In vitro assay to determine inactivation of Toxoplasma gondii in meat samples.

Consumption of raw and undercooked meat is considered as an important source of Toxoplasma gondii infections. However, most non-heated meat products contain salt and additives, which affect T. gondii viability. It was our aim to develop an in vitro method to substitute the mouse bioassay for determining the effect of salting on T. gondii viability. Two sheep were experimentally infected by oral inoculation with 6.5 × 104 oocysts. Grinded meat samples of 50 g were prepared from heart, diaphragm, and four meat cuts. Also, pooled meat samples were either kept untreated (positive control), frozen (negative control) or supplemented with 0.6 %, 0.9 %, 1.2 % or 2.7 % NaCl. All samples were digested in pepsin-HCl solution, and digests were inoculated in duplicate onto monolayers of RK13 (a rabbit kidney cell line). Cells were maintained for up to four weeks and parasite growth was monitored by assessing Cq-values using the T. gondii qPCR on cell culture supernatant in intervals of one week and ΔCq-values determined. Additionally, 500 µL of each digest from the individual meat cuts, heart and diaphragm were inoculated in duplicate in IFNγ KO mice. Both sheep developed an antibody response and tissue samples contained similar concentrations of T. gondii DNA. From all untreated meat samples positive ΔCq-values were obtained in the in vitro assay, indicating presence and multiplication of viable parasites. This was in line with the mouse bioassay, with the exception of a negative mouse bioassay on one heart sample. Samples supplemented with 0.6 %-1.2 % NaCl showed positive ΔCq-values over time. The frozen sample and the sample supplemented with 2.7 % NaCl remained qPCR positive but with high Cq-values, which indicated no growth. In conclusion, the in vitro method has successfully been used to detect viable T. gondii in tissues of experimentally infected sheep, and a clear difference in T. gondii viability was observed between the samples supplemented with 2.7 % NaCl and those with 1.2 % NaCl or less.

Drivers of infection with Toxoplasma gondii genotype type II in Eurasian red squirrels (Sciurus vulgaris).

BACKGROUND: In September 2014, there was sudden upsurge in the number of Eurasian red squirrels (Sciurus vulgaris) found dead in the Netherlands. High infection levels with the parasite Toxoplasma gondii were demonstrated, but it was unclear what had caused this increase in cases of fatal toxoplasmosis. In the present study, we aimed to gain more knowledge on the pathology and prevalence of T. gondii infections in Eurasian red squirrels in the Netherlands, on the T. gondii genotypes present, and on the determinants of the spatiotemporal variability in these T. gondii infections. The presence of the closely related parasite Hammondia hammondi was also determined. METHODS: Eurasian red squirrels that were found dead in the wild or that had died in wildlife rescue centres in the Netherlands over a period of seven years (2014-2020) were examined. Quantitative real-time polymerase chain reaction was conducted to analyse tissue samples for the presence of T. gondii and H. hammondi DNA. Toxoplasma gondii-positive samples were subjected to microsatellite typing and cluster analysis. A mixed logistic regression was used to identify climatic and other environmental predictors of T. gondii infection in the squirrels. RESULTS: A total of 178 squirrels were examined (49/178 T. gondii positive, 5/178 H. hammondi positive). Inflammation of multiple organs was the cause of death in 29 squirrels, of which 24 were also T. gondii polymerase chain reaction positive. Toxoplasma gondii infection was positively associated with pneumonia and hepatitis. Microsatellite typing revealed only T. gondii type II alleles. Toxoplasma gondii infection rates showed a positive correlation with the number of days of heavy rainfall in the previous 12 months. Conversely, they showed a negative association with the number of hot days within the 2-week period preceding the sampling date, as well as with the percentage of deciduous forest cover at the sampling site. CONCLUSIONS: Toxoplasma gondii infection in the squirrels appeared to pose a significant risk of acute mortality. The T. gondii genotype detected in this study is commonly found across Europe. The reasons for the unusually high infection rates and severe symptoms of these squirrels from the Netherlands remain unclear. The prevalence of T. gondii in the deceased squirrels was linked to specific environmental factors. However, whether the increase in the number of dead squirrels indicated a higher environmental contamination with T. gondii oocysts has yet to be established.

