Serum progesterone (P) concentrations using P-EIA kit (Ovucheck, Cambridge Life Science Co., Ltd.) were examined in 8 captive and 7 free-ranging female Hokkaido brown bears (Ursus arctos yesoensis). The intra- and inter-assay coefficients of variation were 8.9%, 12.6% and 16.6%, 22.7%, respectively, bdsed on 2 serum samples. There was a significant correlation between EIA and radioimmunoassay results based on 64 serum samples (r=0.725; p<0.01). Serum P concentrations were examined in 5 pregnant, 2 solitary non-pregnant bears and a lactating non-pregnant bear in captivity. Annual changes of P levels in pregnant bears were observed as a small elevation during the mating season (May-June), a re-elevation in September-October and a sharp elevation in November-December. The sharp elevation was suspected to reflect changes when implantation occurred. Annual changes of P levels in solitary non-pregnant bears were similar to those in pregnant bears. An annual change of P levels in a lactating non-pregnant bear maintained levels under 5 ng/ml. Two of 7 free-ranging bears exhibited P levels over 1 ng/ml and the birth of cubs was confirmed in the following year in 1 of the 2 bears. P concentrations of other free-ranging bears exhibited less than 1 ng/ml, and these bears were considered to be non-pregnant. It was concluded that P-EIA kit was available for measuring P concentrations in Hokkaido brown bears.

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Therapeutic effects of Toxoplasma lysate antigen (TLA) were studied in mice bearing the tumor in the second passage of 20-methylcholanthrene (MC)-induced tumor cells. Intramuscular administration of TLA 7 days after the tumor-cell inoculation caused apparent inhibition of the tumor growth on day 14. The second treatment facilitated the therapeutic effects. Intravenous transfer of spleen cells prepared from TLA-sensitized mice into tumor-bearing mice also represented the growth inhibitory effects. Prominent effects were seen when the transferred cells were prepared 5 days after sensitization of donor animals. The inhibitory effects were absent in the groups transferred only the adherent cells or the non-adherent cells prepared from sensitized mice. The strongest inhibitory effect was observed in the group to which both adherent and non-adherent spleen cells were transferred simultaneously from sensitized mice. In in vitro experiments, spleen cells obtained from sensitized mice showed cytolytic effect on P-815 or YAC-1 cells after the secondary stimulation in vitro with TLA. Large non-adherent cells containing densely packed granules were induced when cultured with the adherent cells obtained from sensitized mice. These results revealed that TLA can inhibit the growth of the chemically-induced transplantable tumors by activation of adherent and non-adherent spleen cells.

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Changes in plasma concentration of lipid composition were analysed in cattle with anemia due to Theileria sergenti infection. Plasma levels of phospholipids, cholesterol, free cholesterol, high density lipoprotein cholesterol, and vitamin E decreased to 40-67% of the pre-infection levels, corresponding to the decrease of PCV due to the infection. However, no definite changes were detected in plasma level of triglyceride. By gradient centrifugation, it was confirmed that lipid components, other than triglyceride, occur in high density lipoprotein (HDL) and these decreases lowered the HDL value. There was no correlation between this phenomenon and liver function. As similar changes in lipid composition were also observed in phlebotomized calves, it was considered that this phenomenon might partially depend on the acceleration of erythropoiesis as a reaction to anemia caused by T. sergenti infection.

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To determine whether β₂-adrenergic stimulant (clenbuterol) would exhibit beneficial effects upon the elimination of night-time calving, 42 Holstein Friesian cows and heifers at the first stage of labour were injected intramuscularly twice with clenbuterol; 300 μg at 18:00 and 210 μg at 22:00. Other 26 cows and heifers of the same breed at the first stage of labour at 18:00 were not treated and served as controls. All of the treated animals, but one, calved after 5:00 in the next morning, showing a peak of parturition between 5:00 and 10:00. In contrast to this, 42% of the control animals calved at night between 22:00 and 5:00. Thus, the double administrations of clenbuterol were shown to be effective for eliminating night-time calving. The cows treated with clenbuterol tended to show lower incidences of dystocia and retained placenta and a higher first insemination conception rate than the control animals. No adverse effects of the treatment on the viability of newborn calves and milk yields were observed.

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Avian reoviruses grew well in the footpad of chickens inoculated with the viruses via the footpad route, resulting in gross footpad lesions of swelling. The gross footpad lesions induced under some different conditions were investigated for 14 days by two methods. In method A, the lesions were observed grossly and graded as lesion scores 0 to 4. In method B, they were expressed as a swelling index assessed by relative thickness of an inoculated footpad to uninoculated. Both methods are successful, and similar results were obtained. Gross footpad lesions were produced in all chickens aged 9 to 310 days, and the younger were the birds at infection, the earlier and severer were the lesions observed. The lesions began to appear late when the virus titer inoculated was low. It has been elucidated that severity of the lesions depends on virus strains, not on the sexes of chickens.

