The colocalization of immunoreactivity to nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP) and tyrosine hydroxylase (TH) in the superior cervical ganglion (SCG) was investigated in the quail. In this bird, a substantial amount of NOS-immunoreactive (IR) cells were consistently found in the SCG without colchicine treatment or nerve ligation. The finding worthy of pointing out was that three-fourths of these NOS-IR cells were positive for TH. VIP-IR cells appeared with markedly low frequency than NOS-IR cells. They were generally small in size and often located in the ganglion peripheral. There were no VIP-IR cells positive for TH or negative for NOS: VIP immunoreactivity always appears in NOS-IR cells negative for TH. Thus, the results of the present study clearly showed the existence of two distinct subpopulations of postganglionic NOS-IR neurons (one is catecholaminergic and negative for VIP, and the other is non-catecholaminergic and positive for VIP). This suggests that nitric oxide (NO) and possibly VIP act as postganglionic neurotransmitters or neuromodulators in the quail SCG. The predominant appearance of the former category of NOS-IR cells must be considered in relation to some specific NO-induced controlling mechanisms of SCG neurons.
Anaplasma marginale is an etiologic agent of bovine anaplasmosis. This study aimed to molecularly detect and characterize A. marginale that is prevalent in Mongolian cattle populations. A highly specific and sensitive nested PCR (nPCR) method based on the Msp5 gene was developed to detect A. marginale (Msp5 nPCR). The method detected A. marginale from the positive DNA samples obtained from different countries, while no amplicons were observed from DNA samples of several other bovine blood pathogens tested. The detection limit of Msp5 nPCR was determined to be 2 copies/μl. The method was tested against field blood DNA samples prepared from 300 Mongolian cattle in 2010. Results indicated a prevalence rate of 8.7% (26 of 300). On the other hand, partial DNA fragments of an Anaplasma sp. closely related to A. ovis (with 95.0% identity) were detected using a different nPCR method based on groEL gene. The phylogenetic analyses based on the Msp5, groEL and 16S rRNA genes demonstrated that A. marginale isolates in Mongolia were not divergent from the isolates distributed in other countries. The present study successfully established a new nPCR assay that can detect A. marginale, and reported the first molecular detection and characterization of A. marginale and an Anaplasma sp. closely related to A. ovis in Mongolian cattle populations.
Escherichia coli strains were isolated from the feces of 130 diarrheic calves at different farms locations in Korea. The presence of the virulence genes, such as fanC, f41, f17a, eaeA, clpG, afa-8D, sta, stx1 and stx2, in each E. coli isolate was examined. Among the 314 isolates, 157 carried one or more of the virulence genes tested in this study. The most prevalent virulence gene was clpG (45.9%), although f17A (36.9%) and afa-8D (21.7%) were also frequently observed. The sta, stx1 and eaeA genes were detected in between approximately 13 and 17% of the isolates, and the fanC and fim41a genes were detected to a lesser extent. Collectively, our data indicated that diarrhea in calves in these locations can be ascribed to various virulence factors, and the pathogenesis may be more related to virulence genes such as, clpG, f17A, and afa-8D.
Canine serum amyloid A (SAA) is a useful diagnostic marker of systemic inflammation. A latex agglutination turbidimetric immunoassay (LAT) was validated for automated measurements. The aim of the study was to evaluate the clinical applicability of SAA measured by the LAT. SAA was measured in 7 groups of dogs with and without systemic inflammation (n=247). Overlap performance was investigated. Diagnostic performance was compared to body temperature and leukocyte markers. Clinical decision limits for SAA were estimated. In dogs with neurological, neoplastic or gastrointestinal disorders (n=143), it was investigated whether a higher proportion of SAA positive dogs could be detected in cases of complications with risk of systemic inflammation. Significantly higher concentrations of SAA were measured in dogs with (range [48.75; 5,032 mg/l]), compared to dogs without systemic inflammation [0; 56.4 mg/l]. SAA was a more sensitive and specific marker of systemic inflammation (area under the receiver-operating characteristic curve (AUC) 1.00), compared to body temperature (0.6) and segmented neutrophils (best performing leukocyte marker, 0.84). A clinical decision limit of 56.4 mg/l was established giving close to perfect discrimination between dogs with and without systemic inflammation. Higher proportions of SAA-positive dogs were observed in dogs with neurological, neoplastic and gastrointestinal disorders with complications known to increase risk of systemic inflammation, compared to uncomplicated cases. The automated LAT makes SAA applicable as a relevant diagnostic marker of systemic inflammation in dogs for routine random-access real-time use in a general clinical setting.
