We expected that the enlarged area of zygomatic arch, one of some skull characters adapted for enlarged eye, has an influence on form and function of muscles of mastication in the Baikal seal (Phoca sibirica). So, in this species, the Mm. masseter, temporalis, pterygoidei, digastricus were observed in the macroscopic level. The skull characters related to these muscles were also compared between the Baikal seal and a close-related species, the ringed seal (Phoca hispida). The Mm. masseter and temporalis were well-developed using the enlarged attachment area of zygomatic arch. In contrast, the M. digastricus is suggested to be not so important in the Baikal seal, because the temporal bone is not so developed as in the ringed seal. It is suggested that the Baikal seal has especially developed the Mm. temporalis and masseter using an enlarged area of zygomatic arch among Pusa species. We also suggest that the robust temporal bone is equipped to have the M. digastricus developed as a main retractor of mandibular body in the ringed seal.

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Aging produces alterations in certain functions of the hypothalamo-pituitary axis that result in sexually dimorphic changes in the somatotrophs. Since quantitative morphological data on these age-associated alterations are scarce, we prompted to make a morphometric immunohistochemical assessment as well as undertake an ultrastructural study of the somatotrophic (GH) cell population in male and female rats of different ages. Young (3-month-old), old (20-month-old), and senescent (29-month-old) Sprague-Dawley rats of both sexes were sacrificed by rapid decapitation, their pituitaries immediately dissected out, and processed for both immunohistochemistry and electron microscopy. Analysis of different morphometric parameters revealed that surface density, volume density, and cell density significantly decreased in old and senescent rats as compared to young animals, with this reduction being clearly more marked in females. Both the GH-cell area and perimeter decreased in senescent male rats, while these parameters increased in senescent females. The ultrastructure of the GH cells from old and senescent animals of both sexes evinced changes suggestive of an immature state, with some somatotrophs having the appearance of cells undergoing an involutive process. We conclude that aging has a differential impact on the GH cells of male and female rats with respect to the immunohistochemical and ultrastructural features of that cell population.

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We measured adult mandibles of Ryukyu wild pig (Sus scrofa riukiuanus) from Tokunoshima Island and compared the osteometrical data with those from six Nansei Islands. The mandibles in Tokunoshima Island were larger than those from Amami-Oshima and Okinawa Islands in some measurements. We concluded that the size cline was not statistically recognized among populations. In the principal component analysis, the size cline was also denied, and the separation could be made among island populations in female. It is suggested that the populations in Tokunoshima and Okinawa Islands may be different from those in Amami-Oshima, Kakeroma, Ishigaki and Iriomote Islands in skull proportion.

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The present study was designed to reveal whether astrocytic activation following axotomy causes a numerical increase of nuclear bodies (NBs) in chicken astrocytes. Astrocytes in the spinal cord were activated by a unilateral spinal nerve transection. The frequency of NBs was calculated at the lateral motor columns of the normal group and at the ipsi- and contralateral sides of operated group. In the growing chickens, NBs of each astrocyte were a few and decreased steadily in number with age. In the operated chickens, the frequency of NBs elevated temporarily both in the ipsi- and contralateral sides, and reached its maximum value by 30 post-operative days. The frequency of NBs increased more prominently in the operated side than in the unoperated side. Thus, the present study provides the first evidence for a significant increase in the number of NBs in the hyperactive astrocytes caused by axotomy.

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Clostridium novyi (C. novyi) Type B alpha-toxin was purified from culture supernatant by column chromatography, and was inactivated by formalin. A purified alpha-toxoid vaccine was prepared by mixing it with an aluminum phosphate gel adjuvant. Guinea pigs immunized twice with 4 μg or more of alpha-toxin survived against challenge with C. novyi Type B spores. Anti-alpha-toxin (antitoxin) titer was measured by toxin neutralization test using Vero cells. All of the guinea pigs having antitoxin titers of 10 units (U) or more at challenge were survived. In another experiment, guinea pigs were immunized with crude alpha-toxoid vaccines prepared by inactivated culture supernatant or by adding broken bacterial cells to the former. In this experiment, 10 U of antitoxin titer was the border of survival or death after challenge. Guinea pigs with antitoxin titers of less than 5 U, 5 U and 10 U died at 2, 3 to 4 and 4 days, respectively, after challenge. These results suggest that C. novyi alpha-toxin was the main protective antigen against challenge exposure to spores in guinea pigs.

