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Microsporidia can cause infection in various animals and humans. To determine the recent prevalence of Encephalitozoon in companion birds in Japan, 364 bird feces and 16 conjunctival exudates, as well as 28 exhibition bird feces, were examined using polymerase chain reaction (PCR). Thirty-five (9.6%) feces and 2 (12.5%) conjunctival exudates from companion birds were PCR positive, and sequence analysis revealed that all detected organisms were Encephalitozoon hellem genotype 1A. The prevalence by region varied from 4.5% in the Shikoku region to 14.3% in the Chugoku region. By age, the prevalence in birds younger than 6 months of age was 13.3%. We also discuss the threat of human infection as a zoonotic disease.

Mammary tumors are the most common tumors in women and non-spayed female dogs. One of the reasons for mammary tumors is mutations of the tumor suppressor gene, BRCA2. BRCA2 participates in homologous recombination repair by interacting with the RAD51 recombinase. BRCA2 has two RAD51-binding domains, consisting of BRC repeats and the C-terminal RAD51-binding domain, respectively. Although several studies have addressed the function of the C-terminal RAD51-binding domain of human BRCA2, the amino acid sequences required for the RAD51-interaction activity remain unclear. In this study, the C-terminal RAD51-binding domains of canine and human BRCA2 were compared; the canine domain displayed a weaker interaction with RAD51. This difference was attributed to the C-terminal portion of the domain via a comparison between canine and human domains. Furthermore, peptides shorter than those previously reported displayed RAD51-interacting activity, and a core motif of this domain consisting of 25 amino acids was identified. Since a mutation (S3323N) was reported in the core motif of this domain, the effect of this mutation was evaluated. The mutant exhibited similar RAD51-binding activity as that of the wild-type protein, suggesting that the mutation was functionally neutral. These data suggested that the C-terminal portion of the BRCA2 C-terminal RAD51-binding domain influenced its RAD51-interaction activity, and a minimum core motif of 25 amino acids was identified in this domain. These data may help clarify BRCA2 function, as well as the tumorigenic effects of BRCA2 mutation.

Although kinship (parent-offspring or siblings) contact has been suggested as a driving factor for sarcoptic mange epizootic in raccoon dogs (Nyctereutes procyonoides), no effect has been reported. In contrast, habitat fragmentation caused by urbanization may result in a high occurrence of sarcoptic mange, because habitat fragmentation may promote contact infection by increasing the population density of raccoon dogs. The habitat distribution of raccoon dogs may therefore influence epizootic sarcoptic mange. The genetic relationship between raccoon dogs was analyzed to examine Sarcoptes scabiei transmission between kin. The relationship between S. scabiei infection and the habitat of raccoon dogs was also investigated. Seventy-five raccoon dogs from Takasaki, Gunma prefecture, were examined from 2012 to 2018; 23 were infested with S. scabiei. The genotypes were determined using 17 microsatellite loci, and the relationships were categorized into four patterns by the ML-Relate software. There was no significant difference between infested pairs and other two pairs (Chi- squared test: χ 2 =0.034, df=1, P=0.85). Although it was difficult to predicate because the mortality rate was unclear in this study, kinship contact does not seem to be an important factor for sarcoptic mange epizootic. S. scabiei infection rates were significantly associated with the location of village sections (OR=1.55, 95% CI=1.11-2.17, P=0.011). It is suggested that direct/indirect contact between individuals living closely together is an important factor for the transmission of S. scabiei.

We analyzed the genotypes of three pregnant females and their litters to investigate the phenomenon of multiple paternity in wild raccoon dogs (Nyctereutes procyonoides) using 17 microsatellite markers. If a female has mated with only one male during estrus, then the maximum number of paternal alleles will not exceed two among littermates with the same father. The results revealed two out of three litters had three or four paternal alleles at one or five microsatellite loci. Therefore, the female had mated with more than one male during estrus. To the best of our knowledge, the present study is the first to report the possibility of multiple paternity in wild raccoon dogs.

