Lorenzo Castigliego

Natural and recombinant bovine somatotropin: immunodetection with a sandwich ELISA

Bovine Somatotropin (bST) is a peptide hormone secreted by the anterior pituitary gland and its recombinant form (rbST) is used for artificially boosting milk yield in cows. Identification of rbST is difficult in that there is little difference from the pituitary bST (pbST). In this work, we further studied the possibility of immunologically discriminating between rbST and pbST. With this purpose, we produced mouse monoclonal antibodies using, as antigen, a peptide mimicking the N-terminus of rbST from Monsanto (rbST-M) conjugated to keyhole limpet haemocyanin (KLH) and polyclonal antibodies in rabbits immunized with the whole bST or rbST-M. Hence, we developed a sandwich ELISA with the obtained antibodies for detection and quantification of bST in serum and compared its performance on the two worldwide commercialized rbSTs: rbST-M and rbST from LG Life Science (rbST-LG). The lowest detection limit of the assay was 0.05 ng/ml for rbST-M, 0.10 ng/ml for rbST-LG and 0.15 ng/ml for pbST. Furthermore, the assay showed the capability to amplify the signal in the presence of rbSTs, recognizing more efficiently rbST-M and rbST-LG than pbST (ECn pbST/ECn rbST: 3 and 1.6 respectively). Its employment for measuring bST levels in sera from bovines administered with rbST LG allowed us to detect exceptional values due to the treatment itself and probably further increased as a consequence of the higher affinity for rbSTs of our monoclonal antibody.

Evolution of the Anisakis risk management in the European and Italian context

Due to the social and legislative implications, the presence of Anisakis spp. larvae in fishery products has become a concern for both the consumers and the official Control Authorities. The issuance of a large number of provisions, aimed at better managing fish products intended to be consumed raw or almost raw and the associated risks, resulted in a very complicate legal framework. In this work, we analyzed the evolution of the normative through an overview on the local and international legislations, focusing on issues that are of practical interest for Food Business Operators (FBOs) in the fishery chain. In addition, we performed a survey across the Department of Prevention of the Italian Local Health Authorities (LHA) and the main fish markets in Italy to collect the operating procedures and the monitoring plans. Overall, we found many differences, due to the absence of a national reference standard for the management of the Anisakis risk. From this examination, it turns clear that only a participation of all the involved institutions, a strategy of synergistic interventions, as well as a correct training of FBOs, can result in an effective risk management and a proper risk communication, which should overcome states of confusion and unnecessary negative impacts on the economy.

On the alterations in serum concentration of somatotropin and insuline-like growth factor 1 in lactating cows after the treatment with a little studied recombinant bovine somatotropin.

A study was performed to delineate bST and IGF-1 variation, over a whole lactation, in cows treated with a nowadays widely commercialised but little studied sustained release formulation of recombinant bST. Total bST levels were found to be exceptionally high in the first days after administration, but decreased rapidly in the second week after injection. The increase in the IGF-1 serum concentration was significant for almost the entire biweekly cycle. Based on this study, the peaks of ST (often above 100 ng/ml) are considered particularly unlikely to be found in non-treated bovines, even under pathological conditions, especially when detected in a number of animals within a herd. Notwithstanding the great heterogeneity of results on this topic, these data suggest that tests against fraud involving the use of rbST in dairy products may be regarded as a feasible possibility.

Two alternative multiplex PCRs for the identification of the seven species of anglerfish (Lophius spp.) using an end-point or a melting curve analysis real-time protocol

Anglerfish (Lophius spp.) is consumed worldwide and is an important economic resource though its seven species are often fraudulently interchanged due to their different commercial value, especially when sold in the form of fillets or pieces. Molecular analysis is the only possible mean to verify traceability and counteract fraud. We developed two multiplex PCRs, one end-point and one real-time with melting curve post-amplification analysis, which can even be run with the simplest two-channel thermocyclers. The two methods were tested on seventy-five reference samples. Their specificity was checked in twenty more species of those most commonly available on the market and in other species of the Lophiidae family. Both methods, the choice of which depends on the equipment and budget of the lab, provide a rapid and easy-to-read response, improving both the simplicity and cost-effectiveness of existing methods for identifying Lophius species.

Pentaplex PCR As Screening Assay for Jellyfish Species Identification in Food Products

Salted jellyfish, a traditional food in Asian Countries, is nowadays spreading on the Western markets. In this work, we developed a Pentaplex PCR for the identification of five edible species (Nemopilema nomurai, Rhopilema esculentum, Rhizostoma pulmo, Pelagia noctiluca, and Cotylorhiza tuberculata), which cannot be identified by a mere visual inspection in jellyfish products sold as food. A common degenerated forward primer and five specie-specific reverse primers were designed to amplify COI gene regions of different lengths. Another primer pair targeted the 28SrRNA gene and was intended as common positive reaction control. Considering the high level of degradation in the DNA extracted from acidified and salted products, the maximum length of the amplicons was set at 200 bp. The PCR was developed using 66 reference DNA samples. It gave successful amplifications in 85.4% of 48 ready to eat products (REs) and in 60% of 30 classical salted products (CPs) collected on the market.

Labeling of ethnic food in the Prato Chinese community.

