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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.

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.

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 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.

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.

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.

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

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.

Two field studies were accomplished on a dairy farm in Saxony to compare different strategies for the control of natural cryptosporidiosis in newborn calves. In the first study, 96 newborn calves were allocated to four different groups immediately after birth. Calves of group H and of group HN were treated orally with 120 μg/kg body weight (BW) of halofuginone lactate daily during the first seven days of life. Calves of group C and of group CN were treated with a same volume of tap water. As an additional measure, the pens of groups HN and CN were disinfected with 3% Neopredisan 135-1(®)(p-chloro-m-cresol), the pens of groups C and H remained non-disinfected. Faeces were examined semi-quantitatively for oocyst excretion using carbolfuchsin-staining and the clinical course was recorded. While disinfection alone (group CN) had no effect on oocyst shedding and diarrhoea, treatment with halofuginone lactate (groups H and HN) reduced oocyst shedding and diarrhoea significantly. Combination of treatment and disinfection (group HN) controlled cryptosporidiosis completely during the first two weeks after birth. However, prevalence of diarrhoea and oocyst shedding was higher in the third week of life in group HN than in any of the other groups. This delayed occurrence of cryptosporidiosis was not seen in study 2 when all calves were similarly protected by treatment and specific disinfection. Oocyst shedding was not observed in peripartal cows either by carbolfuchsin staining, ELISA or PCR.

In the present study, efficacy of the toltrazuril treatment for prevention of coccidiosis and necrotic enteritis was tested. Ninety-six 14-day-old commercial broiler chickens were caged and divided into eight groups (n=12), designated groups 1 to 8. Chickens of groups 1 to 6 were inoculated orally at 18 days of age with 25,000 oocysts of Eimeria tenella and 75,000 oocysts of Eimeria brunetti. At 22 days of age, chickens of groups 1 to 6 were infected with 10(9) colony-forming unit Clostridium perfringens. Chickens of group 1 were treated with 75 parts/10(6) toltrazuril in drinking water for 8 h on two consecutive days up to 12 h before Eimeria infection, while chickens of groups 2 to 5 were treated with the same dose of toltrazuril at 12 h, 36 h, 60 h and 84 h after Eimeria infection, respectively. The non-treated group 6 served as a positive control. Chickens in group 7 were treated with toltrazuril at 17 and 18 days of age, and those of group 8 remained uninfected and non-treated as a negative control. The feed conversion ratio was higher in the positive control compared with other groups. The mortality rates were 16.8% and 41.7% in the late toltrazuril-treated (at 84 h) and infected non-treated chickens, respectively. Lesions scores of necrotic enteritis or coccidiosis in infected, non-treated chickens were significantly more severe compared with negative controls (P

Toxoplasma gondii is one of the most common zoonotic parasites in the world. The parasite causes no or mild symptoms in immunocompetent humans. However, a high potential hazard exists for seronegative pregnant women and immunocompromised patients. The consumption of meat containing tissue cysts or oocyst-contaminated vegetables and fruits or the handling of cat feces poses a high risk of infection with T. gondii. It is known that raw minced meat, raw fresh sausages, and locally produced raw meat products are possible causes of T. gondii infection. The infectivity of T. gondii tissue cysts in meat products depends, among other factors, on the pH and the salt concentration. Therefore, the impact of these two factors on the tissue cysts was examined. For this purpose, dissected musculature and brain from experimentally infected mice (donor mice) were placed in a cell culture medium (RPMI 1640). The medium was adjusted to different pH values (pH 5, 6, and 7) with lactic acid and to different salt concentrations (2.0, 2.5, and 3.0%) with sodium chloride (NaCl) or nitrite-enriched curing salt (NCS) for the various tests. After storage at 4°C for different time periods, the materials were fed to bioassay mice. Later, the brains were examined for presence of T. gondii to assess the infectivity. The data show that T. gondii tissue cysts have a high pH tolerance. Cysts were infectious in the muscle for up to 26 days (pH 5). In contrast to their tolerance to pH, cysts were very sensitive to salt. Muscle cysts survived at an NaCl concentration of up to 2.0% only, and for no longer than 8 days. At NaCl concentrations of 2.5 and 3.0%, the cysts lost their infectivity after 1 day. When NCS instead of NaCl was used under the same conditions, T. gondii muscle cysts retained infectivity for only 4 days at 2.0%. Consequently, NCS (NaCl plus 0.5% nitrite) has a stronger effect on T. gondii cysts than does common table salt. Sausages produced with low NaCl concentration and short contact times pose a potential risk for susceptible individuals.

