Hitoshi Hatai

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.

Detection of Avian leukosis virus genome by a nested polymerase chain reaction using DNA and RNA from dried feather shafts.

The nested polymerase chain reaction (nPCR) using frozen feather pulp is useful for detecting fowl glioma-inducing virus (FGV), which belongs to the Avian leukosis virus family, and it has recently been suggested that FGV has spread to ornamental chickens kept in Japanese zoological gardens. In the current study, the practicality of using DNA and RNA from dried feather shafts as PCR samples was examined to establish a simple method for tissue preservation. Feather shafts were collected from 7 FGV-positive chickens and stored at room temperature for 30 days. DNA and RNA were extracted from these dried materials. All DNA and complementary DNA (cDNA) prepared from the RNA showed positive results for chicken β-actin and FGV, respectively. Screening for FGV was performed on Japanese fowls kept in zoological garden N. Of the feather shafts collected from 57 birds, 1 sample tested positive for FGV according to PCR of DNA and cDNA samples from the dried feather shafts. This positive bird originated from zoological garden A and had brain lesions suggestive of fowl glioma. The results suggest that DNA and RNA from dried feather shafts can be used in nPCR to detect the FGV genome.

Cerebellar hypoplasia associated with an avian leukosis virus inducing fowl glioma

Fowl glioma-inducing virus (FGV), which belongs to subgroup A of avian leukosis virus (ALV), shows tumorigenicity and pathogenicity, mainly in the nervous system, and causes astrocytoma and perineurioma. Apart from these neoplasms, cerebellar anomaly was found in chickens infected with FGV in ovo. The study reported here describes the morphologic characteristics of the affected cerebellum. Specific-pathogen-free chickens (C/O) were inoculated with FGV through the yolk sac on the 7th day of incubation. The cerebellar anomaly included diffuse depletion of granular cells of the internal granular layer (IGL), remnants of the external granular layer (EGL), and disorganization of the Purkinje cell layer. These cerebellar changes were observed in all birds except one. in the infected embryos, the EGL was thicker and had an irregular arrangement with a thin molecular layer (ML) and IGL, compared with the control. The granular cells were immunohistochemically positive for ALV common antigen. immunohistochemical analysis for vimentin revealed disarrangement and decreased number of Bergmanns fibers. Use of the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method and electron microscopy indicated that apoptotic granular cells were frequently observed in the EGL and ML. These results suggested that the cerebellar anomaly was hypoplasia, principally resulting from the apoptosis of granular cells in the EGL and ML caused by FGV infection and that the cell loss induced obstruction of granular cell migration and disarrangement of Bergmanns fibers in the ML.

Epidemiological study of fowl glioma-inducing virus in chickens in Asia and Germany

Fowl glioma-inducing virus (FGV), which belongs to avian leukosis virus (ALV) subgroup A, induces fowl glioma. This disease is characterized by multiple nodular gliomatous growths of astrocytes and has been previously reported in Europe, South Africa, Australia, the United States and Japan. FGV and FGV variants have spread to ornamental Japanese fowl, including Japanese bantams (Gallus gallus domesticus), in Japan. However, it is unclear how and where FGV emerged and whether FGV is related to the past fowl glioma in European countries. In this study, the prevalence of FGV in European, Asian and Japanese native chickens was examined. FGV could not be isolated from any chickens in Germany and Asian countries other than Japan. Eighty (26%) out of 307 chickens reared in Japan were positive by FGV-screening nested polymerase chain reaction and 11 FGV variants with an FGV-specific sequence in their 3′ untranslated region were isolated. In addition, four other ALVs lacking the FGV-specific sequence were isolated from Japanese bantams with fowl glioma and/or cerebellar hypoplasia. These isolates were considered to be distinct recombinant viruses between FGV variants and endogenous/exogenous avian retroviruses. These results suggest that the variants as well as distinct recombinant ALVs are prevalent among Japanese native chickens in Japan and that FGV may have emerged by recombination among avian retroviruses in the chickens of this country.

Encephalitozoon infections in Rodentia and Soricomorpha in Japan.

Encephalitozoon is an obligate intracellular microsporidian parasite that infects a wide range of mammalian hosts. In this study, we used nested PCR to investigate the presence of Encephalitozoon infection in Rodentia and Soricomorpha in Japan. We attempted to amplify and sequence Encephalitozoon-specific DNA from brain and viscera samples of 180 animals collected between 2008 and 2010. Forty-three samples (23.9%) from the orders Rodentia and Soricomorpha were positive for Encephalitozoon. This study is the first report of Encephalitozoon infection in Rodentia and Soricomorpha in Japan, and our findings suggest that these hosts may play a role in the spread of microsporidian spores in the environment.

