Identification and Characterization of Onchocerca volvulus Heat Shock Protein 70 (OvHSP70) as Novel Diagnostic Marker of Onchocerciasis in Human Urine.

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.

Vaginal Microbiota and Proinflammatory Status in Patients with Polycystic Ovary Syndrome: An Exploratory Study.

Background/Purpose: Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disease most common in patients of childbearing age. This pathology is associated with clinical, metabolic, and reproductive complications. We evaluated the diversity of the vaginal microbiota (VM), the vaginal inflammatory reaction (VIR), the proinflammatory state, and the activation of polymorphonuclear neutrophils (PMN) with the production of neutrophil extracellular traps (NETs). Methods: Thirty-three patients who attended a consultation at the Hospital UTPL-Santa Inés, Loja, Ecuador, from May to August 2023 who were diagnosed with PCOS participated in this study. Blood samples, vaginal discharge, and a survey were obtained. Results: A high number of patients, 23/33 (69.7%), presented altered microbiota in clinical variables associated with PCOS phenotypes A and B, sexual partners (>2), and oligomenorrhoea. A significant statistical association was only observed for sexually transmitted infections at sampling (p = 0.023) and insulin (p = 0.002). All eight cases studied with VIR had PMN/NETotic activity. A high frequency of proinflammatory states was observed in all vaginal microbiota states. Conclusions: These results suggest that the PCOS could trigger a proinflammatory state in the vaginal epithelium independently of the state of the vaginal microbiota. Furthermore, the presence of NETs observed in the cases studied could decrease fertility in these PCOS patients.

Toxoplasma gondii modulates the host cell cycle, chromosome segregation, and cytokinesis irrespective of cell type or species origin.

BACKGROUND: Toxoplasma gondii is an apicomplexan intracellular obligate parasite and the etiological agent of toxoplasmosis in humans, domestic animals and wildlife, causing miscarriages and negatively impacting offspring. During its intracellular development, it relies on nutrients from the host cell, controlling several pathways and the cytoskeleton. T. gondii has been proven to control the host cell cycle, mitosis and cytokinesis, depending on the time of infection and the origin of the host cell. However, no data from parallel infection studies have been collected. Given that T. gondii can infect virtually any nucleated cell, including those of humans and animals, understanding the mechanism by which it infects or develops inside the host cell is essential for disease prevention. Therefore, we aimed here to reveal whether this modulation is dependent on a specific cell type or host cell species. METHODS: We used only primary cells from humans and bovines at a maximum of four passages to ensure that all cells were counted with appropriate cell cycle checkpoint control. The cell cycle progression was analysed using fluorescence-activated cell sorting (FACS)-based DNA quantification, and its regulation was followed by the quantification of cyclin B1 (mitosis checkpoint protein). The results demonstrated that all studied host cells except bovine colonic epithelial cells (BCEC) were arrested in the S-phase, and none of them were affected in cyclin B1 expression. Additionally, we used an immunofluorescence assay to track mitosis and cytokinesis in uninfected and T. gondii-infected cells. RESULTS: The results demonstrated that all studied host cell except bovine colonic epithelial cells (BCEC) were arrested in the S-phase, and none of them were affected in cyclin B1 expression. Our findings showed that the analysed cells developed chromosome segregation problems and failed to complete cytokinesis. Also, the number of centrosomes per mitotic pole was increased after infection in all cell types. Therefore, our data suggest that T. gondii modulates the host cell cycle, chromosome segregation and cytokinesis during infection or development regardless of the host cell origin or type.

Toxoplasma gondii infection-induced host cellular DNA damage is strain-dependent and leads to the activation of the ATM-dependent homologous recombination pathway.

