Comparison of Direct and Indirect Detection of Toxoplasma gondii in Ovine Using Real-Time PCR, Serological and Histological Techniques.

Toxoplasma gondii is a zoonotic pathogen and the ingestion of tissue cysts by consumption of lamb or mutton has been identified as a possible cause of infection in humans. Many serological surveys in sheep have been performed, showing relevant serological rates; however, while the detection of antibodies indicates an exposure to T. gondii, this does not necessarily imply the presence of tissue cysts in edible tissue. The current study aims to provide further understanding on the occurrence of T. gondii in sheep muscles and the strength of correlation between serological positivity and presence of the parasite in sheep. From 349 sheep, samples (i.e., blood, heart and diaphragm) were collected and subjected to ELISA tests, real-time PCR and histological tests. Despite the high seroprevalence, T. gondii DNA was detected in the heart and/or the diaphragm from 13 out of the 349 tested sheep (3.7%); all were adults (13/191). Furthermore, the histological tests did not reveal the presence of T. gondii tissue cysts in any of the examined portions of interventricular septum. It should be considered that the likelihood of detecting genetic material of the parasite is probably influenced by the uneven distribution of the tissue cysts in the carcass as well as the methodology applied. The findings of this study support the importance of describing the uncertainty associated with the data used for risk assessment to reduce inaccurate estimation or risk overestimation.

Cross-contamination in the kitchen: A model for quantitative microbiological risk assessment.

A quantitative microbiological risk assessment model for the cross-contamination transmission route in the kitchen (KCC) is presented. Bacteria are transmitted from contaminated (chicken) meat to hands, kitchen utensils, and other surfaces, subsequently contaminating a salad. The model aims to estimate the fraction of bacteria on the meat that is ingested due to cross-contamination, determine the importance of the different transmission routes, and assess the effect of scenarios (interventions) on the fraction ingested. The cross-contamination routes defined, bacterial source-to-recipient transfer fractions as available and derived from literature, and important characteristics (e.g., washing in cold water vs. hot water with soap) shaped the KCC model. With this model, 32 scenarios of an eight-step preparation of a "meat and salad" meal in a domestic kitchen were stochastically simulated. The "cutting board-salad" route proved dominant and the salad plays a major role in the final exposure. A realistic scenario (washing hands, cutting board, and knife with cold water after cutting the meat) estimates that a mean fraction of 3.2E - 3 of the bacteria on the meat is ingested. In the case of "hand washing with hot water and soap" and "cutting board and knife replacement," the mean fraction ingested is 3.6E - 6. For a subsequent meal, where the contaminated sources were kitchen fomites, the estimated mean fraction is 4.3E - 4. In case of hamburger, part of the bacteria is unavailable for cross-contamination, resulting in a mean fraction ingested of about 5.4E - 5. The role of the dishcloth in cross-contamination transmission proved to be minor.

Evaluation of the application for new alternative biodiesel production process for rendered fat including Category 1 animal by-products (BDI-RepCat® process, AT).

A new alternative method for the production of biodiesel from rendered fat, including animal by-product (ABP) Category 1 tallow, was evaluated. The method consists of a conversion phase, based on esterification and transesterification in a single step (at temperature ≥ 200°C, pressure ≥ 70 bar with a retention time ≥ 15 min), using MgO as a catalyst and in the presence of methanol (10-15%), followed by vacuum distillation (at ≥ 150°C, ≤ 10 mbar) of the end-product, biodiesel and the co-product, glycerine. Prions (PrPS c), which are abnormal isoforms of the prion protein, were considered by the applicant to be the most resistant hazard. In accordance with previous EFSA Opinions and current expert evaluation, a reduction in prion infectivity, or detectable PrPS c, of at least 6 log10 should be achieved for the process to be considered equivalent to the processing method laid down in the Regulation (EU) No 142/2011. Published data from an experimental replication of the conversion step of the biodiesel production process under consideration were provided, which showed an at least 6 log10 reduction in detectable PrPS c, by Western blot, in tallow that had been spiked with murine and human prion strains. In addition, it was demonstrated that the presence of methanol does not affect the recovery or detection of PrPS c from a biodiesel substrate. Based on scientific literature, the vacuum distillation step has been shown to be capable of achieving an additional 3 log10 reduction in PrPS c. Therefore, the proposed alternative method is considered to be at least equivalent to the processing method laid down in the legislation for the production of biodiesel from raw materials including Category 1 ABP.

Modelling and magnitude estimation of cross-contamination in the kitchen for quantitative microbiological risk assessment (QMRA).

In the kitchen of the consumer, two main transmission routes are relevant for quantitative microbiological risk assessment (QMRA): the cross-contamination route, where a pathogen on a food product may evade heating by transmission via hands, kitchen utensils and other surfaces, e.g. to non-contaminated products to be consumed raw; and the heating route, where pathogens remain on the food product and are for the most part inactivated through heating. This project was undertaken to model and estimate the magnitude of cross-contamination in the domestic environment. Scientific information from the relevant literature was collected and analyzed, to define the cross-contamination routes, to describe the variability sources and to extract and harmonise the transfer fractions to be included as model parameters. The model was used to estimate the relative impact of the cross-contamination routes for different scenarios. In addition, the effectiveness of several interventions in reducing the risk of food-borne diseases due to cross-contamination was investigated. The outputs of the model showed that the cutting board route presents a higher impact compared to other routes and replacement of the kitchen utensils is more effective than other interventions investigated; the transfer to other surfaces and objects, which can house bacteria in the environment, is also described. Laboratory cross-contamination trials have been performed to estimate bacterial transfer via cutting, from the external surface of the meat to the cutting surfaces and to the knife. The results, obtained from the laboratory trials, show magnitudes of and differences in the bacterial transfer fraction to the knife and the cutting surface in relation to which side of the meat is contaminated. Despite the complexity of factors which influence bacterial transfer, the combination of laboratory work with mathematical modelling enhanced scientific understanding and appreciation of the uncertainty of the estimates. QMRA methodology results in magnitude estimation of cross-contamination in the kitchen and evaluation of intervention strategies.