Reduced neural progenitor cell count and cortical neurogenesis in guinea pigs congenitally infected with Toxoplasma gondii.

Toxoplasma (T.) gondii is an obligate intracellular parasite with a worldwide distribution. Congenital infection can lead to severe pathological alterations in the brain. To examine the effects of toxoplasmosis in the fetal brain, pregnant guinea pigs are infected with T. gondii oocysts on gestation day 23 and dissected 10, 17 and 25 days afterwards. We show the neocortex to represent a target region of T. gondii and the parasite to infect neural progenitor cells (NPCs), neurons and astrocytes in the fetal brain. Importantly, we observe a significant reduction in neuron number at end-neurogenesis and find a marked reduction in NPC count, indicating that impaired neurogenesis underlies the neuronal decrease in infected fetuses. Moreover, we observe focal microglioses to be associated with T. gondii in the fetal brain. Our findings expand the understanding of the pathophysiology of congenital toxoplasmosis, especially contributing to the development of cortical malformations.

Detection of Toxoplasma gondii-specific antibodies in pigs using an oral fluid-based commercial ELISA: Advantages and limitations.

Toxoplasma gondii is a major food-borne parasite and undercooked meat of infected pigs represents an important source of infection for humans. Since infections in pigs are mostly subclinical, adequate diagnostic tests for use at the farm level are pursued. Oral fluid (OF) was shown to be a promising matrix for direct and indirect detection of infections with various pathogens in pigs. The objective of this study was to assess whether T. gondii infections in pigs could be diagnosed using an indirect ELISA kit adapted for OF samples (OF-ELISA). Routine serology and OF-immunoblot (IB) were used as standards for the comparison. For this, serial OF samples from sows (n = 8) and fatteners (n = 3) experimentally inoculated with T. gondii oocysts, individual field samples from potentially exposed sows (n = 9) and pooled OF samples from potentially exposed group-housed fatteners (n = 195 pig groups, including 2,248 animals) were analysed for antibodies against T. gondii by ELISA. For individual animals, OF-ELISA exhibited a relative diagnostic specificity of 97.3% and a relative diagnostic sensitivity of 78.8%. In experimentally infected animals, positive OF-ELISA results were observed from 1.5 weeks post inoculation (pi) until the end of the experimental setup (8 to 30 weeks pi); however, values below the estimated cut-off were occasionally observed in some animals despite constant seropositivity. In potentially exposed individual animals, OF- and serum-ELISA results showed 100% agreement. In group-housed fatteners, antibodies against T. gondii could be reliably detected by OF-ELISA in groups in which at least 25% of the animals were seropositive. This OF-ELISA, based on a commercially available serum-ELISA, may represent an interesting non-invasive screening tool for detecting pig groups with a high exposure to T. gondii at the farm level. The OF-ELISA may need further adjustments to consistently detect individual infected pigs, probably due to variations in OF antibody concentration over time.

Systematic Review and Modelling of Age-Dependent Prevalence of Toxoplasma gondii in Livestock, Wildlife and Felids in Europe.

