The occurrence of Paenibacillus and Clostridium spores in silage is of great concern for dairy producers because their spores can contaminate milk and damage processed milk and semi-hard cheeses. ...Spoiled silage is considered to be the main contamination source of the total mixed ration (TMR), feces of dairy cows, and consequently bulk tank milk via the contamination of cow teats by dirt during milking. The presence of an anaerobic and facultative anaerobic sporeformer population in different matrices (soil, corn silage, other feeds, TMR, feces, and milk) and its transmission pathway has been studied on 49 dairy farms by coupling plate count data with 16S-DNA identification. The different matrices have shown a high variability in the anaerobic and facultative anaerobic spore count, with the highest values being found in the aerobically deteriorated areas of corn silages. Clostridium tyrobutyricum, Paenibacillus macerans, and Paenibacillus thermophilus were detected in all the matrices. The TMR spore count was influenced by the amount of spoiled corn silage in the TMR and by the care taken when cleaning the spoiled silage before feed-out. Most of the farms that prevent the presence of visible moldy silage in the silo and carefully clean to remove molded spots were able to maintain their TMR spore counts below 4.0 log spores/g. When a level of 4.5 log spores/g of TMR was exceeded, the feces presented a greater contamination than 3.0 log spores/g. Moreover, the higher the number of spores in the feces was, the higher the number of spores in the milk. Most of the farms that presented a feces contamination greater than 5.0 log spores/g had a higher milk spore contamination than 1,000 spores/L. Careful animal cleaning and good milking practices have been found to be essential to maintain low levels of contamination in bulk tank milk, but it has emerged that only by coupling these practices with a correct silage management and cleaning during TMR preparation can the contamination of milk by spores be kept at a low level. It has been found that aerobically deteriorated silage has a great capacity to contaminate TMR and consequently to increase the risk of milk spore contamination, even when routine milking practices are adopted correctly.
This study investigates the microbiological conditions of large game animal carcasses following evisceration. Carcasses of animals (N=291) hunted in the Upper Susa Valley (Italian Alps) were analysed ...for pH, Aerobic Viable Count (AVC), Enterobacteriaceae, Yersinia spp., Listeria monocytogenes and Salmonella spp. After shooting, evisceration occurred within 60min in 90.7% of animals and sampling within 90min in 88.3% of animals. Mean pH values (5.97: ruminants; 5.77: wild boar) were similar to those of regularly slaughtered domestic species. AVC values were highest in animals shot in the abdomen. Within species, AVC and Enterobacteriaceae values did not differ across different shooting-evisceration/sampling times. However, these counts exceeded 5 and 2.5 log, respectively, in 18% of wild boar and 39% of ruminants; the highest values were detected in wild boar. No pathogens were detected in any species. These results reveal inadequate hygiene in game meat handling/harvesting, implicating the need for improved practices.
In this study the occurrence of visible anisakid larvae in semi-preserved anchovy products sold on the Italian market was investigated. Totally, 107 ready to eat products (33 salted-ripened, 49 in ...oil and 25 marinated) were sampled. Each sample was digested, then the digested material was observed under natural and UV light. Parasites were counted, collected and microscopically identified to genus level. A representative subset was molecularly identified using the cox2 gene. At least one visible Anisakis sp. larva was found in 54.2% of the total 107 products analysed and totally 1283 dead larvae were collected. Anisakis sp. larvae were found in all the 33 salted products and 1139 (88.8%) larvae were collected, with a range of 1–105 parasites per product. Larval density per gram was 0.13. Anisakis sp. larvae were found in 49.0% of the products in oil and 143 (11.1%) larvae were isolated, with a range of 0–28 and a density of 0.03. Only 1 larva was found in the 25 marinated products (4.0%, density 0.00). A highly significant difference between all the product categories in respect of number of larvae per product, frequency of products contaminated by at least one larva and larval density per gram was found. Within the subset of larvae molecularly analysed (n=122), 92 (75.4%) were identified as A. pegreffii and 30 (24.6%) as A. simplex. This study showed that semi-preserved anchovy products heavily contaminated with Anisakis spp. larvae reach the market. Beyond the negligible risk for anisakidosis, the presence of dead visible parasites may cause immediate rejection in consumers. In addition, the potential risk related to allergic reactions in sensitized individuals needs to be further assessed. In order to avoid commercialization of obviously contaminated products, fresh anchovies' batches intended for the production of such products should be accurately selected by the processing industry applying inspection methods.
•Different kinds of ready to eat anchovy products were analysed by digestion.•At least one visible Anisakis spp. larva was found in 54.2% of the products.•A total of 1283 dead larvae were collected.•The product category influenced the number of larvae and contamination rate.•Salted products were found to be the most contaminated (100%).
