Dr. Klaus Cussler (project leader), Marion Krug, Paul Ehrlich Institut, 63207 Langen, Germany
Prof. Dr. Günter Müller, Martina Ecker, BgVV, 07743 Jena, Germany
Dr. Lukas Bruckner, IVI, 3147 Mittelhäusern, Switzerland
m.wegener@em.uni-frankfurt.de
Keywords: pig; cytokines, growth factors; refinement; diagnostic approaches: vaccines
Duration: 3 years Project Completion: 1999
Background and Aim
Endotoxin (lipopolysaccharide, LPS) is a constituent of the cell wall of gram-negative bacteria. It is found in many vaccines produced from these bacteria. High levels of endotoxin may give rise to a range of adverse reactions often observed in animals following vaccination. For several porcine vaccines the maximum content of endotoxin is limited to 1 x 106 IU/dose by the European Pharmacopoeia (Ph. Eur.); however, very few data are available to justify this limit.
Method and Results
In this, study pigs of various ages were vaccinated with 11 inactivated as well as 2 live attenuated vaccines. The endotoxin content of the vaccines was determined by means of the Limulus Amoebocyte Lysate Test (LAL) using the gel clot method according to the Ph. Eur. The animals were intensively monitored for 24 h after vaccination for the appearance of clinical symptoms: body temperature was measured and blood samples were taken for analysis (including blood cell count) prior to vaccination and 1, 2, 4, 6 and 24 h after injection. The chromogenic kinetic method of the LAL was used to determine the concentration of endotoxin in plasma. Release and pharmacokinetics of interleukin 6 (IL 6) and tumour necrosis factor (TNF) were determined in a bioassay by means of the murine cell line 7TD1 for IL 6 and the porcine kidney cell line PK 15 for TNF. Biologically active TNF was determined in a commercially available ELISA.
Clinical symptoms including upregulation of body temperature and leucocytosis were observed during the first 24 h after inoculation of the vaccines. However, high doses of endotoxin (> 1 x 106 IU/vaccine dose) initially decreased the number of circulating white blood cells. The animals absorbed endotoxin from the vaccine in a dose-dependent manner. Depending on the kind of vaccine (inactivated or live attenuated) we found maximum endotoxin concentrations ranging from 1,40 to 6,5 IU/ml plasma 2h after immunisation. TNF reached a peak in concentration within the first hour (260-7000 U/ml) while maximal concentrations for IL 6 were seen after the second hour (24-500 U/ml). We found large differences in the individual reactions and severity of side effects due not only to the absolute endotoxin content of the vaccine but also to the application route (i.m. or s.c.), the age of the animals and the specifications of the vaccine itself. Therefore it would not be appropriate to establish one single maximum endotoxin limit and apply it to all vaccines. Control pigs never showed any abnormal reactions (see also 3R-Info-Bulletin 18).
Conclusions and Relevance for 3R
The endotoxin limits for pig vaccines in the Ph. Eur. should be re-evaluated on an individual basis. Given the wide variation in the individual reactions, our results clearly demonstrate the need to establish an individually defined endotoxin limit for each porcine vaccine derived from gram negative bacteria. The method presented here offers parameters to investigate possible side effects more effectively. Regulations require that vaccines be "free of local or systemic adverse reactions". The clinical examination parameters presented here will serve to define more precise criteria for the safety evaluation of porcine vaccines. Improved clinical examination may help to optimise test procedures and result in a reduction of animal usage. Calculating possible side-effects in advance may avoid suffering not only of laboratory pigs.
(see also 3R-INFO-BULLETIN Nr. 20)
Published updated Version 2007
(pdf)
References
1. Wegener, M.; (1998) Untersuchungen zur Belastung von Schweinen durch endotoxinhaltige Impfstoffe, Tierlaboratorium 21, 47-54.
2. Ecker, M.; Müller, G.; (1998) Zytokinfreisetzung nach Applikation endotoxinhaltiger Impfstoffe, ALTEX 15, Suppl., 68-71.
3. Wegener, M.; Cussler, K.; (1998) Untersuchungen zur Festlegung tierschutzkonformer Grenzwerte bei der Prüfung endotoxinhaltiger Schweineimpfstoffe, ALTEX 15, Suppl., 65-68.
4. Krug, M.; Cussler, K.; (1999) Endotoxin in porcine vaccines: clinical signs and safety aspects in: Humane Endpoints in Animal Experiments for Biomedical Research (Hendrikson, C.F.M.; Morton, D.B., eds.). Royal Society of Medicine press Limited, London, 114-117.
5. Ecker, M.; Müller, G.; Köhler, H.; Cussler, K.; Krug, M.; Bruckner, L.; (1999) A possible role of tumour necrosis factor and IL-6 for the evaluation of adverse reactions of pigs after immunisation with endotoxin containing vaccines, 3rd World Congress on Alternatives and Animal Use in the Life Sciences, 29 August-02 September 1999, I-Bologna, ATLA Special Issue 27, 374.
6. Krug, M.; Cussler, K.; Ecker, M.; Müller, G.; Bruckner, L.; (1999) Side-effects in pigs after immunisation with endotoxin containing vaccines, 3rd World Congress on Alternatives and Animal Use in the Life Sciences, 29 August-02 September 1999, I-Bologna, ATLA Special Issue 27, 375.