Search

Chicken coccidiosis causes annual losses exceeding GBP 10 billion globally. The most pathogenic species for domestic fowls including Eimeria tenella, E. acervulina, and E. maxima, can lead to gastrointestinal issues ranging from mild to fatal. In this study, stages of E. tenella and freshly isolated chicken heterophils were co-cultured for 180 min. These interactions were analyzed using live 3D holotomographic and confocal microscopy. We observed that E. tenella stages were entrapped by heterophils and heterophil extracellular traps (HETs). Notably, different HET phenotypes, specifically sprHETs and aggHETs, were induced regardless of the stage. Furthermore, the quantification of extracellular DNA release from co-cultures of heterophils and sporozoites (ratio 1:1) for 180 min demonstrated a significantly higher release (p = 0.04) compared to negative controls. In conclusion, research on the chicken innate immune system, particularly fowl-derived HETs, remains limited. More detailed investigations are needed, such as exploring the time-dependent triggering of HETs, to establish a standard incubation time for this pathogen defense mechanism. This will enhance our understanding of its role in parasite survival or death during HET confrontation.

Abstract 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. Graphical Abstract

Besnoitia besnoiti is a cyst-forming apicomplexan parasite and the causal agent of bovine besnoitiosis. During early phase of infection, tachyzoites replicate within host endothelial cells in a host cell cholesterol-dependent process. By applying U18666A treatments, we here evaluated the role of Niemann–Pick type C protein 1 (NPC1) in both, intracellular B. besnoiti replication and host cellular cholesterol distribution. Additionally, B. besnoiti-driven changes in NPC1 gene transcription were studied by qPCR. Overall, U18666A treatments significantly reduced B. besnoiti proliferation and induced cholesterol accumulation in host cytoplasmic dense vesicles. However, NPC1 gene transcription was not affected by B. besnoiti infection.

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.

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.

Abstract 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.

Besnoitia besnoiti is an obligate intracellular apicomplexan parasite and the causal agent of bovine besnoitiosis. Bovine besnoitiosis has a considerable economic impact in Africa and Asia due to reduced milk production, abortions, and bull infertility. In Europe, bovine besnoitiosis is classified as an emerging disease. Polymorphonuclear neutrophils (PMN) are one of the most abundant leukocytes in cattle blood and amongst the first immunological responders toward invading pathogens. In the case of B. besnoiti, bovine PMN produce reactive oxygen species (ROS), release neutrophil extracellular traps (NETs), and show increased autophagic activities upon exposure to tachyzoite stages. In that context, the general processes of NETosis and autophagy were previously reported as associated with AMP-activated protein kinase (AMPK) activation. Here, we study the role of AMPK in B. besnoiti tachyzoite-induced NET formation, thereby expanding the analysis to both upstream proteins, such as the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK), and downstream signaling and effector molecules, such as the autophagy-related proteins ULK-1 and Beclin-1. Current data revealed early AMPK activation (B. besnoiti-exposed and AMPK activator (AICAR)-treated bovine PMN. This finding correlated with upstream responses on the level of CAMKK activation. Moreover, these reactions were accompanied by an augmented autophagic activity, as represented by enhanced expression of ULK-1 but not of Beclin-1. Referring to neutrophil effector functions, AICAR treatments induced both AMPK phosphorylation and NET formation, without affecting cell viability. In B. besnoiti tachyzoite-exposed PMN, AICAR treatments failed to affect oxidative responses, but led to enhanced NET formation, thereby indicating that AMPK and autophagic activation synergize with B. besnoiti-driven NETosis.

Bovine spermatozoa are highly susceptible to oxidative stress (OS), and it is known to affect their cellular functions. The main leukocyte producers of reactive oxygen species (ROS) in mammalian semen are polymorphonuclear neutrophils (PMN). PMN activation can result in the formation of neutrophil extracellular traps (NETs), which have been shown to affect the motility and function of spermatozoa. However, OS effects on bull spermatozoa derived from individual NETs components have not been investigated. The hypothesis of this study was that specific NETs components might generate OS on bull spermatozoa. Bovine sperm cells were incubated with five NETs-associated molecules, including 30 μg/mL histone 2A (H2A), neutrophil elastase (NE), 1 μg/mL myeloperoxidase (MPO), cathepsin G (Cat-G), and cathelicidin LL37 (LL-37), for a time course ranging from 15 to 240 min. Fluorescence microscopy was used to evaluate the coincubation of bovine PMN and sperm cells. Within 15 min, H2A, NE, and LL-37 caused membrane disruption, while MPO and Cat-G caused OS on bull spermatozoa after 1 h of coincubation. NET formation was observed within 15 min of coincubation in co-cultures of bovine PMN/sperm cells. This study is the first to report on the role of cytotoxic OS effects caused by NETs-derived components in bovine sperm in vitro.

