To clarify the functions of the equine guttural pouch, the distribution of various immunoglobulin isotypes and subisotypes in the guttural pouch mucosa were examined in healthy horses.IgGa was present in the mucosa of guttural pouch, mucosal lymph nodules and submucosal lymph nodules.IgM was scattered in the mucosal lymph nodules and in the germinal centers of the submucosal lymph nodules.IgGc was recognized only in the submucosal lymph nodules.These immunoglobulin isotypes and subisotypes were found in lymphocytes and plasma cells.On the other hand, IgA was detected in glandular epithelial cells and the surface layer of the mucosal epithelium, as well as in free cells.This finding suggests that IgA is secreted through the glandular epithelium.Based on the above findings, we conclude that the guttural pouch has phylactic ability.

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The spinal projections of afferent fibers innervating the facet joints between caudal vertebrae were examined by the use of anterograde transport of wheat germ agglutinin−horseradish peroxidase(WGA−HRP).Experiments were performed on 5 adult cats in which spinal dorsal roots below the 2nd sacral segment(S2)on the right side were cut.Injections of WGA−HRP into the caudal facet joints gave rise to extensive cranio−caudal distribution of WGA−HRP positive products along the spinal cord, indicating that many afferent fibers innervating unilateral facet joints terminate bilaterally in laminae I−II, V−VI and X of the thoracic, lumbar, sacral and caudal spinal cord.These afferent fibers may convey a series of sensory information from the caudal facet joints to the spinal cord.

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Tektins are a group of proteins that form filamentous polymers in the walls of ciliary microtubules.The cloning of canine cDNA encoding tektin, was carried out and identified from the testis of beagle dog.Canine tektin cDNA is 1, 523 bp in length, has an open reading frame of 1281 bp nucleotides encoding a protein of 426 deduced amino acids.The predicted amino acid sequence has 77% and 33−50% of homology with the murine tektin and the sea urchin tektins.The amino acid sequence RPNVELCRD and four cysteine residues were conserved in the dog, mouse and sea urchin, suggesting the functional significance of this protein domain and the animo acid residues in the tektin proteins.

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The pathogenicity of an adenovirus isolated from pigeons(Pigeon adenovirus, PA)with inclusion body hepatitis in Taiwan was investigated in specific−pathogen−free(SPF)chicks and in racing pigeons.One−day−old SPF chicks were inoculated subcutaneously with 10⁴ 50% tissue culture infective dose(TCID₅₀, low dose)and 10⁸ TCID₅₀(high dose)of the virus, respectively.The chicks began to die three days post inoculation(DPI)with high dose of the virus and the mortality reached 100%;the chicks began to die 6 DPI and the mortality reached 90% at 14 DPI with low dose.The adult pigeons seemed to be resistant to the PA.However, this virus decreased the production of antibody against Newcastle disease virus in pigeons.It is found that this PA belongs to genetic group D from the restriction patterns produced by BamH I and Hind III.

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The nucleotide sequences of the gene encoding chlamydial heat shock protein 60(cHSP60)of 7 Chlamydia psittaci strains were determined.Comparison of sequences of the cHSP60 gene among chlamydiae showed high identities of the nucleotide sequences by 81.0% or greater and of the deduced amino acid sequences by 92.2% or greater.Comparison of the amino acid sequences between chlamydia and the other bacterial HSP60s resulted in the finding of three highly conserved regions, suggesting that these regions play a role in some function.In addition, 26− or 27−functional residues in the Escherichia coli GroEL out of the 28−residues are conserved in the amino acid sequences of the cHSP60.The data suggest that the function of the cHSP60 may be the same as that of the E.coli GroEL.

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The effects of several factors related to lipids on bovine adipocyte differentiation were investigated in primary culture.Adipocyte differentiation was assessed by development of glycerol−3−phosphate dehydrogenase(GPDH)activity and morphological observation.Addition of triglyceride mixture(Intralipid), caprylic acid and very low−, low− and high−density lipoproteins(VLDL, LDL and HDL)stimulated bovine preadipocyte differentiation in serum−free condition.Especially, VLDL strongly increased both cell protein contents and GPDH activity, suggesting that it stimulated both proliferation and differentiation of bovine preadipocytes.Under Intralipid−induced condition, differentiation of preadipocytes from subcutaneous adipose tissues was more evident than those from omental adipose tissues.However, such depot difference was not observed in medium supplemented with indomethacin, which is a peroxisome proliferator−activated receptor(PPAR)γ agonist.This suggests that the differentiation capacity of bovine preadipocytes was different between depots and such difference is dependent on the ability to utilize lipids as endogenous PPARγ ligands.Therefore, lipid metabolites have the stimulatory effects on bovine adipocyte differentiation in vitro, and lipoproteins, especially VLDL, may play an important role in development of bovine adipose tissues in vivo.

