Microtubule assembly and in vitro development of bovine oocytes with increased intracellular glutathione level prior to vitrification and in vitro fertilization.

Although vitrification is a useful technique for preservation of bovine oocytes, the yield of blastocysts derived from the vitrified oocytes is still low. We have recently reported a new type of cryoinjury, multiple aster formation, by which pronuclear migration and development of vitrified-warmed and in vitro-fertilized bovine oocytes are impaired. The aim of the present study was to investigate the effect of glutathione (GSH) content of vitrified bovine oocytes on multiple aster formation and subsequent in vitro development. Treatment of bovine cumulus-oocyte complexes with ß-mercaptoethanol (ßME) and L-cysteine (Cys) during in vitro maturation resulted in 2.5-fold higher GSH content not only in fresh control but also in vitrified-warmed oocytes. The percentage of normally fertilized zygotes exhibiting sperm aster(s) was >95% in all four groups (with or without ßME/Cys × fresh control or vitrified). The frequency of multiple aster formation in vitrified oocytes (three-fold higher than that in fresh control oocytes) was not affected by the increased level of intracellular GSH with ßME/Cys. Consequently, the migration and development of pronuclei as well as the yield of blastocysts from vitrified-warmed oocytes (17 versus 41%) were not improved. In addition, there was no effect of increased GSH level on the yield of blastocysts in fresh control groups.

Characterization of factors stimulating differentiation of mouse myeloid leukemia cells from a Yoshida sarcoma cell line cultured in serum-free medium.

A clone, YS-T22, of cells from Yoshida sarcoma cell line, YSSF-212T, grown in "serum-free" culture medium produced factors stimulating differentiation of mouse myeloid leukemia cells (M1) to macrophages and granulocytes. The formation of macrophages and granulocytes was accompanied by induction of phagocytosis, locomotive activity, and lysosomal enzyme activities. The rates of induction of these differentiated phenotypes were proportional to the concentration of the factor added and the period of treatment. The factor stimulating differentiation of M1 cells was a heat-labile, nondialyzable proteinaceous substance that was inactivated by trypsin but not by ribonuclease or glycosidases. On diethylaminoethyl cellulose chromatography, the factor stimulating differentiation of M1 cells from conditioned medium of YS-T22 cells was eluted in various fractions with or without activity of the colony-stimulating factor.

Purification and characterization of an endothelial cell growth factor from serum-free culture medium of human diploid fibroblast cells.

Serum-free culture supernatants of human embryo fibroblast cells contain endothelial cell growth factor (f-ECGF) which supports the serial propagation of human umbilical vein endothelial cells in the serum-free culture medium (medium A). This growth-stimulating activity has been partially purified from serum-free culture-conditioned medium. The stability of the activity to acid (pH 4.0-4.5) was utilized for the first step in purification. f-ECGF had a high affinity to heparin-Sepharose CL-6B, and was isolated by the methods of heparin affinity, of ion-exchange and gel filtration chromatography from the serum-free culture-conditioned medium preparation. The purified f-ECGF had an isoelectric point in the pH range 4.5-6, and a molecular weight of approx. 30 kDa determined by either gel filtration or SDS-polyacrylamide gradient gel electrophoresis. The f-ECGF has high affinity for concanavalin A column, and the activity was partially eluted from the column with ethylene glycol and alpha-methylmannose. The results indicate that f-ECGF is an acidic-glyco-protein with heterogeneous sugar chain(s).

Further characterization of porcine plasma fibronectin which contains fucosylated carbohydrate chains.

Porcine plasma fibronectin and its functional four fragments produced by cathepsin B digestion were examined for biological, immunochemical and biochemical properties. Native fibronectin, 150-kDa and 130-kDa fragments exhibited similar cell attachment-promoting activity to each other. In an Ouchterlony double immunodiffusion system, these three polypeptides formed a precipitin line with anti-fibronectin antiserum, while the 50-kDa and 30-kDa fragments did not. The 150-kDa and 130-kDa fragments contained free sulfhydryl(s). The glycopeptide fractions were prepared by pronase digestion of porcine and human plasma fibronectin, and radiolabeled with [14C]acetic anhydride. The results of affinity chromatography with concanavalin A and lentil lectin immobilized on agarose indicated that the porcine glycopeptide fraction was different from the human fraction in that a larger part (58%) of the former was bound to lentil lectin. About 90% of this lentil lectin-reactive glycopeptides lost this reactivity upon alpha-L-fucosidase digestion. The glycopeptide fractions were also prepared from three carbohydrate-containing domains. Less than 30% of the radioactivity of the glycopeptide fractions of 150-kDa and 130-kDa fragments was retained on the lentil lectin-agarose, while about 90% of that from the 50-kDa fragment was retained. These results indicate that porcine plasma fibronectin has characteristics very similar to those of human plasma fibronectin and others, but is unique in that it contains fucosylated carbohydrate chains which unevenly distribute through functional domains.

