Campylobacter spp. have been identified as etiologic agents in outbreaks and sporadic cases of gastroenteritis in developed countries. In developing countries, most reported Campylobacter infections are in children. Previously reported prevalences of Campylobacter spp. in children in Southeast Asia range from 2.9% to 15%. The frequency and pattern of occurrence of Campylobacter spp. differ between developed and developing countries, especially in the number of cases reported in adults and the presence of any seasonal patterns in occurrence. Although the severity of Campylobacter infection in adults was different between developed and developing countries, the clinical symptoms of infection in adults resulting from infection in developing countries was similar to those in developed countries. Many different animal species maintain Campylobacter spp. with no clinical signs. There do not appear to be significantly different colonization rates of Campylobacter in food animals between developed and developing countries. The role of C. jejuni as a primary pathogen in farm animals is uncertain. C. jejuni can be found in feces of diarrheic and healthy calves and piglets. Campylobacter with resistance to antimicrobial agents have been reported in both developed and developing countries, and the situation seems to deteriorate more rapidly in developing countries, where there is widespread and uncontrolled use of antibiotics resistance was observed at high levels in food animals in both developed and developing countries. Studies suggested an association between antimicrobial use in food animals and the development of resistance in human isolates in developed countries.

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To clarify the cellular origin and the fate of M cells, detailed distributions of the epithelial cells were investigated scanning electron microscopically on the follicle-associated epithelia (FAE) of chicken cecal tonsils. The distribution of M cells was closely related with the situation of the crypt orifices in chicken cecal tonsils. In undeveloped cecal tonsils, the intestinal crypts were localized at the periphery of the FAE. In these tonsils, M cells without microvilli (M₀) were predominantly populated in the basal region of the FAE, whereas goblet cells and microvillous epithelial cells (MV) were more distributed in the middle to the apical region of the FAE. A few M cells with short microvilli were dispersed throughout the FAE. Significantly shrunk MV (MVs) clustered together in transitional portions from the lateral face to the roof of the FAE. In well-developed cecal tonsils, the crypts also opened at the lateral surface in addition to the periphery of the FAE. In these tonsils, the M₀ accumulated densely in the small areas around the crypt orifices exclusively. No sign of exfoliation of apoptotic epithelial cells was found in the M₀-accumulated areas and at their peripheral boundaries. The MVs were often clustered in the central regions among the crypt orifices in addition to the roof of the FAE. These findings suggest that M cells are directly derived from the undifferentiated crypt epithelial cells, not fall into apoptotic cell death and further differentiate into MV in the FAE of chicken cecal tonsils.

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Changes in the distribution of retinal ganglion cells (RGCs) were studied using the retrograde labeling of DiI in chicks and chick embryos. The small retinal area filled with labeled RGCs was observed in the retinal fundus on E8. The labeled retinal area expanded radially toward the peripheral retina as the retina grew, and finally occupied a whole retina by P1. The temporal retina was labeled more rapidly than in the nasal retina. The observed-increasing rate of the labeled area was corrected with the growing rate of the retina. Consequently, the corrected-increasing rate of the labeled area was estimated to be about 390% between E8 and E11, and 20-50% after E11. This means that spreading speed of the maturated RGCs lowered until 1/10-1/20 after E11.

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A mixed-antigen agar gel enzyme assay (AGEA) was developed to detect antibodies to poxviruses in chicken and turkey sera. The assay combines the principles of immunodiffusion and enzyme assay. For the detection of antibodies to fowl poxvirus (FP), pigeon poxvirus (PP) and turkey poxvirus (TP) in turkey serum samples, the three antigens were combined to form a mixed-antigen assay. To screen for antibodies to FP and PP in chicken serum samples, the two antigens were combined. When FP and PP viruses were combined as antigens, the sensitivity for chicken sera was 64% but the sensitivity of the agar gel precipitation test (AGPT) was 34% (P<0.001). When antibodies were detected in turkey sera using the mixed antigens, the AGEA had a sensitivity of 66.4% while that of AGPT was 25% (P<0.001).

