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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.

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

Review The neglected zoonosis cystic echinococcosis affects mainly pastoral and rural communities in both low-income and upper-middle-income countries. In Europe, it should be regarded as an orphan and rare disease. Although human cystic echinococcosis is a notifiable parasitic infectious disease in most European countries, in practice it is largely under-reported by national health systems. To fill this gap, we extracted data on the number, incidence, and trend of human cases in Europe through a systematic review approach, using both the scientific and grey literature and accounting for the period of publication from 1997 to 2021. The highest number of possible human cases at the national level was calculated from various data sources to generate a descriptive model of human cystic echinococcosis in Europe. We identified 64 745 human cystic echinococcosis cases from 40 European countries. The mean annual incidence from 1997 to 2020 throughout Europe was 0·64 cases per 100 000 people and in EU member states was 0·50 cases per 100 000 people. Based on incidence rates and trends detected in this study, the current epicentre of cystic echinococcosis in Europe is in the southeastern European countries, whereas historical endemic European Mediterranean countries have recorded a decrease in the number of cases over the time. Key messages: - This study aims to shed light on the unrecognised incidence of cystic echinococcosis in Europe, unveiling its epidemiological effect by providing a quantitative measure of number, incidence, and trends of human cases documented within the period 1997–2021; - Since human cystic echinococcosis cases are generally under-reported and data have uncertainty (partly due to misdiagnosis), data provided in this study should be considered as a conservative estimate of the real impact of this zoonotic infection historically occurring in Europe; - For the years 2017–19, we identified a total number of cystic echinococcosis cases four-fold higher than for The European Surveillance System (TESSy) data; - Decreasing trends have been recorded in most southern Mediterranean and some eastern European countries, where cystic echinococcosis has traditionally been highly prevalent; - Increasing trends have been identified in some eastern and southeastern European countries but, unexpectedly, also in most non-endemic countries of northern and western Europe; - Based on incidence and trends from 2017–19, the current epicentre of cystic echinococcosis in Europe is represented by the Balkan Peninsula; - Cystic echinococcosis in Europe remains a relevant public health issue and findings from this study should be used to support the planning of surveillance and control programmes in Europe according to the WHO 2021–2030 roadmap for neglected tropical diseases. This research was funded by the MEmE project from the EU’s Horizon 2020 Research and Innovation programme under grant agreement number 773830: One Health European Joint Programme. This work was also partially supported by research funding (grant PRG1209) from the Estonian Ministry of Education and Research info:eu-repo/semantics/publishedVersion

Background Alveolar echinococcosis (AE) is a clinically serious zoonosis caused by the fox tapeworm Echinococcus multilocularis. We studied the diversity and the distribution of genotypes of E. multilocularis isolated from foxes in Brandenburg, Germany, and in comparison to a hunting ground in North Rhine-Westphalia. Methods Echinococcus multilocularis specimens from 101 foxes, 91 derived from Brandenburg and 10 derived from North Rhine-Westphalia, were examined. To detect potential mixed infections with different genotypes of E. multilocularis, five worms per fox were analyzed. For genotyping, three mitochondrial markers, namely cytochrome c oxidase subunit 1 (Cox1), NADH dehydrogenase subunit 1 (Nad1), and ATP synthase subunit 6 (ATP6), and the nuclear microsatellite marker EmsB were used. To identify nucleotide polymorphisms, the mitochondrial markers were sequenced and the data were compared, including with published sequences from other regions. EmsB fragment length profiles were determined and confirmed by Kohonen network analysis and grouping of Sammon’s nonlinear mapping with k-means clustering. The spatial distribution of genotypes was analyzed by SaTScan for the EmsB profiles found in Brandenburg. Results With both the mitochondrial makers and the EmsB microsatellite fragment length profile analyses, mixed infections with different E. multilocularis genotypes were detected in foxes from Brandenburg and North Rhine-Westphalia. Genotyping using the mitochondrial markers showed that the examined parasite specimens belong to the European haplotype of E. multilocularis, but a detailed spatial analysis was not possible due to the limited heterogeneity of these markers in the parasite population. Four (D, E, G, and H) out of the five EmsB profiles described in Europe so far were detected in the samples from Brandenburg and North Rhine-Westphalia. The EmsB profile G was the most common. A spatial cluster of the E. multilocularis genotype with the EmsB profile G was found in northeastern Brandenburg, and a cluster of profile D was found in southern parts of this state. Conclusions Genotyping of E. multilocularis showed that individual foxes may harbor different genotypes of the parasite. EmsB profiles allowed the identification of spatial clusters, which may help in understanding the distribution and spread of the infection in wildlife, and in relatively small endemic areas. Graphical Abstract

Background: Besnoitia darlingi, B. neotomofelis and B. oryctofelisi are closely related coccidian parasites with felids as definitive hosts. These parasites use a variety of animal species as intermediate hosts. North American opossums (Didelphis virginiana), North American southern plains woodrats (Neotoma micropus) and South American domestic rabbits (Oryctolagus cuniculus) are intermediate hosts of B. darlingi, B. neotomofelis and B. oryctofelisi, respectively. Based on conserved regions in the Internal Transcribed Spacer-1 (ITS-1) sequence of the ribosomal DNA (rDNA), a real-time PCR for a sensitive detection of these Besnoitia spp. in tissues of intermediate hosts and faeces of definitive hosts has recently been established. Available sequence data suggest that species such as B. akodoni and B. jellisoni are also covered by this real-time PCR. It has been hypothesised that additional Besnoitia spp. exist worldwide, which are closely related to B. darlingi or B. darlingi-like parasites (B. neotomofelis, B. oryctofelisi, B. akodoni or B. jellisoni). Related but not closely related to these species is B. besnoiti, the cause of bovine besnoitiosis.Methods: Faecal samples from two free-ranging cheetahs (Acinonyx jubatus) from Namibia that had previously tested positive for coccidian parasites by coproscopy, were used for this study. A conventional PCR verified the presence of coccidian parasite DNA. To clarify the identity of these coccidia, the faecal DNA samples were further characterised by species-specific PCRs and Sanger sequencing.Results: One of the samples tested positive for B. darlingi or B. darlingi-like parasites by real-time PCR, while no other coccidian parasites including Toxoplasma gondii, Hammondia hammondi, H. heydorni, B. besnoiti, and Neospora caninum were detected in the two samples. The rDNA of the B. darlingi-like parasite was amplified and partially sequenced. Comparison with existing sequences in GenBank revealed a close relationship to other Besnoitia spp., but showed also clear divergences. Conclusions: Our results suggest that a so far unknown Besnoitia species exists in Namibian wildlife, which is closely related to B. darlingi, B. neotomofelis, B. oryctofelisi, B. akodoni or B. jellisoni. The cheetah appears to be the definitive host of this newly discovered parasite, while a prey species of the cheetah may act as intermediate hosts.