A 26-year and 6-month-old male sika deer that was kept at the Showa Park, Tokyo, Japan, collapsed and died of severe disease wasting and severe tabefaction. Grossly, numerous masses, 0.3-1.0 cm diameter, were dispersed throughout the liver. The multiple masses were composed of tumor cells, which had hypochromatic nuclei and abundant faintly eosinophilic cytoplasm, arranged in nests of various sizes. Immunohistochemically, tumor cells were positive for cytokeratin, chromogranin A, synaptophysin and gastrin. Ultrastructurally, the cytoplasm of the tumor cells contained abundant membrane-bound electron-dense granules. A metastatic lesion was observed in the renal, hepatic and pancreatic lymph nodes. On the basis of these findings, this tumor was diagnosed as a neuroendocrine carcinoma with metastases to the lymph nodes.

Cancer consists of heterogeneous cells that contain a small population of cells that possess stem cell properties; these cells, referred to as cancer stem cells (CSCs) or tumor-initiating cells, are involved in tumor progression and metastasis. Using a sphere-forming assay, canine mammary CSCs were found to be similar to human breast CSCs. Metabolic reprogramming has been recognized as a hallmark of various cancers. However, the significance of cellular metabolism in CSCs remains unclear. The aim of this study was to define the metabolic characteristics of CSCs derived from canine mammary tumors and gain an understanding of the maintenance of stemness. We identified metabolite profiles of canine mammary adenocarcinoma cell lines using gas chromatography-mass spectrometry. Metabolites were extracted from both adherent and sphere-forming cells derived from three cell lines. Sphere-forming cells were separated from adherent cells using an orthogonal, partial least-squares discriminant analysis. Sphere-forming cells were found to contain high levels of the amino acids alanine, glycine and proline compared with adherent cells. They also had high levels of palmitoleate, palmitate and dihomo-gamma-linolenic acid compared with adherent cells. In a sphere-forming assay, palmitate increased the number of spheres for all cell lines. These results indicate that the sphere-forming cells derived from canine mammary adenocarcinoma cell lines have specific metabolic profiles that may be useful for the development of CSC-specific therapies targeting metabolic pathways and potential stemness biomarkers; these results also clarify the maintenance of stemness in canine mammary CSCs.

Two guereza colobus monkeys (Colobus guereza) reared in a zoological garden in Japan suddenly died of multifocal fibrinonecrotic gastroenteritis and septicemia associated with infection by Yersinia spp. It was necessary to microbiologically differentiate Yersinia frederiksenii and Y. enterocolitica. We described the pathological findings and discuss the causal agent to emphasize the need to revert to using a combination of multiple examinations for diagnosis.

A 14-year and 8-month-old intact male Amur tiger presented with an enlarged left testis, measuring 5.7 × 5.5 × 4.5 cm. The cut surface was mottled dark red to reddish brown in color. Microscopically, the enlarged left testis comprised round or polygonal neoplastic cells arranged in a diffuse sheet pattern. These neoplastic cells had a hyperchromatic nucleus and an abundant eosinophilic cytoplasm. Immunohistochemically, these neoplastic cells were positive for vimentin, chromogranin A, synaptophysin, melan-A, inhibin-α, and S100 and negative for desmin and WT-1. Based on these morphological and immunohistochemical findings, the tumor was diagnosed as a Leydig cell tumor.

Gliomas are common intracranial neoplasias in dogs. However, the underlying pathogenic mechanisms remain unclear. In humans, isocitrate dehydrogenase 2 (IDH2) is often mutated in gliomas. Although almost human IDH2 mutations have been identified at the Arg172 codon, few studies have reported structural, functional or mutational information for canine IDH2. In this study, we cloned the full-length canine IDH2 (cIDH2) cDNA and substituted wild type Arg174 (cIDH2 WT: corresponding to R172 of human IDH2) with Lys (cIDH2 R174K). The cIDH2 WT and R174K proteins were overexpressed in HeLa cells, and their presence was confirmed using an anti-human IDH2-WT mAb (clone: KrMab-3) and an anti-IDH2-R172K mAb (clone: KMab-1). The IDH2 activity between cIDH2 WT and cIDH2 R174K transfectants was compared by measuring the production of NADH and NADPH. NADPH production was lower for cIDH2 R174K than that for cIDH2 WT transfectants. Finally, we detected increased expression of hypoxia inducible factor-1 alpha (HIF-1α) in cIDH2 R174K transfectants. This indicates that mutations at R174 can potentially induce carcinogenesis in canine somatic cells.

