Food Additives & Contaminants: Part A, 2015 Vol. 32, No. 7, 1089–1098, http://dx.doi.org/10.1080/19440049.2015.1036321
Species identification of processed animal proteins (PAPs) in animal feed containing feed materials from animal origin Sonja Axmann*, Andreas Adler, Agnes Josephine Brandstettner, Gabriela Spadinger, Roland Weiss and Irmengard Strnad Austrian Agency for Health and Food Safety (AGES), Institute for Animal Nutrition and Feed, Linz, Austria (Received 4 February 2015; accepted 23 March 2015) Since June 2013 the total feed ban of processed animal proteins (PAPs) was partially lifted. Now it is possible to mix fish feed with PAPs from non-ruminants (pig and poultry). To guarantee that fish feed, which contains non-ruminant PAPs, is free of ruminant PAPs, it has to be analysed with a ruminant PCR assay to comply with the total ban of feeding PAPs from ruminants. However, PCR analysis cannot distinguish between ruminant DNA, which originates from proteins such as muscle and bones, and ruminant DNA, which comes from feed materials of animal origin such as milk products or fat. Thus, there is the risk of obtaining positive ruminant PCR signals based on these materials. The paper describes the development of the combination of two analysis methods, micro-dissection and PCR, to eliminate the problem of ‘falsepositive’ PCR signals. With micro-dissection, single particles can be isolated and subsequently analysed with PCR. Keywords: microdissection; PAP; animal proteins; feed-ban; real-time PCR
Introduction Bovine spongiform encephalopathy (BSE), commonly known as mad cow disease, is a fatal, neurodegenerative disease in cattle that causes a spongy degeneration in the brain and spinal cord. BSE was first identified in the UK in 1985 and 1986. Epidemiological studies have found that BSE is a feed-borne infection transmitted to animals through BSE-infected meat and bone meal (MBM) in animal feed (Wilesmith et al. 1988). The outbreak of this disease and its relation to the consumption of contaminated animal feed led to the ban of feedingstuffs containing any processed animal proteins (PAPs) including MBM. The only exception was fish meal, which may be fed to fish and non-ruminants (Regulation (EC) 999/2001). Additionally Regulation (EC) No. 1069/2009 (repealing Regulation (EC) 1774/2002) specifies the prohibition of feeding farmed animals with the same species. Feed samples are checked by classical optical light microscopy for the presence of PAPs. With this method it is possible to distinguish fish bones from terrestrial animal bones. The TSE (Transmissible Spongiform Encephalopathy) Roadmap 2 (European Commission 2010) considered that the transmission risk of BSE from non-ruminants to nonruminants is very unlikely. Therefore, the recent easing of the total feed ban has been completed in June 2013 (Commission Regulation (EC) 56/2013). Because of that, PAPs from non-ruminants (pig and poultry) can be added to fish feed, whereas ruminant PAPs remain totally prohibited. Thus, detection of terrestrial PAPs with optical light microscopy provides insufficient information. In fact,
the method can discriminate between fish and terrestrial PAPs, but it cannot distinguish terrestrial PAPs of different species. To guarantee, that fish feed, which contains nonruminant PAPs, is free of ruminant PAPs, it has to be analysed with a ruminant PCR assay either if terrestrial particles are detected with classical microscopy or if PAPs are mentioned in the composition (Commission Regulation (EC) 51/2013). But using PCR analysis, we are faced with the problem that this method cannot distinguish ruminant DNA, which originates from proteins such as muscles and bones, from ruminant DNA, which comes from feed materials of animal origin such as milk products, fat, etc. (Catalogue of feed materials: Commission Regulation (EU) No. 68/2013). Thus, there is the risk of obtaining positive ruminant PCR signals based on these materials. The European Animal Protein Association (EAPA), European Fats Processors and Renderers Association (EFPRA) and European Feed Manufacturers’ Federation (FEFAC) drew attention to this issue at the meeting of the Standing Committee on the Food Chain and Animal Health (SCoFCAH) in December 2013 (2013 letter from EAPA, EFPRA and FEFAC to DG SANCO as a request for discussion at SCoFCAH held in Brussels on 16–17 December 2013; unreferenced). They considered that positive PCR signals are not evidence for the presence of illegal ruminant DNA, as legal products may be the source for positive testing results. Moreover, the European Commission outlined in the TSE Roadmap 2 (2010) that a lifting of the ban on the use of PAPs from non-ruminants in non-ruminant feed could
*Corresponding author. Email: [email protected] © 2015 Australian Agency for Health and Food Safety Ltd. Published by Taylor & Francis.
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be planned, but without lifting the existing prohibition of intra-species recycling (e.g. poultry PAP could only be fed to pigs and pig PAP to poultry). That would mean that, for example, pig feed containing poultry PAP has to be checked with pig PCR assay in order to comply with the intra-species ban and with ruminant PCR assay to meet the total ban on feeding ruminant PAPs. For this case, blood products from non-ruminants may be a problem as well in addition to the aforementioned feed materials. For example, a feeding stuff for pigs contains poultry PAPs, milk powder and blood meal simultaneously. If poultry PAP is mentioned in the composition or terrestrial particles were detected by microscopy, the feed has to be analysed with pig and ruminant PCR assays. Hence, because of the presence of blood and milk, the analysis will lead to positive results with both PCR assays. Therefore, it is of great interest to develop an analytical method for the investigation of compound feed by which the species of PAPs can be determined without getting positive results due to legal feed materials of animal origin. The combination of microdissection and PCR has been found. The microdissection device consists of a light microscope connected to a laser which can isolate small fragments of bones or muscles by catapulting them into the lid of a reaction tube. The origin of the particles up to their species level can be determined with subsequent PCR analysis. A similar analytical technique, the combination of near-infrared microscopy (NIRM) and PCR, has already been demonstrated (Fumière et al. 2010). But the use of NIRM for routine analysis is difficult. The limitation of this method is the number of particles that are manually analysed and isolated. This paper examines the capability of the microdissection technique and the combination of microdissection with PCR. Light microscopy and ruminant PCR analysis are already applied as validated investigation methods also for official control of feeding stuffs in the European Union (Commission Regulation (EC) 51/2013).
Materials and methods Reference materials Pig, sheep, cattle and poultry PAPs were provided by the EURL-AP (European Reference Laboratory for Animal Proteins in Feedingstuffs, Gembloux, Belgium).
Commercial feeds The cattle feed ‘Kuhkorn Kompakt 183’ (composed of wheat bran, corn concentrated feed, corn, grain mash, corn germ extraction meal, dry cuts, rapeseed extraction meal, molasses, wheat, calcium carbonate, cacao shells, sodium chloride and magnesium oxide) was obtained from Garant Tiernahrung GmbH (Pöchlarn, Austria). It was
ground with a Retsch grinding mill ZM200 (Retsch GmbH, Haan, Germany) to obtain particles with a diameter less than 0.5 mm. The calf milk ‘Kälbermilch 19/0’ (composed of whey powder, vegetable oil refined palm/ coconut 4:1, wheat protein hydrolysate, wheat swelling flour and dextrose) was obtained from Inntaler Tiernahrung GmbH (Ingolstadt, Germany).
Processed animal proteins (PAPs) Poultry PAP was obtained from the Styrian animal cadaver utilisation (STKV, Landscha, Austria) and is produced under Commission Regulation (EC) 1069/2009 (133°C and 3 bars for 20 min). Pig PAP was obtained from Garant Tiernahrung GmbH and imported from Spain (ElPozo, Murcia, Spain). PAPs were ground with a Retsch grinding mill ZM200 to obtain particles with a diameter less than 0.5 mm.
Compound feeds Commercial cattle feed, 5% in mass fraction of poultry (compound feed A) or pig PAP (compound feed B) and 5% in mass fraction of calf milk were mixed for 10 min with a Turbula mixer T 2 C (Willy A Bachofen AG, Basel, Switzerland). All mixes were also produced without calf milk.
Aqua feed plus 0.1% cattle PAP Aqua feed (composed of soya meal, wheat, fish meal, sunflower seed, maize gluten, permitted flavour, fish oil, rapeseed oil, vitamins, mono ammonium phosphate, permitted anti-fungal/anti-oxidant, yeasts, minerals, algae, betaine and astaxanthin) containing 0.1% cattle PAP in mass fraction was sent to us in course of an international proficiency test (EURL-AP 2013c). The EURL-AP selected the feed among the EURL-AP sample bank and it was tested to be free of any ruminant traces by ruminant PCR and microscopy.
Extraction and preparation of the sediment The sediment was prepared according to Regulation (EC) 51/2013. Material (10 g) was transferred into a separation funnel, mixed with 50 ml tetrachlorethylene (VWR, Radnor, PA, USA) and shaken vigorously for 30 s. Further, 50 ml tetrachlorethylene were added cautiously to wash down the inside surface of the funnel to remove any adhering particles. The resulting mixture was incubated for at least 5 min before the sediment was separated off by opening the stopcock. The sediment was then dried. If more than 5% of the sediment consisted of particles > 0.5 mm, it was sieved at 0.25 mm.
Food Additives & Contaminants: Part A Extraction and preparation of the flotate The flotate is prepared according to Regulation (EC) 51/ 2013. After recovery of the sediment two phases remain in the separation funnel: the liquid phase (tetrachlorethylene) and the solid phase made of the floating material (flotate). Tetrachloroethylene was poured off completely by opening the stopcock. The remaining flotate was transferred into a Petri dish and air dried in a laboratory fume hood. If more than 5% of the flotate consisted of particles > 0.5 mm, it was sieved at 0.25 mm.
Slide preparation A test portion of the sediment or the flotate was spread on the slide, sprayed with liquid cover glass (LCG) (Carl Zeiss AG, Oberkochen, Germany) and distributed with a fine needle. The slide was then dried for 5–10 min at 60° C. Afterwards two drops of immersion oil were applied and the slide was rotated in a way that the oil covered all particles of the sample. Excess of oil was wiped away and the slide was again dried for 5–10 min at 60°C.
