The transverse sections of radius diaphysis in an 11-year-old giant Holstein cow with dermal dysplasia of a collagen disorder-related skin fragility (Cow 1), probably based on increasing turnover of the dermal collagen as reported previously, were morphologically and physico-chemically investigated. Cow 1 had about one and a half times as much as the body weight of normal Holstein cows, aged 5 to 6.5 years with stabilized growth. The bone samples were compared with those of a 12-year-old Holstein cow as controls (Cow 2). It has been reported that the long-bone diaphysis of young calves and some herbivorous dinosaurs are occupied with laminar bone showing a concentric appositional formation, and that such a laminar bone is characteristically seen during the growing period of some farm animals and large dogs that show very rapid growth rates. Cow 1 had a smaller number of osteons than Cow 2 in the outer-half layer of the diaphysis, and showed an intermediate type between Cow 2 and a 1-year-old Holstein ox in the entire layers, although their bone volumes were similar among them. There were no significant differences in Ca and P concentrations and the Vickers microhardness values between the bone matrix of Cow 1 and Cow 2. The bone-collagen fibrils of Cow 1 showed uneven diameters and a disordered arrangement. Thus, there may be some relation in collagen formation between the bone matrix of Cow 1 and the dermis. From the remaining volume of laminar bone, Cow 1, aged 11 years, had probably shown growth until quite recently, so that we consider that Cow 1 became a giant animal, in the same way as some herbivorous dinosaurs.

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The incisor development of fetal rats on gestation day 19 was well correlated with their fetal weights. The number of odontoblasts in the mandibular incisors, an index of incisor development, increased more than that of the maxillary incisors with increase in fetal body weights. Maternal acetazolamide treatments were observed to suppress the mean fetal weight and to retard incisor development. A smaller incisor size, a thinner predentin layer, and fewer odontoblasts were characteristic of the acetazolamide group. There was also a good correlation between the fetal weights and the number of odontoblasts in the acetazolamide group. From these results, we postulated that the retarded incisor development of the fetal rats caused by the maternal acetazolamide treatment was related to their suppressed fetal weights. However, the regression coefficient of the fetal weights and the number of odontoblasts in the acetazolamide group was smaller than that of the vehicle control group. It may indicate that retarded incisor development in response to maternal acetazolamide treatment is to some extent independent of suppressed fetal weight.

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The process of the disappearance of epithelial cells was examined in chicken cecal villi and follicle-associated epithelium (FAE). The apoptotic epithelial cells with intense DNA-fragmentation and their exfoliation were found in the villous tips. The epithelial cells with weak DNA-fragmentation were seen in the upper portion of the villi and their sparse exfoliations were also found there. Numerous epithelial cells in the intestinal lumen expressed the apoptotic features. A row of apoptotic epithelial cells with DNA-fragmentation was also found in the apical FAE, whereas no M cells exhibited any apoptotic signs. In all cecal regions, CD3⁺, CD8⁺, and TCR2⁺ lymphocytes were predominant in the epithelium at the upper portion of the villi and the FAE. CD4⁺ lymphocytes were mainly seen in the lamina propria. TCR1⁺ lymphocytes were not abundant in comparison with TCR2⁺ lymphocytes in the epithelium. TCR3⁺ T lymphocytes were rarely detected. These results suggest that the chicken cecal epithelial cells exfoliated into the lumen after the induction of the apoptosis, and that the induction may be involved with CD3⁺, CD8⁺, and TCR2⁺ lymphocytes. No death in M cells suggests that M cells may transform into microvillous epithelial cells.

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In addition to the lipoprotein-deficient d >1.25 fraction, haptoglobin was detected in the high-density lipoprotein (HDL) and the very high-density lipoprotein (VHDL) fractions from sera of calves with experimental pneumonia and cows with naturally occurring fatty liver. It was not found in the chylomicrons, very low-density lipoprotein and low-density lipoprotein fractions. Washing of the HDL fraction did not decrease the haptoglobin concentration. Transferrin and immunoglobulin G were immunoblotted to examine the possibility of contamination of the lipoprotein fractions by the d >1.25 fraction. The two serum proteins were detected only in the d>1.25 fraction, not in any lipoprotein fractions. The distribution pattern of haptoglobin in the lipoprotein fractions was distinct from that of serum albumin. Concentrations of haptoglobin in the HDL fractions from pneumonic sera were largely proportional to those in whole sera. Cholesteryl ester concentrations were decreased in sera from calves with pneumonia, as in cows with fatty liver. A protein immunologically related to hemoglobin was also detected in particular in the VHDL fractions from sera of both groups. These results suggest that haptoglobin or a complex with the hemoglobin-like protein may have a role or roles related to the lipid metabolism.

