In order to determine the means of monitoring the immunological status of allograft recipients in miniature swine, an assay was developed to measure interleukin (IL)-2 production in vitro by pretreatment of donor peripheral blood lymphocytes (PBL). Miniature swine were given 0 to 4 weekly intravenous transfusions of 5-10×10⁷ donor PBL incompatible at major histocompatibility complex (MHC) and assayed in vitro for donor specific immune IL-2-like activities. The results are summarized as follows: (1) IL-2-like activity in 24 hr and 48 hr supernatants from mixed lymphocyte cultures (MLC) with MHC-incompatible PBL was detected without pretreatment. The 48 hr MLC supernatant exhibited a high IL-2-like activity compared with the 24 hr; (2) IL-2-like activity after only one transfusion with MHC-incompatible PBL was higher than that without pretreatment; (3) IL-2-like activity in 4 weekly transfusions was detectable slightly earlier than that without pretreatment or three transfusions with MHC-incompatibile PBL.

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A model system capable of investigating immunological changes was first established in Babesia rodhaini infected mice with an aid of a drug, diminazene diaceturate (DD). Intraperitoneal (ip) inoculation with B. rodhaini resulted in acute death in euthymic (nu/+) and athymic (nu/nu) BALB/c mice. Treatment with DD at an early stage of infection saved both mice from acute death. Parasitemia recurred in some of them but resulted in death only in nu/nu mice. A re-challenge with 10⁵ parasitized erythrocytes (PE) on the surviving mice on day 28 post infection revealed resistance in nu/+ but not in nu/nu mice. The results suggested a participation of the thymus in the protective mechanisms. Immunological changes were then observed on nu/+ and nu/nu mice which were inoculated ip with 10⁴PE and treated with the drug, and then challenged with 10⁵PE ip on day 28. An antibody response was measured with immediate reaction by footpad injection of a soluble antigen of B. rodhaini and by ELISA of serum antibody using the antigen and protein A, on day 10 and later, and further a pronounced response was detected after re-challenge in nu/+ mice. No response was detected by ELISA in nu/nu mice. Delayed footpad reaction was seen in nu/+ mice by day 14 and later but it was suppressed after the re-challenge. When spleen cells obtained from drug-treated and re-challenged nu/+ mice on day 8 after the re-challenge were transferred to nu/nu mice and the nu/nu mice were infected with 10⁴PE ip, 3 out of the 5 recipient mice survived and showed a low level of transient parasitemia, whereas remaining 2 mice died in a short period of infection accompanying severe parasitemia. The model system seems make an analysis of effective cells in babesial immunity feasible.

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Cryoglobulin was isolated from a horse which had glomerulo-nephritis and a history of swelling and skin ulcers of the limbs in the winter. The isolated cryoglobulin showed a single peak on a gel permeation chromatography column with an apparent molecular mass (Mr) of 180, 000 which could be divided into two gamma bands by cellulose acetate electrophoresis. Immunoelectrophoretic analysis revealed that the cryoglobulin formed two precipitation lines with anti-horse IgG. Spur formation was observed when the cryoglobulin and the IgG purified from a normal healthy horse were cross-reacted with anti-horse IgG on a double diffusion gel. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis under the reduced conditions showed that the isolated cryoglobulin consisted of two doublets of polypeptides with Mr values of 52, 000 and 50, 000, and 31, 000 and 30, 000, corresponding to the heavy chain and the light chain of the horse IgG molecules, respectively. These results suggest that the isolated cryoglobulin might consist of two different IgG molecules, and that the manifestations such as foot swelling with skin ulcers and renal failures were all induced by the cryoglobulin in the serum.

