Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method.

PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

A ring trial to harmonize Toxoplasma gondii microsatellite typing: comparative analysis of results and recommendations for optimization.

A ring trial among five European laboratories was organized to reach consistency in microsatellite (MS) typing of the zoonotic parasite Toxoplasma gondii. Three sample sets were circulated and analyzed by each laboratory following a previously published method that is based on fragment length polymorphism of 15 MS markers. The first sample set compared typing results in general and focused on effects of DNA concentration; the second sample set focused on the polymorphic fingerprinting markers that can differentiate T. gondii strains within the same archetypal lineage; and the third set focused on non-archetypal genotypes. Methodological variations between laboratories, including the software programs used to determine MS fragment length, were collated using a questionnaire. Overall, lineage-level typing results reached a high level of agreement, especially in samples with the highest DNA concentrations. However, laboratory-specific differences were observed for particular markers. Major median differences in fragment length, of up to 6 base pairs, were related to the fluorophore used to label fragment-specific primers. In addition, primer pairs with identical sequences obtained from different suppliers resulted in fragments of differing length. Furthermore, differences in the way the sequencing profiles were assessed and interpreted may have led to deviating results in fragment length determination. Harmonization of MS typing, for example, by using the same fluorophores or by numerical adjustments applied to the fragment-lengths determined, could improve the uniformity of the results across laboratories. This is the first interlaboratory comparison, providing guidelines (added as a supplement) for the optimization of this technique.

Development and characterization of monoclonal antibodies against Besnoitia besnoiti tachyzoites.

This is the first report on the development and characterization of eight monoclonal antibodies (MABs) generated against whole- and membrane-enriched tachyzoite extracts of the apicomplexan parasite Besnoitia besnoiti. Confocal laser scanning immunofluorescence microscopy was used to localize respective epitopes in B. besnoiti tachyzoites along the lytic cycle. A pattern compatible with dense granule staining was observed with MABs 2.A.12, 2.F.3 and 2.G.4, which could be confirmed by immunogold electron microscopy for MABs 2.A.12 and 2.F.3. In particular, MABs 2.F.3 and 2.G.4 were secreted during early invasion, proliferation and egress phases. MABs 3.10.8 and 5.5.11 labelled the tachyzoite surface, whilst MABs 1.17.8, 8.9.2 and 2.G.A recognized the apical tip, which is reminiscent for microneme localization. Besides, the epitopes recognized by the latter two (MABs 8.9.2 and 2.G.A) exhibited a redistribution from the anterior part across the parasite surface towards the posterior end during invasion. Most MABs developed were genus-specific. Indeed, the MABs cross-reacted neither with T. gondii nor with N. caninum tachyzoites. In summary, we have generated MABs that will be useful to study the key processes in the lytic cycle of the parasite and with additional promising diagnostic value. However, the molecular identity of the antigens recognized remains to be elucidated.

Chicken line-dependent mortality after experimental infection with three type IIxIII recombinant Toxoplasma gondii clones.

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.

Experimental Toxoplasma gondii and Eimeria tenella co-infection in chickens.

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

Isolation and molecular characterization of a new Neospora caninum isolate from cattle in Argentina.

Neospora caninum is one of the most important causes of bovine abortion, but isolation of live parasites from infected tissue is difficult. The aims of the present study were to obtain new isolates of N. caninum from congenitally infected asymptomatic newborn cattle in Argentina and to perform characterization by multilocus-microsatellite analysis. Five clinically normal born calves, with demonstrable N. caninum antibodies in precolostrum serum by indirect fluorescent antibody test, were euthanized and their brain samples were processed for histopathological, immunohistochemical, polymerase chain reaction (PCR) analysis, and for bioassay in γ-interferon knockout (GKO) mice. Although N. caninum DNA was detected in brain from all the calves by PCR, viable N. caninum was isolated in GKO mice from only one calf. Neospora caninum tachyzoites of this Argentinean isolate, designated NC-Argentina LP1, were propagated in VERO cell cultures seeded with tachyzoites from the infected GKO mice tissues. Multilocus-microsatellite typing on DNA derived from cell cultured tachyzoites revealed a unique genetic pattern, different from reported isolates. This is the first bovine isolation and genetic characterization of N. caninum in Argentina.