Background Human African Trypanosomiasis (HAT) also called sleeping sickness is an infectious disease in humans caused by an extracellular protozoan parasite. The disease, if left untreated, results in 100% mortality. Currently available drugs are full of severe drawbacks and fail to escape the fast development of trypanosoma resistance. Due to similarities in cell metabolism between cancerous tumors and trypanosoma cells, some of the current registered drugs against HAT have also been tested in cancer chemotherapy. Here we demonstrate for the first time that the simple ester, ethyl pyruvate, comprises such properties. Results The current study covers the efficacy and corresponding target evaluation of ethyl pyruvate on T. brucei cell lines using a combination of biochemical techniques including cell proliferation assays, enzyme kinetics, phasecontrast microscopic video imaging and ex vivo toxicity tests. We have shown that ethyl pyruvate effectively kills trypanosomes most probably by net ATP depletion through inhibition of pyruvate kinase (Ki = 3.0\(\pm\)0.29 mM). The potential of ethyl pyruvate as a trypanocidal compound is also strengthened by its fast acting property, killing cells within three hours post exposure. This has been demonstrated using video imaging of live cells as well as concentration and time dependency experiments. Most importantly, ethyl pyruvate produces minimal side effects in human red cells and is known to easily cross the blood-brain-barrier. This makes it a promising candidate for effective treatment of the two clinical stages of sleeping sickness. Trypanosome drug-resistance tests indicate irreversible cell death and a low incidence of resistance development under experimental conditions. Conclusion Our results present ethyl pyruvate as a safe and fast acting trypanocidal compound and show that it inhibits the enzyme pyruvate kinase. Competitive inhibition of this enzyme was found to cause ATP depletion and cell death. Due to its ability to easily cross the blood-brain-barrier, ethyl pyruvate could be considered as new candidate agent to treat the hemo-lymphatic as well as neurological stages of sleeping sickness.

The ectoparasites of Milvago c. chimango (Vieillot) in the Ñuble area were identified for qualitative and quantitative descriptive analyses. Three species of Phthiraptera were recorded (prevalences between parentheses): One Amblycera, Aquiligogus maculatus (Menoponidae – 100%), and two Ischnocera, Acutifrons vierai chimango (73.9 ± 9.2%) and Caracaricola chimangophilus (26.1 ± 9.2%) (both, Philopteridae). Moreover, three taxa of Acarina, Gabucinia sp. (Gabuciniidae – 69.6 ± 9.6%), Dubininia accipitrina (Xolalgidae – 52.2 ±10.4%), and mites belonging in Syringophilidae (39.1 ± 10.2%) were encountered. No species of Parasitiformes or Diptera were observed.

Two studies were conducted in the Eimeria zuernii infection model in order to investigate the pathology of E. zuernii coccidiosis and the efficacy of toltrazuril (Baycox 5% suspension) in this infection. For this purpose, a total of 30 calves were infected experimentally with E. zuernii oocysts and faecal samples taken regularly from the rectum and examined for faecal consistency and oocyst excretion. Six of the calves underwent pathological examination at various points in time after infection. Significant macroscopic and microscopic changes were demonstrated and parasitic stages were identified in the intestinal mucosa of infected calves during the late prepatent and patent period. Inflammatory reactions revealed by light microscopy were confirmed by electron microscopical investigations. Treatment of calves with toltrazuril during the late prepatent period resulted in significantly lower frequencies of diarrhoea and levels of oocyst excretion, and weight gain was significantly higher than in shamtreated animals.

A total of 114 quails (Callipepla californica) were collected and the following ectoparasites were isolated in Ñuble Province (Chile), between November 1994 and April 1995 (prevalence in %): Epicolinus elipticus (28.1%), Zlotozyckella stefani (60.5%), Megninia glynglimura (100%), Pseudolichus sp. (43.0%), Amblyomma sp. (13.2%) and a mite from the Trombiculidae family (11.4%). The parasite prevalences presented no correlation with age (juveniles and adults), sex, and capture area (humid and arid). The ectoparasites found represent the first recorded on this host in Chile.

