To obtain the data required for identification of skeletal remains excavated from archaeological sites, histometrical observations were made in the cross sections of the mid-shaft of humerus, radius, femur and tibia of raccoon dogs (Nyctereutes procyonoides) and badgers (Meles meles) captured in Kagoshima Prefecture. There were interspecific differences between both animals in the breadth, the depth and the area of medullary cavity at the mid-shaft of the bones. In badgers, all measurements were greater in male than in female bones. The thickness and the area of compact bones in male raccoon dogs were larger than those of female. No differences in histological structure could be detected among the bones, but an interspecific difference was found in the shape of osteons; round and constant-sized osteons consisting of 3 to 5 lamellae in raccoon dogs, while round or elliptic osteons varying in size from 3 to 8 lamellae in badgers. The ratios, the osteon areas per unit compact bone areas, were higher in all the bones of raccoon dogs. The short diameters of osteons and the ratios were greater in males in both animals. In females, the short diameter of osteons was smaller, and the number of osteons was larger. The results revealed interspecific differences between both animals and sexual dimorphism in each species.

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The monoclonal antibody (MAb), named TSd-1, specific to spermatogenic cells of the common tree shrew (Tupaia glis) was established and characterized using immunohistochemistry and immunoblotting. MAb TSd-1 reacted with elongating and matured spermatids in a stage-dependent manner. TSd-1 recognized a 94 kilodalton (kDa) peptide in the plasma membrane and cytosol. Additionally, an extremely weak 107 kDa band was detected only in the cytosol. The reactions were not detected in round spermatids. In elongating Stage VI spermatids, the plasma membrane and the granular structure within the cytoplasm were intensely positive, and most intense after the appearance of new round spermatids in the lower layer (Stage I). The reactions were observed neither in the other organs of the common tree shrew nor in the testes of other animals, indicating that TSd-1 antigen is specific to the spermatogenic cells of the common tree shrew, and may act on elongating or matured spermatids.

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This work was conducted to determine whether aspirin and ibuprofen, when administered prenatally may potentiate a re-opening of the neonatal ductus arteriosus (DA) induced by PGE₂ after postnatal closure. In the first experiment, a subcutaneous injection of PGE₂ (4 μg) was administered to newborn rats 3 hr after a Cesarean delivery from pregnant females which had been orally given 100 or 300 mg/kg/day of aspirin and 10 or 30 mg/kg/day of ibuprofen on days 18, 19 and 20 of gestation. The ratio of the DA to the pulmonary artery (PA) was determined at intervals after the injection. The DA/PA ratio was significantly higher in newborn rats from mothers who were transplacentally administered these agents than the control. We also examined the hypothesis that maternal treatment with nonsteroidal anti-inflammatory drugs (NSAID), such as aspirin and ibuprofen, inhibits the catabolism of PGE₂ and that the increased re-opening of the DA was partly due to this inhibition. 15-hydroxy prostaglandin dehydrogenase (15-PGDH) in neonatal lungs, the key enzyme involved in catalyzing PGE₂ to convert it to its inactive metabolite 15-keto-PGE ₂, was not affected by maternal treatment with aspirin and ibuprofen. These results suggest that the increased ductal responsiveness to PGE₂ in newborn rats was a common response after maternal NSAID treatment, but the catabolism of PGE₂ in the lungs did not always contribute to this response.

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With a polymerase chain reaction (PCR) method, goose parvovirus (GPV) DNA was detected in Muscovy ducklings inoculated with attenuated GPV strains, IH and IHC. Strain IH that had been passed 20 times in Muscovy duck embryos could be detected in ducklings at 2- to 28-days after oral inoculation by PCR, however, a cell culture adapted strain IHC that had been passed 15 times in Muscovy duck embryos and then successively 50 times in Muscovy duck embryo fibroblasts could not be detected by 6 days post-inoculation by the oral route, but via intramuscular inoculation the virus was detected from 6 dpi. With both strains Muscovy ducklings produced neutralizing antibodies against GPV, but GPV could be recovered from heart muscles even in birds that had high titer of neutralizing antibody. This means that GPV remains in birds for a long period under the presence of high titer of neutralizing antibody in the serum. Recovery of the virus was consistent with PCR results with one exception in which the bird had a neutralizing antibody titer of more than 100,000. After inoculation of these strains, no clinical signs were detected in ducklings. These results suggest that strains IH and IHC can be candidates for live attenuated vaccine for GPV infection.

