Molecular characterization of enterotoxigenic and borderline oxacillin resistant Staphylococcus strains from ovine milk.

Staphylococcal enterotoxins (SEs) produced by Staphylococcus spp. are superantigens responsible for food-poisoning and are associated to mobile genetic elements such as Staphylococcus aureus pathogenicity islands (SaPI). The presence of 13 enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej, sel, sek, seq, and tst) was tested in 15 S. aureus and 24 coagulase-negative Staphylococcus (CNS) multi-resistant strains isolated from ovine milk in Sardinia. All CNS isolates were enterotoxin-negative, whereas co-presence of sec, sel and tst was observed in most of the S. aureus strains. One isolate of S. aureus was characterized by tst alone. A multiplex PCR assay aimed at discriminating between the integrase genes of pathogenicity islands SaPI2, SaPIbov1, and SaPIMW2 was developed. We demonstrated that strains harboring sec, sel and tst were associated with SaPIbov1, whereas the strain positive for tst was associated with SaPI2. Borderline oxacillin resistant S. aureus strains were also detected. RAPD analysis of the Staphylococcus strains showed that clonal relationships were correlated with pathogenic profiles.

A rapid phage assay for detection of viable Mycobacterium avium subsp. paratuberculosis in milk.

Paratuberculosis is an incurable gastroenteritis among ruminants that is promoted by Mycobacterium avium subsp. paratuberculosis (MAP), an acid-fast mycobacterium. To accelerate the detection of viable pathogen, a conventional (peptide mediated magnetic separation: PMS) and novel (phage-bead qPCR: PBQ) phage based assay was optimized. A superior limit of detection (LOD) of 10 MAP per 10 mL milk was suggested for PBQ compared to 100 cells/10 mL for PMS-phage assay. Via PBQ, viable MAP was found in 48.78% out 41 unpasteurized sheep and goat milk samples. Sheep milk samples (n = 29) that were tested by PMS-phage assay contained no viable MAP. The absence of viable MAP in milk collected from 21 of the recent sheep animals was also confirmed by PBQ after a 2-week gap. Although, the two phage assays comparably detected no viable MAP in the milk samples, MAP DNA and antibodies against MAP were recognized in milk and sera of some of these animals within two instances of sampling representing that some sheep animals were MAP shedders. In conclusion, PBQ and PMS-phage could be promising methods for the assessment of MAP viability in milk samples. However, PBQ was privileged over the PMS-phage assay due to the lower LOD, rapidity, higher sensitivity, lack of need to M. smegmatis and consequent virucidal treatment that are essential in PMS-phage assay for making lawn and inactivation of exogenous mycobacteriophages respectively.

Epidemiological survey on the prevalence of Salmonella spp. in the Sardinian pig production chain, using real-time PCR screening method.

The aim of this study was to evaluate the prevalence of Salmonella spp. in the Sardinian pig production chain in order to establish the incidence of monophasic serovariant of Salmonella Typhimurium on isolates with molecular methods (real-time PCR and multiplex PCR). Samples were collected in three EC slaughterhouses, four small slaughterhouses annexed to farmhouses, one meat distribution center, four meat cutting laboratories and four sausage processing plants. A total of 166 samples were collected and analyzed: 46 environmental samples, 48 finishing pigs, 16 piglets, 24 samples of non-processed meat, 28 meat preparations and 4 meat products. All samples were processed with an initial screening using the real-time PCR MicroSEQ® Salmonella spp detection Kit (Applied biosystems, life technologies) and with the TaqMan® Real-time PCR to confirm the kit results. Samples that tested positive for Salmonella spp were confirmed with cultural method using the standard ISO 6579. Positive samples were submitted to phenotypic identification. One colony from each positive sample was serotyped with multiplex PCR method. Salmonella spp was isolated in 7 on 166 samples (4.22 %). Among the positive samples, two came from finishing pigs, two belonged to the category meat preparations, two to meat products, one was an environmental sample. Multiplex PCR confirmed that the collected strains belonged to the species Salmonella Typhimurium (1), Salmonella derby (3) and monophasic serovariant of Salmonella Typhimurium (3).

Salmonella serovar distribution from non-human sources in Italy; results from the IT-Enter-Vet network.

