Shelf life of donkey milk subjected to different treatment and storage conditions.

The aim of this study was to investigate the effects of different treatment conditions on microbiological indicators of donkey milk hygiene and their evolution during shelf life at 4 and 12°C from 3 to 30d, simulating a farm-scale pasteurization and packing system. Four treatment conditions were tested: no treatment (raw milk), pasteurization (65°C × 30 min), high-pressure processing (HPP), and pasteurization plus HPP. The microbiological quality of the raw donkey milk investigated was not optimal; our results highlight the importance of raw milk management with the need for animal hygiene management and good dairy farming practices on donkey farms to improve handling procedures. The raw milk treated with HPP alone showed visible alterations with flocks, making the milk unfit for sale. The microbiological risk posed by consumption of raw donkey milk was significantly reduced by heat treatment but farm-scale packing systems cannot guarantee an extended shelf life. In contrast, the pasteurization plus HPP treatment was the most effective method to maintain microbiological milk quality. Microflora growth had little effect on pH in donkey milk: pH values were significantly different only between raw milk and pasteurized and pasteurized plus HPP milk stored at 12°C for 3d. Alkaline phosphatase activity and furosine could be used as indicators of proper pasteurization and thermal processing in donkey milk. Moreover, the presence and growth of Bacillus cereus in the case of thermal abuse hamper the wide-scale marketing of donkey milk due to the potential consequences for sensitive consumers and therefore further tests with time/temperature/high-pressure protocols associated with B. cereus are needed. Finally, our study shows that an HPP treatment of pasteurized milk after packing extends the shelf life of donkey milk and assures its microbial criteria up to 30d if properly stored at 4°C until opening; therefore, combined heat treatment and storage strategies are recommended to enhance the shelf life of donkey milk.

Microbiological and Chemical Analysis of Food Collected Under Official Control in the Emilia-Romagna Region of Northern Italy, 2014-2019.

This study analyzed data from 6 years (2014-2019) of official controls in the Emilia-Romagna region (northern Italy) to investigate the frequencies of human pathogens and chemical hazards in foods during production and distribution. Campylobacter spp. was the most prevalent pathogen, isolated in 4.4% of the 1,078 food samples examined, followed by Salmonella spp. (2.8%), Shiga toxin-producing Escherichia coli (STEC) (1.9%), and Listeria monocytogenes (0.9%). Salmonella serotyping showed that the isolates belonged to the serotypes most commonly isolated from humans in Emilia-Romagna. These serotypes were as follows: S. Infantis (34.8%), mostly isolated from chicken, monophasic S. Typhimurium (1,4, [5],12:i:-) (12.6%), S. Bredeney (8.9%), and S. Derby (8.6%). No Clostridium botulinum, Yersinia spp., and Shigella spp. were isolated. No positivity was detected for hepatitis A virus, while 5.1% of samples taken in the production phase of the food chain were found to be contaminated with norovirus. The chemical analyses identified environmental contaminants within legal limits (heavy metals, 0.6% positive overall; mycotoxins, 0.4% positive overall), analytes subjected to monitoring (perfluoro-alkyl substances (PFASs), 6.2% positive overall; inorganic arsenic, no positives overall) and process contaminants and additives within legal limits (acrylamide, 9.6% positive overall; permitted or nonpermitted additives, 0.9% positive overall). Only one sample showed dioxins and polychlorinated biphenyls (PCBs) at levels higher than the legal limits. The monitoring by competent authorities (CA) of food contamination can generate useful data that can be used as a basis for estimating the exposure to different food contaminants over time and for evaluating the effects of control measures on the contamination of food.

Effect of time and temperature before chilling on the hygiene of carcasses in wild boar hunted in central Italy.

