Toxoplasma gondii is one of the most prevalent zoonotic protozoan parasites worldwide and affects the vast majority of warm-blooded animal species, including humans. Postnatal infection in humans occurs through the ingestion of sporulated T. gondii oocysts or via the oral intake of parasite tissue cysts during the consumption of raw or undercooked meat. In this regard, given their high exposure to oocysts, chickens (Gallus domesticus) raised on the ground constitute a potential source of T. gondii. For the first time in Spain, a survey was undertaken in commercial retail free-range poultry. A total of 50 thighs from different animals were analysed. The samples were homogenised and an acid pepsin digestion procedure was applied prior to molecular analysis. Toxoplasma gondii DNA was isolated from meat by qPCR. Two sets of primers were used for DNA amplification targeting the specific sequence of a 529 bp repeat element and another set of primers was utilised for the surface antigen protein-1 gene. DNA extracted from 5 out of 50 tissue samples was positive for both genes by qPCR amplification. The 10% prevalence of Toxoplasma infection found in commercial free-range chickens raises public health issues.

Toxoplasma gondii, an obligate intracellular parasite, is the aetiological agent of toxoplasmosis, a disease that affects approximately 25% – 30% of the world’s population. At present, no safe and effective vaccine exists for the prevention of toxoplasmosis. Current treatment options for toxoplasmosis are active only against tachyzoites and may also cause bone marrow toxicity. To contribute to the global search for novel agents for the treatment of toxoplasmosis, we herein report the in vitro activities of previously synthesised benzyltriazole derivatives. The effects of these compounds against T. gondii in vitro were evaluated by using a expressing green fluorescent protein (GFP) type I strain parasite (RH-GFP) and a type II cyst-forming strain of parasite (PruΔku80Δhxgprt). The frontline antitubercular drug isoniazid, designated as Frans J. Smit -isoniazid (FJS-INH), was also included in the screening as a preliminary test in view of future repurposing of this agent. Of the compounds screened, FJS-302, FJS-303, FJS-403 and FJS-INH demonstrated 80% parasite growth inhibition with IC50 values of 5.6 µg/mL, 6.8 µg/µL, 7.0 µg/mL and 19.8 µg/mL, respectively. FJS-302, FJS-303 and FJS-403 inhibited parasite invasion and replication, whereas, sulphadiazine (SFZ), the positive control, was only effective against parasite replication. In addition, SFZ induced bradyzoite differentiation in vitro, whilst FJS-302, FJS-303 and FJS-403 did not increase the bradyzoite number. These results indicate that FJS-302, FJS-303 and FJS-403 have the potential to act as a viable source of antiparasitic therapeutic agents.

The sensitivity and specificity of various serologic tests for antibodies to Toxoplasma gondii were compared in 1,000 naturally exposed sows, using isolation of viable T gondii as the definitive test. Serum samples obtained from heart blood of 1,000 sows from Iowa were examined for T gondii antibodies by use of the modified agglutination test (MAT), latex agglutination test (LAT), indirect hemagglutination test (IHAT), and ELISA. Toxoplasma gondii was isolated from 170 hearts of 1,000 sows by bioassays in mice and cats. The percentage of samples diagnosed as positive for each of the serologic tests was: MAT = 22.2% (titer >greater than or equal to 1:20), IHAT = 6.4% (titer greater than or equal to 1:64), LAT = 10.4% (titer greater than or equal to 1: 64), and ELISA = 24.1% (OD > 0.360). The sensitivity and specificity of these tests were calculated respectively to be: 82.9 and 90.29% for MAT, 29.4 and 98.3% for IHAT, 45.9 and 96.9% for LAT, and 72.9 and 85.9% for ELISA. The dye test was run at 1:20 dilution on only 893 sera because of bacterial contamination and presence of anticomplement substances. Dye test antibodies were found in 17.8% of the sera, and sensitivity and specificity were 54.4 and 90.8%, respectively. Thus, the MAT had the highest sensitivity among all serologic tests used.

The capability of diclazuril, a benzeneacetonitrile anticoccidial agent, to inhibit development of tachyzoites of Toxoplasma gondii was examined in cultured human foreskin fibroblast cells and in Hsd:ICR mice. Treatment of infected cell cultures with 10.0, 1.0, 0.1 or 0.01 micrograms of diclazuril/ml for 3 days resulted in > 99% reduction in tachyzoite counts, compared with controls. Treatment with 0.005 micrograms of diclazuril/ ml resulted in > 97% reduction in tachyzoite counts, compared with controls. Treatment of host cells with 10.0, 1.0, 0.1, and 0.01 micrograms of diclazuril/ml for 24 hours prior to tachyzoite inoculation resulted in 97, 31, 0, and 0% reduction in tachyzoite counts, compared with controls, respectively, 3 days after inoculation. All mice that were treated orally with 10.0 mg of diclazuril/kg of body weight and 80% of mice treated orally with 1.0 mg of diclazuril/kg 1 day prior to and for 10 days after tachyzoite inoculation were protected against acute toxoplasmosis. Mice treated with 10.0 mg of diclazuril/kg did not develop protective immunity, whereas mice treated with 1.0 mg of diclazuril/kg survived challenge exposure.

