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Identification of coagulase-negative staphylococci isolated from ovine milk samples by PCR–RFLP of 16S rRNA and gap genes

Onni, Toniangelo and Sanna, G. and Cubeddu, G. P. and Marogna, Gavino and Lollai, Stefano and Leori, Guido and Tola, Sebastiana (2010) Identification of coagulase-negative staphylococci isolated from ovine milk samples by PCR–RFLP of 16S rRNA and gap genes. Veterinary Microbiology, Vol. 144 (3-4), p. 347-352. eISSN 1873-2542. Article.

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DOI: 10.1016/j.vetmic.2010.01.016

Abstract

The identification of coagulase-negative staphylococci (CNS) causing ovine infections remains problematic, although these bacteria are considered the main etiologic agents of subclinical mastitis 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.

Item Type:Article
ID Code:5658
Status:Published
Refereed:Yes
Uncontrolled Keywords:Coagulase-negative staphylococci, ovine mastitis, 16S rRNA and gap genes, PCR–RFLP, DNA sequencing
Subjects:Area 07 - Scienze agrarie e veterinarie > VET/06 Parassitologia e malattie parassitarie degli animali
Divisions:002 Altri enti e centri di ricerca del Nord Sardegna > Istituto zooprofilattico sperimentale della Sardegna, Sassari
Publisher:Elsevier
eISSN:1873-2542
Deposited On:08 Mar 2011 12:52

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