Angela Di Pinto

A collagenase-targeted multiplex PCR assay for identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus.

A multiplex PCR assay using three collagenase-targeted primer pairs for the species-specific detection of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus was developed. The results highlight the species specificity of the three primer sets designed. Because of the increasing importance of Vibrio spp. in human foodborne diseases, molecular approaches for routine microbial screening and monitoring of clinical, environmental, and food samples also have become more important. The results of this study indicate that the gene coding for collagenase should be used as an alternative molecular target to discriminate among the three Vibrio species.

DNA barcoding for detecting market substitution in salted cod fillets and battered cod chunks.

The Italian Ministry of Agricultural, Food and Forestry Policies (MiPAAF) Decree dated 31 January 2008, which reports the Italian name for fish species of commercial interest, establishes that baccalà can be obtained exclusively from G. macrocephalus (Pacific cod) and G. morhua (Atlantic cod). This paper describes the COI-based DNA identification system to verify the substitution or misbranding of gadoid fish species and, consequently, its concordance with the labels on salted cod fillets shown as baccalà and on battered cod chunks labelled as bocconcini di baccalà. The analysis of interpretable sequences revealed that 55/65 dried salted cod fillet samples were detected as belonging to the family Gadidae, while 10/65 samples appeared to belong to the Lotidae family, while among battered cod chunks labelled as bocconcini di baccalà, the post-sequencing data analysis shows that the labels were completely wrong, with 28/40 samples from Pollachius virens and 12/40 samples from Brosme brosme. The substitution rate for products labelled on the market as baccalà in this study raises significant issues relating to food safety and consumer protection.

Occurence of potentially enterotoxigenic Bacillus cereus in infant milk powder

Considering that powdered infant milk formula effectively supports the growth of numerous pathogens, this study investigates the prevalence of potentially pathogenic Bacillus cereus in dried milk products by evaluating the occurrence of B. cereus and the presence of virulence-associated genes. The approach consisted of enriching, isolating and biochemical identifying isolates, followed by PCR assays aimed at the hbl (C, D, A, B), nhe (A, B, C) and cytK enterotoxin genes coding HBL complex, NHE complex and cytotoxin K, respectively. Among cytK-positive strains, the discrimination of two different forms for cytotoxin K, cytK-1 and cytK-2 was performed. Bacillus cereus was detected in powdered infant milk formula samples. All the strains harbored at least one gene of the cytK, HBL and NHE enterotoxins. Because of an increasing trend in invasive infections by B. cereus in infants and immunocompromised children, our PCR findings highlight the need to implement an adequate control plan in order to guarantee the health of potentially fragile consumers. From a hygiene point of view, intensive and continuous monitoring of potentially pathogenic B. cereus may be crucial for powdered infant milk formula safety and even recommended in order to assess the infant health risk, as proposed by Commission Regulation (EC) no. 1441/2007 on microbiological criteria for foodstuffs. Furthermore, the detection in this study of B. licheniformis, B. subtilis and B. mycoides strains raises significant health issues regarding Bacillus spp. in powdered infant milk formula.

Detection of Brucella spp. in soft cheese by semi-nested polymerase chain reaction.

Brucellae are Gram-negative bacteria with a predilection for the reticuloendothelial system and the reproductive tract of cattle, sheep, goats and pigs. In these species brucellosis is of great economic significance because of the resulting abortion and infertility (Samartino et al. 1993; Corbel, 1997; Altecruse et al. 1998). Human brucellosis, caused by contamination from infected animals (percutaneous, conjunctival or nasal infection), is very common and remains a serious health hazard in Mediterranean Europe, western Asia, and parts of Africa and South America (Boschiroli et al. 2001). Raw milk, butter, ice-cream and unpasteurized cheese are also potential vehicles for the transmission of Brucella .

Occurrence of potentially pathogenic arcobacters in shellfish

Considering that several recent cases of human gastroenteritis have been associated with species from the Arcobacter genus, and that few data are currently available about the occurrence of this genus in Italian shellfish, the aim of the present study was to evaluate the occurrence of Arcobacter spp. and the presence of virulence-associated genes. The approach consisted of cultural and biomolecular (multiplex-PCR and 16S-RFLP) methods identifying isolates, followed by PCR assays aimed at the cadF, ciaB, cjl349, irgA, hecA putative virulence genes. Arcobacter spp. was detected in 16/70 (22.8%) shellfish samples. Specifically, Arcobacter spp. was highlighted in 10/42 (23.8%) mussel and in 6/28 (21.4%) clam samples. Subsequently, biomolecular assays revealed Arcobacter butzleri in 12/16 (75%) and Arcobacter cryaerophilus 1B in 4/16 (25%) isolates. PCRs aimed at the five putative virulence genes demonstrated widespread distribution of these genes among Arcobacter isolates and some differences from the results published by other authors. Our research provides more information regarding the health risks associated with the consumption of raw bivalve molluscs and underlines the need to implement an adequate control plan by performing intensive and continuous monitoring in order to guarantee human health.

