Vet. Pathol. 17: 40-44 (1980) Subclinical Scurvy in the Guinea Pig G. L. CLARKE, A. M. ALLEN, J. D. SMALL and A. LOCK Comparative Pathology Section and Small Animal Section, Veterinary Resources Branch, Division of Research Services, National Institutes of Health, Bethesda, Md. Abstract. Twenty-eight guinea pigs from nine episodes of subclinical scurvy had diarrhea, weight loss and dehydration. The classical signs of scurvy were not seen. Microscopically the epiphyses were attenuated and irregular. The amount of osteoid was less than normal. Many guinea pigs had acute enteritis. In some there were many hemosiderin-laden macrophages in the intestinal lamina propria. All episodes were associated with either autoclaving food without adequate supplementation or other inadequate feed management practices. Of the common laboratory species, the guinea pig and primate require a continuous exogenous source of ascorbic acid for maintenance of good health. The overt classical lesions of ascorbic acid deficiency are readily recognized and consist of hemorrhage in the subperiosteum, skeletal muscles, joints and intestine. Microscopically, there is altered dentin formation and deranged epiphyseal growth centers of long bones with osteoid formation greatly reduced [5, 61. We report a more occult or subclinical form of scurvy [ 10) that is not heralded by the gross hemorrhage in the periosteum, joints or intestinal tract. The syndrome has appeared frequently at our institution in the past 2 to 3 years for a variety of reasons related to food management. We therefore believe it may occur elsewhere and may go unrecognized as the initial cases did here. Overt lesions eventually did appear, and histologic examination then revealed the characteristic bone lesions and others typical of scorbutic enteropathy. Case History Twenty-eight guinea pigs from nine episodes of subclinical scurvy were autopsied. There had been unexpected deaths among guinea pigs previously considered healthy. Guinea pigs often had diarrhea, weight loss and dehydration. Not all in a group were clinically affected, however, and only those with diarrhea or weight loss or both were examined. Results The alimentary canal generally contained a scant amount of ingesta. The gross lesions most frequently seen were confined to the intestinal tract. The jejunum, ileum and colon were atonic and mildly to severely hyperemic. Their content was thin, 40
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Subclinical Scurvy in the Guinea PigSubclinical Scurvy in the Guinea Pig
G. L. CLARKE, A. M. ALLEN, J. D. SMALL and A. LOCK
Comparative Pathology Section and Small Animal Section, Veterinary Resources Branch, Division of Research Services, National Institutes of Health, Bethesda, Md.
Abstract. Twenty-eight guinea pigs from nine episodes of subclinical scurvy had diarrhea, weight loss and dehydration. The classical signs of scurvy were not seen. Microscopically the epiphyses were attenuated and irregular. The amount of osteoid was less than normal. Many guinea pigs had acute enteritis. In some there were many hemosiderin-laden macrophages in the intestinal lamina propria. All episodes were associated with either autoclaving food without adequate supplementation or other inadequate feed management practices.
Of the common laboratory species, the guinea pig and primate require a continuous exogenous source of ascorbic acid for maintenance of good health. The overt classical lesions of ascorbic acid deficiency are readily recognized and consist of hemorrhage in the subperiosteum, skeletal muscles, joints and intestine. Microscopically, there is altered dentin formation and deranged epiphyseal growth centers of long bones with osteoid formation greatly reduced [5, 61.
We report a more occult or subclinical form of scurvy [ 10) that is not heralded by the gross hemorrhage in the periosteum, joints or intestinal tract. The syndrome has appeared frequently at our institution in the past 2 to 3 years for a variety of reasons related to food management. We therefore believe it may occur elsewhere and may go unrecognized as the initial cases did here. Overt lesions eventually did appear, and histologic examination then revealed the characteristic bone lesions and others typical of scorbutic enteropathy.
Case History Twenty-eight guinea pigs from nine episodes of subclinical scurvy were autopsied. There
had been unexpected deaths among guinea pigs previously considered healthy. Guinea pigs often had diarrhea, weight loss and dehydration. Not all in a group were clinically affected, however, and only those with diarrhea or weight loss or both were examined.
Results
The alimentary canal generally contained a scant amount of ingesta. The gross lesions most frequently seen were confined to the intestinal tract. The jejunum, ileum and colon were atonic and mildly to severely hyperemic. Their content was thin,
40
Subclinical Scurvy 41
watery and occasionally blood tinged. Some guinea pigs had irregular areas of depilation but no frank alopecia was seen.
