Virchows Arch. B Cell Path. 21,229--247 (1976) 9 by Springer-Verlag 1976
Nutritional Osteodystrophy in Captive Green Iguanas (Iguana iguana) * Marilyn P. Anderson and Charles C. Capen Department of Veterinary Pathobiology, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio Received April 15, 1976
Summary. Captive lizards fed a variety of vegetarian and carnivorous diets frequently develop severe nutritional osteodystrophy. Five hatchling iguanas which were captive for 5 months and fed a diet low in calcium and phosphorus developed clinical signs and lesions typical of nutritional osteodystrophy in captive lizards. They had tetany with severe osteoporosis, pathologic fractures, and cartilaginous diaphyseal enlargements of long bones which either were associated with fractures or with intact bones. Hatchling and juvenile iguanas fed a similar diet low in calcium (0.1%) and phosphorus (0.2%) under experimental conditions developed hypocalcemia, tetany, osteoporosis with osteomalacia and pathologic fractures. Experimental iguanas fed a diet low in calcium (0.2% ) but adequate in phosphorus (1.1%) developed progressive hypocalcemia and severe osteoporosis with pathologic fractures. Control iguanas fed a diet with adequate calcium (2.7%) and phosphorus (1.1%) had well mineralized bones with wide cortices and thick metaphyseal trabeculae. Iguanas fed low calcium experimental diets whether low or adequate in phosphorus developed hypocalcemia with compensatory secondary hyperparathyroidism resulting in severe osteodystrophy similar to that reported in captive lizards. Key words: Iguanas - - Bone disease - - Dietary calcium - - Hyperparathyroidism. Introduction
One of the most common diseases of captive lizards is an osteodystrophy which is secondary either to nutritional imbalances in calcium, phosphorus and vitamin D or to renal disease [11, 15, 26, 28]. Most lizards are known to be either omnivorous, carnivorous or herbivorous [25] but few nutrient requirements have been established qualitatively [26]. Formulation of adequate diets are difficult since quantitative nutrient requirements are not known for reptiles. Nutritional imbalances in calcium, phosphorus and vitamin D are usually due to high meat diets without adequate calcium supplementation [18, 26]. Nutritional osteodystrophy in lizards is characterized by progressive locomotor difficulty, soft fragile bones, and rapidly forming fusiform enlargements of long bones [15, 26, 28]. Muscular twitching and tetany frequently occur in lizards with osteodystrophy. Similar signs have been reported in lizards and snakes which are hypocalcemic after parathyroidectomy [8, 9, 20, 23]. Most lizards with nutritional osteodystrophy improve clinically with calcium and vitamin D therapy [28]. The objectives of this investigation were (1) to characterize a diet-induced ostcodystrophy in captive hatchling green iguanas, and (2) to investigate the pathogenesis of nutritional osteodystrophy in reptiles by correlating changes in * Supported by grants RR 05463, GM 1052 and RR 00366-05, National Institutes of Health, USPHS
230
M . P . Anderson and C. C. Capen
plasma electrolytes with sequential histopathologic and radiographic alterations in b o n e s of g r e e n i g u a n a s f e d e x p e r i m e n t a l d i e t s w i t h v a r i e d c a l c i u m a n d phosphorus content. M a t e r i a l s a n d Methods
Diet-Induced Osteodystrophy Five hatchling green iguanas (Iguana iguana), 12 g body weight and 7 cm snout-vent length, were obtained as part of an airfreight shipment from a commercial dealer in California. The iguanas were housed in chick incubators at 28 ~ C and with 10 h of light per day. Tap water was available ad libitum and twice a day the iguanas were fed a mixture composed of equal volumes of drained canned corn, peas, applesauce and a high meat baby food 1. Powdered eggshell was added to the diet several weeks after onset of clinical illness and until presentation for necropsy.
Experimental Osteodystrophy Thirty hatchling (12 g body weight, 7 cm snout-vent length) and 40 juvenile (90 g body weight, 12 cm snout-vent length) green iguanas (Iguana iguana) were received by airfreight from a commercial dealer in Florida. Stainless steel pans (28.0 • 45.7 • 17.8 cm) fitted on top with aluminum wire screen (16 • 18 mesh) were used to house 2 to 3 iguanas. Disposable absorbent paper 2 was used to line the bottom of the pans. The iguanas were fed once a day on quartered white paper plates and tap water was available ad libitum in petri dishes. Ambient temperature was maintained at 28~ with a fan driven space heater in a 5 by 10 foot room and 10 h of nonactinic light were provided each day. An adequate humidity was maintained by placing a 25 cm diameter bowl of water in front of the space heater. The hatchling iguanas were randomly divided into 2 experimental dietary groups and 1 control group. The juvenile iguanas were similarly divided into 3 groups. The control diet was equivalent to the experimental low CA-adequate P diet (Table 1) but supplemented with 8.0 g of calcium carbonate to contain 2.7% calcium and 1.1% phosphorus (Table 2). The second experimental diet was composed of equal volumes of applesauce, drained canned corn, drained canned peas, and a high meat baby food. 3 It was low in both calcium and phosphorus (Table 2). The ingredients of the baby food diet were homogenized in a blender. 4 The diets were stored in 18 oz plastic bags 5 and frozen until 12 h before use. All iguanas were serially radiographed at 1 to 3 month intervals using an industrial X-ray unit 6 and nonscreen X-ray film 7 at 5 Ma and 25 to 30 KV for 2 to 15 seconds at a film focal distance of 43 em. Masking tape was used to secure the iguanas in close apposition to the film. Body weight was recorded weekly for all iguanas. Three hatchling and 3 juvenile iguanas from each dietary group were euthanatized at 1 to 3 month intervals by giving lethal doses of ketamine hydrochloride s subcutaneously over the back or intramuscularly in the tail. Plasma samples were collected at the time of necropsy. The neck was cut just posterior to the skull from the dorsum until the carotid arteries were severed. Freely flowing blood was pipetted into heparinized test tubes. The plasma was frozen at --20 ~ C until electrolyte analyses were performed. Phosphorus values were determined by a micromethod [3] and calcium values were determined by atomic absorption spectrophotometry. 9 1 High meat dinner (strained, chicken with vegetables), Gerber Products Co., Fremont, Mich. 49412 2 Anipads, ANCARE Corp., Manhasset, L.I., N.Y. 11030 3 High meat dinner (strained, chicken with vegetables and cereal), Beech-Nut, Inc., New York, N.Y. 10022 4 Model 613-A-S, Hamilton Beach, Division of Scovill Mfg. Co., Waterbury, Ct. 5 Whirl-Pak, NASCO, Fort Atkinson, Wis. 53538 6 Model LC-90, General Electric Co. 7 Monopak, GAF Corp., New York, N.Y. 10020 8 Vetalar, Parke-Davis and Co., Detroit, Mich. 48232 9 Model 303 atomic absorption spectrophotometer, Perkin-Elmer, Norwalk, Ct. 06852
Nutritional 0steodystrophy in Iguanas
231
Table 1. Experimental low calcium-adequate phosphorus diet Ingredient
Amount
Fresh, lean, ground beef heart MnSO 4" H20 KlOa Folic acid L-ascorbic acid Ca (H2PO4)2.H20 CaCOa Glucose Corn oil
500 g 19.1 mg 38 tzg 135 ~g 50 mg 610 mg 100 mg 1.12 g 2.25 g
Table 2. Diet analyses expressed as percent on a dry weight basis Diet
Calcium
Phosphorus Ca:P
Protein
Fat
Control Experimental low CA adequate P Experimental low CA low P
2.7 0.2 0.1
1.1 1.1 0.2
82 82 17
14 14 6
2.4:1 1:5.5 1:2
The left parathyroid gland and left ultimobranchial gland were collected for electron microscopy and 1 ~z sections were stained with toluidine blue for light microscopic evaluation [27]. Iguana iguana have 2 parathyroid glands located at the junction of the common carotid artery with the internal and external carotid arteries and the ductus caroticus. Only 1 ultimobranchial gland, on the left side, was found in the hatchling and juvenile iguanas. I t was situated along the trachea immediately dorsal to the thyroid gland. This location was more cranial than has been reported in other lizards [7, 10, 19, 24]. Sections of femur, humerus and kidney were fixed in 10% neutral buffered formalin. Bone, after demineralization to effect in 10% sodium ethylenediaminetetraacetate, and kidney were sectioned at 6 t~ and stained with hematoxylin and eosin. Femur and humerus also were fixed in 70% ethyl alcohol, embedded in methyl methacrylate [16], sectioned at 6 ~zand stained with Goldner's modified trichrome [14] or Krutsay's modified silver stain [17].
