Atsushi Kodama

Acute neuropathogenicity with experimental infection of equine herpesvirus 9 in common marmosets (Callithrix jacchus).

Background  Equine herpesvirus 9 (EHV‐9) is a new neurotropic equine herpesvirus which induced encephalitis in a variety of animals. However, there was no information on the susceptibility of EHV‐9 in primates.

B-cell intestinal lymphoma with Mott cell differentiation in a 1-year-old miniature Dachshund

A 1-year-old intact female miniature Dachshund was presented with hematochezia, vomiting, and diarrhea of more than 1-week duration. An abdominal mass was palpated, which at exploratory surgery was found to be a 7-cm-long thickened section of ileum. The thickened ileum was resected. Impression smears revealed numerous small- to medium-sized lymphocytes, with a smaller number of cells resembling Mott cells. The Mott-like cells contained multiple pale vacuoles that were positive for periodic acid-Schiff (PAS) in wet-fixed smears, consistent with Russell bodies. Histologic evaluation of the surgically excised ileum revealed 2 populations of neoplastic lymphoid cells. The majority were uniform medium-sized lymphocytes with hyperchromatic oval or round nuclei and inconspicuous nucleoli. The remaining cells resembled Mott cells, which contained several PAS-positive eosinophilic globules in the cytoplasm, occasionally compressing the nucleus. The majority of neoplastic cells stained positively for vimentin, CD20, CD79a, and Pax-5, but were negative for CD3 and lysozyme; 43.5% of cells stained positively for Ki-67. The Mott cells were strongly positive for immunoglobulin but were negative for Pax-5. Using electron microscopy, a homogenous substance of intermediate electron density was observed frequently in the cisternae of rough endoplasmic reticulum in the cytoplasm of the Mott cells, and rarely in the perinuclear cisternae of the lymphoid cells, corresponding to the site of immunoglobulin staining. Monoclonal rearrangement of immunoglobulin heavy-chain (IgH) gene was observed by PCR testing for lymphocyte-antigen receptor rearrangement. The morphologic features, immunophenotype, and IgH gene rearrangement verified the lymphoid cells were neoplastic (mature cell type) and had a B-cell phenotype, with evidence of immunoglobulin production and differentiation into Mott cells. This case was unusual because of the age of the dog and because most intestinal lymphomas are T-cell phenotype. The Mott cell morphology also differed from typical mature B-cell lymphoma types and may be a unique B-cell lymphoma variant.

Constitutive phosphorylation of the mTORC2/Akt/4E-BP1 pathway in newly derived canine hemangiosarcoma cell lines

BackgroundCanine hemangiosarcoma (HSA) is a malignant tumor with poor long-term prognosis due to development of metastasis despite aggressive treatment. The phosphatidyl-inositol-3 kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is involved in its endothelial pathologies; however, it remains unknown how this pathway plays a role in canine HSA. Here, we characterized new canine HSA cell lines derived from nude mice-xenografted canine HSAs and investigated the deregulation of the signaling pathways in these cell lines.ResultsSeven canine HSA cell lines were established from 3 xenograft canine HSAs and showed characteristics of endothelial cells (ECs), that is, uptake of acetylated low-density lipoprotein and expression of canine-specific CD31 mRNA. They showed varied morphologies and mRNA expression levels for VEGF-A, bFGF, HGF, IGF-I, EGF, PDGF-B, and their receptors. Cell proliferation was stimulated by these growth factors and fetal bovine serum (FBS) in 1 cell line and by FBS alone in 3 cell lines. However, cell proliferation was not stimulated by growth factors and FBS in the remaining 3 cell lines. Phosphorylated p44/42 Erk1/2 was increased by FBS stimulation in 4 cell lines. In contrast, phosphorylation of Akt at Ser473, mTOR complex 1 (mTORC1) at Ser2448, and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) at Ser65 was high in serum-starved condition and not altered by FBS stimulation in 6 cell lines, despite increased phosphorylation of these residues in normal canine ECs. This suggested that the mTORC2/Akt/4E-BP1 pathway was constitutively activated in these 6 canine HSA cell lines. After cell inoculation into nude mice, canine HSA tumors were formed from 4 cell lines and showed Akt and 4E-BP1 phosphorylation identical to the parental cell lines.ConclusionsOur findings suggest that the present cell lines may be useful tools for investigating the role of the mTORC2/Akt/4E-BP1 pathway in canine HSA formation both in vivo and in vitro.

Expression of the anti-apoptotic factors Bcl-2 and survivin in canine vascular tumours.

To investigate whether anti-apoptotic factors play a role in the malignant growth of canine haemangiosarcomas (HSAs), 83 HSAs and 22 haemangiomas were examined immunohistochemically for bcl-2 and survivin expression. Additionally, bcl-2 and survivin mRNA expression was quantified by semiquantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). Immunolabelling for bcl-2 was observed in 50 of the 83 HSA samples (60.2%) but in none of the haemangiomas. The average survivin positive index was 24.7% in the HSAs and 0.6% in the haemangiomas. In contrast to the high average value for survivin mRNA expression, which was approximately six times that for the haemangiomas, no significant difference was observed between HSAs and haemangiomas for the average bcl-2 mRNA expression level. The discrepancy between bcl-2 mRNA and bcl-2 protein expression requires further investigation, but the results suggest that malignant proliferation in canine HSAs is associated with bcl-2 and survivin expression.

