The histological disorders related to the focal disappearance of the epiphyseal growth plate were examined histochemically in the proximal tibia of rats administered a high dose of vitamin A. Animals were given 100, 000 IU/100 g body weight/day of vitamin A for 5 days from 4 weeks after birth (VA rats) or given deionized water as control and sacrificed on Day 12 and 19 of the experiment. Tibiae were examined by immunohistochemistry for type I, II and X collagens, lectin-histochemistry for Helix pomatia and backscattered electron imaging. On Day 12, the abnormally developed calcified cartilage matrix was detected within the epiphyseal growth plate in VA rats. The uncalcified cartilage matrix contained type I collagen but lacked type II collagen. In addition, the eroded regions accompanied with numerous osteoclasts and osteoblasts were detected in the epiphyseal growth plate. On day 19, eroded regions penetrated the epiphyseal growth plate to result in its focal disappearances with the eroded surfaces entirely covered with bone tissue in VA rats. These findings suggested that the cartilage matrix of the epiphyseal growth plate was abnormally calcified and showed the phenotypes like bone matrix. The eroded regions of the epiphyseal growth plate seemed to be caused by the invasion of osteoclasts into the altered cartilage matrix and might develop to the focal disappearances by the modeling or remodeling due to action of osteoclasts and osteoblasts.

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Changes in the lactate dehydrogenase (LDH) isozyme pattern of primary culture of neurons treated with botulinum C3 enzyme were examined in order to elucidate the functional changes accompanying the morphological change that follows ADP-ribosylation of Rho protein. Primary neurons were prepared from the cerebrum of ICR mouse embryos on day 15. Neurons were cultured in MEM with 10% fetal calf serum at 37°C. In the neurons treated with C3 enzyme, a typical morphological change was observed after 24 hr, and the LDH isozyme pattern was changed after 72 hr. The ratio of H-subunit to M-subunit in LDH was decreased by C3 treatment, suggesting the induction of a state of lower intracellular oxygen consumption in neurons in the primary cultures.

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This study was performed to isolate a vaccine strain of S. aureus from clinical or subclinical mastitis and to choose the most optimal adjuvant for immune response of alpha toxin and capsular polysaccharide (CPS) of field strain. Of thirty strains of S. aureus isolated from milk of clinical or subclinical mastitis, V112 strain isolated from milk of gangrenous mastitis was used in this vaccine. Twenty one of rabbits were allocated into 5 groups based on adjuvants and immunized twice every 2 weeks for 8 weeks. This vaccine was composed of alpha toxin (10 hemolytic units) and formalinized whole cells (1 × 10¹¹ cells/ml). Five rabbits received PBS solution as a control group. The highest antibody titers against alpha toxin and CPS were observed in dextran sulfate- and aluminium hydroxide-adjuvant group at 8 weeks after immunization, respectively. These results of the study showed that one adjuvant could not induce strong and long-term immune response of alpha toxin and CPS antigens. Therefore, the use of combined adjuvants in subunit vaccine may be useful and feasible.

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Transcranial Doppler ultrasonography (TCD) has been used to confirm changes in cerebral hemodynamics. In this study, we investigated whether the parameters for the basilar artery measured by TCD were correlated with the intracranial and cerebral perfusion pressures in extreme intracranial hypertension. An intracranial hypertension model was produced in seven dogs by inflating a balloon inserted into the epidural space. The resistance index was compared with the corresponding intracranial pressure and cerebral perfusion pressure values during intracranial hypertension. A significant correlation was recognized between the resistance index and cerebral perfusion pressure. Therefore, measurement of the basilar artery by TCD in the dog with intracranial hypertension is useful in estimating the intracranial circulation in cases where the measurement of intracranial pressure is not available or not indicated.

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Babesia caballi merozoites were prepared by combining two improved methods of cultivation and purification of merozoites using Percoll-gradiation, and the protein compositions of merozoites were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The relative molecular masses of the major proteins and protein masses separated by electrophoresis were >94, 80-70, 50-45, 34-30, 30-28 and 18 kDa. By Western blotting, twelve proteins or protein groups were recognized by pooled sera from two horses experimentally infected with B. caballi. Among twelve proteins, five new proteins (54, 30-26, 24, and two 18 kDa) were identified, and the 48 kDa protein was revealed to consist of 2 components in the B. caballi merozoite. One protein (54 kDa) of B. caballi was also recognized by the pooled sera from two horses experimentally infected with B. equi.

