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Toxoplasma gondii is a protozoan parasite of public health importance, infecting all warm-blooded animals, including chickens. Undercooked chicken meat or relevant products such as sausages could lead to human infections. In free-range, organic and slow-growth farming systems where the susceptibility period for chickens is extended, more knowledge about potential risk factors is essential. This study is the first seroepidemiological survey in different regions and types of chicken farms in Greece, using a major tachyzoite surface antigen-based ELISA (TgSAG1), combined with magnetic-capture PCR (mc-PCR) and bioassay for the isolation of strains from the chickens’ tissues. Potential risk factors for T. gondii infection in these hosts were also investigated. Additionally, the co-existence of T. gondii and Eimeria spp. infections was assessed to elucidate epidemiological links between these two protozoan infections. Overall T. gondii seroprevalence was 9.5%. Of the backyard chickens sampled, 41.2% were seropositive and 70% of the organic and free-range layer farms had at least one T. gondii seropositive hen. No serologically positive broilers were found, although mc-PCR revealed a positive sample, highlighting the importance of accurate early-infection direct detection of T. gondii infections to ensure public health. T. gondii isolates obtained by mouse bioassay were genotyped. All belonged to type II (ToxoDB#3) as confirmed also by microsatellite typing. Production system, type of nutrition, and feeding system automation were identified as the most significant risk factors, while no association was found between the presence of cats and T. gondii seropositivity as calculated on both a farm level and per individual bird sampled.

Cryptosporidium is an enteric protozoan parasite which is able to cause severe gastrointestinal disease and is distributed all over the world. Since information about the prevalence of cryptosporidiosis in German dogs is rare, the aim of this study was to examine the occurrence of Cryptosporidium spp. in dogs and the potential zoonotic risk emanating from these infected animals. In total, 349 fecal samples of 171 dogs were collected during the dogs’ first year of life. The samples were examined for Cryptosporidium spp. using PCR, targeting the small subunit ribosomal RNA gene (SSU rRNA). Further analysis of Cryptosporidium parvum and Cryptosporidium canis positive samples was accomplished using the 60 kDa glycoproteine gene (GP60). Overall, 10.0% (35/349) of the specimens were tested positive for Cryptosporidium. Cryptosporidium canis was found in 94.3% (33/35) of these samples and the zoonotic type C. pavum in 5.7% (2/35). Both C. parvum infections were subtyped as IIaA15G2R1. Sixteen of the C. canis positive samples were successfully amplified at the GP60 gene locus. These isolates were identified to belong to the subtype families XXd, XXe, or XXb; however, 2 samples could not be assigned to any of the described subtype families. Considering the close contact between pets and their owners, dogs may act as a potential source of infection for human cryptosporidiosis. The results of this study, in context with other studies from different countries, provide important further insights into the distribution of Cryptosporidium species in dogs and their zoonotic potential.

Radiation-attenuated intracellular parasites are promising immunization strategies. The irradiated parasites are able to invade host cells but fail to fully replicate, which allows for the generation of an efficient immune response. Available radiation technologies such as gamma rays require complex shielding constructions and are difficult to be integrated into pharmaceutical production processes. In this study, we evaluated for the first time low-energy electron irradiation (LEEI) as a method to generate replication-deficient Toxoplasma gondii and Cryptosporidium parvum. Similar to other radiation technologies, LEEI mainly damages nucleic acids; however, it is applicable in standard laboratories. By using a novel, continuous, and microfluidic-based LEEI process, tachyzoites of T. gondii and oocysts of C. parvum were irradiated and subsequently analyzed in vitro. The LEEI-treated parasites invaded host cells but were arrested in intracellular replication. Antibody-based analysis of surface proteins revealed no significant structural damage due to LEEI. Similarly, excystation rates of sporozoites from irradiated C. parvum oocysts were similar to those from untreated controls. Upon immunization of mice, LEEI-attenuated T. gondii tachyzoites induced high levels of antibodies and protected the animals from acute infection. These results suggest that LEEI is a useful technology for the generation of attenuated Apicomplexan parasites and has potential for the development of anti-parasitic vaccines.

Eimeria tenella is the causative agent of cecal coccidiosis in poultry characterized by weight loss, hemorrhagic diarrhea, and high mortality rates. Research into herbal candidates with possible anticoccidial activity has increased lately. As an alternative to animal experiments, an in vitro reproduction inhibition assay (RIA) was previously designed to determine the sensitivity of E. tenella isolates against ionophores. In this study, the RIA was used to test the anticoccidial activity of nutmeg oil, cinnamon oil, and glabridin. The concentration of nutmeg oil used in this study ranged between 1.1 and 139.1 μg/ml. Nutmeg oil exhibited a moderate in vitro inhibitory activity ranging from 35.5 to 49.5%. In contrast, no inhibitory effect was detected when incubating E. tenella sporozoites for 24 h with cinnamon oil at concentrations of 0.3 to 80.5 μg/ml. Glabridin (0.08–41.7 μg/ml) prevented the replication of sporozoites at a rate of 14.1 to 81.7% of inhibition. The calculated minimum concentrations of glabridin needed to inhibit parasite replication by 75%, 50%, and 30% (MIC75, MIC50, and MIC30) were 21.43 μg/ml, 5.28 μg/ml, and 0.96 μg/ml, respectively. Further studies to assess the in vitro efficacy of glabridin were performed by studying mRNA gene expression of stress-induced protein genes (HSP-70, NADPH, and EtPP5) after exposure of E. tenella sporozoites to glabridin at MIC75 for 0.5 h, 1 h, 2 h, and 4 h (a time-dependent experiment). Moreover, a dose-dependent experiment was performed using glabridin at a concentration matching MIC75, MIC50, and MIC30 for 24 h. In the time-dependent experiment, a significant (p

