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The aim of this study was to evaluate the use of light microscopy with differential staining techniques for the discrimination of insect material from marine arthropods - classified as fishmeal. Specifically, three samples of single-species insect material, Hermetia illucens (HI), Bombyx mori (BM) and Tenebrio molitor (TM), and two samples of marine arthropods, shrimp material and krill, were analysed and compared after staining by two reagents to enhance fragment identification. Alizarin Red (AR) and Chlorazol Black (CB), which react respectively with calcium salts and chitin, were tested for their potential efficacy in distinguishing between insect and marine materials. Results indicated that AR failed to stain HI, BM and TM materials. By contrast, the three insect species materials tested were stained by CB. When shrimp fragments and krill were considered, AR and CB stained marine materials reddish-pink and light blue to black, respectively. By combining these results, it can be suggested that CB staining may efficiently be used to mark insect materials; AR does stain shrimp fragments but does not stain the tested insect material, indicating a possible approach for discriminating between insects and marine arthropods. However, since the present study was performed on pure materials and a small set of samples, possible implementation of this technique still needs to be confirmed in complex matrices such as compound feed.

Bone fragments are essential structures for the detection of processed animal proteins (PAPs) in feed by light microscopy for official controls according to Annex VI of European Union Regulation EC/152/2009. The preparation of samples submitted for analysis requires a grinding step to make them suitable for microscopic slide preparation and observation. However, there are no technical guidelines set down for this step despite the fact that it can lead to an increase in bone numbers due to fragmentation. This was demonstrated by an in-house study carried out by the Irish National Reference Laboratory (NRL) for animal protein detection. The present collaborative study investigated the possible effects of three different grinding conditions on the final result for a feed adulterated with 0.05 and 0.01% (w/w) of PAP. The microscopic analysis either combined or not with an Alizarin Red staining was carried out by 10 different laboratories. The results demonstrated that although a large variation in the numbers of bone fragments was noted, five of the six different grinding/staining combinations applied at two levels of PAP adulteration did not significantly (at p = 0.05) differ from one another. The only exception occurred when grinding the feed containing 0.05% of PAP with a rotor mill equipped with a 0.5-mm sieve and combined with a staining which resulted in a greater number of bone fragments by forced fragmentation. Overall, the impact of the grinding/staining combinations on the final results was shown to be negligible when considering the regulatory limit of detection (LOD) requirement for the method and the current rules of implementation of the light microscopic method. From a total of 180 analyses carried out on the feed matrix containing 0.05% of PAP no false-negative result was observed, and at a level of 0.01% PAP only 10 false-negative results occurred.

Near infrared microscopy (NIRM) has been developed as a rapid technique to predict the chemical composition of foods, reduce analytical costs and time and ease sample preparation. In this study, NIRM has been evaluated as an alternative to classical chemical analysis to determine the nitrogen and carbon content of small samples of tomato (Solanum lycopersicum L.) leaf powder. Near infrared spectra were obtained by NIRM for independent leaf samples collected on 216 plants grown under six different levels of nitrogen. From these, 30 calibration and 30 validation samples covering the spectral range of the whole set were selected and their nitrogen and carbon contents were determined by a reference method. The calibration model obtained for nitrogen content proved to be excellent, with a coefficient of determination in calibration (R(2)c) higher than 0.9 and a ratio of performance to deviation (RPDc) higher than 3. Statistical indicators of prediction using the validation set were also very high (R(2)p values > 0.90). However, the calibration model obtained for carbon content was much less satisfactory (R(2)c < 0.50). NIRM appears as a promising and suitable tool for a rapid, non-destructive and reliable determination of nitrogen content of tiny samples of tomato leaf powder.

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., (Copyright © 2010 Wiley-Liss, Inc.)

At present, European legislation prohibits totally the use of processed animal proteins in feed for all farmed animals (Commission Regulation (EC) No. 1234/2003-extended feed ban). A softening of the feed ban for non-ruminants would nevertheless be considered if alternative methods could be used to gain more information concerning the species origin of processed animal proteins than that which can be provided by classical optical microscopy. This would allow control provisions such as the ban of feeding animals with proteins from the same species or intra-species recycling (Regulation (EC) No. 1774/2002). Two promising alternative methods, near-infrared microscopy (NIRM) and real-time polymerase chain reaction (PCR), were combined to authenticate, at the species level, the presence of animal particles. The paper describes the improvements of the real-time PCR method made to the DNA extraction protocol, allowing five PCR analyses to be performed with the DNA extracted from a single particle.

