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Angiostrongylus vasorum is a metastrongyloid lungworm causing severe cardiovascular disease in domestic and wild animals. During its heteroxenous life cycle, A. vasorum requires obligate gastropod intermediate hosts. Little is known about A. vasorum larval organ tropism and development in gastropod intermediate hosts. Thus, the aim of this study was to analyze in vivo development of A. vasorum larval stages in experimentally infected African giant snails (Lissachatina fulica). Adult L. fulica (n = 26) were orally infected with A. vasorum-L1 and thereafter continuously euthanized. Gastropod organs were artificially digested and microscopically analyzed for the presence of A. vasorum larvae. Moreover, paraffin-fixed organs were investigated histologically for snail-borne innate immune response. In the current study, the success of L. fulica oral infection was demonstrated, thereby reaching larval recovery rates of up to 49.7%. During snail infection, an organ tropism of A. vasorum larvae was detected for the lungs and the foot muscular tissue. Overall, A. vasorum-driven gastropod innate immune reactions against larvae varied greatly. In some specimens, larvae were found effectively ensnared by recruited hemocytes, resulting in granuloma formation, whilst in others, hemocyte-mediated reactions were barely observed. Nevertheless, these evidences demand more studies on hemocyte-derived effector mechanisms against A. vasorum.

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.

Hystrichis tricolor is a neglected avian enoplid nematode (superfamiliy Dioctophymatoidea) and known to parasitize various species of the Anatidae (Anas spp. and Mergus spp.) from the northern hemisphere, inducing mainly proventriculitis in domestic and wild waterfowl. Here, we focus on the pathological findings of naturally H. tricholor-infected Egyptian geese (Alopochen aegyptiaca) and a neozoan shelduck (Tandorninae) of Germany. Nowadays, this species is considered the fastest-spreading alien waterfowl species within Western Europe. Additionally, molecular sequencing coupled with phylogenetic characterization of H. tricolor is reported. Post mortem analyses unveiled patent gastric H. tricolor infections in eight of twelve infected birds (8/12; 66.7%), inducing proventriculitis resulting in large visible nodular lesions. Histopathological findings point to chronic host pro-inflammatory immune reactions. These results demonstrate the potential role of Egyptian geese as natural reservoir hosts of H. tricholor and highlight their possible role in parasite transmission (i.e., spillback) into endemic waterfowl species. Due to avian health concerns, the occurrence of hystrichiosis should be monitored in native waterfowl in the future, introducing appropriate management practices in conservation strategies of endemic wild birds not only in Germany but elsewhere in Europe.

Gurltia paralysans is a neglected and re-emerging metastrongyloid angio-neurotropic nematode causing severe chronic meningomyelitis in domestic cats (Felis catus) as well as in free-ranging small wild felids such as kodkods (Leopardus guigna), margays (Leopardus wiedii) and the northern tiger cat (Leopardus triginus) in South America. Within these definitive hosts (DH), adult males and females of G. paralysans parasitize the leptomeningeal veins of the subarachnoid space and/or the meningeal veins of spinal cord parenchyma, inducing vascular alterations. Feline gurltiosis has been associated with progressive thrombophlebitis of the meningeal veins, resulting in ambulatory paraparesis, paraplegia, ataxia, hindlimb proprioceptive deficit, uni- or bilateral hyperactive patellar reflexes, faecal and urinary incontinence, and tail paralysis. The complete life cycle of G. paralysans has not been elucidated yet, but most probably involves gastropods as obligate intermediate hosts (IH). In terms of epidemiology, G. paralysans infections in domestic and wild felids are scattered around various South American countries, with hyperendemic areas in southern parts of Chile. Etiological diagnosis of G. paralysans still represents a challenge for clinicians due to a lack of evidence of the excretion of either eggs or larvae in faeces or in other body fluids. Diagnosis is based on clinical neurological signs, imaging findings through computed tomography (CT), myelography, magnetic resonance imaging (MRI), and post mortem examination. Nonetheless, novel diagnostic tools have been developed, including semi-nested PCR for detecting circulating G. paralysans DNA in the cerebrospinal fluid, serum and blood samples as well as in serological diagnostic kits detecting parasite-derived antigens, but these need validation for routine usage. The hypothetical life cycle of G. paralysans is addressed in this article, including the exogenous stages (i.e., eggs, and first- (L1), second- (L2) and third-stage (L3) larvae) and obligate gastropod IH and/or paratenic hosts (PH), and we propose possible anatomical migration routes of infective L3 that reach the leptomeningeal veins in vivo. Finally, the pro-inflammatory endothelium- and leukocyte-derived innate immune reactions of the host against G. paralysans, which most likely result in thrombophlebitis and meningomyelitis, are briefly touched on.