Immunization with native surface protein NcSRS2 induces a Th2 immune response and reduces congenital Neospora caninum transmission in mice.

NcSRS2, a tachyzoite surface protein of Neospora caninum, is an immunodominant protein with respect to induction of antibody production and has a role in attachment and invasion of host cells. Native NcSRS2 was isolated from whole tachyzoite lysate antigen by affinity chromatography using NcSRS2 specific monoclonal antibody and used to immunize BALB/c mice in a congenital transmission study. NcSRS2 was a highly conserved protein as indicated by comparison of deduced amino acid sequence obtained from NcSRS2 gene sequences of 10 geographically distinct N. caninum isolates. Mice immunized with purified native NcSRS2 produced antigen-specific antibody, primarily of IgG 1 subtype. Following challenge during gestation with 10(7) tachyzoites, immunized mice had a statistically significant decreased frequency of congenital transmission compared to non-immunized mice (P

Effects of extracellular matrices on human keratinocyte adhesion and growth and on its secretion and deposition of fibronectin in culture.

Extracellular matrices (ECMs) play an important role as components of basement membrane of normal human skin and in migrating epidermal cells in wound healing. We investigated the effects of various ECMs on human keratinocyte adhesion and growth as well as on its secretion and deposition of fibronectin (FN) in vitro using a serum-free, low-calcium culture system. Since cell adhesion is the first step of cell growth, we performed cell adhesion assay for 14 h. Human keratinocytes adhered best on FN and less well on types I/III collagen, type IV collagen, and heparan sulfate proteoglycan (HSPG) as compared with bovine serum albumin (BSA) (control) or laminin (La). Cell growth assayed for 7-8 days on the dishes coated with various extracellular matrices revealed significantly increased keratinocyte growth on FN and on types I/III collagen in comparison with that on type IV collagen, HSPG, BSA (control), or La. Morphology of keratinocytes and of their colonies on FN and types I/III collagen was strikingly different from that of the control; the colonies were not so compact as in the control, but rather loose and larger; each keratinocyte was spread out more on these substrata. These morphologic features seemed to correlate with the increased keratinocyte growth on these extracellular matrices. Both immunofluorescence study for FN with keratinocytes in 8-day culture on various extracellular matrices and enzyme-linked immunosorbent assay for FN measurement on substratum or in conditioned medium with keratinocytes in 5-day culture demonstrated that extracellular matrices modulated the secretion and deposition of FN by human keratinocytes in culture; the keratinocyte growth correlated with the amount of FN detected on substratum but not with that in medium. Based on the results of the present investigation, we think that the growth of human keratinocytes depends on the amount of FN on substratum.

Isolation and characterization of human placenta fibronectin.

Fibronectin was isolated from human placenta tissues and compared with human plasma fibronectin. Placenta and plasma fibronectins had similar amino acid compositions, immunological properties, and cell attachment-promoting activities, but differed in apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which could be accounted for at least partly by the difference in carbohydrate composition. Unlike plasma fibronectin, placenta fibronectin failed to form a precipitin line with concanavalin A in a double diffusion system. The non- or low-reactivity of placenta fibronectin with this lectin was also demonstrated by affinity chromatography with concanavalin A-agarose, in which more than 90% of the radiolabeled glycopeptides derived from placenta fibronectin was not retained on the gel. The two fibronectins also differed in the reactivity with Lens culinaris agglutinin of their glycopeptide fractions. These data indicate that placenta and plasma fibronectins are different in their carbohydrate structures and, therefore, suggest the presence of a tissue- or cell-specific mechanism for processing the carbohydrates of this glycoprotein.

A survey of Theileria sergenti infection, daily weight gain and conception proportions in 85 herds of grazing heifers in Japan.

