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Internal transcribed spacer sequence-based rapid molecular identification of Prototheca zopfii and Prototheca
S Marques1, V A R Huss2, K Pfisterer2
1Centro 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.
Abstract:
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.
Insights
Rapid molecular identification of Prototheca zopfii and Prototheca blaschkeae in bovine mastitis is crucial. This study developed a species-specific PCR assay using ribosomal DNA internal transcribed spacer sequences for accurate diagnosis in milk samples.
Area of Science:
- Veterinary Microbiology
- Molecular Diagnostics
- Algal Diseases
Background:
- Prototheca algae are emerging mastitis pathogens in cattle, causing herd problems due to their resistance.
- Prototheca zopfii and Prototheca blaschkeae are the most common species implicated in bovine mastitis.
- Current diagnostic methods may not be sufficiently rapid or efficient for timely herd management.
Purpose of the Study:
- To develop a reliable and rapid molecular method for identifying and differentiating Prototheca zopfii and Prototheca blaschkeae.
- To utilize the internal transcribed spacer (ITS) sequences of ribosomal DNA for species-specific detection.
- To enable accurate diagnosis directly from bovine mastitic milk samples.
Main Methods:
- Sequencing of the complete internal transcribed spacer (ITS) regions of 32 Prototheca isolates.
- Design and application of species-specific PCR primers based on ITS sequence variability.
- Direct application of the developed PCR assay to bovine mastitic milk samples.
Main Results:
- Substantial interspecies and moderate intraspecies variability was observed in the ITS sequences.
- Species-specific PCR primers were successfully designed and validated.
- The PCR method accurately identified Prototheca zopfii and Prototheca blaschkeae directly from milk samples.
Conclusions:
- A rapid and reliable species-specific PCR assay for Prototheca zopfii and Prototheca blaschkeae in bovine mastitis has been developed.
- This molecular method facilitates timely diagnosis and improved herd management strategies.
- No significant correlation was found between intraspecific ITS phylogeny and geographical distribution of isolates.
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