Results
Diet-Induced Osteodystrophy The h a t c h l i n g i g u a n a s were active, gained weight a n d h a d n a t u r a l b o d y conf o r m a t i o n until a b o u t t h e 3rd m o n t h in t h e l a b o r a t o r y . The iguanas c l i m b e d a n d m o v e d a b o u t less t h a n usual a n d w i t h difficulty. W i t h i n t h e following 2 weeks 1 or b o t h femurs d e v e l o p e d oval d i a p h y s e a l enlargments. B y 5 m o n t h s all i g u a n a s h a d d i a p h y s e a l e n l a r g e m e n t s on 1 or m o r e long bones (Fig. 1). T h e femur, humerus, tibia, fibula a n d r a d i u s were m o s t c o m m o n l y affected. Some of t h e p h a l a n g e s were similarly affected. The d i a p h y s e a l e n l a r g e m e n t s were c h a r a c t e r i s t i c a l l y oval a n d firm e x t e n d i n g to t h e p h y s e a l region b u t n o t bridging epiphyses or j o i n t capsules. T h e overlying skin was d a r k , f i r m l y a t t a c h e d a n d s t r e t c h e d t a u t (Fig. 1). P r o g n a t h i s m a n d increased flexibility of t h e m a n d i b l e s were p r o n o u n c e d b u t grossly visible t h i c k e n i n g of m a n d i b u l a r a n d m a x i l l a r y bones was n o t observed. The a p p e t i t e was m a i n t a i n e d u n t i l several d a y s before d e a t h . The iguanas r e q u i r e d assistance to e a t a n d d r i n k during t h e last 1 or 2 weeks of life due to
232
M.P. Anderson and C. C. Capen
Fig. 1. Diaphyseal enlargement of the right tibia and fibula in a hatchling iguana fed a low calcium and low phosphorus diet for 4 months. Note the taut skin over the swollen region (arrow) Fig. 2. Tetany in a hatchling iguana fed a low calcium and low phosphorus diet for 5 months. The right tibia has a characteristic diaphyseal enlargement
difficulties in ambulation and stiffness of the vertebral column. Tctany lasting 15 to 30 seconds was observed sporadically for several days prior to death (Fig. 2). Tetany was characterized by arching of the back and neck with extension of the limbs. Two iguanas died during the night and 3 were euthanatized following tetany. Radiographic examination at necropsy revealed generalized osteopenia with poorly mineralized, thin cortices of long bones including those with diaphyseal enlargements. The actual extent of palpable and grossly visible diaphyseal enlargements was not disclosed radiographically. Fractures of the diaphysis were not consistently associated with the mid-shaft enlargments, but pathologic fractures were frequently seen in the metaphysis of affected bones. Long bones had enlarged medullary cavities and an abnormal rectangular shape due to failure of remodeling in the cut back zone (Fig. 3). Dissection of skin and muscle tissue from limbs at necropsy revealed oval to fusiform, white enlargements which encircled most of the shaft of the long bones (Fig. 4). These enlargements were cartilaginous in appearance and could be sectioned readily with a scalpel.
Nutritional Osteodystrophy in Iguanas
233
Fig. 3. Radiograph illustrating osteopenia with enlarged medullary cavities and oval diaphyseal proliferations of partially mineralized cartilage (arrows) in a hatchling iguana fed a low calcium and low phosphorus diet for 5 months Fig. 4. Bilateral diaphyseal cartilagenous enlargement (arrows) in the femur, tibia and fibula of a hatehling iguana. The skin and skeletal muscle were removed from the leg on the left; only skin was removed from the right leg
Undecalcified histologic sections of affected femurs revealed thin mineralized cortices with a cuff of proliferating cartilage that replaced and/or compressed the overlying skeletal muscle (Fig. 5). Fractures of cortical bone often were not evident in affected long bones. I n addition, the total mass of metaphyseal spongiosa of long bones was deficient due to reduction in number, length and width of trabeculae in the primary and secondary spongiosa. The lesions in long bone varied considerably in severity. The least severe lesion was a proliferation of spindle cells in the periostcum of the diaphysis forming a layer several cells thick over the thin cortices. More advanced lesions were characterized by a marked thinning of cortical bone t h a t was surrounded by consecutive layers of osteoid, hyahne cartilage and primitive mesenchymal tissue. This immature connective tissue invaded as well as displaced overlying skeletal muscle. Mature hyaline cartilage was the predominant component in the diaphyseal
234
M. 1). Anderson and C. C. Capen
O
Z
!7
E> O
~ v
9 ~b
. O
Nutritional Osteodystrophy in Iguanas
235
masses of the most severely enlarged bones and was primarily responsible for the diaphyseal enlargements detected clinically. The cartilage was partially mineralized and had vascular resorption cavities in more central areas. Primary vascular channels in the cartilaginous mass enlarged to form hematopoietic marrow spaces. This often resulted in cartilaginous trabeculae oriented perpendicular to the shaft of the long bone. There appeared to be some direct metaplasia of cartilage to bone as well as endochondral bone formation following chondroclastic remodeling. When fractures occurred in long bones they were usually in the metaphysis and a typical cartilaginous callus was seen along both the endosteal and periosteal surfaces of the cortex [21]. Vertebrae had lesions similar to those of long bones. The dorsal, lateral and ventral laminae encasing the spinal cord were severely thinned. The vertebral bodies often had massive periosteal cartilaginous proliferations ventrally and laterally with compression of subjacent muscle and spinal nerves. There was no compression of the spinal cord by the expanding masses of cartilage.
Experimental Osteodystrophy in Hatehling Iguanas Initially hatchling iguanas in each of the 3 dietary groups were active, alert and ate regularly once a day. The iguanas fed the experimental diet low in calcium and phosphorus had a clinical disease similar to the iguanas with the typical diet-induced osteodystrophy except the course was protracted by several months. In contrast, iguanas fed the experimental low CA-adequate P diet had a shorter clinical course of approximately 4 weeks. Table 3. Body weight gains of hatchling iguanas expressed as percent of initial body weight Months on experiment
Dietary group Control
Experimental low CA-low 1)
1
12
6
2 3 4 5 6
42 58 65 89 102
10 18 34 26 24
Body weights in all groups of control and experimental iguanas increased initially but revealed significant differences later (Table 3). Mean initial and final body weight for the 3 dietary groups were 11.3 g and 27.3 g (control), 12.5 g and 10.5 g (experimental low CA-adequate P), and 14.2 g and 16.9 g (experimental low CA-low P), respectively. Iguanas fed the low CA-adequate P diet had a drop in body weight by the second week. The body weights of iguanas fed the low CA-low P diet decreased after the 4th month. Control iguanas continued to gain weight until the experiment was terminated at 6 months. Plasma calcium levels in experimental iguanas were lower and phosphorus levels were higher than in control iguanas although these differences were not
236
M.P. Anderson and C. C. Capen
Fig. 6. Radiograph illustrating osteopenia of the rear limbs with abnormally rectangular long bones (arrows) in a hatchling iguana fed a low calcium and low phosphorus diet for 6 months Fig. 7. Radiograph illustrating a pathologic fracture (arrow) of the left femur in an osteopenic hatchling iguana fed a low calcium and low phosphorus diet for 5 months Fig. 8. Radiograph illustrating well mineralized long bones in a control iguana fed a diet adequate in calcium and phosphorus for 6 months
statistically significant. Mean plasma calcium in hatchling iguanas fed the experimental low CA-low P diet fell from 8.2 mg/100 ml at 1 month to 7.8 rag/100 ml at 6 months with values decreasing as low as 4.2 mg/100 ml. Mean plasma phosphorus ranged from 1.4 rag/100 ml to 4.5 mg/100 ml during the experimental period. Mean plasma calcium of iguanas fed the experimental low CA-adequate P diet fell
Nutritional Osteodystrophy in Iguanas
237
Fig. 9. Prominent, well mineralized trabeculae (arrow) in the metaphysis of a femur from a control hatchling iguana. The control diet containing adequate calcium and phosphorus was fed for 6 months. H & E, x 125 Fig. 10. Short and narrow trabeculae (arrow) in the primary and secondary spongiosa of a femur from an experimental hatchling iguana. The experimental low calcium diet was fed for I month. H & E, x 125 Fig. 11. Absence of trabecular bone in the metaphysis of a femur from an experimental hatchling iguana. The experimental low calcium and low phosphorus diet was fed for 5 months. H & E, x 125
238
M.P. Anderson and C. C. Capen
from 11.3 mg/100 ml to 7.6 mg/100 ml within 6 weeks and the phosphorus level ranged from 2.6 to 3.7 mg/100 ml during this period. One iguana fed this experimental diet for 4 weeks was euthanatized while in tetany. The plasma calcium value was 7.6 mg/100 ml and phosphorus was 3.7 mg/100 ml. Several other iguanas in this group also had tetanic spasms 1 to 2 days prior to death. Mean plasma calcium in control iguanas ranged from 11.3 to 12.1 mg/100 ml and phosphorus ranged from 1.1 to 1.4 mg/100 ml. Generalized osteopenia was detected radiographically after the 2nd month in iguanas fed the experimental low CA-low P diet. Long bones had thin cortices and short, thin metaphyseal trabeculae. A failure of remodeling in the cut back zone resulted in abnormally rectangular long bones (Fig. 6). After the 4th month the iguanas developed diaphyseal swellings of limbs similar to those seen in the hatchling iguanas with the typical diet-induced osteodystrophy. However, all swellings were associated with pathologic fractures. The fractures were closed and usually occurred near the metaphysis (Fig. 7). Some fractures were oblique and involved the entire diaphysis. A mineralized callus was not evident radiographically after 5 to 6 weeks. However, a firm cartilaginous callus was palpable and grossly visible. Radiographically, long bones of iguanas fed the experimental low CA-adequate P diet had thin cortices. There was no evidence of diaphyseal enlargements of long bones or other gross lesions. In contrast, long bones of control iguanas were well mineralized with thick cortices and normal conformation (Fig. 8). Microscopically, the femur of control iguanas had thick well mineralized cortices with numerous broad trabeculae in the metaphysis (Fig. 9). After 1 to 2 months the experimental iguanas had a striking reduction in the amount of bone in the primary and secondary spongiosa (Fig. 10). After 5 months both primary and secondary spongiosa were absent in the experimental iguanas fed the low CA-low P diet (Fig. 11). The cortices were greatly attenuated and a prominent transverse layer of osteoid covered the metaphyseal surface of the physis. There was no quantitative difference in thickness of the physis between control and experimental iguanas. However, experimental iguanas fed the low CA-low P diet had chondrocytes arranged more in small clumps in the zone of hypertrophic cartilage and not as well aligned as in control iguanas.
