The identification of coagulase-negative staphylococci (CNS) causing ovine infections
remains problematic, although these bacteria are considered the main etiologic agents of
subclinicalmastitis in sheep and goats. In this study, 226 CNS isolates were collected from
2201 milking sarda sheep belonging to 15 flocks with high somatic cell count scores. All
isolates were subjected to identification with the API Staph ID test, and then to the
amplification of staphylococcal 16S rRNA and gap genes by PCR assays. The gap gene was
subjected to restriction fragment length polymorphism analysis with the restriction
endonuclease AluI, whereas the 16S rRNA gene was subjected to ribosomal fingerprinting
with the restriction endonucleases RsaI, PstI and AluI. When PCR–RFLP patterns of CNS
isolates were different from those of their reference strains, gap gene amplicons were
sequenced for definitive identification. The API Staph ID test, in alternative to the
genotypic identification method, produced considerably different results in terms of
species identified within each group. Using the PCR–RFLP assay, most of the isolates
clustered together with the Staphylococcus epidermidis type strain (131, corresponding to
57.9%), followed by S. caprae (34, corresponding to 15%) and S. chromogenes (30,
corresponding to 13.2%). In conclusion, the PCR–RFLP assay of 16S rRNA and gap genes is a
more reliable and reproducible method than the API Staph ID test for the identification of
CNS causing sheep mastitis.
2010 Elsevier
The identification of coagulase-negative staphylococci (CNS) causing ovine infectionsremains problematic, although these bacteria are considered the main etiologic agents ofsubclinicalmastitis in sheep and goats. In this study, 226 CNS isolates were collected from2201 milking sarda sheep belonging to 15 flocks with high somatic cell count scores. Allisolates were subjected to identification with the API Staph ID test, and then to theamplification of staphylococcal 16S rRNA and gap genes by PCR assays. The gap gene wassubjected to restriction fragment length polymorphism analysis with the restrictionendonuclease AluI, whereas the 16S rRNA gene was subjected to ribosomal fingerprintingwith the restriction endonucleases RsaI, PstI and AluI. When PCR–RFLP patterns of CNSisolates were different from those of their reference strains, gap gene amplicons weresequenced for definitive identification. The API Staph ID test, in alternative to thegenotypic identification method, produced considerably different results in terms ofspecies identified within each group. Using the PCR–RFLP assay, most of the isolatesclustered together with the Staphylococcus epidermidis type strain (131, corresponding to57.9%), followed by S. caprae (34, corresponding to 15%) and S. chromogenes (30,corresponding to 13.2%). In conclusion, the PCR–RFLP assay of 16S rRNA and gap genes is amore reliable and reproducible method than the API Staph ID test for the identification ofCNS causing sheep mastitis. 2010 Elsevier
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