In a 5-year survey regarding its prevalence and importance in five German state veterinary laboratories Cryptosporidium was diagnosed annually in 19–36% of faecal samples either submitted to the laboratories or taken post mortem. In approximately half of the cases no other enteropathogens were detected. However, only 73% of 30 laboratories participating in a questionnaire survey routinely tested for this parasite, and the majority of researchers considered cryptosporidiosis to be of minor importance. In a placebo-controlled field study 152 suckling calves were treated daily against cryptosporidiosis either with sulfadimidine or with halofuginone (Halocur®, Intervet) over 1 week. Treatment by oral drench started at the onset of diarrhoea in the herd. Oocyst excretion, faecal consistency and health status were recorded five times for a 3-week period. Oocyst excretion peaked 7–14 days in the placebo group after the onset of diarrhoea, and during that period prevalence and intensity of excretion were significantly lower in the halofuginone-treated group compared to the sulfadimidine and the placebo control groups. The health status (diarrhoea, dehydration) declined in all groups but was significantly (P

The current drugs against sleeping sickness are derived from cancer chemotherapeutic approaches. Herein, we aimed at evaluating the in vitro effect of alcoholic extracts of Artemisia annua (AMR), Rumex abyssinicus (RMA), and Catha edulis Forsk (CEF) on proliferation/viability of 1321N1 astrocytoma, MCF-7 breast cancer, THP-1 leukemia, and LNCaP, Du-145, and PC-3 prostate cancer cells and on Trypanosoma brucei cells. Proliferation of tumor cells was evaluated by WST-1 assay and viability/behaviour of T. brucei by cell counting and light microscopy. CEF was the most efficient growth inhibitor in comparison to AMR and RMA. Nevertheless, in LNCaP and THP-1 cells, all extracts significantly inhibited tumor growth at 3 μg/mL. All extracts inhibited proliferation of T. brucei cells in a concentration-dependent manner. Microscopic analysis revealed that 95% of the T. brucei cells died when exposed to 33 μg/mL CEF for 3 hrs. Similar results were obtained using 33 μg/mL AMR for 6 hrs. In case of RMA, however, higher concentrations were necessary to obtain similar effects on T. brucei. This demonstrates the antitumor efficacy of these extracts as well as their ability to dampen viability and proliferation of T. brucei, suggesting a common mechanism of action on highly proliferative cells, most probably by targeting cell metabolism.

Cryptosporidium parvum is a zoonotic agent that infects humans and animals occasionally causing severe, watery diarrhoea. In immunocompetent hosts, cryptosporidiosis is self-limiting but can have a fatal outcome in immunocompromised individuals. Cryptosporidium is one of the most common causes of waterborne diseases (recreational water and drinking water) in humans, a leading cause of moderate to severe childhood diarrhoea, and a major agent of diarrhoea in calves leading to high economic losses and up to 10% lethality. So far, available treatment options are insufficient for both veterinary and human clinical disease cases. Here, we report for the first time that the novel bumped kinase inhibitor (BKI) 1294 targeting the calcium-dependent protein kinase 1 (CDPK1) of Cryptosporidium is able to reduce the oocyst shedding of C. parvum by calves--its natural host--without obvious side effects.

En la provincia de Ñuble (Chile), entre Noviembre de 1994 y abril de 1995, se examinaron 235 tórtolas (Zenaida auriculata) para detectar endo y ectoparásitos. Los siguientes endoparásitos fueron aislados (prevalencia en %): Killigrewia delafondi (9,4%), Raillietina sp. (1,7%), Echinostoma sp. (0,9%), Heterakis gallinarum (0,4%) y Eimeria labbeana (5,5%). Los ectoparasitos identificados correspondieron a: Falculifer isodontus (55,7%), Diplaegidia columbae (73,2%), Amblyomma sp. (5,1), Columbicola baculoides (64,7%), Bonomiella sp. (8,5%), Hohorstiella sp. (4,7%) y ácaros de la familia Trombiculidae (6,8%). Ácaros de la familia Trombiculidae, el cestodo K. delafondi y el protozoo E. labbeana habían sido registrados previamente en Chile. Los otros nueve parásitos representan el primer registro para tórtolas en Chile

