Immunohistochemical localization of insulin-like growth factor-I (IGF-I) was investigated in the liver of fasted and refed chickens by using an antigen retrieval method. The present study is the first one showing the localization of IGF-I in the chicken liver. Immunoreactivity for IGF-I was detected on the paraffin sections of livers from the fed and refed chickens after the treatment with the antigen retrieval agent. A moderate number of cells showing IGF-I immunoreactivity were scattered in the parenchyma of the liver from fed chickens. These cells were relatively large and polygonal in shape and seemed to be hepatocytes. Reaction products were observed as a granular structure in the cytoplasm of IGF-I-immunoreactive hepatocytes. The number of immunoreactive hepatocytes was increased in the liver from refed chickens compared with fed chickens. Diffuse reaction products as well as granular ones were observed throughout the cytoplasm of IGF-I-immunoreactive hepatocytes of livers from refed chickens. There are, however, no regular patterns of the distribution of immunoreactive hepatocytes in the parenchyma of both fed and refed chickens. In the liver of the fasted chickens, clear immunoreactivity for the peptide was not observed. These data show that IGF-I is located in the chicken hepatocytes and influenced by the nutriture.

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To elucidate the roles of both constitutive endothelial nitric oxide synthase (eNOS) and neuronal NOS (nNOS), and inducible NOS (iNOS) in acute experimental testicular torsion, the expression of iNOS and constitutive eNOS and nNOS were studied in the rat testis with ischemia/reperfusion (I/R) injury. Western blot analysis showed that all three isoforms of NOS increased significantly at 24-48 hr after I/R and declined slightly thereafter. After I/R, immunoreactivity for both iNOS and nNOS was detected, mainly in the interstitial space around damaged tubules, while germ cells in the damaged tubules were immunostained intensely for eNOS. We postulate that increased expression of the three NOS isoforms in the testis after I/R, which might generate nitric oxide, affects delayed germ cell death following I/R via paracrine or autocrine fashion.

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In a previous study, we reported a new method for counting quail blood cells. After quail blood cells were stained with fluorescent lipophilic dye (DiOC₆(3)), absolute counts of erythrocytes, granulocytes, and monocytes were obtained by means of flow cytometry (FC). The FC method has the potential for application to avian blood cells count; however, the method was unable to distinguish between lymphocytes and thrombocytes. In the present study, we improved the FC method to obtain separate counts of lymphocytes using DiOC₅(3). After quail blood cells were stained with DiOC₅(3), the cells were measured with FC. Each blood cell type was distinguished by means of their typical FL-1 (green fluorescence) and SSC (side scatter). Absolute numbers of erythrocytes, granulocytes, monocytes and lymphocytes in whole blood were obtained. The improved FC analysis worked equally well with chicken (Gallus gallus) and goose (Anser cygnoides) blood.

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Lawsonia intracellularis is an obligate intracellular pathogenic bacterium that causes proliferative enteropathy in various animals. The detection of L. intracellularis in clinical and environmental samples is necessary for the diagnosis of infection and epidemiological investigations. For the detection of L. intracellularis in fecal samples, we have developed an immunological method using immunomagnetic separation and ATP bioluminescence. Magnetic beads were coated with an anti-Lawsonia surface antigen (LsaA) antibody in order to capture the L. intracellularis in fecal samples from infected rabbits and the bacteria captured by the immunomagnetic beads were assayed by means of ATP bioluminescence. Our results showed that L. intracelluraris was detected by immunomagnetic separation of bacteria-holding magnetic beads and ATP-based bioluminescence, suggesting that our methods could be useful for the diagnosis of proliferative enteropathy.

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The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene is one of the endogenous matrix metalloproteinase (MMP) inhibitors. It was reported that decreased RECK expression closely correlated with tumor malignancy. We determined the cDNA sequence of the canine RECK gene. The cDNA sequence and deduced amino acid of canine RECK were 2,913 bases and 971 residues, respectively. The predicted amino acid sequence of the protein showed 95.5% and 91.9% homology with human and mouse RECK, respectively. RECK mRNA expression was analyzed in various canine tissues and tumor cell lines by quantitative RT-PCR. The highest RECK expression was detected in lung and testis. In comparison with the tissues, a remarkably low expression level was detected in tumor cell lines. In addition, the RECK gene was transfected in the canine transitional cell carcinoma, and its influence on cell proliferation, migration, and invasion was analyzed. The transfected RECK gene suppressed only canine tumor invasion. These results showed that RECK might play an important role in tumor malignancy in dogs as well as in other mammalians.