Enzyme-linked immunosorbent assays for the detection of Toxoplasma gondii antigen-containing IgM immune complexes (T gondii-specific IgM-IC) and IgG immune complexes (T gondii-specific IgG-IC) in the serum of cats were developed. Serum from clinically ill, naturally infected cats; healthy, naturally infected cats; and healthy cats experimentally inoculated with T gondii was assayed. All combinations of T gondii-specific IgM, IgG, antigens, IgM-IC, and IgG-IC were detected in naturally infected and experimentally infected cats. Clinically ill cats and cats with ocular signs of toxoplasmosis were more likely than healthy cats to have T gondii-specific IC in serum. It was concluded that T gondii-specific IC form in the serum of cats, may play a role in clinical disease development, and affect the results of T gondii-specific IgM, IgG, and antigen serologic assays.

Four-week-old chickens were inoculated orally with 1,000 or 100,000 oocysts of the ME-49 or GT-1 strain of Toxoplasma gondii, and their antibody responses were measured, using the direct modified agglutination test, latex agglutination test, indirect hemagglutination test, ELISA, and the Sabin-Feldman dye test. Antibodies against T gondii were detected by use of the modified agglutination test and ELISA within 2 weeks of oocyst inoculation, and antibodies persisted until termination of the study by postinoculation day 68. The latex agglutination test was insensitive in detecting T gondii antibodies, and antibodies were not detected by use of the dye and indirect hemagglutination tests. Of tissues bioassayed in mice for tissue cysts by pepsin digestion of individual organs of chickens on postinoculation day 68, tissue cysts were found in the brain of all 5, heart of 3, and leg muscles of 2, but not in the liver and breast muscles. None of the birds developed clinical toxoplasmosis.

An ELISA was developed to detect circulating antigens of Toxoplasma gondii in the serum of cats. For the experiment, toxoplasmosis was induced in a group of cats by oral administration of bradyzoites. An ELISA that detects anti-Toxoplasma IgG, an ELISA to detect circulating antigens, and fecal examinations were performed on samples from each cat for 1 year after inoculation. When coupled with IgG-class antibody measurement, antigen detection can aid in the diagnosis of some cases of subclinical feline toxoplasmosis.

Subclinical Toxoplasma gondii infection was induced in young and adult cats by oral administration of tissue cysts. An antibody-capture ELISA to detect anti-Toxoplasma IgM-class antibodies in the serum of cats was developed. The serologic response to experimental infection was followed in the 2 groups of cats by use of anti-Toxoplasma IgM and IgG detection. This study shows that anti-Toxoplasma IgM-class antibody titers develop early in the course of experimental infection in cats and that the combination of IgM- and IgG-class antibody titer measurement can aid in the detection of recent subclinical toxoplasmosis.

To study the distribution of tissue cysts in porcine tissues, 16 pigs were fed oocysts of 4 strains of Toxoplasma gondii (4 pigs/strain). Pigs were euthanatized between postinoculation days 103 and 875 and portions of 5 to 14 organs were bioassayed in mice and/or cats for T gondii. For bioassays, 50- to 100-g portions of tissue were incubated in acidic pepsin solution to free bradyzoites from cysts in parenchyma, and washed sediment from the digests of each specimen was inoculated SC into mice (6 mice/organ). For bioassays in cats, a 500-g portion or whole organ was fed to Toxoplasma-free cats (1 cat/organ). Toxoplasma gondii was recovered from tissues of 14 of the 16 pigs (from the brains of 12, hearts of 11, tongues of 10, and diaphragms of 6). Toxoplasma gondii was isolated from commerical cuts of meat from 5 infected pigs, from the arm picnic and ham of 3, Boston butt, spareribs, and tenderloin of 2, and bacon and tailbone of 1. Regarding the 4 pigs euthanatized between postinoculation days 759 and 865, cats shed T gondii oocysts after the ingestion of hearts of all 4; tongues of 3; bacons, hams, arm picnics, Boston butts, spareribs, and diaphragms of 2; and livers, kidneys, and tenderloins of 1. Toxoplasma gondii was found to be inconsistently distributed among the organs and muscles, but overall, tongue and heart were more heavily infected than were other tissues. Tissue cysts in pork were rendered nonviable at -12 C for 3 days.

Toxoplasma gondii, clinical disease in nonvaccinated dairy goats, protective immunity against clinical toxoplasmosis in dairy goats vaccinated with Hammondia hammondi but not H. heydorni, antibody to T. gondii in vaccinated and challenge exposed goats