Ph. Eur. Reference Standard - LEAFLET
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1 European Directorate for the Quality of Medicines & HealthCare European Pharmacopoeia (Ph. Eur.) 7, Allée Kastner CS 30026, F Strasbourg (France) Tel. +33 (0) Fax (0) For any question: (HelpDesk) Ph. Eur. Reference Standard - LEAFLET SWINE ERYSIPELAS BACTERIA SEROTYPE 1 BRP batch 1 Swine erysipelas bacteria serotype 1 BRP batch 1 is intended for use in the immunogenicity and potency assays of swine erysipelas vaccines (challenge method) as described in the Ph. Eur. monograph swine erysipelas vaccine (inactivated) (0064). The BRP consists of freeze-dried swine Erysipelothrix rhusiopathiae strain A 360 presented in vials with an expected content of 6.5 x 10 7 Cfu 1 per vial. STORAGE Keep vials unopened at +4 C. Do not store at temperatures lower than -20 C to avoid deterioration of the rubber stoppers. USE Allow the vial and content to reach room temperature. Tap vial gently to collect material at the bottom. Using an appropriate syringe reconstitute the reference preparation by injecting a suitable volume of sterile saline (0.9 % NaCl). It is recommended to dissolve the pellet in 1.0 ml to get a bacterial concentration high enough for direct use in the challenge test. Use as soon as possible after reconstitution (direct use in challenge test or culture). CULTURE AND CHALLENGE See annex 1 2. A challenge dose for pigs is 0.1 ml (approx. 6.4 x 10 7 Cfu/ml). The challenge test is performed according to annex 2. 1 Cfu = Colony forming unit 2 It is recommended to perform a bacterial subculture. The resulting working seed will be aliquoted and freeze-dried and directly used in the next challenge tests. Catalogue code: S Revision 06 Date of issue: 28/10/2008 1/7
2 CAUTION Swine erysipelas bacteria serotype 1 BRP batch 1 is not appropriate for administration to humans or animals, other than those included in erysipelas vaccine challenge testing. Erysipelas is a zoonotic disease and safety precautions have to be followed. In case of suspected infection (e.g. self injection), please contact a medical centre or physician immediately. The challenge strain is highly susceptible to penicillin treatment. This preparation must be handled according to the QA system appropriate for biological testing laboratories. Please refer to the corresponding safety data sheet which can be downloaded from the internet web site of the EDQM ( or is delivered upon request. LITERATURE K. Cussler, S. Johannes, M.E. Esposito-Farese. Collaborative study for the establishment of erysipelothrix rhusiopathiae challenge strains as European Pharmacopoeia Biological Reference Preparations for the immunogenicity/efficacy test of swine erysipelas vaccines. Pharmeuropa Bio , , Catalogue code: S Revision 06 Date of issue: 28/10/2008 2/7
3 ANNEX 1 1. MATERIAL PREPARATION OF WORKING SEEDS FROM SWINE ERYSIPELAS BACTERIA BRP (serotype 1/ serotype 2) 1.1. Reagents, solutions and broth media Freeze-dried ampoule of swine erysipelas bacteria, serotype 1, BRP Batch 1 Freeze-dried ampoule of swine erysipelas bacteria, serotype 2, BRP Batch 1 Horse serum bouillon: 1) 25 g standard-i-bouillon (Merck) 2) Dissolve in 1 liter distilled H 2 O 3) Sterilise and recool 4) Add 50 ml horse-serum 5) Sterile filtrate. Feist-bouillon: 1) 6 g glucose 2) 5 g protease pepton No. 2 (Becton Dickinson, Heidelberg, Germany) 3) 5 g yeast extract (Merck, Darmstadt, Germany) 4) 0.5 g Arginin (Merck, Darmstadt, Germany) 5) 0.5 ml Tween 80 6) add 0.2 M phosphate buffer ph 8 to a final volume of 1 liter. Haemacel 35 (ASID, Böblingen, Germany) Sodium chloride 0.9%, sterile Blood agar plates (Merck, Darmstadt, Germany) 1.2. Laboratory equipment Shake flasks Sterilised cotton swab Drigalsky spatula Sterilised glass pipettes Incubator (37 C) Centrifuge (1,500-2,000 g) API-system (api Coryne, biomérieux, Lyon, France) 2. METHOD Catalogue code: S Revision 06 Date of issue: 28/10/2008 3/7