The interest in certified game meat chains highlights the need for the evaluation and the management of factors affecting carcass hygiene along the peculiar steps of the production. The effects of time and temperature before chilling were specifically evaluated on aerobic colony count and Enterobacteriaceae count in hunted wild boar carcasses. Thirty wild boars were considered in two process steps where the hunted animal are still not chilled: after evisceration and just before chilling. Environmental temperature, carcass temperature and the elapse time between the two-step considered were registered. Furthermore, surface microbial loads were analyzed on the inner part of the carcasses. The mean time between the two sampling steps was 6 hours with an average environmental temperature of 20.49°C. A carcass temperature 9.6°C drop was observed during this period. In this lap of time aerobic colony count and Enterobacteriaceae count increased of 0.68 Log CFU/cm2 and 1.01 Log CFU/cm2 respectively, with a moderate correlation with the time but not with the temperature delta. The results reveal that the temperature conditions in central Italy hunting areas were not able to quickly reduce the carcass temperature and therefore the time between carcass evisceration and chilling should not exceed 6 hours.

Antimicrobial Resistance Profile and ExPEC Virulence Potential in Commensal Escherichia coli of Multiple Sources.

We recently described the genetic antimicrobial resistance and virulence profile of a collection of 279 commensal E. coli of food-producing animal (FPA), pet, wildlife and human origin. Phenotypic antimicrobial resistance (AMR) and the role of commensal E. coli as reservoir of extra-intestinal pathogenic Escherichia coli (ExPEC) virulence-associated genes (VAGs) or as potential ExPEC pathogens were evaluated. The most common phenotypic resistance was to tetracycline (76/279, 27.24%), sulfamethoxazole/trimethoprim (73/279, 26.16%), streptomycin and sulfisoxazole (71/279, 25.45% both) among the overall collection. Poultry and rabbit were the sources mostly associated to AMR, with a significant resistance rate (p > 0.01) to quinolones, streptomycin, sulphonamides, tetracycline and, only for poultry, to ampicillin and chloramphenicol. Finally, rabbit was the source mostly associated to colistin resistance. Different pandemic (ST69/69*, ST95, ST131) and emerging (ST10/ST10*, ST23, ST58, ST117, ST405, ST648) ExPEC sequence types (STs) were identified among the collection, especially in poultry source. Both ST groups carried high number of ExPEC VAGs (pandemic ExPEC STs, mean = 8.92; emerging ExPEC STs, mean = 6.43) and showed phenotypic resistance to different antimicrobials (pandemic ExPEC STs, mean = 2.23; emerging ExPEC STs, mean = 2.43), suggesting their role as potential ExPEC pathogens. Variable phenotypic resistance and ExPEC VAG distribution was also observed in uncommon ExPEC lineages, suggesting commensal flora as a potential reservoir of virulence (mean = 3.80) and antimicrobial resistance (mean = 1.69) determinants.

Modeling the behavior of Listeria innocua in Italian salami during the production and high-pressure validation of processes for exportation to the U.S.

A model describing Listeria innocua evolution according to process parameters of 51 Italian salami processes and HPP in 31 companies was developed. A total of 51 challenge tests were performed. During processing a L. innocua reduction of 0.34-4.32 Log10 CFU/g was observed and HPP further reduced the count of 0.48-3.47 Log10 CFU/g; an overall reduction of 1.04-5.68 is reached. PH after acidification/drying process, aw after seasoning, duration of the seasoning and caliber resulted associated (p < 0.05) with L. innocua decrease. HPP efficacy was associated (p < 0.05) with aw and pH of the product: higher the pH and aw after the acidification/drying and seasoning phases, higher resulted the L. innocua reduction after HPP. No significant association was observed between L.innocua and salt, nitrate and starter content and other characteristics of process. The model meets companies and Authorities needs and represents a useful tool to predict L. monocytogenes lethality, giving recommendations to food business operators interested in exportation to the U.S.

Factors affecting the microbiological load of Italian hunted wild boar meat (Sus scrofa).

