Molecular Study of Toxoplasma gondii Isolates Originating from Humans and Organic Pigs in Belgium.

Toxoplasma gondii is a worldwide prevalent, zoonotic parasite of major importance for public health, which can infect any warm-blooded animal species, including humans. Humans can get infected by consumption of meat from a chronically infected animal, by ingestion of sporulated oocysts (resulting from the sexual replication in felids), via contaminated water, soil, or vegetables, and by vertical transmission via the placenta. Infection through meat consumption is estimated to be one of the main sources of human toxoplasmosis cases in developed countries, and more specifically pork is considered to be responsible for 41% of foodborne human toxoplasmosis cases in the United States. To better assess the role of pork as a source of T. gondii infection in humans in Belgium, parasites were isolated from pigs to compare with human clinical isolates in a molecular epidemiological study. A positive result was obtained by magnetic capture-quantitative polymerase chain reaction for T. gondii in 14 out of the 92 hearts sampled during 2016 and 2017 from pigs raised in organic farms. From 9 of these 14 samples, parasites were isolated by mouse bioassay, demonstrating the presence of viable T. gondii in animals intended for human consumption. When genotyped and compared with 15 human isolates obtained during 2015 and 2016, a highly related structured population was demonstrated. Overall, these findings demonstrate the presence of infectious T. gondii in pigs intended for human consumption. Therefore, a potential transmission of T. gondii strains from pigs to humans could occur. However, both species could also be infected via a common source of infection such as oocysts. Furthermore, Belgium does not have an official surveillance program for T. gondii in human cases or food-producing animals; as a consequence, the detection of the infection source of a patient is very rare. Overall, this study reinforces the identification of pork as a potential risk for the consumers.

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.

QuilA-Adjuvanted T. gondii Lysate Antigens Trigger Robust Antibody and IFNγ+ T Cell Responses in Pigs Leading to Reduction in Parasite DNA in Tissues Upon Challenge Infection.

Toxoplasma gondii is an intracellular parasite of all mammals and birds, responsible for toxoplasmosis. In healthy individuals T. gondii infections mostly remain asymptomatic, however this parasite causes severe morbidity and mortality in immunocompromised patients and congenital toxoplasmosis in pregnant women. The consumption of raw or undercooked pork is considered as an important risk factor to develop toxoplasmosis in humans. Since effective therapeutic interventions to treat toxoplasmosis are scarce, vaccination of meat producing animals may prevent T. gondii transmission to humans. Here, we evaluated the elicited immune responses and the efficacy of a potential vaccine candidate, generated by size fractionation of T. gondii lysate proteins, to reduce the parasite burden in tissues from experimentally T. gondii infected pigs as compared to vaccination with total lysate antigens (TLA). Our results show that both the vaccine candidate and the TLA immunization elicited strong serum IgG responses and elevated percentages of CD4+CD8+IFNγ+ T cells in T. gondii infected pigs. However, the TLA vaccine induced the strongest immune response and reduced the parasite DNA load below the detection limit in brain and skeletal muscle tissue in most animals. These findings might inform the development of novel vaccines to prevent T. gondii infections in livestock species and humans.

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.

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.

Transfer of Campylobacter from a Positive Batch to Broiler Carcasses of a Subsequently Slaughtered Negative Batch: A Quantitative Approach.

The present study was conducted to quantify Campylobacter cross-contamination from a positive batch of broiler chicken carcasses to a negative batch at selected processing steps and to evaluate the duration of this cross-contamination. During each of nine visits conducted in three broiler slaughterhouses, Campylobacter levels were determined on broiler carcasses originating from Campylobacter-negative batches processed immediately after Campylobacter-positive batches. Data were collected after four steps during the slaughter process (scalding, plucking, evisceration, and washing) at 1, 10, and 20 min after the start of the slaughter of the batches. Campylobacter levels in ceca of birds from Campylobacter-positive batches ranged from 5.62 to 9.82 log CFU/g. When the preceding positive batch was colonized at a low level, no (enumerable) carcass contamination was found in a subsequent negative batch. However, when Campylobacter levels were high in the positive batch, Campylobacter was found on carcasses of the subsequent negative batch but at levels significantly lower than those found on carcasses from the preceding positive batch. The scalding and the evisceration process contributed the least (< 1.5 log CFU/g) and the most (up to 4 log CFU/ g), respectively, to the Campylobacter transmission from a positive batch to a negative batch. Additionally, the number of Campylobacter cells transferred from positive to negative batches decreased over the first 20 min of sampling time. However, the reduction was slower than previously estimated in risk assessment studies, suggesting that pathogen transfer during crosscontamination is a complex process.