It is known that long-term post-weaning individual housing significantly reduces emissions of 22-kHz calls in male rats. In this study, we assessed post-weaning successive changes in 22-kHz calls emitted by male rats under two different types of post-weaning housing conditions (individually and socially). In addition, we evaluated the critical point at which a significant reduction in 22-kHz calls could be observed in male rats housed individually after weaning. Significantly fewer 22-kHz calls were emitted by individually housed rats compared to socially housed rats at 16 weeks of age, indicating that 13 weeks after weaning may be a critical point for the reduction of 22-kHz calls caused by post-weaning individual housing.
Five novel, canine lymphoma cell lines (Ema, CLC, CLK, Nody-1 and UL-1) were established from dogs suffering from lymphoma and characterized in vitro and in vivo. All cell lines, except CLC, were characterized with T-cell phenotypes, by flow cytometric analysis and polymerase chain reaction for antigen receptor rearrangement. Cell proliferation rates and transcriptional levels of MYC, PTEN, KIT and FLT3 varied between each cell line. Intraperitoneal xenotransplantation of Ema, CLC, Nody-1 and UL-1 lymphoma cell lines into NOD/SCID mice induced ascites, intraperitoneal tumors and severe infiltration of lymphoma cells into the pancreas and mesentery. Establishment of novel canine lymphoma cell lines with different characteristics is critical for elucidating the pathophysiology of canine lymphoma and improving current therapies.
The study aims were (1) to confirm the effects of nutritional improvement in prepartal and postpartal periods, monitored using the serum metabolic profile test (MPT) and reproductive performance, and (2) to clarify regional characteristics of the MPT results within our jurisdiction by using our MPT database. Experiment 1: Among 42 breeding cattle herds in our jurisdiction mainly fed home-pasture roughage, 3 experimental herds showing subnormal blood urea nitrogen (BUN) levels were selected and compared with 1 representative excellent herd. Dietary remedial measures were implemented from feed analysis in each herd. BUN concentration in all 3 herds increased significantly, and open days postpartum in 2 of the herds were significantly reduced, compared with values before dietary supplementation. Experiment 2: Thirty-seven herds within our jurisdiction were grouped into 3 categories (Area 1, 2 and 3) by location and soil condition of the herd pastureland. The MPT and reproductive performance in cows whose blood samples were collected at both prepartum (60–20 days before calving) and postpartum (30–90 days after calving) were compared among the 3 areas. Significant regional differences were found in prepartal albumin, total cholesterol, BUN, and glucose and postpartal BUN, glucose and open days (P<0.05). Overall, the MPT (especially BUN) might be useful for determining the metabolic nutritional status of breeding cattle herds, particularly those fed home-pasture roughage. Additionally, poor/unsatisfactory reproductive performance of beef breeding cattle herds probably reflects inadequate nutritional content of the diet, possibly arising from regional pastureland differences.
We established a homologous sandwich enzyme-linked immunosorbent assay (ELISA) to measure serum levels of canine ferritin. Our assay uses a rabbit anti-canine heart ferritin polyclonal antibody, and canine heart ferritin as a standard. Serum ferritin concentration in healthy dogs (n=163) was 789 ± 284 ng/ml (mean ± standard deviation), a value higher than reported previously. Confidence levels relating to repeatability, dilution and recovery for this method were high. Therefore, we believe our developed sandwich ELISA will be effective in evaluating serum levels of canine ferritin.
