In the present study, we employed flow cytometry to evaluate the level of parasitemia of Babesia gibsoni infecting canine erythrocytes in vivo and in vitro by using fluorescent nucleic acid staining. Peripheral blood samples from a B. gibsoni-infected dog and cultured B. gibsoni parasitizing in canine erythrocytes were stained with a membrane-permeable fluorescent nucleic acid stain, SYTO16. In this study, we utilized normal canine erythrocytes (LK erythrocytes) and canine erythrocytes containing high concentrations of potassium, reduced glutathione, and some free amino acids (HK erythrocytes) as host cells for culture. In vivo parasitized cells were discriminated completely from unparasitized cells and a correlation (r=0.998) between the percentage of SYTO16-positive cells and parasitemia in vivo was observed. On the other hand, in vitro erythrocytes could not be divided clearly into parasitized and unparasitized cells. However, when LK erythrocytes were used as host cells, the percentage of SYTO16-positive cells was the almost same as, and was well correlated (r=0.932) with the level of parasitemia. When HK erythrocytes were used as host cells, the percentage of SYTO16-positive cells was almost half of, but was correlated (r=0.982) with the level of parasitemia. Therefore, we attempted to observe the changes in the percentage of parasitized cells after treatment with antiprotozoal drug or mitochondria inhibitors by using flow cytometry. The changes in the percentage of SYTO16-positive cells corresponded well with the changes of the level of parasitemia when the parasites in HK erythrocytes were cultured with each compound. The present results suggest that flow cytometric detection using SYTO16 is a rapid and reliable method for monitoring parasitemia both in vivo and in vitro.

Bovine nasal and oral discharges were used as samples for bovine viral diarrhea virus (BVDV) gene detection. Viral genes in serum (S), nasal discharge (N) and oral discharge (O) were quantified with real-time polymerase chain reaction using SYBR Green by the relative quantification method, and findings were compared among samples. Although the quantity of the BVDV gene in S was greater than those in N and O, all samples were available to identify persistently infected (PI) cattle with BVDV by reverse transcription polymerase chain reaction (RT-PCR). The swab samples were able to be stored for a few days at 4degC with a little decrease of amplification signal in RT-PCR. Oral swab sampling was easier than nasal swab sampling, and was also less uncomfortable for the cattle than other sampling methods without pain or unnecessary retention. This sampling method can be performed without any special technique and equipment. Therefore, the oral swab sampling method is useful for screening to detect BVDV PI cattle by RT-PCR.

To establish vaccine strains of H5 and H7 influenza viruses, A/duck/Hokkaido/Vac-1/04 (H5N1) [Vac-1/04 (H5N1)], A/duck/Hokhaido/Vac-3/07 (H5N1) [Vac-3/07 (H5N1)], and A/duck/Hokkaido/Vac-2/04 (H7N7) [Vac-2/04 (H7N7)] were generated from non-pathogenic avian influenza viruses isolated from migratory ducks. Vac-1/04 (H5N1) and Vac-3/07 (H5N1) were generated by genetic reassortment between H5N2 or H5N3 virus as an HA gene provider and H7N1 or H6N1 virus as an NA gene provider. Vac-2/04 (H7N7) was a genetic reassortant obtained using H7N7 and H9N2 viruses to give high growth character of the H9N2 virus in chicken embryonated eggs. The results of sequence analyses and experimental infections revealed that these H5N1 and H7N7 reassortant viruses were non-pathogenic in chickens and embryos, and had good growth potential in embryonated eggs. These viruses should be useful to develop vaccines against H5 and H7 highly pathogenic avian influenza viruses.

