Internal transcribed spacer sequence-based rapid molecular identification of Prototheca zopfii and Prototheca blaschkeae directly from milk of infected cows.
The increasing incidence of rare mastitis-causing pathogens has urged the implementation of fast and efficient diagnostic and control measures. Prototheca algae are known to be associated with diseases in humans and animals. In the latter, the most prevalent form of protothecosis is bovine mastitis with Prototheca zopfii and Prototheca blaschkeae representing the most common pathogenic species. These nonphotosynthetic and colorless green algae are ubiquitous in different environments and are widely resistant against harmful conditions and antimicrobials. Hence, the association of Prototheca with bovine mastitis represents a herd problem, requiring fast and easy identification of the infectious agent. The purpose of this study was to develop a reliable and rapid method, based on the internal transcribed spacer (ITS) sequences of ribosomal DNA, for molecular identification and discrimination between P. zopfii and P. blaschkeae in bovine mastitic milk. The complete ITS sequences of 32 Prototheca isolates showed substantial interspecies but moderate intraspecies variability facilitating the design of species-specific PCR amplification primers. The species-specific PCR was successfully applied to the identification of P. zopfii and P. blaschkeae directly from milk samples. The intraspecific ITS phylogeny was compared for each species with the geographical distribution of the respective Prototheca isolates, but no significant correlation was found.
Centro de Investigação em Biodiversidade e Recursos Genéticos Universidade do Porto (CIBIO/UP), InBio Laboratório Associado Campus Agrário de Vairão, Rua Padre Armando Quintas, n 7, 4485-661 Vairão, Portugal; Laboratório de Doenças Infecciosas do Departamento de Clínicas Veterinárias, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto, Rua de Jorge Viterbo Ferreira, n 228, 4050-313 Porto, Portugal. Electronic address: saramarques@mail.icav.up.pt.
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S
Marques
Department of Biology, Molecular Plant Physiology, University of Erlangen-Nürnberg, Staudtstrasse 5, D-91058 Erlangen, Germany.
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Huss
Department of Biology, Molecular Plant Physiology, University of Erlangen-Nürnberg, Staudtstrasse 5, D-91058 Erlangen, Germany.
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K
Pfisterer
Department of Biology, Molecular Plant Physiology, University of Erlangen-Nürnberg, Staudtstrasse 5, D-91058 Erlangen, Germany.
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C
Grosse
Centro de Investigação em Biodiversidade e Recursos Genéticos Universidade do Porto (CIBIO/UP), InBio Laboratório Associado Campus Agrário de Vairão, Rua Padre Armando Quintas, n 7, 4485-661 Vairão, Portugal; Laboratório de Doenças Infecciosas do Departamento de Clínicas Veterinárias, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto, Rua de Jorge Viterbo Ferreira, n 228, 4050-313 Porto, Portugal.
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G
Thompson
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004269:DNA, Bacterial / Q000235:genetics*; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D005260:Female; D005838:Genotype; D008414:Mastitis, Bovine / Q000382:microbiology*; D008892:Milk / Q000382:microbiology*; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011525:Prototheca / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification
D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer
Prototheca spp. bovine mastitis; geographical distribution; internal transcribed spacer (ITS); rapid molecular identification; species-specific PCR (ssPCR)