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.

Omental leiomyosarcoma with unusual giant cells in a Beagle dog - Short communication.

A 10-year-old castrated male Beagle dog was presented with a 2-month history of intermittent vomiting and abdominal pain. The dog was referred to the Veterinary Teaching Hospital at Iwate University for further evaluation, and a splenic tumour was suspected on the basis of ultrasonography and computed tomography. Surgery identified a large, solid, light-pink mass on the greater omentum with blood-coloured ascites in the abdominal cavity, and resection was performed. Microscopically, the mass comprised spindle-shaped tumour cells and scattered osteoclast-like giant cells. Most spindle-shaped cells were positive for vimentin, desmin, and smooth muscle actin (α-SMA), whereas osteoclast-like giant cells were positive only for vimentin. On the basis of histopathological and immunohistochemical findings, a diagnosis of leiomyosarcoma was made. To the best of our knowledge, this represents the first report of leiomyosarcoma associated with osteoclast-like giant cells developing from the greater omentum in a dog.

Gastric duplication: proventricular cystic choristoma on the spleen of a broiler chicken.

Gastric duplication (choristoma) is a very rare anomaly in both humans and other animal species. We report herein a case of proventricular cystic choristoma in a 62-day-old broiler chicken. The broiler chicken was brought from the slaughterhouse to our laboratory after a pale-brown cystic structure closely attached to a thin, compressed spleen was identified. On necropsy, the cystic structure showed a clear mucinous fluid. Histopathologically, the cystic structure was lined by proventricular mucosa presenting in folds and sulci. Submucosal proventricular glands, consisting of secretory units and collecting tubules, occupied parts of the choristomal wall. The gastric mucosa lining the choristomal wall stained positive with periodic acid-Schiff stain, stained negative with Alcian blue staining, and showed a thin layer of smooth muscle fibers and a thick connective tissue layer with Masson trichrome. Ectopic pancreas was noticed attached to the wall of the choristoma. To the best of our knowledge, this represents the first report of choristoma in an avian species.

Pathology of spontaneous tumour lesions in pullets and adult chickens in commercial farms - Short communication.

Twenty pullets and adult chickens, aged 100 to 403 days, from several commercial chicken farms were examined by gross and histopathology. Grossly, all chickens had white-greyish masses in the visceral organs with or without enlargement of the peripheral nerves. Histopathological examination revealed Marek's disease (MD) lymphoma, lymphoid leukosis (LL) and myeloid leukosis (ML) in 14/20, 5/20 and 1/20 of the chickens, respectively. Lesions of the sciatic nerves in chickens diagnosed as having MD lymphoma were various. No neoplastic and/or inflammatory cells were noted in the peripheral nerves of chickens diagnosed as having LL and ML. These results indicated that MD lymphoma could also develop in older chickens; thus, microscopic examination is needed to identify MD in older chickens showing lymphocyte-derived tumours.

Relationship between tumour development and detection of Marek's disease virus in the feather follicular epithelium of older chickens.

To demonstrate the relationship between tumour development and virus replication, eight specific-pathogen-free pullets of line P2 (Group P; 14 weeks old) and five adult chickens (Group A; 96 weeks old) were inoculated with virulent Marek's disease virus (vMDV). Five chickens of Group P died or were euthanised due to moribund condition following the development of neoplastic lesions between days 53 and 91. On histopathological examination, these lesions were characterised by the proliferation of lymphoid cells of variable size. On analysis by polymerase chain reaction (PCR), the MDV meq gene was detected in Group P from day 21, and it was continuously identified in five chickens until they died or were euthanised. Abnormal signs and histopathological changes were not observed in chickens of Group A. The MDV meq gene was temporarily detected in some chickens of Group A, but it remained almost undetectable throughout the experimental period. In older chickens inoculated with vMDV, the onset of MD lymphoma development tended to be delayed as compared with the young chicks. The relationship between MD lymphoma development and virus replication in older chickens has been suggested. Our data might indicate the underlying existence of an age-related resistance to vMDV challenge.

Acidic environments induce differentiation of Proteus mirabilis into swarmer morphotypes.

