Influence of a nanoscale coating on plucking fingers and stainless steel on attachment and detachment of Salmonella Enteritidis, Escherichia coli and Campylobacter jejuni.

Gastroenteritis caused by Campylobacter represents the most common reported foodborne bacterial illness worldwide, followed by salmonellosis. Both diseases are often caused by the consumption of contaminated, insufficiently heated poultry meat. This can result from contamination of the meat during the slaughtering processes. Food contact surfaces like stainless steel or plucking fingers contribute significantly to cross-contamination of poultry carcasses. Modification of these surfaces could lead to a reduction of the bacterial burden, as already proven by successful application in various food industry sectors, such as packaging.In this study, nanoscale silica-coated and uncoated stainless-steel surfaces and plucking fingers were compared on a pilot scale regarding attachment and detachment of Campylobacter jejuni, Salmonella Enteritidis and Escherichia coli.The bacteria did not adhere less to the coated plucking fingers or stainless-steel sections than to the uncoated ones. The coating also did not lead to a significant difference in detachment of Campylobacter jejuni, Salmonella Enteritidis and Escherichia coli from the investigated surfaces compared to the uncoated ones.Our study did not reveal any differences between the coated and uncoated surfaces with regard to the investigated bacteria. In order to achieve a better adaptation of the coating to slaughterhouse conditions, future studies should focus on its further development based on the investigation of specific coating parameters.

Impact of nanoscale silicon dioxide coating of stainless-steel surfaces on Listeria monocytogenes.

High resistance to environmental factors as well as the ability to form biofilms allow Listeria monocytogenes to persist for a long time in difficult-to-reach places in food-producing plants. L. monocytogenes enters final products from contaminated surfaces in different areas of plants and poses a health risk to consumer. Modified surfaces are already used in the food industry to prevent cross-contamination. In this study, stainless-steel surfaces were coated with nanoscale silicon dioxide and the effects on attachment, bacterial growth and detachment of L. monocytogenes were evaluated. Attachment was considered for three different ways of application to simulate different scenarios of contamination. Bacterial growth of L. monocytogenes on the surface was recorded over a period of up to 8 h. Detachment was tested after cleaning inoculated stainless-steel surfaces with heated distilled water or detergent. Coating stainless-steel surfaces with nanoscale silica tends to reduce adherence and increased detachment and does not influence the bacterial growth of L. monocytogenes. Further modifications of the coating are necessary for a targeted use in the reduction of L. monocytogenes in food-processing plants.

Prevalence and molecular characterization of Toxoplasma gondii in different types of poultry in Greece, associated risk factors and co-existence with Eimeria spp.

Toxoplasma gondii is a protozoan parasite of public health importance, infecting all warm-blooded animals, including chickens. Undercooked chicken meat or relevant products such as sausages could lead to human infections. In free-range, organic and slow-growth farming systems where the susceptibility period for chickens is extended, more knowledge about potential risk factors is essential. This study is the first seroepidemiological survey in different regions and types of chicken farms in Greece, using a major tachyzoite surface antigen-based ELISA (TgSAG1), combined with magnetic-capture PCR (mc-PCR) and bioassay for the isolation of strains from the chickens' tissues. Potential risk factors for T. gondii infection in these hosts were also investigated. Additionally, the co-existence of T. gondii and Eimeria spp. infections was assessed to elucidate epidemiological links between these two protozoan infections. Overall T. gondii seroprevalence was 9.5%. Of the backyard chickens sampled, 41.2% were seropositive and 70% of the organic and free-range layer farms had at least one T. gondii seropositive hen. No serologically positive broilers were found, although mc-PCR revealed a positive sample, highlighting the importance of accurate early-infection direct detection of T. gondii infections to ensure public health. T. gondii isolates obtained by mouse bioassay were genotyped. All belonged to type II (ToxoDB#3) as confirmed also by microsatellite typing. Production system, type of nutrition, and feeding system automation were identified as the most significant risk factors, while no association was found between the presence of cats and T. gondii seropositivity as calculated on both a farm level and per individual bird sampled.

