Animal or human protothecosis belongs to rather rare, endemic, pro-inflammatory infections. It is caused by achlorophyllous algae of the genus Prototheca. Especially, P. bovis (formerly P. zopfii genotype 2) is often inflected as a non-bacterial causative agent of dairy cattle mastitis. In this study, we present a multiplex real-time PCR (qPCR) system for rapid and exact Prototheca spp. detection and quantification. Limit of detection, diagnostic sensitivity, and specificity were determined. For the first time, specific sequences of AccD (encoding acetyl CoA reductase) for P. bovis, cox1 (encoding cytochrome C oxidase subunit 1) for P. wickerhamii, cytB (encoding cytochrome B) for P. blashkeae and atp6 (encoding transporting ATPase F0 subunit 6) for P. ciferrii (formerly P. zopfii genotype 1) were used for species identification and quantification together with 28S rRNA sequence detecting genus Prototheca. The developed qPCR assay was applied to 55 individual cow milk samples from a herd suspected of protothecosis, 41 bulk milk samples from different Czech farms, 16 boxed milk samples purchased in supermarkets and 21 environmental samples originating from a farm suspected of protothecosis. Our work thus offers the possibility to diagnose protothecosis in the samples, where bacterial mastitis is the most commonly presumed and thereby assisting adequate corrective measures to be taken.

Dairy Research Institute Prague Ke Dvoru 12a 160 00 Prague 6 Czech Republic

Department of Animal Origin Food and Gastronomic Sciences University of Veterinary and Pharmaceutical Sciences Brno Czech Republic Palackeho 1946 1 Brno 612 42 Czech Republic

Department of Bacteriology State Veterinary Institute Jihlava Rantirovska 93 20 Jihlava Horni Kosov 586 01 Jihlava Czech Republic

Laboratory of Food Microbiology Department of Food and Feed Safety Veterinary Research Institute Hudcova 296 70 621 00 Brno Czech Republic

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