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.

A 7-year-old female domestic rabbit suffered from labored respiration, poor appetite, mild anemia and thrombocytopenia. Radioscopic examination revealed masses in multiple locations including the intrapleural cavity and spleen. Forty-three days after the first visit to a private veterinary clinic, the rabbit died of severe respiratory distress. Microscopically, all of the masses were composed of round to polygonal neoplastic cells with distinct cell borders that were arranged in a sheet pattern. Multinucleated giant neoplastic cells were often observed. Some neoplastic cells had phagocytozed one or more erythrocytes. Immunohistochemical staining revealed that the neoplastic cells expressed vimentin, CD204, Iba-1 and lysozyme, but not CD163. Based on the morphological and immunohistochemical findings, this case was diagnosed as disseminated histiocytic sarcoma with hemophagocytosis.

Cancer stem cells or tumor-initiating cells (TICs) are a small subpopulation of cells that have the capacity to self-renew, differentiate and initiate tumors. These cells may function in tumor initiation, aggression and recurrence. Whether spheres derived from canine rhabdomyosarcoma cells have stem cell-like properties is unclear. We induced sphere formation in the canine rhabdomyosarcoma cell lines, CMS-C and CMS-J, and characterized the spheres in vitro and in vivo. Sphere-forming cells were more resistant to vincristine, mitoxantrone and doxorubicin than adherent cells. Xenograft transplantation demonstrated that 1 × 10 3 sphere-forming cells derived from CMS-C were sufficient for tumor formation. The sphere assay showed that the sphere-forming cells were present in these tumors. These results suggest that the spheres derived from canine rhabdomyosarcoma cells may possess characteristics of TICs. This study provides the foundation for elucidating the contribution of TICs to rhabdomyosarcoma tumorigenesis.

Canine prostate cancer (cPCa) is an untreatable malignant neoplasm resulting in local tissue invasion and distant metastasis. MicroRNAs (miRs) are small non-coding RNAs that function as oncogenes or tumor suppressors. The purpose of this study was to characterize the expression of miRs that are altered in cPCa tissue. The expression levels of 277 mature miRs in prostatic tissue (n=5, respectively) were compared between the non-tumor and tumor groups using real-time PCR. Five miRs (miR-18a, 95, 221, 222 and 330) were up-regulated, but 14 miRs (miR-127, 148a, 205, 299, 329b, 335, 376a, 376c, 379, 380, 381, 411, 487b and 495) were down-regulated specifically in cPCa (P<0.05). These miRs have potential use as early diagnosis markers for cPCa and in miR-based therapy.

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.

A 12-year-old female American shorthair cat presented with a one-month history of hematuria and general lethargy. Abdominal ultrasonography revealed complete thickening of the left uterine wall. At a diagnostic laparotomy, a large mass arising from the left uterine horn was discovered, and ovariohysterectomy was performed. Histological diagnosis revealed a T-cell high-grade lymphoma of the uterus. After the ovariohysterectomy, the patient achieved complete remission and was maintained by combination chemotherapy from 14 days after surgery. However, relapse occurred in the urinary bladder wall on day 287, and the patient died of postrenal acute renal failure on day 310. This is the first report of a feline case of primary uterine lymphoma that was treated with ovariohysterectomy followed by systemic chemotherapy.

We describe a case of human Becker muscular dystrophy (BMD)-like myopathy that was characterized by the declined stainability of dystrophin at sarcolemma in a pig and the immunostaining for dystrophin on the formalin-fixed, paraffin-embedded (FFPE) tissue. The present case was found in a meat inspection center. The pig looked appeared healthy at the ante-mortem inspection. Muscular abnormalities were detected after carcass dressing as pale, discolored skeletal muscles with prominent fat infiltrations and considered so-called "fatty muscular dystrophy". Microscopic examination revealed following characteristics: diffused fat infiltration into the skeletal muscle and degeneration and regeneration of the remaining skeletal muscle fibers. Any lesions that were suspected of neurogenic atrophy, traumatic muscular degeneration, glycogen storage disease or other porcine muscular disorders were not observed. The immunostaining for dystrophin was conducted and confirmed to be applicable on FFPE porcine muscular tissues and revealed diminished stainability of dystrophin at the sarcolemma in the present case. Based on the histological observations and immunostaining results, the present case was diagnosed with BMD-like myopathy associated with dystrophin abnormality in a pig. Although the genetic properties were not clear, the present BMD-like myopathy implied the occurrence of dystrophinopathy in pigs. To the best of our knowledge, this is the first report of a natural case of myopathy associated with dystrophin abnormalities in a pig.

