Two chicken B cell lines, termed MuH1 and MuH4, which are resistant to ouabain, were established as fusion partners for production of chicken monoclonal antibody from the parental cell lines R27H1 and R27H4 which were deficient in thymidine kinase activity. The MuH1 synthesized, but did not secrete μ chain, whereas the MuH4 secreted non-specific IgM. Fusions of the MuH1 and MuH4 with spleen cells from chickens immunized with human IgG resulted in successful preparations of hybridomas secreting antibodies specific to human IgG. Transformed cells insensitive to HAT appeared in many culture wells after cell hybridizations with R27H4, but on hybridization with MuH1 or MuH4 the appearance of such unfavorable cells could be avoided by the inclusion of ouabain in HAT medium. Thus, in comparison with the R27H4 line, the MuH1 and MuH4 lines did not provide higher fusion efficiencies, but gave increased opportunities to obtain hybridomas producing specific antibodies. Among a total of eight hybridomas, four derived from HuM1 and four derived from MuH4, seven secreted both IgG and IgM, and the remaining one derived from MuH1 secreted only IgG. In all eight hybridoma lines. IgG was specifically reactive to human IgG.

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In the Marek's disease virus (MDV) serotype 2 (MDV2) genome, a gene equivalent to the glycoprotein I (gI) of other alphaherpesviruses was identified and sequenced. The primary translation product comprises 355 amino acids with a M_r of 38.4 kDa. The predicted amino acid sequence possesses several characteristics typical of membrane glycoproteins, including a N-terminal hydrophobic signal sequence, C-terminal transmembrane and cytoplasmic domains, and extra-cellular region containing three potential N-linked glycosylation sites. Compared to other MDV serotypes, MDV2 gI showed 49% identity with MDV1 gI, and 36% identity with HVT gI at the amino acid level. In transcriptional analyses, a 3.5 kb mRNA which starts between 56 and 147 bps upstream of the potential translational initiation codon of gI was identified as the gI-specific transcript. By a recombinant baculovirus, this potential gI encoding region was expressed as two specific products 45 and 43 kDa. Both products were susceptible to tunicamycin treatment, indicating that they were glycoprotein. Further, the expressed gI reacted with all chicken-antisera raised to each of the three serotypes of MDV (strains GA, SB-1, and FC126), suggesting that gI is expressed by all three serotypes of MDV in infected cells and conserves common antigenic epitope(s) beyond serotypes.

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The cross-sensitivity of X-ray-hypersensitive lung fibroblasts from LEC strain (LEC) rats to othet DNA-damaging agents was examined. The LEC cells were 2- to 3-fold more sensitive to bleomycin (BLM) that induces DNA double-strand breaks, and to a cross-linking agent, mitomycin C, than the cells from WKAH strain (WKAH) rats, while they were slightly sensitive to alkylating agents, ethyl nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine, but not to UV-irradiation. Although no difference was observed in the initial yields of DNA double-strand breaks induced by BLM between LEC and WKAH cells, the repair process of DNA double-strand breaks was significantly slower in LEC cells than in WKAH cells.

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The culture conditions which are able to support the differentiation of bovine intramuscular (i.m.) stromal-vascular (S-V) cells into adipocytes were investigated. Bovine i. m. S-V cells were able to undergo adipose conversion, assessed by emergence of lipid droplets and induction of glycerol-3-phosphate dehydrogenase (GPDH) activity, when treated with 0.25μM dexamethasone and 0.5 mM 1-methyl-3-isobutylxanthine in a serum-deprived (0.1%) medium containing 850 nM insulin and 1 mM octanoate. Octanoate was essential for morphological differentiation and addition of 25μM of cholesterol with octanoate induced higher GPDH activity. The differentiation of the cells was not observed in the medium containing 10% fetal calf serum which supported the cell proliferation of undifferentiated cells even after confluence. Similarly, both bovine brain extracts and muscle extracts inhibited the differentiation of S-V cells in the serum-deprived condition. These results suggest that the existence of lipids such as octanoate and the process of growth-arrest in preadipocytes and/or other cells are necessary for the differentiation of bovine S-V cells into adipocytes in culture.

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The 2-year prognosis of malignant mammary tumors seen in 175 bitches in the Tokyo metropolitan area was assessed based on their TNM clinical staging and histological classification. The larger the tumor size became (T category), the poorer was the clinical prognosis. The 2-year survival rates of the animals with regional lymph node metastasis of tumor cells (N1, N2 category) and/or distant metastasis (M1 category) were markedly lower than those of the animals without such involvement. As the grade of TNM staging increased, the prognosis was poorer, however, there were no significant differences in survival rates among subtypes of adenocarcinomas (tubular, papillary and papillary cystic) determined by WHO histological classification. It was also noticed that animals having carcinomas without tubular formation or myoepithelial cell proliferation had a lower survival rate than animals having carcinomas with those characteristics; and invasive carcinomas into adjacent skin or lymphatic/vascular vessels implied a poorer prognosis than non-invasive ones. The results suggest that a combined practice of TNM system and our evaluation on the above-mentioned 4 histologic features could be useful for prognostic determination of canine mammary cancers.

