137Cesium (137Cs) assessment in wild boars from northwestern Italy.

Radionuclide contamination is a serious health issue caused by nuclear experiments and plant accidents, as seen for the Chernobyl and Fukushima nuclear plants. Italy has been especially interested in northwestern alpine regions, as have several other nations. The aim of this work was to indagate 134Cs and 137Cs contamination in wild boars, which were considered bioindicators sampled in the Chisone/Germanasca Valley and the Pellice Valley districts (Piedmont, Italy) in two hunting seasons (2014 and 2016). In the 2014 season, only the livers of the animals (n = 48) were sampled, whereas in 2016, five different anatomical sampling sites were sampled for each animal (n = 16). The analyses were conducted in an accredited laboratory (Agenzia Regionale per la Protezione dell'Ambiente-ARPA) by the aid of an HPGe detector (Ortec) with a relative efficiency of 50%. In general, the contamination levels registered in 2014 were under the detection limit for 134Cs and low for 137Cs (Chisone/Germanasca valley: min: 0.0, max: 23.9 median 11.0 Bq/kg vs Pellice valley: min 0, max: 31.7, median: 9.6 Bq/kg) and no health concern can be supposed. In the first-year samples, the liver showed a negative correlation between age and contamination level. In the second year of sampling, low levels were confirmed (min: 3.1 Bq/kg, max: 113.3; median 17.7 Bq/kg). Multiple sampling from the same animal showed that the diaphragm (median = 27.7 Bq/kg) kidney (27.4) and tongue (27.6) were more contaminated than the liver (17.7) and spleen (15.3). Moreover, a linear mixed model revealed a negative organ-by-age interaction, meaning that interorgan differences in contamination level were greater in younger (5-11 months) than in older (18-36 months) animals. Different feeding habits can be the explanation. Our paper shows that muscle sites (diaphragm and tongue) can be useful for radionuclide pollution surveillance in wild boar populations and that younger animals show more interorgan variability in contamination levels than older animals. More investigations are needed to confirm this correlation and to fulfill the request for more data to achieve better risk assessment.

Pet feeding habits and the microbiological contamination of dog food bowls: effect of feed type, cleaning method and bowl material.

BACKGROUND: Safe pet feeding practices and food bowl hygiene measures are important for minimising the risk of microbiological contaminations in the domestic environment. This study compares the practices reported by dog and cat caregivers, and investigates whether cleaning method, feed type or bowl material affects the microbiological contamination of dog food bowls. RESULTS: Data from 351 dog caregivers and 186 cat caregivers were collected via an online survey. The majority of dogs (70.7%) were fed twice daily, whereas cats (43%) were mostly fed ad libitum. The most common material for dog food bowls was metal (67.1%) versus plastic (38.1%) and metal (37.6%) for cats. Dog food bowls were most frequently cleaned after each meal (35.7%); whereas for cats, 21.5% were cleaned after each meal, 22.7% once a day and 19.3% 2-3 times a week. Total mesophilic aerobic bacteria counts (TMABc), Enterobacteriaceae counts and pathogenic bacteria (Salmonella spp., Campylobacter spp., Verotoxigenic E. coli [VTEC]) were assessed for 96 dog food bowls. TMABc were higher in metal vs. plastic bowls (p < 0.001) and in those used for wet food vs. dry food (p = 0.0397). Enterobacteriaceae counts were higher in bowls washed by hand vs. dishwasher (p = 0.0515), whereas no differences were found between hand washing vs. dry wiping. Salmonella spp., Campylobacter spp. or E. coli VTEC contaminations were not detected. CONCLUSIONS: The surveyed Italian dog and cat caregivers reported different habits concerning feeding frequency, food bowl material and cleaning frequency. Wet food and metal bowls were associated with higher levels of microbiological contamination of dog food bowls. Furthermore, in relation to wet washing methods, contaminations were likely to be greater following hand washing than they were following the use of a dishwasher. Practical guidelines for safe feeding practices and hygiene measures are needed to minimise the risk of microbiological contaminations in domestic environments.

A Fibre- vs. cereal grain-based diet: Which is better for horse welfare? Effects on intestinal permeability, muscle characteristics and oxidative status in horses reared for meat production.