Young dogs are particularly susceptible to infections with endoparasites. The occurrence of endoparasites was investigated in young dogs from Central Germany between July 2020 and July 2022. In total, 386 fecal samples originating from 171 dogs were examined for the prevalence of endoparasites using a combined flotation- and sedimentation technique and conventional PCR. Overall, in 41.2% (159/386) of the examined samples, endoparasites were detected. The most frequently occurring endoparasites were Giardia duodenalis (29%), Cryptosporidium spp. (9.1%), Cystoisospora spp. (7.3%), and Toxocara canis (6%). Sequencing of G. duodenalis positive samples showed that most infections belonged to the host-specific assemblages C (38.4% (43/112)) and D (35.7% (40/112)). The zoonotic assemblage A was identified in 8% (9/112) of the samples. Moreover, mixed infections were observed as follows: C/D in 5 (4.5%), D/A in 4 (3.6%), and C/A in 3 (2.7%) samples. All assemblage A infections were assigned to the potentially zoonotic subassemblage AI. Co-infections of G. duodenalis and Cryptosporidium spp. were observed in 3.1% (12/386) of the samples. Analyzing several host factors for their potential association with endoparasitic infection, the origin of dogs, as well as the living environment were identified as the main risk factors for infection with endoparasites. Overall, this study shows a high infection rate with endoparasites, especially G. duodenalis, in young dogs from Germany. The results of this study contribute to further insight into the distribution and potential risk factors associated with endoparasitic infections, as well as the zoonotic potential these parasites may present.

Background Cryptosporidiosis is a parasitic disease associated with potentially fatal diarrhea. The most used method in Cryptosporidium subtyping is based on the glycoprotein gene gp60. Each infection can represent a parasite population, and it is important to investigate the influence on transmission and virulence, as well as any impact on public health investigations. However, an easy-to-use method for detection is lacking. Methods Here we report on the use of the bioinformatic program TIDE for deconvolution of gp60 chromatograms. A combination of single oocyst analysis and cloning successfully confirmed the within-sample parasite population diversity. Retrospective sample analysis was conducted on archived chromatograms. Results For Cryptosporidium parvum, 8.6% multistrain infections (13 of 152) obscured by currently used consensus base calling were detected. Importantly, we show that single oocysts can harbor a mixed population of sporozoites. We also identified a striking dominance of unappreciated polymerase stutter artefacts in all 218 chromatograms analyzed, challenging the uncritical use of gp60 typing. Conclusions We demonstrate the value of a new, easy-to-use analytical procedure for critical characterization of C. parvum and Cryptosporidium hominis in epidemiological investigations, also applicable retrospectively. Our findings illuminate the hidden parasite diversity with important implications for tracing zoonotic and person-to-person transmissions.

Background Toxoplasma gondii is an obligate intracellular parasite with a worldwide distribution. Congenital infection in humans and animals may lead to severe symptoms in the offspring, especially in the brain. A suitable animal model for human congenital toxoplasmosis is currently lacking. The aim of this study is to establish and validate the guinea pig as a model for human congenital toxoplasmosis by investigating the impact of the T. gondii infection dose, the duration of infection and the gestational stage at infection on the seroconversion, survival rate of dams, fate of the offspring, T. gondii DNA loads in various offspring tissues and organs and the integrity of the offspring brain. Methods Pregnant guinea pigs were infected with three different doses (10, 100, 500 oocysts) of T. gondii strain ME49 at three different time points during gestation (15, 30, 48 days post-conception). Serum of dams was tested for the presence of T. gondii antibodies using immunoblotting. T. gondii DNA levels in the dam and offspring were determined by qPCR. Offspring brains were examined histologically. Results We found the survival rate of dams and fate of the offspring to be highly dependent on the T. gondii infection dose with an inoculation of 500 oocysts ending lethally for all respective offspring. Moreover, both parameters differ depending on the gestational stage at infection with infection in the first and third trimester of gestation resulting in a high offspring mortality rate. The duration of infection was found to substantially impact the seroconversion rate of dams with the probability of seroconversion exceeding 50% after day 20 post-infection. Furthermore, the infection duration of dams influenced the T. gondii DNA loads in the offspring and the integrity of offspring brain. Highest DNA levels were found in the offspring brain of dams infected for ≥ 34 days. Conclusion This study contributes to establishing the guinea pig as a suitable model for human congenital toxoplasmosis and thus lays the foundation for using the guinea pig as a suitable animal model to study scientific questions of high topicality and clinical significance, which address the pathogenesis, diagnosis, therapy and prognosis of congenital toxoplasmosis. Graphical abstract

Poultry coccidiosis causes considerable economical losses to the livestock industry. Eimeria parasites are responsible for this disease. On a global scale, E. acervulina and E. tenella are amongst the most common Eimeria spp. infecting broilers. E. tenella is commonly used as infection model in in vivo and in vitro studies. On the other hand, E. acervulina has barely been studied under in vitro conditions. A well established and widely used in vitro model for E. tenella infection is the Madin-Darby bovine kidney cell line (MDBK); however, little is known regarding suitability of MDBK cells as host cells for E. acervulina. We infected MDBK monolayers with two different doses, 5 × 104 and 2 × 105, of E. acervulina sporozoites and evaluated cultures at 24 and 96 h post infection (hpi). For comparison, we ran an identical infection assay using E. tenella sporozoites. To assess parasite reproduction, the number of DNA copies of E. acervulina SCAR marker and E. tenella ITS-1 gene was quantified using real-time quantitative PCR. We found that the number of E. acervulina copies increased significantly at 24 hpi in comparison to E. tenella (p E. acervulina gene copies were considerably reduced while E. tenella continued to multiply (p E. acervulina sporozoite cell invasion. Nevertheless, modifications of in vitro cultivation appear necessary to allow qualitative and quantitative studies over longer periods of parasite reproduction.