Experimental Osteodystrophy in Juvenile Iguanas The iguanas in each dietary group initially were active, alert and ate regularly once a day. Iguanas fed either the control or the experimental low CA-low P diet maintained their daily food consumption, activity and alertness until termination of the experiment at 16 months. After the 8th month iguanas fed the experimental low CA-adequate P diet had mandibles that were slightly more flexible than those of control iguanas. Mandibular pliability was readily detected by the 12th month and at 16 months the mandibles could be easily approximated by medial compression to half the distance of their separation. Excessive loss of teeth was frequently noted in iguanas fed the experimental low CA-low P diet for 12 or more months. By comparison, iguanas fed the experimental low CAadequate P diet had reduced food consumption after 4 months and were eating
Nutritional Osteodystrophy in Iguanas
239
Table 4. Body weight gains of juvenile iguanas expressed as percent of initial body weight Months fed diet
Dietary group Control
Experimental low CA-low P
Experimental low CA-adequate P
2 4 6 8 10 12 14 16
25 47 47 68 66 79 68 78
13 20 21 22 27 32 36 48
6 12 9 12 14 ----
every 2nd or 3rd day. They became progressively less active and alert from the 6th month to the 10th month of feeding the low CA-adequate P diet. Slight mandibular flexibility was detected only after the 8th month in juvenile iguanas fed the experimental low CA-adequate P diet. Excessive tooth loss was not noted in juvenile iguanas fed either the experimental low CA-adequate P diet or control diet. Neither enlargements nor other abnormal conformations were noted in facial bones of iguanas in any of the three groups. Mandibles of control iguanas were well mineralized and inflexible for the 16 month experimental period. Body weight gains of juvenile iguanas in the three dietary groups differed after the 5th month (Table 4). The body weight of iguanas fed the low CA-low P diet plateaued between the 4th and 8th month. The slight increase in body weight after the 8th month was due primarily to the development of ascites and anasarea. The body weight of juvenile iguanas fed the experimental low CAadequate P diet plateaued and did not increase during the experimental period of 10 months. In contrast, iguanas fed the control diet had a progressive gain in body weight, without ascites, during the 16 month experimental period. Mean initial and final body weights of iguanas in the three dietary groups were 96 g and 275 g (control), 92 g and 106 g (experimental low CA-adequate P), and 91 g and 163 g (experimental low CA-low P), respectively. Plasma calcium and phosphorus values were similar for juvenile iguanas in the 3 groups until about the 3rd month (Fig. 12). Both groups of experimental iguanas developed progressive hypocalcemia and hyperphosphatemia although these differences from controls were not statistically significant. The range of plasma calcium and phosphorus values was less variable in the control iguanas compared to the experimental iguanas. Plasma calcium was consistently above 9 rag/100 ml and the plasma phosphorus usually was below 7 mg/100 ml in control juvenile iguanas (Fig. 12). The reason for the higher plasma phosphorus at 16 months is uncertain but it represents a single control iguana that had a moderate interstitial fibrosis of the kidney. Generalized osteopenia was evident radiographieally at 2 to 3 months and increased in severity throughout the experimental period in iguanas fed either of the experimental diets (Fig. 13). Multiple rib fractures usually not associated
240
M.P. Anderson and C. C. Capen
Fig. 12. Mean plasma calcium and phosphorus in juvenile iguanas fed experimental and control diets. ND= no data. C= control diet with adequate CA and P. B= low CA-low P diet. H--low CA-adequate P diet with costochondral junctions were present in iguanas fed either of the experimental diets for 3 or more months (Fig. 13). One iguana fed the experimental low CAadequate P diet had a pathologic fracture in the distal femur at 3 months. Radiographically there was no evidence of callus formation and mineralization after 4 weeks when the iguana was euthanatized. However, a cartilaginous callus was present at necropsy. Ascites was detected radiographically in iguanas fed the experimental low CA-low P diet for 10 to 16 months. Costochondral junctions and physes appeared normal in the three groups of iguanas. Bones of control iguanas were well mineralized and without evidence of osteoporosis throughout the 16 months period of the experiment. The femur of juvenile iguanas fed the experimental low CA-adequate P diet had varying degrees of osteoporosis characterized by thinned cortices and wide medullary cavity with narrow trabeculae in the metaphysis. Occasionally, flattened osteoblasts lined endosteal and periosteal surfaces. Large multinucleated osteoclasts in prominent Howship's lacunae were particularly abundant along endosteal surfaces and metaphyseal trabeculae after feeding the experimental diet for 3 to 6 months. Osteoclasts diminished in number and size from 6 to 10 months and were situated along primary trabeculae. Cortical bone was thin and metaphyseal trabeculae were lined by flattened ostcoblasts. Osteoid was sparse and was present mainly as a thin layer along the base of cartilage columns of the physis. The femurs of iguanas fed the experimental low CA-low P diet had a similar osteoporosis after the 2nd to 3rd month with thin cortices and narrow trabeculae in the metaphysis (Fig. 14). In contrast to the iguanas fed the experimental low
Nutritional Osteodystrophy in Iguanas
241
.~
O
~
.~
c~
~'~ 9 ~
.~,~
CA-adequate P diet, osteoid seams were progressively widened and covered the narrow trabeculae after the 5th month (Fig. 15). Cortical bone was thin and there were wide osteoid seams on endosteal and periosteal surfaces. Osteoblasts varied from flat to plump and were present in moderate numbers at all intervals examined. 0steoclasts were present in Howship's lacunae until the 5th month and were limited mainly to subphyseal and diaphyseal locations because of the osteoid accumulation on other endosteal surfaces. Femurs of control iguanas were characterized by wide cortices and abundant, thick metaphyseal trabeculae (Fig. 16). Osteoblasts varied in shape from flat to
Fig. 14. Osteoporosis in a juvenile iguana fed a low calcium and low phosphorus diet for 16 months. Note the narrow metaphyseal trabeeulae (long arrow) and t h i n cortex (short arrow). H & E, x 50
Fig. 15. Osteomalacia in the femur of an iguana fed a low calcium and low phosphorus diet for 16 months. Note the wide osteoid seams (arrows). Same bone as in Figure 14. Goldner's modified trichrome, X 125
Fig. 16. F e m u r from a control iguana at 16 months. Note the thick metaphyseal trabeculae (long arrows) and wide cortex (short arrow). H & E, X 50
Fig. 17. Metaphyseal trabecula of the femur from a control iguana. Osteoid seams are narrow (arrow). Same bone as in Figure 16. Goldner's modified trichrome, x 125 5 VirchowsArch. B Cell Path.
244
M.P. Anderson and C. C. Capen
plump and usually lined one side of the metaphyseal trabeculae. The opposite sides of the trabeculae were usually irregular in contour due to the presence of Howship's lacunae with or without osteoelasts. Moderate numbers of osteoclasts were present on subphyseal, trabecular and diaphyseal endosteal surfaces. Osteoid was present in small amounts along trabeeulae (Fig. 17) but osteoid seams were narrow compared to the abundant osteoid accumulations in iguanas fed the experimental low CA-low P diet (Fig. 15).