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The immune responses of hypothyroid dogs to canine thyroglobulin (cTg) were evaluated for the proliferative ability of peripheral blood mononuclear cells (PBMC). PBMC from three hypothyroid dogs with high titers of thyroglobulin autoantibody (TgAA) and 3 clinically normal dogs were cultured with 5, 10, or 20 μg/ml of cTg for 72 hr. The proliferative responses of the cells were determined by the level of incorporated BrdU. The numbers of cells expressing Thy-1, CD4, CD8 and IgG in the PBMC were counted by the immunofluorescence method. Proliferative responses to cTg were observed in the cells from hypothyroid dogs. The number of cells expressing IgG and CD8 in the hypothyroid dogs tended to be high compared with the clinically normal dogs. The CD4⁺ cells in cultures from hypothyroid dogs increased depending upon the amount of cTg. There was a significant (P<0.05) positive correlation between the number of CD4⁺ cells and the concentration of cTg in the cultures from hypothyroid dogs. These findings suggest a possible relationship between canine hypothyroidism and cellular immunity. Loss of self tolerance to thyroid antigens in CD4⁺ T cells may play an important role in the development of canine hypothyroidism.

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To elucidate the cause of growth retardation, 14 cattle with renal tubular dysplasia (RTD) were investigated by determining serum growth hormone (GH) and insulin-like growth factor (IGF)-1 concentrations. Compared with 6 healthy cattle, the baseline, maximum, area under the curve and peak amplitude of serum GH concentrations were higher or tended to be higher. Serum IGF-1 concentrations were lower normal or lower in cattle with RTD than in healthy cattle. Serum IGF-1 concentrations correlated significantly with body weight and chest girth, but not with serum GH and creatinine concentrations. Growth retardation in RTD might be associated partially with GH resistance.

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We demonstrated that Demodex canis was transferred to skin xenografts of a dog and a hamster onto severe combined immunodeficiency mice. After the transfer of mites, the number of eggs, larvae, nymphs and adult mites per gram of canine and hamster xenografts increased, whereas no live mites were detected on murine allograft. These results indicate that D. canis proliferates in hair follicles of dog and hamster skins but not in murine allograft. Therefore, D. canis may have host preference but not strict host-specificity.

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Hematological findings during the course of infection and the antibody response in Syrian hamsters infected with Babesia microti were examined. A macrocytic hypochromic anemia with an increase of the reticulocyte count was detected as a rise in the parasitized erythrocyte rate. White blood cell counts also remarkedly increased with the increases of both neutrophils and active-shaped monocytes, and thus they particularly play an important role in eliminating the parasite. In Western blotting with the sera from the hamsters infected with B. microti, a 38 kDa protozoan antigen reacted to the early-term sera, and additionally 28, 32, and 34 kDa antigens also reacted to the medium- and latter-term, and convalescent sera. These antigens were immunodominant and the antibodies against these antigens had also important roles for inhibition of this parasite.

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Among many detrimental injuries, alcohol is implicated in hepatitis, fatty liver, hepatic fibrosis, and cirrhosis. The purpose of this study was to evaluate the protective effect of bio-active ceramic water on alcohol-induced hepatic injury in pigs. Twelve male Landrace pigs were divided into 3 groups. Groups 1, 2, and 3 were fed with bio-active ceramic water + normal liquid diet, bio-active ceramic water + liquid diet containing 15% ethanol, and tap water + liquid diet containing 15% ethanol for 12 weeks, respectively. For serological, histopathological, and immunohistochemical analysis, all pigs were sacrificed at week 12. In group 3, serum ALT and AST levels increased, and mild fatty change and moderate necrosis were detected in the liver. Collagen fibers, myofibroblasts, and CYP2E1 were also increased or activated in group 3. In group 2, there were mild hepatic injuries compared to group 3. However, injuries and activations were not observed in the liver in group 1. We suggest that the bio-active ceramic water used in the present study had protective capability against ethanol-induced hepatic injury and that having no toxic effect on the pig liver. The bio-active ceramic water might be useful as a therapeutic drinking water in patients suffering from alcoholic liver diseases.

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Tumor necrosis factor-alpha (TNF-α) has been reported to be associated with the progression of lymphoproliferative neoplastic diseases and retroviral infections. Hence we examined immunohistochemically the expression patterns of TNF-receptors (TNF-RI and RII) on lymphoma cells derived from the 29 cases of enzootic bovine leukosis (EBL). Lymphomas obtained in 29 animals with EBL were histopathologically classified into three types: diffuse mixed type (10 cases), diffuse large type (9 cases), and diffuse large cleaved type (10 cases). Immunohistochemically using a monoclonal antibody to a bovine lymphocyte surface antigen, the lymphomas were classified into three phenotypes: B-1a (CD5⁺/CD11b⁺), B-1b (CD5^-/CD11b⁺) and B-2 (conventional B) (CD5^-/CD11b^-). Interestingly, the lymphoma cells in all animals expressed TNF-RII, but not TNF-RI. Although, in EBL, lymphoma cells of which the histopathological and immunological property differs has been formed, the expression patterns of TNF-Rs had the universality in all lymphoma cells. TNF-RII, which induces cell proliferation, was expressed but TNF-RI, which induces cell apoptosis was not expressed on all lymphoma cells, suggesting that TNF-Rs play an important role in the malignant proliferation of B cells and formation of lymphomas in EBL.

