A Case of Congenital Hyperostosis in a Newborn Piglet

NOTE Pathology

Koji UETSUKA1)*, Takayuki SUZUKI1), Hirohito OGAWA1), Hiroko SATO1), Kunio DOI1) and Tetsuo NUNOYA1)

1)Nippon Institute for Biological Science, 9–2221–1 Shinmachi, Ome, Tokyo 198–0024, Japan

(Received 21 June 2011/Accepted 18 September 2011/Published online in J-STAGE 30 September 2011)

ABSTRACT. One female newborn piglet showed prominent thickening of both forelimbs and died soon after birth. Histopathologically, thin and woven trabeculae of bone was extending out from the edge of cortical bone in the affected forelimbs, and diagnosed as congenital hyperostosis. The extent of radially proliferated trabeculae was most prominent in radioulna. Many round- to spindle-shaped cells were observed in periosteum, which were considered to be osteoblasts. Around the periosteum, the mesenchymal proliferation was extensive with abundant mucus, and cartilaginous metaplastic changes were observed mainly around the radioulna and humerus. Dila- tation of vessels with fibrin deposition in vessel walls was often observed, which were considered to reflect the localized circulatory disturbance. KEY WORDS: bone, congenital anomaly, congenital hyperostosis, pathology, piglet. doi: 10.1292/jvms.11-0297; J. Vet. Med. Sci. 74(2): 259–262, 2012

Congenital hyperostosis (cortical hyperostosis, diaphy- hindlimbs were collected and fixed in 4% phosphate-buff- seal dysplasia) is a rare disease of newborn pigs. Affected ered paraformaldehyde solution. Four-micron-thick sec- piglets are either stillborn or die within the first few days of tions were prepared from paraffin-embedded tissue blocks life, and show various degrees of thickening of one or more using standard methods and stained with hematoxylin and forelimbs, and sometimes the hindlimbs as well [11]. eosin (HE). In addition, alcian blue (AB)-periodic acid Diseases similar to congenital hyperostosis in newborn Schiff (PAS) double-staining, phosphotungstic acid hema- pigs have been reported in human [4] and other animal spe- toxylin (PTAH) staining, and immunostaining for vimentin cies including monkeys [10], dogs [2, 3], foals [12] and were performed in forelimb sections. chickens [5]. Among them, most intensely investigated one Immunohistochemistry was performed by the labeled is infantile cortical hyperostosis in human, which is also streptavidin biotin (LSAB) method using primary mouse known as Caffey’s disease [4]. Comparing to this, reports in monoclonal antibody against vimentin (Dako, Carpinteria, animal disorders including congenital hyperostosis in pig- CA, U.S.A.). The secondary antibody used was biotinylated lets are scarce [5, 6, 9]. rabbit anti-mouse IgG antibody (KPL, Gaithersburg, MD, As the cause of congenital hyperostosis in pigs, two U.S.A.). The sections were incubated with peroxidase- pathogenesis have been considered. One is due to inherit- labeled streptavidin (Dako, Glostrup, Denmark) and visual- ance as an autosomal recessive trait, and the other is due to ized with 3,3’-diaminobenzidine-tetrahydrochloride as a local circulatory disturbance [11]. In the present case chromogen. Counterstaining was done with hematoxylin. reported here, the genetic background of relevant piglets is For electron microscopy, paraformaldehyde-fixed tissues obscure, therefore, we examined histopathologic character- were refixed in 1% osmium tetroxide, and embedded in istics of affected forelimbs including the local circulatory disturbance as a possible cause. Namely, we compared the histopathologic changes and their distributions among the bones constituting forelimbs, that is, humerus, radioulna and metacarpal. The occurrence of disease was detected in a 170-sow far- row-to-feeder farm located in A prefecture in Japan, and in the farm, the condition has been occasionally observed dur- ing the past years. In the present case, among 10 littermates, prominent thickening of both forelimbs was observed in 5 piglets, of which 3 died soon after birth. One female of these 3 littermates was submitted to the authors’ institute for pathological diagnosis (Fig. 1). Tissue samples of various organs and both forelimbs and