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The pattern of astrocytic gliosis (AG) was examined in 2-month-old to 18-year-old dogs using glial fibrillary acidic protein (GFAP) immunohistochemistry and electron microscopy. Coronal sections from various levels of the central nervous system (CNS) were stained with hematoxylin & eosin, Luxol Fast Blue, Nissl, and Bodian in addition to GFAP. A consistent pattern of dge-related AG was observed in the dogs. The white matter, cortico-medullary junction, and subcortical nuclei in the cerebrum, central nuclei in the cerebellum, various nuclei in the brain stem, and grey matter of the spinal cord showed even and intense GFAP staining. AG was also prominent in the cerebral and cerebellar cortices and thalamus. Moderate AG was observed in the hippocampus and white matter of the cerebellum and spinal cord. Electron microscopy demonstrated increased number of profiles of degenerative neural components in the vicinity of hypertrophic astrocytes in the cerebral cortex of the aged dogs. Moderate to severe AG was consistently shown in the CNS of the aged dogs. In contrast, young normal dogs showed minimum amounts of GFAP-positive astrocytes in the CNS. These findings suggest that the observed AG in the CNS of the dogs is a morphological expression of aging.

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The cell-free antigen (CFA), with highly hemagglutination activity, obtained from the culture supernatant of Bordetella bronchiseptica was compounded with oil adjuvant to make a component vaccine (CFAV). In the immunization trial in mice, the offsprings whose mothers were immunized with CFAV escaped from death when challenged intrapleurally with virulent strain of B. bronchiseptica. The protective indices (difference of LD₅₀ dose of the challenge strain between immunized and control groups) of the offsprings from CFAV-immunized mothers were over 3.0 in common logarithm value. Moreover, about 90% of the offsprings from CFAV-immunized mothers were negative in nasal turbinate atrophy, while over 80% of them from non-immunized mothers showed obvious turbinate atrophy when challenged intranasally with virulent strain. On the one hand, remarkable differences in the number of bacteria recovered from nostrils were observed between both test groups. It was concluded that CFAV is a very effective vaccine against B. bronchiseptica infection in animals.

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Virus subpopulations with variable virulence, immunogenicity, and infectivity to pigs were readily generated by passaging Tengani isolate of African swine fever virus, either biologically cloned or uncloned, in Vero cell cultures. Avirulent virus populations which account for more than 99% of virus in an uncloned preparation of the 27th padsage are laboratory artefacts, perhaps do not exist in nature. Furthermore, attenuation of virulence did not occur uniformly in all subpopulations newly generated, and a continuous modulation of virus populations differing in immunogenicity and virulence took place in the same individuals inoculated with the 27th passage virus. The same virus preparation, appearing to be slightly virulent in pigs, contained at least a virulent subpopulation that was manifested only by further inoculating susceptible pigs with viremic blood collected at various times during the clinical course. A cloned virus after 23 passages in cell cultures generated a subpopulation (99.9%) which induced subclinical infection in pigs; however, the infection did not confer a solid immunity to homologous challenge with Tengani isolate in these pigs. The Tengani isolate contained subpopulations of virus with immunogenicities shared by the Lisbon '60 isolate and also contained at least one subpopulation specific for the Tengani only.

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Body-length frequencies of Dirofilaria immitis collected from the cardio-pulmonary blood vessel of the dogs were studied in Shiga Prefecture. Japan, in October 1983 and in January and May 1984. The frequencies were not distributed normally because they included the populations of shorter-bodied worms, probably the growing younger worms, whereas the distributions of the body length of worms collected in July 1983 were proved to fit the single normal distribution. From the results of body-length distribution analysis, the worms collected in different seasons were each composed of 1 to 3 groups with different mean body-lengths, standard deviations and composition rates and succesive changes were observed in the distribution patterns. According to the analysis of population structures, the infective season was estimated to range from April or May to October or November in 1983 from the respective ages of shorter-bodied worm groups. The annual rates of first infection and re-infection in 1983 determined by the population of shorter-bodied females collected in January were 48% and 93% respectively and the number of newly infecting females per dog ranged from 1 to 27 with a geometric mean of 8.5. The numbers of newly infecting female worms were distributed following the negative binomial and Poisson distributions in newly infected and already infected dogs respectively.

