Research Items (57)
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.
- May 2016
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
- Apr 2016
The research on biomarkers to detect livestock treated with recombinant bovine somatotropin (rbST) is still an open issue. In fact, beyond undertaking confirmation methods, there is the need to develop simple and inexpensive screening tests. In this direction, some proposals have been forwarded, mostly involving the measurement of circulating molecules, whereas the possibility of using biomarkers related to gene expression is a field under investigation. The present study was carried out on sixteen buffalos, eight of which treated with rbST. Blood samples were collected six times during the treatment to investigate on the presence of differentially expressed genes in leukocytes. Analysis with the microarray technique was performed on two sampling moments, in order to obtain a first selection of genes. Further analysis was carried out by real time RT-PCR, in order to create a discriminating linear system. A study on the variation of the error related to the number of samples included in statistics was also performed. Results showed that, including an increasing number of samples to build the discriminating algorithm, the b-error grows and tends to stabilize on 6.5%. This study clearly shows the paramount importance of including a proper number of samples to obtain reliable algorithms.
- Jan 2016
The meat of Ruvettus pretiosus and Lepidocybium flavobrunneum (gemfishes) contains high amounts of indigestible wax esters that provoke gastrointestinal disorders. Although some countries banned the sale of these species, mislabelling cases have been reported in sushi catering. In this work, we 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 specie-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 the DNA samples tested (reference and commercial), provide a rapid and reliable response in identifying the two toxic species and combat health frauds.
- Sep 2015
The presence of anisakid larvae in fish is a public health issue, and effective risk management procedures are needed to avoid that heavily infected products reach the market. Currently, an official sampling plan for fresh fish defining sample size, inspection methods, and criteria to accept or reject the merchandise is lacking at the European and Italian level. In this study, we compared the visual inspection proposed by the sampling plan of the Lombardy Region (Italy) to the UV press method and to an optimized digestion procedure with the aim to assess its ability in detecting visible parasites. Thirty-one batches of Engraulis encrasicolus, each composed of ∼30 specimens, were collected and subsequently analyzed with the three techniques. The mean abundance (MA) was calculated after each procedure and compared on the basis of a threshold value. The results showed that the visual inspection performed similarly to the digestion method, with a sensitivity of 93 %, a specificity of 100 %, and an accuracy of 97 %. Overall, the comparison showed that, in the proposed sampling plan, the visual inspection is effective in rejecting unmarketable anchovies and in preventing the commercialization of unsafe products. This method is simple, less demanding than digestion in terms of time and equipment, and thus suitable as a standardized procedure to be routinely applied by food business operators. The hazard characterization, performed by sequencing the mtDNA cox2 gene, has identified the visible larvae as Anisakis pegreffii in 98 % of the cases, highlighting the zoonotic potential of the parasites found and the need for preventive measures.
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.
The morphological similarity among Sparidae species, which are characterized by a different market price, represents a serious problem for their trade and for stock management, since it encourages frauds for substitution. The most accredited morphological method for their identification is based on the dental-plate, but this approach is not simple and cannot be used for prepared products. When molecular methods are used the DNA degradation induced by cooking is the main drawback. In this work, we collected 314 reference tissues belonging to 75 Sparidae species and we produced a dataset of full (FDB) and mini-barcode (MDB) reference sequences starting from DNA extracted from fresh and ethanolpreserved tissues using universal primes. Moreover, some fresh samples were cooked. The FDB was successfully amplified in 91% (fresh), 50% (cooked) and 81% (ethanol-preserved) samples, while the amplification rates of the MDB were considerably higher in case of cooked (100%) and ethanol-preserved (94%) samples. The same primers were used for the amplification of the DNA obtained from 58 market samples (MS). All the DNA barcodes were compared with BOLD and GenBank using IDs and BLAST analysis. FDB was able to provide unambiguous species-level identifications for 53 (78%) and 44 (64.7%) reference samples analyzed on BOLD and GenBank, respectively. The Mini-DNA barcode (MDB) showed a lower discriminating power with 32 (45.7%) and 29 (41.4%) sequences unambiguously matched to a species on BOLD and GenBank. However, the MDB allowed to identify all the reference sequences as belonging to the Sparidae family. FDB and MDB showed a similar performance in analyzing the MS, allowing to highlight 21 (38%) mislabeled MS. Our study, while confirming the FDB as a reliable tool for fish authentication, proposes the MDB as a promising tool to recover molecular information in case of cooked products.
