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.

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.

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 Toxoplasma 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.

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.

Clinical, epidemiological and cost aspects of contact lens related infectious keratitis in Belgium: results of a seven-year retrospective study.

AIM: Evaluation of the clinical, epidemiological and cost aspects of contact lens related infectious corneal ulcers requiring hospitalisation. METHODS: A retrospective analysis was performed on the files of patients hospitalised for contact lens induced corneal ulcer in the eight Belgian University Hospitals over a seven-year period (January 1997 until December 2003). For all hospitalised patients registration of the diagnosis is compulsory using the International Code of Diagnostics (ICD-9). RESULTS: 107 patients with contact lens related corneal ulcer were included. The great majority, 99 subjects, used soft contact lenses, of which 9 were disposables, 73 planned replacement and 17 conventional lenses. Only 6 patients were night and day wearers. Three patients used daily disposable lenses. The most frequently cultured organisms were Pseudomonas and other Gram-negative germs (70%) and Acanthamoeba (16%). The majority (77%) of the corneal ulcerations were localised centrally which resulted in an average visual loss of 4 lines. In 16 patients a corneal graft was performed and one eye had to be eviscerated. CONCLUSION: Despite important technological improvements in contact lens materials and care systems, the problem of infectious ulceration has all but disappeared. On the contrary, during the study period, the number of patients hospitalised increased from 5 in 1997 to 22 in 2003, which is only partially explained by the increasing prevalence of lens wearers: 3,5% of the Belgian population in 1995 and 6,5% in 2003.

An unusual cause of abdominal pain.

A 36-year-old woman presented to the Emergency Room because of abdominal pain associated with hematuria and red blood blending to stool. On admission, the physical examination revealed abdominal tenderness and diffuse cutaneous hematoma. The laboratory findings showed abnormal clotting tests with high International Normalised Ratio (INR) and prolonged activated partial thromboplastin time. Hemoperitoneum and ureteral hematoma were noted on the abdomen computed tomography. The patient confessed she had ingested difenacoum for several weeks. All the symptoms resolved with fresh frozen plasma perfusion and vitamin K.