Human dendritic cell interactions with the zoonotic parasite Cryptosporidium parvum result in activation and maturation.

Cryptosporidiosis in humans is caused by infection of the zoonotic apicomplexan parasite Cryptosporidium parvum. In 2006, it was included by the World Health Organization (WHO) in the group of the most neglected poverty-related diseases. It is characterized by enteritis accompanied by profuse catarrhalic diarrhea with high morbidity and mortality, especially in children of developing countries under the age of 5 years and in HIV patients. The vulnerability of HIV patients indicates that a robust adaptive immune response is required to successfully fight this parasite. Little is known, however, about the adaptive immune response against C. parvum. To have an insight into the early events of the adaptive immune response, we generated primary human dendritic cells (DCs) from monocytes of healthy blood donors and exposed them to C. parvum oocysts and sporozoites in vitro. DCs are equipped with numerous receptors that detect microbial molecules and alarm signals. If stimulation is strong enough, an essential maturation process turns DCs into unique activators of naïve T cells, a prerequisite of any adaptive immune response. Parasite exposure highly induced the production of the pro-inflammatory cytokines/chemokines interleukin (IL)-6 and IL-8 in DCs. Moreover, antigen-presenting molecules (HLA-DR and CD1a), maturation markers, and costimulatory molecules required for T-cell stimulation (CD83, CD40, and CD86) and adhesion molecules (CD11b and CD58) were all upregulated. In addition, parasite-exposed human DCs showed enhanced cell adherence, increased mobility, and a boosted but time-limited phagocytosis of C. parvum oocysts and sporozoites, representing other prerequisites for antigen presentation. Unlike several other microbial stimuli, C. parvum exposure rather led to increased oxidative consumption rates (OCRs) than extracellular acidification rates (ECARs) in DCs, indicating that different metabolic pathways were used to provide energy for DC activation. Taken together, C. parvum-exposed human DCs showed all hallmarks of successful maturation, enabling them to mount an effective adaptive immune response.

Cryptosporidium parvum-induced neutrophil extracellular traps in neonatal calves is a stage-independent process.

Introduction: Infections with the apicomplexan obligate intracellular parasite Cryptosporidium parvum lead to cryptosporidiosis-a worldwide zoonotic infection. C. parvum is one of the most common diarrheal pathogens in young calves, which are the main reservoir of the pathogen. Cryptosporidiosis leads to severe economic losses in the calf industry and being a major contributor to diarrhea morbidity and mortality in children. Polymorphonuclear neutrophils (PMN) are part of the innate immune system. Their effector mechanisms directed against invasive parasites include phagocytosis, production of antimicrobial molecules as well as the formation of so-called neutrophil extracellular traps (NETs). Like other leukocytes of the innate immune system, PMN are thus able to release chromatin fibers enriched with antimicrobial granular molecules extracellularly thereby immobilizing and partially killing invasive bacteria, viruses, fungi and parasites. Methods: In vitro interactions of neonatal bovine PMN and C. parvum-oocysts and sporozoites were illustrated microscopically via scanning electron microscopy- and live cell imaging 3D holotomographic microscopy analyses. C. parvum-triggered NETosis was quantified via extracellular DNA measurements as well as verified via detection of NET-typical molecules [histones, neutrophil elastase (NE)] through immunofluorescence microscopy analysis. To verify the role of ATP in neonatal-derived NETosis, inhibition experiments were performed with NF449 (purinergic receptor antagonist with high specificity to P2X1 receptor). Results and discussion: Using immunofluorescence- and SEM-based analyses, we demonstrate here for the first time that neonate bovine PMN are capable of forming NETs against C. parvum-sporozoites and oocysts, thus as a stage-independent cell death process. Our data further showed that C. parvum strongly induces suicidal neonatal NETosis in a P2X1-dependent manner, suggesting anti-cryptosporidial effects not only through firm sporozoite ensnarement and hampered sporozoite excystation, but also via direct exposure to NETs-associated toxic components.