This study investigates the microbiological conditions before maturation of wild boar meat (Sus scrofa) processed in approved game handling establishments in Italy. Fillets and legquarters of 37 carcasses were tested to assess Aerobic Colony Count (ACC), Enterobacteriaceae Count (EC) and Salmonella presence. Salmonella was never found and mean values of ACC and EC were 4.67 ±â€¯1.78 SD and 2.60 ±â€¯1.58 SD log CFU/cm2, respectively. Both ACC and EC increased with time between evisceration and skinning, were significantly higher in fillets and when meat was processed by untrained operators. ACC also increased with boars' weight and when carcasses were cleaned with running potable water. Based on limits set by EU Regulation No 1441/2007 for pork meat, most legquarters resulted satisfactory or acceptable (59% for ACC and 70% for EC), while most fillets were unsatisfactory (76% ACC, 78% EC). Results show that the wild game meat supply chain can be a safe process when handling practices reported in European and National regulations are met.

Effect of production process and high-pressure processing on viability of Salmonella spp. in traditional Italian dry-cured coppa.

The aim of the study was to investigate the combined effect of the manufacturing process followed by HPP treatment on the inactivation of Salmonella spp. in artificially contaminated coppa samples, in order to verify the ability of the combined processes to achieve the objective of a 5-log reduction of Salmonella spp. needed for exportation to the U.S. Fresh anatomical cuts intended for coppa production were supplied by four different delicatessen factories located in Northern Italy. Raw meat underwent experimental contamination with Salmonella spp. using a mixture of 3 strains. Surface contamination of the fresh anatomical cuts was carried out by immersion into inoculum containing Salmonella spp. The conditions of the HPP treatment were: pressure 593 MPa, time 290 seconds, water treatment temperature 14°C. Surface and deep samples were performed post contamination (T0), end of the cold phase (T1), end of process (Tend), and after HPP treatment (postHPP) and Salmonella spp. Enumerated. The results of this study show a significant reduction of Salmonella spp. all through the production process (P<0.01) for all companies, followed by an additional reduction of bacterial counts due to HPP treatment (P<0.01), both in superficial and deep contaminations (P<0.01). The superficial overall reduction resulted of 1.58 to 5.04 log CFU/g during the production process. HPP treatment resulted in a significant (P<0.01) superficial and deep decrease in Salmonella spp. enumeration varying from 0.61 to 4.01 log and from 1.49 to 4.13 log. According to the data presented in this study, only the combined approach of coppa manufacturing process followed by HPP treatment always led to a 5-log reduction of Salmonella spp. required by USDA/FSIS guidelines.

Effect of production process and high-pressure processing on viability of Listeria innocua in traditional Italian dry-cured coppa.

In this study the effect of the application of High Pressure Treatment (HPP) combined with four different manufacturing processes on the inactivation of Listeria innocua, used as a surrogate for L. monocytogenes, in artificially contaminated coppa samples was evaluated in order to verify the most suitable strategy to meet the Listeria inactivation requirements needed for the exportation of dry-cured meat in the U.S. Fresh anatomical cuts intended for coppa production were supplied by four different delicatessen factories located in Northern Italy. Raw meat underwent experimental contamination with Listeria innocua using a mixture of 5 strains. Surface contamination of the fresh anatomical cuts was carried out by immersion into inoculum containing Listeria spp. The conditions of the HPP treatment were: pressure 593 MPa, time 290 seconds, water treatment temperature 14°C. Listeria innocua was enumerated on surface and deep samples post contamination, resting, ripening and HPP treatment. The results of this study show how the reduction of the microbial load on coppa during the production process did not vary among three companies (P>0.05) ranging from 3.73 to 4.30 log CFU/g, while it was significantly different (P<0.01) for the fourth company (0.92 log CFU/g). HPP treatment resulted in a significant (P<0.01) deep decrease of L. innocua count with values ranging between 1.63-3.54 log CFU/g with no significant differences between companies. Regarding superficial contamination, HPP treatment resulted significant (P<0.01) only in Coppa produced by two companies. The results highlight that there were processes less effective to inhibit the pathogen; in particular for company D an increase of L. innocua count was shown during processing and HPP alone cannot be able to in reaching the Listeria inactivation requirements needed for exportation of dry-cured meat in the U.S. According to the data reported in this paper, HPP treatment increases the ability of the manufacturing process of coppa in reducing Listeria count with the objective of a lethality treatment.

Reduction of Salmonella spp. populations in Italian salami during production process and high pressure processing treatment: Validation of processes to export to the U.S.

