Induction of NETosis in ovine colostral PMN upon exposure to Neospora caninum tachyzoites.

Colostrum is one of the most important factors influencing the health and development of mammalian neonates. It is well-established that leukocytes, including polymorphonuclear neutrophils (PMN), migrate from the mother to the infant via colostrum uptake. In this study, for the first time, we studied the ability of ovine colostral-derived PMN to extrude neutrophil extracellular traps (NETs) against the abortive apicomplexan parasite Neospora caninum. Although this cell population plays a significant role in the transmission of maternal innate immunity to neonates, little is known about colostral PMN activities in sheep. However, this cell population is a significant source of the transfer of maternal immunity to the neonate. Colostral PMN continues to exert immunological effects even after transitioning into the colostrum. The present study aimed to investigate the extrusion of NETs by ovine colostral PMN exposed to the apicomplexan parasite, N. caninum, which is known to cause devastating reproductive disorders in cattle, small ruminants, wildlife animals, and dogs. The present study is the first to demonstrate that ovine colostral PMN can produce NETs after stimulation with vital N. caninum tachyzoites. Ovine colostrum-derived NETs were detected by chromatin staining and antibody-based immunofluorescence staining of NET-specific structures, including neutrophil elastase (NE) and global histones (H1, H2A/H2B, H3, H4), as well as scanning electron microscopy (SEM) analysis.

Neospora caninum-induced NETosis in canine colostral polymorphonuclear neutrophils.

Neospora caninum represents an obligate intracellular apicomplexan parasite of the family Sarcocystidae causing severe reproductive disorders in cattle, small ruminants, wild animals and canids worldwide. Neutrophil extracellular traps (NETs) were recently described as effective host defense mechanism of polymorphonuclear neutrophils (PMN) derived from cattle, dogs, goats and dolphins against N. caninum tachyzoites. Nonetheless, nothing is known so far on canine colostral PMN immune reactions against N. caninum although breeding bitches represent a susceptible dog cohort and infected bitches may spread tachyzoites through transplacental transmission to their offspring. Thus, isolated colostrum PMN from bitches were assessed for PMN phagocytic activities as well as NETs release against viable N. caninum tachyzoites. In vitro interactions of canine colostrum-derived PMN with tachyzoites were analyzed at different ratios and time spans. Extracellular chromatin staining was applied in order to unveil classical molecules of NETs, such as neutrophil elastase (NE), global histones (H1, H2A/H2B, H3, H4) and myeloperoxidase (MPO), via antibody-based immunofluorescence microscopy analysis. N. caninum tachyzoites induced canine NETs in colostral PMN and scanning electron microscopy (SEM) analysis revealed NETs formation by colostral PMN thereby ensnaring tachyzoites after exposure. In summary, NETs released from canine colostral PMN might represent an early and effective maternal defense mechanism of the definitive host helping neonates to reduce initial intracellular replication of not only parasites but of other invasive pathogens after colostrum consumption.

Canine Angiostrongylus vasorum-Induced Early Innate Immune Reactions Based on NETs Formation and Canine Vascular Endothelial Cell Activation In Vitro.

Due to its localization in the canine blood stream, Angiostrongylus vasorum is exposed to circulating polymorphonuclear neutrophils (PMN) and the endothelial cells of vessels. NETs release of canine PMN exposed to A. vasorum infective stages (third stage larvae, L3) and early pro-inflammatory immune reactions of primary canine aortic endothelial cells (CAEC) stimulated with A. vasorum L3-derived soluble antigens (AvAg) were analyzed. Expression profiles of the pro-inflammatory adhesion molecules ICAM-1, VCAM-1, P-selectin and E-selectin were analyzed in AvAg-stimulated CAEC. Immunofluorescence analyses demonstrated that motile A. vasorum L3 triggered different NETs phenotypes, with spread NETs (sprNETs) as the most abundant. Scanning electron microscopy confirmed that the co-culture of canine PMN with A. vasorum L3 resulted in significant larval entanglement. Distinct inter-donor variations of P-selectin, E-selectin, ICAM-1 and VCAM-1 gene transcription and protein expression were observed in CAEC isolates which might contribute to the high individual variability of pathological findings in severe canine angiostrongylosis. Even though canine NETs did not result in larval killing, the entanglement of L3 might facilitate further leukocyte attraction to their surface. Since NETs have already been documented as involved in both thrombosis and endothelium damage events, we speculate that A. vasorum-triggered NETs might play a critical role in disease outcome in vivo.