Histological examination revealed marked hyperemia of intestinal lamina propria with varying amounts of edema. In the most severely affected guinea pigs there was a mononuclear infiltrate in the lamina propria and submucosa, with either free blood or neutrophils or both in the lumen. In four of 28 guinea pigs there were many hemosiderin-laden macrophages in the lamina propria adjacent to the deep intestinal glands (fig. 1). In the metaphyseal region of the long bones there was a greatly reduced amount of osteoid around the islands and spicules of calcified cartilage (fig. 2). The normal guinea pig epiphysis has numerous osteoid trabeculae extending from the epiphysis into the metaphysis (fig. 3). Sometimes the marrow cavity was practi- cally devoid of bony trabeculae extending from the calcified cartilage on the epiphysis (fig. 4). The epiphyses were generally thin with fewer than normal chondrocytes forming irregular columns. Their sequence of maturation varied greatly. In some there was little evidence of maturation with few hypertrophied chondrocytes. In others there was good evidence of chondrocyte seriation with numerous hypertro- phied chondrocytes undergoing degeneration without ossification. Osteoblastic activ- ity was minimal, and osteoclastic activity was increased in the region of calcified cartilage (fig. 4). These changes are the hallmarks of early ascorbic acid deficiency in the guinea pig with resultant reduction in matrix formation (91. In overt scurvy there are varying degrees of fibroplasia [6] with little or no collagen production in addition to the changes seen in subclinical scurvy (fig. 5) .
Discussion
Histologic examination of long bones of affected guinea pigs showed a marked decrease and in some cases a total absence of osteoid trabeculae in the metaphysis, a specific lesion associated with ascorbic acid deficiency [9]. Although ascorbic acid is required by fibroblasts and osteoprogenitor cells for matrix formation such as dentin, collagen and osteoid, it also plays an important part in the metabolism of amino acids and assimilation of other nutrients. Ascorbic acid is essential for the formation of hydroxyproline and hydroxylysine in the collagen molecule [7, 81, and is necessary for the catabolism of cholesterol to bile acids [2,4]. Deficiency of ascorbic acid results in the accumulation of cholesterol in the liver with concomitant decrease in bile acid formation [2, 41. The reduced secretion of bile acids greatly affects lipid absorption and the assimilation of fat soluble vitamins. Evidence of disease caused by deficiency of fat soluble vitamins was not seen in the cases we report. It is theoretically possible to produce secondary deficiencies of fat soluble vitamins and essential fatty acids through malabsorption although this has not been reported. Guinea pigs receiving diets deficient in ascorbic acid progress to a state of relative inanition. This contributes to weight loss and death 131. The changes in cartilage proliferation and maturation have been reported [4]. The clinical histories of all our outbreaks of subclinical scurvy have shown poor feed management practices in the affected colonies. Two episodes occurred in one laboratory that removed greens from the diet and made a simultaneous change in dry diet. The other cases were either
Fig. 1: Numerous macrophages laden with crystalline hemosiderin surrounding deep intes-
Fig. 2 Epiphysis of young scorbutic guinea pig with thin irregular epiphysis; abnormal
Fig. 3 Epiphysis of normal weanling guinea pig. HE. Fig. 4 Osteoid spicule near epiphysis. Active osteoclasis with less than expected osteoblastic
tinal glands of small intestine. HE.
chondrocyte seriation. Marrow cavity is nearly devoid of osteoid spicules. HE.
activity. HE. 42
Subclinical Scurvy 43
Fig. 5 Diffuse fibroplasia in marrow cavity distal to epiphysis. Overt scurvy.
associated with storage of feed at abnormally high temperatures or autoclaving without adequate supplementation. One episode resulted from improper formulation by the feed manufacturer. The last episode occurred behind a strict barrier facility where all ingoing feed was autoclaved and stored for relatively long periods of time. When the disease was diagnosed, the survivors were given daily doses of purified ascorbic acid and they recovered. The percentage of animals with clinical illness or pathognomonic lesions has varied from few to many; precise figures are unavailable. Marked variation in susceptibility between guinea pigs is in keeping with the findings of investigators who have reported on the individuality of ascorbic acid requirements in guinea pigs [ 1 11. Subclinical scurvy in guinea pigs appears to be a common malady and probably occurs much more frequently than is generally appreciated. When unexpected deaths are preceded by anorexia, weight loss and diarrhea in a guinea pig colony, subclinical scurvy should be included in the differential diagnosis.
