Abstract
Besnoitia besnoiti infection in cattle is an important emerging protozoan disease in Europe causing economic losses and severe clinical signs, such as generalized dermatitis, orchitis, and vulvitis in affected animals. Neutrophil extracellular trap (NET) formation was recently demonstrated as an important effector mechanism of PMN acting against several invading pathogens. In the present study, interactions of bovine PMN with tachyzoites of B. besnoiti were investigated in this respect in vitro. For the demonstration and quantification of NETs, extracellular DNA was stained by Sytox Orange or Pico Green. Fluorescent illustrations as well as scanning electron microscopy analyses (SEM) showed PMN-promoted NET formation rapidly being induced upon contact with B. besnoiti tachyzoites. Co-localization of extracellular DNA with histones, neutrophil elastase (NE) and myeloperoxidase (MPO) in parasite entrapping structures confirmed the classical characteristics of NET. Exposure of PMN to viable, UV attenuated and dead tachyzoites showed a significant induction of NET formation, but even tachyzoite homogenates significantly promoted NETs when compared to negative controls. NETs were abolished by DNase treatment and were reduced after PMN preincubation with NADPH oxidase-, NE- and MPO-inhibitors. Tachyzoite-triggered NET formation led to parasite entrapment as quantitative assays indicated that about one third of tachyzoites were immobilized in NETs. In consequence, tachyzoites were hampered from active invasion of host cells. Thus, transfer of tachyzoites, previously being confronted with PMN, to adequate host cells resulted in significantly reduced infection rates when compared to PMN-free infection controls. To our knowledge, we here report for the first time B. besnoiti-induced NET formation. Our results indicate that PMN-triggered extracellular traps may represent an important effector mechanism of the host early innate immune response against B. besnoiti which may lead to diminishment of initial parasite infection rates during the acute infection phase.
Highlights
Bovine besnoitiosis is an endemic disease mainly in Africa and Asia caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti
scanning electron microscopy analyses (SEM) analyses revealed that exposure of live B. besnoiti tachyzoites to bovine PMN resulted in the formation of a delicate network of thicker and thinner strands of fibres originating from PMN and being firmly attached to the parasites, seemingly trapping them (Fig. 1)
The data emphasize the relevance of this effector mechanism in the defense of B. besnoiti as parasite-triggered Neutrophil extracellular trap (NET) formation actively interferes rate which may have a tremendous impact on subsequent parasite proliferation
Summary
Bovine besnoitiosis is an endemic disease mainly in Africa and Asia caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti. Upcoming with reports on B. besnoiti infections in Portugal in 2005 [1], there is clear evidence for a spread of this disease in Europe since outbreaks were recently described in Spain [2], France [3], Germany [4], Italy [5,6,7] and Switzerland [8]. Since all these European countries had previously been free of bovine besnoitiosis the European Food Safety Authority classified this parasitosis as an emerging disease in the EU in 2010 [9]. This cell type has previously been shown to interact with both Eimeria bovis [10] and closely related apicomplexan parasites such as Toxoplasma gondii [11,12,13] confirming an important role of PMN in innate immune reactions against these parasites
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