This study involved ten enterprises producing Italian salami, 20 different samples of fermented sausages underwent challenge tests to assess and record the following parameters: time, temperature, pH, aw, and Salmonella counts. A linear regression model was used to describe the Salmonella spp. decay: at the end of the process the result of total Salmonella reduction was 0.97-5.84 Log10 CFU/g and it was significantly associated with pH at the end of acidification/drying process, aw at the end of seasoning period, the duration of seasoning, and the caliber of salami respectively. High Pressure Processing (HPP) further reduced the Salmonella level by 2.41-5.84 Log10 CFU/g with an efficacy that resulted inversely associated with aw of salami at the end of seasoning; the objective of 5-Log reduction was always reached in all the cases tested by the production process plus HPP. This model could be a useful tool for enterprises and Authorities to evaluate the efficacy of the processes to reduce Salmonella load for exportation to the U.S.

Shiga Toxin-Producing Escherichia Coii in Slaughtered Pigs and Pork Products.

During the years 2015-2016, 83 faecal samples were collected at slaughter from pigs reared in farms located in Central-Northern Italy. During the years 2014-2016 a total of 562 pork products [465 not-readyto-eat (NRTE) and 97 ready-to-eat (RTE) products] were collected from retail outlets, large retailers and processing plants. The samples were analysed according to ISO TS 13136:2012. Out of 83 swine faecal samples, 77 (92.8%) resulted stx-positive by real time polymerase chain reaction (PCR), 5 stx2+ and 1 stx1+ Shiga toxin-producing Escherichia coli (STEC) strains were isolated. Among the 465 NRTE samples, 65 (14.0%) resulted stx-positive by real time PCR and 7 stx2+ STEC strains were isolated. The stx2 gene was detected more frequently than the stx1 gene both in faecal samples (90.4 vs 8.4%) and in NRTE pork products (13.3 vs 1.3%). All the RTE samples included in the analysis resulted stx-negative. Among the samples resulted positive for stx and eae genes, serogroup-associated genes were detected at high frequency: O26 resulted the most frequent in faecal samples (81.3%) and O145 in pork products (88.1%). The O157 serogroup resulted positive in 83.3 and 78.1% of pork products and faecal samples, respectively. Despite the frequent detection by real time PCR of genes indicating the possible presence of STEC strains belonging to the six serogroups, the bacteriological step did not confirm the isolation of any such strains.

Detection of Food Hazards in Foods: Comparison of Real Time Polymerase Chain Reaction and Cultural Methods.

Foodstuffs should not contain microorganisms or their toxins or metabolites in quantities suggesting an unacceptable risk for human health. The detection of food hazards in foods is performed by several tests that produce results dependent on the analytical method used: an analytical reference method, defined as standard, is associated with each microbiological criterion laid down in Regulation 2073/2005/EC, but, analytical methods other than the reference ones, in particular more rapid methods, could be used. Combined screening methods performed by real time-polymerase chain reaction (RT-PCR) are currently validated as alternative methods according to the ISO 16140:2003 and certified by the Association Française de Normalisation. However, the positive results obtained with these alternative methods, the investigated molecular relations that resulted positive have to be confirmed with cultural methods using the same enrichment media in which the molecular screening was performed. Since it is necessary to assess if these testing schemes provide equivalent guarantees of food safety, the aim of this retrospective study is to analyse the data collected, from 2012 to 2014 by Emilia Romagna Region in the field of Piano Regionale Alimenti (Food Regional Plan) during official controls monitoring food samples of animal and other than animal origin. Records performed by combined methods of molecular screening of Salmonella spp., Listeria monocytogenes and thermophilic Campylobacter and cultural confirmation results were gathered together and the results were compared in order to assess the sensitivity of the methods. A total of 10,604 food samples were considered in this study: the comparison of the data revealed that the RT-PCR method detected Salmonella, L. monocytogenes, and thermophilic Campylobacter in 2.18, 3.85 and 3.73% of the samples, respectively, whereas by using cultural method these pathogens were isolated in 0.43, 1.57 and 1.57% of samples, respectively. In spite of the use of the same enrichment broth, the RT-PCR method disclosed a percentage of positive samples that was negative to cultural examination ranging between 20 and 43%, with a PCR/culture ratio between 2.37 to 5.00. In conclusion, the results of this study pose a doubt about the sensitivity of the official cultural methods regarding the isolation of the three investigated foodborne pathogens. Moreover this study may be a useful tool for veterinary authorities to assess appropriate sampling plans to control the risk relating to the consumption of contaminated foods.