Angiostrongylus vasorum, Crenosoma vulpis, Aelurostrongylus abstrusus and Troglostrongylus brevior can cause severe cardiovascular and pulmonary symptoms in companion animals and wildlife. Recently, these nematodes were reported to spread within Europe and South America. The reasons behind this are still unknown, but obligate gastropod intermediate host populations might play a role. Therefore, lungworm infections in terrestrial slug populations in selected geographic areas of the Federal States of Bavaria and of Baden-Wuerttemberg, Germany, were studied. In total, 517 slugs (462 Arion spp., 51 Deroceras reticulatum, one Limax maximus, and three unknown slug species) were collected in the summer and autumn seasons, artificially digested and microscopically and molecularly analyzed for the presence of metastrongyloid lungworm larvae. Overall, gastropods showed a prevalence of 11.61% (60/517) for A. vasorum, 1.74% (9/517) for A. abstrusus, 0.77% (4/517) for C. vulpis and 0.97% (5/517) for T. brevior infections, respectively. In Obrigheim (Baden-Wuerttemberg), a hyperendemic focus of canine angiostrongylosis was identified. Here, gastropod infection rates rose from 13.60% (17/125) to 62.96% (34/54) within a few months. In total, 25.61% (84/328) of analysed terrestrial gastropods from Baden-Wuerttemberg were positive for metastrongyloids. In contrast, Bavarian gastropods showed a much lower prevalence of 4.76% (9/189). For the first time, the presence of T. brevior was confirmed for Arion spp. in Baden-Wuerttemberg via molecular analyses. Overall, the current data confirm that canine angiostrongylosis occurs in hyperendemic foci in certain geographic areas with high infection rates in intermediate host populations. As a result, the prevalence for a specific region can rise remarkably within a short period of time. Thus, for a better understanding of lungworm epidemiology in Germany and to protect dogs from angiostrongylosis in hyperendemic foci, it seems mandatory to enhance current efforts on Metastrongyloidea-targeted monitoring on a geographical and time span-related level.

Cryptosporidiosis is an intestinal disease that affects a variety of hosts including animals and humans. Since no vaccines exist against the disease till date, drug treatment is the mainstay of disease control. Nitazoxanide (NTZ) is the only FDA-approved drug for the treatment of human cryptosporidiosis. However, its efficacy in immunocompromised people such as those with AIDS, in malnourished children, or those with concomitant cryptosporidiosis is limited. In the absence of effective drugs against cryptosporidiosis, improving the efficacy of existing drugs may offer an attractive alternative. In the present work, we have assessed the potential of the cell-penetrating peptide (CPP) octaarginine (R8) to increase the uptake of NTZ. Octaarginine (R8) was synthetically attached to NTZ in an enzymatically releasable manner and used to inhibit growth of Cryptosporidium parvum in an in vitro culture system using human ileocecal adenocarcinoma (HCT-8) cell line. We observed a significant concentration-dependent increase in drug efficacy. We conclude that coupling of octaarginine to NTZ is beneficial for drug activity and it represents an attractive strategy to widen the repertoire of anti-cryptosporidial therapeutics. Further investigations such as in vivo studies with the conjugate drug will help to further characterize this strategy for the treatment of cryptosporidiosis.

