IN RATS G. W. ASH. Imperial Chemical Industries Ltd, Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire SUMMARY

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1 Laboratory Animals (1971) 5, AN EPIDEMIC OF CHRONIC IN RATS by SKIN ULCERATION G. W. ASH Imperial Chemical Industries Ltd, Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire SUMMARY An outbreak of chronic skin ulceration in a well-established specifiedpathogen-free colony of rats is described. A coagulase-positive staphylococcus was associated with the outbreak, which occurred during winter months and quickly subsided. Various theories are advanced to account for the outbreak, the cause of which was not firmly established. EINE EPIDEMIE VON CHRONISCHER HAUTULCERATION IN RATTEN Man beschreibt einen Ausbruch von chronischer Hautulceration in einer etablierten spezifiziert pathogenfreien Rattenko]onie. Ein coagu]asepositiver Staphy]okokkus war mit diesem Ausbruch verbunden, der wahrend der Wintermonate passierte und bald vori.iberging. Mehrere Theorien werden gepriift, um diesen Ausbruch zu erk]aren, dessen Ursache nicht definitif festgestellt wurde. UNE EPIDEMIE D'ULCERATION CHRONIQUE SUR LA PEAU DES RATS On decrit une eruption d'ulceration chronique de peau dans une colonie bien etablie de rats exempts d'organismes pathogenes specifiques. Un staphy]ocoque coagulase-positif fut associe avec cette eruption qui eclata pendant les mois d'hiver et disparut rapidement. Des theories diverses sont proposees pour expliquer cette epidemie dont la cause n'a pas ete definitivement etablie. The specified-pathogen-free (SPF) Breeding Unit of the Pharmaceuticals Division of Imperial Chemical Industries was established at Alderley Park in ]959, and has been barrier-maintained ever since (Davey, 1959; Ash, 1960). The rats, of Wi star origin, are bred as a closed colony in harems, the females being separated before littering. The young are weaned at 2] days, and the females are returned to their own harem cages immediately after weaning. The young rats are housed in separate sexes, 12 to each mesh bottom cage 250 X 360 mm long (10 X 14 in). Animals are issued by weight from the stock, the number of animals per cage being thereby progressively reduced

116 G. W. ASH so that at 10-12 weeks of age there are 4 or 5 males per cage and 8 females (on average 40-50 g lighter than the males) per cage.

2 116 G. W. ASH so that at weeks of age there are 4 or 5 males per cage and 8 females (on average g lighter than the males) per cage. Watering is automatic and food is given in a hopper at the front of each cage. The colony is divided between 2 floors, maintained as separate self-contained areas-the ground floor housing I 000 and the first floor 500 breeding females. From the founding of the SPF colony, local non-specific hair-loss has been observed in the rats and briefly described (Ash, 1960). Over the last 4 or 5 years the phenomenon has been observed at an increasingly early age, from 6 weeks of age upwards. Microscopical and macroscopical examination of the skin has not established the cause, and the coat grew again after varying lengths of time, usually within 4-6 weeks. The males are weighed at least once a week, and the females are weighed at weaning, at 5-6 weeks and again at 9-14 weeks of age. Animals of suitable weight are issued to the Research Department. While weighing the rats during December] 967 the skin of a few animals was seen to have small areas of ulceration, or small areas covered with a hard scab, particularly in the head and shoulder region. METHODS The affected rats were isolated and kept under observation. In January 1968 the incidence of the ulcers increased and steps were taken to follow the course of the outbreak, and to look for factors which may have caused it, or were conducive to its spread. The following facts were recorded as each case was found: age, sex and number of animals per cage; site and brief description of the lesions; any other observation considered to be relevant. The size of the affected area varied considerably from a small scab a few millimetres wide to an ulcerating area stretching like a collar from one ear to the other, the area of a given lesion sometimes increased quite rapidly, probably due to scratching. Only in a minority of cases were bald patches or thin hair recorded when the lesion was first noticed. When not covered by a hard scab, the lesion consisted of an ulcerated area, clean and moist, the edges of which were not raised nor indurated. The lesions on 21 rats were examined for the presence of bacteria. Each lesion was swabbed and the swab used to inoculate blood agar culture plates, which were incubated for 24 hours at 37 C. Phage typing was carried out on 19 of the strains so isolated. Microscopical examinations were made of sections of tissues from the lesions of 5 rats. The tissues were fixed in Zenker acetic solution and embedded in paraffin wax; some sections were stained with haemotoxylin and eosin; Gram and periodic-acid-schiff methods being employed on others to search for bacteria and fungi. Whole animals were inspected for the presence of external parasites.

