The regional distribution and relative frequency of endocrine cells was studied immunohistochemically (PAP method) in the alimentary tract of the red-bellied frog, Bombina orientalis, using antisera against serotonin, somatostatin, chromogranin (CG), cholecystokinin (CCK)-8, bombesin, secretin, glucagon and pancreatic polypeptide (PP). Eight kinds of endocrine cells were identified in this study. These immunoreactive cells were located in the gastric glands of the stomach regions and in the intestinal or esophageal epithelium with variable frequencies. They were spherical or spindle-shaped. Serotonin- and somatostatin-immunoreactive cells were demonstrated in the whole alimentary tract including esophagus. CG-immunoreactive cells were restricted to the stomach. CCK-8-immunoreactive cells were observed from the antrum to the ileum. Bombesin-immunoreactive cells were restricted to the stomach. Secretin-immunoreactive cells were demonstrated in the pylorus, duodenum and ileum. Glucagon-immunoreactive cells were found in the antrum and duodenum. PP-immunoreactive cells were detected from the antrum to the rectum. In conclusion, throughout the alimentary tract of the red-bellied frog, the different regional distribution and relative frequency of endocrine cells were demonstrated. The regional distributions and relative frequencies of the endocrine cells in the alimentary tract of the red-bellied frog were resembled to those of the other anuran species except for esophagus.

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The pattern of cerebrovascular substance P (SP)- and calcitonin gene-related peptide (CGRP)-immunoreactive (IR) innervation was investigated in the quail. SP- and CGRP-IR nerves were relatively a few in the rostral part of the anterior circulation, and very scanty or lacking in its caudal part and the whole of the posterior circulation. A significant finding was that the anterior circulation in the majority of individuals is furnished with a varying proportion of SP-IR nerves with or without CGRP immunoreactivity. There was a good correlation in the expression of CGRP immunoreactivity between SP-IR cells in the ophthalmic division of the trigeminal ganglion and SP-IR nerves supplying the major cerebral arteries. In the quail, SP- and CGRP-IR fiber bundles are usually present in the internal ethmoidal artery (IEA). From these and other findings, it is most probable that cerebral perivascular SP- and CGRP-IR nerves are mainly derived from the same categories of neurons in the primary sensory ganglion via the IEA. The close association of varicose SP-IR axons to the nerve cells in the pial arteries suggests that these intrinsic neurons may play some vasocontrolling roles through the modulatory effect of their pericellular SP-IR axons.

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The relationship between release properties of the model antigen, bovine serum albumin (BSA), from formulations in vitro and immune response after administration of various oil adjuvanted vaccines containing liquid paraffin was examined in chickens. The vaccine prepared at an hydrophile-lipophile-balance (HLB) number of 4.8 showed slower release of BSA and higher immune response on injected chickens than that with an HLB number of 6.0. Decreases of aqueous volume ratio in the formulation also led to slower release of BSA and higher immune response. The slower release rate of BSA showed higher ELISA antibody titer even at 20 weeks after vaccination. The ELISA antibody titer inversely was related to the constant release rate, k, calculated from the in vitro release test. The correlation coefficient was 0.863. The immune response of oil adjuvanted vaccines containing Haemophilus paragallinarum agreed well with these results with BSA. Our results indicated that a stronger and more prolonged immune response of oil adjuvanted vaccines was achieved by slower release rate of antigen from the formulation. In addition, there was a good correlation between immune response and the value of k.

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The antigenic polypeptides in Salmonella Enteritidis (SE) were chronologically recognized by the chicken immune system, using Western immunoblotting. Broiler chicks challenged at three days of age with SE strain carrying the most prevalent plasmid profile of 14.1 and about (∼) 50 kb were bled at 17, 24, 31, 38 and 45 days of age. Pooled sera of blood collected at each age was reacted by Western immunoblotting with banded polypeptides of three predominant SE strains that acquired the following respective plasmid profiles: 14.1 kb; 14.1 and ∼50.0 kb; and 1.8, 14.1 and ∼50.0 kb. The immunoblots of each pooled sera collected at a specific age against the three SE strains were similar. More specifically, the early immune response at 17 days of age had antibodies recognizing only one polypeptide in the three SE strains namely, the 35.8 kDa. At 24 or 31 days of age, the acquired immunity to infection had antibodies recognizing five similar polypeptides in the three SE strains namely, the 14.4 (fimbriae protein), 21.5 (fimbriae protein), 30.5, 35.8, and 66.2 kDa. At 38 and 45 days of age, the antibodies recognized additional polypeptides namely, the 41.5 and 55.6 kDa, respectively. The recognition of the 41.5 and 55.6 kDa polypeptides at 45 days of age was associated with higher invasiveness of SE to spleens and livers (15.6%) and in higher cecal colonization (59.4%) in comparison to absence of recognition to the two polypeptides at 31 days of age associated with low infectivity to spleens (0%), livers (3.1%), and ceca (9.4%).