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The temporal relation between the luteinizing hormone (LH) surge and the occurrence of two components of sexual behavior was investigated in female goats. Behavior observation and blood sampling were carried out every 2-6 hr from 26 hr before through 116hr after prostaglandin (PG) administration which was given to induce luteolysis during the mid-luteal phase. The preovulatory LH surge appeared 30-100 hr after PG injection, and it continued for 6.5±0.4 hr. Although the interval from PG injection to the onset of the LH surge differed considerably among individuals, the proceptivity (defined as an approach to a male with vigorous tail-swishing) and the receptivity (defined as an acceptance of male's mounting or copulation) occurred well synchronized with the LH surge in each female. The proceptivity and receptivity were observed from - 7.4±1.7 hr to 16.8±1.5 hr and from - 2.6±1.6 hr to 13.0±2.0 hr, respectively, with regard to the LH surge onset (0 hr). Thus the proceptivity appeared first and lasted longest, and the receptivity was always seen around the time of the LH surge suggesting an existance of causal relationship between these two preovulatory events. The present results support the view that the female goat has evolved an efficient behavioral strategy such that it actively attracts males before it becomes fully receptive of male's approach to mate, when the LH surge occurs simultaneously to ensure a high conception rate at the subsequent ovulation.

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The cranial cruciate ligaments (CCL) of 13 dogs with clinical signs of CCL rupture and those of 22 clinically healthy young beagle dogs for laboratory use were examined histopathologically and immunohistopathologically. The most constant changes at an early stage of degenerating ligament tissue in affected dogs were nuclear enlargement and perinuclear halo formation of fibrocytes followed by chondroid metaplasia. These changes were also frequent in apparently healthy young beagles kept under laboratory conditions. PAS and alcian blue positive substance accumulated around activated fibrocytes and within perinuclear halos. S-100 protein was also positive in these cells preceding the morphological change of chondroid metaplasia. Increased mitotic figures and Ki-67 positive cells showed the proliferating nature of these cells at a later stage. Alteration of extracellular matrices from dense collagen fiber type to those of cartilage tissue seemed to predispose dogs to rupture of the CCL along with a degradation in collagen fiber of the primary bundles. Collagen fiber bundles with a parallel fibrillar array never formed in the CCL with degraded primary bundles, whereas activated fibrocytes constantly underwent chondroid metaplasia. The pathogenic mechanism underlying chondroid metaplasia was thought to be nonspecific and attributable to an essential property of activated fibrocytes in the mature tendon tissue.

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The gangliosides of Theileria sergenti piroplasms were isolated and analyzed by thin-layer chromatography (TLC) and TLC immunostaining test. Four species of gangliosides, designated as G-1, G-2, G-3, and G-4, were separated on TLC. G-1, G-2, G-3, and G-4 ganglioside showed the same mobility as GM3, sialosylparagloboside (SPG), i-active ganglioside, and I-active ganglioside on the TLC plate, respectively. In order to characterize the molecular species of gangliosides from T. sergenti, G-1, G-2, G-3, and G-4 gangliosides were purified and tested by TLC immunostaining test with monoclonal antibodies against gangliosides. G-1 ganglioside had reactivity to anti-GM3 monoclonal antibody. G-2 gave reaction with monoclonal antibody to SPG containing N-glycolylneuraminic acid (NeuGc). G-3 showed reactivity to the anti-i-active ganglioside (NeuGc) monoclonal antibody. G-4 was recognized by the monoclonal antibody which reacts with I-active ganglioside (NeuGc). In addition, sialic acid moiety of the gangliosides from T. sergenti piroplasms was also analyzed. N-acetylneuraminic acid-containing gangliosides were hardly detectable in T. sergenti piroplasms. Gangliosides from T. sergenti (G-1, G-2, G-3, and G-4) carried only NeuGc as their sialic acid moiety. These results suggest that G-1, G-2, G-3, and G-4 gangliosides are GM3 (NeuGc) [NeuGcα2-3Galβ1-4Glcβ1-1Cer], SPG (NeuGc) [NeuGcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1Cer], i-active ganglioside (NeuGc) [NeuGcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1Cer], and I-active ganglioside (NeuGc) [NeuGcα2-3Galβ1-4GlcNAcβ1-3 (Galα1-3Galβ1-4GlcNAcβ1-6) Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1Cer], respectively.