Particle isolation with a PALM microbeam– microdissection system The microdissection system (Carl Zeiss) consists of several components: the inverse microscope (Axio Observer. A1), the laser unit (MicroBeam Module Rel. 4.2), the cap holder (CapMover II), the digital microscopy camera (AxioCam Icc1 Rev. 3) and the computer running the software RoboSoftware 4.2 to use the microdissection device. Before starting particle isolation, 30 µl water of molecular biology grade (5 Prime GmbH, Hilden, Germany) were pipetted into the lids of the 0.5 ml reaction tubes. The slide was then clamped in the inverse microscope and was first coarsely scanned. Suspicious particles were marked and checked whether any contaminants adhere to them. Afterwards, the reaction tube was fixed into the cap holder and particles were catapulted with the laser into the lid of the tube. If necessary, the particles can be cut with the laser before catapulting.
DNA extraction DNA extraction of particles To pelletise the particles, the 0.5 ml reaction tubes were centrifuged at 13 000g for 15 min and the water was removed immediately after. For DNA extraction proteinase K solution (Ambion®, Life Technologies, Carlsbad, CA, USA) was added at a concentration of 0.4 µg µl–1 to DirectPCR® lysis reagent for genotyping using crude lysates (Viagen Biotech, Inc., Los Angeles, CA, USA). A total of 7 µl of the prepared extraction buffer were added to the particles. The mixture was then incubated
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overnight at 56°C and subsequently proteinase K was stopped for 50 min at 85°C. Afterwards the mixture was briefly centrifuged and diluted with 20 µl PCR grade water (Roche, Risch, Switzerland). A total of 5 µl of the DNA extract was used for real-time PCR. DNA extraction of compound feed DNA from compound feed was extracted using the Wizard® Magnetic DNA Purification System for Food kit (Promega, Fitchburg, WI, USA). The capability of this kit for compound feed has already been evaluated (Fumière et al. 2006). DNA of feed was prepared according to the standard operation procedure DNA extraction using the Wizard Magnetic DNA Purification System for Food kit, which is published on the webpage of the EURL-AP (EURL-AP 2013a). Real-time PCR All PCR assays were performed with the LightCycler® 480 device (Roche). Ruminant real-time PCR assay Ruminant PCR was performed according to the standard operation procedure ‘Detection of ruminant DNA in feed using real-time PCR’ published on the webpage of the EURL-AP (EURL-AP 2013b). AGES laboratories participated as Austrian National reference laboratory (NRL) to the validation and implementation studies organised by the EURL-AP (Fumière et al. 2012a, 2012b). The cut-off Ct value of this assay was determined at 37.07 cycles in our laboratory. Pig real-time PCR assay For each pig target amplification 17.5 µl LightCycler® 480 Probes Master containing FastStart Taq DNA polymerase, nucleotides and MgCl2 (Roche), 8.5 pmole of forward and reverse primer (Microsynth, Balgach, Switzerland), 8.5 pmole Taqman probe labelled with reporter dye FAM and quencher dye TAMRA (Microsynth), 5 µl template DNA and PCR grade water (Roche) were mixed together to obtain a reaction volume of 35 µl. The thermal programme was defined with an initial denaturation step at 95°C for 10 min followed by 50 cycles consisting of 15 s denaturation at 95°C and 1 min annealing and elongation at 50°C. The pig PCR protocol detects domestic pig target and the sequence information of primer and probe was provided by the EURL-AP. The cut-off Ct value for this assay was determined in our laboratory for our PCR platform at 39.16 cycles. For correct calculation, pig calibrants (Joint Research Centre – Institute for Reference Materials and Measurement, Brussels, Belgium) and a
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calculation sheet for determining the exact copy number and cut-off were used (EURL-AP, Gembloux, Belgium). Poultry real-time PCR assay For each poultry target amplification 12.5 µl LightCycler® 480 Probes Master containing FastStart Taq DNA polymerase, nucleotides and MgCl2 (Roche), 7.65 pmole of forward and reverse primer (Microsynth), 9 pmole Taqman probe labelled with reporter dye FAM and quencher dye TAMRA (Microsynth), 5 µl template DNA and PCR grade water (Roche) were mixed together to obtain a reaction volume of 25 µl. The thermal programme was defined with an initial denaturation step at 95°C for 10 min followed by 50 cycles consisting of 15 s denaturation at 95°C and 1 min annealing and elongation at 50°C. The poultry PCR protocol detects domestic chicken and turkey targets. Sequence information of primer and probe was provided by the EURL-AP. The cut-off value will be determined in the course of the poultry validation study organised by the EURL-AP. Results and discussion In all trials we were confronted with the fact that each sample is unique. Every single tube generated by microdissection contains particles which were different in size and general condition. Thus, there was just one possibility to extract DNA from one tube sample. In addition, we worked with microscopically small particles, and there was always an increased risk of losing these during the extraction process. The first preliminary experiments were performed to show that muscle and bone particles derived from PAP of known origin can be isolated successfully with the microdissection and identified correctly by realtime PCR after DNA extraction. Species-specific identification of separated particles from reference material with real-time PCR The extraction of DNA from particles has already been evaluated. DirectPCR® (Tail) lysis reagent has been demonstrated to be the most effective DNA extraction method for particles isolated by near-infrared microscopy (NIRM) (Fumière et al. 2010). A part of our work was the adaptation of this method to make it suitable for microdissected particles. Due to the high temperatures during the manufacturing process of PAP we were faced with a high degree of DNA degradation. In addition the sediment preparation with tetrachlorethylene and subsequent fixing to the slide could influence the effectiveness of DNA extraction. In the initial experiment slides for microdissection were prepared from sediments of pig, ruminant and poultry PAP reference material. In order to obtain a good
microscopic image, the slides were covered with liquid cover glass. Ten samples from each preparation were shot with microdissection and every tube contained two bone or two muscle fibre fragments. Blank samples containing water, liquid cover glass and immersion oil were also prepared. DNA extraction was carried out with DirectPCR® (Tail) lysis reagent supplemented with proteinase K at a concentration of 0.4 µg µl–1 for an incubation period of 15 min at 56°C. Thereafter, DNA was screened with ruminant, poultry and pig real-time PCR in order to verify species specificity of the assays. For poultry PCR assay Ct values < 35 cycles were considered as positive and Ct values ≥ 35 cycles were considered as negative results. In the case of ruminant and pig real-time PCR assays, we had well-defined cutoff Ct values for our PCR platform. Ct values ≤ 37.07 cycles received with the ruminant PCR assay corresponded to positive results and Ct values > 37.07 cycles indicated negative results. With the pig PCR assay we obtained a cut-off Ct value of 39.16 cycles. Consequently, Ct values ≤ 39.16 cycles were assessed positively and Ct values > 39.16 cycles were assessed negatively. According to the results shown in Table 1, bone samples generally yielded better results than muscle fibre samples. Tubes containing DNA from pig or poultry bones produced satisfying animal-specific positive PCR signals, whereas we sometimes received negative results from DNA of muscle fibres. Just four pig and three poultry muscle fibre samples were positive according to their corresponding species. The results, which were given from ruminant fragments, were insufficient. Two samples containing DNA from ruminant bones were positive and three yielded negative PCR signals close to the cut-off of 37.07 cycles. Samples containing DNA from ruminant muscle fibres failed completely. Nevertheless, we hoped to improve the results by changes in DNA extraction conditions. Therefore, we extended the incubation time with proteinase K from 15 min to overnight. We analysed 30 cattle samples, each containing two particles. Ten cattle bone samples were incubated for 15 min and other 10 bone samples were incubated overnight with proteinase K at 56°C. The remaining 10 tubes containing muscle fibres were incubated overnight as well. The results of the 20 samples containing cattle bones are shown in Table 2. When trying with a 15 min digestion, we obtained three weak positive results (Ct values close to the cut-off 37.07 cycles), while after overnight digestion six out of 10 bone samples gave clearly positive PCR signals (Table 2, indicated in bold). Results yielded from cattle muscle fibres were persistent negative (data not shown). Consequently, in the case of bone samples the extension of the incubation period revealed twice as many positive results with more distinct Ct values. Unfortunately we
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Table 1. Real-time PCR results from DNA of bones and muscle fibres after 15-min incubation with proteinase K. Pig
Poultry
Ruminant
Content
Ct value
Result
Content
Ct value
Result
Content
Ct value
Result
2 bo, pig
33.87 34.27 34.34 32.55 33.65
+ + + + +
2 bo, poultry
31.76 30.83 24.95 31.34 31.08
+ + + + +
2 bo, cattle
35.03 36.32 38.10 37.16 37.20
+ + – – –
2 mf, pig
37.84 40.01 38.37 35.95 34.01
+ – + + +
2 mf, poultry
26.88 42.17 41.66 25.99 24.78
+ – – + +
2 mf, cattle
40.09 40.12 40.04 40.09 39.62
– – – – –
Note: bo, Bone; mf, muscle fibre; +, positive; –, negative.