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The intradermal skin test(IDST)and serologic allergy test(SAT)has been developed for confirming a diagnosis of canine atopy and determining allergens for immunotherapy.To determine the prevalence of causative allergens for canine atopic dermatitis in Japan, IDST and SAT were performed with the CMG Immunodot strips on 95 atopic dogs using 9 allergens.In addition, we compared agreement rate, sensitivity and specificity between them(using IDST as the standard).The allergen most commonly positive in both tests was house dust mites(IDST:69.5%, SAT:48.4%).Moreover, Japanese cedar, mugwort and grass mix were detected as attendant causative allergens.Agreement rates between the two tests ranged from 67.4% to 96.8%;the overall mean agreement rate were 81%.SAT was shown to have sensitivity to IDST ranging from 16.7 to 68.2%.The specificities were very high for all allergens, on the order of 94.9−100%(median=98.7%).Finally, the efficacy of immunotherapy was evaluated on 27 atopic dogs based on IDST(15 dogs)and SAT(12 dogs)results.Overall, 60%(9/15)of the IDST group and 66.8%(8/12)of the SAT group experienced a 50% to 100% reduction in their symptomatology.No significant differences were found in response to immunotherapy during the follow−up period between allergen selection methods.These results indicate the value of serologic tests as an aid to identifying an allergen solution for immunotherapy.

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Using both in vivo and in vitro tests, dogs with atopic dermatitis were examined for sensitization with Japanese cedar(Cryptomeria japonica, CJ)pollen allergen.Ten dogs with clinical manifestation of atopic dermatitis were shown to be sensitized to CJ pollen based on the results of intradermal skin test and serum antigen−specific IgE test.In vitro lymphocyte stimulation test showed blastogenic response after stimulation with crude antigen of CJ pollen in all of the 5 cases examined.The peripheral leukocytes showed increased histamine release after stimulation with crude antigen of CJ pollen in 2 cases examined.These data indicate that a proportion of dogs with atopic dermatitis is sensitized to CJ pollen in a cell−mediated manner and show immediate phase reaction of type I hypersensitivity.

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Chromogranin A(CGA)is a member of a family of highly acidic proteins co−stored and co−released with catecholamines in the adrenal medullary cells as well as in other neurons and paraneurons.The nucleotide sequence encoding equine CGA was determined using RT−PCR and rapid amplification of complementary DNA(cDNA)ends(RACE)techniques.A total 1, 828 bp of the nucleotide sequence reveals that equine CGA is a 448−residue protein preceded by an 18−residue signal peptide.Comparison of the amino acid sequence of equine CGA with those of human, porcine, bovine, mouse, rat and frog CGA showed high conservation at the NH₂−terminal 1−77 amino acids regions(94.8%, 93.5%, 92.2%, 81.8%, 83.1% and 66.2%, respectively)and COOH−terminal 314−430 amino acids regions(90.6%, 81.4%, 90.6%, 80.5%, 83.3% and 39.0%, respectively), as well as a potential dibasic cleavage site, whereas the middle portion showed marked sequence variation(52.5%, 49.1%, 38.9%, 26.6%, 27.9% and 6.2%, respectively).Northern blot analysis and RT−PCR elucidated the tissue distribution of equine CGA mRNA.Its expression was confirmed not only in the adrenal medullary cells but also in other organs(cerebrum, cerebellum, pituitary gland, spinal cord, liver, thyroid gland, striated muscle, lung, spleen, kidney, parotid gland and sublingual gland).Further, in adrenal chromaffin cells and pituitary cells of the anterior−intermediate lobe, the expression was confirmed by in situ hybridization with anti−sense CGA cRNA probe.

