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

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.

Field disinfestation trials against Culicoides in north-west Sardinia.

Bluetongue (BT) first affected Sardinia in August 2000, spreading rapidly across the island causing more than 6,000 outbreaks and significant economic damage. Culicoides imicola Kieffer (Diptera: Ceratopogonidae) was the main vector of the disease and was also found to be the most abundant Culicoides species on Sardinia. During 2002, a field trial was conducted to evaluate the efficacy of an insecticide on local Culicoides populations in north-western Sardinia. A synthetic pyrethroid derivative (Mycrocip, ICF, Cremona, Italy) was used on two farms where outbreaks of BT had been reported; a third farm was used as control. The same treatment was repeated after 15 days. For the collection of Culicoides, two blacklight traps were placed on each farm and operated every second day for two weeks before and after insecticide treatment. Insect collections and data analyses were performed in accordance with the protocols of the Italian National Reference Centre for Exotic Diseases (CESME: Centro Studi Malattie Esotiche). For each collection, the total number of insects, Culicoides spp. and C. imicola was determined. A slight decrease in the number of Culicoides collected on treated farms was recorded for only a few days after treatment. Mycrocip played a secondary role in suppressing insect numbers, but did not reduce the number of Culicoides. Indeed, periodic variations of Culicoides population sizes correlated with significant changes in weather conditions that prevailed, including oscillating temperatures, winds and relative humidity.