Table 2. Real-time PCR results from DNA of cattle bone particles after 15-min and overnight incubation with proteinase K. Ruminant – 15 min Content
2 Bones Cattle
Ruminant – overnight
Ct value
Result
38.28 37.94 39.15 39.08 36.64 37.50 37.11 38.09 36.78 36.99
– – – – + – – – + +
Content
2 Bones Cattle
Ct value
Result
37.21 28.32 41.19 26.16 38.84 34.76 41.20 29.76 30.90 32.79
– + – + – + – + + +
Note: +, Positive; –, negative.
were unable to achieve improvements concerning the muscle fibres. In the following series of studies, we aimed to determine if we could detect different species in the same tube. Therefore, particles from animals of varying origin were combined in the vessels. Investigation tubes were prepared containing either two bones or two muscle fibres of every species. DNA extraction was carried out with the overnight proteinase K digestion step. Thereafter the samples were screened with ruminant, pig and poultry PCR assays. The resulting Ct values of the individual samples are shown in Table 3 and the summary is presented in Table 4. The results of samples containing bone particles were very promising. The average Ct value of samples containing poultry bones was 32.25 cycles. Thus, in case of 15 samples we received 14 positive results and just one sample was interpreted as negative. Pig bone samples yielded an average Ct value of 34.01 cycles. Thirteen samples out of 15 were clearly positive and two were
negative. The average Ct value of ruminant bone samples was 33.78 cycles. Eleven samples were considered positive and the rest was negative. The results of DNA extracted from poultry and pig muscle fibres were also very promising. The average poultry Ct value was 33.58 cycles, whereas 10 samples were clearly positive and five were negative. DNA from pig muscle fibres provided an average Ct value at 35.11 cycles. Thirteen samples were positive showing a Ct value ≤ 39.16 cycles and two samples were negative. The results from ruminant muscle fibres did not come up to our expectations. All 15 samples had a Ct value above 37.07 cycles and thus were negative. Based on our results, we observed that DNA extraction from different tissues worked differently well. Species determination was successfully performed with DNA extracts from bones, whereas muscle fibres did not yield similar outcomes. This has already been proven by Lassen et al. (1994). They compared DNA extracts from ancient bones and mummified soft tissues by PCR analysis and came to the result that skeletal material should be given the preference for DNA investigations. Bones exhibited the least DNA degradation degree, followed by liver then spleen, kidney, thyroid, blood, brain, muscle and lymph nodes. A plausible explanation for this observation could be that the compact, more durable bone surface provides some protection against bacterial and enzymatic degradation. In addition, DNA is bound to an inorganic component (calcium hydroxyapatite) in the bone, which in turn contributes to its stabilisation (Lindahl 1993). These observations might be suitable for our results. We do not work with mummified tissues or ancient bones, but with animal material that has been exposed to very high temperatures, which also amounts to massive degradation of DNA. Since particles were mainly isolated from sediment material, the emphasis will lie in the DNA extraction from bone fragments, because predominantly bones were
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Table 3. Real-time PCR results from DNA of particles of varying animal origin combined in one investigation tube after overnight incubation with proteinase K. Pig Content
Poultry
Summary of the results from Table 3. Positive Negative results Average Ct value (without n.s. results (< 35 (≥ 35 cycles values) cycles) and n.s.)
Ruminant
Ct Ct Ct value Result value Result value Result n.s. 30.98 30.00 35.53 35.83
– + + + +
n.s. n.s. n.s. n.s. n.s.
– – – – –
31.20 29.24 27.76 34.50 38.90
+ + + + –
2 mf, pig and cattle 37.53 41.66 37.37 33.32 34.73
+ – + + +
n.s. n.s. n.s. n.s. n.s.
– – – – –
39.98 39.09 43.11 41.24 45
– – – – –
2 bo, poultry and lamb
n.s. n.s. n.s. n.s. n.s.
– – – – –
31.49 33.29 34.38 29.59 28.66
+ + + + +
34.72 36.69 n.s. 28.56 31.15
+ + – + +
2 mf, poultry and cattle
n.s. n.s. n.s. n.s. n.s.
– – – – –
35.33 31.46 28.35 40.48 34.81
– + + – +
43.85 41.63 45 40.05 40.90
– – – – –
2 bo, pig and poultry
28.43 40.60 33.21 33.77 32.94
+ – + + +
31.27 34.27 30.33 32.30 35.40
+ + + + –
37.72 n.s. 40.95 44.2 n.s.
– – – – –
2 mf, pig and poultry
36.80 35.31 29.36 34.88 35.03
+ + + + +
34.69 34.30 26.98 31.19 33.88
+ + + + +
n.s. 45 41.47 n.s. n.s.
– – – – –
2 bo, pig, poultry and lamb
33.86 38.08 35.17 36.07 32.55
+ + + + +
32.79 32.36 32.79 31.25 33.66
+ + + + +
35.90 44.36 34.40 37.37 30.51
+ – + – +
2 mf, pig, poultry and cattle
33.86 35.18 33.20 34.26 n.s.
+ + + + –
29.18 n.s. 39.77 27.74 41.08
+ – – + –
40.67 45 38.96 40.16 n.s.
– – – – –
2 bo, pig and lamb
Table 4.
Note: bo, bone; mf, muscle fibre; n.s., no signal; +, positive; –, negative.
sedimented whereas most muscle fibres remained in the flotate. Our data indicated that we sometimes have to expect random failures at the level of particle analysis. In some instances we yielded negative results with the animalspecific PCR assay. The reason for that remains unclear until now. We could speculate that we definitely got
Poultry bo Poultry mf
32.25 33.58
14 10
1 5
Positive Negative results results (> cut-off and (≤ cut-off) n.s.) Pig bo Pig mf Ruminant bo Ruminant mf
34.01 35.11 33.78 41.72
13 13 11 0
2 2 4 15
Note: n.s., No signal; bo, bone; mf, muscle fibre.
failures because we were working with microscopically small particles and thus DNA or the fragment itself were lost during the extraction process. But at least we always got three positive results out of five bone particle samples. Similarly, we could show that we could achieve equivalent results, even if particles of different species origin were combined into one sample. Every species was successfully detected in vessels containing combinations of bones derived from two or three different animals.
Isolation and identification of bones from aqua feed In the next experiment we wanted to illustrate that our method is just as suitable for aqua feed. Therefore, we examined a feed sample, which was sent by the EURL-AP in course of an international proficiency test (EURL-AP 2013c). It was composed of aqua feed and 0.1% cattle PAP in mass fraction. Aqua feed basically consists of a high proportion of fish meal. Therefore, the content of muscle fibres derived from fish meal was very high and hence it was statistically unlikely that muscle fibres coming from terrestrial animals were isolated by microdissection when these PAPs were added in low amounts to the aqua feed. Thus, we restricted to the isolation of terrestrial bones. A total of 10 g of the aqua feed were used for sedimentation and then microscopy slides were prepared. Ten tubes each containing two terrestrial bones were produced by microdissection. DNA was extracted with DirectPCR® (Tail) lysis reagent and subsequently analysed with ruminant real-time PCR. As expected, the results were very satisfying. The species origin of the bones was confirmed with ruminant PCR in six out of 10 tubes. The corresponding Ct values were below the ruminant cut-off Ct value of 37.07 cycles and therefore the feed sample was assessed positive for ruminant DNA (Ct values not shown).
Food Additives & Contaminants: Part A Identification of separated particles from feeding stuffs containing feed materials of animal origin (milk powder) The main focus of our work laid in the development of an analytical method by which one can detect PAPs from ruminants and/or non-ruminants in feeding stuffs without being negatively influenced by legal feed materials of animal origin (e.g. milk powder, fat, blood products, etc.). These materials might be the reason for positive animal-specific PCR and thus feeding stuffs would be rejected unjustifiably. For this reason we investigated self-made compound feed A (CFA) containing poultry PAP and compound feed B (CFB) containing pig PAP. All mixtures were prepared once with and once without calf milk. In order to create conditions that were as realistic as possible, we used commercially available PAPs instead of reference material. Moreover, cattle feed and calf milk used for this purpose are generally available products. Previously, these materials were analysed with the animal PCR assays to ensure that they were not contaminated with foreign DNA (Table 5). DNA extraction was carried out with the Wizard Magnetic DNA Purification System for Food using 100 mg portions and following PCR was performed. The expected results were confirmed. Contamination with foreign DNA was detected in none of the starting materials. Ruminant DNA was established exclusively in calf milk, pig DNA in porcine PAP and poultry DNA in poultry PAP. Adjacent, slides from sediments of CFA and CFB were prepared to isolate predominantly bones with the microdissection device. Thirty samples were produced from each feed, 15 samples from the mixtures with calf milk and 15 samples from the mixtures without calf milk. Moreover, control samples were made from CFA and CFB plus milk containing feed particles. Since most of these particles remain in the supernatant during sedimentation, microscopy slides were prepared from the flotate and the feed fragments isolated there from. Every blank sample was supplemented with one corn, two wheat and
Table 5. Real-time PCR results from DNA of commercial feeds and PAPs. Pig
Poultry
Ruminant
Ct Ct Ct value Result value Result value Result Commercial feed Cattle feed Calf milk PAPs Pig PAP Poultry PAP
n.s. n.s.
– –
n.s. n.s.
– –
38.17 23.83
– +
28.41 n.s.
+ –
41.02 21.89
– +
40.96 38.85
– –
Note: n.s., No signal; +, positive; –, negative.
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two rapeseed particles. Further controls were included that contained only water and LCG to ensure contamination-free reagents. DNA extraction of the particles and controls was performed with the DirectPCR® (Tail) lysis reagent from all samples and DNA was subsequently screened with pig or poultry and ruminant real-time PCR assays (Table 6). The results were very auspicious. In the case of CFA containing poultry PAPs, we could generate Ct values < 35 cycles from 19 out of 30 samples. Eleven samples gave Ct values ≥ 35 cycles or no signal. A contamination of the isolated particles with milk could be excluded. In none of the samples was ruminant DNA detected, even when calf milk was added to the feed. All ruminant Ct values lay either above the cut-off 37.07 cycles or the samples gave no fluorescence signal. Comparable outcomes were achieved in the feed containing pig PAPs (CFB). Twenty-six out of 30 samples gave positive pig results. Four samples gave Ct values > 39.16 cycles and thus were negative. All PCR results from the ruminant assay yielded Ct values above the cut-off 37.07 cycles or no fluorescence signal. Hence, a contamination with milk could be excluded, too. Finally, the controls were examined with ruminant, pig or poultry PCR assay (Table 6). All ruminant PCR results were negative. The fluorescence signals rose above the ruminant cut-off value or no signals were observed. Equally, pig or poultry PCR results gave either no fluorescence signal or Ct values above the cut-off. With this key experiment we are able to present a working method for detecting PAPs in compound feed without getting positive results based on feed materials of animal origin. Because of this methodical possibility we can feed samples on particle level. Conclusions Since June 2013 (Commission Regulation (EC) No. 56/ 2013) it has been allowed to add PAP from non-ruminants to fish feed. To ensure that the PAP originates exclusively from non-ruminants, the feed has to be tested with ruminant PCR according to the current legislation situation (Commission Regulation (EC) No. 51/ 2013). But the new official control method is reason for criticism because the number of ‘low level’ positive ruminant results in aqua feed rose (2013 letter from EAPA, EFPRA and FEFAC to DG SANCO as a request for discussion at SCoFCAH held in Brussels on 16–17 December 2013; unreferenced). Until now, it cannot be excluded that legal feed materials of animal origin are the reason for these results. Moreover, a lifting of the ban on the use of PAP from non-ruminants in nonruminant feed without lifting the existing prohibition of intra-species recycling is considered (European Commission 2010). Accordingly, pig meal could be
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Table 6. Real-time PCR results from DNA of bones isolated from sediments of compound feed A (containing poultry PAP) and compound feed B (containing pig PAP) with or without calf milk. Poultry CF Sediment of CFA: 3 bones
Sediment of CFA plus calf milk: 3 bones
Flotation of CFA plus calf milk: 1 corn, 2 wheat, 2 rapeseed LCG + H2O
Ruminant
Ct value Result Ct value Result 30.27 34.87 35.38 30.69 33.95 27.68 30.11 31.21 35.00 33.55 28.33 38.63 33.92 38.27 33.94 30.41 35.14 38.94 n.s. 34.16 31.88 35.16 32.28 34.78 38.43 35.87 40.75 29.17 34.81 34.82 40.19 n.s. n.s. n.s. n.s. n.s. n.s.
