Int. J. Exp. Path. (I992), 73, 95-98

The HID 5o (hypothermia-inducing dose 50): an alternative to the LD5o for measurement of bacterial virulence J.S. Soothill*, D.B. Mortont and A. Ahmad* Departments of *Medical Microbiology and tBiomedical Science and Ethics, The Medical School, University of Birmingham, UK

Received for publication 24 June I99I Accepted for publication 3 October I99I

Summary. A group of 40 mice involved in bacterial LD50 estimations was monitored over a period of 4 days by two teams of observers. One team measured the colonic temperature of the mice, the other assessed them clinically and killed those they judged terminally ill. The temperatures of all mice considered terminally ill dropped before this judgement was made. Measurement of such infection-induced hypothermia provides a more objective, reproducible, and earlier endpoint than that used in conventional LD50 estimation.

Keywords: hypothermia, mice, bacteria, virulence testing, LD50, HID50 The virulence of a bacterial strain can be measured by the lethal dose 50 (LD50). This may be done in mice by injecting doses of bacterial suspension into their peritoneal cavities. In the past, death has been used as an endpoint, but to avoid unnecessary suffering more humane endpoints have been sought. Ideally, infected mice could be observed for minor signs of illness that could be used as endpoints in their own right. However, judgements about such signs are not always objective and their possible transitory nature would make such monitoring difficult. Beynen et al. (I987) used a scoring system based on the presence of clinical signs to assess discomfort (minor severity) in gallstone-bearing mice and found considerable observer variation. More substantial signs of illness have been used in rats to produce useful but approximate measures of the toxicity of chemicals (Van

den Heuvel et al. 1990). Where LD50 measurements are thought to be necessary it is customary in Britain to kill animals that are judged to be terminally ill, but such judgements are usually subjective and some workers may err on the side of killing animals late in order to ensure accuracy. A more objective endpoint is therefore desirable. Gordon and Fogelson (I990) used hypothermia as an indicator of toxicity of metal salts in mice, and Siems (I989) used several signs including a fall in temperature to predict death in infection studies of mice. We decided to investigate whether there was a level of hypothermia that was an accurate early predictor of illness which would be classified in present practice as terminal. Materials and methods LD50s are usually carried out in two stages,

Correspondence: Dr J.S. Soothill, Department of Medical Microbiology, The Medical School, Birmingham B15 2TT, UK.

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an initial dose ranging study and a formal

LD50 measurement. We measured temperature in a second stage study of virulence in mice of Pseudomonas aeruginosa (Ps. aeruginosa), and two first stage studies, one with Staphylococcus aureus (Staph. aureus) the other with Staphylococcus epidermidis, all the studies being done as parts of other projects. All strains of bacteria were human clinical isolates. Adult outbred CBA mice were used, weighing between 20 and 25 g, and were housed two or three to a cage. They were maintained in a conventional manner and given Rat and Mouse Breeding Diet 4iB (Pilsbury's of Birmingham) and water ad libitum. Males were used in the Ps. aeruginosa

LD50 estimation, females in the staphylococcal dose-ranging studies. For the Pseudomonas study, four groups of five mice received doses of bacteria that decreased in I . 5-fold steps between the groups, the highest dose being 6 x io7. For each of the staphylococcal studies, five groups of two mice received doses decreasing in twofold steps. For Staph. aureus the highest dose was 9 x io8 for Staph. epidermidis it was 5 x Io8. The mice received the intraperitoneal injections, the cages were coded to prevent bias and observations were then made by two teams of observers. One team recorded the following clinical signs when present; partial closure of the eyes, fur stand-

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on end, reduced mobility, hunching of the body, and diarrhoea. The other team then measured the colonic temperature of the mice using a probe inserted via the anus into the large intestine, so that its tip was kept at I. 5 cm from the anal margin for After the temperatures had been measured the team monitoring clinical signs, who were unaware of the temperatures of the mice, killed those they considered terminally ill. Temperatures and signs were recorded every 2 hours from 07oo to 2300 h and every 4 hours during the remaining period. Monitoring was continued for 3 days, after which the remaining mice appeared well and no more were killed or died during a further period of 4 days.

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vivors' temperatures did not show such large falls (Figs 2 and 3), the lowest temperature that was measured in a survivor being 3 5°C. The highest temperature at which a mouse was judged to be terminally ill was 3s5.2°C.

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Results room temperature during the experiment ranged from 19.2 to 23.10C with a

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of 20. IC. Three of the 20 (3/20) Ps. aeruginosainfected, 3/IO Staph. aureus-infected, and 9/ Staph. epidermidis-infected mice survived. The data for the Ps. aeruginosa strain were adequate for the LD50 to be estimated by the probit method (Finney I 964). No mouse died spontaneously; all that were dead at the end of the experiment had been killed because they were considered terminally ill; the temmean

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perature of all such mice fell before such a was made (Fig. i), but the sur-

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Discussion

Selection of a temperature to indicate terminal illness is likely to vary with ambient temperature and other factors, but in our system it would have to be less than 3 5sC. To ensure accuracy, a i°C safety margin is probably wise, and thus 340C is a suitable temperature. If this had been used as an endpoint rather than the clinical endpoint the same numbers of mice would have been judged to be terminally ill, and for the Ps. aeruginosa strain the hypothermia-inducing dose 50 (HID50) would have been numerically identical to the LD50. In the three virulence estimations nine of the 25 terminally-ill mice would have been killed earlier using a 340C endpoint than using a clinical endpoint. Three mice were killed earlier using a clinical endpoint than would have been using 340C as an endpoint. Judgements as to which symptoms were present in the mice differed considerably between the obsevers, which illustrates the subjectivity of such judgements. If a specifically trained group of observers were used such differences could probably be reduced. Use of hypothermia as an endpoint would

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which facilitates the monitoring of animals by teams, rather than by single observers. However, regular monitoring of mousetemperatures is time-consuming for the observers and probably uncomfortable for the mice and we suggest that only mice that appear ill should have their temperatures measured. Although we have worked only with bacteria the HID50 may also be applicable in toxicity tests for other agents. Acknowledgements

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reduce the length of time for which laboratory animals suffer in bacterial virulence estimations. In addition, the temperature measurement provides an objective endpoint

We would like to thank Mr P. Townsend, Miss M. Coleman, Miss D. Gunn, Dr J. Smith and Mr T. Sargent for their help in monitoring the animals, and the Wellcome Trust, The British Veterinary Animal Welfare Association, and Medical Research International for financial support.

References BEYNEN A.C., BAUMANS V., BERTENS A.P.M.G., HAVENAAR R., HESP A.P.M. & VAN ZUTPHEN L.F.M. (I987) Assessment of discomfort in gallstone-bearing mice: a practical example of the problems encountered in an attempt to recognise discomfort in laboratory animals. Lab. Anim. 21, 35-42. FINNEY D.J. (I964) Probit Analysis. Cambridge University Press. GORDON C.J. & FOGELSON L. (I990) Hypothermia and hypometabolism: sensitive indices of whole-body toxicity following exposure to metallic salts in the mouse. 1. Toxicol. Environ. Hlth, 29, I85-200. SIEMS J.J. (i 989) Quantitative means to justify the early euthanasia of rodents used in infectious disease studies. MSc thesis. Utah State University, Logan, Utah, USA. VAN DEN HEUVEL M.J., CLARK D.G., FIELDER R.J., KOUNDAKJIAN P.P., OLIVER G.J.A., PELLING D., ToMLINSON N.J. & WALKER A.P. (I990) The international validation of a fixed-dose procedure as an alternative to the classical LD50 test. Food Chem. Toxicol. 28, 469-482.