Development of an in vivo model for Toxoplasma gondii infections in chickens and turkeys simulating natural routes of infection.

Turkeys and chickens were orally infected with tissue cysts (one mouse brain) or oocysts (103, 105 or 106 oocysts) of three T. gondii strains of the clonal types II and III (ME49, CZ-Tiger, NED) to investigate the influence of the applied T. gondii strain and infective doses on the distribution of T. gondii in several organs and tissues and the serologic response of chickens and turkeys. Organ samples from 16 different tissues, including heart, brain, muscles and gizzard were analyzed by PCR. Brain and heart were found most frequently positive for T. gondii DNA in both species, followed by gizzard. Serological analysis with kinetic ELISA for turkey samples and IFAT for chicken samples were performed once a week. In both species a dose-depending serological response was found. Turkeys seroconverted one week after infection with CZ-Tiger strain and medium and high doses of ME49 oocysts. In chickens, infection with medium and high doses of CZ-Tiger led to seroconversion one week p.i. Frequency of T. gondii positive organs showed a trend of a dose-effect in both species after infection with the type II strains. The NED strain showed low virulence in chickens and turkeys, demonstrated by clearly less T. gondii positive organs. Infection with tissue cysts of all three strains revealed T. gondii stages in tissues of turkeys and chickens. In conclusion, our data show a risk for human infection with T. gondii due to consumption of chicken and turkey meat.

Validation of EN ISO method 10273 - Detection of pathogenic Yersinia enterocolitica in foods.

EN ISO 10273 method for the detection of pathogenic Yersinia enterocolitica in foods was validated in the project Mandate M/381 funded by European Commission. A total of 14 laboratories from five European countries participated in the interlaboratory study (ILS) organized during 2013 and 2014. Before the ILS, the method was revised by an international group of experts and the performance of the revised method was assessed in an ILS study. The results are published as a part of the standard EN ISO 10273 revision. The study included three rounds with different sample types; raw milk, iceberg lettuce and minced meat, inoculated with a low and high level of pathogenic Y. enterocolitica strains representing major pathogenic bioserotypes 4/O:3 and 2/O:9. The homogeneity and stability of the samples were verified before dispatching them to the laboratories. The results demonstrated the method sensitivity of 96% in raw milk, 97% in minced meat, and 98% in lettuce at high inoculation level of pathogenic Y. enterocolitica. The specificity was 100% in raw milk, 96% in minced meat, and 98% in lettuce. The level of detection, LOD50, varied between study rounds, being 9.4 CFU/25 ml in raw milk, 9.9 CFU/25 g in minced meat and 63 CFU/25 g in lettuce samples. During the study, confirmation by using real-time PCR method ISO/TS 18867 together with pyrazinamidase testing was also validated, as alternative to conventional biochemical confirmation. When comparing different isolation steps used in the revised method during the study rounds, PSB enrichment and plating on CIN after alkaline (KOH) treatment showed the highest sensitivity (52-92%) in raw milk and minced meat samples. In lettuce samples, however, ITC with KOH treatment before plating on CIN showed higher sensitivity (64% at low level; 82% at high level) than plating on CIN from PSB with KOH treatment (44% at low level; 74% at high level). Statistical analysis of different isolation steps supported the use of two enrichment media, PSB and ITC, in the revised method. Recovery of pathogenic Y. enterocolitica on CIN was most efficient after KOH treatment and, based on the analysis, plating on CIN agar without KOH treatment could be left as optional procedure in the method.

Survival of Toxoplasma gondii tachyzoites in simulated gastric fluid and cow's milk.

The protozoan parasite Toxoplasma gondii is one of the most important food-related pathogens worldwide. Besides contact to oocysts or ingestion of tissue cysts mainly by consumption of raw or undercooked meat from infected animals, raw milk is considered to be a risk factor and possible route of transmission for tachyzoites. This stage of the parasite is usually very sensitive to acidic pH and, therefore, considered unlikely to survive stomach passage. However, tachyzoites were shown to survive for several days in milk and there are also reports on transmission of toxoplasmosis via milk. Thus, the aim of the study was to examine retention of infectivity of tachyzoites in simulated gastric fluid (SGF) of different acidity and to elucidate whether addition of different shares of milk would affect survival of the parasites. Tachyzoites were exposed to SGF of pH 2.0 through 6.0 and their remaining infectivity was examined by cell culture. Furthermore, the impact on survival was investigated in different admixtures of milk to the SGF (25, 50, 75%) as well as in pure milk. Tachyzoites were shown to retain infectivity in SGF of pH 5.0 and 6.0 for at least 90min while they were more sensitive to lower pH values. Admixture of milk resulted in extension of survival. The results support the hypothesis of tachyzoites to survive stomach passage and their retention of infectivity.

Quantitative detection of Toxoplasma gondii in tissues of experimentally infected turkeys and in retail turkey products by magnetic-capture PCR.

Magnetic-capture PCR was applied for the quantitative detection of Toxoplasma gondii in tissues of experimentally infected turkeys and retail turkey meat products. For experimental infection, three T. gondii strains (ME49, CZ-Tiger, NED), varying infectious doses in different matrices (organisms in single mouse brains or 10(3), 10(5), or 10(6) oocysts in buffer) were used. From all animals, breast, thigh, and drumstick muscle tissues and for CZ-Tiger-infected animals additionally brains and hearts were analyzed. Using the magnetic-capture PCR large volumes of up to 100 g were examined. Our results show that most T. gondii parasites are present in brain and heart tissue. Of the three skeletal muscle types, drumsticks were affected at the highest and breast at the lowest level. Type III strain (NED) seems to be less efficient in infecting turkeys compared to type II strains, because only few tissues of NED infected animals contained T. gondii DNA. Furthermore, the number of detected parasitic stages increased with the level of infectious dose. Infection mode by either oocyst or tissue cyst stage did not have an effect on the amount of T. gondii present in tissues. In retail turkey meat products T. gondii DNA was not detectable although a contact with the parasite was inferred by serology.

Prevalence of specific IgG-antibodies against Toxoplasma gondii in domestic turkeys determined by kinetic ELISA based on recombinant GRA7 and GRA8.

The protozoan parasite Toxoplasma (T.) gondii is one of the most common zoonotic infectious agents worldwide. Besides its sexual reproduction in cats, T. gondii can also infect a wide spectrum of other warm-blooded animals. These include animals used for human consumption such as pigs or chickens. Nevertheless, the role of turkeys for the epidemiology of T. gondii infections has not been studied thoroughly. We have established a kinetic ELISA (KELA) for the detection of T. gondii-specific IgG antibodies in turkey serum samples. The test is based on the recombinant dense granule antigens GRA7 and GRA8. These proteins were used as an antigen mixture at a concentration of 0.13 µg per well. The overall sensitivity of the assay was between 92.6% and 100% and the specificity ranged from 78.1% to 100%, depending on the method used to calculate these parameters. Using this KELA we examined 1913 turkey serum samples from 14 turkey farms from different areas of Germany. From these sera, 387 produced a signal in the KELA, corresponding to a true seroprevalence of up to 20.2%. The seropositivity rate in individual fattening cycles at individual farms ranged from 0.0% to 77.1%, whereas the rates were highly variable within the individual farms and individual fattening cycles. Consequently, conditions of animal husbandry could not be associated with particular seroprevalence rates. Although seropositivity cannot be linked directly to infectious tissue cysts in the muscle tissue of commercially produced turkey meat, we state that there is a potential risk of being infected by consuming turkey meat products that were not heat treated.