Reproductive failure in dogs is often due to unknown causes, and correct diagnosis and treatment are not always achieved. This condition is associated with various congenital and acquired etiologies that develop inflammatory processes, causing an increase in the number of leukocytes within the female reproductive tract (FRT). An encounter between polymorphonuclear neutrophils (PMNs) and infectious agents or inflammation in the FRT could trigger neutrophil extracellular traps (NETs), which are associated with significantly decreased motility and damage to sperm functional parameters in other species, including humans. This study describes the interaction between canine PMNs and spermatozoa and characterizes the release of NETs, in addition to evaluating the consequences of these structures on canine sperm function. To identify and visualize NETs, May–Grünwald Giemsa staining and immunofluorescence for neutrophil elastase (NE) were performed on canine semen samples and sperm/PMN co-cultures. Sperm viability was assessed using SYBR/PI and acrosome integrity was assessed using PNA-FITC/PI by flow cytometry. The results demonstrate NETs release in native semen samples and PMN/sperm co-cultures. In addition, NETs negatively affect canine sperm function parameters. This is the first report on the ability of NETs to efficiently entrap canine spermatozoa, and to provide additional data on the adverse effects of NETs on male gametes. Therefore, NETs formation should be considered in future studies of canine reproductive failure, as these extracellular fibers and NET-derived pro-inflammatory capacities will impede proper oocyte fertilization and embryo implantation. These data will serve as a basis to explain certain reproductive failures of dogs and provide new information about triggers and molecules involved in adverse effects of NETosis for domestic pet animals.

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.

Abstract Inhibition of eukaryotic initiation factor 4A has been proposed as a strategy to fight pathogens. Rocaglates exhibit the highest specificities among eIF4A inhibitors, but their anti-pathogenic potential has not been comprehensively assessed across eukaryotes. In silico analysis of the substitution patterns of six eIF4A1 aa residues critical to rocaglate binding, uncovered 35 variants. Molecular docking of eIF4A:RNA:rocaglate complexes, and in vitro thermal shift assays with select recombinantly expressed eIF4A variants, revealed that sensitivity correlated with low inferred binding energies and high melting temperature shifts. In vitro testing with silvestrol validated predicted resistance in Caenorhabditis elegans and Leishmania amazonensis and predicted sensitivity in Aedes sp., Schistosoma mansoni, Trypanosoma brucei, Plasmodium falciparum, and Toxoplasma gondii. Our analysis further revealed the possibility of targeting important insect, plant, animal, and human pathogens with rocaglates. Finally, our findings might help design novel synthetic rocaglate derivatives or alternative eIF4A inhibitors to fight pathogens.

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.

IntroductionThe 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.MethodsThis 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 DiscussionData 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.

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.

Obligatory endoparasitic mites of the genera Halarachne Allman, 1847 and Orthohalarachne Newell, 1947 (Acari: Halarachnidae) parasitize different segments of the respiratory tract of marine mammals, including pinnipeds and sea otters, and infestations can cause asymptomatic to serious respiratory diseases. However, knowledge on biology, pathogenic potential and occurrence of halarachnid mites infesting pinnipeds, especially in captivity, is scarce. A two-year-old South American sea lion (Otaria flavescens Shaw, 1800) male, born and held at the Vienna Zoo, was anesthesized for routine pre-transport examinations, including computed tomography, bronchoalveolar lavage, and blood sampling. During the final phase of general anesthesia, the individual abruptly became apneic and died despite all attempts at resuscitation. At necropsy, 45 highly motile whitish millimeter-sized structures were macroscopically detected in the trachea, bifurcatio tracheae and main bronchi and were identified as adult stages of Orthohalarachne diminuata Doetschman, 1944 following morphological descriptions. After trepanation of the nasal cavity and sinus paranasalis, a total of 407 larval and 3 nymphal specimens distributed in clusters were detected. Macroscopically, sinus mucosa showed hyperemia and multiple petechial hemorrhages. Histopathological analyses of paranasal sinuses revealed mite cross-sections surrounded by sanioserous exudate and epithelial exfoliation. For the first time, O. diminuata was molecularly characterized and phylogenetically analyzed based on its 16S rDNA. Our study constitutes the first record of a severe O. diminuata infestation in captive O. flavescens and one of the few host-parasite records in general. We present clinical data and pathological results, the first scanning electron microscopic images of a O. diminuata larval stage and discuss the etiology of this autochthonous infestation, possible transmission pathways and detrimental effects. Further studies on biology and pathogenic effects of halarachnid mites, as well as on the development of non-invasive sampling techniques are essentially required for a better understanding of (ortho-)halarachnosis in pinnipeds held in zoological gardens.

We report molecular identification of an adult Spirometra mansoni tapeworm retrieved from a crab-eating fox (Cerdocyon thous) in Colombia, confirming presence of this parasite in South America. This tapeworm is the causative agent of human sparganosis, commonly reported from Southeast Asia, and represents the second congeneric species with known zoonotic potential in the Americas.

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.

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.