MCT-Dependent Cryptosporidium parvum-Induced Bovine Monocyte Extracellular Traps (METs) under Physioxia.

The apicomplexan protozoan parasite Cryptosporidium parvum is responsible for cryptosporidiosis, which is a zoonotic intestinal illness that affects newborn cattle, wild animals, and people all over the world. Mammalian monocytes are bone marrow-derived myeloid leukocytes with important defense effector functions in early host innate immunity due to their ATP purinergic-, CD14- and CD16-receptors, adhesion, migration and phagocytosis capacities, inflammatory, and anti-parasitic properties. The formation of monocyte extracellular traps (METs) has recently been reported as an additional effector mechanism against apicomplexan parasites. Nonetheless, nothing is known in the literature on METs extrusion neither towards C. parvum-oocysts nor sporozoites. Herein, ATP purinergic receptor P2X1, glycolysis, Notch signaling, and lactate monocarboxylate transporters (MCT) were investigated in C. parvum-exposed bovine monocytes under intestinal physioxia (5% O2) and hyperoxia (21% O2; most commonly used hyperoxic laboratory conditions). C. parvum-triggered suicidal METs were confirmed by complete rupture of exposed monocytes, co-localization of extracellular DNA with myeloperoxidase (MPO) and histones (H1-H4) via immunofluorescence- and confocal microscopy analyses. C. parvum-induced suicidal METs resulted not only in oocyst entrapment but also in hindered sporozoite mobility from oocysts according to scanning electron microscopy (SEM) analyses. Early parasite-induced bovine monocyte activation, accompanied by membrane protrusions toward C. parvum-oocysts/sporozoites, was unveiled using live cell 3D-holotomographic microscopy analysis. The administration of NF449, an inhibitor of the ATP purinergic receptor P2X1, to monocytes subjected to varying oxygen concentrations did not yield a noteworthy decrease in C. parvum-induced METosis. This suggests that the cell death process is not dependent on P2X1. Additionally, blockage of glycolysis in monocyte through 2-deoxy glucose (2-DG) inhibition reduced C. parvum-induced METosis but not significantly. According to monocyte energetic state measurements, C. parvum-exposed cells neither increased extracellular acidification rates (ECAR) nor oxygen consumption rates (OCR). Lactate monocarboxylate transporters (MCT) inhibitor (i.e., AR-C 141990) treatments significantly diminished C. parvum-mediated METs extrusion under physioxic (5% O2) condition. Similarly, treatment with either DAPT or compound E, two selective Notch inhibitors, exhibited no significant suppressive effects on bovine MET production. Overall, for the first time, we demonstrate C. parvum-mediated METosis as P2X1-independent but as an MCT-dependent defense mechanism under intestinal physioxia (5% CO2) conditions. METs findings suggest anti-cryptosporidial effects through parasite entrapment and inhibition of sporozoite excystation.

ATP Purinergic Receptor P2X1-Dependent Suicidal NETosis Induced by Cryptosporidium parvum under Physioxia Conditions.