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A sandwich ELISA for the bovine IL-1 receptor antagonist (bIL-1ra) was developed using recombinant (r) bIL-1ra produced by Escherichia coli, anti-rbIL-1ra rabbit IgG, its biotinylated one and avidin-peroxidase. This ELISA system enabled detection of rbIL-1ra at a concentration of more than 2 ng/ml. This ELISA was applied to quantitation of bIL-1ra in sera and whey of mastitic and healthy cows. The results indicate that although IL-1ra levels in healthy and mastitic sera and whey were comparable, serum IL-1ra/IL-1?b ratio of euthanized cows was significantly lower than that of the recovered.

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A 32-month-old spayed female Pug was referred for an MRI study due to convulsions. The MRI examination indicated encephalitis. However, echocardiography and pathological examinations revealed that this case had a ventricular septal defect and double chambered right ventricle which is a rare congenital heart disease in the dog. An anomalous muscle bundle crossed the right ventricular outflow tract, dividing the right ventricle into 2 chambers.

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Our previous reports indicated that C57BL/6 mice infected with a lethal variant of Plasmodium yoelii 17X (P. yoelii 17XL) produced high levels of interleukin 10 (IL-10) and interferon-γ (IFN-γ) while mice infected with the nonlethal variant of the parasite did not produce detectable levels of IL-10. In the present study, the involvement of IL-10 and IFN-γ in exacerbation or regulation of blood-stage malaria was investigated by using the lethal variant of P. yoelii 17XL and monoclonal antibodies (mAb) against the cytokines. C57BL/6 mice were injected intraperitoneally with a neutralizing anti-IL-10 mAb or anti-IFN-γ mAb after inoculation with P. yoelii 17XL. Treatment of mice with anti-IL-10 mAb resulted in substantial prolongation of survival and 60% of treated mice survived while 100% of control mice died by day 11. On the contrary, treatment of mice with anti-IFN-γ mAb exacerbated infection and all mice died after an earlier period than those treated with normal rat Ig. No differences in parasitemias were found between treated and untreated mice. To elucidate the involvement of nitric oxide in the host protection or exacerbation, mice were treated with aminoguanidine, an inhibitor of nitric oxide synthetase, after inoculation of P. yoelii 17XL. Neither mortality nor parasitemia was influenced by the treatment. These results indicate that an IFN-γ response is associated with protective immunity in mice infected with P. yoelii 17XL, while an IL-10 response is associated with disease exacerbation during the infection.

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An immunohistochemical study was carried out on the kinetics of Langerhans cells (LCs) at various pathological stages of “Kasen”. Skin lesions of “Kasen” that were collected by biopsy from May to October were classified histopathologically into three stages: initial (Group I, 31 cases), developing (Group II, 50 cases) and regressing (Group III, 13 cases). LCs showed a positive reaction with anti-equine thymocytes (EqT6) monoclonal antibody (MoAb) and anti-major histocompatibility complex (MHC) class II MoAb by immunohistochemical staining. The anti-EqT6 MoAb was intensely positive along the cytoplasmic process. The number of LCs per unit area increased markedly with the passage of time from the initial to the developing stage of the disease, particularly in the epidermo-dermal junction (EDJ). However, the number of LCs tended to decrease in the epidermal layer. In conclusion, the LCs moving into the epidermal layer moved into the EDJ and dermis during the time course of lesion development, and the changes occurring in LCs possibly influenced the progression of the skin lesions of “Kasen”.

