In this study, we investigated the effects of N-acetylserotonin (NAS) on cell proliferation and neuroblast differentiation in the mouse dentate gyrus using anti-Ki67 and anti-doublecortin (DCX) antibodies. Ki67 is expressed in the nucleus or on the surface of chromosomes during all of the active phases of the cell cycle, and DCX is expressed in neuronal precursor cells as well as in immature neurons. At 17 weeks of age, 20 mg/kg of NAS or the same volume of vehicle was intraperitoneally administered once a day for 3 weeks. The animals were sacrificed 2 hr after the last vehicle or NAS treatment. NAS treatment significantly increased the number of Ki67-positive nuclei and DCX-immunoreactive neuroblasts with well-developed dendrites (tertiary dendrites) compared to the vehicle-treated group. However, the number of DCX-immunoreactive neuroblasts without tertiary dendrites was not changed. The administration of NAS also significantly increased the protein levels of brain-derived neurotrophic factor (BDNF) in the dentate gyrus. This result suggests that NAS significantly promotes cell proliferation and the number of differentiating neuroblasts with tertiary dendrites through an increase in BDNF levels in the mouse dentate gyrus.
A mutant strain, PBA322, was constructed by electroporation of a phagemid containing the coding region of antisense RNA of the ompH gene, encoding 39 kDa capsular protein or OmpH, into the parental strain P-1059 (serovar A:3) of Pasteurella multocida, and the pathogenicity was determined in mice and chickens. Grayish colonies of the mutant, indicating loss of capsule synthesis, were observed under a stereomicroscope using obliquely transmitted light, while iridescent colonies were observed for the parental strain. Moreover, strain PBA322 showed a low amount of OmpH compared with the parental strain on SDS-PAGE. Additionally, the capsule of strain PBA322 was thinner than that of the parental strain according to electron microscopy, correlating to the attenuation against chickens. In conclusion, strain PBA322, the mutant of P. multocida strain P-1059, was completely attenuated for chickens.
We collected a total of 206 Haemaphysalis longicornis ticks by flagging in pastures in Yonaguni Island, Okinawa, Japan, in April 2008. Four of the 206 tick DNA samples tested were positive in a polymerase chain reaction (PCR) screening for the 16SrRNA gene of Anaplasmataceae. Partial sequences of 4 PCR products were identical to each other. Longer sequences of the 16SrRNA gene were successfully determined in 2 of the 4 tick samples, and the obtained 1,392 bp and 1,300 bp sequences revealed high similarity to the 16SrRNA gene sequences of the validated Ehrlichia species, including Ehrlichia ewingii, E. chaffeensis, and E. canis (98.3-98.6%). We also sequenced 1,304 bp of the groEL gene from the 2 tick samples, and found that these had the highest similarity to sequences of E. ewingii (94.0-94.4%) in the validated ehrlichial species. Based on the 16SrRNA and groEL gene sequences, the ehrlichial agents detected in this study were similar to the Ehrlichia species detected in Asia and may compose a new Ehrlichia species with other Ehrlichia species detected in Asia.
We examined secondary structures of the ribonuclease P RNA sequences obtained from DNA databases, and identified a determinative prototype of the P12 helix peculiar to each species of hemoplasmas. This key structure will provide a rapid means for species identification of these uncultivable pathogens without making a phylogenetic tree based on alignments of nucleotide sequences. This procedure based on palindromic nucleotide substitutions at the stem portion of the P12 helix provide clear information such as the level of heterogeneity within a species, the relatedness between species, or facilitating the characterization and clustering of specific strains. In conclusion, the PNS analysis is based on the evaluation of only the strategic and highly conserved genomic region in the specificity domain of RNase P RNA.
Bovine mastitis due to Prototheca zopfii leads to reduced milk production and is difficult to cure. Therefore, prevention is the best approach and this is best achieved through the use of effective disinfectants. The aim of this study was to evaluate the in vitro algaecide efficacy of conventional disinfectants against strains of P. zopfii genotype 1 and 2. The minimal algaecide concentration (MAC) of alkyldiaminoethylglycine hydrochloride, chlorhexidine, dioxide chlorine, povidone iodine and sodium hypochlorous acid against 10 isolates and the type strain (SAG2063T) of P. zopfii genotype 1 as well as 10 isolates and the type strain (SAG2021T) of P. zopfii genotype 2 were examined using the micro dilution method. This in vitro study indicated that alkyldiaminoethylglycine hydrochloride, chlorhexidine, povidone iodine and sodium hypochlorous acid, but not dioxide chlorine, are effective against both genotypes of P. zopfii.
