In order to obtain the basic data to identify the skeletal remains from the archaeological sites, morphological and morphometrical studies were carried out on skulls of living raccoon dogs (35 males and 45 females) and badgers (16 males and 8 females) from Kagoshima Prefecture. Macroscopically, the sexual differences were observed in badgers for the parts of the zygomatic process of the temporal bone and the occipital squama, but were not in raccoon dogs. Among 24 cranial measurements, significant sexual differences were found in five measurement items in raccoon dogs, while 12 items in badgers. Mandibles showed significant sexual differences in both species. Raccoon dogs had significantly larger values than badgers in most of the items concerning length of cranium and most mandibular measurements. The discrimination efficiencies of discriminant formulae between both sexes were lower in raccoon dogs, but higher in badgers, and the efficiencies between both species were obtained 100%. In the regression formulae for estimating skull length, some formulae showed high coefficients of determination in both species. These observations represented interspecific and sexual differences in the skulls of raccoon dogs and badgers.

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Lectin binding patterns in spermatogonia of Syrian hamsters in gonadally active and inactive states were examined by light and electron microscopy. After exposure to a short day cycle (SD), the testis weight and the diameter of seminiferous tubules decreased, reaching the minimum at 13 weeks. At that time, spermatogenesis was severely disrupted. In the animals kept exposed to an SD, spermatogenesis reinitiated spontaneously after 23 weeks. In the animals transferred to a long day cycle (LD) after exposure to an SD for 13 weeks, spermatogenesis reinitiated only 4 weeks later. As to the lectin binding, Dolichos biflorus agglutinin (DBA) bound specifically to spermatogonia. The number of DBA-positive spermatogonia per one seminiferous tubule increased until 13 weeks after exposure to an SD, and then gradually decreased. DBA bound to only type A spermatogonia in active testes, whereas it bound to type A, intermediate and type B spermatogonia in inactive testes. Moreover, SDS-PAGE and Western blot analysis of testes in active and inactive states indicated that DBA-binding bands (115 kDa, 76 kDa) in inactive testes were intense compared with those in active testes. The 82 kDa band was detected only in inactive testes. These results supported the finding obtained from lectin histochemistry. DBA-binding glycoprotein was detected in all types of spermatogonia in inactive testes, suggesting that this glycoprotein way concern the active/inactive state of spermatogenesis. The present study also indicated that DBA is a useful marker for spermatogonia in inactive testes of Syrian hamster.

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To determine a serotype of Marek's disease virus (MDV) persistently infected in a lympohoid leukosis (LL)-cell line, LSCC-BK3, clone A (BK3A), and to examine the pathogenicity of the virus, an attempt was made to isolate MDV from culture fluid of the LL-cell line, using chick embryo fibroblast cultures resistant to infection with subgroup A avian leukosis virus (ALV) to eliminate subgroup A ALV. The MDV isolate, serologically identified as serotype 2 MDV and designated as the 2H strain, was free from ALV and reticuloendotheliosis virus. A serotype 2-specific antigen of MDV was detected in 44% of BK3A, but antigens of serotypes 1 and 3 were not detected in this cell line, by the indirect immunofluorescent antibody test using serotype-specific monoclonal antibodies. MDV antigens were undetectable in LSCC-BK3, clone 2C; both cell lines were established from the same chicken. Neither clinical signs nor macroscopic lesions were observed in any of 19 chicks inoculated with the 2H strain. Three out of 19 chicks histopathologically examined had no lesions. These results suggest that serotype 2 MDV can persist in B cells transformed by ALV without cytopathic effect at a high rate, and the isolate may become a candidate for MD vaccine strains.