Toxoplasma gondii is a zoonotic parasite of importance to both human and animal health. The parasite has various transmission routes, and the meat of infected animals appears to be a major source of human infections in Europe. We aimed to estimate T. gondii prevalence in a selection of animal host species. A systematic literature review resulting in 226 eligible publications was carried out, and serological data were analyzed using an age-dependent Bayesian hierarchical model to obtain estimates for the regional T. gondii seroprevalence in livestock, wildlife, and felids. Prevalence estimates varied between species, regions, indoor/outdoor rearing, and types of detection methods applied. The lowest estimated seroprevalence was observed for indoor-kept lagomorphs at 4.8% (95% CI: 1.8-7.5%) and the highest for outdoor-kept sheep at 63.3% (95% CI: 53.0-79.3%). Overall, T. gondii seroprevalence estimates were highest within Eastern Europe, whilst being lowest in Northern Europe. Prevalence data based on direct detection methods were scarce and were not modelled but rather directly summarized by species. The outcomes of the meta-analysis can be used to extrapolate data to areas with a lack of data and provide valuable inputs for future source attribution approaches aiming to estimate the relative contribution of different sources of T. gondii human infection.

Toxoplasma gondii and Alternaria sp.: An Original Association in an Immunosuppressed Dog with Persistent Skin Lesions.

Dogs and cats may suffer from a variety of diseases, mainly immune mediated, that require the administration of immunosuppressive drugs. Such therapies can cause adverse effects either by the toxicity of the drugs or as a consequence of immune suppression and associated opportunistic infections. Here we present an, yet unknown, association of Toxoplasma gondii and Alternaria fungus, within cutaneous lesions in a dog under long-term immunosuppressive therapy. The diagnosis of such infections is laborious and not obvious at first glance, since the clinical signs of cutaneous toxoplasmosis, neosporosis or alternariosis are not specific. A further laboratory confirmation is needed. Therefore, we currently recommend that dogs and cats should undergo serologic testing for toxoplasmosis or neosporosis prior to immunosuppressive therapy and a regular dermatological evaluation during the immunosuppressive therapy.

Prevalence and molecular characterization of Toxoplasma gondii in different types of poultry in Greece, associated risk factors and co-existence with Eimeria spp.

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.

Fulminant Pneumonia Due to Reactivation of Latent Toxoplasmosis in a Cat-A Case Report.

Toxoplasma (T.) gondii is an obligate intracellular parasite with felids, including domestic cats, as definitive hosts. In immunocompetent individuals, T. gondii infection is usually asymptomatic. However, under immunosuppression, it may have severe pathological impacts, which often result from the reactivation of a chronic infection. In this case study, a 21-month-old female domestic shorthair cat-diagnosed with primary immune-mediated hemolytic anemia three months prior and treated with cyclosporine and prednisolone-presented with acute tachypnea, dyspnea, diarrhea, and anorexia. Thoracic radiography suggested severe pneumonia. Testing for Mycoplasma spp., Anaplasma spp., Ehrlichia spp., and lungworm infection was negative. Serology for T. gondii revealed seroconversion of IgG, but not of IgM, indicating previous exposure to T. gondii. The cat remained stable but tachypneic for three days, followed by an acute onset of dyspnea and clinical deterioration, after which euthanasia was elected. Numerous protozoa were present in a postmortem transtracheal bronchoalveolar lavage and fine-needle aspiration of the lung. Microsatellite typing classified the extracted DNA as T. gondii type II variant TgM-A. This case demonstrates that T. gondii reactivation, leading to fulminant pneumonia, can be a sequela of immunosuppressive treatment in cats and should, therefore, be considered as a differential diagnosis in immunosuppressed cats with acute-onset respiratory signs. Rapid diagnosis may prevent fatal consequences.

Validation of a commercial version of a competitive enzyme linked immunosorbent assay for the detection of antibodies to Besnoitia besnoiti.

