田中 良和

The geneLEAD VIII is a fully-automated nucleic acid extraction/quantitative PCR equipment developed by Precision System Science Co. Ltd, (PSS). To take advantage of its capability, we developed a quantitative assay system to measure growth of animal viruses. The system was used to assay one of the Chinese herbal extracts whose anti-malarial activities were previously reported and demonstrated its dose-dependent anti-viral activity against feline infectious peritonitis virus (FIPV), a feline coronavirus causing the fatal diseases in cats, and relatively low cell toxicity. The assay developed in this study is useful to screen antiviral drugs and the anti-FIPV activity of the herbal extract identified have a potential to lead to development of new drugs against FIPV and other coronaviruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Due to the high incidence of mammary tumors in dogs, it is important to elucidate the pathogenesis of these tumors in veterinary medicine. Radiation therapy is often used to treat mammary tumors that target DNA lesions. RAD51 is a key molecule that repairs DNA damage via homologous recombination. We examined the relationship between RAD51 expression and radiosensitivity in mammary tumor cell lines. CHMp and CHMm from the same individual were selected based on the differences in RAD51 expression. The radiosensitivity of both cell lines was examined using MTT and scratch assays; CHMm, which has high RAD51 expression, showed higher sensitivity to radiation than CHMp. However, the nuclear focus of RAD51 during DNA repair was formed normally in CHMp, but not in most of CHMm. Since irradiation resulted in the suppression of cell cycle progression in CHMp, the expression of p21, a cell cycle regulatory factor, was detected in CHMp after 15 Gy irradiation but not in CHMm. These results indicate that functional expression is more important than the quantitative expression of RAD51 in canine mammary tumor cells in response to DNA damage.

Feline coronaviruses (FCoVs) infect cats worldwide and cause severe systemic diseases, such as feline infectious peritonitis (FIP). FIP has a high mortality rate, and drugs approved by the Food and Drug Administration have been ineffective for the treatment of FIP. Investigating host factors and the functions required for FCoV replication is necessary to develop effective drugs for the treatment of FIP. FCoV utilizes an endosomal trafficking system for cellular entry after binding between the viral spike (S) protein and its receptor. The cellular enzymes that cleave the S protein of FCoV to release the viral genome into the cytosol require an acidic pH optimized in the endosomes by regulating cellular ion concentrations. Ionophore antibiotics are compounds that form complexes with alkali ions to alter the endosomal pH conditions. This study shows that ionophore antibiotics, including valinomycin, salinomycin, and nigericin, inhibit FCoV proliferation in vitro in a dose-dependent manner. These results suggest that ionophore antibiotics should be investigated further as potential broad-spectrum anti-FCoV agents.

Hemangiosarcoma (HSA) is a malignant neoplasm that occurs in humans and canines with a poor prognosis owing to metastatic spread, despite effective treatment. The frequency of spontaneous HSA development is higher in canines than in humans. Therefore, canine HSA is a useful model of intractable human disease, which requires early detection and an effective therapeutic strategy. A high frequency of the p110α phosphatidylinositol‑4,5‑bisphosphate 3‑kinase catalytic subunit alpha (PIK3CA) mutations is detected in a comprehensive genome‑wide analysis of canine cases of HSA. The present cloned the full‑length cDNA of canine PIK3CA and identified a mutation in codon 1047 from canine cases of HSA and cell lines that were established from these. The enforced expression of the 1047th histidine residue (H1047)R or L mutants of canine PIK3CA in HeLa cells enhanced epidermal growth factor receptor (EGFR) signaling via Akt phosphorylation. PIK3CA mutant canine HSA cell lines exhibited the hyperphosphorylation of Akt upon EGF stimulation as well. Alpelisib, a molecular targeted drug against PIK3CA activating mutations, exerted a significant antitumor effect in canine PIK3CA‑mutated HSA cell lines. By contrast, it had no significant effect on canine mammary gland tumor cell lines harboring PIK3CA mutations. On the whole, the findings of the present study suggest that alpelisib may be highly effective against PIK3CA mutations that occur frequently in canine HSA.