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MARC-145 cell monolayers infected with PRRS virus were fixed in 3% neutral buffered formalin and embedded in paraffin. The sections were stained by avidin-biotin complex immunoperoxidase (ABC) method. Test sera were applied to sections as primary antibodies. The positive reactions were detected by ABC method and indirect immunofluorescent assay (IFA). There was good correlation between ABC and IFA, and the titers in ABC were higher than those in IFA. The present results indicate that the immunohistochemical staining is a useful test for the detection and quantitation of PRRS virus antibody in swine sera as well as IFA.

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We investigated 12 anesthetized normal dogs using transesophageal echocardiography to understand the effects of respiration on the pulmonary venous flow. Additionally, we observed whether the diameter of the pulmonary vein changes with the heart beat. The pulsed Doppler wave form of pulmonary venous flow predominantly demonstrated two backward flows, with one peak occurring during ventricular systole and another during ventricular diastole. Sometimes a small forward flow occurred during left atrial contraction. In comparison with expiration, the peak velocity and velocity-time integral of the flow wave under inspiration occurred during both systole and diastole were significantly smaller. The diameter of the pulmonary vein decreased during left atrial contraction and increased during left ventricular systole and diastole.

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Laparotomy was performed on seven thoroughbreds to attach a force transducer to the proximal jejunum, distal jejunum, and ileum, as well as to the serous membrane of the cecum. Following observation of intestinal motility in conscious horses, they were intravenously injected with motilin (0.6 μg/kg) to examine its effect on intestinal motility. Strong contractions peculiar to horses were observed in small intestine. Further, motilin caused strong contractions in the proximal jejunum. The results suggested the involvement of motilin in the regulation mechanism of intestinal motility.

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Glutathione S-transferase fusion proteins of bovine interleukin-2 (IL-2), IL-4, IL-6 and interferon-γ (IFN-γ) were expressed in Escherichia coli. Complementary DNA (cDNA) for open reading frame of each cytokine without signal peptide encoding region was amplified by reverse transcriptional polymerase chain reaction method and was subcloned into pGEX-5X-1. In result, IL-6 and IFN-γ fusion proteins in bacteria were soluble, but IL-2 and IL-4 fusion proteins were insoluble. The insoluble IL-2 fusion protein successfully refolded by urea became soluble. The recombinant IL-2, IL-6 and IFN-γ could be obtained by the batch method using Glutathione Sepharose 4B and Factor Xa digestion, which may be useful for preparation of antisera as antigens and functional studies.

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A 24 kd protein from Rhipicephalus sanguineus (Rs24p) which was common to larvae, nymphs, male and female whole body and salivary gland extract of males and female was detected specifically in the serum from dogs after repeated infestation with adult R. sanguineus. The duration of antibodies against Rs24p in dogs infested with adults was examined by Western blotting analysis. Anti-Rs24p antibody was detected in two of 4 dogs during the period of 40 days in the first infestation. In the second infestation, all dogs showed positive reaction against Rs24p, but the duration of the antibodies varied greatly among the animals.

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We performed a pathologic examination of the brains of three dogs in an epileptic beagle colony. Histologically, all the cases had diffuse astrocytosis in the cerebral cortex and basal ganglia as well as the hippocampus, whereas they showed acute nerve cell change in the hippocampus and some other areas of the cerebrum. One of these animals showed laminar myelin pallor associated with the presence of many vacuoles in the IV to VI layers of the bilateral motor cortices. Most of the vacuoles contained fine granules stained with luxol-fast-blue stain. Ultrastructural examination revealed that some oligodendrocytes and perineuronal satellite oligodendrocytes in the bilateral cerebral motor cortices of the two affected dogs had many vacuoles surrounded by myelin-like lamellar structures. These findings suggest a possibility that astrocytosis in the cerebrum and vacuolar degeneration of oligodendrocytes in the cerebral motor cortex may be, at least in part, related to the occurrence or development of seizures.