Introduction Hammondia hammondi and Toxoplasma gondii are closely related protozoan parasites, but only T. gondii is zoonotic. Both species use felids as definitive hosts and cannot be differentiated by oocyst morphology. In T. gondii, a 529-base pair (bp) repetitive element (TgREP-529) is of utmost diagnostic importance for polymerase chain reaction (PCR) diagnostic tests. We identified a similar repetitive region in the H. hammondi genome (HhamREP-529). Methods Based on reported sequences, primers and probes were selected in silico and optimal primer probe combinations were explored, also by including previously published primers. The analytical sensitivity was tested using serial dilutions of oocyst DNA. For testing analytical specificity, DNA isolated from several related species was used as controls. The newly established TaqMan PCR (Hham-qPCR1) was applied to tissues collected from H. hammondi-infected gamma-interferon gene knockout (GKO) mice at varying time points post-infection. Results Ten forward and six reverse primers were tested in varying combinations. Four potentially suitable dual-labelled probes were selected. One set based on the primer pair (Hham275F, Hham81R) and the probe (Hham222P) yielded optimal results. In addition to excellent analytic specificity, the assay revealed an analytical sensitivity of genome equivalents of less than one oocyst. Investigation of the tissue distribution in GKO mice revealed the presence of parasite DNA in all examined organs, but to a varying extent, suggesting 100- to 10,000-fold differences in parasitic loads between tissues in the chronic state of infection, 42 days post-infection. Discussion The use of the 529-bp repeat of H. hammondi is suitable for establishing a quantitative real-time PCR assay, because this repeat probably exists about 200 times in the genome of a single organism, like its counterpart in T. gondii. Although there were enough sequence data available, only a few of the primers predicted in silico revealed sufficient amplification; the identification of a suitable probe was also difficult. This is in accord with our previous observations on considerable variability in the 529-bp repetitive element of H. hammondi. Conclusions The H. hammondi real-time PCR represents an important novel diagnostic tool for epidemiological and cell biological studies on H. hammondi and related parasites. Graphical Abstract

Background Besnoitia darlingi, B. neotomofelis and B. oryctofelisi are closely related coccidian parasites with felids as definitive hosts. These parasites use a variety of animal species as intermediate hosts. North American opossums (Didelphis virginiana), North American southern plains woodrats (Neotoma micropus) and South American domestic rabbits (Oryctolagus cuniculus) are intermediate hosts of B. darlingi, B. neotomofelis and B. oryctofelisi, respectively. Based on conserved regions in the internal transcribed spacer-1 (ITS1) sequence of the ribosomal DNA (rDNA), a real-time PCR for a sensitive detection of these Besnoitia spp. in tissues of intermediate hosts and faeces of definitive hosts has recently been established. Available sequence data suggest that species such as B. akodoni and B. jellisoni are also covered by this real-time PCR. It has been hypothesised that additional Besnoitia spp. exist worldwide that are closely related to B. darlingi or B. darlingi-like parasites (B. neotomofelis, B. oryctofelisi, B. akodoni or B. jellisoni). Also related, but not as closely, is B. besnoiti, the cause of bovine besnoitiosis. Methods Faecal samples from two free-ranging cheetahs (Acinonyx jubatus) from Namibia that had previously tested positive for coccidian parasites by coproscopy were used for this study. A conventional PCR verified the presence of coccidian parasite DNA. To clarify the identity of these coccidia, the faecal DNA samples were further characterised by species-specific PCRs and Sanger sequencing. Results One of the samples tested positive for B. darlingi or B. darlingi-like parasites by real-time PCR, while no other coccidian parasites, including Toxoplasma gondii, Hammondia hammondi, H. heydorni, B. besnoiti and Neospora caninum, were detected in the two samples. The rDNA of the B. darlingi-like parasite was amplified and partially sequenced. Comparison with existing sequences in GenBank revealed a close relationship to other Besnoitia spp., but also showed clear divergences. Conclusions Our results suggest that a so far unknown Besnoitia species exists in Namibian wildlife, which is closely related to B. darlingi, B. neotomofelis, B. oryctofelisi, B. akodoni or B. jellisoni. The cheetah appears to be the definitive host of this newly discovered parasite, while prey species of the cheetah may act as intermediate hosts. Graphical Abstract

Background Free-ranging chickens are often infected with Toxoplasma gondii and seroconvert upon infection. This indicates environmental contamination with T. gondii. Methods Here, we established a bead-based multiplex assay (BBMA) using the Luminex technology for the detection of T. gondii infections in chickens. Recombinant biotinylated T. gondii surface antigen 1 (TgSAG1bio) bound to streptavidin-conjugated magnetic Luminex beads served as antigen. Serum antibodies were detected by a fluorophore-coupled secondary antibody. Beads of differing color codes were conjugated with anti-chicken IgY or chicken serum albumin and served for each sample as an internal positive or negative control, respectively. The assay was validated with sera from experimentally and naturally infected chickens. The results were compared to those from reference methods, including other serological tests, PCRs and bioassay in mice. Results In experimentally infected chickens, the vast majority (98.5%, n = 65/66) of birds tested seropositive in the BBMA. This included all chickens positive by magnetic-capture PCR (100%, n = 45/45). Most, but not all inoculated and TgSAG1bio-BBMA-positive chickens were also positive in two previously established TgSAG1-ELISAs (TgSAG1-ELISASL, n = 61/65; or TgSAG1-ELISASH, n = 60/65), or positive in an immunofluorescence assay (IFAT, n = 64/65) and in a modified agglutination test (MAT, n = 61/65). All non-inoculated control animals (n = 28/28, 100%) tested negative. In naturally exposed chickens, the TgSAG1bio-BBMA showed a high sensitivity (98.5%; 95% confidence interval, CI: 90.7–99.9%) and specificity (100%; 95% CI: 85.0–100%) relative to a reference standard established using ELISA, IFAT and MAT. Almost all naturally exposed chickens that were positive in bioassay or by PCR tested positive in the TgSAG1bio-BBMA (93.5%; 95% CI: 77.1–98.9%), while all bioassay- or PCR-negative chickens remained negative (100%; 95% CI: 85.0–100%). Conclusions The TgSAG1bio-BBMA represents a suitable method for the detection of T. gondii infections in chickens with high sensitivity and specificity, which is comparable or even superior to other tests. Since assays based on this methodology allow for the simultaneous analysis of a single biological sample with respect to multiple analytes, the described assay may represent a component in future multiplex assays for broad serological monitoring of poultry and other farm animals for various pathogens.

Concerted evolution is often observed in multigene families such as the CEA gene family. As a result, sequence similarity of paralogous genes is significantly higher than expected from their evolutionary distance. Gene conversion, a “copy paste” DNA repair mechanism that transfers sequences from one gene to another and homologous recombination are drivers of concerted evolution. Nevertheless, some gene family members escape concerted evolution and acquire sufficient sequence differences that orthologous genes can be assigned in descendant species. Reasons why some gene family members can escape while others are captured by concerted evolution are poorly understood. By analyzing the entire CEA gene family in cattle (Bos taurus) we identified a member (CEACAM32) that was created by gene duplication and cooption of a unique transmembrane domain exon in the most recent ancestor of ruminants. CEACAM32 shows a unique, testis-specific expression pattern. Phylogenetic analysis indicated that CEACAM32 is not involved in concerted evolution of CEACAM1 paralogs in ruminants. However, analysis of gene conversion events revealed that CEACAM32 is subject to gene conversion but remarkably, these events are found in the leader exon and intron sequences but not in exons coding for the Ig-like domains. These findings suggest that natural selection hinders gene conversion affecting protein sequences of the mature protein and thereby support escape of CEACAM32 from concerted evolution.