Trypanosoma brucei brucei Induces Polymorphonuclear Neutrophil Activation and Neutrophil Extracellular Traps Release.

Trypanosoma brucei brucei trypomastigotes are classical blood parasites of cattle, these stages might become potential targets for circulating polymorphonuclear neutrophils (PMN). We here investigated NETs extrusion and related oxygen consumption in bovine PMN exposed to motile T. b. brucei trypomastigotes in vitro. Parasite exposure induced PMN activation as detected by enhanced oxygen consumption rates (OCR), extracellular acidification rates (ECAR), and production of total and extracellular reactive oxygen species (ROS). Scanning electron microscopy (SEM) showed that co-cultivation of bovine PMN with motile trypomastigotes resulted in NETs formation within 120 min of exposure. T. b. brucei-induced NETs were confirmed by confocal microscopy demonstrating co-localization of extruded DNA with neutrophil elastase (NE) and nuclear histones. Immunofluorescence analyses demonstrated that trypomastigotes induced different phenotypes of NETs in bovine PMN, such as aggregated NETs (aggNETs), spread NETs (sprNETs), and diffuse NETs (diffNETs) with aggNETs being the most abundant ones. Furthermore, live cell 3D-holotomographic microscopy unveiled detailed morphological changes during the NETotic process. Quantification of T. b. brucei-induced NETs formation was estimated by DNA and nuclear area analysis (DANA) and confirmed enhanced NETs formation in response to trypomastigote stages. Formation of NETs does not result in a decrease of T. b. brucei viability, but a decrease of 26% in the number of motile parasites. Referring the involved signaling pathways, trypomastigote-induced NETs formation seems to be purinergic-dependent, since inhibition via NF449 treatment resulted in a significant reduction of T. b. brucei-triggered DNA extrusion. Overall, future studies will have to analyze whether the formation of aggNETs indeed plays a role in the outcome of clinical disease and bovine African trypanosomiasis-related immunopathological disorders, such as increased intravascular coagulopathy and vascular permeability, often reported to occur in this disease.

Histone H2A and Bovine Neutrophil Extracellular Traps Induce Damage of Besnoitia besnoiti-Infected Host Endothelial Cells but Fail to Affect Total Parasite Proliferation.

Besnoitia besnoiti tachyzoites infect and develop in bovine endothelial cells in vivo and trigger the release of neutrophil extracellular traps (NETs) from bovine polymorphonuclear neutrophils (PMN). The purpose of this study was to analyze if pure B. besnoiti tachyzoite-triggered NETs would damage endothelial host cells and subsequently influence intracellular development and proliferation of B. besnoiti tachyzoites in primary bovine endothelial cells. For comparison purposes, isolated A23187-induced NETs were also used. Thus, we here evaluated endothelial host cell damage triggered by histone 2A (H2A) and B. besnoiti tachyzoite-induced NET preparations and furthermore estimated the effects of PMN floating over B. besnoiti-infected endothelium under physiological flow conditions on endothelial host cell viability. Overall, all treatments (H2A, B. besnoiti-triggered NETs and floating PMN) induced endothelial cell death of B. besnoiti-infected host cells. However, though host cell damage led to significantly altered intracellular parasite development with respect to parasitophorous vacuole diameter and numbers, the total proliferation of the parasite over time was not significantly affected by these treatments thereby denying any direct effect of NETs on intracellular B. besnoiti replication.

Rumen perforation caused by horn injury in two cows.

Post-operative complications of trocarisation and rumenotomy are the most common causes of peritonitis associated with a rumen disorder. Since horn injury leading to rumen perforation has not previously been reported in the literature, two cows with this condition are reported. Small superficial skin lesions were observed in one of the cows and the other had a perforating skin lesion in the left abdomen. Both cows had signs of hypovolaemic shock. Ultrasonography revealed hypoechoic fluid, echoic lesions and occasional fibrinous septa caudoventral to the reticulum. Caudally the fluid extended to the left flank fold and occupied about one third of the peritoneal cavity. The area of the skin perforation in the left abdomen was swollen and the muscle layers could not be differentiated using ultrasonography. Diffuse fibrino-purulent peritonitis was diagnosed in both cows, and because of a poor prognosis, they were euthanased and necropsied. Perforation of the abdominal wall and rumen with diffuse fibrino-purulent peritonitis was present. Ultrasonography is a suitable tool to characterise the inflammatory lesions between the rumen and left abdominal wall and objectify the interpretation of clinical findings. Horn injury should be included in the rule outs for cattle with left abdominal skin wounds and diffuse peritonitis.