Species Identification by Means of Mitochondrial Cytochrome <i>b</i>DNA Sequencing in Processed Anchovy, Sardine and Tuna Products

Identifying the contents of processed food products is essential to correct labelling. In processed foodstuffs, species identification through morphological analysis is difficult. Several factors hinder the identification of fish species in processed foods: proteins or other materials subjected to analysis may be denatured during heat treatments;the presence of other ingredients (e.g., olive and other vegetable oils) may interfere with the analysis. Consequently, possible frauds perpetrated by replacing valuable species with less precious ones may go undetected. In most processed samples (e.g. canned products), DNA is degraded into small fragments, which considerably reduces the sensitivity of molecular analysis. The main goal of our research was to develop an analytical method able to identify fish species in highly processed products, such as canned fish. The assay was developed by combining an effective method of DNA recovery from samples with the detection of small-sized sequences of the mitochondrial Cytb gene. This method appears particularly suitable when morphological characterization is difficult, to carry out such as in canned products where DNA is degraded or present in small quantities. We have analyzed 60 samples of seafood commercial products identifying 3 different genera and five different species. All analyzed samples revealed a correct species declaration, for one sample we highlighted important commercial fraud. We also used bio-informatic identification systems for the Sequence Alignment and the construction of phylogenetic tree to better confirm the revealed fraud.

Isolation of <i>Bacillus cereus</i>in a Facility Preparing School Meals

Food safety is a fundamental requirement in mass catering, as large numbers of meals are served each day to potentially vulnerable consumers, such as children. Food Business Operators implement plans for the microbiological monitoring of the meals prepared and served in the catering sector, and for the swab-sampling of surfaces. From January 2018 to June 2019, our laboratory analyzed both food and swab samples from four catering facilities. Considering the EFSA 2018 data, we specifically focused on samples analyzed for Bacillus cereus. Our data substantially showed episodic contamination due to a piece of equipment that is not usually subjected to microbiological control, thus suggesting that every aspect should be scrutinized in order to identify critical points. While Bacillus cereus is widespread in nature and common in soil, it is adapted for growth in the intestinal tracts of insects and mammals. It is often present in a variety of foods, and may cause an emetic or a diarrheal type of food-associated illness. B. cereus produces several toxins. Multiplex PCR enables seven toxin genes to be detected (hblC, hblD, hblA, nheA, nheB, nheC and cytK).

Monitoring Plan on PCDD/Fs and DL-PCBs Milk Contamination in Campania Region (Italy)

During the carrying out of the monitoring plan as part of the Regional Law n. 3/2005 emerged, in 2008, the well known “buffalo milk crisis” that led to the enactment of other monitoring plans targeted to the research of dioxins in food. From the last monitoring plan on PCDD/Fs and DL-PCBs in Campania Region, named as “Surveillance plan 2011-2014”, all dairy products from N. 50 farms intended to the production of bovine, buffalo and ovine milk, resulted compliant to the maximum levels fixed by EU in Regulation 1259/2011, suggesting the effectiveness of the restrictive measures on farming practices applied after the sanitary emergency of 2008. On the other hand, the action limits reported in EU Recommendations 516/2011 and 711/2013 were exceeded in two sheep farms and two buffalo farms, to represent the critical issues that nowadays exist.

The Role of Ozone Carried by Liposomes in the Therapy of Infectious and Skin-Regenerating Ocular Surface