Rhipicephalus appendiculatus serpin-3 (RAS-3), R. appendiculatus serpin-4 (RAS-4) and a 36kDa immuno-dominant protein of R. appendiculatus (RIM36) were reported as candidate antigens for the anti-tick vaccine to control ixodid ticks. In the present study, we generated recombinant proteins of RAS-3 (rRAS-3), RAS-4 (rRAS-4) and RIM36 (rRIM36), and assessed their potency as an anti-tick cocktail vaccine in cattle model. RT-PCR analysis showed that RAS-3, RAS-4 and RIM136 transcripts were detected in both adult male and female ticks during feeding. Immunization of cattle with the combination of rRAS-3, rRAS-4 and rRIM36 had raised antibodies against all recombinants and anti-sera had reacted with the molecules from the tick salivary gland extract. Tick infestation challenge demonstrated protective immunity against female ticks, resulting in mortality rates of 39.5 and 12.8 % for the vaccinated and control groups, respectively. Moreover, the mortality rate of Theileria parva-infected female ticks was 48.5 and 10.8 % in the vaccinated and control group, respectively. In order to evaluate the levels of pathogen transmission capacity by T. parva-infected ticks fed on immunized cattle, the occurrence of T. parva in the bovine parotid lymph node and peripheral blood was also determined and quantified by real-time PCR. Although the infection with T. parva could not be protected by the vaccine, the occurrence of pathogen in peripheral blood was delayed 1 to 2 days after the infestation challenge in vaccinated group. These results suggest that this cocktail vaccine plays a role in the prevention of tick infestation.

This study was performed to examine whether the brain activities induced by noxious algesic chemical substances in anesthetized animals could be detected by blood oxygen-level-dependent functional magnetic resonance imaging (BOLD-fMRI). Multislice gradient echo images of the primary somatosensory cortex were obtained using a 7.05 T superconducting system and a one-turned surface coil centered over the primary somatosensory cortex of the 1.0%-isoflurane-anesthetized rat. The Z-score t-map of BOLD signals and its time-course analysis revealed that subcutaneous injection of formalin into the left forepaw immediately induced an early response in the contralateral primary sensory cortex lasting for a few minutes, followed by a late response until 20 min after stimulation. In contrast, injection of capsaicin into the left forepaw evoked only the early response. Furthermore, pretreatment with morphine completely abolished these responses induced by the chemical algesic substances. Thus BOLD-fMRI is a useful method to analyze the brain activities of painful stimulation in anesthetized animals.

The health hazards of individual organophosphorus insecticides have been characterized by their acute toxicity, mainly by investigating their cholinesterase inhibition. However, the chronic effects of most of these toxicants on the drug-metabolizing enzymes have not been investigated. Profenofos (O-4-bromo-2-chlorophenyl O-ethyl S-propyl phosphorothioate) is an organophosphorus pesticide widely used in cotton cultivation. In the present study, we investigated the effect of profenofos on male-specific cytochrome P450 (CYP) enzymes in adult Wistar rats. We orally administered 17.8 mg/kg body weight, twice weekly for 65 days. Profenofos downregulated levels of hepatic and testicular CYP2C11 and CYP3A2 mRNA and protein expression. Testicular aromatase (CYP19A) mRNA was decreased in the profenofos-treated rats compared to controls. Overall, the present study suggests that profenofos acts as an endocrine disruptor of male-specific CYP enzymes and affects testosterone concentration, which implicates its deleterious effects on animal or human males chronically exposed to organophosphorus pesticide.

Nigella sativa (family Ranunculaceae ) is an annual plant that has been traditionally used on the Indian subcontinent and in Middle Eastern countries. In this study, we investigated the effect of N. sativa oil on the drug-metabolizing cytochrome P450 (CYP) enzymes and whether it has a protective effect against the acute hepatotoxicity of CCl4. Intraperitoneal injection of rats with CCl4 drastically decreased CYP2E1, CYP2B, CYP3A2, CYP2C11, and CYP1A2 mRNA and protein expressions. Oral administration of 1 ml/kg N. sativa oil every day for one week prior to CCl4 injection alleviated CCl4-induced suppression of CYP2B, CYP3A2, CYP2C11, and CYP1A2. Moreover, CCl4 increased iNOS and TNFalpha mRNA, while N. sativa oil administration for one week prior to CCl4 injection downregulated the CCl4-induced iNOS mRNA and up-regulated IL-10 mRNA. These results indicate that N. sativa oil administration has a protective effect against the CCl4-mediated suppression of hepatic CYPs and that this protective effect is partly due to the downregulation of NO production and up-regulation of the anti-inflamnatory IL-10.

