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

Improvement of sequences in whole-body MRI at highfield (3.0Tesla)

Bildgebende Verfahren spielen in der Diagnostik und Therapie verschiedenster Erkrankungen eine entscheidende Rolle. Die Magnetresonanztomographie als Ganzkörper- Modalität ist eine gut verträgliche Untersuchungsmethode ohne Einwirkung von Strahlen oder Verwendung jodhaltiger Kontrastmittel. Ziel dieser Studie ist es, die Wertigkeit einer Ganzkörper- MRT bei einer höheren Feldstärke von 3.0 Tesla unter Verwendung optimierter Sequenzparameter zu evaluieren. Bei allen 48 Patienten wurde ein standardisiertes Untersuchungsprotokoll verwendet, welches sowohl eine T1-w GRE- Sequenz, eine STIR- Sequenz als auch eine T1-w 3D-GRE-fatsat- Sequenz beinhaltete. Die Auswertung der Bilder erfolgte durch zwei MRT- erfahrene Radiologen; neben der Anzahl der Läsionen wurden auch deren Größe und Lokalisation sowie ggf. relevante Nebenbefunde notiert. Diese Ergebnisse wurden mit denen der - im Rahmen der Diagnostik bereits durchgeführten – Referenzuntersuchungen (CT- Thorax, -Abdomen, Skelettszintigraphie) verglichen. Der Vergleich mit den Referenzuntersuchungen ergab ein gemischtes Bild. Die Konkordanzraten variierten von 36,4% (im Vergleich MRT/ CT- Thorax) über 51,7% (MRT/ Skelettszintigraphie) bis hin zu 85,7% (MRT/ CT- Abdomen). Das bedeutet, dass es vor allem auf dem Gebiet der MRT- Lungenbildgebung noch Defizite gibt, während die MRT- Leberbildgebung mittlerweile den direkten Vergleich mit der Computertomographie anstreben kann. Bei unseren Auswertungen war die verwendete 3D-GRE-fatsat- Sequenz (LAVA) den anderen eindeutig überlegen, sowohl in Bezug auf die Anzahl der erkannten Läsionen als auch in Hinblick auf qualitative Darstellbarkeit. Die Sensitivität der 3D- GRE-fatsat- Sequenz betrug im intersequenziellen Vergleich 96,7%.Body-imaging procedures – such as MRI and CT – play a determining role in the diagnostics and therapy of different illnesses. The magnet resonance imaging as a whole-body modality is a well acceptable examination method without needing X-rays or iodine-containing contrast media. The aim of this study is to evaluate the valency of whole-body MRI using a field strength of 3.0 Tesla and optimised sequence parameters. A standardised examination protocol was used for all 48 patients, including a T1w GRE-sequence, a STIR-sequence as well as a T1w 3D-GRE-fatsat-sequence. The images where analysed by 2 MRI- experienced radiologists; beside the number of the lesions there were taken their size and localisation as well. These results were compared to those of CT and scintigraphy. The comparison with the different examinations proved a mixed result. The concordance rates varied from 36.4% (compared to thoracic CT) up to 51.7% (scintigraphy) and 85.7% (abdominal CT). This means that there are still deficits concerning MRI of the lung, while abdominal MRI meanwhile achives results comparable to CT. Concerning the intersequential comparison the T1w 3D-GRE-fatsat-sequence (LAVA) delivered unambiguously better results, according to the number of recognised lesions as well as in view of qualitative presentability. Using the T1w 3D-GRE fatsat sequence there was demonstrated a sensitivity of 96.7% within the intersequential comparison

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

Seroprevalence of Neospora caninum-specific antibodies in German breeding bitches

Abstract 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

<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

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