Intrathecal (IT) immunization involves injecting antigens directly into the intraventricular or subarachnoid spaces, or brain, to induce antigen-specific antibodies (Ab) in the cerebrospinal fluid (CSF). In the present study, rabbits were immunized IT with inactivated rabies virus to investigate the origins of CSF Ab. The time course of Ab induction and tumor necrosis factor-alpha expression suggested the possibility that the CSF Ab originated in the serum. In addition, Ab-producing cells infiltrated around the blood vessels of the brain, suggesting local production of Ab within the central nervous system (CNS). Furthermore, subcutaneous (SC) immunization prior to IT immunization induced a rapid and magnified Ab response in the CSF compared with IT immunization alone. These results were confirmed by the fact that mice immunized SC prior to IT were more resistant to intracerebral challenge with rabies virus than mice immunized via the IT route alone. Taken together, these results suggest that combined SC and IT immunization is a more effective vaccination protocol for prophylaxis and treatment of rabies.

To evaluate the efficacy of intracerebral (IC) immunization, mice were immunized with a rabies vaccine by the subcutaneous (SC), intramuscular (IM) or IC route, and 10-fold the 50% lethal dose of rabies virus was inoculated into the hindleg of the immunized or non-immunized mice. The antibody titer in serum was elevated and boosted by additional immunization via all routes, but highest after the IC immunization followed by the IM and SC routes, in this order. Intracerebrally immunized mice were completely protected from death and the neurological signs of infection, whereas the IM or SC immunization only partly protected the mice. In mouse models, IC immunization is more effective at inducing a protective immune response against the transneural spread of rabies virus than IM or SC immunization.

Swine hemagglutinating encephalomyelitis virus (HEV) causes encephalomyelitis or vomiting and wasting disease in suckling piglets. Neurotoropism of the virus has been demonstrated in previous in vivo studies. In the present study, we investigated the infectivity and propagation of HEV in comparison with those of pseudorabies virus (PRV), another neurotropic virus, using dorsal root ganglia cells of newborn mice containing nerve cells and non-neuronal cells. HEV infected nerve cells but did not infect non-neuronal cells, whereas PRV infected both cell types. By using cytoskeletal inhibitors, it was suggested that propagation of HEV and PRV within and among nerve cells depended on microtubules and intermediate filaments of nerve cells, indicating that the viruses may be transported between the cell body and axonal terminals of neurons by fast axonal flow.

Many small wild birds died in the 2005-2006 wintertime in Hokkaido. Thirteen birds were pathologically examined and it was attempted to detect West Nile and influenza viruses from their organs. Consecutive pathological changes were fresh hemorrhage and acute circulatory failure. Viral detections were negative. Selective occurrence in wintertime, literature review and the results of pathological and virological examinations suggested chemical deicer poisoning as the cause of wild bird death. Chicks treated orally with deicer showed acute death and their pathological changes were similar to those of the wild birds. Because the chicks showed significant elevation of plasma Na concentration, plasma electrolyte analysis of the affected wild birds might be crucial to confirm our tentative diagnosis.