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Southern blot hybridization of DNA samples from 9 primary tumors of avian lymphoid leukosis (LL) rapidly induced byALV infection 27-74 days post inoculation was carried out to search for rearrangement of the c-myc gene with human c-myc gene exon III as a probe. Rearrangement of the c-myc gene was detected by appearance of new EcoRI fragments in 7 out of 9 tumors examined. The size of the fragments ranged from 3.1 to 4.0 kilobases (kb). In addition to these fragments, two fragments (9.0 kb and 13 kb) were observed in one tumor, and a faint fragment (3.5 kb) was observed in another tumor. Rearrangement of the c-myc gene was not detected in the remaining two tumors kept in unsuitable condition. These results suggest that rearrangement of c-myc gene was induced even in rapidly induced LL as well as that induced after long incubation period. This is the first report of involvement of c-myc gene in rapidly induced B-cell lymphoma (LL).

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We compared the effects of Pasteurella multocida toxin (PMT) with Bordetella bronchiseptica dermonecrotizing toxin (DNT) at a cellular level under same conditions. Both PMT and DNT cause actin stress fiber formation in MC3T3-E1 cells which is known to be regulated by the small GTP-binding protein Rho. DNT induced mobility shifts of Rho on SDS-polyacrylamide gel electrophoresis, indicating direct modification as reported elsewhere. In contrast, no alternations in the electrophoretic mobility of Rho were found in lysates from PMT-treated cells. PMT but not DNT increased the intracellular level of inositol phosphates, indicating the elevation of phospholipase C (PLC) activity in the PMT-treated cells. These results indicate that PMT does not have Rho as a target but activates PLC. The formation of actin stress fiber by PMT seems to be stimulated through the indirect activation of Rho, which resides downstream of PLC. PMT and DNT seem to elicit similar toxic effects, at least in part, through the activation of Rho.

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Effects of the deleted form of hepatocyte growth factor (dHGF) on serum hyaluronate levels, an index for liver cirrhosis, were studied in rats. The levels of serum hyaluronate increased in rats with dimethylnitrosamine- or carbontetrachloride-induced cirrhotic liver with prolongation of prothrombin time, which indicates disorder of liver function. Daily intravenous injection of dHGF reduced the elevated serum hyaluronate levels with improvement of the prolonged prothrombin time. These results suggest that the amelioration of hepatic function disorder by dHGF leads to a reduction of the increased serum hyaluronate levels.

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Previous studies have shown that lactoferrin induces growth inhibitory effects in mouse macrophages against intracellular Toxoplasma gondii, and these effects were not mediated by the oxygen-dependent and inorganic nitrogen-dependent pathway. To clarify the mechanism of anti-Toxoplasma gondii activity induced by lactoferrin, we examined whether lactoferrin promoted the phosphorylation of tyrosine residues in macrophage proteins. In immunoblotting assays using anti-[phosphorylated tyrosine] monoclonal antibody, phosphorylation of tyrosine residues was detected in protein(s) of approximately 30 kDa in macrophages incubated with lactoferrin. Inhibition of the lactoferrin-induced tyrosine-phosphorylation by genistein led to loss of the lactoferrin-induced growth inhibitory effect against the parasites. These findings suggest that lactoferrin induces tyrosine-phosphorylation in macrophages, and the tyrosine-phosphorylation seems to be associated with the induction of the growth inhibitory activity exerted against intracellular Toxoplasma gondii.