*CORRESPONDENCE TO: UETSUKA, K., Nippon Institute for Biological Science, 9–2221–1 Shinmachi, Ome, Tokyo 198–0024, Japan. e-mail: [email protected] Fig. 1. Gross appearance of the affected piglet. There is a ©2012 The Japanese Society of Veterinary Science prominent thickening of forelimbs and cervicofacial areas. 260 K. UETSUKA ET AL. epoxy resin. Ultrathin sections were double stained with uranyl acetate and lead citrate, and observed using a trans- mission electron microscope (H600, Hitachi, Tokyo, Japan). Since the virus-induced osteopetrosis, one of similar diseases to congenital hyperostosis in newborn pigs, is known in chickens [1], microbiological examinations were performed on some organ samples including the forelimb to exclude the involvement of virus as well as other infectious agents. In the bacterial examination, Vagococcus fluvialis and Staphylococcus sp. were isolated from the lungs and heart, respectively. However, no pathogenic bacteria were isolated from any samples including the forelimb. The virological test was performed according to the multiple PCR method described by Ogawa et al. [8] on the brain, lung, lung-associated lymph node and forelimb. However, there were no positive reactions to type 2 porcine circovirus, Aujeszky’s disease virus, rotavirus, transmissi- ble gastroenteritis virus, getah virus, porcine reproductive Fig. 2. (A) The transverse cut surface of affected forelimb. With and respiratory syndrome virus, Japanese encephalitis virus periosteal edema and proliferation of the mesenchymal tissues, and porcine epidemic diarrhea virus. obvious osteophytic bone formation is observed from the radius At necropsy, there was a prominent thickening of fore- and ulna. (B) The transverse cut surface of radius and ulna. The radial formation of osteophytic bone is obvious. (C) The trans- limbs and cervicofacial areas. The affected forelimbs verse cut surface of radius. Radiating trabeculae of woven bone became swollen up to twice their normal size and rigid with extend from the surface of apparently normal cortical bone. difficult movement. The transverse cut surface of the Bar=500 m. HE. affected forelimb bones at the diaphyses showed an obvious osteophytic bone formation radially from the radius and ulna with periosteal edema and proliferation of mesenchymal tissues (Fig. 2A and 2B). The other organs and tissues including hindlimbs appeared grossly normal. Histopathologically, the affected forelimbs revealed radial formation of thin and woven trabeculae of bone which extended out from the edge of cortical bone with normal appearance beneath the thickened periosteum (Fig. 2C). As these changes were consistent with the previous report [5, 6, 9], the present case was diagnosed as congenital hyperostosis in newborn piglet. Congenital hyperostosis in piglets have been reported only in a few papers, and histopathological findings were scarcely detailed. Therefore, in this report, we examined histopathological changes furthermore. The extent of radially proliferated trabeculae was compared among bones constituting the forelimb, that is, humerus, radioulna and metacarpal at their diaphysis. The proliferation was most prominent in radioulna, moderate in humerus, and just mild in metacarpal. The periosteum continuous with the inner trabecular bone and the outer surrounding tissue showed thick membranous appearance, which consisted of crude fibrous component and many round- to spindle-shaped cells (Fig. 3A). These Fig. 3. (A) The periosteum of radius. Crude fibrous component round- to spindle-shaped cells were considered as osteo- and many round to spindle cells are observed. Bar=120 m. blasts in the previous report [5]. In addition, many fine HE. (B) The anti- vimentin immunostaining of the periosteum granules of irregular dimensions were observed in this area. of radius. The proliferating cells show positive staining. These granules were described as cell fragments or granular Bar=120 m. (C) The ultrastructure of proliferating cell. Many products of degenerating osteoprogenitor cells in the previ- rough endoplasmic reticula are observed in the cytoplasm. ous report [5]. The round- to spindle-shaped cells prolifer- Bar=2 m. ated around the periosteum were immunohistochemically CONGENITAL HYPEROSTOSIS IN A PIGLET 261