Horses reared for meat production are fed high amounts of cereal grains in comparison with horses raised for other purposes. Such feeding practice may lead to risk of poor welfare consequences. The aim of this study was to investigate the effects of two feeding practices on selected metabolic parameters and production aspects. Nineteen Bardigiano horses, 14.3 ± 0.7 months of age, were randomly assigned to two groups-one fed with high amounts of cereal grains (HCG; n = 9; 43% hay plus 57% cereal grain-based pelleted feed) vs. one fed with high amounts of fibre (HFG; n = 10; 70% hay plus 30% pelleted fibrous feed)-for 129 days. At slaught on abattoir, biological and tissue samples were collected to evaluate the microbiological contamination of mesenteric lymph nodes and liver; selected meat quality traits (chemical composition and fatty acid profile of the Longissimus thoracis et lumborum muscle); and the oxidative status of the horse. A linear mixed model was used: dietary treatment and sex were fixed effects and their interaction analysed on production and metabolic parameters as dependent variables. Results showed an increased intestinal permeability in the horses fed HCG compared to HFG, according to the significant increased total mesophilic aerobic bacteria counts in mesenteric lymph nodes (p = 0.04) and liver samples (p = 0.05). Horses in HCG showed increased muscle pH (p = 0.02), lighter muscle colour (L) (p = 0.01), increased intramuscular fat concentrations (p = 0.03), increased muscle glutathione peroxidase and superoxide dismutase activities (p = 0.01 and p = 0.03, respectively). Moreover, horses in HCG had lower muscle water holding capacity at interaction with sex (p = 0.03, lower in female), lower muscle protein content (p = 0.01), lower concentration of muscle PUFAs (p = 0.05) and lower plasma catalase activities (p = 0.05). Our results showed that feeding a high cereal grains diet can have global effects on horse physiology, and thus represents a threat for their welfare.

Mycobiota Composition of Robiola di Roccaverano Cheese along the Production Chain.

Robiola di Roccaverano is a Protected Designation of Origin (PDO) cheese from the Piedmont region of Italy. In this study, the mycobiota occurring during Robiola di Roccaverano production was elucidated. Samples of milk, Natural Milk Cultures (NMC), curd, 5- and 15-days ripened cheese were collected from one dairy plant and the mycobiota was analyzed by the metataxonomic approach. Milk samples showed a high diversity and Cladosporium, Kluyveromyces marxianus, Geotrichum candidum and Debaryomyces hansenii were found with higher relative abundance. This mycobiota remains quite stable in NMC and curd matrices although the relative abundance of K. marxianus and G. candidum yeasts increased significantly and shaped the fungal composition of 5- and 15-day ripened cheese.

The Bacterial and Fungal Microbiota of "Robiola di Roccaverano" Protected Designation of Origin Raw Milk Cheese.

Robiola di Roccaverano is an artisanal Protected Designation of Origin (PDO) soft cheese made with raw goat's milk and by the addition of Natural Milk Culture (NMC) to drive the fermentation process. Cheeses collected from five different dairy plants were analyzed for their bacterial and fungal microbiota diversity. Lactococcus lactis and Leuconostoc mesenteroides were the main bacterial population, while Galactomyces candidum and Kluyveromyces marxianus constituted the core mycobiota but many other minor taxa were observed, suggesting a high level of complexity in fungal composition by these cheeses compared to bacteria population.

Safety assessment of traditional Plaisentif cheese.

Traditional foods are gaining more and more market due to consumers' increasing willingness to buy products linked to national cultures: among these products, cheese plays an important role. Plaisentif is a traditional Piedmont cheese, only made during violets blooming season. The aim of this work is to evaluate the safety of this cheese, taking into account the EU Regulations. Microbiological hazards as well chemical, biogenic amines and mycotoxins, analysis were investigated. Salmonella spp. and Listeria monocytogenes were never detected in cheeses after ripening. Biogenic amines were present in very low quantities. Ochratoxin A was never detected and patulin was detected in over one cheese during the two years of sampling. This is the first attempt to characterize traditional Plaisentif cheese from a safety point of view. All the information acquired can be held as a necessary basis for reinforcing the culture of traditional products, for economic opportunities in mountainous regions and for safeguarding traditions and cultural identities.

Short communication: High-throughput sequencing approach to investigate Italian artisanal cheese production.

