(2)

Part D, of Chapter II, point 3 is replaced by the following: ‘3. Procedure I. For complete pools (100 g of samples at a time) (a) 16 ± 0,5 ml of 25 % hydrochloric acid (0,2 % final) is added to a 3 litre beaker containing 2,0 litres ± 200 ml of tap water, preheated to 46 to 48 °C; a stirring rod is placed in the beaker, the beaker is placed on the preheated plate and the stirring is started. (b) 10 ± 1 g of powder pepsin (or 30 ± 3 ml of liquid pepsin) is added. (c) 100-115 g of samples collected in accordance with point 2 are chopped in the blender, with 150 ± 15 ml of preheated digestion buffer. (d) The chopped meat is transferred to the 3 litre beaker containing the water, pepsin and hydrochloric acid. (e) The mincing insert of the blender is immersed repeatedly in the digestion fluid in the beaker and the blender bowl is rinsed with a small quantity of digestion fluid to remove any meat still adhering. (f) The beaker is covered with aluminium foil. (g) The magnetic stirrer must be adjusted so that it maintains a constant temperature of 44 to 46 °C throughout the operation. During stirring, the digestion fluid must rotate at a sufficiently high speed to create a deep whirl without splashing. (h) The digestion fluid is stirred until the meat particles disappear (approximately 30 minutes). The stirrer is then switched off and the digestion fluid is poured through the sieve into the sedimentation funnel. Longer digestion times may be necessary (not exceeding 60 minutes) in the processing of certain types of meat (tongue, game meat, etc.). (i) The digestion process is considered satisfactory if not more than 5 % of the starting sample weight remains on the sieve. (j) The 20 microns nylon mesh filter is placed on the filtration support. The conical filtration steel funnel is fixed to the support with the block system and the steel sieve of 180 microns mesh size is placed on the funnel. The vacuum pump is connected with the filtration support and with the metal or plastic tank, to collect the digestive fluid. (k) Stirring is stopped and the digestion fluid is poured into the filtration funnel through the sieve. The beaker is rinsed with approximately 250 ml of warm water. The rinsing liquid is poured into the filtration ramp after the digested fluid has been successfully filtrated. (l) The filtration membrane is taken with the forceps, holding it by an edge. The filtration membrane is folded (minimal) in four and put in the 15 ml conical tube. The choice of conical tube must be adapted to the pestle. (m) The filtration membrane is pushed at the bottom of the 15 ml conical tube with the help of the pestle and strongly pressed by doing approximately 20 successive back and forth movements with the pestle which should be positioned inside the filtration membrane folding according to the manufacturer’s instructions. (n) 0,5 ± 0,01 ml of sample diluents is added into the 15 ml conical tube by pipette and the filtration membrane is homogenised with the pestle by doing successive low amplitude back and forth movements for approximately 30 seconds, avoiding abrupt movements to limit liquid splashes according to the manufacturer’s instructions. (o) Each sample, the negative control, and the positive control, are dispensed into different fields of the agglutination card by pipettes, according to the manufacturer’s instructions. (p) The latex beads are added into each field of the agglutination card by a pipette, according to the manufacturer’s instructions, without making them come into contact with the sample/s and controls. In each field, the latex beads are then gently mixed with a disposable stick until the homogeneous liquid covers the entire field. (q) The agglutination card is put on the 3D rocker and is rocked for 10 ± 1 minutes according to the manufacturer’s instructions. (r) After the time established by the manufacturer’s instructions, the rocking is stopped and the agglutination card is put on a plane surface and the reaction results are read immediately, according to the manufacturer’s instructions. In the case of a positive sample, the beads aggregates must appear. In the case of a negative sample, the suspension remains homogeneous without beads aggregates. II. Pools of less than 100 g as set out in Chapter I(3)(II) For pools of less than 100 g, the procedure set out in Chapter I(3)(II) must be followed. III. Positive or doubtful results Where examination of a collective sample produces a positive or uncertain latex agglutination result, a further 20 g sample is taken from each swine in accordance with Chapter I(2)(a). The 20 g samples from five swine are pooled and examined using the method described in Section I. In this way samples from 20 groups of five swine must be examined. When a positive latex agglutination is obtained from a group of five swine, further 20 g samples are collected from the individuals in the group and each is examined separately using the method described in Section I. When a positive or uncertain latex agglutination result is obtained, at least 20 g of swine muscle must be sent to the national reference laboratory for confirmation using one of the methods described in Chapter I. Parasite samples must be kept in 90 % ethyl alcohol for conservation and identification at species level at the EU or national reference laboratory. After parasite collection, positive fluids must be decontaminated by heating to at least 60 °C. IV. Cleaning and decontamination procedure after a positive or doubtful result. When the examination of a collective or individual sample produces a positive or doubtful latex agglutination result, all material in contact with meat (blender bowl, beaker, stirring rod, temperature sensor, conical filtration funnel, sieve and forceps) must be carefully decontaminated by soaking for few seconds in warm water (65 to 90 °C). Meat residues or inactivated larvae that could remain on their surface may be removed with a clean sponge and tap water. If required, a few drops of detergent can be added for degreasing equipment. It is then recommended to rinse each piece thoroughly to remove all traces of detergent.’