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In this study, we determined the effects of transcranial electrical stimulation (TCES) on the anaesthetic requirements of thiopental and the analgesic requirements of remifentanil, in rats. The experiments were performed on 120 albino male Wistar rats, which were randomly allocated to four groups (n=30). (Thiopental, Thiopental+TCES, Thiopental+Remifentanil, and Thiopental+Remifentanil+TCES). Animals were anaesthetized with thiopental, and 15 min later, remifentanil was injected to rats in the Remifentanil groups. TCES was started in the stimulated groups 20 min after thiopental administration. Rats were stimulated 5 times for this experiment. The times for recovery, herein called Cognition Recovery Time and Motion Recovery Time were measured. Cognition Recovery and Motion Recovery Times were not affected by the stimulation. Analgesia was assessed using the wet tail-flick latency (TFL). In the Thiopental group, the analgesia level returned to control values on the 35th min. In the Thiopental+Remifentanil group, the analgesia level returned to control values on the 50th min. In the Thiopental+ TCES group, the analgesia level reached the peak value on the 65th min. In the Thiopental+Remifentanil+TCES group, the analgesia level reached the peak value on the 35th min and analgesia remained high during the 90 min after cessation of the stimulation. The analgesic potency for the Thiopental+Remifentanil+TCES group was increased by 30-40% when compared with the prior TFL values, 160% when compared with the control group, and 50-75% when compared with Thiopental+ TCES group on the 35th min (P<0.001). In conclusion, TCES markedly decreases the anaesthetic and analgesic requirements for thiopental and remifentanil in rats.

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A new valved conduit was developed using a canine aortic valve. The bioprosthetic valve was fixed with glutaraldehyde and epoxy compound (Denacol-EX313/810). A vascular graft composed of ultra-fine polyester fiber (10 mm in diameter, 200 mm in length) was used. Four dogs underwent apico-aortic valved conduit (AAVC) implantation and aortic banding (bypass group, BG), while another 4 dogs underwent aortic banding without AAVC implantation (control group, CG). Cardiac catheterization and angiocardiography were performed for assessment of hemodynamics 2 weeks and 6 months after surgery. Left ventricular systolic pressure, left ventricular end-diastolic pressure and the left ventricular-aortic pressure gradient differed significantly (P<0.01) between the BG and CG dogs. Left ventricular angiocardiography showed patency of the valved conduit in all the BG dogs. Echocardiography was performed before and 2, 4 and 6 months after surgery, and showed that while pressure overload caused concentric myocardial hypertrophy in the CG dogs, the left ventricle dilated eccentrically in the BG dogs. Furthermore, relief of left ventricular pressure overload by AAVC was maintained.

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The anesthetic sparring and cardiovascular effects produced by midazolam 0.8 mg/ml-ketamine 40 mg/ml-medetomidine 0.05 mg/ml (0.025 ml/kg/hr) drug infusion during sevoflurane in oxygen (MKM-OS) anesthesia was determined in healthy horses. The anesthetic sparring effects of MKM-OS were assessed in 6 healthy thoroughbred horses in which the right carotid artery was surgically relocated to a subcutaneous position. All horses were intubated and ventilated with oxygen using intermittent positive pressure ventilation (IPPV). The end-tidal concentration of sevoflurane (ET_SEV) required to maintain surgical anesthesia was approximately 1.7%. Heart rate and mean arterial blood pressure averaged 23-41 beats/min and 70-112 mmHg, respectively. All horses stood between 23-44 min after the cessation of all anesthetic drugs. The cardiovascular effects of MKM-OS anesthesia were evaluated in 5 healthy thoroughbred horses ventilated using IPPV. Anesthesia was maintained for 4 hr at an ET_SEV of 1.7%. Each horse was studied during left lateral (LR) and dorsal recumbency (DR) with a minimum interval between evaluations of 1 month. Cardiac output and cardiac index were maintained between 70-80% of baseline values during LR and 65-70% of baseline values during DR. Stroke volume was maintained between 75-85% of baseline values during LR and 60-70% of baseline values during DR. Systemic vascular resistance was not different from baseline values regardless of position. MKM-OS anesthesia may be useful for prolonged equine surgery because of its minimal cardiovascular depression in both of lateral and dorsal recumbency.

