Strategies of Newcastle disease (ND) vaccination were demonstrated in a commercial ostrich farm in Japan. Three of 13 seven-month-old ostriches kept in a pen were vaccinated with a live ND vaccine by eye dropping for the 1st and 2nd vaccinations and spraying for the 3rd to 5th vaccinations. Antibodies against ND virus (NDV) were detected in all of the unvaccinated ostriches by virus neutralization test. At 2.5 months post final vaccination, 2 ostriches introduced into the pen raised antibodies against NDV. These data indicate that NDV may be transmitted from vaccinated to unvaccinated ostriches in the flock and that the virus may be sustained for a certain period in the flock. These data may be helpful for ND vaccination management in ostrich farms.

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Porphyromonas gulae, a gram-negative black-pigmented anaerobe, is a pathogen for periodontitis in dogs. An approximately 41-kDa fimbrial subunit protein (FimA) encoded by fimA is regarded as associated with periodontitis. In the present study, the fimA genes of 17 P. gulae strains were sequenced, and classified into two major types. The generation of phylogenetic trees based on the deduced amino acid sequence of FimA of P. gulae strains along with sequences from several strains of Porphyromonas gingivalis, a major cause of human periodontitis, revealed that the two types of FimA (types A and B) of P. gulae were similar to type I FimA and types II and III FimA of P. gingivalis, respectively. A PCR system for classification was established based on differences in the nucleotide sequences of the fimA genes. Analysis of 115 P. gulae-positive oral swab specimens from dogs revealed that 42.6%, 22.6%, and 26.1% of them contained type A, type B, and both type A and B fimA genes, respectively. Experiments with a mouse abscess model demonstrated that the strains with type B fimA caused significantly greater systemic inflammation than those with type A. These results suggest that the FimA proteins of P. gulae are diverse with two major types and that strains with type B fimA could be more virulent.

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The objective of the present study was to characterize Erysipelothrix sp. strains from recent erysipelas outbreaks in Japan. Eighty-three (100%) strains were identified as E. rhusiopathiae, based on serotyping and spaA PCR. Fifty (60.3%), 5 (6.0%), and 28 (33.7%) strains were isolated from animals with acute, subacute and chronic outbreaks, respectively, of which 79 (95.2%), 1 (1.2%), and 3 (3.6%) belonged to serotypes 1a, 2a, and untypeable, respectively. Fifteen strains (including 3, 2, and 10 from acute, subacute, and chronic cases, respectively) were sensitive to acriflavine, and showed high levels of virulence in mice; of which strains from acute cases, and from subacute and chronic cases killed 100%, and 80 to 100% mice, respectively at challenge doses of 10² CFU per mouse. Based on sequence analysis of a 432-bp hypervariable region in spaA gene, 83 strains could be divided into 3 groups: (i) group 1 (3 strains of serotype 1a) had Ala-195 and Ile-203; (ii) group 2 (76 strains of serotype 1a and 3 of untypeable) had Asp-195 and Met-203; and (iii) group 3 (one strain of serotype 2a) had Asn-195 and Ile-203. The results of the present study suggest that the serotype 1a strains belonging to the group 2 might be widespread in pig populations in Japan.

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Leptospira interrogans serovar Manilae strain UP-MMC was inoculated into miniature pigs to assess its pathogenicity. Leptospires were recovered from the whole blood, kidneys, and livers in the acute phase without showing any clinical signs. Under immunosuppressive conditions by dexamethasone, leptospires were recovered from the kidneys and their genes were detected from the urine in the chronic phase. These results indicate that leptospires persisted in the kidneys until the chronic phase, and excretion of leptospires in the urine was enhanced under immunosuppressive conditions, resulting in horizontal transmission among pigs on farms.

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In this study, we report hematocrit and plasma chemistry values for adult captive collared scops owls (Otus lettia) and crested serpent eagles (Spilornis cheela hoya). In particular, we address the gender-specific differences within these values. We measured hematocrit (HCT) and plasma chemistry values for uric acid (UA), plasma urea nitrogen (BUN), total protein (TP), albumin (ALB), glucose (GLU), cholesterol (CHO), triglyceride (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), total bilirubin (TBIL), creatine (CRE), creatine phosphokinase (CPK), amylase (AMY), calcium (CA), ionic phosphorous (IP) and sodium (NA), potassium (K) and chloride ions (CL) in 37 adult captive collared scops owls and 39 adult captive crested serpent eagles. Significant differences between the sexes were found for UA, GLU and CPK in the collared scope owls. UA and GLU concentrations were significantly higher (P<0.01 and P<0.05) among males than females, while the CPK concentration was significantly lower (P<0.05) in males. There were no significant differences in of all of the measured parameters between male and female eagles. These finding suggested that HCT and plasma chemistry values of raptors vary individually according to species and sex. Our results provide the 1st available reference data for ranges of plasma values in adult captive collared scops owls and crested serpent eagles, making them a potentially useful complementary diagnostic tool for veterinary care of individuals for both species in captivity.

