Analysis of Clonal Type-Specific Antibody Reactions in Toxoplasma gondii Seropositive Humans from Germany by Peptide-Microarray

BACKGROUND: Different clonal types of Toxoplasma gondii are thought to be associated with distinct clinical manifestations of infections. Serotyping is a novel technique which may allow to determine the clonal type of T. gondii humans are infected with and to extend typing studies to larger populations which include infected but non-diseased individuals. METHODOLOGY: A peptide-microarray test for T. gondii serotyping was established with 54 previously published synthetic peptides, which mimic clonal type-specific epitopes. The test was applied to human sera (n = 174) collected from individuals with an acute T. gondii infection (n = 21), a latent T. gondii infection (n = 53) and from T. gondii-seropositive forest workers (n = 100). FINDINGS: The majority (n = 124; 71%) of all T. gondii seropositive human sera showed reactions against synthetic peptides with sequences specific for clonal type II (type II peptides). Type I and type III peptides were recognized by 42% (n = 73) or 16% (n = 28) of the human sera, respectively, while type II-III, type I-III or type I-II peptides were recognized by 49% (n = 85), 36% (n = 62) or 14% (n = 25) of the sera, respectively. Highest reaction intensities were observed with synthetic peptides mimicking type II-specific epitopes. A proportion of the sera (n = 22; 13%) showed no reaction with type-specific peptides. Individuals with acute toxoplasmosis reacted with a statistically significantly higher number of peptides as compared to individuals with latent T. gondii infection or seropositive forest workers. CONCLUSIONS: Type II-specific reactions were overrepresented and higher in intensity in the study population, which was in accord with genotyping studies on T. gondii oocysts previously conducted in the same area. There were also individuals with type I- or type III-specific reactions. Well-characterized reference sera and further specific peptide markers are needed to establish and to perform future serotyping approaches with higher resolution

Development of a multiplex real time PCR to differentiate Sarcocystis spp. affecting cattle

Cattle are intermediate hosts of Sarcocystis cruzi, Sarcocystis hirsuta and Sarcocystis hominis which use canids, felids or primates as definitive hosts (DH), respectively, and in addition of Sarcocystis sinensis from which the DH is unknown. The aims of the present study were to develop and optimize a multiplex realtime PCR for a sensitive and specific differentiation of Sarcocystis spp. affecting cattle and to estimate the prevalence of Sarcocystis spp. in Argentinean cattle. The 18S rRNA genes from individual sarcocysts were amplified and cloned to serve as controls. For the amplification of bovine Sarcocystis spp. a total of 3 primers were used in combination with specific individual probes. Each assay was evaluated and optimized individually and subsequently combined in a multiplex assay (BovSarcoMultiplex real time PCR). The analytical specificity of the multiplex assay was assessed using 5 ng of DNA of heterologous Sarcocystis spp. and other apicomplexan parasites, and no positive reactions were observed other than for the species the PCR targeted. The analytical sensitivity ranged between 0.0125 and 0.125 fg of plasmid DNA (equivalent to the DNA of 2–20 plasmid DNA copies) or resembling DNA of 0.1–0.3 bradyzoites. A total of 380 DNA loin samples from Argentina were tested and 313, 29, 14 and 2 were positive for S. cruzi, S. sinensis, S. hirsuta and S. hominis, respectively. S. sinensis was the most prevalent species among thick walled Sarcocystis spp. in Argentinean cattle. Mixed infections were detected in 8.9% of all samples. Diagnostic sensitivity and specificity for the BovSarcoMultiplex real time PCR relative to previous microscopic examination for thin and thick-walled cyst were 91.5% and 41.7%, 36.3% and 95.9% respectively. Improved DNA extraction methods may allow to further increase the specific and sensitive detection of Sarcocystis spp. in meat samples.Fil: Moré, Gastón Andrés. Institute of Epidemiology. Federal Research Institute for Animal Health. Friedrich-Loeffler-Institute; Alemania. 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. Centro Cientifico Tecnológico La Plata; ArgentinaFil: Schares, Susann. Institute of Epidemiology. Federal Research Institute for Animal Health. Friedrich-Loeffler-Institute; AlemaniaFil: Maksimov, Aline. Institute of Epidemiology. Federal Research Institute for Animal Health. Friedrich-Loeffler-Institute; AlemaniaFil: Conraths, Franz J.. Institute of Epidemiology. Federal Research Institute for Animal Health. Friedrich-Loeffler-Institute; AlemaniaFil: Venturini, Maria Cecilia. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; ArgentinaFil: Schares, Gereon. Institute of Epidemiology. Federal Research Institute for Animal Health. Friedrich-Loeffler-Institute; Alemani

Serotyping of Toxoplasma gondii in cats (Felis domesticus) reveals predominance of type II infections in Germany.

BACKGROUND Cats 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. METHODOLOGY To 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). FINDINGS Positive 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. CONCLUSIONS Cats 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

<it>Toxoplasma gondii</it> 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

<p>Abstract</p> <p>Tachyzoite clones obtained from a single <it>Toxoplasma gondii</it> 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 <it>T. gondii</it> infections showing combinations of type II and type III alleles at different loci. Forty-five individual clones were obtained from all mixed <it>T. gondii</it> tachyzoite cell cultures by limiting dilution. Sixteen <it>T. gondii</it> 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 <it>T. gondii</it> 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 <it>T. gondii</it> 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 <it>T. gondii</it> occur under natural conditions and result in the emergence of clones with increased virulence in mice.</p

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. Keywords: Besnoitia tarandi, In vitro isolation, Multilocus microsatellite typing, Serological assa

In <i>Toxoplasma gondii</i> type I-infected cats reactions against type I, I/II and I/III specific peptides are strongest and are overrepresented in number.

