Neuropathogenicity of newly isolated avian leukosis viruses from chickens with osteopetrosis and mesenchymal neoplasms.

ABSTRACT The prototype fowl glioma-inducing virus (FGVp) causes fowl glioma and cerebellar hypoplasia in chickens. In this study, we investigated whether a strain of avian leukosis virus (ALV), associated with avian osteopetrosis and mesenchymal neoplasms, is able to induce fowl glioma. We encountered avian osteopetrosis and mesenchymal neoplasms, including myxosarcoma and rhabdomyosarcoma, in Japanese native chickens used for both egg-laying and meat production. These birds were also affected by non-suppurative encephalitis and glioma in their brains. Four ALV strains (GifN_001, GifN_002, GifN_004, GifN_005) were isolated, and a phylogenic analysis of envSU showed that these isolates were classified into different clusters from FGVp and the variants previously reported. Whereas the envSU shared a high identity (94.7%) with that of Rous sarcoma virus (strain Schmidt-Ruppin B) (RSV-SRB), the identity between envTM of GifN_001 and that of FGVp was high (94.5%), indicating that GifN_strains may emerge by recombination between FGVp and other exogenous ALVs. Specific-pathogen-free chickens inoculated in ovo with GifN_001 revealed fowl glioma and cerebellar hypoplasia. These results suggest that the newly isolated strains have acquired neuropathogenicity to chickens.

Tracheal stenosis in a yellow-crowned parrot (Amazona ochrocephala) due to diffuse ossification and osteopetrosis of tracheal rings.

Tracheal luminal stenosis can cause clinical respiratory distress in wild birds. We describe a case of tracheal stenosis due to diffuse ossification with osteopetrosis of tracheal rings in a yellow-crowned parrot (Amazona ochrocephala) with a history of chronic respiratory distress and death after development of marked dyspnoea. An ante-mortem radiographic examination revealed that the tracheal rings were radiopaque and that there were multiple areas of osteopenic change in long bones. At necropsy, there was stenosis of the tracheal rings characterized by complete replacement of cartilage by thickened compact bone with osteopetrosis and bone necrosis. The clinical respiratory distress and death of the parrot were associated with tracheal luminal stenosis due to thickening of the tracheal rings by diffuse ossification with osteopetrosis.

Neuropathology and craniofacial lesions of osteopetrotic Red Angus calves.

Inherited osteopetrosis was identified in cattle herds in Wyoming, Nebraska, and Missouri in 2008 to 2010. Ten affected Red Angus calves were examined to characterize lesions in brain, teeth, and skull. Six affected aborted or stillborn calves were homozygous for the recently characterized deletion mutation in SLC4A2. Four affected calves were heterozygous for the SLC4A2 mutation and survived 1 to 7 days after birth. Gross lesions were similar in all 10 calves. Brains were rectangular and dorsoventrally compressed, with concave depressions in the parietal cortex owing to thickened parietal bone. Cerebellar hemispheres were compressed with herniation of the cerebellar vermis into the foramen magnum. Moderate bilateral chromatolysis affected multiple cranial nerve nuclei and, in some calves, the red nucleus. There was loss of retinal ganglion cells with severe atrophy of optic nerves. Periventricular corpora amylacea were in the thalamus, caudate nucleus, and midbrain. Vessels and neuropil in the dorsomedial aspect of the thalamus were mineralized. Dysplastic change in premolar and molar teeth comprised intra-alveolar intermingling of dentin, enamel, cementum, and bone, contributing to dental ankylosis. Changes in the heads of osteopetrotic calves are similar to those in children with malignant forms of homozygous recessive osteopetrosis.

Bovine viral diarrhea virus cyclically impairs long bone trabecular modeling in experimental persistently infected fetuses.