Two miniature dachshunds, a 7-year-old neutered male and an 8-year-old male, presented with chronic hematochezia and tenesmus. A solitary pedunculated or multiple diffuse colorectal polyps were identified by colonoscopy and resected by polypectomy. Inflammatory colorectal polyps (ICRPs) were diagnosed according to histopathological findings. Both cases were treated with immunosuppressive therapy, and the clinical signs were resolved, although the colorectal polyps remained to some extent. Several months after the initial diagnosis, both cases presented with recurrence of hematochezia and enlargement of the polyps. A second colonoscopic polypectomy was performed, and adenoma was diagnosed histopathologically in both cases. ICRPs are a nonneoplastic disease, but their long-term prognosis is unknown. Careful follow-up seems to be important, and repetitive biopsy is recommended when growth of polyps is identified in miniature dachshunds with ICRPs.
Puberty onset in mammals is tightly coupled to the animal’s nutritional and metabolic state. In the present study, we evaluated the effects of a high-fat diet on leptin and adiponectin levels, leptin mRNA expression and puberty onset in female rats. On day 21, female rats were divided into 2 groups, normal food (NF) and high-fat food (HF). The HF group showed a significantly earlier (P<0.001) date of vaginal opening and lower body weight (P<0.001) than the NF group. The rats fed the HF food had a significantly heavier uterus (P<0.05) than those fed the NF food, whereas the serum leptin and adiponectin levels and leptin mRNA expression were not significantly different between the NF and HF groups. We speculate that the fat-induced nutritional imbalance in young females may lead to neuroendocrine dysfunction during adolescence.
Veterinary x-ray photography and examinations of synovial fluid, blood and urine were conducted on a Cynomolgus Monkey from China (5 years old) which exhibited macroscopically visible systemic joint swelling after the quarantine period. The presence of inflammatory cells in the synovial fluid obtained by arthrocentesis, high counts of neutrophils, monocytes and large unstained cells and the elevated serum CRP level suggested that the lesions in this animal were due to polyarthritis.
Tritrichomonas suis (=T. foetus) is a protozoan parasite of pigs, cattle and cats. Based on host range and genetic differences, T. suis has been divided into a ‘cat genotype’ and a ‘cattle genotype’, with the latter genotype capable of infecting both cattle and pigs. Since no information is currently available on the genetic characteristics of T. suis from pigs in Japan, we conducted a molecular survey of T. suis using fecal DNA from pigs in Japan. Of the 64 pigs examined, nested PCR revealed that 36 (56.3%) were positive for T. suis. Sequence analysis of 8 positive samples showed that 7 of the pig isolates belonged to the ‘cattle genotype’ and the remaining isolate belonged to the ‘cat genotype’. The findings revealed that T. suis infection is common in pigs in Japan and that pigs can be infected by both genotypes.
We previously investigated the hereditary cerebellar cortical abiotrophy in littermates at postnatal day (PD) 25–31 delivered from a pair of rabbits. To estimate the onset time and incipient lesions associated with the cerebellar cortical abiotrophy of the cases, we mated the same pair again and examined early stages of the disease in F1 rabbit showing ataxia (PD 15), finding evidence that the ataxia is passed to subsequent generations via autosomal recessive inheritance. Clinical signs of the affected rabbit showed early-onset dysstasia and ataxia. The affected rabbit showed apoptotic granular cells before and after migration completion, degeneration (swelling) of parallel fiber terminals, abnormal junction (invaginated junction) of the parallel fiber-Purkinje cell synapses and irregular orientation of the Purkinje dendritic arbor in the molecular layer. Additionally, a reduced number of synaptic junctions between parallel fibers and Purkinje cells were detected, as well as at PD 25–31. Secondary changes, such as reduction or degeneration of Purkinje cells and granular cells were not yet observed at early stages. As synapse abnormality preceded the degeneration or reduction of Purkinje and granular cells at early stages, we concluded that the pathogenesis of the present cerebellar lesion was caused by failed synaptogenesis during postnatal cerebellar development.