Near-infrared microscopy (NIRM) has been proved to be a powerful tool for the detection of banned meat and bone meal (MBM) in feed. The identification of MBM traces and its ability to differentiate animal from vegetable feed ingredients is based on the evaluation of near-infrared spectra obtained from individual particles present in the sample. This evaluation is supported by appropriate decision rules for the absorbances at specific wavelengths. Here we show that the method and the corresponding decision rules can be successfully transferred from the laboratory which constructed the decision rules to two independent laboratories that were not involved in the calibration process of the method. The analytical results from blind feed samples containing MBM (positive samples) and feed samples without MBM (negative samples) revealed a very good agreement between the three laboratories, thus demonstrating the transferability of the method.

In this paper we present an alternative method for detection of meat and bone meal (MBM) in feedstuffs by near-infrared microscopic (NIRM) analysis of the particles in the sediment fraction (dense fraction (d >1.62) from dichloroethylene) of compound feeds. To apply this method the particles of the sediment fraction are spread on a sample holder and presented to the NIR microscope. By using the pointer of the microscope the infrared beam is focussed on each particle and the NIR spectrum (1112-2500 nm) is collected. This method can be used to detect the presence of MBM at concentrations as low as 0.05% mass fraction. When results from the NIRM method were compared with the classical microscopic method, a coefficient of determination (R2) of 0.87 was obtained. The results of this study demonstrated that this method could be proposed as a complementary tool for the detection of banned MBM in feedstuffs by reinforcement of the monitoring of feeds.

The near-infrared microscope (NIRM) is an instrument that has demonstrated its great ability to detect contaminants not by analyzing a single spectrum but rather by analyzing hundreds or thousands of spectra from individual particles, while at the same time being a non-destructive and easy-to-use technique. It has been used for the detection of meat-and-bone meal (MBM) in compound feeds within the context of the European bovine spongiform encephalopathy (BSE; commonly known as "mad cow disease") crisis. This study describes, for the first time, an application of NIRM instrument standardization using a measurement cell in an inter-laboratory study conducted within the framework of a qualitative determination of animal proteins in compound feeds, based on spectra obtained with eight instruments. The standardization cell was assessed for its ability to produce good optical matching of the instruments and/or to evaluate instrument performance.

The European Union Reference Laboratory for the detection of animal proteins in feedingstuffs (EURL-AP) has developed an online micrographs collection supporting its network activities within the European Union for the detection of prohibited animal by-products in feed. So far, the only official method for detecting these by-products is light microscopy, which is highly dependent on the skills of a microscopist because it relies on particle recognition. In order to help the microscopist network to achieve high proficiency levels, it was necessary to create an online reference tool based on micrographs and accessible via an Intranet platform. Members of the National Reference Laboratories for animal proteins in feedingstuffs (NRL-AP) and the International Association for Feedingstuff Analysis – Section Feedingstuff Microscopy (IAG) have access to this micrographs collection. This paper describes how the online collection was created and what conditions had to be taken into account in creating such a tool. It also describes how information are periodically updated and managed within the context of the large amount of information included in each micrograph. The need for a robust back-office system as the foundation for all the research activities in this project is also covered, and the evaluation of the use of the online collection is discussed.

Abstract: Recent developments in spectroscopy have led to the use of spectroscopic imaging instruments for the control and monitoring of food and feed products. This kind of instruments offers the possibility of collecting thousands of spectra of particles being the result of the grinding of compound feedstuffs. The major advantages are that the recognition of feed ingredients is independent on the expertise of the analyst and that it is possible to automate all procedures and to analyse more samples per unit of time than classical microscopy or NIR microscopy. The objective of this study is the development of a new method for a rapid, precise and reliable screening of compound feeds. For that, a classification tree was built by sorting the particles in a dichotomist way where each node constitutes a discriminating step. These steps are completed by discriminant equations created from the hyperspectral databases obtained with a near infrared (NIR) camera for each class of raw materials. Discriminant equations were constructed using Support Vector Machines (SVM). For a new sample the aim is to determine its composition by using the classification tree. As general conclusion, hyperspectral data in combination with SVM as classification technique is a promised methodology for the determination of open formulations. [Copyright &y& Elsevier]

Species-specific detection and detection of groups of species such as ruminants is required according to European legislation dealing with the safe use of animal by-products in animal nutrition. Various methods are applied to the analysis of feed samples for the presence of banned processed animal proteins (PAPs) including meat and bone meal (MBM). Classical microscopy as described in the Commission Directive EC/2003/126 is the only official method to detect the presence of constituents of animal origin in feed, nevertheless some deviating protocols allowed under the old Directive (EC/88/1988) claim to gain comparable results. An inherent limitation of the microscopic method is the lack of species specificity. Immunoassays showed the most promising potential in research projects or intercomparison studies being able to detect ruminant PAPs at a concentration level of 0.5%. The aim of this paper is to present the results of the intercomparison study conducted on behalf of European Commission's Directorate General for Health and Consumer Protection (SANCO) in 2004 to establish whether the two-solvent method would gain comparable results to the current European Method and to evaluate the current capability of immunoassays of determining the species in PAPs present in feed. [ABSTRACT FROM AUTHOR]