BACKGROUND: Several reports suggest a further spread of besnoitiosis to countries in which Besnoitia besnoiti-infected bovine herds have not been noticed yet. Cattle infected without clinical signs may represent reservoirs. Serological analyses in affected herds or animals from endemic regions are necessary to identify subclinical or inapparent infections and stop transmission to naïve animals or herds. The Monoscreen AbELISA Besnoitia besnoiti (BIO K 466) is based on a previously published in-house competitive ELISA, the Bb-cELISA1, but has a different test architecture. The present study aimed to use sera from a previous evaluation of Bb-cELISA1 to assess whether BIO K466 shows identical results. In addition, further well-characterized positive and negative samples were analysed to estimate diagnostic sensitivity and specificity. METHODS: A first set of sera consisted of a total of 305 bovine sera, collected from German herds infected by B. besnoiti, Neospora caninum or Sarcocystis spp. Sera had been characterized by reference serological tests (i.e. immunoblot, immunofluorescence antibody test and an in-house indirect ELISA). A second set consisted of 200 confirmed B. besnoiti-positive sera from French herds. Negative cattle sera (n = 624) originated from Norway and The Netherlands, countries in which bovine besnoitiosis has not been reported yet. RESULTS: Using the first set of sera, the BIO K466 showed an estimated diagnostic sensitivity of 97.9% (95% CI: 91.9%-99.6) and a diagnostic specificity of 99.5% (95% CI: 96.9%-100%) relative to reference serological tests. A direct comparison of the results revealed an almost perfect agreement between the results of the in-house Bb-cELISA1 and the commercialized version (kappa 0.98; 95% CI: 0.95-1). The validation using positive bovine sera from France and negative sera from other European countries revealed a diagnostic sensitivity of 97.5% (95% CI: 93.9%-99.1%) and specificity of 99.5% (95% CI: 98.5%-99.9%). CONCLUSION: In conclusion, BIO K 466 appears to be a suitable tool to diagnose bovine besnoitiosis, but needs further validation especially in cases of inconclusive, suspected false-positive or -negative results in other serological tests.

Switzerland-wide Neospora caninum seroprevalence in female cattle and identification of risk factors for infection.

Introduction: Neospora caninum is an important cause of abortion in cattle worldwide. Infection in cattle occurs horizontally by ingestion of oocysts shed by canids or vertically, from an infected dam to the fetus, and may result in abortion, stillbirth, or birth of seropositive offspring. The control of bovine neosporosis is difficult and costly. The objectives of this study were to estimate the current nationwide seroprevalence of N. caninum infections in Swiss cattle and to assess risk factors for infection with this parasite. Methods: We conducted a cross-sectional study with cattle farms randomly selected and stratified according to population size, resulting in a sample of 780 female cattle. The cattle originated from 161 farms distributed over all Switzerland. The serum samples were tested for antibodies against N. caninum using a commercial ELISA and if inconclusive, retested using an in-house immunoblot technique. To collect farm parameters relevant to N. caninum transmission and prevention, farm owners were mailed a questionnaire which addressed topics putatively related to N. caninum infection such as husbandry, history of abortion, and presence of dogs on farm. Univariate analysis by generalized linear mixed model (with animal seropositivity as outcome variable) and logistic regression modeling (with farm seropositivity as outcome variable) was conducted on farm parameters investigated in the questionnaire. Results: By ELISA and immunoblot, 4.2% (33/780) of cattle sera yielded positive results. At the farm level, 16.2% (26/161) of the sampled farms had at least one seropositive animal. The return rate of the valid questionnaires was 54.0%. At the animal level, odds for farm seropositivity were 3.8 times higher when rodents had been recorded by the farmer as a problem on the farm. At the farm-level, two protective factors were identified: rearing of replacement heifers and feeding of concentrated feed. Conclusion: We recorded a low seroprevalence of N. caninum in a random sample of Swiss cattle representative for the years 2017-2018. Based on a questionnaire survey, we could identify risk and protective factors for infection with N. caninum, however their biological relevance needs to be confirmed in further studies.

Immunomagnetic separation of Toxoplasma gondii and Hammondia spp. tissue cysts generated in cell culture.

