Trichinella spiralis a new alien parasite in Italy and the increased risk of infection for domestic and wild swine.

In Europe, Trichinella spiralis, the most dangerous species for humans of the genus Trichinella, has a patchy distribution with important foci in Eastern countries and Spain. This zoonotic pathogen was apparently not circulating among wild and domestic animals of Italy. In 2016, muscle larvae belonging to this nematode species were detected in a red fox (Vulpes vulpes) shot in the Piacenza province (Northern Italy). This parasite may have been introduced into northern Italy from eastern Europe by hunters, by a hunting dog, or by immigrants, who illegally carried infected meat in their personal baggage. In the same year, T. spiralis infected sausages illegally introduced by personal baggage into Italy from Romania, were inadequately disposed of in the garbage of a central Italian town. Even though these two episodes may not be connected in time and space, they represent an increased risk of infection for domestic and wild swine, which are highly susceptible to this pathogen. In these animals, T. spiralis shows a higher larval burden and a longer survival time than other Trichinella species. Since most of the Italian pig production plants are in northern Italy, the circulation of T. spiralis should be strictly monitored in wildlife living in these areas.

Estimation of Mycobacterium avium subsp. paratuberculosis load in raw bulk tank milk in Emilia-Romagna Region (Italy) by qPCR.

Consumption of milk and dairy products is considered one of the main routes of human exposure to Mycobacterium avium subsp. paratuberculosis (MAP). Quantitative data on MAP load in raw cows' milk are essential starting point for exposure assessment. Our study provides this information on a regional scale, estimating the load of MAP in bulk tank milk (BTM) produced in Emilia-Romagna region (Italy). The survey was carried out on 2934 BTM samples (88.6% of the farms herein present) using two different target sequences for qPCR (f57 and IS900). Data about the performances of both qPCRs are also reported, highlighting the superior sensitivity of IS900-qPCR. Seven hundred and eighty-nine samples tested MAP-positive (apparent prevalence 26.9%) by IS900 qPCR. However, only 90 of these samples were quantifiable by qPCR. The quantifiable samples contained a median load of 32.4 MAP cells mL(-1) (and maximum load of 1424 MAP cells mL(-1) ). This study has shown that a small proportion (3.1%) of BTM samples from Emilia-Romagna region contained MAP in excess of the limit of detection (1.5 × 10(1) MAP cells mL(-1) ), indicating low potential exposure for consumers if the milk subsequently undergoes pasteurization or if it is destined to typical hard cheese production.

Evaluation of viable Mycobacterium avium subsp. paratuberculosis in milk using peptide-mediated separation and Propidium Monoazide qPCR.

The causative agent of paratuberculosis in ruminants, Mycobacterium avium subsp. paratuberculosis (MAP), although still a matter of debate, has been linked with Crohn's and other human diseases. The availability of rapid methods for assessing the viability of MAP cells in food, in particular milk, could be of great use for risk management in food safety. MAP viability is generally assessed using culture techniques that require prolonged incubation periods for the growth of MAP. To differentiate between viable and nonviable MAP cells in milk samples, this study explores the combination of two already described techniques: peptide magnetic bead separation followed by Propidium Monoazide qPCR. Using an Ordinal Multinomial Logistic Regression model to analyze the results obtained after spiking milk samples with mixtures containing different percentages of viable/dead cells, we were able to assess the probability of the viability status of MAP found in milk. This model was applied to contaminated pasteurized milk to ascertain the efficacy of heat treatment in MAP killing. The method reported herein can potentially be used for direct detection of MAP viability in milk.

High-resolution melting for analysis of short sequence repeats in Mycobacterium avium subsp. paratuberculosis.

Analysis of micro- and minisatellite loci is widely used in sub-typing of Mycobacterium avium subsp. paratuberculosis. Microsatellite (short sequence repeat, SSR) loci have shown highest discriminatory power, but direct sequencing of amplicons is required for correct assignment of the repeat number. We developed an alternative method to sequencing, focusing on the SSR8 locus (constituted by GGT triplets from three to six repeats). The approach is based on asymmetric quantitative PCR, followed by high-resolution melting analysis with unlabelled probes (UP-HRM). Data showed perfect concordance between direct sequencing and UP-HRM, which is faster, simpler and more cost effective.

Effectiveness of combination of Mini-and Microsatellite loci to sub-type Mycobacterium avium subsp. paratuberculosis Italian type C isolates.

BACKGROUND: Mycobacterium avium subsp. paratuberculosis (Map) is the etiological agent of paratuberculosis. The aim of our study was to combine Mini-and Microsatellite loci analysis in order to explore the effectiveness of this sub-typing method in a group of Map isolates. For this purpose, 84 Italian Type C Map isolates, each from a different cattle herd, were submitted to MIRU-Variable-Number Tandem-Repeats (VNTRs) typing and Short Sequence repeats (SSRs) sequencing. Moreover, the method was used to analyse the variability inside 10 herds (from three to 50 isolates per herd). RESULTS: The molecular sub-typing, carried out using three SSR and 10 MIRU-VNTR loci, differentiated the 84 isolates into 33 clusters, reaching a Simpson's Discriminatory Index (SID) value of 0.952 (0.933 to 0.972, 95% confidence intervals). Among all considered loci, six (SSR2, MIRU2, SSR1, SSR8, VNTR3527 and VNTR1067) showed relevant allelic variability. Thirty-eight% of the isolates were clustered into four genotypes, differing from each other for the SSR2 locus. The other isolates, characterised by differences in two or more loci, were spread among the rest of the clusters. The intra-herd analysis revealed more than one genotype in most herds with a similar distribution of clusters. CONCLUSIONS: Our results revealed the advantage of using both Mini-and Microsatellite approaches for successfully discriminating among Map Type C isolates from the same geographic area, host species and herd. These data suggest that the combination of loci here proposed could be a useful molecular tool for regional epidemiological studies.