Early Kinetics of Intestinal Infection and Immune Responses to Two Toxoplasma gondii Strains in Pigs.

Toxoplasma gondii is an obligate intracellular parasite, able to infect all homeothermic animals mostly through ingestion of (oo)cysts contaminated food or water. Recently, we observed a T. gondii strain-specific clearance from tissues upon infection in pigs: while the swine-adapted LR strain persisted in porcine tissues, a subsequent infection with the human-isolated Gangji strain cleared parasites from several tissues. We hypothesized that intestinal immune responses shortly after infection might play a role in this strain-specific clearance. To assess this possibility, the parasite load in small intestinal lymph node cells and blood immune cells as well as the IFNγ secretion by these cells were evaluated at 2, 4, 8, 14, and 28 days post oral inoculation of pigs with tissue cysts of both strains. Interestingly, at day 4 post inoculation with the LR strain the parasite was detected by qPCR only in the duodenal lymph node cells, while in the jejunal and ileal lymph node cells and PBMCs the parasite was detected from day 8 post inoculation onwards. Although we observed a similar profile upon inoculation with the Gangji strain, the parasite load in the examined cells was much lower. This was reflected in a significantly higher T. gondii-specific serum IgG response in LR compared to Gangji infected pigs at day 28 post inoculation. Unexpectedly, this was not reflected in the IFNγ secretion upon re-stimulation of the cells where almost equal IFNγ secretion was observed in both groups. In conclusion, our results show that T. gondii first enters the host at the duodenum and then probably disseminates from this site to the other tissues. How the early immune response influences the clearance of parasite from tissues needs further study.

Pork as a source of transmission of Toxoplasma gondii to humans: a parasite burden study in pig tissues after infection with different strains of Toxoplasma gondii as a function of time and different parasite stages.

Toxoplasma gondii is an ubiquitous apicomplexan parasite which can infect any warm-blooded animal including humans. Humans and carnivores/omnivores can also become infected by consumption of raw or undercooked infected meat containing muscle cysts. This route of transmission is considered to account for at least 30% of human toxoplasmosis cases. To better assess the role of pork as a source of infection for humans, the parasite burden resulting from experimental infection with different parasite stages and different strains of T. gondii during the acute and chronic phases was studied. The parasite burden in different tissues was measured with a ISO 17025 validated Magnetic Capture-quantitative PCR. A high burden of infection was found in heart and lungs during the acute phase of infection and heart and brain were identified as the most parasitised tissues during the chronic phase of infection, independent of the parasite stage and the strain used. Remarkably, a higher parasite burden was measured in different tissues following infection with oocysts of a type II strain compared with a tissue cyst infection with three strains of either type II or a type I/II. However, these results could have been affected by the use of different strains and euthanasia time points. The parasite burden resulting from a tissue cyst infection was not significantly different between the two strains.

Seroprevalence of anti-Toxoplasma gondii antibodies in Egyptian sheep and goats.

BACKGROUND: Toxoplasmosis is a zoonotic disease that affects a wide range of animals, including small ruminants. Sheep and goats are considered as biological indicators for the contamination of the environment with Toxoplasma gondii oocysts. In addition, in countries such as Egypt, where sheep and goat meat is frequently consumed, T. gondii infection in small ruminants may also pose a public health risk. To establish baseline estimates of the prevalence of T. gondii infection in Egyptian small ruminants, we used an indirect immunofluorescence assay (IFA) and an enzyme-linked immunosorbent assay (ELISA) to assess the seroprevalence in 398 sheep from four Egyptian governorates (Cairo, Giza, Dakahlia and Sharkia) and in 100 goats from Dakahlia. The positive and negative agreements of both tests were calculated and the true prevalence was estimated using a Bayesian approach. RESULTS: The true prevalence of antibodies to T. gondii as determined by both tests was higher in Egyptian goats (62%) than in sheep for each province (between 4.1 and 26%). Sheep slaughtered at the Cairo abattoir had the lowest true prevalence (4.1%), while true prevalences in Dakahlia, Giza and Sharkia governorates (26%, 23% and 12%, respectively) were substantially higher. CONCLUSIONS: The high prevalence of antibodies to T. gondii may indicate an important role of goat and sheep in the transmission of human toxoplasmosis in Egypt, given the habit of eating undercooked grilled mutton.

A more sensitive, efficient and ISO 17025 validated Magnetic Capture real time PCR method for the detection of archetypal Toxoplasma gondii strains in meat.