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On March 22, 1998, a mature, male, hyposthenic Pacific striped dolphin (Lagenorhynchus obliquidens) was stranded at Aoshima Beach in Miyazaki prefecture, Japan. A necropsy performed 14 hr after death revealed mild diffuse congestion and edema of the leptomeninges and mild pulmonary atelectasis. Histopathologically, non-purulent inflammatory were observed throughout the cerebrum, thalamus, midbrain, pons, medulla oblongata, and spinal cord. Hematoxylin and eosin stain revealed no viral inclusion bodies. Immunohistochemistry using a monoclonal antibody against nucleoprotein of canine distemper virus (CDV-NP) revealed a number of CDV-NP-positive granular deposits in the cytoplasm and cell processes of the degenerating or intact neurons. The present paper is a first report of spontaneously occurred morbillivirus infection in a dolphin at the Pacific Ocean around Japan.

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Pathological studies were carried out on the lungs of guinea pigs intratracheally inoculated with 4.6 × 10^6-8 colony forming units (CFU)/head of Actinobacillus pleuropneumoniae serovar 1. All animals in the highest dose group died within 24 hr post inoculation (hpi) and showed pulmonary lesions being hemorrhagic in nature while all animals in the lowest dose group were killed as scheduled at 11 days post inoculation (dpi) and showed only hyperplasia of peribronchial lymphoid tissues. In the middle dose group, two died within 24 hpi, two died at 9 dpi, and the remaining one was killed at 11 dpi. Two guinea pigs which died at 9 dpi showed fibrinonecrotic pleuropneumonia which is the most characteristic acute pulmonary lesion in swine, and has not yet been reproduced in laboratory animals up to the present time. This suggests that guinea pigs may be a useful laboratory animal for studying the pathogenesis of Actinobacillus pleuropneumoniae infection in swine.

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A female Shetland sheep dog died suddenly with hemorrhagic diarrhea and vomitting, and was examined pathologically and microbiologically. Gross pathological change was restricted to the intestinal tract. The intestine contained watery, blood-stained fluid. Histopathologically, the principal intestinal lesion was superficial mucosal hemorrhagic necrosis at the jejunoileum. Many Gram-positive bacilli were found adhering to the necrotic mucosal surface in parts of the intestinal tract. Clostridium perfringens in pure culture were isolated from jejunal contents by anaerobic culture. These results suggested that the typical lesion of this case coincided with canine hemorrhagic enteritis and enterotoxemia due to C. perfringens infection could be the cause of sudden death.

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Genomic DNAs of 14 mammary tumors were analyzed by Southern blot hybridization using a human c-yes-1 oncogene probe. The amplification was successful in half of the cases (7 adenocarcinomas). The degree of amplification was approximately 4-fold, and a high proportion was seen in malignant tumors. In addition, DNA polymorphism was detected in two adenocarcinomas.

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A malignant basal cell tumor was found in the skin of the abdomen of a female Djungarian hamster of unknown age and weighing 40 g. Histologically, the tumor mass was composed of cells resembling the basal cells of the epidermis, and these cells exhibited solid type proliferation. In the tumor tissue and necrotic foci, horn cysts were observed. Immunohistochemically, cytokeratin was present in the tumor cells and horncysts. By electron microscopic examination, the tumor cells had scanty cell organelles and a few desmozomes. This paper describes a rare malignant basal cell tumor in a Djungarian hamster.

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The aim of the present study was to examine the signaling pathways for a low dose of angiotensin II (ANG II) on Na⁺ uptake of primary cultured rabbit renal proximal tubule cells (PTCs) in hormonally defined serum-free medium. The results were as follows; ANG II (10^-11 M) stimulated the proliferation of PTCs. 10^-11 M ANG II stimulated Na⁺ uptake by 20%, whereas 10^-9 M ANG II inhibited it by 20% (p<0.05). The stimulatory effect of 10^-11 M ANG II on Na⁺ uptake was inhibited by amiloride (10^-3 M) and by losartan (ANG II receptor subtype 1 antagonist, 10^-8 M) but not by PD123319 (ANG II receptor subtype 2 antagonist, 10^-8 M). Pertussis toxin (PTX, 50 ng/ml) prevented the ANG II-induced stimulation of Na⁺ uptake (p<0.01). 8-Bromoadenosine 3', 5'-cyclic monophosphate (8-Br-cAMP, 10^-6 M) did not affect Na⁺ uptake. SQ 22536 (adenylate cyclase inhibitor, 10^-6 M) also did not change the ANG II-induced stimulation of Na⁺ uptake. ANG II did not stimulate cAMP production. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.01 ng/ml) produced significant increase in Na⁺ uptake. When ANG II and TPA were added together to the PTCs, there was no additive effect on Na⁺ uptake. Staurosporine (calcium-dependant protein kinase C inhibitor, 10^-6 M) led to a complete inhibition of ANG II-induced stimulation of Na⁺ uptake. ANG II-treatment resulted in a 26% increase in total protein kinase C (PKC) activity. However, 10^-11 M ANG II did not change [Ca²⁺]_i mobilization and [³H]-AA release while 10^-9 M ANG II increased both of them. In conclusion, the PTX-sensitive PKC pathway may be the main signaling cascade in the stimulatory effects of low dose of ANG II (10^-11 M) on Na⁺ uptake in the primary cultured rabbit renal proximal tubule cells in hormonally defined serum-free medium.