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Monoclonal antibodies to the major outer membrane protein (MOMP) of Chlamydia psittaci derived from a parrot were established for antigenic analysis of avian C. psittaci. With 17 monoclonal antibodies to MOMP, 19 reactivity patterns were identified on 112 strains of C. psittaci, C. pneumoniae and C. trachomatis, which were isolated from birds, mammals and humans in Japan, U.S.A., Canada and Taiwan, from 1938 to 1987. Immunological reactivity of budgerigar-derived strains to the monoclonal antibodies was different from that of pigeon-derived strains. Imported bird-derived strains were distinguishable from domestic bird-derived strains by the reactivity to the monoclonal antibodies. A close relationship between the subtypes and geographic origins was indicated on budgerigar-derived strains. On the contrary, various reactivity patterns were shown in pigeon-derived strains isolated in a narrow area. The monoclonal antibodies established in the present work may be useful probes for ecological study of avian C. psittaci.

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Pathogenesis of anemia in the calves infected with Theileria sergenti was investigated from the viewpoint of erythrocyte survival decrease in the circulating blood. For investigation of erythrocyte survival a method of erythrocyte labelling with non-radioactive chromium (⁵⁰Cr) was utilized. It was found that (1) the erythrocyte survival decreased markedly in the T. sergenti-infected calves compared with that in the uninfected calves; the survival rate of 25.7% for infected calves and 86.0% for uninfected ones on the fourth day after re-introduction of the labelled erythrocytes into the original donors, and that (2) the survival of non-parasitized erythrocytes in the infected calves was also decreased, which indicates no obvious relationship between parasitism and decrease in survival of erythrocytes.

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We conducted a seroepidemiological survey for antibodies to bovine leukemia virus (BLV), by using agar gel immunodiffusion technique, in dairy cows, water buffaloes, and yellow cattle throughout Taiwan. The positive reactors were 8.4% (376/4, 459) in 1985 and 5.8% (1, 277/22, 190) in 1986, in 15 prefectures and 7 cities. Relatively high infection rate appeared in the northern and southern areas of Taiwan. Positive reactors increased gradually with age. The incidence of positive antibodies was 2 to 3 times higher in pasture-style farms than in housed-style farms. Among the 6, 313 imported cattle, 302 (4.8%) showed positive reaction. Between 1985 and 1987, 5 cattle showed enzootic bovine leukosis among 351 sero-positive reactors in four highly positive prefectures. Survey of 134 water buffaloes and yellow cattle showed no positive reactors. This survey demonstrated that BLV-infection has increased over the years and spread throughout Taiwan.

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An enzyme-linked immunosorbent assay (ELISA) for detecting avian leukosis virus (ALV) antigens was developed with rabbit anti-ALV serum. The ELISA detected purified ALV of subgroups A and B at a concentration of 0.4ng/well and about 10³ infectious units/well estimated by a resistance-inducing factor (RIF) test, and antigens in culture fluids from chicken embryo fibroblasts infected with subgroups A, B or E of ALV. These results showed that common antigens among the subgroups were detected by the ELISA. When virus titration was performed, virus infectivity could be determined by the ELISA within 7 days after cultivation. The titer was similar to that obtained by the RIF test on 19 days after 3 subcultures. These results indicate that the ALV-isolation test by the ELISA was superior to the RIF test in rapidity and applicability to large-scale field trials. Four specific pathogen-free (SPF) chicken lines maintained in this laboratory were examined for endogenous ALV antigens by the ELISA. Sera from laying hens had considerably high absorbance (A) values, whereas albumen samples showed low A values except for some samples (7/40 hens). Although most of sera from 1-day-old SPF chicks showed lower A values than those from laying hens, some sera showed A values as high as those from viremic chicks in 2 lines. Endogenous ALV was isolated from sera from laying hens (6/40) and their albumens (4/7) with high A values. Two SPF Chicken lines were found to produce endogenous virus at a high frequency.

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Between 1982 and 1988, ascarid nematodes were found in the feces of Japanese calves in Kyushu and Okinawa districts. Seven males and 21 females of the ascarids were observed morphologically for identification of species. Male and female ascarids were 15.64 (14.0-18.0) cm and 25.75 (16.5-34.0) cm in average length, respectively. Eggs were 81.6μm and 71.8μm in large and small diameters, respectively. The body of ascarids was translucent and soft. The boundary between the enlabium and prelabium of lip was clearly visible and the esophageal ventriculus was also observed. The vulva was situated at a distance of about 1/7∼1/9 of body length from the anterior end of body. The surface of egg shell was relatively smooth, without rugose albuminous coat. These morphological features coincided with those of Toxocara vitulorum.