Histologic Evaluation o/Parathyroid and Ultimobranchial Glands and Kidney /rom Hatehling and Juvenile Iguanas with Experimental Osteodystrophy Parathyroid and ultimobranchial glands of hatchling and juvenile iguanas did not differ grossly in size, shape, color or consistency between control and experimental iguanas during the experimental periods of 6 months (hatchling) and 16 months (juveniles). Neither parathyroid nor ultimobranchial glands had evidence of cyclic degeneration as has been reported for lizards collected from the wild at various times of the year [22]. Chief cells of the parathyroid gland were closely packed together, oval or elongate and were subdivided into several groups by fine strands of fibrous connective tissue and capillaries. Chief cells of iguanas fed the experimental diets had a greater cytoplasmic to nuclear ratio than control iguanas especially after the 2nd month for the hatchling iguanas and after the 3rd month for the juvenile iguanas. Ultimobranchial glands were composed mainly of c-cell containing follicles lined with a columnar to pseudostratified columnar epithelium. Small clumps of c-cells were interspersed in connective tissue between the follicles. There were no differences detectable by light microscopy between the experimental and control iguanas [2]. Lesions were not present in kidneys of hatchling or juvenile iguanas throughout the experimental period except for 1 control iguana which had moderate interstitial fibrosis. Discussion Iguanas fed low calcium diets develop hypocalcemia, secondary hyperparathyroidism, and severe bone loss that shared many features with the disease in mammals and birds [18]. Hatchling iguanas fed the experimental low CAadequate P diet developed osteoporosis within 1 to 2 months whereas from 3 to 6 months were required for the bone lesions to develop in juvenile iguanas. The degree of diet-induced hypocalcemia varied in severity but neuromuscular tetany occasionally developed shortly before death. In addition, iguanas fed the low calcium and low phosphorus diets for 4 to 8 months developed severe osteomalacia superimposed on the osteoporosis. The osteoporosis and osteomalacia developed more rapidly in hatchlings compared to juvenile iguanas. The relatively low protein content of the low calcium--low phosphorus diet was reflected by ascites, anasarca, and may have contributed to the osteopenia that developed in iguanas fed this diet. The metabolic bone disease produced in iguanas by feeding the experimental diet low in calcium but adequate in phosphorus was similar to lesions reported
Nutritional Osteodystrophy in Iguanas
245
in toads (Xenopus laevis) which were fed all-liver diets [6]. Bone lesions in toads were present by 5 months and were characterized by thin, fragile long bones with pathologic fractures that had a poorly mineralized callus. Tetanic spasms of limbs and muscular twitching occurred shortly before death and there was radiographic evidence of generalized demineralization of the skeleton. Young toads were more severely affected than adults. Although adult toads were maintained for 15 months on the liver diet without clinical evidence of bone disease, radiographic examination revealed a progressive development of generalized osteoporosis. The toads developed a striking prognathism similar to that observed in hatchling iguanas with diet-induced osteodystrophy. The diaphyseal enlargements on long bones were observed more frequently in iguanas with the typical diet-induced osteodystrophy than in experimental iguanas housed in a more confined environment. This suggested that in iguanas which were in a negative calcium balance and had developed progressive osteoporosis, the physico-chemical stresses exerted by running and climbing stimulated the periosteum to reinforce the weakened bones structurally by the formation of concentric layers of osteoid, cartilage, and immature fibrous connective tissue. Experimental iguanas confined in small laboratory pens were less active and developed a similar severe degree of osteoporosis but with considerably less periosteal proliferation. Pathologic fractures developed occasionally in experimental iguanas with severe osteoporosis during periods of vigorous physical activity. The term "osteoperiostitis" has been used by other investigators to describe a proliferative lesion of this type in the diaphysis of long bones [11]. The anatomic location and histologic composition of a bone lesion reported in a monitor lizard (Varanus) appeared to be similar to the diaphyseal enlargements and rib lesions described in iguanas [13]. Hypercallosis has been reported in long bones of children with osteogenesis imperfecta [4, 5, 12]. This bone lesion is remarkably similar both grossly and microscopically to the diaphyseal enlargements seen in the hatchling iguanas with the typical diet-induced osteodystrophy. Supracortical masses composed of cartilage, woven bone, and ehondroid and myxomatous tissue encroached upon the adjacent skeletal muscle and stretched the overlying skin. The etiology of the hypercallosis in children is unknown, although hypovitaminosis C and mineralization of fracture hematomas have been suggested as possible causes [12]. However, there was no evidence of subperiosteal hemorrhages in iguanas of this study and the lesions were not consistently associated with fractures. Since the diaphyseal enlargements in iguanas were associated with severe osteoporosis, physico-chemical stresses to the periosteum of weakened bones should be considered in the pathogenesis of hypercallosis in children. The proliferative changes in the diaphysis of iguanas also shared certain features with the bone lesions of pulmonary hypertrophic osteoarthropathy, but there was not a space-occupying lesion in the thorax or pulmonary infection. The results of this investigation demonstrated that dietary deficiency of calcium results in secondary hyperparathyroidism in iguanas as it does in mammals and birds. Ultrastructural studies revealed that chief cells in the parathyroids were primarily in the active stage of the secretory cycle [2]. The rough endoplasmie reticulum was hypertrophied and often present as extensive circular arrays that nearly filled the cytoplasmic area. Golgi complexes were prominent
246
M.P. Anderson and C. C. Capen
in t h e perinuclear region. Chief cells of p a r a t h y r o i d glands were d e g r a n u l a t e d of m a t u r e s e c r e t o r y granules a n d C-cells in t h e u l t i m o b r a n c h i a l g l a n d a c c u m u l a t e d storage granules in response to t h e l o n g - t e r m d i e t - i n d u c e d h y p o c a l c e m i a . Conc o m i t a n t d i e t a r y p h o s p h o r u s deficiency in iguanas resulted in t h e d e v e l o p m e n t of a p r o m i n e n t o s t e o m a l a c i a in a d d i t i o n to t h e osteoporosis. W i d e osteoid seams were p r e s e n t in t h e periosteal zone a n d n u m e r o u s large osteoblasts were embedd e d in t h e t h i c k l a y e r of osteoid [1]. L a c u n a e of d e e p - s e a t e d cortical osteocytes h a d i r r e g u l a r l y roughened borders a n d occasionally a d j a c e n t lacunae a p p e a r e d to coalesce due to osteocytic osteolysis. I n active i g u a n a s t h e slowly developing osteoporosis led to h y p e r p l a s i a of t h e p e r i o s t e u m in long bones with differentiation of p r i m i t i v e m e s e n c h y m a l cells into cartilage, osteoid, a n d fibrous connective tissue to form striking d i a p h y s e a l enlargements. The authors gratefully acknowledge the valuable technical assistance of Robert Ashleman, Anita Burkey, Richard Gallagher, Owen Kinding, Sally Lause, Michael Ott, Dorothy Roof, and Barbara Speidel.
References 1. Anderson, M. P., Capen, C. C.: Fine structural changes of bone cells in experimental nutritional osteodystrophy of green iguanas. Virchows Arch. Abt. B 20, 169-184 (1976) 2. Anderson, M. P., Capen, C. C.: Ultrastructural evaluation of parathyroid and ultimobranchial glands in iguanas with experimental nutritional osteodystrophy. Gen. comp. Endocr. (1976): (In press) 3. Baginski, E., Zak, B. : Micro-determination of serum phosphate and phospholipids. Clin. chim. Acta 5, 843-848 (1960) 4. Baker, S. L.: Hyperplastic callus simulating sarcoma in two cases of fragilitas ossium. J. Path. Bact. 58, 609-623 (1946) 5. Banta, J. V., Schreiber, R. R., Kulik, W. J. : Hyperplastic callus formation in osteogenesis imperfecta simulating osteosarcoma. J. Bone J t Surg. A 53, 115-122 (1971) 6. Bruce, H. M., Parkes, A. S. : Rickets and osteoporosis in Xenopus laevis. J. endocr. 7, 64-81 (1950) 7. Clark, N. B. : Parathyroid glands in reptiles. Amer. Zool. 7, 869-881 (1967) 8. Clark, N. B. : Effect of parathyroidectomy in the lizard, Anolis carolinensis. Gen. comp. Endocr. 10, 99-102 (1968) 9. Clark, N. B.: Function of the parathyroid gland of the snake, Thamnophis sirtalis. J. exp. Zool. 178, 9-14 (1971) 10. Clark, N. B. : The ultimobranchial body of reptiles. J. exp. Zool. 178, 115-124 (1971) 11. Cowan, D. F.: Diseases of captive reptiles. J. Amer. vet. med. Ass. 153, 848-859 (1968) 12. Fairbank, H. A. T., Baker, S. L. : Hyperplastic callus formation, with or without evidence of a fracture, in osteogenesis imperfecta. Brit. J. Surg. 36, 1-16 (1948) 13. Frye, F. L., Dutra, F. : Multiple osteocartilagenous exostoscs in a monitor lizard. VM/SAC, 68, 1414-1416 (1973) 14. Goldner, J. A. : A modification of the Masson trichromc technique for routine laboratory purposes. Amer. J. Path. 14, 237-243 (1938) 15. Ippen, R. : Considerations on the comparative pathology of bone disease in reptiles. Zbl. allg. Path. path. Anat. 108, 424434 (1966) 16. Jowsey, J., Kelly, P. J., Riggs, B. L., Bianeo, A. J., Jr., Scholz, D. A., Gershon-Cohen, J. : Quantitative microradiographic studies of normal and osteoporotic bone. J. Bone J t Surg. A 47, 785-806 (1965) 17. Krutsay, M.: Methode zur Darstellung einiger Kalziumverbindungen in histologischen Schnitten. Acta histochem. (Jena) 15, 189-191 (1963) 18. Miller, R. M. : Nutritional secondary hyperparathyroidism (a review of etiology, symptomatology and treatment in companion animals). VM/SAC 64, 400-408 (1969)