+ + – + + + + + – + + – + – + + – – – + + – + + – – – + + + – – – – – – –
n.s. 44.04 42.31 40.87 40.56 39.17 41.43 40.79 40.88 40.32 45.00 41.56 43.31 45.00 41.35 41.13 40.42 44.43 44.76 41.50 43.72 38.43 41.00 40.74 39.51 45.00 42.71 40.67 n.s. 42.11 41.86 42.63 41.45 44.36 n.s. 45.00 42.22
– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
Pig
Ruminant
CF
Ct values
Result
Ct value
Result
Sediment of CFB: 3 bones
34.16 35.81 38.52 34.40 33.46 35.85 35.81 38.88 35.34 41.63 38.57 34.84 35.92 34.55 33.57 40.66 37.54 38.13 36.37 40.31 41.54 33.74 32.90 36.55 35.23 31.12 34.76 33.38 35.13 33.17 n.s. 40.09 40.57 n.s. 41.43 n.s. n.s.
+ + + + + + + + + – + + + + + – + + + – – + + + + + + + + + – – – – – – –
38.95 n.s. 45.00 n.s. 40.38 45.00 40.05 n.s. 41.46 39.32 45.00 41.45 n.s. n.s. 45.00 n.s. 45.00 39.64 45.00 41.17 41.26 n.s. 40.84 n.s. 45.00 n.s. n.s. n.s. n.s. n.s. 42.12 41.48 39.86 n.s. 41.99 45 n.s.
– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
Sediment of CFB plus calf milk: 3 bones
Flotation of CFA plus calf milk: 1 corn, 2 wheat, 2 rapeseed LCG + H2O
Note: n.s., No signal; CFA, compound feed A; CFB, compound feed B; CF, compound feed; +, positive; –, negative.
fed to poultry and poultry meal to pigs. This would mean that feed samples also have to be tested with pig or poultry PCR in addition to ruminant PCR to comply with the intra-species feed ban. The possible easing justifies an even greater demand to solve the analytical problem with legal feed materials of animal origin. The situation will become more complex solely because blood products from non-ruminants can lead to positives results. Our technique makes it possible to offer a solution for this problem. We present a system that allows us to examine individual particles of a feeding stuff. This method is a combination of microscopy, microdissection and real-time PCR. First, microscopy preparations are made from the sediment of a feed. After
identification of animal fragments of terrestrial origin with the microscope, they will be isolated from the slide by microdissection with the help of a laser. Thereafter, DNA is extracted from the particles and then analysed by PCR. In the course of our preliminary investigations we demonstrated that we could determine the corresponding species to particles isolated from ruminant, pig and poultry PAP reference material. Bone fragments gave better results than muscle fibre fragments. An explanation might be that due to the high temperatures during the manufacturing process of PAPs DNA from muscle fibres is more degraded than that from bones. However sporadic, we had failures. The reason for that remains unclear until
Food Additives & Contaminants: Part A now. But it might be speculated that there was an increased risk of losing particles or DNA during the extraction process because we worked with microscopically small particles. The analysis for feeding stuffs, which should meet the intra-species feed ban and/or the total ban of feeding ruminants, has to be based on zero tolerance. Theoretically, only one positive PCR result from an isolated fragment out of a feed sample has to be sufficient to reject the feed. In practice, the result must be reproducible. Therefore, it is necessary to isolate and investigate several tubes containing particles from one feed sample. In further experiments bone particles from feed mixtures containing simultaneously PAPs (from pig or poultry) and calf milk were isolated. It was shown that most of the separated fragments could be assigned to their respective animal species (pig or poultry) without obtaining positive ruminant results due to the milk. During our investigations we obtained single random failures on the level of particle analysis. Nevertheless, in the worst result at the feed level we received at least eight positive PCR signals out of 15 investigated particle samples. This number should be more than enough to make a definitive statement. Thus, we can demonstrate that at the level of feed sample analysis we never got false results. Calf milk supplement in the feed did not lead to ‘false positive’ ruminant PCR results. The results of our research allowed us to apply the method for aqua feed, which has to be examined for ruminant DNA due to the easing of the feed ban (Commission Regulation (EU) No. 56/2013). Particles of feed samples with ‘low level’ positive ruminant results could be investigated by the combination of microdissection and real-time PCR. Thereby, we would be able to achieve a higher LOD. The LOD of the ruminant PCR system amounts 0.1% PAP in feed, while the LOD of microscopy amounts 0.0025%. Finally, we can summarise that we have developed an analysis method with which the problem of ‘low level’ positive ruminant results in fish feed can be prevented effectively. In addition, the presented technique is suitable to face a possible further easing of the feed ban in relation to pig and poultry PAP feed for nonruminants.
Disclosure statement No potential conflict of interest was reported by the authors.
References Commission Regulation (EU) No. 51/2013 of 16 January 2013 amending Regulation (EC) No. 152/2009 as regards the methods of analysis for the determination of constituents of
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animal origin for the official control of feed [Internet]. 2013. [cited 2014 Sep 19]. Available from: http://eurl.craw.eu/img/ page/Legislation/51-2013_EN.pdf Commission Regulation (EU) No. 56/2013 of 16 January 2013 amending Annexes I and IV to Regulation (EC) No. 999/ 2001 of the European Parliament and of the Council laying down rules for the prevention, control and eradication of certain transmissible spongiform encephalopathies [Internet]. 2013. [cited 2014 Sep 22]. Available from: http://eurlex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L: 2013:021:0003:0016:EN:PDF Commission Regulation (EU) No. 68/2013 of 16 January 2013 on the Catalogue of feed materials [Internet]. 2013. [cited 2015 Mar 16]. Available from: http://eur-lex.europa.eu/ LexUriServ/LexUriServ.do?uri=OJ:L:2013:029:0001:0064: EN:PDF EURL-AP: EURL-AP PCR Proficiency Test 2013c – Final Version [Internet]. 2013c. [cited 2014 Sep 23]. Available from: http://www.eurl.craw.eu/img/page/proficiency/EURLAP%20PCR%20ILS%202013%20final%20version.pdf EURL-AP: EURL-AP Standard Operating Procedure - Detection of ruminant DNA in feed using real-time PCR [Internet]. 2013b. [cited 2014 Sep 19]. Available from: http://eurl.craw. eu/img/page/sops/EURL-AP%20SOP%20Ruminant%20PCR %20V1.0.pdf EURL-AP: EURL-AP Standard Operating Procedure - DNA extraction using the ‘Wizard® Magnetic DNA purification system for Food’ kit [Internet]. 2013a. [cited 2014 Sep 19]. Available from: http://eurl.craw.eu/img/page/sops/EURL-AP %20SOP%20DNA%20extraction%20V1.0.pdf European Commission: The TSE (Transmissible Spongiform Encephalopathy) roadmap 2 [Internet]. 2010. [cited 2014 Sep 18]. Available from: http://ec.europa.eu/food/food/biosafety/tse_bse/docs/roadmap_2_en.pdf Fumière O, Dubois M, Baeten V, Von Holst C, Berben G. 2006. Effective PCR detection of animal species in highly processed animal byproducts and compound feeds. Anal Bioanal Chem. 385:1045–1054. Fumière O, Marien A, Berben G. 2012a. Validation study of a real-time PCR method developed by TNO Triskelion bv for the detection of ruminant DNA in feedingstuffs [Internet]. [cited 2014 Sep 19]. Available from: http://www.eurl.craw. eu/img/page/interlaboratory/Ruminant_Validation_Study_ draft_ver_15_06_2012.pdf Fumière O, Marien A, Berben G. 2012b. EURL-AP PCR Implementation Test 2012. Final version [Internet]. [cited 2014 Sep 19]. Available from: http://www.eurl.craw.eu/ img/page/proficiency/EURL-AP%20PCR%20ILS%202012 %20final%20version%2006-6-2012.pdf Fumière O, Marien A, Fernández Pierna JA, Baeten V, Berben G. 2010. Development of a real-time PCR protocol for the species origin confirmation of isolated animal particles detected by NIRM. Food Addit Contam: Part A. 27:1118–1127. Lassen C, Hummel S, Herrmann B. 1994. Comparison of DNA extraction and amplification from ancient human bone and mummified soft tissue. Int J Legal Med. 107:152–155. Lindahl T. 1993. Instability and decay of the primary structure of DNA. Nature. 362:709–715. Regulation (EC) No. 1069/2009 of the European Parliament and of the Council of 21 October 2009 laying down health rules as regards animal by-products and derived products not intended for human consumption and repealing Regulation (EC) No.
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1774/2002 (Animal by-products Regulation) [Internet]. 2009. [cited 2014 Sep 19]. Available from: http://eur-lex.europa.eu/ LexUriServ/LexUriServ.do?uri=OJ:L:2009:300:0001:0033:EN: PDF Regulation (EC) No. 999/2001 of the European Parliament and of the Council of 22 May 2001 laying down rules for the prevention,
control and eradication of certain transmissible spongiform encephalopathies [Internet]. 2001. [cited 2014 Sep 19]. Available from: http://ec.europa.eu/food/fs/bse/bse36_en.pdf Wilesmith JW, Wells GA, Cranwell MP, Ryan JB. 1988. Bovine spongiform encephalopathy: Epidemiological studies. Vet Rec. 123:638–644.
