Toxoplasma gondii infections in chickens - performance of various antibody detection techniques in serum and meat juice relative to bioassay and DNA detection methods.

Chickens, especially if free-range, are frequently exposed to Toxoplasma gondii, and may represent an important reservoir for T. gondii. Poultry products may pose a risk to humans, when consumed undercooked. In addition, chickens are regarded as sensitive indicators for environmental contamination with T. gondii oocysts and have been used as sentinels. The aim of the present study was to determine the suitability of commonly used antibody detection methods, i.e. the modified agglutination test (MAT), IFAT and ELISA to detect T. gondii-infected chickens. Samples of experimentally and naturally infected chickens were used. The infection state of all chickens was determined by Magnetic-Capture (MC-) real-time PCR (RT PCR). Naturally exposed chickens were additionally examined by mouse bioassay and conventional RT PCR on acidic pepsin digests (PD-RT PCR). Blood serum and meat juice of various sources were tested for antibodies to T. gondii. In naturally infected chickens, there was substantial agreement between the mouse bioassay and MC-RT PCR or the mouse bioassay and conventional PD-RT PCR. PD-RT PCR was slightly more sensitive than MC-RT PCR, as all (26/26) bioassay-positive chickens also tested positive in at least one of the tissues tested (heart, drumstick). By MC-RT PCR, 92.3% (24/26) of the naturally infected bioassay-positive chickens were positive. The diagnostic sensitivity of MC-RT PCR was clearly related to the organ examined. Based on a quantitative assessment of the MC-RT PCR results in experimentally infected chickens, brain and heart tissues harbored an at least 100 times higher parasite concentration than breast, thigh or drumstick musculature. In naturally infected chickens, only three out of 24 birds, which were MC-RT PCR-positive in heart samples, also tested positive in drumstick musculature. Under experimental conditions, the agreement between MC-RT PCR and the serological techniques revealed 100% diagnostic sensitivity and specificity. Under field conditions, examinations of sera by ELISA, IFAT and MAT showed good performance in identifying chickens that were positive in either a mouse bioassay, MC-RT PCR, or PD-RT PCR as illustrated by diagnostic sensitivities of 87.5%, 87.5% and 65.2%, respectively, and diagnostic specificities of 86.2%, 82.8% and 100%, respectively. The examination of meat juice samples from breast, drumstick or heart musculature revealed similar or even better results in the ELISA. The results in the MAT with meat juice from breast musculature were less consistent than those of ELISA and IFAT because a number of negative chickens tested false-positive in the MAT. The MAT performed similar to ELISA and IFAT when applied to test meat juice samples collected from heart, thigh or drumstick musculature.

Experimental Toxoplasma gondii and Eimeria tenella co-infection in chickens.

The widespread apicomplexan parasites Toxoplasma gondii (T. gondii) and Eimeria tenella (E. tenella) are important pathogens with high prevalence in poultry. The aim of our study was the investigation of mutual influences in co-infected chickens, focusing on immune response and course of infection. Two separate trials were performed using in total 96 1-day-old chickens, divided into four study groups: group NC (negative control, uninfected), group PC-T (oral or intramuscular infection with T. gondii oocysts (trial 1) or tachyzoites (trial 2), respectively), group PC-E (oral infection with E. tenella (trial 1) or E. tenella and Eimeria acervulina (trial 2)), and group TE (co-infection). T. gondii and Eimeria infections were validated by different parameters, and cytokine expression in the gut and spleen was investigated. T. gondii-specific antibodies were detected earliest 4 days post infection (p.i.) by immunoblot and direct DNA detection was possible in 22.1% of all tissue samples from infected chickens. Eimeria spp. merogony seemed to be enhanced by co-infection with T. gondii, interestingly without marked differences in oocyst excretion between co-infected and Eimeria spp. mono-infected chickens. An increase of messenger RNA (mRNA) expression of Th1- (IFN-γ, IL-12, TNF-α) and Th2-related cytokines (IL-10) mainly in groups PC-E and TE was observed, however, without statistically significant differences between co-infection and single infection with Eimeria. In conclusion, most of the measurable immune response could be attributed to Eimeria infection. To the best of our knowledge, this is the first report on co-infection experiments of T. gondii with Eimeria spp. in chickens.

Persistence of Toxoplasma gondii tissue stages in poultry over a conventional fattening cycle.

Toxoplasma gondii is a widely spread protozoon in humans, mammals and poultry. Regarding the latter, nothing is known yet about the duration of T. gondii persistence and distribution over a conventional fattening cycle of turkeys and chickens. Twenty-four turkeys and 12 broiler chickens were infected intravenously with 1×10(6) T. gondii tachyzoites (strain NED). Serum antibody levels were determined weekly by ELISA (turkeys) or immunofluorescent antibody test (chickens). Turkeys were slaughtered at 4, 8, 12 and 16 weeks post-infection (p.i.), and chickens 5 or 10 weeks p.i. (n = 6 per group). Sixteen different tissue samples per bird were analysed for T. gondii by PCR. All infected animals showed seroconversion. In turkeys, 15.9% of all samples were tested positive for T.-gondii-DNA. Among the edible tissues (drumstick, thigh, breast muscle, heart, liver and gizzard) 7.8% tested positive. Among poultry slaughtered after different periods of time after infection no significant differences (P>0.05) regarding the number of positive samples were observed. Only 4 out of 192 samples (2.1%) from infected chickens contained detectable T. gondii DNA.The PCR findings suggested that T. gondii may persist in poultry. Particularly in turkey it was shown that edible tissues stay infected for at least 16 weeks p.i. which indicates a potential risk for consumers of undercooked turkey meat whereas chickens appear less susceptible to T. gondii infection.