* Departamento Ciencias Pecuarias, Facultad de Medicina Veterinaria, Universidad de Concepcion, Casilla 537,Chillan, Chile.** Instituto de Parasitologia, Escuela de Med. Veterinaria, Universidad de Leipzig, Leipzig, an der Tierklinik 33,D-04103 Leipzig, Alemania.*** Instituto de Investigaciones Silvestres, Escuela de Med Veterinaria de Hannover, Bunteweg 17, D-30559,Hannover, Alemania.**** Museo Nacional de Historia Natural de Turinguen, An der Brucke 3, D-07407 Rudolstadt, Alemania.DANIEL GONZALEZ*, ARWID DAUGSCHIES**, KLAUS POHLMEYER***, LUIS RUBILAR*,OSCAR SKEWES * y EBERHARD MEY****

In order to evaluate the prevalence of Isospora suis in conventional piglet production in Germany, pooled faecal samples from 327 pig litters from 18 pig production units (20-320 sows each) were examined. At least 10 litters from each farm were investigated. I. suis was present on 83% of the farms and 42.5% of the litters, the infection rate being highest in the third week of age (48.2%). I. suis was found more frequently in samples of diarrhoea than in firm faeces (49.2% compared to 22.2%). Twenty naturally infected piglets from six of these farms underwent examination post mortem, including histology, virology and bacteriology. Histological examination revealed atrophy of the villi in various degrees, mild crypt hyperplasia, fusion of the villi, metaplastic epithelium, erosions and necrosis, especially in the medium and the posterior jejunum and in the ileum. Asexual and sexual developmental stages of the parasite were found in varying numbers in the epithelium of the whole of the small intestine. Bacteria and viruses were mostly excluded as the cause of diarrhoea, and it was concluded that I. suis was the primary pathogen inducing distinct changes and clinical symptoms of diarrhoea.

Angiostrongylus (A.) vasorum is a nematode that causes angiostrongylosis in domestic and wild canids. Red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) are suspected of providing a wildlife reservoir for A. vasorum infections in pet dogs. To obtain data on the occurrence of A. vasorum in wildlife, red fox and raccoon dog carcasses (hunted or found dead) were collected from January to September 2009 in the Federal State of Brandenburg, Germany. Lung tissue samples were subjected to DNA extraction and examined for the presence of A. vasorum DNA by means of real-time PCR. A. vasorum DNA was detected in 11 out of 122 (9.0 %) lungs of red foxes and in none of the lung samples of raccoon dogs. These data suggest that red foxes are a reservoir of A. vasorum infections for pet dogs in this area.

Filarial infections of dogs are attracting attention across Europe because of the risk of spread into previously non-endemic areas (e.g. Dirofilaria repens with Culicidae as vectors) and as emerging zoonotic agents. The occurrence of filarial infections in German dogs has been analysed based on 8,545 samples collected either from imported animals or following travel into endemic regions. All samples were tested by means of modified Knott's test and heartworm antigen assay within the period 2008 - 2010. Heartworm antigen was detected in 127 samples (1.49 %; 95 % CI: 1.25 - 1.77 %), but only 38 dogs also had microfilariae in their blood samples. On the other hand, 125 animals (1.46 %; 95 % CI: 1.23 - 1.74 %) were only positive in the Knott's test. For discrimination by means of PCR and sequencing a total of 73 blood samples as well as two samples of adult worms were included, which have been sent by veterinarians during 2008 - 2010. A mono-infection caused by D. repens was detected in 35 cases, while D. immitis was proven in 15 samples, with 6 of these showing a combination of D. immitis and D. repens. Imported Dipetalonema dracunculoides (transmitted by Rhipicephalus sanguineus or Hippobosca longipennis) or Acanthocheilonema reconditum (fleas and lice serve as intermediate hosts) infections were diagnosed in 24 cases and in a single sample a co-infection of A. reconditum and D. repens was evident. D. repens was the most common filarial infection imported and it was introduced into Germany from eleven European countries. Slovenia and Hungary are reported for the first time as endemic for D. repens and A. reconditum, respectively. Furthermore this study reports, to the best of our knowledge, for the first time import of D. dracunculoides from the Canary Islands, A. reconditum from Majorca, D. immitis from Corfu and a co-infection of D. repens and A. reconditum from Spain as well as mixed infections of D. repens and D. immitis from Corfu, Sardinia and Bulgaria. Co-infections with other arthropod-borne infections as well as therapeutical follow-up were also considered. Selamectin (as spot-on formulation) was not able to clear microfilaraemia in dogs infected with either D. repens, A. reconditum or D. dracunculoides, whereas a topical moxidectin/imidacloprid formulation was able to eliminate microfilariae in one dog infected with A. reconditum.