In this study, high-throughput sequencing (HTS) was used to investigate the microbiota of Robiola di Roccaverano production, an artisanal Protected Designation of Origin soft cheese made with raw goat milk by addition of a natural milk starter (NMS), from the Piedmont region of Italy. Different steps of production of Robiola di Roccaverano cheese at one artisanal dairy were monitored. Matched samples of milk, NMS, curd, and 5-d and 15-d matured cheeses were collected at different periods of the year. The DNA sequences obtained by HTS belonged to 5 phyla: Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Tenericutes. In milk, Proteobacteria and Firmicutes were mainly found, and several operational taxonomic units (OTU) belonging to contaminant bacteria such as Pseudomonas, Serratia, and Staphylococcus were observed. However, in NMS, curd, and 5- and 15-d cheeses, Firmicutes were principally observed where OTU of Lactococcus lactis were predominant, followed by Leuconostoc mesenteroides OTU. The results of the analysis showed high bacterial diversity in milk samples compared with NMS, curd, and 5- and 15-d cheeses, suggesting strong action of NMS in driving the characteristics of the final products.

Microbial characterization of an artisanal production of Robiola di Roccaverano cheese.

Robiola di Roccaverano, from the Piedmont region of Italy, is a Protected Designation of Origin soft cheese made with raw goat milk. The peculiarity of this cheese is that during the manufacturing process, a natural starter culture (NC) is added to raw milk. This study examined the viable microorganisms of technological interest, including lactic acid bacteria and fungal populations, in samples of raw milk, NC, and fresh and ripened cheese collected from one dairy using culture-dependent techniques. First, the isolated colonies were analyzed using random amplification of polymorphic DNA (RAPD) PCR, and strains with similar fingerprints were clustered together. Further, representative isolates of each group were subjected to 16S or 26S ribosomal DNA sequencing. Finally, species-specific PCR was conducted to distinguish the Lactococcus lactis ssp. lactis and Lc. lactis ssp. cremoris. Among the studied lactic acid bacteria, 13 RAPD profiles were obtained, corresponding to 9 different bacterial species or subspecies. Concerning mold and yeast isolates, 5 species were found that coincided with 5 RAPD types. Observing the strains isolated in the study, Lc. lactis was the most prevalent species in raw milk and NC samples, and Leuconostoc mesenteroides was the predominant species identified in 5- and 15-d cheese isolates. Furthermore, whereas only these 2 species were detected in NC, Enterococcus and Lactobacillus genera were found in raw milk and cheese, respectively. Concerning the mold and yeast isolates, in NC Kluyveromyces spp. was mainly found, and in cheese samples the representative species were Geotrichum candidum and Yarrowia lipolytica. Finally, raw milk and cheese safety were evaluated, and the samples complied with the standard required by European Commission regulation number 2073/2005.

Microbiological analysis of the Robiola di Roccaverano cheese by means of traditional culturing methods.

Robiola di Roccaverano is a Protected Designation of Origin soft cheese made with goat's milk, produced in Piedmont region (Italy). The peculiarities of this cheese are: i) the use of the raw milk, ii) the addition of a Natural Milk Starter, iii) the application of traditional techniques of production and iv) the localization of the dairies in rural area. All these aspects influence the microbial flora of final product and make interesting its investigation. Samples were collected at different moment of the cheese making process and during the different seasons. In this preliminary study, the safety and the hygiene parameters of the production were evaluated. Lactic acid bacteria, moulds and yeasts involved in cheese-making process were also enumerated. Pathogens were not found in all samples and the counts of coagulase positive staphylococci were within the standard of law. The enumeration of microorganisms of technological interest demonstrated that, nevertheless the artisanal manufacturing process applied, the dairy was able to standardize the final products.

Cross-contamination in canine and feline dietetic limited-antigen wet diets.