Fowl glioma-inducing virus (FGV), which belongs to subgroup A of avian leukosis virus (ALV), is tumorigenic in the nervous system. In a zoological garden in Japan, approximately 40% of chickens, including Japanese fowls, were infected with FGV. Because this zoological garden plays a role as a major supplier of Japanese fowl for other zoological gardens, FGV infection is suspected to have spread among ornamental chickens. In this study, the prevalence of the disease was examined in a total of 129 chickens in three other zoological gardens by nested polymerase chain reaction (PCR), reverse transcription nested PCR and enzyme-linked immunosorbent assay. Twenty-six to 56 percent of the fowls in each of the examined gardens were positive by nested PCR. The phylogenetic analysis revealed that the 3' untranslated region, including the specific sequence of FGV, of the 14 isolated ALVs showed high sequence identity and a close relationship with FGV. In addition, the env gene of the isolates frequently showed mutations and deletions of nucleotides. These results suggest that FGV is prevalent among ornamental chickens kept in zoological gardens in Japan.

Rabbits were immunized with inactivated influenza virus via the subarachnoidal (SA) or subcutaneous (SC) route, and the antibody titers in cerebrospinal fluid (CSF) and serum were assayed. There were no nervous signs or morphological lesions related to SA immunization. In the SC group, the antibody titer was elevated in serum, but not elevated in CSF. In the SA group, the antibody titer was significantly elevated in serum and even in CSF, and their antibody titers were much greater than in the SC group. The present results suggest that intrathecal immunization is more effective than SC immunization at inducing a protective immune response against the transneural spread of viruses.

Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL) is a nuclear IkappaB protein that is also known as interleukin-1-inducible nuclear ankyrin repeat protein and inhibitor of nuclear factor kappaBzeta (IkappaBzeta). We previously observed that MAIL-deficient mice were affected by atopic dermatitis-like skin lesions and demonstrated the importance of MAIL in the skin. In this study, we investigated MAIL expression in mouse keratinocytes. MAIL mRNA was constitutively expressed in the skin epidermis. MAIL expression was also confirmed in primary keratinocytes and the PAM212 keratinocyte cell line. The inhibitors of nuclear factor kappaB (NF-kappaB)-Bay11-7082 and the IkappaBalphaM supersuppressor-considerably downregulated MAIL expression in the keratinocytes. Immunoreactivity for NF-kappaB components was localized in the cytoplasm and nucleus of normal unstimulated keratinocytes. The expression level of MAIL in the skin did not change following lipopolysaccharide (LPS) administration to mice. Interestingly, in accordance with the in vivo findings, the MAIL expression level did not change following LPS stimulation even in primary keratinocytes; however, MAIL expression was strongly increased by interleukin-1 stimulation. These results collectively suggest that the constitutive expression of MAIL in keratinocytes is controlled, at least in part, by NF-kappaB and that there may be LPS-specific repressive mechanisms that inhibit MAIL induction.

Mammary tumors are the most common tumor type in women as well as in female dogs. The BRCA2 gene encodes a large nuclear protein that is involved in DNA repair, and mutations in the human BRCA2 confer an increased risk of female mammary tumors. The BRCA2 protein acts as a tumor suppressor, and inactivation of BRCA2 by loss of heterozygosity is implicated in mammary carcinogenesis. In this study, to establish an appropriate polymorphic marker for loss of heterozygosity analysis of the canine BRCA2, we analyzed the genomic sequences of the exon 27 regions of 30 mammary-tumor-bearing and 21 tumor-free dogs. In addition to 10204ins/delAAA, which is the only polymorphism previously identified for the canine BRCA2 locus, we discovered four novel single nucleotide polymorphisms. The analysis of these five polymorphisms revealed the presence of four allele types. Since 10204ins/delAAA was the most common of the five polymorphisms identified, we developed a PCR-based assay method to assay for this polymorphism. We believe that this method is valuable for loss of heterozygosity analysis of the canine BRCA2 gene in tumor pathogenesis.

Odontogenic cysts, which showed cystic radiolucency in the jaw bone by radiographic examination and computed tomography, were enucleated by operation in 3 dogs. One dog had a odontogenic keratocyst in the incisive bone of the right maxilla and another 2 cases revealed dentigerous cysts in the mandible. These cyst walls were enucleated or transpired by semiconductor laser. Afterwards, osteogenesis was confirmed at the defective part of jaw bone by extirpation of the cyst in all cases, and no recurrence has been noted in any cases. Odontogenic cyst is a disease which should be treated by surgical extirpation or transpiration.