The Eurasian lynx (Lynx lynx) represents an endangered wild felid species. In Germany, it currently occurs in three isolated populations in and around the Harz Mountains, the Palatinate Forest and the Bavarian Forest. Lynx parasitic infections affect animal health and might have an influence on population performance. Therefore, we investigated the protozoan and helminth fauna of free-ranging Eurasian lynx of the Harz population with emphasis on zoonotic parasites. Individual scat samples (n = 24) were collected from wild animals between 2019 and 2021 in the Harz National Park and surrounding areas. In total, 15 taxa of endoparasites were detected, including seven nematodes (i.e., Aelurostrongylus abstrusus, Angiostrongylus spp., Uncinaria stenocephala, Toxascaris leonina, Toxocara cati, Cylicospirura spp. and Capillaria spp.), one cestode (Diphyllobothriidae) and one trematode (Heterophylidae) as well as six protozoans (i.e., Cystoisospora rivolta, Cystoisospora felis, Toxoplasma gondii/Hammondia spp., Sarcocystis spp., Giardia intestinalis and Cryptosporidium spp.). Moreover, first-stage larvae (L1) of spurious lungworm, Protostrongylus pulmonalis, originating from lagomorph preys were identified. This work represents the first report on patent A. abstrusus and Angiostrongylus spp. infections in wild German Eurasian lynxes. Some of the identified parasites represent relevant pathogens for lynxes, circulating between these carnivorous definitive hosts and a variety of mammalian and invertebrate intermediate hosts, e.g., Sarcocystis spp., T. gondii/Hammondia spp., T. cati, T. leonina, A. abstrusus and Angiostrongylus spp., while others are considered exclusively pathogenic for wild felids (e.g., Cylicospirura spp., C. rivolta, C. felis). This study provides insights in the occurrence of zooanthroponotically relevant metazoan (i.e., T. cati and U. stenocephala) and protozoan (i.e., G. intestinalis) species in free-ranging lynx. The present work should be considered as a baseline study for future monitoring surveys on endoparasites circulating in wild Eurasian lynx for appropriate management practices in lynx conservation strategies in Europe.

Cryptosporidium parvum is an important diarrhoea-associated protozoan, which is difficult to propagate in vitro. In 2017, a report described a continuous culture of C. parvum Moredun strain, in the oesophageal squamous cell carcinoma cell line COLO-680N, as an easy-to-use system for C. parvum propagation and continuous production of oocysts. Here, we report that—using the Köllitsch strain of C. parvum—even though COLO-680N cells, indeed, allowed parasite invasion and early asexual parasite replication, C. parvum proliferation decreased after the second day post infection. Considering recurring studies, reporting on successful production of newly generated Cryptosporidium oocysts in the past, and the subsequent replication failure by other research groups, the current data stand as a reminder of the importance of reproducibility of in vitro systems in cryptosporidiosis research. This is of special importance since it will only be possible to develop promising strategies to fight cryptosporidiosis and its ominous consequences for both human and animal health by a continuous and reliable methodological progress.

Gurltia paralysans and Aelurostrongylus abstrusus are neglected metastrongyloid nematode species which infect domestic and wild cats in South American countries and in Chile, but no epidemiological studies on concomitant infections have been conducted in Chile so far. The aim of this study was not only to evaluate the occurrence of concomitant infections, but also to identify epidemiological risk factors associated with of G. paralysans and A. abstrusus infections in urban domestic cats (Felis catus) from Southern Chile. Blood samples from clinically healthy domestic cats from three cities of Southern Chile—Temuco, Valdivia, and Puerto Montt—were analyzed by an experimental semi-nested PCR protocol. A total of 171 apparently healthy domestic cats in Temuco (n = 68), Valdivia (n = 50), and Puerto Montt (n = 53) were sampled and analyzed. A total of 93 domestic cats (54.4%) were positive for G. paralysans, and 34 (19.9%) were positive for A. abstrusus infections. From those animals, 34 (19.9%) were co-infected. Cats positive with G. paralysans were found in all three cities; 47.2% in Puerto Montt, 48% in Valdivia, and 64.7% in Temuco. Levels of infection for A. abstrusus in the population under study were 4% (Valdivia), 10% (Puerto Montt), and 32.4% (Temuco). The present large-scale epidemiological study confirmed the presence of these neglected nematodes in domestic cat populations in Southern Chile, and described the possible risk factors associated with feline gurltiosis and aelurostrongylosis.

