Toxoplasma gondii sexual cross in a single naturally infected feline host: generation of highly mouse-virulent and avirulent clones, genotypically different from clonal types I, II and III.

Tachyzoite clones obtained from a single Toxoplasma gondii oocyst field sample were genotyped and characterized regarding mouse virulence. PCR-RFLP genotyping of tachyzoites initially isolated from interferon-γ-knockout (GKO) mice, BALB/c mice and VERO cell culture using the nine independent, unlinked genetic markers nSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico revealed mixed T. gondii infections showing combinations of type II and type III alleles at different loci. Forty-five individual clones were obtained from all mixed T. gondii tachyzoite cell cultures by limiting dilution. Sixteen T. gondii clones showed type III alleles at all loci and 29 clones displayed a combination of type II and type III alleles at different loci. Five clone groups were identified in total, four of which include T. gondii clones that showed a non-canonical allele pattern and have never been described in natural infections before. All tested clones, except two, were highly virulent in BALB/c mice. The isolation of different non-canonical T. gondii clones originating from an oocyst sample of a single naturally infected cat demonstrate that sexual recombination as well as re-assortment of chromosomes via a sexual cross of T. gondii occur under natural conditions and result in the emergence of clones with increased virulence in mice.

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

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

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

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

A real-time quantitative polymerase chain reaction for the specific detection of Hammondia hammondi and its differentiation from Toxoplasma gondii.

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.

Sensitive, quantitative detection of Besnoitia darlingi and related parasites in intermediate hosts and to assess felids as definitive hosts for known and as-yet undescribed related parasite species.

Besnoitia darlingi, B. neotomofelis and B. oryctofelisi are closely related coccidian parasites with cats as definitive hosts. While B. darlingi uses opossums as intermediate hosts, B. neotomofelis and B. oryctofelisi have been described in Southern Plains woodrats (Neotoma micropus) from the USA and in domestic rabbits from Argentina, respectively. A comparison of the Internal Transcribed Spacer-1 (ITS-1) region of the ribosomal DNA (rDNA) of these Besnoitia spp. showed only a few differences. The present study aimed at developing a real-time PCR to detect B. darlingi, B. neotomofelis and B. oryctofelisi in tissues of intermediate and in faeces of definitive hosts in order to support studies of these organisms' epidemiology and pathogenesis. The established PCR was based on primer regions distinct from the ITS-1 sequences of ungulate Besnoitia spp. and made use of a Besnoitia universal probe. To monitor inhibition, a heterologous internal control was established based on the enhanced green fluorescent protein gene. The real-time PCR reacted with B. darlingi, B. neotomofelis and B. oryctofelisi, while the novel PCR did not recognize ungulate Besnoitia spp. (B. besnoiti, B. bennetti, B. tarandi). DNA of Apicomplexa ascribed to other Besnoitia-related genera, including other gut parasites of cats (Cryptosporidium parvum, Giardia duodenalis, Tritrichomonas foetus), was not recognized. The real-time PCR had an analytic sensitivity of less than 1 tachyzoite per reaction. In feline faeces spiked with B. darlingi oocysts, the limit of detection was a DNA amount equivalent to 1 oocyst per PCR reaction. In B. darlingi infected ɣ-interferon knock-out mice, the lung was identified as the predilection organ. In conclusion, this real-time PCR should advance further studies on these parasites and may inspire research on related species, not only in the Americas, but also in other parts of the world.

Serological survey of Neospora spp. and Besnoitia spp. in horses in Portugal.