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Male copulatory behavior and the function of the hypothalamo-hypophysial-gonadal axis in hypothyroid male rats were investigated in the present study. Hypothyroidism was induced by thyroidectomy or thiouracil. In male copulatory behavior test, intromission latencies in hypothyroid rats were significantly longer than those in euthyroid rats and ejaculation frequencies were reduced in hypothyroid male rats compared to control rats without reduction of plasma concentrations of testosterone. These changes in copulatory behavior in hypothyroid male rats were restored to control levels by administration of T4 (5 μg/rat). Hypothyroidism decreased adrenal weights, and basal and peak concentrations of corticosterone during diurnal variation, whereas it increased peak concentrations of ACTH in adult male rats. These results indicate that hypothyroidism causes adrenal dysfunction directly and results in hypersecretion of ACTH. The adrenal disturbance observed in hypothyroid rats may affect male copulatory behavior.

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A monoclonal antibody, 6E9 established from mice injected with dolphin peripheral blood lymphocytes (PBLs) was characterized. In addition to its reactivity against 89.4% of dolphin PBLs, 6E9 reacted with 33.1% of bovine PBLs of which 22% were CD5 ⁺, 11.1% were CD5^-. 6E9 recognized a 34 kD protein on the surface of dolphin and bovine PBLs. Analysis of the protein's N-terminal amino acid sequence indicated that 6E9 recognizes bovine major histocompatibility complex (MHC) class II antigens. These results suggested that 6E9 recognized MHC class II antigens on bovine PBLs. As we have already produced an anti-dolphin MHC class I monoclonal antibody, analysis of immune system using these monoclonal antibodies will advance our understanding of the evolution of the mammalian immune system.

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A variety of virulence factors (VFs) such as type 1 fimbriae, pilus associated with pyelonephritis, S fimbriae, afimbrial adhesin, α-hemolysin, aerobactin and cytotoxic necrotizing factor 1 are associated with uropathogenic Escherichia coli. In this study, 80 uropathogenic E. coli strains in 50 dogs and 30 cats suffering from UTI. In addition, 60 E. coli strains were isolated from fecal samples from 30 each of healthy dogs and cats. The distribution of VFs of uropathogenic E. coli strains isolated from dogs and cats suffering from urinary tract infections (UTI) were examined by the colony hybridization test with seven DNA probes specific for VFs, and the results were compared with those obtained in the studies on strains from humans with UTI. In uropathogenic E. coli strains isolated from dogs and cats suffering from UTI, VFs were detected as frequently as in the strains isolated from humans with UTI. Although less frequently, genes encoding these VFs especially pap, sfa, hly and cnf 1 genes were also associated with E. coli strains isolated from feces of healthy cats, in contrast to the distribution pattern of uropathogenic E. coli observed in humans. Furthermore, all VFs except pil were significantly more frequently detected in strains isolated from urine of animals with cystitis than in those isolated from feces of healthy humans. These results indicate that VFs of E. coli contribute to the pathogenesis of UTI in dogs and cats.

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A single injection method for estimating glomerular filtration rate (GFR) by measuring plasma inulin and creatinine clearances was evaluated in 10 healthy cats. GFRs were estimated from the plasma clearance (PC) by dividing the injected dose of an indicator by the area under the plasma disappearance curve (AUC). AUC was determined by 2 common pharmacokinetic analyses, the two-compartment model and the trapezoidal rule. AUCs determined by these two methods were significantly correlated both in inulin (r=0.993) and creatinine (r=0.959). To minimize errors, GFR was estimated by PC only if AUC/10 was greater than the area under the curve from the final sampling time to infinitive (A2). GFRs determined by PC of inulin at final sampling time of 180 and 240 min were 3.61 ± 0.64 and 3.63 ± 0.67 ml/min/kg of body weight (mean ± SD), respectively. These values corresponded to the reference range reported for normal cats. In contrast, when creatinine was used as a maker, A2 was always greater than AUC/10 at any final sampling time and GFRs estimated using these AUCs of creatinine were significantly greater than those of inulin, suggesting creatinine may not be suitable indicator for the single injection method.