Immunohistochemical Analysis of the Akt/mTOR/4E-BP1 Signalling Pathway in Canine Haemangiomas and Haemangiosarcomas

The specific signalling pathways that are deregulated in canine endothelial tumours have not yet fully elucidated. Therefore, the aim of the present study was to examine activation of the Akt/mammalian target of rapamycin (mTOR)/eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) signalling pathway in spontaneously arising canine haemangiomas (HAs) and haemangiosarcomas (HSAs) in order to identify novel molecular targets for treatment. Surgically-resected samples of HA (n = 27), HSA (n = 37), granulation tissue (n = 4) and normal skin (n = 4) were investigated by immunohistochemistry. Approximately 80% of the HSA samples had moderate to intense expression of phosphorylated Akt at Ser473 (p-Akt Ser473), p-Akt Thr308, p-4E-BP1 Thr37/46 and eukaryotic initiation factor 4E, which was significantly higher than in the HAs and was similar to the expression in activated endothelial cells (ECs). Although p-mTOR complex1 (p-mTORC1) Ser2448 was expressed by most of the activated ECs, only 35% of the HSA samples had weak to moderate expression. Because mTORC2 and phosphorylates Akt Ser473 was activated in HSA samples, the present findings suggest that the mTORC2/Akt/4E-BP1 pathway, regulated independently of mTORC1, may be important for targeting therapy in canine HSAs.

Avian poxvirus infection in a white-tailed sea eagle (Haliaeetus albicilla) in Japan

An adult female white-tailed sea eagle (Haliaeetus albicilla), over 12 years old, was found moribund and sent to the Wildlife Rescue Center in Kushiro, Japan. Grossly, the bird had multifocal yellow to black nodules in the beak, tongue, mucosa of the oral cavity, eyelids, and legs. Histologically, the cutaneous nodules revealed severe epidermal hyperplasia. The thickened epithelium, from prickle cell layer to horny layer, consisted of swollen keratinocytes containing frequent eosinophilic intra-cytoplasmic inclusions, Bollinger bodies. Ultrastructurally, the epidermal cells had cytoplasmic viral particles with characteristics of poxvirus. Furthermore, the 4b core gene sequence of an avian poxvirus was detected in a DNA sample prepared from the nodular lesions by polymerase chain reaction. The nucleotide sequence of the polymerase chain reaction product showed 78 to 95% similarities to the sequences of other avian poxviruses. Phylogenetic analysis showed that the sequence is clustered in clade A but distant from all the subclades previously reported. The results imply that it is a novel avian poxvirus. To our knowledge this is the first report of avian poxvirus infection in white-tailed sea eagles.

Immunohistochemical Demonstration of Angiogenesis-Associated Homeobox Proteins in Canine Vascular Tumours

Angiogenic homeobox genes regulate the behaviour of endothelial cells (ECs) during angiogenesis, so the aim of this study was to determine whether expression of these genes may be a determinant of malignancy in canine haemangiosarcoma (HSA). Homeobox proteins were evaluated immunohistochemically in tissue samples from canine HSAs (n=78), haemangiomas (HAs; n=30) and samples of granulation tissue (n=8). Active ECs in granulation tissue were positively labelled by antisera specific for HoxA9, HoxB3, HoxD3, HoxB7, Pbx1 and Meis1. Quiescent ECs in granulation tissue did not express HoxD3 and Pbx1. There were significantly more neoplastic cells positively labelled for HoxA9, HoxB3, HoxD3 and Pbx1 in HSA compared with HA. Almost all tumours were positive for HoxB7 and Meis1. HoxB3, HoxD3, Pbx1 and Meis1 proteins were detected in 80-90% of the HSAs, but in <20% of the HAs. Overall, homeobox protein expression in HSA appears to have a phenotype similar to that of active ECs in angiogenesis. The expression of homeobox genes associated with angiogenesis might be associated with the malignant growth of HSA.

Cynomolgus monkeys (Macaca fascicularis) may not become infected with equine herpesvirus 9.

Background  It was suggested that Equine herpesvirus 9 (EHV‐9) could be transmitted to higher non‐human primates.

Destructive Cholangitis in an Adult Jack Russell Terrier

A 4-year-old female Jack Russell terrier dog exhibited vomiting and severe jaundice of the visible mucous membranes and skin. Ultrasonography revealed diffuse areas of high echogenicity and focal areas of low echogenicity in the left lobe of the liver. On macroscopic observation of the biopsied liver specimen, many scattered irregularly shaped red spots were observed on the liver surface and on the cut surface. Histopathologically, there was loss of the interlobular bile duct and cholangitis accompanied by infiltration of pigment-laden macrophages in the Glisson’s capsule. Therefore, in the present case the dog was diagnosed with destructive cholangitis.

Equine Herpesvirus 9 (EHV-9) Induced Encephalitis in Nonhuman Primates

Equine Herpesvirus 9 (EHV-9) is a new member of the equine herpesviruses which was isolated from Thomson’s gazelles (Gazella thomsoni) that died of fulminant encephalitis in a Japanese zoo (Fukushi et al., 1997; Yanai et al., 1998). Previously, experimental infections of EHV-9 were conducted in various species of animals other than primates to clarify the infectivity and virulence of this virus and to assess the emerging aspects of EHV-9 in zoo and domestic animal populations. EHV-9 caused fatal infections with fulminant encephalitis characterized by neuronal degeneration and necrosis as well as intra-nuclear inclusion bodies in rodents (Fukushi et al., 1997; Fukushi et al., 2000), goats (Taniguchi et al., 2000), pigs (Narita et al., 2000, 2001), dogs (Yanai et al., 2003a) and cats (Yanai et al., 2003b). Based on several experimental studies of EHV-9 involving various domestic animals such as dogs and cats often found in close proximity to humans, there were grave concerns that EHV-9 could be transmitted to humans through contact with affected animals or zebras through certain routes. In order to assess the risk of EHV-9 to humans, we tried to determine the infectivity of EHV-9 in non-human primates, including common marmosets (Callithrix jacchus) and cynomolgus macaques (Macaca fasciocularis), which have strong similarities to humans, using the nasal route.