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The nucleotide sequence of cDNA coding the 38-amino acid of the C-terminal domain of insulin-responsive glucose transporter (GLUT4) was determined by the method of reverse transcription-polymerase chain reaction in the sheep, goat and pig, and compared with that of bovine which have been shown to have a unique amino acid conversion of Asn508 to His. The deduced amino acid sequence was completely identical in the three species, and did not have the amino acid conversion at position 508. Western blot analysis confirmed that an antiserum raised against a rat C-terminal peptide cross-reacted efficiently with GLUT4 of these livestock mammals.

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Chicken monoclonal antibodies (mAbs) were developed against bovine prion protein (PrP) peptide. Chickens immunized with bovine PrP peptide B204 (amino acid residues 204-220) coupled to keyhole limpet hemocyanin produced specific antibodies to the peptide as determined by an enzyme-linked immunosorbent assay (ELISA) using the B204 peptide coupled to ovalbumin as target antigen. From a fusion experiment using the chicken fusion partner cell line MuH1 and immune spleen cells, 19 mAbs reactive with B204 were generated. These mAbs were subdivided into five groups based on competitive ELISA using B204 and four 10-amino acid peptides. These five groups included all combinations expected based on comparison of amino acid sequences among the five species, bovine, mouse, human, sheep and hamster, examined. These results indicate that the chicken mAb system is a suitable technique for immunological analysis of PrP in mammals.

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Serological tests were performed to investigate extent of tick-borne diseases in dogs infested with Rhipicephalus sanguineus at a kennel in Okayama Prefecture. Three of 22 dogs (13.6%) were positive for Ehrlichia canis. Two of 19 dogs (10.5%) were positive for Rickettsia japonica. Three of 22 dogs (13.6%) were positive for Babesia gibsoni. None of these animals were positive for Coxiella burnetii or Hepatozoon canis. A microfilaria was detected in a drop smear of hemolymph from an engorged female tick, however species was not determined. It is possible that these ticks can transmit pathogens to domestic dogs which are rare in Japan.

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In order to determine the sex of carcass remains of the Sika deer (Cervus nippon), we improved a polymerase chain reaction (PCR) technique for amplification of the Sika deer Sry, a male-specific DNA region on the mammalian Y chromosome. From the nucleotide sequence of the Sry region obtained here, PCR primers, MT1 and MT2, capable of amplifying a shorter Sry region were newly designed, and a microsatellite locus was used as a positive control. Using these primers, 96 of 109 sex-unknown fawns (88%, 96/109) were successfully sexed (46 males and 50 females) regardless of the conditions of carcasses found in the field. The results and the methodology could greatly contribute to the study of the mortality pattern of the Sika deer population.

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Major histocompatibility complex (MHC) of pigs is known as swine leukocyte antigen (SLA). The cDNA encoding a new allele of SLA class II DQ β-chain was successfully isolated from a CSK miniature pig (derived from Göttingen strain) and characterized by sequence analyses. SLA-DQB cDNA fragment encoding β₂-domain was amplified by reverse transcriptase-polymerase chain reaction using the sequences preserved in a various vertebrates as primers. Using non-radioisotope technique with the PCR product as a probe, cDNA clone G01 was isolated from a spleen cDNA library, and nucleotide sequence of this clone was determined. This clone encompassed a whole SLA-DQ β-chain coding region, containing a total length of 1161 nucleotides with an open reading frame (ORF) of 786 nucleotides, 5' untranslated region of 15 nucleotides, and 3' untranslated region of 360 nucleotides ending with a canonical polyadenylation signal, followed by a poly A tail. Sequence comparisons of the ORF of this clone with those of known SLA-DQB genes confirmed that this clone is a new allele (SLA-DQB^*G01). Phylogenetic analysis of the nucleotide sequences of swine, human, and murine MHC class II genes indicated that SLA-DQB was more similar to HLA-DQB1 than H-2Aβ. Comparison of the nucleotide and deduced amino acid sequences among SLA-DQB alleles showed that the SLA-DQ β-chain polymorphism was found almost in β₁-domain which contains the antigenic peptide binding sites.