Neotropical wild felids (NWF) are obligate carnivore species present in Central and South America, and some are considered endangered due to constantly decreasing populations. NWF can become infected by a wide range of protozoan and metazoan parasites, some of them affecting their health conditions and others having anthropozoonotic relevance. Parasitological studies on NWF are still very scarce, and most data originated from dead or captive animals. On this account, the current study aimed to characterize gastrointestinal parasites of free-ranging jaguars (Panthera onca), pumas (Puma concolor), ocelots (Leopardus pardalis), and jaguarundis (Herpailurus yagouaroundi), i.e., four out of six NWF species endemic to Colombia. Fecal samples from jaguars (n = 10) and ocelots (n = 4) were collected between 2012 and 2017 as part of the Jaguar Corridor Initiative from six geographic locations in Colombia. In addition, cestode specimens were obtained during puma and jaguarundi necropsies. Scat samples were processed by standardized sodium acetate-acetic acid-formalin (SAF), sedimentation, and flotation techniques and by carbol fuchsin-stained fecal smears. Morphological evaluation of feces showed the presence of one cestode (Spirometra sp.), a nematode (Toxocara cati), an acanthocephalan (Oncicola sp.), and one cyst-forming coccidian (Cystoisospora-like oocysts). Feces oocysts were submitted to a Toxoplasma gondii-specific PCR for species identification, but no product was amplified. The cestodes isolated from a puma and jaguarundi were molecularly characterized by sequencing cytochrome c oxidase subunit I, identifying them as Taenia omissa and as a T. omissa sister lineage, respectively. These results collectively demonstrate the potential role of NWF as natural reservoir hosts for neglected zoonotic parasites (e.g., Spirometra sp., T. cati) and highlight their possible role in parasite transmission to human communities. Due to public health concerns, the occurrence of these parasites should be monitored in the future for appropriate zoonotic management practices in conservation strategies and wild felid health management programs.

Apicomplexan parasites are obligatory intracellular protozoa. In the case of Toxoplasma gondii, Neospora caninum or Besnoitia besnoiti, to ensure proper tachyzoite production, they need nutrients and cell building blocks. However, apicomplexans are auxotrophic for cholesterol, which is required for membrane biosynthesis. P-glycoprotein (P-gp) is a transmembrane transporter involved in xenobiotic efflux. However, the physiological role of P-gp in cholesterol metabolism is unclear. Here, we analyzed its impact on parasite proliferation in T. gondii-, N. caninum- and B. besnoiti-infected primary endothelial cells by applying different generations of P-gp inhibitors. Host cell treatment with verapamil and valspodar significantly diminished tachyzoite production in all three parasite species, whereas tariquidar treatment affected proliferation only in B. besnoiti. 3D-holotomographic analyses illustrated impaired meront development driven by valspodar treatment being accompanied by swollen parasitophorous vacuoles in the case of T. gondii. Tachyzoite and host cell pre-treatment with valspodar affected infection rates in all parasites. Flow cytometric analyses revealed verapamil treatment to induce neutral lipid accumulation. The absence of a pronounced anti-parasitic impact of tariquidar, which represents here the most selective P-gp inhibitor, suggests that the observed effects of verapamil and valspodar are associated with mechanisms independent of P-gp. Out of the three species tested here, this compound affected only B. besnoiti proliferation and its effect was much milder as compared to verapamil and valspodar.

Although Aelurostrongylus abstrusus infections in domestic cats (Felis silvestris catus) have sporadically been reported in Colombia, there is still no data available on epidemiology nor on the biology of this neglected lungworm parasite. Thus, this epidemiological study aimed to evaluate the occurrence of patent A. abstrusus infections in domestic cats from the Colombian Federal State of Antioquia. In total, 473 fecal samples of indoor/outdoor domestic cats were collected and analyzed thereafter by the Baermann funnel migration technique for the presence of A. abstrusus first stage larvae 1 (L1). The occurrence of A. abstrusus was confirmed in 0.4% (2/473) of investigated cats. Due to the presence of patent A. abstrusus infections in investigated cats, it is unfailing to include this lungworm within differential diagnoses of feline pulmonary disorders. Despite the fact that the Baermann funnel technique is currently the cheapest and the gold standard diagnostic tool for feline aelurostrongylosis, this technique is still unknown by Colombian veterinary surgeons and rarely utilized in small animal veterinary clinics. The current survey intends to generate awareness on this neglected parasitosis and to be considered as a baseline study for future surveys monitoring feline aelurostrongylosis not only in domestic/stray cats but also in endemic wild felid species of Colombia.

