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

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.

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<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.

Neospora caninum and Toxoplasma gondii in brain tissue of feral rodents and insectivores caught on farms in the Netherlands.

We investigated the presence of both Neospora caninum and Toxoplasma gondii in 250 brain tissue samples from 9 species of feral rodents and insectivores caught on 10 organic farms in the Netherlands in 2004. Collected samples were conserved in 4% paraformaldehyde solution and analysed by real-time PCR. For N. caninum, 31 samples originating from 6 species tested positive (12.4%): common shrews (33.3%), wood mice (17.6%), harvest mice (16.7%), house mice (15.4%), white-toothed shrews (10.8%) and common voles (4.2%). For T. gondii, the overall contamination level was 4%, and only three species were found to be positive: house mice (9.0%), common voles (4.2%) and white-toothed shrews (2.0%). Most N. caninum infected samples (27/31; 87%) were found on farms where dogs were present. Due to the observation that rodents and insectivores can contract both parasites, they might function as indicator species for the parasitic load on farms.

IFN-γ expression and infectivity of Toxoplasma infected tissues are associated with an antibody response against GRA7 in experimentally infected pigs.

Toxoplasma gondii, an obligate intracellular parasite, can be transmitted to humans via the consumption of infected meat. However, there are currently no veterinary diagnostic tests available for the screening of animals at slaughter. In the current work, we investigated whether cytokine responses in the blood, and antibody responses against recombinant T. gondii GRA1, GRA7, MIC3 proteins and a chimeric antigen EC2 encoding MIC2-MIC3-SAG1, are associated with the infectivity of porcine tissues after experimental infection with T. gondii. Two weeks after experimental infection of conventional 5-week-old seronegative pigs, an IFN-γ response was detected in the blood, with a kinetic profile that followed the magnitude of the GRA7 antibody response. Antibody responses to GRA1, MIC3 and EC2 were very weak or absent up to 6 weeks post infection. Antibodies against GRA7 occurred in all infected animals and were associated with the presence of the parasite in tissues at euthanasia a few months later, as demonstrated by quantitative real-time PCR and isolation by bio-assay. Remarkably, although brain and heart tissue remained infectious, musculus gastrocnemius and musculus longissimus dorsi were found clear of infectious parasites 6 months after experimental infection. Seropositive response in a GRA7 ELISA indicates a Toxoplasma infection in pigs and is predictive of the presence of infectious cysts in pig heart and brain. This new ELISA is a promising tool to study the prevalence of Toxoplasma infection in pigs. Clearance of the infection in certain pig tissues suggests that the risk assessment of pig meat for human health needs further evaluation.

Toxoplasma gondii and Neospora caninum in wildlife: common parasites in Belgian foxes and Cervidae?

Sera from Cervidae were tested for the presence of antibodies against Neospora caninum using ELISA; and against Toxoplasma gondii using SAG1-ELISA and a commercially available agglutination test. The T. gondii seroprevalence was 52% (38/73) in roe deer (Capreolus capreolus), 0% in bred fallow deer (0/4) (Dama dama) and red deer (0/7) (Cervus elaphus). We found 2.7% of the roe deer samples and none of the bred deer samples positive for N. caninum. Brain samples from wild roe deer, red deer and red foxes (Vulpes vulpes) were tested for the presence of T. gondii and N. caninum DNA using multiplex real-time PCR. We detected T. gondii in 18.8% (57/304) of the red foxes and in 1 of the 33 deer samples. N. caninum was found in 6.6% of the red foxes and in 2 roe deer samples. Twenty-six of the T. gondii positive DNA extracts from the red fox samples were genotyped. Twenty-five were type II and only one was found to be type III.

10th survey of antimicrobial resistance in noninvasive clinical isolates of Streptococcus pneumoniae collected in Belgium during winter 2007-2008.

OBJECTIVES: The aim of the study was to evaluate the antibiotic resistance in noninvasive clinical isolates of Streptococcus pneumoniae collected in Belgium during winter 2008-2007. METHOD: Four hundred and forty eight unduplicated isolates collected by 15 laboratories were tested by microdilution following CLSI. RESULTS: Insusceptibility rates (I+R) were as follows: penicillin G (PEN) 11.6% (4.0% R), ampicillin 11.4% (4.0% R), amoxicillin+/-clavulanic acid 0, cefaclor 10.3% (9.6% R), cefuroxime 9.2% (8.7% R), cefuroxime-axetil 8.7% (7.8% R), cefotaxime, ceftazidime and cefepime 2.0% (0% R), imipenem 2.5% (0% R), ciprofloxacin and ofloxacin 5.1% (0.4% R), levofloxacin 0.7% (0.4% R), moxifloxacin 0.4% (0.2% R), erythromycin (ERY) 29.7% (29.2% R), azithromycin 29.7% (28.8% R), telithromycin 0%, clindamycin 26.3% (25.4% R) and tetracycline (TET) 21.9% (16.5% R). From 2001 to 2008, a significant decrease in penicillin-insusceptibility (21.0% to 11.6%), penicillin-resistance (9.7% to 4.0%) and ciprofloxacin-insusceptibility (11.2% to 5.1%) was found. Cross-resistance between penicillin and other betalactams in penicillin-insusceptible isolates was incomplete: all these isolates remained fully susceptible to amoxicillin. Erythromycin-insusceptibility was significantly higher in children than in adults (43.9%/27.4%), while penicillin-insusceptibility significantly higher in Brussels than in the Flanders (22.9%/8.1%). The commonest resistance phenotype was ERY-TET (12.7%) followed by ERY (7.4%) and PEN-ERY-TET (5.8%). Capsular types 19 (25%), 14 (19.3%), 23 (15.4%) and 15 (13.5%) were the most important in penicillin-insusceptible. CONCLUSION: We noted a decrease in resistance to the majority of the compounds. Insusceptibility rates were higher in children than in adults and the difference between the north and the south of Belgium became less marked.

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

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.

GRA7 provides protective immunity in cocktail DNA vaccines against Toxoplasma gondii.

In a previous study, single-gene vaccination with GRA1, GRA7 or ROP2 was shown to elicit partial protection against Toxoplasma gondii. In this study, the contribution of each antigen in the evoked humoral and cellular immune responses was evaluated after vaccination with plasmid mixtures containing GRA1, GRA7 and ROP2. Cocktail DNA vaccinated mice developed high antibody titers against the antigens from two-gene DNA vaccine cocktails, but lower titres when immunized with the three-gene cocktail. High numbers of IFN-gamma secreting splenocytes were generated predominantly against GRA7. Brain cyst burden was reduced by 81% in mice vaccinated with the three-gene mixture and they were completely protected against acute toxoplasmosis. Similar high levels of brain cyst reductions were obtained after vaccination with cocktails composed of GRA1 and GRA7 (89% reduction), or GRA7 and ROP2 (79% reduction), but not with the cocktail composed of GRA1 and ROP2. In low dose single-gene vaccinations, IFN-gamma and strong protection could only be elicited by GRA7. Hence, the presence of GRA7 in the DNA vaccine formulation was important for optimal protection and this was correlated with GRA7-specific IFN-gamma production. We propose GRA7 as a main component in cocktail DNA vaccines for vaccination against T. gondii.