Naturally acquired bovine besnoitiosis: histological and immunohistochemical findings in acute, subacute, and chronic disease.

The pathogenesis of bovine besnoitiosis, a disease of increasing concern within Europe, is still incompletely understood. In this study, disease progression after natural infection with the causative apicomplexan Besnoitia besnoiti was monitored in histological skin sections of 5 individual female cattle over time. High-frequency skin sampling of 2 cattle with mild and 2 with severe acute, subacute, and chronic besnoitiosis, as well as from 1 animal during subclinical disease, enabled documentation from the beginning of the disease. Skin sections were stained with hematoxylin and eosin and Giemsa, periodic acid-Schiff reaction, and anti-Besnoitia immunohistochemistry. In all 4 clinically affected animals, tachyzoite-like endozoites could be detected for the first time by immunohistochemistry, and tissue cyst evolution was monitored. Besnoitiosis-associated lesions were not detected in the animal showing the subclinical course. Because of the inconsistency of the nomenclature of Besnoitia tissue cyst layers in the literature, a new nomenclature for B. besnoiti cyst wall layers is proposed: tissue cysts consist of a hypertrophied host cell with enlarged nuclei, an intracytoplasmic parasitophorous vacuole with bradyzoites, a sometimes vacuolated inner cyst wall, and an outer cyst wall in more developed cysts. Inner and outer cyst walls can be readily distinguished by using special stains. In 1 animal, extracystic B. besnoiti zoites were immunohistochemically detected during the chronic stage. At necropsy, the 2 severely affected cows displayed large numbers of B. besnoiti cysts in a variety of tissues, including the corium of the claws, contributing mainly to the development of chronic laminitis in these 2 cases.

Besnoitia besnoiti infection in cattle and mice: ultrastructural pathology in acute and chronic besnoitiosis.

Current knowledge on bovine besnoitiosis, caused by the emerging apicomplexan pathogen Besnoitia besnoiti, is still fragmentary. So far, studies dealing with ultrastructural pathology focused mainly on the easily accessible chronic stage, whereas ultrastructural investigations of tachyzoites were confined to in vitro studies. In the study presented here, the ultrastructural pathology of naturally B. besnoiti-infected cattle in the acute and chronic disease stages and experimentally B. besnoiti-infected mice was monitored. Further, the ultrastructure of tachyzoites and bradyzoites was investigated. Skin samples of two adult Limousin cows and one adult Limousin bull naturally infected with B. besnoiti and liver and skin samples of gamma-interferon knockout mice infected with B. besnoiti were examined in semithin sections stained with toluidine blue and safranin and in ultrathin sections contrasted with uranyl acetate and lead citrate. Samples of vessel walls of the bull and nasal mucosa of one cow were examined by scanning electron microscopy. Few tachyzoites-like endozoites were detected for the first time in bovine skin, and large numbers of tachyzoites were detected in murine skin and liver. Within tissue cysts in bovine skin, numerous bradyzoites were observed displaying signs of degeneration. Tachyzoites had apicomplexan endozoite ultrastructure. B. besnoiti tachyzoites and bradyzoites differed in shape and the number of amylopectin granules. Transmission and scanning electron microscopy confirmed the presence of two different cyst wall layers, and the present results on cyst wall ultrastructure were in accordance with those previously obtained by histological sections.

Persistence of Toxoplasma gondii tissue stages in poultry over a conventional fattening cycle.

