A rapid colorimetric assay has been applied to functional studies of monkey lymphocytes such as mitogen-induced blastogenesis, complement-dependent cytotoxicity, interleukin 2, and interferon assays, in which a tetrazolium dye is reduced to a formazan salt by viable cells but not by dead cells, accompanying with color change from yellow to purple. The amount of formazan dye generated is easily read on a ELISA reader and is directly proportional to the number of viable target cells, depending on the degree of cell activation. This assay has several advantages over conventional methods with respects to rapidity, precision and no requirement of radioisotopes.

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The present study was designed to evaluate the immunostimulatory and fibrogenic factor(s) from alveolar macrophages (AM) after the in vitro exposure to silica or asbestos particles. Interleukin 1(IL-1)-like thymocyte proliferation activity was detected in the supernatants from rat AM cultures stimulated in vitro with silica or asbestos particles in a dose-dependent manner. In addition to this thymocyte proliferation activity, those supernatants from silica-or asbestos-stimulated AM cultures also exhibited fibroblast proliferation activity. Both of these activities were enhanced by addition of lipopolysaccharides (LPS) to silica- or asbestos-stimulated AM cultures. It was shown that both of the activities in AM culture supernatants were produced by AM during the 24 hr-incubation, and that these AM products were non-dialyzable, rather heat-stable, and trypsin-sensitive in nature. The gel filtration of AM culture supernatants revealed that the major peaks of both activities ranged in the low molecular weight (18000-25000 daltons), and also in the high molecular weight (75000-100000 daltons) fractions, which appeared to be the aggregates of the low molecular weight components. These results indicate at least that fibroblast proliferation activity in AM culture supernatants is partly attributable to IL 1-like factor. This provides an important implication regarding the pathogenic role of IL 1 from AM in pulmonary disorders after exposures to mineral dusts.

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Light microscopic and ultrastructural observations, using immunoperoxidase staining for myoglobin and actin were performed on pleomorphic rhabdomyosarcoma in an ICR-male mouse aged 98 weeks, which arose in the striated muscle of the lumbar region. The tumor consisted of three types of cells showing varying degrees of myofibrinogenesis. The small round or elongated mononucleated cells (type I), which predominated in number, were immature mesenchymal cells. Some of these cells contained small amount of perinuclear intermediate filaments (9-11 nm in diameter). The eosinophilic plump cells (type II) exhibited one or two irregular nuclei and broad cytoplasm containing both types of myofilaments, actin and myosin filaments, not arranged to form myofibrils. The multinucleated giant cells (type III) alone possessed a Well developed element of tumoral myofibrils, Z-band. C-type virus like particles were rarely found in cytoplasmic vesicles or intercellular spaces of all types of cells. Myoglobin-positive staining was detected in most of the type III cells, and a few of the type II cells, while actin-positive staining was seen in most of both types cells. However, the type I cells were consistently negative for these two markers.

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Several factors affecting serum total cholesterol level of lactating Holstein cows were statistically evaluated. One thousand and sixty-three lactating Holstein cows were examined in a period from July, 1982 to April, 1983. Data from each cow were classified according to farms, number of calvings, health condition, daily milk yield and lactation day. Serum total cholesterol was significantly influenced by all these factors, especially daily milk yield and lactation day. However, no significant difference in total cholesterol level was observed between the summer and winter periods when other factors such as the number of calvings, health condition and lactation day were excluded.

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Distribution of thermophilic campylobacter was investigated in the total of 700 wild birds living in urban areas of Japan and mountainous environments around the base of Mt. Fuji. Thermophilic campylobacters were isolated from 55 (7.9%) of the 700 birds. By species, they were detected in 51(13.5%) of 378 feral pigeons, 2 (2.4%) of 82 pintails and 3 (60.0%) of 5 sparrows. However, campylobacters were not isolated from other 235 wild birds consisting of swallows, sparrows caught at Sagamihara, black-tailed gulls, pintails caught at Tokyo, eastern turthledoves caught at Ebina and grey starling, brown thrush, narcissus flycatcher, etc. caught at Mt. Fuji. In pigeons, large differences were demonstrated in the detection rates of this bacterium depending on the site of capture and even among the groups captured in the same area, varying from zero to 50.0%. All the strains isolated from pigeons and sparrows were C. jejuni. Two strains isolated from pintails were identified to be C. coli, while no C. laridis was detected. Seventeen (34.7%) of 49 strains of C. jejuni isolated from feral pigeons and 2 strains (100.0%) from sparrows were serotyped into some of the author's serogroup system (TCK 1-TCK 32) by typing with slide agglutination. Especially TCK 20 was frequently found in feral pigeons. Two strains of C. coli isolated from pintails were not serotyped by any of the antisera to C. jejuni.

