Radu Blaga

Experimental Toxoplasma gondii infections in pigs: Humoral immune response, estimation of specific IgG avidity and the challenges of reproducing vertical transmission in sows

Ten pregnant sows were experimentally inoculated per os with T. gondii in order to investigate vertical and galactogenic transmission of the parasite and the evolution and maturation of the specific IgG humoral response in the sows and piglets. Five seronegative sows received 104T. gondii (CZ isolate clone H3) sporulated oocysts during late-pregnancy (Exp. 1), three sows received 104 oocysts during mid-pregnancy (Exp. 2) and three sows from Exp. 1 (and two seronegative sows) were re-inoculated with 105 oocysts during a further pregnancy (late-pregnancy) (Exp. 3). Besides, six 4.5 week-old piglets inoculated per os with 5×103 oocysts were also included in the serological investigations. All animals seroconverted (PrioCHECK Toxoplasma Ab porcine ELISA, Prionics, Switzerland) by 2-3 weeks post inoculation (wpi) and remained seropositive for at least 38 weeks or until euthanasia. Four chronically infected sows from Exp. 1 and 2 were serologically monitored during a further pregnancy and no reactivation, but a decrease of the antibody levels was observed at farrowing (Exp. 4). In all experiments, the specific IgG-avidity was initially low, increased during the course of infection and after re-inoculations. An avidity index (AI) ≥40% could be used to rule out recent infections (<8 weeks) in most (15 of 16) animals. In some piglets (18.6% of 70) delivered by inoculated sows (Exp. 1 and 2), maternal antibodies were still detectable at 2 months (but not by 3 months) of age, with constant high avidity values, comparable to those of the dams at farrowing. In all experiments, the sows remained asymptomatic and delivered non-infected offspring at term. A total of 208 normal and 5 stillborn piglets delivered by the inoculated sows (Exp. 1-4) tested serologically negative before colostrum uptake. Placentas (n=88) from all sows and tissues (brain, liver, lung, heart, and masseter muscle) from 56 delivered piglets were analysed histopathologically and by real-time PCR for T. gondii with negative results. Colostrum and milk samples from all sows were negative by real-time PCR for T. gondii DNA. In addition, no seroconversion was observed in 16 piglets from seronegative dams that were transferred to infected dams one day after birth to detect a possible infection through colostrum or milk during the suckling period. Although vertical transmission of T. gondii was demonstrated in naturally infected pigs, many factors involved in the outcome of vertical transmission and congenital toxoplasmosis in pigs are still unknown.

Mini-FLOTAC for counting Toxoplasma gondii oocysts from cat feces--comparison with cell counting plates.

Oocysts of Toxoplasma gondii represent one of the most common environmental contaminants causing the zoonotic infection toxoplasmosis. The aim of the present study was to compare the Mini-FLOTAC device with traditional cell counting plates (Kova Slide) for the detection of T. gondii oocysts from feline feces. Two types of experiments were performed: (i) purified oocysts were counted in different dilutions and (ii) specific pathogen free T. gondii-negative cat feces was inoculated with numbers of purified oocysts and counting was performed directly from feces. Our analysis showed a thousand times higher sensitivity of Mini-FLOTAC (5 × 10(2) oocysts) compared to Kova Slide (5 × 10(5) oocysts). Also, when compared by McNemars test, counting of the purified oocysts showed a higher sensitivity of Mini-FLOTAC compared to Kova Slide, for a dilution of 10(3) oocysts/ml (chi(2) = 6.1; P < 0.05). A better sensitivity was also found with Mini-FLOTAC in dilutions of 10(5) and 10(4) oocysts/ml, when counted from feces (chi(2) = 4.2 and 8.1, respectively, P < 0.05). Our results show that Mini-FLOTAC is more sensitive than traditional methods of T. gondii oocysts detection and quantification is more accurate. Furthermore, Mini-FLOTAC simplicity and cost effectiveness allow it to be used with light microscopes in any laboratory or field conditions. We therefore recommend its use for regular screening. Further studies are needed to validate Mini-FLOTAC for the detection of oocysts in soil and water samples in field conditions.

The first isolation and molecular characterization of Toxoplasma gondii from horses in Serbia

BackgroundConsumption of undercooked or insufficiently cured meat is a major risk factor for human infection with Toxoplasma gondii. Although horsemeat is typically consumed rare or undercooked, information on the risk of T. gondii from infected horse meat to humans is scarce. Here, we present the results of a study to determine the presence of T. gondii infection in slaughter horses in Serbia, and to attempt to isolate viable parasites.MethodsThe study included horses from all regions of Serbia slaughtered at two abattoirs between June 2013 and June 2015. Blood sera were tested for the presence of specific IgG T. gondii antibodies by the modified agglutination test (MAT), and samples of trypsin-digested heart tissue were bioassayed in mice. Cyst-positive mouse brain homogenates were subjected to DNA extraction and T. gondii strains were genotyped using 15 microsatellite markers (MS).ResultsA total of 105 slaughter horses were sampled. At the 1:6 cut-off 48.6% of the examined horses were seropositive, with the highest titre being 1:400. Viable parasites were isolated from two grade type mares; both parasite isolates (RS-Eq39 and RS-Eq40) were T. gondii type III, and both displayed an increased lethality for mice with successive passages. These are the first cases of isolation of T. gondii from horses in Serbia. When compared with a worldwide collection of 61 type III and type III-like strains, isolate RS-Eq39 showed a combination of MS lengths similar to a strain isolated from a duck in Iran, and isolate RS-Eq40 was identical in all markers to three strains isolated from a goat from Gabon, a sheep from France and a pig from Portugal. Interestingly, the source horses were one seronegative and one weakly seropositive.ConclusionsThe isolation of viable T. gondii parasites from slaughter horses points to horsemeat as a potential source of human infection, but the fact that viable parasites were isolated from horses with only a serological trace of T. gondii infection presents further evidence that serology may not be adequate to assess the risk of toxoplasmosis from horsemeat consumption. Presence of T. gondii type III in Serbia sheds more light into the potential origin of this archetypal lineage in Europe.

Animaux réservoirs de Toxoplasma gondii : état des lieux en France

Resume La toxoplasmose est une maladie largement distribuee a travers le monde. Elle est due a Toxoplasma gondii , un protozoaire ayant les felides comme hote definitif (le chat) et les vertebres homeothermes comme hotes intermediaires. L’infection humaine se fait principalement par voie orale, soit par l’ingestion d’oocystes excretes par voie fecale par les chats, soit par l’ingestion de kystes tissulaires qui peuvent etre presents dans une grande variete de produits carnes. La transmission directe a partir d’un chat a son proprietaire n’est probablement pas frequente, mais les populations de chats sont plutot a l’origine d’une contamination environnementale large et durable. En France, des plans de surveillance dans les viandes ovines, bovines et porcines, destinees a la consommation humaine ont permis de mettre en evidence une seroprevalence de Toxoplasma gondii qui se situe, selon l’espece et l’origine des viandes, entre 2,46% pour les porcelets hors-sol et 69,5% pour les ovins adultes d’origine francaise. Le role des animaux sauvages en tant qu’hotes intermediaires du parasite est egalement a prendre en compte dans l’epidemiologie de la toxoplasmose, principalement comme facteur de dispersion du parasite et, secondairement, comme agent de contamination humaine. En Guyane, l’hypothese de l’existence d’un cycle sauvage de Toxoplasma gondii est evoquee depuis quelques annees, mettant en jeu les felides sauvages et leurs proies, mammiferes et oiseaux, dont certaines especes peuvent etre des produits de chasse pour les humains.