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Recombinant baculoviruses were constructed to express the putative proteins VP1, VP2 or VP3 of the chicken anemia virus (CAV). The recombinant VP1, VP2 or VP3 were detected by SDS-PAGE, and their molecular weights were 50, 30/27 and 16 kDa, respectively. The VP2 and VP3 reacted with sera from CAV-infected chickens in Western blot analysis and when used as an enzyme-linked immunosorbent assay (ELISA) antigen, but VP1 did not. Antibodies to CAV were detected, by ELISA using crude insect cell lysates containing VP2 or VP3, from 2 to 20 weeks or 2 to 7 weeks after CAV infection, respectively. These findings indicate that recombinant VP2 and VP3 expressed in the baculovirus vector system can be used as antigens to detect anti-CAV antibodies in ELISA.

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Lipopolysaccharides (LPSs) of 8 isolates of Coxiella burnetii from a variety of clinical and geographical sources could be divided into four groups based on molecular heterogeneity in silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles in the region of the 10 to 17 kDa. The lipopolysaccharide of group 1 was identified on isolates from acute Q fever patient, milk and tick. The three remaining groups were primarily found on isolates from human cases of chronic Q fever. These LPSs shared many antigenic epitopes, as determined by immunoblotting with mouse anti-C. burnetii antisera.

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The condition of an electroporation method was re-evaluated for the introduction of foreign plasmid DNA into Rhodococcus equi. The method is based on an electroporation of the bacteria made competent by culturing in a broth containing glycine and by heat shock at 50°C. Transformation of R. equi could be achieved with a chloramphenicol-resistant shuttle vector originating from Rhodococcus fascians at an efficiency of about 10⁴ transformants/ μg DNA. The bacteria were also shown to become competent when they were incubated with a chemical transformation buffer prior to washing with an electroporation buffer.

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A 14 kilodalton (kDa) serum amyloid A (apoSAA) protein was purified from cow serum. Rabbit antiserum to the 14 kDa apoSAA recognized, in addition to the 14 kDa protein, a 7.5-9.0 kDa protein and a protein having a molecular mass of less than 6.5 kDa (<6.5 kDa protein). The possibility that the two proteins were contaminants was excluded by results showing that the two proteins detected in early stages of purification procedures were not found in the purified 14 kDa apoSAA fraction, as revealed by immunoblot analysis. As in the 14 kDa apoSAA, the 7.5-9.0 kDa protein was localized in the high-density lipoprotein fraction, while the <6.5 kDa protein was in the low-density lipoprotein fraction. In calves with pneumonia induced by inoculation to the lungs of Pasteurella haemolitica, the serum concentration of the 14 kDa apoSAA was increased, whereas those of the 7.5-9.0 kDa and the <6.5 kDa proteins were conversely decreased. The time-course study indicated that the increase in concentration of the 14 kDa apoSAA and decrease in that of the <6.5 kDa protein occurred almost simultaneously. These results suggest that the 14 kDa apoSAA and the immunologically related 7.5-9.0 kDa and <6.5 kDa proteins act as positive and negative acute phase reactants, respectively, and also that concentrations of the three proteins are regulated in concert in acute phase plasma.

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Granulocyte transfusion (GT) was performed in an 8-month-old heifer with leukocyte adhesion deficiency (BLAD) to monitor the changes in transfused CD18-positive neutrophils and associated neutrophil chemiluminescent (CL) response in β₂-integrin-deficient host. The CD18-positive neutrophils were detected in blood from the BLAD heifer during the first 3 hr after 2.6 × 10⁹ cells were infused by GT, and disappeared by 5 hr after GT. The CL response of neutrophils was increased 1.7 to 2.8-fold in the BLAD heifer during the first 3 hr after GT, thereafter CL response decreased gradually from 2 to 5 hr after GT.

