Processed Animal Proteins In Feed Research Articles

A validated real-time PCR test for simultaneous detection of Gallus gallus and Meleagris gallopavo in feed instead of an impossible poultry test

Description of the subject. Use of all processed animal proteins (PAPs) in animal feeds was banned in the EU due to the outbreak of bovine spongiform encephalopathy (BSE). This total feed ban was progressively lifted for non-ruminant PAPs from 2013 onwards. Use of ruminant PAPs in feed remains totally prohibited. In order to extend the reintroduction of poultry PAPs in pig feed, and of pig PAPs in poultry feed, as recently decided, poultry species and pig detection tests were required to check that there is no intra-specific recycling. Objectives. In this study, we describe the official EURL-AP real-time PCR method for the simultaneous detection of chicken (Gallus gallus L.) and turkey (Meleagris gallopavo L.), the two most widely used poultry species in PAPs. Method. Classic quality criteria for the validation of a PCR method are considered. A cut-off is required for the interpretation of results. Transferability was tested through an interlaboratory study. Results. The developed PCR assay amplifies an 84 bp fragment encompassing the mitochondrial DNA on 12S ribosomal RNA, tRNA-Val and 16S ribosomal RNA sequences. The qualitative method was successfully assessed for several performance criteria: specificity, sensitivity, efficiency and robustness. The applicability of the test was verified on poultry PAPs and on compound feed containing 0.1% in mass fraction of poultry PAPs. An interlaboratory validation study demonstrated that the proposed real-time PCR method is transferable. Conclusions. The combined chicken-turkey real-time PCR assay is fit for the purpose of detecting poultry PAPs.

Processed animal proteins (PAPs) in animal nutrition: Assessment of the chemical risk of essential and non-essential elements

BackgroundProcessed animal products (PAPs) could be a great alternative to common protein supplements and represent a good example of recycling and valorization of by-products. Due to the reintroduction of certain types of PAPs in feed, a deeper knowledge of these heterogeneous matrices is needed. Thus, the aim of this study is to evaluate the levels of essential elements and inorganic contaminants in 55 PAPs considered as potential alternatives to common protein supplements. MethodsPAPs samples were analysed for essential (cobalt, nickel, chromium, copper, zinc, iron and manganese) and non-essential elements (arsenic, cadmium, lead and mercury) by Inductively Coupled Plasma Mass Spectrometer (ICP-MS), Graphite Furnace Atomization Atomic Absorption Spectrometer (GF-AAS) and dual cell Direct Mercury Analyzer spectrometer (DMA-80). ResultsEssential elements were found with the following decreasing order iron>zinc>copper>manganese>chromium>nickel>cobalt (mg kg−1). Only one sample was found non-compliant to lead concentration according to the European Union Regulation while negligible values of others non-essential elements were found. ConclusionsThis study suggests that PAPs could be a useful supplement for animal diet due to their natural content of essential elements. A careful monitoring of chemical elements should be required and eventually guidelines have to be drafted for a correct use of PAPs to ensure a safe and sustainable feed production.

Application of Mass Spectrometry-Based Immunoassays for the Species- and Tissue-Specific Quantification of Banned Processed Animal Proteins in Feeds

Processed Animal Proteins (PAPs) are considered as a sustainable protein source to improve the nutritional profile of feed for livestock and aquaculture. However, the use of these proteins is strongly regulated since the bovine spongiform encephalopathy (BSE) crisis. The reintroduction of nonruminant PAPs for use in aquaculture in 2013 has driven the need for alternative analytical methods to determine the species origin as well as the tissue source (legal or not). The current official methods, light microscopy and polymerase chain reaction, do not fulfill these requirements. Furthermore, future methods need to be quantitative, because the pending zero-tolerance-concept is planned to be replaced by accurate thresholds. Here, we developed a 7-plex mass spectrometry-based immunoassay that is capable of quantifying 0.1% (w/w) ruminant PAP in feed in a tissue- and species-specific way. The workflow comprises a 2 h tryptic digestion of PAPs in suspension, an immunoaffinity enrichment of peptides, and LC-MS/MS-based quantification. In combination with a previously published assay for species identification, we were able to confirm the species and tissue origin of sixring trial samplesobtained in former PCR and microscopy proficiency tests. The sensitive, quantitative, species- and tissue-specific character of the developed assays meets the requirements for new methods for PAP detection and can be used in future feed authentication studies.

Mass Spectrometry-Based Immunoassay for the Quantification of Banned Ruminant Processed Animal Proteins in Vegetal Feeds.