<div><p>Intensities (MSIVs) by which clonal type I-infected cats reacted with individual peptides were analyzed using ANOVA and the Least Significant Difference (LSD)-Post-Hoc-Test (A). Whiskers represent 95% confidence intervals of the means of MSIV (bars). The differences between the means of MSIVs were regarded as statistically significant, when they were equal or higher than the LSD values. Different letters above the whiskers indicate significant differences between the mean intensities in the LSD-Post-Hoc-Test. </p> <p>To evaluate whether positive or negative serum reactions against clonal type-specific peptide cohorts were over- or underrepresented in cats infected with <i>T. gondii</i> clonal type I, a log-linear model analysis was used and the results presented in a mosaic plot (B). The size of each box in the mosaic plot corresponds to the observed frequencies of positive (Pos) and negative (Neg) peptide reactions as well as the number of analyzed peptides within each peptide cohort. Pearson residuals represent standardized deviations of observed from expected values. The Pearson residuals 0-2 with solid blue line indicate that the number of positive or negative reactions is higher, but not statistically significantly higher than expected (Pearson chi-squared p-value < 0.1). Blue scale shadings suggest the statistically significant rejection of the null hypothesis, i.e. overrepresentation of reactions against particular peptide groups (Pearson residuals (>2), Pearson chi-squared p-value < 0.05). Dashed red lines indicate an underrepresentation of positive or negative peptide reactions which is not statistically significant. Red scale shadings suggest a statistically significant rejection of the null hypothesis, i.e. underrepresentation of peptide reactions within the analyzed peptide group (Pearson residuals (<-2) Pearson chi-squared p-value, 0.05).</p></div

In cats infected with non-canonical <i>Toxoplasma gondii</i> strongest reactions were observed against type I/III specific peptides and the number of reactions against these peptides were overrepresented.

<div><p>Intensities (MSIVs) by which non-canonical type-infected cats reacted with individual peptides were analyzed using ANOVA and the Least Significant Difference (LSD)-Post-Hoc-Test (A). To evaluate whether positive or negative serum reactions against clonal type-specific peptide cohorts were over- or underrepresented in cats infected with atypical <i>T. gondii</i>, a log-linear model analysis was used and the results presented in a mosaic plot (B).</p> <p>Detailed explanations of [A] and [B] are already given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080213#pone-0080213-g001" target="_blank">Figure 1</a>. </p></div

Naturally <i>Toxoplasma gondii</i> seropositive cats and cats with a known clonal type II infection recognized similar peptide patterns.

<p>To explore whether there are particular patterns of anti-peptide reactions among all serologically positive <i>T. gondii</i> cat sera, a XY-fused Selforganizing Kohonen Network analysis (XYF-SKN) was performed. Figure (A) presents the clustering of various groups of <i>T. gondii</i> positive cat sera in a Y-map. Most of the sera derived from cats infected with non-canonical <i>T. gondii</i> types (A), infected by clonal types I, II or III (I, II, or III), or naturally infected cats (N) clustered in the grid nodes <i>1</i> to <i>8</i> either together (type III- and atypical type-infected [node <i>4</i>] as well as the type II and naturally infected cats [nodes <i>1</i> to <i>3</i> and <i>6</i> to <i>8</i>]) or separately (cats infected with type I <i>T. gondii</i> [node <i>5</i>]). Three cat groups were predicted using XYF-SKN, e.g. a cat group infected with non-canonical and type III <i>T. gondii</i> strains (<i>A</i>), a clonal type I infected group (<i>I</i>) and a naturally-infected cat group (<i>N</i>). Predicted cat groups are presented by different background colours of grid nodes, e. g <b><i>A</i></b> by red, <b><i>I</i></b> by green and <b><i>N</i></b> by blue. Figure (B) shows the number of group-specific sera within predicted clusters (<i>A</i>, <i>I</i> and <i>N</i>), further confirming that sera of naturally infected cats clustered mainly with type II reference sera. Figure (C) shows the number of group-specific sera within eight grid nodes in Figure (A).</p

In a <i>Toxoplasma gondii</i> type III-infected cat type III and I/III specific peptides were recognized strongest and were overrepresented in number.

<div><p>Intensities (MSIVs) by which clonal type III-infected cats reacted with individual peptides were analyzed using ANOVA and the Least Significant Difference (LSD)-Post-Hoc-Test (A). To evaluate whether positive or negative serum reactions against clonal type-specific peptide cohorts were over- or underrepresented in a cat infected with <i>T. gondii</i> clonal type III, a log-linear model analysis was used and the results presented in a mosaic plot (B).</p> <p>Detailed explanations of [A] and [B] are already given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080213#pone-0080213-g001" target="_blank">Figure 1</a>. </p></div

In <i>Toxoplasma gondii</i> type II-infected cats reactions against type II and I/II specific peptides are strongest and overrepresented in number.

<div><p>Intensities (MSIVs) by which clonal type II-infected cats reacted with individual peptides were analyzed using ANOVA and the Least Significant Difference (LSD)-Post-Hoc-Test (A). To evaluate whether positive or negative serum reactions against clonal type-specific peptide cohorts were over- or underrepresented in cats infected with <i>T. gondii</i> clonal type II, a log-linear model analysis was used and the results presented in a mosaic plot (B).</p> <p>Detailed explanations of [A] and [B] are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080213#pone-0080213-g001" target="_blank">Figure 1</a>. </p></div