1
2
References BONUCCI, E.: The fine structure of epiphyseal cartilage in experimental scurvy. J Pathol 102219-227, 1970 HORING, D.; WEISER, H.: Ascorbic acid and cholesterol: effect of graded oral intakes on cholesterol conversion to bile acids in guinea pigs. Experientia 32687-689, 1976
44 Clarke et al
3 HUNT, C.E.: Personal commun., 1978 4 JENKINS, S.A.: Hypovitaminosis C and cholelithiasis in guinea pigs. Biochem Biophys Res
Commun 71103-105, 1977 5 NAVA, J.M.; HUNT, C.E.: Nutrition, nutritional disease and nutritional research application
in The Biology of the Guinea Pig, ed. Wagner and Manning, pp. 247-250; Academic Press, New York, 1976
6 SMITH, H.A.; JONES, T.C.; HUNT, R.D.: Veterinary Pathology, p. 990, 4th ed.; Lea & Febiger, Philadelphia, 1972
7 STONE, N.; MEISTER, A.: Function of ascorbic acid in the conversion of proline to collagen hydroxyproline. Nature 194555-557, 1962
8 UDENFRIEND, S.: Formation of hydroxyproline in collagen. Science 1521335-1 340, 1966 9 VILTER, R.E.: Effects of deficiency in animals in The Vitamins, ed. Sebrell and Hams, p.
10 VITALE, J.J.: Deficiency diseases in Pathologic Basis of Disease, ed. Robbins, pp. 492-494;
11 WILLIAMS, R.J.; DEASON, D.: Individuality in vitamin C needs. Proc Natl Acad Sci 51
481, 2nd ed.; Academic Press, New York, 1967
Saunders, Philadelphia, 1974
1638-1641, 1967
G. L. CLARKE, A. M. ALLEN, J. D. SMALL and A. LOCK
Comparative Pathology Section and Small Animal Section, Veterinary Resources Branch, Division of Research Services, National Institutes of Health, Bethesda, Md.
Abstract. Twenty-eight guinea pigs from nine episodes of subclinical scurvy had diarrhea, weight loss and dehydration. The classical signs of scurvy were not seen. Microscopically the epiphyses were attenuated and irregular. The amount of osteoid was less than normal. Many guinea pigs had acute enteritis. In some there were many hemosiderin-laden macrophages in the intestinal lamina propria. All episodes were associated with either autoclaving food without adequate supplementation or other inadequate feed management practices.
Of the common laboratory species, the guinea pig and primate require a continuous exogenous source of ascorbic acid for maintenance of good health. The overt classical lesions of ascorbic acid deficiency are readily recognized and consist of hemorrhage in the subperiosteum, skeletal muscles, joints and intestine. Microscopically, there is altered dentin formation and deranged epiphyseal growth centers of long bones with osteoid formation greatly reduced [5, 61.
We report a more occult or subclinical form of scurvy [ 10) that is not heralded by the gross hemorrhage in the periosteum, joints or intestinal tract. The syndrome has appeared frequently at our institution in the past 2 to 3 years for a variety of reasons related to food management. We therefore believe it may occur elsewhere and may go unrecognized as the initial cases did here. Overt lesions eventually did appear, and histologic examination then revealed the characteristic bone lesions and others typical of scorbutic enteropathy.
Case History Twenty-eight guinea pigs from nine episodes of subclinical scurvy were autopsied. There
had been unexpected deaths among guinea pigs previously considered healthy. Guinea pigs often had diarrhea, weight loss and dehydration. Not all in a group were clinically affected, however, and only those with diarrhea or weight loss or both were examined.
Results
The alimentary canal generally contained a scant amount of ingesta. The gross lesions most frequently seen were confined to the intestinal tract. The jejunum, ileum and colon were atonic and mildly to severely hyperemic. Their content was thin,
40
Subclinical Scurvy 41
watery and occasionally blood tinged. Some guinea pigs had irregular areas of depilation but no frank alopecia was seen.
Histological examination revealed marked hyperemia of intestinal lamina propria with varying amounts of edema. In the most severely affected guinea pigs there was a mononuclear infiltrate in the lamina propria and submucosa, with either free blood or neutrophils or both in the lumen. In four of 28 guinea pigs there were many hemosiderin-laden macrophages in the lamina propria adjacent to the deep intestinal glands (fig. 1). In the metaphyseal region of the long bones there was a greatly reduced amount of osteoid around the islands and spicules of calcified cartilage (fig. 2). The normal guinea pig epiphysis has numerous osteoid trabeculae extending from the epiphysis into the metaphysis (fig. 3). Sometimes the marrow cavity was practi- cally devoid of bony trabeculae extending from the calcified cartilage on the epiphysis (fig. 4). The epiphyses were generally thin with fewer than normal chondrocytes forming irregular columns. Their sequence of maturation varied greatly. In some there was little evidence of maturation with few hypertrophied chondrocytes. In others there was good evidence of chondrocyte seriation with numerous hypertro- phied chondrocytes undergoing degeneration without ossification. Osteoblastic activ- ity was minimal, and osteoclastic activity was increased in the region of calcified cartilage (fig. 4). These changes are the hallmarks of early ascorbic acid deficiency in the guinea pig with resultant reduction in matrix formation (91. In overt scurvy there are varying degrees of fibroplasia [6] with little or no collagen production in addition to the changes seen in subclinical scurvy (fig. 5) .
