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.

Primary chondrosarcoma in the liver of a dog.

Primary chondrosarcoma was found in the quadrate lobe of the liver of a 6-year-old, intact, male Golden Retriever. At 6 months after partial hepatectomy, recurrence in the liver occurred. The dog died of its systemic metastases 10 months thereafter. Histologically, the hepatic mass revealed neoplastic chondrocytes with abundant chondroid matrix, and there were few myxoid areas where the cellularity and pleomorphism of the neoplastic cells were more prominent. The neoplastic cells were positive for periodic acid-Schiff and were immunohistochemically positive for vimentin and S-100 protein; the matrix was deeply stained for alcian blue and was metachromatic for toluidine blue stain. This tumor might be derived from pluripotent mesenchymal cells in the connective tissue of the liver. To the best of our knowledge, in all mammalians, including humans, this is the first report of extraskeletal chondrosarcoma primarily arising in the liver.

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

Regression of idiopathic thrombocytopenic purpura after endoscopic mucosal resection of gastric mucosa associated lymphoid tissue lymphoma.

Recent reports have suggested an association between Helicobacter pylori infection and both gastric mucosa associated lymphoid tissue (MALT) lymphoma and thrombocytopenic purpura. Although treatments eradicating H pylori lead to regression of these diseases in some cases, the exact mechanisms are still controversial. This case report describes a patient with thrombocytopenic purpura accompanied by an early stage gastric MALT lymphoma. Endoscopic mucosal resection of the lesion in this patient led to dramatic regression of thrombocytopenic purpura, and t(11;18)(q21;q21), which means resistance more likely to H pylori eradication therapy, was confirmed by fluorescence in situ hybridisation. There is no evidence of recurrence and his platelet count is within normal limits after 24 months of follow up. This is the first case report describing regression of thrombocytopenic purpura after mucosal resection of a gastric MALT lymphoma. We suggest that while some cases of thrombocytopenic purpura may be induced by H pylori, others may be due to an autoreactive antibody produced by MALT lymphoma B cells.

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 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.

Genetic and biological diversity among isolates of Neospora caninum.

Neospora caninum is a protozoan parasite that causes bovine abortion. The epidemiology of N. caninum is poorly understood and little is known about the genetic diversity of the parasite, or whether individual isolates differ in virulence. Such diversity may, among other factors, underlie the range of pathologies seen in cattle. In this study we analysed biological and genetic variation in 6 isolates of N. caninum originating from canine and bovine hosts by measurement of growth rate in vitro, Western blotting and random amplification of polymorphic DNA (RAPD). This comparative analysis of intra-species diversity demonstrated that heterogeneity exists within the species. The relative growth rate in vitro, as assessed by 3[H]uracil uptake, showed significant variation between isolates. However, no significant differences were detected between the antigenic profiles of each isolate by Western blotting. RAPD-PCR was performed on DNA from the 6 Neospora isolates; 3 strains of Toxoplasma gondii, Sarcocystis sp. and Cryptosporidium parvum were also analysed. Twenty-six RAPD primers gave rise to 434 markers of which 222 were conserved between all the Neospora isolates and distinguished them from the other Apicomplexa. An additional 54 markers were unique for Neospora but were polymorphic within the species and able to differentiate between the individual isolates. The RAPD data were subjected to pair-wise similarity and cluster analysis and showed that the Neospora isolates clustered together as a group, with T. gondii as their nearest neighbour. N. caninum isolates showed no clustering with respect either to host or geographical origin. The genetic similarity between Neospora isolates from cattle and dogs suggests that these hosts may be epidemiologically related, although further analysis of bovine and canine field samples are required. The genetic and biological diversity observed in this study may have important implications for our understanding of the pathology and epidemiology of neosporosis.

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.

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.

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 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.

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.

Properties of delay-period neuronal activity in the monkey dorsolateral prefrontal cortex during a spatial delayed matching-to-sample task.

The dorsolateral prefrontal cortex (PFC) has been implicated in visuospatial memory, and its cellular basis has been extensively studied with the delayed-response paradigm in monkeys. However, using this paradigm, it is difficult to dissociate neuronal activities related to visuospatial memory from those related to motor preparation, and few studies have provided evidence for the involvement of PFC neurons in visuospatial memory of a sensory cue, rather than in motor preparation. To extend this finding, we examined neuronal activities in the dorsolateral PFC while a rhesus monkey performed a spatial delayed matching-to-sample (SDMTS) task, which allows us to adequately access visuospatial memory independent of any sensorimotor components. The SDMTS task required the subject to make a lever-holding NOGO response or a lever-releasing GO response when a visuospatial matching cue (white spot, one of four peripheral locations, 15 degrees in eccentricity) matched or did not match a sample cue (physically the same as the matching cue) that had been presented prior to a delay period (3 s). Thus, the SDMTS task requires the subject to remember visuospatial information regarding the sample cue location during the delay period and is suitable for accessing visuospatial memory independent of any sensorimotor components, such as motor preparation, for directed movements. Of a total of 385 task-related neurons, 184 showed a sustained increase in activity during the delay period ("delay-period activity"). Most of these neurons (n = 165/184, 90%) showed positional delay-period activity, i.e., delay-period activity where the magnitude differed significantly with the position of the sample cue. This activity appears to be involved in visuospatial memory and to form a "memory field." To quantitatively examine the properties of positional delay-period activity, we introduced a tuning index (TI) and a discriminative index (DI), which represent the sharpness of tuning and the discriminative ability, respectively, of positional delay-period activity. Both TI and DI varied among neurons with positional delay-period activity and were closely related to the time from the onset of the sample cue to the onset of positional delay-period activity; positional delay-period activity with sharper tuning and a greater discriminative ability had a slower onset. Furthermore, at the population level, both TI and DI were increased during the delay period in the neuronal population with a high DI value. These results extend previous findings to suggest that integrative, convergent processes of neuronal activities for increasing the accuracy of visuospatial memory may occur in the dorsolateral PFC. Thus, a critical role of the dorsolateral PFC in visuospatial memory may be to sharpen it to guide behaviors/decisions requiring accurate visuospatial memory.

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.

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.

Pathological and immunological findings of athymic nude and congenic wild type BALB/c mice experimentally infected with Neospora caninum.

Neospora is a cyst-forming coccidian parasite that causes abortions and neuromuscular disorders in a wide variety of mammals. Japanese bovine isolate JPA1 was inoculated intraperitoneally into BALB/c nu/ nu (athymic nude) and BALB/c (congenic wild type) female mice to examine the distribution of parasites and resistance mechanisms to Neospora infection. All the athymic nude mice died within 28 days after intraperitoneal injection of 2 x 10(5) JPA1 tachyzoites, whereas all the congenic wild type mice survived without exhibiting any clinical signs. Tachyzoites were identified in the uterus and pancreas and later spread to many other organs. Most tachyzoites identified in the necrotic foci were localized in the epithelium of the venules and capillaries. Nude mice developed high level of serum interferon-gamma and interleukin-6 as infection proceeded. Inflammatory response to Neospora infection might be mediated by Th1-type dependent cellular immunity.

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.