Isocitrate dehydrogenase 1 (IDH1) mutations are common in gliomas, acute myeloid leukemia, and chondrosarcoma. The mutation 'hotspot' is a single arginine residue, R132. The R132H mutant of IDH1 produces the 2-hydroxyglutarate (2-HG) carcinogen from α-ketoglutarate (α-KG). The reduction of α-KG induces the accumulation of hypoxia-inducible factor-1α subunit (HIF-1α) in the cytosol, which is a predisposing factor for carcinogenesis. R132H is the most common IDH1 mutation in humans, but mutations at the R132 residue can also occur in tumor tissues of dogs. The current study reported the discovery of a novel Tyr208Cys (Y208C) mutation in canine IDH1 (cIDH1), which was isolated from 2 of 45 canine chondrosarcoma cases. As the genomic DNA isolated from chondrosarcoma tissue was mutated, but that isolated from blood was not, Y208C mutations were considered to be spontaneous somatic mutations. The isocitrate dehydrogenase activity of the Y208C mutant was attenuated compared with that of wild-type (WT) cIDH1, but the attenuation of Y208C was less intense than that of the R132H mutation. The induction of HIF-1α response element activity and cell retention of HIF-1α were not increased by Y208C overexpression. In silico and cell biological analysis of IDH1 dimerization revealed that the Y208C mutation, but not the R132H mutation, attenuated binding activity with WT cIDH1. These data suggested that the attenuation of dimerization by the Y208C mutation may cause tumorigenesis through different mechanisms other than via 2-HG production by the IDH1 R132 mutation.

In order to elucidate the relationship between migration period and immunity related to susceptibility, we conducted research on Black-headed gulls (Chroicocephalus ridibundus). We captured 260 gulls and collected their peripheral blood. Their leukocyte (WBC) count, percentages of heterophils (Het) and lymphocytes (Lym), heterophil and lymphocyte ratio (H/L ratio), and CD4 and CD8α expression levels (CD4 and CD8α, respectively) were quantitatively analyzed over three migration periods (Autumn migration, Wintering, Spring migration). In Adult gulls, WBC counts and CD4 levels significantly increased. Moreover, the Het and H/L ratio decreased from the Autumn migration to Wintering. Conversely, only WBC counts and CD4 levels measurements significantly decreased from Wintering to Spring migration (P<0.05). The tested parameters of the Tokyo-bay population show a greater significant difference than the measurements of immunity of the Mikawa-bay population. This study suggests that the migratory period has a negative effect on an aspect of the immune system. Including the period-difference in the immune systems in the local population, it is necessary to investigate the relationship between the ecology of migratory birds and their immunity.

Feline histiocytic sarcoma (HS) is an aggressive and uncommon tumor originating from dendritic cells/macrophages. Here, a feline HS cell line, FHS-1, was established from a case of feline HS and characterized. Immunohistochemically, FHS-1 cells were positive for vimentin and Iba-1, and negative for MHC class II and CD163. FHS-1 cells were positive for α-naphthyl butyrate esterase staining, which was clearly inhibited by sodium fluoride. FHS-1 cells had phagocytic and antigen uptake/processing activities. Moreover, FHS-1 cells were tested for susceptibility to feline infectious peritonitis virus (FIPV) strain 79-1146 however, this cell line was not susceptible to this viral strain. Although FHS-1 cells lost the expression of MHC class II and CD163, our findings indicate that FHS-1 is a feline HS cell line that retains functional properties of dendritic cells/macrophages in terms of phagocytic and antigen uptake/processing activities. While FHS-1 cells are not suitable for in vitro study of FIP using strain 79-1146, they may be applicable for studies aimed at developing new diagnostic and therapeutic strategies for feline HS.

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.

Feline coronavirus (FCoV) causes the fatal disease feline infectious peritonitis, which is currently incurable by drug treatment, and no effective vaccines are available. Cyclosporin A (CsA), a cyclophilin (Cyp) inhibitor, inhibits the replication of FCoV in vitro and in vivo as well as the replication of human and animal coronaviruses. However, the mechanism underlying the regulation of coronavirus replication by CsA is unknown. In this study, we analysed the role of Cyps in FCoV replication using knockdown and knockout cells specific to Cyps. Inhibition of CypA and CypB reduced FCoV replication, with replication in knockout cells being much less than that in knockdown cells. Furthermore, the proteins expressed by CypA and CypB harbouring mutations in their respective predicted peptidyl-prolyl cis-transisomerase active sites, which also alter the affinities between Cyps and CsA, inhibited FCoV replication. These findings indicate that the peptidyl-prolyl cistransisomerase active sites of Cyps might be required for FCoV replication.