Effect of the ozonated oil in liposomes: they act against Staphylococcus aureus and Pseudomonas aeruginosa is among the most frequent eye pathogens and causes acute and chronic infections of the ocular surface. Staphylococcus aureus and Pseudomonas aeruginosa were tested in this study. The bacterial suspension of Staphylococcus aureus was diluted to obtain 150 CFU/ml. The bacterial suspension of Pseudomonas aeruginosa was diluted to obtain 15 CFU/ml. Various volumes of liposome-vehiculated ozonated oil were added (400 μl, 200 μl, 100 μl and 50 μl) to 100 μl of bacterial suspension;both of Staphylococcus aureus and Pseudomonas aeruginosa are incubated at 37?C for 10 minutes. The cytotoxicity of liposome-vehiculated ozonated oil was analysed at the University of Campania “Luigi Vanvitelli”, Department of Precision Medicine. The HaCaT epidermal keratinocyte cell line (ATCC, USA) was grown in Dulbecco’s Modified Eagle Medium (Euroclone) with the addition of 10% foetal bovine serum (FBS) (Euroclone), 2 mM of L-glutamine (Euroclone) and antibiotics (100 U/ml penicillin, 100 g/ml streptomycin) (Euroclone). The microbiological results clearly show the antimicrobial efficacy of liposome-vehiculated ozonated oil against bacterial strains such as Staphylococcus aureus and Pseudomonas aeruginosa. Furthermore, the studies carried out in vitro on the keratinocyte line showed how ozonated oil in liposomes does not evidence any cell toxicity, and that after 3 days of treatment, it promotes cell growth compared to the positive control.

Recent Epidemic-Like Anthrax Outbreaks in Italy: What Are the Probable Causes?

In Italy, in an area of about 50 km2 between Basilicata and Campania Southern regions, 28 outbreaks of anthrax occurred from August 28th to September 27th 2011. Different species were affected: laboratory tests confirmed anthrax in cattle, horses and sheep. The genetic analysis of strains isolated from infected animals indicated that outbreaks occurring in the two regions were not correlated. Intriguingly, the incidence was highly significant in horses compared to that of cattle and sheep or goats, which were prevalent species in the animal population. In addition, allinfected horses and many cattle developed a fatal, sub-acute form of anthrax, characterized by the presence of massive edema, usually absent in hyperacute forms. The characteristics of these outbreaks suggested a possible role of tabanids as vectors in the transmission of B. anthracis like-epidemic infection.

Inter-Laboratory Ring Trial to Evaluate Reverse Transcription Polymerase Chain Reaction Methods Used for <i>Dolphin Morbillivirus</i>Detection in Italy

Dolphin Morbillivirus (DMV) is one of the most frequently detected pathogens in stranded cetacean specimens worldwide as well as in Italy. Due to the persistence of DMV in the Mediterranean Sea and to the lack of information about the efficiency of the available diagnostic techniques, the Italian National Reference Centre for diagnostic activities on dead stranded marine mammals (C.Re.Di.Ma) performed the first inter-laboratory ring trial with the aim to standardize a diagnostic biomolecular approach for DMV in Italy. Viral isolation is usually considered the “gold standard” for the definitive diagnosis of most pathogens, but it is not often feasible in DMV diagnosis, due to the poor preservation of virus-targeted tissues in stranded cetacean carcasses, as well as to the lack of appropriate sensitivity of cell lines towards DMV variability. Therefore direct viral detection on tissues by means of reverse transcription-PCR (RT-PCR) represents a valuable option for DMV infection’s diagnosis. For detecting DMV in cetacean die-offs occurred in the Mediterranean basin since 2013, C.Re.Di.Ma developed an RT-PCR based method targeting to a 287 bp fragment of DMV nucleoprotein (N) gene. With the purpose to evaluate its performances in terms of accuracy (Se = sensitivity and Sp = specificity) and precision (reproducibility), it was submitted to a ring trial. So, 12 Public Laboratories belonging to the Italian dead stranded marine mammals diagnostic network were asked to analyze a panel of 40 samples (positive and negative for DMV, using different dilutions of a viral suspension obtained from a cell culture supernatant of a DMV strain) with the aforementioned technique. Furthermore, we also aimed at comparing the accuracy of other 7 molecular methods routinely applied for DMV detection in Italy. For this purpose, the second panel of identical 40 DMV +ve and -ve samples was provided to Laboratories that routinely used DMV detection methods other than those developed by C.Re.Di.Ma, in order to be analyzed simultaneously with the method they usually applied. The C.Re.Di.Ma technique showed high accuracy [mean Se = 97.8% (95% CI 84.2% - 99.3%), mean Sp = 98.1% (95% CI 72.5% - 99.9%)] and very good precision [k combined equal to 0.91 (95% CI 0.87 - 0.95)]. In conclusion, this study highlighted a satisfactory reliability of most of the molecular methods used in Italy for DMV detection.