Osteodystrophic diseases of sheep

J.

COMP.

PATH. 1966.

VOL.

159

76.

OSTEODYSTROPHIC II.

DISEASES

RICKETS

IN YOUNG

OF

SHEEP

SHEEP

BY D. I. NISBET,

E. J. BUTLER* Moredun

Research Institute,

and B. S. W. SMITH Edinburgh

and J. M. ROBERTSON Vet&my

Investigation

Department,

and C. C. BANNATYNE West of Scotland Agricultural

College

INTRODUCTION Comparatively few accounts are to he found in the literature concerning the natural occurrence of rickets in young sheep in this country. Innes (1934-35) stated that “no fully authenticated cases of rickets in sheep have yet been recorded in England, and, therefore, its existence as a disease entity in this animal in this country is still in doubt,” and later Ewer (1950), describing the prevention of rickets in sheep in East Anglia, said that the condition was apparently not common in Britain. Innes was referring to the absence of histological confirmation in any cases which had been described as rickets, while Ewer was referring to clinical evidence of rickets. Little further investigation of the histopathology of rickets in hoggs (young growing sheep 5 to 12 months old) in this country appears to have been carried out in the last 30 years, and much information that has been recorded on both natural and experimentally-produced cases is the result of clinical, biochemical and radiological observations (Ewer, 1950, 1951a, 1951b; Crowley, 1961; McRoberts, 1961; McRoberts and Hill, 1962). Th ese findings, of course, have the advantage that results can be obtained at intervals throughout the course of the experimental or natural disease, while histopathological examination necessitates the death of the animal and hence provides only one result at one stage in the course of the disease for any given animal. Rickets? however, is one of the diseases in which the histopathologist is able to provide m all cases, but more particularly in the milder type, a more accurate diagnosis than the clinician or the radiologist, and attention has already been drawn to this by Fitch (1943), Follis, Jackson, Elliott and Park (1943) and Fitch and Ewer (1944). The biochemist, by analysis of blood and bones, provides invaluable additional evidence, but the conclusive diagnosis is the histopathological one. This paper records the results of investigations into the occurrence of rachitic disease in young sheep on four selected farms using both histological and analytical methods. MATERIALS AND METHODS A general description of the way in which material was obtained for these investigations has already been published (Nisbet, Butler, Bannatyne and Robertson, 1962), but in this procedure the frequency of sampling and acquisition of sheep for post-mortem examination depend on the co-operation of the flock-owner, and so * Present address: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge.

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are limited. In order to provide a better selection of sheep for blood sampling and post-mortem examination we grazed small flocks of our own sheep on two pastures (Farms H and I) were stiffness and dental malocclusion (Shanks and Donald, 1955; McRoberts, 1961; Duckworth, Benzie, Cresswell, Hill, Dalgarno, Robinson and Robson, 1961) had been recognised in hoggs grazed durmg the winter months from November to April. This enabled us to obtain such samples as were considered necessary at appropriate intervals and to select a representative number of sheep for postmortem examinations. Pathological investigations. In all 18 hoggs aged 9 to 12 months, including 8 clinical cases of rickets, from 4 farms were submitted to post-mortem examination : 8 were of the South Country Cheviot breed, 7 were Blackface, 2 were North Country Cheviots, and 1 was a Greyface. Material for histological examination was taken from the thoracic and abdominal organs and from the skull, mandible, lumbar vertebrae, ribs, costochondral junctions and limb bones. All tissues were fixed in 10 per cent. form01 saline neutralised by saturation with MgCO,. Bone blocks were decalcified in 5 per cent. nitric acid by the method described by Clayden (1952), while some duplicate bone blocks were prepared using the method described by Ball (1957), cut without prior decalcification and stained by von Kossa’s method. For bone sections Harris’s haematoxylin was found to give superior results to that of Mayer. Chemical investigations. Blood samples were collected for chemical analysis from 28 clinical cases of rickets from the 4 farms mentioned above. The samples were analysed for inorganic phosphate, calcium, alkaline phosphatase, magnesium, haemoglobin and protein. The 4th rib and tibia from the right side were taken for ash and mineral element analysis from 12 of the above cases when they were slaughtered. For comparison similar bones were also collected from 10 normal sheep of the same breed and age, in which no abnormalities were found on histological examination. The preparation of the samples and the analytical methods used have been described previously (Nisbet et al., 1962). RESULTS