Toxoplasma gondii is a globally occurring apicomplexan parasite that infects humans and animals. Globally, different typical and atypical haplotypes of T. gondii induce varying pathologies in hosts. As an obligate intracellular protozoon, T. gondii was shown to interfere with host cell cycle progression, leading to mitotic spindle alteration, chromosome segregation errors and cytokinesis failure which all may reflect chromosomal instability. Referring to strain-dependent virulence, we here studied the potential of different T. gondii strains (RH, Me49 and NED) to drive DNA damage in primary endothelial host cells. Utilizing microscopic analyses, comet assays and γ-H2AX quantification, we demonstrated a strain-dependent induction of binucleated host cells, DNA damage and DNA double strand breaks, respectively, in T. gondii-infected cells with the RH strain driving the most prominent effects. Interestingly, only the NED strain significantly triggered micronuclei formation in T. gondii-infected cells. Focusing on the RH strain, we furthermore demonstrated that T. gondii-infected primary host cells showed a DNA damage response by activating the ATM-dependent homologous recombination (HR) pathway. In contrast, key molecules of the nonhomologous DNA end joining (NHEJ) pathway were either not affected or downregulated in RH-infected host cells, suggesting that this pathway is not activated by infection. In conclusion, current finding suggests that T. gondii infection affects the host cell genome integrity in a strain-dependent manner by causing DNA damage and chromosomal instability.

Toxoplasma gondii Me49 and NED strains arrest host cell cycle progression and alter chromosome segregation in a strain-independent manner.

Toxoplasma gondii is an obligate intracellular parasite that modulates a broad range of host cell functions to guarantee its intracellular development and replication. T. gondii includes three classical clonal lineages exhibiting different degrees of virulence. Regarding the genetic diversity of T. gondii circulating in Europe, type II strains and, to a lesser extent, type III strains are the dominant populations, both in humans and animals. Infections with the type I strain led to widespread parasite dissemination and death in mice, while type III is considered avirulent. Previously, we demonstrated that primary endothelial cells infected with the T. gondii RH strain (haplotype I) were arrested in the G2/M-phase transition, triggering cytokinesis failure and chromosome missegregation. Since T. gondii haplotypes differ in their virulence, we here studied whether T. gondii-driven host cell cycle perturbation is strain-dependent. Primary endothelial cells were infected with T. gondii Me49 (type II strain) or NED (type III strain), and their growth kinetics were compared up to cell lysis (6-30 h p. i.). In this study, only slight differences in the onset of full proliferation were observed, and developmental data in principle matched those of the RH strain. FACS-based DNA quantification to estimate cell proportions experiencing different cell cycle phases (G0/1-, S-, and G2/M-phase) revealed that Me49 and NED strains both arrested the host cell cycle in the S-phase. Cyclins A2 and B1 as key molecules of S- and M-phase were not changed by Me49 infection, while NED infection induced cyclin B1 upregulation. To analyze parasite-driven alterations during mitosis, we demonstrated that both Me49 and NED infections led to impaired host cellular chromosome segregation and irregular centriole overduplication. Moreover, in line with the RH strain, both strains boosted the proportion of binucleated cells within infected endothelial cell layers, thereby indicating enhanced cytokinesis failure. Taken together, we demonstrate that all parasite-driven host cell cycle arrest, chromosome missegregation, and binucleated phenotypes are T. gondii-specific but strain independent.

Transmission electron microscopy reveals the presence of SARS-CoV-2 in human spermatozoa associated with an ETosis-like response.