Nutritional Osteodystrophy in Iguanas
247
19. Moseley, J. M., Matthews, E. W., Breed, R. H., Galante, L., Tse, A., MacIntyre, I.: The ultimobranchial origin of calcitonin. Lancet 108-110 (1968) 20. Oguro, C. : Parathyroid gland of the snake (Elaphe quadrivirgata] with special reference to parathyroidectomy. Gen. comp. Endocr. 15, 313-319 (1970) 21. Pritchard, J. J., Ruzicka, A. J.: Comparison of fracture repair in the frog, lizard and rat. J. Anat. (Lond.) 84, 236-261 (1950) 22. Sidky, Y. A.: Histological studies on the parathyroid glands of lizards. Z. Zellforsch. 65, 760-769 (1965) 23. Sidky, Y.A.: Effect of parathyroidectomy in lizards. Gen. comp. Endocr. 7, 22-26 (1966) 24. Sidky, u A. : The carotid sinus in lizards with an anatomical survey of the ventral neck region. J. Morph. 121, 311-322 (1967) 25. Smith, H. M. : Handbook of lizards. Ithaca, N.Y. : Comstock 1946 26. Wallach, J . D . : Environmental and nutritional diseases of captive reptiles. J. Amer. vet. med. Ass. 159, 1632-1643 (1971) 27. Weisbrode, S. E., Capen, C. C. : Effects of uremia and vitamin D on bone and the ultrastructure of thyroid parafollicular cells and parathyroid chief cells in the rat. Virchows Arch. Abt. B 16, 231-241 (1974) 28. Zwart, P., Van de Watering, C. C. : Disturbance of bone formation in the common iguana (Iguana iguana L.): pathology and etiology. Acta Zool. Path. Anat. 48, 333-356 (1969) Marilyn P. Anderson, DVM, PhD Charles C. Capen, DVM, PhD Department of Veterinary Pathobiology College of Veterinary Medicine The Ohio State University Co]umbus, Ohio 43210, USA
Nutritional Osteodystrophy in Captive Green Iguanas (Iguana iguana) * Marilyn P. Anderson and Charles C. Capen Department of Veterinary Pathobiology, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio Received April 15, 1976
Summary. Captive lizards fed a variety of vegetarian and carnivorous diets frequently develop severe nutritional osteodystrophy. Five hatchling iguanas which were captive for 5 months and fed a diet low in calcium and phosphorus developed clinical signs and lesions typical of nutritional osteodystrophy in captive lizards. They had tetany with severe osteoporosis, pathologic fractures, and cartilaginous diaphyseal enlargements of long bones which either were associated with fractures or with intact bones. Hatchling and juvenile iguanas fed a similar diet low in calcium (0.1%) and phosphorus (0.2%) under experimental conditions developed hypocalcemia, tetany, osteoporosis with osteomalacia and pathologic fractures. Experimental iguanas fed a diet low in calcium (0.2% ) but adequate in phosphorus (1.1%) developed progressive hypocalcemia and severe osteoporosis with pathologic fractures. Control iguanas fed a diet with adequate calcium (2.7%) and phosphorus (1.1%) had well mineralized bones with wide cortices and thick metaphyseal trabeculae. Iguanas fed low calcium experimental diets whether low or adequate in phosphorus developed hypocalcemia with compensatory secondary hyperparathyroidism resulting in severe osteodystrophy similar to that reported in captive lizards. Key words: Iguanas - - Bone disease - - Dietary calcium - - Hyperparathyroidism. Introduction
One of the most common diseases of captive lizards is an osteodystrophy which is secondary either to nutritional imbalances in calcium, phosphorus and vitamin D or to renal disease [11, 15, 26, 28]. Most lizards are known to be either omnivorous, carnivorous or herbivorous [25] but few nutrient requirements have been established qualitatively [26]. Formulation of adequate diets are difficult since quantitative nutrient requirements are not known for reptiles. Nutritional imbalances in calcium, phosphorus and vitamin D are usually due to high meat diets without adequate calcium supplementation [18, 26]. Nutritional osteodystrophy in lizards is characterized by progressive locomotor difficulty, soft fragile bones, and rapidly forming fusiform enlargements of long bones [15, 26, 28]. Muscular twitching and tetany frequently occur in lizards with osteodystrophy. Similar signs have been reported in lizards and snakes which are hypocalcemic after parathyroidectomy [8, 9, 20, 23]. Most lizards with nutritional osteodystrophy improve clinically with calcium and vitamin D therapy [28]. The objectives of this investigation were (1) to characterize a diet-induced ostcodystrophy in captive hatchling green iguanas, and (2) to investigate the pathogenesis of nutritional osteodystrophy in reptiles by correlating changes in * Supported by grants RR 05463, GM 1052 and RR 00366-05, National Institutes of Health, USPHS
230
M . P . Anderson and C. C. Capen
plasma electrolytes with sequential histopathologic and radiographic alterations in b o n e s of g r e e n i g u a n a s f e d e x p e r i m e n t a l d i e t s w i t h v a r i e d c a l c i u m a n d phosphorus content. M a t e r i a l s a n d Methods
Diet-Induced Osteodystrophy Five hatchling green iguanas (Iguana iguana), 12 g body weight and 7 cm snout-vent length, were obtained as part of an airfreight shipment from a commercial dealer in California. The iguanas were housed in chick incubators at 28 ~ C and with 10 h of light per day. Tap water was available ad libitum and twice a day the iguanas were fed a mixture composed of equal volumes of drained canned corn, peas, applesauce and a high meat baby food 1. Powdered eggshell was added to the diet several weeks after onset of clinical illness and until presentation for necropsy.
Experimental Osteodystrophy Thirty hatchling (12 g body weight, 7 cm snout-vent length) and 40 juvenile (90 g body weight, 12 cm snout-vent length) green iguanas (Iguana iguana) were received by airfreight from a commercial dealer in Florida. Stainless steel pans (28.0 • 45.7 • 17.8 cm) fitted on top with aluminum wire screen (16 • 18 mesh) were used to house 2 to 3 iguanas. Disposable absorbent paper 2 was used to line the bottom of the pans. The iguanas were fed once a day on quartered white paper plates and tap water was available ad libitum in petri dishes. Ambient temperature was maintained at 28~ with a fan driven space heater in a 5 by 10 foot room and 10 h of nonactinic light were provided each day. An adequate humidity was maintained by placing a 25 cm diameter bowl of water in front of the space heater. The hatchling iguanas were randomly divided into 2 experimental dietary groups and 1 control group. The juvenile iguanas were similarly divided into 3 groups. The control diet was equivalent to the experimental low CA-adequate P diet (Table 1) but supplemented with 8.0 g of calcium carbonate to contain 2.7% calcium and 1.1% phosphorus (Table 2). The second experimental diet was composed of equal volumes of applesauce, drained canned corn, drained canned peas, and a high meat baby food. 3 It was low in both calcium and phosphorus (Table 2). The ingredients of the baby food diet were homogenized in a blender. 4 The diets were stored in 18 oz plastic bags 5 and frozen until 12 h before use. All iguanas were serially radiographed at 1 to 3 month intervals using an industrial X-ray unit 6 and nonscreen X-ray film 7 at 5 Ma and 25 to 30 KV for 2 to 15 seconds at a film focal distance of 43 em. Masking tape was used to secure the iguanas in close apposition to the film. Body weight was recorded weekly for all iguanas. Three hatchling and 3 juvenile iguanas from each dietary group were euthanatized at 1 to 3 month intervals by giving lethal doses of ketamine hydrochloride s subcutaneously over the back or intramuscularly in the tail. Plasma samples were collected at the time of necropsy. The neck was cut just posterior to the skull from the dorsum until the carotid arteries were severed. Freely flowing blood was pipetted into heparinized test tubes. The plasma was frozen at --20 ~ C until electrolyte analyses were performed. Phosphorus values were determined by a micromethod [3] and calcium values were determined by atomic absorption spectrophotometry. 9 1 High meat dinner (strained, chicken with vegetables), Gerber Products Co., Fremont, Mich. 49412 2 Anipads, ANCARE Corp., Manhasset, L.I., N.Y. 11030 3 High meat dinner (strained, chicken with vegetables and cereal), Beech-Nut, Inc., New York, N.Y. 10022 4 Model 613-A-S, Hamilton Beach, Division of Scovill Mfg. Co., Waterbury, Ct. 5 Whirl-Pak, NASCO, Fort Atkinson, Wis. 53538 6 Model LC-90, General Electric Co. 7 Monopak, GAF Corp., New York, N.Y. 10020 8 Vetalar, Parke-Davis and Co., Detroit, Mich. 48232 9 Model 303 atomic absorption spectrophotometer, Perkin-Elmer, Norwalk, Ct. 06852
Nutritional 0steodystrophy in Iguanas
231
Table 1. Experimental low calcium-adequate phosphorus diet Ingredient
Amount
Fresh, lean, ground beef heart MnSO 4" H20 KlOa Folic acid L-ascorbic acid Ca (H2PO4)2.H20 CaCOa Glucose Corn oil
500 g 19.1 mg 38 tzg 135 ~g 50 mg 610 mg 100 mg 1.12 g 2.25 g
Table 2. Diet analyses expressed as percent on a dry weight basis Diet
Calcium
Phosphorus Ca:P
Protein
Fat
Control Experimental low CA adequate P Experimental low CA low P
2.7 0.2 0.1
1.1 1.1 0.2
82 82 17
14 14 6
2.4:1 1:5.5 1:2
The left parathyroid gland and left ultimobranchial gland were collected for electron microscopy and 1 ~z sections were stained with toluidine blue for light microscopic evaluation [27]. Iguana iguana have 2 parathyroid glands located at the junction of the common carotid artery with the internal and external carotid arteries and the ductus caroticus. Only 1 ultimobranchial gland, on the left side, was found in the hatchling and juvenile iguanas. I t was situated along the trachea immediately dorsal to the thyroid gland. This location was more cranial than has been reported in other lizards [7, 10, 19, 24]. Sections of femur, humerus and kidney were fixed in 10% neutral buffered formalin. Bone, after demineralization to effect in 10% sodium ethylenediaminetetraacetate, and kidney were sectioned at 6 t~ and stained with hematoxylin and eosin. Femur and humerus also were fixed in 70% ethyl alcohol, embedded in methyl methacrylate [16], sectioned at 6 ~zand stained with Goldner's modified trichrome [14] or Krutsay's modified silver stain [17].
