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.

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.

Cryptosporidiosis remains a major cause of diarrhea-related childhood death, particularly in developing countries. Although effective anti-retroviral therapy has significantly reduced the cryptosporidiosis burden in western nations, the situation in developing countries remains alarming due to limited therapeutic options and a lack of preventive measures. To better control disease transmission and develop effective prevention strategies, a thorough understanding of the genetic diversity of circulating species is crucial. While cryptosporidiosis has previously been reported in Cameroon, information on the genetic diversity of parasite strains is lacking. In a large cross-sectional study conducted between March 2020 and March 2021 in four regions of Cameroon, Southwest, Littoral, Center and West, a total of 1119 fecal samples of children (n = 1119) were collected and genetically analyzed. This study aimed to assess the genetic diversity of Cryptosporidium strains circulating in this patient cohort in Cameroon. Using modified Ziehl–Neelsen fecal smear staining, an overall prevalence of 8.5% (96/1119) was recorded. PCR analysis revealed a prevalence of 15.4% in the Center, 7.2% in the Littoral, 10.5% in the West, and 13.1% in the Southwest regions. Molecular analysis identified Cryptosporidium. hominis and Cryptosporidium parvum as circulating species, with all subtype families suggesting anthroponotic transmission. No zoonotic subtypes of C. parvum were detected. These findings confirm that cryptosporidiosis transmission in Cameroon is primarily anthroponotic. Nonetheless, much larger epidemiological surveys, including other patient cohorts, are necessary for final confirmation of this statement.

Despite several decades of mass drug administration and elimination-related activities, human onchocerciasis still represents a major parasitic threat in endemic regions. Among the challenges encountered by the elimination program is the lack of a suitable diagnostic tool that is accurate and non-invasive. Currently used methods are either invasive or not suitable for monitoring large numbers of patients. Herein, we describe the identification and characterization of Onchocerca volvulus heat shock protein 70 (OvHSP70) as a novel diagnostic biomarker for human onchocerciasis, which can directly be detected in urine samples of infected patients. This nematode-specific antigen was identified through LC-MS after differential SDS-PAGE using urine-derived protein extracts from O. volvulus-infected patients in Cameroon. Polyclonal antibodies generated in rabbits after cloning and expression of OvHSP70 in Escherichia coli reliably differentiated between urine samples from infected- and uninfected patients in a hypoendemic area of human onchocerciasis. These results provide an excellent basis for further development of a non-invasive and scalable diagnostic assay for human onchocerciasis using urine samples. Such a urine-based diagnostic assay will be of major importance for the elimination program of human onchcerciasis in endemic countries.

The transfection of Cryptosporidium represents a major challenge, and current protocols are based on electroporation of freshly excysted sporozoites using a rather large amount of plasmid DNA which typically has a very poor yield. In this study, we report a fast and simple protocol for transfection of Cryptosporidium parvum that takes advantage of the DNA condensing power of the poly cationic polymer polyethylenimine (PEI) and the gene delivery property of the short cell-penetrating peptide octaarginine. Our novel protocol requires a very low amount of plasmid DNA and does not necessitate special laboratory equipment to be performed. Transfection appears to be more efficient in oocysts just triggered for excystation than the excysted sporozoites. Altogether, the application of octaarginine with PEI allows efficient transfection. To the best of our knowledge, this is the first report on an electroporation-free protocol for transfection of sporozoites of a Cryptosporidium species.