Cryptosporidiosis is a zoonotic intestinal disease that affects humans, wildlife, and neonatal cattle, caused by Cryptosporidium parvum. Neutrophil extracellular traps (NETs), also known as suicidal NETosis, are a powerful and ancient innate effector mechanism by which polymorphonuclear neutrophils (PMN) battle parasitic organisms like protozoa and helminths. Here, C. parvum oocysts and live sporozoites were utilized to examine suicidal NETosis in exposed bovine PMN under both 5% O2 (physiological conditions within small intestinal tract) and 21% O2 (normal hyperoxic conditions in research facilities). Both sporozoites and oocysts induced suicidal NETosis in exposed PMN under physioxia (5% O2) and hyperoxia (21% O2). Besides, C. parvum-induced suicidal NETosis was affirmed by total break of PMN, co-localization of extracellular DNA decorated with pan-histones (H1A, H2A/H2B, H3, H4) and neutrophil elastase (NE) by means of confocal- and immunofluorescence microscopy investigations. C. parvum-triggered NETs entrapped sporozoites and impeded sporozoite egress from oocysts covered by released NETs, according to scanning electron microscopy (SEM) examination. Live cell 3D-holotomographic microscopy analysis visualized early parasite-induced PMN morphological changes, such as the formation of membrane protrusions towards C. parvum while undergoing NETosis. Significant reduction of C. parvum-induced suicidal NETosis was measured after PMN treatments with purinergic receptor P2X1 inhibitor NF449, under both oxygen circumstances, this receptor was found to play a critical role in the induction of NETs, indicating its importance. Similarly, inhibition of PMN glycolysis via 2-deoxy glucose treatments resulted in a reduction of C. parvum-triggered suicidal NETosis but not significantly. Extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) were not increased in C. parvum-exposed cells, according to measurements of PMN energetic state. Treatments with inhibitors of plasma membrane monocarboxylate transporters (MCTs) of lactate failed to significantly reduce C. parvum-mediated NET extrusion. Concerning Notch signaling, no significant reduction was detected after PMN treatments with two specific Notch inhibitors, i.e., DAPT and compound E. Overall, we here describe for the first time the pivotal role of ATP purinergic receptor P2X1 in C. parvum-mediated suicidal NETosis under physioxia (5% O2) and its anti-cryptosporidial properties.

A four year epidemiological and chemotherapy survey of babesiosis and theileriosis, and tick vectors in sheep, cattle and goats in Dehgolan, Iran

Babesia and Theileria are two protozoa belonging to the phylum Apicomplexa, which result in babesiosis and theileriosis in different hosts, cause considerable problems in domestic animals and as a consequence economic losses. These two diseases are transmitted by ticks. This survey was carried out due to the lack of studies and information regarding tick vectors and Theileria and Babesia species in the Dehgolan area of Iran, which is a center of agriculture and animal holding industry. This study was conducted during a four-year period, between 2012 and 2016. Infection with Theileria and Babesia was observed throughout the year while in contrast to cattle and sheep, infection with Babesia in goats was not observed between December and February. Infection with Babesia and Theileria reached the highest peak in July and this was more considerable for Theileria in cattle and sheep with a prevalence of 37­47% and 46­79%, respectively. The infection rate in goats for both diseases was 20%. The results in this study showed that Imidocarb Diproprionate and Buparvaquone are effective treatments for Babesia and Theileria, respectively. In this survey, the ticks of Rhipicephalus spp. and Hyalomma anatolicum had the highest infection rate with Babesia and Theileria, respectively. Considering the significant relationship between the prevalence of Theileria and Babesia with tick infection in this study and other studies, more studies on climate changes and tick vector prevalence are necessary.

Orf virus infection in human ecthyma contagiosum: a report of two cases in the West of Iran.

Ecthyma contagiosum is caused by the orf virus, a member of the genus Parapoxvirus in the family Poxviridae. Humans acquire the infection from contact with infected or recently vaccinated animals in conjunction with skin trauma. In this study, we report two cases of orf infection in two women who had contact with animals. Diagnosis was based, apart from the clinical signs, on histological examination, virus isolation using fetal bovine esophagus cells, electron microscopy and PCR.

Molecular and morphological characterization of Parabronema skrjabini of sheep and goats at three different geographical zones in Iran.

Parabronema skrjabini is a spirurid nematode of the family Habronematidae that lives in the abomasum of ruminants such as sheep and goats. The purpose of this study was to investigate the molecular and morphological aspects of Parabronema skrjabini in sheep and goats in Iran. The worms were collected from these animal species from three different regions. An internal transcribed spacer 2 ribosomal DNA (ITS2-rDNA) fragment of Parabronema skrjabini was amplified by polymerase chain reaction (PCR) using a pair of specific primers (Para-Ir-R and Para-Ir-F). Morphological studies based on the body length, the frontal shield, spicules of male and egg dimensions were performed. ITS2-rDNA sequences were between 167 and 299 bp in different isolates. ITS2 homology in different isolates was between 68% and 77% compared with the sequence data in GenBank. Morphological results showed that the average length of male and female worms in sheep were 16.5 mm and 36 mm and in goats 16 mm and 35.5 mm, respectively. The average length of the small and large spicules in sheep were 657.5 µm and 304.07 µm and in goats 653.08 µm and 302.66 µm, respectively. To our knowledge, this is the first study in the world exploring the genetic diversity of Parabronema in sheep and goats. Add this sentence in discussion: the low ITS2-rDNA identity in different isolates from Iran as compared to the reference sequence in GenBank (68-77%) raise questions regarding the species identity of the parasites isolated in Iran.