Abstract The seal heartworm Acanthocheilonema spirocauda (Nematoda: Onchocercidae) parasitizes the heart and pulmonary arteries of various phocid seals of the Northern Hemisphere. Over many decades, potential vectors of this parasite have been discussed, and to this date, the life cycle is not fully known. The seal louse Echinophthirius horridus (Anoplura: Echinophthiriidae) is an obligatory, permanent and haematophagous ectoparasite of phocids that has been hypothesized to function as obligate intermediate host for A. spirocauda. We examined 11 adult E. horridus specimens collected from stranded harbour seals (Phoca vitulina) in rehabilitation at the Sealcentre Pieterburen by X-ray microCT imaging, aiming to illustrate larval A. spirocauda infection sites in situ. In three of these specimens, thread-like larvae were detected in insect organs. Detailed imaging of the most infected louse revealed a total of 54 A. spirocauda larvae located either in fat bodies or the haemocoel. Histological analysis of the same specimen illustrated nematode cross-sections, confirming X-ray microCT data. The current data strongly suggest that E. horridus is a natural intermediate host for A. spirocauda. Moreover, we demonstrate the potential of X-ray microCT-based imaging as a non-destructive method to analyze host-parasite interactions, especially in the neglected field of marine mammal parasitology.

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.

Neutrophil extracellular traps (NET) formation is one important host innate defense mechanism elicited by polymorphonuclear neutrophils (PMN). NETs are composed by chromatin and proteins with microbicidal and signaling activity. So far, there is one report on Toxoplasma gondii-triggered NETs in cattle, however, exact mechanisms, including signalling pathways and dynamics governing this reaction remain largely unknown. Recently, involvement of cell cycle proteins was demonstrated for phorbol myristate acetate (PMA)-triggered human PMN-derived NETs. Here, we studied the involvement of cell cycle proteins in T. gondii-induced NETs in exposed bovine PMN. Through confocal and transmission electron microscopy we discovered that Ki-67 and lamin B1 signals are upregulated and relocated during T. gondii-induced NETosis. Nuclear membrane disruption was also observed as a hallmark of NET formation in bovine PMN confronted with viable T. gondii tachyzoites, mimicking some steps of mitosis. However, we did not observe centrosome duplication as previously described for human PMN-derived NET formation stimulated with PMA.

The protozoan parasite Eimeria bovis is the causative agent of bovine coccidiosis, an enteric disease of global importance that significantly affects cattle productivity. Previous studies showed that bovine NETosis—an important early host innate effector mechanism of polymorphonuclear neutrophil (PMN)—is elicited by E. bovis stages. So far, the metabolic requirements of E. bovis-triggered NET formation are unknown. We here studied early glycolytic and mitochondrial responses of PMN as well as the role of pH, distinct metabolic pathways, P2 receptor-mediated purinergic signaling, and monocarboxylate transporters 1 and 2 (MCT1, MCT2) in E. bovis sporozoite-induced NET formation. Seahorse-based experiments revealed a rapid induction of both neutrophil oxygen consumption rate (OCR) and early glycolytic responses, thereby reflecting immediate PMN activation and metabolic changes upon confrontation with sporozoites. The impact of these metabolic changes on NET formation was studied via chemical inhibition experiments targeting glycolysis and energy generation by the use of 2-fluor-2-deoxy-D-glucose (FDG), 6-diazo-5-oxo-L-norleucin (DON), sodium dichloroacetate (DCA), oxythiamine (OT), sodium oxamate (OXA), and oligomycin A (OmA) to block glycolysis, glutaminolysis, pyruvate dehydrogenase kinase, pyruvate dehydrogenase, lactate dehydrogenase, and mitochondrial ATP-synthase, respectively. Overall, sporozoite-induced NET formation was significantly diminished via PMN pretreatments with OmA and OXA, thereby indicating a key role of ATP- and lactate-mediated metabolic pathways. Consequently, we additionally studied the effects of extracellular pH, MCT1, MCT2, and purinergic receptor inhibitors (AR-C141900, AR-C155858, theobromine, and NF449, respectively). Pretreatment with the latter inhibitors led to blockage of sporozoite-triggered DNA release from exposed bovine PMN. This report provides first evidence on the pivotal role of carbohydrate-related metabolic pathways and purinergic receptors being involved in E. bovis sporozoite-induced NETosis.

Graphical Abstract

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.