Direct detection and quantification of Toxoplasma gondii in meat samples from feral raccoons (Procyon lotor) in Germany by magnetic-capture real-time PCR.

Because the number of wild raccoons in Germany is increasing constantly, it appears to be economic reasonable to use their meat as food. For this purpose, it is essential to generate data regarding the pathogen load of the meat to be consumed and handled. It is known that raccoons, particularly in Germany, show a high seroprevalence of Toxoplasma gondii. Because serological data only indicates contact of a host to a parasite additional direct detection is needed to prove presence of parasitic stages in particular tissues. Therefore, a total of 150 samples from raccoons with known serostatus were tested and quantified using magnetic-capture real-time PCR for Toxoplasma gondii. As it represents potentially consumption-relevant parts of raccoons, meat from forelimb and hindlimb was examined. Samples were stratified into three groups based on the animals' serostatus (each 50 negative, low positive, and high positive). All samples from seronegative animals were found negative by MC-PCR as well. In a total of 56 meat samples from 100 seropositive animals, T. gondii DNA was detected. Statistically significant more samples were positive by MC-PCR in the high positive than in the low positive serostatus group (38/50 vs. 18/50, p < 0.0001). Furthermore, samples from the former group were also found to have statistically significant higher DNA equivalent values compared to samples from the low positive serostatus group (p < 0.0001). These results suggest that meat from seropositive raccoons may contain considerable numbers of T. gondii presenting a potential public health risk for humans whilst handling and consumption.

Toxoplasma gondii in raccoons (Procyon lotor) in Germany: a serosurvey based on meat juice.

Toxoplasma gondii seroprevalence was determined in meat juice samples of 820 free-living raccoons from Germany. The animals were collected between December 2017 and April 2021. Using a commercial enzyme linked immunosorbent assay (ELISA), the overall seroprevalence was found to be 48.5%. Statistical analysis revealed significant seroprevalence differences between seasons, sex, and weight of analysed raccoons. The prevalence in late winter/spring (57.7%) was significantly higher than in autumn (38.4%) (p < 0.0003). Male raccoons (50.5%) were more often seropositive than females (41.0%) (p = 0.028). Increasing animal weight had a significant impact on the relative probability of a positive serostatus (odds ratio: 1.783, p < 0.0001). Furthermore, we found regional differences in seroprevalence, but there was no statistically significant difference resulting from animal age, degree of habitat urbanization and hunting year. Meat juice is a suitable medium for serological surveys for T. gondii in meat producing animals, as sampling is even possible after slaughter or during meat inspection when blood is no longer available. The observed high seroprevalence indicates that T. gondii infection is widespread among the German raccoon population providing a potentially relevant source of T. gondii transmission to humans upon consumption or handling of animal products.

Fluorescent bead-based serological detection of Toxoplasma gondii infection in chickens.