This study was conducted to evaluate the changes in acute-phase proteins and cytokine concentrations in dairy cows with naturally occurring peracute Klebsiella pneumoniae (K. pneumoniae) mastitis and their association with the outcome of the disease. Seventeen Holstein cows with K. pneumoniae mastitis from 8 dairy farms were divided on the basis of outcome after local and systemic therapy into 2 groups comprising 8 euthanized cows and 9 that recovered. Changes in acute-phase proteins and cytokine concentrations in cows with K. pneumoniae mastitis were evaluated at the onset of the disease (day 0) and at days 3, 7 and 14 after therapy and compared with those of 13 healthy dairy cows. The concentrations of haptoglobin (Hp) and interleukin (IL)-6 in serum and α1-acid glycoprotein and IL-1β in serum and whey on day 0 were significantly (P<0.05) higher in the euthanized cows than in those that recovered and the healthy cows. A correlation (r=0.90, P<0.01, n=17) was found between IL-6 and Hp concentrations in sera from recovered and euthanized cows at day 0. This indicated that serum concentrations of Hp and IL-6 at the initial examination were prognostic factors for survival, and the cutoff values were 2,020 μg/ml and 32 ng/ml, respectively. These results suggest that IL-6 and Hp concentrations are involved in the manifestation of K. pneumoniae mastitis and may be possible indicators of the prognosis of peracute K. pneumoniae mastitis.
This study was performed to evaluate the cross section of the thymus in 46 clinically healthy Holstein calves and heifers from birth to 100 weeks of age and 8 age-matched affected animals with chronic bronchopneumonia by ultrasonography. The cross sectional area of the thymus increased in healthy calves and heifers from birth to 31 weeks of age, peaked around 35 weeks of age, and then decreased until 100 weeks after birth. A positive correlation (r= 0.74, P<0.05) was found between the area in cross section and the weight of the whole thymus in calves and heifers with chronic bronchopneumonia. The cross- sectional areas of the thymus were significantly (P<0.05) lowered in animals with chronic bronchopneumonia. The cross sections of thymus in clinically healthy calves at 2 weeks of age and heifers at 34 weeks and 87 weeks of age were hypoechoic, moderately hyperechoic and hyperechoic, respectively. The observation of thymus by ultrasonography seems to be an effective tool for estimating the size of the thymus objectively in Holstein calves and heifers from birth to 100 weeks of age. The results of this study prove the approach to be useful and applicable to monitor the growth of thymus in Holstein calves and heifers and their association with chronic pneumonia.
A 14-year-old, mixed breed dog was presented with acute pain and paresis of the hindlimbs. Ultrasonography revealed an intraluminal mass and an abrupt halt of blood flow signal in the distal abdominal aorta. The mass had homogeneous hyperechoic echotexture compared with blood flow. Although clinical presentation suggested a thromboembolism and pituitary dependent hyperadrenocorticism was suspected as a predisposing cause based on adrenal function tests and ultrasonography, an aortic chondrosarcoma originating from the distal abdominal aorta was diagnosed with histologic examination. Primary aortic sarcoma is extremely rare, and extraskeletal chondrosarcoma is only reported in 2 cases previously. Aortic neoplasm should be included in differential diagnosis when an intraluminal aortic mass is observed on ultrasonography and acute paresis of hindlimbs is shown.
Neonatal Japanese Black (JB) calves show a high incidence of diarrhea. The objective of this study was to analyze the immune cell populations of neonatal JB calves in detail and examine its correlation with the incidence of diarrhea immediately after birth. Understanding the immune cell populations is helpful in clinics in order to determine the condition of the immune system for prevention of diseases. Blood samples were obtained from JB calves on the day of birth. The peripheral leukocyte populations were analyzed separately for calves that had diarrhea within 2 weeks after birth (diarrhea group; n=26) and for calves without diarrhea (control group; n=74). The numbers of the peripheral blood CD3+TcR1-N12+ and CD8+ T cells were significantly lower in the diarrhea group compared with the control group. These findings suggest that the congenital lower peripheral γδ and CD8+ T cells results in a high risk of diarrhea in neonatal JB calves.