Abstract Background Belonging to the anopluran family Echinophthiriidae, Echinophthirius horridus, the seal louse, has been reported to parasitise a broad range of representatives of phocid seals. So far, only a few studies have focused on the vector function of echinophthiriid lice, and knowledge about their role in pathogen transmission is still scarce. The current study aims to investigate the possible vector role of E. horridus parasitising seals in the Dutch Wadden Sea. Methods E. horridus seal lice were collected from 54 harbour seals (Phoca vitulina) and one grey seal (Halichoerus grypus) during their rehabilitation period at the Sealcentre Pieterburen, The Netherlands. DNA was extracted from pooled seal lice of individual seals for molecular detection of the seal heartworm Acanthocheilonema spirocauda, the rickettsial intracellular bacterium Anaplasma phagocytophilum, and the cell wall-less bacteria Mycoplasma spp. using PCR assays. Results Seal lice from 35% of the harbour seals (19/54) and from the grey seal proved positive for A. spirocauda. The seal heartworm was molecularly characterised and phylogenetically analysed (rDNA, cox1). A nested PCR was developed for the cox1 gene to detect A. spirocauda stages in seal lice. A. phagocytophilum and a Mycoplasma species previously identified from a patient with disseminated ‘seal finger’ mycoplasmosis were detected for the first time, to our knowledge, in seal lice. Conclusions Our findings support the potential vector role of seal lice in the transmission of A. spirocauda and reveal new insights into the spectrum of pathogens occurring in seal lice. Studies on vector competence of E. horridus, especially for bacterial pathogens, are essentially needed in the future as these pathogens might have detrimental effects on the health of seal populations. Furthermore, studies on the vector role of different echinophthiriid species infecting a wide range of pinniped hosts should be conducted to extend the knowledge of vector-borne pathogens.

Apicomplexan parasites are considered as defective in cholesterol synthesis. Consequently, they need to scavenge cholesterol from the host cell by either enhancing the uptake of extracellular cholesterol sources or by upregulating host cellular de-novo biosynthesis. Given that Eimeria bovis macromeront formation in bovine lymphatic endothelial host cells in vivo is a highly cholesterol-demanding process, we here examined host parasite interactions based on host cellular uptake of different low-density lipoprotein (LDL) types, i.e., of non-modified (LDL), oxidized (oxLDL), and acetylated LDL (acLDL). Furthermore, the expression of lipoprotein-oxidized receptor 1 (LOX-1), which mediates acLDL and oxLDL internalization, was monitored throughout first merogony, in vitro and ex vivo. Moreover, the effects of inhibitors blocking exogenous sterol uptake or intracellular transport were studied during E. bovis macromeront formation in vitro. Hence, E. bovis-infected primary bovine umbilical vein endothelial cells (BUVEC) were treated with inhibitors of sterol uptake (ezetimibe, poly-C, poly-I, sucrose) and of intracellular sterol transport and release from endosomes (progesterone, U18666A). As a read-out system, the size and number of macromeronts as well as merozoite I production were estimated. Overall, the internalization of all LDL modifications (LDL, oxLDL, acLDL) was observed in E. bovis-infected BUVEC but to different extents. Supplementation with oxLDL and acLDL at lower concentrations (5 and 10 µg/ml, respectively) resulted in a slight increase of both macromeront numbers and size; however, at higher concentrations (25–50 µg/ml), merozoite I production was diminished. LOX-1 expression was enhanced in E. bovis-infected BUVEC, especially toward the end of merogony. As an interesting finding, ezetimibe treatments led to a highly significant blockage of macromeront development and merozoite I production confirming the relevance of sterol uptake for intracellular parasite development. Less prominent effects were induced by non-specific inhibition of LDL internalization via sucrose, poly-I, and poly-C. In addition, blockage of cholesterol transport via progesterone and U18666A treatments resulted in significant inhibition of parasite development. Overall, current data underline the relevance of exogenous sterol uptake and intracellular cholesterol transport for adequate E. bovis macromeront development, unfolding new perspectives for novel drug targets against E. bovis.

Infectious vaginitis is a microbiological syndrome of great importance in public health that affects millions of women worldwide. However, no studies have explored the phenomenon of the production of the neutrophil extracellular traps (NETs) that are released into the female reproductive tract in these pathologies. This study aimed to determine the presence of NETosis in vaginal discharges of women with bacterial vaginosis, candidiasis, and trichomoniasis by characterizing NETs. Extracellular DNA with neutrophil elastase and citrullinated histones was identified to confirm the NET components (n = 10). The concentration, phenotypes of NETs, and number of NETotic cells were determined. The results showed an increase in NETotic cells in women with Candida albicans (CA) and Trichomonas vaginalis (TV) and an increase in NETs in TV-induced vaginitis. Samples of CA- and TV-infected women showed different NET phenotypes (diffNETs, sprNETs, and aggNETs); diffNETs were found in high concentrations in samples with CA and were increased in three types of NETs in TV infections. Samples with intermediate microbiota and bacterial vaginosis showed increased NETotic cells while the intermediate microbiota presented a higher concentration of NETs. Therefore, alterations in the microbiota and the presence of fungal and parasitic infections are important stimuli for the activation and induction of NETosis, and their cytotoxic effects could enhance tissue damage.