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Fifty-four Japanese strains of Mycoplasma hyosynoviae isolated from porkers during 1980 to 1995, and 107 Japanese strains of M. hyorhinis isolated from piglets with respiratory disease during 1991 to 1994 were investigated for the in vitro activities of 13 antimicrobial agents [josamycin, tylosin, spiramycin, kitasamycin, erythromycin, lincomycin (LCM), kanamycin (KM), chloramphenicol(CP), thiamphenicol (TP), tiamulin (TML), oxytetracycline (OTC), chlortetracycline (CTC), and enrofloxacin (ERFX)] by the agar dilution method. Of the drugs tested TML showed the highest activity with minimum inhibitory concentration (MIC) of 0.013 to 0.1μg/ml (MIC₉₀ 0.05μg/ml) against strains of M. hyosynoviae, and 0.2 to 0.78μg/ml (MIC₉₀ 0.39μg/ml) against strains of M. hyorhinis. ERFX, LCM, most of the 16-membered macrolide antibiotics and tetracyclines also showed low MICs against both mycoplasma species. The susceptibility of KM, CP and TP to the mycoplasmas was considered to be of a secondary grade. Two of 54 strains of M. hyosynoviae, and 11 of 107 strains of M. hyorhinis showed resistance to all 14- and 16-membered macrolide antibiotics tested. Tetracyclines (OTC and CTC) showed a relatively broad MIC distribution from 0.1 to 6.25μg/ml against the M. hyosynoviae strains tested. All of the strains isolated during 1980 to 1984 were susceptible at the concentration of 0.78μg/ml or less (MIC₉₀ 0.78μg/ml) to OTC and 1.56μg/ml or less (MIC₉₀ 1.56μg/ml) to CTC, while the susceptibility of strains isolated recently, during 1994 to 1995, was more than 0.78μg/ml (MIC₉₀ 3.13μg/ml) to OTC, and more than 1.56μg/ml (MIC₉₀ 6.25μg/ml) to CTC.

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A rare case of ameloblastoma with prominent stromal ossification in an 8-year-old female dog was studied. A bony mass recurred rapidly in the right mandible at the first molar region. Histopathologic examination revealed the lesion to be an atypical variant of ameloblastoma. Epithelial cells showed marked cell atypia, and mitotic figures were rather common. The collagenous stroma was abundant, with prominent formation of bone trabecular rimmed by active osteoblasts. The tumor was highly proliferative and aggressive, and thought to be malignant in nature.

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The hooves of a racehorse which were affected with white line disease and hoof wall disorders on both forelimbs were histopathologically investigated using thin ground section and standard paraffin section techniques. On both hooves, large quantities of fungus were found to have invaded the white line tissues, especially in the terminal horn which were markedly damaged. The fungus was also present among the cellular debris in the fissures of horny tissues. The morphological characteristics of the fungus were brown (its natural color), PAS-positive, mold-like shape with septa inside the tissues, and unicellular spores outside the tissues. These findings suggest that onychomycosis was a primary and/or secondary cause of white line disease in this subject.

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Quantitative comparisons between serum components and somatic cells in 202 bovine quarter milks collected at 3 dairy farms were conducted. Detection of serum components was carried out by an immunological technique, and cell counts were calculated by Breed's method. A high correlation (r=0.77) between cell counts and γglobulin contents was demonstrated, while correlation between cell counts and serum albumin was not close (r=0.45). In a comparison between cell counts and γglobulin contents, 19 (65.4%) out of 29 milks containing over 500, 000 cells/ml had γglobulin less than 1.138 mg/ml, which was also seen in 97% of 172 milks having less than 500, 000 cells/ml. From these results, it was suggested that the γglobulin content of milk might be of use for differentiating normal milk from abnormal ones.

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Long-Evans Cinnamon (LEC) rats show a novel maturational arrest from CD4⁺8⁺ to CD4⁺8^- thymocytes but a cause of this mutation is not identified. The candidate for this mutation is a defect in the function of CD4 or major histocompatibility complex (MHC) class II because gene-disrupted mice defective for CD4 or MHC class II molecules show a specific defect in CD4⁺ T cells. Previously, we showed that MHC class II is not a cause of this maturational arrest. Therefore, in this study, we focus on the function of CD4 molecules in LEC rat thymocytes. CD4 molecules on LEC rat thymocytes associated with protein tyrosine kinase, p56^1ck, normally. Furthermore, cross-linking of CF4 molecules by anti-rat CD4 mAb elicited the elevation of intracellular calcium concentrations ([Ca²⁺]_i) in LEC rat thymocytes, suggesting that CD4 molecules can deliver the signal normally. These results indicate that function of CD4 is normal and the maturational blockade of CD4⁺8^- thymocytes in LEC rats is not caused by specific lymphocyte molecules that have been shown in gene-disrupted mice.