Pancreatitis is a common exocrine pancreatic disease in dogs, and the pancreatic lipase immunoreactivity (PLI) test is used for diagnosis. Enzyme catalytic assay is thought to have low specificity, but a lipase activity assay with increased specificity has been developed in human clinical chemistry. We measured serum lipase activity of 65 client-owned dogs using the newly developed FUJI DRI-CHEM slide and compared the results with their PLI concentrations. The results showed a good correlation (r=0.91), and the normal and pancreatitis dogs identified based on the PLI values were correctly separated based on lipase activity. The present study suggests that FUJI DRI-CHEM lipase activity would be helpful for diagnosis of pacreatitis in dogs and, in particular, that it can be used as a patient-side assay and contributes to immediate treatment.
A 9-year old female spayed Rottweiler was diagnosed with cryptogenic epilepsy and started on zonisamide monotherapy (8.3 mg/kg, PO, q 12 hr). Three weeks after the 1st dose of zonisamide the dog presented for vomiting, inappetence and icterus. Serum biochemistry showed marked elevation of liver enzymes, consistent with hepatocellular damage and cholestasis. No underlying cause for liver disease was identified and a drug-induced hepatopathy was suspected. Zonisamide was discontinued and replaced by potassium bromide. Supportive therapy consisted of intravenous fluids, antiemetics, antibiotics and hepatoprotectants. The dog made a complete recovery and serial serum biochemical examinations showed complete normalisation of liver parameters 8 weeks after discontinuation of zonisamide. Based on a human Drug-induced Liver Injury Diagnostic Scale, the likelihood for zonisamide-induced hepatopathy was classified as "possible". Veterinary practitioners and owners should be educated about the potential for an idiosyncratic drug reaction to zonisamide. If signs of hepatotoxicity are recognised early and zonisamide is discontinued, complete recovery is possible.
An epidemiological survey on a Theileria parasite infection of cattle in Northeast China was carried out using allele-specific PCR and DNA sequence analysis of the major piroplasm surface protein (MPSP) gene. The results showed that 14 of 104 blood samples were positive for Theileria by PCR. Among the positive cases, co-infection with various combinations of C- and I-type parasites was detected in 12 samples; no B- and Thai-type parasites were detected by allele-specific PCR. Phylogenetic analysis based on the MPSP gene sequences revealed that Theileria parasites with the MPSP types 1, 2, and 4 were distributed in Northeast China.
We studied the prevalence of avian Plasmodium in 509 mosquitoes of 9 species collected from the Ishigaki and Iriomote islands in the Yaeyama Archipelago, located southwest from the mainland of Japan. Two identical avian Plasmodium lineages were detected from Culex (Culiciomyia) nigropunctatus. Detected lineages were phylogenetically classified into different clade to avian Plasmodium lineages from birds and mosquitoes in the mainland of Japan but identical to a lineage detected from a resident bird, White-breasted Waterken (Amaurornis phoenicurus). This is the first detection of avian Plasmodium DNA from mosquitoes in the Yaeyama Archipelago and suggested that resident birds might have been infected with an avian Plasmodium lineage specific to the studied area and C. nigropunctatus could be the candidate vector mosquito species.
To clarify the morphological characteristics of the cynomolgus monkey immune system, we analyzed quantitative data on their lymphoid organs. Spleens, major lymph nodes and Peyer's patches were sampled from cynomolgus monkeys, and the lymphoid follicle and germinal center areas and percentages of CD3- and CD20-positive areas were calculated. All the organs analyzed showed large interindividual variations in the sizes of lymphoid follicles and germinal centers. Lymphoid follicle in the spleen, submandibular lymph nodes and Peyer's patches showed no marked difference in size. Germinal center size in the mesenteric lymph nodes and Peyer's patches were significantly smaller than those in the spleen. Areas containing T cells were largest in the lymph nodes, while those containing B cells were largest in the spleen and Peyer's patches. The mean size of the splenic lymphoid follicle in cynomolgus monkeys is larger than that in rats and similar to that in humans. Based on the large individual variation and the characteristics of lymphoid organs, it is important to use cynomolgus monkeys in standard toxicity studies. Taking advantage of the characteristics of each species enables reliable evaluation of the immunologic system in standard toxicity studies.
Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder of cattle characterized by accumulation of the disease-associated prion protein (PrPSc) in the central nervous system (CNS). The immunohistochemical patterns and distribution of PrPSc were investigated in the CNS, brains, and spinal cords of 7 naturally occurring BSE cases confirmed by the fallen stock surveillance program in Japan. No animals showed characteristic clinical signs of the disease. Coronal slices of 14 different brain areas in each case were immunohistochemically analyzed using an anti-prion protein antibody. Immunolabeled PrPSc deposition was widely observed throughout each brain and spinal cord. Intense PrPSc deposition was greater in the thalamus, brainstem, and spinal cord of the gray matter than in the neocortices. The topographical distribution pattern and severity of PrPSc accumulation were mapped and plotted as immunohistochemical profiles of the different brain areas along the caudal-rostral axis of the brain. The distribution pattern and severity of the immunolabeled PrPSc in the CNS were almost the same among the 7 cases analyzed, suggesting that the naturally occurring cases in this study were at the preclinical stage of the disease. Immunohistochemical mapping of the PrPSc deposits will be used to clarify the different stages of BSE in cattle.
A mass was located in the small intestine of a slaughtered 6-month-old male Landrace-cross pig that had no clinical abnormalities. This egg-shaped well-circumscribed mass was situated in the submucosal and muscular tissue layers and protruded into the lumen. Histopathologically, the tumor comprised discrete or aggregated ganglion and schwannian cells in neuropil-like tissue. Some ganglion cells contained Nissl substance in their cytoplasm. The ganglion cells stained positive for neuron-specific enolase, class III β-tubulin, neurofilament, and synaptophysin; the schwannian cells stained positive for vimentin, S-100 protein, and glial fibrillary acidic protein. The tumor was diagnosed as a ganglioneuroma in accordance with these findings. Here, we have reported detailed immunohistochemical findings in addition to the histopathological features of a swine ganglioneuroma.
Cytochrome P450 3A (CYP3A) is the major subfamily of CYP, one of the most important metabolizing enzymes for drugs in humans and other mammals. We found two novel CYP3A genes, CYP3A131 and CYP3A132 in domestic cats (Felis catus). Both feline CYP3A proteins consist of 504 deduced amino acids and show high identity with canine CYP3A homologues and those of some artiodactyls. CYP3A131 transcripts were expressed predominantly in liver and small intestine, and to a negligible extent in other tissues, including brain, heart, kidney and lung. CYP3A132 expression was only detected in liver with much lesser amount. These results suggest the possible major role of CYP3A131 in xenobiotic metabolism including first-pass effects in domestic cats.
Calcium transporter genes, such as transient receptor potential cation channel subfamily V members 5/6 (TRPV5/6), Na+/Ca2+ exchanger 1 (NCX1), and plasma membrane calcium-transporting ATPase 1b (PMCA1b), are essential for maintaining homeostasis and metabolizing Ca2+ ions. The TRPV5 and TRPV6 proteins play an important role in Ca2+ absorption, and NCX1 and PMCA1b are both critical for intracellular calcium homeostasis. In this study, the tissue-specific mRNA and protein expression of these calcium transporter genes in the duodenum, kidney and heart of the horse (Equus caballus) was examined using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. The tissue localization of these calcium transporters was also investigated using immunohistochemistry. The results showed that TRPV5 mRNA was highly expressed in the kidney but was scarce in the duodenum and heart. TRPV6 mRNA levels were similar in all the tissues. NCX1 and PMCA1b were both highly expressed in the heart, but no difference in NCX1 and PMCA1b mRNA expressions was observed in the duodenum and kidney. The aspect of protein expression was similar with mRNA expression data. Localization of calcium transporter genes were detected enterocytes in duodenum, the distal convoluted tubules in the kidney, and within the cardiac muscle cells of the heart. Based on these results, calcium transport genes appear to be expressed in horse tissues at levels similar to those observed in other vertebrates.
