Topography of ganglion cells in the retina of the horse(Thoroughbred)was analyzed in the wholemount retina stained with cresyl violet, and a total number of ganglion cells were estimated.Sizes of ganglion cells were also measured and size spectra were analysed.The main results showed that:(1)a common point in 4 wholemount retinae, based on cell densities and retinal locations, was that a retina could be divided into 5 regions, namely visual streak, nasal, temporal, dorsal and ventral region to the visual streak.A maximum cell density of 4, 000 cells/mm² was found in the visual streak.And a total number of ganglion cells was estimated in a range of 398×10³−469×10³, with a mean of 441×10³±31×10³(n=4).(2)cell sizes were measured as the mean lengths of the major and minor axes of the somas, and were in a range of 5−53.8μm.The lowest mean diameter was 14.0μm(±3.7)in the visual streak and the highest was 25.9μm(±7.6)in the ventral region.Cell size spectra were unimodal and positively skewed.It is expected that these analyses will provide an anatomical and physiological background for further study of the visual system in the horse.

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Although many mast cells locate under the endothelial layer alond the sublobular veins in canine liver, the cell function remains to be fully defined.To establish the nature of the canine mast cell, the mast cells were examined by electron microscopy.A few monocytes contacted with luminal surface of endothelial cells under which mast cells situated.To confirm the chemotaxis of monocyte by hepatic mast cells, the hepatic venous vessels were treated with a histamine releaser(compound 48/80).The monocytes invaded into the subendothelial layer and extended their pseudopodium to the degranulated mast cells.It presumes that some mediators within the mast cell granules might act as a chemotactic substance to the monocyte.On the contrary, mast cells were migrating from subendothelial layer to venous lumen under normal condition.The migrating mast cell showed strong acid phosphatase reaction in their granules.It suggests that the granules of migrating mast cell became visible to acid phosphatase activity by a physical force such as contact stimulation, and that a part of mast cells remigrate from the venous wall to other places by the blood flow.Furthermore, hepatic mast cells were revealed to contain both endothelin−1 and histamine in their granules by immunocytochemistry.As these substances have an activity of stronger venous constriction, it seems that the mast cells play an important role in the blood flow regulation of the canine liver.

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Effects of recombinant human granulocyte colony−stimulating factor(rhG−CSF)on the number and blastogenesis of lymphocytes were evaluated in clinically healthy dogs treated subcutaneously with rhG−CSF at a dose of 2.5 μg/kg for 3 days.Significant increases in the number of leukocytes and segmented neutrophils were observed after the administration of rhG−CSF.The number of lymphocytes also increased on days 1 and 2 after the treatment.Activities of phytohemagglutinin, concanavalin A, and pokeweed mitogen−induced lymphocyte blastogenesis(LB)were augmented to twice the pretreatment levels by the administration of rhG−CSF.These results suggested that administration of rhG−CSF activated lymphocyte functions such as LB in healthy dogs.

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The purpose of this study is to attempt the induction of early immunopotentiation of antibodies specific to fimbriae of Salmonella enterica serovar Enteritidis(SE), by administering thymulin and zinc to SE−vaccinated chicken breeders, and the improvement of protection against a controlled−live challenge by SE.The first two groups of breeders were administered subcutaneously at 15 and 19 weeks of age a killed SE vaccine.Breeders of the third and fourth groups were left unvaccinated.Breeders of the first group, immunopotentiated by thymulin and zinc, were able to induce the earliest antibodies in their pooled sera at 2 weeks post the first SE−vaccination, specific to fimbriae(21 KDa)of SE.However, the second group that was only vaccinated with the same SE−vaccine produced specific antibodies to fimbriae at 3 weeks following the second vaccination(22 weeks of age).Breeders of the third group, that were neither SE−vaccinated nor immunopotentiated by thymulin and zinc, but were challenged by live SE at 22 weeks of age, were able to show specific antibodies to fimbriae at 3 weeks post challenge(25 weeks of age).The fourth group that was deprived of SE−vaccination, immunopotentiators, and challenge didn’t show any background antibodies specific to SE−fimbriae.The presence of the earliest antibody−immunopotentiation to fimbriae of SE in breeders of the first group, administered thymulin and zinc, was associated with the lowest frequency of SE−infected ceca(10%)among the challenged groups.In addition, breeders of the first group were the only challenged birds resulting in absence of SE infection in their cecal tonsils.The first group−vaccinated, immunopotentiated, and challenged, and the second group−vaccinated and challenged only resulted in breeders with absence of SE infection in their oviducts and spleens.In conclusion, immunopotentiation of chicken breeders by thymulin and zinc induces the earliest specific antibodies to fimbriae of SE associated with the lowest frequency of SE−infected ceca, and absence of SE infection from cecal tonsils, oviducts and spleens.