Dentostomella translucida is an oxyurid nematode that was first discovered in the Mongolian gerbil but has also been detected in other wild and housed rodents. In conventional laboratory animals, oxyurid nematode parasites are widespread infections. A proven treatment strategy for pinworm eradication is the oral application of benzimidazoles, such as fenbendazole. In general, this drug is regarded as safe with minimal side effects. Nevertheless, in Sprague Dawley rats, a significantly reduced litter size could be seen after longer treatment with fenbendazole. Even though Dentostomella translucida was already described in Syrian golden hamsters (Mesocricetus auratus), data on treatment with fenbendazole and its effects on reproduction is lacking. Therefore, the main purposes of the study were (1) the verification of the effectiveness of fenbendazole as medicated feed (150 ppm) against this parasite in naturally infected Syrian golden hamsters in conventional husbandry and (2) monitoring of possible effects on reproduction during the treatment. Results show that fenbendazole treatment was highly effective against Dentostomella translucida, as numbers of pinworm eggs in the faeces were significantly reduced already after the first week of treatment in all animals. After four weeks of treatment, eggs were eradicated entirely. Interestingly, the average weaning weight was significantly reduced during treatment, but the litters were in good health.

Mixed infections of Toxoplasma gondii and Eimeria tenella are likely to occur frequently due to the high prevalence of both pathogens in free-ranging chickens. In this study, we investigated the co-occurrence of the two parasites in the same immune-competent host cell towards altered patterns of parasite-host interactions. Chicken blood monocyte–derived macrophages were co-infected with T. gondii RH tachyzoites and E. tenella Houghton sporozoites in vitro for 24 h. Through monitoring the uptake of pH-sensitive pHrodo™ Zymosan BioParticles (“Zymosan”) by macrophages, we created a three-dimensional model and to analyze quantitatively phagocytosis using confocal laser scanning microscopy. Assessments of parasite populations were performed by qPCR at 2, 6, 12, and 24 h post-infection (hpi). At 6 hpi, phagocytosis was inhibited in the E. tenella–infected cultures while no inhibition of phagocytosis was observed due to T. gondii. Phagocytosis activity revealed more complex interactions during co-infection. At 12 and 24 hpi, phagocytosis response to “Zymosan” was distinctly weaker in co-infected cells than in all other groups except for cells mono-infected with high doses of E. tenella at 24 hpi. By qPCR, significantly reduced numbers of both intracellular parasites were recorded (10-fold) in all infected groups at 2 hpi. At 12 hpi, the T. gondii population reached lowest values but dramatically increased by 24 hpi. Our data confirm that macrophage phagocytosis is involved in the control of invasion by apicomplexan parasites in chicken which particularly applies to E. tenella infection and it was able to be altered by the co-existing parasites.

Trichomonas gallinae are parasitic flagellates of importance in wild and domestic birds. The parasite is worldwide distributed, and Columbine birds are its main host. Current research focuses mostly on epidemiological and phylogenetic studies. However, there is still a lack of knowledge regarding parasite-host interaction or therapy development. Real-time PCR is a useful tool for diagnostic and quantification of gene copies in a determined sample. By amplification of a 113-bp region of the 18S small subunit ribosomal RNA gene, a SYBR green-based real-time PCR assay was developed. A standard curve was prepared for quantification analysis. Assay efficiency, linearity, and dissociation analysis were successfully performed. Specificity, sensibility, and reproducibility analysis were tested. This assay could be a useful tool not only for diagnostic purposes but also for future in vivo and in vitro T. gallinae studies.

The parasite Cryptosporidium parvum Tyzzer 1912 destroys parts of the intestinal brush border membrane which is important for the uptake of nutrients like glucose. In this study, glucose transport mechanisms of the host cells (IPEC-J2 cells) infected by C. parvum were investigated. The mRNA expression levels of glucose transporters (GLUT) 1 and 2 and Na+-coupled glucose transporter (SGLT) 1 were compared in infected and uninfected cells over an infection time of 24-96 h by RT-qPCR. Furthermore, the protein expression of SGLT 1 and GLUT 2 was quantified in western blot studies. While the protein expression of SGLT 1 was not altered in infected cells, mRNA expression of SGLT 1 and GLUT 1 was significantly increased 24 h p. i. and decreased 96 h p. i. The mRNA expression of GLUT 2 was significantly decreased 24 h, 72 h, and 96 h p. i. and also correlated significantly with the infection dose at 72 h p. i. In contrast to that, the protein expression of GLUT 2 was significantly increased 48 h p. i., associated with a significantly higher intracellular glucose level in infected cells compared with control cells at that time point of infection. This points to an adaptation of the host cells' glucose uptake taking place in the acute phase of the infection. A better understanding of these molecular mechanisms following a C. parvum infection may probably lead to an improvement of therapy strategies in the future.

The protozoan Cryptosporidium parvum is one of the major causative pathogens of diarrhoea in young ruminants; therefore, it causes economic losses and impairs animal welfare. Besides C. parvum, there are many other non-infectious and infectious factors, such as rotavirus, Escherichia coli, and Giardia duodenalis, which may lead to diarrhoeic disease in young livestock. Often, more than one infectious agent is detected in affected animals. Little is known about the interactions bet-ween simultaneously occurring pathogens and their potential effects on the course of disease. In this review, a brief overview about pathogens associated with diarrhoea in young ruminants is presented. Furthermore, information about coinfections involving Cryptosporidium is provided.

Cryptosporidium parvum is a main cause of severe diarrhea in young calves leading to economic loss and animal suffering. Little is known about the epidemiology of the genetic subtypes that may differ in their infectiousness and zoonotic potential. The present study analyzes the distribution of Glycoprotein 60 (GP60) variants in C. parvum positive samples obtained from young calves in Germany where individual fecal samples of 441 pre-weaned calves collected from 98 German dairy herds were evaluated for cryptosporidiosis (Göhring et al., 2014). DNA was extracted for Restriction Fragment Length Polymorphism (RFLP) analysis from 268 samples that were positive for Cryptosporidium by coproantigen EIA originating from 91 farms confirming C. parvum as the only species occurring. GP60 subtype analysis revealed 12C. parvum subtypes, all belonging to the zoonotic allele family IIa. The most widespread subtype was IIaA15G2R1 (71.4%). Other subtypes occurred sporadically: IIaA16G2R1, IIaA16G3R1 and IIaA17G2R1 (6.2%), IIaA13G1R2 (3.5%), IIaA14G1R1 (3.1%), IIaA14G2R1 (1.3%), IIaA17G1R1 (0.9%) and IIaA13G2R1, IIaA16G1R2, IIaA17G1R2 and IIaA24G1R1 (0.4%). Altogether a high diversity of subgenotypes was found with IIaA15G2R1 clearly dominating irrespective of geographic region. All subgenotypes are considered zoonotic underscoring that pre-weaned calves are likely to be a reservoir for zoonotic C. parvum and thus may be a risk for animal-to-human transmission.