Anaerobiosis, critical for successful ensilage, constitutes a challenge in baled silages. The loss of complete anaerobiosis causes aerobic deterioration and silages undergo dry matter and nutrient ...losses, pathogen growth, and mycotoxin production. Silage may represent an ideal substrate for Listeria monocytogenes, a pathogen of primary concern in several cheeses. The aim of this research was to investigate the occurrence of Listeria in baled silage fed to cows producing milk for a protected designation of origin cheese, and to characterize isolates by repetitive sequence-based PCR. Listeria spp. were detected in 21 silages and L. monocytogenes in 6 out of 80 of the analyzed silages; 67% of positives were found in molded zones. Results of the PCR typing showed genotypic homogeneity: 72.9 and 78.8% similarity between strains of Listeria spp. (n=56) and L. monocytogenes (n=24), respectively. Identical profiles were recovered in molded and nonmolded areas, indicating that contamination may have occurred during production. The application of PCR allowed the unambiguous identification of Listeria isolated from baled silages, and repetitive sequence-based PCR allowed a rapid and effective typing of isolates. Results disclose the potential of the systematic typing of Listeria in primary production, which is needed for the understanding of its transmission pathways.
This study aimed at selecting effective histological indicators of the freezing process, for the discrimination of fresh and frozen common octopus
Octopus vulgaris
. Histological indices of freezing ...process were selected in mantle and arm muscle and axial nerve tissues. Seven histological parameters were chosen: overall muscle tissue structural organization (a); gaping among muscle bundles (b); presence of optically empty spaces between and within muscle bundles (c); white spaces percentage between and within muscle bundles (d); overall nerve structural organization (e); presence of linear fissures and/or empty spaces within neuropil and axonal tract (f); presence of empty spaces within connective tissue matrix surrounding the nerve (g); empty space percentage within arm axial nerve region (h). The parameters were assessed on 150 mantle muscle sections (a, b, c, d), 150 arm muscle (b, c) and nervous (e, f, g, h) tissue sections belonging to 20 fresh exemplars further subdued to conventional freezing procedure at − 20 °C, 25 fresh curled exemplars, 25 exemplars industrially frozen at − 80 °C, and 20 thawed, curled, and individually quick frozen exemplars. Overall structural organization (a, e), gaping (b), and presence of optically empty spaces (c, f) confirmed significant morphological freezing indices in mantle and arm sections. In mantle, two d values (d < 25% and 25% < d < 33%) were proposed as complementary thresholds to be applied in association with b and c indices for freezing discrimination. These parameters appear eligible to set a method for discriminating fresh/thawed octopus products to be applied both in official control and self-check activities.
The European anchovy (Engraulis encrasicolus), one of the most important pelagic fish resources in the Mediterranean Sea, is frequently infected by anisakid larvae. Food Business Operators (FBOs) ...should use appropriate sampling plans and analytical methods to avoid commercialization of massively infected batches and reduce the risk of transmission of viable zoonotic larvae. In this study, performed at FishLab (Department of Veterinary Sciences of the University of Pisa) during 2016, an official sampling plan was associated with a digestion protocol for the inspection of anchovies. Considering that anisakid larvae are usually located in the fish visceral cavity and in the adjacent muscles (VM), this part was analyzed. In particular, we assessed the reliability of the digestion of a subsample of 150g (±30g) of VM, randomly collected from 29 specimens, in estimating the marketability of the anchovies' batch. Fifty-seven samples of 29 anchovies were collected. Each anchovy was sectioned to separate VM. All the subsamples were digested, and visible larvae counted. A high correlation between the number of larvae in VM regions and in the total batch was observed, indicating a very significant contribution of the VM region on total number of parasites. The Mean Abundance (MA) was used to assess the batch marketability according to a threshold calculated on the basis of the maximum number of nematodes tolerated per sample. Considering that the MA can be calculated only when the number of examined specimens is known, the number of visible Larvae per gram of tissue (LpG) was calculated on 150g (±30g) of VM subsamples. A LpG marketability threshold was calculated dividing the maximum number of tolerated nematodes by the average weight of a sample of 29 anchovies calculated considering data available in literature. To evaluate the diagnostic performance of the LpG threshold, the marketability of 57 batches assessed on the basis of the MA threshold was assumed as the gold standard. The proposed LpG showed very high Specificity and Sensitivity. These findings suggest that the analysis of VM is representative of the overall infestation of the batch, both when considering the absolute number of parasites and the LpG, and may represent a valid alternative to the whole anchovy digestion. In particular, the use of an automated digestive method, coupled with the aforesaid sampling plan, could allow the procedure to be used by FBOs in operational conditions.
•Visible parasites in viscera/adjacent muscle (VM) were used as a predictor of infection.•Anchovies' marketability was assessed according to Larvae per gram (LpG) of VM tissue.•LpG showed good diagnostic performances when compared to the gold standard.•The method is an easy tool for inspecting anchovies timely as they reach the market.•The method will prevent the commercialization of massively infected fish.