Although feline coronavirus (FCoV) causes feline infectious peritonitis (FIP), which is a fatal infectious disease, there are no effective therapeutic medicines or vaccines. Previously, in vitro studies have shown that cyclosporin (CsA) and FK506 inhibit virus replication in diverse coronaviruses. CsA and FK506 are targets of clinically relevant immunosuppressive drugs and bind to cellular cyclophilins (Cyps) or FK506 binding proteins (FKBPs), respectively. Both Cyp and FKBP have peptidyl-prolyl cis-trans isomerase (PPlase) activity. However, protein interacting with NIMA (Finn a member of the parvulin subfamily of PPlases that differs from Cyps and FKBPs, is essential for various signaling pathways. Here we demonstrated that genetic silencing or knockout of Pin1 resulted in decreased FCoV replication in vitro. Dipentamethylene thiuram monosulfide, a specific inhibitor of Pin1, inhibited FCoV replication. These data indicate that Pin1 modulates FCoV propagation. (C) 2015 Elsevier B.V. All rights reserved.

OBJECTIVE To determine whether thioredoxin (TRX)-1 can be used as a valid biomarker for oxidative stress in dogs. ANIMALS AND SAMPLES 10 Beagles and Madin-Darby canine kidney cells. PROCEDURES Madin-Darby canine kidney cells were used to verify antigen cross-reactivity between human and canine anti–TRX-antibodies. Dogs were assigned to receive 21% or 100% O2 (5 dogs/group) via an artificial respirator during a 3-hour period of isoflurane anesthesia (starting at 0 hours). Blood and urine samples were collected before (baseline) and at 6, 12, 24, and 48 hours after commencement of inhalation anesthesia. Concentrations of TRX-1 and 8-hydroxy-2’-deoxyguanosine (8-OHdG) in plasma and urine samples were analyzed urine concentrations were reported as ratios against urine creatinine concentration. RESULTS Canine TRX-1 was recognized by monoclonal human anti-TRX-1 antibodies (clones of adult T-cell leukemia-derived factor [ADF]-11 and ADF21) by western blot analysis. Results of an ELISA indicated that plasma TRX-1 concentration and urine TRX-1-to-creatinine concentration ratio increased rapidly after the 3-hour period of hyperoxia with maximal peaks at 12 and 6 hours, respectively. Urine 8-OHdG-to-creatinine concentration ratio also increased significantly after hyperoxia induction. However, unlike the rapid increase in urine TRX-1-to-creatinine concentration ratio, maximal urine 8-OHdG-to-creatinine concentration ratio was attained at 48 hours after hyperoxia induction. These variables remained unchanged from baseline in the control group. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that human anti-TRX monoclonal antibodies cross-reacted with canine TRX, and plasma TRX-1 concentrations were rapidly increased in dogs following an oxidative stress challenge. Thus, TRX may be a valuable clinical biomarker for detecting oxidative stress more rapidly than 8-OHdG in dogs.

Background: Feline infectious peritonitis is a fatal disease of cats caused by infection with feline coronavirus (FCoV). For detecting or genotyping of FCoV, some RT-PCR plus nested PCR techniques have been reported previously. However, referring to the whole genome sequences (WGSs) registered at NCBI, there are no detection methods that can tolerate the genetic diversity among FCoV population. In addition, the quasispecies nature of FCoV, which consists of heterogeneous variants, has been also demonstrated; thus, a universal method for heteropopulations of FCoV variants in clinical specimens is desirable. Results: To develop an RT-PCR method for detection and genotyping of FCoV, we performed comparative genome analysis using WGSs of 32 FCoV, 7 CCoV and 5 TGEV strains obtained from NCBI. As the PCR target, we focused on the nsp14 coding region, which is highly conserved and phylogenetically informative, and developed a PCR method targeting nsp14 partial sequences. Among 103 ascites, 45 pleural effusion and 214 blood specimens from clinically ill cats, we could detect FCoV in 55 (53.4%), 14 (31.1%) and 19 (8.9%) specimens using the present method. Direct sequencing of PCR products and phylogenetic analysis allowed discrimination between type I-and II-FCoV serotypes. Our nsp14 amino acid sequence typing (nsp14 aa ST) showed that the FCoV clone with sequence type (ST) 42, which was the most predominant genotype of WGS strains, was prevalent in domestic cats in Japan. Conclusions: Our nsp14 PCR scheme will contribute to virus detection, epidemiology and ecology of FCoV strains.