Toxoplasma gondii is a widely spread protozoon in humans, mammals and poultry. Regarding the latter, nothing is known yet about the duration of T. gondii persistence and distribution over a conventional fattening cycle of turkeys and chickens. Twenty-four turkeys and 12 broiler chickens were infected intravenously with 1×10(6) T. gondii tachyzoites (strain NED). Serum antibody levels were determined weekly by ELISA (turkeys) or immunofluorescent antibody test (chickens). Turkeys were slaughtered at 4, 8, 12 and 16 weeks post-infection (p.i.), and chickens 5 or 10 weeks p.i. (n = 6 per group). Sixteen different tissue samples per bird were analysed for T. gondii by PCR. All infected animals showed seroconversion. In turkeys, 15.9% of all samples were tested positive for T.-gondii-DNA. Among the edible tissues (drumstick, thigh, breast muscle, heart, liver and gizzard) 7.8% tested positive. Among poultry slaughtered after different periods of time after infection no significant differences (P>0.05) regarding the number of positive samples were observed. Only 4 out of 192 samples (2.1%) from infected chickens contained detectable T. gondii DNA.The PCR findings suggested that T. gondii may persist in poultry. Particularly in turkey it was shown that edible tissues stay infected for at least 16 weeks p.i. which indicates a potential risk for consumers of undercooked turkey meat whereas chickens appear less susceptible to T. gondii infection.

Comparison of host cell invasion and proliferation among Neospora caninum isolates obtained from oocysts and from clinical cases of naturally infected dogs.

In a previous study we have shown that the in vitro invasion rate (IR) and tachyzoite yield (TY) are associated with the virulence phenotypes of Neospora caninum isolates of bovine origin. In addition, we recently observed marked differences in virulence when canine isolates were compared in a pregnant BALB/c mouse model. In this study, we investigated whether invasion and proliferation capacities could be used as virulence-related N. caninum phenotypic traits. Of the isolates compared in mice, four canine isolates obtained from oocysts (Nc-Ger2, Nc-Ger3, Nc-Ger-6, Nc-6 Arg) had shown a low-moderate virulence, and two further isolates obtained from dogs with neurological signs (Nc-Bahia, Nc-Liv) were highly virulent. The IR for each isolate was determined by a plaque assay and the counting of immunofluorescence-labeled parasitophorous vacuoles at 3 days post-inoculation (p.i.). The TY was determined by the quantification of tachyzoites at 56 h p.i. by real-time PCR. Most of the canine isolates showed similar IR values under controlled invasion conditions for 4h and 72 h p.i., indicating a limited time period for invasion similar to that observed for bovine isolates. The Nc-Ger3, Nc-Bahia, and Nc-Liv isolates showed a significantly higher IR and TY than the Nc-Ger2 and Nc-Ger6 isolates (P<0.0001). A correlation was found between the IRs and TY (ρ>0.885, P<0.033), as well as between the TY and both dam morbidity (ρ=0.8452, P<0.033) and pup mortality (ρ>0.8117, P<0.058) in mice. These results demonstrate the importance both the invasive and proliferative capacities have on the virulence of canine N. caninum isolates.

Sarcocystis sinensis is the most prevalent thick-walled Sarcocystis species in beef on sale for consumers in Germany.

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.

Antimicrobial effects of murine mesenchymal stromal cells directed against Toxoplasma gondii and Neospora caninum: role of immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs).

Mesenchymal stromal cells (MSCs) have a multilineage differentiation potential and provide immunosuppressive and antimicrobial functions. Murine as well as human MSCs restrict the proliferation of T cells. However, species-specific differences in the underlying molecular mechanisms have been described. Here, we analyzed the antiparasitic effector mechanisms active in murine MSCs. Murine MSCs, in contrast to human MSCs, could not restrict the growth of a highly virulent strain of Toxoplasma gondii (BK) after stimulation with IFN-γ. However, the growth of a type II strain of T. gondii (ME49) was strongly inhibited by IFN-γ-activated murine MSCs. Immunity-related GTPases (IRGs) as well as guanylate-binding proteins (GBPs) contributed to this antiparasitic effect. Further analysis showed that IFN-γ-activated mMSCs also inhibit the growth of Neospora caninum, a parasite belonging to the apicomplexan group as well. Detailed studies with murine IFN-γ-activated MSC indicated an involvement in IRGs like Irga6, Irgb6 and Irgd in the inhibition of N. caninum. Additional data showed that, furthermore, GBPs like mGBP1 and mGBP2 could have played a role in the anti-N. caninum effect of murine MSCs. These data underline that MSCs, in addition to their regenerative and immunosuppressive activity, function as antiparasitic effector cells as well. However, IRGs are not present in the human genome, indicating a species-specific difference in anti-T. gondii and anti-N. caninum effect between human and murine MSCs.