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One litter (Group A) of three unacquainted groups of littermates (4 piglets/litter), 64.0 ± 0.8 days old, was moved to the pen of another litter (Group B) and they were housed together for 19 days after grouping (phase 1). The pigs in Group B violently attacked all the pigs in Group A for 9 hr after grouping. The remaining group was not grouped and used as controls. The plasma cortisol concentrations 1 hr after grouping were significantly higher than those 1 hr before and 24 hr after grouping, and the suppression of lymphocyte blastogenesis of peripheral blood mononuclear cells (PBMC) induced by mitogens was observed on 3, 8 and 19 days after grouping. After phase 1 ended, the pigs in Group A were returned to their own pen for 7 days, and then they were regrouped with the pigs in Group B and reared together for a further 14 days. Neither agonistic behavior nor change of plasma cortisol after regrouping was seen. Though the lymphocyte blastogenesis of PBMC induced by the mitogens on day 0 after regrouping was significantly lower in the pigs of Groups A and B compared to those in control pigs, a significant difference in lymphocyte blastogenesis among three groups was not seen on 7 and 14 days after regrouping. These findings indicate that fighting after grouping unacquainted litters increases plasma cortisol, and suppresses lymphocyte blastogenesis for 26 days after grouping.

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To clarify the relationship between uric acid urolithiasis and purine catabolites in newborn piglets, the incidence of uric acid urolithiasis and the plasma concentrations of xanthine, hypoxanthine, uric acid and allantoin were examined in 32 piglets. The newborn piglets were divided into two groups: normal (over 1.2 kg, n=18, group N) and low body weight (below 0.9 kg, n=14, group L). The animals in both groups were given water (non-nutrition, n=11, treatment W), artificial milk (normal nutrition, n=12, treatment M), or a combination of water and allopurinol (prophylactic treatment for the urolithiasis, n=9, treatment A), during the first 60-hr of birth. At necropsy, the incidence of urolithiasis was higher in the piglets that received treatment W than those in the treatment M or A in both the N and L groups. In group L, the plasma xanthine, hypoxanthine and uric acid concentrations were markedly increased in the piglets that underwent treatment W compared with the treatment M. In both the N and L groups, the plasma allantoin concentration was higher in the treatment W piglets as compared with the treatment M piglets. These results suggested that the occurrence of uric acid urolithiasis in the newborn piglets is attributable to increased purine catabolites due to a starvational condition after birth.

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The proportion of S-phase cells in a WKAH rat cell population decreased and that of G1-phase cells increased at 8 and 18 hr post-incubation following UV-irradiation, although no significant change was observed in the ratio of the proportion of S-phase to G1-phase cells in a LEC rat cell population. Thus, UV-radiation-induced delay in the progression from the G1 to S phase was observed in WKAH rat cells but was not apparent in LEC rat cells. The fraction of LEC rat cells containing a sub-G0 DNA content increased with post-incubation time after UV-irradiation, but not that of WKAH rat cells. The proportion of the sub-G0 fraction in LEC rat cells increased with increasing doses of UV-rays. Low molecular weight DNAs extracted from UV-irradiated LEC rat cells exhibited an intense DNA ladder pattern at 18 and 24 hr post-irradiation by electrophoretic analysis, but not those from UV-irradiated WKAH rat cells. These results showed a higher sensitivity of LEC rat cells in induction of apoptosis than that of WKAH rat cells to UV-irradiation, although there was no difference in the survival curves among the cell lines from LEC and WKAH rats after UV-irradiation.

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A 246-base pair (bp) retroviral sequence, which was homologous to a long terminal repeat of avian erythroblastosis virus (AEV), was detected and cloned from Md5 strain (Md5) of Marek's disease virus type 1 (MDV1) by representational difference analysis (RDA). The retroviral sequence was thought to be located in the border region of short unique region (U_S) and short terminal repeat (TR_S), but did not exist in the border region of U_S and the inverted short repeat (IR_S) of the Md5 genome. A cloned fragment of the US/TR_S border region of the Md5 genome showed a construction of U-E'-R-U'-E-TR_S with the regions designated as follows: E, expanded TR_S reported by Jones et al. [Proc. Natl. Acad. Sci. U.S.A. 90, 3855, 1993]; E', a partial copy of the expanded TR_S; R, the retroviral sequence detected in Md5 genome; U, TR_S-end sequence of U_S; U', a partial copy of TR_S-end sequence of U_S. The sequence unit indicated as E'-R-U' was thought to be heterogeneously repeated in the Md5 genome. Since this retroviral sequence reportedly did not exist in the original stock of Md5, the retroviral sequence is thought to be inserted in the Md5 genome without experimental co-infection of avian cells with retrovirus and MDV1. These results suggest that RDA could be useful for the detection of retroviral sequences in the herpesvirus genome.