Noise assessment in slaughterhouses by means of a smartphone app.

Regulation (EC) No. 1099/2009 on the protection of animals at the time of killing establishes an obligation to spare animals any avoidable suffering or stress prior to their slaughter. Although it has been pointed out that stressors also include noise, which can cause suffering and affect the quality of the meat, current legislation does not set a limit for environmental noise in slaughterhouses. This study was conducted in three slaughterhouses in central Italy to assess the environmental acoustic level using a smartphone app. The selected, medium-sized slaughterhouses for pigs and cattle were subjected to measurements using a sound-level meter (Noise Meter for iOS) during working hours at the unloading area and lairage, along the chute to the restraining pen, at the time of stunning and at the slaughter hall. For the bovine lines the average values expressed in dB ranged from 76.33 (SD 2.08) to 93.00 (SD 2.14) for abattoir 1, from 75.00 (SD 1.87) to 92.33 (SD 4.89) for abattoir 2 and from 75.67 (SD 7.09) to 88.83 (SD 4.79) for abattoir 3. For the pig lines the average values expressed in dB ranged from 77.50 (SD 3.11) to 100.33 (SD 1.53) for abattoir 1, from 83.00 (SD 2.00) to 99.75 (SD 2.63) for abattoir 2 and from 71.20 (SD 6.49) to 99.50 (SD 1.31) for abattoir 3. Data show that the pig slaughter line was always noisier than the cattle line and the slaughter hall always showed the highest values (i.e. 100 dB), when compared to the unloading area (i.e. 79 dB).

Analysis of the causes of the seizure and destruction of carcasses and organs in a slaughterhouse in central Italy in the 2010-2016 period.

Meat safety and hygiene are fundamental issues for producers and endusers. To achieve these goals, the inspection of carcasses and organs at slaughter is critical. The results of post-mortem inspection are the basis for planning preventive measures against consumer risks and for limiting economic losses. In this retrospective study, the causes of the seizure and destruction of carcasses and organs were analysed at a slaughterhouse in central Italy from 2010 to 2016. 436,646 slaughtered animals were taken into consideration, for a total of 61,799 seizures (73.29% pigs, 23.87% cattle, 2.77% sheep and 0.07% horses). The organs or groups of organs that most frequently showed lesions in pigs were liver (72.38%), heart (10.77%) and pluck (10.20%); in cattle, lungs (64.86%), liver (31.20%) and stomachs (11.63%); in sheep, liver (77.15%), pluck (18.70%) and lung (3.80%); in horses, liver (75.56%), kidney (68.89%) and lung (31.11%). Among the diagnoses, parasitic diseases of the liver (ascariasis and distomatosis) were especially frequent in all species, followed by pericarditis and polyserositis in pigs and diseases affecting the respiratory system in cattle. The data obtained show that postmortem inspection is of fundamental importance for limiting risks for the consumer and ensuring meat safety. It is also evident, even more than ten years after the coming into force of the so-called hygiene package regulations, that the slaughterhouse can still act as an epidemiological observatory to provide the data needed for the development of plans for the control and eradication of the most frequent diseases in the area.

Prevalence and characterisation of shigatoxigenic Escherichia coli isolated from beef cattle fed with prebiotics.

Ten Holstein Friesian calves were divided into two groups of five: one group was given prebiotics in their food, while the other group served as the control group. Every two weeks from birth up to 18 months, samples of feces were taken from the rectal ampulla to determine the concentration of E. coli. At each sampling session, three aliquots per sample were collected. The arithmetic mean was calculated and all values (converted into logs) were analysed with GraphPad InStat for analysis of variance, followed by the Tukey-Kramer test. A total of 69 E. coli strains were detected, 29 (42.03%) from treated animals and 40 (57.97%) from the control group. The isolates were analysed by PCR for the presence of the stx-1, stx-2, hly and eae genes and by the Kirby Bauer test for susceptibility to the most commonly used antimicrobials in cattle breeding. Hierarchical clustering of the isolates was done using Ward's method. Thirty samples were positive for the stx-1 gene, 18 for stx- 2, 12 for both stx-1 and stx-2, 8 for hly, and 10 for eae. 4.3% were resistant to sulfamides, 8.6% to tetracycline, 1.4% to gentamicin, 94.6% to cephalothin, 2.8% to chloramphenicol, 13% to ampicillin, 13% to amoxicillin/clavulanic acid, 7.2% to sulphonamides, 4.3% to ceftriaxone, 5.7% to nalidixic acid, 34.7% to ticarcillin, 88.5% to erythromycin, and 5.7% to streptomycin. The isolates from the samples taken from day 210 to day 300 were grouped into a single cluster. Bacteriological examinations showed a reduction in the concentration of E. coli in the feces of the treated animals compared to the control group. The presence of strains with shigatoxigenic Escherichia coli virulence profiles and the reduction of these in the treated animal group demonstrated that diet can play an important role in reducing E. coli prevalence in cattle.