应用DNA环介导恒温扩增技术快速检测空肠弯曲菌

【目的】空肠弯曲菌是一种重要的人畜共患病致病菌,主要经被污染的水源、乳制品、生禽肉进行传播,建立快速、准确的诊断方法是防控空肠弯曲菌感染的重要措施。【方法】本文引入一种新型的核酸检测技术--DNA环介导恒温扩增 (loop-mediated isothermal amplification, LAMP),以空肠弯曲菌ORF-C序列为检测靶基因,建立了空肠弯曲菌LAMP快速检测方法。【结果】利用本LAMP方法对21种病原菌进行检测,仅空肠弯曲菌为阳性结果,说明该方法具有高度的特异性。该LAMP方法对空肠弯曲菌纯培养菌的检测灵敏度达6CFU/反应管,对污染材料中空肠弯曲菌的检测灵敏度为9CFU/反应管。【结论】本研究建立的空肠弯曲菌LAMP检测方法操作简便、检测快速,具有良好的实用性。      

Isolation of Campylobacter spp. from Client‐Owned Dogs and Cats,and Retail Raw Meat Pet Food in the Manawatu,New Zealand

Campylobacter causes acute gastroenteritis in people worldwide and is frequently isolated from food, animals and the environment. The disease is predominately food‐borne but many routes of transmission and sources of infection have been described, including contact with pets. The prevalence of Campylobacter spp. in dogs and cats varies widely, and data on New Zealand pets are limited. This study aimed to investigate the prevalence of Campylobacter spp. in dogs, cats and retail raw meat pet food products in New Zealand and to characterize Campylobacter jejuni isolates using multilocus sequence typing (MLST). Ninety dogs and 110 cats examined at the Massey University Veterinary Teaching Hospital for elective procedures, and fifty locally purchased retail raw meat pet diets were sampled. Two culture protocols combining Bolton broth enrichment and mCCDA and CAT agars in a microaerobic atmosphere at 42°C and 37°C with species identification using PCR were performed. The prevalence of Campylobacter spp., C. jejuni, Campylobacter upsaliensis and Campylobacter helveticus was 36%, 13%, 23% and 1% in dogs and 16%, 5%, 5% and 7% in cats, respectively. One dog had Campylobacter lari confirmed, and three dogs and one cat had multiple Campylobacter spp. detected. Significantly more animals tested positive using CAT than mCCDA agar (P < 0.001). Being neutered, vaccinated for Bordetella bronchiseptica, fed dry diets and brought in for neutering were protective factors for dogs, whereas attendance for dental treatment was a risk factor for cats. Campylobacter spp. were isolated from 28%, C. jejuni 22%, C. lari 6% and Campylobacter coli 6% of food samples. Six isolates positive by Campylobacter genus PCR were identified as Arcobacter butzleri. Poultry meat was more likely to be positive than non‐poultry meat (P = 0.006). Of the 13 C. jejuni pet isolates with full MLST profiles, eight were of different sequence types (ST) and all nine food isolates were of different STs.     

Campylobacter fetus Bacteremia in a Healthy Patient Returning from a Trip to the Ecuadorian Amazonia

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.     

Development and evaluation of an indirect enzyme-linked immunosorbent assay for the detection of antibodies against Campylobacter fetus in cattle

Campylobacteriosis is a zoonosis that occurs worldwide. Infection with Campylobacter fetus (C. fetus) causes infertility and abortion in sheep and cattle. The current study focuses on the SapA gene of C. fetus that encodes surface array proteins and plays an important role in the virulence of C. fetus. The SapA-N (1398 bp) and SapA-C (1422 bp) fragments were amplified from the C. fetusSapA gene using polymerase chain reaction (PCR), and the corresponding recombinant proteins rSapA-N and rSapA-C were expressed in Escherichia. coli BL21 cells. Results of Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the immunological activity of rSapA-N was higher than that of rSapA-C (P < 0.05). Therefore, rSapA-N was selected to establish an indirect ELISA for detecting antibodies against C. fetus. The diagnostic criteria were as follows: S/P ? 0.45: positive; S/P < 0.4: negative; 0.45 > S/P ? 0.4: suspected. The specificity and sensitivity of our method were 94.3% and 88.6%, respectively. Moreover, no cross-reactions were observed between rSapA-N and serum samples that were positive for other bovine bacterial pathogens diseases such as Mycobacterium avium subspecies paratuberculosis. One hundred and two serum samples from cows that had experienced abortion were tested. Four and 2 C. fetus-positive serum samples were found among the 70 bovine brucellosis-positive samples and the 32 infectious bovine rhinotracheitis (IBR)-positive samples, respectively. The findings suggest that the rSapA-N-based ELISA method has immense potential in future applications.     

Evaluation of a PCR assay for identification and differentiation of Campylobacter fetus subspecies

Objective To evaluate a polymerase chain reaction assay for identification of Campylobacter fetus and differentiation of the defined subspecies.
Design Characterisation of bacterial strains by traditional phenotyping, polymerase chain reaction, a probabilistic identification scheme and macrorestriction profiling using pulsed field gel electrophoresis.
Procedure The results of identification of 99 bacterial strains as determined by conventional phenotyping or by poly-merase chain reaction were compared. Two of these were type strains of C fetus subsp fetus and C fetus subsp venerealis ; the remaining strains were field isolates putatively identified as C fetus . In cases where the subspecies identity was disputed, isolates were identified by means of a probabilistic identification scheme and by macrorestriction profiling.
Results The agreement between strain identities initially suggested by traditional phenotypic methods and the PCR assay was found to be 80.8%. The polymerase chain reaction proved to be a reliable technique for the species and subspecies identification of C fetus ; equivocal results were obtained in only two instances. Initial misidentifications by conventional phenotyping methods were attributed to methodological differences used in various laboratories.
Conclusion Our results indicate that misidentification of C fetus i n routine diagnostic laboratories may be relatively common. The PCR assay evaluated gave rapid and reproducible results and is thus a valuable adjunctive method for the identification of C fetus and subsequent subspecies differentiation.     