Nowadays, pet food claiming high-valued fish among ingredients is largely available on the market. Unfortunately, the modifications induced by processing make species identification by visual inspection difficult and hinder the enforcement of the legislation on traceability. In this work, after aligning 819 sequences of Clupeidae, Engraulidae, Salangidae and Scombridae families, we developed new universal primers for the amplification and sequencing of 2 short fragments (±118 and ±213) of the mitochondrial 16s ribosomal RNA (16S rRNA) gene. Once tested on 130 DNA reference samples, these primers were used in the analysis of highly degraded DNA extracted from 43 canned cat food containing whole minnows (whitebait) (M) and tuna, or bonito or mackerel fillets (F). Three M and 2 F samples were analyzed for each can. A BLAST and a FINS analysis, the latter performed only on the 118 bp fragment, were performed separately on the sequences obtained from M and F samples. All the M samples were identified at the species or genus level by both BLAST and FINS analysis. This allowed to highlight an impressive rate of mislabeling (100%). F samples, for which FINS was less performing in species identification, resulted mislabeled in 40% of the products.
- Mar 2015
The number of seafood species sold on Western markets is constantly growing and many unconventional species are sold in ethnic food outlets. In this work, 68 ethnic seafood products variously processed were collected from the Italian market and a molecular analysis was performed by sequencing a full cytochrome c oxidase (COI) DNA barcode (FDB, ∼655bp) or a mini COI DNA barcode (MDB, ∼139bp) using universal primers. Barcodes were then compared with sequences available in BOLD and GenBank. In addition, the label information was assessed according to the European legislation. By using the IDs analysis on BOLD a maximum species identity ≥98% was retrieved for 84% of the sequences. Of these, 67% were unambiguously identified at species level (51.3% of the FDB and 74% of the MDB). Using NCBI BLAST, 74% of the sequences scored a maximum species identity ≥98%, of which 73% were identified at species level (52% of the FDB and 61% of the MDB). Both databases performed better in mollusk identification. Overall, 45 products (66%) were not correctly labelled according to the European requirements. Finally, the comparison between the molecular and the label analysis highlighted that 48.5% of the products presented discrepancies between labeling and molecular identification. In particular, health implications were highlighted for 2 samples labeled as squid but identified as Lagocephalus spp., a poisonous puffer fish species banned from the EU market. The present results confirm DNA barcoding as a reliable tool for protecting health and economic interests of the consumers.
- Jan 2015
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.
- Nov 2014
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.
The morphological similarity among Sparidae species, which are characterized by a different market price, represents a serious problem for their trade and for stock management, since it encourages fraud for substitution. The most accredited morphological method for their identification is based on the dental-plate, but this approach is not simple and cannot be used for prepared products. When molecular methods are used the DNA degradation induced by cooking is the main drawback. In this work, we collected 314 reference tissues belonging to 75 Sparidae species and we produced a dataset of full (FDB) and mini-barcode (MDB) reference sequences starting from DNA extracted from fresh and ethanol-preserved tissues using universal primes. Moreover, some fresh samples were cooked. The FDB was successfully amplified in 91% (fresh), 50% (cooked) and 81% (ethanol-preserved) samples, while the amplification rates of the MDB were considerably higher in case of cooked (100%) and ethanol-preserved (94%) samples. The same primers were used for the amplification of the DNA obtained from 58 market samples (MS). All the DNA barcodes were compared with BOLD and GenBank using IDs and BLAST analysis. FDB was able to provide unambiguous species-level identifications for 53 (78%) and 44 (64.7%) reference samples analyzed on BOLD and GenBank, respectively. Mini-DNA barcode (MDB) showed a lower discriminating power with 32 (45.7%) and 29 (41.4%) sequences unambiguously matched to a species on BOLD and GenBank. However, the MDB allowed to identify all the reference sequences as belonging to the Sparidae family. FDB and MDB showed a similar performance in analyzing the MS, allowing to highlight 21 (38%) mislabeled MS. Our study, while confirming the FDB as a reliable tool for fish authentication, proposes the MDB as a promising tool to recover molecular information in case of cooked products.
