3R-INFO-BULLETIN 18

September 2001

The Authors

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.

Marion Krug kruma@pei.de
and
Klaus Cussler cuskl@pei.de

Paul-Ehrlich- Institut Bundesamt für Sera und Impfstoffe
63207 Langen
Germany

Editor
Peter Maier, Scientific Adviser of the 3R Research Foundation

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.

Table 1
Table 1: Vaccines tested, range of endotoxin concentration and number of tested

Fig 1
Fig.1: Body temperature (1a), white blood cell count (WBC) (1b), endotoxin concentration in the plasma (1c), after application of vaccines containing different amounts of endotoxin [<1x10⁶ IU/dose (n=44), >1x10⁶ IU/dose (n=25).

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).

Table 2
Table 2: Animals (%) showing clinical signs after application of vaccines containing different amounts of endotoxin [<1x10⁶ IU/dose (n=44), >1x10⁶ IU/dose (n=25)].

Fig 2
Fig. 2: Pigs during investigation.

Important consequences

1. Each type of vaccine needs its own endotoxin limit:
The general limit of 1x10⁶ IU endotoxin/dose is no guarantee for safety. The systemic responses of the animals towards the various vaccines containing endotoxin showed a great variability. This is not surprising given the differences in vaccine compositions (species of bacteria, choice of adjuvants, etc.). Our results demonstrate the need to define the individual endotoxin limit for each porcine gram-negative bacterial vaccine separately.

2. Revision of the regulations:
Based on the results of this project, Requests for Revision concerning the endotoxin content and safety aspects of all vaccines containing gram-negative bacteria were submitted to the European Directorate for the Quality of Medicines by the German Pharma-copoeia Commission in September 2000 (6). These Requests for Revision have been endorsed by Expert Group 15V of the Ph.Eur. As a result, revised standards will be published soon. 3.Standardized clinical examination parameters reduces the number of animals required for vaccine testing: Clinical examination parameters developed in this project will serve to define more precise criteria for the safety evaluation of porcine vaccines. Improved clinical examination may help to optimise test procedures and avoid suffering. With the help of a standardised score to interpret test results, a reduction in animal numbers for vaccine testing might be achievable.

References:

1. Culbertson, Jr.R.; Osburn, B.I. (1980): The Biologic Effects of Bacterial Endotoxin: a short Review. Veterinary Scientific Communications 4, 3-14
Cort, N.; Kindahl,H. (1980): Endotoxin-Induced Abortion in Early Pregnant Gilts and its Prevention by Flunixin-Meglumine. Acta Veterinaria scandinavia 31, 347-358
Ecker, M.; Müller, G. (1998): Zytokinfreisetzung nach Applikation endo-toxinhaltiger Impfstoffe. ALTEX 15, Suppl., 68-71
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
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
Requests for Revision of the Ph.Eur. monographs "Neonatal piglet colibacillosis vaccine (inactivated)" (1997:0962) "Porcine actinobacillosis vaccine (inactivated)" (1999:1360) "Porcine progressive atrophic rhinitis vaccine (inactivated)" (1999:1361)

¹:

Endotoxin and side effects after vaccination
Free endotoxins (lipopolysaccharides, LPS) are potent pyrogens (fever-causing substances) produced by the lysis of the bacterial cell wall. As they resist most common inactivation methods (heating, chemical detergents etc.), they can be found in varying amounts in many vaccines derived from gram-negative germs. LPS in low amounts have an immunostimulatory effect whereas high concentrations of free LPS cause severe adverse reactions such as hyperthermia, anorexia, circulatory disorders, changes in blood parameters, abortion and shock symptoms (1, 2). Many of these reactions are caused by cytokines such as TNF alpha and interleukin 6 (IL-6), which are released into the blood even after exposure to low LPS concentrations. Due to current mass breeding and housing conditions, observations on individual animals are often not possible. Side effects after mass immunisation programs (in Europe up to 108 pigs are vaccinated each year) often go unnoticed, or are put down to the high susceptibility of pigs towards stress.