Prototheca zopfii Genotype II induces mitochondrial apoptosis in models of bovine mastitis

Prototheca zopfii is an alga increasingly isolated from bovine mastitis. Of the two genotypes of P. zopfii (genotype I and II (GT-I and II)), P. zopfii GT-II is the genotype associated with acute mastitis and decreased milk production by unknown mechanisms. The objective was to determine inflammatory and apoptotic roles of P. zopfii GT-II in cultured mammary epithelial cells (from cattle and mice) and murine macrophages and using a murine model of mastitis. Prototheca zopfii GT-II (but not GT-I) invaded bovine and murine mammary epithelial cells (MECs) and induced apoptosis, as determined by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay. This P. zopfii GT-II driven apoptosis corresponded to mitochondrial pathways; mitochondrial transmembrane resistance ({Delta}{Psi}m) was altered and modulation of mitochondrion-mediated apoptosis regulating genes changed (increased transcriptional Bax, cytochrome-c and Apaf-1 and downregulated Bcl-2), whereas caspase-9 and -3 expression increased. Apoptotic effects by P. zopfii GT-II were more pronounced in macrophages compared to MECs. In a murine mammary infection model, P. zopfii GT-II replicated in the mammary gland and caused severe inflammation with infiltration of macrophages and neutrophils and upregulation of pro-inflammatory genes (TNF-, IL-1{beta} and Cxcl-1) and also apoptosis of epithelial cells. Thus, we concluded P. zopfii GT-II is a mastitis-causing pathogen that triggers severe inflammation and also mitochondrial apoptosis. Author summaryBovine mastitis (inflammation of the udder) reduces milk production and quality, causing huge economic losses in the dairy industry worldwide. Although the alga Prototheca zopfii is a major cause of mastitis in dairy cows, mechanisms by which it damages mammary tissues are not well known. Here, we used cell cultures and a mouse model of mastitis to determine how Prototheca caused inflammation and cell death in mammary tissues. Prototheca invaded mammary gland cells, from cattle and mice, as well as macrophages (white cells that take up and kill pathogens) and caused cell death by interfering with mitochondria. Furthermore, Prototheca causes severe inflammation and tissue damage when injected into the mammary glands of mice. Although there are two genotypes of P. zopfii, only genotype II causes tissue damage, whereas gentotype I, common in farm environments, does not damage mammary tissues. Since P. zopfii is an alga and not a bacterium, antibiotic treatments, frequently used to treat mastitis in cattle, are not effective against this organism. Understanding how P. zopfii damages mammary tissue and causes mastitis is important new knowledge to promote future development of evidence-based approaches to prevent and treat mammary gland infections with this organism.