Although swarmer morphotypes of Proteus mirabilis have long been considered to result from surfaced-induced differentiation, the present findings show that, in broth medium containing urea, acidic conditions transform some swimmer cells into elongated swarmer cells. This study has also demonstrates that P. mirabilis cells grown in acidic broth medium containing urea enhance virulence factors such as flagella production and cytotoxicity to human bladder carcinoma cell line T24, though no significant difference in urease activity under different pH conditions was found. Since there is little published data on the behavior of P. mirabilis at various hydrogen-ion concentrations, the present study may clarify aspects of cellular differentiation of P. mirabilis in patients at risk of struvite formation due to infection with urease-producing bacteria, as well as in some animals with acidic or alkaline urine.

A novel cyclic peptide (Naturido) modulates glia-neuron interactions in vitro and reverses ageing-related deficits in senescence-accelerated mice.

The use of agents that target both glia and neurons may represent a new strategy for the treatment of ageing disorders. Here, we confirmed the presence of the novel cyclic peptide Naturido that originates from a medicinal fungus (Isaria japonica) grown on domestic silkworm (Bombyx mori). We found that Naturido significantly enhanced astrocyte proliferation and activated the single copy gene encoding the neuropeptide VGF and the neuron-derived NGF gene. The addition of the peptide to the culture medium of primary hippocampal neurons increased dendrite length, dendrite number and axon length. Furthermore, the addition of the peptide to primary microglial cultures shifted CGA-activated microglia towards anti-inflammatory and neuroprotective phenotypes. These findings of in vitro glia-neuron interactions led us to evaluate the effects of oral administration of the peptide on brain function and hair ageing in senescence-accelerated mice (SAMP8). In vivo analyses revealed that spatial learning ability and hair quality were improved in Naturido-treated mice compared with untreated mice, to the same level observed in the normal ageing control (SAMR1). These data suggest that Naturido may be a promising glia-neuron modulator for the treatment of not only senescence, but also Alzheimer's disease and other neurodegenerative diseases.

Comparative susceptibility of some avian and mammalian lymphoblastoid cell lines to chicken anemia virus.

Although MSB1 is the most-commonly used lymphoblastoid cell line for isolation of chicken anemia virus (CAV), some researchers have reported a few biological drawbacks. None of them were supported by the results of the present study. Another four avian (HP1, HP2, BK3 and CU10) and two mammalian (BTL-26 and KO-1) cell lines were investigated for susceptibility to the TK-5803 and AH-9409 strains. Both strains caused CPE on BK3 like MSB1. The mean number of positive cells for each strain in MSB1 and BK3 were not significantly different. The majority of the HP2, CU10 and HP1 cells showed no CPE. The virus titers of both strains were higher in MSB1 and BK3 (10(6.5-7.5) TCID(50)/0.1 ml) than in HP2, CU10 and HP1 (10(3.5-4.5) TCID(50)/0.1 ml). BTL-26 and KO-1 were resistant to CAV. BK3 could be used for isolation of CAV.

Intestinal volvulus with coagulative hepatic necrosis in a chicken.

A 7-week-old SPF chicken inoculated at 4 weeks of age with chicken anemia virus was puffed up depressed and had ruffled feathers and a good body condition. Intestinal volvulus involving the jejunum and part of the duodenum forming two loops with one knob was observed. Microscopically, venous infarction of the obstructed loops, periportal and sublobular multifocal coagulative hepatic necrosis and granulomatous inflammation of the cecal tonsils were observed. Gram staining revealed no bacteria in hepatic tissue; however, gram-positive bacilli were detected in the necrotic debris in the intestinal lumen. Immunosuppression might have predisposed the chicken to intestinal and cecal tonsil infection that then progressed to volvulus. Loss of the mucosal barrier in infarction might allow bacterial toxins and vasoactive factors to escape into the systemic circulation (toxemia) and be responsible for the hepatic necrosis.

Late Marek's disease in adult chickens inoculated with virulent Marek's disease virus.