Results
Diet-Induced Osteodystrophy The h a t c h l i n g i g u a n a s were active, gained weight a n d h a d n a t u r a l b o d y conf o r m a t i o n until a b o u t t h e 3rd m o n t h in t h e l a b o r a t o r y . The iguanas c l i m b e d a n d m o v e d a b o u t less t h a n usual a n d w i t h difficulty. W i t h i n t h e following 2 weeks 1 or b o t h femurs d e v e l o p e d oval d i a p h y s e a l enlargments. B y 5 m o n t h s all i g u a n a s h a d d i a p h y s e a l e n l a r g e m e n t s on 1 or m o r e long bones (Fig. 1). T h e femur, humerus, tibia, fibula a n d r a d i u s were m o s t c o m m o n l y affected. Some of t h e p h a l a n g e s were similarly affected. The d i a p h y s e a l e n l a r g e m e n t s were c h a r a c t e r i s t i c a l l y oval a n d firm e x t e n d i n g to t h e p h y s e a l region b u t n o t bridging epiphyses or j o i n t capsules. T h e overlying skin was d a r k , f i r m l y a t t a c h e d a n d s t r e t c h e d t a u t (Fig. 1). P r o g n a t h i s m a n d increased flexibility of t h e m a n d i b l e s were p r o n o u n c e d b u t grossly visible t h i c k e n i n g of m a n d i b u l a r a n d m a x i l l a r y bones was n o t observed. The a p p e t i t e was m a i n t a i n e d u n t i l several d a y s before d e a t h . The iguanas r e q u i r e d assistance to e a t a n d d r i n k during t h e last 1 or 2 weeks of life due to
232
M.P. Anderson and C. C. Capen
Fig. 1. Diaphyseal enlargement of the right tibia and fibula in a hatchling iguana fed a low calcium and low phosphorus diet for 4 months. Note the taut skin over the swollen region (arrow) Fig. 2. Tetany in a hatchling iguana fed a low calcium and low phosphorus diet for 5 months. The right tibia has a characteristic diaphyseal enlargement
difficulties in ambulation and stiffness of the vertebral column. Tctany lasting 15 to 30 seconds was observed sporadically for several days prior to death (Fig. 2). Tetany was characterized by arching of the back and neck with extension of the limbs. Two iguanas died during the night and 3 were euthanatized following tetany. Radiographic examination at necropsy revealed generalized osteopenia with poorly mineralized, thin cortices of long bones including those with diaphyseal enlargements. The actual extent of palpable and grossly visible diaphyseal enlargements was not disclosed radiographically. Fractures of the diaphysis were not consistently associated with the mid-shaft enlargments, but pathologic fractures were frequently seen in the metaphysis of affected bones. Long bones had enlarged medullary cavities and an abnormal rectangular shape due to failure of remodeling in the cut back zone (Fig. 3). Dissection of skin and muscle tissue from limbs at necropsy revealed oval to fusiform, white enlargements which encircled most of the shaft of the long bones (Fig. 4). These enlargements were cartilaginous in appearance and could be sectioned readily with a scalpel.
Nutritional Osteodystrophy in Iguanas
233
Fig. 3. Radiograph illustrating osteopenia with enlarged medullary cavities and oval diaphyseal proliferations of partially mineralized cartilage (arrows) in a hatchling iguana fed a low calcium and low phosphorus diet for 5 months Fig. 4. Bilateral diaphyseal cartilagenous enlargement (arrows) in the femur, tibia and fibula of a hatehling iguana. The skin and skeletal muscle were removed from the leg on the left; only skin was removed from the right leg
Undecalcified histologic sections of affected femurs revealed thin mineralized cortices with a cuff of proliferating cartilage that replaced and/or compressed the overlying skeletal muscle (Fig. 5). Fractures of cortical bone often were not evident in affected long bones. I n addition, the total mass of metaphyseal spongiosa of long bones was deficient due to reduction in number, length and width of trabeculae in the primary and secondary spongiosa. The lesions in long bone varied considerably in severity. The least severe lesion was a proliferation of spindle cells in the periostcum of the diaphysis forming a layer several cells thick over the thin cortices. More advanced lesions were characterized by a marked thinning of cortical bone t h a t was surrounded by consecutive layers of osteoid, hyahne cartilage and primitive mesenchymal tissue. This immature connective tissue invaded as well as displaced overlying skeletal muscle. Mature hyaline cartilage was the predominant component in the diaphyseal
234
M. 1). Anderson and C. C. Capen
O
Z
!7
E> O
~ v
9 ~b
. O
Nutritional Osteodystrophy in Iguanas
235
masses of the most severely enlarged bones and was primarily responsible for the diaphyseal enlargements detected clinically. The cartilage was partially mineralized and had vascular resorption cavities in more central areas. Primary vascular channels in the cartilaginous mass enlarged to form hematopoietic marrow spaces. This often resulted in cartilaginous trabeculae oriented perpendicular to the shaft of the long bone. There appeared to be some direct metaplasia of cartilage to bone as well as endochondral bone formation following chondroclastic remodeling. When fractures occurred in long bones they were usually in the metaphysis and a typical cartilaginous callus was seen along both the endosteal and periosteal surfaces of the cortex [21]. Vertebrae had lesions similar to those of long bones. The dorsal, lateral and ventral laminae encasing the spinal cord were severely thinned. The vertebral bodies often had massive periosteal cartilaginous proliferations ventrally and laterally with compression of subjacent muscle and spinal nerves. There was no compression of the spinal cord by the expanding masses of cartilage.
Experimental Osteodystrophy in Hatehling Iguanas Initially hatchling iguanas in each of the 3 dietary groups were active, alert and ate regularly once a day. The iguanas fed the experimental diet low in calcium and phosphorus had a clinical disease similar to the iguanas with the typical diet-induced osteodystrophy except the course was protracted by several months. In contrast, iguanas fed the experimental low CA-adequate P diet had a shorter clinical course of approximately 4 weeks. Table 3. Body weight gains of hatchling iguanas expressed as percent of initial body weight Months on experiment
Dietary group Control
Experimental low CA-low 1)
1
12
6
2 3 4 5 6
42 58 65 89 102
10 18 34 26 24
Body weights in all groups of control and experimental iguanas increased initially but revealed significant differences later (Table 3). Mean initial and final body weight for the 3 dietary groups were 11.3 g and 27.3 g (control), 12.5 g and 10.5 g (experimental low CA-adequate P), and 14.2 g and 16.9 g (experimental low CA-low P), respectively. Iguanas fed the low CA-adequate P diet had a drop in body weight by the second week. The body weights of iguanas fed the low CA-low P diet decreased after the 4th month. Control iguanas continued to gain weight until the experiment was terminated at 6 months. Plasma calcium levels in experimental iguanas were lower and phosphorus levels were higher than in control iguanas although these differences were not
236
M.P. Anderson and C. C. Capen
Fig. 6. Radiograph illustrating osteopenia of the rear limbs with abnormally rectangular long bones (arrows) in a hatchling iguana fed a low calcium and low phosphorus diet for 6 months Fig. 7. Radiograph illustrating a pathologic fracture (arrow) of the left femur in an osteopenic hatchling iguana fed a low calcium and low phosphorus diet for 5 months Fig. 8. Radiograph illustrating well mineralized long bones in a control iguana fed a diet adequate in calcium and phosphorus for 6 months
statistically significant. Mean plasma calcium in hatchling iguanas fed the experimental low CA-low P diet fell from 8.2 mg/100 ml at 1 month to 7.8 rag/100 ml at 6 months with values decreasing as low as 4.2 mg/100 ml. Mean plasma phosphorus ranged from 1.4 rag/100 ml to 4.5 mg/100 ml during the experimental period. Mean plasma calcium of iguanas fed the experimental low CA-adequate P diet fell
Nutritional Osteodystrophy in Iguanas
237
Fig. 9. Prominent, well mineralized trabeculae (arrow) in the metaphysis of a femur from a control hatchling iguana. The control diet containing adequate calcium and phosphorus was fed for 6 months. H & E, x 125 Fig. 10. Short and narrow trabeculae (arrow) in the primary and secondary spongiosa of a femur from an experimental hatchling iguana. The experimental low calcium diet was fed for I month. H & E, x 125 Fig. 11. Absence of trabecular bone in the metaphysis of a femur from an experimental hatchling iguana. The experimental low calcium and low phosphorus diet was fed for 5 months. H & E, x 125
238
M.P. Anderson and C. C. Capen
from 11.3 mg/100 ml to 7.6 mg/100 ml within 6 weeks and the phosphorus level ranged from 2.6 to 3.7 mg/100 ml during this period. One iguana fed this experimental diet for 4 weeks was euthanatized while in tetany. The plasma calcium value was 7.6 mg/100 ml and phosphorus was 3.7 mg/100 ml. Several other iguanas in this group also had tetanic spasms 1 to 2 days prior to death. Mean plasma calcium in control iguanas ranged from 11.3 to 12.1 mg/100 ml and phosphorus ranged from 1.1 to 1.4 mg/100 ml. Generalized osteopenia was detected radiographically after the 2nd month in iguanas fed the experimental low CA-low P diet. Long bones had thin cortices and short, thin metaphyseal trabeculae. A failure of remodeling in the cut back zone resulted in abnormally rectangular long bones (Fig. 6). After the 4th month the iguanas developed diaphyseal swellings of limbs similar to those seen in the hatchling iguanas with the typical diet-induced osteodystrophy. However, all swellings were associated with pathologic fractures. The fractures were closed and usually occurred near the metaphysis (Fig. 7). Some fractures were oblique and involved the entire diaphysis. A mineralized callus was not evident radiographically after 5 to 6 weeks. However, a firm cartilaginous callus was palpable and grossly visible. Radiographically, long bones of iguanas fed the experimental low CA-adequate P diet had thin cortices. There was no evidence of diaphyseal enlargements of long bones or other gross lesions. In contrast, long bones of control iguanas were well mineralized with thick cortices and normal conformation (Fig. 8). Microscopically, the femur of control iguanas had thick well mineralized cortices with numerous broad trabeculae in the metaphysis (Fig. 9). After 1 to 2 months the experimental iguanas had a striking reduction in the amount of bone in the primary and secondary spongiosa (Fig. 10). After 5 months both primary and secondary spongiosa were absent in the experimental iguanas fed the low CA-low P diet (Fig. 11). The cortices were greatly attenuated and a prominent transverse layer of osteoid covered the metaphyseal surface of the physis. There was no quantitative difference in thickness of the physis between control and experimental iguanas. However, experimental iguanas fed the low CA-low P diet had chondrocytes arranged more in small clumps in the zone of hypertrophic cartilage and not as well aligned as in control iguanas.
