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Listeria monocytogenes has been identified on minimally processed fruit and vegetables, and can transmit a severe foodborne disease through the consumption of ready-to-eat (RTE) fresh-cut produce, mostly if the storage conditions are incorrect, and the cold chain is not abided to. The aim of the present work was to evaluate the behaviour of L. monocytogenes in RTE fresh-cut papaya and melon, two low-acid fruits stored in temperature-abuse conditions (10 and 8°C, respectively) during the shelf-life indicated by the producer (7 d). A microbiological challenge test was performed to assess the maximum growth rate (MGR) and the duplication time (Td) of the pathogen, testing three strains separately. We also assessed the behaviour of the indigenous microflora of the fruits. Results demonstrated that papaya and melon were two favourable substrates for L. monocytogenes. The MGR was 0.044 log CFU/g/h with a Td of 6.77 h for papaya, and 0.029 log CFU/g/h with a Td of 10.24 h for melon. The storage of RTE fresh-cut papaya and melon at an abusive temperature above 8°C could encourage the growth of L. monocytogenes with a possible risk to public health. [ABSTRACT FROM AUTHOR]

Abstract: Aims: To evaluate the survival of Mycobacterium avium subsp. paratuberculosis (MAP) during anaerobic digestion (AD), we studied two different biogas plants loaded with manure and slurry from paratuberculosis‐infected dairy herds. Methods and Results: Both plants were operating under mesophilic conditions, the first with a single digester and the second with a double digester. Mycobacterium avium subsp. paratuberculosis detection was performed by sampling each stage of the process, specifically the prefermenter, fermenter, liquid digestate and solid digestate stages, for 11 months. In both plants, MAP was isolated from the prefermenter stage. Only the final products, the solid and liquid digestates, of the one‐stage plant showed viable MAP, while no viable MAP was detected in the digestates of the two‐stage plant. Conclusions: Mycobacterium avium subsp. paratuberculosis showed a significant decrease during subsequent steps of the AD process, particularly in the two‐stage plant. We suggest that the second digester maintained the digestate under anaerobic conditions for a longer period of time, thus reducing MAP survival and MAP load under the culture detection limit. Significance and Impact of the Study: Our data are unable to exclude the presence of MAP in the final products of the biogas plants, particularly those products from the single digester; therefore, the use of digestates as fertilizers is a real concern related to the possible environmental contamination with MAP. [ABSTRACT FROM AUTHOR]

Paratuberculosis of ruminants is characterised by chronic enteritis but, at advanced stages of the disease, a systemic dissemination of Mycobacterium avium subsp. paratuberculosis (MAP) in tissues and organs can occur. MAP has been recovered from lymph nodes and muscles of clinical and sub-clinical cows. In most countries, dairy and beef cattle infected with paratuberculosis are routinely sent to slaughter and the consumption of their meat could be a possible route of human exposure to MAP. However, few studies on MAP in ground beef are currently available. During the period November 2013–March 2014 we carried out a survey on the ground beef produced in an industrial meat processing plant. One-hundred and forty samples of ground meat were analysed by IS900-qPCR and culture (VersaTrek System®). The limit of detection (LOD) of qPCR was 630 MAP cells/g (107 CFU/g) while the LOD for culture was 170–230 MAP cells/g (62–115 CFU/g). No samples were positive by direct IS900 qPCR, while two samples were positive by liquid culture. Our data suggest that the presence of live MAP in raw minced meat is possible. In order to avoid exposure for humans through the consumption of contaminated meat, proper cooking of meat is recommended. [ABSTRACT FROM AUTHOR]

Abstract: Paratuberculosis is a chronic granulomatous infection caused by Mycobacterium avium subsp. paratuberculosis (MAP) that affects multiple ruminant species causing important economic losses. Therefore, control programmes at herd and regional levels have been established worldwide and prevalence estimates are needed for their implementation. Although different herd-level prevalence estimations for paratuberculosis have been reported in Europe, very few studies provided comparable and interpretable values, due to poor study designs and lack of knowledge about the accuracy of the diagnostic tests used. To overcome these problems we applied a latent class analysis to the results of two prevalence studies carried out in two neighbouring Northern Italian regions (Lombardy and Veneto) that account for over 50% of the Italian dairy cattle population. Serum samples from a randomly selected number of farms in the two regions were analyzed by different ELISA tests. The herd-level Apparent Prevalences (AP) were 48% (190/391) for Lombardy and 65% (272/419) for Veneto. Median within-herd APs were 2.6% and 4.0% for Lombardy and Veneto, respectively. Posterior estimates for the herd-level True Prevalences (TP) based on a Bayesian model were very similar between the two regions (70% for Lombardy and 71% for Veneto) and close to previous estimates of infected herds in Europe. The two 95% credibility intervals overlap each other, virtually showing only one distribution of the herd-level true prevalence for both regions. On the contrary, estimates of the within-herd TP distributions differed between the two regions (mean values: 6.7% for Lombardy and 14.3% for Veneto), possibly due to the different age distribution within the herds from the two regions. [Copyright &y& Elsevier]