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The Cry proteins produced by Bacillus thuringiensis are considered to be highly specific insecticidal proteins. Judged to be safe for humans and farm animals due to their insect-oriented selective toxicity, the proteins have been utilized as a biological pesticide and introduced into genetically modified plants. However, some critical fundamental characters of the Cry proteins remain unclear, and the direct effects of activated Cry proteins on mammalian cells have not yet been fully confirmed. Therefore, in this study we employed primary cultured bovine hapatocytes as a model system to determine if Cry1Ab, a Cry protein, affects mammalian cells. There were no significant changes in the secretion of albumin or the morphology of the Cry1Ab-treated cells. The LDH release showed a tendency to increase after the administration of Cry1Ab, but not significantly. Taking these results on bovine hepatocytes into consideration, Cry1Ab has little acute toxicity on mammalian cells.

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Apolipoprotein E (apoE) is a protein constituent of lipoproteins, and acts as a receptor-binding ligand. Although the existence of bovine apoE in lipoprotein fractions has already been reported, quantitative studies on the changes of apoE in plasma and lipoprotein fractions are lacking. In the present study, an increase of a 38 kDa protein in the very low-density lipoprotein (VLDL) fraction obtained from fasted calves was detected. This 38 kDa protein was identified as bovine apoE by determination of the N-terminal amino acid sequence. Bovine apoE was purified and an enzyme-linked immunosorbent assay (ELISA) was developed. Using this system, the effect of fasting on the concentration of apoE in plasma and the distribution of apoE in lipoprotein fractions were investigated. After 3 days of fasting, the concentration of plasma apoE increased significantly (p<0.05) by 280 %, and was returned to the basal level by 3 days of refeeding. The lipoprotein fractions obtained from before and after fasting was separated by ultracentrifugation. ApoE was significantly increased in VLDL, low-density lipoprotein (LDL) and non-lipoprotein fractions by fasting (p<0.05). On the other hand, in high-density lipoprotein (HDL) fractions obtained from both before and after fasting, the level of apoE was very low compared to the other fractions. These results suggested that bovine apoE contents in triglyceride-rich lipoproteins are modulated by nutritional treatment and closely associated with triglyceride-rich lipoprotein metabolism.

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In this report, a hybrid baculovirus expression system, which means a hybrid virus of the Autographa californica nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus, was used for the large-scale production of porcine mature interleukin-18 (IL-18) in silkworms. Two recombinant hybrid baculoviruses containing cDNA of the porcine precursor IL-18 and the porcine caspase-1 were constructed and were used to infect silkworm larvae. After the co-infection of the two viruses, porcine mature IL-18 was efficiently produced in the haemolymph. The concentration of IL-18 in the haemolymph was 80-100 μg/ml, as determined by porcine IL-18 specific ELISA. This yield was twenty-times more than that of the insect cell expression system described previously. The porcine mature IL-18 produced by the silkworms strongly induced interferon-γ (IFN-γ) production from porcine PBMC. An insect factory system for the large-scale production of useful cytokines for livestock animals will be available in the near future.