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The involvement of apoptosis was evaluated in lesions of endotoxemic piglets. A single injection with E. coli O111:B4 lipopolysaccharide (LPS) induced foci of coagulative necrosis in the liver and kidneys. No significant change was observed in these organs at 1.5 hr after LPS injection, but at 6 hr, epithelial cells with chromatin condensation or fragmentation and apoptotic bodies were visible. Foci of coagulative necrosis were formed within 24 hr after LPS inoculation. In and adjacent to the necrotic foci, dead hepatocytes with nuclear condensation or fragmentation were scattered. These dead cells were positively stained by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) methods. Electronmicroscopy revealed apoptotic cells with condensed or fragmented homogeneous nuclear chromatin, and necrotic cells with irregularly destroyed nuclei and cytoplasmic membranes. Apoptotic cell death were also observed in parietal cells of the stomach and lymphocytes in the lymphatic system. DNA ladders with approximately 200-bp multimers were observed in hepatic, renal and thymic samples prepared after 6 and 24 hr of LPS injection by agarose gel electrophoresis. These results suggest that apoptosis is involved in the pathology of swine endotoxemia.

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An epizootiological survey with histopathological methods was conducted for porcine circovirus in 220 diseased pigs (1-200 days old) in 49 farms from 1985 to 1999. Histopathological lesions containing PCV antigen were detected mainly in the lymphoid tissues from 42 of 189 diseased pigs (22.2%) in 4 of 45 farms (8.9%) from 1990 to 1999. The rate of positive pigs gradually increased from 1997 onward and PCV infection was found in 50% of diseased pigs in 1999. Histopathologically, the lesions in the lymphoid tissues (including lymph nodes, Peyer's patches, tonsil and spleen) were highly correlated with the presence of numerous spherical basophilic intracytoplasmic inclusion bodies with PCV antigen, and consisted of lymphocellular depletion and infiltration of macrophages. Although most affected cells showed cytoplasmic reactivity for PCV, intranuclear antigen was also seen in the lymphocytes, macrophages and ileal epithelial cells. Ultrastructurally, macrophages and giant cells contained electron-dense, round to ovoid lysosomal bodies, in which there were concentric circle or paracrystalline arrays of small nonenveloped icosahedral viral particles, approximately 15-17 nm in diameter. Other consistent infectious agents were present in 90.5% of cases, and porcine reproductive and respiratory syndrome virus infection was in 52.4% of the cases with PCV. The histopathological findings suggested that PCV induced systemic immunosuppression in the infected pigs and made them more susceptible to infection of the organisms. Because of the presence of PCV antigens in the intestinal epithelium, feces may play a significant role in dissemination of PCV.

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A wild raccoon dog (Nyctereutes procyonoides) that manifested severe illness and died was examined. Necropsy revealed severe emaciation, systemic icterus and petechial hemorrhages on the mucous membranes. Histopathologically, necroses were seen in the liver and brain stem associated with meningitis. Eosinophilic intranuclear inclusion bodies were observed in the spleen and intestinal mucosa, and eosinophilic intracytoplasmic inclusion bodies were seen in transitional epithelium in the bladder. Listeria monocytogenes 4b was isolated from the liver, spleen, kidneys and lungs, and the pathogen was also detected in the liver and brain stem immuno-histopathologically. The disease was diagnosed as listeriosis associated with canine distemper virus infection in a raccoon dog.

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A 9-month-old male Shih-Tzu dog had a right mandibular tumor composed of strands, or nest-like proliferation of epithelial cells with abundant fibrous stroma characterized by spheroid to large nodular deposition of amyloid with Congo-red stain. Globule calcification was also seen throughout the tumor tissue and the spheroid depositions often had a concentrically laminated structure (Liesegang rings). The case was diagnosed as amyloid-producing odontogenic tumor in a dog.

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The aim of this study was to determine the effect of perineural capsaicin (CAPS) treatment on cardiopulmonary reflexes elicited by topical laryngeal instillation of CAPS and distilled water (DW) in sevoflurane-anesthetized dogs. Cardiopulmonary reflexes elicited by CAPS (10 μg/ml, 10 ml) were attenuated by perineural CAPS treatment to the superior laryngeal nerves (SLNs) (P<0.05), whereas those by DW (10 ml) remained unaffected (P>0.05). The reflex responses to DW that remained even after the perineural CAPS treatment were eliminated by laryngeal anesthesia with lidocaine. These results suggest that cardiopulmonary reflexes from the laryngeal mucosa elicited by CAPS instillation can be blocked by perineural CAPS treatment to the SLNs, which may result from inhibition of the laryngeal CAPS-sensitive C-fiber afferents.