The DNA repair protein Ku70 is a key player in chemoresistance to anticancer agents (e.g., etoposide) or radioresistance. The responses of different organs to radiation vary widely and likely depend on the cell population in the organs. Previously, we established and characterized Ku70-deficient murine lung epithelial (Ku70 -/- MLE) cells and found that these cells are more sensitive than Ku70 +/- MLE cells (control cells) to X-irradiation, as determined by clonogenic survival assay; however, the mechanism underlying this sensitivity remains unclear. In this study, we examined the mechanism by which X-irradiation triggers the death of Ku70 -/- MLE cells. Our results showed that Ku70 -/- MLE cells were more sensitive to radiation-induced apoptosis than control cells, although X-irradiation activated caspase-3 and caspase-7, and cleaved PARP in both cell lines. We also examined the expression level of phosphorylated H2AX (γH2AX), which is a marker of DSB, and observed the phosphorylation of H2AX and the elimination of γH2AX in both cell lines after X-irradiation. The elimination in Ku70 -/- MLE cells was slower than that in control cells, suggesting that DSB repair activity in the Ku70 -/- MLE cells is lower than that in control cells. These findings suggest that Ku70 might play a key role in the inhibition of apoptosis through the DSB repair pathway in lung epithelial cells. Our findings also suggest that these cell lines might be useful for the study of Ku70 functions and the Ku70-dependent DSB repair pathway in lung epithelial cells.
A quantitative assay method using LC/ESI-MS/MS for simultaneous determination of MCP in cattle plasma was developed and validated. Chromatographic separation was carried out using a multimode column (2 × 150 mm, 3 μm) with gradient elution (0.05% formic acid/methanol with 0.05% formic acid). MCP and levosulpiride (internal standard) were analyzed in the precursor/product ion pair of m/z 300.1/226.9 and 342.0/112.0, respectively. Linear calibration curves were obtained in the range of 2.5–500 ng/ml (R2>0.999) with a lower limit of quantification of 0.05 ng/ml. Mean recoveries were 96–103%, and the coefficient of variation was less than 6.5%. Plasma MCP concentrations after intravenous administration at 0.4 mg/kg to 12 cattle were determined by the LC-MS/MS method.
Fluoroquinolone resistance is mainly caused by mutations in quinolone resistance-determining regions of DNA gyrase and topoisomerase IV in Escherichia coli. The AcrAB-TolC efflux pump contributes to resistance against fluoroquinolone and other antimicrobials. In this study, we investigated a high-level mechanism of fluoroquinolone resistance in E. coli that was isolated from human clinical samples and canine fecal samples. E. coli strains with high levels of fluoroquinolone resistance have been found to be frequently resistant to cephalosporins. Strains with high-level fluoroquinolone resistance exhibited lower intracellular enrofloxacin (ENR) concentrations, higher expression of AcrA, and a greater reduction in the fluoroquinolone minimum inhibitory concentration for treatment with an efflux pump inhibitor. The frequency of strains with enhanced ENR resistance selection and the survival rate of E. coli in the presence of ENR in vitro were correlated well with AcrA protein expression levels in the parental strains. These results suggest that AcrAB-TolC efflux pump over-expression is related to high-level fluoroquinolone resistance and the selection of strains with enhanced fluoroquinolone resistance.