Background: Infections with eggs of Echinococcus granulosus sensu lato (s.l.) can cause cystic echinococcosis in intermediate host animals and humans. Upon ingestion of viable eggs, oncospheres hatch from the eggs and subsequently develop into fluid-filled larval cysts, most frequently in the liver or the lungs. The slowly growing cysts progressively interfere with organ function. The risk of infection is determined by the host range of the parasite, its pathogenicity and other epidemiologically relevant parameters, which differ significantly among the five species within the E. granulosus s.l. complex. It is therefore essential to diagnose the correct species within E. granulosus s.l. to help understand specific disease epidemiology and to facilitate effective implementation of control measures. For this purpose, simple, fast and cost-effective typing techniques are needed.Methods: Mitochondrial genome sequences from the respective members of E. granulosus s.l. complex were used to identify target regions for the detection and genotyping. The selected primer pairs were first tested in a SYBR-green assay. In the next step, primers discriminating between the genotypes (according to post-amplification melting curves) were tested together with the respective probes in a TaqMan® Real-Time Polymerase Chain Reactions (qPCRs). For the analysis of analytical sensitivity and specificity of the real-time PCRs (SYBR-green and TaqMan), a panel of reference DNAs from various Echinococcus spp. was used.Results: We developed quantitative qPCRs and corresponding sequence-specific hydrolysis DNA probes to target polymorphic regions in the mitochondrial genome of E. granulosus s.l.. In a single-step typing approach, we distinguished E. granulosus s.l. members in four epidemiologically relevant subgroups. These were E. granulosus sensu stricto (G1, G3), E. equinus (G4), E. ortleppi (G5) and the E. canadensis cluster (G6 to G8 and G10). The technique also allowed identification and differentiation of these species from other Echinococcus or Taenia taxa for samples isolated from cysts or faeces. Conclusions: Single-step genotyping techniques for the molecular diagnosis of Echinococcus spp. by qPCRs may not only improve diagnostic performance, but also our knowledge on the epidemiology of the parasites and help controlling the various agents of cystic echinococcosis.

Serological assays are commonly used to determine the prevalence of Toxoplasma gondii infection in livestock, but the predictive value of seropositivity with respect to the presence of infective tissue cysts is less clear. The present study aimed at the identification of seropositive and seronegative free-range laying hens from organic and backyard farms, and the relationship with the presence of viable tissue cysts. In addition, potential risk and protective factors on the selected farms were investigated. An in-house T. gondii surface antigen (TgSAG1, p30, SRS29B) ELISA was validated with sera from experimentally infected chickens and used to examine 470 serum samples collected from laying hens from large organic and small backyard farms at the end of their laying period. A total of 11.7% (55/470) of all chickens tested positive, and another 18.9% (89/470) of test results were inconclusive. The highest seroprevalences were observed on small backyard farms with 47.7% (41/86) of chickens being seropositive while another 20.9% (18/86) of test results were inconclusive. Twenty-nine seropositive, 20 seronegative and 12 laying hens which yielded inconclusive ELISA results, were selected for further examination. Hearts and limb muscles of these hens were examined for T. gondii tissue cysts in a bioassay with IFNɣ-knockout or IFNɣ-receptor-knockout mice. Viable T. gondii was isolated from 75.9% (22/29) of the seropositive, 25.0% (3/12) of the inconclusive, and 5.0% (1/20) of the seronegative chickens. All 26 chickens tested positive in heart samples, while drumstick muscles (i.e. limb muscles) tested positive only in three. Data on putative risk and protective factors were collected on the farms using a standard questionnaire. Generalised multilevel modelling revealed farm size, cat related factors ('cats on the premise', 'cats used for rodent control'), hen house/hall related factors ('size category of hen house/hall', 'frequency category of cleaning hen house/hall', 'service period') as significantly associated with seropositivity to T. gondii in hens. The final model, which included the age of the birds as an effect modifier and farm as a random effect variable, revealed that the use of cats for rodent control and an area available per hen in the chicken run of ≥10sqm were statistically significant risk factors for T. gondii seropositivity. Overall this study showed that exposure to T. gondii is common in small backyard farms but is rare on large organic farms with a high density of free-range hens, even when cats were present on the premises.

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.

Oral uptake of infectious Echinococcus multilocularis eggs shed by canids with their faeces may lead to development of alveolar echinococcosis in humans, which is clinically similar to a malignant infiltrative tumor and may be fatal if left untreated. E. multilocularis is therefore regarded as one of the most important and neglected metazoan parasites in the Northern hemisphere. The diagnosis of this tapeworm in the final host plays a key role in the epidemiology of E. multilocularis. The diagnostic performance of a magnetic-capture (MC) DNA extraction protocol in combination with a minor groove-binder real time PCR (MC-MGBqPCR) for the detection of E. multilocularis eggs was determined relative to a highly sensitive variant of the Intestinal Scraping Technique (IST) using faecal samples of foxes. In addition, we compared results obtained by MC-MGBqPCR with those of a previously validated protocol (QIAamp Fast DNA Stool Mini Kit (QT) combined with a TaqMan qPCR). Furthermore, a workflow using the NucleoMagVet DNA extraction kit (NM) in combination with MGBqPCR and TaqMan-qPCR was also included in the comparisons. To estimate the analytical sensitivity, phosphate-buffered saline and fox faecal samples were spiked with different numbers of eggs and tested in defined combinations of DNA extraction and PCR protocols. To assess the diagnostic sensitivity of the different workflows, samples were used that had been collected from the ampulla recti or the rectum of 120 foxes hunted in Brandenburg, Germany. The samples represented five IST categories formed according to the E. multilocularis worm burden of the foxes. For DNA extraction by MC or using two other commercial extraction kits, the supernatants obtained from 3 g of bead-beaten faecal samples were used. The extracted DNAs were then processed in the respective PCR protocols. The MC-MGBqPCR showed the highest diagnostic sensitivity (93%; 95% Confidence Interval (CI): 86–97%) relative to IST. The QT extraction protocol in combination with TaqMan-qPCR had the second highest sensitivity (89%; 95% CI: 80–94%), followed by NM with MGBqPCR (86%; 95% CI: 77–93%) in comparison to IST. The lowest diagnostic sensitivity was found for the NM combined with the TaqMan-qPCR protocol (72%; 95% CI: 62–82%). In conclusion, the MC-MGBqPCR seems to represent a suitable alternative to IST. However, applied to 3 g faecal samples, the less costly QT-TaqMan-qPCR workflow yielded a similar diagnostic sensitivity relative to IST. However, differences between these two workflows were not statistically significant., Highlights • PCR based protocols can be used for the detection of E. multilocularis in faeces of final hosts after careful validation • A DNA capture method seems to represent a suitable alternative to the Intestinal Scraping Technique • The QT-TaqMan-qPCR workflow yielded also a similar diagnostic sensitivity relative to the Intestinal Scraping Technique • None of the DNA extraction kits was able to remove faecal PCR inhibitors completely