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To examine substrate specificity and susceptibility to lead, erythrocyte 5'-nucleotidase was measured in dogs, cats, cattle and humans, and its relationship to the reticulocyte count in these species was determined. The reticulocyte count in dogs was similar to that in humans, but the count in cats was higher than that in humans. Reticulocytes were not observed in cattle. The activities of canine erythrocyte 5'-nucleotidase measured using cytidine and uridine 5'-monophosphates, which are preferentially catalyzed by one of the human pyrimidine 5'-nucleotidase isozymes (P5N-I), were similar to those of the human enzyme. The canine enzyme preferentially catalyzed thymidine 3'-monophosphate, which is catalyzed only by human P5N-II, more strongly than the human enzyme. This suggests that canine erythrocytes have two isozymes similar to human P5N-I and P5N-II, and a higher P5N-II-like activity than human erythrocytes. Feline erythrocytes had the highest level of P5N-I-like activity among the species examined, and the bovine enzymic activities including those of P5N-I and II were the lowest among these species. According to these observations, the reticulocyte count was approximately proportional to the P5N-I-like activity in these species. Therefore, the P5N-I-like activity may be involved in the morphological maturation of mammalian erythrocytes. The canine and feline erythrocytes had markedly high activity and preferentially catalyzed purine 5'-monophosphate suggesting the presence of a purine-specific 5'-nucleotidase as in human erythrocytes. In addition, the canine and feline P5N-I-like activity showed less susceptibility to lead than the human P5N-I. This may be a reason why there are few case reports of lead-induced anemia in dogs and cats.

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The present study was conducted to determine the clinical and clinico-pathologic characteristics of Shiba dogs with GM1 gangliosidosis, which is due to an autosomal recessively inherited deficiency of lysosomal acid β-galactosidase activity. Clinical and clinico-pathological features were investigated in 10 homozygous Shiba dogs with GM1 gangliosidosis. The age at onset was 5 to 6 months and the dogs manifested progressive neurologic signs including loss of balance, intermittent lameness, ataxia, dysmetria and intention tremor of the head. The dogs were unable to stand by 10 months of age due to a progression of ataxia and spasticity in all limbs. Corneal clouding, a visual defect, generalized muscle rigospasticity, emotional disorder and a tendency to be lethargic were observed at 9 to 12 months. The dogs became lethargic from 13 months of age. The survival period seemed to be 14 to 15 months. As a clinico-pathologic feature, lymphocytes with abnormally large vacuoles were observed in peripheral blood (30 to 50% of total lymphocytes) through the lifetime of the dogs. The clinical and clinico-pathologic characteristics of this animal model are useful for not only the development and testing of potential methods of therapy, but also the diagnosis of affected homozygous Shiba dogs in veterinary clinics.

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Fluoroscopy (FS)- or transesophageal echocardiography (TEE)-guided heartworm removal was carried out using flexible alligator forceps to compare the rate of worm removal. As a result, the worm removal rates were similar between the two procedures. However, the TEE-guided procedure does not involve radiation exposure, and facilitates observation of worms in the cardiac chamber and pulmonary artery. Therefore, the TEE-guided procedure is thought to be more useful than the FS-guided procedure in clinical setting.

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For the direct detection of dermatophytes in skin scrapings and hairs from animals, a primer pair specific to the chitin synthase 1 (CHS1) gene of dermatophytes was constructed. By PCR analysis with the primer pair, dermatophyte DNA could be diagnosed directly and rapidly in clinical skin samples.

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Thymus and activation-regulated chemokine (TARC) is a member of CC chemokine and plays an essential role in recruitment of CC chemokine receptor 4 positive Th2 cells to allergic lesion. To investigate the association of TARC in allergic inflammation of cats, a TARC cDNA was cloned from feline thymus by RT-PCR with 3' rapid amplification of cDNA ends (RACE) method. The feline TARC clone contained a full length open reading frame encoding 99 amino acids which shared 80.8%, 72.5%, 65.6% and 67.8% homology with dog, human, mouse and rat homologues, respectively. Expression of TARC mRNA was detected not only in thymus but also in spleen, lung, lymph node, kidney, small intestine, colon and skin of the normal cat tissues examined. Furthermore, it was found that TARC mRNA was strongly expressed in lesional skin of cats with eosinophilic plaque. The present results demonstrated that TARC might be involved in the pathogenesis of eosinophilic plaque in cats.