The effects of transport for 5 hr by road and lairage for 48 hr on acute phase protein in Limousine feedlot beef cattle (n=10, 14-16 months old, body weight 620 ± 70 kg) were examined. Blood was collected at before loading, immediately after unloading and 12, 24 and 48 hr after the initiation of transportation. Serum was collected for assay of haptoglobin (Hp) and serum amyloid A (SAA), plasma was obtained for assay fibrinogen (Fb) and the white blood cell count (WBC) was determined in whole blood at each time point. A significant effect of experimental conditions on Hp, SAA and WBC was observed. In particular, this effect was found 24 hr after transport for Hp and SAA and after 48 hr for WBC. Application of a linear regression model showed a high correlation between WBC and Hp and SAA. Lairage in a slaughterhouse represents a stressful condition that can compromise animal welfare and meat quality. Monitoring of SAA with the highest sensitivity and specificity could be a useful marker of welfare condition in this period.
Bartonella henselae is the causative agent of cat scratch disease (CSD) in humans. Cats are the main reservoir of this bacterium and may infect humans through scratches and bites. The purpose of this study was to determine the B. henselae seroprevalence in cats in Turkey. A total of 298 cats blood samples were collected from six different provinces of Turkey. Sera were tested for the presence of anti-B. henselae IgG antibodies by indirect fluorescent antibody test (IFA). The seroprevalence of B. henselae was 27.9% (83/298) for the cats examined in this study. The seroprevalence of cats by province was significantly higher in Bursa (41.3%), Adana (33.9%), Aydin (27.5%) and Burdur (32.3%) than in Kayseri (17.9%) and Istanbul (12.5%). Statistically significant differences were not observed between cat sexes and living conditions of cats. The results revealed that B. henselae is an important zoonotic pathogen in Turkey.
Sow lifetime performance and by-parity performance were analyzed using a 3 by 3 factorial design, comprising 3 herd productivity groups and 3 sow efficiency groups. Data was obtained from 101 Japanese herds, totaling 173,526 parity records of 34,929 sows, for the years 2001 to 2006. Sows were categorized into 3 groups based on the lower and upper 25th percentiles of the annualized lifetime pigs born alive: low lifetime efficiency sows (LE sows), intermediate lifetime efficiency sows or high lifetime efficiency sows. Herds were grouped on the basis of the upper and lower 25th percentiles of pigs weaned per mated female per year, averaged over 6 years: high-, intermediate- or low-performing herds. Mixed-effects models were used for comparisons. LE sows in high-performing herds had 57.8 fewer lifetime nonproductive days and 0.5 earlier parity at removal than those in low-performing herds (P<0.05). The number of pigs born alive of LE sows continuously decreased from parity 1 to 5, whereas those of high lifetime efficiency sows gradually increased from parity 1 to 4 before decreasing up to parity ≥ 6 (P<0.05). In conclusion, the LE sows have a performance pattern of decreasing number of pigs born alive across parity. The present study also indicates that high-performing herds culled potential LE sows earlier than the other herds.
The present study determined the association among the expression of COX-2, stages of endometritis, and types and number of local immune cells infiltrating into the gilts' endometrium. The uterine tissues from 24 Landrace x Yorkshire gilts identified as acute endometritis (n=7), chronic endometritis (n=7), and normal endometrium (n=10) were included. The tissues were prepared for both histological and immunohistochemical investigations. The immunoexpression of COX-2 in every layer of the gilts' endometria was appraised by avidin-biotin-peroxidase complex method via image analysis; and was reported as percentage of positive area and staining index. The results revealed that the immunoexpression of COX-2 was found only in the surface epithelial layer. The gilts with acute endometritis possessed higher both percentage of positive area (68.99% versus 4.50% and 3.43%, P<0.001) and staining index (1.13 versus 0.05 and 0.04, P<0.001) than those with chronic endometritis and normal endometrium, respectively. Positive correlations between the number of surface epithelial neutrophils and percentage of COX-2 positive area (r=0.47, P=0.022), as well as mean staining index (r=0.44, P=0.032) were observed. In conclusion, the immunoexpression of COX-2 was found strongest in the gilts with acute endometritis, meanwhile it was not different between those with chronic endometritis and normal endometrium. This suggested that the expression of COX-2 might be dependent not only on the infiltration of local immune cells in the endometrium, but also on the duration of exposure with inflammatory agents.