5.8S rRNA Sequence and Secondary Structure in Parabronema skrjabini and Related Habronematidae Species.

BACKGROUND: Genomic DNA was isolated from Parabronema skrjabini. rRNA region was amplified and sequenced. METHODS: The RNA secondary structure was predicted using mfold software ( http://mfold.rit.albany.edu ). The secondary structure with bulge, hairpins, helices, interior, external and multi loops was predicted for 5.8srDNA of our sequence of P. skrjabini and a sequence of P. skrjabini and two species of Habronema (H. microstoma and H. muscae) in GenBank. RNA motifs were predicted by MEME program version 4.10.2. RESULTS: The length of 5.8S rRNA sequence for P. skrjabini#1, P. skrjabini#2, H. microstoma and H. muscae was 158, 156, 127 and 127bp, and the DG required for the formation of the secondary structure was -70.50, -56.40, -41.50 and -41.40 kcal/Mol, respectively. Common structural elements were initially recognized with the help of mfold by screening for thermodynamically optimal and suboptimal secondary structures (default settings, with T = 37 °C). The energy levels of the presumptive secondary structures were then calculated with mfold at the DNA level. Both motifs and the sequence of P. skrjabini#1 were completely different from the other analyzed samples. This difference might be due to the differences in host and geographical area. CONCLUSION: This is the first molecular study of P. skrjabini in sheep, which could be further used in the structure modeling across Habronematidae.

Molecular characterization of the first internal transcribed spacer of rDNA of Parabronema skrjabini for the first time in sheep.

Parabronema skrjabini is a spirurid nematode of the family Habronematidae that lives in the abomasum of ruminants such as sheep and goats. The purpose of this study was to investigate the molecular aspects of Parabronema skrjabini in sheep. The worms were collected from sheep in Sanandaj (west of Iran). The first internal transcribed spacer (ITS) of ribosomal DNA (rDNA) nucleotide fragments of Parabronema skrjabini were amplified by polymerase chain reaction (PCR) using two pairs of specific primers (Para-Ir-R and Para-Ir-F). ITS1 homology in the sequence of this study was 69% compared with the sequence data in GenBank. To our knowledge, this is the first study in the world exploring the genetic diversity of P. skrjabini in sheep based on ITS1.

Buxtonella spp. like infection in cattle in Sanandaj province, Iran.

Buxtonellosis is a disease caused by the ciliated protozoan Buxtonella sulcata (Jameson, 1926). B. sulcata is a common protozoan of ruminants and may result in subclinical infection or clinical disease including diarrhea. This study examined the prevalence of B. sulcata in cattle from Sanadaj province, Iran. Faecal samples were collected from cattle in the province from May 2013 to June 2014. A total of 217 cattle were selected randomly according to the age, sex, health, management system and season. In total, 99 cattle (45.63%) were found to be infected with B. sulcata. In adults, prevalence of B. sulcata infection (51.64%) was higher than calves (28.58%) and young cattle (40.55%). The prevalence of B. sulcata infection was also found to be higher in female (47.32%) than male (38.46) cattle. B. sulcata infection was significantly (p<0.05) higher in poor health cattle (body condition and weight) (79.54%) than healthy cattle (24.47%). This study demonstrates that cattle are highly susceptible to B. sulcata infection under a variety of housing and environmental conditions in this region of Iran. The study also demonstrates a significant effect of infection on cattle health.