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Primary culture of bovine brain cells was examined for its susceptibility to Neospora caninum infections, and this model was used to investigate the effects of bovine interferon gamma (IFN-γ) and tumor necrosis factors alpha (TNF-α) on tachyzoite growth. Tachyzoites of N. caninum grew well in this culture, and tachyzoite growth in astroglia and microglia were confirmed by immunocytochemical staining. IFN-γ inhibited the tachyzoite growth, and this inhibition was not reversed by the addition of nitric oxide antagonist. TNF-α, to a lesser extent, also inhibited the tachyzoite growth. Th-1 type cytokines may play an important role in host defense mechanisms in N. caninum infection.

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To identify surface proteins of eimerian sporozoite, Eimeria tenella sporozoites were labeled with biotin-LC-hydrazide, and analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and Western blotting. More than 200 sporozoite proteins were recognized with silver staining after 2D-PAGE. Among them, 7 biotin-labeled proteins were detected as surface molecules after Western blotting and reaction with horseradish peroxidase conjugated streptavidin. Their molecular masses ranged from 23 to 117 kilo daltons and their isoelectric points from 4.3 to 6.3. It appears that the biotin-labeling technique can be used to analyze of surface proteins of eimerian parasites.

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Recently, cholangiocellular carcinoma (CCC) was successfully induced in the hamster by infecting with Clonorchis sinensis following hepatocarcinogen treatment and has been proposed as a suitable model to study the pathogenesis of human CCC. In this hamster model, oval cells are suggested to be cells of origin of CCC. More direct analysis of histogenesis of CCC would become possible if large numbers of highly purified oval cells of hamster origin are obtained. In this study, we describe successful isolation of highly purified oval cells from hamsters. Oval cells were induced by diethylnitrosamine and 2-acetylaminofluorene treatment under choline deficient diet and isolated by centrifugal elutriation method. This isolated cells were highly homogenous in size (10.9 +/- 1.1 μm in diameter) and had a high nuclear to cytoplasmic ratio, an oval-shaped nucleus and a few cytoplasmic organelles. Immunocytochemically, 85.4 +/- 1.6%, 75.1 +/- 2.0%, 62 +/- 1.5% and 25.6 +/- 2.7% of the isolated cells were positive for cytokeratin 19, OV-6, albumin and alpha-fetoprotein, respectively, indicating that these cells had phenotypic characteristics of both hepatocytes and bile duct epithelium. The isolated cells were therefore considered to be hamster oval cells.

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We established a cell line (MHB-2) from a hepatoblastoma (HB) induced by diethylnitrosamine (DEN) and sodium phenobarbital (PB) in male B6C3F₁ mice and examined the biological characteristics of MHB-2. MHB-2 cells grew as monolayers in culture and showed a spindle or polygonal shape. Immunohistochemically, the original tumor cells and MHB-2 cells were negative for keratin, alpha-fetoprotein and albumin. Electron microscopically, MHB-2 cells had irregular-shaped nuclei with prominent nucleoli, abundant free ribosomes, myelinosomes, desmosomes and surface microvilli. Growth of this cell line was significantly accelerated by hepatocyte growth factor (HGF) and expression of its receptor c-met was confirmed by the reverse transcription-polymerase chain reaction (RT-PCR). MHB-2, however, was not found to be tumorigenic when transplanted into the subcutaneous tissue of syngeneic, nude or scid mice. To our knowledge, this is the first report on the establishment of a cell line derived from a mouse HB. MHB-2 would be useful for further studies to clarify the biological characteristics of mouse HB.

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Desmin has been suggested as a possible histopathological marker for hypertrophic cardiomyopathy (HCM) in humans. To test whether a similar pattern of desmin staining applies to HCM in cats, we conducted an immunohistochemical study on myocardial samples from 13 cats (HCM 4, other cardiomyopathies (OCM) 4, and control 5). The pattern of staining for desmin in HCM cats was not the same as that reported in humans, but was weaker than in OCM cats and controls. This suggested that desmin may be a possible histochemical marker for feline HCM, but our data was insufficient to clearly confirm this.

