Laboratory Animals (1979) 13,257-261

The influence of Bacillus piliformis (Tyzzer) infections on the reliability of pharmacokinetic experiments in mice A. SCHAICH


Department of Veterinary Virology and Immunology, Department of Pathology, and Department of Pharmacology and Toxicology, Royal Veterinary and Agricultural University of Copenhagen, 13Biilowsvej, DK-1870 Copenhagen V, Denmark Summary The half-lives of warfarin and trimethoprim were significantly longer in mice acutely infected with Bacillus piliformis and in mice which had clinically recovered from previous experimental infection with the organism. The volume of distribution of trimethoprim but not of warfarin was significantly greater in infected mice than in controls. Body clearance of warfarin was significantly reduced in both disease states. For trimethoprim this parameter was only reduced in the acute state of the disease. The importance of careful control of Tyzzer's disease in laboratory animals for use in pharmacological research is stressed.

Bacillus pili/armis infections are widely distributed in colonies of mice and rabbits (Fries, 1977a, 1978) and of rats (Fries & Svendsen, 1978) even including specified-pathogen-free (SPF) colonies. Subclinical B. pili/ormis infections are also common, and may be activated when the animals are stressed, for example by travelling or by treatment with toxic doses of drugs, resulting in disease and death. The infection may be transmitted transplacentally (Fries, 1978, 1979b) and thus maintained even in a colony where animals are obtained by hysterectomy. The pharmacokinetics of drugs may be changed by disease if the organs involved in drug elimination are affected (Wilkinson & Schenker, 1975). As B. piliformis is found within hepatocytes and causes liver necroses it was of interest to study the influence of infection on the disposition of drugs. The purpose of the present experiment was to investigate the influence of an acute B.pili/ormis infection on the half-life (T!ei)' the volume of distribution (V d) and the body clearance (ClbOdY) of warfarin and trimethoprim (TMP) in mice. In many species both warfarin and TMP are metabolized mainly in the liver (Aggeler, O'Reilly, Koller, Duckert & Streuli, 1966; Rieder & Schwartz, 1975). In addition, the same parameters were determined in mice which had clinically recovered from a previous experimental infection.


26 October 1978. Accepted 12 March 1979.

Material and methods


300 NMRI!BOM/SPF male mice, 5 weeks old, weighing 20-25 g, were obtained from the breeding colony and were housed in a conventional animal room at 22 ± 1°C and 55 ± 5% relative humidity. The mice were given a standard diet and tapwater from bottles ad libitum (Fries, 1977a).

Infective materials and experimental infection A B. piliformis strain isolated from a spontaneous

case of Tyzzer's disease in a mouse and maintained by serial passages in mice was employed (Fries, 1977b): serial passages 463-487 were used. For the acute experiments, to ensure severe infection, mice of 6 weeks of age were inoculated intravenously with 0·2 ml 2% (w/v) homogenates prepared from infected mouse liver (Fries, 1977b). In the experiment on the clinically-recovered infected animals, the mice were inoculated with 0·2 ml 0·2-0·6% homogenates, when they were 5 weeks old.

Drug administration and blood sampling Warfarin (6 mg/kg bodyweight) or TMP (40 mg/kg) dissolved in distilled water were injected intravenously in a tail vein. The volume given corresponded to 1% of the bodyweight. The experiment was performed 2 days (44 ± 2 h) after inoculation with B. pili/ormis in the . acutely-infected mice, and 8 days after inoculation in the clinically-recovered mice. Mice were exsanguinated at 30 min-IO h (warfarin) and 10 min-4 h (TMP) after drug administration. The animals were anaesthetized with ether and the blood was collected in syringes from a pocket in the axilla region formed by a cut with scissors through the skin, muscles and brachial vessels. In this way it is possible to obtain 1·0-1· 5 ml blood. Blood samples were stabilized with heparin and centrifuged to separate the plasma.

Grading of liver lesions After exsanguination of the mice their livers were examined macroscopically. Lesions on the liver surface were graded according to number and severity: 0; + « 10); ++ (10-20); +++ (20-50); ++++ (>50).

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Fries & Ladefoged

258 Determination of warfarin and TMP in plasma The plasma concentration of warfarin was determined by a spectroftuorimetric method (Corn & Berberich, 1967), while TMP was estimated by the spectroftuorimetric method of Schwartz, Koechin & Weinfeld (1969).

JJ9/ml 20



10 5

Calculation ofT!el' Vd and Clbody

Assuming the I-compartment model, half-life and volume of distribution were calculated by means of a computer program (BMD 03R) for linear regression analysis. Body clearance was calculated according to the equation:

1 20


10 5



k•. V d'

where k. = elimination rate constant = 0·693/T!'I. The statistical evaluation of the differences between the pharmacokinetic parameters was done by com-

1 20 10

parison of 2 regression lines (Diem& Lentner, 1970).

Results Some mice inoculated with 0·2 ml 2% homogenate showed ruffled haircoat and depression within 44 h of inoculation. The liver necroses of the acutely-infected, drugtreated mice, were of pinpoint size. Those of the clinically-recovered mice sacrificed on day 8 were fewer but consistently larger (0·5-2 mm diameter). Most mice inoculated with 0·2 ml 0·2-0·6% homogenate developed signs of disease, and about half of them died (Fries, I979a), 3-5 days after inoculation. At autopsy, numerous pin-point necroses were found in the livers. Almost all the surviving mice recovered clinically before the drug administration. The pharmacokinetic parameters of warfarin and TMP in the acutely infected and the recovered mice are shown in Table 1. The half-lives of both drugs were significantly longer in both acutely infected and recovered mice (Figs 1-2) than in the control animals. The distribution volume of warfarin was not altered in any of the groups of mice, compared with the values found in controls, but TMP had a significant greater distribution volume in infected mice.



600 min


Fig. I. Plasma concentrations of warfarin (6 mg/kg body weight, intravenous) in normal mice (a), and those acutely infected with Baciltus piliformis (b) and infected but clinically recovered (c). Each point represents the result of the analysis of the plasma from a single mouse.

The body clearance of warfarin was significantly reduced in both groups of infected mice, and that of TMP in the acutely infected mice (Table 1). Grouping the mice according to the number of liver necroses revealed a correlation between changes in pharmacokinetic parameters and the severity of liver disease (Table 2). The most distinct alteration was seen with TMP in acutely-infected mice, where the half-life in the severely affected mice was 3-4 times longer than that in the less affected mice or controls. The distribution volume of TMP was also significantly greater in the severely affected than in the less affected or control animals.

Table I. Pharmacokinetic parameters of warfarin and TMP in mice with Tyzzer's disease Drug Warfarin (6 mg/kg) TMP (40 mg/kg)

Number mice


T!el (min)


Clbody (lim in x kg)

68 49 66 30 39 39

none acute clinically recovered none acute clinically recovered

170 381 257 10 41 13

0·40 0·41 0·40 2·30 5·38 3·20

0·00161 0·00074 0·00106 0·158 0·092 0·171


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Differencefrom controls (p) Vd Cloody T!el

The influence of Bacillus piliformis (Tyzzer) infections on the reliability of pharmacokinetic experiments in mice.

257 Laboratory Animals (1979) 13,257-261 The influence of Bacillus piliformis (Tyzzer) infections on the reliability of pharmacokinetic experiments...
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