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Abstract
Nocardia is one of the causing agents of bovine mastitis and increasing prevalence of nocardial mastitis in shape of serious outbreaks has been reported from many countries. However, the mechanisms by which this pathogen damages the bovine mammary epithelial cells (bMECs) is not yet studied. Therefore, this study was designed with the aim to evaluate the apoptotic effects elicited by Nocardia and to investigate the pathway by which the Nocardia induce apoptosis in bMECs. Clinical Nocardia cyriacigeorgica strain from bovine mastitis was used to infect the bMECs for different time intervals, viz. 1, 3, 6, 12, and 18 h, and then the induced effects on bMECs were studied using adhesion and invasion assays, release of lactate dehydrogenase (LDH), apoptosis analysis by annexin V and propidium iodide (PI) double staining, morphological, and ultrastructural observations under scanning electron microscope (SEM) and transmission electron microscope (TEM), mitochondrial transmembrane potential (ΔΨm) assay using flow cytometry, and the protein quantification of mitochondrial cytochrome c and caspase-9 and caspase-3 by western blotting. The results of this study showed that N. cyriacigeorgica possessed the abilities of adhesion and invasion to bMECs. N. cyriacigeorgica was found to collapse mitochondrial transmembrane potential, significantly (p < 0.05) release mitochondrial cytochrome c and ultimately induce cell apoptosis. Additionally, it promoted casepase-9 (p < 0.01) and casepase-3 (p < 0.05) levels, significantly (p < 0.01) increased the release of LDH and promoted DNA fragmentation which further confirmed the apoptosis. Furthermore, N. cyriacigeorgica induced apoptosis/necrosis manifested specific ultrastructure features under TEM, such as swollen endoplasmic reticulum, cristae degeneration, and swelling of mitochondria, vesicle formation on the cell surface, rupturing of cell membrane and nuclear membrane, clumping, fragmentation, and margination of chromatin. The present study is the first comprehensive insight into patho-morphological ultrastructural features of apoptosis/necrosis induced by N. cyriacigeorgica, which concluded that the clinical N. cyriacigeorgica induced apoptotic changes in the bMECs through mitochondrial-caspase dependent apoptotic pathway.
Highlights
Nocardia species are gram-positive, aerobic, saprophytic, and widespread environmental actinomycetes, which have been reported as an opportunistic intracellular pathogen of human and animals (Sullivan and Chapman, 2010; Conville and Witebsky, 2011)
In the infected groups at 12 and 18 h, distinct typical DNA ladder bands were observed. These results indicated that N. cyriacigeorgica could induce apoptosis/necrosis in bovine mammary epithelial cells (bMECs) and the apoptotic DNA fragment was observed after 12 h post-infection
The quantitative analysis of adhesion and invasion of N. cyriacigeorgica and visualized results of Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) showed that N. cyriacigeorgica possess the adhesion and penetration abilities
Summary
Nocardia species are gram-positive, aerobic, saprophytic, and widespread environmental actinomycetes, which have been reported as an opportunistic intracellular pathogen of human and animals (Sullivan and Chapman, 2010; Conville and Witebsky, 2011). The common manifestations of nocardiosis are pulmonary nocardiosis, central nervous system (CNS) nocardiosis, extrapulmonary nocardiosis, cutaneous, subcutaneous or lymphocutaneous nocardiosis, and nocardial bacteremia (Ambrosioni et al, 2010; Al Akhrass et al, 2011; Wilson, 2012) Whereas, in cattle, it is associated with farcy, abortion, pulmonary, and systemic nocardiosis (Beaman and Sugar, 1983; Bawa et al, 2010; Hamid, 2012). Nocardial bovine mastitis is the most important manifestation of nocardiosis and it has been reported from many countries (Dohoo, 1989; Hamid et al, 1998; Cook and Holliman, 2004; Brown et al, 2007; Pisoni et al, 2008; Ribeiro et al, 2008; Condas et al, 2013). Its huge economic losses are mostly due to decrease milk production and culling of dairy cows (Cook and Holliman, 2004; Condas et al, 2013)
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