(2)

Chapter II of Annex I is amended as follows: (a) in point A.1: (i) point (k) is replaced by: ‘(k) A thermometer accurate to 0,5 °C within the range 20 °C to 70 °C.’; (ii) point (o) is replaced by: ‘(o) Petri dishes of approximately 90 mm in diameter, gridded with squares of approximately 1 cm, or equivalent equipment for larval counting as in point 6.14 of the ISO 18743:2015.’; (iii) point (q) is replaced by: ‘(q) Pepsin with the following strength: — if powder or granular: 1:10 000 NF (US National Formulary) corresponding to 1:12 500 BP (British Pharmacopoeia) and to 2 000 FIP (Fédération internationale de pharmacie), or — if liquid: stabilised liquid pepsin with minimum 660 European Pharmacopoeia units/ml. Other pepsin activities can be used, provided the final activity in the digest fluid is equivalent to the activity of 10 g of 1:10 000 NF as stipulated in point 5.3 of the ISO 18743:2015.’; (iv) point (s) is replaced by: ‘(s) Calibrated scale, for weighing samples and/or pepsin (accuracy ± 0,1 g).’; (b) point A.2 is replaced by the following: ‘2. Collecting of specimens and quantity to be digested As stipulated in point 4.2 of the ISO 18743:2015 (see also Annexes A and B thereto for further details).’; (c) in point A.3, points III and IV are replaced by the following: ‘III. Recovery of larvae by sedimentation — Ice (300 to 400 g of ice flakes, scaly ice or crushed ice) is added to the digestion fluid to bring its volume up to about 2 litres. The digestion fluid is then stirred until the ice has melted. In the case of smaller pools (see Section II(b)), the amount of ice must be reduced correspondingly. — The chilled digestion fluid is transferred to a 2 litre separation funnel, equipped with a vibrator in an extra clamp. — Sedimentation is allowed to proceed for 30 minutes, during which time the sedimentation funnel is vibrated intermittently, i.e. one minute vibration followed by a one-minute pause. — After 30 minutes, a 60 ml sample of the sediment is quickly run off into a 100 ml measuring cylinder (the funnel is rinsed with detergent solution after use). — The 60 ml sample is allowed to stand for at least 10 minutes, after which time the supernatant is withdrawn by suction to leave a volume of 15 ml, to be examined for presence of larvae. — For suction, a disposable syringe, equipped with a plastic tube, can be used. The length of the tube must be such that 15 ml remains in the measuring cylinder when the flanges of the syringe rest on the cylinder’s rim. — The remaining 15 ml is poured into a petri dish or equivalent equipment for larval counting, and examined using a trichinoscope or stereo-microscope. — The measuring cylinder is washed with 5 to 10 ml of tap water and the washings are added to the sample. — Digests are to be examined as soon as they are ready. Under no circumstances is examination to be postponed until the following day. Where the digests are unclear, they must be clarified as follows: — the final sample of 60 ml is poured into a measuring cylinder and allowed to stand for 10 minutes; 45 ml of supernatant fluid is then removed by suction and the remaining 15 ml is made up to 45 ml with tap water, — after a further settling period of 10 minutes, 30 ml of supernatant fluid is removed by suction and the remaining 15 ml is poured into a petri dish or equivalent equipment for larval counting and examined using a trichinoscope or stereo-microscope. — the measuring cylinder is washed with 10 ml of tap water and these washings are added to the sample in the petri dish or equivalent equipment for larval counting and examined using a trichinoscope or stereo-microscope. IV. Positive or doubtful results Where examination of a collective sample produces a positive or uncertain result, a further 20 g sample is taken from each pig, as stipulated in point 4.2 of the ISO 18743:2015 (see also Annexes A and B thereto for further details). The 20 g samples from five pigs are pooled and examined using the method described in this Chapter. In this way samples from 20 groups of five pigs will be examined. When Trichinella is detected in a pooled sample from five pigs, further 20 g samples