Eimeria bovis is an intracellular apicomplexan parasite that causes considerable economic losses in the cattle industry worldwide. During the first merogony, E. bovis forms large macromeronts with >140,000 merozoites I in host endothelial cells. Because this is a high-energy demanding process, E. bovis exploits the host cellular metabolism to fulfill its metabolic requirements. We here analyzed the carbohydrate-related energetic metabolism of E. bovis–infected primary bovine umbilical vein endothelial cells during first merogony and showed that during the infection, E. bovis–infected culture presented considerable changes in metabolic signatures, glycolytic, and mitochondrial responses. Thus, an increase in both oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were found in E. bovis–infected host cells indicating a shift from quiescent to energetic cell status. Enhanced levels of glucose and pyruvate consumption in addition to increased lactate production, suggesting an important role of glycolysis in E. bovis–infected culture from 12 days p.i. onward. This was also tested by glycolytic inhibitors (2-DG) treatment, which reduced the macromeront development and diminished merozoite I production. As an interesting finding, we observed that 2-DG treatment boosted sporozoite egress. Referring to mitochondrial activities, intracellular ROS production was increased toward the end of merogony, and mitochondrial potential was enhanced from 12 d p. i. onward in E. bovis–infected culture. Besides, morphological alterations of membrane potential signals also indicated mitochondrial dysfunction in macromeront-carrying host endothelial culture.

Abstract Toxoplasma gondii is a zoonotic and intracellular parasite with fast proliferating properties leading to rapid host cell lysis. T. gondii modulates its host cell on numerous functional levels. T. gondii was previously reported to influence host cellular cell cycle and to dampen host cell division. By using primary endothelial host cells, we show for the first time that T. gondii tachyzoite infections led to increased host cell proliferation and to an enhanced number of multi-nucleated host cells. As detected on DNA content level, parasite infections induced a G2/M cell cycle arrest without affecting expression of G2-specific cyclin B1. In line, parasite-driven impairment mainly concerned mitotic phase of host cells by propagating several functional alterations, such as chromosome segregation errors, mitotic spindle alteration and blockage of cytokinesis progression, with the latter most likely being mediated by the downregulation of the Aurora B kinase expression.

Abstract Background Antillean manatees (Trichechus manatus manatus) are large herbivorous aquatic mammals living in limited areas of South, Central and North America. As with other aquatic mammals, Antillean manatees can be infected by a variety of protozoan and metazoan parasites, some of them with zoonotic potential, which affect not only their welfare but also population health status. Therefore, we conducted the first epidemiological survey in Colombian free-ranging Antillean manatees to estimate their actual gastrointestinal parasite status. Results In total, 69 faecal samples were collected from free-ranging individual manatees during ecology field studies in the rivers Carare and San Juan and in two associated wetlands in the Andean region of Colombia. Parasite diversity encompassed six different endoparasite species. The highest prevalence was found for protozoan infections with Eimeria nodulosa (47.8%) and Eimeria manatus-like species (type A, B; 43.4%), followed by Entamoeba sp. (14.49%) and Giardia sp. (1.4%) infections. In addition, infections with the trematode Chiorchis fabaceus were detected at a high prevalence (33.3%). Molecular characterization of sirenian Eimeria species led to the distinction of three species, E. nodulosa and two E. manatus-like species (type A, B). Phylogenetic analyses indicated a host-specific adaptation of sirenian Eimeria species as previously reported for Eimeria species from other mammalian hosts. Conclusions This study provides the first record of Antillean manatee infection with Giardia and Entamoeba species in Colombia, representing two important anthropozoonotic parasite genera. This survey should serve as a baseline investigation for future monitoring on parasitic zoonoses in this mammal and encourage for investigations on their impact on both public health and wild manatee welfare.

Angiostrongylus vasorum is a cardiopulmonary nematode, causing several clinical manifestations in dogs, e.g., severe respiratory signs, coagulopathy, and gastrointestinal or neurological signs. In the last decades, this parasite has been described to spread and emerge in Europe and North America. Scant studies on A. vasorum occurrence in South America exist. Recently, A. vasorum was detected in gastropod intermediate hosts in Colombia, where data on definitive host prevalence, e.g., dogs and wild canids, are still limited. Therefore, the sera of 955 dogs, varying in age and breed from seven different departments all over Colombia, were collected and analysed for A. vasorum antigens and parasite-specific antibodies by ELISA. In total, 1.05 % (n = 10; 95 % CI 0.40–1.69) of the samples were antigen-positive and 2.62 % (n = 25; 95 % CI 1.61–3.63) were antibody-positive. These results confirm the presence of A. vasorum in Colombia, although positive results in antigen and antibody reactions in the same dog were not detected. This study is the first large-scale survey on A. vasorum seroprevalences in dogs from Colombia.

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.