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To observe pulmonary venous flow in dogs, the echocardiographic imaging planes and the techniques for examination, and the validations of anatomic location were investigated. Then, the velocity pattern of pulmonary venous flow was recorded in normal conscious dogs. Six imaging planes were available for observing the right or left caudal lobe pulmonary venous flow with two-dimensional or pulsed Doppler echocardiography. Of these, the left lateral apical 4-chamber view can be applied as standard view, because the pulmonary venous flow and transmitral flow could be recorded in this view simultaneously with small sampling angle. The velocity pattern of pulmonary venous flow demonstrated two forward waves in 19 of 20 dogs examined, with one peak occurring during ventricular systole and another during ventricular diastole. A reversed flow during atrial contraction was also seen in 11 dogs. In the two forward waves, the mean peak velocity and velocity-time integral of ventricular diastolic forward flow were significantly higher than those of systolic forward flow (46.49 ± 6.79 vs. 31.13 ± 4.92 cm/s, p<0.0001 and 8.18 ± 1.84 vs. 5.14 ± 0.82 cm, p<0.0001, respectively). The deceleration time of diastolic forward flow shortened with the increase of heart rate (r=^-0.87, p<0.0001). Pulmonary venous flow in dogs can be observed under transthoracic two-dimensional or pulsed Doppler echocardiography.

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The Otsuka Long-Evans Tokushima Fatty (OLETF) rat has been recently established as the best model of non-insulin-dependent diabetes mellitus (NIDDM) with mild obesity. In this study, we found that the F₁ progeny produced from the crosses of OLETF and F344 rats exhibit a reciprocal cross effect on NIDDM-relevant phenotypes, fasting and postprandial glucose levels and body weight, suggesting the existence of X-linked locus affecting susceptibility to NIDDM. We thus examined the linkage between 7 X-linked microsatellite markers and NIDDM-relevant phenotypes, using 160 (OLETF × F344)F ₂ progeny. Suggestive evidence for a X-linked locus affecting glucose levels at 120 min after glucose administration was found in a region near X-linked marker, DXMgh4. The identified locus also showed significant effects on fasting glucose levels and body weight.

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The morphology of the uterine wall in the postpartum Chinese hamster (Cricetulus griceus) was studied with particular reference to the changes in the arterioles in the mesometrial endometrium. At day 0 postpartum, the endometrial arterioles were characterized by the appearance of trophoblastic giant cells plugging ruptured arterioles. On days 1 to 3 postpartum, the giant cells were replaced by epitheloid cells. On days 4 to 5 postpartum, the epitheloid cell mass was invaded by fibroblasts to occlude the arterial lumens. On days 6 to 7 postpartum, endometrial regeneration was completed. The giant cell might play a role in hemostasis immediately after parturition.

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Sarcocystis suihominis was detected for the first time in Japan from the heart and diaphragm of 5 out of 600 older culled breeding pigs slaughtered in Saitama Prefecture, Japan. Fresh cysts were 1,080-2,040 × 106-170 μm in size. Bradyzoites measured 15 × 4 μm on average. The cyst wall was usually observed thick, 4-6 μm, and striated, but occasionally thin and smooth according to the difference in sectioning angle and in portion of cysts. Scanning electron microscopy showed that many palisade-like villar protrusions, 6-7 × 0.3-0.5 μm in size, were closely folded onto the surface of cyst. A small number of microtubules were seen in the core of protrusion. No dogs nor domestic cats fed with 20 fresh cysts each excreted oocysts or sporocysts in the feces throughout the experimental period of 30 days.

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To study the neurophysiological functions of metallothioneins (MTs), localization of MT-I and -II was examined immunohistochemically in a variety of brain lesions in dogs, including infarct, laminar cortical necrosis, hemorrhage, invasive growth of tumour, inflammatory lesions in granulomatous meningoencephalitis and distemper encephalitis. MT-I and -II were demonstrated in both nucleus and cytoplasm of hypertrophic astrocytes in most brain lesions examined regardless of the type, size, localization and duration of the lesions. In addition, MT expression was stronger in a population of hypertrophic astrocytes localizing inside of the surviving brain tissue rather than those localizing at the boundary between the surviving brain tissue and necrotic area, where severe inflammatory changes were developing. These results suggest that MT-I and -II may play roles not only in protection of neurons from metals and free radicals ubiquitous in the inflammatory lesions but also in repair of injured neural tissues.

