Prototheca zopfii is an achlorophyllic algae that causes bovine mastitis, resulting in a reduction in milk production and the secretion of thin, watery milk with white flakes. The aim of the present study was to evaluate the bacterial flora in the udder environment in protothecal mastitis. We used metagenomic next-generation sequencing (NGS) analysis to identify 16S rRNA genes from bacterial flora present in milk samples from protothecal mastitic dairy cows. Seven clinical strains of P. zopfii genotype 2 were isolated from 7 milk samples from 7 cases (Holstein cow) of protothecal mastitis; another 9 milk samples were obtained from 9 normal Holstein cows. The samples were collected in 2017 from cows in one dairy located in the Kushiro region in Hokkaido, Japan, which had a history of protothecal mastitis infection. The NGS produced 10,000 to 15,000 sequences in each DNA sample. To facilitate comparison, we grouped the sequencing results according to the culture-based protothecal mastitis diagnosis; sequences derived from the milk samples obtained from healthy cows were grouped separately. Sequences classified as Streptococcus spp., Pseudomonas spp. and Sphingomonas spp., Caulobacter segnis, Macrococcus caseolyticus, Methylobacterium tarhaniae, and Sphingomonas leidyi were the main sequences detected in the groups of samples from cows characterized by culture as having protothecal mastitis. Notably, Calothrix desertica (a cyanobacterium) sequences showed higher prevalence in these samples. To our knowledge, this is the first study to report that C. desertica sequences, effectively absent in the samples derived from healthy cows, are detected at high prevalence in samples from protothecal mastitic animals.
Interactions between virulence factors of pathogens and host responses play an important role in the establishment of infection by microbes. We focused on interactions between Cryptococcus neoformans proteins and heparin, which is abundant on host epithelial cells. Surface proteins were extracted and analyzed. Fractions from anion-exchange column chromatography interacted with heparin in surface plasmon resonance analyses. Heparin-binding proteins were purified and then separated by gel electrophoresis; and were identified as transaldolase, glutathione-disulfide reductase, and glyoxal oxidase. These results imply that multifunctional molecules on C. neoformans cells, such as those involved in heparin binding, may play roles in adhesion that trigger responses in the host.