Acute ruminal acidosis (ARA) occurs after an excessive intake of rapidly fermentable carbohydrates and is characterized by the overproduction of D-lactate in the rumen that reaches the bloodstream. Lameness presentation, one of the primary consequences of ARA in cattle, is associated with the occurrence of laminitis and aseptic polysynovitis. Fibroblast-like synoviocytes (FLS) are predominant cells of synovia and play a key role in the pathophysiology of joint diseases, thus increasing the chances of the release of pro-inflammatory cytokines. Increased D-lactate levels and disturbances in the metabolism of carbohydrates, pyruvates, and amino acids are observed in the synovial fluid of heifers with ARA-related polysynovitis prior to neutrophil infiltration, suggesting an early involvement of metabolic disturbances in joint inflammation. We hypothesized that D-lactate induces metabolic reprogramming, along with an inflammatory response, in bovine exposed FLS. Gas chromatography-mass spectrometry (GC-MS)-based metabolomics revealed that D-lactate disrupts the metabolism of bovine FLS, mainly enhancing glycolysis and gluconeogenesis, pyruvate metabolism, and galactose metabolism. The reverse-transcription quantitative PCR (RT-qPCR) analysis revealed an increased expression of metabolic-related genes, including hypoxia-inducible factor 1 (HIF-1)α, glucose transporter 1 (Glut-1), L-lactate dehydrogenase subunit A (L-LDHA), and pyruvate dehydrogenase kinase 1 (PDK-1). Along with metabolic disturbances, D-lactate also induced an overexpression and the secretion of IL-6. Furthermore, the inhibition of HIF-1, PI3K/Akt, and NF-κB reduced the expression of IL-6 and metabolic-related genes. The results of this study reveal a potential role for D-lactate in bFLS metabolic reprogramming and support a close relationship between inflammation and metabolism in cattle.

Neutrophil extracellular traps (NETs) play a key role in fertilisation by eliminating microorganisms and entrapping spermatozoa in the female reproductive tract (FRT). The deleterious effects of NETs on spermatozoa have been previously described; however, individual exposure to NET-derived components in bull spermatozoa has not been explored. The aim of this study was to evaluate the effects of the main NET-derived proteins, histone 2A (H2A), neutrophil elastase (ELA), myeloperoxidase (MPO), pentraxin 3 (PTX), cathepsin G (Cat-G), and cathelicidin LL37 (LL-37), at concentrations of 1, 10, and 30 μg/mL, on sperm parameters. Sperm were selected and incubated with different NET-derived proteins for 4 h. Membrane and acrosome integrity, lipoperoxidation, and membrane phospholipid disorders were also evaluated. Bovine polymorphonuclear neutrophil (PMN)/sperm co-cultures were evaluated by scanning electron microscopy and immunofluorescence. All NET-derived proteins/enzymes resulted in a reduction in membrane integrity, acrosome integrity, and lipoperoxidation at a concentration of 30 μg/mL. Bovine PMN/sperm co-cultures showed marked NET formation in the second hour. In conclusion, all NET-derived proteins/enzymes exerted cytotoxic effects on bull sperm, and this effect should be considered in future investigations on the uterine microenvironment and the advancement of spermatozoa in the FRT.

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.

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.

Since late 1970s, the southern Chilean city Valdivia constitutes home for a unique bachelor group of South American sea lions (Otaria flavescens), initially descendant from colonies at the Pacific coast, but now directly living in a freshwater habitat in close proximity to human population and a vast amount of wild and domestic animal species. In the framework of a parasitological monitoring program, 115 individual fecal samples were collected from synanthropic South American sea lions between March and May 2018. For comparative reasons, 79 individual fecal samples from two free-living O. flavescens colonies at the Pacific coast were also sampled. Coproscopical analyses revealed the presence of nine different parasite taxa in individual fecal samples, including two protozoan (Cryptosporidium spp. and Giardia spp.) and seven metazoan parasites (Anisakidae gen. spp., Diphyllobothriidae gen. spp., Ogmogaster heptalineatus, Trematoda indet. type 1, Trematoda indet. type 2, Otostrongylus circumlitus, and Parafilaroides spp.), and morphological and molecular characterizations of adult helminths confirmed identification of following species: Anisakis simplex/A. pegreffi, Pseudoterranova cattani, Contracaecum ogmorhini, and Adenocephalus pacificus. For the first time, the results of the current study show the presence of zoonotic relevant Giardia- and Cryptosporidium-infections in two free-ranging colonies of South American sea lions apart from human settlement. Furthermore, a detailed literature search of previous publications on the endoparasite fauna of South American sea lions was conducted, revealing reports of at least 50 protozoan and metazoan parasite taxa including findings of the current study. Thereby, at least 25 of reported taxa (50%) have been recorded to bear zoonotic potential. The present study illustrates a successful application of non-invasive screening methods and their applicability in the field of marine mammal parasitology, bringing new insights into the endogenous parasite fauna of South American sea lions in Southern Chile, including anthropozoonotic protozoan and metazoan taxa.