BACKGROUND: Severe acute syndrome coronavirus 2 can invade a variety of tissues, including the testis. Even though this virus is scarcely found in human semen polymerase chain reaction tests, autopsy studies confirm the viral presence in all testicular cell types, including spermatozoa and spermatids. OBJECTIVE: To investigate whether the severe acute syndrome coronavirus 2 is present inside the spermatozoa of negative polymerase chain reaction-infected men up to 3 months after hospital discharge. MATERIALS AND METHODS: This cross-sectional study included 13 confirmed moderate-to-severe COVID-19 patients enrolled 30-90 days after the diagnosis. Semen samples were obtained and examined with real-time polymerase chain reaction for RNA detection and by transmission electron microscopy. RESULTS: In moderate-to-severe clinical scenarios, we identified the severe acute syndrome coronavirus 2 inside spermatozoa in nine of 13 patients up to 90 days after discharge from the hospital. Moreover, some DNA-based extracellular traps were reported in all studied specimens. DISCUSSION AND CONCLUSION: Although severe acute syndrome coronavirus 2 was not present in the infected men's semen, it was intracellularly present in the spermatozoa till 3 months after hospital discharge. The Electron microscopy (EM) findings also suggest that spermatozoa produce nuclear DNA-based extracellular traps, probably in a cell-free DNA-dependent manner, similar to those previously described in the systemic inflammatory response to COVID-19. In moderate-to-severe cases, the blood-testes barrier grants little defence against different pathogenic viruses, including the severe acute syndrome coronavirus 2. The virus could also use the epididymis as a post-testicular route to bind and fuse to the mature spermatozoon and possibly accomplish the reverse transcription of the single-stranded viral RNA into proviral DNA. These mechanisms can elicit extracellular cell-free DNA formation. The potential implications of our findings for assisted conception must be addressed, and the evolutionary history of DNA-based extracellular traps as preserved ammunition in animals' innate defence might improve our understanding of the severe acute syndrome coronavirus 2 pathophysiology in the testis and spermatozoa.

Paraparesis due to angio-neurotropic Gurltia paralysans in a domestic cat (Felis catus) and retrospective study on feline gurltiosis cases in South America.

Introduction: The nematode Gurltia paralysans is a neglected angio-neurotropic parasite causing chronic meningomyelitis in domestic cats (Felis catus) as well as wild felids of the genus Leopardus in South America. Adult G. paralysans nematodes parasitize the leptomeningeal veins of the subarachnoid space and/or meningeal veins of the spinal cord parenchyma. The geographic range of G. paralysans encompasses rural and peri-urban regions of Chile, Argentina, Uruguay, Colombia and Brazil. Methods: This case report presents clinical and pathological findings of a G. paralysans-infected cat suffering from severe thrombophlebitis and meningomyelitis resulting in ambulatory paraparesis. Neurological examination of affected cat localized the lesions at the thoracolumbar (T3-L3) and lumbosacral (L4-Cd4) segments. Molecular and morphological characteristics of extracted nematodes from parasitized spinal cord veins confirmed G. paralysans. Additionally, data obtained from a questionnaire answered by cat owners of 12 past feline gurltiosis cases (2014-2015) were here analyzed. Questionnaire collected data on age, gender, geographic location, type of food, hunting behavior, type of prey, and other epidemiological features of G. paralysans-infected cats. Results and Discussion: Data revealed that the majority of cats originated from rural settlements thereby showing outdoor life styles with hunting/predatory behaviors, being in close contact to wild life [i.e. gastropods, amphibians, reptiles, rodents, birds, and wild felids (Leopardus guinia)] and with minimal veterinary assistance. Overall, this neglected angio-neurotropic G. paralysans nematode still represents an important etiology of severe thrombophlebitis and meningomyelitis of domestic cats living in endemic rural areas with high biodiversity of definitive hosts (DH), intermediary (IH), and paratenic hosts (PH). The intention of this study is to generate awareness among veterinary surgeons as well as biologists on this neglected feline neuroparasitosis not only affecting domestic cats but also endangered wild felid species of the genus Leopardus within the South American continent.

Metaphylactic strategies using toltrazuril against coccidiosis in goat kids.