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To detect allergen-specific IgE in dogs with allergic diseases, we developed a recombinant canine high affinity IgE receptor α chain (FcεRIα)-based IgE detection system. Using the recombinant protein of canine FcεRIα expressed by an Escherichia coli expression system, we could detect house dust mite (Dermatophagoides farinae) allergen-specific IgE in sera from dogs naturally and experimentally sensitized to this allergen with ELISA and western blotting. The IgE binding activity of recombinant canine FcεRIα on ELISA was impaired by heat treatment of these sera. The specificity of this recombinant canine FcεRIα-based IgE detection system was confirmed by inhibition assays with canine IgE. The recombinant canine FcεRIα-based IgE detection system established in this study offers an alternative tool to measure allergen-specific IgE in dogs.

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A cat was presented with severe progressive anemia despite marked erythroblastosis. The cat was negative for feline leukemia virus antigen and feline immunodeficiency virus antibody. Bone marrow cytology revealed an excess of erythroid cells with a predominance of prorubricytes and basophilic rubricytes. No response to immunosuppressive therapy was obtained, and a tentative diagnosis of myelodysplastic syndrome was made. The cat showed a partial response to low-dose cytarabine (20 mg/m² subcutaneously q24) but died 51 days after the 1st admission. Histopathological examination revealed fibrosis in the bone marrow and marked infiltration of erythroid cells into other organs.

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A full-length cDNA sequence of canine L-type amino acid transporter 1 (Lat1) was determined from a canine brain. The sequence was 1828 bp long and was predicted to encode 485 amino acid polypeptides. The deduced amino acid sequence of canine Lat1 showed 93.2% and 91.1% similarities to those of humans and rats, respectively. Northern blot analysis detected Lat1 expression in the cerebellum at 4 kb, and Western blot analysis showed a single band at 40 kDa. RT-PCR analysis revealed a distinct expression of Lat1 in the pancreas and testis in addition to the cerebrum and cerebellum. Notably, Lat1 expression was observed in the tissues of thyroid cancer, melanoma and hemangiopericytoma. Although the cancer samples examined were not enough, Lat1 may serve as a useful biomarker of cancer cells in veterinary clinic.

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To evaluate the relationship among immune status and increased morbidity and mortality, peripheral blood lymphocytes (CD3⁺, CD4⁺, CD8⁺ and CD21⁺ cells) from 32 healthy dogs over 8 years of age were analyzed. Twenty-five of the 32 dogs were followed-up for 3 years after the analysis; and 14 dogs were found to be diseased, and nine dogs died. There was no notable difference between the ages of the dogs that died compared with the ones that survived. The relative percentage of CD4⁺ and the CD4⁺:CD8⁺ ratio decreased notably in dogs falling ill compared with healthy dogs. The relative percentage of CD3⁺ lymphocytes showed a notable decrease in dogs that died within 3 years in comparison with dogs that survived. In a discriminant analysis of morbidity and mortality, most patients were correctly classified as diseased or not and surviving or dead, respectively. These results indicate that the immunophenotypes of peripheral blood lymphocytes in older dogs offer promise as parameters for evaluating mortality and morbidity.

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In this study, we demonstrated growth curves and reference values for hematological and serum biochemical parameters of Microminipigs, the world smallest experimental minipigs. In both male and female animals, the body weights (BWs) at 3 and 6 months of age were <5 kg and <10 kg, respectively, and growth curve revealed almost plateau (approximately 20 kg BW) after 18 months of age. Major hematological and serum biochemical parameters showed no gender differences and the values were very similar to those in Göttingen and Yukatan minipigs. The values obtained in this study can serve as fundamental reference, and thereby facilitate the use of Microminipig in life science research.