3 EPIDEMIC SKIN ULCERATION IN RATS 117 Infected animals not used for these investigations were either painlessly destroyed without further examination if the lesions were extensive, or isolated and observed for recovery. Since all the lesions looked alike, and there were no other syndromes recognised at the time to confuse the picture, examinations of all the lesions were not carried out. The possibility that the occurrence and spread of the ulcers was in some way connected with population density in the cage was considered, but it was soon realised that the number of rats present in the cage at the time of recording an affected rat was not significant, because the cage population was rapidly and constantly changing. A way had to be found of setting up different conditions, the results of which might throw light on this point. This had to be done within the existing system-it was not possible to set aside space or rats to carry out any separate investigation. From early in March male and female weaners were put in the cages in groups of 8 and 11 alternately, so that half the rats started off in the stock colony at 8 per cage, the other half at 11 per cage. This was discontinued in June, after 16 weeks, at the same time as the general recording. Numbers RESULTS of rats affected Records were kept from 29 January to 22 June 1968, a total of 21 weeks, by which time the incidence of the disease was very low (Fig. I). During 11 > 10 Q) Q) 9 "0 c :J 8.2 Males o Females....., 7 "0 6 u 5 c 4 '0.c 3 E 2 :J <- [ Ie JJJJ[J JJ 0 ) [101\ 24, } b" 6, J \ 2b b 4, 18 25,,\ 1'5 i2 1 February March April May June Week ending Fig. 1. Numbers of rats with lesions recorded weekly.

4 118 G. W. ASH this period 102 rats were found with lesions-78 males and 24 females-and a study of the sites of the lesions showed that in 93 % of the males and 96 % of the females they occurred in the anterior region, around the head and shoulders. Table 1 shows the distribution as percentages within the sexes. It is not easy to find a meaningful way of expressing the number of rats at risk. The rat population at anyone time would only be estimated, because the duration of maintenance in stock varied from 1 day to 14 weeks, depending on stock levels and demand at the time. During the 21 weeks of recording, the average stock levels were of the order of rats at anyone time. In this period, a total of males and females were weaned and put into stock, later to develop lesions at rates of 6.82 and 2.02 per respectively. Table 1. Percentage distribution of skin lesions in 102 affected rats (78 rj rj, 24 ). Males Females Around ears Dorsal region of neck 15 4 Shoulder region Dorsal and other areas The rats caged at the experimental densities produced a smaller number of affected animals because less were at risk. From the group with 8 per cage, 7 males and 1 female were recovered (1.65 and 0.23 per 1 000) while from the group with 11 per cage, the numbers were 12 males and 3 females (2.84 and 0.68 per 1 000). Bacteriology 20 cultures yielded coagulase-positive staphylococci (Staphylococcus aureus); in 7 the organism was present in pure culture, and in the remainder it was the predominant species. 1 rat yielded a coagulase-negative staphylococcus. Of the 19 strains which were phage-typed, 11 were type 42E, and 8 were 6/47/53/54/75/77 /83A/84/85. These strains of human origin were, therefore, similar to those identified from rats and mice in the Breeding Unit in 1965 (Wheater, 1965). Histopathology The sections showed ulceration of the epithelium and chronic inflammation of the dermis. A few Gram-positive cocci were observed on the surface of the ulcerated areas. Fungal infection was not detected, nor was there any evidence of parasitic invasion.

5 EPIDEMIC SKIN ULCERATION IN RATS ]]9 Recovery times Because of the higher incidence of lesions among the males, 45 males but only ] 3 females were isolated and observed for recovery. The lengths of time taken were all within the same limits, and of course depended to some extent on the severity of the lesion and its age when first found. The times taken varied from 7 to 38 days, and these periods, related to the week of onset, are shown in Fig. 2, the sexes being considered together. III OJ 8 c: o ';;;,.2 '"> III "tl '0 OJ.0 E :J Z 15 ZZ, June Week ending Fig. 2. Healing time related to week of onset. DISCUSSION The method of issuing rats leads to all the males being weighed at least once a week, but the females less frequently, and this difference in frequency of weighing, and therefore in extent of examination of the sexes, must be borne in mind when studying the incidence by age (Fig. 3). Because the males are weighed more often, these figures should give a truer picture of the age at which their lesions occurred, and it can be seen that the most susceptible age is 7-9 weeks, with a reasonably high incidence in the weeks immediately before and after.