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.
Over the years, the European Union has developed a comprehensive legal framework to ensure seafood traceability. In fact, in this sector, where the complexity of the marketing patterns has reduced the efficiency of controls, frauds are becoming widespread. Moreover, the rapid growth of Chinese communities has led to an increase of importations from Asia, which sometimes do not fully respect rules on traceability. In this study, we performed a survey on seafood products collected from the market of the Chinese community of Prato (Italy), to assess the frequencies and types of non-compliance in the light of the requirements established by the European traceability legislation on fisheries and aquaculture. Examination of labels and contents of Chinese seafood products imported to Italy found that 83% did not meet EU requirements for traceability. Overall, this survey put into light the difficulties of the ethnic communities to conform to the European rules, the need to adapt the control system to the fast developing trade reality at all levels of the chain and the advisability to create standards that could be adopted worldwide.
In the fish food sector, due to a growing globalization of the market, where intentional and unintentional frauds reach alarming levels, the molecular analysis is increasingly used by both official agencies, to enforce the law on traceability, and private companies, to verify the quality of goods. DNA extraction represents a necessary and critical step for all types of DNA analysis. Among the drawbacks associated with this procedure, there are handling of toxic materials, low DNA yield, and low throughput, due to time-consuming manual procedures. In this work, to overcome some of these problems, we developed an alternative method based on a bead-milling procedure without proteinase K digestion. The new method was then compared with both a salting-out protocol, developed in a previous work, and a commercial kit. Yield, spectrophotometric purity, electrophoretic degradation pattern, and amplificability of the extracted DNA were assessed. In particular, DNA amplificability was evaluated by comparing the band intensity on the gel, after amplification of the 16S rRNA and cytochrome oxidase I genes with a conventional PCR, and the take-off cycles, after amplification of the 16S rRNA gene with a real-time PCR. The results showed that the bead-based method allowed to obtain acceptable amounts of DNA, with good purity and good characteristics of amplificability. Although the salting-out method remains the most effective protocol in terms of pure performances, the bead-milling procedure can be considered a valid alternative, in the light of its lower demand in terms of labor and costs
- Nov 2013
The jellyfish belonging to the Rhizostomeae order are a typical Asian seafood and are nowadays also marketed in the Western countries. The jellyfish species used for food cannot be identified due to the loss of the morphological characteristics during processing. In this work, a pre-extraction treatment consisting of desalting under running water for 48 h, followed by incubation in 400 mM EDTA solution for 1 h, was developed for products containing high concentration of salts. Moreover, considering the DNA degradation observed in both classical (CPs) and ready to eat (RE) commercial samples, five sets of primers for the amplification of fragments with different lengths belonging to the mitochondrial COI gene were designed and used to obtain the sequences from 54 reference specimens and 70 market samples. A phylogenetic analysis was then performed using the neighbor-joining method. It was found that a fragment of 142 bp was enough informative to allow identification at the species level. The results showed that most of the products were made of Nemopilema nomurai (94% of RE and 45.5% of CPs). The analysis permitted to uncover an alarming level of mislabeling which reaches 79% for CPs and 100% for ready-to-eat products.
Question - Does anyone have experience with the samples or the MW marker not using all of the gel, when running sds page gel?
With 15% acrylamide gels it is normal that dye runs much faster than proteins. Reduce acrylamide content to 12%.
- Nov 2012
The problems concerning the recognition based on morphology of fish products have led to search for alternative methods to ensure proper identification, especially for those products that are more liable to substitution frauds, due to the type of processing. In this paper, through the development of a method for the amplification and sequencing of portions of the mitochondrial DNA, we examined fish fillets purchased as Greenland Halibut (Reinhardtius hippoglossoides) by a Tuscan school canteen. These fillets were reported by the cooks to have an unusual reaction to cooking. The phylogenetic analysis, performed on fragments of the genes 16srRNA and cytb, has identified the product as Pacific Rassera, thus demonstrating the existence of a commercial fraud, already suspected by the employees of the canteen. While confirming the great potential of the methods based on DNA analysis as a tool for control and traceability, the results of this study highlight the importance of proceeding through a strategy that, from time to time, consider the nature and type of the product and the availability of information on the molecular databases.
