Distribution, virulence, genotypic characteristics and antibiotic resistance of Listeria monocytogenes isolated over one-year monitoring from two pig slaughterhouses and processing plants and their fresh hams.

Listeria monocytogenes contamination in raw pork and ready to eat foods is an important food safety concern, also for the increasing detection of antimicrobial-resistant isolates. Data on L. monocytogenes occurrence, persistence, distribution and genetic characterization in two different plants, namely in continuum from slaughtered pigs, environment and unfinished products (fresh hams) were observed by one-year monitoring and were integrated with their antimicrobial resistance patterns. A total of 98 samples out of the overall 1131 (8.7%) were positive for L. monocytogenes, respectively 2.6% and 13.2% in plants A and B: only three serotypes were identified, 1/2c (50%), 1/2b (36.7%) and 1/2a (13.27%), and strains were classified in 35 pulsotypes and 16 clusters by PFGE; a unique P-type was highlighted according to the detection of virulence genes. The contamination flow of L. monocytogenes has a low occurrence in slaughterhouse (Plant A = 1.1%, Plant B: 3.1%; p > 0.05) and increased throughout the processing chain with trimming area as the most contaminated (Plant A: 25%, Plant B: 57%; (p < 0.05)), both in the environment and in unfinished products (80% in hams before trimming in plant B). The dominant role of environmental contamination in post-slaughter processing is confirmed to be a significant cause of meat contamination by L. monocytogenes. Very high levels of resistance were observed for clindamycin (57%) and high resistance levels (>20-50%) to ciprofloxacin, oxacillin, levofloxacin and daptomycin, confirming the L. monocytogenes resistance trend to a wide range of antimicrobial agents. A total of 11 L. monocytogenes isolates were multidrug resistant and 7 out of them were isolated from slaughtered pigs. An interesting significant (p < 0.05) statistical correlation has been found between resistance to some antimicrobial agents and lineage/serotypes. Microbiological sampling of food and environments after sanitization are commonly used as verification procedure for the absence of L. monocytogenes in food plants and to give assurance of food safety, but strains characterization is necessary for industries to target specific control measures, like the enforcement of the hygiene program and of the control of operator activities, at least for permanent strains. The only presence of L. monocytogenes could not be considered as the conclusive assessment of a potential risk for public health, also in terms of emerging and emerged antimicrobial resistances.

Development and evaluation of a new Real-Time RT-PCR assay for detection of proposed influenza D virus.

The occurrence of virus belonging to the putative genus Influenzavirus D, has been demonstrated all-around the world arousing interest within the scientific community. Most of the published virological surveys are based on the first described Real-Time PCR method, designed on the PB1 gene of the first isolate. The necessity of extending investigation to different animal species and geographic areas, requires a continuous update of molecular tests, considering newly sequenced strains. Moreover, the availability of an alternative assay, is essential either to confirm data, or for ensuring the detection of the widest number of strains. A new Real-Time PCR, specific for influenza D virus (IDV), was developed and evaluated. The target sequences of primers and probe are highly conserved among IDV strains currently known. The specificity of the method was demonstrated in silico by BLAST, and in vitro with a huge panel of common swine and bovine respiratory pathogens. The analytical sensitivity of the Real-Time PCR was estimated through synthetic RNA molecules and the limit of detection was about 20 copies/µL. The assay was assessed in field and proved to be a valuable tool for the detection of IDV strains.

Effects of DNA extraction method on Porcine circovirus-2 real-time polymerase chain reaction quantification in swine lymph node samples.

Quantitative real-time polymerase chain reaction (PCR) has become an important tool for Porcine circovirus-2 (PCV-2) research and diagnosis. However, significant differences in detection limit and quantification data, among laboratories and quantitative real-time PCR methods, have been demonstrated. New efforts are required for providing more accurate and comparable results. The current study is an evaluation of the effects of DNA extraction procedures on PCV-2 quantification in lymph node samples. Differences, greater than 1 log(10) copies/g, were shown among PCV-2 loads detected after different extraction procedures. The work highlighted the critical role of the DNA extraction method in PCV-2 quantification by quantitative real-time PCR. This important aspect should be evaluated when comparing data from different laboratories or different studies. The PCV-2 quantification data should not be considered comparable before demonstrating the equivalence of the DNA extraction methods performed.