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In order to study the differentiation level of two chicken monocytic cell lines, IN24 and LSCC-NP1 cells, alpha-naphthyl acetate esterase (ANAE) was examined by ultrastructural cytochemical technique. Morphologically and functionally, IN24 cells showed immature character compared to LSCC-NP1 cells. In IN24 and LSCC-NP1 cells, ANAE activity was localized on the external face of the plasma membrane and coated vesicles, but not in lysosomes and phagocytic vacuoles. ANAE reactivity showed a dense linear reaction product on the entire cell surface in IN24 cells and granular reaction product in LSCC-NP1 cells. On treatment with interferon-γ (IFN-γ), ANAE reactivity of IN24 cells became similar to that of LSCC-NP1 cells. IN24 cells treated with lipopolysaccharide (LPS) had less amounts of ANAE surface product, and reactivity was partially dispersed on the cell surface.

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Three murine monoclonal antibodies (MAbs) were prepared against an enterotoxin (ET) produced by Bacillus cereus. Although these MAbs were found to react with the ET, their specificities appeared to be different in competitive binding assays. One of the MAbs (D-8), which was highly reactive with the ET, will be useful in developing immunological methods to detect crude ET and to isolate the ET in high yield.

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The three glycoproteins each of feline herpesvirus type 1 (FHV-1) and canine herpesvirus (CHV) were purified by affinity chromatography using glycoprotein-specific monoclonal antibodies and used individually or in combination in immunizing mice to determine their relative immunogenicity. All the glycoproteins induced detectable virus neutralizing antibodies to the homologous virus but FHV-1 gp143/108 and its cross-reacting counterpart, CHV gp145/112, elicited the highest titers not only to the homologous virus but to the heterologous virus as well. The production of ELISA antibodies after glycoprotein immunization was variable, while hemagglutination-inhibiting antibodies were produced by only 1 out of 10 FHV-1 gp60-inoculated mice. In general, the antibody titers induced by CHV glycoproteins were lower than those by FHV-1 glycoproteins. These results indicate that these glycoproteins may be useful as subunit vaccines against FHV-1 and CHV infections.

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Fecal and nasal samples were collected from 180 calves with diarrhea and 36 clinically normal co-habitants, and tested for virus using HRT-18 cell cultures derived from human rectal adenocarcinoma. A cytopathic virus was isolated from 5 fecal and 56 nasal samples obtained from diarrheic calves. All calves in which the virus was isolated from diarrheic feces were positive for virus isolation from nasal swabs. The virus was also isolated from the nasal swabs of 10 clinically normal calves that were co-habitants with diarrheic calves. Because they were morphologically similar to coronavirus, agglutinated mouse erythrocytes and serologically identical with the Nebraska calf diarrhea coronavirus, new isolates were identified as bovine coronavirus. The demonstration of viral antigens in nasal epithelial cells by a direct immunofluorescence was in close agreement with the virus isolation in HRT-18 cell cultures. This is the first report on the isolation of bovine coronavirus from newborn calves with diarrhea in Japan. The evidence that the virus was frequently isolated from nasal swabs is of great interest for understanding the pathogenesis of bovine coronavirus infection.

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An avian thymidine kinase deficient (TK^-) fibroblast cell line (QTTK^-) was established from a Japanese quail cell line, QT35, and characterized the biological properties. QTTK^- could grow in the presence of 5-bromo-2'-deoxyuridine (BUdR, 100μg/ml) and not in the growth medium with hypoxanthine-aminopterin-thymidine. Compared to QT35 cells, the ³H-thymidine incorporation of the QTTK^- cells and the TK activity of the cell extract significantly decreased to 0.3% and 0.5%, respectively. In the thymidylate synthetase activity, the karyotype, and the sensitivities to either fowlpox virus (FPV) or herpesvirus of turkeys (HVT), QTTK^- cells were similar to the parental QT35 cells. Since QTTK^- cells were permissive to FPV and HVT infections and these viruses could not grow in the presence of 50 to 75μg/ml of BUdR, QTTK^- cells may be useful for the construction of recombinant FPV and HVT that have foreign genes within the TK gene of the virus genome.