- Oct 2012
The rising demand for seafood and trade globalization has brought about a rapid increase in the number of fish species traded. Consequently, the occurrence of mislabelling is growing as well, reaching levels of concerns in USA, Canada and Europe. In this light, the evolving consciousness of consumers and the new exigencies of commerce call for greater safety and quality requirements. These factors have made urgent the need for efficient traceability systems, aimed to ensure transparency on the identity and origin of the traded products and the compliance with the regulations concerning illegal fisheries and labelling. Moreover, greater efforts are necessary to create a list of market names that can be recognized both locally and internationally, in order to overcome the confusion regarding fish denominations. In this context, molecular analysis represents the most promising challenge to verify and support traceability in the seafood chain. Nowadays, the three mitochondrial genes cytb, COI and 16srRNA are the most targeted for this purpose and, among the available procedures, the DNA bar coding is the most commonly applied to verify the labelling compliances, also at the official level. In this review, the most important issues relating to these topics have been reported and discussed.
- Sep 2012
The accident at the nuclear plant of Fukushima on 11 March 2011 has raised many questions regarding the contamination of food products, including fishery products, in relation to the discharge of contaminated water into the sea. Despite the European Union responding to this emergency by issuing Regulations that impose analytical controls on products from Japan, the spreading of alarmist information has created some concerns among consumers. The purpose of this work was to investigate how many fishery products coming from the area considered at risk, actually reach the European markets. The analyzed data show that imports from Japan represent only a small percentage in the Italian import-export context and that only few species are sold on our markets. Increased attention should instead be directed to those products that are caught in the ocean areas most at risk, but come from countries other than lapan.
An investigation has been performed on the fraudulent use of anabolic substances in the Region of Molise. One hundred fourty-four bovines (12-24 months old, 123 males and 21 females) have been included in the survey. Ante-mortem assessment on their behaviour and clinical analysis on some target organs were carried out. After slaughtering, samples of prostate, bulbo-urethral glands, Bartholin’s glands, mammary gland, ovaries, thymus and thyroid were collected and processed for an anatomo-histopathological evaluation, as suggested in the guidelines of the Italian National Plan for Residues (PNR) 2009. Overall, the 15% of the subjects analysed have been classified as “suspect”, whereas the 44% as “uncertain” and the remaining 59% as “negative”. The lesion most frequently found was a serious atrophy of the thymic parenchyma with fat infiltration (15% of males and 14% of females), suggesting a prevalence of an illegal use of cortisonic drugs.
The seven AnglerWsh species, which belong to the genus Lophius, have a different value on the market, worldwide. If whole fishes can be identified by their morphological characteristics, they become indistinguishable when prepared or processed. In this study, a rapid method based on polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) was developed for the authentication of the seven Lophius species, using a cytochrome b gene fragment of 566 bp. After a genus-specific PCR, a fast digestion with the restriction enzyme BfaI, followed by agarose gel electrophoresis, allowed a clear species identification by producing specific restriction patterns. The total time required was as low as 6 h, DNA extraction included. The method was then used to analyse 48 commercial samples, whose phylogenetic analysis confirmed the PCR–RFLP response at 100 %. Results showed that mislabelling occurs on the market regardless the kind of processing.
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. Ante-mortem assessment on their behaviour and clinical examination was performed. After slaughtering, 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 examined animals have been classified as "suspect," whereas 44.4% as "uncertain" and the remaining 40.3% 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.
The jellyfish is a popular seafood in South-East Asia. It can also be found throughout Europe in many Chinese shops, which despite being well-organized from a commercial perspective, often disregard rules on product traceability. Therefore because jellyfish has still not been officially defined by the European Legislation on Food Hygiene, we performed a survey on fifty-six jellyfish products sold on the Italian market. This was done by assessing label compliance with European law and with the new Chinese Food Safety Law of 2009. Our survey found many incongruences and shortfalls including the presence of a trade name referring to vegetables or a lack of an unequivocal specification of ingredients. Focus is therefore needed on the possible hazards associated with the consumption of jellyfish products. In addition, regulations need adjusting in order to create more transparent marketing and sales practices.