Persistent infection (PI) with bovine viral diarrhea virus (BVDV) has been associated with osteopetrosis and other long bone lesions, most commonly characterized as transverse zones of unmodeled metaphyseal trabeculae in fetuses and calves. This study was undertaken to characterize the morphogenesis of fetal long bone lesions. Forty-six BVDV-naïve pregnant Hereford heifers of approximately 18 months of age were inoculated with noncytopathic BVDV type 2 containing media or media alone on day 75 of gestation to produce PI and control fetuses, respectively, which were collected via cesarean section on days 82, 89, 97, 192, and 245 of gestation. Radiographic and histomorphometric abnormalities were first detected on day 192, at which age PI fetal long bone metaphyses contained focal densities (4 of 7 fetuses) and multiple alternating transverse radiodense bands (3 of 7 fetuses). Day 245 fetuses were similarly affected. Histomorphometric analysis of proximal tibial metaphyses from day 192 fetuses revealed transverse zones with increased calcified cartilage core (Cg.V/BV, %) and trabecular bone (BV/TV, %) volumes in regions corresponding to radiodense bands (P < .05). Numbers of tartrate resistant acid phosphatase positive osteoclasts (N.Oc/BS, #/mm(2)) and bone perimeter occupied (Oc.S/BS, %) were both decreased (P < .05). Mineralizing surface (MS/BS, %), a measure of tissue level bone formation activity, was reduced in PI fetuses (P < .05). It is concluded that PI with BVDV induces cyclic abnormal trabecular modeling, which is secondary to reduced numbers of osteoclasts. The factors responsible for these temporal changes are unknown but may be related to the time required for osteoclast differentiation from precursor cells.

Forensic investigation of a 1986 outbreak of osteopetrosis in commercial brown layers reveals a novel avian leukosis virus-related genome.

Avian leukosis virus (ALV) is known to cause several neoplastic conditions in chickens, such as B-cell lymphomas, myelocytomas, erythroblastosis, and other types of neoplasia including osteopetrosis. We describe herein the identification of unique ALV-related proviral DNA sequences in an archived chicken bone affected with osteopetrosis. The osteopetrotic bone was obtained from an affected 46-week-old brown layer during an outbreak of osteopetrosis in Costa Rica in 1986. Analysis of proviral DNA in the 23-year-old osteopetrotic bone revealed unique exogenous ALV-related sequences that were named CR-1986 (Costa Rica, 1986). The 5' and 3' long terminal repeats (LTR) in the proviral DNA were identical to each other. The U3 regions in the LTRs were most similar to equivalent sequences in ALV-J, while U5 was identical to known endogenous ALV-E sequences. The predicted CR-1986 envelope protein was most similar to the envelope of myeloblastosis associated virus type 1 (MAV-1), although the percentage of amino acid sequence similarity to MAV-1 was low (90.4%). The variable and hypervariable regions of gp85 displayed several mutations compared to representative strains of ALV. The gp37 (transmembrane or TM) envelope protein showed three leucine to serine mutations that may represent important changes in the conformation of this protein, a finding that is currently being investigated. Several recombination events may have contributed to the emergence of CR-1986 because each analyzed segment was similar to a different ALV. CR-1986 may represent a unique ALV based on distinctive characteristics of its predicted envelope protein in comparison to previously reported ALVs.

Osteopetrosis-like disease in a cat with respiratory distress.

Magnetic resonance (MR) and computed tomography (CT) were performed in an 8-year-old, spayed female cat with chronic effort respiration at the inspiration phase and stertor. Increased bone opacity in the areas of the head, neck and thorax were observed on radiography. MR images showed no signal intensity on both transverse T1WI and T2WI of the nasal cavity. CT revealed increased bone density and hypertrophy of the nasal turbinate and a narrowed nasal passage. From these results, we concluded this case had osteopetrosis-like disease, and that the respiratory distress was caused by hypertrophy of the nasal turbinate.

Association of persistent synthesis of viral DNA with macrophage accessory cell dysfunction induced by avian retrovirus myeloblastosis-associated virus of subgroup B inducing osteopetrosis in chickens.

This investigation concentrates on a regenerative anemia and immunosuppression occurring in the absence of osteopetrosis. Polyclonal activation of T-cells was used as an in vitro test system to study immunosuppression induced by the avian myeloblastosis-associated virus of Subgroup B inducing osteopetrosis [MAV-2(O)]. T-cell unresponsiveness in vitro was attributed to a defect in an accessory cell function of the macrophage. Counterflow centrifugation fractionation followed by mixing experiments indicated that the T-cell population from immunosuppressed chickens responded to mitogen stimulation when added to control macrophage cultures. In addition, lymphocyte fractions from uninfected chickens were unresponsive when added to macrophage cultures isolated from MAV-2(O)-infected chickens. Cultured splenic macrophages isolated from infected chickens contained high levels of both integrated and unintegrated viral DNA and formed syncytia by 21 days in culture. The macrophages remained viable and exhibited mature functional characteristics during mitogen stimulation assays. Therefore, it was speculated that the persistent synthesis of retrovirus DNA might be involved in the inability of infected macrophages to function as accessory cells.

Osteopetrosis in a neonatal donkey.