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It has been speculated that populations of aspermic Fasciola flukes in Korea and Japan have a close phylogenetic relationship. To evaluate this, we analyzed 33 Korean aspermic Fasciola flukes on the basis of nuclear ribosomal internal transcribed spacer 1 (ITS1) and mitochondrial NADH dehydrogenase subunit 1 (nad1) and cytochrome c oxidase 1 (cox1) sequences. Fh, Fg, and Fh/Fg types were detected in the ITS1 region and displayed the fragment patterns of F. hepatica, F. gigantica, and both species, respectively by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Additionally, three concatenated haplotypes of nad1 and cox1(nad1/cox1) were detected, and 2 of these, Kor1/Kor1 (Fsp1/Fsp1) haplotype and Kor2a/Kor2 (Fsp2/Fsp2) haplotype, were shared by Korean and Japanese aspermic flukes. The Fst value (0.019), calculated using the concatenated sequences, indicated that Korean and Japanese aspermic Fasciola populations were genetically undifferentiated. Interestingly, a combination of the Fh/Fg type and Kor1/Kor1 haplotype was found at the highest frequency in Korean aspermic flukes, whereas the Fg type and Fsp2/Fsp2 haplotype combination was found at a conspicuously high frequency in Japanese aspermic flukes. This indicates that a founder effect caused by the introduction of infected hosts may have played a key role in the introduction of aspermic Fasciola flukes from Korea into Japan.

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The goal of this research was to identify mechanisms responsible for the spongy change induced in rats after repeated hexachlorophene (HCP) or cuprizone (CPZ) dosing. Rats were dosed with 35 mg/kg HCP for 5 days followed by drug withdrawal for 7 days suffered spongy changes to the white matter of the cerebrum, cerebellum, medulla oblongata, and spinal cord that were accompanied by degeneration of oligodendroglia. The severity of both lesions increased prominently on day 5; however, the spongy change decreased and degeneration of oligodendroglia reversed on day 12 (7 days after dosing ceased). On day 12, cerebral cortex oligodendroglia were stained strongly by anti-CNPase. Other rats were fed for 8 days with powdered chow containing 1% (w/w) CPZ, which was then withdrawn for 16 days. The rats exhibited the spongy change in the white matter of the cerebrum and cerebellum as well as oligodendroglial cell death from day 3. The severity of both lesions increased prominently on day 8. Cerebral cortex oligodendroglia were stained strongly by anti-CNPase on days 3 to 8 and decreased to the control levels by day 24 (16 days after dosing ceased). Electron microscopy revealed that oligodendroglia frequently displayed apoptotic morphology. These findings suggest that CNPase expression was induced in the course of restoration following HCP-induced insults to oligodendroglia and the myelin sheath, and in the course of demyelination by CPZ-induced damage to oligodendroglia. However, the role of CNPase on both courses is unclear.

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A 5-day-old Huacaya alpaca cria (Vicugna pacos) was euthanized due to deteriorating health. At birth, the cria had ophthalmologic abnormalities, but had appropriate mentation. At 2 days of age, the cria gradually stopped suckling and began to circle. At 5 days old, the owner elected euthanasia due to declining clinical condition. Grossly, the right iris had a scalloped pupillary margin, and the right olfactory bulb was malformed. Histopathology revealed persistent hyperplastic primary vitreous bilaterally and iridal abnormalities, as well as aplasia of the olfactory ventricle, olfactory tract, and olfactory foramen on the right side.

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Excessive proliferation of mesangial cells is observed in various types of glomerular disease including glomerulonephritis (GN), which is progressive in nature and eventually results in end-stage renal disease (ESRD). Vitamin K₁ 2,3-epoxide reductase (VKOR) and the vitamin K-dependent growth arrest-specific gene 6/Axl pathway play a key role in mesangial cell proliferation in GN. In the present study, we indicate the potential of a VKOR inhibitor, 3-acetyl-5-methyltetronic acid (AMT), to prevent the proliferation of glomerular mesangial cells and suppress the progression of GN. AMT was administered intravenously to rats once daily for 12 days and a mouse anti-Thy1 monoclonal antibody was injected intravenously after the AMT treatment on Day 6. Creatinine clearance (CCr) significantly increased and the albumin-to-creatinine ratio (ACR) significantly decreased in the AMT-treated group of the Thy-1 GN rats. In addition, glomerular and tubular damage was improved histopathologically in the AMT-treated group. AMT did not affect blood coagulation due to its unique pharmacokinetic properties. The concentration of AMT reached the IC₅₀ for VKOR in kidney, but not in liver. A novel VKOR inhibitor, AMT, reduced renal mesangial cell proliferation and could be a supportive treatment for GN.