A survey of Theileria sergenti infections, daily weight gain and conception proportion was conducted in 85 herds of grazing heifers in Japan. Basic information and epidemiological data from participating herds were obtained by mailed questionnaires, which were completed by field veterinarians. The average cumulative incidence and proportion of treated animals in the participating herds were 25.7 and 21.1%, respectively. The average daily weight gain and conception proportion were 0.51 kg per day and 56.9%, respectively. The basic information and epidemiological data had a large range and standard deviation, which reflect the wide diversity of the grazing herds in Japan. Herds with heavy tick infestation had significantly higher cumulative incidence and proportion of treated animals, therefore, this factor can be a good estimator to predict the occurrence and loss by theileriosis of the herds. The present questionnaire survey was useful for obtaining information about herds in different regions, and this survey method can be applied to the research of other animal diseases in Japan.

Reduction in tick numbers (Haemaphysalis longicornis), mortality and incidence of Theileria sergenti infection in field-grazed calves treated with flumethrin pour-on.

The effectiveness of the pour-on formulation of flumethrin was tested on grazing cattle. Flumethrin was applied once a month from April to October from 1990 to 1995 to cattle grazing in the Aso area of Kumamoto Prefecture in Japan. Both the number of ticks in the field and the number of ticks feeding on cattle decreased remarkably in relation to the number of years flumethrin was applied. Ticks in the field were not detected in 1994 and 1995, and ticks feeding on cattle decreased to 4% in 1995. Mortality due to Theileria sergenti infection also decreased significantly after more than 3 years of flumethrin pour-on application, although overall mortality did not change. At the end of the trial the incidence of T. sergenti had decreased to one-fifth of the pretrial value, although total incidence of disease had not changed. These results indicated that multiple-year seasonal application of flumethrin pour-on to grazing cattle effectively decreased the number of ticks and decreased both mortality and incidence of T. sergenti.

Seroprevalence of Neospora caninum in dairy cattle in Bahia, Brazil.

Sera collected from 447 dairy cattle on 14 dairy farms were tested for Neospora caninum antibodies by use of an immunofluorescent antibody technique. Positive reactions with titres > or =1:200 were found in 63 (14.09%) of animals. Neospora positive sera were also tested for Toxoplasma gondii antibodies by using a commercial latex agglutination test. Antibodies to T. gondii were detected in 3 (4.76%) of 63 N. caninum positive sera. These results indicate that N. caninum infection is widespread among dairy cattle in Bahia state.

Seroprevalence of antibodies to Neospora caninum and associated abortion in dairy cattle from central Thailand.

A total of 904 sera from dairy cattle in 11 provinces of central Thailand were tested for antibodies to Neospora caninum employing the indirect fluorescent antibody test (IFAT). Fifty four (6%) cattle were positive in IFAT, titres of 1:200 (16 cattle), 1:400 (9 cattle), 1:800 (14 cattle), 1:1600 (7 cattle), 1:3200 (6 cattle) and two positives. No significant difference was observed among the provinces. The seropositivity for Toxoplasma gondii by a commercial latex agglutination test was 4% (2 out of 50) in positive sera, 2.9% (2 out of 69) in negative sera for anti-Neospora antibodies and 3.4% (4 out of 119) in total. The results of the IFAT were not associated with the presence of antibodies to T. gondii in bovine sera. Furthermore, the cause of abortions experienced in neighbouring three areas in the northeast, where pregnant heifers were newly introduced into small-scale farms from the central region, was investigated. The positive rates for anti-N. caninum antibody were 12, 28 and 44% at a cut-off titre of 1:200, and cattle were suspected to be infected after the introduction. In the area with the highest rate, seven out of eight aborting cattle were positive for antibodies to N. caninum while other two areas had similar abortion rates in both negative and positive cattle. However, in the latter two areas, positive rates for Trypanosoma evansi antigen along with parasitaemic animals were observed by an antigen-detection ELISA, but not for the former area. Considering the endemic diseases of the areas, Neospora was presumed to be responsible for the abortions in the former area while the examination results pointed out T. evansi as the most probable cause in the latter two areas. This is the first report of Neospora-associated abortion in Southeast Asia.

In vitro isolation and characterization of bovine Neospora caninum in Korea.

The brains of nine aborted bovine fetuses and two newborn calves born from dams suspected to be infected with Neospora caninum were homogenized and inoculated into Vero cells. All fetuses and calves were from cows determined as seropositive to N. caninum by an IFA test. Sera and thoracic fluids of all fetuses and calves also revealed high antibody titer to N. caninum by IFAT ranging from 1:800 to 1:3200. N. caninum was isolated from the brains of one aborted fetus and one newborn calf when the brain homogenates were grown continuously in Vero cell culture. N. caninum tachyzoites, giemsa-positive, were first observed on Days 45 and 56 postinoculation in the newborn calf and the aborted fetus, respectively. The isolates (KBA-1 and KBA-2) were morphologically and ultrastructurally similar to previously published Neospora isolates. The isolated parasites were confirmed as N. caninum by means of the antigenic reactivities, immunostaining, PCR and southern blotting, and electron microscopy.

