Bovine blood biomarkers as a way of processed animal proteins detection in feedingstuffs

peer reviewedThe prohibition of using animal by-products in feedingstuffs depends on two factors: their nature defined by the tissue/cell type and the species of origin, and on their destination (pets, fur animals or other farmed animals). Proteomics is particularly well-suited to the purpose of PAPs detection as it is a tissue and species-specific method. The aim of this study was the identification and the selection of specific peptide biomarkers using tandem mass spectrometry for the detection of bovine blood products and blood meals in animal feed. Twenty-nine samples of blood meals and blood products (plasma or haemoglobin powder) of porcine, poultry and bovine origin as well as three milk products and two fish meals were analysed using a Q TOF mass spectrometer. Vegetal feed samples adulterated with 1% or 10% of bovine plasma powder, haemoglobin powder or blood meal were also analysed to evaluate the applicability of the method. Four proteins of interest were highlighted: Alpha-2-macroglobulin, apolipoprotein A-1, serotransferrin and haemoglobin (α and β chains). From these proteins, sixteen peptides were identified as potential bovine blood biomarkers in feedingstuffs. Nine of them could be used for the detection of plasma powder and seven of them for haemoglobin powder or blood meal. The evaluation of these peptides by a search against NCBInr database revealed that some of them could also be used to detect other ruminant bloods such as ovine or caprine ones. These preliminary results are promising. Efforts are now focused to improve the protocol in order to increase the sensitivity of the method as regards the selected proteins

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

The Azolla leaf cavity pore : a highly differentiated protective structure between the leaf cavity and the environment

Doctorat en sciences - UCL, 200

Specific detection of blood derived products in animal feed using UPLC-MS/MS

peer reviewedBlood derived products are valuable animal products used in feed for their nutritional value and their positive effects on growth and health. Nevertheless, since the BSE crisis, their use is strictly regulated. Blood meal and blood products (hemoglobin powder and plasma powder) of bovine origin are totally prohibited. Blood meal and blood products of porcine origin are authorised in aquafeed, whereas only blood products are allowed to be used in feed intended for other non-ruminant. The detection of the type of protein and the species of origin is therefore crucial to ensure feed safety. With the current official methods for the detection of PAPs, light microscopy and PCR, it is not always possible to specifically identify this type of protein source. The objective of our work was to set-up a routine mass spectrometry method for a qualitative detection of blood meal and hemoglobin powder of bovine and porcine origin in feed. The method was based on the detection of species-specific peptides biomarkers identified in a previous study by a non-targeted approach. All biomarkers were peptides of α and β hemoglobin subunits. Proteins were extracted using a TCA/acetone protein precipitation protocol followed by purification with a 2-D Clean-Up Kit (GE Healthcare, USA). After an in-solution trypsin digestion step, analyses were performed by liquid chromatography (Acquity system, Waters, UK) coupled with a triple quadrupole mass spectrometer (Xevo TQS, Waters, UK) with electrospray ionisation. The acquisition and processing of data was carried out by MassLynx software (v. 4.1, Waters) and multiple reaction monitoring (MRM) design was made using the open-source software Skyline (https://skyline.gs.washington.edu/labkey/project/home/software/Skyline/begin.view). Reference hemoglobin powder was used to select MRM transitions for each peptide biomarkers and to optimise their collision energy. Commercial feed material and compound feed containing or free from blood derived products were then analysed. Selected transitions were present in materials or feed known to contained blood derived product of the same origin and absent in the others. Artificially contaminated feed with various contamination levels were also analysed in order to evaluate the influence of matrix composition and to experimentally determine the limit of detection. A first estimation of the LOD was around 0.05 % w/w which was below the LOD imposed by the EC for animal proteins detection method

Innovative methods for the determination of the taxonomic origin of processed animal proteins in feed.

