Tiziana Civera

A multiplex polymerase chain reaction for the identification of cows’, goats’ and sheep's milk in dairy products

Abstract A multiplex PCR able to identify cows’, goats’ and sheeps milk in dairy products was developed. Specific primers were designed in the mitochondrial 12s and 16s rRNA genes so as to generate fragments of different length. The assay was applied to 19 cheeses from the retail trade in order to verify the label statements. The multiplex PCR results were confirmed by PCR-restriction fragment length polymorphism. The proposed multiplex PCR represents a rapid and sensitive method applicable on a routine basis. In fact it enables to detect, in a single step, goats’, sheeps and cows’ milk in dairy products with a good sensitivity threshold (0.5%).

Listeria monocytogenes in Gorgonzola: Subtypes, diversity and persistence over time

L. monocytogenes represents a primary concern in the production of Gorgonzola, a Protected Designation of Origin (PDO) Italian blue-veined cheese produced only in the Piedmont and Lombardy regions. L. monocytogenes isolates (N=95) obtained from Gorgonzola rinds, paste, and production/ripening environments were serotyped and then genotyped using Pulsed Field Gel Electrophoresis (PFGE). The goal of this study was to investigate the variability of L. monocytogenes PFGE-types across different PDO Gorgonzola manufacturers (N=22). The majority of the strains (88%) were serotyped as 1/2a. PFGE identified 2 major pulse-types grouping 62 strains, detected from different plants and years, suggesting the presence of persistent and niche-adapted L. monocytogenes. In 9 plants, environmental strains shared the same pulse-types with strains from rinds or paste, suggesting a possible transmission pathway. Encouragingly, L. monocytogenes was retrieved from only 1 paste, indicating that production processes were under control in 21 plants. In the remaining plant, un-effective pasteurization or cross-contamination during production processes could be the cause of the contamination. Consequently, it is imperative that producers operate under the total respect of the Good Manufacturing Practices and following the principles of the Hazard Analysis Critical Control Point plans, in order to contain contamination throughout the whole processing.

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.

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.

Identification of Four Tuna Species by Means of Real-Time PCR and Melting Curve Analysis

Dalmasso, A., Fontanella, E., Piatti, P., Civera, T., Secchi, C. and Bottero, M.T., 2007. Identification of four tuna species by means of Real-Time PCR and melting curve analysis. Veterinary Research Communications, 31(Suppl. 1), 355–357

Authentication of meat from game and domestic species by SNaPshot minisequencing analysis.

The aim of the present study is to develop an assay for the specific identification of meat from Capreolus capreolus, Cervus elaphus, Capra ibex, Rupicapra rupicapra, targeting sequences of the cytochrome b (cyt b) gene of mitochondrial DNA. The assay is also intended to enable differentiation between meat from these wild species as well as Ovis aries, Capra hircus, Bubalus bubalis, Bos taurus and Sus scrofa domestic species. The primers used in the preliminary PCR were designed in well conserved regions upstream and downstream of the diagnosis sites. They successfully amplified a conserved 232bp region from the cyt b gene of all the species taken into consideration. The sites of diagnosis have been interrogated using a minisequencing reaction and capillary electrophoresis. All the results of the multiplex PER (primer extension reaction) test were confirmed by fragment sequencing. The assay offers the possibility of discriminating nine species at the same time.

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

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.

Microbial ecology of Gorgonzola rinds and occurrence of different biotypes of Listeria monocytogenes.

In this study we investigated the microbiota of Gorgonzola rinds and maturing shelf swabs collected in 5 different maturing cellars in the Northwest part of Italy, in association with the detection and characterization of Listeria monocytogenes. Culture-dependent and -independent methods were performed in order to profile the main microbial populations present on the rinds and in the maturing shelves and species-specific PCR and Pulsed Field Gel Electrophoresis (PFGE) were used to identify and type L. monocytogenes isolates. The microflora was predominated by lactic acid bacteria and coagulase negative cocci, while enterococci and yeasts were very variable between the samples. Arthrobacter sp., Carnobacterium sp., Staphylococcus sp. and Brevibacterium linens, as bacteria, and Debaryomyces hansenii, as yeast, were detected by Denaturing Gradient Gel Electrophoresis (DGGE). Cluster analysis of the DGGE profiles clearly highlighted a cellar-specific microflora. L. monocytogenes was isolated in 11.1% of the rinds and 29.4% of the swabs and the molecular characterization of the isolates suggests a route of contamination from the maturing shelves to the rinds. No correlation was found between DGGE profiles and presence or absence of L. monocytogenes.

Multiplex primer-extension assay for identification of six pathogenic vibrios

A multiplex Primer-Extension Reaction (PER) assay, was specifically designed for the identification, of the major human pathogenic Vibrio species (V. cholerae, V. parahaemolyticus, V. vulnificus, V. mimicus, V. alginolyticus and V. fluvialis) in fishery products. The assay, directed towards the rpoA gene, was tested on a total of 287 samples representing six Vibrio species and ten non-Vibrio species. The primers used in the preliminary PCR, designed in well conserved regions upstream and downstream of the diagnosis sites, successfully amplified a 284 bp fragment. The diagnosis sites were simultaneously interrogated using a multiplex PER and the results were confirmed by fragment sequencing. The proposed test provides an appropriate tool to monitor the presence of these human pathogenic species in seafood samples and to evaluate the potential hazard for consumers.

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.