The ban of processed animal proteins (PAPs) in feed for farmed animals introduced in 2001 was one of the main EU measures to control the bovine spongiform encephalopathy (BSE) crisis. Currently, microscopy and polymerase chain reaction (PCR) are the official methods for the detection of illegal PAPs in feed. However, the progressive release of the feed ban, recently with the legalization of nonruminant PAPs for the use in aquaculture, requires the development of alternative methods to determine the species origin and the source (legal or not). Additionally, discussions about the need for quantitative tests came up, particularly if the zero-tolerance-concept is replaced by introducing PAP thresholds. To address this issue, we developed and partially validated a multiplex mass spectrometry-based immunoassay to quantify ruminant specific peptides in vegetal cattle feed. The workflow comprises a new sample preparation procedure based on a tryptic digestion of PAPs in suspension, a subsequent immunoaffinity enrichment of the released peptides, and a LC-MS/MS-based analysis for peptide quantification using isotope labeled standard peptides. For the very first time, a mass spectrometry-based method is capable of detecting and quantifying illegal PAPs in animal feed over a concentration range of 4 orders of magnitude with a detection limit in the range of 0.1% to 1% (w/w).

A quantitative real time polymerase chain reaction approach for estimating processed animal proteins in feed: preliminary data

Lifting of the ban on the use of processed animal proteins (PAPs) from non-ruminants in non-ruminant feed is in the wind, avoiding intraspecies recycling. Discrimination of species will be performed through polymerase chain reaction (PCR), which is at a moment a merely qualitative method. Nevertheless, quantification of PAPs in feed is needed. The aim of this study was to approach the quantitative determination of PAPs in feed through Real Time (RT)-PCR technique; three different protocols picked up from the literature were tested. Three different kind of matrices were examined: pure animal meals (bovine, chicken and pork); one feed sample certified by the European reference laboratory on animal proteins (EURL AP) in feed spiked with 0.1% bovine meal; and genomic DNAs from bovine, chicken and pork muscles. The limit of detection (LOD) of the three protocols was set up. All the results obtained from the three protocols considered failed in the quantification process, most likely due to the uncertain copy numbers of the analytical targets chosen. This preliminary study will allow us to address further investigations, with the purpose of developing a RT-PCR quantitative method.

Scientific Opinion on the revision of the quantitative risk assessment (QRA) of the BSE risk posed by processed animal proteins (PAPs)

The cattle Bovine Spongiform Encephalopathy (BSE) risk posed by bovine derived Processed Animal Proteins (PAPs) in feed was estimated, the diagnostic methods and their sensitivity to detect animal proteins in feed and compared different risk assessment methods for animal proteins in feed was reviewed. It was concluded that the current global limit of detection for PAPs in feed is still considered to be 0.1%. Data related to BSE monitoring and PAP production in the European Union (EU) were considered. A model (EFSA QRA PAP model) was developed to study the magnitude of the total BSE infectivity in PAPs and in ruminant feed under a certain scenario assuming some specific cross-contaminations. On the basis of the 2009 BSE surveillance data in the EU, assuming a 0.1% contamination (the limit of detection for PAP in feed) with non-ruminant PAPs and according to the EFSA QRA PAP model, the total BSE infectivity load that could enter in cattle feed per year in the EU would be equivalent to 0.2 Cattle oral Infectious Dose 50% (Co ID50) (9 x 10-5 - 1.3 CI95%) (that would mean that less than one additional BSE infected cattle could be expected in the EU cattle population per year with an upper 95% confidence). The specific scenario described by the model and the related assumptions and uncertainties are discussed in this scientific opinion.

An overview of the legislation and light microscopy for detection of processed animal proteins in feeds

From the first cases of bovine spongiform encephalopathy (BSE) among cattle in the United Kingdom in 1986, the route of infection of BSE is generally believed by means of feeds containing low level of processed animal proteins (PAPs). Therefore, many feed bans and alternative and complementary techniques were resulted for the BSE safeguards in the world. Now the feed bans are expected to develop into a "species to species" ban, which requires the corresponding species-specific identification methods. Currently, banned PAPs can be detected by various methods as light microscopy, polymerase chain reaction, enzyme-linked immunosorbent assay, near infrared spectroscopy, and near infrared microscopy. Light microscopy as described in the recent Commission Regulation EC/152/2009 is the only official method for the detection and characterization of PAPs in feed in the European Union. It is able to detect the presence of constituents of animal origin in feed at the level of 1 g/kg with hardly any false negative. Nevertheless, light microscopy has the limitation of lack of species specificity. This article presents a review of legislations on the use of PAPs in feedstuff, the detection details of animal proteins by light microscopy, and also presents and discusses the analysis procedure and expected development of the technique.