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The effect of ozone treatment on the development and viability of Toxocara canis eggs was studied. Despite treatment with ozone, unembryonated T. canis eggs could develop into viable second-stage larvae when assayed by larvae recovery after oral inoculation into mice. The viability of second stage larvae of T. canis was also not affected by ozone treatment. No significant difference was observed in the larvae recovery count and migratory pattern of the ozone-treated larvae and the untreated control because the majority of the larvae were recovered from the liver and lungs on day 2 postinoculation. However, scanning electron microscopy of the ozone treated T. canis eggs showed many blebs on the surface of the protein coat at the basement of the honeycomb-like structures. The honeycomb-like structures on the egg surface were also observed to be distorted after ozone treatment. Thus, in spite of inducing some surface morphological changes on the egg, ozone was observed to have no effect on the viability of the embryonated second stage larvae of T. canis.

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Benign Theileria species distributed in China and Korea were characterized by allele-specific polymerase chain reaction (PCR), based on the sequences of major immunodominant piroplasm surface protein genes. In China, all the isolates contained Chitose (C) type parasites. One out of 5 isolates tested was a mixed population of Ikeda (I), C and B-2 types, whereas, all the isolates from Korea consisted of I type parasites. Except for 4 isolates, 29 isolates from Korea consisted of more than two types of parasites. The present data showed that benign Theileria species distributed in these countries were mixed parasite populations.

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The protective effect of lactoferricin against Toxoplasma gondii infection was examined in experimental murine toxoplasmosis. All mice orally administered 5.0 mg of lactoferricin, and challenged with cysts of T. gondii at a dose of LD₉₀ survived until the end of experiment (35 days post challenge). Intraperitoneal administration of 0.1 mg of lactoferricin also prevented death in 100% of treated mice challenged with T. gondii cysts. In contrast, 80% of untreated mice died of acute toxoplasmosis within 14 days post challenge. In the mice treated perorally with lactoferricin, the number of cysts in the brain was significantly lower than that in untreated mice. Levels of interferon-r in the serum of infected mice treated perorally with lactoferricin showed a tendency to lower than those in the infected mice without treatment. These results demonstrate that oral administration of lactoferricin induces resistance to T. gondii infection in mice.

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An survey of Theileria parasite infection in cattle in Taiwan was carried out by polymerase chain reaction (PCR). A total of 491 blood samples, 105 from southern area and 386 from northern area, were collected from bovine in 16 different farms. From northern area, Theileria piroplasms could be seen in only 4 of 105 blood samples microscopically. However, when p32/34 genes (encoding immunodominant piroplasm surface proteins) were amplified by PCR, 15 blood samples were detected positive. They were analyzed by using allele-specific primers of 3 allelic forms of p32/34 and all contained C type of T. sergenti. Four blood samples were found infected with both C and B (T. buffeli) type parasites. Examination of 386 blood samples from southern area of Taiwan did not reveal any Theileria parasite microscopically, as well as by PCR amplification.

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The anthelmintic efficacy of milbemycin oxime against dog whipworm, Trichuris vulpis, was evaluated. A total of 21 T. vulpis positive dogs were divided into 3 groups, one (5 dogs) for control and the other two (8 dogs each) for anthelmintic treatment with oral administration of milbemycin oxime. Milbemycin oxime showed mean efficacies of 96.0% and 98.6%, at doses of 0.5 mg and 1.0 mg base/kg of body weight, respectively.

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Coxiella burnetii was isolated by inoculation into cyclophosphamide-treated mice from 9 of 29 (31%) vaginal swabs collected from feline clients at veterinary clinics in Shizuoka city.