Farms A description is given of the four farms from which we received material. Farm H. This farm situated in Midlothian comprised 600 acres of permanent grazing varying from 200 to 800 feet in altitude. There was a history of progressive loss of condition in hoggs grazed throughout winter with little or no supplementary feeding, followed in late winter and early spring by the occurrence of stiffness, lameness and dental malocclusion in many of these hoggs. We therefore selected 20 South Country Cheviot wether hoggs, 6 months old, from a group of 120 at Moredun Institute, which had just been purchased at a local market. In order to establish as far as possible that they were normal they were examined clinically, and blood and faeces samples were taken for chemical analysis and worm egg counts respectively. The 20 hoggs were dosed with phenothiazine, and were then allowed to graze this pasture from 6.12.58 until 20.3.59 when circumstances prevented them being kept on for longer. They were given no supplementary feeding and during this time they were examined clinicaIly and blood samples were collected on 5 occasions. At the conclusion of the grazing period, 8 of these hoggs, including 3 clinical cases of rickets, were killed and examined post-mortem. Farm I. The farm situated in Lanarkshire was composed of 45 acres arable grazing and 150 acres rough moorland grazing at an altitude of 800 feet. There was a similar history of loss of condition, stiffness, lameness and dental malocclu-

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sion in late winter and early spring in Blackface hoggs grazed in previous years with no supplementary feeding. One hogg had shown symptoms of stiffness and lamness which were accounted for at post-mortem examination by lesions of rickets. In October 1958 we selected 15 Blackface ewe hoggs, 6 months old, from the group of 120 at Moredun Institute. Clinical examination, blood analyses and worm eggs counts confirmed that they were normal as far as could be determined, and they were put out to graze on Farm I from 22.10.58 until 7.4.59. No supplementary feeding was given during this period, during which they were examined clinically and blood sampled on 4 occasions. One of the hoggs died on 27.2.59. On 7.4.59, 5 more, including one clinical case of rickets, were killed and examined post-mortem. Farm J. This arable farm situated in Ayrshire was on low ground. Stiffness had been observed in hoggs over a period of about 7 years, but only in alternate years. Out of a group of 60 Greyface (Border Leicester X Blackface) hoggs 30 were affected in February 1959. They had been receiving *lb. per head of a concentrate mixture daily from November 1958 onwards. One of the affected hoggs was obtained for post-mortem examination. Farm K. This arable farm in Dumfriesshire was also on low ground. No stiffness or lameness was observed until early February 1959 when a group of 120 North Country Cheviot hoggs folded on mangolds started to lose condition and became lame and stiff in their forelegs. Apart from lack of access to grass they had been fed generously from December 1958, getting as many mangolds as they would eat together with about llb. concentrate mixture and also some cake daily. About 30 out of the 120 hoggs were affected. The owner stated that when purchased in August 1958 the animals were in poorer condition than similar lambs obtained in previous years from the same sources. Two affected hoggs were obtained for post-mortem examination. Clinical

Features

These fell into 2 groups, which were related to the type of farm and the method of husbandry. Thus the cases on the hill farms, H and I showed features which were less severe and dramatic than those on the arable farms J and K. Farms H and I. Loss of condition was the first indication of disease and was noticed towards the end of January, becoming progressively more marked during the next 2 months, until by March all the hoggs on both farms were in poor condition. Stiffness of gait became apparent early in February in a number of hoggs, 6/20 on Farm H and l/15 on Farm I, and signs of cappi (osteoporosis) were detected in most of these and in a few others by pressure on the frontal bone with the thumb. There was no evidence of dental malocclusion in any of these animals. Farms J and K. Symptoms were first noted at the beginning of February when a high proportion of the hoggs showed lack of appetite with resultant loss of condition. They became listless and were stiff or even lame, preferring to spend much of the time lying down. When standing, all four legs were kept together underneath the belly like a foundered horse. The forelimbs were most severely affected and when walking these were hardly flexed at all but moved in one piece. When made to run the sheep tended to collapse on the ground with their forelimbs and