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Species identification of processed animal proteins (PAPs) in animal feed containing feed materials from animal origin Sonja Axmann*, Andreas Adler, Agnes Josephine Brandstettner, Gabriela Spadinger, Roland Weiss and Irmengard Strnad Austrian Agency for Health and Food Safety (AGES), Institute for Animal Nutrition and Feed, Linz, Austria (Received 4 February 2015; accepted 23 March 2015) Since June 2013 the total feed ban of processed animal proteins (PAPs) was partially lifted. Now it is possible to mix fish feed with PAPs from non-ruminants (pig and poultry). To guarantee that fish feed, which contains non-ruminant PAPs, is free of ruminant PAPs, it has to be analysed with a ruminant PCR assay to comply with the total ban of feeding PAPs from ruminants. However, PCR analysis cannot distinguish between ruminant DNA, which originates from proteins such as muscle and bones, and ruminant DNA, which comes from feed materials of animal origin such as milk products or fat. Thus, there is the risk of obtaining positive ruminant PCR signals based on these materials. The paper describes the development of the combination of two analysis methods, micro-dissection and PCR, to eliminate the problem of ‘falsepositive’ PCR signals. With micro-dissection, single particles can be isolated and subsequently analysed with PCR. Keywords: microdissection; PAP; animal proteins; feed-ban; real-time PCR
Introduction Bovine spongiform encephalopathy (BSE), commonly known as mad cow disease, is a fatal, neurodegenerative disease in cattle that causes a spongy degeneration in the brain and spinal cord. BSE was first identified in the UK in 1985 and 1986. Epidemiological studies have found that BSE is a feed-borne infection transmitted to animals through BSE-infected meat and bone meal (MBM) in animal feed (Wilesmith et al. 1988). The outbreak of this disease and its relation to the consumption of contaminated animal feed led to the ban of feedingstuffs containing any processed animal proteins (PAPs) including MBM. The only exception was fish meal, which may be fed to fish and non-ruminants (Regulation (EC) 999/2001). Additionally Regulation (EC) No. 1069/2009 (repealing Regulation (EC) 1774/2002) specifies the prohibition of feeding farmed animals with the same species. Feed samples are checked by classical optical light microscopy for the presence of PAPs. With this method it is possible to distinguish fish bones from terrestrial animal bones. The TSE (Transmissible Spongiform Encephalopathy) Roadmap 2 (European Commission 2010) considered that the transmission risk of BSE from non-ruminants to nonruminants is very unlikely. Therefore, the recent easing of the total feed ban has been completed in June 2013 (Commission Regulation (EC) 56/2013). Because of that, PAPs from non-ruminants (pig and poultry) can be added to fish feed, whereas ruminant PAPs remain totally prohibited. Thus, detection of terrestrial PAPs with optical light microscopy provides insufficient information. In fact,
the method can discriminate between fish and terrestrial PAPs, but it cannot distinguish terrestrial PAPs of different species. To guarantee, that fish feed, which contains nonruminant PAPs, is free of ruminant PAPs, it has to be analysed with a ruminant PCR assay either if terrestrial particles are detected with classical microscopy or if PAPs are mentioned in the composition (Commission Regulation (EC) 51/2013). But using PCR analysis, we are faced with the problem that this method cannot distinguish ruminant DNA, which originates from proteins such as muscles and bones, from ruminant DNA, which comes from feed materials of animal origin such as milk products, fat, etc. (Catalogue of feed materials: Commission Regulation (EU) No. 68/2013). Thus, there is the risk of obtaining positive ruminant PCR signals based on these materials. The European Animal Protein Association (EAPA), European Fats Processors and Renderers Association (EFPRA) and European Feed Manufacturers’ Federation (FEFAC) drew attention to this issue at the meeting of the Standing Committee on the Food Chain and Animal Health (SCoFCAH) in December 2013 (2013 letter from EAPA, EFPRA and FEFAC to DG SANCO as a request for discussion at SCoFCAH held in Brussels on 16–17 December 2013; unreferenced). They considered that positive PCR signals are not evidence for the presence of illegal ruminant DNA, as legal products may be the source for positive testing results. Moreover, the European Commission outlined in the TSE Roadmap 2 (2010) that a lifting of the ban on the use of PAPs from non-ruminants in non-ruminant feed could
*Corresponding author. Email: [email protected] © 2015 Australian Agency for Health and Food Safety Ltd. Published by Taylor & Francis.
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be planned, but without lifting the existing prohibition of intra-species recycling (e.g. poultry PAP could only be fed to pigs and pig PAP to poultry). That would mean that, for example, pig feed containing poultry PAP has to be checked with pig PCR assay in order to comply with the intra-species ban and with ruminant PCR assay to meet the total ban on feeding ruminant PAPs. For this case, blood products from non-ruminants may be a problem as well in addition to the aforementioned feed materials. For example, a feeding stuff for pigs contains poultry PAPs, milk powder and blood meal simultaneously. If poultry PAP is mentioned in the composition or terrestrial particles were detected by microscopy, the feed has to be analysed with pig and ruminant PCR assays. Hence, because of the presence of blood and milk, the analysis will lead to positive results with both PCR assays. Therefore, it is of great interest to develop an analytical method for the investigation of compound feed by which the species of PAPs can be determined without getting positive results due to legal feed materials of animal origin. The combination of microdissection and PCR has been found. The microdissection device consists of a light microscope connected to a laser which can isolate small fragments of bones or muscles by catapulting them into the lid of a reaction tube. The origin of the particles up to their species level can be determined with subsequent PCR analysis. A similar analytical technique, the combination of near-infrared microscopy (NIRM) and PCR, has already been demonstrated (Fumière et al. 2010). But the use of NIRM for routine analysis is difficult. The limitation of this method is the number of particles that are manually analysed and isolated. This paper examines the capability of the microdissection technique and the combination of microdissection with PCR. Light microscopy and ruminant PCR analysis are already applied as validated investigation methods also for official control of feeding stuffs in the European Union (Commission Regulation (EC) 51/2013).
Materials and methods Reference materials Pig, sheep, cattle and poultry PAPs were provided by the EURL-AP (European Reference Laboratory for Animal Proteins in Feedingstuffs, Gembloux, Belgium).
Commercial feeds The cattle feed ‘Kuhkorn Kompakt 183’ (composed of wheat bran, corn concentrated feed, corn, grain mash, corn germ extraction meal, dry cuts, rapeseed extraction meal, molasses, wheat, calcium carbonate, cacao shells, sodium chloride and magnesium oxide) was obtained from Garant Tiernahrung GmbH (Pöchlarn, Austria). It was
ground with a Retsch grinding mill ZM200 (Retsch GmbH, Haan, Germany) to obtain particles with a diameter less than 0.5 mm. The calf milk ‘Kälbermilch 19/0’ (composed of whey powder, vegetable oil refined palm/ coconut 4:1, wheat protein hydrolysate, wheat swelling flour and dextrose) was obtained from Inntaler Tiernahrung GmbH (Ingolstadt, Germany).
Processed animal proteins (PAPs) Poultry PAP was obtained from the Styrian animal cadaver utilisation (STKV, Landscha, Austria) and is produced under Commission Regulation (EC) 1069/2009 (133°C and 3 bars for 20 min). Pig PAP was obtained from Garant Tiernahrung GmbH and imported from Spain (ElPozo, Murcia, Spain). PAPs were ground with a Retsch grinding mill ZM200 to obtain particles with a diameter less than 0.5 mm.
Compound feeds Commercial cattle feed, 5% in mass fraction of poultry (compound feed A) or pig PAP (compound feed B) and 5% in mass fraction of calf milk were mixed for 10 min with a Turbula mixer T 2 C (Willy A Bachofen AG, Basel, Switzerland). All mixes were also produced without calf milk.
Aqua feed plus 0.1% cattle PAP Aqua feed (composed of soya meal, wheat, fish meal, sunflower seed, maize gluten, permitted flavour, fish oil, rapeseed oil, vitamins, mono ammonium phosphate, permitted anti-fungal/anti-oxidant, yeasts, minerals, algae, betaine and astaxanthin) containing 0.1% cattle PAP in mass fraction was sent to us in course of an international proficiency test (EURL-AP 2013c). The EURL-AP selected the feed among the EURL-AP sample bank and it was tested to be free of any ruminant traces by ruminant PCR and microscopy.
Extraction and preparation of the sediment The sediment was prepared according to Regulation (EC) 51/2013. Material (10 g) was transferred into a separation funnel, mixed with 50 ml tetrachlorethylene (VWR, Radnor, PA, USA) and shaken vigorously for 30 s. Further, 50 ml tetrachlorethylene were added cautiously to wash down the inside surface of the funnel to remove any adhering particles. The resulting mixture was incubated for at least 5 min before the sediment was separated off by opening the stopcock. The sediment was then dried. If more than 5% of the sediment consisted of particles > 0.5 mm, it was sieved at 0.25 mm.
Food Additives & Contaminants: Part A Extraction and preparation of the flotate The flotate is prepared according to Regulation (EC) 51/ 2013. After recovery of the sediment two phases remain in the separation funnel: the liquid phase (tetrachlorethylene) and the solid phase made of the floating material (flotate). Tetrachloroethylene was poured off completely by opening the stopcock. The remaining flotate was transferred into a Petri dish and air dried in a laboratory fume hood. If more than 5% of the flotate consisted of particles > 0.5 mm, it was sieved at 0.25 mm.
Slide preparation A test portion of the sediment or the flotate was spread on the slide, sprayed with liquid cover glass (LCG) (Carl Zeiss AG, Oberkochen, Germany) and distributed with a fine needle. The slide was then dried for 5–10 min at 60° C. Afterwards two drops of immersion oil were applied and the slide was rotated in a way that the oil covered all particles of the sample. Excess of oil was wiped away and the slide was again dried for 5–10 min at 60°C.