Fig. 4. The proliferationg mesenchyme of affected forelimb. The Fig. 5. The vessels of affected forelimb. Fibrin deposition is cartilaginous metaplastic changes are observed. Bar=100 m. obvious around the dilated vessel. Bar=250 m. PTAH stain- AB-PAS double-staining. ing. positive for vimentin (Fig. 3B) and ultrastructurally had bone trabeculae and cartilaginous metaplasia of prolifera- many rough endoplasmic reticula in their cytoplasm (Fig. tive mesenchyme were observed in forelimbs, and these 3C). Taking the previous reports [5] into consideration, pathological changes was prominent especially around the these findings suggest that they would be osteoblasts. humerus and radioulna, but they were not present in other The mesenchymal proliferation was extensive around the bone. Such localized distribution of pathological changes periosteum. By AB-PAS double-staining, the proliferated suggests the possibility of some circulatory disturbance due mesenchymal tissue was positively stained by AB but not by to vascular changes. In the present case, although vascular PAS, suggesting an existence of abundant mucus containing changes defining the circulatory disturbance were not able acid mucopolysaccharide. Mucus was not evenly distrib- to be identified, vascular changes relating to localized uted in the proliferated mesenchyme, and cartilaginous hypertensive changes were observed. The local circulatory metaplastic changes were observed in some area, mainly disturbance caused by vascular changes would influence the around the radioulna and humerus (Fig. 4). Such metaplas- distribution and extent of pathological changes in forelimbs tic lesion is of interest because there is no description about of newborn piglet. Therefore, as a pathogenesis of the this in the previous reports to our knowledge. present hyperostosis in a newborn piglet, a certain local cir- Dilatation of veins with fibrin deposition in vessel walls culatory disturbance may be more likely than genetic etiol- was often observed in the area of mesenchymal proliferation ogy. and the region surrounding tendon sheath or bone (Fig. 5). As the genetic etiology, the involvement of specific male These vascular changes were considered to reflect the exist- pig in the farm might be suspected according to the situation ence of a localized circulatory disturbance. However, no of disease occurrence of this case. However, it is difficult to distinct causal relation could be found between the distribu- evaluate the possibility of genetic etiology, because there tion of vascular changes and that of edematously prolifer- are little utilizable information to confirm the extensive ated mesenchyme in the forelimb. records of disease occurrence and family history of the dis- Congenital hyperostosis in pigs is etiologically unknown ease. and explained as an autosomal recessive trait by some Further investigation would be necessary in many more researchers [7], but this has not been proven. Actually, the cases to clarify the distinct relation between vascular case described by Baker and Lewis did not fit the genetic changes and circulatory disturbance. etiology [3]. Although the pathogenesis in the present case is not known, the periosteal reaction is consistent with that REFERENCES in previous cases, and edema caused by a local circulatory disturbance is suggesting the possibility of localized hyper- 1. Baker, J. R. 1965. Calcified Tissues (Richelle., L. J. and Dalle- tensive changes as a cause. Arteriosclerotic lesions in small magne, M. J. eds.), University of Liege, Belgium. arteries and arterioles have been identified in vessels sup- 2. Baker, J. R. 1979. pp. 240. In: Spontaneous Animal Models of plying the radioulnar region in affected piglets, supporting a Human Diseases, Vol. 2 (Andrews, I. J., Ward, B. C., Altman, N. H. and Kitchen, H. eds.), Academic Press, New York. possibility of localized hypertensive changes, perhaps due 3. Baker, J. R. and Lewis, D. G. 1975. Bone disease in a dog to abnormal intrauterine positioning of the fetus [9]. similar to infantile cortical hyperostosis (Caffey’s disease). In the present case, periosteal proliferation of radiating Vet. Rec. 97: 74–75. 262 K. UETSUKA ET AL.

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