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.

Anemia mielotísica por osteopetrose em um cão: relato de caso / Mielotisic anemia caused by osteopetrosis in a dog: case report

A osteopetrose é uma doença rara, caracterizada pelo aumento generalizado da densidade óssea. Tem como característica principal, a reabsorção osteoclástica defeituosa, resultando no acúmulo de massa óssea. Além disso, pode ocorrer retardo do crescimento, desnutrição progressiva, anemia e caquexia. O presente relato descreve o caso de uma cadela, com aproximadamente nove meses de idade, sem raça definida, com histórico de apatia e disorexia. Hemogramas seriados demonstraram pancitopenia persistente. Vários exames laboratoriais foram realizados para excluir doenças como erliquiose, leishmaniose e cinomose, porém todos foram negativos. O mielograma constatou hipocelularidade relativa por provável aplasia/hipoplasia medular. Exames radiográficos evidenciaram o aumento da radiopacidade óssea e hipertrofia da região cortical, e a necropsia confirmou a redução do canal medular. O exame histopatológico confirmou a osteopetrose. Conclui-se que a osteopetrose pode causar comprometimento na produção de células sanguíneas, sendo uma causa rara de anemia mielotísica, além de alterações neurológicas secundárias à má-formação dos ossos do crânio.

Osteopetrosis is a rare disease characterized by generalized increase in bone density. The defective osteoclastic resorption results in the accumulation of bone mass. Furthermore, there may be growth delay, progressive malnutrition, anemia and cachexy. This report describes the case of a nine month old, mixed breed, female dog presented with apathy and disorexia. Laboratory tests were performed to rule out Ehrlichiosis, Leishmaniasis, and Canine distemper, but all were negative. Hemogram showed persistent pancytopenia and myelogram showed relative hypocellularity, probably due to spinal cord aplasia/hypoplasia. Radiographs showed increased bone opacity and hypertrophy of the cortical region, and the necropsy confirmed the reduction of the medullary canal. Histopathological examination confirmed osteopetrosis. In conclusion, osteopetrosis can compromise blood cell production, and is a rare cause of mielotisic anemia. It can also cause neurological deficits due to cranium bone deformation.

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.

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.

Familial osteopetrosis in Agouti paca: report of nine cases / Osteopetrose familiar em Agouti paca: relato de nove casos

Nine cases of familial osteopetrosis were studied in Agouti paca rodents maintained in captivity. Animals were distributed in three groups depending on the severity of their skeletal lesions. Based upon clinical, radiological, and microscopic findings, it was concluded that one animal had level I lesions, three animals had level II lesions, and five animals had level III osteopetrosis and osteonecrosis. Throughout the entire axial and appendicular skeleton, there was an increased amount of both trabecular and cortical bone tissue. All analyzed bones showed thickened cortex and reduced medullary canals. Bone trabeculae were thick and confluent. Cortex showed a narrowing of Haversian canals. Numerous cementing lines resulted in typical mosaic patterns. Osteocytes were pycnotic. Osteonecrosis was characterized by the disappearance of osteocytes and bone matrix decomposition.

Descreveram-se nove casos de osteopetrose familiar em Agouti paca mantidas em cativeiro. Os animais foram distribuídos em três grupos de acordo com a gravidade das lesões do esqueleto. Com base nos exames clínico, radiológico e microscópico, foi concluído que um animal apresentou lesões de nível I, três animais tiveram lesões de nível II e cinco animais tiveram osteopetrose de nível III. Por todo o esqueleto axial e apendicular, a quantidade de osso trabecular e osteônico estava aumentada. Todos os ossos analisados mostraram córtex espesso e canais medulares reduzidos. As trabéculas ósseas eram espessas e confluentes. No córtex, verificou-se um estreitamento de canais de Havers. Numerosas linhas de cimentação resultaram em um padrão de mosaico típico. Osteócitos estavam picnóticos e a osteonecrose foi caracterizada pela morte dos osteócitos, com desintegração da matriz óssea.

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.

Pathogenic potential of myeloblastosis-associated viruses.

Myeloblastosis-associated viruses (MAV) are replication competent avian retroviruses responsible for the induction of lymphoid leukosis, osteopetrosis, and nephroblastoma. Although both the route of infection and the strain of host used has been reported to be a critical factor in determining the outcome of viral infection, genetically distinct strains of MAV that exhibit a multiple pathogenic potential have been molcularly cloned. Osteopetrosis is a proliferative disease of the bones and nephroblastoma is a kidney cancer. Both diseases occur in chickens a few weeks after MAV injection. In both cases, the nature of the target cells and mechanisms of transformation induced by MAV remain to be established. Molecular cloning and sequencing of three MAV proviral genomes inducing both osteopetrosis and nephroblastoma or only nephroblastoma have allowed the identification of viral determinants essential for osteopetrosis induction. For the last decade we have focused our attention on the MAV-induced nephroblastoma because it is a unique animal model of the human Wilms' tumor. Studies that we have conducted to understand the molecular basis of MAV tumorigenic potential have led to the identification of viral sequences required for tumor induction and to the discovery of a new cellular gene (nov) likely to play a critical role in avian and human nephroblastoma development.