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The anticholinergic effects of 7 benzodiazepines, bromazepam, camazepam, chlordiazepoxide, diazepam, lorazepam, medazepam and triazolam, were compared by examining their inhibitory effects on the acetylcholine receptor-operated potassium current (I_K.ACh) in guinea-pig atrial myocytes. All of these benzodiazepines (0.3–300 μM) inhibited carbachol (1 μM)-induced I_K.ACh in a concentration-dependent manner. The ascending order of IC₅₀ values for carbachol-induced I_K.ACh was as follows; medazepam, diazepam, camazepam, triazolam, bromazepam, lorazepam and chlordiazepoxide (>300 μM). The compounds, except for bromazepam, also inhibited I_K.ACh activated by an intracellular loading of 100 μM guanosine 5’-[γ-thio]triphosphate (GTPγS) in a concentration-dependent manner. The ascending order of IC₅₀ values for GTPγS-activated I_K.ACh was as follows; medazepam, diazepam, camazepam, lorazepam, triazolam chlordiazepoxide (>300 μM) and bromazepam (>300 μM). To clarify the molecular mechanism of the inhibition, IC₅₀ ratio, the ratio of IC₅₀ for GTPγS-activated I_K.ACh to carbachol-induced I_K.ACh, was calculated. The IC₅₀ ratio for camazepam, diazepam, lorazepam, medazepam and triazolam was close to unity, while it for chlordiazepoxide could not be calculated. These compounds would act on the GTP binding protein and/or potassium channel to achieve the anticholinergic effects in atrial myocytes. In contrast, since the IC₅₀ ratio for bromazepam is presumably much higher than unity judging from the IC₅₀ values (104.0 ± 30.0 μM for carbachol-induced I_K.ACh and >300 μM for GTPγS-activated I_K.ACh), it would act on the muscarinic receptor. In summary, benzodiazepines had the anticholinergic effects on atrial myocytes through inhibiting I_K.ACh by different molecular mechanisms.

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Although there has been extensive research on plasma amino acid profiles of mammals, there is currently a lack of information on seasonal differences in the concentrations of plasma amino acids specifically in cetaceans. The present study examined the response of the plasma amino acids to seasonal changes in the culture environment after controlling for the effect of sex and age. Significant seasonal changes in plasma carnosine (P=0.012), cystine (P=0.0014), isoleucine (P=0.0042), methionine (P=0.002), ornithine (P=0.0096), and taurine (P=0.032) were observed. These amino acids were mainly related to capacity for exercise, ammonia detoxification, thermoregulation, and osmoregulation. We proposed that optimizing plasma amino acids levels by supplementation of amino acids should be of considerable benefit for aquarium-maintained bottlenose dolphins. This study constitutes a first step towards improving our understanding of the metabolism of aquarium-maintained bottlenose dolphins. We also revealed that the ratio of tryptophan to large neutral amino acids significantly declined (P=0.0076), suggesting reduction in serotonin synthesis in winter and autumn. Although further studies are needed, this finding implied that bottlenose dolphins could produce behavioral changes seasonally by the alteration of serotonin activity. To better understand the metabolic machinery for amino acids that facilitate the adaptation of marine mammals to their environments, it is essential to continue monitoring of and further investigations into relationships between plasma amino acids and specific environmental factors.

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In this study, we evaluated the applicability of cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR for the direct detection and identification of Campylobacter jejuni, C. coli and C. fetus from stool specimens of patients with gastroenteritis in comparison to culture methods. A total of 711 stool specimens were examined for the isolation or detection of campylobacters by using Skirrow's selective agar culture plates, a filtration method and the multiplex PCR assay. Forty-one and 36 C. jejuni strains were isolated by culture and filtration methods, respectively. In addition, 2 and 3 C. coli strains were isolated by Skirrow and the filtration methods, respectively. However, when the multiplex PCR was employed, the cdtB genes of C. jejuni and C. coli were detected in 45 and 4 stool samples, respectively, and 9 C. jejuni PCR-positive samples by multiplex PCR were negative by culture method. Sequence analysis of the PCR products obtained from 8 stool specimens from which campylobacters were not isolated by culture method but the sequences exactly matched with that of the cdtB gene of C. jejuni strain 81–176. None of the remaining stool samples which were culture negative for campylobacters produced any amplicon. Stool samples were defined as Campylobacter-positive if detected by any method. The sensitivity of the multiplex PCR was 83%, which was higher than Skirrow (74%) and filtration method (66%). These data indicate that cdtB gene-based multiplex PCR is a rapid and more sensitive method to identify the most important species of Campylobacter for human diseases. (248)