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Seven adult free−ranging sika deer(Cervus nippon yesoensis)were examined by histology, immunohistochemistry and electron microscopy for intestinal spirochetal infection.Histologically epithelial and goblet cell hyperplasia and edema of the lamina propria mucosa with macrophage and lymphocyte infiltration were observed in the cecum and colon in 6 of the 7 deer.Numerous argyrophilic spirochetes were present in the crypts and some had invaded epithelial and goblet cells and caused degeneration.Immunohistochemically the organisms stained positively with polyclonal antisera against Brachyspira(Serpulina)hyodysenteriae and B.pilosicoli.Ultrastructurally they were 6−14 μm long, 0.2−0.3 μm wide and had 4−6 coils and 13 axial filaments per cell;such features were closely similar to those in the Brachyspira species.These results showed that the spirochetes were capable of inducing enteritis in deer and this intestinal spirochete infection might already be prevalent among wild sika deer in Japan.There is a possibility that this spirochetal colitis is a new syndrome in sika deer and that the same and/or similar spirochetes have infected ruminants, including sika deer and cattle.

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Ultrastructural features of Langerhans cells(LCs)of equine “Kasen” were studied.Electron microscopic observation revealed that LCs were dendritic and had irregular nuclear membranes.A number of Birbeck granules(Bgs)of various types were observed in the cytoplasm of LCs.In LCs in the upper stratum spinous, many Bgs were observed(Type 2 LC).LCs in the epidermo−dermal junction(EDJ)had a few Bgs, vesicles(multivesicular bodies)and highly electron−dense granular endosomes in the cytoplasm(Type 3 LC).Inactive LCs were also observed between the keratinocytes(Type 1 LC).Various types of LCs observed in the skin lesions of equine “Kasen” were interpreted as representing those that recognize, intake and process antigens.

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The cardiovascular effects of losartan, a non−peptidic angiotensin II(ANG II)receptor antagonist, were studied in sheep.Eight normotensive, conscious sheep were tested twice:first under normal conditions and second when subjected to water and electrolytic depletion(furosemide 5 mg/kg twice a day for 3 days).Intravenous injection of 30 mg/kg losartan lowered the mean arterial blood pressure(MABP)in both control and water− and electrolyte−depleted sheep alike.The maximal decrease in MABP was significantly greater in diuretic−treated sheep than in controls(20.0±2.7 vs 9.3±1.1 mmHg)and occurred earlier(8.0±3.3 min vs 12.1±2.9 min).The decrease in blood pressure was associated with tachycardia in both controls and diuretic−treated sheep(+5.5±1.8 vs +11.3±3.9 beats/min).The vasopressor response to 0.1 μg/kg ANG II administered 30 min after losartan was completely antagonized.Two hours after losartan administration, MABP was on the increase in all animals and ANG II receptor blockade was partially obliterated in control sheep.The more marked cardiovascular effects recorded in diuretic−treated sheep as compared to control animals were associated with an increased activation of the renin−angiotensin system(plasma renin concentration:6.51±1.33 vs 1.42±0.37 ng angiotensin I/ml/hr).

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An 11−year−old male Shetland sheepdog displayed epistaxis and nasal discharge from the left nasal foramen.Cytological examination of a smear sample obtained by rhinotomy revealed neoplastic mast cells in the nasal cavity, a definitive diagnostic sign of mast cell tumor.The case was treated by surgery combined with radiotherapy and chemotherapy.Eighteen days after the last treatment, marked enlargement of the mandibular lymph nodes and facial edema developed, and the dog was euthanized at the owner’s request.At necropsy, metastatic proliferation of mast cells was confirmed in the lymph nodes and liver, but no neoplastic mast cells were observed in the nasal cavity.

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Eighty−five thoroughbred racehorses with various types of fracture were subjected to arthroscopic surgery(44 horses)or internal fixation(41 horses)under sevoflurane anesthesia.The mean end−tidal sevoflurane concentration during anesthesia ranged from 2.5 to 2.8%.PaCO₂ was maintained between 50 and 65 mmHg by controlled ventilation.The mean arterial blood pressure was maintained above 65 mmHg by infusion of dobutamine and fluids, however, heart rate significantly increased with time.Recovery from anesthesia was calm and smooth in almost all cases.No apparent complication was observed during and after anesthesia in all cases.Therefore, sevoflurane anesthesia is considered to be safe and useful for orthopedic surgery in racehorses.

