Monoamine oxidase B catalytically oxidizes biogenic amines such as phenylethylamine and dopamine, and its activity is presumed to be related to particular behavioral traits. In this study, we first identified a single nucleotide polymorphism (T199C) located on the putative third exon of the canine monoamine oxidase B gene, which causes an amino acid substitution from cysteine to arginine. We then examined the allelic frequencies in five dog breeds (Golden Retriever, Labrador Retriever, Maltese, Miniature Schnauzer, and Shiba) and found significant variation among them. The present results suggest that analysis of the monoamine oxidase B polymorphism could be a useful means of elucidating the genetic background of breed-specific behavioral characteristics in dogs.
We have developed a method by which llama cytokine mRNAs can be quantified using real-time reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted from lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) of llama, reverse transcribed to cDNA, and cytokine profiles for interleukin (IL)-1α, IL-1β, IL-6 and tumor necrosis factor (TNF) α were quantified by real-time PCR. The expressions of mRNAs of inflammatory cytokines IL-1α, IL-1β, IL-6 and TNFα were upregulated upon stimulation with LPS in a dose- and time-dependent manner. Incubation of PBMCs with 100 and 1,000 pg/ml of LPS for 3 to 6 hr resulted in the acceleration of the mRNA levels of inflammatory cytokines. Here, we describe a highly sensitive and reproducible method to quantify the transcription of llama cytokine mRNAs by real-time RT-PCR with the double-stranded DNA-binding dye SYBR Green I.
In this study, we examined oral administration with recombinant R7 (rR7) antigen expressed in Escherichia coli using chicken leucocytozoonosis subunit vaccine (LV)-vaccinated and unvaccinated chickens. Only LV-vaccinated chickens showed re-induction of anti-second-generation schizont (2GS) antibody. Also, LV-vaccinated chickens whose anti-2GS antibody titer was middle-level showed increases of the antibody titer compared to vaccinated-control chickens (P>0.01, >0.05) after oral administration with rR7 antigen.
We isolated and sequenced a 480 bp cDNA encoding mature goat interleukin-18 (gIL-18) from alveolar macrophages and splenocytes activated with LPS by RT-PCR. The gIL-18 gene was cloned into pET32a (+) vectors and sequenced. Nucleotide sequence of gIL-18 shares high homology with cattle. Fusional expression with pET32a (+) of gIL-18 of about 38kD was obtained by SDS-PAGE analysis after induction by IPTG in the E. Coli BL21 expression system. The recombinant protein can induce IFN-γ production in PBMC. The IL-18 mRNA was constitutively detected in goat alveolar macrophages with or without LPS, While, enhanced expression was detected in splenocytes and liver cells if treated by LPS, and can be weakly detected in Peripheral blood mononuclear cells (PBMCs) treated by activators. Significant deference of IL-18 mRNA level may reflect the capacity to produce mature IL-18 in such tissues.
To clarify the effect of nutritive conditions on changes in immune cells in Japanese Black (JB) calves during the growth period, leukocyte populations were analyzed in ten healthy JB calves managed in one herd. The calves were divided into two groups: five calves in Group 1 were given insufficient nutrition, and the other five calves in Group 2 received adequate nutrition. The levels of serum total cholesterol and glucose were significantly lower in Group 1 than in Group 2 at 1 month. The numbers of CD3+, CD4+ and CD8+ cells tended to be lower in Group 1 than in Group 2 at months 1 and 2, and the difference in CD4+ was significant at month 2. The number of MHC class-II+high cells was significantly lower in Group 1 than in Group 2 at months 1 and 2. These results suggest that adequate nutrition might stimulate an increase in immune cells in calves during the growth period.
The serum concentration of non-essential amino acid (NEAA) was measured in ten Holstein dairy cows grouped as low production (n=5) and high production (n=5) from one month pre-partum through four months post-partum and the relationship between production and amino acid concentrations was studied. The glycine (Gly)/NEAA ratio and the glycine/alanine ratio of the high production group were significantly higher than the low production group (p<0.01). The observed decrease of the alanine (Ala)/NEAA ratio was more remarkable in the high production group than in the low production group. Measurement of Gly/Ala ratio in serum may be useful for evaluating the nutritional status of peri-parturient dairy cow.