6 120 G. W. ASH 32 Males 32 Females '"c 0 "u; " '" -0 *" , <3 Age in weeks o.,....., Age in weeks Fig. 3. Incidence of skin lesions relative to age. Since the age of onset is apparently (in the males at least) fairly well defined, it seemed interesting further to attempt to relate the incidence to the dates of weaning (Fig. 4). It is seen that rats weaned during January and the first half of February appear to have the higher incidence of lesions. From the standpoint of a conclusive answer, the re-arrangement to compare the effects of different cage densities came a little too late, as by the end of March the incidence was found to be falling (Fig. 1) and the numbers of affected rats subsequently counted were too small to enable firm conclusions to be drawn. The indications were that a higher proportion of cases was observed from the cages which originally had 11 than from those which had 8. The outbreak occurred mainly during January and February 1968, in stock rats of both sexes, although more males than females were affected. The male cases clearly occurred mostly between 7 and 10 weeks of age: the pattern among females could not be identified because of less frequent examination. No similar outbreak has been experienced before, although the organism isolated from the lesions has been present in the Unit for nearly 8 years, both in rats and mice. This organism, Staphylococcus aureus, was first isolated

7 EPIDEMIC SKIN ULCERATION IN RATS MALES 17 o FEMALES '" c 13 0 'U; 12 2! ' on 9 '0 8 'if!, IJ JJJJ JJ I' I' I I I, I I I I I I I I I, I I I,,,2 9 I 2330,, ,, , Nov December January February March April May Weaned during week ending Fig. 4. Incidence of skin lesions according to the date of weaning. from facial abscesses in mice in 1960, having been introduced, it was thought, by a human carrier among the Breeding Unit staff, The incidence of these abscesses in mice has since slowly declined practically to nil, but it is possible that the strain of staphylococcus became more virulent for rats giving rise to a number of cases of tissue invasion, leading to ulceration. The fact that the incidence of lesions was higher in the males suggested fighting as a pre-disposing factor, but this was ruled out for 2 reasons. This strain of rat has been bred for over 25 years, and in that time fighting has never been observed to any degree; furthermore there was no evidence of real fighting in the form of blood on the rats or on the cage, such as has been seen on the very rare occasions when a fight has occurred. The young rats do 'play', however, and at night they have been observed to roll each other over, often by pseudo-attacks on the head and shoulder region, and the possibility exists that minor abrasions so produced became infected. Areas of skin deficient in hair have been present on rats in the Unit for a long time, and although not always directly associated with a lesion when found, it is possible

122 G. W. ASH that these hairless patches contributed to the genesis of the ulceration, but this could not be proved. The recovery times showed a tendency to drop from February to June (Fig.

8 122 G. W. ASH that these hairless patches contributed to the genesis of the ulceration, but this could not be proved. The recovery times showed a tendency to drop from February to June (Fig. 2), although peaks did occur at some time in each month. It is tempting to think of this time-reduction as a result of increased host reaction, decreased virulence of an invading organism, or a combination of both. The incidence has continued to decline, and only 3 cases have been found in the past 17 months, including 2 winters, suggesting a sub-clinical infection on the way to a state of equilibrium. Blackmore & Francis (1970) described an outbreak of disease associated with coagulase-positive staphylococci corresponding to human phage types. The SPF animal population involved consisted of mice, rats, guinea-pigs, rabbits and cats, and infections, some of which were skin lesions, occurred in members of all species except the cats. The rabbits constituted a problem because it was thought at one time that the infection was being spread, not only from human to rabbit, but from rabbit to rabbit. The possibility that the outbreak at Alderley Park was of dietary origin was considered. The diet used is made by a commercial manufacturer to a formula supplied by Imperial Chemical Industries, and has been in use for many years. The cases occurred during winter months at a time when old food stocks were running out (to be replaced by newly-harvested material). It is possible that resistance could be lower at this time of the year, providing a contributory factor. Despite the maintenance of relatively stable physical conditions inside the Unit, there is evidence that at least one of the species of rodent still shows seasonal variation in certain characteristics. The cause of the outbreak has not been clearly identified, and although the number of animals involved was not great, it is thought that the incident should be recorded for its potential significance to others engaged in the management of SPF or 'clean' colonies of rodents. ACKNOWLEDGEMENTS I would like to express my thanks to Dr T. D. Hennessey for his help with the bacteriological work, and to Miss M. Tucker for the histopathological investigations. REFERENCES Ash, G. W. (1960). A progress report on the Alderley Park Breeding Unit. Journal of the Animal Technicians Association 11, 10. Blackmore, D. K. & Francis, R. A. (1970). The apparent transmission of staphylococci of human origin to laboratory animals. Journal of Comparative Pathology 80, 645. Davey, D. G. (1959). Establishing and maintaining a colony of specific pathogen free mice, rats and guinea pigs. Laboratory Animals Centre Collected Papers 8, 17. Wheater, D. W. F. (1965). The bacterial flora of an S,P.F. colony of mice, rats and guinea pigs. In The husbandry of laboratory animals (cd. M. L. Cona1ty), p London & New York: Academic Press.

EXOSTOSIS OF THE MANDIBLE OF THE CHICKEN

EXOSTOSIS OF THE MANDIBLE OF THE CHICKEN COMPLICATING EDEMA OF THE WATTLES GEORGE MILTON SMITH, M.D.1 (AnutomioaZ Laboratory, Yale School of Medicine, New Haven, Connecticut) During the past year opportunity