Abstract: It is generally accepted that the most likely route of infection of cattle with bovine spongiform encephalopathy (BSE) is by consumption of feeds containing low levels of processed animal proteins (PAPs). This likely route of infection resulted in feed bans, which were primarily aimed at ruminant feeds, and were later extended to all feeds for farmed animals. The feed bans were expected to develop into a future enforcement of the “species-to-species” ban, which prohibits only the feeding of animal-specific proteins to the same species. The species-to-species ban requires support of species-specific identification methods. In the European Union, microscopic evaluation is currently the only accepted method for the detection and characterization of PAPs in feeds, since it is possible to detect contaminations at the requested level of 1g/kg with hardly any false negative nor positive results. This method is predominantly focused on the presence and characteristics of bone fragments, although other structures, e.g. muscle fibres, may provide circumstantial evidence of the respective animal types. Recent developments are the identification of bone fragments at the level of classes (mammal versus bird versus fish), supported by image analysis of bone characteristics. Detection of DNA and specific proteins are additional methods that can be applied for the identification of PAPs in feeds. DNA is known to be very specific for animal species and breeds, whereas proteins can also indicate the type of tissue. The latter aspect is important to differentiate between proteins that are authorised in animal nutrition from banned proteins. Improvements can be noted in recent years for both methods. For a proper application of polymerase chain reaction (PCR) to detect specific sequences of DNA, primer sets have been developed which amplify a DNA sequence shorter than approximately 100 nucleotides. Specific antibodies have been developed for protein detection of ruminant or bovine material. Recent results of various studies indicate that specific DNA and protein detection methods can detect PAPs at a contamination level of 1g/kg. However, full validation of these methods still needs to be carried out. Other methods such as near-infrared spectroscopy (NIRS), near-infrared microscopy (NIRM), near-infrared imaging, liquid chromatography (LC) and olfactometry techniques can and will be applied for the detection of PAPs. NIRS is a non-destructive method that can be applied on-line in feed production plants. Generally, the detection limit is still too high to be applied in official control laboratories. Nevertheless, industrial application is feasible. NIRM and near-infrared imaging are techniques that allow collection of near-infrared spectra from individual particles. The level of detection is lower than 1g/kg since it is based on the microscopic technique, in combination with the option of identification of the individual particles. LC is based on the detection and, if present, the ratio of different polypeptides. For example, carnosine is mainly present in mammals and anserine mainly found in birds. Olfactometry is based on detection of volatile non-specific agents. It is a non-destructive and fast technique. For both LC and olfactometry it appears that the presence of fish material masks the detection of proteins of land animals, even at a contamination level of 5g/kg. Since 2003 five different proficiency studies and ring trials have been organized. The first proficiency study, allowing the participants to apply their own protocol, revealed that correct microscopic detection of 1g/kg of mammalian PAP in the presence of 50g fish meal/kg was realised in 0.44 of the cases. However, a bench mark study organized in the same year showed that a microscopic detection of 0.98 can be reached provided the application of an optimal protocol and a sufficient level of expertise. More recent studies showed that training, the application of a decision support system and use of an improved microscopy protocol resulted in a higher sensitivity. An attractive approach is the combination of the very low detection level of microscopy with identification by other methods. Several strategies for a combination of screening and confirmation methods are discussed in the present paper. The new developments in methodology will support current or new legislation (e.g. species-to-species ban, general application of fish meal). [Copyright &y& Elsevier]

The ban of processed animal proteins (PAPs) in feed for farmed animals led to a significant reduction of the number of bovine spongiform encephalopathy cases. Presently, optical microscopy remains the only reference method for the detection of PAPs to be applied for official control as required by Commission Directive 2003/126/EC. The legislation also foresees that other methods may be applied in addition to classical microscopy, if – for instance – they provide more information about the origin of the animal constituents. Therefore, alternative and complementary techniques were developed as such or in combination. The most promising ones seem to be PCR (Polymerase Chain Reaction), near infrared microscopy and imaging, as well as immunology. Within the framework of a PAP ban regardless of its species origin (total feed ban), most of the studies were mainly focused on the ability of the techniques to detect the presence of PAPs at 0.1% (mass percentage of constituents of animal origin in feed) as indicated as limit of detection in the official method protocol. A possible modification of the legislation requires that the techniques are also able to determine their species origin and to quantify them. The present paper gives a state of the art of the different methods.