Assessment of the influence of NaF or 1,25-(OH)2 vitamin D3 on the proliferative bone effect of an avian osteopetrosis virus, MAV-2(O).

1,25-(OH)2 vitamin D3 (D3) and sodium fluoride (NaF) were given to chicken embryos and newly hatched chickens infected with a slow onset strain of avian osteopetrosis-inducing virus [MAV-2(O)] to determine if either agent influenced MAV-2(O)-induced proliferation of bone. Embryos were administered MAV-2(O) and treated with: 1) up to 240 micrograms NaF or up to 100 ng D3 as embryos; 2) up to 1.8 g NaF/kg or up to 9.5 micrograms D3/kg after hatching: or 3) 240 micrograms NaF as embryos and up to 1.8 g NaF/kg after hatching. Administration of MAV-2(O) alone resulted in expansion of the cortical diameter of bone. Coadministration of NaF or D3 with MAV-2(O) did not influence the change in cortical diameter seen with MAV-2(O) alone at 18 days of incubation, and 3 and 6 weeks after hatching. Increased osteoid relative to bone (hyperosteoidosis), with NaF and MAV-2(O) compared to MAV-2(O) alone, and NaF compared to untreated controls reflected delayed mineralization of osteoid, a known fluoride effect. We conclude that the administration of NaF or D3 did not influence the incidence, severity or time of onset of the MAV-2(O)-induced proliferative changes of bone.

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.

Long terminal repeat (LTR) sequences, env, and a region near the 5' LTR influence the pathogenic potential of recombinants between Rous-associated virus types 0 and 1.

A series of recombinants between Rous-associated virus type 0 (RAV-0), RAV-1, and a replication-competent avian leukosis virus vector (RCAN) have been tested for disease potential in day-old inoculated K28 chicks. RAV-0 is a benign virus, whereas RAV-1 and RCAN induce lymphoma and a low incidence of a variety of other neoplasms. The results of the oncogenicity tests indicate that (i) the long terminal repeat regions of RAV-1 and RCAN play a major role in disease potential, (ii) subgroup A envelope glycoproteins are associated with a two- to fourfold higher incidence of lymphoma than subgroup E glycoproteins, and (iii) certain combinations of 5' viral and env sequences cause osteopetrosis in a highly context-dependent manner. Long terminal repeat and env sequences appeared to influence lymphomogenic potential by determining the extent of bursal infection within the first 2 to 3 weeks of life. This would suggest that bursal but not postbursal stem cells are targets for avian leukosis virus-induced lymphomogenesis. The induction of neutralizing antibody had no obvious influence on the incidence of lymphoma.

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.

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.

Chicken strain G-B1 exhibits a relative resistance to avian osteopetrosis.

The disease induced by the avian myeloblastosis associated virus MAV-2-O in the susceptible chicken strains Brown Leghorn (BLH) and Prague CB (CB) was compared with that induced in the resistant G-B1 strain. Osteopetrosis, stunting and lymphoid organ atrophy were more severe in BLH than in CB chickens. G-B1 animals remained superficially normal until the end of the experiment. In contrast to the other two strains, the histopathological changes were very mild and there was no sign of immunosuppression. After 4 months, however, nephroblastomas could be detected in more than 50 per cent of the infected G-B1 chickens. Similar tumors were also found in CB birds kept for up to 5 months. Antibodies against MAV-2-O specific viral proteins were detected in plasma from infected G-B1 chickens but the titers were less than in plasma of convalescent birds. Virus could be demonstrated in peripheral blood until the end of the experiment (at 8 weeks). Therefore the resistance of the G-B1 strain is due neither to a restriction at the receptor level nor the result of a humoral immune reaction, but represents a new type of relative resistance at the cellular level. From (CC X G-B1)F1 and (CC X G-B1)F2 crosses the resistant phenotype is determined by a single genetic factor. This gene is not linked to the major histocompatibility complex. There is also a sex-dependent factor, possibly hormonal, involved in the resistant phenotype.

MAV-2-O replicates at a reduced rate in cells from the osteopetrosis resistant G-B1 chicken.

The replication of the avian osteopetrosis virus MAV-2-O was compared in chick embryo fibroblasts from two strains of chicken. These were G-B1 which is relatively resistant to MAV-2-O and CB which is susceptible. The production of MAV-2-O was delayed in G-B1 cells (compared with CB cells). The same result was observed after infection with Rous sarcoma viruses of subgroups B, C, and D. In addition, the transforming viruses induced foci on G-B1 fibroblasts 24 to 48 hours later than on CB fibroblasts. In G-B1 cells there was also a delayed kinetics of intracellular viral RNA production. Integrated and linear unintegrated MAV-2-O DNA species were also present in lower amounts in G-B1 than in CB fibroblasts at 3 days postinfection. In vivo studies confirmed the in vitro situation. There was a marked difference in the amount of virus present in the osteoid bone matrix and the osteocytic lacunae of osteopetrotic bones from susceptible and G-B1 chickens. In contrast to the bone lesions from susceptible animals, budding virus particles were not detectable in lesions from G-B1 chickens. There was no difference in the amount of virus in osteopetrotic and non-osteopetrotic bone of susceptible chickens suggesting that virus replication alone is not sufficient for induction of osteopetrosis and that an additional specific virus-cell interaction is required. The relative resistance of strain G-B1 may therefore, be a consequence of a reduced frequency of this interaction. Its basis may be the lower amount of integrated, as well as unintegrated, viral DNA.