A conventional and a realtime multiplex PCR were developed to detect fraudulent substitutions of Bianchetto (juvenile form of Sardina philcardus) and Rossetto (Aphia minuta) with Icefish (Neosalanx spp.), which show similar morphological characteristics. Since it is the major by-catch species, Engraulis encrasicolus was also included in the analytical procedure. A common reverse primer and forward species-specific primer were designed on the mitochondrial cytochrome b gene to amplify sequences of different lengths by conventional PCR. Specific peaks were therefore also provided after melting temperature analysis in real-time PCR, thus enabling each species to be clearly differentiated. The two PCR methods were validated on fresh and processed products after preparing four typical dishes: two marinades (from raw or lightly boiled fish), a pasta sauce, and batter-fried fish cakes. All samples were correctly identified, although there was some DNA degradation after processing.
- Dec 2011
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.
- Sep 2011
Abstract The use of recombinant bovine growth hormone (rbGH) to increase milk yield in cows is banned in some countries. In others, where it is authorized, 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 buffalos, which are sometimes treated with rbGH and represent 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 buffalos for ten weeks with a sustained-release formulation of rbGH and analysed the response of twenty somatotropic axis genes in leukocytes by real-time PCR. Overall changes in gene expression levels were of low magnitude and sometime 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 leukocytes is little helpful for discrimination of rbGH treated buffalos, but do not exclude that another array of genes could provide useful patterns of variation.
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.
- Jun 2011
In this study we developed a sandwich enzyme-linked immunosorbent assays (ELISA) with good performance, low cost and simplicity of execution, to facilitate the measurement of insulin-like growth factor 1 (IGF-1) in buffalo milk. This assay requires a pre-treatment method that consists of an acidification step to separate IGF-1 from its binding proteins (IGFBPs) and a subsequent pH re-equilibration, with a buffer containing excess insulin-like growth factor 2 (IGF-2), which blocks the IGFBPs and enables the IGF-1 molecules to remain free in solution. The limit of quantification was about 0.32 ng mL−1 of milk. The within- and between-assay coefficients of variation were 6.3% and 13%, respectively. Linearity and accuracy were acceptable. The comparison with the classic acid–ethanol extraction method applied to colostrum samples showed a high correlation of results, indicating a similar level of efficiency. The assay developed in this study can thus be applied for measuring IGF-1 in milk.
The chinese catering, which has developed in Italy since the 50's, is nowadays widely represented throughout the country. The management of the health standards for these restaurants has often been shown to be problematic regarding compliance with the requirements of the existing legislation on food safety. These difficulties have increased in the light of the recent trend of Chinese restaurants to offer typical Japanese cuisine, which is more popular with consumers but also more critical for hygiene management. It is therefore considered appropriate to identify specific control and formative systems, with the help of native speakers for a greater effectiveness.
Neosalanx spp and Protosalanx spp are two genuses belonging to the fish family of Salangidae that are caught in China and commercialised abroad with the name of Ice fish or silver fish. Because of their morphological resemblance to some valuable fish products sold on our national market (know as Bianchetto and Rossetto), the Icefish is sometimes involved in commercial frauds. The number of species of the aforesaid genuses are several and sometimes difficult to identify. However, the deep exploitation of the Salangids in China brought to a sharp prevalence of the specie Neosalanx taihuensis on the others, which should be the main if not the only specie imported in Italy. In this work, we analysed the DNA sequences of the cytochrome b of a number of Icefish samples collected both in Italy and in China, to evaluate the species which are actually imported, in order to develop, in the future, a single step molecular analysis for the identification of substitution frauds."
Recently, many changes have been introduced in the food safety control organization of the People’s Republic of China, pushed by important international events to be held within the Country and some big scandals, such as the “melamine milk crisis”. The old “law on food hygiene” of 1995 has been replaced by the “law for food safety” in 2009, to reorganize in particular, the arrangement and responsibilities of the Institutions that operate in this field. In particular, the National Council for Food Safety has been created. It represents the highest authority in the matter, for legislation and investigation on food concerns and coordination of the aforesaid Institutions. However, notwithstanding the many steps ahead, many efforts remain to be performed especially in terms of formation of the food business operators and to solve complex problems, such as the big fragmentation of the primary production, which, at present, would need a huge extent of human and financial resources for the control system.