Suckling calves are prone to Cryptosporidium infection. The variable degree of clinical disease is influenced by keeping conditions and immune status of the host, but diversity of isolate virulence may also contribute. The aim of the current study was to evaluate the cytopathogenic effects of 26 C. parvum field isolates by using a MTT assay in HCT-8 cell monolayers. Cell viability of monolayers inoculated with oocysts of the field isolates varied considerably with values of 17.7% (± 5.1%) to 99.5% (± 7.1%). A standard deviation of 18.6% was detected for cell viability of the in house reference strain, which were tested alongside in every assay. Field isolates were grouped in three categories of cytopathogenicity. Probably the length of storage has an effect on the level of the cell destruction category detected post infection in vitro. The applied tool may help to better understand the variable course of cryptosporidiosis in the field.

Toxoplasma gondii has the ability to infect various nucleated cell types in different hosts. The aim of the present study was to investigate which chicken blood cells were targeted by T. gondii in a mixed blood cell culture similar to in vivo conditions and to evaluate parasite-host cell interactions. The study consisted of two subsequent experiments. In experiment 1, we applied T. gondii tachyzoites (ME49) at a multiplicity of infection of 1 tachyzoite per blood cell and examined parasite replication, cytokine, and inducible nitric oxide synthase (iNOS) mRNA expression between 1 h and 48 h post-infection (p.i.) by quantitative PCR. By using T. gondii RH-GFP tachyzoites expressing the green fluorescent protein (GFP) in experiment 2, we aimed for visualizing infected cells by confocal laser scanning microscopy (CLSM) and flow cytometric analysis at 24 h p.i. The parasite replication curve showed a massive decrease of parasite stages until 24 h p.i. followed by an approximately plateau phase. We observed mainly significantly increased iNOS mRNA expression levels in T. gondii-infected culture compared to uninfected cells. Flow cytometry and CLSM data confirmed monocytes/macrophages as main target cells for T. gondii. Moreover, different lymphocytes like B cells and cytotoxic T cells seem to be targeted to a low extent. Our findings indicate that monocytes/macrophages play a key role during T. gondii infection in chicken as host cells and triggering of immune response. To the best of our knowledge, this is the first report of a mixed chicken blood cell culture experimentally infected with T. gondii.

In vitro and in vivo studies were performed to assess whether Eimeria tenella (E. tenella) oocysts, exposed to low energy electron irradiation (LEEI), might be considered potential vaccine candidates against cecal coccidiosis. Sporulated oocysts were exposed to LEEI of 0.1 kGy to 10.0 kGy. Reproduction inhibition assays (RIA) were performed in MDBK cells to assess infectivity of sporozoites excysted from irradiated and non-irradiated oocysts. LEEI of 0.1 kGy or 0.5 kGy resulted in 73.2% and 86.5% inhibition of in vitro reproduction (%IRIA), respectively. Groups of 12 one day old (D1) chicken were orally inoculated with Paracox®-8 (G1), 2.0 × 103 non-irradiated oocysts (G2) or 1.0 × 104 irradiated oocysts exposed to LEEI of 0.1 kGy (G3, G4) or 0.5 kGy (G5). Chicken of groups G1, G2, G4 and G5 were challenged 3 weeks later (D21) by a single inoculation of 7.5 × 104 non-attenuated oocysts of the same strain while G3 remained unchallenged. All chickens were subject to necropsy 7 days after challenge (D28) to estimate lesion scores (LS) and oocyst index (OI). A positive control (PC, non-vaccinated, challenged) and a negative control (NC, non-vaccinated, non-challenged) were kept in parallel. Chicken of group G5 had similar weight gain as the Paracox®-8 group (G1) after challenge and higher weight gains as compared to the other vaccinated groups. Feed conversion ratio (FCR) did not differ between chickens inoculated with oocysts irradiated with 0.5 kGy (G5) and negative control (NC) before challenge (1.25–1.52). After challenge FCR was 1.99 (G5) to 2.23 (G4) in the vaccinated chicken compared to 1.76 in group NC. LS and OI were significantly lower in all vaccinated groups as compared to group PC. Progeny oocysts collected from the feces of chickens following vaccination with irradiated oocysts exhibited lower in vitro infectivity/reproduction in MDBK cells with %IRIA of 89.7% and 82.4% for progeny of oocysts irradiated with 0.5 kGy and 0.1 kGy, respectively, suggesting hereditary attenuation by LEEI treatment. Seroconversion was demonstrated by ELISA before challenge (D21) in all vaccinated groups, however, chicken inoculated with irradiated oocysts displayed higher antibody levels than those inoculated with precocious oocysts (G1). In Western blot analysis chicken vaccinated with virulent (G2) or 0.1 kGy-irradiated E. tenella oocysts (G3, G4) showed more protein bands compared to G5 (0.5 kGy). We conclude that LEEI could be a promising technology for production of attenuated oocyst vaccines.