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Oxidative damage to the membrane in canine erythrocytes with inherited high Na, K-ATPase activity (HK cells) was compared with that in normal canine cells (LK cells). When 30 mM β-acetylphenylhydrazine (APH) was applied to HK and LK cells, lipid peroxidation and hemoglobin denaturation occurred. Lipid peroxidation determined from malondialdehyde (MDA) formation was significantly lower in HK than in LK cells so far as endogenous glutathione (GSH) concentration was maintained at appropriate levels. With the depletion of GSH, MDA formation was accelerated and difference between HK and LK cells was not significant. Denatured hemoglobin bound to the membrane protein was less in HK than in LK cells. During incubation with APH, osmotic fragility increased markedly in LK cells, while HK cells showed very little change. The amounts of total lipid, total and free cholesterol, glycolipid, phospholipid and fatty acids were essentially the same in both cell types. Fatty acid compositions showed very small differences. The membrane of HK cells thus appear to have greater protection against oxidative damage induced by APH, owing to the presence of excess GSH in HK cells. The capability of HK cells to withstand oxidative damage would not be due to differences in membrane lipid compositions.

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Zymosan-induced and luminol-aided chemiluminescence (CL) of whole blood from beagle dogs was estimated for the function of polymorphonuclear leucocytes (PMNs). Whole blood (0.1 ml) was examined directly and results were obtained within 20 min. A phagocytic function of PMNs can be estimated from the peak CL counts and the number of PMNs in a specimen, and the opsonic activity can also be estimated by the peak time showing peak CL after the addition of non-opsonized zymosan. The optimal temperatures to keep diluted whole blood for the CL measurement was around 13°C. Thus, this method offers information concerning the functions of phagocytic cells in whole blood.

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Ligated intestines of rabbits, mice, rats and chickens were used to examine the penetration of newly excysted juvenile flukes of Japanese Fasciola sp. in vitro. In rabbit intestines, the penetration rate was relatively high in the rectum and duodenum. Penetration rates in the jejunum, ileum, cecum and colon were comparable to those in the rectum and duodenum, although it was lower in the appendix. In the case of mouse, juvenile flukes penetrated the duodenum, jejunum, cecum, and rectum at considerably high rates. In rat intestine, penetration by flukes was less in the duodenum and rectum, although flukes were detected in the jejunum. In chicken intestine, flukes barely penetrated the duodenum, jejunum and rectum. Consequently, newly excysted flukes of Fasciola sp. seem to penetrate any region of the intestine in rabbits and mice. In rats, the middle small intestine may be the site suitable for flukes to penetrate. In chickens, the difficulty in penetration of the intestinal wall may be one of the reasons why chickens are scarcely infected with Fasciola sp.

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Attempts were made to clarify the factors contributing to the resistance of chickens to infection with Japanese Fasciola sp. Infection was not successfully established in chickens by oral inoculation of metacercariae, nor by inoculation of excysted juvenile flukes into the body cavity or to the liver surface. Many metacercarial cysts were detected within two days in the feces of orally inoculated chickens. In the in vitro excystation test with chicken bile at 42°C, metacercariae emerged successfully. These results indicate that the major resistant factors may not act during the migration from the mouth to the liver. Histopathological examination of the liver of experimental chickens could not prove the effect of a resistant factor. Excysted flukes were cultivated at 37-42°C in RPMI1640 supplemented with calf serum, with the result that the survival rate of flukes fell with higher temperatures. When chicken serum was used instead of calf serum, flukes survived for a long period of time at 37°C, while all died within four days at 42°C. The higher body temperature of chickens than that of other mammalian hosts is considered to be the major factor contributing to the resistance of chickens to infection with Fasciola sp.

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Characteristics of signal transduction systems mediated by protein kinase-C (Ca²⁺/phospholipid-dependent protein kinase) and estradiol receptors in cow placenta and caruncle were conducted by evaluating protein kinase-C activity and estradiol receptor concentrations and by exploring substrate proteins for the enzyme. The enzyme activity was detected in cytosolic and total particulate fractions of both tissues. The activity levels in these fractions were comparable to those in other cow tissues such as liver and mammary gland. The enzyme activity was inhibited by palmitoylcarnitine, gossypol and adriamycin, known phospholipid-interacting inhibitors of the enzyme. Phosphorylation by the enzyme and subsequent autoradiography revealed that only 125K protein in placental cytosol and two low molecular weight proteins in caruncular cytosol were found to be substrates for protein kinase-C. Ca²⁺ acts as inhibitor of the phosphorylation of several phosphoproteins other than the substrates. Estradiol receptor concentrations in cytosolic and nuclear fractions were similar in both tissues, and the cytosolic concentrations were also comparable to those in pregnant uterus. However, the nuclear concentrations were extremely low when compared to those in the uterus. The signal transduction systems mediated by protein kinase-C and by estradiol receptors seem to be concertedly suppressed in cow placenta and caruncle.