The protozoan parasite Toxoplasma gondii is a zoonotic parasite that can be transmitted from animals to humans. Felids, including domestic cats, are definitive hosts that can shed oocysts with their feces. In addition to infections that occur by accidental oral uptake of food or water contaminated with oocysts, it is assumed that a large proportion of affected humans may have become infected by consuming meat or other animal products that contained infective parasitic stages of T. gondii. Since farm animals represent a direct source of infection for humans, but also a possible reservoir for the parasite, it is important to control T. gondii infections in livestock. Moreover, T. gondii may also be pathogenic to livestock where it could be responsible for considerable economic losses in some regions and particular farming systems, e.g. in areas where the small ruminant industry is relevant. This review aims to summarize actual knowledge on the prevalence and effects of infections with T. gondii in the most important livestock species and on the effects of toxoplasmosis on livestock. It also provides an overview on potential risk factors favoring infections of livestock with T. gondii. Knowledge on potential risk factors is prerequisite to implement effective biosecurity measures on farms to prevent T. gondii infections. Risk factors identified by many studies are cat-related, but also those associated with a potential contamination of fodder or water, and with access to a potentially contaminated environment. Published information on the costs T. gondii infections cause in livestock production, is scarce. The most recent peer reviewed reports from Great Britain and Uruguay suggest annual cost of about 5–15 million US $ per country. Since these estimates are outdated, future studies are needed to estimate the present costs due to toxoplasmosis in livestock. Further, the fact that T. gondii infections in livestock may affect human health needs to be considered and the respective costs should also be estimated, but this is beyond the scope of this article., Graphical abstract Unlabelled Image, Highlights • Brief overview on T. gondii prevalence in livestock • Natural and experimental T. gondii infections • Risk factors for T. gondii infection of livestock • Economic impact of toxoplasmosis in livestock

Highly pathogenic avian influenza virus (HPAIV) infection in poultry caused devastating mortality and economic losses. HPAIV of subtypes H5 and H7 emerge from precursor viruses of low pathogenicity (LP) by spontaneous mutation associated with a shift in the susceptibility of the endoproteolytic cleavage site of the viral hemagglutinin protein from trypsin- to furin-like proteases. A recently described natural pair of LP/HP H7N7 viruses derived from two spatio-temporally linked outbreaks in layer chickens was used to study how a minority of mutated HP virions after de novo generation in a single host might gain primacy. Co-infection experiments in embryonated eggs and in chickens were conducted to investigate amplification, spread and transmissionof HPAIV within a poultry population that experiences concurrent infection by an antigenically identical LP precursor virus. Simultaneous LPAIV co-infection (inoculum dose of 106 egg-infectious dose 50% endpoint (EID50)/0.5 mL) withincreasing titers of HPAIV from 101 to 105.7 EID50/0.5 mL) had a significant impeding impact on HP H7 replication, viral excretion kinetics, clinical signs and histopathological lesions (in vivo) and on embryo mortality (in ovo). LP/HP co-infected chickens required a hundredfold higher virus dose (HPAIV inoculum of 105 EID50) compared to HPAIV mono-infection (HPAIV inoculum of 103 EID50) to develop overt clinical signs, mortality and virus spread to uninfected sentinels. Escape and spread of HP phenotypes after de novo generation in an index host may therefore be highly precarious due to significant competition with co-circulating LP precursor virus.

Due to their ground-feeding behaviour, free-ranging chickens and turkeys are exposed to oocysts and are good indicators of the presence of Toxoplasma gondii in the environment. In addition, poultry may become infected by ingestion of tissues of infected intermediate hosts such as small rodents. Free-ranging poultry are considered an important source of T. gondii infection in humans, especially in developing countries. Knowledge on T. gondii genotypes in infected animals and humans is important for understanding the epidemiology of T. gondii infections. The aim of the present study was to analyse the ability of experimentally infected turkeys and chickens to develop a T. gondii clonal type-specific antibody response (IgY) after i.v. inoculation with tachyzoites of three T. gondii clonal lineages, types I, II and III. A peptide microarray displaying a panel of 101 different synthetic peptides was used for serotyping. Peptide sequences were derived from polymorphic regions of 16 T. gondii proteins (GRA1, GRA3-7, SAG1, SAG2A, SAG3, SAG4, SRS1, SRS2, ROP1, NTPase I and NTPase III and BSR4). The array was probed with 120 sera from experimentally infected chickens and turkeys inoculated with different doses of T. gondii tachyzoites (104, 103 and 102) collected from isolates representative for T. gondii clonal types I (RH), II (ME49) or III (NED) and uninfected controls. After screening of the peptides with reference sera from chickens and turkeys, and evaluation of data by Receiver Operating Characteristics analysis, 41 and 40 peptides were identified that appeared suitable to detect type-specific reactions with sera collected at 2, 5, 7 and 9 weeks p.i. Selected peptides allowed the identification of T. gondii clonal types, until 9 week p.i., which the chickens or turkeys had been inoculated with. At 9 weeks p.i., a high proportion of the experimentally infected chickens (67% (12/18)) and turkeys (61% (11/18)) no longer reacted with the selected peptides. Serotyping of the infection in individual chickens or turkeys was only possible when the whole peptide panel was applied. Clonal type-specific antibody responses were dynamic in both poultry species and depended on the individual animal and the time after infection.

Chickens, especially if free-range, are frequently exposed to Toxoplasma gondii, and may represent an important reservoir for T. gondii. Poultry products may pose a risk to humans, when consumed undercooked. In addition, chickens are regarded as sensitive indicators for environmental contamination with T. gondii oocysts and have been used as sentinels. The aim of the present study was to determine the suitability of commonly used antibody detection methods, i.e. the modified agglutination test (MAT), IFAT and ELISA to detect T. gondii-infected chickens. Samples of experimentally and naturally infected chickens were used. The infection state of all chickens was determined by Magnetic-Capture (MC-) real-time PCR (RT PCR). Naturally exposed chickens were additionally examined by mouse bioassay and conventional RT PCR on acidic pepsin digests (PD-RT PCR). Blood serum and meat juice of various sources were tested for antibodies to T. gondii. In naturally infected chickens, there was substantial agreement between the mouse bioassay and MC-RT PCR or the mouse bioassay and conventional PD-RT PCR. PD-RT PCR was slightly more sensitive than MC-RT PCR, as all (26/26) bioassay-positive chickens also tested positive in at least one of the tissues tested (heart, drumstick). By MC-RT PCR, 92.3% (24/26) of the naturally infected bioassay-positive chickens were positive. The diagnostic sensitivity of MC-RT PCR was clearly related to the organ examined. Based on a quantitative assessment of the MC-RT PCR results in experimentally infected chickens, brain and heart tissues harbored an at least 100 times higher parasite concentration than breast, thigh or drumstick musculature. In naturally infected chickens, only three out of 24 birds, which were MC-RT PCR-positive in heart samples, also tested positive in drumstick musculature. Under experimental conditions, the agreement between MC-RT PCR and the serological techniques revealed 100% diagnostic sensitivity and specificity. Under field conditions, examinations of sera by ELISA, IFAT and MAT showed good performance in identifying chickens that were positive in either a mouse bioassay, MC-RT PCR, or PD-RT PCR as illustrated by diagnostic sensitivities of 87.5%, 87.5% and 65.2%, respectively, and diagnostic specificities of 86.2%, 82.8% and 100%, respectively. The examination of meat juice samples from breast, drumstick or heart musculature revealed similar or even better results in the ELISA. The results in the MAT with meat juice from breast musculature were less consistent than those of ELISA and IFAT because a number of negative chickens tested false-positive in the MAT. The MAT performed similar to ELISA and IFAT when applied to test meat juice samples collected from heart, thigh or drumstick musculature.