The study summarises the results obtained over the period 2002-2013 by the Italian IT-Enter-Vet network, aimed at collecting data on Salmonella isolates from non-human sources. A total of 42,491 Salmonella isolates were reported with a progressive decrease over the years. S. Typhimurium was the most frequent serovar up to 2011, but then, it was overtaken by S. 4,[5],12,:i:-, S. Derby, S. Livingstone and S. Enteritidis alternated as the third most commonly isolated serovars. With regard to the sources of isolation, S. Typhimurium was distributed ubiquitously among the animal species. On the contrary, S. 4,[5],12,:i:- and S. Derby were strictly associated with pigs, whereas S. Livingstone, S. Enteritidis and S. Infantis were clearly related to poultry. Intriguingly, when the frequency of serovar distribution along the food chain was considered, it was evident that S. Typhimurium and S. Derby tended to persist along the chain, as they were isolated even more frequently from foods than from animals. A similar distribution was found for S. Enteritidis and S. Hadar. Despite limitations related to non-mandatory participation of laboratories in the network, the data presented are valuable to obtain a picture of the evolution of Salmonella from non-human sources over time in Italy.

Intramammary infusion of a live culture of Lactococcus lactis in ewes to treat staphylococcal mastitis.

PURPOSE: Alternatives to antibiotic therapy for mastitis in ruminants are needed. We present an evaluation, in two trials, of the efficacy of an intramammary infusion of a live culture of Lactococcus lactis for the treatment of subclinical and clinical mastitis in ewes. METHODOLOGY: In total, 67 animals were enrolled: 19 lactating ewes (study 1), including healthy (N=6) and coagulase-negative staphylococci (CNS)-infected ewes (N=13); and 48 lactating ewes (study 2) with either CNS mastitis (N=32), or Staphylococcus aureus mastitis (N=16), for a total of 123 mammary glands. Intramammary infusions were performed with either L. lactis or PBS for 3 (study 1) or 7 (study 2) consecutive days. Antibiotic-treated and untreated control glands were included. Milk samples for microbiology, somatic cell analysis and milk production were collected before and after treatment.Results/Key findings.L. lactis rapidly activated the mammary glands' innate immune response and initiated an inflammatory response as evidenced by the recruitment of polymorphonuclear neutrophils and increased somatic cell counts. But while leading to a transient clearance of CNS in the gland, this response caused mild to moderate clinical cases of mastitis characterized by abnormal milk secretions and udder inflammation. Moreover, S. aureus infections did not improve, and CNS infections tended to relapse. CONCLUSION: Under our experimental conditions, the L. lactis treatment led to a transient clearance of the pathogen in the gland, but also caused mild to moderate clinical cases of mastitis. We believe it is still early to implement bacterial formulations as alternatives in treating mastitis in ruminants and further experimentation is needed.

Genetic and pathological characteristics of Cryptococcus gattii and Cryptococcus neoformans var. neoformans from meningoencephalitis in autochthonous goats and mouflons, Sardinia, Italy.

In this study, we examined in Sardinia the brain of 555 autochthonous sheep, 50 goats, and 4 mouflons which were found affected by neurological signs. We found 6 goats and one mouflon with meningoencephalitis caused by Cryptococcus sp. There was no evidence of cryptococcal infections in any of the examined sheep. MLST genotyping on Cryptococcus sp. isolates identified Cryptococcus gatti genotype AFLP4/VGI and Cryptococcus neoformans var. neoformans genotype AFLP2/VNIV. Phylogenetically, all Cryptococcus gattii isolates fell within the autochthonous animal, human and environmental Mediterranean isolate cluster, forming a distinct branch along with environmental strains from Alicante, in the southern Mediterranean coast of Spain.

Validation of a serological test for the diagnosis of canine rickettsial disease.

Canine vector borne diseases include a variety of illnesses affecting domestic dogs worldwide. Clinical abnormalities are often nonspecific during rickettsial infections, and coinfections caused by other tick-transmitted agents may be common. The aim of this study was to validate a differential serological assay for the diagnosis of rickettsial infections by the indirect fluorescent antibody (IFA) test. Sensitivity (DSe), specificity (Dsp), accuracy (Acc), positive and negative predictive values (PPV and NPV), positive and negative likelihood ratios (LR+ and LR-), Cohen's Kappa agreement, Youden's J index and odds values were calculated in order to define the positive and negative post-test probability and to establish a link between clinical signs compatible with a rickettsial infections and serological confirmation.