BACKGROUND: Adverse food reactions (AFRs) are defined as abnormal responses to an ingested food or food additive. Diagnosis and treatment of AFRs consist of the complete elimination of these ingredients in the dietary trial. Previous studies have demonstrated the presence of undeclared ingredients in commercial limited-antigen dry food diets that can compromise the results and efficacy of dietary elimination trails. The aim of this study was to assess a selection of commercial canine and feline dietetic limited-antigen wet foods for the potential cross-contamination of animal proteins from origins not mentioned on the label. RESULTS: Eleven canine and feline dietetic limited-antigen wet foods (9 novel animal protein foods, 1 vegetarian and 1 hydrolyzed) were analyzed by polymerase chain reaction (PCR) to detect the presence DNA of animal and vegetal origins. PCR analysis confirmed the contamination of 6 of the 11 (54.5%) limited-antigen wet diets with undeclared animal protein. One of these 6 diets was solely composed of animal protein sources completely unrelated to those declared on the label. None of the foods containing horse meat or fish were contaminated, and neither were the vegetarian or the hydrolyzed food products. Moreover, the results show that had zoological class primers only been used to check for cross-class contaminations, as are generally used in the pet food industry for in-house checks, the apparent contamination rate would have been significantly underestimated: less than 20% (3/11), instead of the actual rate of 54.7% using species-specific primers. CONCLUSION: This study reveals a high rate of cross-contamination in dietetic limited-antigen wet canine and feline foods, as previously described for dietetic dry limited-antigen foods (reported to be more than 80%). These results add new fuel to the discussion about the potential causes underlying the failure of elimination diets, since animal protein contaminants may actually be present in the commercial dietetic limited-antigen diets. AFRs may therefore occur as a result of inadequate practices in the pet food industry.

Characterization of bacterial communities of donkey milk by high-throughput sequencing.

The interest in donkey milk (DM) is growing because of its functional properties and nutritional value, especially for children with allergies and food intolerances. However, most of the available reports of DM microbiota are based on culture-dependent methods to investigate food safety issues and the presence of lactic acid bacteria (LAB). The aim of this study was to determine the composition of DM bacterial communities using a high-throughput sequencing (HTS) approach. Bulk milk samples from Italian donkey dairy farms from two consecutive years were analysed using the MiSeq Illumina platform. All sample reads were classified into five phyla: Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Verrucomicrobia. The most prevalent genera-Pseudomonas, Ralstonia, Acinetobacter, Cupriavidus, Citrobacter and Sphingobacterium-were Gram-negative bacteria. The core microbiota was composed of genera that comprise commonly associated milk bacteria, LAB and species normally found in soil, water and plants. Reads assigned to LAB genera-Streptococcus, Lactococcus, Enterococcus, Leuconostoc, Lactobacillus, and Carnobacterium-corresponded on average to 2.55% of the total reads per sample. Among these, the distribution of reads assigned to coccus- and bacillus-shaped LAB was variable between and within the farms, confirming their presence and suggesting a complex population of these bacteria in DM. The present study represents a general snapshot of the DM microbial population, underlining its variability and motivating further studies for the exploitation of the technological potential of bacteria naturally present in DM.

Distinguishing industrial meat from that of indigenous chickens with molecular markers.

The aim of investigation was to evaluate a traceability system to detect industrial chicken meat among indigenous products, considering issues that could affect assignment accuracy. The dataset included 2 Italian indigenous meat breeds, namely Bionda Piemontese (2 ecotypes) and Bianca di Saluzzo, one broiler line, and 3 layer lines. Assignment tests were performed using a standard panel of 28 microsatellite loci. To evaluate effects of inbreeding and substructure on assignment accuracy, a simulated dataset was prepared. Broilers and layers belong to homogeneous populations and never enter the clusters of indigenous breeds. Ambiguity or misallocation are expected between the Bionda ecotypes and between the 2 indigenous breeds, but it is unlikely that niche products provided by Bionda and Bianca will compete with one another. Non-random mating reduces accuracy, but only populations having weak genetic differentiation are involved, namely those that are less interesting to discriminate. The dataset can be used as a reference population to distinguish commercial meat from indigenous meat with great accuracy. Misallocations increase as number of loci decreases, but only within or between the indigenous breeds. A subpanel of the most resolving 14 loci keeps sufficient informative content to provide accuracy and to correctly allocate additional test samples within the reference population. This analytical tool is economically sustainable as a method to detect fraud or mislabeling. Adoption of a monitoring system should increase the value of typical products because the additional burden of molecular analyses would improve commercial grade and perception of quality.

Isolation and characterisation of lactic acid bacteria from donkey milk.