Neospora caninum NC-6 Argentina induces fetopathy in both serologically positive and negative experimentally inoculated pregnant dams.

Neospora caninum infection is a major cause of abortion in cattle. The objectives of this study were to genetically characterize the N. caninum NC-6 Argentina isolate using a multilocus microsatellite analysis approach and to study its biological behavior by experimental inoculations into seronegative and seropositive pregnant cattle, evaluating the humoral and cellular immune response elicited and the occurrence of transplacental transmission and fetopathy. Pregnant cows (65 days of gestation) seropositive and seronegative to N. caninum were intravenously inoculated with tachyzoites of the NC-6 Argentina N. caninum strain and slaughtered at 108 ± 2 days of gestation. Serum samples were analyzed for N. caninum antibodies by indirect fluorescent antibody test. The cellular immune response was analyzed by detection of gamma interferon (γIFN) production in blood cells. Tissue samples from dams, fetuses, and placental cotyledons were processed by histopathological and immunohistochemical techniques and examined for N. caninum DNA by PCR. Positive DNA samples were further analyzed by multilocus microsatellite typing for N. caninum. Inoculated animals had significantly higher N. caninum antibody titers and γIFN production than control animals. One seropositive inoculated cow aborted, one seronegative cow had a non-viable fetus, and the remaining fetuses from the experimentally inoculated dams had histopathologic lesions. The PCR was positive in 3/4 fetuses from seronegative inoculated cows and in 2/3 fetuses from seropositive inoculated cows. Multilocus microsatellite analysis revealed that the N. caninum DNA present in fetuses and placentas had an identical pattern to NC-6 Argentina strain. The NC-6 Argentina strain proved to be able to cross the placenta and to induce fetopathy in both the seropositive and seronegative dams.

First isolation of Neospora caninum from the faeces of a dog from Portugal.

We report the in vitro isolation of Neospora caninum from the faeces of a naturally infected 8-year-old male stray boxer from Portugal. Vero cell cultures were infected using parasite stages obtained after oral inoculation of gamma-interferon knockout mice with 10(2) sporulated oocysts. The isolate was identified by microscopical examination, as well as histological, immunological and molecular methods including a DNA-microsatellite-based typing technique, and was subsequently named NC-P1. The DNA-microsatellite pattern observed in the NC-P1 isolate was not previously reported for any N. caninum isolate. To our knowledge, this is the first isolation of N. caninum from the faeces of a naturally infected dog from Portugal.

Molecular comparison of Neospora caninum oocyst isolates from naturally infected dogs with cell culture-derived tachyzoites of the same isolates using nested polymerase chain reaction to amplify microsatellite markers.

Neospora caninum infection is an important cause of bovine abortion. The infection can be transmitted transplacentally or by ingestion of oocysts shed by definitive hosts. There are few reports of dogs naturally shedding N. caninum oocysts and only some oocyst isolates were transferred into cell culture. The aim of the present study was to analyse N. caninum oocysts from the faeces of naturally infected dogs using a microsatellite-based typing technique and to compare them with cell culture-derived tachyzoites of the same isolates. To this end, N. caninum oocysts from six naturally infected dogs were inoculated into gamma-interferon knockout mice. After these mice had developed disease, tissue samples or peritoneal washings from necropsied mice were transferred into cell culture. Nested-PCR techniques were developed for the sensitive and specific amplification of N. caninum microsatellite-containing regions (MS1B, MS2, MS3, MS4, MS5 and MS10). DNA was extracted from oocysts and cell culture tachyzoites of each isolate, followed by amplification and sequence analysis of microsatellite-containing regions. Each parasite isolate examined yielded a unique microsatellite genotype, while no differences were revealed when data for N. caninum oocysts were compared with cultured tachyzoites of the same isolate. Our technique may allow the typing of clinical samples and different strains of N. caninum at the molecular level. This method may prove useful for the identification of infection sources in molecular epidemiological studies.

Isolation and molecular characterization of Toxoplasma gondii from captive slender-tailed meerkats (Suricata suricatta) with fatal toxoplasmosis in Argentina.