Composite milk samples from 548 cows, and samples from feces, feed, bedding, water, liners (before and after milking), and the postdipping product were aseptically collected from 2 Italian dairy herds from February to November of 2006. Prototheca zopfii was isolated from 11.9% of milk samples, 15% of feces, and 33.3% of bedding samples. No viable cells of P. zopfii were observed in water before washing procedures, whereas 25 to 28.6% of samples from water used for washing both refrigeration tanks and milking equipment were contaminated with this yeast-like microalga. Analogously, the presence of P. zopfii was detected only on swabs collected from the liners after milking. Interestingly, in 1 of the 2 herds, water from the drinking trough was contaminated by viable cells of both P. zopfii and the related environmental species Prototheca stagnora. No viable cells were observed in cow feed. On the basis of the results presented herein, P. zopfii seemed to be widespread throughout the environments of dairy herds where outbreaks of bovine mastitis had occurred. [ABSTRACT FROM AUTHOR]

Abstract: In this study, the isolation of 52 mycobactin-independent fast growing mycobacteria from 631 bulk milk samples (8.2%), is reported. These strains, isolated during a bulk milk survey for Mycobacterium avium subsp. paratuberculosis (Map), strongly affected Map detection both by PCR and by culture, as they gave a positive IS900 PCR signal and resulted to totally inhibit the growth of Map when spotted on HEYM slants already inoculated with 200μl of 10-fold dilutions containing from 5×10 to 5×103 Map cells/ml. 16S rRNA gene sequencing, using the MicroSeq 500 16S rDNA Bacterial Sequencing Kit (Applied Biosystems), was performed on a subset of six strains, identifying Mycobacterium porcinum with 100% homology in all six cases. The 52 strains were characterized by PCR-restriction fragment length polymorphism (RFLP) analysis of the hsp65 gene, which confirmed the identification of M. porcinum for all the isolates. Using specific primers designed on the Map-IS900 sequence and on the M. porcinum sequence determined in this study, a 1385bp sequence from the M. porcinum genome was characterized. This IS900-like sequence showed 82% homology with Map IS900. From our findings the following results emerged: (a) any culture showing one or more M. porcinum colonies represents a potential “false negative” result and should therefore be considered as contaminated; (b) IS900-like elements could be more widespread than was previously thought; (c) IS900 PCR positive results should be interpreted cautiously, as confirmed by the evidence that the primer pair used in this study resulted not to be specific. [Copyright &y& Elsevier]

• Phylogenetic analysis disclosed the within-herd population structure of MAP. • Bayesian phylodynamic inference was feasible despite a low substitution rate. • Multiple isolates recovered from a single animal originate from different donors. Mycobacterium avium subsp. paratuberculosis (MAP) is characterized by a low genomic rate of mutation. Current subtyping tools, such as Mini-Micro-satellite analyses, do to have not sufficient discriminatory power to disclose MAP's evolution on small spatial and temporal scales. The aim of the study was to investigate the population structure of MAP inside a single dairy herd using whole genome sequencing (WGS) approaches. For this purpose, the genomes of 43 field isolates, recovered from the faeces of 36 cows of the same dairy herd from 2012 to 2016, were sequenced by WGS. The isolates' genomes showed a low number (43) of polymorphic sites (SNPs), confirming the clonal origin of the herd infection. However, despite the limited genomic diversity found in WGS, the phylogenetic analysis was discriminatory enough to detect the presence of different genomic clades and sub-clades inside the herd population. In addition, the phylodynamic reconstruction showed the existence of an ancestor clade from which the other clades and sub-clades originated. Moreover, by reconstructing the putative within-herd transmission networks using WGS data, we demonstrated that: (i) in a herd where MAP is endemic, multiple isolates recovered from a single animal and differing from each other by few (three/four) SNPs can originate from different transmission or passive shedding events and not from intra-host evolution; and (ii) variability of minisatellites coupled with a few microsatellites does not represent reliable tracers of within-herd infection chains. Our findings show that WGS, coupled with relevant epidemiological information, represents a valuable tool to work out fine epidemiological and micro-evolutionary relationships such as those at herd-level scale. [ABSTRACT FROM AUTHOR]

Abstract: The most important animal disease caused by yeast-like algae belonging to the genus Prototheca is bovine mastitis. Although the infection can be caused by both Prototheca zopfii genotype 2 and Prototheca blaschkeae, the bulk of prevalence of bovine protothecal mastitis has been so far attributed to the former, being P. blaschkeae only sporadically isolated. However, we report here the first outbreak of bovine mastitis caused by P. blaschkeae in an Italian dairy herd. One hundred and four individual milk samples, three bulk tank milk and 16 environmental samples within the herd were screened for the presence of Prototheca: five, one and four positive samples, were respectively observed. Molecular analysis revealed that, with the sole exception of one environmental isolate belonging to P. zopfii genotype 2, all Prototheca strains were identified as P. blaschkeae. Our results might suggest that even P. blaschkeae can induce mastitis outbreaks, while it is not clear if the higher incidence of P. zopfii genotype 2 as causative agent of protothecal mastitis could reflect an intrinsic higher pathogenicity or it could be simply the consequence of its, so far observed, higher diffusion in worldwide dairy herd ecosystems. [Copyright &y& Elsevier]