Goat coccidiosis compromises animal welfare, reduces productivity and may cause mortality and delayed growth rates in goat kids around the weaning period worldwide. This field study was conducted to evaluate the efficacy of metaphylactic treatments with two doses of toltrazuril (20 or 40 mg/kg body weight - BW, p. o.), at different timing, in kids naturally infected with Eimeria spp. A total of 97 healthy goat kids (Majorera milk aptitude breed) were divided into five groups, depending on the age of treatment (2 or 7 weeks). One group remained untreated as a negative control until the end of the study. Faecal oocyst shedding, faecal consistency, and body weight of the animals were monitored at day 0 and at weekly intervals. Counts of oocysts per gram of faeces (OPG) were determined by a modified McMaster technique. Morphometric identification of Eimeria species was carried out on individual faecal samples from each experimental group after oocyst sporulation. Goat kids treated at two weeks of age maintained OPG values close to zero during the 5 weeks post-treatment and, overall, had lower faecal oocyst counts than untreated control animals. No significant differences were observed between the two doses of toltrazuril used in two-week-old treated animals. By contrast, when treatment was carried out at seven weeks of age, the dose of 40 mg/kg BW of toltrazuril reduced oocyst levels for longer and to a greater extent than the 20 mg/kg dose. Irrespectively of the treatment and dose, toltrazuril delayed the appearance of pathogenic Eimeria species, i. e. Eimeria ninakohlyakimovae and Eimeria arloingi. As a whole, Eimeria christenseni, with a rather moderate pathogenicity, was highly predominant throughout the study period, including the untreated control group, which was probably the reason why clinical signs of coccidiosis were barely observed throughout the experiment. Under these circumstances, the positive effect of toltrazuril on body weight condition observed in some treated groups was difficult to correlate to the timing and doses. Metaphylactic treatments with 20 mg/kg BW toltrazuril given at two weeks of age are sufficient to control oocyst excretion in goat kids; whereas if administered later in 7-week-old animals, thereby coinciding with the frequently observed peak of oocyst elimination in goat kids under field conditions, a higher dose might be advisable to prevent environmental contamination with infectious oocysts.

Prevalence of Eimeria spp. infections and major histocompatibility complex class II DRA diversity in Mongolian Bactrian camels (Camelus bactrianus).

Introduction: The two-humped Bactrian camel (Camelus bactrianus) is a large, even-toed ungulate native to the steppes of Central Asia. Domestic Bactrian camels are economically important in Mongolia and other Central Asian countries. These animals are used for transport, milk and meat production, and camel racing which is a great culture of nomads. Eimeriosis, also known as coccidiosis, is considered as an economically important parasitic diseases in Bactrian camels. There is still considerable lack of data concerning the spectrum of monoxenous Eimeria species, their epizootiology as well as their precise life cycles in Bactrian camels. This study was performed to determine the prevalence of Eimeria species in camelids from southern part of Mongolia. Methods: A total of 536 fresh camel fecal samples (n = 536) collected from herds located in five different Aimags (provinces) of Mongolia were examined. Eimeria spp. oocysts were isolated using the sugar flotation technique, and after sporulation, oocysts were identified by morphometric evaluation. Results: We identified the most common Eimeria species infecting Mongolian Bactrian camels: Eimeria cameli (22.3%), Eimeria rajasthani (37.3%) and Eimeria dromedarii (27.7%). Interestingly, mixed infections were detected in 24.8% (n = 133) of the samples, while 39.0% (n = 209) were negative for coccidian stages. To investigate the immunogenetic response of the Mongolian Bactrian camels to Eimeria spp. infection, we screened the genetic diversity in a functional important immune response gene of the major histocompatibility complex (MHC). We detected two polymorphic sites in the MHC class II DRA exon 2, which translated into one non-synonymous and one synonymous amino acid (aa) change. Discussion: The resulting aa alleles were not significantly associated with any of the three detected Eimeria species infections, nor could we show heterozygote advantage in non-infected Mongolian Bactrian camels. Further investigations on molecular epidemiology, in vitro culture, pathogenicity and host-parasite interactions will be necessary to better understand the impact of eimeriosis in Bactrian camels.

Role of Free-Ranging Synanthropic Egyptian Geese (Alopochen aegyptiaca) as Natural Host Reservoirs for Salmonella spp. in Germany.

