Withdrawal periods after treatment of pigs with oxytetracycline in- and outside the European Union

Withdrawal periods are used to avoid animals being delivered to slaughter before the concentration of the antimicrobial has declined to values below the maximum residue limit (MRL). This paper characterises the withdrawal periods in force for oxytetracycline 100 mg/ml for intramuscular use in pigs. We investigated the variation in duration of the withdrawal period between 68 oxytetracycline products from 29 countries in- and outside the European Union. More specifically, we tested whether there is a regional difference, a difference between major and minor pig meat exporting countries, whether the product is long-acting or not, and whether year of market authorisation correlated with the withdrawal period. The results showed a large variation in duration of the withdrawal periods, ranging from 5 to 40 days. Variation was observed both between and within countries. Moreover, major exporting countries were associated with a longer withdrawal period than minor exporting countries (P = 0.00099). There were no regional differences, and the year of market authorisation had no impact, but long-acting products had a shorter withdrawal period than short-acting products (P = 0.048). The variation in withdrawal periods observed questions the utility of using compliance with the withdrawal period as a means of assessing whether the meat is safe for consumption. This is particularly relevant when a pig producer unintentionally delivers pigs for slaughter before the withdrawal period has expired and, aware of this, informs the abattoir. The findings call for further harmonisation in determining the withdrawal periods for all veterinary medicinal products (VMP). Until this happens, if animals are prematurely sent to slaughter, we suggest that the concentration of the VMP at the time of slaughter is calculated and compared with the MRL to determine meat safety