Background: Bovine polymorphonuclear neutrophils (PMN) constitutively express the Toll-like receptors (TLRs) TLR2 and TLR4 and have been shown to generate Neutrophil extracellular traps (NETs) upon exposure to Eimeria bovis. The present work investigated the role of TLR2 and TLR4 in the recognition and uptake of E. bovis sporozoites, IL-8 production and neutrophil extracellular trap (NET) formation. Methods: TLR expression was performed by flow cytometric analysis on PMN exposed to live carboxyfluorescein succinimidyl ester (CFSE)-stained sporozoites. Supernatants of PMN exposed to different E. bovis sporozoite preparations and antigens in the absence or presence of TLR antibodies were assessed for IL-8 secretion. Cells were exposed to sporozoite preparations and assessed for the activation of transcription factor NF-κB using a luciferase reporter assay. Immunofluorescence analysis was done to investigate TLR2 and TLR4 surface expression and NET formation on bovine PMN exposed to vital sporozoites. Results: we observed significantly increased TLR2 and TLR4 expression with a mean increase in expression that was greater for TLR2 than TLR4. This upregulation neither inhibited nor promoted sporozoite phagocytosis by bovine PMN. Live sporozoites together with anti-TLR2 mAb resulted in a significant enhancement of IL-8 production. NF-κB activation was more strongly induced in TLR2-HEK cells than in TLR4/MD2-HEK cells exposed to heat-killed sporozoites and antigens. Immunofluorescence analysis showed TLR-positive signals on the surface of PMN and concomitant NET formation. Conclusions: This is the first report on E. bovis-induced concomitant TLR2 and TLR4 expression during bovine PMN-derived NETosis.

Gurltia paralysans is a metastrongyloid nematode which belongs to the Angiostrongylidae family and presents tropism for veins of the subarachnoid space in vivo of domestic and wild felids causing a progressive and chronic clinical manifestation of paraparesis/paraplegia. The geographic distribution of G. paralysans includes rural and periurban areas of South America and was recently reported in Europe. To date, a definitive diagnosis of feline gurltiosis is still conducted by post-mortem examination of the spinal cord in affected animals. A presumptive diagnosis of feline gurltiosis can also be achieved based on data of compatible clinical signs and associated epidemiological risk factors. The aim of this preliminary study was to evaluate the commercial serological test Angio Detect TM® (IDEXX Laboratories) as a possible diagnostic method of feline gurltiosis in vivo. For the study, 10 domestic felines (Felis catus) which originated from a high endemic area of Southern Chile, were analyzed. All felines presented chronic paraparesis or severe paraplegia. Subsequently, commercial Angio Detect TM® serological tests for the detection of closely related Angiostrongylus vasorum in canids were performed according to manufacturer’s instructions. Conducted serological tests were positive in seven of ten felines showing paraplegia/paraparesis and presumably infected with G. paralysans, and four of them were additionally necropsied, and presented macroscopic findings compatible with feline gurltiosis. Furthermore, the presence of adult female and male G. paralysans specimens at the level of the subarachnoid vasculature in affected spinal cord segments were observed during necropsy. Histopathology demonstrated severe eosinophilic meningomyelitis, coagulopathies with thrombosis in G. paralysans-parasitized leptomeningeal veins. Results of this preliminary study suggest a cross-reaction between A. vasorum-specific antigens, which also parasitize blood vessels in vivo, and G. paralysans when using an Angio Detect TM® test, which suggests its helpful use as a new diagnostic method for feline gurltiosis in live domestic felines. Additional specific antigen research will be required in order to better understand the sensitivity and specificity of A. vasorum antigens used in this test and for existing cross-reactivity with G. paralysans-derived antigens for future a suitable intra vitam immunodiagnosis of neglected feline gurltiosis.