Transgenic research on cattle embryos has been developed to date using viral or plasmid DNA delivery systems. In this study, a different gene delivery system, piggybac transposition, was employed to investigate if it can be applied for producing transgenic cattle embryos. Green or red fluorescent proteins (GFP or RFP) were transfected into donor fibroblasts, and then transfected donor cells were reprogrammed in enucleated oocytes through SCNT and developed into pre-implantation stage embryos. GFP was expressed in donor cells and in cloned embryos without any mosaicism. Induction of RFP expression was regulated by doxycycline treatment in donor fibroblasts and pre-implantational stage embryos. In conclusion, this study demonstrated that piggybac transposition could be a mean to deliver genes into bovine somatic cells or embryos for transgenic research.
The purposes of this study were 1) to examine changes in the indocyanine green (ICG) clearance by feeding and 4-day fasting in dry cows, and (2) to investigate the relationship between ICG clearance and blood chemistry profile in postpartum cows. In 3 dry cows, post-feeding ICG half-life (T1/2) was shorter than the pre-feeding value, and post-fasting T1/2 was longer than pre-feeding and post-feeding values. In 16 lactating cows, T1/2 value at 2 weeks postpartum showed positive correlations with AST, T-Bil and γ-GTP. These results suggested that ICG clearance correlated with T-Bil and liver enzymes can be sensitive and accurate diagnostic marker for impaired liver function in dairy cows. In addition, ICG clearance is greatly affected by feeding and fasting.
Tests for acute oral toxicity, eye irritation, corrosion and dermal toxicity of colloidal silver nanoparticles (AgNPs) were conducted in laboratory animals following OECD guidelines. Oral administration of AgNPs at a limited dose of 5,000 mg/kg produced neither mortality nor acute toxic signs throughout the observation period. Percentage of body weight gain of the mice showed no significant difference between control and treatment groups. In the hematological analysis, there was no significant difference between mice treated with AgNPs and controls. Blood chemistry analysis also showed no differences in any of the parameter examined. There was neither any gross lesion nor histopathological change observed in various organs. The results indicated that the LD50 of colloidal AgNPs is greater than 5,000 mg/kg body weight. In acute eye irritation and corrosion study, no mortality and toxic signs were observed when various doses of colloidal AgNPs were instilled in guinea pig eyes during 72 hr observation period. However, the instillation of AgNPs at 5,000 ppm produced transient eye irritation during early 24 hr observation time. No any gross abnormality was noted in the skins of the guinea pigs exposed to various doses of colloidal AgNPs. In addition, no significant AgNPs exposure relating to dermal tissue changes was observed microscopically. In summary, these findings of all toxicity tests in this study suggest that colloidal AgNPs could be relatively safe when administered to oral, eye and skin of the animal models for short periods of time.
Surveillance of jaagsiekte sheep retrovirus (JSRV) infection was performed by polymerase chain reaction (PCR) of blood DNA samples collected from 40 sheep and goats in 10 different flocks in Hokkaido, the northern island of Japan. No exogenous (oncogenic) JSRV sequence was detected by PCR in these samples, while the ovine endogenous retrovirus sequence was successfully amplified in all samples. Our paper is the first demonstration of JSRV surveillance in Japan and shows no evidence of oncogenic JSRV infection in sheep and goats in Hokkaido.
In Japan, most pig populations are now free from pseudorabies virus (PRV) due to the recent success of an extensive eradication program. However, PRV infection persists in Japanese wild boars (Sus scrofa leucomystax), representing another potential reservoir for the virus in Japan. In this study, the seroprevalence of PRV in wild boars captured in three different prefectures was ascertained. A virus neutralization (VN) test showed that 6 of 173 serum samples (3%) were positive for VN antibody; glycoprotein E-ELISA revealed infection with the wild-type, but not the available vaccine strain, PRV. These results indicate that PRV has continued to spread among wild boars in Japan.
ERRATUM (Printer's correction)
(Vol. 73, No. 10, p. 1261, 2011)
A legend of Fig. 4 was printed incorrectly. Error: (a) P<0.05 vs. 3 M mice control; (b) P<0.05 vs. 3 M mice control (n≥4). Correction: a P<0.05 vs. 3 M mice control; b P<0.05 vs. 18 M mice control (n≥4).