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heads shaking and shivering. The worst affected sheep lay on their sides to remove all weight from their limbs, with the forelimbs stretched straight out from the body as if flexing them caused discomfort. Some of the affected hoggs showed enlargement of the carpal and fetlock joints (Fig. 1). There was no clinical evidence of dental malocclusion. Anatomical

Findings

The carcases of the hoggs from farms H and I were in much poorer condition than those from J and K, since they had received no supplementary feeding throughout the winter. Farm H. Of the 8 hoggs examined only 2 were in good condition, 3 were poor and the remaining 3 were emaciated. Four showed macroscopic lesions of rickets and these included the 3 which had shown stiffness when clinically examined previously. In all 4 the lesions involved the distal ends of the metacarpal and metatarsal bones, but similar lesions were also present in other sites such as the proximal ends of the femur and tibia and the distal end of the radius. The lesion consisted of a thickening of the epiphyseal cartilage which was sometimes irregular, but was more commonly uniform, the cartilage being up to 3 to 4 times the normal thickness. Another common finding in the distal ends of the metacarpal and metatarsal bones was a white linear zone closely applied to the metaphyseal aspect of the epiphyseal cartilage. One of the 2 hoggs in good condition showed no macroscopic skeletal abnormalities. All 6 hoggs in poor or emaciated condition showed additionally signs of cappi with marked thinning of the frontal bone and deficiency of cortical and cancellous bone in long bones such as the femur, easily demonstrable in longitudinal section. Rib fractures were also common. Farm I. All 7 hoggs examined were in poor condition with signs of osteoporosis. Macroscopic lesions of rickets were present in 5 of the 7. In one, which on clinical examination had been stiff and had shown enlargement of both fore fetlock joints, the distal ends of the metacarpal bones were expanded. Another of the 5 had also shown signs of stiffness during life. The lesions of rickets were similar to those described above, the distal ends of the metacarpal and metatarsal bones invariably showing thickening, either uniform or irregular, of the epiphyseal cartilage, with a white linear zone on its metaphyseal aspect in 3 of the 5 cases (Fig. 2). In the animal with expansion of the distal metacarpal bones the distal epiphysis could be separated from the shaft quite readily with only slight pressure. Similar thickening of epiphyseal cartilages was also found in the distal radius and the proximal and distal tibia of these hoggs (Fig. 3). In 4 hoggs there were also macroscopic abnormalities in the mandible. The coronoid processes were more horizontal than normal and the vertical parts of the mandibular rami were shortened as if compressed. The horizontal parts of the rami in the region of the 5th cheek tooth were thickened but felt very spongy, and the at-tic&r condyles were small and flat. Farm J. The one hogg examined showed lesions of rickets in the costochondral junctions, the distal ends of the humerus and radius and the proximal and distal ends of the tibia. Other bones were not available for examination. The epiphyses were expanded and the epiphyseal cartilages were 4 or 5 times the normal thick-