peer reviewedThe use of animal by-products in feed depends on their nature defined by the type of tissue or body parts and the species of origin. Currently, the detection of unauthorised processed animal proteins (PAPs) is based on light microscopy and PCR methods. Light microscopy identifies structures on the basis of their morphology and enables identification of particles (such as bones, cartilages, muscle fibres,…) while PCR is able to detect and identify the presence of specific animal DNA in feed. Nevertheless, for some scenarios, even combined, these methods do not succeed in determining the taxonomic origin of the PAPs. A typical example is that of an aquafeed containing authorised porcine PAP together with dairy products: the analysis will conclude of the potential presence of ruminant PAP. Therefore, there is a need for developing methods allowing a taxonomic characterisation of visual structures such as bones fragments and muscle fibres. For the characterisation of bones, NIRM has yet demonstrated its potential. However the limitation of NIRM is when the presence of bones is reduced or absent. This study investigated the potential of NIRM for the determination of the taxonomic origin of muscle fibres. The NIRM was experimented on 2 porcine PAPs vs. 6 ruminant PAPs and 7 fishmeals all of industrial origin. Results showed that NIRM allows differentiating muscle fibres from different taxonomic origins: fish, ruminant and pig. In addition to this taxonomic classification, results also reveal differences inside taxonomic clusters of PAPs (e.g among different ruminant PAPs and porcine PAPs). The results obtained on this type of meals are promising and offer new perspectives. Tests on adulterated feeds need to be performed by NIRM prior to validation

The pore of the leaf cavity of Azolla species: teat cell differentiation and cell wall projections.

The differentiation of the specialized secretory teat cells of the leaf cavity pore of Azolla species was investigated at the ultrastructural level with emphasis on their peculiar cell wall projections. The results indicated that the projections are formed as soon as the teat cells complete their differentiation and that their production is principally associated with changes in endoplasmic reticulum profiles. The number of projections increases with the teat cell age and is stimulated under salt and P deficiency stresses. Salt stress also promotes their emergence on Azolla species that under normal conditions do not produce projections. Cytochemical tests on different Azolla species showed that the projection composition is almost identical: proteins, acidic polysaccharides, and pectin are always detected. This study revealed that Azolla teat cell projections differ fundamentally from other types of hitherto described cell wall projections that are considered as remnant structures from cell separation. In contrast, in Azolla teat cells projections are actively produced and compounds are excreted by an exocytotic mechanism. The possible role of the projections in the symbiosis of Azolla spp. with Anabaena azollae is discussed

The pore of the leaf cavity of Azolla: Interspecific morphological differences and continuity between the cavity envelopes

Interspecific morphological differences between mature leaf cavity pores of the different Azolla species were investigated by light and low temperature scanning electron microscopy. Results indicated that the pore morphology constitutes a novel taxonomic criterion allowing us to separate the Azolla and Rhizosperma sections. Scanning documents also gave further insight into the pore function: it ensures gaseous exchanges between the environment and the cavity, defends the cavity from external ingress and contributes to the maintenance of the endosymbionts within this cavity. Transmission electron microscopy of the cavity in the pore region indicated that the previously described one-layered inner envelope and three-layered outer envelope, both limiting the mucilage containing the endosymbionts, are not separate entities: they join together at the pore periphery and the inner envelope is actually constitutive of one of the layers of the outer envelope. Our observations allowed us to explain for the first time the possible origin and nature of each of the three constitutive layers of the latter envelope

Identification of specific bovine blood biomarkers with a non-targeted approach using HPLC ESI tandem mass spectrometry

Animal by-products are valuable protein sources in animal nutrition. Among them are blood products and blood meal, which are used as high-quality material for their beneficial effects on growth and health. Within the framework of the feed ban relaxation, the development of complementary methods in order to refine the identification of processed animal proteins remains challenging. The aim of this study was to identify specific biomarkers that would allow the detection of bovine blood products and processed animal proteins using tandem mass spectrometry. Seventeen biomarkers were identified: nine peptides for bovine plasma powder; seven peptides for bovine haemoglobin powder, including six peptides for bovine blood meal; and one peptide for porcine blood. They were not detected in several commercial compound feed or feed materials, such as blood by-products of other animal origins, milk-derived products and fish meal. These biomarkers could be used for developing a species-specific and blood-specific detection method