Equine neosporosis is regarded to be caused either by Neospora hughesi or Neospora caninum and equine besnoitiosis is caused by Besnoitia bennetti, both of which are apicomplexan parasites. N. caninum is the only known Neospora species in Europe, where equine N. caninum infections have been reported as being associated to abortion and reproductive failure. N. hughesi is prevalent in North America and was predominantly linked to neurological disorders. B. bennetti is considered an emergent disease in donkeys in North America and evidence for B. bennetti infection was recently reported in Europe. Though N. caninum and Besnoitia besnoiti are prevalent in cattle in Portugal, little is known about neosporosis in horses and, to the best of our knowledge, no information was hitherto available for Besnoitia spp. The aim of this study was thus to carry out a serological survey to determine the seroprevalence of these parasites in naturally exposed horses in Portugal. A total of 385 animals were screened by the Indirect Fluorescent Antibody Test at the cut-off value 1:50 and positive results were confirmed by Western blot. Exposure to Neospora spp. and Besnoitia spp. was confirmed in 9.1% (95% Confidence Interval [CI]: 6.6-12.4%) and 0.3% (95% CI: 0.0-1.5%) of horses, respectively. Considering the putative economic and animal health impact of neosporosis in horses and the consequences of a possible spread of equine besnoitiosis in Europe and elsewhere, more comprehensive studies are needed to characterize the species detected in serological surveys, evaluate the geographical distribution and assess possible risk factors that could favor transmission.

First highly sensitive and specific competitive ELISA for detection of bovine besnoitiosis with potential as a multi-species test.

Serological cross-reactions represent a serious problem in some currently available tests to diagnose Besnoitia infections in many species including cattle, caribou and donkeys. False-positive results are due to the low positive-predictive value of these serological tests for besnoitiosis. These tests therefore have clear limitations if large herds are screened in areas with low prevalence, since increased numbers of false-positive reactions require confirmatory testing by alternative serological methods, e.g. immunoblotting, which are time-consuming and create extra costs. To overcome this problem, we aimed to develop a highly sensitive and specific competitive ELISA (cELISA) using a panel of 12 monoclonal antibodies raised against the tachyzoite stage of Besnoitia besnoiti. A cELISA set up with one of these antibodies (Bb-cELISA1) was screened with a large panel of B. besnoiti-positive bovine sera to estimate the diagnostic sensitivity of the test. Sera from herds with Neospora caninum- or Sarcocystis spp.-infected cattle were used to estimate its diagnostic specificity. Relative to a reference standard, which combined the results obtained in a previously established highly sensitive and specific ELISA, in the immunofluorescence antibody test and in B. besnoiti tachyzoite and bradyzoite immunoblots, the new Bb-cELISA1 revealed a diagnostic sensitivity of 99.2% (95% confidence interval: 97.1-99.9%) and a diagnostic specificity of 99.9% (95% confidence interval: 97.7-100%). This novel assay was tested on a variety of proven Besnoitia-positive sera from other species, including B. besnoiti-infected cats, rabbits or Besnoitia bennetti-infected donkeys or Besnoitia tarandi-infected caribou. The results obtained with the new Besnoitia-cELISA for these animal species also corresponded almost perfectly with those of the reference tests, which included immunoblot and immunofluorescence antibody tests. In conclusion, the novel Besnoitia-cELISA represents a valuable tool for the diagnosis and control of bovine besnoitiosis and for studies on the epidemiology of Besnoitia infections in a variety of host species, including naturally exposed wildlife and experimental hosts.

Besnoitia tarandi in Canadian woodland caribou - Isolation, characterization and suitability for serological tests.

In the present study, we report the first in vitro isolation of Besnoitia tarandi from North America and the second of B. tarandi at all. The parasite was isolated directly from the skin of a Canadian woodland caribou from the migratory ecotype. The animal belonged to the Leaf River Herd, in Northern Quebec, Canada. The isolate was designated Bt-CA-Quebec1. Sequencing of the 3'-end of the 18S rRNA gene, the complete sequence of the ITS1 and the 5'-end of the 5.8S rRNA gene of Bt-CA-Quebec1 revealed only minor differences to rDNA gene fragments of B. besnoiti. In contrast, the patterns for the microsatellite loci Bt-20 and Bt-21 varied substantially from those reported for B. besnoiti and B. bennetti. Surprisingly, the typing results in the loci Bt-6 and Bt-7 differed between Bt-CA-Quebec1 and results obtained for skin samples from caribou of the Canadian regions of Nunavut and the Northwest Territories reported by other investigators. This indicates that differences might exist among B. tarandi in caribou from different regions in Canada. Mice (γ-interferon knockout) intraperitoneally inoculated with 1.2 × 106 or 1.5 × 106 bradyzoites mechanically released from skin tissue cysts fell ill 8, 9 or 18 days post inoculation. GKO mice inoculated with 3.0 × 104 tachyzoites isolated from the peritoneal cavity of a bradyzoites-inoculated mouse became ill earlier, i.e. 5 days post inoculation. Lung was the predilection site in all mice. Bt-CA-Quebec1 tachyzoites rapidly grew in MARC-145 cells and were used for antigen production. Comparative Western blot analyses revealed only a few differences between B. tarandi Bt-CA-Quebec1 and B. besnoiti Evora antigen when probed with sera collected from chronically infected caribou. Due to its fast growth in vitro, the Bt-CA-Quebec1 isolate may represent an interesting antigen source to establish B. tarandi-specific serological tools and to study the biology of this parasite species further.