Risk factors for Campylobacter infection in Norwegian cats and dogs

Rectal swabs from healthy cats and dogs, and from dogs and cats with clinical diarrhoea were collected approximately every third month from May 2000 to June 2001 from six small-animal practices throughout Norway. A questionnaire was filled in for each animal. Of the 301 healthy cats sampled, 54 (18%) were positive for Campylobacter, compared to 5 out of 31 (16%) cats with diarrhoea. Campylobacter jejuni was isolated from 11 (3%), C. upsaliensis from 42 (13%) and C. coli from 2 (0.6%) of the cats sampled. Isolates from four cats (1%) could not be specified. Of the 529 healthy dogs, 124 (23%) were positive for Campylobacter, compared to 18 of 66 (27%) dogs with diarrhoea. C. jejuni was isolated from 20 (3%) and C. upsaliensis from 117 (20%) of the dogs sampled. Isolates from five dogs (0.8%) could not be specified. Eighteen out of the 20 investigated C. upsaliensis samples were resistant to streptomycin. The clinically healthy animals were included in the analysis to identify factors associated with Campylobacter prevalence. The cat model had low classification ability. The dog-data model indicated increased odds of infection with Campylobacter for dogs ≤1 year, and in dogs sampled during the spring. No difference was observed between the prevalence of Campylobacter infections in cats and dogs with diarrhoea and healthy animals.     

吖啶橙免疫荧光菌团法快速检测空肠弯曲菌的研究

本研究建立了适用于快速检测空肠弯曲菌的吖啶橙免疫荧光苗团培养沉淀法。用该法可检出每毫升接种约40个菌,增菌24h的培养物或每毫升合约10万个菌的菌悬液,不与水弧菌、大肠杆菌等13种杂菌发生交叉反应。对貉、水貂、獭兔和熊等经济动物310份肛拭样本进行了检测,其阳性率分别为66％、19％、21％、29％,与常规培养法均相差不显苦(P>0.05),可在27h内报告结果,而常规培养法则需要5～7d。该法适用于大样本的检测和基层单位应用。     

食品中常见病原菌多重PCR检测方法的建立

根据基因库中沙门氏菌、空肠弯曲杆菌、单核细胞增生李斯特菌和大肠杆菌O157：H7的invA、MapA、hlyA和O gene cluster基因分别设计了4对引物,通过对反应条件的优化,建立了同时检测4种病原菌的多重PCR方法。结果表明,该多重PCR方法可扩增出四条特异性条带,并且任意两条产物片段长度相差大于20％。多重PCR反应体系检测四种病原菌混合模板最低含量为100 CFU。该多重PCR检测方法具有快速、准确和特异性强的优点,可用于快速检测食品中的病原菌。     

Isolation of a Campylobacter lanienae‐like Bacterium from Laboratory Chinchillas (Chinchilla laniger)

Routine necropsies of 27 asymptomatic juvenile chinchillas revealed a high prevalence of gastric ulcers with microscopic lymphoplasmacytic gastroenteritis and typhlocolitis. Polymerase chain reaction (PCR) analysis using Campylobacter genus‐specific partial 16S rRNA primers revealed the presence of Campylobacter spp. DNA in the faeces of 12 of 27 animals (44.4%). Species‐specific partial 16S rRNA PCR and sequencing confirmed that these animals were colonized with Campylobacter lanienae, a gram‐negative, microaerophilic bacterium that was first identified on routine faecal screening of slaughterhouse employees and subsequently isolated from faeces of livestock. Campylobacter lanienae was isolated from the faeces of six PCR‐positive animals and identified with species‐specific PCR and full 16S rRNA sequencing. Phylogenetic analysis showed that these isolates clustered with C. lanienae strain NCTC 13004. PCR analysis of DNA extracted from gastrointestinal tissues revealed the presence of C. lanienae DNA in the caecum and colon of these chinchillas. Gastrointestinal lesions were scored and compared between C. lanienae‐positive and C. lanienae‐negative animals. There was no correlation between colonization status and lesion severity in the stomach, liver, duodenum, or colon. Possible routes of C. lanienae infection in chinchillas could include waterborne transmission and faecal–oral transmission from wild mice and rats or livestock. Based on these findings, the authors conclude that C. lanienae colonizes the lower bowel of chinchillas in the absence of clinical disease. This is the first report of C. lanienae in any rodent species. Campylobacter lanienae isolates from different mammalian species demonstrate heterogeneity by 16S rRNA sequence comparison. Analysis using rpoB suggests that isolates and clones currently identified as C. lanienae may represent multiple species or subspecies.     

不同培养条件下空肠弯曲菌胞外多糖合成研究

为探讨空肠弯曲菌生物膜耐药机理,采用硫酸-蒽酮法测定了浮游菌和生物膜菌胞外多糖的产量。结果表明,两种状态细菌合成的水溶性胞外多糖量,随着时间的延长,呈现出先增加后下降的趋势。在同一时间点,生物膜菌黏附状态均显著低于浮游状态（P ＜0．05）;水不溶性胞外多糖除了黏附状态合成量高于浮游状态外,不具有明显的规律。不同状态细菌合成胞外多糖差异显著,生物膜所处的酸性环境、营养物质的耗尽等可能是产生差异的原因。