Alessandra Dalmasso

Animal species identification in food products: Evolution of biomolecular methods

Species identification in food has increasingly acquired importance due to public health, economic and legal concerns. Traditional methods have relied on the identification of morphological traits, but this does not lead to accurate identification of those species used in many types of processed food. As a result, laboratory techniques have been devised using electrophoretic and immunological methods focussing on protein profiles and, more recently, biomolecular techniques have been developed. However, these techniques also present problems and difficulties, especially in the case of matrices that are heterogeneous or have been subjected to severe treatments during processing.

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

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.

Novel Multiplex Single Nucleotide Polymorphism-Based Method for Identifying Epidemic Clones of Listeria monocytogenes

ABSTRACT A novel primer extension-based, multiplex minisequencing assay targeting six highly informative single nucleotide polymorphisms (SNPs) in four virulence genes correctly identified and differentiated all four epidemic clones (ECs) of Listeria monocytogenes and 9 other strains initially misclassified as non-ECs. This assay allows rapid, accurate, and high-throughput screening for all known ECs of L. monocytogenes.

Ruminant Rhombencephalitis-Associated Listeria monocytogenes Strains Constitute a Genetically Homogeneous Group Related to Human Outbreak Strains

ABSTRACT Listeriosis is a disease that causes significant economic losses at the farm level because of high morbidity and mortality in ruminants. This study was performed to investigate the role of ruminants in the epidemiology of listeriosis in northern Italy and the possible association of animal-adapted strains of Listeria monocytogenes with strains associated with human disease. Twenty ruminant rhombencephalitis isolates previously confirmed as L. monocytogenes by bacteriology and PCR were characterized by serotyping, pulsed-field gel electrophoresis, multi-virulence-locus sequence typing (MVLST), and multiplex single nucleotide polymorphism (mSNP) typing for the detection of epidemic clones. Subtyping results were subsequently compared with those obtained from human, food, and environmental isolates of L. monocytogenes, including 311 isolates from the University of Turin, Grugliasco, Italy, and 165 isolates representing major human listeriosis outbreaks worldwide, in addition to other unrelated isolates. Both mSNP typing and MVLST showed that 60% of the isolates analyzed belonged to epidemic clone I (ECI), which has been epidemiologically linked to several human outbreaks of listeriosis. In particular, the 1981 Canada outbreak was linked to the use of sheep manure and the 1985 California outbreak was linked to the use of raw cows milk. In our study, ECI isolates were collected from different ruminant species on geographically and temporally distinct occasions for the last 13 years. Our results support the hypothesis that ruminants represent possible natural reservoirs of L. monocytogenes strains capable of causing epidemics of listeriosis in humans.

Omics integrating physical techniques: Aged Piedmontese meat analysis

Piedmontese meat tenderness becomes higher by extending the ageing period after slaughter up to 44 days. Classical physical analysis only partially explain this evidence, so in order to discover the reason of the potential beneficial effects of prolonged ageing, we performed omic analysis in the Longissimus thoracis muscle by examining main biochemical changes through mass spectrometry-based metabolomics and proteomics. We observed a progressive decline in myofibrillar structural integrity (underpinning meat tenderness) and impaired energy metabolism. Markers of autophagic responses (e.g. serine and glutathione metabolism) and nitrogen metabolism (urea cycle intermediates) accumulated until the end of the assayed period. Key metabolites such as glutamate, a mediator of the appreciated umami taste of the meat, were found to constantly accumulate until day 44. Finally, statistical analyses revealed that glutamate, serine and arginine could serve as good predictors of ultimate meat quality parameters, even though further studies are mandatory.

Development of a Biomolecular Assay for the Identification of Listeria at Species Level

In this article, we developed a biomolecular assay specifically designed for the identification of Listeria at species level. The proposed test was based on (i) a duplex PCR targeting a specific fragment for L. monocytogenes (plcA gene) and a common fragment for all the Listeria (16S rRNA gene) and (ii) a minisequencing of the common fragment to detect diagnostic sites for the differentiation of the other five species: L. innocua, L. welshimeri, L. seeligeri, L. ivanovii, and L. grayi. The specificity of the assay was first tested on a total of 25 certified strains representing 6 Listeria species and 14 non-Listeria strains as negative control and validated on 124 wild isolates obtained from food samples. The proposed assay provides an appropriate tool for rapid identification of Listeria at species level and it should be of great benefit to the food industry as well as to regulatory or public health laboratories engaged in establishing the safety of food products and the management of listeriosis.

Development of a biomolecular assay for postmortem diagnosis of Taenia saginata cysticercosis.

Bovine cysticercosis is caused by the larval stage of the human tapeworm Taenia saginata. According to European data on meat inspection, the prevalence ranges from 0.007% to 6.8%, but the real prevalence is considered to be at least 10 times higher. Laboratory confirmation of the etiological agent is based on gross, stereomicroscopic, and histological examination of submitted specimens. False identifications may occur, possibly because of death and degeneration of cysts, or because taeniid larvae and other tissue parasites, such as Sarcocystis spp., may cause similar macroscopic morphological lesions. Therefore, tests that can warrant sure identification of taeniid lesions and calcified cysts in the muscle are needed. The focus of our study was to develop a suitable postmortem test that could be applied on putative lesions by T. saginata cysticerci, as ambiguously diagnosed after routine meat inspection. In particular, we proposed a biomolecular assay targeting the mitochondrial cytochrome c oxidase subunit I gene (COI). For developing the polymerase chain reaction assay, viable cysts of Cysticercus bovis (n = 10) were used as positive reference samples, and those of Echinococcus granulosus (n = 3), Cysticercus tenuicollis (n = 3), and Sarcocystis spp. (n = 4) as reference negative controls. Further, to evaluate the applicability of the proposed assay, 171 samples of bovine muscular tissue, obtained from local slaughterhouses and containing lesions recognized as T. saginata cysticerci by macroscopic examination, were tested. The proposed test confirmed the diagnosis at postmortem inspection in 94.7% (162/171) of samples. In conclusion, the assay developed in this study, amplifying a short fragment from the mitochondrial gene COI, showed to be suitable for samples containing both viable and degenerating T. saginata cysticerci, yielding an unequivocal diagnosis.

Biomolecular Approaches for the Identification of Tuna Species

Species identification in seafood is extremely important both for fresh products, often commercialised as fillets or half-processed, and for those products submitted to techniques which modify the characteristics related to the species. The aim of this research is the application of biomolecular assays to differentiate some species of great commercial interest, such as Thunnus thynnus, Thunnus albacares, Thunnus alalunga and Katsuwonus pelamis. The differentiation of tuna fish is particularly difficult as they are phylogenetically very close, thus frustrating the attempts made using electrophoretic and biomolecular techniques. In biomolecular techniques restriction enzymes have largely been used (Ram et al., 1996; Pardo and Pérez-Villareal, 2004); however these cannot be employed when mixed products are considered. This paper describes the design of species-specific primers able to discriminate between the mentioned above tuna species by means of a PCR assay.