We report a complete genome sequence of the methicillin-resistant Staphylococcus schleiferi strain TSCC54, isolated from the skin of a dog in Tokyo, Japan.

Coronaviruses infect a variety of mammalian and avian species and cause serious diseases in humans, cats, mice, and birds in the form of severe acute respiratory syndrome (SARS), feline infectious peritonitis (FIP), mouse hepatitis, and avian infectious bronchitis, respectively. No effective vaccine or treatment has been developed for SARS-coronavirus or FIP virus, both of which cause lethal diseases. It has been reported that a cyclophilin inhibitor, cyclosporin A (CsA), could inhibit the replication of coronaviruses. CsA is a well-known immunosuppressive drug that binds to cellular cyclophilins to inhibit calcineurin, a calcium-calmodulin-activated serine/threonine-specific phosphatase. The inhibition of calcineurin blocks the translocation of nuclear factor of activated T cells from the cytosol into the nucleus, thus preventing the transcription of genes encoding cytokines such as interleukin-2. Cyclophilins are peptidyl-prolyl isomerases with physiological functions that have been described for many years to include chaperone and foldase activities. Also, many viruses require cyclophilins for replication; these include human immunodeficiency virus, vesicular stomatitis virus, and hepatitis C virus. However, the molecular mechanisms leading to the suppression of viral replication differ for different viruses. This review describes the suppressive effects of CsA on coronavirus replication.

In this study, the Japanese strain of type I feline infectious peritonitis virus (FIPV), C3663, was found to have a large deletion of 735 bp within the gene encoding the spike (S) protein, with a deduced loss of 245 aa of the N-terminal region of the S protein. This deletion is similar to that observed in porcine respiratory coronavirus (PRCoV) when compared to transmissible gastroenteritis virus, which correlates with reduced virulence. By analogy to PRCoV, we expected that the pathogenicity of C3663 may be attenuated in cats. However, two of four cats inoculated with C3663 died of FIP, and a third C3663-inoculated cat showed FIP lesions at 91 days after challenge. These results indicate that the 5'-terminal region of the S gene is not essential for the development of FIP.

We determined the population genetic structures of feline and canine Staphylococcus aureus strains in Japan by multilocus sequence typing (MLST). Ecological analyses suggested that multiple feline-related S. aureus clones, including ST133, naturally occur as commensals and can cause endogenous infections in felines. In contrast, S. aureus populations do not likely include any clone that exhibits tropism in domestic dogs. Even if S. aureus infections occur in dogs, the pathologies are likely exogenous infections.

The feline infectious peritonitis virus (FIPV) is a member of the feline coronavirus family that causes FIP, which is incurable and fatal in cats. Cyclosporin A (CsA), an immunosuppressive agent that targets the nuclear factor pathway of activated T-cells (NF-AT) to bind cellular cyclophilins (CyP), dose-dependently inhibited FIPV replication in vitro. FK506 (an immunosuppressor of the pathway that binds cellular FK506-binding protein (FKBP) but not CyP) did not affect FIPV replication. Neither cell growth nor viability changed in the presence of either CsA or FK506, and these factors did not affect the NF-AT pathway in fcwf-4 cells. Therefore, CsA does not seem to exert inhibitory effects via the NF-AT pathway. In conclusion, CsA inhibited FIPV replication in vitro and further studies are needed to verify the practical value of CsA as an anti-FIPV treatment in vivo.

In veterinary medicine, coagulase-positive staphylococci (CoPS) other than Staphylococcus aureus have frequently been misidentified as being S. aureus strains, as they have several phenotypic traits in common. There has been no reliable method to distinguish among CoPS species in veterinary clinical laboratories. In the present study, we sequenced the thermonuclease (nuc) genes of staphylococcal species and devised a multiplex-PCR (M-PCR) method for species identification of CoPS by targeting the nuc gene locus. To evaluate sensitivity and specificity, we used this M-PCR method on 374 staphylococcal strains that had been previously identified to the species level by an hsp60 sequencing approach. We could successfully distinguish between S. aureus, S. hyicus, S. schleiferi, S. intermedius, S. pseudintermedius, and S. delphini groups A and B. The present method was both sensitive (99.8%) and specific (100%). Our M-PCR assay will allow the routine species identification of CoPS isolates from various animal species for clinical veterinary diagnosis.