To investigate the effect of antipyretics on the murine and poultry models of influenzal encephalitis, we injected large quantities of antipyretics, acetylsalicylic acid (aspirin) and dicrofenac sodium (voltaren). The effect of antipyretic treatment on the murine encephalitis model was unremarkable histologically and immunohistochemically. Whereas in chicks, CNS lesions consisting of perivascular cuffing and gliosis appeared only in those animals treated with the antipyretics and viral antigen was detected mainly in the nuclei of glial cells or vascular endothelia of voltaren-treated animals. We here demonstrate that antipyretic treatment aggravated the hematogenous spread of the influenza virus to the CNS in chicks, but did not affect the transneural infection in mice.

Mammary tumors are common in cats. As mutations in human Brca2 confer an increased risk of breast cancer, the full-length cDNA of the feline homologue of Brca2 was sequenced to obtain a basis for studying the relationship between its function and susceptibility to mammary tumors. The feline Brca2 cDNA is 10 kb long, and encodes 3,371 amino acids. The amino acid sequence of feline Brca2 shares low homology with the Brca2 of other mammals, e.g., 53% homology with the murine protein. Analysis of the expression pattern of the feline Brca2 gene revealed that, as previously reported for other mammals, it is transcribed in various tissues, including the mammary gland.

Western blotting was performed to analyze Neospora caninum tachyzoite antigens recognized by mouse IgG at different stages of infection including recrudescence. At the early stage of infection, a 36-38 kDa antigen was clearly recognized by the mouse antisera. After day 48 postinoculation, the signal of the 36-38 kDa antigen gradually weakened. Meanwhile, a 43 kDa antigen was intensely and continuously recognized from 48 to 125 days postinoculation. This 43 kDa antigen was clearly detectable with the antisera from the mice under immunosuppression. Sera from naturally infected cattle strongly reacted with the 43 kDa antigen. Therefore, the 43 kDa antigen may be useful for immunological reactions to detect infected animals except in the early stage of the infection.

The present study was conducted to determine the clinical and clinico-pathologic characteristics of Shiba dogs with GM1 gangliosidosis, which is due to an autosomal recessively inherited deficiency of lysosomal acid beta-galactosidase activity. Clinical and clinico-pathological features were investigated in 10 homozygous Shiba dogs with GM1 gangliosidosis. The age at onset was 5 to 6 months and the dogs manifested progressive neurologic signs including loss of balance, intermittent lameness, ataxia, dysmetria and intention tremor of the head. The dogs were unable to stand by 10 months of age due to a progression of ataxia and spasticity in all limbs. Corneal clouding, a visual defect, generalized muscle rigospasticity, emotional disorder and a tendency to be lethargic were observed at 9 to 12 months. The dogs became lethargic from 13 months of age. The survival period seemed to be 14 to 15 months. As a clinico-pathologic feature, lymphocytes with abnormally large vacuoles were observed in peripheral blood (30 to 50% of total lymphocytes) through the lifetime of the dogs. The clinical and clinico-pathologic characteristics of this animal model are useful for not only the development and testing of potential methods of therapy, but also the diagnosis of affected homozygous Shiba dogs in veterinary clinics.

Intracerebral inoculation of field-isolates as well as established strains of equine herpesvirus-1 (EHV-1) in suckling mice results in viral replication in neurons and glial cells and induces encephalitis. By intraperitoneal (i.p.) inoculation, no histological lesion was observed in the central nervous system (CNS) in suckling mice with the EHV-1 HH1 strain (HH1), whereas a neuroadapted variant (NHH1) produced by serial passage of HH1 in the mouse brain caused severe encephalomyelitis after i.p. inoculation. The purpose of this study was to determine the route of neuroinvasion after i.p. inoculation of NHH1 and to clarify the effects of the brain passage on viral neuroinvasion. NHH1, but not HH1, targeted splenic and pulmonary macrophages and omental fat cells on days 1 and 2 post-inoculation (p.i.). From days 1 to 3 p.i., cell-associated viremia was occurred in NHH1-infected mice, but not in HH1-infected mice. On day 4 p.i., viral antigen was detected in a few endothelial cells, perivascular glial cells and neurons in the CNS in NHH1-infected mice. The number of viral antigen-positive cells increased markedly after day 5 p.i. In contrast, no viral antigen-positive cell was detected in the CNS in HH1-infected mice, except for a few nerve cells in the thoracic cord on day 4 p.i. These results suggest that NHH1 neuroinvasion is hematogenous and is correlated with enhanced extraneural virus growth.