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For a serological diagnostic test for Borna disease (BD), we developed a capture ELISA with specificity and sensitivity based on detection of antibodies against BD virus (BDV) p40 protein. Using our capture ELISA system, the antibody response of rats inoculated intracerebrally with BDV at 4 weeks after birth showed a sharp increase from 1 to 4 weeks postinoculation (p.i.) and a steady level after 5 weeks p.i. To investigate prevalence of BDV infection among wild rats, we examined sera of Rattus norvegicus in Kami-iso town, Oshima district, Hokkaido, suggesting that rats in this area had not been infected by BDV.

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Hyaluronidase, acrosin and N-acetylhexosaminidase activities were examined in sperm collected from 12 beagle dogs and in culture medium after 0.5 hr and 7 hr of sperm incubation. The activities of the three enzymes were significantly higher at 7 hr than at 0.5 hr (P<0.05, 0.01), and the increases were associated with sperm capacitation. It was considered that the three enzymes in the dog sperm are related to fertilization by reason of the findings of the release of these enzymes from the sperm into the medium after 7 hr of incubation.

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The in vivo toxic-hemolytic studies using small experimental animals are complicated by difficulties in preventing hemolysis during repeated collection of blood specimens and in measuring hemoglobin concentration in small amounts of plasma sample. To solve these problems we tried to develope the new techniques for the in vivo hemolysis studies using guinea pigs. The hemolysis accident was minimized to 2.75 mg/dl by collecting the blood directly into heparinized microhematocrit tubes by small longitudinal incision in the auricular artery. The hemoglobin in a small amount of sample (10 μl) was determined by the new analytical system using a microflow spectrophotometer with a modified cyanmethemoglobin method. The standard curve of the hemoglobin concentration in the system revealed a line of Y=1.8X + 0.79 (r=0.999), CV<1% with a minimum detectable concentration of 1.25 mg/dl. By using the new techniques, it was found that the plasma hemoglobin concentration in normal animals were 7.27±0.44 mg/dl (mean±S.E.). The in vivo hemolytic activity of saponin was observed dose-dependently at doses of 30-50 mg/kg, i.v. in the guinea pigs. It is concluded that the present techniques are useful for in vivo hemolytic studies in small experimental animals such as guinea pigs.

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This study was conducted to elucidate the hemolytic activity of a new toxic substance in bracken fern. A crude extract (CE) was prepared from the methanol extracts of bracken by the column chromatography. When the CE was injected subcutaneously in guinea pigs, the hemoglobinuria and hemolysis were observed within 6 hr, and 3 days later edema and hemorrhages in the urinary bladder were observed. The CE was then fractionated by high performance liquid chromatography (HPLC), and three (HF, BF and CF) of the fractions showed the toxic activities in guinea pigs. The HF caused the hemolysis, whereas both the BF and the CF caused the hemorrhagic cystitis without any hemolytic activities. The HF was further fractionated by the HPLC, resulting of the 3 fractions (HF-I, II and III). The hemolysis was caused only with the HF-II, and HF-II as well as HF did not cause the hemorrhagic cystitis. HPLC analysis revealed that both BF and CF contains braxin B and braxin C, respectively, and both HF and HF-II do not contain braxin A, B or C. These facts suggest that bracken fern contains a new toxic substance (hemolysin) which induces the acute hemolysis in guinea pigs.

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The seroepidemiological survey of cats conducted in northern part of Taiwan in 1998 revealed that the positive rate of feline immunodeficiency virus (FIV)-infection was 21.9% (7/32) and the rate was much higher than those of previous reports. We succeeded in isolation of three strains of FIV from peripheral blood mononuclear cells of the blood samples. Nucleotide sequences of the env variable V3 to V5 region of the strains revealed that the isolates from distinct areas belong to subtype C. These data together with our previous report (Inada et al. 1997. Arch. Virol., 142: 1459-1467) indicate that FIV subtype C is prevalent in northern part of Taiwan.

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