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The authors investigated bacteriologically the prevalence of Bartonella infection among 690 pet cats derived from 10 private animal hospitals in six cities (Sapporo, Hokkaido Prefecture; Sendai, Miyagi Prefecture; Joetsu, Niigata Prefecture; Fujisawa, Kanagawa Prefecutre; Kyoto, Kyoto Prefecture; Sanda, Hyogo Prefecutre) and 4 counties (Mishima, Osaka Prefecture; Hikawa, Shimane Prefecture; Aira, Kagoshima Prefecture; Shimajiri, Okinawa Prefecture) located from the north to the south of Japan. Bartonella species were isolated from 7.2% (50/690) of all the cats examined. No Bartonella species were isolated from the cats in Sapporo or Sendai. The isolation rate varied from 2% in Joetsu and Sanda to 20% in Shimajiri. Bartonella clarridgeiae was isolated from two of 50 cats in Kyoto, three of 50 in Mishima and one of 50 in Shimajiri, but not in cats from the other cities or counties. Though the cats of Joetsu, Fujisawa, Kyoto, Sanda, Aira and Shimajiri were infected with either B. henselae or B. clarridgeiae, one of eight infected cats in Mishima was harboring both Bartonella species. Type I of 16S rRNA gene was the predominant type among the isolates of B. henselae, but only one isolate derived from Shimajiri was found to be of type II. Prevalence of B. clarridgeiae and the 16S rRNA gene type of B. henselae among cats in Japan was demonstrated for the first time in this investigation.

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Seven sika deer (Cervus nippon) in a herd of 30 deer in a park died. Upon examination of three dead deer, Salmonella Typhimurium was isolated from the organs and intestinal contents. Histopathological examination revealed catarrhal enteritis and focal necroses in the liver. Immunohistochemically, Salmonella antigen of O4 was detected in the enteric lesions. The case was diagnosed as S. Typhimurium infection in the sika deer. Because of the importance of Salmonella in public health, fecal and soil samples were continuously collected from the paddock. However, no Salmonella was isolated from any samples collected after medication of the deer and thorough disinfection of the immediate environment.

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The anesthetic and cardiovascular effects of a combination of continuous intravenous infusion using a mixture of 100 g/L guaifenesin-4 g/L ketamine-5 mg/L medetomidine (0.25 ml/kg/hr) and oxygen-sevoflurane (OS) anesthesia (GKM-OS anesthesia) in horses were evaluated. The right carotid artery of each of 12 horses was raised surgically into a subcutaneous position under GKM-OS anesthesia (n=6) or OS anesthesia (n=6). The end-tidal concentration of sevoflurane (EtSEV) required to maintain surgical anesthesia was around 1.5% in GKM-OS and 3.0% in OS anesthesia. Mean arterial blood pressure (MABP) was maintained at around 80 mmHg under GKM-OS anesthesia, while infusion of dobutamine (0.39 ± 0.10 μg/kg/min) was necessary to maintain MABP at 60 mmHg under OS anesthesia. The horses were able to stand at 36 ± 26 min after cessation of GKM-OS anesthesia and at 48 ± 19 minutes after OS anesthesia. The cardiovascular effects were evaluated in 12 horses anesthetized with GKM-OS anesthesia using 1.5% of EtSEV (n=6) or OS anesthesia using 3.0% of EtSEV (n=6). During GKM-OS anesthesia, cardiac output and peripheral vascular resistance was maintained at about 70% of the baseline value before anesthesia, and MABP was maintained over 70 mmHg. During OS anesthesia, infusion of dobutamine (0.59 ± 0.24 μg/kg/min) was necessary to maintain MABP at 70 mmHg. Infusion of dobutamine enabled to maintaine cardiac output at about 80% of the baseline value; however, it induced the development of severe tachycardia in a horse anesthetized with sevoflurane. GKM-OS anesthesia may be useful for prolonged equine surgery because of its minimal cardiovascular effect and good recovery.

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The role of keratan sulphate (KS) as a marker of cartilage metabolism was evaluated by using an in vitro model of equine articular cartilage. Articular cartilage was harvested from clinically healthy 6-month-old foals (n=3). Chondrocytes were centrifuged and cultured as pellets. Chondrocyte pellets were stimulated by insulin-like growth factor (IGF)-Iα or interleukin (IL)-1α for 2 weeks. The sulfated glycosaminoglycans (GAG) and antigenic KS concentrations in the culture media were measured by a 1, 9-dimethyl-methylene blue (DMMB) colorimetric assay and an inhibition ELISA using a 1/14/16H9 antibody, respectively. Concentration of GAG was significantly increased in the media of pellets stimulated by both IGF-Iα and IL-1α. Antigenic KS concentration was significantly increased in those stimulated by IL-1α, while no significant change was found in those stimulated by IGF-Iα. A high correlation between GAG and antigenic KS concentrations was found in the media of pellets stimulated by IL-1α (r=0.87), but not in those stimulated by IGF-Iα (r=0.43). The results suggest that the concentration of antigenic KS reacting to 1/14/16H9 mirrors the GAG concentration during the stage of cartilage catabolism, but not during the cartilage anabolic stage. The concentration of antigenic KS reacting to 1/14/16H9 antibody in biological fluids could therefore be a useful marker to further understand principally the catabolic and slightly the anabolic process of articular cartilage metabolism.