Simple Summary Cryptosporidium parvum is one of the causal agents of cryptosporidiosis. This water-borne disease is responsible for around one million human deaths every year and the only approved anti-cryptosporidial drug for humans, i.e., nitazoxanide, lacks efficacy in immunocompromised patients. We here present, to the best of our knowledge, the first analyses of C. parvum impact on the metabolism of its host-cells under physiological oxygen conditions. Moreover, based on the here presented metabolic evidence, we blocked specific metabolic pathways in order to find new anti-cryptosporidial targets. Our findings besides confirming glycolysis as anti-cryptosporidial target prove glutaminolysis and lactate release as necessary for parasite replication. Abstract Cryptosporidium parvum is an apicomplexan zoonotic parasite recognized as the second leading-cause of diarrhoea-induced mortality in children. In contrast to other apicomplexans, C. parvum has minimalistic metabolic capacities which are almost exclusively based on glycolysis. Consequently, C. parvum is highly dependent on its host cell metabolism. In vivo (within the intestine) infected epithelial host cells are typically exposed to low oxygen pressure (1–11% O2, termed physioxia). Here, we comparatively analyzed the metabolic signatures of C. parvum-infected HCT-8 cells cultured under both, hyperoxia (21% O2), representing the standard oxygen condition used in most experimental settings, and physioxia (5% O2), to be closer to the in vivo situation. The most pronounced effect of C. parvum infection on host cell metabolism was, on one side, an increase in glucose and glutamine uptake, and on the other side, an increase in lactate release. When cultured in a glutamine-deficient medium, C. parvum infection led to a massive increase in glucose consumption and lactate production. Together, these results point to the important role of both glycolysis and glutaminolysis during C. parvum intracellular replication. Referring to obtained metabolic signatures, we targeted glycolysis as well as glutaminolysis in C. parvum-infected host cells by using the inhibitors lonidamine [inhibitor of hexokinase, mitochondrial carrier protein (MCP) and monocarboxylate transporters (MCT) 1, 2, 4], galloflavin (lactate dehydrogenase inhibitor), syrosingopine (MCT1- and MCT4 inhibitor) and compound 968 (glutaminase inhibitor) under hyperoxic and physioxic conditions. In line with metabolic signatures, all inhibitors significantly reduced parasite replication under both oxygen conditions, thereby proving both energy-related metabolic pathways, glycolysis and glutaminolysis, but also lactate export mechanisms via MCTs as pivotal for C. parvum under in vivo physioxic conditions of mammals.

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

Background: Both Eimeria (E.) tenella and Toxoplasma (T.) gondii are common apicomplexan parasites in chickens. Host cell invasion by both protozoans includes gliding motility, host cell attachment and active penetration. Chicken macrophages as phagocytic cells participate in the innate host immune response against these two parasites. Methods: In this study, primary chicken monocyte-derived macrophages (MMs) were infected with both pathogens to investigate mutual and host-parasite interactions. MMs cultures were assigned to groups that were infected with E. tenella, T. gondii, or both. In co-infected cultures, MMs were first exposed to E. tenella sporozoites for 2 hours. Afterwards, T. gondii tachyzoite infection was performed. Live-cell imaging was carried out to observe cell invasion and survival of T. gondii by single parasite tracking over a period of 20 hours post infection (hpi). Quantitative analysis for parasite replication was performed at 2, 6, 12 and 24 hpi by real-time PCR (qPCR).Results: We found that during early co-infection T. gondii tachyzoites adhered for more than 4 hours to macrophages. Although they displayed high motility, ability to penetrate the cell membrane of the potential host cell was reduced. qPCR results confirmed that significantly less T. gondii entered E. tenella-activated MMs at 2 hpi, and a reduced proportion of intracellular T. gondii survived and replicated in these cells at 24 hpi. Conclusions: We conclude that E. tenella modulates host cell responses to another apicomplexan agent, T. gondii, reducing active invasion and multiplication in chicken primary macrophages.

This guideline is intended as an aid in the design, implementation and interpretation of studies for the assessment of drug efficacy against Eimeria in cattle and sheep, Cystoisospora in pigs and dogs, and Cryptosporidium in cattle. It deals with the most important aspects of how to conduct both experimental and field studies for dose determination, dose confirmation and assessment of field effectiveness. Also, guidance on the selection of animals, diagnostic techniques, statistical evaluation and methods for the preparation, maintenance and use of parasites is provided. The specific management conditions that may influence the course of natural infections and consequently determine treatment schemes are mentioned and suggestions for best practice in sampling and evaluation of data prior to conducting of efficacy studies are given. The guideline is also intended to assist investigators in carrying out specific studies, provide relevant information for registration authorities involved in the decision-making process, assist in the approval of anticoccidial drugs in the target species, and facilitate the world-wide adoption of standard procedures. Although currently not implemented, issues of drug resistance testing and alternative methods for drug testing are also discussed as future issues in drug testing against mammalian coccidia.

Toxoplasma gondii is known to be able to infect any nucleated cell including immune cells like macrophages. In addition, it is assumed that macrophages serve as trojan horse during distribution in hosts. The underlying causes of parasite host interaction remain yet not fully understood. The aim of the present study was to investigate susceptibility of chicken macrophages to infection with T. gondii and the process of infection in avian cells in comparison to cells of mammalian origin. Primary avian blood monocyte-derived macrophages were infected with tachyzoites of type II (ME49) and III (NED) strains. Long term observations of parasite replication in primary macrophages were compared to data obtained in an avian macrophage cell line (HD11) and a standard cultivation mammalian cell line (VERO). Furthermore, we assessed the immune response of the primary macrophages by long-term investigation of gene expression of IL-1 beta, IL-12p40, Lipopolysaccharide induced TNF-alpha factor (LITAF) and inducible nitric oxide synthase (iNOS) comparing viable and heat-inactivated tachyzoites of the ME49 strain. Albeit, we found no differences between both strains, replication of tachyzoites in avian primary macrophages was significantly different from immortalized cell lines HD11 and VERO. The crucial period of parasite replication was between 8 and 24 h post-infection coinciding with the upregulation of gene expression of cytokines and iNOS revealing an active macrophage response at this period. Gene expression in macrophages was higher after infection with viable tachyzoites than by exposure of cells to heat-inactivated tachyzoites. Hence, we conclude that the process of penetration is pivotal for host cell response to the parasite both in avian as in mammalian cells.