Experimental Osteodystrophy in Juvenile Iguanas The iguanas in each dietary group initially were active, alert and ate regularly once a day. Iguanas fed either the control or the experimental low CA-low P diet maintained their daily food consumption, activity and alertness until termination of the experiment at 16 months. After the 8th month iguanas fed the experimental low CA-adequate P diet had mandibles that were slightly more flexible than those of control iguanas. Mandibular pliability was readily detected by the 12th month and at 16 months the mandibles could be easily approximated by medial compression to half the distance of their separation. Excessive loss of teeth was frequently noted in iguanas fed the experimental low CA-low P diet for 12 or more months. By comparison, iguanas fed the experimental low CAadequate P diet had reduced food consumption after 4 months and were eating
Nutritional Osteodystrophy in Iguanas
239
Table 4. Body weight gains of juvenile iguanas expressed as percent of initial body weight Months fed diet
Dietary group Control
Experimental low CA-low P
Experimental low CA-adequate P
2 4 6 8 10 12 14 16
25 47 47 68 66 79 68 78
13 20 21 22 27 32 36 48
6 12 9 12 14 ----
every 2nd or 3rd day. They became progressively less active and alert from the 6th month to the 10th month of feeding the low CA-adequate P diet. Slight mandibular flexibility was detected only after the 8th month in juvenile iguanas fed the experimental low CA-adequate P diet. Excessive tooth loss was not noted in juvenile iguanas fed either the experimental low CA-adequate P diet or control diet. Neither enlargements nor other abnormal conformations were noted in facial bones of iguanas in any of the three groups. Mandibles of control iguanas were well mineralized and inflexible for the 16 month experimental period. Body weight gains of juvenile iguanas in the three dietary groups differed after the 5th month (Table 4). The body weight of iguanas fed the low CA-low P diet plateaued between the 4th and 8th month. The slight increase in body weight after the 8th month was due primarily to the development of ascites and anasarea. The body weight of juvenile iguanas fed the experimental low CAadequate P diet plateaued and did not increase during the experimental period of 10 months. In contrast, iguanas fed the control diet had a progressive gain in body weight, without ascites, during the 16 month experimental period. Mean initial and final body weights of iguanas in the three dietary groups were 96 g and 275 g (control), 92 g and 106 g (experimental low CA-adequate P), and 91 g and 163 g (experimental low CA-low P), respectively. Plasma calcium and phosphorus values were similar for juvenile iguanas in the 3 groups until about the 3rd month (Fig. 12). Both groups of experimental iguanas developed progressive hypocalcemia and hyperphosphatemia although these differences from controls were not statistically significant. The range of plasma calcium and phosphorus values was less variable in the control iguanas compared to the experimental iguanas. Plasma calcium was consistently above 9 rag/100 ml and the plasma phosphorus usually was below 7 mg/100 ml in control juvenile iguanas (Fig. 12). The reason for the higher plasma phosphorus at 16 months is uncertain but it represents a single control iguana that had a moderate interstitial fibrosis of the kidney. Generalized osteopenia was evident radiographieally at 2 to 3 months and increased in severity throughout the experimental period in iguanas fed either of the experimental diets (Fig. 13). Multiple rib fractures usually not associated
240
M.P. Anderson and C. C. Capen
Fig. 12. Mean plasma calcium and phosphorus in juvenile iguanas fed experimental and control diets. ND= no data. C= control diet with adequate CA and P. B= low CA-low P diet. H--low CA-adequate P diet with costochondral junctions were present in iguanas fed either of the experimental diets for 3 or more months (Fig. 13). One iguana fed the experimental low CAadequate P diet had a pathologic fracture in the distal femur at 3 months. Radiographically there was no evidence of callus formation and mineralization after 4 weeks when the iguana was euthanatized. However, a cartilaginous callus was present at necropsy. Ascites was detected radiographically in iguanas fed the experimental low CA-low P diet for 10 to 16 months. Costochondral junctions and physes appeared normal in the three groups of iguanas. Bones of control iguanas were well mineralized and without evidence of osteoporosis throughout the 16 months period of the experiment. The femur of juvenile iguanas fed the experimental low CA-adequate P diet had varying degrees of osteoporosis characterized by thinned cortices and wide medullary cavity with narrow trabeculae in the metaphysis. Occasionally, flattened osteoblasts lined endosteal and periosteal surfaces. Large multinucleated osteoclasts in prominent Howship's lacunae were particularly abundant along endosteal surfaces and metaphyseal trabeculae after feeding the experimental diet for 3 to 6 months. Osteoclasts diminished in number and size from 6 to 10 months and were situated along primary trabeculae. Cortical bone was thin and metaphyseal trabeculae were lined by flattened ostcoblasts. Osteoid was sparse and was present mainly as a thin layer along the base of cartilage columns of the physis. The femurs of iguanas fed the experimental low CA-low P diet had a similar osteoporosis after the 2nd to 3rd month with thin cortices and narrow trabeculae in the metaphysis (Fig. 14). In contrast to the iguanas fed the experimental low
Nutritional Osteodystrophy in Iguanas
241
.~
O
~
.~
c~
~'~ 9 ~
.~,~
CA-adequate P diet, osteoid seams were progressively widened and covered the narrow trabeculae after the 5th month (Fig. 15). Cortical bone was thin and there were wide osteoid seams on endosteal and periosteal surfaces. Osteoblasts varied from flat to plump and were present in moderate numbers at all intervals examined. 0steoclasts were present in Howship's lacunae until the 5th month and were limited mainly to subphyseal and diaphyseal locations because of the osteoid accumulation on other endosteal surfaces. Femurs of control iguanas were characterized by wide cortices and abundant, thick metaphyseal trabeculae (Fig. 16). Osteoblasts varied in shape from flat to
Fig. 14. Osteoporosis in a juvenile iguana fed a low calcium and low phosphorus diet for 16 months. Note the narrow metaphyseal trabeeulae (long arrow) and t h i n cortex (short arrow). H & E, x 50
Fig. 15. Osteomalacia in the femur of an iguana fed a low calcium and low phosphorus diet for 16 months. Note the wide osteoid seams (arrows). Same bone as in Figure 14. Goldner's modified trichrome, X 125
Fig. 16. F e m u r from a control iguana at 16 months. Note the thick metaphyseal trabeculae (long arrows) and wide cortex (short arrow). H & E, X 50
Fig. 17. Metaphyseal trabecula of the femur from a control iguana. Osteoid seams are narrow (arrow). Same bone as in Figure 16. Goldner's modified trichrome, x 125 5 VirchowsArch. B Cell Path.
244
M.P. Anderson and C. C. Capen
plump and usually lined one side of the metaphyseal trabeculae. The opposite sides of the trabeculae were usually irregular in contour due to the presence of Howship's lacunae with or without osteoelasts. Moderate numbers of osteoclasts were present on subphyseal, trabecular and diaphyseal endosteal surfaces. Osteoid was present in small amounts along trabeeulae (Fig. 17) but osteoid seams were narrow compared to the abundant osteoid accumulations in iguanas fed the experimental low CA-low P diet (Fig. 15).