- Nov 2010
a b s t r a c t The Salangidae fish family includes the genera Neosalanx and Protosalanx, known as icefish or silverfish, which are imported frozen from China to Italy. The variable morphological characteristics and the small size make their particular species extremely difficult to identify. As a consequence it is not feasible to verify the precise identity of the product on the market. This means that Chinese fish can be used to fraudulently replace some much more expensive Italian species, which are similar in shape and size. Ten specimens of Neosalanx taihuensis directly collected from Lake Taihu and two hundred specimens of icefish collected from forty markets (twenty-seven from Italy and thirteen from China) were examined by direct sequencing of a portion of mitochondrial DNA belonging to the cytochrome b (cytb) gene in order to identify them at a specie level and to investigate any potential mislabelling. A BLAST analysis of the obtained sequences identified 90% of market samples as N. taihuensis, which rose to 93% when only products collected in Italy were considered. A phylogenetic tree was also constructed and the high bootstrap values obtained further supported our findings. Our results are also in agreement with some reports by Chinese authors, who describe this species as the most exploited of the Salangidae family in China in terms of internal consumption and exports. Furthermore, these findings corroborate the documentation collected from all over Italy, which reports N. taihuensis as being the only regularly imported species of icefish. The other species of salangids identified were Neosalanx anderssoni and Protosalanx chinensis. Overall, 15% of samples collected on the Italian market were mislabelled, thus confirming the existence of commercial frauds. By verifying the presence of one main icefish species on the Italian market (N. taihuensis), this study has contributed in identifying a precise target for inexpensive and rapid molecular analytical methods aimed at detecting and preventing fraud involving icefish.
- Jul 2010
This study focused on the expression of somatotropic axis genes in the skeletal muscle of dairy cattle. A slow-release recombinant bovine growth hormone (GH) (rbGH) formulation was administered to 5 cows, and saline solution (control) was administered to another 5 cows every 2 wk for a total of 10 wk, starting from the peak of lactation. Tissue and blood samples were collected on days 2 and 14 after each rbGH injection. As target genes insulin-like growth factor (IGF)-1, IGF-2, IGFBPs (1, 2, 3, 4, 5, 6), acute labile subunit (ALS), IGF-1 receptor (IGF-1R), GH receptor (GHR), and the known GHR 5'-UTR variants were selected as target genes, and their relative expression was measured using real-time polymerase chain reaction. In GH-treated cows, an increase in expression was observed for GHR 5'-UTR variant 1I on day 14 (P < 0.05), whereas a significant down-regulation of GHR (P < 0.05) was found after comparing values of treated cows between day 2 and day 14. However, only IGF binding proteins (BP)-5 was found to be appreciably up-regulated in GH-treated cows (P < 0.001), which may indicate the importance of this gene in the overall molecular response to GH administration. Our study indicated that GH treatment did not affect the expression of most somatotropic axis genes, despite the marked increase in GH and IGF-1 in blood (P < 0.001). Nor did it have a large impact on the proportion of GHR 5'-UTR variants in the skeletal muscle of lactating cows. Finally, although we observed a significant variation in the expression of some genes, it would appear that the differences between GH-treated cows and controls were not great enough to be considered as reliable indirect indicators of GH treatment in dairy cattle.
- Jun 2010
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.
- Apr 2010
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.
Nowadays, six of the seven species belonging to the Genus Lophius have an important commercial value in the national and international markets. Usually they are sold beheaded and for this reason they are called tails. This kind of preparation is a limit for the specie-identification by means of the morphological characteristics. The mitochondrial cytochrome b (Cyt b) gene is considered a useful genetic marker to identify fish species. In this work, after obtaining the Cyt b complete sequence of the Lophius species that were missed in the databases, we set up a method based on PCR-RFLP able to identify the six species of Lophius with a commercial denomination in the Italian market.
A microbiological survey was performed on ten brined jellyfish products, sampled in Italy from Chinese food markets. In general, the microbiological conditions were good and respected the standards contemplated in the regulations CE 2073/2005 e 1441/2007. The presence of inhibiting substances and the absence of aerobic mesophilic bacteria in two samples suggest a treatment to preserve the product.