Biopreservation of poultry meat and products from pathogenic microorganisms

This study was focused on the protection of chicken meat from pathogenic microorganisms (Salmonella spp. and Listeria monocytogenes) by using bioprotective cultures of lactic acid bacteria originating from their indigenous microflora. The study was divided into 6 main stages: Stage 1: The prevalence and antimicrobial resistance of Salmonella isolates recovered from chicken carcasses and the environment of 4 slaughterhouses of Northern Greece was investigated. Stage 2: The prevalence and antimicrobial resistance of Listeria monocytogenes recovered from chicken carcasses and the environment of the same 4 slaughterhouses of Northern Greece was investigated. Stage 3: Lactic acid bacteria originating from chicken carcases were isolated, identified and evaluated for their inhibitory activity against strains of Salmonella spp. and Listeria monocytogenes. Stage 4: Cells of lactic acid bacteria (LAB) isolated from the neck skin of poultry carcasses were cultured in BHI broth along with cells of Salmonella spp. and Listeria monocytogenes in order to determine their antagonistic activity against the pathogens during storage at 7°C. Stage 5: The LAB strain (Lactobacillus salivarius) with the best inhibitory activity against both Salmonella and L. monocytogenes observed in broths, was chosen to examine its action against the same pathogens on chicken skin. Stage 6: The antimicrobial action of the LAB strain (Lactobacillus salivarius) against the same pathogens was also examined on chicken meat. Salmonella spp. were present in 56 (37%) of the poultry samples tested. All isolates were resistant to 4 antimicrobials (Penicillin, Erythromycin, Vancomycin and Clindamycin). Listeria spp. were present in 99 of the poultry samples tested (99%) and 38 yielded L. monocytogenes (38%). All isolates were found to be resistant to nalidixic acid and oxolinic acid, whereas the majority of them was susceptible to all other antimicrobials. After the molecular identification of lactic acid bacteria, all isolates were found to belong to 5 different species: Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus johnsonii, Pediococcus acidilactici, and Lactobacillus paralimentarius. The antimicrobial activity of lactic acid bacteria (5 species) against Salmonella spp. and Listeria monocytogenes was then examined in BHI broth and Lactobacillus salivarius was found to be the effective LAB against these pathogens. On chicken skin, the growth reduction at the 6th day caused by L. salivarius was lower and did not exceed the 0.54 log cfu/cm² for Salmonella spp. and 0.71 log cfu/cm² for Listeria monocytogenes. The reduction on chicken meat was slightly lower for both pathogens, while differences among chicken skin and meat were not found statistically significant (P>0.05). In conclusion, numerous lactic acid bacteria isolates from poultry carcasses presented sufficient inhibitory activity against Salmonella and Listeria. The results of these experiments suggested that L. salivarius has a potential to be used as protective culture, in combination with other microbial hurdles, to improve the safety and extent the shelf life of chicken meat.Η ερευνητική αυτή εργασία είχε ως κύριο στόχο την προστασία του κρέατος των ορνιθίων από παθογόνους μικροοργανισμούς (Salmonella spp. και Listeria monocytogenes) με τη χρησιμοποίηση προστατευτικών καλλιεργειών οξυγαλακτικών βακτηρίων που προέρχονται από την αυτόχθονη χλωρίδα τους. Το πειραματικό μέρος της έρευνας περιλαμβάνει έξι φάσεις: Στην 1η φάση και 2η φάση έγινε απομόνωση, ταυτοποίηση και έλεγχος της αντιμικροβιακής ευαισθησίας στελεχών Salmonella spp., Listeria spp. και Listeria monocytogenes από τα σφάγια των ορνιθίων και από το περιβάλλον τεσσάρων πτηνοσφαγείων της Β. Ελλάδας. Στην 3η φάση έγινε απομόνωση και ταυτοποίηση στελεχών οξυγαλακτικών βακτηρίων από τα σφάγια των ορνιθίων και αξιολόγησή τους προκειμένου να χρησιμοποιηθούν ως βιοπροστατευτικές καλλιέργειες. Στην 4η φάση έγινε ενοφθαλμισμός των παθογόνων Salmonella spp. και Listeria monocytogenes και των αποτελεσματικότερων οξυγαλακτικών βακτηρίων σε ζωμό ΒΗΙ, μελέτη της εξέλιξης του πληθυσμού των παθογόνων και των οξυγαλακτικών βακτηρίων και στη συνέχεια αξιολόγηση της βιοπροστατευτικής τους ικανότητας με στόχο την εύρεση της αποτελεσματικότερης οξυγαλακτικής καλλιέργειας. Στην 5η φάση και 6η φάση έγινε τεχνητή μόλυνση του δέρματος και τμημάτων (φιλέτο) των σφαγίων των πουλερικών με Salmonella spp. και Listeria monocytogenes και ταυτόχρονα ενοφθαλμισμός με την αποτελεσματικότερη βιοπροστατευτική οξυγαλακτική καλλιέργεια και στη συνέχεια, μελέτη της εξέλιξης του πληθυσμού των παθογόνων και των οξυγαλακτικών βακτηρίων. Τα αποτελέσματα έδειξαν ότι: Salmonella spp. βρέθηκαν σε 56 (37%) από τα 150 ορνίθεια δείγματα που εξετάστηκαν. Όλα τα στελέχη βρέθηκαν ανθεκτικά σε 4 αντιβιοτικά (πενικιλλίνη, ερυθρομυκίνη, βανκομυκίνη και κλινδαμυκίνη). Listeria spp. και Listeria monocytogenes απομονώθηκαν σε ποσοστό 99% και 38%, αντιστοίχως, του συνόλου των εξετασθέντων δειγμάτων σφαγίων ορνιθίων. Όλα τα στελέχη βρέθηκαν ανθεκτικά σε δύο αντιμικροβιακούς παράγοντες (ναλιδιξικό και οξολινικό οξύ), ενώ το μεγαλύτερο ποσοστό των στελεχών βρέθηκαν να είναι ευαίσθητα στους υπόλοιπους. Με βάση τη μοριακή μέθοδο ταυτοποιήθηκαν πέντε διαφορετικά είδη οξυγαλακτικών βακτηρίων: Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus johnsonii, Lactobacillus paralimentarius και Pediococcus acidilactici. Στη συνέχεια, ελέγχθηκε η ανασταλτική δράση των οξυγαλακτικών βακτηρίων εναντίον των Salmonella spp. και Listeria monocytogenes σε ζωμό BHI και προέκυψε ότι ο Lactobacillus salivarius παρουσίασε την εντονότερη ανασταλτική δράση τόσο έναντι των Salmonella spp. όσο και της Listeria monocytogenes. Η στατιστική ανάλυση των αποτελεσμάτων της ανασταλτικής δράσης του Lactobacillus salivarius στο δέρμα και στο κρέας των ορνιθίων έδειξε τα εξής: α) Στο δέρμα των ορνιθίων ο Lactobacillus salivarius προκάλεσε μείωση της αύξησης του πληθυσμού των Salmonella spp. και της Listeria monocytogenes κατά 0,54 log cfu/cm² και 0,71 log cfu/cm², αντιστοίχως, την 6η μέρα του πειραματισμού. β) Στο κρέας των ορνιθίων η μείωση της αύξησης των παθογόνων ήταν ελαφρώς μικρότερη. Συμπερασματικά, η αξιολόγηση της αντιμικροβιακής δράσης των οξυγαλακτικών βακτηρίων σε ζωμό ΒΗΙ, και του αποτελεσματικότερου από αυτά στελέχους στο δέρμα και στο κρέας ορνιθίων έδειξε ότι το είδος Lactobacillus salivarius είναι δυνατόν να χρησιμοποιηθεί ως προστατευτική καλλιέργεια, σε συνδυασμό με άλλα μικροβιακά εμπόδια, για τη βελτίωση της ασφάλειας και την επιμήκυνση του χρόνου συντήρησης του ορνιθείου κρέατος

