Molecular differentiation of cattle Sarcocystis spp. by multiplex PCR targeting 18S and COI genes following identification of Sarcocystis hominis in human stool samples.

Sarcocystis spp. are protozoan parasites which can infect a wide range of vertebrates, including humans; the latter can act as definitive hosts for two cattle Sarcocystis spp.: Sarcocystis hominis and Sarcocystis heydorni. Reports of intestinal sarcocystosis are well documented in the literature, but PCR-based methods have been scarcely used to identify Sarcocystis species in human stools, and have been limited to the molecular analysis of 18S ribosomal RNA (18S rRNA) gene sequences. Since the mitochondrial cytochrome c oxidase subunit I (COI) gene is one of the most promising tools for distinguishing between closely related Sarcocystis spp., and taking into account the lack of publicly available S. hominis COI sequences, in the present study we obtained the first partial COI sequence of S. hominis from human stool samples of patient with gastrointestinal symptoms. We designed specific COI primers to develop a multiplex PCR method for the identification of Sarcocystis spp. in cattle. The submission of the COI sequence described herein and the unambiguous identification of S. hominis through the application of the new multiplex PCR is important for determining the prevalence of this zoonotic Sarcocystis spp. in meat and the risk for consumers.

Enterotoxin gene profiles of Staphylococcus aureus isolated from milk and dairy products in Italy.

UNLABELLED: Staphylococcal foodborne intoxication, occurring after consumption of staphylococcal enterotoxins (SEs) in food, is considered one of the most common forms of bacterial foodborne outbreaks worldwide. Milk and dairy products account for 5% of all the incriminated foods in staphylococcal outbreaks, referring to Europe. The distribution of genes encoding for enterotoxins in Staphylococcus aureus strains is highly variable, with some carried on stable regions of the chromosome and others carried on mobile genetic elements. The aim of this study was to analyse the distribution of genes encoding for SEs in Staph. aureus strains isolated from milk and dairy products. In the period from January 2010 to June 2011, a total of 1245 dairy samples (848 of raw milk and 397 of dairy products) were collected and analysed for detection of genes encoding for 11 SEs and SEls (SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, SER SElJ and SElP) according to the procedures of the Italian National Reference Laboratory for coagulase-positive Staphylococci including Staph. aureus. Staphylococcus aureus strains were isolated in 481 (39%) samples. Of the 481 isolates of Staph. aureus tested, 255 (53%) were positive for one or more SE genes, and thirty-five different enterotoxin gene profiles were distinguished among the isolates. ser gene, found in 134 (28%) of the isolates, was the most frequent, followed by sed (25%) and selj genes (25%). The identification of new SEs increased the isolation frequency of enterotoxigenic staphylococci, thus suggesting that the pathogenic potential of Staph. aureus may be of greater importance than previously thought. Further studies are needed to quantify the expression of these new enterotoxins, and to assess their contribution to foodborne disease burden. SIGNIFICANCE AND IMPACT OF THE STUDY: The analyses targeted 11 staphylococcal enterotoxins genes and 35 different enterotoxin gene profiles were distinguished among the isolates. A total of 255 Staph. aureus isolates were positive for one or more SE genes while ser gene was the most prevalent. In 93% of the isolates bearing genes located on the enterotoxin gene cluster (n = 89), both seg and sei genes were present.

The microbiological conditions of carcasses from large game animals in Italy.

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 60 min in 90.7% of animals and sampling within 90 min 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.

Detection of Salmonella in finishing pigs on farm and at slaughter in Piedmont, Italy.

Salmonella is one of the most common causes of human gastroenteritis often associated with pork consumption. The aims of this cross-sectional study were to collect preliminary data on the presence of Salmonella enterica in pigs in Piedmont (Italy), through sampling on farm and at slaughter and to gather pilot data on serotypes and phagetypes present in the sampled area and distribution of anti-microbial resistance among isolated strains. Salmonella was detected through culture and identified with Salmonella spp. and Salmonella Typhimurium PCR; positive samples were serotyped, phagetyped and tested for antibiotic susceptibility. Positive samples (from 9% of faeces up to 29% of tonsils) were found in 64% of the herds. Salmonella spp. was retrieved also from scalding water. Most of the isolates were Salmonella Derby, Salmonella Typhimurium and Salmonella 4,5,12:i:-. The results of Salmonella Typhimurium specific PCR suggested that Salmonella 4,5,12:i:- might be unrecognized by serotyping. Anti-microbial resistance was recorded in 75-100% of the isolates. Phagetyping allowed the identification of DT104B and DT46A strains. These results set the bases for further research studies that would aim to estimate the real herd prevalence in Piedmont and the diffusion of serotypes and anti-microbial resistant strains within the same region.

Real-time subtyping via PFGE reveals potential epidemiological relatedness among human salmonellosis cases in Northern Italy.