The widespread apicomplexan parasites Toxoplasma gondii (T. gondii) and Eimeria tenella (E. tenella) are important pathogens with high prevalence in poultry. The aim of our study was the investigation of mutual influences in co-infected chickens, focusing on immune response and course of infection. Two separate trials were performed using in total 96 1-day-old chickens, divided into four study groups: group NC (negative control, uninfected), group PC-T (oral or intramuscular infection with T. gondii oocysts (trial 1) or tachyzoites (trial 2), respectively), group PC-E (oral infection with E. tenella (trial 1) or E. tenella and Eimeria acervulina (trial 2)), and group TE (co-infection). T. gondii and Eimeria infections were validated by different parameters, and cytokine expression in the gut and spleen was investigated. T. gondii-specific antibodies were detected earliest 4 days post infection (p.i.) by immunoblot and direct DNA detection was possible in 22.1% of all tissue samples from infected chickens. Eimeria spp. merogony seemed to be enhanced by co-infection with T. gondii, interestingly without marked differences in oocyst excretion between co-infected and Eimeria spp. mono-infected chickens. An increase of messenger RNA (mRNA) expression of Th1- (IFN-γ, IL-12, TNF-α) and Th2-related cytokines (IL-10) mainly in groups PC-E and TE was observed, however, without statistically significant differences between co-infection and single infection with Eimeria. In conclusion, most of the measurable immune response could be attributed to Eimeria infection. To the best of our knowledge, this is the first report on co-infection experiments of T. gondii with Eimeria spp. in chickens.

Toxoplasmosis is a worldwide zoonosis caused by the obligate intracellular apicomplexan parasite Toxoplasma gondii (T. gondii). Chickens are ground-feeders and represent, especially if free-range, important intermediate hosts in the epidemiology of toxoplasmosis and are used as sentinels of environmental contamination with T. gondii oocysts. Until now, little is known about the burden and regional distribution of T. gondii cysts in the chicken brain. It was therefore the aim of this study to investigate the abundance and specific distribution of T. gondii cysts within the chicken brain following chronic infection with a type II strain (76 K) of T. gondii. A total of 29 chickens were included in the study and divided into control group (n = 9) and two different infection groups, a low dose (n = 10) and a high dose (n = 10) group, which were orally inoculated with 1500 or 150,000 T. gondii oocysts per animal, respectively. Seroconversion was detected in the majority of chickens of the high dose group, but not in the animals of the low dose and the control group. Moreover, T. gondii DNA was detected most frequently in the brain and more frequently in the heart than in liver, spleen, thigh and pectoral muscle using qPCR analysis. The number of T. gondii cysts, quantified in the chicken brain using histological analysis, seems to be considerably lower as compared to studies in rodents, which might explain why T. gondii infected chickens very rarely, if at all, develop neurological deficits. Similar to observations in mice, in which no lateralisation for T. gondii cysts was reported, T. gondii cysts were distributed nearly equally between the left and right chicken brain hemispheres. When different brain regions (fore-, mid- and hindbrain) were compared, all T. gondii cysts were located in the forebrain with the overwhelming majority of these cysts being present in the telencephalic pallium and subpallium. More studies including different strains and higher doses of T. gondii are needed in order to precisely evaluate its cyst burden and regional distribution in the chicken brain. Together, our findings provide insights into the course of T. gondii infection in chickens and are important to understand the differences of chronic T. gondii infection in the chicken and mammalian brain.

Infections with arthropod-borne pathogens are an increasing threat world-wide that requires heightened vigilance from veterinary and medical practitioners, especially when they involve new or unusual organisms. A dog was presented to a local veterinary clinic in Germany with malaise, pale mucous membranes and stiff joints. Clinical assessment revealed pyrexia, leukopenia and thrombocytopenia. On suspicion of a tick-borne infection, blood samples were examined for clinical and biochemical parameters and subjected to a Anaplasma phagocytophilum -, Borrelia spp.- and Ehrlichia canis -specific real-time PCR. Additionally, a sample of the pre-therapeutic buffy coat was co-cultured with the Ixodes scapularis cell-line ISE6 for 20 days. Only the PCR specific for A. phagocytophilum DNA yielded a positive result, and furthermore, Anaplasma morulae were visible in granulocytes and tick cells. After co-culturing, extracellular trypomastigote and epimastigote stages of Trypanosoma sp. with an average length of 29.7 μm were observed, featuring a pointed posterior end. Sequence analysis of a 2080 bp fragment of the 18S rRNA gene showed 99% identity to the 18S rRNA gene of Trypanosoma pestanai , previously described from a European badger ( Meles meles ) in France. The dog's condition improved rapidly in response to doxycycline treatment for three weeks. The clinical status normalized and clinical blood parameters were found to be within the reference ranges. To our knowledge this is the first description of T. pestanai infection in a dog, the first detection of T. pestanai in Germany and the first documented co-infection with these two pathogens. Co-infections with unusual opportunistic vector-borne pathogens should be considered, if acute canine granulocytic anaplasmosis is evident.

A cat with multiple subcutaneous nodules suggesting a soft tissue sarcoma by physical and computed tomographic examination was diagnosed as being affected by subcutaneous filariosis based on cytologic and ultrasonographic assessments. Nodules were surgically removed and extracted nematodes were identified by PCR as Dirofilaria repens. Furthermore, DNA of Dipetalonema dracunculoides (syn. Acantocheilonema dracunculoides) was detected by PCR, with no evidence of circulating microfilariae. To the best of the authors' knowledge, this represents the first report describing adults of D repens in multiple subcutaneous nodules in a cat. Cytopathologic examination allowed characterization of the parasitic nature of the nodules. Veterinary practitioners should be aware of the possible nodular presentation of D repens in cats and should include D repens in the differential diagnosis of subcutaneous neoformations in the cat.

