Molecular characterization of Polish Prototheca zopfii mastitis isolates and first isolation of Prototheca blaschkeae in Poland.

Bovine mastitits caused by the colorless, yeast-like alga Prototheca zopfii is a serious and complex condition that results in heavy economic losses in the dairy industry, both through a substantial reduction in milk production and culling of infected animals. Based on the 18S rDNA sequence analysis, genotype-specific PCR assays have recently been developed to differentiate within the species P. zopfii three distinct P. zopfii genotypes (1-3), of which P. zopfii genotype 3 has been considered a new species P. blaschkeae sp. nov. The purpose of this study was to employ the newly-devised molecular approach for the detection of the two P. zopfii genotypes and P. blaschkeae sp. nov. among bovine mastitis isolates from Poland. This study is the first to provide molecular characterization of Polish P. zopfii mastitis isolates. It also gives the first description of bovine mammary protothecosis due to P. blaschkeae in Poland, as evidenced by genotypical, microbiological, and electron microscopy findings.

Establishment of a matrix-assisted laser desorption ionization time-of-flight mass spectrometry database for rapid identification of infectious achlorophyllous green micro-algae of the genus Prototheca.

The possibility of using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid identification of pathogenic and non-pathogenic species of the genus Prototheca has been recently demonstrated. A unique reference database of MALDI-TOF MS profiles for type and reference strains of the six generally accepted Prototheca species was established. The database quality was reinforced after the acquisition of 27 spectra for selected Prototheca strains, with three biological and technical replicates for each of 18 type and reference strains of Prototheca and four strains of Chlorella. This provides reproducible and unique spectra covering a wide m/z range (2000-20 000 Da) for each of the strains used in the present study. The reproducibility of the spectra was further confirmed by employing composite correlation index calculation and main spectra library (MSP) dendrogram creation, available with MALDI Biotyper software. The MSP dendrograms obtained were comparable with the 18S rDNA sequence-based dendrograms. These reference spectra were successfully added to the Bruker database, and the efficiency of identification was evaluated by cross-reference-based and unknown Prototheca identification. It is proposed that the addition of further strains would reinforce the reference spectra library for rapid identification of Prototheca strains to the genus and species/genotype level.