Masahito Kubo

The first report of Hepatozoon species infection in leopard cats (Prionailurus bengalensis) in Korea.

Abstract Hepatozoon species infections were detected in 4 of 5 heart samples from leopard cats (Prionailurus bengalensis) in Korea examined by light microscopy and PCR. Microscopically, the schizonts of the Hepatozoon species were observed within the parasitophorous vacuoles of unidentified host cells that were located between myocytes. The schizonts were approximately 31 ± 4 × 19 ± 3 µm in size; 3 mature schizonts contained approximately 24, 25, and 35 merozoites. In analyses of the partial 18S rRNA gene sequence, the Hepatozoon species infecting the Korean leopard cats was closely related to Brazilian (99.2–99.8% identical) and Spanish (97.0–98.2% identical) isolates of Hepatozoon felis.

Oncolytic Reovirus in Canine Mast Cell Tumor

The usage of reovirus has reached phase II and III clinical trials in human cancers. However, this is the first study to report the oncolytic effects of reovirus in veterinary oncology, focusing on canine mast cell tumor (MCT), the most common cutaneous tumor in dogs. As human and canine cancers share many similarities, we hypothesized that the oncolytic effects of reovirus can be exploited in canine cancers. The objective of this study was to determine the oncolytic effects of reovirus in canine MCT in vitro, in vivo and ex vivo. We demonstrated that MCT cell lines were highly susceptible to reovirus as indicated by marked cell death, high production of progeny virus and virus replication. Reovirus induced apoptosis in the canine MCT cell lines with no correlation to their Ras activation status. In vivo studies were conducted using unilateral and bilateral subcutaneous MCT xenograft models with a single intratumoral reovirus treatment and apparent reduction of tumor mass was exhibited. Furthermore, cell death was induced by reovirus in primary canine MCT samples in vitro. However, canine and murine bone marrow-derived mast cells (BMCMC) were also susceptible to reovirus. The combination of these results supports the potential value of reovirus as a therapy in canine MCT but warrants further investigation on the determinants of reovirus susceptibility.

Hepatozoon ursi n. sp. (Apicomplexa : Hepatozoidae) in Japanese black bear (Ursus thibetanus japonicus)

Morphological and genetic features of a new Hepatozoon species, Hepatozoon ursi n. sp., in Japanese black bear (Ursus thibetanus japonicus) were studied. Schizogonic developmental stages were observed in the lungs of Japanese black bears. The schizonts were sub-spherical in shape and 45.7+/-4.6 x 42.7+/-4.5 microm in size. Each mature schizont contained approximately 80-130 merozoites and 0-5 residual bodies. The merozoites were 7.0+/-0.7 x 1.8+/-0.3 microm in size. Intraleukocytic gametocytes were slightly curved, cigar-like in shape and had a beak-like protrusion at one end. The size of the gametocytes was 10.9+/-0.3 x 3.3+/-0.2 microm. The analyses of the18S rRNA gene sequences supported the hypothesis that H. ursi n. sp. is different from other Hepatozoon species. Mature Hepatozoon oocysts were detected in two species of ticks (Haemaphysalis japonica and Haemaphysalis flava) collected on the bears infected with H. ursi n. sp. Two measured oocysts were 263.2 x 234.0 microm and 331.8 x 231.7 microm, respectively. The oocysts contained approximately 40 and 50 sporocysts, respectively. The sporocysts were sub-spherical in shape and 31.2+/-2.5 x 27.0+/-2.9 microm in size. Each sporocyst contained at least 8-16 sporozoites, with the sporozoites being 12.2+/-1.4 x 3.5+/-0.5 microm in size. H. ursi n. sp. is the first Hepatozoon species recorded from the family Ursidae.

Muscular sarcocystosis in wild carnivores in Honshu, Japan

A total of 65 free-living carnivores collected on Honshu Island, Japan were examined for muscular Sarcocystis species infections. Among them, 12 Japanese raccoon dogs (Nyctereutes procyonoides viverrinus), one Japanese red fox (Vulpes vulpes japonica), three Japanese martens (Martes melampus melampus), and two Japanese badgers (Meles meles anakuma) were found to have sarcocysts in their muscles. No inflammatory reactions associated with sarcocysts were observed. Ultrastructurally, the sarcocysts detected in the Japanese raccoon dogs, Japanese red fox, and Japanese martens were similar to each other, with the sarcocyst wall being thin and exhibiting minute undulations. On the other hand, the sarcocysts detected in the Japanese badgers had a thick cyst wall with numerous finger-like protrusions which contained microtubules. The species of Sarcocystis in Japanese carnivores remain to be determined. This is the first published report on muscular sarcocystosis in Japanese carnivores.