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A thyroid gland tumor, showing unusual histology, was identified in a 13-year-old male Andalusian horse. Microscopically, the tumor consisted of neoplastic proliferation of C-cell (parafollicular cell) with cytoplasmic fine granules, containing diffusely distributed, variously sized colloid-containing follicles. Immunohistochemically, the neoplastic C-cell were positive for calcitonin and follicle-forming epithelial cells showed a positive reaction for thyroglobulin. Ultrastructurally, membrane-bound secretory granules up to 250 nm in diameter were found in the cytoplasm of the parafollicular cells, whereas the follicular epithelial cells had microvilli, junctional complex, and well-developed endoplasmic reticulum.

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Raw milk samples from bulk tanks of a total of 943 farms, which corresponded to approximately 60% of all dairy farms in Nagano Prefecture were examined for Listeria species between December 1990 and April 1991. Listeria spp. were isolated from 29 (3.1%) of 943 milk specimens. In the southern, central, eastern and northern areas of the prefecture, Listeria spp. were isolated from 6.1% (22/362), 1.5% (4/272), 1.4% (2/143) and 0.6% (1/166) of samples, respectively. Listeria monocytogenes was isolated from three (0.3%) bulk tanks in the southern area: two of the strains isolated from two different farm bulk tanks were serovar 4b, and the other one was 1/2a. Besides, between February 1991 and January 1992, 504 samples of raw milk from farm bulk tanks were collected nine times from 56 farms in the southern area, where the prevalence of Listeria spp. was the highest, and examined for the seasonal variation in the presence of Listeria spp. The prevalence of Listeria spp. was higher in spring (14.3%) than in autumn (4.8%). The 56 farms were divided into three groups according to the prevalence of Listeria spp., namely, three farms in Group 1 gave a high contamination rate (50%≤), 14 farms in Group 2 a low contamination rate and the remaining 39 farms in Group 3 no recovering of Listeria spp. Sixteen strains of L. monocytogenes serovar 4b were isolated from four farms.

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In vitro matured bovine oocytes were co-incubated with sperm for 18 hr in a droplet of fertilization medium under a gas atmosphere of 5% CO₂ with 5 or 20% O₂. After removing the cumulus cells, they were fixed to examine their fertilization rate, or cultured for another 154 hr in a chemically defined medium under 5% O₂ to determine their development to the blastocyst stage. There was no difference between the 5 and 20% O₂ groups in the fertilization rate. However, the percentage of inseminated oocytes which developed to the blastocyst stage was higher when in vitro insemination was conducted under 5% O₂ compared with that under 20% O₂ (34.4 vs. 24.7%, P<0.05).

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To determine whether pseudorabies virus (PRV) infection increases the severity of pneumonia by Mycoplasma hyopneumoniae, 18, 10-week-old Cesarean-derived, colostrum-deprived pigs were randomly assigned to 3 groups of 6 pigs each. Pigs in groups A and C were inoculated intranasally with M. hyopneumoniae at 10-week-old. At 11-week-old, pigs in groups B and C were inoculated intranasally with PRV. All pigs were initially seronegative for M. hyopneumoniae and PRV. Three pigs of each group were euthanized at 12-week-old, and remaining pigs at 14-week-old. At necropsy, gross lesions in the lung were observed in the pigs of groups A and C. On post-inoculation-week (PIW) 2 with M. hyopneumoniae (at 12-week-old), lung lesions were recognized in one of the 3 pigs in group A and all the pigs in group C. The mean percentage of the lung lesions were 0.1% in group A and 9.8% in group C. M. hyopneumoniae was isolated from broncho-alveolar lavage fluids (BALF) of pigs in group A with titer of 10² to 10³ CCU/0.2 ml and in group C with titer of 10⁵ to 10⁶ CCU/0.2 ml. On PIW 4 (at 14-week-old), lung lesions were observed in all the pigs in groups A and C, and the mean percentage of the lung lesions were 8.3% in group A and 17.2% in group C. M. hyopneumoniae was isolated from BALF in group A with titer of 10⁴ to 10⁷ CCU/0.2 ml and in group C with titer of 10⁶ to 10⁷ CCU/0.2 ml. PRVs were isolated from nasal swab and tissue samples in groups B and C. After inoculation, antibody against M. hyopneumoniae was detected in groups A and C, and against PRV in groups B and C. Under the present experimental conditions, PRV infection appear to have effect on the severity of experimentally induced acute mycoplasmal pneumonia in young pigs.

