Seroprevalence of Toxoplasma gondii and immune responses in Belgian sheep

In het eerste deel van de thesis worden de parasiet, zijn levenscyclus en infecties bij schapen uitvoerig belicht. Volgens de Scientific Opinion of the Panel on Biological Hazards heeft toxoplasmosis de hoogste humane incidentie van de parasitaire zoönosen. Risicoanalyses, uitgevoerd door hetzelfde panel, beklemtonen daarenboven het gebrek aan kwantitatieve data op Belgisch niveau. Bovendien is er, tot op heden, zeer weinig gekend over de immuniteit in het schaap tijdens een T. gondii infectie. De belangrijkste doelstellingen, geformuleerd in het tweede deel van de thesis, zijn dan ook het nagaan van de prevalentie bij schapen en het verwerven van inzicht in de acute fase van de infectie bij schapen. Deel 3 bestaat uit 3 experimentele studies. In de eerste studie werd een zeer hoge T. gondii-seroprevalentie bij Belgische schapen waargenomen, gemiddeld 87,4% is seropositief. Dit is te wijten aan hoge prevalenties in Oost-Vlaanderen (96,6 %), West-Vlaanderen (96,8 %), Limburg (97,3%) en Vlaams Brabant (97,8%). De seroprevalenties in Antwerpen (65,2 %) en de Waalse regio liggen significant lager (68,6%). Deze waarden tonen aan dat het eten van onvoldoende doorbakken Belgisch schapenvlees een hoog potentieel risico vormt op infectie met T. gondii. Om meer inzicht te krijgen in de parasiet - gastheer interactie, werd in een volgende studie de acute fase van de infectie bestudeerd. Hierbij werden - de eerste 3 weken na infectie - de serum antistoffenrespons, alsook de hoeveelheid parasitair DNA en de parasiet-geïnduceerde interferon-γ en interleukine-12 responsen in het intestinaal weefsel en de drainerende lymfeknopen onderzocht. Reeds de eerste dagen na infectie dringen tachyzoïeten, vooral craniaal, de dunne darmmucosa binnen. Dit gaat gepaard met een interferon-γ en IL-12 respons die het meest uitgesproken is in de mesenteriale lymfeknopen. Veertien dagen na infectie is er een sterke afname tot zelfs afwezigheid van IFN-γ en IL-12 in de Peyerse Platen, de perifere bloed mononucleaire cellen en de popliteus lymfeknoop. Deze daling gaat gepaard met de opkomst van parasiet-specifiek IgG. Gelijktijdig wordt T. gondii DNA uit de meeste mucosale en systemische weefsels geïsoleerd. Echter, één week later is er geen parasitair DNA meer detecteerbaar in de onderzochte weefsels. Dit zou erop kunnen wijzen dat de parasiet deze weefsels reeds verlaten had of eruit verwijderd werd, onder invloed van de opgebouwde immuunrespons. De derde experimentele studie onderzocht de cytokine mRNA expressie door bloed mononucleaire cellen, de antistoffenrespons en de parasitaire load, tijdens een subacute T. gondii infectie van schapen en varkens. Beide species werden met verschillende stammen geïnfecteerd. Hoewel de parasitaire load in weefsels voor beide species vergelijkbaar was, zagen we bij schapen een antistoffenrespons tegen alle 5 geteste T. gondii antigenen, terwijl bij de varkens enkel rGRA7-specifieke antilichamen konden worden aangetoond. Ook de cytokine respons leek meer uitgesproken bij schapen dan bij varkens. De resultaten suggeren dat rGRA7-specifieke serumantilichamen bij levende schapen en varkens en bio-assay en qPCR op hartspierweefsel van geslachte dieren de beste testen zijn om besmetting met T. gondii te detecteren

Evaluation of parasite antigens in Elisa for the detection of toxoplasma infection in pigs

One-third of the human world population is infected with the protozoan parasite Toxoplasma gondii Toxoplasmosis is an old disease but is still very underreported and neglected disease

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

Protective Th1 immune responses against chronic toxoplasmosis induced by a protein-protein vaccine combination but not by its DNA-protein counterpart