A stochastic-modeling evaluation of the foot-and-mouth-disease survey conducted after the outbreak in Miyazaki, Japan in 2000.

When foot-and-mouth-disease (FMD) was identified in Miyazaki prefecture in March 2000, Japan conducted an intensive serological and clinical survey in the areas surrounding the index herd. As a result of the survey during the 21 days of the movement-restriction period, two infected herds were detected and destroyed; there were no other cases in the months that followed. To evaluate the survey used for screening the disease-control area and surveillance area, we estimated the herd-level sensitivity of the survey (HSe) through a spreadsheet model using Monte-Carlo methods. The Reed-Frost model was incorporated to simulate the spread of FMD within an infected herd. In the simulations, 4, 8 and 12 effective-contact scenarios during the 5-day period were examined. The estimated HSes of serological tests (HSeE) were 71.0, 75.3 and 76.3% under the 4, 8 and 12 contact scenarios, respectively. The sensitivity analysis showed that increasing the number of contacts beyond 12 did not improve HSeE, but increasing the number of sampled animals and delaying the dates of sampling did raise HSeEs. Small herd size in the outbreak area (>80% of herds have <20 animals) seems to have helped in maintaining HSeE relatively high, although the serological inspection was carried out before sero-positive animals had a chance to increase in infected herds. The estimated herd-level specificity of serological tests (HSpE) was 98.6%. This HSpE predicted 224 false-positive herds (5th percentile estimate was 200 and 95th percentile was 249), which proved close to the 232 false-positive herds actually observed. The combined-test herd-level sensitivity (serological and clinical inspections combined; CTHSe), averaged 85.5, 87.6 and 88.1% for the 4, 8 and 12 contact scenarios, respectively. Using these CTHSes, the calculated probability that no infected herd was overlooked by the survey was > or =62.5% under the most-conservative, four-contact scenario. The probability that no more than one infected herd was overlooked was > or =89.7%.

A clinical trial to evaluate the effects of flumethrin or ivermectin treatment on hemoparasites, gastrointestinal parasites, conception and daily weight gain in a dairy farm in Japan.

A clinical trial was performed to compare the effects of flumethrin and ivermectin treatments of grazing heifers at one farm in central Japan. 64 heifers were randomly allocated into two groups. Flumethrin (1 mg/kg pour on) was applied approximately once every 3 weeks to heifers in one group and heifers in the second group were injected approximately once every month with ivermectin (200 microg/kg; id). Between groups, no significant differences were detected in the proportions of animals that showed parasitemia of Theileria sergenti and conception risks. Significantly lower average log-transformed nematode-egg counts and higher average daily weight gain were observed in the ivermectin-treated group. Animals with higher body weight at the start of grazing and lower log-transformed total nematode-egg and coccidia-oocyst counts had higher odds of conceiving. Animals with ivermectin treatment, lower body weight at the start of grazing and lower log-transformed coccidia-oocyst count had higher daily weight gain. Ivermectin may be more useful in this farm because of the higher productivity for cattle and lower cost for its usage.

Clinical, anatomic, and immunopathologic characterization of Babesia gibsoni infection in the domestic dog (Canis familiaris).

The pathology associated with acute, chronic, and recrudescent Babesia gibsoni infections was characterized in a group of 6 naturally or experimentally infected, spleen-intact and splenectomized dogs. All experimentally infected dogs became acutely parasitemic, lethargic, anemic, thrombocytopenic, and hemoglobinuric. Anatomic lesions associated, with the disease included diffuse nonsuppurative periportal and centrilobular hepatitis, multifocal necrotizing arteritis, membranoproliferative glomerulonephritis, reactive lymphadenopathy, diffuse erythrophagocytosis, and extramedullary hematopoiesis. The density of CD3+ lymphocytes within the liver sinusoids was markedly increased. Aggregates of large mononuclear cells with immunohistochemical features of activated macrophages were demonstrated in the central veins of the liver. Kupffer cells throughout the hepatic sinusoids appeared hypertrophic and prominent. The density of sinusoidal T lymphocytes, macrophages in central veins, and the degree of Kupffer cell hypertrophy were greatest in the splenectomized dogs. Multifocal deposits of IgM antibody were immunohistochemically demonstrated within the walls of inflamed arteries and renal glomeruli. The results of this study suggest that intense immunostimulation resulting in activation and expansion of T and B lymphocyte populations, macrophage recruitment and activation, vasculitis, glomerulonephritis and anemia contribute to the pathology associated with B. gibsoni infections.