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Recently, canine frozen semen has been attracting attention for breeding purposes, and methods of judging ovulation and optimum timing for insemination have become important. As methods of predicting the canine ovulation, vaginal smear, plasma sex hormone levels and ultrasonographic diagnosis system (US) have been investigated in combination, but a standard technique has not yet been established. Therefore, we investigated a method of predicting canine ovulation in dogs by US, and by measuring plasma LH and progesterone (P) levels three times a day. Ovulation could be observed by detecting irregularly shaped ovarian follicles by US in six of 11 dogs (54.5%). In these dogs, the time between the LH peak and ovulation was 24-48 hr, 38.0 hr on average. The P level on the ovulation day was 1.88-2.81 ng/ml, 2.34 ng/ml on average. A value of 1.88 ng/ml was detected in one dog, but the other five dogs showed P levels of 2 ng/ml or higher. The P level on the day before ovulation was 0.8-1.56 ng/ml, 1.12 ng/ml on average. Assuming that ovulation occurred two days after the LH peak in the 11 experimental dogs, the P level was 2.12-4.06 ng/ml, 2.78 ng/ml on average. The period of a high LH level, not less than 10 ng/ml, continued for 12 hr around the LH peak. Based on these findings, to predict ovulation using US and LH level, it would be necessary for the tests to be performed several times a day. In contrast, it was shown that the day on which a plasma P level of 2 ng/ml or higher was detected by the test performed once a day corresponded to the ovulation day.

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The objective of the experiment was to improve the multifollicle stimulation technique and the ovarian response examination in prepubertal swamp buffalo calves. Six animals were stimulated by gonadotropin hormone 7 days after a progesterone ear-implant. The first stimulation was done by giving 24 mg FSH + 100 μg GnRH (FSH+GnRH) and the second, one month after by giving 2, 000 IU PMSG + 100 μg GnRH (PMSG+GnRH). Twenty-four hours after GnRH, the ovarian responses were checked using rectal palpation and real-time B mode ultrasonography. Five out of six animals (83.3%) responsed to both treatments and were selected for oocyte collection. The oocytes were aspirated directly following a caudal midline laparotomy. The results of ovarian responses to FSH+GnRH and PMSG+GnRH averaged 17.6 ± 12.1 (L=9.8 ± 8.7, R=7.8 ± 6.2) and 17.4 ± 5.6 (L=9.4 ± 2.9, R=8.0 ± 3.7), respectively. The average number of recovered oocytes per animal was 9.0 ± 6.4 and 8.4 ± 1.1, respectively which represented a recovery rate of 56.3 (± 9.2)% and 51.9 (± 10.3)%. More than eighty percent of the recovered oocytes were in an immature stage with more than 2-3 layers of compact cumulus mass. The present study showed that the oocytes were collected successfully in prepubetal buffalo calves after the FSH+GnRH or PMSG+GnRH stimulation and most of the recovered oocytes were immature, which made them suitable for in vitro maturation and fertilization.

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Oocytes were recovered from bitch ovaries at various stages of the estrous cycle by the slicing method. The proportion of Grade A oocytes (darkly pigmented and surrounded in part, or whole, by dense layers of cumulus cells) were counted. Only Grade A oocytes were cultured in TCM199 supplemented with 5% fetal calf serum for evaluation of meiotic competence. There were no significant differences in the total number of oocytes or the proportion of Grade A oocytes that were recovered from bitches at various stages of the estrous cycle. Only 11% of the oocytes reached metaphase II (MII) at 72 hr after initiation of maturation culture. However, the proportions of oocytes reaching MII did not increase with culturing for up to 120 hr.