Recently, excessive losses from Marek's disease (MD) have been noted in adult laying flocks over the age of 40 weeks. We defined these late outbreaks in adult chickens as "late MD", and experimentally reproduced the disease in adult SPF P2 line (50-week-old) or commercial line (74-week-old) chickens inoculated with a virulent strain of Marek's disease virus (MDV). Commercial line chickens were given MDV vaccines (HVT and CVI 988) at hatch. The occurrence of MD was evaluated periodically by the evidence of neurologic signs such as paralysis, torticollis, ataxia, and/or nervous tics, as well as histopathological examination. In P2 line chickens, neurologic signs and MD lymphoma were observed from day 21 onward, and they tended to increase in a time-dependent manner. Meanwhile, in commercial line chickens, only one chicken exhibited MD lymphoma on day 70 post inoculation, but its pathogenesis was questionable. No regression of MD lymphoma was noted in either case. The lesions in the visceral organs, thymus, peripheral nerves, and feather pulps of P2 line chickens were characterized by proliferation of variably sized lymphoid cells. In the feather follicle epithelium, numerous inclusion bodies were noted on day 21 post-inoculation, which tended to decrease afterwards. The morphological findings obtained resembled late MD in field cases. In conclusion, our results demonstrate that adult SPF P2 line chickens are susceptible to virulent MDV, and would be useful for investigation of late MD.

Changes in the gene expression and enzyme activity of hepatic cytochrome P450 in juvenile Sprague-Dawley rats.

The developmental changes in the hepatic cytochrome P450 (CYP) content, mRNA expression of 12 hepatic CYP subtypes, and the enzyme activities of 5 hepatic CYP subfamilies in rats were investigated using non-treated male and female Sprague-Dawley rats of ages postnatal day (PD) 4, 16, 30 and 8 and 12 weeks. The hepatic proliferation kinetics was also determined by using the phospho-histone H3 (p-histon)-labeled hepatocyte index. The developmental changes in the enzyme activities of hepatic expression of CYP1A and CYP3A in rats were similar to those in humans, although there is no fetal-neonatal dominant CYP3A subtype in rat livers unlike human CYP3A7. On the other hand, the developmental pattern of expression of the CYP2C subfamily differed between humans and rats. Enzyme activity and mRNA expression of each hepatic CYP subtype in rats on PD 30 was similar to that after 8 weeks of age, except in the case of sex-dependent CYP subtypes. The p-histon-labeled hepatocyte index was approximately 10-fold higher in PD 30 rats than in 8-week-old rats. Therefore, the livers of juvenile rats, which have high hepatocellular proliferation activity and a sufficient amount of metabolic enzymes such as CYP, may be more sensitive to the cytotoxic and carcinogenic effects of chemicals than the livers of adult rats. Thus, our results on developmental difference of hepatic CYPs in juvenile rats are useful to identify underlying age-dependent susceptibility of chemical-induced toxicity, and to understand developmental change of chemical disposition.

Detection of initiation activity of 1,2-dimethylhydrazine in in vivo medium-term liver initiation assay system using 4-week-old rats without hepatocellular proliferative stimuli during the test chemical treatment period.

We have developed an in vivo medium-term liver initiation assay system to detect initiation activities of chemicals on multi-organ carcinogenesis. However, cell proliferation stimuli during the test chemical treatment period, required in the previously used assay models using adult rats, are laborious; moreover, those cause decrease of hepatic metabolic enzymes and psychological and physical discomfort to animals resulting in inaccurate interpretation. Therefore, we investigated the utility of another in vivo medium-term liver initiation assay model using 4-week-old rats without the cell proliferation stimuli. In this study, we confirmed that 4-week-old and 4.5-week-old male rats have high hepatocyte proliferation activity and similar enzyme activities of hepatic Cytochrome P450 subtypes as compared with 8-week-old male rats. Next, the in vivo medium-term liver initiation assay model using 4-week-old rats without cell proliferation stimuli was evaluated for the detection of the initiation activity of 1,2-dimethylhydrazine (DMH), which is a well-known genotoxic carcinogen. Four-week-old rats were orally administered DMH (single dose, 4 or 16 mg/kg; or 4-day repeat, 1 or 4 mg/kg); subsequently, these rats were treated promotion treatment consisted of administration of 2-acetylaminofluorene and carbon tetrachloride. Four weeks after the first DMH administration, the glutathione S-transferase placental form (GST-P)-positive foci induced by DMH in the liver was measured immunohistochemically. The inductions of GST-P-positive foci in all DMH-treated groups were dose-dependent, duration-dependent and significantly higher than that in non-DMH-treated group. From these results, our assay model was detected the initiation activity of DMH simply, and would be useful to evaluate the carcinogenicity of chemicals.

Gallbladder agenesis in a Chihuahua.