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ness and showed considerable irregularities. There was no macroscopic evidence of osteoporosis. Farm K. Both the hoggs examined had obvious lesions of rickets and enlarged carpal joints had been seen during life. The distal extremity of the radius was expanded and the epiphyseal cartilage was about 4 times the normal thickness. Similar changes were present in the co&o-chondral junctions, the tibia, humerus, metacarpal and metatarsal bones. Histopathology Lesions of rickets were found in 6 out of the 8 hoggs examined from Farm H, the 3 with the most extensive lesions having shown stiffness in life; and in all the other 10 hoggs examined from Farms I, J and K. The lesions seen in the hoggs from Farms J and K were in general more severe and extensive than those from Farms H and I. The mildest cases showed only osteoid margins on the trabecular bone at the costo-chondral junctions or in the mandible. The more severe cases had, in addition, epiphyseal cartilages in many of the long bones (the tibia, radius, metacarpal and metatarsal bones, in particular) which showed deficient provisional calcification, with a resultant increase in the numbers of mature cartilage cells due to failure of removal. Although the epiphyseal cartilages were increased to 4, 5 or more times the normal thickness, the normal columnar pattern of the cartilage cells was maintained in all but the most severe lesions. In these the metaphyseal zones contained masses of uncalcified osteoid tissue, islands of cartilage cells and traumatic damage with areas of haemorrhage at the junction of cartilage and shaft, causing partial separation of the epiphysis and pressure necrosis of mature cartilage cells. The marrow in the region of such lesions often appeared to contain much fibrous tissue (Figs. 4, 5 and 6). Defective provisional calcification of the epiphyseal cartilages and the presence of osteoid seams on trabecular bone were readily demonstrable in undecalcified material stained by the von Kossa method using neutral red or safranin as counterstain (Figs. 7 and 8), but osteoid could also be recognised as stated by Meyer (1956) in decalcified bone stained by Harris’s haematoxylin and eosin. Histological examination also confirmed that the white zones seen post-mortem on the metaphyseal aspect of the epiphyseal cartilage of the distal ends of the metacarpal and metatarsal bones in many of these hoggs consisted of uncalified osteoid. Further description of the rachitic lesions is unnecessary since they conformed closely to the comprehensive account of rickets in young sheep in New Zealand provided by Fitch (1943). Histological evidence of osteoporosis was found in 6 of the 8 hoggs from Farm H and in all 7 hoggs from Farm I, the changes being marked in 3 of the latter animals. On the other hand only very slight evidence of osteoporosis was seen in the hoggs from Farms J and K. In addition to osteoporosis, transverse lines representing periods of arrested growth were found in the metaphyses of the proximal ends of the tibia and humerus of 4 hoggs from Farm H and in 2 from Farm I, but not in any of those examined from Farms J or K. No significant abnormalities were found in tissue other than bone in any of these sheep.

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Biochemical Results The results obtained for the blood samples from 28 clinical cases of rickets are summarised in Table 1, where they are compared with the normal values in 15 cases of osteoporosis (cappi) of the same breeds and age, and with the normal ranges used for diagnostic purposes. The osteoporotic sheep were described in a previous paper (Nisbet et al., 1962) and the results have been used for comparison because of the similarity in breed and age, and because histological examination had excluded the presence of rickets or other concurrent bone disease. They provide normal values because in uncomplicated osteoporosis there is no alteration of the mineral content of the blood. A distinction has been made between the values for rachitic sheep from Farm K and those from the other farms since significant differences were found in the inorganic phosphate and calcium levels of the two groups. It can be seen that the latter showed the classical blood picture of rickets in that they had abnormally low phosphate and calcium levels and high alkaline phosphatase. The development of this blood picture was shown by the samples which were taken at various intervals from the sheep on Farms H and I before the appearance of clinical abnormalities and these results will form the subject of a separate communication. The serum calcium values for the clinical cases from Farm K were even lower than those for the cases from the other farms, but their blood inorganic phosphate levels were very much higher. In view of these differences only the results for sheep from Farms H, I and J are included in Table 2 which summarises the information obtained on the chemical composition of the bones. This shows that the phosphorus, calcium, magnesium and ash contents of the ribs are all significantly lower than the corresponding values for normal sheep. Differences in this direction are also shown by some of the results obtained for the tibia, but since this bone has a more static composition the differences are less marked. Bones from one sheep from Farm K were analysed and the results are similar to those for the above cases with the exception of the figures for the phosphorus, calcium and ash content of the tibia (10.2, 24.8 and 55.5 per cent w/w respectively) which are slightly more than two standard deviations below the corresponding mean values for the cases from Farms H, I and J. DISCUSSION

Since the original investigations of Elliot and Crichton (1926) on the nature and cause of “Bent-leg” in sheep, research on rachitic conditions of sheep in Britain has been carried out by Auchinachie and Fraser (193 l), Duckworth, Godden and Thomson (1943), Ewer (1950, 1951a, 1951b), Benzie, Boyne, Dalgarno, Duckworth, Hill and Walker (1960), McRoberts (1961), McRoberts (1963). These papers are mainly concerned and Hill (1962) and Hemingway with experiments designed to reproduce the disease by means of diets deficient in calcium, phosphorus or vitamin D. The clinical, biochemical and pathological aspects of this disease, as it occurs under natural conditions of sheep husbandry, have received much less attention. Our investigations have shown the presence of rickets, confirmed by histological examination and by chemical analyses of blood and bones, in 16 young growing sheep, 8 of which showed clinical symptoms, from 4 widely scattered farms in