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An epidemiological investigation of 120 canine otitis externa cases in 1, 370 dogs was done on the incidence rate, ear pinna shapes, breeds and their relationships.Eighty−five cases(12.6%)in 672 dogs with pendulous ears and 35 cases(5.0%)in 698 dogs with erect ears had otitis externa, and the difference between them was significant(P<0.05).Ninety−five auditory cerumen specimens were cultured for Malassezia pachydermatis(M.pachydermatis)and analyzed for concentrations of major fatty acids.Although rates of cases positive for M.pachydermatis in both ear pinna shapes were almost the same, i.e.55.2% in the pendulous group and 53.6% in the erect group, the average total fatty acid level of the pendulous ear group was significantly(P<0.05)higher than that in the erect ear group after dismissing extraordinary levels in the Siberian husky.Isolated M.pachydermatis strains were examined for the effects of fatty acid supplementation on their growth.The majority of the strains utilized fatty acids and grew faster in fatty acid supplemented broth.These results suggest that M.pachydermatis, the predominant causative agent of canine otitis externa, prefers the auditory canal of dogs with lipid−rich earwax and grows fast, but growth strongly depends upon the canine breed.

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To investigate the relation between the canine pigmented epidermal nevus(PEN)and cutaneous papillomavirus, we cloned and sequenced the L1 gene of papillomavirus from the canine pigmented epidermal nevus(PEN).Amplification of DNA sample with the L1 consensus primers yielded an expected fragment of approximately 450−bp.The nucleotide sequences of the fragment showed about 64% of sequence similarity to the L1 region of human papillomavirus isolate CP6108 and less than 57% sequence similarity to those of canine oral papillomavirus(COPV).In situ hybridization determined the presence of papillomavirus DNA mainly in the upper stratum granulosum of skin in this case.The results indicated that the canine cutaneous papillomavirus from the PEN lesion was genetically close to human papillomavirus rather than COPV.

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Six strains of Escherichia coli, isolated from urine of dogs with urinary tract infection(UTI), were examined to assess of urovirulence factors(UVFs)in the pathogenesis of UTI in an experimental pyelonephritis mouse model.From the results of ID₅₀ and LD₅₀, isolates having different UVFs in the same O serotypes varied in pathogenicity, and isolates having the same UVFs in different O serotypes had nearly the same pathogenicity.Histopathogenic examination revealed that the presence of pap, hly and cnfl contributed greatly to the development of upper UTI.It has also been suggested that hly and cnfl significantly related to the LD₅₀ of the strain in the mouse model, confirming that UVFs are closely related to the pathogenicity of canine UTI.

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To evaluate the function of the constrictor urethrae of dogs that had urinary incontinence and were diagnosed as having ectopic ureter, the urethral pressure profile(UPP)was measured by means of a microchip catheter transducer.The UPPs(14.5±3.3 mmHg)of the four dogs suffering from ectopic ureter were much lower(p<0.001)than the UPPs of clinically healthy female dogs(35.3±5.7 mmHg).In the cases of ectopic ureter, it was shown that the UPP was lowered, and it was suggested that this would lead to the expression of urinary incontinence.