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The susceptibility of chicken embryos to highly virulent infectious bursal disease virus (hv IBDV), strains F539 and DV86, was examined with respect to three inoculation routes and compared with the classical type of IBDV, strain G691. Death patterns of embryos infected with strains F539 and DV86 of hvIBDV were observed constantly and most of the infected embryos died; however, the death pattern associated with the strain G691 of classical IBDV was erratic and not related to the virus titer inoculated. The chorioallantoic membrane (CAM) route was the most sensitive route of infection, while the allantoic sac (AS) route was the least, regardless of the strain. The difference in titer of hvIBDV between the CAM and yolk sac (YS) route was less than that of classical IBDV. Constant lethality to embryos seems to be distinctive characteristic of hvIBDV.

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Staphylococcus aureus isolates from mastitic cow's milk and farm bulk milk were examined for toxic shock syndrome toxin-1 (TSST-1) gene (tst gene) by the polymerase chain reaction (PCR). The 179 bp band of tst gene was observed in almost all the bovine isolates which showed TSST positive in a latex agglutination, as well as in human strain FRI 1169, but was not observed in bovine isolates of TSST negative. The lowest detectable threshold of the PCR for tst gene was 1.2×10³ cells/ml. When 125 bovine milk samples were cultured selectively for staphylococci and examined by PCR, the tst gene was detected in 10 of the 35 culture fluids, in which staphylococci were recognized by Gram's staining.

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Effect of brefeldin A (BFA) on influenza virus-infected cells was examined by measuring cell viability, microscopic examination, and DNA fragmentation analysis. When Madin-Darby canine kidney (MDCK) cells were infected with influenza A virus together with BFA treatment, virus-induced cell death was observed earlier than in the virus-infected cells without BFA treatment. By microscopic examination, the mode of cell death in virus-infected cells with BFA treatment appeared different from that of the cells without BFA treatment. Because influenza virus causes apoptosis in the host cells, extent of DNA fragmentation was compared between virus-infected cells with and without BFA treatment. BFA treatment resulted in inhibition of DNA fragmentation of virus-infected cells. Treatment of virus-infected cells with anti-oxidant (10 mM N-acetyl-L-cysteine) also inhibited DNA fragmentation of influenza virus-infected cells. A possible mechanism of BFA affecting influenza virus-induced apoptosis is discussed.

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Immunological cross-reactivity between feline and canine serum amyloid A protein (SAA) was studied to establish enzyme-linked immunosorbent assay (ELISA) with heterologous antibody. Purified feline SAA formed a precipitin line in immunogel double diffusion with anti-canine SAA antibody. Immunological cross-reactivity was similarly observed in ELISA. In sandwich ELISA with anti-canine SAA antibody, a dose-response curve was obtained over the range of 2μg/ml to 123μg/ml of purified feline SAA. From the present findings, the sandwich ELISA is found to have high repeatability for quantitation of purified feline SAA and may be applicable to determine the serum concentration of feline SAA.

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The adherent site of "attaching and effacing Escherichia coli" (AEEC; O103: H-, SK-1 strain) on the intestinal epithelial cells of chicks infected naturally and experimentally was ultrastructurally investigated. The eaeA gene was detected by polymerase chain reaction in the SK-1 strain of E. coli isolated from the intestinal content of a chick infected naturally, however, the bundle-forming pilus (bfp) gene could not be detected. The SK-1 strain (bfp-) of AEEC could attach to the intestinal epithelial cell and induce attaching-effacing lesions in the intestine of chicks. Transmission electron microscopy revealed numerous pilus-like microfilaments in the space between colibacilli and the membranes of the intestinal epithelial cells. The present study suggests that SK-1 strain (eaeA+, bfp-) may attach closely to the intestinal epithelial cells by a novel adhesin different from bfp.

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Density gradient centrifugation using Ficoll-Conray solution was attempted to isolate viable canine transmissible sarcoma (CTS) cells. The viability of isolated tumor cells increased from about 50% to >90%, and the yield of CTS cells was >50% with over 99% purity, in when an isolation solution density of 1.05 was used.

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Transovarial transmission of a newly isolated large intraerythrocytic parasite, Babesia sp.1 by Haemaphysalis longicornis was experimentally demonstrated. Larvae of H. longicornis were transovarially infected with the parasite by feeding as adults on the calf which had been experimentally infected with B. sp.1. Piroplasms of B. sp.1 were observed in peripheral blood of the calf which was infested with the parasite-infected larvae. Based on the transmissibility of the parasite with vector ticks, this parasite was suggested to be a variety of B. ovata. Thus, we propose a new variety name for B. sp.1 as B. ovata oshimensis n. var.

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