Puumala virus (PUUV), a causative agent of hemorrhagic fever with renal syndrome (HFRS), is prevalent in Europe and European Russia. No vaccine has been developed for PUUV-associated HFRS, primarily because of the low viral yield in cultured cells. A PUUV strain known as DTK/Ufa-97 was isolated in Russia and adapted for growth in Vero E6 cells maintained in serum-free medium. The DTK/Ufa-97 strain produced a higher viral titer in serum-free medium, suggesting that it may prove useful in the development of an HFRS vaccine. When PUUV-infected Vero E6 cells were grown in serum-free medium, the DTK/Ufa-97 strain yielded more copies of intracellular viral RNA and a higher viral titer in the culture fluid than did the Sotkamo strain. Phylogenetic analysis revealed that PUUVs can be classified into multiple lineages according to geographical origin, and that the DTK/Ufa-97 strain is a member of the Bashkiria-Saratov lineage. The deduced amino acid sequences of the small, medium, and large segments of the DTK/Ufa-97 strain were 99.2% to 100%, 99.3% to 99.8%, and 99.8% identical, respectively, to those of the Bashkirian PUUV strains and 96.9%, 92.6%, and 97.4% identical, respectively, to those of the Sotkamo strain, indicating that the PUUVs are genetically diverse. However, DTK/Ufa-97 and other strains of PUUV exhibited similar patterns of binding to a panel of monoclonal antibodies against Hantaan virus. In addition, diluted antisera (i.e., ranging from 1:160 to 1:640) specific to three strains of PUUV neutralized both homologous and heterologous viruses. These results suggest that the DTK/Ufa-97 strain is capable of extensive growth and is antigenically similar to genetically distant strains of PUUV.

Although mammals produce either sperm or eggs depending on their sex, newborn MRL/MpJ male mice contain oocytes within their testes. In our previous study, the testicular oocyte appears as early as day 0 afterbirth and has morphological characteristics as an oocyte such as zona pellucida and follicular epithelial cells. Based on the observation of F1 between MRL/MpJ and C57BL/6, one of the genes causing the appearance of testicular oocyte exists on the Y chromosome. In the present study, we found testicular oocytes within newborn AKR mice. We have also analyzed the Sry genes from several inbred mouse strains and identified a shortened glutamine repeat near the C-terminal region that is unique to MRL and AKR. These results suggest that polymorphism of glutamine repeat within SRY correlates with the appearance of testicular oocyte and this phenotype is derived from AKR, one of the original strains of MRL mice.

Information on steroid hormone receptor distribution in the uterus is essential to understand the roles of their ligands in pregnancy. This study examined the spatio-temporal localization of estrogen receptor alpha (ERalpha) and progesterone receptor (PR) in the uterus of sika deer (Cervus nippon) to determine the estrogen and progesterone action site during pregnancy. Ovaries and uteri were collected from 21 pregnant sika deer with single fetus and two corpora lutea, ranging from Day 20 to Day 207 of pregnancy. In addition, genital organs were also collected from three sika deer whose gestational status was unknown: one female had only one developing corpus luteum: =Day 4 (metestrus) and two females had two corpora lutea, one of which was at the developing stage equivalent to diestrus or early pregnancy: Day 7 (diestrus). Staining of ERalpha and PR was clear in all cell types during metestrus. During diestrus, the presence of ERalpha was also clear in deep glandular epithelium, stroma and myometrium, whereas it was suppressed in luminal epithelium and shallow glandular epithelium. Staining of PR was suppressed in luminal epithelium but was detectable in other cell types. Staining of ERalpha in all cell types and PR in luminal epithelium and glandular epithelium became undetectable by Day 28. PR was presented in stroma and myometrium throughout pregnancy. The distribution pattern of ERalpha and PR was different during diestrus from that in a ruminant. This could be attributed to estrogen secretion from the maturing and ovulating follicles in the presence of developed corpus luteum.