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

Future feed control – Tracing banned bovine material in insect meal

In the present study, we assessed if different legacy and novel molecular analyses approaches can detect and trace prohibited bovine material in insects reared to produce processed animal protein (PAP). Newly hatched black soldier fly (BSF) larvae were fed one of the four diets for seven days; a control feeding medium (Ctl), control feed spiked with bovine hemoglobin powder (BvHb) at 1% (wet weight, w/w) (BvHb 1%, w/w), 5% (BvHb 5%, w/w) and 10% (BvHb 10%, w/w). Another dietary group of BSF larvae, namely *BvHb 10%, was first grown on BvHb 10% (w/w), and after seven days separated from the residual material and placed in another container with control diet for seven additional days. Presence of ruminant material in insect feed and in BSF larvae was assessed in five different laboratories using (i) real time-PCR analysis, (ii) multi-target ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), (iii) protein-centric immunoaffinity-LC-MS/MS, (iv) peptide-centric immunoaffinity-LC-MS/MS, (v) tandem mass spectral library matching (SLM), and (vi) compound specific amino acid analysis (CSIA). All methods investigated detected ruminant DNA or BvHb in specific insect feed media and in BSF larvae, respectively. However, each method assessed, displayed distinct shortcomings, which precluded detection of prohibited material versus non-prohibited ruminant material in some instances. Taken together, these findings indicate that detection of prohibited material in the insect-PAP feed chain requires a tiered combined use of complementary molecular analysis approaches. We therefore advocate the use of a combined multi-tier molecular analysis suite for the detection, differentiation and tracing of prohibited material in insect-PAP based feed chains and endorse ongoing efforts to extend the currently available battery of PAP detection approaches with MS based techniques and possibly δ13CAA fingerprinting.publishedVersio

Future feed control – Tracing banned bovine material in insect meal

In the present study, we assessed if different legacy and novel molecular analyses approaches can detect and trace prohibited bovine material in insects reared to produce processed animal protein (PAP). Newly hatched black soldier fly (BSF) larvae were fed one of the four diets for seven days; a control feeding medium (Ctl), control feed spiked with bovine hemoglobin powder (BvHb) at 1% (wet weight, w/w) (BvHb 1%, w/w), 5% (BvHb 5%, w/w) and 10% (BvHb 10%, w/w). Another dietary group of BSF larvae, namely *BvHb 10%, was first grown on BvHb 10% (w/w), and after seven days separated from the residual material and placed in another container with control diet for seven additional days. Presence of ruminant material in insect feed and in BSF larvae was assessed in five different laboratories using (i) real time-PCR analysis, (ii) multi-target ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), (iii) protein-centric immunoaffinity-LC-MS/MS, (iv) peptide-centric immunoaffinity-LC-MS/MS, (v) tandem mass spectral library matching (SLM), and (vi) compound specific amino acid analysis (CSIA). All methods investigated detected ruminant DNA or BvHb in specific insect feed media and in BSF larvae, respectively. However, each method assessed, displayed distinct shortcomings, which precluded detection of prohibited material versus non-prohibited ruminant material in some instances. Taken together, these findings indicate that detection of prohibited material in the insect-PAP feed chain requires a tiered combined use of complementary molecular analysis approaches. We therefore advocate the use of a combined multi-tier molecular analysis suite for the detection, differentiation and tracing of prohibited material in insect-PAP based feed chains and endorse ongoing efforts to extend the currently available battery of PAP detection approaches with MS based techniques and possibly δ13CAA fingerprinting.</p

Species-Specific Discrimination of Insect Meals for Aquafeeds by Direct Comparison of Tandem Mass Spectra

Insect protein has the potential to become a sustainable feed ingredient for the rapidly growing aquaculture industry. In the European Union, insect derived protein is placed under the same legislation as processed animal proteins (PAP). It is therefore of interest to develop methods for regulatory use, which unambiguously identify the species origin of insect-based ingredients. We performed (i) total protein quantification of insect samples using the traditional nitrogen-to-protein conversion factor of 6.25 and the sum of anhydrous amino acids, (ii) quantitative amino acid profiling and (iii) high-throughput tandem mass spectrometry to describe and differentiate 18 different commercial-grade insect meal samples derived from Hermetia illucens (8), Tenebrio molitor (5), Alphitobius diaperinus (3) and Acheta domesticus (2). In addition, we investigated and compared different protein extraction and digestion protocols for proteomic analysis. We found that irrespective of sample preparation, shotgun proteomics in combination with direct spectral comparison were able to differentiate insect meal according to their taxonomic classification. The insect specific spectral libraries created in the present work can in future be used to develop more sensitive targeted methods of insect PAP identification and quantification in commercial feed mixtures