Toxoplasmosis is an important zoonosis transmitted from animals to humans world-wide. In order to determine Toxoplasma gondii genotypes in individuals living in Germany and to compare findings with those in animals, we analysed nine independent and unlinked genetic markers (nSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) by PCR-RFLP in 83 archived T. gondii-positive DNA samples from patients with ocular toxoplasmosis (n = 35), toxoplasmic encephalitis (n = 32), systemic toxoplasmosis after bone-marrow transplantation (n = 15) and congenital toxoplasmosis (n = 1). In 46 of these 83 samples the presence of T. gondii DNA was confirmed by conventional end-point PCR. Among these, 17 T. gondii-positive samples were typed at all nine loci. The majority (15/17, 88.2%) of these samples were of T. gondii type II (i.e., including both, the Apico type II and Apico type I variants). In addition, in one sample a T. gondii type II/type III allele combination and in another sample a T. gondii genotype displaying type III alleles at all markers was observed. In the remaining 11 samples in which T. gondii could only be partially typed exclusively type II (n = 10) or type III (n = 1) alleles were observed. Results of the present study suggest that the majority of patients in Germany are infected with type II T. gondii regardless of the clinical manifestation of toxoplasmosis. This finding is in accord with the predominance of type II T. gondii in oocysts isolated from cats and in tissues of other intermediate hosts in Germany.

Three genetically different clones of Toxoplasma gondii, also different in mouse virulence, were studied by experimental infection in chickens. For the experiments, four chicken lines were used, which differed in phylogenetic origin and performance level: two white egg layer lines, one with high laying performance (WLA), one with low (R11) and two brown layer lines, also displaying high (BLA) and low (L68) egg number. Chickens were intraperitoneally infected with three different T. gondii isolates representing type IIxIII recombinant clones, i.e. showing both, type II- and type III-specific alleles. These clones (K119/2 2C10, B136/1 B6H6, K119/2 A7) had exhibited virulence differences in a mouse model. In chickens, a significantly higher mortality was observed in white layer lines, but not in brown layer lines, suggesting that differences in the phylogenetic background may influence the susceptibility of chickens for toxoplasmosis. In addition, antibody (IgY) levels varied in surviving chickens at 31 days post infection. While low to intermediate antibody levels were observed in white layers, intermediate to high levels were measured in brown layers. Infection with a T. gondii clone showing low chicken virulence resulted in higher antibody levels in all chicken lines compared to infection with T. gondii clones of intermediate or high chicken virulence. This was in agreement with the parasite load as determined by real-time PCR. Overall, results show that progeny resulting from natural sexual recombination of T. gondii clonal lineages, may differ in their virulence for mice and chickens., Facultad de Ciencias Veterinarias

Orthobunyaviruses are enveloped viruses that can cause human and animal diseases. A novel and major member is the Schmallenberg virus (SBV), the etiological agent of an emerging disease of ruminants that has been spreading all over Europe since 2011. The glycoproteins Gn and Gc of orthobunyaviruses mediate the viral entry, and specifically Gc is a major target for the humoral immune response. For example, the N terminal subdomain of the SBV glycoprotein Gc is targeted by neutralizing monoclonal antibodies that recognize conformational epitopes. Here, we determined the structural features of the N terminus of Gc, and analysed its interaction with monoclonal antibodies. We were able to demonstrate that one of two N-glycosylation sites is essential for secretion and interaction with a subset of Gc-specific monoclonal antibodies. Furthermore, four disulfide bonds (S–S) were identified and the deletion of the third S–S blocked reactivity with another subset of mAbs with virus-neutralizing and non-neutralizing activity. The mutagenesis of the N-glycosylation sites and the disulfide bonds strongly indicated the independent folding of two subdomains within the SBV Gc N terminus. Further, the epitopes recognized by a panel of mAbs could be grouped into two clusters, as revealed by fine mapping using chimeric proteins. Combining the disulfide bonding and epitope mapping allowed us to generate a structural model of the SBV Gc N-terminus. This novel information about the role and structure of the amino terminal region of SBV Gc is of general relevance for the design of antivirals and vaccines against this virus.

Serological methods are the most commonly used diagnostic tools to detect Toxoplasma gondii infections in pigs . In the absence of a readily available ‘gold standard’, an estimation of diagnostic accuracy is dif- ficult to assess. A commercial ELISA (PrioCHECK Toxoplasma Ab porcine ELISA, Prionics, Schlieren, Switzerland) for the diagn osis of T. gondii infection in pigs was evaluated in naturally infected animals from two distinct populations; indoor and outdoor living animals. An assessment of diagnostic accuracy, using a Bayesian latent class approach with adjustm ent for within indoor and outdoor farm clust ering using random effects, was performed. Tests used for comparison were: IFAT; ELISA using native affinity-puri- fied P30 (SAG1) T. gondii tachyzo ite surface antigen (TgSAG1-ELISA); and Western blot with T. gondii tachyzoites lysate. The data set comprised 297 pig serum samples across outdoor (n = 149) and indoor (n = 148) farms in Argentina. The estimated sensitivity and specificity for the comme rcial ELISA were 98.9% (95% credible interval: 96.2; 100) and 92.7% (95% credible interval: 87.7; 96.6), respectively. The analysis of sera and plasma from pigs (n = 6) experimentally inoculated with 5,000 T. gondii oocysts revealed a pronounced antibody response beginning 2 weeks p.i. until the end of the observation period (11 weeks p.i.) in all animals. Meat juice obtained from inoculated animals after euthanasia also tested positive. These results suggest that the PrioCHECK Toxoplasma Ab porcine ELISA may be a useful tool to perform serological diagn osis of T. gondii infections in pigs to control Toxoplasma infection in pigs and humans. Fil: Basso, Walter Ubaldo. Universitat Zurich; Suiza. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Hartnack, Sonja. Universitat Zurich; Suiza Fil: Pardini, Lais Luján. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Maksimov, Pavlo. Friedrich-Loeffler-Institut; Alemania Fil: Koudela, Bretislav. University of Veterinary and Pharmaceutical Sciences Brno; República Checa Fil: Venturini, María Cecilia. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Schares, Gereon. Friedrich-Loeffler-Institut; Alemania Fil: Sidler, Xaver. Universitat Zurich; Suiza Fil: Lewis, Fraser I.. Universitat Zurich; Suiza Fil: Deplazes, Peter. Universitat Zurich; Suiza