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Seveal factors were evaluated to investigate the cause of variability of the ovarian response to superovulation treatment in 312 Holstein heifers and cows. Data from each donor were classified into 8 variables: places, parity, months, days since calving, gonadotropins used, superovulation treatment days in the estrous cycle, ovulation stimulating preparations and serum total cholesterol. Comparison of the number of embryos between levels of each qualitative variable was performed in animals which had a serum total cholesterol level of 90mg/dl and over in heifers and of 130mg/dl and over in cows. Comparison between ovulation stimulating preparations was also performed in animals which had a serum total cholesterol level of less than 90mg/dl in heifers and of less than 130mg/dl in cows. The percentages of heifers and cows in which over 3 transferable embryos were obtained were higher in the heifers with a serum total cholesterol level of 90mg/dl and over and in the cows with 130mg/dl and over, respectively, as compared to the heifers with less than 90mg/dl and to the cows with less than 130mg/dl. Significant difference in the number of recovered and transferable embryos was observed between the ovulation stimulating preparations. However, in the present study, there were no significant differences between the levels in the other qualitative variables.

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The incidence of retained fetal membranes (RFM) in Holstein cows from records of 1695 calvings in southern Osaka, and plasma levels of certain hormones and vitamins in cows calving during January and March were determined. The RFM incidence was significantly higher (P<0.02) in winter (January and February) and early summer (June and July) than in other seasons (spring and autumn). Daily changes in plasma concentrations of progesterone, estrone and estradiol were similar between two groups with RFM and without RFM (NRFM). Progesterone decreased from a few days before parturition, whereas estrone and estradiol increased from 10-6 days to 4 days before parturition, and decreased near parturition in both groups. While there was no difference between groups in changes in vitamin A, the RFM group had a lower level of β-carotene than the NRFM group near parturition. These results indicated that lower concentrations of plasma β-carotene were associated with the higher RFM incidence in dairy cows in winter.

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Inoculation of Fusobacterium necrophorum into the portal venous system of cattle resulted in the formation of microabscesses and disseminated microthrombi in the liver. The microabscesses expanded with multiplication of F. necrophorum, resulting in the formation of coagulative necrosis. The areas were encapsulated with granulation tissues. The development of hepatic lesions was partially associated with intravascular coagulation in the liver.

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Eight of 85 microfilaria positive dogs developed dirofilarial hemogloginuria (caval syndrome) after oral administration of milbemycin D. The onset was confirmed from 3 to 24 hr after administration. These dogs recovered whithout treatment from 21 to 117 hr after the onset. The findings at the time of the onset were systolic cardiac murmur, positive jugular pulse, irregular heart beat, decrease of heart rate, fall of blood pressure, occurrence of filarial echoes at the tricuspid valve orifice, ventricular to atrial premature heart beat, decrease of circulating microfilariae, increases of serum enzyme activities and plasma hemoglobin concentration and hemoglobinuria. The number of adult heartworms ranged from 10 to 39, and the number of worms per kg of body weight from 1.00 to 8.67. It did not always follow that a large number of heartworms caused the onset of dirofilarial hemoglobinuria. Serum and erythrocyte lipid findings were similar to those in chronic serious dirofilariasis, suggesting a tendency to be easily injured in erythrocytes already at the time of the onset. It was suggested that the suppression of cardiac functions may cause the migration of heartworms from the pulmonary arteries towards the venae cavae.

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A fimbriated and non-hemagglutinating (HA^-) mutant strain was derived from a fimbriated, HA⁺ parent strain of Fusobacterium necrophorum, biovar A. An experiment was carried out to detect any difference in pathogenicity in mice between the two strains. After the intraperitoneal inoculation of the parent strain, 9 out of 10 mice died with hepatic abscess. Organisms with HA⁺ activity were recovered from the abscess, other parts of the livers, spleens, kidneys and lungs. In contrast, all the mice that received the HA^- mutant survived, and HA^- organisms were isolated only from the hepatic abscessess found in 6 out of 10 mice. The results indicate that the mere presence of the fimbriae is insufficient for F. necrophorum to cause pathogenicity in mice, or HA activity. These biological activities may be controlled by some qualitative character of the fimbriae or by some surface structures of the organism other than fimbriae.