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Antigenic properties of two representative allelic products of the major piroplasm surface protein (MPSP) of Theileria sergenti were studied. Sera from cattle infected with either of Ikeda and Chitose types of the parasite reacted strongly with homologous but weakly with heterologous recombinant antigens in immunoblotting. Monoclonal antibodies (MoAbs) produced against the both allelic products of MPSP parasites reacted only to the immunizing antigen. These results suggested that crossreactivity between two allelic products is very low inspite of relatively high homology in their amino acid sequences. Double staining of parasitized erythrocyte smear using type-specific MoAbs by an indirect immunofluorescent assay revealed that the set of MoAbs was useful for quantitative and differential detection of each type of parasite in mixed population.

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Canine monocytes from peripheral blood were stimulated by acetyl low density lipoprotein (LDL) to detect receptor-mediated uptake of lipoproteins and to clarify the role of monocytes involving in atherosclerotic lesions. Monocytes collected from peripheral blood which were treated with phorbol 12-myristate 13-acetate (PMA) took up acetyl LDL and differentiated into macrophage-like cells (foamy macrophages). Uptake of acetyl LDL was inhibited specifically by maleyl canine serum albumin (MSA) depending on its concentration. Monocytes treated with PMA did not take up native LDL. These results suggest that canine monocytes express the receptor-mediated uptake of acetyl LDL by a specific pathway (the so-called scavenger receptor) and that they play a crucial role at the initial stage of canine atherosclerotic lesions.

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A 10-year-old dog, which had been treated for mitral insufficiency, died suddenly. Transmural myocardial infarction secondary to thromboembolic occlusion of the subsinuosal interventricular branch of the left circumflex artery was noted in the posterior lower half of the left ventricular wall, involving the interventricular septum and a part of right ventricular wall. The mitral valve leaflets were markedly thickened (valvular endocardiosis). There were a patchy area of jet lesion and several mural thrombi on the left-atrial endocardium. The embolus in the subsinuosal interventricular branch was composed of mostly platelets and fibrin, showing the same histologic features as those of the mural thrombi on the left-atrial endocardium. From these findings, it was concluded that dislodgement of part of the mural thrombi on the left-atrial endocardium caused thromboembolism of the subsinuosal interventricular branch.

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Effects of topical application of a capsaicin analogue, nonylic acid vanillylamide (NVA, 0.032-10.0 mM) on the arterial diameter in the ear skin were examined in conscious rabbits using a precise dial caliper. In addition, the possibility of nitric oxide (NO) participating in a vasodilatation induced by low concentrations of NVA was tested by an NO synthase inhibitor. At the lowest concentration of NVA (0.032 mM), no significant change in the diameter was observed after external application of the NVA ointment. At concentrations of 0.32 mM or more, NVA produced a significant vasodilator response. However, at higher concentrations of 3.2 and 10.0 mM, NVA induced substantial shrinkage in the arterial diameter and oedema formation, which was not affected by L-NAME (N^G-nitro-L-arginine methyl ester, 3 mg/kg, i.v.), suggesting fluid leakage induced by oedema from the vessels might suppress the vasodilatation. Thus, the concentration-response curve for NVA was bell-shaped. NVA (0.32 mM)-induced vasodilatation was not significantly affected by atropine (1 mg/kg, i.v.) or propranolol (80 μg/kg, i.v.). However, the NVA-induced vasodilatation was completely suppressed by an NO synthase inhibitor, L-NAME (3 mg/kg, i.v.) which had no influence on the resting diameter, but not by an inactive stereoisomer, D-NAME (3 mg/kg, i.v.). These findings suggest that vanilloid receptor activation results in the release of sensory neuropeptides, which in turn stimulate the synthesis of endothelial NO which is responsible for the vasodilatation.