Diagnosis of acute bovine besnoitiosis is a major diagnostic problem. We developed diagnostic tests to serologically diagnose and differentiate acute and chronic cases of bovine besnoitiosis using affinity purified antigens of Besnoitia besnoiti tachyzoites in immunoblots and in both, a conventional ELISA and an avidity ELISA. Sera of acutely and chronically infected cattle were investigated using these tests. Acutely infected cattle initially recognised an antigen of 74 kDa relative molecular mass, followed by reactions with increasing intensity against 81 and 28 kDa antigens. In addition, faint reactions against antigens with 36, 37, 39 and 42 kDa molecular mass started soon after seroconversion and increased over time. An antigen of 45 kDa molecular mass was transiently recognised early after infection but not or only weakly in the chronic stage. At least two antigens, the 39 and the 42 kDa antigens, seem to be located on the surface of B. besnoiti tachyzoites as determined by biotinylation. Affinity purified antigen was used to establish an APure-BbELISA which showed excellent sensitivity (100%) relative to a serological reference system in naturally, most likely chronically, infected cattle. Specificity was also high (99.8%) as determined in cattle from herds with Neospora caninum-associated abortions. The antibody levels in APure-BbELISA were correlated with the parasite load in the skin or the mucous membrane of the vestibulum vaginae as determined by real-time PCR. In acute cases of bovine besnoitiosis (confirmed by the detection of low avidity IgG in the APure-BbELISA) first specific antibodies were detected by ELISA in all animals except one, at the same time or earlier than in the serological reference system. The detection of parasite DNA in skin by real-time PCR was clearly superior to serological analysis in detecting infected cattle during acute besnoitiosis.

Toxoplasma gondii is an apicomplexan protozoan parasite which is able to infect a large variety of warm-blooded animals. Raw or undercooked pork has been regarded as an important source of infection for humans. The aim of this study was to evaluate an in-house enzyme-linked immunosorbent assay to diagnose natural T. gondii infection in swine using native affinity chromatography-purified T. gondii surface protein-1 (TgSAG1-ELISA) as antigen, comparing its performance to that of indirect fluorescent antibody test (IFAT) and immunoblotting (IB). To obtain a panel of sera showing the evolution of the antibody response in the time course 12 pigs were experimentally inoculated intravenously (iv) with tachyzoites of the T. gondii strains RH (clonal type I), ME49 (clonal type II) and NED (clonal type III) and serologically monitored for a period of 11 weeks. Both IFAT and ELISA showed a similar time course of antibody response to T. gondii; but by IFAT this response was characterized by rapidly rising titers with peaks at two weeks post inoculation (wpi), while the ELISA indices increased slowly and reached a maximum in most animals at five wpi. Three-hundred randomly selected sera from a total of 602 pigs of different ages derived from outdoor and indoor farms from Argentina were analyzed. Serum samples testing either positive or negative by both IFAT and IB were considered as "relative standards of comparison" (RSC). Sensitivity and specificity of TgSAG1-ELISA were obtained by a Receiver Operating Characteristics (ROC) analysis and statistical agreement among serological tests was evaluated. Antibodies to T. gondii were detected in 160 of 300 sera (53.3%) by IB, in 133 of 300 (44.3%) by IFAT and in 123 of 300 sera (41%) by TgSAG1-ELISA. One hundred and eleven sera tested positive and 118 sera tested negative by both IFAT and IB (RSC); 103 of 111 positive RSC sera tested positive by TgSAG1-ELISA, and 116 of 118 negative RSC sera tested negative by TgSAG1-ELISA. Agreement observed between RSC and TgSAG1-ELISA was almost perfect ( k = 0.9124, p=0.05) and between IFAT and IB was moderate ( k = 0.53, p=0.05). Relative sensitivity and specificity of the TgSAG1-ELISA using a cut-off index of 0.204 were of 92.8% and 98.3%, respectively. ROC analysis revealed that TgSAG1-ELISA was highly accurate (AUC = 0.983) relative to the RSC. According to the results in this study, the ELISA based on affinity purified T. gondii surface antigen TgSAG1 was useful for the specific and sensitive detection of antibodies to this protozoan parasite in naturally infected pigs.

Effective and sensitive methods for the molecular detection of Echinococcus multilocularis in faecal samples of final hosts are crucial for the prevention and control of human alveolar echinococcosis and for studies on the epidemiology of the parasite. Little is known about the suitability of commercial test kits for isolating DNA of E. multilocularis from fox faeces and the performance of standard Polymerase Chain Reaction (PCR) protocols in relation to the quality of DNA extracted by these kits. We compared four different kits: ZR Faecal DNA MiniPrep™ (Zymo Research), FastDNA® SPIN Kit for Soil (MP Biomedicals), QIAamp® Fast DNA Stool Mini Kit (QIAGEN) and NucleoSpin® Soil Kit (Macherey-Nagel) for the extraction of DNA from E. multilocularis eggs present in faeces of foxes. Negative faecal samples were spiked with 600, 300, 150, 75, 37, 18, 9, 5 or 2 E. multilocularis eggs, and each egg concentration was tested 10 times with each of the DNA extraction kits. Each extracted DNA sample was amplified using three PCR protocols: i. conventional PCR (cPCR, Platinum®Taq, Invitrogen), ii. qPCR with the iQ™ Supermix (Bio-Rad) and iii. qPCR with the QuantiTect® Multiplex-Master Mix (QIAGEN). The highest analytical sensitivities for molecular detection of E. multilocularis eggs in spiked fox faeces were observed when combining either the QIAamp® Fast DNA Stool Mini Kit or the ZR Faecal DNA MiniPrep™ kit with the qPCR using the QuantiTect® Multiplex-Master Mix (Sensitivities 97% and 94%, respectively). Combinations including the remaining test kits (NucleoSpin® Soil Kit and FastDNA® SPIN Kit for Soil) showed a markedly lower analytical sensitivity for PCR examinations. The results of the present study indicate that it is of utmost importance to select suitable DNA extraction kits in combination with robust PCR methods or reagents to achieve acceptable analytical sensitivity in the molecular detection of E. multilocularis eggs in fox faecal samples.