BACKGROUND: Free-ranging chickens are often infected with Toxoplasma gondii and seroconvert upon infection. This indicates environmental contamination with T. gondii. METHODS: Here, we established a bead-based multiplex assay (BBMA) using the Luminex technology for the detection of T. gondii infections in chickens. Recombinant biotinylated T. gondii surface antigen 1 (TgSAG1bio) bound to streptavidin-conjugated magnetic Luminex beads served as antigen. Serum antibodies were detected by a fluorophore-coupled secondary antibody. Beads of differing color codes were conjugated with anti-chicken IgY or chicken serum albumin and served for each sample as an internal positive or negative control, respectively. The assay was validated with sera from experimentally and naturally infected chickens. The results were compared to those from reference methods, including other serological tests, PCRs and bioassay in mice. RESULTS: In experimentally infected chickens, the vast majority (98.5%, n = 65/66) of birds tested seropositive in the BBMA. This included all chickens positive by magnetic-capture PCR (100%, n = 45/45). Most, but not all inoculated and TgSAG1bio-BBMA-positive chickens were also positive in two previously established TgSAG1-ELISAs (TgSAG1-ELISASL, n = 61/65; or TgSAG1-ELISASH, n = 60/65), or positive in an immunofluorescence assay (IFAT, n = 64/65) and in a modified agglutination test (MAT, n = 61/65). All non-inoculated control animals (n = 28/28, 100%) tested negative. In naturally exposed chickens, the TgSAG1bio-BBMA showed a high sensitivity (98.5%; 95% confidence interval, CI: 90.7-99.9%) and specificity (100%; 95% CI: 85.0-100%) relative to a reference standard established using ELISA, IFAT and MAT. Almost all naturally exposed chickens that were positive in bioassay or by PCR tested positive in the TgSAG1bio-BBMA (93.5%; 95% CI: 77.1-98.9%), while all bioassay- or PCR-negative chickens remained negative (100%; 95% CI: 85.0-100%). CONCLUSIONS: The TgSAG1bio-BBMA represents a suitable method for the detection of T. gondii infections in chickens with high sensitivity and specificity, which is comparable or even superior to other tests. Since assays based on this methodology allow for the simultaneous analysis of a single biological sample with respect to multiple analytes, the described assay may represent a component in future multiplex assays for broad serological monitoring of poultry and other farm animals for various pathogens.

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.

Distribution of Alaria spp. mesocercariae in waterfrogs.

The distribution of Alaria-spp.-mesocercariae within the host is relevant for the examination via Alaria spp. mesocercariae migration technique (AMT) regarding predilection sites and may indicate an interaction between parasite and host. Naturally Alaria-exposed frogs of Pelophylax species (n = 13) were examined for systemic distribution and localization-specific parasite density of Alaria spp. mesocercariae. The frogs were necropsied and their body was divided into the following localizations: inner organs, head, torso, forelimbs, and hind limbs. The localizations were analyzed individually and in toto using Alaria spp. mesocercariae migration technique. Our results showed neither statistical differences concerning the number of mesocercariae in the different localizations nor in respect of the rate of positive localizations. Therefore, an accumulation in a particular predilection site seems unlikely. Further research on a representative sample is necessary before final conclusions can be drawn.

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.

Atmospheric pressure plasma jet treatment of Salmonella Enteritidis inoculated eggshells.

Contamination of eggshells with Salmonella Enteritidis remains a food safety concern. In many cases human salmonellosis within the EU can be traced back to raw or undercooked eggs and egg products. Atmospheric pressure plasma is a novel decontamination method that can reduce a wide range of pathogens. The aim of this work was to evaluate the possibility of using an effective short time cold plasma treatment to inactivate Salmonella Enteritidis on the eggshell. Therefore, artificially contaminated eggshells were treated with an atmospheric pressure plasma jet under different experimental settings with various exposure times (15-300s), distances from the plasma jet nozzle to the eggshell surface (5, 8 or 12mm), feed gas compositions (Ar, Ar with 0.2, 0.5 or 1.0% O2), gas flow rates (5 and 7slm) and different inoculations of Salmonella Enteritidis (101-106CFU/cm2). Atmospheric pressure plasma could reduce Salmonella Enteritidis on eggshells significantly. Reduction factors ranged between 0.22 and 2.27 log CFU (colony-forming units). Exposure time and, particularly at 104CFU/cm2 inoculation, feed gas had a major impact on Salmonella reduction. Precisely, longer exposure times led to higher reductions and Ar as feed gas was more effective than ArO2 mixtures.

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.

Effect of temperature on the survival of Alaria alata mesocercariae.