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The interleukin 2 (IL-2) activity was found in the culture supernatants of feline lymphocytes stimulated with concanavalin A (Con A), as detected by the cell proliferation of a IL-2 dependent murine T cell line (CTLL-2). Feline IL-2 closely resembled rat and human IL-2 in the molecular weight of approximately 16000 daltons, as was estimated from Sephadex G-100 gel filtration chromatography studies. Feline IL-2 was labile to trypsin treatment and was rather sensitive to heating at 70°C for 15 min, the incubation at pH 3.2 and pH 10.5, and the treatment with urea (2, 4 and 8 M), as compared to those of human and murine IL-2. The maximal IL-2 activity in the supernatants from cultures in which lymphocytes were stimulated with Con A (10-100μg/ml) appeared 6-12 hr after initiation of the culture. The addition of exogenous feline IL-2 resulted in the enhanced lymphocyte proliferation in response to Con A, whereas IL-2 alone did not have potent proliferative effect. The feline IL-2 activity was removed by a 4-hr incubation with lymphocytes stimulated with Con A for 48 hr, but not with freshly isolated unstimulated lymphocytes, indicating that the absorption of IL-2 activity results from the binding of IL-2 molecules to IL-2 receptors on Con A-stimulated lymphocytes.

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An outbreak of esophagectasis associated with transit impairment of food through the cardia was observed in F344 rats used in 12 long-term dietary studies. Symptoms initiated with slight influenza like signs lasting largely more than 10 weeks, and progressed to frequent sneezing with eruption of food and marked emaciation. An attempt to insert a stomach tube was rejected to pass through the cardiac ostium. Bacteriological and immunological checks for causative agents of pneumonia were negative. At autopsy, markedly dilated esophagus with pulpified powdered chow, and atelectatic areas on the lung were prominently observed. No histological abnormalities corresponding to the symptoms were demonstrated by ordinary light microscopic examinations in the peripheral nerve as well as central nervous system, although suppurative or foreign body pneumonia, otitis media and rhinitis were frequently seen. Females were more vulnerable to the disease than males. Morbidity of the disease varied between each lot of the animals of both sexes. In the highest case 23 out of 50 females died of the disease. The majority of the disease occurred in the animals older than 70 weeks of age.

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To investigate the contribution of intestinal creatine kinase (CK) to serum CK activity and its isoenzymes, neostigmine, an anticholinesterase agent, was injected into 8 dogs to stimulate intestinal peristalsis. Serum CK activity was increased 3 hr after injection and had a similar isoenzyme pattern to that of intestine. CK activity decreased to normal range within 24 or 48 hr. Serum CK activities did not change in dogs injected both with neostigmine and stropine, or only atropine. Thus hyperperistalsis of the interstine might increase the activity of serum CK and altere its isoenzyme pattern. Intestinal CK could contribute to serum CK activity and its isoenzyme pattern.

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Small nuclear RNAs (snRNAs) and small nuclear ribonucleoprotein particles (snRNPs) were purified from the cat placenta as an initial step in the analysis of the regulatory mechanism(s) of gene expression in the placenta. For visual observations, snRNA species including 7S, U2, U1, U4, X-I, X-II, 5S, U5, U6 and X-III were stained with acrydine orange, following the extraction of RNAs with phenol from cat placental cell nuclei and their fractionation by electrophoresis on a 10% polyacrylamide gel containing 7 M urea. The small nuclear RNA U1 from the cat placenta showed approximately the same mobility as that of mouse (129/sv) liver used as a reference on the gel. The logarithms of the nucleotide lengths of cat placenta snRNAs were inversely related to their mobilities. The approximate number of snRNA molecules in the cat placenta cell nucleus was also calculated. Highly purified U1-snRNP which comprises U1 snRNA and eight polypeptide components ranging in molecular weight from 68, 000 to 9, 500 was immunochemically purified using systemic lupus erythematosus-anti(U1)RNP antibody column chromatography. Any polypeptide component involved in the U1-snRNP did not immunoreact with normal human IgG. No immunoreactive materials with anti(U1)RNP antibodies could be detected in cat amniotic fluid. The U1-snRNP and U2-snRNP rich fractions were separated biochemically on a DEAE Sepharose column.