The direct effects of three steroid hormones (progesterone, estradiol-17beta and corticosterone) on the growth of Neospora caninum (N. caninum) tachyzoite were examined in Vero cells. Subsequently, ovariectomized BALB/c mice infected with N. caninum were treated with physiological concentrations of the steroid hormones for 1 or 2 weeks. These hormones had no direct effect on the parasite growth in vitro. In the infected mice, there was no significant difference in the parasite distribution and histopathological changes between the hormone-injected and control groups. No mice showed parasitemia at the time of autopsy. These results suggest that physiological levels of steroid hormones (progesterone, estradiol-17beta and corticosterone) do not reactivate N. caninum in mice.

C/O specific pathogen-free White Leghorn chickens were intracerebrally inoculated at one day of age with a brain homogenate of Japanese bantams (Gallus gallus domesticus) affected with fowl glioma. Histologically, six of eight inoculated chickens developed nonsuppurative meningoencephalitis in cerebrum and two of them had the characteristic lesions of fowl glioma. Hyperplastic lymphoid foci concomitantly developed in many organs of these birds, especially in the heart. Apart from these lymphoid foci, lymphocytic myocarditis was observed in all inoculated birds. Matrix inclusions were also noted in myocardial cells. Immunohistochemically, avian leukosis virus antigens were detected in reticular cells in the lymphoid foci, mesangial cells of the kidney, smooth muscle cells of the blood vessels, and myocardial cells. Of these tissues, the myocardium of all inoculated birds consistently showed strong reactivity for this antigens. The matrix inclusions were also positive for the antigens. These results suggest that the causal virus of fowl glioma has a high propensity to replicate, especially in myocardium and nonsuppurative myocarditis occurs associated with so-called fowl glioma.

Mammary tumors are the most common neoplasm in female dogs, Canis canis, and in women. Mutations in human Brca2 confer an increased risk of female breast cancer. Previous studies have shown that the Brca2 tumor suppressor protein interacts with the recombinational repair protein Rad51. We cloned the full-length cDNA of the canine homologues of Brca2 and Rad51 to obtain a basis for studying their relationship with susceptibility to mammary tumors. The canine Brca2 and Rad51 cDNAs are 11 and 1.5 kb long, encoding 3.471 and 339 amino acids, respectively. The amino acid sequence of canine Brca2 showed 68% homology with the human protein, and 58% homology with a murine protein. There were highly conserved regions in the C-terminus of all three proteins, where the Rad51 interacting domain and putative nuclear localization signals are located. Comparing with the partial genomic sequence previously reported, we found possible nuclear polymorphisms in exon 11, some of which result in amino acid substitutions. On the other hand, canine Rad51 protein had extremely high homology (99%) to the human and murine proteins. Expression of both Brca2 and Rad51 was detected in the mammary gland, suggesting that these two genes interact in the canine mammary gland.

The Ito cell (fat-storing cell) lies in perisinusoidal space of liver and has a variety of functions. We investigated the immunohistochemistry and ultrastructure of Ito cells in normal and cholestatic livers of broiler chickens. Immunohistochemistry demonstrated that Ito cells expressed HHF35 muscle actin, vimentin, desmin, glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), chromogranin A and cytokeratins in normal livers. These cells were diffusely scattered throughout the lobules. Livers treated with extrahepatic bile duct ligation (BDL) showed cholestasis, fibrosis, proliferation of biliary ductules and Ito cells. The Ito cells were frequently found in fibrotic areas and were larger in size with more extensive immunoreactivity than those of normal livers. Ultrastructural study demonstrated that Ito cells were closely associated with the production of collagen fibers in BDL livers. These findings suggest that Ito cells actively react against hepatocytic injuries and play a major role in the hepatic fibrogenesis of cholestatic livers of chickens.