are collected from the individual pigs in the group and each is examined separately using the method described in this Chapter. Parasite samples are to be kept in 70-90 % (final concentration) ethyl alcohol for conservation and identification at species level at the EU or national reference laboratory. For decontamination procedure, see point 12 of the ISO 18743:2015.’; (d) point B.2 is replaced by the following: ‘2. Collecting of specimens As stipulated in point 4.2 of the ISO 18743:2015 (see also Annexes A and B thereto for further details).’; (e) point B.3 is amended as follows: (i) point III (h) is replaced by the following: ‘(h) After three minutes, the plastic bag, complete with filter disc and rennilase solution, is removed from the Stomacher and opened with scissors. The liquid contents are poured into a petri dish or equivalent equipment for larval counting. The bag is washed out with 5 to 10 ml of water, which is then added to the petri dish or equivalent equipment for larval counting and examined using a trichinoscope or stereo-microscope.’; (ii) point IV is replaced by the following: ‘IV. Positive or doubtful results As laid down in point IV of Section A(3).’; (f) point C.1 is amended as follows: (i) point (f) is replaced by the following: ‘(f) Pepsin with the following strength: — if powder or granular: 1:10 000 NF (US National Formulary) corresponding to 1:12 500 BP (British Pharmacopoeia) and to 2 000 FIP (Fédération internationale de pharmacie), or — if liquid: stabilised liquid pepsin with minimum 660 European Pharmacopoeia units/ml. Other pepsin activities can be used, provided the final activity in the digest fluid is equivalent to the activity of 10 g of 1:10 000 NF as stipulated in point 5.3 of the ISO 18743:2015.’; (ii) point (g) is replaced by the following: ‘(g) Calibrated scale, for weighing samples and/or pepsin (accuracy ± 0,1 g).’; (iii) point (m) is replaced by the following: ‘(m) A thermometer accurate to 0,5 °C within the range 20 to 70 °C.’; (g) point C.2 is replaced by the following: ‘2. Collecting of specimens As stipulated in point 4.2 of the ISO 18743:2015 (see also Annexes A and B thereto for further details).’; (h) in point C.3, point VI is replaced by the following: ‘VI. Positive or doubtful results As laid down in point IV of Section A(3).’; (i) point D.1 is amended as follows: (i) point (m) is replaced by the following: ‘(m) Pepsin with the following strength: — if powder or granular: 1:10 000 NF (US National Formulary) corresponding to 1:12 500 BP (British Pharmacopoeia) and to 2 000 FIP (Fédération internationale de pharmacie), or — if liquid: stabilised liquid pepsin with minimum 660 European Pharmacopoeia units/ml. Other pepsin activities can be used, provided the final activity in the digest fluid is equivalent to the activity of 10 g of 1:10 000 NF as stipulated in point 5.3 of the ISO 18743:2015.’; (ii) point (o) is replaced by the following: ‘(o) Calibrated scale, for weighing samples and/or pepsin (accuracy ± 0,1 g).’; (iii) point (u) is replaced by the following: ‘(u) A thermometer accurate to 0,5 °C within the range 20 to 70 °C.’; (j) point D.2 is replaced by the following: ‘2. Collecting of specimens As stipulated in point 4.2 of the ISO 18743:2015 (see also its Annexes A and B for further details).’; (k) in point D.3, points II and III are replaced by the following: ‘II. Pools of less than 100 g as set out in in point 8 of the ISO 18743:2015 Where needed, up to 15 g can be added to a total pool of 100 g and examined together with these samples in accordance with Section I. More than 15 g must be examined as a complete pool. For pools of up to 50 g, the digestion fluid and the ingredients may be reduced to 1 litre of water, 8 ml of hydrochloric acid and 5 g of pepsin. 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 point 4.2 of the ISO 18743:2015 (see also Annexes A and B thereto for further details). 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 the ISO 18743:2015 or one of the equivalent methods as described above. Parasite samples must be kept in 70-90 % (final concentration) 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.’.