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Development and viability of Day-7 and -8 bovine embryos cultured in a simple defined medium using a test-tube system were determined. Day-7 embryos were cultured for 72 hr in BMOC-3 with either the test-tube or microdroplet systems in a gas atmosphere of 5% CO₂, 5% O₂ and 90% N₂. There was no difference between the two systems in their ability to support embryonic development. In the second experiment, Day-7 and -8 embryos were cultured for 72 hr using the test-tube system in four media: BMOC-3, BMOC-3+FCS, HF-10+FCS and MPBS+FCS. The development of Day-7 embryos was significantly reduced when they were cultured in MPBS+FCS in air. However, there was no difference in the development of embryos among the other three media. As for the Day-8 embryos, no difference in development was found among the four media. Embryonic development was reduced when Day-7 embryos were cultured for more than 48 hr and Day-8 embryos were cultured for more than 24 hr respectively. Viability of the cultured embryos was examined further by nonsurgical transfer to recipient animals. Three out of eleven heifers which received Day-7 embryos cultured for 48 hr and five out of eleven which received Day-8 embryos cultured for 24 hr became pregnant. The present results indicated that Day-7 bovine embryos can be cultured in vitro in a simple defined medium for 48 hr, while the culture length of Day-8 embryos in this medium should be limited to 24 hr. The developmental capacity of Day-7and -8 embryos may be accurately evaluated when they are cultured within the confines of these culture lengths.

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Praziquantel was examined for the therapeutic effect on Pharyngostomum cordatum infection in domestic cats. Thirty cats were orally inoculated with 50 metacercariae of P. cordatum each and were divided into 5 groups of 6 cats each. Two groups were subcutaneously injected with praziquantel in a dose of 6mg/kg and 30mg/kg of body weight, respectively on day 40 after infection to examine the effect on mature flukes, and another 2 groups in same doses as above on day 10 after infection to examine the effect on immature flukes. The remaining placebo group was injected with physiological saline in a dose of 0.53ml/kg on days 10 and 40 after infection. Praziquantel was completely efficacious against both the mature and immature flukes in a dose of 30mg/kg, but the efficacy was insufficient in a dose of 6mg/kg.

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A dose of 1mg/kg or 5mg/kg of milbemycin D, a new prophylactic agent for Dirofilaria immitis (heartworm), was given to 201 dogs orally and the reaction was investigated by symptomatic, hematological and biochemical examinations. Dogs were divided into 3 groups: uninfected by heartworm (uninfected group), infected without circulating microfilaria (mf negative group), and infected with circulating mf (mf positive group). The incidence and degree of clinical signs were not clearly different with either dose. Vomiting and arrhythmia, which were noted to show about the same incidence in 3 groups, were seen in a total of 5 (2%) and 22 (11%) dogs, respectively. Body temperature and blood pressure fell slightly, and RBC count, serum total protein concentration, osmolality and sodium concentration were also slightly decreased 3 and/or 6 hr after administration in the uninfected and mf negative groups. Almost all of these changes remained within the normal ranges, and tended to recover 24 hr after administration. No other reactions were seen in the uninfected group. Loss of appetite, pale visible mucous membranes, shock-like reaction and dirofilarial hemoglobinuria (caval syndrome) were noted in a total of 5 (2%), 9 (5%), 9 (5%) and 8 (4%) dogs, respectively, which were seen mainly in the mf positive group. A significant decrease of circulating mf count, rise in body temperature, serum GOT, GPT and ALP activities were also observed 24 hr after administration in the mf positive group. Shock-like reaction was seen in a dog of the mf negative group, but in this case a small number of microfilariae were detected in the liver and lungs on histopathological examination. The effects of dead mf might be involved in the reaction of the mf positive group. From these results, it is considered that clinical application of milbemycin D for uninfected and mf negative dogs presents no risks, but adverse reactions may occur in dogs with microfilaremia.

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Genetic variations of the enzymes of chicken coccidia were examined by starch-gel electrophoresis. Six species of chicken coccidia examined could be characterized by zymographs of both lactate dehydrogenase (LDH) and glucose phosphate isomerase (GPI). Two of six field isolates of Eimeria tenella had GPI of different mobility from the others including the reference (laboratory) strain, although their LDH were identical, indicating that there existed at least two phenotypes of E. tenella strains in Japanese chicken flocks.

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Adjuvant effect for antibody production was observed in different strains of mice sensitized with Theileria sergenti lysate antigen and sheep erythrocytes. By means of an improved Jerne's plaque assay the adjuvant effect was evaluated in the number of plaques as well as in the size of plaque. RR and C3H/He mice responded more efficiently than NC and C57BL/6 mice. C3H/He male mice responded more efficiently than C3H/He female mice.

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A 5-year-old Holstein-Friesian cow which had previously given birth to 2 normal calves was delivered of a stillborn anomalous female calf weighing 27kg after prolonged gestation (372 days). Anencephaly, anophthalmia, persistent truncus arteriosus, duodenal atresia of the blind-end type and many other defects were observed. Pathogenesis of these abnormalities was briefly discussed from an embryological point of view. This was a rather rare case of anencephaly associated with several defects.