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Eight periparturient Holstein Friesian cows were examined for plasma tartrate-resistant acid phosphatase (TRAP) activity to assess the degree of bone metabolic activity and to evaluate the association between the change in calcium (Ca) concentration and bone metabolism during the periparturient period. Milk fever occurred in 1 of 8 cows just after parturition. Plasma TRAP activities did not markedly change in 5 of 8 cows during the experimental period. The changing rate of Ca between preparturition and just after parturition was under -20% in 3 of 8 cows, and low TRAP activities were observed in 2 of these 3 cows. This study suggests that cows with a low TRAP activity are at risk of developing milk fever in comparison to cows with high TRAP activity. Temporary increases of parathyroid hormone were observed in 7 cows, but not in the cow with milk fever.

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Hydroxyurea (HU), an anticancer drug, inhibits ribonucleoside diphosphate reductase and reduces pool sizes of deoxyribonucleoside triphosphate (dNTP). The reduction of dNTP results in inhibition of DNA replication. The cytotoxic effect of HU was investigated using fibroblast cell lines from LEC rats. LEC rat cells showed significantly higher sensitivity to HU than did cell lines from control WKAH rats. No significant differences were observed between the percentages of apoptotic cells in either LEC or WKAH rat cells that had been treated with HU and those that had not been treated with HU. LEC rat cells also showed significantly higher sensitivity to aphidicolin, which blocks DNA synthesis by inhibiting DNA polymerase α, than did WKAH rat cells. In both LEC and WKAH rat cells, intensified bands of p53 protein were observed immediately after treatment with HU. Although the high level of p53 protein persisted in WKAH rat cells until 6 hr post-incubation time after treatment with HU, the level of p53 protein had decreased at 6 hr post-incubation time in LEC rat cells. When the cells were X-irradiated in the absence or presence of HU, the ratio of the surviving fraction without HU to that with HU only slightly increased after X-irradiation in WKAH rat cells. In contrast, the ratio in LEC rat cells significantly increased after X-irradiation in a dose-dependent manner.

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Age-related changes in the canine substantia nigra, were examined using immunostaining for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), neurofilament (NF), ubiquitin, single stranded DNA (ssDNA), and alpha-synuclein (αSN). Brain sections from 34 necropsied dogs, ranging from 2 months to 18 years old, were used for this study. On general histological examinations, several age-related changes, including lipofuscin deposition, polyglucosan bodies, amorphous basophilic inclusions and eosinophilic crystal inclusions, were found in the aged dogs. Immunohistochemically, TH-positive neurons were located only in the substantia nigra. The number of TH-positive neurons was well preserved in all dogs examined, however, the ratio of TH-positive neurons to GFAP-positive glial cells tended to show slight decrease in aged dogs. By ssDNA immunostaining for apoptotic cells, there were no significant results in the number of ssDNA-positive neurons. The number of ubiquitin- and NF-positive swollen neurites was increased markedly in aged dogs. Ubiquitin immunostaining revealed a small number of basophilic and eosinophilic inclusions, although both types of inclusions were negative for NF. By αSN immunostaining, no neurons were immunoreactive and no basophilic or eosinophilic intracytoplasmic inclusions were revealed. These results indicate that in the substantia nigra of aged dogs the dopaminergic neurons are well preserved, but intracytoplasmic inclusions and ubiquitin-positive degenerative neurites are commonly found.

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Nest polymerase chain reaction (PCR), in situ hybridization (ISH), in situ PCR, in situ PCR/hybridization (PCR-ISH) and in situ nest PCR were compared for the detection and localization of intracellular viral DNAs in paraffin sections. MDBK cells were infected with alcelapine herpesvirus 1 ranging from 10¹ to 10⁵ 50% tissue culture infected doses (TCID₅₀), incubated 18 hr, then fixed and processed into paraffin blocks. Sections of the cell preparation were subjected to nest PCR, ISH, in situ PCR, PCR-ISH and in situ nest PCR using specific oligonucleotide primers or probes directed against the viral open reading frame 50. In situ nest PCR and nest PCR were found to be capable of detecting the viral DNA in the cells infected with the lowest virus titer. As compared with other molecular biological methods for the detection of the virus, in situ nest PCR was found to be more sensitive than ISH, in situ PCR and PCR-ISH. In situ nest PCR has wide applications for sensitive localization of low copy viral sequences within cells to investigate the role of viruses in a variety of clinical conditions.