Particle isolation with a PALM microbeam– microdissection system The microdissection system (Carl Zeiss) consists of several components: the inverse microscope (Axio Observer. A1), the laser unit (MicroBeam Module Rel. 4.2), the cap holder (CapMover II), the digital microscopy camera (AxioCam Icc1 Rev. 3) and the computer running the software RoboSoftware 4.2 to use the microdissection device. Before starting particle isolation, 30 µl water of molecular biology grade (5 Prime GmbH, Hilden, Germany) were pipetted into the lids of the 0.5 ml reaction tubes. The slide was then clamped in the inverse microscope and was first coarsely scanned. Suspicious particles were marked and checked whether any contaminants adhere to them. Afterwards, the reaction tube was fixed into the cap holder and particles were catapulted with the laser into the lid of the tube. If necessary, the particles can be cut with the laser before catapulting.
DNA extraction DNA extraction of particles To pelletise the particles, the 0.5 ml reaction tubes were centrifuged at 13 000g for 15 min and the water was removed immediately after. For DNA extraction proteinase K solution (Ambion®, Life Technologies, Carlsbad, CA, USA) was added at a concentration of 0.4 µg µl–1 to DirectPCR® lysis reagent for genotyping using crude lysates (Viagen Biotech, Inc., Los Angeles, CA, USA). A total of 7 µl of the prepared extraction buffer were added to the particles. The mixture was then incubated
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overnight at 56°C and subsequently proteinase K was stopped for 50 min at 85°C. Afterwards the mixture was briefly centrifuged and diluted with 20 µl PCR grade water (Roche, Risch, Switzerland). A total of 5 µl of the DNA extract was used for real-time PCR. DNA extraction of compound feed DNA from compound feed was extracted using the Wizard® Magnetic DNA Purification System for Food kit (Promega, Fitchburg, WI, USA). The capability of this kit for compound feed has already been evaluated (Fumière et al. 2006). DNA of feed was prepared according to the standard operation procedure DNA extraction using the Wizard Magnetic DNA Purification System for Food kit, which is published on the webpage of the EURL-AP (EURL-AP 2013a). Real-time PCR All PCR assays were performed with the LightCycler® 480 device (Roche). Ruminant real-time PCR assay Ruminant PCR was performed according to the standard operation procedure ‘Detection of ruminant DNA in feed using real-time PCR’ published on the webpage of the EURL-AP (EURL-AP 2013b). AGES laboratories participated as Austrian National reference laboratory (NRL) to the validation and implementation studies organised by the EURL-AP (Fumière et al. 2012a, 2012b). The cut-off Ct value of this assay was determined at 37.07 cycles in our laboratory. Pig real-time PCR assay For each pig target amplification 17.5 µl LightCycler® 480 Probes Master containing FastStart Taq DNA polymerase, nucleotides and MgCl2 (Roche), 8.5 pmole of forward and reverse primer (Microsynth, Balgach, Switzerland), 8.5 pmole Taqman probe labelled with reporter dye FAM and quencher dye TAMRA (Microsynth), 5 µl template DNA and PCR grade water (Roche) were mixed together to obtain a reaction volume of 35 µl. The thermal programme was defined with an initial denaturation step at 95°C for 10 min followed by 50 cycles consisting of 15 s denaturation at 95°C and 1 min annealing and elongation at 50°C. The pig PCR protocol detects domestic pig target and the sequence information of primer and probe was provided by the EURL-AP. The cut-off Ct value for this assay was determined in our laboratory for our PCR platform at 39.16 cycles. For correct calculation, pig calibrants (Joint Research Centre – Institute for Reference Materials and Measurement, Brussels, Belgium) and a
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calculation sheet for determining the exact copy number and cut-off were used (EURL-AP, Gembloux, Belgium). Poultry real-time PCR assay For each poultry target amplification 12.5 µl LightCycler® 480 Probes Master containing FastStart Taq DNA polymerase, nucleotides and MgCl2 (Roche), 7.65 pmole of forward and reverse primer (Microsynth), 9 pmole Taqman probe labelled with reporter dye FAM and quencher dye TAMRA (Microsynth), 5 µl template DNA and PCR grade water (Roche) were mixed together to obtain a reaction volume of 25 µl. The thermal programme was defined with an initial denaturation step at 95°C for 10 min followed by 50 cycles consisting of 15 s denaturation at 95°C and 1 min annealing and elongation at 50°C. The poultry PCR protocol detects domestic chicken and turkey targets. Sequence information of primer and probe was provided by the EURL-AP. The cut-off value will be determined in the course of the poultry validation study organised by the EURL-AP. Results and discussion In all trials we were confronted with the fact that each sample is unique. Every single tube generated by microdissection contains particles which were different in size and general condition. Thus, there was just one possibility to extract DNA from one tube sample. In addition, we worked with microscopically small particles, and there was always an increased risk of losing these during the extraction process. The first preliminary experiments were performed to show that muscle and bone particles derived from PAP of known origin can be isolated successfully with the microdissection and identified correctly by realtime PCR after DNA extraction. Species-specific identification of separated particles from reference material with real-time PCR The extraction of DNA from particles has already been evaluated. DirectPCR® (Tail) lysis reagent has been demonstrated to be the most effective DNA extraction method for particles isolated by near-infrared microscopy (NIRM) (Fumière et al. 2010). A part of our work was the adaptation of this method to make it suitable for microdissected particles. Due to the high temperatures during the manufacturing process of PAP we were faced with a high degree of DNA degradation. In addition the sediment preparation with tetrachlorethylene and subsequent fixing to the slide could influence the effectiveness of DNA extraction. In the initial experiment slides for microdissection were prepared from sediments of pig, ruminant and poultry PAP reference material. In order to obtain a good
microscopic image, the slides were covered with liquid cover glass. Ten samples from each preparation were shot with microdissection and every tube contained two bone or two muscle fibre fragments. Blank samples containing water, liquid cover glass and immersion oil were also prepared. DNA extraction was carried out with DirectPCR® (Tail) lysis reagent supplemented with proteinase K at a concentration of 0.4 µg µl–1 for an incubation period of 15 min at 56°C. Thereafter, DNA was screened with ruminant, poultry and pig real-time PCR in order to verify species specificity of the assays. For poultry PCR assay Ct values < 35 cycles were considered as positive and Ct values ≥ 35 cycles were considered as negative results. In the case of ruminant and pig real-time PCR assays, we had well-defined cutoff Ct values for our PCR platform. Ct values ≤ 37.07 cycles received with the ruminant PCR assay corresponded to positive results and Ct values > 37.07 cycles indicated negative results. With the pig PCR assay we obtained a cut-off Ct value of 39.16 cycles. Consequently, Ct values ≤ 39.16 cycles were assessed positively and Ct values > 39.16 cycles were assessed negatively. According to the results shown in Table 1, bone samples generally yielded better results than muscle fibre samples. Tubes containing DNA from pig or poultry bones produced satisfying animal-specific positive PCR signals, whereas we sometimes received negative results from DNA of muscle fibres. Just four pig and three poultry muscle fibre samples were positive according to their corresponding species. The results, which were given from ruminant fragments, were insufficient. Two samples containing DNA from ruminant bones were positive and three yielded negative PCR signals close to the cut-off of 37.07 cycles. Samples containing DNA from ruminant muscle fibres failed completely. Nevertheless, we hoped to improve the results by changes in DNA extraction conditions. Therefore, we extended the incubation time with proteinase K from 15 min to overnight. We analysed 30 cattle samples, each containing two particles. Ten cattle bone samples were incubated for 15 min and other 10 bone samples were incubated overnight with proteinase K at 56°C. The remaining 10 tubes containing muscle fibres were incubated overnight as well. The results of the 20 samples containing cattle bones are shown in Table 2. When trying with a 15 min digestion, we obtained three weak positive results (Ct values close to the cut-off 37.07 cycles), while after overnight digestion six out of 10 bone samples gave clearly positive PCR signals (Table 2, indicated in bold). Results yielded from cattle muscle fibres were persistent negative (data not shown). Consequently, in the case of bone samples the extension of the incubation period revealed twice as many positive results with more distinct Ct values. Unfortunately we
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Table 1. Real-time PCR results from DNA of bones and muscle fibres after 15-min incubation with proteinase K. Pig
Poultry
Ruminant
Content
Ct value
Result
Content
Ct value
Result
Content
Ct value
Result
2 bo, pig
33.87 34.27 34.34 32.55 33.65
+ + + + +
2 bo, poultry
31.76 30.83 24.95 31.34 31.08
+ + + + +
2 bo, cattle
35.03 36.32 38.10 37.16 37.20
+ + – – –
2 mf, pig
37.84 40.01 38.37 35.95 34.01
+ – + + +
2 mf, poultry
26.88 42.17 41.66 25.99 24.78
+ – – + +
2 mf, cattle
40.09 40.12 40.04 40.09 39.62
– – – – –
Note: bo, Bone; mf, muscle fibre; +, positive; –, negative.