This study aimed at isolating and genotyping Toxoplasma gondii from serologically positive free-range chickens from Argentina, and to evaluate the use of sentinel animals during a short time period of exposure to determine environmental contamination with T. gondii oocysts. Two groups of chickens on six farms were compared in this study: (i) young, 2-3 month-old broiler-type chickens reared as sentinel animals on the farms and (ii) adult chickens reared on the same farms for more than one year. Seroconversion rates of 7.0% or 5.7% were observed in sentinel broiler chickens reared for a period of 74 days (January-April 2010) or 88 days (August-November 2010) respectively, as shown by a T. gondii specific immunofluorescent antibody test. Fifty-three percent (17 of 32) of adult chickens were positive and showed higher titres than sentinel animals. Isolation of T. gondii from tissues (brain and heart) of serologically positive chickens was achieved from six of seven free-range adult birds with IFAT titres of 200 and higher. The isolated parasites were analysed by multi-locus polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The isolated T. gondii showed three different genotypes: two genotypes consisted in atypical allele combinations, and the remaining genotype had exclusively clonal type II alleles. All isolates obtained at a single farm, corresponded to the same genotype. The T. gondii genotypes observed are identical to those described in cats, dogs, chickens and capybaras elsewhere in South America. Two isolates, which showed different allele combinations in PCR-RFLP, were characterized in a mouse virulence assay. While one isolate showed a low virulence a second isolate was of intermediate virulence to mice.

Besnoitia besnoiti is an obligate intracellular and emerging coccidian parasite of cattle that mainly infects host endothelial cells during acute infection. We here analyzed early innate immune reactions of B. besnoiti-infected primary bovine umbilical vein endothelial cells (BUVEC). B. besnoiti infections significantly activated BUVEC since the gene transcripts of several adhesion molecules (P-selectin, intercellular adhesion molecule 1(ICAM-1)), chemokines (CXCL1, CXCL8, CCL5), and of COX-2 were significantly upregulated during in vitro infection. Overall, the highest upregulation of most transcripts was observed at 24 or 48 h post infection (p.i.). Enhanced adhesion molecule expression in infected host cells was confirmed by PMN adhesion assays being performed under physiological flow conditions revealing a significantly increased PMN adhesion on B. besnoiti-infected BUVEC layers at 24 h p.i. Furthermore, we were able to illustrate neutrophil extracellular traps (NETs) being released by PMN under physiological flow conditions after adhesion to B. besnoiti-infected BUVEC layers. The present study shows that B. besnoiti infections of primary BUVEC induce a cascade of pro-inflammatory reactions and triggers early innate immune responses.

To obtain estimates for the prevalence of Toxoplasma gondii infection in ducks and geese in Germany, enzyme-linked immunosorbent assays (ELISA) were established based on affinity-purified T. gondii tachyzoite surface antigen 1 (TgSAG1) and used to examine duck and goose sera for T. gondii-specific antibodies. The results of 186 sera from 60 non-infected ducks (Anas platyrhynchos) and 101 sera from 36 non-infected geese (Anser anser) as well as 72 sera from 11 ducks and 89 sera from 12 geese inoculated experimentally with T. gondii tachyzoites (intravenously) or oocysts (orally) and positive in a T. gondii immunofluorescent antibody test (IFAT) were used to select a cut-off value for the TgSAG1-ELISA. Sera obtained by serial bleeding of experimentally inoculated ducks and geese were tested to analyze the time course of anti-TgSAG1 antibodies after inoculation and to assess the sensitivity of the assays in comparison with IFAT. In ducks, IFAT titres and ELISA indices peaked 2 and 5 weeks p.i with tachyzoites, respectively. Only three of six geese inoculated with tachyzoites at the same time as the ducks elicited a low and non-permanent antibody response as detected by the IFAT. In the TgSAG1-ELISA, only a slight increase of the ELISA indices was observed in four of six tachyzoite-inoculated geese. By contrast, inoculation of ducks and geese with oocysts led to an increase in anti-TgSAG1 antibodies within 1 or 2 weeks, which were still detectable at the end of the observation period, i.e. 11 weeks p.i. Inoculation of three ducks and three geese with oocysts of Hammondia hammondi, a protozoon closely related to T. gondii, resulted in a transient seroconversion in ducks and geese as measured by IFAT or TgSAG1-ELISA. Using the newly established TgSAG1-ELISA, sera from naturally exposed ducks and geese sampled in the course of a monitoring program for avian influenza were examined for antibodies to T. gondii; 145/2534 (5.7%) of the ducks and 94/373 (25.2%) of the geese had antibodies against TgSAG1. Seropositive animals were detected on 20 of 61 duck and in 11 of 13 goose farms; the seroprevalences within positive submissions of single farms ranged from 2.2% to 78.6%. Farms keeping ducks or geese exclusively indoors had a significantly lower risk (odds ratio 0.05, 95% confidence interval 0.01-0.3) of harboring serologically positive animals as compared with farms where the animals had access to an enclosure outside the barn.

A serologic survey was conducted among 130 swine slaughtered in the public slaughterhouse of the city of Patos, Paraíba State, Northeastern Brazil, to determine the prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies, and to verify possible associations between sex of the animals and antibody prevalence. The sera were analyzed by indirect antibody tests, considering 1:64 (T. gondii) and 1:50 (N. caninum) dilutions as cut-off points. The prevalence of anti-T. gondii antibodies was 36.2% (47/130) (95% CI = 27.9 - 45.0%) with reciprocal titers ranging from 64 to 2,048, and of anti-N. caninum antibodies was 3.1% (4/130) (95% CI = 0.8 - 7.7%) with reciprocal titers ranging from 50 to 6,400. Three of the four N. caninum-positive samples were also positive for T. gondii antibodies. All Neospora and Toxoplasma IFAT-positive animals were also positive for confirmatory immunoblotting techniques using total and purified N. caninum and T. gondii tachyzoite antigens, i.e., p38 (NcSRS2) and p30 (TgSAG1). There was no association between sex of animals and prevalence of anti-T. gondii and anti-N. caninum antibodies. This is the first indication of N. caninum natural infection in pigs from Brazil. Foi conduzido um estudo sorológico em 130 suínos abatidos no matadouro público do município de Patos, Estado da Paraíba, Nordeste do Brasil, com o objetivo de determinar a prevalência de anticorpos anti-Toxoplasma gondii e anti-Neospora caninum, e verificar possíveis associações entre o sexo dos animais e a prevalência de anticorpos. Os soros foram analisados pelo testes de imunofluorescência indireta, considerando as diluições 1:64 (T. gondii) e 1:50 (N. caninum) como pontos de corte. A prevalência de anticorpos anti-T. gondii foi de 36,2% (47/130) (IC 95% = 27,9 - 45,0%) com títulos variando de 64 a 2.048, e anti-N. caninum de 3,1% (4/130) (IC 95% = 0,8 - 7,7%) com títulos variando de 50 a 6.400. Três das quatro amostras positivas para anticorpos anti-N. caninum também foram positivas para anticorpos anti-T. gondii. Todos os animais positivos na RIFI para Neospora e Toxoplasma também foram positivos nas técnicas confirmatórias de immunoblotting usando antígenos totais e purificados de taquizoítos de N. caninum e T. gondii, ou seja, p38 (NcSRS2) e p30 (TgSAG1). Não houve associação entre o sexo dos animais e a prevalência de anticorpos anti-T. gondii e anti-N. caninum. Essa é a primeira indicação de infecção natural por N. caninum em suínos do Brasil.