In this study, the diagnosis of fatal disseminated toxoplasmosis in three captive slender-tailed meerkats (Suricata suricatta) in the zoo of La Plata, Argentina and the invitro isolation and molecular characterization of Toxoplasma gondii are reported. The animals showed depression, dyspnea and hypothermia, and also ataxia in one case, and died within 1-5 days. The main histopathological lesions included interstitial pneumonia, non-suppurative inflammatory changes and focal necrosis in liver, spleen, kidney and brain. Tachyzoites or tissue cysts were present in lung, liver, spleen, brain, striated muscle, kidney, intestine and mesenteric lymph node sections, and stained strongly with T. gondii antiserum in immunohistochemical analysis. T. gondii was isolated in Swiss mice and in bovine monocytes cultures from tissues of one of the meerkats. The isolate was cryopreserved and it was named TG-Suricata-1. T. gondii DNA was demonstrated in tissues of all three animals and in tachyzoites isolated in cell cultures. The PCR-RFLP analysis of markers based in the loci 3'-SAG2, 5'-SAG2, BTUB, GRA6, SAG3, c22-8, L358, PK1, c29-2 and Apico of T. gondii produced patterns corresponding to the clonal type III. Type III strains of T. gondii possess no or only little virulence in the mouse model, however their association with virulence in other animal species is uncertain. In the present case, T. gondii of the clonal lineage III was responsible for fatal cases in S. suricatta. To our knowledge, this is the first report of isolation and genotyping of T. gondii from S. suricatta.

Validation of PCR-based protocols for the detection of Echinococcus multilocularis DNA in the final host using the Intestinal Scraping Technique as a reference.

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.

Toxoplasma gondii infection and toxoplasmosis in farm animals: Risk factors and economic impact.

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.

The relationship between the presence of antibodies and direct detection of Toxoplasma gondii in slaughtered calves and cattle in four European countries.

In cattle, antibodies to Toxoplasma gondii infection are frequently detected, but evidence for the presence of T. gondii tissue cysts in cattle is limited. To study the concordance between the presence of anti-T. gondii IgG and viable tissue cysts of T. gondii in cattle, serum, liver and diaphragm samples of 167 veal calves and 235 adult cattle were collected in Italy, the Netherlands, Romania and the United Kingdom. Serum samples were tested for anti-T. gondii IgG by the modified agglutination test and p30 immunoblot. Samples from liver were analyzed by mouse bioassay and PCR after trypsin digestion. In addition, all diaphragms of cattle that had tested T. gondii-positive (either in bioassay, by PCR on trypsin-digested liver or serologically by MAT) and a selection of diaphragms from cattle that had tested negative were analyzed by magnetic capture quantitative PCR (MC-PCR). Overall, 13 animals were considered positive by a direct detection method: seven out of 151 (4.6%) by MC-PCR and six out of 385 (1.6%) by bioassay, indicating the presence of viable parasites. As cattle that tested positive in the bioassay tested negative by MC-PCR and vice-versa, these results demonstrate a lack of concordance between the presence of viable parasites in liver and the detection of T. gondii DNA in diaphragm. In addition, the probability to detect T. gondii parasites or DNA in seropositive and seronegative cattle was comparable, demonstrating that serological testing by MAT or p30 immunoblot does not provide information about the presence of T. gondii parasites or DNA in cattle and therefore is not a reliable indicator of the risk for consumers.

Neospora caninum infection in Greek dairy cattle herds detected by two antibody assays in individual milk samples.

A survey to demonstrate the presence and the extent of Neospora caninum infection in dairy cattle was carried out in Greece. Seven hundred and seventy-seven (777) individual milk samples from all milking cows in 10 dairy herds were tested using an ELISA (p38-milk-ELISA) and immunoblot analysis. The herd prevalence was 80.0% and 100.0%, while the overall prevalence was 15.2% (ranging between 0.0% and 38.9%) and 27.9% (ranging between 3.4% and 61.6%) based on the results of the ELISA and the immunoblot test, respectively. In conclusion, N. caninum infection is present among dairy cattle in Greece, deserving more studies to determine its contribution to abortion problems in Greek dairy herds.