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An experiment was carried out to clarify the humoral antibody and cell mediated immune responses of calves experimentally infected with Babesia ovata or Theileria sergenti. Two Holstein calves were inoculated with erythrocytes containing B. ovata or T. sergenti, respectively. They were also splenectomized 12 weeks after inoculation. Throughout the experimental period, they were subjected to general blood tests and examined for macrophage migration inhibitory factor (MIF) activity, IgM and IgG antibody titers, and the microbicidal activity of macrophages. As a result, strong humoral antibody and cell mediated immune responses were noticed 1-3 weeks after inoculation with both protozoa. Of the humoral antibodies, IgM antibody was detected throughout the observation period in the experiment.

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The hemagglutinating and hemolytic activities of Fusobacterium necrophorum biovar A strains were stable during 100 subcultures on agar medium. Therefore, the term "biovar" should be used instead of "phase" for F. necrophorum.

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The process of spore formation was observed by electron microscopy with three strains of Tyzzer's Organisms, "Bacillus piliformis" having a high frequency of sporulation. The sporulation process was morphologically similar to those of other Bacillus species except for the absence of mature spore forms separated from the mother bacterial cells.

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PAS positive deposits in sympathetic ganglia were detected in two adult dogs. The deposits were light microscopically observed in perikarya and nerve fibers. They were clearly digested by α amylase. Ultrastructurally the deposits mainly consisted of excessive glycogen accumulation and mitochondria in enlarged axons. Glycogen particles mostly showed low electron-density. Intracytoplasmic deposits at the light microscopy were identical with axonal ones which caved in the perikaryon.

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A real-time ultrasound scanner was used for pregnancy diagnosis in guinea pigs. The images of fluid-filled round uteri were recognized first on the echograph after day 16 of gestation. Accuracy of the diagnosis approached 100% on day 19 of gestation and the high accuracy was maintained throughout pregnancy period. After day 34 the fetal spine and the beating heart could be observed, and the death of fetuses was able to diagnose. Gestational age could be estimated by measuring the uterine diameter. Litter size also could be estimated with the accuracy of 81%. A real-time ultrasound might be used for the diagnosis of guinea pig pregnancy as an accurate, rapid and easy method.

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A large number of both Demodex criceti and D aurati (Acarina: Demodicidae) were isolated from scrapings of the skin lesion of an aged golden hamster (Mesocricetus auratus) sent to a pet clinic, Takikawa, Hokkaido in December, 1976.

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The serum of guinea pigs infected with Metastrongylus apri (M. apri) promoted the adherence of peritoneal eosinophils, obtained from normal and infected animals, to the third-stage larvae in vitro. The promotion of eosinophil adherence was first detected in the serum 14 days after infection followed by marked increase in the activity as the infection progresses. In such sera, immunoglobulin G capable of reacting with the cuticular surface of the larvae was demonstrated. The antibody-dependent eosinophil adherence reduced the infectivity of the larvae, suggesting that eosinophils may play a protective role against M. apri in vivo.

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A 6-month-old puppy died after exhibiting high fever, diarrhea, and respiratory symptoms and was histologically diagnosed as nocardiosis. Necropsy revealed nodular lesions in the thoracic and visceral organs. Histologically, granulomatous lesions were seen in the liver, cerebrum, cerebellum, pancreas, spleen, myocardium, kidneys, diaphragm, gastrocolic omentum, and lungs. They were characterized by central necrosis surrounded by epithelioid cells, lymphocytes, and neutrophils. Gram-positive, mildly acid-fast, filamentous, branching organisms were detected in the lesions.

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Levels of colostrum-derived antibodies in piglets born to sows naturally infected with Aujeszhy's disease virus were determined, and isolation of the virus from the unweaned piglets with the antibodies was carried out. The antibodies were diminished at constant rates approximatelly similar among different litters and reached negative levels at 45- to 60-day-old. The viruses were isolated from kidneys and brains of the neonatal piglets at 2-day-old. These results indicate that piglets with colostrum-derived antibodies undergo subclinical Aujeszky's disease virus infection.

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IgA-monoclonal gammopathy associated with Bence Jones proteinuria was diagnosed in a 5-year-old female Persian cat. Serum electrophoresis gave a monoclonal peak in the slow γ-globulin region. Serum IgA level was extreamly high more than 10 times of normal value by single radial immunodiffusion. In the urine, two narrow and high peaks were found in the fast γ-globulin region by electrophoresis, and they were confirmed as Bence Jones (B-J) protein. Many tumor nodules were found on the peritoneum with ascites, though no excessive multiplication of plasma cells was observed in bone marrow.

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