The project was carried out by Dr. Marion Krug in the laboratories of the Paul-Ehrlich-Institut in Langen (Germany) at the Bundesamt für Sera und Impfstoffe under the quidance of Dr. Klaus Cussler, Animal Welfare Officer of the Institute. For this work, Dr. Krug was awarded the Tierschutz-Förderpreis 2000 of the Erna-Graff-Stiftung, Berlin.
Paul-Ehrlich- Institut Bundesamt für Sera und Impfstoffe
Prevention of adverse effects in pigs after vaccination
This project on the improvement of the correct definition of harmless endotoxin levels in porcine vaccines was funded by the 3R Research Foundation Switzerland. The results obtained will lead to revised standards in Europe concerning the safety aspect of vaccines for pigs and to a reduction in animal numbers for vaccine testing.
Vaccination of livestock and side effects
Vaccination is a major tool in veterinary medicine for the prevention of infectious diseases in livestock. In particular, bacterial vaccines prepared from gram-negative germs are extensively used. These may contain varying amounts of lipopolysaccharides (LPS) from the cell walls of the bacteria. At high doses these LPS, also known as endotoxins1, can induce many harmful biologic effects (1,2).
Acceptable LPS levels in veterinary vaccines
Before vaccines for veterinary use are licensed, their safety has to be thoroughly demonstrated. Safety tests ensure the lack of adverse reactions in the animals for which they are designed (target species) after the application of a single or repeated single doses, and after an overdose. Effects on the reproductive and immune systems must likewise be ruled out. After licensing, every batch of the vaccine also has to pass a further safety test in the target species: two animals receive a double dose of the vaccine (or tenfold dose for live vaccines) by the recommended administration route. The animals are observed for a certain time period and the rectal temperature is taken prior to vaccination and 2 h, 4, 6, 24 and 48 h after the injection. Neither of the animals should get a fever above 2°C over basal values or show any signs of abnormal local or systemic reactions.
Current standards of the European Pharmacopia (Ph.Eur.) limit the endotoxin content of a vaccine to 1 x 106 IU/dose. A higher dose might be acceptable for a specific vaccine if the safety test has proven the absence of adverse reactions.
Do current endotoxin limits guarantee no adverse effects?
Very little data are available to justify the endotoxin limits published in the Ph.Eur. for pig vaccines. In particular, a quantitative relationship between endotoxin content and possible systemic or local reactions in the animals has yet to be established. The aims of this project were 1) to measure the precise endotoxin concentration in vaccine batches whose safety has been demonstrated in animal tests and 2) to improve the evaluation of the clinical reaction of the pigs after injection, in order to 3) define safe endotoxin levels in vaccines for pigs.
In total, 11 different inactivated vaccines and two live attenuated vaccines were tested in pigs of various ages (Table 1). The vaccines were administered by the route recommended by the manufacturer (intramuscular (i.m.) or subcutaneous (s.c.)).
During the observation period (24 h after injection), the animals showed a large variety of serious clinical signs: hyperthermia (Fig.1a), loss of appetite, lethargy, accelerated respiratory rate and circulatory disorders (Table 2).
Vaccines with high levels of endotoxins (above the limit stated in Ph.Eur.) caused a decrease in the number of white blood cells (Fig.1b).
The animals absorbed the endotoxin from the vaccine in a dose-dependent manner, as we were able to show by plasma analysis using the Limulus Amoebocyte Lysate Test (see also 3R Bulletin 17) (Fig.1c). The absorption rate of the endotoxins was much higher following i.m. than after s.c. injection. The time course of the release of two the cytokines IL-6 and TNF alpha underscored the causal relationship between the administration of the endotoxin-containing vaccine and the appearance of the visible clinical disorders (3). However, large variations in the quantitative reactions were observed between individuals. The control pigs in our experiments showed no abnormal reactions due to the sham vaccination or bleeding, and thus the effects noted in vaccinated animals are almost certainly related to the endotoxin content of the vaccines (4, 5).
1. Each type of vaccine needs its own endotoxin limit:
2. Revision of the regulations:
Endotoxin and side effects after vaccination