Neospora caninum (BT-2 strain) that originated from the brain of a Holstein calf was serially passaged through 10 generations of BALB/c nude mice by intraperitoneal inoculation. Histological examination of the mice revealed that numerous clusters of tachyzoites appeared in the pancreas, stomach and small intestine as well as in the central nervous system (CNS) and skeletal muscles. Intestinal contents of the infected mice were inoculated intraperitoneally into uninfected nude mice and 3 of the 17 inoculated mice showed clinical signs at post inoculation days 3 to 10. The present experiments demonstrated a proliferation of N. caninum tachyzoites in the mucosa of the alimentary tract and pancreas of the nude mice and the intestinal contents of the mice were infective to other nude mice.

A plasmid pLTR-DT which contained a gene for diphtheria toxin A-chain (DT-A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) (BLV-LTR) in the multicloning site of pUC-18 was entrapped in cationic liposomes composed of N-(alpha-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG), dioleoyl phosphatidylethanolamine (DOPE) and dilauroyl phosphatidylcholine (DLPC) (1:2:2, molar ratio) (TMAG-liposome), and their antitumor effect on BLV-infected tumor cells was examined in vivo. The cationic TMAG-liposome containing pLTR-DT was successively injected into the tumor transplanted to nude mice. The growth of tumor was significantly inhibited by the injection of cationic TMAG-liposome containing pLTR-DT. On the other hand, TMAG-liposome containing pUC18 plasmids showed no such effect. These results suggest that a DT-A expression plasmid under the control of BLV-LTR is highly toxic to the BLV-infected tumor cells, and that the cationic liposomes, such as TMAG-liposome, may be efficient transfection reagent for BLV-infected tumor cells and can be utilized for DT-A gene delivery into BLV-infected tumor cells in vivo.

Typical Pneumocystis carinii pneumonia was found in a male thoroughbred foal (46 days old) suffering from diarrhea and hypogammaglobulinemia after birth. The characteristics of the organisms were demonstrated by Grocott methenamine silver staining, and scanning and transmission electron microscopy. The present focal had no histological lesions suggesting immunodeficiency. However, he could not get adequate colostrum from his dam, because she died of hemorrhagic shock due to immuno-mediated thrombocytopenia at delivery. This condition was considered to have predisposed the foal to the pneumonia.

A 72-day-old, female, Golden Retriever dog showed anorexia, coughing, nasal discharge, diarrhea and hematochezia, and died on the 15th clinical day. Pathological examination revealed dual infection with canine distemper virus (CDV) and canine adenovirus (CAV). CAV inclusion bodies occurred only in the liver, and biliary and respiratory system, whereas CDV inclusions were demonstrated in the visceral organs systematically. The CAV inclusions were associated with multifocal hepatocellular necrosis and edematous swelling of the wall of the gall bladder, suggesting infectious canine hepatitis virus (canine adenovirus type 1) infection.

Among the sera from 9 rabbits spontaneously infected with Encephalitozoon cuniculi, a serum which revealed a high titer for E. cuniculi by indirect protein A-gold (IPAG) immunohistochemistry and reacted with the outer layer of the shell of E. cuniculi spores on immunoelectron microscopical examination, was biotinylated. The biotinylated rabbit anti-E. cuniculi IgG reacted immunohistochemically with E. cuniculi, but not with other protozoa tested, namely Neospora caninum, Toxoplasma gondii and sarcocystis. The direct avidin-biotin-peroxidase complex (ABC) immunohistochemistry using biotinylated rabbit anti-E. cuniculi IgG in this study is a useful tool for the diagnosis and study of encephalitozoonosis.

Changes of body weights and hepatic acetyl-CoA carboxylase activities were measured in voles and mice treated with monosodium-L-aspartate (MSA). MSA was administrated subcutaneously to neonates at 4 mg/g. The MSA-treated mice showed remarkable obesity, associated with the increase in the plasma insulin concentrations and acetyl-CoA carboxylase activities. The activity of acetyl-CoA carboxylase of control voles was very low; under half that of mice. In the MSA-treated voles, although the plasma insulin concentrations also increased, acetyl-CoA carboxylase activities were not elevated and signs of obesity were not observed.