Hygiene and Safety of Hard Cheese Made from Raw Cows’ Milk

This study was conducted to evaluate the microbiological status of cheese made from unpasteurized cows’ milk, to examine the safety of the cheese and to observe the changes that occurred in its microbial community during ripening and storage. Furthermore, the pH, the moisture and salt concentration were also monitored throughout processing, ripening and storage. Seven cheesemaking trials took place along with the microbiological and physicochemical analysis of the milk, curd and cheese produced. The milk used for the cheesemaking, two curd samples before the heating and two after the heating, two cheese samples at days 3, 7, 15, 30, 60 and 90 were subjected to microbiological analysis for total mesophilic bacterial count (for milk only), Enterobacteriaceae, E. coli, Staphylococcus, Salmonella, Listeria, and Clostridium. The microbiological quality of raw milk was found to be good. It was initially slightly above the EU limit but improvements associated with farm biosecurity and milking equipment hygiene led to a significantly improved milk quality. A small increase in the prevalence of indicator microorganisms in curd and cheese samples was observed for the first few days, followed by a relatively stable condition as manufacturing proceeded and throughout the ripening of the final product. In two cheesemaking trials, Clostridium perfringens and Salmonella spp. were detected, the first originating from the milk and the second from the environment. The use of good-quality raw milk under sanitary conditions, the application of good manufacturing practices and a maturation period in a controlled environment were found to be the necessary prerequisites for the production of safe raw cheese products

Determination of Aflatoxin M1 in Raw Milk Using an HPLC-FL Method in Comparison with Commercial ELISA Kits—Application in Raw Milk Samples from Various Regions of Greece

The highly toxic Aflatoxin M1 (AFM1) is most often detected in milk using an Enzyme-Linked-Immunosorbent Assay (ELISA) for screening purposes, while High-Performance Liquid Chromatography with Fluorescence Detector (HPLC-FL) is the reference method used for confirmation. The aim of the present study was the comparison between three commercially available ELISA kits and a newly developed HPLC-FL method for the determination of the AFM1 in milk samples. The developed HPLC-FL method was validated for the AFM1 and Aflatoxin M2 (AFM2), determining the accuracy, precision, linearity, decision limit, and detection capability with fairly good results. All three ELISA kits were also validated and showed equally good performance with high recovery rates. Moreover, the Limit Of Detection (LOD) and Limit Of Quantification (LOQ) values were found to be significantly lower than the Maximum Residue Limit (MRL) (50 ng kg−1). After the evaluation of all three commercial kits, the ELISA kit with the optimum performance along with the HPLC method was used for the determination of AFM1 in raw cow’s, goat’s, and sheep’s milk samples (396) obtained from producers in different regions of Greece. The evaluation of both methods showed that this ELISA kit could be considered as a faster and equally reliable alternative method to HPLC in routine analysis for the determination of AFM1 in milk

Indigenous Lactic Acid Bacteria Isolated from Raw Graviera Cheese and Evaluation of Their Most Important Technological Properties

The aim of the present study was to characterize LAB isolates from raw-milk cheeses, to evaluate some of their technological properties and to select a few ‘wild’ LAB strains that could potentially be used as starter cultures. LAB strains were isolated and identified from raw milk, curd, and cheese at 30, 60, and 90 days of ripening. A total of 100 strains were isolated, 20 from each phase of ripening. All isolates were tested for acidification ability, curd formation, and aroma production at 32 °C and 42 °C after 24 and 48 h. Following the acidification test, 42 strains were selected for identification and characterization of their technological properties. A high proportion of lactic acid bacteria and Gram + cocci were found throughout the cheese-making process. Enterococci reached their maximum proportion on the 7th day of ripening while Lactobacilli increased significantly during the first month of ripening. Forty-two strains were identified by phenotypic, biochemical, and molecular techniques. Lactococci were predominant in raw milk and curd while Lactobacilli in the ripening of the cheese. Four LAB strains including one Leuconostoc pseudomenteroides, two Lacticaseibacillus paracasei subsp. paracasei and one Enterococcus hirae, were proposed for their potential use as starters or secondary cultures

Accidental delivery of pigs for slaughter prior to end of withdrawal period for antimicrobial treatment- Ways of handling