4 2.1. Resuspend the freeze-dried strain with 2 ml sterile saline (0.9% NaCl). Dispense 500 µl on each of four blood agar plate and cross it out with a Drigalsky spatula. The agar plates are incubated at 37 C for 48 hours Wash off each plate with 1 ml horse serum bouillon. Place these bacterial suspensions in a shake flask containing 50 ml horse serum bouillon. Incubate the flask at 37 C for at least 24 hours (max. 48 hours) Cross out this medium on two blood agar plates with a sterile cotton swab. This culture is for identification in the API-system, gram-staining and serotyping The culture (of step 2.3.) is centrifuged at 1,500-2,000 g for 15 min. The supernatant is centrifuged a second time in the same manner. The pellets are resuspended in 50 ml Feist-bouillon Perform also bacterial counting of the solution of step Add 50 ml Haemacel to 50 ml Feist-bouillon and mix well to obtain an homogeneous solution Under constant stirring transfer 2 ml of the solution into ampoules and freeze-dry Label and store the ampoules at 5 ± 3 C Perform bacterial plate counting on a representative number of ampoules. Check that all colonies grow in s-(smooth) form 3. N.B. The difference between the bacterial count of step 2.5. and 2.9. reveals the decrease of live bacteria due to the freeze-drying process. 3 If this criteria is not satisfied, the corresponding material can no longer be used as a reference challenge strain. Catalogue code: S Revision 06 Date of issue: 28/10/2008 4/7
5 ANNEX 2 IMMUNOGENICITY / POTENCY TEST OF ERYSIPELAS VACCINES IN PIGS (serotype 1 / serotype 2) 1. AIM Demonstration of immunogenicity / potency of erysipelas vaccines in pigs by intradermal challenge with virulent Erysipelothrix rhusiopathiae strains. 2. MATERIAL Syringes and needles for vaccination ( mm) Syringes and needles for challenge ( mm) Clinical thermometer Water resistent black pencil. 3. ANIMALS 15 pigs of the same origin older than 12 weeks and free of erysipelas antibodies REAGENTS Skin disinfectant Erysipelas vaccine, inactivated Swine erysipelas bacteria, Serotype 1, BRP Batch 1 Swine erysipelas bacteria, Serotype 2, BRP Batch 1 5. TEST PERFORMANCE 5.1. Randomisation Pigs are randomly distributed in two groups (10 vaccinated and 5 controls) and marked for identification Immunisation Administration of the vaccine according to the recommended schedule Preparation of the challenge strain See Annex 1. 4 The use of a validated in-house or commercial serological methodology is required Catalogue code: S Revision 06 Date of issue: 28/10/2008 5/7
6 5.4. Challenge The animals are challenged intradermally on one of the flanks 3 weeks after vaccination. 2 different injection sites should be used for challenge with the serotype 1 and 2 respectively. Depending on the size of the animal, the 2 injection sites should have a minimum distance of 20 to 30 cm. For clear observation of the skin reaction it is recommended to shave the inoculation sites. The injection site is disinfected and marked with a water-resistant pencil. The needle is inserted 2-3mm deep, under the epidermis, the needle being almost parallel to skin surface, at a minimal distance of 5 cm of the marking. Injection into the dermis is validated by the appearance of a visible papule at the injection site. The bacterial inoculum (0.1 ml) should contain Cfu/ml. 6. OBSERVATION The animals are monitored for clinical signs at least twice a day over a 7 day period. 7. THERAPY For animal welfare, pigs with typical erysipelas signs (diamond skin lesions) should be treated with penicillin and/or immunoserum. 8. EVALUATION The pass/fail criteria are laid down in the monograph LITERATURE T. G. White: Type specificity in the vaccination of pigs with killed erysipelothrix rhusiopathiae. Am. J. Vet. Res., 23, , R. L. Wood: Specifity in response of vaccinated swine and mice to challenge exposure with strains of erysipelothrix of various serotypes. Am. J. Vet. Res., 36, , EU Council Directive 90/679/EEC. On the protection of workers from risks related to exposure to biological agents at work (seventh individual directive within the meaning of Article 16(1) of Directive 89/361/EEC), OJ No L 374, , , R. L. Wood: Erysipelas. In: Disease of swine. Leman et al. (eds.), Iowa State University Press, Ames, Iowa (USA), , N. Klein, R. Goddard and C. Pugh: Zuverlässiger Schutz von Schweinen vor Parvovirose und Rotlauf. Der Praktische Tierarzt, 77 (9), , Swine Erysipelas Vaccine (Inactivated), European Pharmacopoeia, 4 th Edition (2002: 0064). PA/PH/Exp 15V/T (96) 45, ANP; Pharmeuropa, 9 (4), , Catalogue code: S Revision 06 Date of issue: 28/10/2008 6/7
7 S. Johannes, U. Rosskopf-Streicher, D. Hausleithner, H. Gyra and K. Cussler: Proposal for a standard method for pig challenge tests. Pharmeuropa Biological Special Issue, BIO 98-1, 39-44, Catalogue code: S Revision 06 Date of issue: 28/10/2008 7/7
Ph. Eur. Reference Standard - LEAFLET
European Directorate for the Quality of Medicines & HealthCare European Pharmacopoeia (Ph. Eur.) 7, Allée Kastner CS 30026, F-67081 Strasbourg (France) Tel. +33 (0)3 88 41 20 35 Fax. + 33 (0)3 88 41 27
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