Seventy-five previously frozen Northern Lapwings, Vanellus vanellus (Linnaeus, 1758) were dissected and examined for trematodes. The available birds were euthanized after suffering severe injuries from a hail storm. Trematode specimens were found in the air sacs, body cavities and kidneys. They were cleared, stained in Alaun-Carmine and examined via light microscopy. Trematodes were assigned to two different families and three different species: Uvitellina vanelli and Selfcoelum sp. from the family Cyclocoelidae and Tanaisia valida of the family Eucotylidae. A prevalence of 12% (for both, Cyclocoelid and Eucotylid infections) was detected. While trematodes of the respiratory system have been discovered in V. vanellus before, the present study describes renal trematodiasis in this bird species for the first time. Furthermore, the first recovering of a trematode of the species Selfcoelum sp. in the Northern Lapwing is described. Full measurements and figures of trematodes are given. Criteria and critical points in specifying and recovering the parasites are discussed.

Toxoplasma (T.) gondii is able to infect various cell types in different hosts. The replication of this parasite within different peripheral mononuclear blood cell populations in chicken has not yet been fully understood. Aim of the present study was to investigate the impact of chicken erythrocytes and thrombocytes as potential host cells for T. gondii. Cultures of primary avian erythrocytes and thrombocytes were inoculated with tachyzoites of T. gondii type II strain ME49. Parasite replication was detected by a quantitative real-time PCR at different times postinoculation until 24 or 48 h, respectively, displaying long-term investigations for the chosen cultures. The parasite replication curve showed a continuous decrease of parasite stages in erythrocytes and thrombocytes. Observations by light microscopy showed massive destruction for both cell populations. Few macrophages in between the infected thrombocytes were viable during the investigation period and showed internalised tachyzoites by confocal laser scanning microscopy. These findings show that T. gondii is not capable of replication in chicken erythrocytes and thrombocytes; therefore, both cannot be considered as potential host cells. In further consequence, monocyte-derived macrophages seem to be the key to the dissemination mechanisms for T. gondii in chicken.

Alveolar echinococcosis (AE) is a parasitic zoonosis occurring in most European countries and also emerging in parts of Asia and North America. AE is caused by the larval stage of Echinococcus multilocularis in intermediate and also in accidental hosts. The principal definitive host is the red fox, but domestic dogs and cats are also potential definitive hosts. Several species of rodents serve as intermediate hosts of this parasite. However, there are also some species acting as accidental intermediate hosts, among them dogs. Since the late 1980s cases of canine AE have been diagnosed. Here, we present a case of canine AE in a two-year old female intact German spaniel from Thuringia, Central Germany. The dog was used as a hunting dog and presented to a small animal clinic for subacute lethargy and inappetence. Abdominal ultrasound and contrast computed tomography (CT) scan were performed and revealed intrahepatic lesions. Multinodular changes of the liver and the greater omentum were demonstrated by exploratory laparotomy. After euthanasia, a necropsy was performed and histological sections of representative tissue samples were prepared. PCR followed by sequencing was conducted with DNA extracted from tissue samples of the liver, hepatic lymph nodes and greater omentum. The sequence herein obtained showed very high similarity with other partial nad2 sequences of E. multilocularis from the GenBank database by BLASTn analysis and was analysed using the maximum likelihood method. The presented case combines the clinical presentation and pathological, parasitological and phylogenetic analyses.

Turkeys and chickens were orally infected with tissue cysts (one mouse brain) or oocysts (103, 105 or 106 oocysts) of three T. gondii strains of the clonal types II and III (ME49, CZ-Tiger, NED) to investigate the influence of the applied T. gondii strain and infective doses on the distribution of T. gondii in several organs and tissues and the serologic response of chickens and turkeys. Organ samples from 16 different tissues, including heart, brain, muscles and gizzard were analyzed by PCR. Brain and heart were found most frequently positive for T. gondii DNA in both species, followed by gizzard. Serological analysis with kinetic ELISA for turkey samples and IFAT for chicken samples were performed once a week. In both species a dose-depending serological response was found. Turkeys seroconverted one week after infection with CZ-Tiger strain and medium and high doses of ME49 oocysts. In chickens, infection with medium and high doses of CZ-Tiger led to seroconversion one week p.i. Frequency of T. gondii positive organs showed a trend of a dose-effect in both species after infection with the type II strains. The NED strain showed low virulence in chickens and turkeys, demonstrated by clearly less T. gondii positive organs. Infection with tissue cysts of all three strains revealed T. gondii stages in tissues of turkeys and chickens. In conclusion, our data show a risk for human infection with T. gondii due to consumption of chicken and turkey meat.

The efficacy and safety of an injectable combination product containing toltrazuril and gleptoferron (Forceris® – CEVA) for the control of coccidiosis due to Cystoisospora suis was investigated in neonatal piglets. The study was carried out on five European commercial pig farms in France, Germany and Spain and 122 litters consisting of 1508 piglets were selected and randomly allocated to one of two treatment groups. The first group received a single intramuscular injection per piglet of the test product, containing 45 mg toltrazuril and 200 mg iron and this was compared with a control group, which was administered a single intramuscular treatment of iron at 200 mg per piglet only. Body weights, faecal scores and oocysts counts, recorded as oocysts per gram of faeces, (OPG) were observed for 21 days. Only 1138 piglets were actually exposed to coccidiosis and data sets of these animals were selected for statistical analysis. The efficacy of the test product in the control of coccidiosis was shown in higher body weight gains, a lower percentage of animals with diarrhoea, fewer samples with positive oocysts counts as well as a lower excretion peak and a reduced area under the curve of OPG from study day (SD) 4 – SD 21. The combination product of toltrazuril and gleptoferron provided an effective alternative approach to current conventional separate treatment for the prevention of iron deficiency anaemia and coccidiosis in neonatal piglets. It reduced the numbers of potentially stressful interventions and work time. Keywords: Piglet, Coccidiosis, Cystoisospora suis, Toltrazuril, Forceris®, Natural infection, Diarrhoea