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A 2-year-old Labrador Retriever developed atrophy of the right temporal muscle, subsequently showed generalized seizure and died 2 months after the clinical onset. Postmortem examination revealed the tumor masses in the right mandibulopharyngeal area, nasopharynx and intracranial space. Histopathologically, these tumor masses were composed of small round neoplastic cells and neuropil-like stroma separated by fibrovascular septa. In the neoplastic masses, small neoplastic cells with round to oval hyperchromatic nuclei and scanty cytoplasm predominated, and angulated neoplastic cells with larger nuclei and moderate cytoplasm were scattered. Immunohistochemically, neoplastic cells were positive for neuron specific enorase, neurofilament protein, chromogranin A, synaptophysin and tyrosine hydroxylase. Based on these findings, this case was diagnosed as peripheral neuroblastoma, presumably originated from the sympathetic ganglion, maybe right craninal cervical ganglion.

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Endothelin (ET), derived from the endothelium of blood vessels, is a potent vasoactive peptide. Although it has been reported to be involved in cardiovascular diseases, such as hypertension, the mechanism by which ET evokes vasoconstriction is still unclear. On the other hand, p42/p44 mitogen-activated protein kinase (MAPK) and p38 MAPK are activated by a variety of growth factors and cellular stresses, respectively. However, the role of p42/p44 MAPK and p38 MAPK on the ET-1-induced vasoconstriction is not fully understood. This study was undertaken to determine whether p42/p44 MAPK and p38 MAPK participate in the regulation of vascular smooth muscle contraction by ET-1. The isometric vasoconstriction and intracellular Ca²⁺ ([Ca²⁺]_i) were simultaneously measured using CAF-100. Phosphorylation of myosin light chain (MLC) and p42/p44 MAPK, p38 MAPK were determined by Western blots. In rat thoracic aorta, ET-1 induced a sustained contraction. In contrast, [Ca²⁺]_i was decreased with time. Both PD98059, an inhibitor of p42/p44 MAPK, and SB203580, an inhibitor of p38 MAPK, partially attenuated ET-1-induced contractions in concentration-dependent manners. ET-1 increased phosphorylation of both p42/p44 MAPK and p38 MAPK, and PD98059 and SB203580 completely decreased phosphorylation of p42/p44 MAPK and p38 MAPK in response to ET-1 stimulation, respectively. On the other hand, PD98059 and SB203580 did not affect MLC phosphorylation in response to ET-1 stimulation. These results indicate that p38 MAPK, as well as p42/p44 MAPK, may partially regulate the ET-1-induced contraction through a MLC phosphorylation-independent pathway.

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The expression of activin and inhibin has been demonstrated in the hypothalamus, but their physiological roles in the brain remain to be elucidated. In the present study, involvement of activin and inhibin in the regulation of food and water intake was examined. Male rats were deprived of food or water for 12 and 60 hr, and mRNA levels of activin/inhibin α, βA and βB subunits in the hypothalamus were estimated by RT-PCR. Gene expression of α subunit transiently decreased at 12 hr of food deprivation, while it did not change during water deprivation. Food and water deprivation for 60 hr increased mRNA levels of βA and βB subunits, respectively. These results indicated that gene expression of each subunit was independently regulated. Injection of activin A (0.5 and 4.0 μg) into the third ventricle decreased food intake. Water intake was suppressed by 4.0 μg, but not 0.5 μg, of activin A. Intracerebroventricular injection of inhibin A (0.5 and 4.0 μg) decreased water intake in a dose dependent manner without affecting food intake, suggesting that inhibin could act independently of activin. Taken together, it is suggested that activin and inhibin take part in the central regulation of nutrient and fluid balance, though further study is needed to determine precise molecular species involved.