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Japanese cedar pollinosis is a type I allergic disease mediated by immunoglobulin E(IgE)antibodies to Japanese cedar(Cryptomeria japonica)pollen antigen(CPAg).By using 22 dogs consisting of 20 dogs aged 3 months and 2 dogs aged 3 years, immunization was performed by subcutaneous injections of CPAg with aluminum hydroxide gel.Variable levels of CPAg−specific IgE antibody response were detected in 21 of the 22 immunized dogs two weeks after the second immunization.This study provided an experimental sensitization system with CPAg in dogs, which will be useful for further immunological studies on Japanese cedar pollinosis.

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The nucleotide sequence of canine α−lactalbumin cDNA from canine mammary tissue was determined by polymerase chain reaction with degenerate primers.A 742 base pairs nucleotide sequence cloned was similar to the size of mRNA in Northern blot analysis.The cDNA encodes 142 amino acid residues containing the conserved sequence motif of α−lactalbumin, demonstrating the highest homology with pig(73% identity−82% similarity)among the known amino acid sequences of α−lactalbumin.The canine cDNA also showed 71% identity−78% similarity with human, 58−73% with mouse, 60−74% with rat, 67−77% with goat, 66−77% with cattle, and 67−76% with sheep, respectively.

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Sarcocystis sp.was detected from cattle slaughtered in Saitama Prefecture, Japan.The cysts were 3, 400−4, 400×198−238μm in size and had the thick cyst wall which was 7 to 10 μm thick and provided with finger−like villar protrusions.The protrusions were 8−9.5×2−2.5 μm in size and had microtubules in the core.

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Antibodies against Cryptosporidium parvum in the saliva and sera of three calves experimentally infected with this parasite were examined by an indirect immunofluorescence antibody test and immunoblotting.Salivary anti−C.parvum IgA antibody appeared on day 12 post−challenge and had a tendency to increase transiently between days 15 and 30 post−challenge.Salivary anti−C.parvum IgG antibody levels showed a gradual increase along with the change in IgA antibody levels during the infection.In contrast, serum anti−C.parvum IgA antibody levels showed only a slight increase between days 15 and 30 post−challenge.Serum anti−C.parvum IgG antibody levels rose on day 12 post−challenge and one calf maintained relatively high level up to the end of the experiment.In immunoblotting, an antigen with a molecular mass of 15 kDa was found to react strongly to salivary IgA antibody and a 27 kDa antigen to react to serum IgG antibody.

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To disclose the mechanism of cellular injury following porcine circovirus(PCV)infection, 12 pigs were examined by the terminal deoxynucleotidyl transferase−mediated dUTP−nick end labeling(TUNEL)method and immunohistochemistry.Histologically, the lymphoid tissues were characterized by marked apoptosis of lymphocytes, lymphocyte depletion, and macrophages and giant cells containing numerous inclusion bodies with or without apoptotic bodies.Immunohistochemically, there were many lysozyme−positive macrophages in the lymphoid follicles, while the number of CD79a−positive B lymphocytes was scanty.Apoptotic cells, which were proved to be TUNEL positive, revealed CD79a positivity.Although detectable mainly in the cytoplasm of macrophages, PCV antigens were found also in the nuclei of macrophages and apoptotic lymphocytes.Ultrastructurally, the presence of PCV virions was confirmed in apoptotic bodies phagocytosed by macrophages.These findings suggested that lymphocyte depletion with apoptotic death of B lymphocytes was caused by PCV, and that some of the inclusion bodies were phagolysosomes derived from the apoptosis.Thus, PCV may trigger the development of wasting disease syndrome by producing an immunocompromised state in pigs.

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Ultrastructural alteration of anionic sites(ASs)in the glomerular basement membrane(GBM)was studied in glomerulonephritis characterized by linear capillary IgG deposition in four dogs infected with Dirofilaria immitis and two normal control dogs using polyethyleneimine.ASs were identified as small dense particles distributed regularly in the lamina rara externa(LRE), but there were no ASs in the lamina densa(LD)of the GBM of the control dogs.In the glomeruli of the infected dogs, ASs were distributed regularly or irregularly in the thickened LD.ASs were in addition localized over the characteristic continuous bands of subendothelial dense deposits.The number of ASs of the LRE increased in all four infected cases as compared to the controls(p<0.01).