Occurrence of emerging food-borne pathogenic Arcobacter spp. isolated from pre-cut (ready-to-eat) vegetables.

Given that changes in consumer food behaviours have led to an increase in the demand for pre-cut ready-to-eat (RTE) vegetables, and that few data are currently available on the occurrence of Arcobacter spp. in such foods, the aim of the present study was to assess the occurrence of Arcobacter spp. that carry virulence-associated genes on pre-cut RTE vegetables, using cultural and molecular methods. Arcobacter was detected using biomolecular identification methods in 44/160 (27.5%) of the samples, of which 40/44 (90.9%) isolates corresponded to A. butzleri and 4/44 (9.1%) to A. cryaerophilus. Studying the incidence of 9 virulence-associated genes revealed the widespread distribution of these genes among the Arcobacter isolates tested. The results obtained in our research provided plenty of information on the health risks associated with the direct consumption of raw vegetables, and highlight the need to implement further studies at each level of the production chain, in order to obtain further information to help protect human health.

Virus infections of honeybees Apis Mellifera

The health and vigour of honeybee colonies are threatened by numerous parasites (such as Varroa destructor and Nosema spp.) and pathogens, including viruses, bacteria, protozoa. Among honeybee pathogens, viruses are one of the major threats to the health and well-being of honeybees and cause serious concern for researchers and beekeepers. To tone down the threats posed by these invasive organisms, a better understanding of bee viral infections will be of crucial importance in developing effective and environmentally benign disease control strategies. Here we summarize recent progress in the understanding of the morphology, genome organization, transmission, epidemiology and pathogenesis of eight honeybee viruses: Deformed wing virus (DWV) and Kakugo virus (KV); Sacbrood virus (SBV); Black Queen cell virus (BQCV); Acute bee paralysis virus (ABPV); Kashmir bee virus (KBV); Israeli Acute Paralysis Virus (IAPV); Chronic bee paralysis virus (CBPV). The review has been designed to provide researchers in the field with updated information about honeybee viruses and to serve as a starting point for future research.

Occurrence of Ochratoxin A in the Wild Boar (Sus scrofa): Chemical and Histological Analysis

Ochratoxins are fungal secondary metabolites that may contaminate a broad variety of foodstuffs, such as grains, vegetables, coffee, dried fruits, beer, wine and meats. Ochratoxins are nephrotoxins, carcinogens, teratogens and immunotoxins in rats and are also likely to be in humans. In 2009/2010, a survey of the presence of Ochratoxin A (OTA) in regularly hunted wild boars in the Calabria region of southern Italy detected OTA in 23 animals in the kidney, urinary bladder, liver and muscles: 1.1 ± 1.15, 0.6 ± 0.58, 0.5 ± 0.54 and 0.3 ± 0.26 μg/kg, respectively. Twelve tissue samples showed levels of OTA higher than the guideline level (1 μg/kg) established by the Italian Ministry of Health. In five wild boars, gross-microscopic lesions were described for the organs displaying the highest concentrations of OTA determined by HPLC-FLD analysis, i.e., the kidney, liver and urinary bladder.

RNA extraction method for the PCR detection of hepatitis A virus in shellfish.

Viruses are the leading cause of foodborne illness associated with the consumption of raw or slightly-cooked contaminated shellfish. This study evaluated the E.Z.N.A. Mollusc RNA extraction and purification kit for the detection of HAV in shellfish. The E.Z.N.A. method, based on the cationic detergent, cetyltrimethyl ammonium bromide, in conjunction with a selective RNA binding silica matrix, efficiently isolated viral RNA with a detection limit of 1TCID(50)/ml by hemi-nested PCR. This method proved to be faster and less expensive than PEG precipitation-based procedures. It is also technically undemanding, requiring no extensive processing steps or excess manipulation, minimizing RNA degradation and ensuring the absence of PCR inhibitors. The E.Z.N.A. method applied to HAV screening of shellfish samples from the Apulian region, revealed a high level of contamination. These results confirm that conventional faecal indicators are unreliable for demonstrating the presence or absence of viruses.

Detection of Arcobacter spp. in Mytilus galloprovincialis samples collected from Apulia region

The aim of the study was to evaluate the occurrence of Arcobacter spp. in 20 samples of Mytilus galloprovincialis purchased at fish markets in Apulia region. The detection of Arcobacter spp. was performed, after selective enrichment, on modified charcoal cefoperazone deoxycholate (mCCD) agar supplemented with Cefoperazone, Amphotericin B and Teicoplanin (CAT). In 6 out of the 20 tested samples the presence of Arcobacter spp. was found and confirmed by genus-based polymerase chain reaction. All the isolates were identified as belonging to the species Arcobacter butzleri using 16S rDNA sequencing and BLAST online. The results represent the first report in Italy of A. butzleri detection in marketed Mytilus galloprovincialis. The survey underlines the epidemiological importance of A. butzleri as an emerging pathogen, and highlights that mussels should be considered as a potential cause of foodborne disease outbreak.