Clostridial diseases are zoonoses and are classified as soil-borne diseases. Clostridium chauvoei and Clostridium tetani cause blackleg disease and tetanus, respectively. Since bacteria and spores are re-distributed by floods and then, subsequently, contaminate soils, pastures and water; the case numbers associated with clostridial diseases usually increase after floods. Because Taiwan is often affected by flood damage during the typhoon season, possible threats from these diseases are present. Thus, this study’s aim is to apply a combination of a commercial nucleic acid extraction kit and PCR to assess the prevalence of Clostridia spp. in soil and to compare the positivity rates for farms before and after floods. The minimum amounts of Clostridium tetanus and Clostridium chauvoei that could be extracted from soils and detected by PCR were 10 and 50 colony forming units (cfu), respectively. In total, 76 samples were collected from the central and southern regions of Taiwan, which are the areas that are most frequently damaged by typhoons. Noteworthy, the positive rates for Clostridium tetanus and Clostridium chauvoei in Pingtung county after the severe floods caused by a typhoon increased significantly from 13.73 and 7.84% to 53.85 and 50.00%, respectively. This study for the first time provides the evidence from surveillance data that there are changes in the environmental distribution of Clostridium spp. after floods. This study indicates that screening for soil-related zoonotic pathogens is a potential strategy that may help to control these diseases.
A total of 250 fecal content samples were collected from 25 farrow-to-finish pig farms and examined for the prevalence of Clostridium difficile by using ethanol treatment followed by plating onto selective media-cycloserine-cefoxitin-mannitol agar-for the isolation of Clostridium difficile. Two specimens (0.8%, 95% confidential interval: 0–2.9%) were positive for C. difficile. One isolate was only positive for toxin B, and the other isolate was negative for both toxins A and B. Thus, prevalence of Clostridium difficile was found to be low among finishing pigs in Japan.
To investigate the prevalence and characterization of foodborne pathogens [Campylobacter spp., Shiga toxin-producing Escherichia coli (STEC), Listeria monocytogenes and Salmonella spp.] in dairy cows, rectal content grab samples were collected from 250 dairy cows reared on 25 dairy farms in eastern Japan from December 2010 through February 2011. Campylobacter jejuni was isolated from 106 (42%) cows on 23 (92%) farms, STEC O157 from three cows on one farm, L. monocytogenes from three cows on another three farms and Salmonella enterica subsp. enterica serovar Typhimurium from eight cows on another farm. STEC O26 was not isolated from any of the dairy farms investigated. The results suggest that C. jejuni is widespread in dairy farms in eastern Japan.
The aim of this study was to investigate the correlations of severity of osteoarthritis (OA) and serum biomarkers including keratan sulfate (KS), hyaluronic acid (HA) and chondroitin sulfate (CS) 846 epitope. We also investigated the effect of glucosamine and fish collagen peptide (FCP) on OA. OA was induced in 12 rabbits (12 weeks of age) by anterior cruciate ligament transection (ACLT). After the surgery, the rabbits were orally administered FCP (F group), glucosamine (G group) or FCP and glucosamine (FG group) for 4 weeks. The control group was provided water ad libitum (C group). Blood was collected before surgery (pre-ACLT) and before euthanasia (post-ACLT) for serum marker measurement. Biomarker levels were measured by using commercial kits. We evaluated OA severity both macroscopically and histologically. Macroscopic evaluation showed mildly eroded condylar surfaces in the C group. Histological findings were significantly different from the FG and other groups. There were no significant differences between each group at post-ACLT in terms of serum KS, HA and CS 846. Histological assessment and serum biomarker measurements performed at post-ACLT showed a significant correlation between HA concentration and OA severity. Variations in the CS 846 concentration at pre-ACLT and post-ACLT were significantly correlated with OA severity. Administration of glucosamine and FCP had chondroprotective effects in the ACLT model. Serum biomarker concentrations were significantly correlated with cartilage injury. Serum biomarker measurement would be useful for monitoring articular cartilage damage in the clinical setting.