Bioassay-guided fractionation of the boiled extract from the stems of Arcangelisia flava led to the isolation of palmatine (1), berberine (2), jatrorrhizine (3), dihydroberberine (4) and 20-hydroxyecdysone (5). The chemical structures of these compounds were elucidated on the basis of their chemical and spectral evidence. The isolated compounds were evaluated for their growth inhibiting effects on Babesia gibsoni in culture for a week. Compounds (1-4) showed significant inhibitions at concentrations from 100 to 1.0 μg/ml, while compound 5 at a concentration of 100 μg/ml, only.
When Syrian hamsters (Mesocricetus auratus) are bred in a cold and short-day environment, most animals go into hibernation after a certain period of time. However, to date it has not been possible to predict which hamster will enter hibernation. In this study, we subcutaneously implanted thermo-loggers in hamsters bred in the cold environment, and recorded the subcutaneous temperature at short intervals until they went into hibernation. A time series analysis of temperature disclosed that a fall of 0.4 to 0.8°C in subcutaneous temperature was seen 5 to 16 days before entering hibernation, and this phenomenon continued for three days or more. No hamster went into the hibernation without displaying this signal. Although the mechanism by which this phenomenon takes place is not clear, it is a sign from the body, which is useful for indicating if a hamster will enter hibernation shortly.
During the attempt to seek T. congolense species-specific diagnostic antigens, we discovered one cDNA clone (P74) encoding 74 kDa putative abc1 protein (p74) from T. congolense PCF cDNA library. It has been suggested that members of the abc1 family are novel chaperonins and essential for both the mitochondrial electron transfer in the bc 1 complex and the coenzyme Q biosynthesis. Although abc1 protein in yeast has a nuclear or mitochondrial subcellular location, neither nuclear localization signal nor mitochondrial targeting signal was found within p74. Northern blot analysis revealed that the transcription level of P74 mRNA in bloodstream form (BSF) cells were 4 times higher than that in procyclic form cells. Western blot analysis also indicated that p74 was only expressed in T. congolense BSF cells, and revealed that molecular mass of native p74 was not 74 kDa but 56 kDa. This indicates extensive post-translational modification in p74. Although further characterization of p74 will be required, our findings provide implications for CoQ biosynthesis pathway in T. congolense.
The therapeutic effect of clindamycin on Eimeria pragensis (E. pragensis) infection in C57BL/6 mice was demonstrated by suppression of oocyst production and the appearance of degenerated endogenous stages of parasite in the intestine. Short-term clindamycin treatment, from 1 to 4 days or 4 to 8 days post infection (pi) at a dose of 800 mg/kg/day was effective to reduce clinical symptoms, oocyst production and schizogonic development. Interestingly, the short-term treatment schedules allowed the development of a measurable degree of protective immunity to challenge infection in the treated mice. In contrast, clindamycin treatment for the full 12 days period, which almost completely inhibited clinical symptoms and oocyst output, prevented the full development of protective immunity in the treated mice. All these data indicate that clindamycin is efficacious as an anti-eimerian agent and that both early and late endogenous developmental stages of E. pragensis exert a deep influence on the development of effective immunity to challenge infection.
The kidneys of 37 Japanese Black calves aged 2 to 65 months diagnosed with Claudin 16 (CL-16) defect by the DNA-based test were examined pathologically. The animals exhibited clinical symptoms such as growth impairment, renal failure, overgrowth of hooves, and anemia at a young age. There was no correlation between the time of onset and age. Kidney weights relative to body weight were similar to those in normal animals, but both kidney net weights and size were reduced due to atrophy in animals that showed severe renal dysfunction. Histopathological examination of the kidneys showed reduction in the number of glomeruli, compensatory hypertrophy of glomeruli and tubules, and glomerular and tubular atrophy accompanied by interstitial fibrosis and lymphocytic infiltration. Glomeruli were clearly less in number in the kidneys of CL-16-defective animals than those of normal animals even in the cases with mild lesions. A small number of immature glomeruli and tubules were also detected, suggesting that there were fewer nephrons developed at birth in CL-16 -defective animals. It was suggested that a defect of the CL-16 gene is involved in the "abnormal development of nephrons". Immunohistopathological examination of the kidneys showed that the epithelium of thick ascending limb of Henle was stained with anti-CL-16 antibody in the control animals, but not in the affected animals, suggesting a defect of CL-16 in the epithelium of renal tubules in the affected animals.