Osteopetrosis is a rare disorder characterised by a defect in osteoclastic bone resorption. This report describes osteopetrosis in a neonatal donkey that suffered a displaced tibial fracture. Radiographic examination identified generalised reduction in medullary cavity size, thickened mid-diaphyseal cortices and conical metaphyseal bone extending toward the mid-diaphysis of long bones. Postmortem examination identified additional fractures and brittle bones. Histologically, osteoclasts were absent in multiple bone sections. Diaphyseal cortices consisted of concentric bone lamellae with marrow tissue infiltration. Large wedges of secondary spongiosa extended from the metaphyseal growth plate. Clinical and histopathological features were similar to an osteoclast-deficient, autosomal recessive form of osteopetrosis in humans.

Transient benign osteopetrosis in a calf persistently infected with bovine virus diarrhoea virus.

A two-day-old Simmental calf was admitted suffering from a fracture of the right femur. The radiographs showed striking changes in all bones, evident as alter-noting zones of dense and less dense tissue (bone-in-bone) in the right femur and striped densities in the vertebral bodies. A stainless steel plate was used to repair the fracture, which healed well. The calf developed normally but was diagnosed as persistently infected with bovine virus diarrhoea (BVD) virus. It was kept in isolation and examined physically and radiographically during the following 13 months. The radiographic changes diminished during the first three months and at 13 months were barely visible. The animal was euthanatized, and immunohistochemistry revealed BVD virus antigen in numerous tissues. The radiographic abnormalities seen in this case are similar to those of the transient form of osteopetrosis in humans. Osteopetrosis in humans is currently thought to have a genetical cause, whereas it appears to be associated with viral disease in animals.

Localization of the mutation responsible for osteopetrosis in the op rat to a 1.5-cM genetic interval on rat chromosome 10: identification of positional candidate genes by radiation hybrid mapping.

Osteopetrosis is caused by a heterogenous group of bone diseases that result in an increase in skeletal mass because of inadequate osteoclastic bone resorption. In the op osteopetrotic rat, the disease has been linked to a single genetic locus located at the proximal end of rat chromosome 10. In this study, we identified a 1.5-cM genetic interval that contains the mutation. We then generated an improved radiation hybrid (RH) map of this region to identify potential functional and positional candidates for the op gene. Using the rat genome radiation hybrid panel, we mapped 57 markers including 24 genes (14 that have not yet been mapped in the rat) and 10 expressed sequence tag markers. Included in the mapped genes are several candidate genes that might significantly influence the biochemical pathways involved in osteopetrosis. These include genes involved in osteoclast differentiation, apoptosis, and the functional capabilities of mature osteoclasts to resorb bone. Further analysis of the genes and expressed transcripts mapped to this region may yield important insights into the multifactorial control of osteoclast function and the mechanisms of failed bone homeostasis in diseases such as osteopetrosis, osteoporosis, and rheumatoid arthritis in which failed bone homeostasis is an instigating or exacerbating circumstance of the disease process.

Parathyroid hormone as a possible causal factor in osteopetrosis of the tl rat.

The possibility of parathyroid dysfunction as a causal factor in the osteopetrosis of the tl rats was explored by evaluating serum calcium (Ca) and immunoreactive parathyroid hormone (iPTH) concentrations in this strain of rats, as compared with those of phenotypically normal littermate (LM) and non-littermate (NLM) control groups. The mean serum Ca concentration in the tl rat was not significantly different from that of the NLM group, although it was less than that of the LM group. However, all Ca values were within the normal range. The mean serum iPTH concentration in the tl rats was not significantly different from those of either control group. The data indicate that the adult tl rat has adequate but not excessive PTH secretion. Therefore, osteopetrosis and its manifestations in the adult tl are not caused by an abnormality of parathyroid function.

A sequential study of bone lesions caused by isolates of an avian osteopetrosis virus, MAV-2(0).

The pathogenesis of avian osteopetrosis caused by rapid and slow-onset isolates of myeloblastosis associated virus, MAV-2(0), was studied by inoculation of 10-day-old chick embryos with virus. Femur and calvarium were examined at 15, 17 and 19 days in ovo and 7 and 25 days after hatching by histologic and immunoperoxidase techniques. Femur and calvarium were also examined by electron microscopy at 17 and 19 days in ovo and at 7 days after hatching. Avian osteopetrotic bone lesions were characterized by exuberant periosteal proliferation; the time of onset varied with different virus isolates. In the femur virus was first associated with osteoprogenitor cells, then with osteoblasts and finally with osteocytes as the cells progressed through normal sequences of differentiation. The amount of virus produced by these cells did not correlate with onset of periosteal proliferation. Slow onset isolates provoked early virus production, but proliferative lesions did not develop until later. Conversely, the rapid onset isolate induced little early virus production, although lesions were present. Periosteal proliferation was associated with and preceded by perivascular edema and perivascular cell necrosis within the bone cortex following infection by all isolates. However, the rapid onset isolate caused more severe lesions than other isolates. These lesions included vascular thrombosis, capillary necrosis and focal bone necrosis. The relationship between early vascular lesions and late periosteal proliferation seen with the slow onset isolates is not as clear as with the rapid onset isolate. Calvarial bone, a representative flat bone, was found to have virus present, but at a level less than the femur. Vascular lesions were rarely seen in the calvarium and bone proliferation did not occur at this site.