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In ultrasonographic diagnosis of ovarian disorders and the estrous cycle in sows, transverse observation of the uterus yielded more characteristic findings than observation of sagittal sections. Transverse ultrasonography revealed that the low progesterone (P) type uterus showed a round structure, while the high P type uterus showed a flattened structure. These results corresponded well with rectal palpation findings: the low P type uterus had a hard, pipe-like structure and the high P type a soft, balloon-like structure. For gilts, we employed a minimum convex type transrectal prostate probe that had an approximately 18 cm insertion handle. The images of the uterus obtained thereby were a similar to those obtained from sows. The above results suggest that it should be possible to diagnose and treat many ovarian disorders in sows and gilts based only on the ultrasonograhic findings. In short, ultrasonograhic findings of a round structure of the uterine wall might be an indication for PMSG (pregnant mare serum gonadotropin) treatment, while findings of a flattened structure might be an indication for PGF_{2 α} administration.

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The effects of ethylene glycol monoethyl ether (EGEE) on testicular cell populations in rats were investigated by a flow cytometric method. Rats were administered by gavage with EGEE at the various doses of 0 (saline alone), 100, 200, 400, and 800 mg/kg body weight/day for 4 weeks. The treatment of EGEE caused decreases in the weight of testis and epididymis and in the number of testicular cells. Histopathologically, exfoliation of germ cells into the tubular lumen was observed at the doses of above 200 mg/kg. The treatment of EGEE at the dose of 400 mg/kg caused moderate testicular degeneration. A significant depletion of haploid cells and a disproportionate ratio of diploid and tetraploid cells were observed as determined by flow cytometric analysis. These results indicate that the toxic effect of EGEE on the male reproductive system may be strongly associated with the disproportion of testicular germ cells.

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Bovine viral diarrhea virus (BVDV) has been segregated into two genotypes, type 1 and type 2. To determine the efficacy of the commercially available bovine viral diarrhea type 1 vaccine used in Japan against BVDV type 2, calves were infected with BVDV type 2 strain 890 4 weeks after administration of the vaccine. The vaccinated calves did not develop any clinical signs and hematological changes such as observed in unvaccinated calves after the challenge. Furthermore, the challenge virus was not recovered from the vaccinated calves throughout the duration of the experiment, whereas it was recovered from all unvaccinated calves. The bovine viral diarrhea vaccine used in Japan is efficacious against infection with BVDV type 2 strain 890.

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Human immunodeficiency virus (HIV) infects lymphocytes and macrophages via CD4 and chemokine receptors. In this study, the infectivity of a chimeric simian and human immunodeficiency virus (SHIV) having a CCR5-specific HIV-1 envelope gene was examined. A SHIV strain termed SHIV-JRFL could enter cells via CD4 with a chemokine receptor CCR5, not CXCR4, and the viral replication was suppressed by recombinant human RANTES, one of β-chemokines. The intravenous inoculation of SHIV-JRFL into two rhesus macaques resulted in a systemic infection, though it was rather weak. During the early infection, the production of RANTES from Con A-stimulated PBMCs of the infected monkeys increased. These results suggested that β-chemokine has the potential to limit the infectivity of an R5-type SHIV.

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Serological survey of bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV) infection was conducted in dairy cattle from 10 different regions of Hokkaido, Japan. Among 390 cattle, 11.0% of cattle were BIV-seropositive and 3.3% were BLV-seropositive. Moreover, in two dairy farms, where bovine leukosis has been reported, prevalence of BIV infections were 6.4 and 9.1%, respectively. In contrast, among 150 beef cattle, 16.6% were BIV-seropositive while none was BLV-seropositive. Dual infections with BLV and BIV in dairy cattle were tested by using 107 BLV-seropositive sera, and 20 sera were found BIV-positive (18.7%). These results indicate that BIV infection was widespread in Hokkaido.

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