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Three specific pathogen-free cats experimentally infected with feline immunodeficiency virus (FIV) strains Petaluma, TM1 and TM2, respectively were observed for over 8 years. Without showing any significant clinical signs of immunodeficiency syndrome (AIDS) for 8 years and 4 months of asymptomatic phase, the Petaluma-infected cat exhibited severe stomatitis/gingivitis, anorexia, emaciation, hematological and immunological disorders such as severe anemia, lymphopenia, thrombocytopenia, and decrease of CD4/CD8 ratio to 0.075, and finally died with hemoperitoneum at 8 years and 8 months post-infection. Histopathological studies revealed that the cat had systemic lymphoid atrophy and bone marrow disorders indicating acute myelocytic leukemia (aleukemic type). Plasma viral titer of the cat at AIDS phase was considerably high and anti-FIV antibody titer was slightly low as compared with the other FIV-infected cats. In addition, immunoblotting analysis using serially collected serum/plasma samples of these cats revealed that antibodies against FIV proteins were induced in all the infected cats, however in the Petaluma-infected cat anti-Gag antibodies disappeared during the asymptomatic period. These results suggested that plasma viral load and anti-FIV Gag antibody response correlated with disease progression, and supported FIV-infected cats as a suitable animal model of human AIDS.

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We previously constructed a simian immunodeficiency virus/human immunodeficiency virus type 1 (HIV-1) chimeric virus, NM-3rN to generate a pathogenic HIV-1 in macaque monkeys. During the in vivo passage of this virus in several monkeys, a viral strain, R43-56 was obtained which acquired a better replication ability in vivo. MM121, one of the three monkeys inoculated with the R43-56, showed weight loss, diarrhea and a rapid and continuous decrease in CD4⁺ lymphocytes at the moribund stage. An autopsy revealed generalized lymphadenopathy, dehydration, and ileocecal intussusception. In situ hybridization showed that the virus infection was in systemic lymphoid organs. We are presently monitoring the survivors to obtain candidates for a more virulent virus. R43-56 may be a better challenge virus and useful tool for human acquired immunodeficiency syndrome research.

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Analysis of the molecular properties of fusion (F) proteins of field isolates of canine distemper virus (CDV) by immunoprecipitation analysis revealed an identical molecular mass of F protein of 3 field isolates as well as the Onderstepoort laboratory strain. Sequencing showed that the F gene of a field isolate (the Yanaka strain) shared 90.1% and 95.7% identities with the Onderstepoort strain at nucleotide and amino acid levels, respectively. All of the 13 cysteine residues and 4 potential asparagine-linked glycosylation sites were completely conserved amongst these strains. These results indicate that the F proteins is much less heterogeneous than that observed in the hemagglutinin proteins of CDV.

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This trial was performed in a 350-sow farrow-to-finish pig farm in which animals had been vaccinated against Aujeszky's disease with glycoprotein gC deletion (gC(-)) vaccine. The prevalence of antibodies against field pseudorabies virus (PRV) in serum samples collected from sows and fattening pigs during the period March 1995 to May 1997 was determined by using a commercial assay kit. The frequencies with which PRV-positive sows were found were 28.9% and 25.0% in March and October of 1995, 12.5% and 2.5% in April and October of 1996, and 0% in May of 1997. The fattening pigs tested at these times had no antibody to field PRV. These results thus indicate the probability that the PRV infection at the farm was eliminated by the gC(-) vaccine.

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