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In isolating skeletal muscle satellite cells, sometimes a problem is encountered in removing contaminating nonmyogenic cells.In the present study, we constructed a novel vector, pSKA−EGFP, which achieves the expression of enhanced green fluorescent protein(EGFP)exclusively in myogenic cells under the control of skeletal α−actin promoter when transfected to primary cultured cells from skeletal muscle.Cells from rat skeletal muscle positive for EGFP after transfecting with pSKA−EGFP were all positive for desmin and none of the nonmyogenic cells expressed EGFP, indicating that the expression of EGFP is specific to myogenic cells.Among the cells positive for EGFP were proliferating cells, presumably satellite cells.In addition, EGFP positive cells derived from horse skeletal muscle after transfecting pSKA−EGFP in vitro formed multinuclear myotubes, indicating that myogenic expression of EGFP driven by skeletal α−actin was achieved also in the equine cells.These results indicated that pSKA−EGFP vector will be useful in identifying and following up the satellite cells in real time, and also permit us to isolate satellite cells in combination with fluorescence−activated cell sorting(FACS).

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Clostridium botulinum type B neurotoxin cleaves VAMP(vesicle−associated membrane protein)/synaptobrevin into two fragments, which results in inhibition of neurotransmitter release.The induced fragment did not react to the antibody raised against the synthetic peptide of the amino−terminal 20 residues of VAMP−2, suggesting that the toxin treatment has caused antigenical alteration in the amino−terminal region of VAMP−2.In rat brain synaptosomes, type B neurotoxin was reduced presumably with sulfhydryls in the membrane and detected in the synaptic vesicle fraction which involved the degradation of VAMP−2 and the inhibition of neurotransmitter release.The light chain in a free form was present in the cytosol fraction.These findings suggest a possibility that type B neurotoxin endocytoses into synaptic vesicles by the recycling pathway and the light chain is penetrable through synaptic vesicle membrane.However, the amount of type B neurotoxin entrapped into synaptic vesicles appears to be extremely small, which may be attributed to a lower sensitivity of the toxin to brain synaptosomes than to peripheral nerve endings.

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Cattle have been recognized as a principal reservoir of Escherichia coli O157:H7.This organism appears to be confined to the gastrointestinal tract and is shed in feces.A probiotic product containing lactic acid−producing Streptococcus bovis LCB6 and Lactobacilus gallinarum LCB12 isolated from adult cattle was developed, and a preliminary experiment was conducted to evaluate its effect on the elimination of E.coli O157 from experimentally infected calves.Eight 4−month−old Holstein calves were orally challenged with E.coli O157 and the probiotic product was administered against four calves continued fecal shedding of E.coli O157 by the 7th day after infection.Fecal shedding of E.coli O157 was completely inhibited and re−shedding was not detected in any of the animals.Remarkable increase of VFAs, especially that of acetic acid in feces after the administration of probiotic bacteria correlated with the diminution of E.coli O157.Four calves that had spontaneously ceased fecal shedding of E.coli O157 by the 7th day exhibited a high concentration of VFAs in feces before and after experimental infection.Although our results are preliminary and obtained from calves under limited conditions, the possible application of probiotic product to reduce fecal shedding of E.coli O157 from cattle is suggested.

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A seven−month−old, male Shih Tzu dog weighing 3.7 kg had an immature cataract in its left eye.A biomicroscopic examination revealed numerous vacuolations in the posterior cortices with nucleus cataracts, covered by an intact anterior lens capsule.The changes observed by ocular sonographic examination(OSG)of the left eye were hyperechoic, and a funnel−cone shape was observed posteriorly with cortex hyperechogenicity in the lens.The left eye was diagnosed as having a posterior lenticonus with congenital cataract.Phacoemulsification was performed on the left eye as diagnostic treatment of the posterior lenticonus and cataract.Postoperative OSG on the left eye revealed a V−shaped linear echo that was indicative of a posterior capsule of the lens.Moreover, it was confirmed that hyperechoic cataract material inside the lens had disappeared.

