T. gondii strains and their dosage influence the parasitic load in tissues of experimentally infected pigs

One of the major routes of a Toxoplasma gondii infection in humans is the consumption of raw or undercooked meat. In the present study, we compared the parasitic load induced by 2 different T. gondii strains in the tissues of experimentally infected 6 weeks old pigs. In the first experiment, pigs were orally infected with 3000 tissue cysts of IPB-Gangji strain. The pigs were euthanized 2 and 6 months after infection, and the following samples were tested by bio-assay and qPCR: brain, heart and several skeletal muscles. Two months after infection, all samples tested positive with both tests. Remarkably, after 6 month no cysts were detected in tenderloin and ham, while brain and heart tissue remained infectious. In the second experiment, pigs were infected orally with a low (700 cysts) and a high (6000) dose of T. gondii IPB-Gangji cysts and euthanized after 4 months. The parasitic load was much higher in the low dose group than in the high dose group, as determined by qPCR. In most animals various samples tested negative in both groups, with the exception of the intercostals muscles. Last experiment was repeated with a low and a high dose of the T. gondii IPB-LR strain. Here, all samples remained infectious with no significant difference in parasitic load between both groups. The parasitic load was higher in brain and heart tissue compared to the skeletal muscles. In bio-assay, numerous mice died from the inoculated samples from pigs infected with the IPB-Gangji strain. Ascites and lungs tested T. gondii positive by qPCR. When inoculated with samples from pigs infected with the IPB-LR strain, no mice died from acute T. gondii infection

T. gondii strains and their dosage influence the parasitic load in tissues of experimentally infected pigs

One of the major routes of a Toxoplasma gondii infection in humans is the consumption of raw or undercooked meat. In the present study, we compared the parasitic load induced by 2 different T. gondii strains in the tissues of experimentally infected 6 weeks old pigs. In the first experiment, pigs were orally infected with 3000 tissue cysts of IPB-Gangji strain. The pigs were euthanized 2 and 6 months after infection, and the following samples were tested by bio-assay and qPCR: brain, heart and several skeletal muscles. Two months after infection, all samples tested positive with both tests. Remarkably, after 6 month no cysts were detected in tenderloin and ham, while brain and heart tissue remained infectious. In the second experiment, pigs were infected orally with a low (700 cysts) and a high (6000) dose of T. gondii IPB-Gangji cysts and euthanized after 4 months. The parasitic load was much higher in the low dose group than in the high dose group, as determined by qPCR. In most animals various samples tested negative in both groups, with the exception of the intercostals muscles. Last experiment was repeated with a low and a high dose of the T. gondii IPB-LR strain. Here, all samples remained infectious with no significant difference in parasitic load between both groups. The parasitic load was higher in brain and heart tissue compared to the skeletal muscles. In bio-assay, numerous mice died from the inoculated samples from pigs infected with the IPB-Gangji strain. Ascites and lungs tested T. gondii positive by qPCR. When inoculated with samples from pigs infected with the IPB-LR strain, no mice died from acute T. gondii infection

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-gamma production by TLA-stimulated PBMCs was correlated with the infection dose and predominantly brought about by CD3+ CD4-CD8 alpha bright 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

A two-step lyssavirus real-time polymerase chain reaction using degenerate primers with superior sensitivity to the fluorescent antigen test

A generic two-step lyssavirus real-time reverse transcriptase polymerase chain reaction (qRT-PCR), based on a nested PCR strategy, was validated for the detection of different lyssavirus species. Primers with 17 to 30% of degenerate bases were used in both consecutive steps. The assay could accurately detect RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, and ABLV. In silico sequence alignment showed a functional match with the remaining lyssavirus species. The diagnostic specificity was 100% and the sensitivity proved to be superior to that of the fluorescent antigen test. The limit of detection was <= 1 50% tissue culture infectious dose. The related vesicular stomatitis virus was not recognized, confirming the selectivity for lyssaviruses. The assay was applied to follow the evolution of rabies virus infection in the brain of mice from 0 to 10 days after intranasal inoculation. The obtained RNA curve corresponded well with the curves obtained by a one-step monospecific RABV-qRT-PCR, the fluorescent antigen test, and virus titration. Despite the presence of degenerate bases, the assay proved to be highly sensitive, specific, and reproducible

Early kinetics of intestinal infection and immune responses to two Toxoplasma gondii strains in pigs

Toxoplasma gondiiis an obligate intracellular parasite, able to infect all homeothermicanimals mostly through ingestion of (oo)cysts contaminated food or water. Recently,we observed aT. gondiistrain-specific clearance from tissues upon infection in pigs:while the swine-adapted LR strain persisted in porcine tissues, a subsequent infectionwith the human-isolated Gangji strain cleared parasites from several tissues. Wehypothesized that intestinal immune responses shortly after infection might play a rolein this strain-specific clearance. To assess this possibility, the parasite load in smallintestinal lymph node cells and blood immune cells as well asthe IFNγsecretion bythese cells were evaluated at 2, 4, 8, 14, and 28 days post oralinoculation of pigs withtissue cysts of both strains. Interestingly, at day 4 post inoculation with the LR strainthe parasite was detected by qPCR only in the duodenal lymph node cells, while in thejejunal and ileal lymph node cells and PBMCs the parasite wasdetected from day 8post inoculation onwards. Although we observed a similar profile upon inoculation withthe Gangji strain, the parasite load in the examined cells was much lower. This wasreflected in a significantly higherT. gondii-specific serum IgG response in LR comparedto Gangji infected pigs at day 28 post inoculation. Unexpectedly, this was not reflected inthe IFNγsecretion upon re-stimulation of the cells where almost equal IFNγsecretion wasobserved in both groups. In conclusion, our results show thatT. gondiifirst enters thehost 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 needsfurther study