Bone surface morphology reflects local skeletal metabolism.

The metabolic activity of bone cells is faithfully reflected in the surface topography of mineralized bone surfaces, and this can be easily detected by scanning electron microscopy (SEM). Forming bone surfaces exhibit knobby projections which represent foci of mineralization, resorbing surfaces are scalloped, and resting surfaces undergoing neither activity are smooth, as shown by Boyde and Hobdell 25 years ago. These phenomena are illustrated in vivo by tooth eruption, a local activity in alveolar bone where resorption and formation are polarized around an erupting tooth, and osteopetrosis, a metabolic bone disease characterized by a congenital reduction or absence of bone resorption. The ability to analyze bone metabolism over large areas of the skeleton by SEM offers a convenient and powerful microscopic technique to assess regional and global bone cell activity in an era where the investigative focus is increasingly molecular.

Bone density and ash studies in avian osteopetrosis.

Bone density and ash were measured in diaphyses of osteopetrotic and normal chicken tibiotarsi. In lesions of less than one year's duration, osteopetrotic bone was less dense than disease-free bone. The new osteopetrotic bone yielded approximately the same ash content per unit bone dry weight, as did disease-free bone of comparable age, but there was consistently less ash per unit bone wet volume in the diseased bones. This indicates that, in its early stages, osteopetrosis is characterized by spongy bone that lacks dense mineral deposition.

Reduced in vivo nonspecific resistance to Listeria monocytogenes infection during avian retrovirus-induced immunosuppression.

Ten-day-old chickens infected with an avian osteopetrosis virus [MAV-2(O)] were more susceptible to challenge with Listeria monocytogenes than virus-free chickens, as demonstrated by reduced bacterial clearance from their spleens. Reduced clearance of L. monocytogenes was observed throughout a 26-day period after MAV-2(O) infection.

Midpalatal suture of osteopetrotic (op/op) mice exhibits immature fusion.

The midpalatal suture was observed histologically in both toothless osteopetrotic (op/op) and normal (control) mice. The normal mice had a mature sutural structure, which consists of a well-developed cartilage cell zone and palatal bone. In contrast, the thickness of the cartilage cell zone was substantially greater in the op/op mice than that in the controls. Moreover, the cartilage cells in the op/op mice were frequently found in the palatal bone as well as in the sutural space, exhibiting an imperfect fusion. It seems that immature fusion at the sutural interface in the op/op mice is related to a decrease in biting or masticatory force accompanied by the failure of tooth eruption in addition to an essential defect in osteoclast differentiation, which is a congenital symptom in op/op mice.

Congenital osteopetrosis in white-tailed deer (Odocoileus virginianus).

Inferior brachygnathia in neonatal fawns occurred sporadically over a 10 yr period in a captive herd of white-tailed deer (Odocoileus virginianus) in southern Ontario. Two fawns submitted for necropsy had marked inferior brachygnathia, protruding tongues, and fractured long bones. Radiographs of the limbs revealed longitudinal striations of relatively translucent immature woven bone that caused loss of distinction between medullary cavities and cortices. Microscopically, there was failure of remodelling of the primary spongiosa and filling of the medulla by cone-shaped chondro-osseous cores. The findings supported a diagnosis of osteopetrosis, usually a hereditary disease characterized by absence of marrow cavities as a result of defective bone remodelling. Osteopetrosis has not been reported previously in deer.

Anemia and osteopetrosis in a dog.

A 1-year-old, male Australian Shepherd Dog with consanguineous parents was discovered to have severe nonregenerative anemia associated with osteopetrosis. Diagnosis of the bone abnormality was established by skeletal radiography and microscopic examination of a rib biopsy specimen. The anemia was attributed to failure to develop normal marrow cavities combined with failure of extramedullary erythropoiesis. Although blood transfusions sustained the dog's life for 15 months, the dog died of a hemolytic transfusion reaction.