Vaccine-induced protection against toxoplasmosis is correlated with cellular immune responses to Taxoplasma gondii, both in animals and man. The goal of the current study was to evaluate whether the combination of a recombinant protein and a plasmid DNA vaccine could offer an advantage over the protein mixture, and protect outbred mice against infection with T gondii. To this purpose, the chimeric protein rEC2, encoding antigenic fragments of surface-associated proteins MIC2, MIC3 and SAG1, was combined with pGRA7 plasmid DNA or rGRA7 protein. High levels of antibodies were elicited by both vaccine formulations. The protein-DNA vaccine elicited a polarized Th1/Th2 immune response, characterized by IFN-gamma and IL-10, and afforded low protection (24%) against brain cyst formation. In contrast, the protein-protein vaccine elicited a Th1-focused immune response, characterized by IFN-gamma and IL-2 production, conferring a strong protection (79%) against brain cyst formation in chronic toxoplasmosis. We show here that GERBU adjuvanted protein vaccines confer better protection against toxoplasmosis than the protein-DNA heterologous vaccine. Crown Copyright (C) 2008 Published by Elsevier Ltd. All rights reserved

Seroprevalence of Toxoplasma gondii in domestic sheep in Belgium

Even though infected sheep are a potential source of Toxoplasma gondii infection in humans, information is lacking concerning the seroprevalence of T. gondii infection in sheep in Belgium. We examined 3170 serum samples for anti-Toxoplasma IgG in sheep by total lysate antigen (TLA) enzyme-linked immunosorbent assay (ELISA). IgG to T. gondii was demonstrated in 87.4% of the tested sheep and in 96.2% of the 209 tested flocks. The seroprevalences in Antwerp (65.2%) and Wallonia (68.6%) are statistically lower than in the other regions in Belgium (96.7-97.8%) (P&lt;0.05). The present study is the first report that analyzed the prevalence of T. gondii infection in sheep in Belgium and confirms the high prevalence of Toxoplasma-specific IgG antibodies in the sheep population</p

An enhanced GRA1-GRA7 cocktail DNA vaccine primes anti-Toxoplasma immune responses in pigs36804

The protozoan parasite Toxoplasma gondii is the causative agent of a worldwide zoonosis and high prevalencies can be found both in animals and humans. An important source of human contamination with T. gondii is the consumption of raw or undercooked meat products. In this study, we evaluated whether DNA vaccination against T. gondii in pigs is able to generate immune responses known to be protective against tissue cyst formation. A GRA1-GRA7 DNA vaccine cocktail was enhanced by codon optimization of the encoding antigens and addition of heat labile enterotoxin expressing vectors as genetic adjuvant. Pigs vaccinated intradermally with this enhanced GRA1-GRA7 DNA vaccine cocktail developed high antibody levels against GRA1, GRA7 and a T. gondii lysate, and lymphocyte proliferation and production of IFN-gamma could be detected in these animals after challenge with the parasite. These results indicate that pigs can be efficiently primed against T. gondii infection by means of a DNA vaccine</p

Interferon-gamma expression and infectivity of Toxoplasma infected tissues in experimentally infected sheep in comparison with pigs

&lt;p&gt;Livestock animals are a potential risk for transmission of toxoplasmosis to humans. Sheep and pigs still remain an important source because their meat is often eaten undercooked which has been regarded as a major route of infection in many countries. Moreover, porcine tissues are processed in many food products. In the current study, the IFN-gamma (T-helper 1 cells), IL-4 (Th2 cells) and IL-10 mRNA (Treg cells) expression by blood mononuclear cells, and the serum antibody response against Toxoplasma gondii total lysate antigen, recombinant T. gondii GRA1, rGRA7, rMIC3 and rEC2, a chimeric antigen composed of MIC2, MIC3 and SAG1, was studied in sheep the first two months after a T. gondii infection and compared with these responses in pigs. At the end of this period, the parasite distribution in heart, brain and two skeletal muscles in sheep was compared with this in pigs. Whereas the parasite distribution was similar in sheep and pigs, the antibody response differed considerably. In sheep, antibodies appeared against all tested T. gondii antigens, but mainly against rGRA7, rMIC3234307 and TLA whereas in pigs only rGRA7-specific antibodies could be demonstrated. Also, the cytokine response differed. Both in sheep and pigs an IFN-gamma response occurred which seemed to be a slightly more pronounced in sheep. In sheep, also IL-10 and IL-4 mRNA expression showed an increase, but later than IFN-gamma and with more variation. However, in pigs no such increase was seen. As concerning diagnosis, results indicate that serum antibodies against GRA7 in live sheep and pigs and heart tissue for bioassay and qPCR in slaughtered animals are the best targets to demonstrate presence of T. gondii infection.&lt;/p&gt;</p