Effects of pH, sodium chloride, and curing salt on the infectivity of Toxoplasma gondii tissue cysts.

Toxoplasma gondii is one of the most common zoonotic parasites in the world. The parasite causes no or mild symptoms in immunocompetent humans. However, a high potential hazard exists for seronegative pregnant women and immunocompromised patients. The consumption of meat containing tissue cysts or oocyst-contaminated vegetables and fruits or the handling of cat feces poses a high risk of infection with T. gondii. It is known that raw minced meat, raw fresh sausages, and locally produced raw meat products are possible causes of T. gondii infection. The infectivity of T. gondii tissue cysts in meat products depends, among other factors, on the pH and the salt concentration. Therefore, the impact of these two factors on the tissue cysts was examined. For this purpose, dissected musculature and brain from experimentally infected mice (donor mice) were placed in a cell culture medium (RPMI 1640). The medium was adjusted to different pH values (pH 5, 6, and 7) with lactic acid and to different salt concentrations (2.0, 2.5, and 3.0%) with sodium chloride (NaCl) or nitrite-enriched curing salt (NCS) for the various tests. After storage at 4°C for different time periods, the materials were fed to bioassay mice. Later, the brains were examined for presence of T. gondii to assess the infectivity. The data show that T. gondii tissue cysts have a high pH tolerance. Cysts were infectious in the muscle for up to 26 days (pH 5). In contrast to their tolerance to pH, cysts were very sensitive to salt. Muscle cysts survived at an NaCl concentration of up to 2.0% only, and for no longer than 8 days. At NaCl concentrations of 2.5 and 3.0%, the cysts lost their infectivity after 1 day. When NCS instead of NaCl was used under the same conditions, T. gondii muscle cysts retained infectivity for only 4 days at 2.0%. Consequently, NCS (NaCl plus 0.5% nitrite) has a stronger effect on T. gondii cysts than does common table salt. Sausages produced with low NaCl concentration and short contact times pose a potential risk for susceptible individuals.

Experimental Toxoplasma gondii oocyst infections in turkeys (Meleagris gallopavo).

Toxoplasma (T.) gondii is a protozoan parasite with a broad range of intermediate hosts. Humans are often infected by ingestion of tissue cysts in raw or undercooked meat or meat products. Turkeys as food-producing animals can also serve as intermediate hosts. The aim of the present study was to investigate occurrence and predilection sites of T. gondii infection in turkeys after oral infection with oocysts. Experimental infections with different doses of T. gondii oocysts were performed in 36 turkeys to mimic natural infection. Systemic distribution of parasitic stages was investigated by screening 14 different tissues including the edible tissues heart, liver, thigh, breast and drumstick muscle. Parasite detection was based on a conventional nested polymerase chain reaction (PCR). Animals were sacrificed 6-12 weeks after infection. Results demonstrated parasite spreading over the whole organism after oral infection by oocysts. Most frequently affected tissues were brain (47.2% of all brains were positive for T. gondii) and thigh muscle (25.0% positive samples). Other muscles were regularly T. gondii-positive, all other sampled tissues were positive at least once. Thus, edible tissues are one of the predilection sites of T. gondii in turkeys which renders raw or undercooked turkey meat a potential risk for parasite transmission to humans. Data were compared to results from previous parenteral turkey infections with tachyzoites. With the exception of brain, liver and breast muscle affection, no significant differences were observed between both infection routes. Both infection models could be used for research purposes with certain advantages and disadvantages.

Tissue tropism of Toxoplasma gondii in turkeys (Meleagris gallopavo) after parenteral infection.

Turkeys are known to be natural hosts for the zoonotic protozoan parasite Toxoplasma gondii. The objective of the present study was to gain further knowledge of possible predilection sites of T. gondii infection in this species after parenteral application of tachyzoites. A total of 38 turkeys were infected with different doses of T. gondii tachyzoites. Birds were killed either 6 to 8 or 10 to 12 weeks after the experimental infection. Fourteen different tissues per bird were investigated by a nested polymerase chain reaction (PCR) for the presence of the parasites' DNA. T. gondii DNA was found in any type of tissue analysed; in 86.1 % of all infected birds, at least one sample was tested positive. Over all intravenously infected birds, 15.4 % of all analysed samples contained T. gondii DNA. Most frequently affected tissues were liver (43.3 % positive samples), breast muscle (26.7 % positive samples) and heart (20.0 % positive samples), while the brain was less frequently positive (6.7 %). The number of positive tissues varied from zero to seven tissues per animal with at least one T. gondii-positive edible tissue sample in 80 % of all intravenously infected birds. Still, the results did not indicate defined target tissues or a cyst distribution pattern. Nonetheless, edible organs were most frequently parasitised. The number of positive findings did not differ between the early and the late examination time points. Therefore, a persistence of the tissue stages until the end of the study (12 weeks after infection) is concluded.