Table 2. Real-time PCR results from DNA of cattle bone particles after 15-min and overnight incubation with proteinase K. Ruminant – 15 min Content
2 Bones Cattle
Ruminant – overnight
Ct value
Result
38.28 37.94 39.15 39.08 36.64 37.50 37.11 38.09 36.78 36.99
– – – – + – – – + +
Content
2 Bones Cattle
Ct value
Result
37.21 28.32 41.19 26.16 38.84 34.76 41.20 29.76 30.90 32.79
– + – + – + – + + +
Note: +, Positive; –, negative.
were unable to achieve improvements concerning the muscle fibres. In the following series of studies, we aimed to determine if we could detect different species in the same tube. Therefore, particles from animals of varying origin were combined in the vessels. Investigation tubes were prepared containing either two bones or two muscle fibres of every species. DNA extraction was carried out with the overnight proteinase K digestion step. Thereafter the samples were screened with ruminant, pig and poultry PCR assays. The resulting Ct values of the individual samples are shown in Table 3 and the summary is presented in Table 4. The results of samples containing bone particles were very promising. The average Ct value of samples containing poultry bones was 32.25 cycles. Thus, in case of 15 samples we received 14 positive results and just one sample was interpreted as negative. Pig bone samples yielded an average Ct value of 34.01 cycles. Thirteen samples out of 15 were clearly positive and two were
negative. The average Ct value of ruminant bone samples was 33.78 cycles. Eleven samples were considered positive and the rest was negative. The results of DNA extracted from poultry and pig muscle fibres were also very promising. The average poultry Ct value was 33.58 cycles, whereas 10 samples were clearly positive and five were negative. DNA from pig muscle fibres provided an average Ct value at 35.11 cycles. Thirteen samples were positive showing a Ct value ≤ 39.16 cycles and two samples were negative. The results from ruminant muscle fibres did not come up to our expectations. All 15 samples had a Ct value above 37.07 cycles and thus were negative. Based on our results, we observed that DNA extraction from different tissues worked differently well. Species determination was successfully performed with DNA extracts from bones, whereas muscle fibres did not yield similar outcomes. This has already been proven by Lassen et al. (1994). They compared DNA extracts from ancient bones and mummified soft tissues by PCR analysis and came to the result that skeletal material should be given the preference for DNA investigations. Bones exhibited the least DNA degradation degree, followed by liver then spleen, kidney, thyroid, blood, brain, muscle and lymph nodes. A plausible explanation for this observation could be that the compact, more durable bone surface provides some protection against bacterial and enzymatic degradation. In addition, DNA is bound to an inorganic component (calcium hydroxyapatite) in the bone, which in turn contributes to its stabilisation (Lindahl 1993). These observations might be suitable for our results. We do not work with mummified tissues or ancient bones, but with animal material that has been exposed to very high temperatures, which also amounts to massive degradation of DNA. Since particles were mainly isolated from sediment material, the emphasis will lie in the DNA extraction from bone fragments, because predominantly bones were
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Table 3. Real-time PCR results from DNA of particles of varying animal origin combined in one investigation tube after overnight incubation with proteinase K. Pig Content
Poultry
Summary of the results from Table 3. Positive Negative results Average Ct value (without n.s. results (< 35 (≥ 35 cycles values) cycles) and n.s.)
Ruminant
Ct Ct Ct value Result value Result value Result n.s. 30.98 30.00 35.53 35.83
– + + + +
n.s. n.s. n.s. n.s. n.s.
– – – – –
31.20 29.24 27.76 34.50 38.90
+ + + + –
2 mf, pig and cattle 37.53 41.66 37.37 33.32 34.73
+ – + + +
n.s. n.s. n.s. n.s. n.s.
– – – – –
39.98 39.09 43.11 41.24 45
– – – – –
2 bo, poultry and lamb
n.s. n.s. n.s. n.s. n.s.
– – – – –
31.49 33.29 34.38 29.59 28.66
+ + + + +
34.72 36.69 n.s. 28.56 31.15
+ + – + +
2 mf, poultry and cattle
n.s. n.s. n.s. n.s. n.s.
– – – – –
35.33 31.46 28.35 40.48 34.81
– + + – +
43.85 41.63 45 40.05 40.90
– – – – –
2 bo, pig and poultry
28.43 40.60 33.21 33.77 32.94
+ – + + +
31.27 34.27 30.33 32.30 35.40
+ + + + –
37.72 n.s. 40.95 44.2 n.s.
– – – – –
2 mf, pig and poultry
36.80 35.31 29.36 34.88 35.03
+ + + + +
34.69 34.30 26.98 31.19 33.88
+ + + + +
n.s. 45 41.47 n.s. n.s.
– – – – –
2 bo, pig, poultry and lamb
33.86 38.08 35.17 36.07 32.55
+ + + + +
32.79 32.36 32.79 31.25 33.66
+ + + + +
35.90 44.36 34.40 37.37 30.51
+ – + – +
2 mf, pig, poultry and cattle
33.86 35.18 33.20 34.26 n.s.
+ + + + –
29.18 n.s. 39.77 27.74 41.08
+ – – + –
40.67 45 38.96 40.16 n.s.
– – – – –
2 bo, pig and lamb
Table 4.
Note: bo, bone; mf, muscle fibre; n.s., no signal; +, positive; –, negative.
sedimented whereas most muscle fibres remained in the flotate. Our data indicated that we sometimes have to expect random failures at the level of particle analysis. In some instances we yielded negative results with the animalspecific PCR assay. The reason for that remains unclear until now. We could speculate that we definitely got
Poultry bo Poultry mf
32.25 33.58
14 10
1 5
Positive Negative results results (> cut-off and (≤ cut-off) n.s.) Pig bo Pig mf Ruminant bo Ruminant mf
34.01 35.11 33.78 41.72
13 13 11 0
2 2 4 15
Note: n.s., No signal; bo, bone; mf, muscle fibre.
failures because we were working with microscopically small particles and thus DNA or the fragment itself were lost during the extraction process. But at least we always got three positive results out of five bone particle samples. Similarly, we could show that we could achieve equivalent results, even if particles of different species origin were combined into one sample. Every species was successfully detected in vessels containing combinations of bones derived from two or three different animals.
Isolation and identification of bones from aqua feed In the next experiment we wanted to illustrate that our method is just as suitable for aqua feed. Therefore, we examined a feed sample, which was sent by the EURL-AP in course of an international proficiency test (EURL-AP 2013c). It was composed of aqua feed and 0.1% cattle PAP in mass fraction. Aqua feed basically consists of a high proportion of fish meal. Therefore, the content of muscle fibres derived from fish meal was very high and hence it was statistically unlikely that muscle fibres coming from terrestrial animals were isolated by microdissection when these PAPs were added in low amounts to the aqua feed. Thus, we restricted to the isolation of terrestrial bones. A total of 10 g of the aqua feed were used for sedimentation and then microscopy slides were prepared. Ten tubes each containing two terrestrial bones were produced by microdissection. DNA was extracted with DirectPCR® (Tail) lysis reagent and subsequently analysed with ruminant real-time PCR. As expected, the results were very satisfying. The species origin of the bones was confirmed with ruminant PCR in six out of 10 tubes. The corresponding Ct values were below the ruminant cut-off Ct value of 37.07 cycles and therefore the feed sample was assessed positive for ruminant DNA (Ct values not shown).
Food Additives & Contaminants: Part A Identification of separated particles from feeding stuffs containing feed materials of animal origin (milk powder) The main focus of our work laid in the development of an analytical method by which one can detect PAPs from ruminants and/or non-ruminants in feeding stuffs without being negatively influenced by legal feed materials of animal origin (e.g. milk powder, fat, blood products, etc.). These materials might be the reason for positive animal-specific PCR and thus feeding stuffs would be rejected unjustifiably. For this reason we investigated self-made compound feed A (CFA) containing poultry PAP and compound feed B (CFB) containing pig PAP. All mixtures were prepared once with and once without calf milk. In order to create conditions that were as realistic as possible, we used commercially available PAPs instead of reference material. Moreover, cattle feed and calf milk used for this purpose are generally available products. Previously, these materials were analysed with the animal PCR assays to ensure that they were not contaminated with foreign DNA (Table 5). DNA extraction was carried out with the Wizard Magnetic DNA Purification System for Food using 100 mg portions and following PCR was performed. The expected results were confirmed. Contamination with foreign DNA was detected in none of the starting materials. Ruminant DNA was established exclusively in calf milk, pig DNA in porcine PAP and poultry DNA in poultry PAP. Adjacent, slides from sediments of CFA and CFB were prepared to isolate predominantly bones with the microdissection device. Thirty samples were produced from each feed, 15 samples from the mixtures with calf milk and 15 samples from the mixtures without calf milk. Moreover, control samples were made from CFA and CFB plus milk containing feed particles. Since most of these particles remain in the supernatant during sedimentation, microscopy slides were prepared from the flotate and the feed fragments isolated there from. Every blank sample was supplemented with one corn, two wheat and
Table 5. Real-time PCR results from DNA of commercial feeds and PAPs. Pig
Poultry
Ruminant
Ct Ct Ct value Result value Result value Result Commercial feed Cattle feed Calf milk PAPs Pig PAP Poultry PAP
n.s. n.s.
– –
n.s. n.s.
– –
38.17 23.83
– +
28.41 n.s.
+ –
41.02 21.89
– +
40.96 38.85
– –
Note: n.s., No signal; +, positive; –, negative.
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two rapeseed particles. Further controls were included that contained only water and LCG to ensure contamination-free reagents. DNA extraction of the particles and controls was performed with the DirectPCR® (Tail) lysis reagent from all samples and DNA was subsequently screened with pig or poultry and ruminant real-time PCR assays (Table 6). The results were very auspicious. In the case of CFA containing poultry PAPs, we could generate Ct values < 35 cycles from 19 out of 30 samples. Eleven samples gave Ct values ≥ 35 cycles or no signal. A contamination of the isolated particles with milk could be excluded. In none of the samples was ruminant DNA detected, even when calf milk was added to the feed. All ruminant Ct values lay either above the cut-off 37.07 cycles or the samples gave no fluorescence signal. Comparable outcomes were achieved in the feed containing pig PAPs (CFB). Twenty-six out of 30 samples gave positive pig results. Four samples gave Ct values > 39.16 cycles and thus were negative. All PCR results from the ruminant assay yielded Ct values above the cut-off 37.07 cycles or no fluorescence signal. Hence, a contamination with milk could be excluded, too. Finally, the controls were examined with ruminant, pig or poultry PCR assay (Table 6). All ruminant PCR results were negative. The fluorescence signals rose above the ruminant cut-off value or no signals were observed. Equally, pig or poultry PCR results gave either no fluorescence signal or Ct values above the cut-off. With this key experiment we are able to present a working method for detecting PAPs in compound feed without getting positive results based on feed materials of animal origin. Because of this methodical possibility we can feed samples on particle level. Conclusions Since June 2013 (Commission Regulation (EC) No. 56/ 2013) it has been allowed to add PAP from non-ruminants to fish feed. To ensure that the PAP originates exclusively from non-ruminants, the feed has to be tested with ruminant PCR according to the current legislation situation (Commission Regulation (EC) No. 51/ 2013). But the new official control method is reason for criticism because the number of ‘low level’ positive ruminant results in aqua feed rose (2013 letter from EAPA, EFPRA and FEFAC to DG SANCO as a request for discussion at SCoFCAH held in Brussels on 16–17 December 2013; unreferenced). Until now, it cannot be excluded that legal feed materials of animal origin are the reason for these results. Moreover, a lifting of the ban on the use of PAP from non-ruminants in nonruminant feed without lifting the existing prohibition of intra-species recycling is considered (European Commission 2010). Accordingly, pig meal could be
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Table 6. Real-time PCR results from DNA of bones isolated from sediments of compound feed A (containing poultry PAP) and compound feed B (containing pig PAP) with or without calf milk. Poultry CF Sediment of CFA: 3 bones
Sediment of CFA plus calf milk: 3 bones
Flotation of CFA plus calf milk: 1 corn, 2 wheat, 2 rapeseed LCG + H2O
Ruminant
Ct value Result Ct value Result 30.27 34.87 35.38 30.69 33.95 27.68 30.11 31.21 35.00 33.55 28.33 38.63 33.92 38.27 33.94 30.41 35.14 38.94 n.s. 34.16 31.88 35.16 32.28 34.78 38.43 35.87 40.75 29.17 34.81 34.82 40.19 n.s. n.s. n.s. n.s. n.s. n.s.
