Abstract
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, although its pathogenesis is not well known. 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 (ΔΨm) 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-α, IL-1β 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.
Highlights
Bovine mastitis, caused by infection with pathogenic microorganisms and destruction of milk-synthesizing tissues[1], reduces milk production and quality and is an important financial threat to the dairy industry[2]
Prototheca zopfii genotype II (GT-II) replicated in the murine mammary gland as it was recovered by culture in greater amounts at 4 dpi compared to the initial inoculum
Pathogenesis of protothecal mastitis and virulence of P. zopfii GT-II isolated from bovine milk were uncertain
Summary
Bovine mastitis (inflammation of the udder), caused by infection with pathogenic microorganisms and destruction of milk-synthesizing tissues[1], reduces milk production and quality and is an important financial threat to the dairy industry[2]. Prototheca zopfii GT-II induced oxidative stress and apoptotic death in cultured bovine mammary epithelial cells (bMECs), Prototheca zopfii GT-II is more pathogenic than P. zopfii GT-I, commonly isolated as an enviromental apathogenic microbe[21,22]. Another study reported that mammary gland infected with P. zopfii GT-I had no clinical signs[14], but pathogenesis of protothecal mastitis due to P. zopfii GT-II remains elusive. We aim to determine inflammatory and apoptotic roles of Prototheca zopfii GT-II in cultured mammary epithelial cells (from cattle and mice) and murine macrophages and using a murine model of mastitis
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