A 4-year-old neutered male Chihuahua was presented with a history of anorexia and vomiting. Continuous elevation of liver enzymes was found on repeated blood examinations and the dog was referred to us for further evaluation. The absence of gallbladder was suspected on ultrasonography. Exploratory laparotomy and retrograde cholangiography confirmed gallbladder agenesis and a possible hypoplasia of the right medial and lateral liver lobes. Histologically, proliferation of bile ductules associated with portal fibrosis and pseudolobular formation were apparent in the liver lobes.

Pathological and immunohistochemical study of chickens with co-infection of Marek's disease virus and chicken anaemia virus.

Chicken anaemia virus (CAV) is the most important confounding pathogen in Marek's disease virus (MDV) infection. The effect of CAV co-infection at 4 weeks of age after inoculation of virulent MDV (vMDV, KS strain) or very virulent MDV (vvMDV, Md/5 strain) in 1-day-old chicks was investigated by pathological and immunohistochemical studies. CAV increased the mortality rates induced by vMDV or vvMDV. The packed cell volume was reduced significantly in vMDV-CAV infection; however, no reduction or non-significant reduction was observed in vMDV infection. Bone marrow hypoplasia was related to CAV co-infection and none of the birds inoculated with vMDV or vvMDV had hypoplasia. Severe atrophy of the thymus and bursa of Fabricius was observed in the vvMDV-CAV and vvMDV groups. Complete regeneration of the thymus cortex and bursa of Fabricius in the vMDV group was noted and was in contrast to sequential lymphoid depletion after CAV inoculation in the vMDV-CAV group. The spleen was either regenerated, lymphoid depleted or had lymphoproliferative lesions. Lymphoid depletion in the spleen was not detected in the vMDV group; however, it was prominent in the vMDV-CAV and vvMDV-CAV groups during the first 2 weeks after CAV inoculation. CAV inclusions and antigens were detected in the thymus cortex and spleen of vMDV-CAV and vvMDV-CAV groups during the experiment. Severe depletion of CD8(+) T cells was observed in depleted spleen and thymus. The neoplastic foci appeared around splenic arterioles and venules, and stained mainly by CD4 antibody; however, CD8(+) T cells were singly dispersed or were present in clusters. It could be concluded that CAV was responsible for bone marrow hypoplasia, severe anaemia and hindrance of lymphoid organ regeneration in MDV-CAV co-infection.

A recombinant avian leukosis virus associated with fowl glioma in layer chickens in Japan.

Fowl glioma is characterized by multiple nodular growth of astrocytes, and fowl glioma-inducing virus belonging to avian leukosis virus has been isolated from Japanese bantam as a causal agent. Subcutaneous neoplasms of the head and neck have been reported in layer chickens since 2003 in Japan, and fowl glioma concurred in these affected layers. In the present study, the histopathology of 240 layers, including 18 layers with subcutaneous neoplasms and 222 layers kept with the affected layers, was performed to clarify the characteristics of fowl glioma in layers. Microscopically, 103 layers showed non-suppurative encephalitis, and four layers had locally extensive proliferation or multiple nodules of astrocytes. Gliomas concurred in 11 layers with subcutaneous neoplasms and occurred independently in three layers. In addition, two layers had locally extensive proliferation of small, round cells in the cerebrum. The fowl glioma-inducing virus genome was not detected in the affected brains by nested polymerase chain reaction. Ten isolates were obtained from the affected brains. By nucleotide sequencing of the env gene, SU coding regions of these isolates were most closely related to myeloblastosis-associated virus-like viruses, but TM regions showed the highest similarity to endogenous viral (ev) loci. The genome of one isolate mainly consisted of ev loci and contained several parts of other avian leukosis/sarcoma viruses. These results show that the causal avian leukosis virus of fowl glioma is not just fowl glioma-inducing virus and that different avian leukosis virus strains having oncogenicity in the central nervous system by recombination are spread in layers in Japan.

Total glutathione and glutathione reductase in bovine erythrocytes and liver biopsy.