During the last years the interest in donkey milk has increased significantly mainly because of its compelling functional elements. Even if the composition and nutritional properties of donkey milk are known, its microbiota is less studied. This Research Communication aimed to provide a comprehensive characterisation of the lactic acid bacteria in raw donkey milk. RAPD-PCR assay combined with 16S rDNA sequencing analysis were used to describe the microbial diversity of several donkey farms in the North West part of Italy. The more frequently detected species were: Lactobacillus paracasei, Lactococcus lactis and Carnobacterium maltaromaticum. Less abundant genera were Leuconostoc, Enterococcus and Streptococcus. The yeast Kluyveromyces marxianus was also isolated. The bacterial and biotype distribution notably diverged among the farms. Several of the found species, not previously detected in donkey milk, could have an important probiotic activity and biotechnological potential. This study represents an important insight to the ample diversity of the microorganisms present in the highly selective ecosystem of raw donkey milk.

Apoptosis in muscle-to-meat aging process: The omic witness.

Meat derives from a muscle that undergoes a great number of biochemical and physiological changes. The anoxic condition established from the moment of animal sacrifice forces muscle cells to a sort of reaction, resulting in methodical programmed cell death to avoid necrosis. The duality autophagy and/or apoptosis is at the center of the scientific debate about the biological processes driving the muscle to meat conversion. Here we report an omic time course overview carried on proteome, phosphoproteome and metabolome of Piedmontese longissimus thoracis muscle searching for clues helping us to extricate through the dilemma. The survey depicts a progressive physiological impairing and our evidences push towards the apoptotic behavior: the proteomic time course trend of annexin A2, RKIP, HSPB6, αB crystalline, adenylate kinase, DJ-1 and 31kDa actin fragment; the 0-1day increased phosphorylation of myosin 2 and synaptopodin and the metabolomic time course trend of key metabolic indicators, like GSH/GSSG ratio, taurine and nitrotyrosine. The employed techniques provide strong indications about the likely apoptotic behavior of aging meat in muscle-to-meat conversion process. BIOLOGICAL SIGNIFICANCE: Our work underlines compelling evidences of the apoptotic behavior of Piedmontese beef muscle cells undergoing the muscle-to-meat process, whereas no autophagic clues are inferred from this omic investigation.

Omics integrating physical techniques: aged Piedmontese meat analysis.

Piedmontese meat tenderness becomes higher by extending the ageing period after slaughter up to 44 days. Classical physical analysis only partially explain this evidence, so in order to discover the reason of the potential beneficial effects of prolonged ageing, we performed omic analysis in the Longissimus thoracis muscle by examining main biochemical changes through mass spectrometry-based metabolomics and proteomics. We observed a progressive decline in myofibrillar structural integrity (underpinning meat tenderness) and impaired energy metabolism. Markers of autophagic responses (e.g. serine and glutathione metabolism) and nitrogen metabolism (urea cycle intermediates) accumulated until the end of the assayed period. Key metabolites such as glutamate, a mediator of the appreciated umami taste of the meat, were found to constantly accumulate until day 44. Finally, statistical analyses revealed that glutamate, serine and arginine could serve as good predictors of ultimate meat quality parameters, even though further studies are mandatory.

Application of DNA Barcoding for Controlling of the Species from Octopus Genus.

The DNA barcoding proposes the use of a particular sequence from a single genomic region as the base for an identifying system capable to determine all animal species. This methodology comprises the analysis of a 655 base-pair region from the mithocondrial cytochrome C oxidase gene (COI). Its application in the species identification of fishery products has been very promising. However, in the last years some doubts about its usage have emerged. In this work, we make use of the DNA barcoding for the identification of some of the octopus species with higher commercial interest (Octopus membranaceus, Octopus vulgaris, Octopus aegina, Octopus cyanea) focusing the attention on the reliability and completeness of the available information on the databases. The study looked over 51 individuals apparently belonging to the Octopus genus. For the identification of O.aegina, O.cyanea, O.vulgaris species no particular problems were found. On the other hand, most of the samples of O.membranaceus, though they clearly presented the morphological characteristics of the species, were not identified with the biomolecular analyses.

Analysis of Information on Food Chain in Europe and Piedmont Region, Italy.