Toxoplasma gondii is commonly transmitted among animals and humans by ingestion of infected animal tissues or by consumption of food and water contaminated with environmentally-resistant oocysts excreted by cats. Tissue cysts and oocysts have different walls, whose structures and compositions are poorly known. Herein, we describe an immunomagnetic separation (IMS) method that was successfully used for purification of T. gondii tissue cysts generated in cell culture. We used an IgG monoclonal antibody (mAb) that reacts against antigens in tissue cyst walls. Many in vitro produced cysts were obtained by this IMS; >2,000 T. gondii cysts were isolated from a single culture flask of 25 cm2. Tissue cysts from two Hammondia spp., H. hammondi, and H. heydorni, produced in cell culture were also separated using this method. As a reference, purification of tissue cysts by Percoll gradients was used. Percoll was able to separate T. gondii tissue cysts produced in mice but was not suitable for purifying T. gondii tissue cysts produced in vitro. The IMS described here should favor proteomic studies involving tissue cysts of T. gondii.

Seroprevalence and Risk Factors for Toxoplasma gondii and Neospora caninum in Cattle in Portugal.

Neospora caninum has a worldwide economic impact as an important cause of abortion in cattle, while Toxoplasma gondii, another abortifacient pathogen, is globally a major foodborne zoonotic threat. The study aimed to evaluate the seroprevalence and risk factors for the two parasites in cattle in Portugal. A total of 612 sera from 35 farms were tested by an in-house p30 ELISA for T. gondii and p38 ELISA for N. caninum. T. gondii positive and suspicious sera were confirmed by p30 Western blot or IFAT. T. gondii and N. caninum animal seroprevalence was 9.2% (95%CI 7.1-11.7) and 17.2% (95% CI 14.4-20.4) and herd seroprevalence was 51.4% (95% CI 35.6-67.0) and 68.6% (95% CI 52.0-81.5), respectively. At the univariable level, climate area and precipitation of wettest month, driest month, driest quarter, and warmest quarter were significant predictors of seropositivity for both. N. caninum seropositivity was more likely in the region Norte, densely populated areas, and intensive production, and the probability of T. gondii seropositivity decreased with herd size. Results confirm the need to consider neosporosis in the differential diagnosis of cattle reproductive disorders in Portugal and may be valuable to inform source attribution models for human toxoplasmosis.

Toxoplasma gondii and Neospora caninum infections in sheep and goats in Switzerland: Seroprevalence and occurrence in aborted foetuses.

Toxoplasma gondii and Neospora caninum infections are important causes of abortion in ruminants. Besides, meat from T. gondii infected animals represent a major infection source for humans. The occurrence of these protozoan parasites in Switzerland was investigated both, in a nationwide cross-sectional serological survey, and by molecular methods in aborted sheep and goat foetuses. A total of 653 sheep from 143 farms and 748 goats from 164 farms were tested by commercial ELISAs and inconclusive results were defined by immunoblot. Besides, a risk factor analysis for seropositivity was performed. The observed seroprevalences for T. gondii in sheep and goats were 66.3% and 50.5% at the animal level, and 90.9% and 81.1% at the farm level, respectively. For N. caninum, the detected seroprevalences in sheep and goats were 0.8% and 0.9% at the animal level, and 2.8% and 1.8% at the farm level, respectively. Older small ruminants, and sheep (vs. goats) had a higher risk of being seropositive to T. gondii. Alpine grazing in summer was identified as a protective factor for seropositivity to T. gondii in both animal species. Toxoplasma gondii and N. caninum DNA were detected in 6.1% and 2.4% (n = 82), and in 6.8% and 1.4% (n = 73) of the tested ovine and caprine foetuses, respectively. These results suggest the involvement of these parasites in abortions and reveal a high prevalence of T. gondii and lower prevalence of N. caninum infections in small ruminants in Switzerland. They also suggest that consumption of undercooked meat from T. gondii infected sheep and goats may represent a risk for public health.

Clustering of Cryptosporidium species infections among sheep and cattle but not children in remote highland communities of Madagascar.

