Serum responsiveness to recombinant bovine somatotropin in buffalo: a three-month lactation study using an acid-stripping ELISA for screening.

The misuse of recombinant bovine somatotropin (rbST) to increase milk yield involves buffalo not just cows. Screening methods to identify rbST-treated cattle have already been proposed. However, there have been no studies on prolonged periods with a high number of animals. In this study, we developed a new enzyme-linked immunosorbent assay (ELISA) to measure the serum responsiveness towards rbST, based on an acid-stripping procedure and relatively simple integral calculation dilution curves. We also applied the analysis to 640 serum and 96 milk samples collected from 16 buffalo treated with rbST and 16 controls, over a period of approximately three months. Its suitability as a screening method, in compliance with EU law, was also assessed. A bi-factorial approach was also evaluated, including the measurement of insulin-like growth factor 1 concentration in serum. Results showed that our ELISA could be used on its own for screening purposes, without the need to assess other biomarkers. Copyright © 2016 John Wiley & Sons, Ltd.

A LC-MS-MS method to detect recombinant bovine somatotropin misuse in buffalos.

Recombinant bovine somatotropin (rbST) is a peptide hormone used to increase milk yield in cows and buffalos. In Europe, its use has been banned. However, rbST is sometimes illegally included in zootechnical practices for profit purposes, undermining the fair trade and the law prescriptions. For this reason, efficient and reliable analytical techniques are required to contrast rbST misuse. A few LC-MS-MS methods have been developed to detect, in cow serum, methyonil-rbST, one of the two main rbST forms available on the market. The other form, which is widespread, is identical to the most abundant variant of bovine somatotropin (bST) and differs from the buffalo somatotropin for one amino acid in the N-terminus. For this reason, it is technically possible to distinguish both rbST forms in serum of buffalos. In this work, we describe a novel LC-MS-MS-based method, capable to quantify, with a high sensitivity and selectivity, the methyonil-rbST and the other bST-identical recombinant form in buffalo serum, previously purified using a solid-phase extraction procedure. The method was internally validated and used to analyse 152 serum samples, collected from eight buffalos administered with rbST for a period of 3 months, according to conventional protocols. The obtained results confirmed the suitability of the method in the detection of illegal hormonal treatments. Graphical abstract ᅟ.

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.

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.

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.

A histologic study on growth promoter target organs of slaughtered beef in Molise Region (Italy).

A gross pathology and histological investigation was carried out on bovine target organs of anabolic substances in the Molise Region (Italy). One hundred forty-four bovines (12-24 months old, 123 males and 21 females) were included in the survey. An antemortem assessment of their behavior and clinical examination were performed. After slaughter, samples of prostate, Cowper's glands, Bartholin's glands, mammary gland, ovaries, thymus and thyroid were collected, inspected and processed for histopathology, as suggested in the guidelines of the Italian national program for residue surveillance (PNR). Overall, 15.3% of the examined animals were classified as "suspect," 44.4% were classified as "uncertain," and the remaining 40.3% were classified as "negative." The most frequent lesion was a severe thymus atrophy with fat infiltration (15.4% of males and 14.3% of females), strongly suggesting the illegal use of corticosteroids.

Somatotropic gene response to recombinant growth hormone treatment in buffalo leucocytes.

The use of recombinant bovine growth hormone (rbGH) to increase milk yield in cows is banned in some countries. In others, where it is authorised, it has triggered harsh debates on labelling of dairy products. If many studies have been performed on bovines, there is a lack of information on buffaloes, which are sometimes treated with rbGH and re-present an important economical resource for dairy products in some countries. Analytical methods with legal value for surveillance of rbGH treatments do not yet exist. Research on gene expression biomarkers is one of the most promising approaches to this purpose. For this reason, we treated five buffaloes for 10 weeks with a sustained-release formulation of rbGH and analysed the response of 20 somatotropic axis genes in leucocytes by real-time polymerase chain reaction. Overall changes in gene expression levels were of low magnitude and sometimes affected by the 'time' factor. Only the IGFBP-1 gene showed a significant under-expression (about two-fold; p <0.001) in treated animals. Taken together, these results give evidence that expression analysis of the somatotropic axis genes in leucocytes is little helpful for discrimination of rbGH-treated buffaloes, but do not exclude that another array of genes could provide useful patterns of variation.

Hormone variations in serum and milk of buffaloes (Bubalus bubalis) as potential indicators of treatment with recombinant bovine somatotropin.

Recombinant bovine somatotropin (rbST) is used to increase milk yield in cows, but it has been forbidden in some countries and in the EU. However, rbST misuse represents a concern in both bovine and buffalo dairy production. A number of studies on rbST treatment have been performed on bovines, but there are few data on buffaloes. In this study, we treated eight lactating buffaloes with biweekly injections of a slow-release formulation of rbST, for five cycles of administration, and analysed total ST and insulin-like growth factor 1 (IGF-1) variations in serum and IGF-1 in milk. The aim was to assess their power as potential indicators of rbST-treatment. Blood was collected on days 2, 5, 9 and 14 of each cycle, and milk on days 2, 9 and 14 of cycles 2 and 5. Results showed an extraordinary increase in ST levels on day 2 in treated animals, followed by a rapid decrease over the following days, while a significant increase in IGF-1 was observed both in serum and in milk throughout most of each cycle. These results suggest that serum ST levels are a good indicator of treatment. However, the rapid decrease after the peak limits the useful period of sample collection.

Selecting reference genes in the white blood cells of buffalos treated with recombinant growth hormone.

In this study, we assessed the expression stability of eight genes (GADPH, ACTB, 18S, YWHAZ, SDHA, HMBS, SF3A1, and EEF1A) in the white blood cells of lactating buffalos and their possible use as reference genes for studies on growth hormone (GH)-treated animals. All of the genes showed acceptable stability according to the threshold values suggested by some of the software that was used to analyze them, although the differences between the most stable (SF3A1 and ACTB) and the least stable (18S) were considerable. GH treatment did not influence their expression levels.

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.