Pathological and phylogenetic features of prevalent canine distemper viruses in wild masked palm civets in Japan

Ten wild masked palm civets infected with canine distemper virus (CDV), captured in Japan from 2005 to 2007, were histopathologically and phylogenetically analyzed. Phylogenetic analysis based on the amino acid sequences of the H protein of two CDV isolates from masked palm civets revealed that the two isolates were classified into the clade of recent isolates in Japan. Histopathologically marked lesions of virus encephalitis were present in the brain, whereas gastrointestinal lesions were absent or at a mild degree. The distribution of the lesions resembles that of recent CDV cases in dogs. Therefore, recent CDV infections in masked palm civets could be caused by recently prevalent CDV in dogs. The possibility of the masked palm civet as a spreader of CDV among wildlife is also discussed.

A novel budgerigar-adenovirus belonging to group II avian adenovirus of Siadenovirus

Five budgerigars in the same breeding facility died or showed ruffled feathers. To determine the cause, five dead or euthanized budgerigars were examined. Splenomegaly was observed at necropsy in all birds examined. Histopathology of the spleen revealed a slight-to-moderate deletion of lymphocytes and increase of macrophages. Concurrent congestions in several tissues such as liver, lung, kidney, and/or brain and basophilic intranuclear inclusion bodies in the epithelial cells of renal tubules were found in all the birds examined. Psittacine adenoviral DNA was detected in the kidney of one of the five budgerigars by PCR. Sequencing and phylogenetic analysis of the hexon gene revealed that the adenovirus gene detected in the budgerigar was derived from an unknown adenovirus belonging to the genus Siadenovirus. Using a new pair of primers based on the obtained sequence, we confirmed the presence of the newly found adenovirus in all five birds. The newly found unknown adenovirus is designated as Budgerigar Adenovirus 1.

Characterization of squamate olfactory receptor genes and their transcripts by the high-throughput sequencing approach.

The olfactory receptor (OR) genes represent the largest multigene family in the genome of terrestrial vertebrates. Here, the high-throughput next-generation sequencing (NGS) approach was applied to characterization of OR gene repertoires in the green anole lizard Anolis carolinensis and the Japanese four-lined ratsnake Elaphe quadrivirgata. Tagged polymerase chain reaction (PCR) products amplified from either genomic DNA or cDNA of the two species were used for parallel pyrosequencing, assembling, and screening for errors in PCR and pyrosequencing. Starting from the lizard genomic DNA, we accurately identified 56 of 136 OR genes that were identified from its draft genome sequence. These recovered genes were broadly distributed in the phylogenetic tree of vertebrate OR genes without severe biases toward particular OR families. Ninety-six OR genes were identified from the ratsnake genomic DNA, implying that the snake has more OR gene loci than the anole lizard in response to an increased need for the acuity of olfaction. This view is supported by the estimated number of OR genes in the Burmese pythons draft genome (∼280), although squamates may generally have fewer OR genes than terrestrial mammals and amphibians. The OR gene repertoire of the python seems unique in that many class I OR genes are retained. The NGS approach also allowed us to identify candidates of highly expressed and silent OR gene copies in the lizards olfactory epithelium. The approach will facilitate efficient and parallel characterization of considerable unbiased proportions of multigene family members and their transcripts from nonmodel organisms.

A Case of Chondrosarcoma in a Free-flying Great Egret

A free-flying Great Egret (Ardea alba) captured in Gifu, central Japan, in May 2006 had a large mass on the right carpal joint. The tumor was diagnosed as chondrosarcoma by histopathologic examination.

Parasitological and Molecular Features of the Hepatozoon Species in the Myocardium of Japanese Martens (Martes melampus melampus)

Abstract The Hepatozoon species in the myocardium of Japanese martens (Martes melampus melampus) was characterized by histological and molecular methods. Histologically, granulomatous nodules with Hepatozoon sp. merozoites/gametocytes, or schizonts, or both were observed in the hearts of Japanese martens. The most frequently observed forms were merozoites/gametocytes within phagocytes; each host cell included a zoite, which was not microscopically identifiable as a merozoite or gametocyte. Schizonts were oval in shape and 36.9 ± 5.7 × 28.9 ± 3.4 µm in size; each schizont had approximately 20–60 nuclei. The size of the merozoites could not be measured because no mature schizonts were observed. In the analyses of the partial 18S rRNA gene sequence, it was strongly suggested that the Hepatozoon sp. in Japanese marten and the Hepatozoon sp. in pine marten (Martes martes) in Scotland were the same species.

Meningoencephalitis associated with Sarcocystis spp. in a free-living Japanese raccoon dog (Nyctereutes procyonoides viverrinus).

A free-living, young adult, male Japanese raccoon dog (Nyctereutes procyonoides viverrinus) was rescued in Gifu, Japan in March 2009. The animal was weak and emaciated with neurological signs that included head tilt, tremor and tic. The brain showed no gross abnormality at necropsy, but microscopically there was severe meningoencephalitis associated with protozoa, which were morphologically consistent with the asexual developmental stage of Sarcocystis spp. The protozoa were immunohistochemically negative for Toxoplasma gondii and Neospora caninum, but reacted weakly with antiserum specific for Sarcocystis cruzi. Analysis of the partial 18S rRNA gene sequence revealed that the protozoa were most closely related to an unidentified Sarcocystis species that was isolated from the white-fronted goose (Anser albifrons).