Identification of Prototheca Species Responsible for Bovine Mastitisby New Multiplex PCR Method

摘要

The genus Prototheca consists of achlorophyllous algae closely related to the green algae of the genus Chlorella. Species of the genus Prototheca exist in the environment as ubiquitous detritus inhabitants and contaminants of various substrates. Untilnow, within the known Prototheca spp., only P. zopfii genotype 2, P. wickerhamii and P. blaschkeae have been associated with pathology in humans and animals (Hosaka 2004). In dairy cattle, Prothoteca spp. were considered rare pathogens in the past, andassociated to infection in presence of predisposing factor, such as poor environmental conditions and insufficient milking hygiene (Janosi 2001). Nowadays, however, it has attracted considerable attention because cases of acute to chronic mastitis were recognized increasingly to be endemic worldwide (Ricchi et al., 2010; Gao et al., 2012). Different molecular techniques have been proposed to identify these pathogens, however such of these methods are unable to differentiate among different Prototheca species or are time consuming, demanding more than a PCR reaction. In the present study, a multiplex PCR reaction, based on specific amplification of mithocondrial and chloroplastic sequences, able to identify in a single reaction the three Prototheca spp.associated to bovine mastitis, has been developed.