AIM: This study aimed to assess the applicability of a combined approach of traditional and molecular epidemiology in order to detect salmonellosis outbreaks in the Piedmont region (Italy), characterized by high Salmonella prevalence. METHODS AND RESULTS: Pulsed field gel electrophoresis (PFGE) was used in real-time and in combination with clinical surveillance to assess the relatedness of salmonellosis human cases; subsequently, PFGE profiles of clinical isolates were compared with those of isolates from food items collected during the same study period to identify putative food sources of Salmonella. The real-time subtyping approach allowed the identification of an outbreak (21 isolates), which was undetected by epidemiological surveillance. CONCLUSIONS: Traditional epidemiological investigation did not allow the formulation of hypotheses on food items possibly associated with the outbreak owing mainly to patients' difficulties in remembering foods they ate, and the tendency of health-care professionals to direct patient's suspicion towards specific food items. SIGNIFICANCE AND IMPACT OF THE STUDY: This finding highlighted the value of real-time molecular subtyping in salmonellosis outbreak identification. In order to improve national epidemiological investigations implementing public health agency network and planning, information campaigns for health-care professionals are required.

Development of a multiplex PCR assay for the identification of pathogenic genes of Escherichia coli in milk and milk products.

A multiplex PCR for the simultaneous detection of some pathogenic genes of enteropathogenic, enterotoxigenic and verocytotoxin-producing Escherichia coli was developed. In this study primers found in literature as well as primers to the purpose designed were used. In this way, it was possible to generate specific fragments of 96, 170, 229, 285, 348, 414 and 510 bp for Hlya, St, EaeA, Lt, Vt1, UidA and Vt2 genes, respectively. When applied to bacterial strains experimentally inoculated in milk and milk products, the proposed PCR showed a detection limit of 5 x 10(4)CFU/ml for Hyla, St, Eaea, Vt1 primers, while for Lt and Vt2 primers the limit resulted of 10(6)CFU/ml.

A multiplex PCR assay for the identification of animal species in feedstuffs.

A multiplex Polymerase Chain Reaction (PCR) assay was applied to feedstuff analysis for the identification of the most used species in rendering plants (ruminant, poultry, fish and pork materials). Primers were designed in different regions of mitochondrial DNA (12S rRNA, tRNA Val and 16S rRNA) after alignment of the available sequences in the GenBank database. The primers generated specific fragments of 104-106, 183, 220-230 and 290 bp length for ruminants, poultry, fish and pork, respectively. The detection limit was 0.004% for fish primers and 0.002% for ruminants, poultry and pork primers. The multiplex PCR proposed in this study can be considered a valid alternative to the microscopic method for the detection of animal derived materials banned by a European Union Regulation as a preventive measure against the spread of Bovine Spongiform Encephalopathy.

Development of a PCR assay for the detection of animal tissues in ruminant feeds.

The European Community ban on use of meat and bone meal in ruminant feed, as a consequence of the spread of bovine spongiform encephalopathy in Europe, has prompted a number of investigations about the possibility of detecting animal tissues in feedstuff. In this paper, a study on vertebrate primers, designed in the 16S rRNA gene of mitochondrial DNA, is described. These primers were able to amplify fragments that contained between 234 and 265 bp. The fragments were specific for bovine, porcine, goat, sheep, horse, rabbit, chicken, trout, and European pilchard and were confirmed by sequence analysis amplicons. The primers were used in a PCR assay applied to five samples of meat and blood meals of different species and subjected to severe rendering treatments (134.4 to 141.9 degrees C and 3.03 to 4.03 bar for 24 min). The presence of vertebrate tissues was detected in all samples. The assay proved to be rapid and sensitive (detection limit 0.0625%). It can be used as a routine method to detect animal-derived ingredients in animal feedstuff.

Identification of cow's milk in "buffalo" cheese by duplex polymerase chain reaction.

A duplex polymerase chain reaction (PCR) was developed to identify the milk of bovine and buffalo species in cheese products, particularly in mozzarella cheese, a typical Italian cheese made from buffalo's milk. Two sets of primers were designed on the basis of the alignment of the sequence codifying mitochondrial cyt b available in the GenBank database. The primers proved to be species-specific, giving rise to 279-bp (bovine) and 192-bp (buffalo) amplified fragments. Since the amplification conditions for bovine and buffalo primers were identical, a duplex PCR was successfully applied to identify the two species in a single reaction step. This technique, when used to test cheese products from the retail trade, allowed the detection of partial or even total substitution of cow's milk for buffalo's milk, in some cases in samples of cheese misleadingly labeled "pure buffalo" mozzarella.

Lipolysis in a Belgian sausage: Relative importance of endogenous and bacterial enzymes.

The importance of bacterial and meat enzymes in lipolysis and carbonyl formation was evaluated during dry sausage ripening. Sausages were prepared with and without addition of an antibiotic-antimicotic mixture. In some experiments, an extra inoculum of Micrococcaceae was added and in two experiments, glucose was omitted. Total viable bacterial counts after 21 days were lowered by at least 2 log units in the presence of antibiotics. Free fatty acid productions after 3 and 21 days, in the presence of antibiotics were not significantly lower than observed in the control sausages. Total carbonyl compounds (benzidine reaction compounds) were significantly lowered by the presence of antibiotics compared to the control sausages except when glucose was omitted from the recipe. The data suggest that lipolysis is almost exclusively brought about by muscle and fat tissue. Polyunsaturated fatty acids are liberated from the polar lipid fraction and their specific liberation is higher than for monounsaturated and saturated fatty acids. Carbonyl production from lipids seems to be independent of bacterial activity.