Epithelial cells are shed into milk during lactation, and although they generally reflect the cellular characteristics of terminally differentiated luminal cells, previously the detection of more ...primitive cells was described in human milk where a cell population of epithelial lineage was detected expressing markers typical of progenitor cells. In this investigation, we report the development of flow cytometry analysis to allow multiparametric assessment of mammary epithelial cells observed in milk. Cells collected from milk samples of 10 healthy dairy cows were directly analyzed for 6 different markers: CD45, CD49f, cytokeratin 14, cytokeratin 18, presence of nucleus, and cell viability. Milk samples were collected in 3 different periods of lactation: early lactation (EL=d 0–30), mid-lactation (ML=d 90–120), and late lactation (LL=210–250). Here we identify the differential expression of precursor or differentiated cell markers (or both) in mammary epithelial cells present in bovine milk. Myoepithelial cells, as indicated by cells staining positively for cytokeratin 14+/cytokeratin 18−, were observed to increase from EL to LL with a high correlation with nuclear staining inferring potential proliferative activity. Furthermore, a significant increase in CD49f+ and cytokeratin 14+/cytokeratin 18+ positive cells was observed in LL. This assay is a sensitive approach for evaluating the variations in the frequency and features of living epithelial cells, whose reciprocal balance may be significant in understanding mammary gland cellular function throughout lactation. These observations suggest that mammary epithelial cell immunophenotypes could be investigated as biomarkers for mammary gland function in dairy cows.
In this study the results of a systematic review and meta-analysis on mislabeling in seafood products sold on the Italian market are presented The aim was especially targeted to answer the research ...question “What is the mislabeling rate in seafood products sold on the Italian market?“. Scientific papers (SPs), were filtered using pre-determined inclusion criteria and data related to sampling and mislabeling was analysed. Samples were categorized according to their taxon (species, family order) or generic market group (MG), market form (unprocessed/processed), distribution channel and geographical area. Samples were considered mislabeled when the species found by molecular analysis did not comply the information indicated in the label. The mislabeling rate (m. r.) was weighted on the sample size and provided overall and for each category. In the 51 selected SPs (published from 2005 to 2022) the most sampled taxa were fish (83.8%): mackerels, cods, herrings, flatfishes and jacks were the most represented. Unprocessed fillet/slice was the most analysed market form (61.4%), and samples were especially collected at retails (76.5%). Ten regions were sampled, especially Tuscany and Apulia. The overall weighted m. r. was 28.4% (CI 26%–30%), falling within the m. r. range found at international level (Luque & Donlan, 2019). M. r. over the CI (>30%) were observed in 1) jellyfishes, European perch, European grouper, Atlantic mackerel and samples labeled as “spinarolo”, “baccalà” or “palombo”; 2) Unprocessed fresh, processed salted and highly processed samples; 3) small distribution channel; 4) Southern regions. Significative differences in m. r. concerned taxa, distribution channels and geographical areas. Despite some bias of the SPs may affect the results (lack of sampling plans; poor data on molluscs and crustaceans; no standardization in m. r. interpretation) this is the first systematic review and meta-analysis that, synthesizing evidence on Italian seafood mislabeling, can support policy making for minimizing frauds impacts.
To investigate the genetic relatedness between Lactococcus garvieae strains isolated from fish and dairy samples collected in northern Italy, using random-amplified polymorphic DNA (RAPD)-polymerase ...chain reaction (PCR), Sau-PCR and amplified fragment length polymorphism (AFLP). Eighty-one isolates from bovine and caprine dairy products (n = 53) and from diseased rainbow trouts and other fishes (n = 28) were examined. All methods showed a typeability of 100%, repeatability ranging from 84·4% to 97·5% and discriminatory powers from 0·798 to 0·986. Dairy and fish strains revealed a low genetic relatedness as they are often grouped into distinct clusters. RAPD analysis discriminated 52 genotypes when primer M13 was used, whereas with primer P5 only 27 genotypes were identified. When Sau-PCR was performed, 13 genotypes were detected while AFLP analysis allowed the differentiation of 32 genotypes. L. garvieae strains isolated from dairy samples are generally not related to those collected from fish lactococcosis outbreaks. L. garvieae strains exhibit a genetic diversity related to the specific animal host they colonize. RAPD M13 fingerprinting proved to be a molecular tool for comparing isolates, whereas Sau-PCR and AFLP analyses were useful techniques to investigate the distribution of L. garvieae populations in the environment.
A multiplex PCR able to identify cows’, goats’ and sheep's milk in dairy products was developed. Specific primers were designed in the mitochondrial 12s and 16s rRNA genes so as to generate fragments ...of different length.
The assay was applied to 19 cheeses from the retail trade in order to verify the label statements. The multiplex PCR results were confirmed by PCR-restriction fragment length polymorphism.
The proposed multiplex PCR represents a rapid and sensitive method applicable on a routine basis. In fact it enables to detect, in a single step, goats’, sheep's and cows’ milk in dairy products with a good sensitivity threshold (0.5%).
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