In vitro isolation and characterisation of a bovine Neospora species in Japan.

Eleven aborted bovine fetuses and five calves suspected as having neosporosis were necropsied and tissues from these animals were inoculated into bovine cardiopulmonary aortic endothelial cells and monkey kidney cells and maintained at 37 degrees C with 5 per cent CO2. Neospora tachyzoites were observed in one cell 49 days after inoculation. The isolated parasite (JPA1) was morphologically identical to the previously reported bovine Neospora species (BPA1) and confirmed by its strong antigenic reactivity with bovine control antisera to Neospora species and its lack of reactivity with Toxoplasma gondii and Sarcocystis cruzi antisera. This is the first bovine Neospora species isolate in Asia and further studies with this isolate are now expected.

Feline embryo transfer during the non-breeding season.

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

Serological survey of antibody to Neospora caninum in Japanese dogs.

Prevalence of antibody to Neospora caninum (NC) in Japanese dogs were examined. The antibody was positive in 15 of 48 dogs (31.3%) reared in the dairy farms that had case of the abortions due to NC infection or had the cattle seropositive to NC, whereas the prevalence was 7.1% (14 of 198 dogs) among the dogs kept in urban areas. In one dog breeder, all 17 Shetland sheepdogs older than 7 months were seropositive, and one pup was diagnosed as neosporosis 2 months before the first serological examination. The antibody titers of the dogs kept at this breeder were almost unchanged for 1.5 years. Serological evidence of the dogs in the dairy farms and urban areas indicates the transmission of NC between dogs and cattle. Also serological results of the dogs in one breeder may suggest the potential horizontal transmission among dogs.

The inhibitory effect of interferon gamma and tumor necrosis factor alpha on intracellular multiplication of Neospora caninum in primary bovine brain cells.

Primary culture of bovine brain cells was examined for its susceptibility to Neospora caninum infections, and this model was used to investigate the effects of bovine interferon gamma (IFN-gamma) and tumor necrosis factors alpha (TNF-alpha) on tachyzoite growth. Tachyzoites of N. caninum grew well in this culture, and tachyzoite growth in astroglia and microglia were confirmed by immunocytochemical staining. IFN-gamma inhibited the tachyzoite growth, and this inhibition was not reversed by the addition of nitric oxide antagonist. TNF-alpha, to a lesser extent, also inhibited the tachyzoite growth. Th-1 type cytokines may play an important role in host defense mechanisms in N. caninum infection.

Evaluation of the indirect fluorescent antibody test for diagnosis of Babesia gibsoni infections in dogs.

We determined the extent of the serologic cross-reactivity in the indirect fluorescent antibody (IFA) test for Babesia gibsoni, and the optimal cut-off titer for seropositivity in the test. The lowest titer to B gibsoni detected in a dog with naturally acquired clinical babesiosis was 1,280, and 7 of 12 dogs had titer between 10,240 and 20,480. Two experimentally infected normosplenic dogs developed high titer (40,960 to 81,920) to B gibsoni, and the same sera reacted in IFA tests for B canis (titer < or = 640), Toxoplasma gondii (titer < or = 2,560), and Neospora caninum (titer < or = 10,240). Dogs that were experimentally infected with B canis and T gondii had titer < or = 160 to B gibsoni. Dogs that were experimentally infected with N caninum had titer (80 to 10,240) to N caninum, but failed to have serologic reactivity to B gibsoni. Serologic titer of healthy dogs from Australia, a country where B gibsoni is not known to exist, was < or = 160 to B gibsoni. On the basis of these findings, a cut-off titer of 320 was considered to be appropriate for serodiagnosis of B gibsoni in dogs with clinical signs of babesiosis. A more conservative titer of 1,280 was established as the cut-off titer for seroepidemiologic studies based on relative costs and benefits of false-positive results and failure to isolate B gibsoni parasites after splenectomy and immunosuppression from a clinically normal dog with B gibsoni titer of 640.(ABSTRACT TRUNCATED AT 250 WORDS)