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Each of 5 drugs, i.e., 4 different vasodilator drugs (captopril, enalapril, hydralazine and prazosin) and a cardiotonic drug (digoxin), was administered to dogs with mitral regurgitation (MR) for 1-72 days in order to quantitatively evaluate the influence of therapeutic agents on blood flow in heart disease. Hemodynamic changes were assessed before and after administration of each drug by determining mitral regurgitant jet mapping area (MRMA) and aortic forward flow mapping area (AFMA), which were displayed by the color Doppler method, and the ratio of MRMA to AFMA (MRMA/AFMA) as parameters. When the four vasodilator drugs were used appropriately, MRMA and MRMA/AFMA decreased in all cases, compared with the values before the administration. These two parameters showed dose-dependent changes after administration of captopril, enalapril and hydralazine. When the cardiotonic drug was used, MRMA and MRMA/AFMA increased in 4 of 5 cases. The MRMA/AFMA values were slightly more reproducible than the MRMA values, whereas the AFMA values showed no constant tendency when any vasodilator drug or the cardiotonic drug was used. These results suggest that the efficacy of cardiotonic and vasodilator drugs in MR can be quantitatively evaluated by determining MRMA/AFMA in particular, and MRMA.

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Three eyes in two Siberian husky dogs were clinically diagnosed as persistent hyperplastic primary vitreous (PHPV) by means of ophthalmoscopy and ultrasonography (USG). Examination of mildly affected PHPV eyes with an ophthalmoscope showed the axial part of the posterior capsule to be opaque. The central lesion of the posterior capsule in severely affected eyes had been opaque with many blood vessels. Echographic changes in mild cases of PHPV were outside of the lens, linearly hyperechoic, parallel to the posterior lens capsule. In a severely affected eyeball, funnel-shaped hyperechoic change was noted in the retrolental space. Two months later, phacoemulsification was performed for diagnostic treatment of PHPV since progressive cataract was observed in this eye.

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Four estrous beagles were inseminated with 1 × 10⁸ sperm into both the right and left uterine horns, and the uterine horn and oviduct on one side were removed under anesthesia after 7 hr and 24 hr, respectively. The lumen of the uterine horns and oviducts was flushed with canine capacitation medium (CCM), and movement of the sperm in CCM was assessed by phase-contrast microscopy. In a second experiment, ejaculated sperm obtained from 5 normal beagles was incubated in CCM supplemented with oviductal flush fluid (OF-CCM) at 38°C with 5% CO₂ in air. Motility of sperm, and percentages of hyperactivated sperm (%HA) and acrosome-reacted sperm (%AR) among freely swimming (FS) sperm were investigated until 24 hr after the start of incubation. After 7 hr of incubation the sperm was coincubated with canine oocytes in OF-CCM for 2 hr, and the number of zona pellucida-binding (zona-binding) sperm was then counted. The %HA among the sperm in the oviductal flush fluid both 7 hr (mean ± S.E.; 15.0 ± 2.4%) and 24 hr (77.5 ± 5.2%) after intrauterine insemination were significantly higher than in the uterine flush fluid (P<0.05, 0.01, respectively). The motility and %HA among FS-sperm in OF-CCM were higher than in the control medium without oviductal fluid. However, there was no difference in the %AR between OF-CCM and control medium. The number of zona-binding sperm in OF-CCM (8 ± 1) was significantly greater than in control medium (5 ± 1) (P<0.05). These results suggest that oviductal fluid in the estrous bitch maintains sperm motility and induces sperm capacitation.

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In a cow diagnosed as having ovarian cysts, we observed changes in the ovarian structures by ultrasonography for 71 days and examined plasma concentrations of sex hormones. The cow had 2 regressing cysts at the start of this study and 3 new follicles subsequently developed into cysts. With regression of these cysts, 2 new follicles developed and ovulated spontaneously, followed by the formation of 2 corpora lutea. On the day prior to ovulation, a preovulatory luteinizing hormone (LH) surge was detected. With regression of the corpora lutea, a new follicle developed and underwent atresia. Meantime, another follicle developed and became a cyst without ovulation. No preovulatory LH surge was observed during the period from regression of the corpora lutea to cyst formation. The results indicate that absence of the preovulatory LH surge is associated with occurrence of ovarian cysts and this endocrine aberration is reversible.