The Oxytricha trifallax Mitochondrial Genome

The Oxytricha trifallax mitochondrial genome contains the largest sequenced ciliate mitochondrial chromosome (∼70 kb) plus a ∼5-kb linear plasmid bearing mitochondrial telomeres. We identify two new ciliate split genes (rps3 and nad2) as well as four new mitochondrial genes (ribosomal small subunit protein genes: rps- 2, 7, 8, 10), previously undetected in ciliates due to their extreme divergence. The increased size of the Oxytricha mitochondrial genome relative to other ciliates is primarily a consequence of terminal expansions, rather than the retention of ancestral mitochondrial genes. Successive segmental duplications, visible in one of the two Oxytricha mitochondrial subterminal regions, appear to have contributed to the genome expansion. Consistent with pseudogene formation and decay, the subtermini possess shorter, more loosely packed open reading frames than the remainder of the genome. The mitochondrial plasmid shares a 251-bp region with 82% identity to the mitochondrial chromosome, suggesting that it most likely integrated into the chromosome at least once. This region on the chromosome is also close to the end of the most terminal member of a series of duplications, hinting at a possible association between the plasmid and the duplications. The presence of mitochondrial telomeres on the mitochondrial plasmid suggests that such plasmids may be a vehicle for lateral transfer of telomeric sequences between mitochondrial genomes. We conjecture that the extreme divergence observed in ciliate mitochondrial genomes may be due, in part, to repeated invasions by relatively error-prone DNA polymerase-bearing mobile elements

ALADIN laser frequency stability and its impact on the Aeolus wind error

The acquisition of atmospheric wind profiles on a global scale was realized by the launch of the Aeolus satellite, carrying the unique Atmospheric LAser Doppler INstrument (ALADIN), the first Doppler wind lidar in space. One major component of ALADIN is its high-power, ultraviolet (UV) laser transmitter, which is based on an injection-seeded, frequency-tripled Nd:YAG laser and fulfills a set of demanding requirements in terms of pulse energy, pulse length, repetition rate, and spatial and spectral beam properties. In particular, the frequency stability of the laser emission is an essential parameter which determines the performance of the lidar instrument as the Doppler frequency shifts to be detected are on the order of 10^8 smaller than the frequency of the emitted UV light. This article reports the assessment of the ALADIN laser frequency stability and its influence on the quality of the Aeolus wind data. Excellent frequency stability with pulse-to-pulse variations of about 10 MHz (root mean square) is evident for over more than 2 years of operations in space despite the permanent occurrence of short periods with significantly enhanced frequency noise (> 30 MHz). The latter were found to coincide with specific rotation speeds of the satellite's reaction wheels, suggesting that the root cause are micro-vibrations that deteriorate the laser stability on timescales of a few tens of seconds. Analysis of the Aeolus wind error with respect to European Centre for Medium-Range Weather Forecasts (ECMWF) model winds shows that the temporally degraded frequency stability of the ALADIN laser transmitter has only a minor influence on the wind data quality on a global scale, which is primarily due to the small percentage of wind measurements for which the frequency fluctuations are considerably enhanced. Hence, although the Mie wind bias is increased by 0.3 m/s at times when the frequency stability is worse than 20 MHz, the small contribution of 4 % from all Mie wind results renders this effect insignificant (< 0.1 m/s) when all winds are considered. The impact on the Rayleigh wind bias is negligible even at high frequency noise. Similar results are demonstrated for the apparent speed of the ground returns that are measured with the Mie and Rayleigh channel of the ALADIN receiver. Here, the application of a frequency stability threshold that filters out wind observations with variations larger than 20 or 10 MHz improves the accuracy of the Mie and Rayleigh ground velocities by only 0.05 and 0.10 m/s, respectively, however at the expense of useful ground data

Overexpression of DNA Polymerase Zeta Reduces the Mitochondrial Mutability Caused by Pathological Mutations in DNA Polymerase Gamma in Yeast