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A rapid and readily available DNA probe kit was developed for the detection of Salmonella spp. This kit utilized the colorimetric DNA/rRNA sandwich hybridization method in microtiter wells. Within 3 hr Salmonella spp. in selective enrichment broth cultures were detected by the DNA probe kit. The kit effectively identified all of 187 strains of Salmonella tested and yielded no false-positive reactions in the examination of 674 pure cultures of non-salmonellae. The DNA probe kit could detect 10⁵ cfu/ml in pure culture. A total of 379 naturally contaminated samples (raw chicken meat, liquid egg, animal feeds, poultry feces and frozen foods) were tested, both by the standard culture method and the DNA probe kit. The 169 of these samples were culture positive and 210 were culture negative. The sensitivity of the DNA probe kit was 98.2% (166/169) and the specificity was 99.5% (209/210). These results show that the DNA probe kit is a useful tool to examine a large number of various samples for contamination by Salmonella spp. in food and livestock industry.

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The seroprevalence of Bartonella henselae and Toxoplasma gondii among apparently healthy individuals, mainly blood donors, in Thailand was investigated by an indirect fluorescent antibody technique and by a latex agglutination test, respectively. Of 163 serum samples examined, 9 (5.5%) were found to be positive for B. henselae-IgG, 2 (1.2%) for B. henselae-IgM, and 5 (3.1%) for the T. gondii antibody. No significant difference was observed between male and female samples in the serological test with either B. henselae or T. gondii. The age of individuals with B. henselae-IgG was distributed from the 20s to the 70s, and B. henselae-IgM was found in the individuals of the 30s and 60s. The age of T. gondii positive samples ranged from the 20s to the 60s. In this study, the prevalence of B. henselae infection among healthy individuals in Thailand was serologically demonstrated for the first time.

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Between 1991 and 1993, the intestinal contents and feces of wild animals in Japan were examined for the presence of Listeria. The wild animals examined included 623 mammals (11 species) and 996 birds (18 species). Listeria species were isolated from 38 (6.1%) of the 623 mammalian samples and 133 (13.4%) of 996 bird samples. The highest incidence of Listeria in the mammals was found in Japanese monkeys (20.0%) and that in birds was found in crows (43.2%). The incidence of Listeria in Japanese monkeys varied from 0 to 40.0% depending on the capture area. L. monocytogenes was isolated from 11 of these positive samples. Serovars 1/2a and 4b predominated in eight serotyped L. monocytogenes isolates.

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Our previous report indicated that addition of Orvus ES Paste (OEP) to the extender of frozen canine semen protected acrosomes and maintained sperm motility after thawing. In this study, artificial insemination (AI) using the frozen semen was carried out. The frozen semen was prepared using egg yolk Tris-fructose citrate, and the final concentrations of glycerol and OEP were 7% (v/v) and 0.75% (v/v), respectively. AI was performed during the optimal mating period predicted from the peripheral plasma progesterone level. In intrauterine insemination (IUI), the bitches were laparotomized and 1 × 10⁸ spermatozoa were infused into one of the uterine horns. In insemination of non-OEP supplemented semen, 3 × 10⁸ spermatozoa were inseminated. In intravaginal insemination (IVI), 10-40 × 10⁸ spermatozoa were inseminated. Conception was obtained in nine of 10 bitches (90.0%) that underwent IUI. The number of newborns was from 1 to 7 (mean 3.6 ± 0.9). The mean ratio of the number of puppies to the number of ovulations in the inseminated uterine horn was 71.8%. The number of puppies did not exceed the number of ovulation in the inseminated uterine horn. Conception using non-OEP supplemented frozen semen was unsuccessful in all four bitches. In IVI, conception was not obtained in any of the six bitches that received insemination of 10 × 10⁸ or 40 × 10⁸ spermatozoa, but two of three bitches that received insemination of 20 × 10⁸ spermatozoa were fertilized. It was shown that a high conception rate can be obtained by IUI using OEP-supplemented frozen canine semen. Developmenmt of a non-surgical method of IUI and a method of freezing canine sperm applicable to IVI is necessary.

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To characterize factors affecting the number of bovine oocytes recovered transvaginally, a regression analysis was performed between the responsiveness to multiple-ovulation treatment and the number of oocytes recovered transvaginally. The number of embryos recovered following multiple-ovulation treatment and the number of oocytes recovered transvaginally increased when the number of follicles to be aspirated transvaginally increased (P<0.05, P<0.01). The number of cumulus-oocyte complexes recovered transvaginally also increased when the number of oocytes to be aspirated transvaginally increased (P<0.001). However, the number of viable embryos that recovered following multiple-ovulation treatment had no relation to the number of cumulus-oocyte complexes recovered transvaginally. These results suggested that more oocytes can be recovered from donors that have a high responsiveness to multiple-ovulation treatment.

