail chicken. Therefore, contact with backyard poultry or their faecal material is a potential additional infection pathway outside of exposure to the established pathways associated with the consumption of Campylobacter-contaminated commercial meat or foods cross-contaminated from contaminated poultry. 相似文献
18.
根据番鸭细小病毒(MDPV)和鹅细小病毒(GPV)基因组的非同源序列各设计了1对引物MDPVF1/R1和GPVF1/R1,建立了一种PCR方法,用该PCR方法分别对GPV、MDPV、鸭瘟病毒(DPV)、鸭肝炎病毒(DHV)、鸭呼肠孤病毒(DRV)、犬细小病毒(CPV)和猫泛白细胞减少症病毒(FPLV)的病毒培养物及其核酸进行扩增。结果,引物MDPVF1/R1仅特异性扩增出MDPV的900bp核酸片段,引物GPVF1/R1仅特异性扩增出GPV的465bp核酸片段。表明,建立的PCR方法可用于GPV和MDPV的鉴别诊断。 相似文献
19.
A rapid screening assay forCampylobacter fetus in bull semen was developed using the polymerase chain reaction (PCR) and restriction endonuclease analysis (REA) to complement isolation by culture. An oligonucleotide primer pair (C1/C2) from the hypervariable region of 16S rRNA ofC. fetus was used to amplify a 362 base pair fragment by PCR. The PCR/REA assay, which is completed in 10 hours, detected as few as threeC. fetus subsp.venerealis cells in experimentally infected raw bull semen and in semen diluted with milk or egg yolk Tris (EYT). All the strains tested, of both subspecies ofC. fetus, were amplified, as were some otherCampylobacter species. Restricting the amplified products byAluI differentiatedC. fetus from the other organisms. There was no visible product generated by PCR fromC. sputorum subsp.bubulus, a saprophytic organism found in the prepuce of bulls, or from seven other species of bacteria found in semen. A modification of the PCR assay, using another primer pair (C3/C2) and two temperature PCR cycling conditions, increased the probability of detectingC. fetus subsp.venerealis. PCR amplification followed by REA could be used to screen bovine semen rapidly forC. fetus. In most cases, sequencing of C1/C2 PCR generated products would be preferable for distinguishing between the two subspecies ofC. fetus.Abbreviations AI
artificial insemination
- bp
base pair
- Cff
Campylobacter fetus subsp.fetus
- Cfv
Campylobacter fetus subsp.venerealis
- EYT
egg yolk Tris
- MH
Mueller-Hinton
- PCR
polymerase chain reaction
- REA
restriction endonuclease analysis
- TE
Tris-EDTA 相似文献
20.
Evaluation of a serum-based PCR assay for the diagnosis of canine monocytic ehrlichiosis 总被引:1,自引:0,他引:1
Mathios E. Mylonakis Victoria I. Siarkou Leonidas Leontides Eleftheria Bourtzi-Hatzopoulou Vassilios I. Kontos Alexander F. Koutinas 《Veterinary microbiology》2009,138(3-4):390-393
The aim of this study was to estimate the relative diagnostic sensitivity and specificity of a polymerase chain reaction (PCR) assay in the serum of dogs with naturally occurring non-myelosuppressive canine monocytic ehrlichiosis (CME), and to investigate the association between PCR positivity and immunofluorescence antibody (IFA) titres for Ehrlichia canis. Serum samples obtained from 38 dogs with non-myelosuppressive CME and 12 healthy dogs were analyzed retrospectively. Each serum sample was analyzed in triplicate using an E. canis-specific nested PCR assay targeting a 389 bp sequence of the 16S rRNA gene. E. canis DNA was amplified in 24 of 38 (63.1%) affected dogs; all samples from healthy dogs were negative. A high level of agreement was found among the PCR replicates (P < 0.0001). Median IFA titre of the 24 PCR-positive dogs was significantly lower than that of the PCR-negative infected dogs (P = 0.0029), indicating that E. canis DNA may circulate prior to the development of a high antibody titre. Serum-based PCR analysis is suggested for the early diagnosis of CME when whole blood samples are not available. 相似文献