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The relationship between ejaculation intervals and semen quality in 4 male cats aged 3−5 years was investigated in this study.Semen was collected 10 times at intervals of every day, every other day, and every three days using an artificial vagina.Semen was collected consecutively twice on the day of semen collection, and the semen quality was examined.In semen collected every day, the number of sperm in the first collection decreased, and the frequency of immature sperm rapidly increased after the 4th day.In semen collected every other day and every three days, although the semen volume markedly varied among the animals on both first and second collections, the volume remained stable for each animal, the number of sperm was similar in the first and second collections, but was clearly larger in the first collection(p<0.01).Sperm motility and abnormality were stable among the various intervals and between the first and second collections in each animal.

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To clarify the sperm count required for fertilization by artificial intravaginal insemination(AIVI), twenty−nine female cats were examined.Six male cats aged 2−12 years with normal semen quality, copulation capability, and fertility were used.In AIVI, animals received administration of 250 iu hCG once or 100 iu twice on days 2−4 of estrus to induce ovulation, and were inseminated 15, 20, or 30 hr after the initial hCG administration.The success of ovulation was judged by elevation of the peripheral progesterone level after hCG administration.AIVI was investigated at three sperm counts, 20×10⁶(Experiment 1), 40×10⁶(Experiment 2), and 80×10⁶(Experiment 3), with semen collected by the artificial vagina method.Semen was infused in the vagina under general anesthesia by advancing a 9 cm−long nylon probe with 1.5 mm diameter connected to a 1 ml syringe in the vagina for 3−4cm.Ovulation was induced in 43 of 45 animals(95.6%).One of 16 animals was fertilized(conception rate:6.6%)by AIVI in Experiment 1.In Experiments 2 and 3, conception was obtained in six of 18 animals(33.3%)and seven of nine animals(77.8%), respectively, and the mean numbers of kits were 4.0±0.4 and 3.3±0.5, respectively, and the mean numbers of kits were 4.0±0.4(SE)and 3.3±0.5, respectively, showing no significant difference.There were no differences in the time of insemination after hCG administration and the conception rate among these groups.Our findings showed that the number of sperm required for fertilization by AIVI of fresh semen in cats was 80×10⁶.

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To obtain normal kits by embryo treansfer(ET)during the non−breeding season, maintenance of pregnancy was carried out by administration of sustained action progesterone(P₄)in queens.Embryos were recovered six days after mating from five donor queens in which ovulation was induced by administration of eCG and hCG.The number of embryos recovered ranged from 24 to 53(mean:37.2±6.4)per animal and most embryos were compacted morulae.The yield of embryos was 49.0−93.3%(mean:73.8±9.6%).As for recipients, porcine pituitary gland preparation and hCG were administered to 19 queens and estrus and ovulation were induced in 18 queens(94.7%).These queens underwent intrauterine ET of five compacted morulae and 17 cats(94.4%)were impregnated.The number of implantations was 2−5(mean:3.7±0.3).Among these impregnated queens, 15 cats received P₄ adminstration starting on day 24 of gestation and 1−5 newborns(mean:3.4±0.3)were obtained by normal delivery or caesarean section on day 64−69 of gestation.However, two animals that were not treated with P₄ underwent spontaneous abortion about the mid gestational period.Therefore, it is possible to obtain normal kits from queens in the non−breeding season by ET with maintenance of pregnancy by P₄ administration.

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Pseudorabies virus(PRV)infection in resistant swine caused immunosuppression which sometimes resulted in secondary infection by other viruses or bacteria.However the mechanism of the immunosuppression is not well understood.In this study, the effect of PRV on the immune system was examined in the mouse model.Splenocytes or lymphocytes prepard from the spleen of BALB/c mice were incubated in vitro with mitogen, and the ability of cells to proliferation was measured.When the cells were incubated with PRV, the ability of cells to proliferate was inhibited, although PRV did not multiply in the lymphocytes.UV−inactivated PRV also suppressed the proliferation of mice splenocyte.This result suggests that the structural component of PRV virion might cause the immunosuppression.

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