- Jun 2009
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.
Besides immunochemical approaches, biomolecular studies can be carried out in order to discover a greater number of biological indicators to be exploited for the identification of bovines treated with recombinant somatotropin (rbST). With this aim, we analysed the expression of a number of genes related to the somatotropic axis in leucocytes from rbST treated cows and non-treated animals. Significant differences were observed in the genes IGF-1,IGFBP-1, IGFBP-4 and the I- 5’UTR variant of the GHR gene.
The Genus Lophius, belonging to the Lophiidae Family and commercially called anglerfish, has an important commercial value in the national and international markets. The seven species of the Genus are very similar and it is difficult to identify them by means of the traditional taxonomic methods also because fish are often commercialized decapitated. DNA analysis is one of the most useful tools for identifying animal species. In this work, in order to provide some more information on the DNA sequence of the aforesaid species, we use some universal primers to amplify the mitochondrial cytochrome b gene of Lophius vomerinus (Valenciennes, 1837) and obtained the complete sequence by sequencing of PCR products.
- Sep 2007
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
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.
The color of food, especially meat and meat products, is a parameter that strongly influences consumer choice. In Italy, repeated cases of darkening in deboned thigh meat of male turkeys packaged in modified atmosphere (MAP; 80% 02, 20% CO2) have been reported. The pH, lipid oxidation (TBARS), heme proteins, and iron content were investigated in MAP samples of turkey males, females, and in oxygen-permeable film-packaged males. Furthermore, the absorbance spectrum (400 to 700 nm) of the meat extracts was analyzed to better delineate the evolution and characteristics of the darkening process. Results showed that darkening occurred only in males with higher content of total iron, independently of the content of heme proteins, which differs only between males and females. Furthermore, pH was higher in muscles taken as controls, with respect to muscles involved in the darkening, as well as in females. Finally, TBARS values were found to be higher in darkened regions than in not darkened ones, as well as in MAP samples with respect to oxygen-permeable film-packaged samples. These findings suggest that darkening occurrence might depend on kind of muscle, sex, and individual characteristics of the animals raised under the same breeding conditions.
- Nov 2004
In this work the issues associated to the diagnostic methods for the detection of algal biotoxins are described. Algal biotoxins presence in water, and the subsequent accumulation in several fish products, imply a remarkable hazard both for public health and environmental protection. In particular, it is here described an overview of the studies performed with the aim to develop new analytical systems, of the obtained results and perspectives, Such alternative systems are needed in order to overcome some of the technical and time-related limits, as well as the ethic controversies, characterising the traditional biological methods.
RIASSUNTO Il problema legato al benessere animale è una questione dai risvolti etici che negli ultimi anni ha notevolmente condizionato la ricerca scientifica coinvolgendo sempre di più alcuni settori della medicina veterinaria. Nei laboratori di ricerca, infatti, si fa spesso uso di animali per la sperimentazione, come nel caso della produzione di anticorpi. Le metodiche impiegate possono talvolta causare sofferenze agli animali e, per questo motivo, da anni sono allo studio metodi alternativi a quelli più frequentemente utilizzati. L’immunizzazione di galline con antigeni di natura proteica consente di ottenere grandi quantità di anticorpi dai tuorli delle uova. Questo tipo di procedura comporta alcuni vantaggi sia dal punto di vista del benessere animale, in quanto scongiura i prelievi ematici per la valutazione del titolo anticorpale dell’animale in vita ed il salasso finale per la raccolta degli anticorpi, sia da quello economico dati i minori costi gestionali rispetto ai mammiferi da laboratorio di medie dimensioni e la maggiore resa finale di anticorpi. In questo lavoro sono stati prodotti anticorpi policlonali nelle galline contro una delle somatotropine bovine ricombinanti (rbST) esistenti in commercio, al fine di operare un confronto con anticorpi policlonali precedentemente prodotti nel coniglio contro la stessa molecola. Il raffronto delle curve ottenute tramite un test ELISA sandwich, messo a punto in precedenza per la quantificazione di bST nel siero e ripetuto utilizzando le due diverse specie anticorpali, ci ha permesso di valutare l’effettiva validità dell’immunizzazione di galliformi per la produzione di grandi quantità di immunoglobuline. SUMMARY Concerns about animal welfare are connected with ethical issues that affect scientific research, involving many veterinary fields. As a matter of facts, animals are often employed for experimental purposes, such as the production of antibodies. Applied methods could sometimes cause suffering to animals and for this reason several studies on alternative methods for antibody production are in progress. Immunisation of hens against proteic antigens allows to obtain a great amount of antibodies from the yolk of treated chicken eggs. This sort of practice brings many advantages for animal welfare, since blood collection is not necessary. In addition, some more advantages come either from the reduced breeding costs with respect to medium-size mammals or the higher antibodies yield. In this work, we have produced polyclonal antibodies in hens against one of the commercialised recombinant bovine somatotropins (rbST), with the aim to make a comparison with rabbit polyclonal antibodies against the same molecule, already produced in an earlier work. With this purpose, we performed a sandwich ELISA previously developed for bST measurement in serum and then compared the curves obtained by using the two different kind of polyclonal antibodies. This allowed us to estimate the worth of poultry immunisation for the production of a great amount of specific antibodies.