Ethnic food consumption is a quickly growing reality within Chinese communities, which have a well-organized “internal” food market for both Asian and ethnic foods produced in the European Union. The main problems associated with these markets are related to hygienic conditions, certifications of accomplishment, and personnel management. Moreover, controls and identification of the products are difficult because of cultural and linguistic barriers. In this study, five markets managed by the Chinese were visited, and the conformity of the reported label information found on different kinds of food (prepackaged or loose) was assessed by a collaboration between the Local Authorities of Control of the Prato territory, which hosts the largest Chinese community in Italy, and of native speakers of Chinese. All visited markets presented products (n = 75) with non-conformities: lack of translation (6%) and incomplete/mistaken translation of the commercial name (72%) and place of production (12%). In addition to the legal implications of the observed non-compliances, certain sanitary issues were taken into consideration. In fact, a number of the products that belong to risk categories could be misclassified in a non-risk category. Lastly, missing ingredients or complete alteration of their commercial names may represent health threats in cases of allergen ingestion by allergic or intolerant consumers.

Universal Primers Used for Species Identification of Foodstuff of Animal Origin: Effects of Oligonucleotide Tails on PCR Amplification and Sequencing Performance

M13 universal non-homologous oligonucleotide tails incorporated into universal primers have been shown to improve amplification and sequencing performance. However, a few protocols use these tails in the field of food inspection. In this study, two types of M13 tails (by Steffens and Messing) were selected to assess their benefits using universal cytochrome oxidase subunit I (COI) and 16S ribosomal RNA gene (16SrRNA) primers in standard procedures. The primer characteristics were tested in silico. Then, using 20 DNA samples of edible species (birds, fishes, and mammals), their performance during PCR amplification (band recovery and intensity) and sequencing (sequence recovery, length, and Phred score) was assessed and compared. While 16SrRNA tailed and non-tailed primers performed similarly, differences were found for COI primers. Messing’s tails negatively affected the reaction outputs, while Steffens’ tails significantly improved the band intensity and the length of the final contigs based on the individual bidirectional read sequence. This different performance could be related to a destabilization effect of certain tails on primers with unfavorable mismatches on the annealing region. Even though our results cannot be generalized because the tail performances are strictly dependent on laboratory conditions, they show that appropriate tails can improve the overall throughput of the analysis, supporting food traceability.

A Conventional Multiplex PCR Assay for the Detection of Toxic Gemfish Species (Ruvettus pretiosus and Lepidocybium flavobrunneum): A Simple Method To Combat Health Frauds

The meat of Ruvettus pretiosus and Lepidocybium flavobrunneum (gemfishes) contains high amounts of indigestible wax esters that provoke gastrointestinal disorders. Although some countries have banned the sale of these species, mislabeling cases have been reported in sushi catering. This work developed a simple conventional multiplex PCR, which discriminates the two toxic gemfishes from other potentially replaced species, such as tunas, cod, and sablefish. A common degenerate forward primer and three species-specific reverse primers were designed to amplify cytochrome oxidase subunit I (COI) gene regions of different lengths (479, 403, and 291 bp) of gemfishes, tunas, and sablefish, respectively. A primer pair was designed to amplify a fragment (193 bp) of the cytb gene of cod species. Furthermore, a primer pair targeting the 16S rRNA gene was intended as common positive control (115 bp). The method developed in this study, by producing the expected amplicon for all of the DNA samples tested (reference and commercial), provides a rapid and reliable response in identifying the two toxic species to combat health frauds.

Immunodiscrimination between native and recombinant somatotropin: a possible pathway?

Somatotropin (ST) is a peptidic hormone produced by the pituitary gland in mammals which performs regulative functions in growth and lactation in ruminants. The recombinant form of bovine ST (rBST) is used to increase milk yield in cows. This kind of treatment, which is authorised in many countries, has been definitively banned by the E.U. because of its potential hazard to animal health (Kronfeld, 2001; Macrı̀, 1999). There are currently no analytical methods able to reveal rBST administration. However, there are circulating molecules which can be considered as analytical targets and indirect indicators for the abovementioned treatment of bovines, since their concentration is proportional to the stimulative action of ST. The most famous of these is Insulin-like Growth Factor 1 (IGF-1), a hormone mainly secreted by the liver (Prosser et al., 1989) that mediates the biological action of ST on several tissues. Almost all seric IGF-1 circulates bound to specific IGF-binding proteins (IGFBPs); for this reason the current analytical methods used to measure it are still imprecise (Breier et al., 1991; Twigg et al., 2000). On the other hand, the direct analytical approach is very difficult because the primary sequence of recombinant BST differs from the native form for only one aminoacid at the N-terminal position. The aim of our work is to evaluate the possibility of producing antibodies capable of direct discrimination, between BST and rBST. We have used the murine immune system as our model. We produced polyclonal antibodies against a synthetic peptide that mimics the N-terminal region of rBST. Immunoenzymatic tests of the sera derived from immunised animals allowed us to evaluate their eventual different reactivity against the two forms of the protein, suggesting the possibility of producing monoclonal antibodies

An Immunoenzymatic Method to Measure IGF-1 in Milk

Guidi, A., Castigliego, L., Iannone, G., Armani, A. and Gianfaldoni, D., 2007. An immunoenzymatic method to measure igf-1 in milk. Veterinary Research Communications, 31(Suppl. 1), 373–376