Non-esterified fatty acids (NEFAs) such as oleic acid (OA) and linoleic acid (LA) are associated with a higher incidence of infectious diseases such as metritis and mastitis during the bovine peripartum. Fatty acids can induce an increase in the release of ATP, and changes in the expression levels of purinergic receptors in bovine polymorphonuclears (PMN) during peripartum have also been reported. PMN respond to inflammatory processes with production of ROS, release of proteolytic and bactericidal proteins, and formation of neutrophil extracellular traps (NETs). NETs formation is known to require ATP production through glycolysis. Studies have shown that the above-mentioned metabolic changes alter innate immune responses, particularly in PMN. We hypothesized that NEFAs induce the formation of NETs through ATP release by Pannexin 1 and activation of purinergic receptors. In this study, we found that OA and LA induce NET formation and extracellular ATP release. Carbenoxolone, a pannexin-1 (PANX1) inhibitor, reduced OA- and LA-induced ATP release. We also found that P2X1, P2X4, P2X5, P2X7, and PANX1 were expressed at the mRNA level in bovine PMN. Additionally, NEFA-induced NET formation was completely abolished with exposure to NF449, a P2X1 antagonist, and partially inhibited by treatment with etomoxir, an inhibitor of fatty acid oxidation (FAO). Our results suggest that OA and LA induce NET formation and ATP release via PANX1 and activation of P2X1. These new data contribute to explaining the effects of NEFA high concentrations during the transition period of dairy cattle and further understanding of pro-inflammatory effects and outcome of postpartum diseases.

Abstract Background Exotic reptiles such as tortoises, have become increasingly common domestic pets worldwide and are known to host different gastrointestinal parasites. Some of these parasites bear zoonotic potential. In the present survey, we parasitologically examined tortoise faecal samples (n = 1005) from 19 different species held as pets in private German households and German zoological gardens. Methods Saline faecal smears were used to generate prevalence data for potentially health-compromising gastrointestinal parasites. In addition, we performed complete parasitological dissections of dead tortoises (n = 49) to estimate endoparasite burdens precisely. Results Analysed tortoise faecal samples contained a broad spectrum of endoparasites. We detected ten taxa of endoparasites; oxyurid nematodes (e.g. Tachygonetria spp.) were the most prevalent parasites in faecal samples (43.18%), followed by ascarids (Angusticaecum spp.) (0.01%), Hexamita spp. (0.007%), Balantidium spp. (0.007%), trichomonads (0.004%), Strongyloides spp. (0.003%), Entamoeba spp. (0.005%), Hartmanella spp. (0.001%), Blastocystis spp. (0.002%), heterakids (0.001%) and Trimitus spp. (0.001%). Additionally, we investigated dead tortoise individuals (n = 49; of 10 different species) for aetiological diagnosis and estimation of endoparasite burden. Of these individuals, 38 (77.6%) were infected with parasites and 14 (28.6%) of them died most probably due to severe parasitic infection. Oxyurid infections correlated positively with calcium deficiency and metabolic bone disease (MBD) as well as nephrosis/nephritis, mainly occurring in juvenile tortoises (< 5 years of age). Conclusions The saline faecal smear technique proved to be efficient in detecting different metazoan and protozoan parasite stages in tortoise faeces. The prevalence of oxyurid infections was particularly high. In combination with pathological findings in clinical oxyuridosis obtained from necropsied animals, our findings call for further, detailed investigations on pathogenesis and immunology of oxyurids in pet reptiles. Coprological analyses for parasite detection should be mandatory before tortoises are transferred to a new owner, animal group, or public and private enclosures such as zoos. We advocate for regular health screenings in pet tortoises and, if parasitic infections are diagnosed, adequate medication or alternative hygiene management should be considered to improve and maintain individual and population health.

Abstract The gastropod-borne nematodes Angiostrongylus vasorum and Aelurostrongylus abstrusus are global causes of cardio/pulmonary diseases in dogs and cats. In the last decade, the number of reports on canine and feline lungworms has increased in several areas of Europe and North America. The unspecific clinical signs and prolonged course of these diseases often renders diagnosis challenging. Both infections are considered as emerging and underestimated causes of disease in domestic pets. In South America, little information is available on these diseases, apart from occasional reports proving the principle presence of A. vasorum and A. abstrusus. Thus, the purpose of this review is to summarize reports on infections in both domestic and wildlife animals in South America and to increase the awareness on gastropod-borne metastrongyloid parasites, which also include important zoonotic species, such as A. cantonensis and A. costaricensis. This review highlights the usefulness of diagnostic tools, such as the Baermann funnel technique, serology and PCR, and proposes to include these routinely on cases with clinical suspicion for lungworm infections. Future national epidemiological surveys are recommended to be conducted to gain a deeper insight into the actual epidemiological situation of gastropod-borne parasitoses in South America.