Recent findings of Alaria alata mesocercariae in wild boars and other animals in Europe reinforced the concern about the public health risk posed by this parasite especially if the game meat is insufficiently heated during preparation. Cooking and freezing are effective methods for the inactivation of parasites in meat whereas refrigeration is considered as an essential part of the Good Hygiene Practice. Additionally, microwave dielectric heating may represent an equally effective tool for parasite inactivation. Therefore, isolated vital mesocercariae were examined with respect to their resilience against heating, refrigeration, freezing, and microwave heating. A. alata mesocercariae stored in Ringer's solution do not survive heating temperatures that exceed 60.0 °C. Similarly, exposure to microwave heating ensured an inactivation of all parasite developmental stages after 90 s of treatment. In contrast, the parasites' tolerance towards cold is far higher as the mesocercariae survived refrigeration temperatures (4.0 ± 2 °C) in Ringer's solution for up to 13 days. An effective inactivation by cold is therefore only guaranteed if the infested game meat is frozen to a core temperature of -13.7 °C for a minimum of 2 h at least. Game meat should be handled with the same or even higher caution than meat of husbandry animals since wild animals may be infected with parasites or other zoonotic agents that are not common in livestock. It is therefore of crucial importance that appropriate temperature time protocols are used for the reliable inactivation of these zoonotic agents.

Effects of in vitro conditions on the survival of Alaria alata mesocercariae.

The aim of this study was to determine the effect of different concentrations of table salt (NaCl) and ethanol (v/v) solutions on the viability of Alaria alata mesocercariae. Furthermore, the survival of A. alata mesocercariae during simulated human gastric digestion was evaluated. For this purpose, A. alata mesocercariae migration technique (AMT) was used for the isolation of the parasite from high-positive A. alata mesocercariae meat from wild boar, raccoon, raccoon dog, and badger meat. In total, we have studied the behavior of 582 larvae under different conditions (NaCl, ethanol, and artificial gastric juice) in three independent in vitro experiments. The larvae survived at a NaCl concentration of up to 2.0% until day 21 with a median survival time of 11 days. At 3.0% NaCl concentration, the larvae lost their vitality after less than 24 h. In addition, it was found that ethanol concentrations from 8.0 to 70.0% were effective at reducing survival of A. alata mesocercariae within a short period of time (<1 min). Finally, our studies have revealed that it required 120 min to reliably inactivate all A. alata mesocercariae within HCl-pepsin digestion solution with a pH of 1.5-2.0 at 37°C. Consequently, the results showed that 3.0% is the minimum concentration of NaCl in meat products recommended for human consumption because at lower NaCl concentration the parasite survived for a substantial period of time. Finally, the common concentrations of ethanol used for the disinfection of surfaces in household and/or laboratory, are sufficient for the inactivation of A. alata mesocercariae.

Humoral immune responses in chickens and turkeys after infection with Toxoplasma gondii by using recombinant antigens.

Toxoplasma gondii is a parasite which can be transmitted to humans via the consumption of contaminated meat products derived from different animal species, e.g., poultry. In Europe, the consumption rate of poultry meat is high and may pose a risk for humans. However, little is known about the prevalence and immune response against T. gondii in these animals. Based on these circumstances, we experimentally infected 18 turkeys and 16 chickens with the parasite. Turkeys were infected either with tachyzoites on different routes or with various amounts of oocysts. In contrast, chickens were only infected with different doses of oocysts. The immunoglobulin (Ig) Y humoral immune responses of these animals were investigated in a lineblot assay against the recombinant T. gondii antigens rGRA1, rGRA6, rGRA9, rSAG1, and rSUB1. By using the recombinant antigens rGRA6, rGRA9, and rSUB1 in the lineblot assay, we found a correlation between the humoral immune response and the parasite stage in turkeys. Thereby, an infection with oocysts induced a stronger, permanent long-lasting antibody response compared to tachyzoite-infected animals. Only a minor relation between the oocyst infection dose and the manifestation of the immune response in chickens was found 7 days post infection (dpi) by using rGRA1 and rGRA9. However, an inconstant detection of antigen-specific IgY antibodies in the lineblot assay seems not to be a sufficient method for the identification of a Toxoplasma infection in chickens. In contrast, the detection of anti-rGRA6, anti-rGRA9, and anti-rSUB1 IgY antibodies showed potential for the identification of an infection in turkeys.

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.