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In male hypogonadic mutant rat (hgn/hgn), gonocytes degenerate and peritubular cells form multiple layers around seminiferous tubules during early postnatal testicular development. Alkaline phosphatase (AP) activity has been used as not only a tracer for the primordial germ cells (PGCs) but also a histochemical marker for the peritubular myoid cells. In the present study, we examined the localization of AP activity during the postnatal testicular development in the hgn/hgn and phenotypically normal (+/+ or +/hgn) rat. In the normal testis, high AP activity was located in the surface of the PGCs on 3 days of age. As the PGCs differentiated into spermatogonia, the AP activity drastically decreased in intensity on 7 days and was completely lost by 12 days. In the hgn/hgn, the PGCs showing high AP activity occupied the inside of dilated seminiferous tubules on 3 and 7 days of age. The luminal AP activity declined gradually by 18 days and disappeared on 21 days, when the germ cells completely degenerated in the hgn/hgn testis. In the normal, high AP activity emerged in a layer of the peritubular cells surrounding the tubules on 7 days and afterwards, indicating that the peritubular cells were differentiating into myoid cells. In the hgn/hgn, the peritubular cells formed multiple layers around the tubules and showed weak AP activity on 3 to 18 days of age. On 21 days of age, high AP activity emerged in a single layer of the peritubular cells directly attached to the basement membrane in the hgn/hgn. These results indicate that the PGCs showing AP activity kept remained at later stage in the hgn/hgn and that the single layer of mature myoid cells showing high AP activity appeared much later in the hgn/hgn testis than in normal.

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Through serological surveillance of wild animals by enzyme linked immunosorbent assay with protein G (PG-ELISA), we obtained epidemiological data on Lyme borreliosis in Japanese wild animals. In this study, we carried out serological surveillance for Lyme borreliosis in wild Japanese serows (Capricornis crispus). Forty-six of 200 (23%) serum samples were positive, indicating that Lyme borreliosis is prevalent in wild Japanese serows. This positive rate was relatively higher than that of other animals and was similar/identical to that in other important hosts worldwide. Our results suggest that Japanese serows may be one of the important hosts of Lyme borreliosis in the central mountainous and forested areas of Japan.

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A total of 40 strains of Listeria monocytogenes which have been demonstreted to be serovar 1/2a, 1/2b, and 4b were genotyped by pulsed-field gel electrophoresis (PFGE) after separate digestion with Apa I, Asc I, Sma I, and Sse 8387 I. Twenty-seven unrelated strains including four representative strains showed distinctly different genotypes according to their PFGE profiles. Then nine strains isolated from shredded cheese of different lots and four strains isolated from the cheese-processing environment were shown to display the same genotype. Therefore, it is suggested that the Listeria was spread in cheese by cross-contamination from the cheese-processing environment. Thus, PFGE analysis has a good typeability and excellent discriminatry power, and has provided a useful tool for investigation of the source of Listeria contamination.

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Equine respiratory patterns during swimming were examined in five normal horses. The experiment included a preliminary warming-up stage and 6 circuits of swimming around an annular pool of a 50-meter-circumference. The horses were examined for respiratory rates, intratracheal pressures, inspiratory time (T_I), expiratory time (T_E), respiratory cycle (T; T_I+T_E), heart rates, blood lactate concentrations, hematocrit and blood gases. The respiratory rates were maintained around 25/min. Blood gas values changed significantly during swimming. The intratracheal pressures during expiration and inspiration increased significantly with exercise duration compared to the immediately after the warming-up stage. The duty ratio (T_I/T) averaged 0.33, which implied that the expiratory time was roughly doubled the inspiratory time. We considered that a longer expiratory time may limit sudden collapse of airways by water pressure during swimming and prevent a radical decrease of air space volume, thus maintains buoyancy.