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Orchiopexy of the cryptorchid (CR) testis and castration of the scrotal testis were performed in three unilaterally CR beagles at six months of age. Induction rates for ejaculated sperm hyperactivation (HA) and the acrosome reaction (AR) in vitro in these orchiopexied dogs were compared with five those in normal beagles one year later. Canine spermatozoa were incubated for 9 hr at 38°C under 5% CO₂ in air in canine capacitation medium at a concentration of 30×10⁶ sperm/ml. HA was observed using high-speed videomicrography. The AR spermatozoa were evaluated by the triple stain technique. As a result, there was no significant difference between 'the CR dogs after orchiopexy' (CDO) and the normal dogs (ND) with respect to sperm motility just after ejaculation. However, sperm motility of CDO decreased markedly during incubation. There was a significant difference in sperm motility between CDO (Mean±SD; 47±12%) and ND (80±9%) after three hours of incubation (p<0.01). No significant difference was observed between CDO and ND with respect to the HA rate of motile spermatozoa throughout the incubation period. The peak of HA rate was found in both CDO (58±5%) and ND (61±16%) after seven hours of incubation. The AR rate of spermatozoa in CDO was lower than that in ND after six hours of incubation. The AR rate of CDO (26±4%) was significantly lower than ND (46±5%) after eight hours of incubation (p<0.01). It is assumed that there might be relation between a rapid decrease of motility and low AR rate in spermatozoa of CDO during incubation.

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In situ hybridization (ISH) technique with a biotin-labeled probe was established for detecting feline interleukin 1 (IL-1)α mRNA in necropsied specimens. Homology between human IL-1α cDNA used as a probe and feline IL-1α mRNA was confirmed by means of dot blot hybridization using the biotin-labeled probe. Hence, we tried by this biotinylated probe to detect mRNA of IL-1α in paraffin-embedded sections. The following results were obtained for the routine procedures: 1) coating slides with poly-L-lysine and/or heating at 60°C at least for 6 hours gave an excellent result for the adhesion of the tissue sections, 2) 10μg/ml solution of proteinase K treatment for 30 minutes or 50 to 100μg/ml solution of proteinase K treatment for 10 to 30 minutes at 37°C gave the good results in the detection of ISH signal, 3) suitable denaturation time of probes at 70 to 90°C was 5 to 15 minutes, and 4) effective hybridization was obtained by incubation for 24 hours at 4°C, for 18 to 24 hours at 25°C or for 5 to 24 hours at 37°C

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Seasonally anestrous Suffolk ewes (n=28) were randomly divided into 5 groups and treated with varying doses of melatonin as follows; Groups C (n=4), M1 (n=6), M2 (n=6), M3 (n=6), M4 (n=6) of ewes were fed pellets containing 0, 1, 2, 3 and 4 mg melatonin, respectively, daily for 60 days from May 17 (Day 0). Following feeding of the pellets at 13.00 hr plasma levels of melatonin rapidly increased reaching the peak values within 30 min, which ranged from 92.0 to 292.7 pg/ml and were highly correlated with the dose of melatonin administered (r=0.986, P<0.01). Maximum dose of melatonin (4 mg) produced an increase of plasma melatonin similar in magnitude to nocturnal peaks of endogenous secretion. The onset of ovulatory cyclicity, assesed from plasma progesterone profiles, was advanced by melatonin administration. The mean ± SEM intervals from the commencement of melatonin treatment until the onset of ovulatory cyclicity were 53.0±5.8, 53.6±2.5, 42.0±5.6 and 44.3±4.3 days for the Groups M1, M2, M3 and M4, respectively, which were shorter (M1, P<0.05; M2, M3 & M4, P<0.01) than that for the Group C (72.5±1.4 days). The melatonin treatment also suppressed, in a dose related manner, the rise in plasma prolactin under the lengthening photoperiod. We conclude that the dose-related efficacy of melatonin could be ascribed to the difference in the diurnal profiles of circulating melatonin.