Third dose of COVID-19 mRNA vaccine closes the gap in immune response between naïve nursing home residents and healthy adults.

peer reviewed[en] BACKGROUND: Nursing home residents, a frail and old population group, respond poorly to primary mRNA COVID-19 vaccination. A third dose has been shown to boost protection against severe disease and death in this immunosenescent population, but limited data is available on the immune responses it induces. METHODS: In this observational cohort study, peak humoral and cellular immune responses were compared 28 days after the second and third doses of the BNT162b2 mRNA COVID-19 vaccine in residents and staff members of two Belgian nursing homes. Only individuals without evidence of previous SARS-CoV-2 infection at third dose administration were included in the study. In addition, an extended cohort of residents and staff members was tested for immune responses to a third vaccine dose and was monitored for vaccine breakthrough infections in the following six months. The trial is registered on ClinicalTrials.gov (NCT04527614). FINDINGS: All included residents (n = 85) and staff members (n = 88) were SARS-CoV-2 infection naïve at third dose administration. Historical blood samples from 28 days post second dose were available from 42 residents and 42 staff members. Magnitude and quality of humoral and cellular immune responses were strongly boosted in residents post third compared to post second dose. Increases were less pronounced in staff members than in residents. At 28 days post third dose, differences between residents and staff had become mostly insignificant. Humoral, but not cellular, responses induced by a third dose were predictive of subsequent incidence of vaccine breakthrough infection in the six months following vaccination. INTERPRETATION: These data show that a third dose of mRNA COVID-19 vaccine largely closes the gap in humoral and cellular immune response observed after primary vaccination between NH residents and staff members but suggest that further boosting might be needed to achieve optimal protection against variants of concern in this vulnerable population group

Toxoplasma gondii, a successful and underestimated foodborne parasite: development of detection methods and their use for the screening of animal reservoirs

Toxoplasma gondii is een wereldwijd voorkomende parasiet die in staat is om bijna alle warmbloedige dieren te infecteren. Ongeveer 50% van de Belgische bevolking heeft antilichamen tegen deze parasiet en is dus latent geïnfecteerd. Hoewel deze parasiet endemisch is in ons land, is slechts weinig gekend over de aanwezigheid ervan bij wilde en huisdieren. Dit doctoraal werk had als doel de nodige technieken te ontwikkelen om de prevalentie van T. gondii te onderzoeken in dierlijke reservoirs. In hoofdstuk 1 wordt de literatuur in verband met de verscheidene biologische en epidemiologische aspecten van T. gondii overlopen. Hoofdstuk 2 behandelt de belangrijkste serologische en directe methoden voor de detectie van T. gondii in humane, dierlijke en omgevingsstalen. In hoofdstuk 3 worden verschillende serologische methoden geëvalueerd voor het testen van kattensera op de aanwezigheid van anti-T. gondii antilichamen. In deze studie, werd de seroprevalentie in de Belgische huiskatten populatie geëvalueerd op 25%. We vonden een 6,25% kans voor een seronegatieve kat om positief te worden in haar volgende levensjaar. In hoofdstuk 4 wordt de ontwikkeling van een ELISA voor de detectie van anti-T. gondii antilichamen bij hertachtigen beschreven. Ook wordt de aanwezigheid van anti-Neospora caninum antilichamen bij deze dieren onderzocht. Bij wilde reeën werd een seroprevalentie gevonden van respectievelijk 52% en 2.7% voor T. gondii en N. caninum. Verder wordt de ontwikkeling van een Multiplex qPCR methode beschreven voor de simultane detectie van zowel T. gondii als N. caninum DNA. Deze methode werd toegepast voor het screenen van vossen en wilde hertachtigen. De gemiddelde prevalentie bij de vossen was 18,8% en 6,6% voor respectievelijk T. gondii en N. caninum; terwijl slechts 1 en 2 reeën positief waren voor respectievelijk, T. gondii en N. caninum. Om de transmissie route van zowel T. gondii en N. caninum verder te onderzoeken, werd de aanwezigheid van beide parasieten bij wilde kleine zoogdieren, gevangen op verscheidene locaties op of rond 9 biologische varkensbedrijven en een biologisch pluimveebedrijf in Nederland onderzocht (hoofdstuk 5). De gevangen dieren vertegenwoordigden 7 soorten knaagdieren en 2 soorten insectivoren. T. gondii DNA werd gevonden in 4% van de stalen, terwijl 12,4% positief was voor N. caninum DNA. De meeste met N. caninum geïnfecteerde stalen (27/31 of 87%) waren afkomstig van bedrijven waar honden werden gehouden. Deze studie vervolledigt een voorgaand onderzoek naar de rol van kleine zoogdieren in de transmissie van T. gondii op biologische varkensbedrijven (hoofdstuk 6). Onze resultaten tonen aan dat niet enkel kleine knaagdieren maar ook insectivoren een belangrijke rol kunnen spelen in de transmissie van zowel T. gondii als N. caninum naar hun respectievelijke eindgastheer en naar vee. Deze kleine zoogdieren zouden bijgevolg ook kunnen dienen als indicatoren voor de aanwezigheid van beide parasieten op landbouwbedrijven. Dit onderzoek toont ook het belang aan van een effectieve plaagdieren bestrijding, bijvoorbeeld in het kader van de productie van T. gondii- vrij vlees. In hoofdstuk 7 worden de ontwikkelde methoden en de behaalde resultaten besproken en vergeleken met de bestaande literatuur. Ook wordt besproken hoe onze resultaten bijdragen tot een betere kennis van de (ver)spreiding van T. gondii en N. caninum in de Belgische dierenpopulatie in het algemeen en meer specifiek in biologische landbouwbedrijven