Food chain information (FCI) is an innovation of the new European regulation. Its purpose is to enhance the concept of food security. FCI includes specifications such as: health status, information on treatments and diseases, analytical reports on control plans, zoonoses or environmental contaminants, production performance, etc. The aim of this article is to compare the different European guidelines and analyse the situation in Piedmont in order to assess potential problems and propose solutions. European guidelines are similar one another, but they have been tailored to the epidemiological situations of each state. Except for Spain and Germany, FCI models are different for each species and the poultry sector is the most detailed. Unfortunately, Italy has not provided guidelines yet, and this has generated considerable differences. Overall, the number of FCI models with incomplete information is the largest group compared to the models not completed for each entry. The main deficiencies are related to pharmacological treatments. The health status of the farm is listed consistently regarding the compulsory eradication plans, but other national voluntary or accreditation plans are rarely mentioned. The situation is similar in other European countries. In conclusion, FCI is an effective tool if applied with consistency and reason. Only in this way the collection of data will be effective and representative of the food chain.

Multiplex primer-extension assay for identification of Yersinia species.

A multiplex primer-extension reaction (PER) assay, was specifically designed for the identification of ten Yersinia species. The assay, directed towards the tufA (elongation factor Tu) gene, was tested on a total of 42 samples representing Yersinia species and non-Yersinia species. The primers used in the preliminary PCR, designed in highly conserved regions upstream and downstream of the diagnosis sites, successfully amplified a 587 bp fragment. The diagnosis sites were simultaneously interrogated using a multiplex PER and the results were confirmed by fragment sequencing. The proposed test provides an appropriate tool to monitor the presence of Yersinia spp. in food samples and to evaluate the potential hazard for consumers.

Degenerative myelopathy in German Shepherd Dog: comparison of two molecular assays for the identification of the SOD1:c.118G>A mutation.

Degenerative myelopathy (DM) is a late-onset, slowly progressive degeneration of spinal cord white matter which is reported primarily in large breed dogs. The missense mutation SOD1:c.118G>A is associated with this pathology in several dog breeds, including the German Shepherd Dog (GSD). The aims of the present study were to develop a tool for the rapid screening of the SOD1 mutation site in dogs and to evaluate the association of the polymorphism with DM in the German Shepherd breed. Two different techniques were compared: a minisequencing test and a real-time pcr allelic discrimination assay. Both approaches resulted effective and efficient. A sample of 47 dogs were examined. Ten subjects presented the symptoms of the illness; for one of them the diagnosis was confirmed by postmortem investigations and it resulted to be an A/A homozygote. In another clinically suspected dog, heterozygote A/G, the histopathological examination of the medulla showed moderate axon and myelin degenerative changes. GSD shows a frequency of the mutant allele equal to 0.17, quite high being a high-risk allele. Because canine DM has a late onset in adulthood and homozygous mutant dogs are likely as fertile as other genotypes, the natural selection is mild and the mutant allele may reach high frequencies. A diagnostic test, easy to implement, may contribute to control the gene diffusion in populations. The SOD1:c.118G>A mutation could be a useful marker for breeding strategies intending to reduce the incidence of DM.

Ruminant rhombencephalitis-associated Listeria monocytogenes strains constitute a genetically homogeneous group related to human outbreak strains.

Listeriosis is a disease that causes significant economic losses at the farm level because of high morbidity and mortality in ruminants. This study was performed to investigate the role of ruminants in the epidemiology of listeriosis in northern Italy and the possible association of animal-adapted strains of Listeria monocytogenes with strains associated with human disease. Twenty ruminant rhombencephalitis isolates previously confirmed as L. monocytogenes by bacteriology and PCR were characterized by serotyping, pulsed-field gel electrophoresis, multi-virulence-locus sequence typing (MVLST), and multiplex single nucleotide polymorphism (mSNP) typing for the detection of epidemic clones. Subtyping results were subsequently compared with those obtained from human, food, and environmental isolates of L. monocytogenes, including 311 isolates from the University of Turin, Grugliasco, Italy, and 165 isolates representing major human listeriosis outbreaks worldwide, in addition to other unrelated isolates. Both mSNP typing and MVLST showed that 60% of the isolates analyzed belonged to epidemic clone I (ECI), which has been epidemiologically linked to several human outbreaks of listeriosis. In particular, the 1981 Canada outbreak was linked to the use of sheep manure and the 1985 California outbreak was linked to the use of raw cow's milk. In our study, ECI isolates were collected from different ruminant species on geographically and temporally distinct occasions for the last 13 years. Our results support the hypothesis that ruminants represent possible natural reservoirs of L. monocytogenes strains capable of causing epidemics of listeriosis in humans.