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A sex determination method using DNA extracted from feces has been developed for sika deer (Cervus nippon). We determined a partial sequence of the amelogenin gene of sika deer, which exists on both X and Y chromosomes with a deletion region on the Y chromosome. Based on the sexually dimorphic sequences, we designed a pair of primers which could amplify DNA fragments the lengths of which are different between males and females. PCR products were detected in 34 out of 37 fecal samples collected from captured deer and the sexes estimated by the present method were perfectly matched with the actual sexes.

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To determine drug-induced hyperfunction of marmoset thyroids due to inhibition of synthesis or enhancement of metabolic elimination of thyroid hormones, males were orally administered 10 and 30 mg/kg/day methimazole (MMI), 30 and 100 mg/kg/day spironolactone (SPL), or 50 mg/kg/day phenobarbital (PB) for 4 weeks. MMI caused marked hypertrophy of follicular epithelial cells in accordance with a significant decrease in the plasma thyroxin (T₄) level. Hypertrophied epithelial cells were filled with dilated rough endoplasmic reticulum and reabsorbed intracellular colloids, and the luminal surface was covered with abundant microvilli. The colloid included vacuoles positive to anti T₄ immuno-staining. SPL and PB also caused similar histomorphological changes, although they were less severe than those due to MMI and were not clearly associated with decrease in the plasma T₄ levels. Hepatic T₄ UDPGT activities tended to increase due to SPL and PB treatment, however, which were not so significant as increases in microsomal cytochrome P-450 contents. Some animals treated with SPL and PB showed marked increases in thyroid weights due to inactive dilated follicles. In conclusion, hyperactivity of thyroid follicles was induced in marmosets not only due to inhibition of T₄ synthesis produced by MMI but also because of enhancement of hepatic T₄ elimination produced by SPL and PB. However, hypertrophic effects of SPL and PB were less severe than MMI, because plasma T₄ levels were maintained at almost pretreatment or control levels after SPL or PB treatment.

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N-glycosylation and glucose trimming of the influenza virus hemagglutinin (HA) and neuraminidase (NA) were studied by using glycosylation inhibitor (tunicamycin; TM) and glucosidase inhibitors. TM treatment of MDCK cells infected with a reassortant virus NWS-N8 resulted in reduced transport of the viral glycoproteins to the cell surface. The degree of the effects differed between the HA and the NA (80% reduction for the HA and 97% reduction for the NA), indicating a difference in dependency on N-glycosylation between these glycoproteins. Differential dependency on glucose trimming was clearly demonstrated when the surface transport of the glycoproteins was compared after treatment of the virus-infected cells with glucosidase inhibitors. Fluorescence-activated cell sorting (FACS) analysis revealed that the surface transport of the NA reduced to 50% after castanospermine (CST) treatment but not did that of the HA. An anti-viral effect of a glucosidase inhibitor on the NWS-N8 strain was also demonstrated. The correlation between the expression of the NA on the cell surface and virus yield suggests that CST may interfere with virus release through its effect on the NA.

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Equine arteritis virus (EAV) was readily isolated in RK-13 cell monolayers by plaque assay from seminal plasma of experimental carrier stallions when they contained high titers of virus regardless of the presence of non-viral cytotoxicity in the seminal plasma. The cytotoxicity interfered with virus isolation from seminal plasma which contained virus at titers less than 10 PFU/ml. However, it was possible to detect the virus in seminal plasma pretreated with PEG (#6000). EAV was consistently identified by RT-PCR from crude seminal plasma which contained virus at titers of more than 10^2.7 PFU/ml. In vitro detection of EAV by virus isolation supplemented with RT-PCR using seminal plasma was proved to be an effective alternative to the standard test mating as a diagnostic method for carrier stallions.

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The susceptibilities of culture cells to twelve avian influenza virus strains were determined with ten established cell lines including MDCK and ESK cells and three primary culture cells. The established cell lines derived from embryonic swine kidney (ESK) and chicken kidney (CK) primary culture cells were more sensitive to the avian influenza viruses than the other eleven cells. The ESK cell had a particularly higher infective titer than the MDCK cell with and without trypsin supplement in culture medium, and dispersion of the infective titers was narrower than that of the MDCK cell. The ESK cell is a suitable candidate for routine work on avian influenza viruses in laboratories.

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