To reclassify phenotypically identified Staphylococcus intermedius strains, which might include true S. intermedius strains and novel species such as Staphylococcus pseudintermedius and Staphylococcus delphini, we analyzed molecular phylogenies and phenotypic characteristics of 117 S. intermedius group (SIG) strains tentatively identified as being S. intermedius by the Rapid ID32 Staph assay. From phylogenetic analyses of sodA and hsp60 sequences, the SIG strains were divided into three clusters, which belonged to S. pseudintermedius LMG 22219(T), S. intermedius ATCC 29663 T, and S. delphini LMG 22190(T). All the SIG strains from dogs, cats, and humans were identified as being S. pseudintermedius. The wild pigeon strains, except one, were identified as being S. intermedius, and strains from all domestic pigeons, one wild pigeon, horses, and a mink were identified as being S. delphini. In addition, a phylogenetic analysis of nuc genes revealed that S, delphini strains were divided into two clusters: one was the cluster (S. delphini group A) that belonged to S. delphini LMG 22190T, and the other was the cluster (S. delphini group B) that was more related to S. pseudintermedius LMG 22219T than S. delphini LMG 22190T. The DNA-DNA hybridization results showed that S. delphini group B strains were distinguished from S. delphini group A, S. intermedius, and S. pseudintermedius strains. S. intermedius is distinguishable from S. pseudintermedius or S. delphini by positive arginine dihydrolase and acid production from beta-gentiobiose and D-mannitol. However, phenotypical characteristics to differentiate S. delphini group A, S. delphini group B, and S. pseudintermedius were not found. In conclusion, SIG strains were reclassified into four clusters with three established and one probably novel species.

We surveyed methicillin-resistant coagulase-positive staphylococcus (MRCPS) strains from 57 (26 inpatient and 31 outpatient) dogs and 20 veterinary staff in a veterinary teaching hospital. From the staff, three MRCPS strains were isolated, and two were methicillin-resistant Staphylococcus aureus (MRSA). In contrast, 18 MRCPS strains were detected in both inpatient (12 of 26 [46.2%]) and outpatient (6 of 31 [19.4%]) dogs. Among them, only one strain was MRSA. Using direct sequencing of sodA and hsp60 genes, the 18 MRCPS strains other than MRSA from a staff and 17 dogs, were finally identified as Staphylococcus pseudintermedius, a novel species of Staphylococcus from a cat. All of the methicillin-resistant S. pseudintermedius (MRSP) strains were multidrug resistant to erythromycin, clindamycin, trimethoprim-sulfamethoxazole, and levofloxacin. Most of the MRSP strains showed high-level resistance to oxacillin (&gt;= 128 mu g/ml, 15 of 18 [83.3%]), and 10 of 15 (66.7%) high-level oxacillin-resistant MRSP strains carried type III SCCmec. DNA fingerprinting of MRSP strains by pulsed-field gel electrophoresis yielded eight clusters: clone A with four subtypes, clone B with four subtypes, clone C with three subtypes, and five other different single clones. MRSP strains from the staff and some inpatient and outpatient dogs shared three major clones (clones A, B, and Q, but the strains of the other five different clusters were distributed independently among inpatient or outpatient dogs. This genetic diversity suggested that the MRSP strains were not only acquired in this veterinary teaching hospital but also acquired in primary veterinary clinics in the community. To our knowledge, this is the first report of MRSP in dogs and humans in a veterinary institution.

Plasma metabolite and immunoreactive insulin concentrations and activities of enzymes related to energy metabolism in peripheral leukocytes were measured in growing Holstein calves. A ratio of girth of abdomen divided by girth of thorax (A/T ratio) of calves was significantly elevated after weaning, and the A/T ratio maybe a good indicator to evaluate rumen development. Plasma glucose and free fatty acid concentrations were changed in calves accompanying change in feeding. Activities of lactate dehydrogenase with pyruvate as substrate (LDH-P) and hexokinase (HK) in cytosolic fractions of peripheral leukocytes decreased significantly after weaning the calves reflecting the change of energy source from milk replacer with high percentages of fat and glucose and lactose as absorbable carbohydrate to pelleted feed containing starch as less absorbable carbohydrate and roughage. Some peripheral leukocyte enzymes such as LDH and HK may be good indicators to evaluate changes in energy metabolism of growing calves. (c) 2005 Elsevier Ltd. All rights reserved.