In yeast, DNA polymerase zeta (Rev3 and Rev7) and Rev1, involved in the error-prone translesion synthesis during replication of nuclear DNA, localize also in mitochondria. We show that overexpression of Rev3 reduced the mtDNA extended mutability caused by a subclass of pathological mutations in Mip1, the yeast mitochondrial DNA polymerase orthologous to human Pol gamma. This beneficial effect was synergistic with the effect achieved by increasing the dNTPs pools. Since overexpression of Rev3 is detrimental for nuclear DNA mutability, we constructed a mutant Rev3 isoform unable to migrate into the nucleus: its overexpression reduced mtDNA mutability without increasing the nuclear one

Développement de méthodes complémentaires pour la détection de produits d’origine animale dans les aliments pour animaux d’élevage

Since the mid-1980s, several transmissible spongiform encephalopathies have been reported in humans and animals. Bovine spongiform encephalopathy was first identified in cattle in 1986 and, in subsequent years in other animal species. Consumption by cattle of meat and bone meal containing carcasses of infected animals was incriminated. The use of such animal by-products was first banned in feed for ruminants and then, with few exceptions such as the use of fish meal for non-ruminants, extended to all feeds for farmed animal. Thanks to prevention and control efforts, a gradual lifting of the feed ban is now possible. The reintroduction of non-ruminant processed animal proteins into the feed of aquaculture animals was adopted in 2013. In this context of dynamic relaxation of the ban, the challenge remains the development of complementary methods or the adaptation of official methods aimed at refining the identification of feed materials. Indeed, the combination of light microscopy and PCR does not differentiate among certain animal by-products and this can make the results uninterpretable. This PhD thesis focused on (1) the evaluation of current legislation and available methods of analysis for the detection of animal proteins; (2) the development of a fluorescence in situ hybridisation method combining the advantages of microscopy (localisation and identification of type of tissues) and molecular biology techniques (identification of the species by DNA); (3) the identification of specific peptides from blood products by high resolution mass spectrometry and (4) the development of a sensitive and routinely applicable tandem mass spectrometry method paving a new way to the detection of processed animal proteins. The results obtained allowed to (1) highlight current and foreseen analytical gaps as part of the feed ban relaxation dynamics; (2) propose a method for a specific detection of processed bone particles of ruminant origin independently of contamination by other by-products such as milk powder; (3) identify 9 biomarker peptides of plasma powder of bovine origin and 7 biomarker peptides of bovine haemoglobin powder, 6 of which also allow the detection of blood meal of bovine origin and (4) develop a UHPLC MS/MS multi-target method allowing the simultaneous detection of several by-products of ruminant origin (blood meal, blood products and dairy products) at a limit of detection (LOD) of 0.1% (w/w). Finally, new analytical schemes incorporating the results of this thesis as well as potential sources of improvement for future analytical developments have been proposed.EURL-A

Blood meal and blood products detection using Synchronous fluorescence spectroscopy

peer reviewedIn Europe, ruminant processed animal proteins (PAPs) and blood products are not allowed to be used in feed for farmed animal. In contrast, blood meal and blood products of porcine origin are both authorised in aqua feed, whereas only porcine blood products are allowed to be used in feed intended for other non-ruminants. Besides official methods (light microscopy and PCR), complementary methods are developed in order to refine the by-products identification. By-products derived from blood are one of these products for which additional information are needed. Indeed, these prohibited materials sometimes cannot be distinguished from those authorised (e.g. milk powder) using official methods. The aim of this work was to develop a fast and easy method to detect blood meal and blood products. This study was based on the detection of hemoglobin in animal feed by synchronous fluorescence spectroscopy (SFS). To achieve this goal, preliminary tests were carried out on reference material (hemoglobin and albumin purchased from Sigma Aldrich) in order to determine appropriate conditions (solvent) and parameters (offset values) for hemoglobin detection. Selected settings were then applied to analyse protein extracts of commercial feed material derived from blood. The results obtained on blood meal and blood products (hemoglobin powder and plasma powder) showed fluorescence spectral bands that characterise hemoglobin. In order to determine whether the method was fit for the purpose, real commercial compound feeds known to contain or to be free from blood products or blood meal were analysed. The Principal Component Analysis (PCA) applied to these spectra showed that it was possible to discriminate samples containing hemoglobin from those that do not contain. This result confirmed that SFS is a promising screening method for the detection of hemoglobin in animal feed