BACKGROUND: The aim of this study was to identify local transmission patterns of Cryptosporidium spp. infections among livestock and humans in four extremely rural and remote highland communities in Madagascar. METHODS: In this cross-sectional study, households were randomly sampled throughout a 1-year study period, with one feces sample collected from each child (≤ 5 years old), sheep and cattle. Cryptosporidium spp. were identified using a nested PCR assay targeting the 18S ribosomal RNA gene. All samples positive for Cryptosporidium hominis were further subtyped by sequencing the 60-kDa glycoprotein gene (gp60). Spatial clustering methods were applied to analyze potential transmission patterns. RESULTS: In total, 252 households participated in the study, and samples from 197 children, 862 cattle and 334 sheep were collected and included in the study. Of the samples collected, 11 (5.6%) from children, 30 (3.5%) from cattle and 42 (12.6%) from sheep tested positive for Cryptosporidium spp. Very little overlap in the species distribution between human and animal infections was found. Global (overall) and local (spatially defined) clustering was observed for Cryptosporidium spp. infections in sheep and for Cryptosporidium xiaoi/bovis infections among sheep and cattle. DISCUSSION: The results of this analysis do not support the occurrence of defined disease outbreaks, rather they point to a continuous series of transmission events that are spatially aggregated. Despite the close coexistence between humans, sheep and cattle in the study area, mutual transmission was not observed. Hence, the study underlines the importance of sustained sanitation and hygiene measures to prevent cryptosporidiosis transmission among infants, since asymptomatic children serve as an infection reservoir. Similarly, the study highlights the importance of improving hygiene to reduce the transmission of Cryptosporidium spp. in livestock, an infection with serious consequences, especially in newborn calves.

First Expert Elicitation of Knowledge on Drivers of Emergence of Bovine Besnoitiosis in Europe.

Bovine besnoitiosis (BB) is a chronic and debilitating parasitic disease in cattle caused by the protozoan parasite Besnoitia besnoiti. South European countries are affected and have reported clinical cases of BB. However, BB is considered as emerging in other countries/regions of central, eastern and northern Europe. Yet, data on drivers of emergence of BB in Europe are scarce. In this study, fifty possible drivers of emergence of BB in cattle were identified. A scoring system was developed per driver. Then, the scoring was elicited from eleven recognized European experts to: (i) allocate a score to each driver, (ii) weight the score of drivers within each domain and (iii) weight the different domains among themselves. An overall weighted score was calculated per driver, and drivers were ranked in decreasing order of importance. Regression tree analysis was used to group drivers with comparable likelihoods to play a role in the emergence of BB in cattle in Europe. Finally, robustness testing of expert elicitation was performed for the seven drivers having the highest probability to play a key role in the emergence of BB: i.e., (i) legal/illegal movements of live animals from neighbouring/European Union member states or (ii) from third countries, (iii) risk of showing no clinical sign and silent spread during infection and post infection, (iv) as a consequence, difficulty to detect the emergence, (v) existence of vectors and their potential spread, (vi) European geographical proximity of the pathogen/disease to the country, and (vii) animal density of farms. Provided the limited scientific knowledge on the topic, expert elicitation of knowledge, multi-criteria decision analysis, cluster and sensitivity analyses are very important to prioritize future studies, e.g., the need for quantitative import risk assessment and estimation of the burden of BB to evidence and influence policymaking towards changing (or not) its status as a reportable disease, with prevention and control activities targeting, firstly, the top seven drivers. The present methodology could be applied to other emerging animal diseases.

Toxoplasma gondii Genotyping: A Closer Look Into Europe.

Toxoplasma gondii is a major zoonotic agent which may cause harmful effects mainly in pregnant and immunocompromised hosts. Despite many efforts on its genetic characterization, an entirely clear picture of the population structure in Europe has not been achieved yet. The present study aimed to summarize the available genotyping information and to map the distribution of circulating strains. There is consensus on type II T. gondii genotypes prevailing in Europe, but the absence of harmonization in the use of typing methods limits detailed knowledge. Standardized, high-end typing tools and integrative strategies are needed to fill the gaps and complete an accurate image of the T. gondii genetic population in Europe.