Seroprevalence of Neospora caninum-specific antibodies in German breeding bitches.

BACKGROUND: Neospora caninum is an intracellular obligate apicomplexan parasite responsible for multisystemic lesions in dogs. Being definitive hosts and reservoirs, dogs excrete environmentally resistant oocysts. Breeding bitches represent a susceptible dog group and infected bitches may spread this parasite through transplacental transmission. RESULTS: A total of 218 serum samples of German breeding bitches were collected to determine the presence of N. caninum. Antibodies were detected in 16 (7.33%) bitches using a commercial indirect enzyme-linked immunosorbent assay (ELISA). Immunoblotting analysis confirmed all seropositive samples detected by ELISA, proving that the animals were infected with N. caninum. The owners were interviewed regarding breed, age, environment, type, vaccine status, feeding habits and the presence of reproductive disorders. Seropositive animals were between the ages of two to seven years; three of them were kept in kennels while the others were household dogs, one of which was additionally a hunting dog. Owners of four seropositive bitches reported one gestation, while multiple pregnancies had been recorded for the other twelve bitches. Fourteen bitches were regularly vaccinated and six were fed with fresh raw meat. CONCLUSIONS: Although the results confirmed a low incidence of N. caninum seropositive German breeding bitches, further epidemiological and surveillance studies are required to complement our findings regarding the current situation of neosporosis in this specific canine population of Germany.

Detection of specific antibodies to Neospora caninum and Toxoplasma gondii in naturally infected alpacas (Lama pacos), llamas (Lama glama) and vicunas (Lama vicugna) from Peru and Germany.

Sera of an experimentally Neospora caninum infected llama and a non-infected control llama were used to establish an immunoblot, an ELISA and an IFAT to detect antibodies against N. caninum tachyzoites. Subsequently, serum samples collected from a total of 871 South American Camelids (SAC: Lama glama, Lama pacos, Lama vicugna) of two farms in Peru and from 32 SAC of a farm in central Germany were examined for antibodies against N. caninum and Toxoplasma gondii. Based on the recognition of specific bands in the immunoblot, sera of SAC from Peru were differentiated into N. caninum-positive (n = 18) and T. gondii-positive (n = 30) samples and into samples negative or inconclusive for both parasites. Using the immunoblot results as the reference, a modified version of the p38-ELISA and the IFAT were evaluated for detecting N. caninum antibodies in SAC sera. Applying a cut-off as determined by two graph-receiver operating characteristic analysis both, the ELISA and the IFAT, exhibited a sensitivity and specificity of about 95% in the SAC sera from Peru. Serological testing confirmed that SAC may become infected with N. caninum under field conditions in Peru. In addition to alpacas and llamas also 114 wild living vicunas had been examined for antibodies against N. caninum. However, only the alpacas and llamas but no vicunas were found N. caninum-positive. In contrast, T. gondii-seropositive animals were detected in all three SAC species. The lack of N. caninum-seropositive vicunas indicates that in the study area in Peru wild canids might not serve as definitive hosts of N. caninum while for T. gondii a life cycle including wild felids is likely. On the German farm no N. caninum- but only T. gondii-seropositive SAC (n = 14) were detected. The seroprevalence of T. gondii infection was significantly higher in adult SAC (alpacas in Peru, llamas in Germany) than in crias (i.e. < 12 months old foals) indicating that the predominant route of infection is post natal. Since the present study was restricted to a few farms, the seroprevalences determined are not representative. However, our results confirm natural infections with N. caninum and T. gondii in SAC. Whether these infections are linked to any disease, e.g. reproductive losses, has to be clarified in further studies.