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South Scotland. The clinical manifestations varied ,from unthriftiness to recumbency with enlargement of carpal and fetlock joints. It was confirmed that the metacarpal and metatarsal bones of the sheep are particularly susceptible to rickets, and that examination of the distal epiphyseal cartilages of these bones can provide a reliable indication of the presence of the disease, a point previously noted by Fitch and Ewer (1944). The method of husbandry practised was not uniform and indicates the involvement of two main factors in the aetiology viz. a deficiency of phosphorus, possibly coupled with a vitamin D deficiency, and growth stimulation by the rapid growth of pasture or by supplementary feeding. On farms H and I the sheep were out-wintered on natural hill pasture and were given no supplementary feeding. An analysis of the soil and pasture on these farms indicated that large areas contained unusually low levels of phosphorus (Purves, 1958; Voss, 1958), suggesting that a deficiency of this element was involved in the production of the disease. Although thii interpretation provides the most likely explanation for the occurrence of the disease and is one which would be given first priority in any further investigation, two basic difficulties are encountered when one attempts to obtain more conclusive evidence that dietary deficiencies of calcium, phosphorus and/or vitamin D are responsible for the development of rickets in young sheep at pasture. In the first place the dietary requirements of the growing sheep for these factors in order to produce an acceptable rate of weight gain are not accurately known; in the second, pasture analyses do not give a true reflection of the mineral element or vitamin D intake of the sheep, whose selective grazing habits may provide quite a different intake from that indicated by the analyses. Thus, Field (1965) has found IJO relationship between the dietary intake of calcium, magnesium and potassium and the corresponding concentrations of these elements in lowland pasture throughout a period of two years. This state of af%irs will also apply to sheep grazing hill pastures, and particularly so in winter and early spring when rickets occurs. Rickets is essentially a disease of the immature animal and it has been shown that the severity of the lesions increases with the rate of growth (Duckworth et al., 1943 ; Fitch, 1943 ; Follis, 1958; Benzie et al., 1960). All the caseswe observed occurred in the late winter or early spring when the first flush of pasture herbage stimulated growth in the sheep. When climatic conditions produce a plentiful supply of herbage in the spring a higher incidence of rickets may be expected. During the winter the amount of herbage available is sufficient to support only a slow rate of growth at the most and in many instances growth is completely arrested. The winter conditions, together with the fact that the lamb has only one-fifth of adult leg length to achieve between the time of weaning and maturity (Benzie et d., 1960), explain why rickets does not occur in hill sheep during the winter despite their deficient diet. It has been shown experimentally that when growth is arrested by partial starvation rachitogenic diets do not have an adverse effect (Duckworth et al., 1943). On the other hand growth stimulation would appear to have been a much more important aetiological factor on farms J a& K which were low ground arable farms. There the sheep were given concentrate mixtures during the winter and it is possible that these failed to satisfy the increased requirement for calcium, phosphorus or vitamin D. D

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Vitamin D deficiency due to lack of solar radiation is likely to play an important part in the causation of rickets in young grazing sheep under all conditions of husbandry in Britain, an effect which may be slightly more accentuated in Scotland which lies between latitudes 55” and 59”N. Quarterman, Dalgarno and McDonald (1961) have shown that the summer increase in the blood levels of vitamin D due to sunlight in Scotland is not maintained beyond the end of autumn, and Ewer (1950) h as stated that over much of Britain during the winter months the level of vitamin D synthesis from sunlight will be suboptimal. Ewer (1953) has reviewed the literature on the vitamin D requirements of sheep and he concluded that much more experimental work was necessary before these requirements could be determined for grazing sheep. In addition, experiments quoted by Ewer have shown that there can be considerable individual variation in the response of sheep to lack of vitamin D.