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One-month-old Syrian hamsters of the APA and Std: golden strains were inoculated intraperitoneally with 10⁵ PFU/head of the D variant of encephalomyocarditis (EMC) virus and examined virologically and pathologically up to 7 days after inoculation. APA hamsters developed apparent hyperglycemia due to pancreatic islet cell damage while Std: golden hamsters did not. Hamsters of both strains showed clear histopathologic changes in the testis with prominent viral replication as well as in the brain, heart and exocrine pancreas. The susceptibility to EMC virus-infection was higher in males than in females and in APA than in Std: golden hamsters.

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A mouse lethal toxin (MLT) produced by Bacillus cereus isolated in vomiting-type food poisoning was purified by chromatography on DEAE-Sephadex A-25 followed by gel filtration on Sephadex G-75. Purified MLT possessed a molecular weight of 33, 000-34, 000. It showed mouse lethality and hemolytic (HL) activity on sheep and rabbit erythrocytes; the latter erythrocytes were more weakly hemolyzed than the former ones. However, fluid accumulation in mouse ligated intestinal loops was not induced by purified MLT at the highest concentration used. Both MLT and HL activities were stable at pH 6-9, during storage at -20°C for 8 weeks, and resistant to papain, cholesterol, lecithin, and dithiothreitol treatments. Most activity was lost during storage at 4°C or 25°C for 2 weeks or upon treatment with trypsin, trypanblue, or ethanol. The activities were resistant to heating at 37°C for 5 min, less resistant at 98°C for 5 min, and sensitive at 60°C for 5 min. It can be concluded from the results that MLT is different from the diarrheagenic toxin produced by B. cereus isolated in diarrheal-type food poisoning, but is similar to, if not identical, hemolysin II.

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Water buffalo (Murrah) oocytes were collected from ovaries obtained from the slaughter house. They were classified according to the character of the cumulus cells under a stereomicroscope, and cultured in 25 mM Hepes buffered Tissue Culture Medium-199 (TCM-199) supplemented with 5% estrous water buffalo serum in an atmosphere containing 5% CO₂ in air at 39°C. After 20-24 hr of in vitro maturation, the oocytes were fertilized using capacitated sperm obtained from 4 different bulls. For cleavage the oocytes were cultured at 39°C in TCM-199 supplemented with 1% estrous water buffalo serum and in an atmosphere containing 5% CO₂ in air. The good oocytes, with compact and dense cumulus cells cleaved significantly higher (p<0.01, 67.3%, 33/49), than those of fair, partially naked oocytes with thin cumulus layers (27.5%, 25/91) or small remnants of cumulus cells and poor naked oocytes (3/100). A substantial variation in fertilization and developmental rates (16.0% to 43.8%) was observed among 4 different bulls. Late non-surgically into 14 buffalo recipients on day 6 or 7 of their estrous cycle. One recipient was diagnosed to be pregnant by rectal palpation on day 60 and confirmed to be so on day 90 post-estrus.

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From fresh faeces of a wild bird (Melanitta fusca), a virus that showed granular cytopathic effects (CPE) on chicken kidney cell (CKC) cultures was isolated. By indirect immunofluorescence analyses (IFA), this isolate reacted with an antiserum against a bovine rotavirus. The isolate produced clear plaques on CKC by conventional techniques, without trypsin. Three virus plaques were selected by plaque size (small, medium, and large), and cloned by three successive plaque cloning. In the SDS-PAGE analyses, dsRNA bands showed a typical profile of avian rotavirus and quite different from that of avian reovirus. With dsRNA patterns, IFA results, CPE, and a morphological property, the clones were identified as avian rotaviruses of group A rotavirus. The clones killed chicken embryos, when they were inoculated to yolk sac.

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