Shiga Toxin-Producing Escherichia coli in Meat and Vegetable Products in Emilia Romagna Region, Years 2012-2013.

In 2012-2013 Emilia-Romagna Region introduced a monitoring plan for Shiga toxin-producing Escherichia coli (STEC) in foodstuff. Six hundred eighty-nine meat samples and 273 fruit and vegetable products were analyzed according to ISOTS13136. Pre-enriched samples were tested by multiplex real time PCR targeting the virulence genes eae, stx1 and stx2. Stx2 positive samples were investigated for the presence of serogroup O104 associated gene. O103, O111, O145, O157, O26 associated genes were tested on samples positive for stx in association with eae gene. Isolation of E. coli strains was attempted from samples positive for serogroup-associated genes. Thirty-four meat products (4.9%) resulted positive for stx1 and/or stx2 genes and 46 (6.7%) for stx1 and/or stx2 genes in association with eae gene. Forty-five (6.5%) samples resulted positive at least at one serogroup. Serogroup O103, O104, O111, O145, O157 and O26 genes were detected respectively in 1.3, 0.3, 0.1, 3.9, 2.9 and 2.5% samples; 0.6% samples resulted positive for STEC isolation (2 E. coli O103 and 2 E. coli O157). It is worth noting that STEC virulence genes were detected at high frequency (19%) in fresh pork meat sausages. Four (1.5%) vegetable samples were positive for stx1 and/or stx2 genes and 1 (0.4%) for stx1 and/or stx2 genes in association with eae gene; none resulted positive for the tested serogroups. Only a low number of samples positive by molecular methods were confirmed by cultural isolation. It is therefore of the uttermost importance for appropriate risk management, to be fully aware of the meaning of the analytical result.

Fecal shedding of thermophilic Campylobacter in a dairy herd producing raw milk for direct human consumption.

Factors affecting the fecal shedding of thermophilic Campylobacter in Italian dairy farms were investigated in a 12-month longitudinal study performed on a dairy farm authorized to sell raw milk in Italy. Fifty animals were randomly selected from 140 adult and young animals, and fecal samples were collected six times at 2-month intervals. At each sampling time, three trough water samples and two trough feed samples also were collected for both adult and young animals. Samples were analyzed with real-time PCR assay and culture examination. Overall, 33 samples (9.7%) were positive for thermophilic Campylobacter by real-time PCR: 26 (9.2%) of 280 fecal samples, 6 (16.6%) of 36 water samples, and 1 (4.2%) of 24 feed samples. Campylobacter jejuni was isolated from 6 of 280 samples; no other Campylobacter species was isolated. A higher (but not significantly) number of positive fecal samples were found in younger animals (11.33 versus 6.92% of adult animals), and a significantly higher number of positive water samples were collected from the water troughs of young animals. A distinct temporal trend was observed during the study period for both cows and calves, with two prevalence peaks between November and December and between May and July. Several factors such as calving, housing practices, herd size, management practices forcing together a higher number of animals, and variations in feed or water sources (previously reported as a cause of temporal variation in different farming conditions) were excluded as the cause of the two seasonal peaks in this study. The factors affecting the seasonality of Campylobacter shedding in the dairy herds remain unclear and warrant further investigation. The results of the present study indicate that special attention should be paid to farm hygiene management on farms authorized to produce and sell raw milk, with increased surveillance by the authorities at certain times of the year.

Temporal Variation of Faecal Shedding of Escherichia coli 0157:H7 in A Dairy Herd Producing Raw Milk for Direct Human Consumption.