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Axial correction was performed surgically in two miniature dachshunds presenting with lateral patellar dislocation and limping caused by pes varus. Pes varus had resulted from asymmetric closure of the physis of the distal tibia. Prior to surgery, osteotomy was simulated by measuring X-ray films to determine the distance required for the wedge opening. Transverse-opening wedge osteotomy was performed on the medial side of the distal tibia, and beta-tricalcium phosphate (β-TCP) was inserted in a wedge shape into the area created by the cuneiform osteotomy. Finally, the tibia was fixed by a veterinary 1.5/2.0-mm T-plate. Both dogs were able to walk a few days after surgery, and the lateral dislocation of the patella normalized almost completely in about one month. At two months, X-ray films showed that the implant had remained in position without any dislocation, and the β-TCP had fused with the surrounding bone.

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Carcinogenicity and chronic toxicity of biphenyl was examined in the male and female BDF₁ mice fed a diet containing biphenyl at 667, 2,000 or 6,000 ppm for 2 years. There was no difference in survival rate between any biphenyl-containing diet-fed group of either sex and the respective control. Body weights of the males and females fed 6,000 ppm diet were significantly lower than the respective control. Incidences of hepatocellular carcinomas and hepatocellular adenomas in the females fed diets containing biphenyl were significantly increased in a dose-related manner, and exceeded a range of the historical control data in the Japan Bioassay Research Center. Incidences of basophilic cell foci in the liver were increased in the females fed 2,000 and 6,000 ppm diets. There was no increase in tumor or tumor-related lesion in the males fed diets containing biphenyl. Chronic toxicity of biphenyl was characterized by increased incidences of urothelial desquamation in the renal pelvis in males and females and mineralization in the inner stripe of renal outer medulla in females, as well as changes in serum levels of BUN, ALP and some electrolytes in males and females. In conclusion, the 2-year oral administration of biphenyl-containing diets induced pre-neoplastic and neoplastic lesions in the liver of females and non-neoplastic lesions in the kidney of males and females. Causative factors for the biphenyl-induced hepatocarcinogenicity were discussed in light of our published finding of peroxisome proliferation.

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Pneumotropic virus invasion of parenchymal organs in mice with immunosuppressants is worth studying from an etiopathogenetic viewpoint as an animal model of a compromised host. This study analyzed the invasion by Sendai virus (SeV) of mouse organs in immunosuppressive states induced by cortisone (CO) and cyclophosphamide (CY). After subcutaneous inoculation of CO or CY, or both, in mice infected intra-nasally with SeV, the SeV invasion was investigated by detecting viremia and viral antigen in organs. SeV Nagoya strain and one treatment of CO or CY caused viremia at 18 hr or 3 d, respectively, with infection in tracheal lymph nodes, but not in untreated mice. SeV invaded hepatocytes and splenolymphocytes on days10 and 5-10, respectively, after infection. CO or CY treatment, three times, induced viral invasion of brain tissues or serious respiratory infection, respectively, but progressive invasion of abdominal parenchymal organs was not different between treated groups. One CO and two CY treatments intensified viral invasion into many organs, maintaining seronegativity. Cerebral blood vessels had the highest incidence of viral antigen in the brain. Astroglial sheets, choroid plexuses, pia maters, and ventricular epithelia tested positive; test-positive neurons were few. SeV MN strain caused progressive invasion of the brain with gliosis and neuronophagy. Blood-brain barrier disruption was caused by virulence of the MN strain. Half the infected mice in two groups treated with CO once and CY twice succumbed to delayed hypersensitivity, suggested by cerebro-microvascular nodulation.

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Bovine ephemeral fever (BEF), a vector-borne disease of cattle, is caused by the Ephemerovirus of the family Rhabdoviridae. In the past 40 years, Taiwan has had seven BEF epizootics, and we have previously reported the first five. This study summarizes the 2001 and 2002 epizootics; conducted case-control serologic studies on 10 herds involved in the 2001 epizootic; determined whether the recent BEF viruses have varied significantly; and discusses the relationship between epizootic patterns and possible variant BEF viruses. For mature cows that had received at least 2 doses of vaccine before the study, a negative correlation between the prevaccinated (the 3rd dose and after) serum neutralization antibody (SNA) titers and their postvaccinated peak rates was found. When prevaccinated SNA levels were at ≤ 32, their postvaccinated SNA levels increased significantly faster (P<0.01) than for those at ≥ 32. The glycoprotein gene of isolates from 1999, 2001, and 2002 had a 99.2-99.9% homology, without consistent amino acid variations in the neutralization sites. Phylogenetic analysis of Taiwanese isolates revealed 2 distinct clusters, the 1983-1989 and 1996-2002 isolates. Cross-neutralization tests confirmed the glycoprotein gene sequence analysis results. In conclusion, annual boosters at SNA levels > 32, at more than 2 doses, or at intervals shorter than 6 months are not advisable. The occurrence of frequent small epizootics implies the dominance of BEF virus over host immunity, but not a variant virus.

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