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Spleen cells from Toxoplasma lysate antigen (TLA)-sensitized BALB/c mice showed the strong cytotoxic activity against both natural killer (NK)-sensitive cells (YAC-1 and RL♂-1) and NK-insensitive cells (P-815), when incubated with TLA or recombinant human IL-2 (rhIL-2). The increment of TLA concentration in culture medium increased the cytotoxic activity. Treatment of effector cells; spleen cells from TLA-sensitized mice incubated with TLA, with anti-asialo GM1 or anti-Thy-1 plus complement inhibited the cytotoxic activity of effector cells, whereas treatment with anti-mouse Lyt-2.2 serum plus complement had no effect on the cytotoxic activity. Treatment of spleen cells from TLA-sensitized mice with anti-asialo GM1 and/or anti-Thy-1 plus complement inhibited cytotoxic activities of effector cells. These results suggested that spleen bells sensitized with TLA both in vivo and in vitro were asialo GM1 positive and Thy-1 positive; and the majority of cytotoxic cells induced by TLA were similar to lymphokine-activated killer (LAK)cells induced by IL-2.

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Thirteen beef cows were superovulated using 4, 000 i.u. of pregnant mare serum gonadotrophin (PMSG) on days 9 to 14 of the estrous cycle, followed by two injections of 500 pg prostaglandin F_2α analogue (PGF₂<2α> 48 and 55 hrs later. Seven of them were injected intramuscularly with bovine anti-PMSG serum 12 hrs after the first signs of estrus. The remaining 6 cows were served as controls and received no antiserum. Peripheral blood concentrations of progesterone (P) and estradiol-17β (E₂) were compared in relation to the superovulatory responses. The injection of anti-PMSG serum did not significantly affect the numbers of the corpora lutea (CL), the anovulatory follicles and the transferable embryos at 7 to 8 days after superovulatory estrus, but increased the ratio of embryos classified as excellent or good quality. Although the plasma P concentration showed no significant differences between the anti-PMSG-treated and control cows, the plasma E₂ concentration displayed a characteristic difference, suppressing the second E₂ peak in the anti-PMSG-treated cows. It is concluded that the use of bovine anti-PMSG serum for PMSG/PGF_2α-treated cows at 12 hrs after the beginning of the estrus improves the quality of embryos recovered, probably due to inhibition of high estrogenic environment following ovulation.

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The efficacy of the albumen test for infectious avian leukosis virus (ALV) was examined in detecting congenitally transmitting hens. Seventy-three White Leghorn non-viremic hens with antibody to ALV were used. Eleven of the hens shed infectious ALV into their egg albumen, whereas only 7 of the 11 ALV-positive hens shed ALV antigens. The egg albumen test for infectious ALV was shown to be more effective in detecting the congenitally transmitting hens than that for ALV antigens. Then, twenty of the 62 hens which shed no infectious ALV into the albumen were studied for transmission of ALV to their embryos and for discharging ALV into the oviduct and vagina. Six of the 50 embryos from 4 hens were found to be infected with ALV but all of the 227 embryos from remaining 16 hens were free from the infection. Discharge of the virus into the oviduct and vagina was found both in the 4 transmitting hens and in 6 of the 16 non-transmitting hens. These results suggest that the hens discharging ALV into the oviduct, even though they do not shed ALV into egg albumen, may transmit the virus sporadically to their embryos.

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Seven Holstein-Friesian cows showing chronic nephropathy were studied by renal microangiography and its correlated histopathology. In cases of pyelonephritis associated with severe pathological lesions such as thickening of arterial walls, narrowing of the arterial and arteriolar lumen, and interstitial inflammation and abscess formation, patchy loss of the peritubular capillary plexus from the cortex to the medulla was clearly demonstrated by microangiography. Interlobular arteries were tortuous and attenuated or truncated. Opacification in the vasa rectae and interstitial capillaries was increased. Extensive non-perfused regions could be detected in the cortex. In cases of mild interstitial nephritis and moderate pyelonephritis, microangiography showed focal changes in the renal vasculature. Microangiography is thus shown to clearly demonstrate changed in the renal vasculature corresponding to the severity of the histopathological lesions.