The objective of this study was to analyse over time the evolution of E. coli O157:H7 faecal shedding in a dairy herd producing raw milk for direct human consumption. The study was performed between October 2012 and September 2013 in an average size Italian dairy farm where animals are housed inside the barn all over the year. The farm housed about 140 animals during the study - 70 cows and 70 calves and heifers. Twenty-six animals were randomly selected from both the cows and young animals group, and faecal sampling was performed rectally six times two months apart in each animal. Eleven animals were culled during the study and a total of 285 faecal samples were collected. At each faecal sampling, three trough water samples and two trough feed samples were also collected for a total of 36 water samples and 24 feed samples. Samples were analysed by real time polymerase chain reaction (RT-PCR) and culture. Overall, 16 (5.6%) faecal samples were positive for E. coli O157 by RT-PCR. Cultural examination found 9 (3.1%) samples positive for E. coli O157; all the isolates were positive for stx1, stx 2 and eae genes. One (4.1%) feed sample was positive for E. coli O157 by RT-PCR; none of the water samples was positive for E. coli O157. The model highlighted a general significant reduction of the number of positive samples observed during the study from the first to the sixth sampling (P=0.000) and a positive relation between the presence of positive samples and average environmental temperature (P=0.003). The results of the study showed that in an Italian dairy farm housing animals all year, faecal shedding of E. coli O157 followed the same temporal trend reported for other types of farming. The enhanced faecal shedding during warmer months may have a significant impact on environmental contamination and the safety of raw milk and its byproducts.

Visual evaluation of cattle cleanliness and correlation to carcass microbial contamination during slaughtering.

The aim of the study was to establish whether the visual cleanliness of cattle slaughtered was correlated to hide and carcass contamination as indicated by aerobic colony count (ACC), Enterobacteriaceae count (EC) and Escherichia coli count (ECC). Cattle in a slaughterhouse were visually inspected and assigned to a category from 1 (very clean) to 5 (very dirty) based on cleanliness. Fifteen animals for each category were randomly selected, hide and carcass sampled and analyzed for ACC, EC and ECC. Results showed that increasing dirt on cattle was associated with higher ACC, EC and ECC on hide and carcasses. Carcass ACC and ECC belonging to animals classified in cleanliness categories 3, 4 or 5 have a higher probability of exceeding the limits set by the Reg. EU 2073/2005. The study supports the conclusion that the pre-slaughter visual evaluation of animal cleanliness and application of corrective actions can be an effective aid to reduce carcass contamination.

Development of a minor groove binding probe based real-time PCR for the diagnosis and quantification of Leishmania infantum in dog specimens.

A new quantitative real-time PCR (qPCR) assay based on Taqman® technology and minor groove binding (MGB) probe was developed for the diagnosis of leishmaniosis and quantification of Leishmania infantum DNA in infected dogs. This method was based on the amplification of a 122bp fragment of the highly conserved kDNA minicircles of L. infantum. The reaction was performed using the StepOnePlus™ system with StepOne software™. This assay was able to detect the presence of protozoan parasite DNA in amounts as low as 0.03 parasites per reaction. The standard curve designed for the quantification of parasites showed linearity over seven log DNA concentration range with a correlation coefficient >0.999 and both intra- and inter-assay variability demonstrated the high efficiency and reproducibility of the assay. The qPCR also proved to be successfully applicable to different clinical samples including blood, bone marrow, lymph node aspirates and conjunctival swabs.

First reports of Trichinella pseudospiralis in wild boars (Sus scrofa) of Italy.

Trichinella pseudospiralis is a non-encapsulated species infecting both mammals and birds. In Italy, this parasite was reported only in two night-birds of prey of Central Italy. In January 2010, Trichinella larvae were detected in three wild boars (Sus scrofa) of two regions of Northern Italy by enzymatic digestion. The parasites were identified as T. pseudospiralis by multiplex-PCR. The first infected wild boar was hunted in the Emilia Romagna region and the other two infected wild boars were bred outdoors in a small family farm of the Friuli Venezia Giulia region. These new epidemiological data reinforce the role of the wild boar as the main reservoir of T. pseudospiralis in Europe.