We investigated the correlation of plasma amino acid concentrations and the effects of glucosamine and fish collagen peptide (FCP) on osteoarthritis (OA). OA was induced according to the rabbit anterior cruciate ligament transection (ACLT) model. After surgery, the rabbits were orally administered FCP (F group), glucosamine (G group) or both (FG group) for 4 weeks. The control group (C group) was not administered anything. Blood was collected before surgery (pre-ACLT) and before euthanasia (post-ACLT). Changes in the alanine, threonine and methionine concentrations were significant between pre- and post-ACLT. The correlation between the histological assessment and arginine concentration post-ACLT was significant. These findings indicate that measurement of plasma amino acids is useful for evaluation of the efficacy of intervention for OA.
L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities such as cellular growth, proliferation and maintenance. This amino acid transporter recently has received attention because of its preferential and up-regulated expression in a variety of human tumors, in contrast to its limited distribution and low-level expression in normal tissues. In this study, to explore the feasibility of using LAT1 expression as a molecular marker in mammary gland tumors (MGT), we performed a comparative study of LAT1 expression at the mRNA and protein levels in normal mammary gland cells and tumor cells. Conventional RT-PCR and Western blotting were performed on MGT tissues from 16 dogs and normal organs from nine healthy dogs. LAT1 expression was detected in ten of the 16 MGT patients. As is the case in human tissues, LAT1 showed limited expressional distribution in normal canine organs. For quantitative expressional comparison, extensive real-time RT-PCR was performed on mRNA samples from 53 MGT patients. The comparison demonstrated that LAT1 mRNA levels from MGT tissues were 20 times higher than those in normal mammary gland tissues. Additionally, histologically invasive MGT showed a higher expression of LAT1 than non-invasive tumors. These findings suggest that LAT1 could be a clinical marker and therapeutic target for invasive malignant MGT.
Quail, like chickens, are susceptible to H5N1 subtype highly pathogenic avian influenza virus (HPAIV). Both birds experience high mortality, but quail usually survive a few more days than chicken. To understand why, we monitored quail and chickens after inoculation with 106 fifty-percent egg infectious doses of HPAIV A/whooper swan/Aomori/1/2008 (H5N1). The clinical course initiated as depression at 48 hr post inoculation (h.p.i.) in quail and at 36 h.p.i. in chicken, and all infected birds died. Mean death time of quail (91 hr) was significantly longer than that of chicken (66 hr). The virus titers of most tissue samples collected before death were not significantly different. At 24 h.p.i., interferon gamma (IFN-γ) mRNA expression in peripheral blood mononuclear cells (PBMC) was up-regulated in the quail but down-regulated in the chicken, although TLR-7 and seven other cytokines showed no significant differences between quail and chicken. The viral load in quail PBMC was significantly lower than that in chickens. These results suggest that the induction of IFN-γ after HPAIV infection in quail is related to lower titer of HPAIV. In conclusion, the different clinical courses observed between quail and chicken infected with H5N1 HPAIV might be caused by different IFN-γ responses against the HPAIV infection.
Beak and feather disease virus (BFDV) is a causative agent of psittacine beak and feather disease (PBFD), which shows a characteristic feather disorder in psittacine birds. In the present study, the subclinical infection rate of PBFD in imported and domestically bred psittacine birds was investigated by polymerase chain reaction. As a result, 126 of 402 birds (31.3%) were found to be BFDV positive. The DNA sequences of the part of open reading frame (ORF) C1 were determined for 16 BFDV-positive randomly selected samples. One of 16 samples was found to have a mixed infection, and 5 different BFDV sequences were obtained from a single African grey parrot. In phylogenic analysis, almost BFDV sequences included in each genetic cluster of phylogenic tree belonged to the same psittacine subfamily. BFDV derived from African grey parrot was closely related to the BFDV derived from cockatoos by way of exception. The natural habitat of the African grey parrot and cockatoos is different, and therefore, the possibility of interspecies cross infection through the bird trade is suggested from the exceptional BFDV sequences.