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.

Microbiological Safety and Sensory Quality of Cultivated Mushrooms (Pleurotus eryngii, Pleurotus ostreatus and Lentinula edodes) at Retail Level and Post-Retail Storage.

In this study, the microbiological and sensory quality of cultivated mushrooms (Pleurotus ostreatus and eryngii and Lentinula edodes) available at the Austrian retail level were determined. Aerobic mesophilic bacteria (AMC), Enterobacteriaceae (EB), Pseudomonadaceae (PS), lactic acid bacteria (LAB), yeast, moulds and presumptive Bacillus cereus were enumerated at the day of purchase and after storage at 4 °C for 7 or 12 days. Additionally, the presence of Salmonella spp. and Listeria monocytogenes was investigated. Isolates of presumptive spoilage bacteria were confirmed by partial 16S rRNA sequencing. At the day of purchase, 71.2% of the samples were of high microbiological quality and grouped into the low contamination category (AMC < 5.0 log cfu/g), while the sensory quality of 67.1% was categorized as "very good or good". After storage, the number of samples with high microbial quality was 46.6%, and only 37.0% of the samples scored as "very good or good". The most abundant species across all mushroom samples were the Pseudomonas fluorescens species complex (58.4%) and the potential mushroom pathogen Ewingella americana (28.3%). All mushroom samples tested negative for Salmonella spp., L. monocytogenes and Bacillus cereus. The microbiological and sensory quality of the analysed mushrooms at the day of purchase and after storage was considered to be good overall. Longer transport distances were found to have a significant influence on the microbiological and sensory quality.

Pulsed-Light Treatment of Dried Parsley: Reduction of Artificially Inoculated Salmonella and Impact in Given Quality Parameters.

ABSTRACT: Dried parsley is regularly contaminated with foodborne pathogens, especially Salmonella. Application of contaminated ingredients in ready-to-eat dishes without further thermal treatment represents a considerable health risk. This study examined the suitability of pulsed light as a novel decontamination method of Salmonella in dried parsley, along with the impact on selected quality parameters (chlorophyll content, phenolic compounds, color, and odor) and product characters (temperature and water activity value). Samples were inoculated with one of three Salmonella isolates (Salmonella Cerro or one of two isolates of Salmonella Agona) at two contamination levels of 103 or 107 CFU/g and treated under various experimental factors, including distance to the light source and exposure time, resulting in fluences in the range of 1.8 to 19.9 J/cm2. At selected parameter settings (9.8 and 13.3 J/cm2), the effect of prolonged storage time (48 h) of inoculated samples before treatment on the reduction of Salmonella Cerro was examined. Samples treated at the same fluences were also stored for 35 days at 22 to 25°C. The three Salmonella isolates were significantly reduced by pulsed light (P < 0.05). Reduction factors ranged between 0.3 and 5.2 log CFU with varying sensitivities of the isolates. In general, increasing fluences (depending on exposure time and distance to the light source) resulted in increasing reductions of Salmonella. However, on closer examination, exposure time and distance to the light source had a varying influence on the reduction of the different Salmonella isolates. Decreasing reduction factors were observed by increasing the contamination level and prolonging the storage time of inoculated samples before treatment. No undesirable changes in quality parameters and sensory analysis were detectable at fluences of 9.8 and 13.3 J/cm2, indicating that pulsed light may be a suitable alternative for the decontamination of dried parsley.

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.

Impact of Selected Factors on the Occurrence of Contact Dermatitis in Turkeys on Commercial Farms in Germany.

In a long term research project in Germany the influence of husbandry on the health of fattening turkeys (Study 1) as well as the influence of practiced rearing conditions on the health of turkey poults (Study 2) was examined in 24 farms and at the meat processing plant. In all examined rearing farms, litter samples for the determination of litter moisture were taken. This paper summarizes the results obtained by our working group from 2007 until 2012. The results elucidate the universal problem of foot pad dermatitis (FPD). Nearly 100% of the observed turkeys showed a clinically apparent FPD at the meat processing plant. Furthermore, skin lesions of the breast, especially breast buttons were diagnosed, particularly at the slaughterhouse. FPD was detected in the first week of the rearing phase. Prevalence and degree showed a progressive development up to the age of 22-35 days, whereas 63.3% of the poults had foot pad alterations. As even mild alterations in the foot pad condition can be indicators for suboptimal design of the rearing environment, especially high litter moisture, it is important to focus on the early rearing phase.

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.

[Examination of the prevalence of skin injuries in debeaked fattened turkeys]. / Untersuchungen zur Prävalenz von Hautverletzungen bei schnabelkupierten Mastputen.

Within the framework of a cooperative research project, turkey health as well as numerous aspects of animal welfare were examined in various intensive commercial farms with varying rearing forms. The present study demonstrates the prevalence of skin injuries concerning living animals as well as carcasses of slaughtered turkeys of both sexes. Although all turkeys were debeaked, prevalences of skin injuries at a value of 12.8% with male turkeys and 13.8% with female turkeys could be found at the age of 16 weeks. Within the scope of medically inspections skin injuries were mainly established in the head region, especially the snood as well as in the region of the back. By means of their clinical picture pecking wounds and scratch injuries could be distinguished. Skin lesion produced primary as a result of scratching the skin surface, could be enlarged by the animal itself or by conspecifics by beak pecking, especially after bleeding, in spite of debeaked upper beak. Injuries in the head region, especially of the snood, could be explained mainly as a result of pecking by conspecifics by reason of their clinical picture and were found mainly in male turkeys. Skin injuries in the region of the back, especially at the coxal tuberosity, were identified primarily as scratch marks. They were found mainly in female turkeys. A statistically significant correlation of the injury prevalence to particular husbandry parameters discussed in the literature as "predisposing" or "limiting" factors (e.g., population density, light regime or offer of employment material) could not be established in this study. At the meat inspection fresh scratching injuries at the chest and the hind legs were diagnosed most often. Especially fresh injuries of the carcass point to a misguided behaviour of the staff concerning animal handling during loading and transport which is not conform to animal welfare.

[Examination of rearing standards and health status in turkeys in Germany]. / Erhebungen zur Haltung und Gesundheit bei Mastputen in Deutschland.

Within the framework of a cooperative research project, sponsored by the Federal Ministry of Food, Agriculture and Consumer Protection in Germany (BMELV), turkey health as well as numerous aspects of animal welfare in various intensive commercial farms with varying rearing forms were examined. For this purpose extensive documentation of flock management and health status information was conducted over the past two years at the farms. Additional information was ascertained from the carcasses at the slaughterhouse. The first results of this study, which look at flock management, will be presented here. Upon reviewing the questionnaires, it is clear that on one hand almost all farmers orientate there stocking rate upon the allowed limits, on the other hand, however, when considering national general requirements, which are very important parameters thatargely influence animal health (i. e. litter quality and care of sick/injured animals) farmer compliance lacks. Especially the litter quality, which, among other parameters, plays a very important role in food pad health, proved to be unsatisfactory. Unfortunately, no instruments are available that measure the litter quality, yet. The status of footpad health was a major problem in all the rearing systems. However, the prevalence of epithelial necrosis and deep skin lesions varied immensely among different flocks of the same age.