Salmonella is one of the most common and pathogenic bacteria worldwide, causing severe enteritis in humans and representing a relevant intestinal illness in One Health for young, old and immunosuppressed patients. Various Salmonella serovars have been described to be responsible for human Salmonellosis. Birds represent natural carriers of different zoonotic-relevant Salmonella serovars and Anseriformes can not only transmit Salmonella spp. to humans but also manifest clinical Salmonellosis. In this study, 138 scat samples (n = 138) of free-ranging Egyptian geese (EG; Alopochen aegyptiaca) were collected in Germany, including 83 scat samples from city parks, 30 samples from 14 public swimming pools and 25 fresh caecal samples of dead EG. Collected EG scat samples were examined for the presence of Salmonella spp. according either to the ISO 6579 (2017) norm or to a combination of bacterial pre-enrichment and specific PCR for detection of Salmonella DNA. All 138 analysed EG faecal samples resulted Salmonella-negative. Furthermore, the survival of Salmonella enterica subsp. enterica Serovar Anatum in spiked EG droppings was tested in four different concentrations of chlorinated pool water. In vitro testing demonstrated that S. Anatum-spiked EG droppings were still infectious for up to six hours in chlorinated pool water according to current German regulations for public swimming pools. This study is to be considered as a baseline investigation to clarify the role of synanthropic EG as natural carriers of zoonotic Salmonella in cities; nonetheless, large-scale epidemiological studies, including higher numbers of samples as well as more urban locations, are needed for final conclusions on the occurrence of this intestinal bacteria in neozootic EG.

Fasciola hepatica soluble antigens (FhAg) induce ovine PMN innate immune reactions and NET formation in vitro and in vivo.

Fasciola hepatica causes liver fluke disease, a worldwide neglected and re-emerging zoonotic disease, leading to hepatitis in humans and livestock. In the pathogenesis, flukes actively migrate through liver parenchyma provoking tissue damage. Here, parasites must confront leukocytes of the innate immune system in vivo. Polymorphonuclear neutrophils (PMN) are the most abundant granulocytes and first ones arriving at infection sites. PMN may display neutrophil extracellular traps (NETs), consisting of nuclear DNA, decorated with histones, enzymes, and antimicrobial peptides. We investigated for the first time whether F. hepatica soluble antigens (FhAg) can also trigger NETosis and innate immune reactions in exposed ovine PMN. Thus, isolated PMN were co-cultured with FhAg and NET formation was visualized by immunofluorescence and scanning electron microscopy analyses resulting in various phenotypes with spread NETs being the most detected in vitro. In line, NETs quantification via Picogreen®-fluorometric measurements revealed induction of anchored- and cell free NETs phenotypes. Live cell 3D-holotomographic microscopy revealed degranulation of stimulated PMN at 30 min exposure to FhAg. Functional PMN chemotaxis assays showed a significant increase of PMN migration (p = 0.010) and intracellular ROS production significantly increased throughout time (p = 0.028). Contrary, metabolic activities profiles of FhAg-exposed PMN did not significantly increase. Finally, in vivo histopathological analysis on F. hepatica-parasitized liver tissue sections of sheep showed multifocal infiltration of inflammatory cells within liver parenchyma, and further fluorescence microscopy analyses confirmed NETs formation in vivo. Overall, we hypothesized that NET-formation is a relevant host defence mechanism that might have a role in the pathogenesis of fasciolosis in vivo.

Effect of different sperm populations on neutrophils extracellular traps (NETs) formation in cattle.

In cattle, clinical and subclinical inflammation in the bovine female reproductive tract (FRT) significantly reduces fertility. PMN participate in this FRT-associated inflammation by eliminating pathogens by eliciting various defense mechanisms, with the release of neutrophil extracellular traps NETs) being the latest process discovered. Consistently, human-, bovine- and porcine-derived spermatozoa induce release of NETs in exposed PMN of the same species origin, and thereby decreasing sperm motility through NETs-mediated entrapment. The release of NETs in the presence of different sperm sub-populations is evaluated in this work. Cryopreserved bovine sperm were selected and different sperm populations were used: viable sperm, sperm with oxidative stress, capacitated sperm, and sperm with loss of viability. Isolated PMN of dairy cows were co-incubated with these sperm populations for 4 h. Neutrophil elastase (NE) and DNA were detected by fluorescence microscopy analysis. It was noted that exposed bovine PMN released NETs in the presence of sperm. Moreover, sperm-triggered NETosis resulted different phenotypes of NETs, i. e. spread NETs (sprNETs), diffused NETs (diffNETs) and aggregated NETs (aggNETs). Viable/motile spermatozoa induced a higher proportion of NETotic cells at 15, 60 and 120 min in comparison to controls. In conclusion, all bovine sperm populations in co-culture with PMN generated NETs extrusion while viable sperm activated NETotic cells to a greater extent. With this being an early event in the activation of bovine PMN.