Abstract Background Angiostrongylus vasorum, Crenosoma vulpis and Eucoleus aerophilus are a source of increasing concern, potentially causing significant pulmonary and severe cardiac/systemic diseases in domestic dogs and wild canids, especially red foxes (Vulpes vulpes). To investigate the prevalence and geographical distribution of these parasites in central Germany, a total of 569 foxes were examined by dissection. Methods Pluck (heart and lung) and faecal samples of red foxes were collected from three regions of Germany. Lungs, hearts and adjacent vessels were processed for adult nematode detection. Parasitological diagnoses of faecal samples were performed by SAF technique, Giardia- and Cryptosporidium-Coproantigen-ELISAs and by a duplex copro-PCR for the detection of A. vasorum and C. vulpis DNA. Results Foxes originated from three Federal States of central Germany: Thuringia (n = 359); Rhineland-Palatinate (n = 121) and Hesse (n = 89). High prevalences for all three nematodes were detected, with E. aerophilus (69.4%; 395/569), followed by C. vulpis (32.3%; 184/569) and A. vasorum (14.1%; 80/569). In case of A. vasorum, prevalences varied significantly between Federal States, with the highest prevalence of 27.3% in Rhineland-Palatinate, followed by 19.1% and 8.4% in Hesse and Thuringia, respectively. The presence of A. vasorum in fox populations showed a rather patchy distribution, increasing from north-eastern to south-western regions. Analyses on C. vulpis revealed prevalences of 35.1%, 30.3% and 25.6% (Thuringia, Hesse and Rhineland-Palatinate, respectively). The most prevalent lungworm nematode was E. aerophilus, with a prevalence of 75.2%, 71.9% and 66.9% (Rhineland-Palatinate, Hesse and Thuringia, respectively) and an almost area-wide equal distribution. Significant differences for single parasite prevalences within geographical regions of the Federal States could be detected whilst no correlation between age or gender and parasite occurrence was estimated. Weak seasonality for the winter months for A. vasorum, stronger correlation to spring and late summer for C. vulpis and no correlation to any season for E. aerophilus were detected. The method of dissection revealed a significantly higher sensitivity for C. vulpis when compared with the results of the duplex copro-PCR. Conclusions A sylvatic cycle was confirmed for all three lungworm nematodes in the examined area. The prevalences for all three lungworm nematodes are some of the highest recorded so far in German foxes. The data suggest that A. vasorum might be spreading from south-western to north-eastern parts of Germany.

Abstract Background Neospora caninum is an intracellular obligate apicomplexan parasite responsible for multisystemic lesions in dogs. Being definitive hosts and reservoirs, dogs excrete environmentally resistant oocysts. Breeding bitches represent a susceptible dog group and infected bitches may spread this parasite through transplacental transmission. Results A total of 218 serum samples of German breeding bitches were collected to determine the presence of N. caninum. Antibodies were detected in 16 (7.33%) bitches using a commercial indirect enzyme-linked immunosorbent assay (ELISA). Immunoblotting analysis confirmed all seropositive samples detected by ELISA, proving that the animals were infected with N. caninum. The owners were interviewed regarding breed, age, environment, type, vaccine status, feeding habits and the presence of reproductive disorders. Seropositive animals were between the ages of two to seven years; three of them were kept in kennels while the others were household dogs, one of which was additionally a hunting dog. Owners of four seropositive bitches reported one gestation, while multiple pregnancies had been recorded for the other twelve bitches. Fourteen bitches were regularly vaccinated and six were fed with fresh raw meat. Conclusions Although the results confirmed a low incidence of N. caninum seropositive German breeding bitches, further epidemiological and surveillance studies are required to complement our findings regarding the current situation of neosporosis in this specific canine population of Germany.

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.

Coccidian parasites are obligate intracellular pathogens that affect humans and animals. Apicomplexans are defective in de novo synthesis of cholesterol, which is required for membrane biosynthesis and offspring formation. In consequence, cholesterol has to be scavenged from host cells. It is mainly taken up from extracellular sources via LDL particles; however, little is known on the role of HDL and its receptor SR-BI in this process. Here, we studied effects of the SR-BI-specific blocker BLT-1 on the development of different fast (Toxoplasma gondii, Neospora caninum, Besnoitia besnoiti) and slow (Eimeria bovis and Eimeria arloingi) replicating coccidian species. Overall, development of all these parasites was significantly inhibited by BLT-1 treatment indicating a common SR-BI-related key mechanism in the replication process. However, SR-BI gene transcription was not affected by T. gondii, N. caninum and B. besnoiti infections. Interestingly, BLT-1 treatment of infective stages reduced invasive capacities of all fast replicating parasites paralleled by a sustained increase in cytoplasmic Ca++ levels. Moreover, BLT1-mediated blockage of SR-BI led to enhanced host cell lipid droplet abundance and neutral lipid content, thereby confirming the importance of this receptor in general lipid metabolism. Finally, the current data suggest a conserved role of SR-BI for successful coccidian infections.

Drosophila suzukii is a neobiotic invasive pest that causes extensive damage to fruit crops worldwide. The biological control of this species has been unsuccessful thus far, in part because of its robust cellular innate immune system, including the activity of professional phagocytes known as hemocytes and plasmatocytes. The in vitro cultivation of primary hemocytes isolated from D. suzukii third-instar larvae is a valuable tool for the investigation of hemocyte-derived effector mechanisms against pathogens such as wasp parasitoid larvae, bacteria, fungi and viruses. Here, we describe the morphological characteristics of D. suzukii hemocytes and evaluate early innate immune responses, including extracellular traps released against the entomopathogen Pseudomonas entomophila and lipopolysaccharides. We show for the first time that D. suzukii plasmatocytes cast extracellular traps to combat P. entomophila, along with other cell-mediated reactions, such as phagocytosis and the formation of filopodia.