Bovines are intermediate hosts of Sarcocystis cruzi, Sarcocystis hirsuta, and Sarcocystis hominis, which use canids, felids, or primates as definitive hosts, respectively. Cattle represent also intermediate hosts of Sarcocystis sinensis, but the definitive hosts of this parasite are not yet known. Sarcocystosis in cattle is frequently asymptomatic. The infection is characterized by the presence of thin-walled (S. cruzi) or thick-walled muscle cysts or sarcocysts (S. hominis, S. sinensis, and S. hirsuta). Recent reports suggest high prevalence of the zoonotic S. hominis in beef in Europe. We therefore aimed at differentiating Sarcocystis spp. in beef offered to consumers in Germany using molecular and microscopical methods, focusing on those species producing thick-walled sarcocysts. A total of 257 beef samples were obtained from different butcheries and supermarkets in Germany and processed by conventional and multiplex real-time PCR. In addition, 130 of these samples were processed by light microscopy and in 24.6 % thick-walled cysts were detected. Transmission electron microscopical analysis of six of these samples revealed an ultrastructural cyst wall pattern compatible with S. sinensis in five samples and with S. hominis in one sample. PCR-amplified 18S ribosomal DNA (rDNA) fragments of 28 individual thick-walled cysts were sequenced, and sequence identities of ≥98 % with S. sinensis (n = 22), S. hominis (n = 5) and S. hirsuta (n = 1) were observed. Moreover, nine Sarcocystis sp. 18S rDNA full length gene sequences were obtained, five of S. sinensis, three of S. hominis, and one of S. hirsuta. Out of all samples (n = 257), 174 (67.7 %) tested positive by conventional PCR and 179 (69.6 %) by multiplex real-time PCR for Sarcocystis spp. Regarding individual species, 134 (52 %), 95 (37 %), 17 (6.6 %), and 16 (6.2 %) were positive for S. cruzi, S. sinensis, S. hirsuta, and S. hominis, respectively. In conclusion, S. sinensis is the most prevalent thick-walled Sarcocystis species in beef offered for consumption in Germany. Further studies are needed to identify the final host of S. sinensis as well as the potential role of this protozoan as a differential diagnosis to the zoonotic species S. hominis., Facultad de Ciencias Veterinarias, Laboratorio de Inmunoparasitología

BackgroundCats are definitive hosts of Toxoplasma gondii and play an essential role in the epidemiology of this parasite. The study aims at clarifying whether cats are able to develop specific antibodies against different clonal types of T. gondii and to determine by serotyping the T. gondii clonal types prevailing in cats as intermediate hosts in Germany.MethodologyTo establish a peptide-microarray serotyping test, we identified 24 suitable peptides using serological T. gondii positive (n=21) and negative cat sera (n=52). To determine the clonal type-specific antibody response of cats in Germany, 86 field sera from T. gondii seropositive naturally infected cats were tested. In addition, we analyzed the antibody response in cats experimentally infected with non-canonical T. gondii types (n=7).FindingsPositive cat reference sera reacted predominantly with peptides harbouring amino acid sequences specific for the clonal T. gondii type the cats were infected with. When the array was applied to field sera from Germany, 98.8% (85/86) of naturally-infected cats recognized similar peptide patterns as T. gondii type II reference sera and showed the strongest reaction intensities with clonal type II-specific peptides. In addition, naturally infected cats recognized type II-specific peptides significantly more frequently than peptides of other type-specificities. Cats infected with non-canonical types showed the strongest reactivity with peptides presenting amino-acid sequences specific for both, type I and type III.ConclusionsCats are able to mount a clonal type-specific antibody response against T. gondii. Serotyping revealed for most seropositive field sera patterns resembling those observed after clonal type II-T. gondii infection. This finding is in accord with our previous results on the occurrence of T. gondii clonal types in oocysts shed by cats in Germany.

Toxoplasma gondii infections occur worldwide in humans and animals. In immunocompromised or prenatally infected humans, T. gondii can cause severe clinical symptoms. The identification of specific epitopes on T. gondii antigens is essential for the improvement and standardization of the serological diagnosis of toxoplasmosis. We selected 20 peptides mimicking linear epitopes on GRA1, GRA2, GRA4, and MIC3 antigenic T. gondii proteins in silico using the software ABCpred. A further 18 peptides representing previously published epitopes derived from GRA1, SAG1, NTPase1, and NTPase2 antigens were added to the panel. A peptide microarray assay was established to prove the diagnostic performance of the selected peptides with human serum samples. Seropositive human serum samples ( n = 184) were collected from patients presenting with acute toxoplasmosis ( n = 21), latent T. gondii infection ( n = 53), and inactive ocular toxoplasmosis ( n = 10) and from seropositive forest workers ( n = 100). To adjust the cutoff values for each peptide, sera from seronegative forest workers ( n = 75) and patients ( n = 65) were used. Univariate logistic regression suggested the significant diagnostic potential of eight novel and two previously published peptides. A test based on these peptides had an overall diagnostic sensitivity of 69% (100% in ocular toxoplasmosis patients, 86% in acutely infected patients, 81% in latently infected patients, and 57% in seropositive forest workers). The analysis of seronegative sera performed with these peptides revealed a diagnostic specificity of 84%. The results of our study suggest that the use of a bioinformatic approach for epitope prediction in combination with peptide microarray testing is a powerful method for the selection of T. gondii epitopes as candidate antigens for serological diagnosis.

Sheep and goats are highly susceptible for infections with Toxoplasma gondii and may play a major role in the transmission of toxoplasmosis to humans. The aim of this study was to obtain up-to-date data on T. gondii infection in small ruminants and to identify putative risk factors in sheep and goats reared under dairy husbandry systems most commonly applied in Greece. To this end, ELISA tests were established for the examination of sheep and goat sera based on the use of TgSAG1, a major surface antigen of T. gondii tachyzoites. Serum samples from 2-4 years old small ruminants, 1501 from sheep and 541 from goats were examined. These samples had been collected on 69 farms in a mountainous and in a costal environment of Northern Greece from September 2008 to January 2009. In addition to farms containing only sheep (n = 28) and farms containing only goats (n = 9) also mixed farms with both animal species (n = 32) were sampled. A standardized questionnaire was used to obtain information on putative risk factors. Sheep showed a higher seroprevalence (48.6% [729/1501]) for T. gondii than goats (30.7% [166/541]). Univariate multi-level modelling assuming random effects by the factor "farm" revealed that goats were statistically significantly less often seropositive than sheep in (OR 0.475 [95% CI: 0.318-0.707]). No statistically significant regional differences in seroprevalence were observed. Risk factor analysis using univariate multi-level modelling revealed that sheep and goats that were kept under intensive (OR 4.30 [95% CI: 1.39-13.27]) or semi-intensive (OR 5.35 [95% CI: 2.33-12.28]) conditions had significantly higher odds of being seropositive. Further significant risk factors were "feeding concentrate" (OR 3.88 [95% CI: 1.81-8.29]) and providing "water from the public supply" (OR 1.67 [95% CI: 4.56-12.39]) to small ruminants.