Canine distemper virus (CDV) growth and the morphological characterization were examined in a cell line established from a canine malignant histiocytosis (CCT cell line). The susceptibility of the CCT cells to 3 CDV strains, FXNO, YSA-TC and MD-77 was shown by detection of the antigen in the indirect fluorescent assay. After passaging 4 and 9 times through the CCT cells, only FXNO strain could produce the syncytia where demonstrated the antigens. Titers of 9 passaged viruses through the CCT cells showed slightly higher in the CCT cells than those in Vero cells. Morphological characterization of karyorrhexis and specific DNA ladder by extracted DNA electrophoresis indicated apoptosis in the CDV infected CCT cells.
A total of 88 Staphylococcus and 61 Streptococcus isolates from diseased animals throughout Japan were examined in 2000 for the minimum inhibitory concentrations of 24 different antimicrobials by the agar dilution method standardized by the Japanese Society of Chemotherapy. The resistance rates to aminobenzylpenicillin (36.4%) and benzylpenicillin (35.2%) were high in Staphylococcus isolates, and those to oxytetracycline (45.9%) and kanamycin (21.3%) were high in Streptococcus isolates. Two isolates resistant to oxacillin harbored the mecA gene. One was Staphylococcus epidermidis derived from a pig with arthritis, and the other Staphylococcus cohnii from a head of cattle with mastitis.
An 8-year-old Beagle dog had exophthalmos of the left eye for the last two past months. On ophthalmoscopy, the intraocular lesion could not be evaluated due to the opacity of the cornea. Ultrasonography revealed that the eyeball was distorted in shape and shifted in position, however, the precise lesion could not be identified. On magnetic resonance (MR) imaging, the lesion was observed as hyperintense on T1-weighted and hypointense on T2-weighted images, similar to those reported in human melanoma. The lesion was histologically diagnosed to be malignant intraocular melanoma. Though this is only a case report, canine ocular melanoma may show the similar characteristic MR images as in human uveal malignant melanoma.
The recurrence of estrus and fertility after removal of a subcutaneous chlormadinone acetate implant (CMA-I) administered to prevent estrus for 4 years, was investigated in 8 female dogs and the results compared with those for 4 untreated female dogs (control group). The sex hormones present during the estrous cycle were also investigated. There were no significant differences in the estrous cycle after removal of the implant between the CMA-I-treated group and the control group. However, although conception was achieved after mating and no uterine diseases developed in the control group, only 5 (4 dogs, 41.7%) of the 12 cases (6 dogs) in which mating took place at the second to fourth estrus after the removal of CMA-I resulted in pregnancy in the CMA-I-treated group. Furthermore, 6 (75.0%) of the 8 dogs in the CMA-I-treated group developed uterine diseases including pyometra or hydrometra. There were no significant differences in plasma progesterone, LH and prolactin levels between the non-pregnant and pregnant dogs in the CMA-I-treated group or control group. These results suggest that long-term implantation of CMA-I affects fertility after the implant is removed.
Genetic and antigenic analyses of bovine respiratory syncytial virus were conducted on 12 field strains from Tohoku and Hokuriku districts in Japan during from 2002 to 2004. On the phylogenetic tree of the nucleotide sequences of the glycoprotein region, the examined strains fell in the same cluster as the strain isolated in Nebraska and were classified as the subgroup III. The examined strains were subdivided into 2 lineages (A, B). Isoleucine 200 of the epitope domain was replaced by threonine as a feature of the lineage B strains. The examined strains showed the nucleotide sequence homologies of 88.3-93.3% with the known Japanese strains classified as the subgroup II and of 86.1-96.6% with those in the subgroup III. No significant difference was found on the neutralization index between the examined strain and the 52-163-13 phylogenetically similar to the Japanese vaccine one. The results suggest that the subgroup III strains have existed in Japan and that epidemics of the strains could be protected due to the present vaccination.
Although winter dysentery (WD) has been suspected to occur frequently in Korea, to date the exact epidemiology of WD has remained unknown. Therefore, we investigated the causative agents of WD by using electron microscopy, ELISA, RT-PCR, and nested PCR from 97 fecal samples of 32 WD-affected herds collected from 8 provinces during 2002-2004. The bovine coronavirus (BoCV) was consistently detected in all herds with WD. Of other pathogens, only coccidian oocyts were inconsistently but concurrently detected with BoCV. Ten isolates were identified as BoCV by immune electron microscope, immunofluorescent test and ELISA with antiserum to BoCV, and RT-PCR. From these results, it is concluded that WD caused by BoCV occurred in relatively high frequency and was widespread in Korea. The results provide important epidemiological data for the control and establishment of a surveillance system for WD in Korea.