Serosurveillance for Francisella tularensis Among Wild Animals in Japan Using a Newly Developed Competitive Enzyme-Linked Immunosorbent Assay

Tularemia, a highly infectious zoonotic disease caused by Francisella tularensis, occurs sporadically in Japan. However, little is known about the prevalence of the disease in wild animals. A total of 632 samples obtained from 150 Japanese black bears, 142 Japanese hares, 120 small rodents, 97 rats, 53 raptors, 26 Japanese monkeys, 21 Japanese raccoon dogs, 20 masked palm civets, and three Japanese red foxes between 2002 and 2010 were investigated for the presence of antibodies to F. tularensis by competitive enzyme-linked immunosorbent assay (cELISA) and the commonly used microagglutination (MA) test. Seropositive cELISA and MA results were obtained in 23 and 18 Japanese black bears, three and two Japanese raccoon dogs, and two and one small rodents, respectively. All MA-positive samples (n=21) were also positive by cELISA. Six of seven samples that were only positive by cELISA were confirmed to be antibody-positive by western blot analysis. These findings suggest that cELISA is a highly sensitive and useful test for serosurveillance of tularemia among various species of wild animals. Because this is the first study to detect F. tularensis-seropositive Japanese raccoon dogs, these could join Japanese black bears as sentinel animals for tularemia in the wild in Japan. Further continuous serosurveillance for F. tularensis in various species of wild animals using appropriate methods such as cELISA is important to assess the risks of human exposure and to improve our understanding of the ecology of F. tularensis in the wild.

Pathogenicity of avian leukosis viruses related to fowl glioma-inducing virus

Fowl glioma-inducing virus (FGV), which belongs to avian leukosis virus subgroup A, causes the so-called fowl glioma and cerebellar hypoplasia in chickens. In the present study, the complete nucleotide sequences of four isolates (Tym-43, U-1, Sp-40 and Sp-53) related to the FGV prototype were determined and their pathogenicity was investigated. Phylogenetic analysis showed that the 3′-long terminal repeat of all isolates grouped together in a cluster, while sequences of the surface (SU) proteins encoded by the env gene of these viruses had 85 to 96% identity with the corresponding region of FGV. The SU regions of Tym-43, U-1 and FGV grouped together in a cluster, but those of Sp-40 and Sp-53 formed a completely separate cluster. Next, C/O specific-pathogen-free chickens were inoculated in ovo with these isolates as well as the chimeric virus RCAS(A)-(FGVenvSU), constructed by substituting the SU region of FGV into the retroviral vector RCAS(A). The four variants induced fowl glioma and cerebellar hypoplasia and the birds inoculated with Sp-53 had the most severe lesions. In contrast, RCAS(A)-(FGVenvSU) provoked only mild non-suppurative inflammation. These results suggest that the ability to induce brain lesions similar to those of the FGV prototype is still preserved in these FGV variants.

A Canine Case of Primary Intra-Right Atrial Paraganglioma

ABSTRACT An 11-year-old mixed breed dog was presented with signs of anorexia. Radiographic and ultrasound examinations revealed a large mass in the heart, between the right atrium and the right ventricle. Upon gross inspection, a multilobulated tumor arising from the right atrial wall and occupying the right atrium was identified. Microscopical analysis demonstrated that this tumor consisted of neoplastic cells with granular cytoplasm, which were separated into nests by fine fibrovascular stroma and were negative for Grimelius’s method. Immunohistochemical examinations revealed that the neoplastic cells expressed chromogranin A, synaptophysin and neuron specific enolase. Electron microscopy revealed that the cytoplasm of the neoplastic cells held secretory granules. Based on these pathological findings, the tumor was diagnosed as a paraganglioma. This report is a rare case of primary paraganglioma deriving from the right atrium and provides a detailed characterization of its morphological features.

Hepatic myelolipoma and amyloidosis with osseous metaplasia in a swan goose (Anser cygnoides).

An adult swan goose (Anser cygnoides) kept in a zoological garden had gross hepatic enlargement with extensive ill-defined white foci. Microscopically, the hepatic lesions were composed of a mixture of adipocytes and myeloid cells. The goose was also affected with systemic amyloidosis and there were areas of osseous metaplasia associated with deposition of amyloid within the liver.

Naturally Occurring Multiple Perineuriomas in a Chicken (Gallus domesticus)

A 2-year-old, male Japanese native fowl (Gallus gallus domesticus) was presented with an inability to feed and torticollis. At a necropsy, there were cylindrical enlargements and yellow discoloration of multiple peripheral nerves, including nerves of the lumbosacral plexus, brachial plexus, and spinal ganglia. On histologic examination, these lesions consisted of diffuse proliferations of spindle cells with characteristic onion bulb-like structures around residual axons. The spindle cells were immunohistochemically positive for glucose transporter 1 (GLUT1) and negative for S-100 α/β proteins. On the basis of microscopic, histologic, and immunohistochemical findings, the tumors were diagnosed as multiple perineuriomas.