Besnoitia besnoiti-induced neutrophil clustering and neutrophil extracellular trap formation depend on P2X1 purinergic receptor signaling.

Bovine besnoitiosis is a re-emerging cattle disease caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti. Neutrophil extracellular trap (NET) formation represents an efficient innate immune mechanism of polymorphonuclear neutrophils (PMN) against apicomplexan parasites, including B. besnoiti. PMN purinergic signaling was proposed as a critical factor for NET formation. One important purinergic ligand is ATP, which is recognized as a danger signal and released into the extracellular space acting as an autocrine/paracrine signaling molecule. ATP-driven effects on PMN via the nucleotide P2 receptor family include chemotaxis, reactive oxygen species (ROS) production, and NET formation. So far, data on both PMN ATP concentrations and the role of ATP as a key modulator of purinergic signaling in B. besnoiti tachyzoite-triggered bovine NETosis is scarce. Current data showed that B. besnoiti tachyzoite exposure to bovine PMN neither changed total PMN ATP nor extracellular ATP quantities even though it significantly triggered NET formation. Moreover, B. besnoiti tachyzoite-exposed PMN revealed enhanced oxygen consumption rates (OCR) as quantified by the Seahorse metabolic analyzer. Exogenous supplementation of ATP or non-hydrolizable ATP (ATPγS) led to increased extracellular acidification rates (ECAR) but failed to alter tachyzoite-induced oxidative responses (OCR) in exposed PMN. In addition, exogenous supplementation of ATPγS, but not of ATP, boosted B. besnoiti tachyzoite-induced anchored NET formation. Referring to purinergic signaling, B. besnoiti tachyzoite-triggered anchored NET formation revealed P2X1 purinergic as receptor-dependent since it was blocked by the P2X1 inhibitor NF449 at an IC50 of 1.27 µM. In contrast, antagonists of P2Y2, P2Y6, P2X4, and P2X7 purinergic receptors all failed to affect parasite-driven NETosis. As an interesting finding, we additionally observed that B. besnoiti tachyzoite exposure induced PMN clustering in a P2X1-dependent manner. Thus, we identified P2X1 purinergic receptor as a pivotal molecule for both B. besnoiti tachyzoite-induced PMN clustering and anchored NET formation.

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.

Mass Spectrometry Imaging of In Vitro Cryptosporidium parvum-Infected Cells and Host Tissue.

Cryptosporidium parvum is a zoonotic-relevant parasite belonging to the phylum Alveolata (subphylum Apicomplexa). One of the most zoonotic-relevant etiologies of cryptosporidiosis is the species C. parvum, infecting humans, cattle and wildlife. C. parvum-infected intestinal mucosa as well as host cells infected in vitro have not yet been the subject of extensive biochemical investigation. Efficient treatment options or vaccines against cryptosporidiosis are currently not available. Human cryptosporidiosis is currently known as a neglected poverty-related disease (PRD), being potentially fatal in young children or immunocompromised patients. In this study, we used a combination of atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) and liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to determine and locate molecular biomarkers in in vitro C. parvum-infected host cells as well as parasitized neonatal calf intestines. Sections of C. parvum-infected and non-infected host cell pellets and infected intestines were examined to determine potential biomarkers. Human ileocecal adenocarcinoma cells (HCT-8) were used as a suitable in vitro host cell system. More than a thousand different molecular signals were found in both positive- and negative-ion mode, which were significantly increased in C. parvum-infected material. A database search in combination with HPLC-MS/MS experiments was employed for the structural verification of markers. Our results demonstrate some overlap between the identified markers and data obtained from earlier studies on other apicomplexan parasites. Statistically relevant biomarkers were imaged in cell layers of C. parvum-infected and non-infected host cells with 5 µm pixel size and in bovine intestinal tissue with 10 µm pixel size. This allowed us to substantiate their relevance once again. Taken together, the present approach delivers novel metabolic insights on neglected cryptosporidiosis affecting mainly children in developing countries.