Prevalence, risk factor and diversity of Cryptosporidium in cattle in Latvia.

The epidemiology of Cryptosporidium spp. in Latvia was investigated by testing fecal samples from 926 animals aged from one day to 24 years for the presence of Cryptosporidium spp. oocysts. The samples were collected from 87 cattle farms and from four slaughterhouses, and analyzed by conventional and fluorescent microscopy, followed by Cryptosporidium species and C. parvum subtype differentiation. Moreover, using a questionnaire, we surveyed factors that could be relevant as risk factors of Cryptosporidium spp. infection on the farms. Cryptosporidium spp. were shed by 33.8% of the investigated cattle and at least one shedding animal was found on 77.8% of the farms. In the present study, all four Cryptosporidium species reported to commonly infect cattle and two additional Cryptosporidium species (C. scrofarum and C. ubiquitum) were identified. In addition, mix infections of C. parvum/C. bovis, C. bovis/C. ryanae, C. parvum/C. ryanae, C. parvum/C. andersoni and C. bovis/C. andersoni were observed. C. parvum and C. bovis was mostly prevalent in young animals (0-3 months old) and in addition, diarrhea associated with C. parvum infection was observed only in very young animals. Cryptosporidium andersoni and C. ryanae in age group 0-3 months was observed in low prevalence, while a higher proportion of animals with diarrhea associated with C. andersoni infection was observed in very young animals and with C. ryanae in animals age group 4-24 months. Eight previously described C. parvum subtypes were observed. The majority of the subtypes were in the IIa subtype family, while one subtype was identified from the IId subtype family. The most common subtype was IIaA15G2R1, which was found in 34.2% of the C. parvum successfully subtyped samples. The probability of Cryptosporidium spp. associated diarrhea in cattle decreased significantly with the age of the animals and a prolonged period during which calves were fed with milk.

New Insights into Gastrointestinal and Pulmonary Parasitofauna of Wild Eurasian lynx (Lynx lynx) in the Harz Mountains of Germany.

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

Establishment and validation of a guinea pig model for human congenital toxoplasmosis.

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.

Comparison of Direct and Indirect Toxoplasma gondii Detection and Genotyping in Game: Relationship and Challenges.

The importance of game as a source of Toxoplasma gondii (T. gondii) infection in humans is largely unknown. New data on the presence of T. gondii in game hunted in the Federal State of Brandenburg, Germany, were obtained by direct and indirect detection (ELISA). DNA extracted either directly (5 g heart or foreleg muscle, DE) or after acid pepsin digestion (50 g heart, PD) or enriched by magnetic capture (50 g heart, MC) was examined by real-time PCR (qPCR). ELISA revealed seroprevalences of 20% in wild boar (Sus scrofa), 11% in roe deer (Capreolus capreolus) and 6% in red deer (Cervus elaphus). T. gondii DNA was detected by at least one direct detection method in 12% of wild boar, 6% of roe deer, 2% of fallow deer (Dama dama) and 2% of red deer. In both, positive wild boar and roe deer, T. gondii type II specific alleles were the most prevalent, as assessed by PCR-restriction fragment length polymorphism. The highest proportion of positive animals was detected by MC qPCR, followed by PD qPCR with a similar proportion of positive findings. Investigation of 50 g of heart muscle revealed a significantly higher proportion of positive qPCR results than analysis of 5 g (p = 0.048). An association between seropositivity and direct detection was evident in wild boar and roe deer (p < 0.001). Infectivity of T. gondii DNA-positive samples was confirmed by bioassay (4/4), providing evidence that game could represent a relevant source of viable T. gondii posing a risk for human infection.