Cryptosporidiosis is a common protozoan parasitic infection that causes diarrhoea in neonatal calves. The high shedding of environmentally resistant oocysts facilitates outbreaks of cryptosporidiosis in humans. In total, 58 farms (512 calves) in Germany (Saxony and Brandenburg) were visited three times each. Faecal samples of pre-weaned calves were microscopically examined for oocysts of Cryptosporidium spp. using Heine staining and were scored with regard to their consistency. Overall, 88.9% of calves tested microscopically positive for Cryptosporidium spp. in at least one sample, and the excretion of oocysts was significantly (P

Whitish roundworms were found in the faeces of a female (3 months old) and a male (1 month old) European bison calf from an animal park in Central Germany. Since the faeces samples were not assignable to the individual animals, it is not certain if both calves were infected. The worms were identified as Toxocara vitulorum by morphological and molecular analysis, and a treatment with doramectin was initiated. To monitor the success of the therapy, faecal samples were examined before and after treatment by combined flotation/sedimentation technique, followed by a quantitative McMaster analysis in case of positive results. Additionally, stages of several Eimeria species and Trichuris sp. were found. Eggs of T. vitulorum were detected one week after application of doramectin, so, a second treatment was performed. However, no eggs of T. vitulorum were found at the time of the second treatment. Because of the difficulty of T. vitulorum eradication in infected herds, further investigations in the following year will be conducted.

Cystic echinococcosis (CE) is endemic in many parts of sub-Saharan Africa. In contrast to the eastern part of the continent, very little data exists on the current disease situation in southern Africa including Zambia. This study determined frequency and species identity of Echinococcus spp. circulating in livestock and dogs in the Western Province of Zambia. Cysts were collected in slaughterhouses at meat inspection (cattle) and during examination of home slaughtered pigs, while dog faecal samples were collected per-rectum and examined microscopically for the presence of taeniid eggs. Individual taeniid eggs from faecal samples and individual protoscoleces from cysts were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and/or sequencing of the NADH-dehydrogenase subunit 1 (nad1) and cytochrome C oxidase 1 (cox1) gene. Fifty-four of 2000 cattle (2.7%) were found infected with a total of 65 cysts, predominantly fertile lungs cysts; all cysts were identified as Echinococcus ortleppi. Two out of 52 home-slaughtered pigs (3.8%) were infected with a fertile lung cyst each; both cysts were also identified as E. ortleppi. Microscopic examination revealed 10/289 dog faecal samples to contain taeniid eggs, of which four samples (two each) contained Echinococcus canadensis (G6/7) or Taenia hydatigena, respectively. This is the first insight in the Echinococcus species circulating in Zambia providing premises for further studies into transmission dynamics of CE in the southern African region.

Magnetic-capture PCR was applied for the quantitative detection of Toxoplasma gondii in tissues of experimentally infected turkeys and retail turkey meat products. For experimental infection, three T. gondii strains (ME49, CZ-Tiger, NED), varying infectious doses in different matrices (organisms in single mouse brains or 10(3), 10(5), or 10(6) oocysts in buffer) were used. From all animals, breast, thigh, and drumstick muscle tissues and for CZ-Tiger-infected animals additionally brains and hearts were analyzed. Using the magnetic-capture PCR large volumes of up to 100 g were examined. Our results show that most T. gondii parasites are present in brain and heart tissue. Of the three skeletal muscle types, drumsticks were affected at the highest and breast at the lowest level. Type III strain (NED) seems to be less efficient in infecting turkeys compared to type II strains, because only few tissues of NED infected animals contained T. gondii DNA. Furthermore, the number of detected parasitic stages increased with the level of infectious dose. Infection mode by either oocyst or tissue cyst stage did not have an effect on the amount of T. gondii present in tissues. In retail turkey meat products T. gondii DNA was not detectable although a contact with the parasite was inferred by serology.

The aim of the present study was to evaluate alternatives for inactivating Cryptosporidium parvum under experimental conditions. Disinfectants against this protozoan are usually based on cresols and often difficult to handle in laboratories. Four different substances (ethanol, denatured ethanol, sodium hypochlorite and peroxide) at different concentrations were tested for several exposure times (30 min, 2 h, 4 h, 12 h and 24 h). The results show an inactivation over 99% by using 10% H2O2 at an exposure time over 2 h as well as 3 and 6% NaOCl after 12 h of exposure. Furthermore, the ability of UV-C light to inactivate oocysts on smooth surfaces (e.g., laminar flow) was evaluated. To mimic laboratory conditions, oocysts were given on germ carriers. Best results (>99%) were achieved at an exposure time of 30 min (100.8 mJ/cm(2)).

Highlights • Efficacy of Forceris® against piglet coccidiosis successfully demonstrated. • Treated piglets show reduced diarrhoea and oocysts excretion. • Forceris® treatment is safe and improves body weight gain. • Combination of two active agents in one product reduces the numbers of stressful interventions and work time., The efficacy and safety of an injectable combination product containing toltrazuril and gleptoferron (Forceris® – CEVA) for the control of coccidiosis due to Cystoisospora suis was investigated in neonatal piglets. The study was carried out on five European commercial pig farms in France, Germany and Spain and 122 litters consisting of 1508 piglets were selected and randomly allocated to one of two treatment groups. The first group received a single intramuscular injection per piglet of the test product, containing 45 mg toltrazuril and 200 mg iron and this was compared with a control group, which was administered a single intramuscular treatment of iron at 200 mg per piglet only. Body weights, faecal scores and oocysts counts, recorded as oocysts per gram of faeces, (OPG) were observed for 21 days. Only 1138 piglets were actually exposed to coccidiosis and data sets of these animals were selected for statistical analysis. The efficacy of the test product in the control of coccidiosis was shown in higher body weight gains, a lower percentage of animals with diarrhoea, fewer samples with positive oocysts counts as well as a lower excretion peak and a reduced area under the curve of OPG from study day (SD) 4 – SD 21. The combination product of toltrazuril and gleptoferron provided an effective alternative approach to current conventional separate treatment for the prevention of iron deficiency anaemia and coccidiosis in neonatal piglets. It reduced the numbers of potentially stressful interventions and work time.