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Aminoglycoside antibiotics are generally accepted to accumulate in renal proximal tubule cells from the luminal surface and show toxic effects on the cells.The binding affinity and membrane permeability of aminoglycoside antibiotics are different at the brush border membrane(BBM)and the basolateral membrane(BLM)of proximal tubule cells.This study was performed, therefore, to investigate the differential effects of the aminoglycoside antibiotic gentamicin(GM)on cultured LLC−PK₁ cells, a pig kidney proximal epithelial cell line, after addition to the BBM or the BLM side.LLC−PK₁ cells were cultured on microporous membranes until forming confluent monolayers, and then GM was added to either the BBM or the BLM side.GM caused release of enzymes from the organelles, with a higher level of release observed following addition to the BBM side than that to the BLM side.Patterns of [³H]GM uptake by the cells differed in a manner dependent on whether it was added to the BBM or the BLM side.That is, the cellular uptake from the BBM side increased with incubation time, while that from the BLM side showed rapid saturation.These results suggested that aminoglycoside antibiotics show differential effects on cultured proximal epithelial cells and have differential patterns of cellular uptake when added to the BBM or the BLM side.

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To clarify the mechanism of cephalosporin nephrotoxicity, the cytotoxic effects of cephaloridine(CER), a nephrotoxic cephalosporin antibiotic, on the pig kidney proximal tubular epithelial cell line(LLC−PK₁)were studied in culture.CER increased the content of hydrogen peroxide and decreased the activity of catalase in the treated cells, followed by an increase in the content of lipid peroxide and decreases in both glutathione peroxidase activity and in the non−protein sulfhydryl content.The levels of NADPH−dependent hydrogen peroxide and superoxide anion production by microsomes prepared from LLC−PK₁ cells, and by NADPH−cytochrome P−450 reductase purified from the rat renal cortex were significantly increased by paraquat.The production of these molecules was antagonized by ρ−chloromercuribenzoate, an inhibitor of NADPH−cytochrome P−450 reductase.On the other hand, CER did not significantly affect the production of hydrogen peroxide or superoxide anions.These results suggested that the cytotoxic effect of CER on cultured LLC−PK₁ cells was due to the increases in hydrogen peroxide and lipid peroxide levels and not microsomal oxygen radical production, and that the mechanism of this cytotoxicity is very different from that of paraquat which induces microsomal oxygen radical production.

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Porcine epidemic diarrhea virus(PEDV)is the causative agent of neonatal diarrhea in piglets, which causes high mortality rates.In this study, the immunoprophylactic effects of chicken egg yolk immunoglobulin(Ig Y)against PEDV were investigated in neonatal pigs.Ig Y was found to reduce the mortality in piglets after challenge exposures.The field application of Ig Y also revealed significant differences in survival rates of piglets given Ig Y, as compared with placebo or control.The results in this study indicated that Ig Y against PEDV could be an alternative way of supplementing prophylactic measures like colostral antibodies from sows.

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Canine herpesvirus 1(CHV−1)has a relatively narrow host cell range when compared to other alphaherpesviruses.The early events of CHV−1 infection in a permissive Madin−Darby canine kidney(MDCK)and non−permissive cell lines.In order to quantify attachment and penetration, were investigated quantitative competitive PCR(QCPCR)method was established for quantitation of CHV−1 DNA.In all non−permissive cells tested, no significant decrease in viral attachment was observed.When CHV−1 was treated with heparin, viral attachment to MDCK cells was reduced by 25% of the input CHV−1 attached to MDCK cells even in the presence of 50 μg/ml heparin.However, the attachment of CHV−1 to non−permissive cells was severely impaired by heparin treatment.In permissive MDCK cells, about 80% of attached CHV−1 penetrated into cells.However, only 4−10% of CHV−1 attached to non−permissive cells penetrated into cells.Our data indicated that CHV−1, like other herpesviruses, attached to permissive MDCK cells through two mechanisms:the first one is through the interaction mediated by heparan sulfate(HS)on the cell surface and the second involves unidentified viral component and the cellular receptor.In contrast, the non−permissive cells lacked the cellular receptor for the second attachment mechanism and the defect in viral penetration into non−permissive cell might be related to the lack of the cellular receptor.

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