Abstract
Besnoitia besnoiti is an obligate intracellular apicomplexan protozoan parasite, which causes bovine besnoitiosis. Recently increased emergence within Europe was responsible for significant economic losses in the cattle industry due to the significant reduction of productivity. However, still limited knowledge exists on interactions between B. besnoiti and host innate immune system. Here, B. besnoiti bradyzoites were successfully isolated from tissue cysts located in skin biopsies of a naturally infected animal, and we aimed to investigate for the first time reactions of polymorphonuclear neutrophils (PMN) exposed to these vital bradyzoites. Freshly isolated bovine PMN were confronted to B. besnoiti bradyzoites. Scanning electron microscopy (s.e.m.)- and immunofluorescence microscopy-analyses demonstrated fine extracellular networks released by exposed bovine PMN resembling suicidal NETosis. Classical NETosis components were confirmed via co-localization of extracellular DNA decorated with histone 3 (H3) and neutrophil elastase (NE). Live cell imaging by 3D holotomographic microscopy (Nanolive®) unveiled rapid vital NETosis against this parasite. A significant increase of autophagosomes visualized by specific-LC3B antibodies and confocal microscopy was observed in B. besnoiti-stimulated bovine PMN when compared to non-stimulated group. As such, a significant positive correlation (r = 0.37; P = 0.042) was found between B. besnoiti-triggered suicidal NETosis and autophagy. These findings suggest that vital- as well as suicidal-NETosis might play a role in early innate host defence mechanisms against released B. besnoiti bradyzoites from tissue cysts, and possibly hampering further parasitic replication. Our data generate first hints on autophagy being associated with B. besnoiti bradyzoite-induced suicidal NETosis and highlighting for first time occurrence of parasite-mediated vital NETosis.
Highlights
Besnoitia besnoiti, an obligate intracellular apicomplexan parasite, was firstly described in 1912 (Besnoit and Robin, 1912)
Cysts containing numerous typical banana-shaped B. besnoiti bradyzoites had a three-layered wall (Fig. 1B and C, thickness of 10–30 μm thick): the outer wall composed of compressed collagen type I fibres (Fig. 1B and C, black arrows), the middle layer representing a thick hyaline capsule composed of extracellular matrix (Fig. 1B and C, clear arrows) and the inner layer composed of a small rim of host cell cytoplasm with often multiple flattened nuclei containing the PV (Fig. 1B and C, arrowheads)
Bovine besnoitiosis is caused by the parasite B. besnoiti which is a cyst forming apicomplexan parasite closely related to Toxoplasma gondii and Neospora caninum (Ellis et al, 2000)
Summary
An obligate intracellular apicomplexan parasite, was firstly described in 1912 (Besnoit and Robin, 1912). Besides Europe, bovine besnoitiosis is a vastly endemic disease in the Middle East, Asia, South America (Trujillo and Benavides, 2011; Vogelsang and Gallo, 1941) and Africa (Bigalke and Prozesky, 2014; Cortes et al, 2014) causing significant economic losses in cattle industry due to significant reduction of productivity (Jacquiet et al, 2010; Maqbool et al, 2012). B. besnoiti-infected cattle present pyrexia, intensive respiratory disorders, increased heart rates, subcutaneous oedema, anasarca, swollen joints, conjunctivitis, nasal discharge, photophobia, reduced milk yield and orchitis associated with permanent infertility in bulls (Bigalke, 1981; Álvarez-García et al, 2013; Cortes et al, 2014).
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