Non-invasive detection of Orthohalarachne attenuata (Banks, 1910) and Orthohalarachne diminuata (Doetschman, 1944) (Acari: Halarachnidae) in free-ranging synanthropic South American sea lions Otaria flavescens (Shaw, 1800).

Respiratory mites of the genera Orthohalarachne and Halarachne (Acari: Halarachnidae) are causative agents of nasopharyngeal/nasopulmonary acariasis in pinnipeds and sea otters. Until now, these endoparasitic mites were mainly diagnosed via necropsies and invasive procedures. So far, non-invasive diagnostic techniques have neither been developed nor applied in free-ranging pinnipeds. In the current study, we aimed to evaluate the halarachnid mite infestation status of free-ranging "urban" South American sea lions Otaria flavescens in the city of Valdivia, Chile. Therefore, non-invasive sampling methods were used in the current study, e. g. by observation-based sampling of freshly expectorated nasal mucus in the animal environment. Further, collection devices were developed for target-oriented sampling of sneezed nasal mucus, including sterile petri dishes and stretched clingfilms mounted on telescopic rods. Applying these techniques, 26 individual sputum samples were collected. 11.5% of sputum samples proved positive for halarachnid larvae (in total, n = 7), which were morphologically identified as Orthohalarachne attenuata (n = 2) or Orthohalarachne diminuata (n = 5). In one of the individual sea lion mucus samples, both Orthohalarachne species were detected, thereby confirming a patent co-infestation in vivo. 16S rDNA-based molecular identification of individual Orthohalarachne spp. larvae confirmed morphological findings. For the first time, we here molecularly characterized Orthohalarachne spp. on the basis of three gene regions [18S, 28S and the internal transcribed spacer 1 (ITS1)]. Overall, current data include the successful application of non-invasive techniques to sample halarachnid mites from free-ranging synanthropic pinnipeds and contribute to the current knowledge on respiratory mites infesting South American sea lions by combining morphological and molecular methods to overcome challenges in species identification. This study should further serve as baseline study and calls for more research on occurrence, biology and health implications of orthohalarachnosis in free-living as well as captive pinnipeds.

Neglected zoonotic helminthiases in wild canids: new insights from South America.

The global threat of neglected tropical diseases (NTDs) constitutes a public health issue in underdeveloped countries. Zoonotic helminthiases are the most common human NTD agents in developing countries in sub-Saharan Africa, Asia, and the Americas, causing a global burden of disease that exceeds that of more recognized infectious diseases such as malaria and tuberculosis. Wild canids are well-known mammals that act as natural reservoirs of zoonotic-relevant helminthiasis worldwide, thus playing a pivotal role in their epidemiology and transmission to humans. Here we evaluate the occurrence of zoonotic gastrointestinal helminths in two Neotropical wild canid species from the Amazonian and Andean regions of Colombia, i.e., the bush dog (Speothos venaticus) and the crab-eating fox (Cerdocyon thous). We recovered tapeworm proglottids from bush dog fecal samples and identified them molecularly as the canine-specific lineage of Dipylidium caninum by using cytochrome c oxidase subunit I (cox1) gene sequences. Moreover, examination of a crab-eating fox during necropsy revealed the presence of non-embryonated eggs of the neglected nematode Lagochilascaris cf. minor, in addition to eggs and gravid proglottids of the cestode Spirometra mansoni. These findings represent the first report of zoonotic-relevant cestodes, i.e., D. caninum ("canine genotype"), S. mansoni, and the nematode L. cf. minor, in bush dogs and crab-eating foxes as final hosts. The occurrence of these zoonotic helminthiases in wild canid species calls for regular monitoring programs to better understand the epidemiology and transmission routes of neglected dipylidiasis, lagochilascariosis, and sparganosis in South America.

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.

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.