+ + – + + + + + – + + – + – + + – – – + + – + + – – – + + + – – – – – – –
n.s. 44.04 42.31 40.87 40.56 39.17 41.43 40.79 40.88 40.32 45.00 41.56 43.31 45.00 41.35 41.13 40.42 44.43 44.76 41.50 43.72 38.43 41.00 40.74 39.51 45.00 42.71 40.67 n.s. 42.11 41.86 42.63 41.45 44.36 n.s. 45.00 42.22
– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
Pig
Ruminant
CF
Ct values
Result
Ct value
Result
Sediment of CFB: 3 bones
34.16 35.81 38.52 34.40 33.46 35.85 35.81 38.88 35.34 41.63 38.57 34.84 35.92 34.55 33.57 40.66 37.54 38.13 36.37 40.31 41.54 33.74 32.90 36.55 35.23 31.12 34.76 33.38 35.13 33.17 n.s. 40.09 40.57 n.s. 41.43 n.s. n.s.
+ + + + + + + + + – + + + + + – + + + – – + + + + + + + + + – – – – – – –
38.95 n.s. 45.00 n.s. 40.38 45.00 40.05 n.s. 41.46 39.32 45.00 41.45 n.s. n.s. 45.00 n.s. 45.00 39.64 45.00 41.17 41.26 n.s. 40.84 n.s. 45.00 n.s. n.s. n.s. n.s. n.s. 42.12 41.48 39.86 n.s. 41.99 45 n.s.
– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
Sediment of CFB plus calf milk: 3 bones
Flotation of CFA plus calf milk: 1 corn, 2 wheat, 2 rapeseed LCG + H2O
Note: n.s., No signal; CFA, compound feed A; CFB, compound feed B; CF, compound feed; +, positive; –, negative.
fed to poultry and poultry meal to pigs. This would mean that feed samples also have to be tested with pig or poultry PCR in addition to ruminant PCR to comply with the intra-species feed ban. The possible easing justifies an even greater demand to solve the analytical problem with legal feed materials of animal origin. The situation will become more complex solely because blood products from non-ruminants can lead to positives results. Our technique makes it possible to offer a solution for this problem. We present a system that allows us to examine individual particles of a feeding stuff. This method is a combination of microscopy, microdissection and real-time PCR. First, microscopy preparations are made from the sediment of a feed. After
identification of animal fragments of terrestrial origin with the microscope, they will be isolated from the slide by microdissection with the help of a laser. Thereafter, DNA is extracted from the particles and then analysed by PCR. In the course of our preliminary investigations we demonstrated that we could determine the corresponding species to particles isolated from ruminant, pig and poultry PAP reference material. Bone fragments gave better results than muscle fibre fragments. An explanation might be that due to the high temperatures during the manufacturing process of PAPs DNA from muscle fibres is more degraded than that from bones. However sporadic, we had failures. The reason for that remains unclear until
Food Additives & Contaminants: Part A now. But it might be speculated that there was an increased risk of losing particles or DNA during the extraction process because we worked with microscopically small particles. The analysis for feeding stuffs, which should meet the intra-species feed ban and/or the total ban of feeding ruminants, has to be based on zero tolerance. Theoretically, only one positive PCR result from an isolated fragment out of a feed sample has to be sufficient to reject the feed. In practice, the result must be reproducible. Therefore, it is necessary to isolate and investigate several tubes containing particles from one feed sample. In further experiments bone particles from feed mixtures containing simultaneously PAPs (from pig or poultry) and calf milk were isolated. It was shown that most of the separated fragments could be assigned to their respective animal species (pig or poultry) without obtaining positive ruminant results due to the milk. During our investigations we obtained single random failures on the level of particle analysis. Nevertheless, in the worst result at the feed level we received at least eight positive PCR signals out of 15 investigated particle samples. This number should be more than enough to make a definitive statement. Thus, we can demonstrate that at the level of feed sample analysis we never got false results. Calf milk supplement in the feed did not lead to ‘false positive’ ruminant PCR results. The results of our research allowed us to apply the method for aqua feed, which has to be examined for ruminant DNA due to the easing of the feed ban (Commission Regulation (EU) No. 56/2013). Particles of feed samples with ‘low level’ positive ruminant results could be investigated by the combination of microdissection and real-time PCR. Thereby, we would be able to achieve a higher LOD. The LOD of the ruminant PCR system amounts 0.1% PAP in feed, while the LOD of microscopy amounts 0.0025%. Finally, we can summarise that we have developed an analysis method with which the problem of ‘low level’ positive ruminant results in fish feed can be prevented effectively. In addition, the presented technique is suitable to face a possible further easing of the feed ban in relation to pig and poultry PAP feed for nonruminants.
Disclosure statement No potential conflict of interest was reported by the authors.
References Commission Regulation (EU) No. 51/2013 of 16 January 2013 amending Regulation (EC) No. 152/2009 as regards the methods of analysis for the determination of constituents of
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animal origin for the official control of feed [Internet]. 2013. [cited 2014 Sep 19]. Available from: http://eurl.craw.eu/img/ page/Legislation/51-2013_EN.pdf Commission Regulation (EU) No. 56/2013 of 16 January 2013 amending Annexes I and IV to Regulation (EC) No. 999/ 2001 of the European Parliament and of the Council laying down rules for the prevention, control and eradication of certain transmissible spongiform encephalopathies [Internet]. 2013. [cited 2014 Sep 22]. Available from: http://eurlex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L: 2013:021:0003:0016:EN:PDF Commission Regulation (EU) No. 68/2013 of 16 January 2013 on the Catalogue of feed materials [Internet]. 2013. [cited 2015 Mar 16]. Available from: http://eur-lex.europa.eu/ LexUriServ/LexUriServ.do?uri=OJ:L:2013:029:0001:0064: EN:PDF EURL-AP: EURL-AP PCR Proficiency Test 2013c – Final Version [Internet]. 2013c. [cited 2014 Sep 23]. Available from: http://www.eurl.craw.eu/img/page/proficiency/EURLAP%20PCR%20ILS%202013%20final%20version.pdf EURL-AP: EURL-AP Standard Operating Procedure - Detection of ruminant DNA in feed using real-time PCR [Internet]. 2013b. [cited 2014 Sep 19]. Available from: http://eurl.craw. eu/img/page/sops/EURL-AP%20SOP%20Ruminant%20PCR %20V1.0.pdf EURL-AP: EURL-AP Standard Operating Procedure - DNA extraction using the ‘Wizard® Magnetic DNA purification system for Food’ kit [Internet]. 2013a. [cited 2014 Sep 19]. Available from: http://eurl.craw.eu/img/page/sops/EURL-AP %20SOP%20DNA%20extraction%20V1.0.pdf European Commission: The TSE (Transmissible Spongiform Encephalopathy) roadmap 2 [Internet]. 2010. [cited 2014 Sep 18]. Available from: http://ec.europa.eu/food/food/biosafety/tse_bse/docs/roadmap_2_en.pdf Fumière O, Dubois M, Baeten V, Von Holst C, Berben G. 2006. Effective PCR detection of animal species in highly processed animal byproducts and compound feeds. Anal Bioanal Chem. 385:1045–1054. Fumière O, Marien A, Berben G. 2012a. Validation study of a real-time PCR method developed by TNO Triskelion bv for the detection of ruminant DNA in feedingstuffs [Internet]. [cited 2014 Sep 19]. Available from: http://www.eurl.craw. eu/img/page/interlaboratory/Ruminant_Validation_Study_ draft_ver_15_06_2012.pdf Fumière O, Marien A, Berben G. 2012b. EURL-AP PCR Implementation Test 2012. Final version [Internet]. [cited 2014 Sep 19]. Available from: http://www.eurl.craw.eu/ img/page/proficiency/EURL-AP%20PCR%20ILS%202012 %20final%20version%2006-6-2012.pdf Fumière O, Marien A, Fernández Pierna JA, Baeten V, Berben G. 2010. Development of a real-time PCR protocol for the species origin confirmation of isolated animal particles detected by NIRM. Food Addit Contam: Part A. 27:1118–1127. Lassen C, Hummel S, Herrmann B. 1994. Comparison of DNA extraction and amplification from ancient human bone and mummified soft tissue. Int J Legal Med. 107:152–155. Lindahl T. 1993. Instability and decay of the primary structure of DNA. Nature. 362:709–715. Regulation (EC) No. 1069/2009 of the European Parliament and of the Council of 21 October 2009 laying down health rules as regards animal by-products and derived products not intended for human consumption and repealing Regulation (EC) No.
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1774/2002 (Animal by-products Regulation) [Internet]. 2009. [cited 2014 Sep 19]. Available from: http://eur-lex.europa.eu/ LexUriServ/LexUriServ.do?uri=OJ:L:2009:300:0001:0033:EN: PDF Regulation (EC) No. 999/2001 of the European Parliament and of the Council of 22 May 2001 laying down rules for the prevention,
control and eradication of certain transmissible spongiform encephalopathies [Internet]. 2001. [cited 2014 Sep 19]. Available from: http://ec.europa.eu/food/fs/bse/bse36_en.pdf Wilesmith JW, Wells GA, Cranwell MP, Ryan JB. 1988. Bovine spongiform encephalopathy: Epidemiological studies. Vet Rec. 123:638–644.
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