Withdrawal periods after treatment with antimicrobials are set to minimise the frequency and concentration of residues in meat from treated pigs. Still, by mistake, pigs can be sent for slaughter too early. How should the abattoir respond when a pig producer contacts the abattoir to inform them of such a mistake? To address this, two questionnaire surveys were undertaken during spring of 2022, targeting the competent authority (CA) and the food business operator (FBO) from countries in- and outside the European Union. The results covering answers from 78 respondents from 27 countries show that most countries have procedures in place, but also various ways of responding, between CA and FBO, as well as between countries with a large export volume versus countries with a small export volume of pig meat. We developed a best practice model for handling such events, which covers stages before and after the pig is slaughtered and is subjected to official meat inspection and resulting decisions in accordance with relevant legislation. The model involves a quantitative exposure risk assessment, which should be undertaken by the FBO and verified by the CA. The assessment produces estimates of the concentrations of residues at the time of slaughter. If higher than the maximum residue limit, the expected use of the carcasses should be considered. Hereby, the consumer exposure risk can be assessed using the acceptable daily intake (ADI) value, and a risk-based management decision about the safe handling of the meat can be made. This approach would compensate for the huge variation in withdrawal periods for the same substances in force in the European countries

Investigating the Association Between the Caecal Microbiomes of Broilers and Campylobacter Burden

One of the major transmission routes for the foodborne bacterial pathogen Campylobacter is undercooked poultry meat, contaminated from intestinal contents during processing. In broilers, Campylobacter can grow to very high densities in the caeca, and is often considered to be a commensal or an opportunistic pathogen in poultry. Reduction of caecal loads of Campylobacter may assist in lowering incidence rates of Campylobacter food poisoning. To achieve this, there needs to be a better understanding of the dynamics of Campylobacter colonization in its natural niche, and the effect of the local microbiome on colonization. Previous studies have shown that the microbiome differed between Campylobacter colonized and non-colonized chicken intestinal samples. To characterize the microbiome of Campylobacter-colonized broilers, caecal samples of 100 randomly selected birds from four farms were analyzed using amplified 16S rRNA gene sequences. Bacterial taxonomic analysis indicated that inter-farm variation was greater than intra-farm variation. The two most common bacterial groups were Bacteroidetes and Firmicutes which were present in all samples and constituted 29.7–63.5 and 30.2–59.8% of the bacteria present, respectively. Campylobacter was cultured from all samples, ranging from 2 to 9 log10 CFU g-1. There was no clear link between Campylobacter counts and Firmicutes, Bacteroidetes, or Tenericutes levels in the 16S rRNA operational taxonomic unit (OTU)-based analysis of the caecal microbiome, but samples with high Campylobacter counts (>9 log CFU g-1) contained increased levels of Enterobacteriaceae. A decrease in Lactobacillus abundance in chicken caeca was also associated with high Campylobacter loads. The reported associations with Lactobacillus and Enterobacteriaceae match changes in the intestinal microbiome of chickens and mice previously reported for Campylobacter infection, and raises the question about temporality and causation; as to whether increases in Campylobacter loads create conditions adverse to Lactobacilli and/or beneficial to Enterobacteriaceae, or that changes in Lactobacilli and Enterobacteriaceae levels created conditions beneficial for Campylobacter colonization. If these changes can be controlled, this may open opportunities for modulation of chicken microbiota to reduce Campylobacter levels for improved food safety

Accidental delivery of pigs for slaughter prior to end of withdrawal period for antimicrobial treatment- Ways of handling

Withdrawal periods after treatment with antimicrobials are set to minimise the frequency and concentration of residues in meat from treated pigs. Still, by mistake, pigs can be sent for slaughter too early. How should the abattoir respond when a pig producer contacts the abattoir to inform them of such a mistake? To address this, two questionnaire surveys were undertaken during spring of 2022, targeting the competent authority (CA) and the food business operator (FBO) from countries in- and outside the European Union. The results covering answers from 78 respondents from 27 countries show that most countries have procedures in place, but also various ways of responding, between CA and FBO, as well as between countries with a large export volume versus countries with a small export volume of pig meat. We developed a best practice model for handling such events, which covers stages before and after the pig is slaughtered and is subjected to official meat inspection and resulting decisions in accordance with relevant legislation. The model involves a quantitative exposure risk assessment, which should be undertaken by the FBO and verified by the CA. The assessment produces estimates of the concentrations of residues at the time of slaughter. If higher than the maximum residue limit, the expected use of the carcasses should be considered. Hereby, the consumer exposure risk can be assessed using the acceptable daily intake (ADI) value, and a risk-based management decision about the safe handling of the meat can be made. This approach would compensate for the huge variation in withdrawal periods for the same substances in force in the European countries