Discussion
Histologic examination of long bones of affected guinea pigs showed a marked decrease and in some cases a total absence of osteoid trabeculae in the metaphysis, a specific lesion associated with ascorbic acid deficiency [9]. Although ascorbic acid is required by fibroblasts and osteoprogenitor cells for matrix formation such as dentin, collagen and osteoid, it also plays an important part in the metabolism of amino acids and assimilation of other nutrients. Ascorbic acid is essential for the formation of hydroxyproline and hydroxylysine in the collagen molecule [7, 81, and is necessary for the catabolism of cholesterol to bile acids [2,4]. Deficiency of ascorbic acid results in the accumulation of cholesterol in the liver with concomitant decrease in bile acid formation [2, 41. The reduced secretion of bile acids greatly affects lipid absorption and the assimilation of fat soluble vitamins. Evidence of disease caused by deficiency of fat soluble vitamins was not seen in the cases we report. It is theoretically possible to produce secondary deficiencies of fat soluble vitamins and essential fatty acids through malabsorption although this has not been reported. Guinea pigs receiving diets deficient in ascorbic acid progress to a state of relative inanition. This contributes to weight loss and death 131. The changes in cartilage proliferation and maturation have been reported [4]. The clinical histories of all our outbreaks of subclinical scurvy have shown poor feed management practices in the affected colonies. Two episodes occurred in one laboratory that removed greens from the diet and made a simultaneous change in dry diet. The other cases were either
Fig. 1: Numerous macrophages laden with crystalline hemosiderin surrounding deep intes-
Fig. 2 Epiphysis of young scorbutic guinea pig with thin irregular epiphysis; abnormal
Fig. 3 Epiphysis of normal weanling guinea pig. HE. Fig. 4 Osteoid spicule near epiphysis. Active osteoclasis with less than expected osteoblastic
tinal glands of small intestine. HE.
chondrocyte seriation. Marrow cavity is nearly devoid of osteoid spicules. HE.
activity. HE. 42
Subclinical Scurvy 43
Fig. 5 Diffuse fibroplasia in marrow cavity distal to epiphysis. Overt scurvy.
associated with storage of feed at abnormally high temperatures or autoclaving without adequate supplementation. One episode resulted from improper formulation by the feed manufacturer. The last episode occurred behind a strict barrier facility where all ingoing feed was autoclaved and stored for relatively long periods of time. When the disease was diagnosed, the survivors were given daily doses of purified ascorbic acid and they recovered. The percentage of animals with clinical illness or pathognomonic lesions has varied from few to many; precise figures are unavailable. Marked variation in susceptibility between guinea pigs is in keeping with the findings of investigators who have reported on the individuality of ascorbic acid requirements in guinea pigs [ 1 11. Subclinical scurvy in guinea pigs appears to be a common malady and probably occurs much more frequently than is generally appreciated. When unexpected deaths are preceded by anorexia, weight loss and diarrhea in a guinea pig colony, subclinical scurvy should be included in the differential diagnosis.
1
2
References BONUCCI, E.: The fine structure of epiphyseal cartilage in experimental scurvy. J Pathol 102219-227, 1970 HORING, D.; WEISER, H.: Ascorbic acid and cholesterol: effect of graded oral intakes on cholesterol conversion to bile acids in guinea pigs. Experientia 32687-689, 1976
44 Clarke et al
3 HUNT, C.E.: Personal commun., 1978 4 JENKINS, S.A.: Hypovitaminosis C and cholelithiasis in guinea pigs. Biochem Biophys Res
Commun 71103-105, 1977 5 NAVA, J.M.; HUNT, C.E.: Nutrition, nutritional disease and nutritional research application
in The Biology of the Guinea Pig, ed. Wagner and Manning, pp. 247-250; Academic Press, New York, 1976
6 SMITH, H.A.; JONES, T.C.; HUNT, R.D.: Veterinary Pathology, p. 990, 4th ed.; Lea & Febiger, Philadelphia, 1972
7 STONE, N.; MEISTER, A.: Function of ascorbic acid in the conversion of proline to collagen hydroxyproline. Nature 194555-557, 1962
8 UDENFRIEND, S.: Formation of hydroxyproline in collagen. Science 1521335-1 340, 1966 9 VILTER, R.E.: Effects of deficiency in animals in The Vitamins, ed. Sebrell and Hams, p.
10 VITALE, J.J.: Deficiency diseases in Pathologic Basis of Disease, ed. Robbins, pp. 492-494;
11 WILLIAMS, R.J.; DEASON, D.: Individuality in vitamin C needs. Proc Natl Acad Sci 51
481, 2nd ed.; Academic Press, New York, 1967
Saunders, Philadelphia, 1974
1638-1641, 1967
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