CONCLUSIONS

Rickets was diagnosed by histological and biochemical examinations in 16 sheep, aged 9 to 12 months, which were obtained from two arable and two hill farms in the South of Scotland. The clinical manifestations varied from unthriftiness to recumbency, and included stiffness of gait and enlarged carpal and fetlock joints. Since these manifestations are often slight and non-specific, the true incidence of rickets in sheep may be considerably higher than is generally realised. There was some indication that deficiencies of phosphorus and vitamin D were involved in the aetiology, and on the two arable farms these deficiencies appeared to be accentuated by a stimulation of growth which was produced by supplementary feeding. ACKNOWLEDGMENTS

Our thanks are due to Dr. D. Purves of the Edinburgh School of Agriculture and Mr. R. C. Voss of the West of Scotland Agricultural College for the collection and analyses of soil samples. We are also indebted to Mr. J. B. Dow, F.I.M.L.T., for the preparation of histological specimens, and to Mr. C. S. Munro, A.I.M.L.T., and Mrs. M. Walker of the Biochemistry Department at Moredun Institute, for technical assistance. REFERENCES

Auchinachie, D. W., and Fraser, A. H. H. (1931). J. ugric. Sci., 22, 560. Ball, J. (1957). J. An. Path., 10, 281. Benzie, D., Boyne, A. W., Dalgarno, A. C., Duckworth, J., Hill, R., and Walker, D. M. (1960). J. ugric. Sci., 54, 202. Clayden, Crowley,

E. C. (1952). J. med. Lab. Technol., J. P. (1961). Vet. Rec., 73, 295.

10, 103.

Duckworth, J., Benzie, D., Cresswell,E., Hill, R., Robinson, J. F., and Robson, H. W. (1961). Res. vet. Sci., 2, 375. Duckworth, J., Godden, W., and Thomson, W. (1943). J. ugric. Sci., 33, 190. Elliot, Ewer,

W., and Crichton, A. (1926). Ibid., 16, 65. T. K. (1950). Vet. Rec., 62, 603; (1951a). Bit. 300; (1953). Aust. vet. J., 29, 310.

J. Nutr., 5, 287; (1951b).

Ibid.,

I). I. NISHIC’L (21 al.

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Fig. 5.

Costa-chondral junction. Rachitic metaphysis xvith massesof cartilage, some showing conversion to p‘ seudo-osteoid’. H & E : 50. Fig. 6. Distal radius. Rachitic metaphysis with retention of columnar pattern of mature cartilage cells. Ostroid can be seen. H & tC 80. Fig. 7. Section of undecalcified rachitic costo-chondral junction illustrating tlcfcctivr provisional calcification in the upper half and massesof ostroid in thr louw half. \‘o n Kossa and satianin. /: 50.

Fig. 8.

Section of undecalcifcd rib shaft. I‘ wralxcular bone \vith thick ostroitl borders. Van Kossa and safranin. ,X 120.

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Field, A. C. (1965). Personal communication. Fitch, L. W. N. (1943). Aust. vet J., 19, 2. Fitch, L. W. N., and Ewer, T. K. (1944). Ibid., 20, 220. Follis, R. H. (1958). Deficiency Disease. C. C. Thomas; Illinois. Follis, R. H., Jackson, D., Eliot, M., and Park, E. A. (1943). Amer. J. dis. Child., 66, 1. Hemingway, R. G. (1963). Proc. Nutr. Sot., 22, xvi. Innes, J. R. M. (1934-35). Fourth Rep. Dir. Inst. anim. Path. Camb., p. 206. McRoberts, M. R. (1961). Proc. Nutr. Sot., 20, xxxvii. McRoberts, M. R., and Hill R. (1962). Nature, London, 194, 92. Meyer, P. C. (1956). J. Path. Bact., 71, 325. Nisbet, D. I., Butler, E. J., Bannatyne, C. C., and Robertson, J. M. (1962). J. camp. Path., 72, 270. Purves, D. (1958). Personal communication. Quarterman, J., Dalgarno, A. C., and McDonald, I. (1961). Proc. ,Yutr. Sot., 20, xxviii. Shanks, P. L., and Donald, L. G. (1955). Vet. Rec., 67, 312. Voss, R. C. (1958). Personal communication. [Received

for publication,

June

16t1z, 19651