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Effective constituents for renal disease were partially purified from a herbal prescription. The efficacy on renal diseases was examined using an experimental model system of glomerular lesion in mice induced by Agkistrodon acutus venom (Ac₁-P). The extract of boiling water of the herbal prescription (P-3) was first fractionated by ether into an acidic fraction and a mixture of basic and neutral fractions. The acidic fraction was proved to be more effective and then further examined by thin layer chromatography and spectrophotometry. One of the main components was confirmed to be caffeic acid which had inhibitory effect on renal failure in mice by Ac₁-P. This effect was considered to be caused by caffeic acid inhibiting the proteolytic enzyme activity of Ac₁-P. Caffeic acid should thus have prophylactic or inhibitory effect on glomerular disease in which proteolytic enzymes may have pathogenic roles.

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The cardiac musculature of intrapulmonary veins was examined to elucidate its distribution pattern in the musk shrew, the primitive mammalian type, using by both light and electron microscopies. The musculature was distributed extensively from the hilum to the small veins less than 50 μm in diameter, suggesting that the phylogenic origin of this musculature may be traceable to the primitive mammalian types. Ultrastructure of cardiac myocyte in the intrapulmonary venous walls was essentially similar to that of left atrial myocyte. This suggests that the cardiac musculature may contribute to the regulation of pulmonary blood circulation. The abundance of large lipid droplets in both atrial and pulmonary myocytes was also observed and seemed to be the characteristic ultrastructure related to the physiological condition in this animal.

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A simple and reproducible assay method for UDP-glucuronyltransferase (GT) towards 5-hydroxytryptamine (5-HT) was developed. It consists of the removal of unconjugated 5-HT by 0.6 N NH₄OH-saturated n-amyl alcohol and the colorimetric estimation of 5-HT glucuronide. Using this assay method, some properties of the enzyme activity in rat liver microsomes were studied. Simple Michaelis-Menten kinetics was followed with respect to 5-HT and the apparent Km value for 5-HT was 0.1 mM. However, the deviation from this kinetics was observed with respect to UDP-glucuronic acid (UDPGA). The apparent Km values for UDPGA were 0.6 mM and 5 mM. The enzyme activity was stimulated by divalent cations. For Mg²⁺, the enzyme did not obey this kinetics, and the apparent Km values for Mg²⁺ were 1 mM and 10 mM. In the presence of Mg²⁺, the apparent Km value for 5-HT did not change but the V_max value increased. On the other hand, the addition of a low concentration of Mg²⁺ decreased the apparent Km value for UDPGA and increased the V_max value. The addition of a high concentration of Mg²⁺ did not change the apparent Km value for UDPGA but increased the V_max value. These results indicate that the enzyme activity is stimulated by the formation of Mg²⁺-UDPGA complex which showed higher affinity for the enzyme than UDPGA at the low concentration of Mg²⁺ and further stimulated by the formation of Mg²⁺-enzyme complex at the higher concentration of Mg²⁺.

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Changes of body weights and hepatic acetyl-CoA carboxylase activities were measured in voles and mice treated with monosodium-L-aspartate (MSA). MSA was administrated subcutaneously to neonates at 4 mg/g. The MSA-treated mice showed remarkable obesity, associated with the increase in the plasma insulin concentrations and acetyl-CoA carboxylase activities. The activity of acetyl-CoA carboxylase of control voles was very low; under half that of mice. In the MSA-treated voles, although the plasma insulin concentrations also increased, acetyl-CoA carboxylase activities were not elevated and signs of obesity were not observed.

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The distributions of senile plaques (SP) and cerebrovascular amyloidosis (CA) were studied by employing thioflavin S and modified Bielschowsky stains, and β-protein immunohistochemistry on serial sections of the brains of aged dogs older than 10 years. Mature and perivascular plaques, both of which contained compact amyloid deposits, always showed a close topographic relationship to CA. In contrast, the majority of diffuse plaques showed no topographic relationship to CA. Cell bodies of neurons and/or glia were almost always involved in the diffuse plaques. In addition, β-protein immunohistochemistry demonstrated amyloid deposits on the periphery of occasional neurons. These findings suggest that different mechanisms may be involved in the development of the different subtypes of SP in the brains of aged dogs.

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The extracellular low-molecular-mass protein A isolated from Staphylococcus aureus KS 1034 was analyzed for its amino acid contents and terminal amino acid sequences. The C-terminal and the N-terminal amino acid sequences of the low-molecular-mass protein A were -Asn-Ala-Phe and Ala-Gln-His-Asp-Glu-Ala-Gln-, respectively. For immunochemical properties, the low-molecular-mass protein A was similar to protein A isolated from S. aureus Cowan I. Extracellular protease activity of KS 1034 strain was considerably lower than S. aureus C-30 isolated from chicken that produced the extracellular low-molecular-mass protein A.

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