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Two new canine osteosarcoma cell lines were established. One (OOS) was established from a 10-year-old female maltese dog with mandibular osteosarcoma and the other (HOS) from a 7-year-old male mongrel dog with scapular osteosarcoma. Histopathological types of OOS and HOS were mixed and fibroblastic cell type, respectively. Transmission electron microscopic features of HOS revealed prominent rough endoplasmic reticulum, suggesting higher malignancy comparing to OOS. Doubling time of OOS and HOS were 45.0 ± 0.5 hr and 42.0 ± 0.1 hr, respectively. Alkaline phosphatase activities of OOS and HOS were quite low. Histological features of tumor tissues produced by transplantation of these cells into nude mice were identical to those of original osteosarcomas.

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Plasma LH and testosterone (T) levels and semen quality after a single intramuscular injection of 1,000 IU hCG were investigated in a Beagle dog with azoospermia and a Beagle dog with poor semen quality. The plasma LH levels of both dogs did not change after the treatment. Although the plasma T levels of the dog with azoospermia increased temporarily, no sperm were detected in its semen. In the dog with poor semen quality high levels of plasma T were maintained for 2 weeks after hCG treatment and its semen quality was temporarily improved between 3 and 4 weeks after treatment. These findings indicate that the semen quality of dogs with oligozoospermia can be temporarily improved after a single injection of hCG.

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In order to investigate the possible mechanisms for caffeine-induced ocular hypertension, the intraocular pressure (IOP) and the outflow through the trabecular meshwork were measured in beagle dog eyes after dosing with intravenous caffeine (30 mg/kg) alone or in combination with the topical β-blocker befunolol [applied as 100 μl of a 1% (w/v) solution] which inhibits aqueous humor formation in the ciliary body. Intravenous injections of caffeine significantly increased the IOP at 0.25 and 1 hr after a single dose. The ocular hypertension recovered within 2 hr following dosing. Over time, there were no differences in the outflow between the caffeine and control groups. The instillation of befunolol lowered outflow and produced ocular hypotension. The levels of the IOP and outflow in dogs treated with caffeine and befunolol in combination were almost the same as those in dogs treated with befunolol alone. Single-dose and combination-dose studies demonstrate that intravenous caffeine increases the IOP in normal beagle dogs possibly by increasing aqueous humor formation and not by the inhibition of aqueous humor drainage through the trabecular meshwork.

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We previously reported the attenuation of thymidine kinase (TK) deficient mutant (C7301dlTK) of feline herpesvirus type 1 (FHV-1) in cats and the construction of a recombinant FHV-1 (C7301dlTK-Cap) inserted a precursor capsid gene of feline calicivirus (FCV) into the TK deletion locus of the C7301dlTK. In this study, we constructed a further improved recombinant FHV-1 (dlTK(gCp)-Cap) carrying a putative FHV-1 gC promoter sequence upstream of the FCV precursor capsid gene of the C7301dlTK-Cap. Growth kinetics of the dlTK(gCp)-Cap in cell cultures was similar to those of C7301dlTK and C7301dlTK-Cap. A strong expression of FCV immunogenic antigen by dlTK(gCp)-Cap was confirmed by indirect immunofluorescence and enzyme-linked immunosorbent assays. In addition, one vaccination with dlTK(gCp)-Cap protected cats more effective against subsequent virulent FCV challenge than that with C7301dlTK-Cap.

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Reactivities of feline calicivirus (FCV) field isolates with monoclonal antibodies (MAbs) were examined by enzyme-linked immunosorbent assay (ELISA). The reactivities of the viruses in ELISA were different from our previous results using the neutralization tests (NT). Many isolates were positive in ELISA with MAbs which recognized neutralizing epitope 3B and/or 4. However, most were negative in NT in our previous study. After absorption of two FCV strains with host cells, the non-infectious virus fluid still reacted with MAb, which recognized epitope 3B and/or 4 in ELISA. These results indicated the possibility that neutralizing epitopes are expressed on non-infectious virus particles or exist as proteinaceous molecules in virus fluid.

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