McDonald W L, Fry B N, Deighton M A
School of Applied Sciences, RMIT University, Bundoora Campus, Bundoora, Vic. 3083, Australia.
Vet Microbiol. 2005 Dec 20;111(3-4):241-6. doi: 10.1016/j.vetmic.2005.10.012. Epub 2005 Nov 10.
Traditional microbiological methods for identification of Streptococcus spp. causing bovine mastitis have been demonstrated to be less than highly reliable. PCR-RFLP analysis of 16S-23S ribosomal DNA was used to characterise seven reference strains of streptococcal mastitis pathogens as well as four reference strains of other gram-positive, catalase-negative cocci of bovine origin to allow comparative identification of field isolates. RFLP analysis of PCR products, using a combination of two restriction endonucleases in single reactions (HaeIII and AluI, HaeIII and RsaI or AluI and RsaI) generated unique patterns for species of Streptococcus, Enterococcus and Lactococcus. One hundred field isolates of Streptococcus spp. collected from cows with clinical or subclinical mastitis were tested. Fifty-seven isolates, classified by conventional tests as S. uberis, were identified as 47 S. uberis and six S. parauberis by their unique RFLP patterns. The remaining four isolates had RFLP patterns distinct from the reference strains and two of these were identified as closely related to S. iniae and two to Aerococcus viridans by 16S rRNA sequencing. Conventional identification of 17 S. agalactiae and 26 S. dysgalactiae subsp. dysgalactiae was confirmed by RFLP. Use of a combination of restriction enzymes in a single tube enabled the rapid, accurate, cost effective and easily performed identification of all major streptococcal mastitis pathogens.
已证明，用于鉴定引起牛乳腺炎的链球菌属的传统微生物学方法可靠性不高。采用16S - 23S核糖体DNA的PCR - RFLP分析对7株链球菌性乳腺炎病原体参考菌株以及4株源自牛的其他革兰氏阳性、过氧化氢酶阴性球菌参考菌株进行特征分析，以便对现场分离株进行比较鉴定。在单个反应中使用两种限制性内切酶组合（HaeIII和AluI、HaeIII和RsaI或AluI和RsaI）对PCR产物进行RFLP分析，可为链球菌属、肠球菌属和乳球菌属产生独特的图谱。对从患有临床或亚临床乳腺炎的奶牛中收集的100株链球菌属现场分离株进行了检测。通过常规检测分类为乳房链球菌的57株分离株，根据其独特的RFLP图谱鉴定为47株乳房链球菌和6株副乳房链球菌。其余4株分离株具有与参考菌株不同的RFLP图谱，其中2株通过16S rRNA测序鉴定为与海豚链球菌密切相关，另外2株与绿色气球菌密切相关。通过RFLP证实了17株无乳链球菌和26株停乳链球菌停乳亚种的常规鉴定。在单个试管中使用限制性酶组合能够快速、准确、经济高效且易于操作地鉴定所有主要的链球菌性乳腺炎病原体。
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