Avian leukosis virus-induced osteopetrosis is associated with the persistent synthesis of viral DNA.

DNAs from 19 cases of avian leukosis virus-induced osteopetrosis have been analyzed for viral sequences. Among these were instances of rapid, intermediate, and slow onset osteopetrosis. The DNAs from osteopetrotic bone contained no evidence for osteopetrosis being caused by proviral insertions into or viral transductions of a host protooncogene. Instead, DNAs from osteopetrotic bone displayed evidence for osteopetrosis being associated with the persistent synthesis of viral DNA. Each of the 19 DNAs contained unintegrated as well as integrated viral DNA. Rapid onset osteopetrosis contained about 3X more viral and proviral DNA than intermediate or late onset osteopetrosis. Unintegrated viral DNA could not be detected in DNAs extracted from the bursa bone marrow of osteopetrotic chickens or in DNA extracted from the normal bones of an avian leukosis virus-infected chicken. Thus, the persistent synthesis of unintegrated viral DNA was observed in osteopetrotic but not normal tissues of avian leukosis virus-infected chickens.

[Morphological study of congenital osteopetrosis in op rats]. / Etude morphologique de l'ostéopétrose congénitale du rat "op".

Osteopetrosis appears spontaneously on the "op" rat (Fatty/ORL). At the confirmed stage, description of histologic lesions is made. The attention is attracted by an endocavitary tissue observed at later stage of disease. This one does not seem to be the result of a metaplasia but rather the image of a persistent osteoblastic activity.

Congenitally osteopetrotic (oplop) mice are not cured by transplants of spleen or bone marrow cells from normal littermates.

Congenital mammalian osteopetrosis is characterized by a generalized skeletal sclerosis due to reduced bone resorption by osteoclasts. This condition can be cured in several mutant strains of mice and rats by transplantation of spleen or bone marrow cells from normal littermates. The ability of this regimen to cure osteopetrosis in oplop mice was examined in 23 mice treated with spleen or bone marrow cells from normal littermates and followed for up to 80 days. In no instance was there radiographic or histologic evidence of removal of the excessive skeletal mass characteristic of the disease. These data show that spleen or bone marrow cells do not cure osteopetrosis in this mutation. Recent demonstrations that not all children with congenital osteopetrosis are cured by bone marrow transplants from HLA-matched donors suggest that the oplop mouse mutation may be a useful model system in which to develop alternate treatments.

Rapid induction of osteopetrosis by subgroup E recombinant viruses.

Avian osteopetrosis is a proliferative bone disorder initiated at high frequency by MAV-2(O), a subgroup B avian myeloblastosis-associated virus. To examine the role of the MAV-2(O) genome in osteopetrosis induction, a series of recombinant viruses between MAV-2(O) and RAV-O was constructed. Recombinant viruses were selected for rapid growth and subgroup E envelopes. The T1 oligonucleotide fingerprint patterns of viruses selected in this manner demonstrated that they were recombinants and were clonally pure because they had oligonucleotides from each parent, and each oligonucleotide was present in single molar yield. When injected into 10-day-old chicken embryos, approximately 50% of the recombinant viruses induced osteopetrosis within 3 weeks after hatch. Therefore, subgroup E envelope did not inhibit osteopetrosis induction. The osteopetrosis that was induced varied from slight to severe, but none of the recombinant viruses induced osteopetrosis as severe as the MAV-2(O) parent.

Electron microscopy of avian osteopetrosis induced by retrovirus MAV.2-O.

Diaphyseal tibial bone of 12.5-13-day and 19-day-old embryos and 20-day-old hatched chicks infected with retrovirus MAV.2-O were examined by transmission electron microscopy. The viruses were associated with lining osteoblasts and osteocytes. Whereas the infection of the osteoblast layer seemed to be a transient stage, virus association with osteocytes was a constant and main ultrastructural feature. The viruses were found either in the osteoid or in the periosteocytic space of the bone lacunae. They arose from dense cytoplasmic areas located near the cell plasmalemma via a budding process. The newly budded virus particles often had a large tail or a fine stalk-like process lost in the extracellular space. The viruses underwent calcification by deposition of inorganic material and were incorporated in the bone trabeculae. No production of virus was observed in typical osteoclasts with well-differentiated ruffled borders. The viral-induced avian osteopetrosis seemed to result from increased bone deposition through stimulation of osteoblast and osteocyte activities, whereas osteoclastic bone resorption seemed to be undisturbed.