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Recently, the authors have shown that marked necrosis and fibrosis of myocardium were observed in rats given alkaline ionized water (AKW). To clarify the cause of myocardial lesions, the activities of myosin ATPase, actomyosin ATPase and creatine kinase (CK) in myocardium of rats given AKW at 15 weeks-old were compared with those in myocardium of rats given tap water (TPW). Furthermore, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of myocardiac myosin and isoelectric focusing (IEF) of myocardiac CK were performed which revealed a distinct difference between AKW and TPW groups. The activities of myosin ATPase and actomyosin ATPase in the AKW group were higher than those in the TPW group, and these elevated activities were caused by the degradation of myosin in the AKW group judging from the SDS-PAGE pattern of myosin. On the other hand, the activity of CK in the AKW group was lower than that in the TPW group, and the IEF pattern of CK showed leakage of myocardiac CK. These results indicate that increases in actomyosin ATPase activity and myosin ATPase activity, plus the decrease in CK activity caused the disorder of coupled reaction in male rats given AKW at 15 weeks-old. It is concluded that this disorder of coupled reaction may cause marked myocardiac necrosis and fibrosis in rats given AKW.

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A monoclonal antibody (MAb) reactive with 36 field isolates and 2 laboratory strains of feline calicivirus (FCV) was produced by immunizing mice with the mixture of FCVs. The MAb (4D7) reacted with FCVs in an enzyme-linked immunosorbent assay (ELISA), but had no neutralizing activity against the F4 strain of FCV. MAb 8G1, previously produced against the FCV F4 strain, also reacted in ELISA with all FCVs used in the present study. However, the epitopes recognized by 4D7 and 8G1 were different. Using these two MAbs and a polyclonal rabbit antibody, we attempted to develop a sandwich ELISA for detection of FCV antigen. The combination of 4D7 and the polyclonal rabbit IgG was most sensitive. Using this system, all the field isolates of FCV cultured in vitro were detected. However, among the 36 swab samples, from which FCV was isolated, 4 were negative.

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The nucleotide sequences of the glycoprotein I (gI) and E (gE) genes of equine herpesvirus type 4 (EHV-4) strain TH20 were determined. The predicted region encoding the EHV-4 gI gene is 1,263 nucleotides, corresponding to a polypeptide of 420 amino acids in length. The predicted region encoding the EHV-4 gE gene is 1,647 nucleotides, corresponding to a polypeptide of 548 amino acids in length. The EHV-4 gI and gE genes show 74% and 85% identity at the amino acid level with those of equine herpesvirus type 1 (EHV-1), respectively. Furthermore, we have found an open reading frame homologous to the EHV-1 gene 75, which overlaps in part with the 3' end of EHV-4 gE gene. These sequence data will be useful for development of a modified live vaccine against equine herpesvirus type 1 and 4 infections.

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The emergence of virulent avian influenza viruses in poultry is unpredictable. To gain insight into the mechanism of this event, we previously examined the possible role of older (14-day-old) embryonated eggs, in which virulent mutants were preferably selected (Horimoto and Kawaoka, Virology 206: 755-759, 1995). However, it is unknown why virulent mutants replicate predominantly in older eggs. In the present study, we compared protease activities responsible for cleavage activation of the hemagglutinin (HA) in allantoic fluids in 10-day and 14-day-old eggs. In vitro assays showed that the protease activities were stronger in the14-day-old than 10-day-old eggs. The allantoic fluids with strong protease activity degraded HA. These results indicate that replication of avirulent viruses is hampered in older eggs, while that of virulent viruses whose HAs are activated by other intracellular proteases was not, possibly leading to a replicative advantage for virulent mutants in the older eggs.

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