The goal of the present study was to measure the total glutathione level and glutathione reductase activity in bovine erythrocytes and liver biopsy. Five cows were injected intraperitoneally with DL-ethionine (12.5 mg/kg B.W.), and two control cows were injected with normal saline (0.9% NaCl). Ultrasonography guided liver biopsy, and blood samples were collected at 0, 4, 7 and 10 days after injection. The hepatic total glutathione level was significantly increased on Days 7 (p<0.05) and 10 (p<0.01), and hepatic glutathione reductase activity was significantly increased on Days 4 (p<0.05), 7 (p<0.01) and 10 (p<0.01). There were insignificant changes in the erythrocytic total glutathione level. The present study demonstrated that liver biopsy is a valuable tool for detecting oxidative stress and for diagnosing hepatic dysfunction in cattle from the viewpoint of the status of glutathione and glutathione reductase.

Outbreak of pasteurellosis in captive Bolivian squirrel monkeys (Saimiri boliviensis).

In September 2012, five Bolivian squirrel monkeys housed in a zoological park died within sequential several days without obvious clinical signs. In a necrospy, one monkey presented swelling of the kidney with multifocal white nodules in the parenchyma, and other two had pulmonary congestion. Histopathologically, multifocal bacterial colonies of gram-negative coccobacillus were found in the sinusoid of the liver in all monkeys examined (Nos.1-4). Additionally, purulent pyelonephritis, pneumonia and disseminated small bacterial colonies in blood vessels were observed. Immunohistochemically, the bacterial colonies from two monkeys were positive for P. multocida capsular serotype D. Based on these findings, these monkeys were diagnosed as septicemia caused by acute P. multocida infection.

Mandibular Reconstruction by Using a Liquid Nitrogen-Treated Autograft in a Dog with an Oral Tumor.

A 10 yr old intact female German shepherd dog presented with a large peripheral odontogenic fibroma and malignant melanoma on her lower jaw. The tumor was resected with a unilateral subtotal rostral hemimandibulectomy. After the mandible was removed, it was devitalized intraoperatively by freezing it in liquid nitrogen. It was subsequently reimplanted. New bone tissue formed in the gap between the frozen bone and the host bone. The regenerated bone contained osteocytes, osteoblasts, and blood vessels. The cosmetic appearance of the dog was preserved. The dog had normal mastication. The malignant melanoma recurred rostral of the left canine tooth at 159 days after the reconstruction surgery. A subtotal hemimandibulectomy was consequently performed. This is the first reported case of mandibular reconstruction using a liquid nitrogen-treated autograft in a dog with oral tumors.

Extralobar pulmonary sequestration in two pinniped species.

Two cases of extralobar pulmonary sequestrations from a walrus (Odobenus rosmarus) and a Steller sea lion (Eumetopias jubatus) are described in the present study. Grossly, an independent, soft unilocular cystic mass was found within the abdominal cavities of both animals, adherent to the diaphragm in O. rosmarus and attached to the cardia of the stomach in E. jubatus. Histopathologically, the cysts were lined by pseudostratified ciliated columnar epithelium with abundant goblet cells, while the wall comprised of glands, hyaline cartilage, bronchiole- and alveolus-like structures, smooth muscles, and large, well-developed elastic and muscular arteries. The pinniped cases presented are exceptionally rare and to the best of the authors' knowledge, marks the first descriptions of this congenital anomaly in wildlife.

Persistence of chicken anemia virus antigen and inclusions in spontaneous cases of Marek's disease visceral lymphomas in broiler chickens at slaughterhouses.

The chicken anemia virus (CAV) and Marek's disease virus (MDV) infect chickens worldwide; a single or dual infection by these viruses has a great impact on poultry production. In the present study, we examined the existence of CAV antigen and its inclusions in Marek's disease (MD) lymphomas in chickens in the slaughterhouses of Iwate prefecture, Japan. Forty-nine spleens and 13 livers with different degrees of nodular lesions were histopathologically examined at our laboratory. Grossly, the tested organs showed various sizes and anatomical architectures. Based on the cellular morphology and the infiltrative nature of the neoplastic lymphocytes, MD was confirmed in 76% (37/49) of the spleens and 92% (12/13) of the livers. The lesions of MD, according to the pattern of lymphocytic accumulation in the affected organs, were divided into multifocal, coalesced and diffuse. CAV intranuclear inclusion bodies were detected within the small and the large bizarre lymphocytes of the MD lymphomas in 2 livers and 9 spleens, and the immunostaining test for CAV confirmed the persistence of CAV antigens and inclusions in the neoplastic cells. This study demonstrated the persistence of CAV infection within the neoplastic cells of naturally occurring MD lymphomas in chickens.