Histologic Evaluation o/Parathyroid and Ultimobranchial Glands and Kidney /rom Hatehling and Juvenile Iguanas with Experimental Osteodystrophy Parathyroid and ultimobranchial glands of hatchling and juvenile iguanas did not differ grossly in size, shape, color or consistency between control and experimental iguanas during the experimental periods of 6 months (hatchling) and 16 months (juveniles). Neither parathyroid nor ultimobranchial glands had evidence of cyclic degeneration as has been reported for lizards collected from the wild at various times of the year [22]. Chief cells of the parathyroid gland were closely packed together, oval or elongate and were subdivided into several groups by fine strands of fibrous connective tissue and capillaries. Chief cells of iguanas fed the experimental diets had a greater cytoplasmic to nuclear ratio than control iguanas especially after the 2nd month for the hatchling iguanas and after the 3rd month for the juvenile iguanas. Ultimobranchial glands were composed mainly of c-cell containing follicles lined with a columnar to pseudostratified columnar epithelium. Small clumps of c-cells were interspersed in connective tissue between the follicles. There were no differences detectable by light microscopy between the experimental and control iguanas [2]. Lesions were not present in kidneys of hatchling or juvenile iguanas throughout the experimental period except for 1 control iguana which had moderate interstitial fibrosis. Discussion Iguanas fed low calcium diets develop hypocalcemia, secondary hyperparathyroidism, and severe bone loss that shared many features with the disease in mammals and birds [18]. Hatchling iguanas fed the experimental low CAadequate P diet developed osteoporosis within 1 to 2 months whereas from 3 to 6 months were required for the bone lesions to develop in juvenile iguanas. The degree of diet-induced hypocalcemia varied in severity but neuromuscular tetany occasionally developed shortly before death. In addition, iguanas fed the low calcium and low phosphorus diets for 4 to 8 months developed severe osteomalacia superimposed on the osteoporosis. The osteoporosis and osteomalacia developed more rapidly in hatchlings compared to juvenile iguanas. The relatively low protein content of the low calcium--low phosphorus diet was reflected by ascites, anasarca, and may have contributed to the osteopenia that developed in iguanas fed this diet. The metabolic bone disease produced in iguanas by feeding the experimental diet low in calcium but adequate in phosphorus was similar to lesions reported
Nutritional Osteodystrophy in Iguanas
245
in toads (Xenopus laevis) which were fed all-liver diets [6]. Bone lesions in toads were present by 5 months and were characterized by thin, fragile long bones with pathologic fractures that had a poorly mineralized callus. Tetanic spasms of limbs and muscular twitching occurred shortly before death and there was radiographic evidence of generalized demineralization of the skeleton. Young toads were more severely affected than adults. Although adult toads were maintained for 15 months on the liver diet without clinical evidence of bone disease, radiographic examination revealed a progressive development of generalized osteoporosis. The toads developed a striking prognathism similar to that observed in hatchling iguanas with diet-induced osteodystrophy. The diaphyseal enlargements on long bones were observed more frequently in iguanas with the typical diet-induced osteodystrophy than in experimental iguanas housed in a more confined environment. This suggested that in iguanas which were in a negative calcium balance and had developed progressive osteoporosis, the physico-chemical stresses exerted by running and climbing stimulated the periosteum to reinforce the weakened bones structurally by the formation of concentric layers of osteoid, cartilage, and immature fibrous connective tissue. Experimental iguanas confined in small laboratory pens were less active and developed a similar severe degree of osteoporosis but with considerably less periosteal proliferation. Pathologic fractures developed occasionally in experimental iguanas with severe osteoporosis during periods of vigorous physical activity. The term "osteoperiostitis" has been used by other investigators to describe a proliferative lesion of this type in the diaphysis of long bones [11]. The anatomic location and histologic composition of a bone lesion reported in a monitor lizard (Varanus) appeared to be similar to the diaphyseal enlargements and rib lesions described in iguanas [13]. Hypercallosis has been reported in long bones of children with osteogenesis imperfecta [4, 5, 12]. This bone lesion is remarkably similar both grossly and microscopically to the diaphyseal enlargements seen in the hatchling iguanas with the typical diet-induced osteodystrophy. Supracortical masses composed of cartilage, woven bone, and ehondroid and myxomatous tissue encroached upon the adjacent skeletal muscle and stretched the overlying skin. The etiology of the hypercallosis in children is unknown, although hypovitaminosis C and mineralization of fracture hematomas have been suggested as possible causes [12]. However, there was no evidence of subperiosteal hemorrhages in iguanas of this study and the lesions were not consistently associated with fractures. Since the diaphyseal enlargements in iguanas were associated with severe osteoporosis, physico-chemical stresses to the periosteum of weakened bones should be considered in the pathogenesis of hypercallosis in children. The proliferative changes in the diaphysis of iguanas also shared certain features with the bone lesions of pulmonary hypertrophic osteoarthropathy, but there was not a space-occupying lesion in the thorax or pulmonary infection. The results of this investigation demonstrated that dietary deficiency of calcium results in secondary hyperparathyroidism in iguanas as it does in mammals and birds. Ultrastructural studies revealed that chief cells in the parathyroids were primarily in the active stage of the secretory cycle [2]. The rough endoplasmie reticulum was hypertrophied and often present as extensive circular arrays that nearly filled the cytoplasmic area. Golgi complexes were prominent
246
M.P. Anderson and C. C. Capen
in t h e perinuclear region. Chief cells of p a r a t h y r o i d glands were d e g r a n u l a t e d of m a t u r e s e c r e t o r y granules a n d C-cells in t h e u l t i m o b r a n c h i a l g l a n d a c c u m u l a t e d storage granules in response to t h e l o n g - t e r m d i e t - i n d u c e d h y p o c a l c e m i a . Conc o m i t a n t d i e t a r y p h o s p h o r u s deficiency in iguanas resulted in t h e d e v e l o p m e n t of a p r o m i n e n t o s t e o m a l a c i a in a d d i t i o n to t h e osteoporosis. W i d e osteoid seams were p r e s e n t in t h e periosteal zone a n d n u m e r o u s large osteoblasts were embedd e d in t h e t h i c k l a y e r of osteoid [1]. L a c u n a e of d e e p - s e a t e d cortical osteocytes h a d i r r e g u l a r l y roughened borders a n d occasionally a d j a c e n t lacunae a p p e a r e d to coalesce due to osteocytic osteolysis. I n active i g u a n a s t h e slowly developing osteoporosis led to h y p e r p l a s i a of t h e p e r i o s t e u m in long bones with differentiation of p r i m i t i v e m e s e n c h y m a l cells into cartilage, osteoid, a n d fibrous connective tissue to form striking d i a p h y s e a l enlargements. The authors gratefully acknowledge the valuable technical assistance of Robert Ashleman, Anita Burkey, Richard Gallagher, Owen Kinding, Sally Lause, Michael Ott, Dorothy Roof, and Barbara Speidel.
References 1. Anderson, M. P., Capen, C. C.: Fine structural changes of bone cells in experimental nutritional osteodystrophy of green iguanas. Virchows Arch. Abt. B 20, 169-184 (1976) 2. Anderson, M. P., Capen, C. C.: Ultrastructural evaluation of parathyroid and ultimobranchial glands in iguanas with experimental nutritional osteodystrophy. Gen. comp. Endocr. (1976): (In press) 3. Baginski, E., Zak, B. : Micro-determination of serum phosphate and phospholipids. Clin. chim. Acta 5, 843-848 (1960) 4. Baker, S. L.: Hyperplastic callus simulating sarcoma in two cases of fragilitas ossium. J. Path. Bact. 58, 609-623 (1946) 5. Banta, J. V., Schreiber, R. R., Kulik, W. J. : Hyperplastic callus formation in osteogenesis imperfecta simulating osteosarcoma. J. Bone J t Surg. A 53, 115-122 (1971) 6. Bruce, H. M., Parkes, A. S. : Rickets and osteoporosis in Xenopus laevis. J. endocr. 7, 64-81 (1950) 7. Clark, N. B. : Parathyroid glands in reptiles. Amer. Zool. 7, 869-881 (1967) 8. Clark, N. B. : Effect of parathyroidectomy in the lizard, Anolis carolinensis. Gen. comp. Endocr. 10, 99-102 (1968) 9. Clark, N. B.: Function of the parathyroid gland of the snake, Thamnophis sirtalis. J. exp. Zool. 178, 9-14 (1971) 10. Clark, N. B. : The ultimobranchial body of reptiles. J. exp. Zool. 178, 115-124 (1971) 11. Cowan, D. F.: Diseases of captive reptiles. J. Amer. vet. med. Ass. 153, 848-859 (1968) 12. Fairbank, H. A. T., Baker, S. L. : Hyperplastic callus formation, with or without evidence of a fracture, in osteogenesis imperfecta. Brit. J. Surg. 36, 1-16 (1948) 13. Frye, F. L., Dutra, F. : Multiple osteocartilagenous exostoscs in a monitor lizard. VM/SAC, 68, 1414-1416 (1973) 14. Goldner, J. A. : A modification of the Masson trichromc technique for routine laboratory purposes. Amer. J. Path. 14, 237-243 (1938) 15. Ippen, R. : Considerations on the comparative pathology of bone disease in reptiles. Zbl. allg. Path. path. Anat. 108, 424434 (1966) 16. Jowsey, J., Kelly, P. J., Riggs, B. L., Bianeo, A. J., Jr., Scholz, D. A., Gershon-Cohen, J. : Quantitative microradiographic studies of normal and osteoporotic bone. J. Bone J t Surg. A 47, 785-806 (1965) 17. Krutsay, M.: Methode zur Darstellung einiger Kalziumverbindungen in histologischen Schnitten. Acta histochem. (Jena) 15, 189-191 (1963) 18. Miller, R. M. : Nutritional secondary hyperparathyroidism (a review of etiology, symptomatology and treatment in companion animals). VM/SAC 64, 400-408 (1969)
Nutritional Osteodystrophy in Iguanas
247
19. Moseley, J. M., Matthews, E. W., Breed, R. H., Galante, L., Tse, A., MacIntyre, I.: The ultimobranchial origin of calcitonin. Lancet 108-110 (1968) 20. Oguro, C. : Parathyroid gland of the snake (Elaphe quadrivirgata] with special reference to parathyroidectomy. Gen. comp. Endocr. 15, 313-319 (1970) 21. Pritchard, J. J., Ruzicka, A. J.: Comparison of fracture repair in the frog, lizard and rat. J. Anat. (Lond.) 84, 236-261 (1950) 22. Sidky, Y. A.: Histological studies on the parathyroid glands of lizards. Z. Zellforsch. 65, 760-769 (1965) 23. Sidky, Y.A.: Effect of parathyroidectomy in lizards. Gen. comp. Endocr. 7, 22-26 (1966) 24. Sidky, u A. : The carotid sinus in lizards with an anatomical survey of the ventral neck region. J. Morph. 121, 311-322 (1967) 25. Smith, H. M. : Handbook of lizards. Ithaca, N.Y. : Comstock 1946 26. Wallach, J . D . : Environmental and nutritional diseases of captive reptiles. J. Amer. vet. med. Ass. 159, 1632-1643 (1971) 27. Weisbrode, S. E., Capen, C. C. : Effects of uremia and vitamin D on bone and the ultrastructure of thyroid parafollicular cells and parathyroid chief cells in the rat. Virchows Arch. Abt. B 16, 231-241 (1974) 28. Zwart, P., Van de Watering, C. C. : Disturbance of bone formation in the common iguana (Iguana iguana L.): pathology and etiology. Acta Zool. Path. Anat. 48, 333-356 (1969) Marilyn P. Anderson, DVM, PhD Charles C. Capen, DVM, PhD Department of Veterinary Pathobiology College of Veterinary Medicine The Ohio State University Co]umbus, Ohio 43210, USA