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Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) are a promising source of cells for bone tissue engineering. Matrigel is a basement membrane extract containing multiple extracellular components. This mixture may promote the osteogenic differentiation of MSCs and provide a more appropriate microenvironment for transplanted cells. Here, we investigated the effect of Matrigel on the osteogenic potential of Ad-MSCs. Canine Ad-MSCs were cultured in 2D and 3D matrices and implanted into subcutaneous pouches of dogs either with or without Matrigel. Culture mineralization, cell adhesion efficiency, cell proliferation, osteoid matrix production and alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase activities were quantified and compared. Ad-MSCs grown in 2D cultures with Matrigel showed higher levels of calcium deposition and ALP activity than those grown in the absence of Matrigel under osteogenic conditions. In 3D cultures, the cells cultivated with Matrigel showed greater attachment, proliferation and osteogenic differentiation than those grown without Matrigel. In vivo, Ad-MSCs implanted with Matrigel showed higher osteogenic potential than those without Matrigel. In conclusion, these data suggest that the use of Matrigel can increase the osteogenic potential of canine Ad-MSCs.

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A 12-year-old neutered male shih tzu developed progressive pelvic limb paraparesis. Computed tomography showed a radiolucent mass lesion in the spinal canal at the left side of the 11th thoracic vertebra. The mass was not enhanced by intravenous contrast medium injection. It was hyperintense on both T1- and T2-weighted magnetic resonance images. The signal intensity of the mass was decreased with a fat suppression technique, indicating a fatty origin. After removal of the mass via T11–T12 hemilaminectomy, chronic panniculitis was confirmed by histopathological examination. This case demonstrates the utility of computed tomography and magnetic resonance imaging for the diagnosis of spinal canal pyogranulomatous inflammation.

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We examined the proliferation capacity and neuronal differentiation potency of canine bone marrow stromal cells (BMSCs). In addition, the microstructures of neuron-like cells after neuronal differentiation were observed under a scanning electron microscope. Canine BMSCs grew to confluency at 10.0 ± 2.5 days, and 3.8 ± 2.1 × 10⁶ BMSCs were collected in one passage. Approximately 65% of canine BMSCs changed to neuron-like morphology after neuronal differentiation, and nearly all neuron-like cells stained positive against neuron-specific enolase. In addition, microstructures such as the cellular organelles, filaments and growth cones of these cells bore a close resemblance to those of the original mature neurons. These results suggested that canine BMSCs might be capable of differentiating into neurons.

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The purpose of this study was to observe the changes in body temperature before parturition using a wireless temperature monitoring device (WTMD) and to evaluate the usefulness of body temperature measurements using a digital rectal thermometer (DRT) and a microchip transponder thermometry device (MTTD) for predicting parturition in mares. The body temperatures using a WTMD at 0 hr and -1 hr were significantly different from those at the same time on Days 1–5 (P<0.01). The temperature differences between the morning of Day 0 and at -3 hr, -2 hr, -1 hr and 0 hr using the DRT and MTTD showed a significant drop compared with the temperature differences between the morning and evening of Days 1–5 (P<0.05). Furthermore, when the cutoff value of the temperature differences between the morning and other times was set to ≤0, the sensitivities of the DRT and MTTD in the evening of Day 0 and at -3 hr were 43% and 100% and 71% and 86%, respectively. The results suggested that monitoring the body temperature differences between morning and within 3 hr before the time of parturition is a valuable method for predicting parturition in mares. Conversely, this method would be more useful in predicting parturition when used in combination with other observations such as the mammary gland size and waxing of the teat ends because it has nearly a 20% probability of false-positive results prior to the day of parturition.

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We have developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detecting H7N7 equine influenza virus (EIV). The detection limit of the RT-LAMP assay was a virus dilution of 10^-4, which was 10 times more sensitive than that of Espline Influenza A&B-N and the same as that of reverse transcription polymerase chain reaction. The RT-LAMP assay specifically amplified H7N7 EIV strains but did not amplify several pathogens related to equine respiratory disease including H3N8 EIV strains. Because it provides ease of manipulation, the RT-LAMP assay is suitable for large-scale surveillance for H7N7 EIV. In addition, the combination of the H3N8 RT-LAMP assay, which was developed previously, with the H7N7 RT-LAMP assay should be useful to discriminate between H3N8 and H7N7 EIVs in clinical laboratories.

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