Toxoplasma gondii is a globally prevalent, zoonotic parasite of major importance to public health. Various indirect and direct methods can be used for the diagnosis of toxoplasmosis. Whereas serological tests are useful to prove contact with the parasite has occurred, the actual presence of the parasite in the tissues of a seropositive animal is not demonstrated. For this, a bioassay is still the reference method. As an alternative, various PCR methods have been developed, but due to the limited amount of sample that can be tested, combined with a low tissue cyst density, those have proved to be insufficiently sensitive. A major improvement of the sensitivity was achieved with magnetic capture-based DNA extraction. By combining the hybridization of specific, biotinylated probes with the capture of those probes with streptavidin-coated paramagnetic beads, T. gondii DNA can selectively be "fished out" from a large volume of meat lysate. Still, several studies showed an insufficient sensitivity compared with the mouse bioassay. Here we present a method that is more sensitive (99% limit of detection: 65.4 tachyzoites per 100g of meat), economical and reliable (ISO 17025 validated) by adding a non-competitive PCR inhibition control (co-capture of cellular r18S) and making the release of the target DNA from the streptavidin-coated paramagnetic beads UV-dependent. The presented results demonstrate the potential of the modified Magnetic Capture real time PCR as a full alternative to the mouse bioassay for the screening of various types of tissues and meat, with the additional advantage of being quantitative.

Strain- and Dose-Dependent Reduction of Toxoplasma gondii Burden in Pigs Is Associated with Interferon-Gamma Production by CD8+ Lymphocytes in a Heterologous Challenge Model.

Toxoplasma gondii is a worldwide prevalent parasite of humans and animals. The global infection burden exceeds yearly one million disability-adjusted life years (DALY's) in infected individuals. Therefore, effective preventive measures should be taken to decrease the risk of infection in humans. Although human toxoplasmosis is predominantly foodborne by ingestion of tissue cysts in meat from domestic animals such as pigs, the incidence risk is difficult to estimate due to the lack of screening of animals for infection and insights in location and persistence of the parasite in the tissues. Hence, experimental infections in pigs can provide more information on the risk for zoonosis based on the parasite burden in meat products intended for human consumption and on the immune responses induced by infection. In the present study, homo- and heterologous infection experiments with two distinct T. gondii strains (IPB-LR and IPB-Gangji) were performed. The humoral and cellular immune responses, the presence of viable parasites and the parasite load in edible meat samples were evaluated. In homologous infection experiments the parasite persistence was clearly strain-dependent and inversely correlated with the infection dose. The results strongly indicate a change in the amount of parasite DNA and viable cysts in porcine tissues over time. Heterologous challenge infections demonstrated that IPB-G strain could considerably reduce the parasite burden in the subsequent IPB-LR infection. A strong, however, not protective humoral response was observed against GRA7 and TLA antigens upon inoculation with both strains. The in vitro IFN-γ production by TLA-stimulated PBMCs was correlated with the infection dose and predominantly brought about by CD3+CD4-CD8αbright T-lymphocytes. The described adaptive cellular and humoral immune responses in pigs are in line with the induced or natural infections in mice and humans. Previous studies underscored the heterogeneity of T. gondii strains and the corresponding virulence factors. These findings suggest the potential of the IPB-G strain to elicit a partially protective immune response and to reduce the parasite burden upon a challenge infection. The IPB-G strain could be used as a promising tool in limiting the number of viable parasites in edible tissues and, hence, in lowering the risk for human toxoplasmosis.

Seroprevalence of zoonotic parasites in pigs slaughtered in the Kathmandu Valley of Nepal.

For several years, the demand for pork has been on the rise in Nepal. To assess the importance of pork as a carrier of zoonotic agents, we performed a cross-sectional study in the Kathmandu Valley of Nepal, in which we serologically determined the infection status of slaughtered pigs with regard to three of the most important parasites transmitted through pork consumption: Trichinella spp., Taenia solium cysticerci, and Toxoplasma gondii. From 2007 to 2010, 742 pigs were sampled at slaughter, of which 0.1% (95% confidence interval [CI] 0.0-0.7%) were found positive for Trichinella infection, 13.8% (95% credibility interval [CrI] 0.8-28.5%) for T. solium cysticercosis, and 11.7% (95% CI 5.2-17.5%) for Toxoplasma infection. Further monitoring of the related animal and human disease burden and strengthening of food safety protocols throughout the pork production chain are strongly recommended.