We used recombinant human IGF-1 and goat polyclonal antibodies against human IGF-1. The antigen–antibody interaction was monitored in real time using a low-cost biosensor. The instrument is a microbalance system based on resonant quartz crystals covered with a gold film. This active surface is able to capture particular types of molecules (in our case proteins), forming reversible chemical bonds. The molecules that directly bind the active surface or those that complex with bound molecules load the quartz, modifying its resonance properties. Such modifications interfere with the
- May 2003
In the last years there has been a rapid development of the biosensors field, even within the area of food control. In fact, if at first the biosensors have been restricted to an academic concern, they currently are beginning to find an employment even in the practice, either in analysis laboratories or in process control. In particular, the technical features of these appliances make them proper for the monitoring of hygienic and technological quality of food-stuff.
RIASSUNTO Le Aflatossine, appartenenti al più ampio gruppo delle micotossine, sono metaboliti tossici prodotti da Aspergillus spp., la cui importanza ispettiva è legata alla contaminazione di alcune derrate alimentari e mangimi animali. Tali molecole sono responsabili di molti effetti biologici pericolosi per l’uomo e per gli animali. Si capisce quindi l’importanza di un controllo efficace e costante e la necessità di un continuo miglioramento delle metodologie di rilevamento grazie anche agli avanzamenti della ricerca biotecnologica. In questo lavoro sono riportate le fasi preliminari della messa a punto di un metodo immunologico, eventualmente applicabile al settore biosensoristico, per il rilevamento della Aflatossina B1 in vari tipi di substrati. In particolare, sono stati prodotti anticorpi policlonali di coniglio e di topo e monoclonali di topo che, dai test effettuati, hanno rivelato una buona affinità ed un’elevata specificità nei confronti di tale antigene, caratteristiche fondamentali per un possibile utilizzo su immuno-biosensori, oltre che per lo sviluppo di metodiche classiche. SUMMARY Mycotoxyns are a structurally diverse group of secondary metabolites produced by different genera of fungi. They include Aflatoxins derived from Aspergillus spp. Such toxins are found world-wide in food and feed and exhibit a wide array of biological effects (genotoxic, carcinogenic, embryotoxic and teratogenic) in humans and animals. From here the importance of a continuos and efficient survey. In this work, with the aim to set up a rapid and sensitive immunological method, suitable for biosensors, we present the results obtained from the production and the characterization of anti-Aflatoxin B1 antibodies. Polyclonal antibodies against Aflatoxin B1 were produced in rabbits and mice by immunisation with Aflatoxin B1 covalently bound to Bovine Serum Albumin (BSA). Monoclonal antibodies specific for Aflatoxin B1, were obtained after fusion of mouse myeloma cells with splenocytes of immunised mice. In order to characterise our antibodies we have performed classical immunoenzymatic test (ELISA). Results shows that both polyclonal and monoclonal antibodies are able to bind the antigen with high selectivity and specificity. Particularly, our monoclonal antibodies are capable to discriminate Aflatoxin B1 from its hydroxylated form (M1), with a very good repeatability and then showing all the essential features to set up immunological analysis either classical or implying the use of immunosensors.