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Fecal and plasma E₁S of a sow with mummified fetuses, was compared with normal delivery cases. Fecal and plasma fluctuation patterns in E₁S were similar. In the sow with fetal mummification both fecal and plasma E₁S concentration rapidly decreased after day 80-90 compared to normal farrowing sows. This coincided with the estimated time of fetal death.

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The in vitro development and the quality of blastocysts produced from the nuclear transfer (NT) embryos reconstituted from primary cultured cumulus cells (NT-cumulus) were examined compared to in vitro fertilized embryos (IVF) and NT embryos reconstituted from the embryonic blastomeres (NT-blastomere). The cleavage rate, and the development to blastocyst were the same for all three sets of embryos. The time required for blastocoel formation starting from the time of the initial cleavage was shorter for NT embryo groups than IVF ones. All experimental groups produced morphologically similar and normal blastocysts containing the same cell number. The percentage of the blastocysts with normal chromosomal complements were the same for NT-cumulus and IVF.

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Serotype 1 strains of Marek’s disease virus (MDV1), except attenuated vaccine strains, are known to cause lymphomas in visceral organs of infected chickens. To know additional genetic differences between oncogenic and nononcogenic MDV1, polymerase chain reaction (PCR) was performed to amplify the meq gene of the viral genome. In addition to the 1, 062-bp band including the native meq open reading frame (ORF), a 1.2-kb band was amplified from the DNA sample prepared from chick embryo fibroblast infected with an attenuated strain, CVI988, but not with oncogenic strains. Sequence analysis of the 1.2-kb band showed that a 178-bp sequence was inserted to the meq ORF of CVI988. This ORF could encode for the Meq protein with a different transactivator domain. Southern blot analysis also confirmed the insertion of the 178-bp sequence in the meq ORF of CVI988. This insertion of 178-bp sequence may explain the reason why CVI988 is not oncogenic.

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The complete nucleotide sequence of a cDNA clone representing the M5 RNA segment of epizootic hemorrhagic disease virus Japan serotype 2 (EHDV-2), Ibaraki virus, was determined. The M5 segment is 1641 base pairs long with the single open reading frame which predicts a polypeptide of 527 amino acids. The comparison of the amino acid sequence of the VP5 with those of EHDV-1, bluetongue virus serotype 10, and African horse sickness virus serotype 4 revealed that the protein shared 67%, 57% and 42% homologies, respectively. In addition, the VP5 protein was expressed in insect cells by recombinant baculovirus, which could be recognized by the mouse anti-EHDV-2 sera at a position of the expected 59 kDa on immunoblot analysis.

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The complete nucleotide sequence of cDNA clones representing the L2 dsRNA from Japan isolate of epizootic hemorrhagic disease serotype 2 (EHDV-2JPN) was determined. The EHDV-2JPN L2 gene is 3002 base pairs long with a single open reading frame of 2949 bp which predicts a polypeptide of 982 amino acid residues. Comparison of VP2 sequence between Japan and North American Isolates of EHDV-2 showed a 72% homology in spite of the same serotype, although those among the North American isolates showed a high genetic identity (>97%).

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A survey of antibodies to bovine immunodeficiency virus (BIV) known as bovine lentivirus and bovine leukemia virus (BLV) was conducted with samples from water buffalo and cattle populations in Pakistan. A total of 370 water buffaloes and 76 cattle were tested, and l0.3% and 15.8%, respectively, were found positive for anti-BIV p26 antibodies determined by Western blotting, while 0.8% of water buffaloes and no cattle were positive for anti-BLV antibodies determined by immunodiffusion test. BIV-seropositive water buffaloes and cattle were found to have BIV proviral DNA in the peripheral blood mononuclear cells determined by nested polymerase chain reaction. This is the first report of BIV infections in water buffaloes.

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Several diagnostic methods including immunofluorescence, enzyme-linked immunosorbent assay, polymerase chain reaction and immunohistochemistry have been developed for the detection of porcine epidemic diarrhea virus (PEDV). An immunohistochemical method using a new recombinant antibody produced by a phage antibody system (PAS16) kit was investigated and compared with that using a monoclonal antibody for PEDV detection in PEDV-infected piglets. In both the immunohistochemical methods, PEDV antigens were detected in the cytoplasm of villous enterocytes and in the macrophages infiltrated in the lamina propria at 18 to 110 hr post inoculation. The positive signals with the recombinant PAS16 antibody were similar to those with the monoclonal antibody. This result suggests that the recombinant PAS16 antibody can be applicable for the rapid immunohistochemical diagnosis of PEDV infection.

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