We investigated the effects of lipopolysaccharide (LPS)-induced endotoxemia on the expression of aquaporin-4 (AQP4) in the rat anterior pituitary gland, using the real-time polymerase chain reaction and immunohistochemistry. After intraperitoneal injection of LPS, the level of AQP4 mRNA doubled at 2, 4 and 8 hr. Immunohistochemical analysis showed an increase with time in AQP4 immunostaining in folliculo-stellate cells following LPS injection; the intensity of immunoreactivity peaked at 8 hr. At the same time, some cyst-like structures, formed by AQP4-positive cells, were observed. These findings indicate that LPS induces the expression of AQP4 in the anterior pituitary gland. The present results should provide an important key to elucidate the pathogenesis of the anterior pituitary gland during endotoxemia.
The inactivation effect of a novel photocatalyst on polyethylene terephthalate film on goose parvovirus (GPV), avian influenza virus (AIV) and Qβ phage was evaluated. Under a light emitting diode (LED) light (range 410–750 nm), GPV was inactivated by irradiation at 1,000 lux for 6 hr, while AIV and Qβ phage were inactivated by irradiation at 150 lux for 2 hr. These data suggest that this new photocatalyst can potentially be used as one of the materials to inactivate viruses in the indoor environment and help us to prevent viral infectious diseases through indirect contact.
We investigated the contribution of quinolone resistance-determining region (QRDR) mutations to fluoroquinolone (enrofloxacin, orbifloxacin and danofloxacin) susceptibility in 58 Mycoplasma bovis isolates from dairy cattle in Japan. Fluoroquinolone non-resistant isolates (fluoroquinolone-MICs≤2 μg/ml) possessed no QRDR mutations (19 isolates) or Ser83Leu in GyrA (32 isolates). Fluoroquinolone-resistant isolates (fluoroquinolone-MICs≥4 μg/ml) possessed Ser83Leu in GyrA and Ser81Pro in ParC (3 isolates) or Ser83Phe in GyrA and Ser80Ile in ParC (4 isolates). Laboratory-derived fluoroquinolone-resistant mutants selected from 2 isolates (possessing Ser83Leu in GyrA) had an amino acid substitution in ParC at the same position (Ser80Ile or Ser81Tyr) as fluoroquinolone-resistant isolates, suggesting a concurrent amino acid substitution in ParC (Ser80 or Ser81) is important in fluoroquinolone resistance in M. bovis isolates.
A total of 159 Thai isolates of Mycoplasma hyopneumoniae isolated from pneumonic lungs of pigs during 2006–2011 were investigated for their in vitro susceptibility to 12 antimicrobial agents. Low activity of chlortetracycline was indicated by the MIC range from 3.12–100 μg/ml and MIC90 of 50 μg/ml. Seventy-six isolates showed resistance to enrofloxacin, whereas 2 isolates showed resistance to macrolides and lincomycin. In addition, a point mutation at A2058G was revealed by sequence analysis of 23S ribosomal RNA in both isolates. The present results confirmed the rapid increase of resistant M. hyopneumoniae isolates against chlortetracycline, enrofloxacin, macrolides and lincomycin in Thailand. Selection of drugs to control swine diseases in Thailand must be done more prudently in consideration of reducing the antimicrobial resistance.
We hypothesized that fattening Wagyu cattle fed conventional low-vitamin fattening diets are exposed to oxidative stress. In this experiment, we studied the plasma concentrations of 8-isoprostane and the fat depot-specific effects of the diet-induced adipogenic (C/EBPβ, C/EBPδ, C/EBPα and PPARγ2) and adipokine (VEGF, FGF-2, leptin and adiponectin) gene expressions in fattening Wagyu steers. Animals were fed a high-vitamin (α-tocopherol and β-carotene) diet (HV) or a control diet (CT) during the fattening period (from 10 to 30 months of age). The plasma concentrations of 8-isoprostane, a marker of oxidative stress, were significantly lower in the HV group than in the CT group. In mesenteric adipose tissue, the expressions of the adipogenic and adipokine genes in the HV group were significantly lower than those in the CT group. In contrast, there were no differences in the expression of the adipogenic and adipokine genes in subcutaneous adipose tissue between groups. These results suggest that higher intake of dietary α-tocopherol and β-carotene affects the expression patterns of adipogenic and adipokine genes in a fat depot-specific manner with the reduction of plasma 8-isoprostane concentrations.
Bacterial and mammalian ferritins are known to bind heme. The use of α-casein and biotinylated hemin could be applicable to detection of protein-bound heme and of proteins with heme-binding capacity, respectively. Although commercial horse spleen ferritin and purified horse spleen ferritin (L:H subunit ratio=4) bound to an α-casein-coated plate, and this binding could be inhibited by hemin, recombinant iron-binding protein (rDpr), derived from heme-deficient Streptococcus mutans and expressed in Escherichia coli, did not bind to an α-casein-coated plate. Both horse spleen ferritins bound to α-casein-immobilized beads. Commercial horse spleen ferritin and rDpr showed direct binding to hemin-agarose beads. After preincubation of commercial horse spleen ferritin or rDpr with biotinylated hemin, they showed indirect binding to avidin-immobilized beads through biotinylated hemin. These results demonstrate that α-casein is useful for detection of heme-binding ferritin and that both hemin-agarose and the combination of biotinylated hemin and avidin-beads are useful for detection of the heme-binding capacity of ferritin. In addition, this study also revealed that Dpr, a decameric iron-binding protein, from heme-deficient cells binds heme.
Urea breath test (UBT) using an infrared spectral analyzer is widely used for non-invasive and rapid detection of gastric Helicobacter spp. in human, but not veterinary medicine. The main purposes of this study were to determine the reference range of the UBT in dogs and to evaluate its clinical usefulness. To address the first aim, 6 healthy laboratory beagles were subjected to UBT and upper gastrointestinal endoscopy. Gastric endoscopic biopsy samples from the antrum, corpus and fundus were examined for Helicobacter spp. by polymerase chain reaction (PCR) testing, rapid urease test (RUT), histology and cytology. Amoxicillin, metronidazole and omeprazole were given to infected dogs for 14 days, and dogs that became Helicobacter-negative were used to determine the reference range for UBT. To address the second aim, 32 canine patients underwent UBT before upper gastrointestinal endoscopy, and the sensitivity and specificity of UBT were calculated based on our newly determined reference range using PCR as the gold standard for detection of Helicobacter spp. Initially, all 6 laboratory beagles were infected in all gastric regions and became uninfected after eradication. The mean ± 2 SD UBT value after eradication was 0.6 ± 1.8‰, and the reference range for UBT was determined to be less than 2.5‰. UBT was completed successfully in 27 patients. Using our reference range, UBT displayed 89% (16/18) sensitivity and 89% (8/9) specificity, indicating that UBT was quite useful for the detection of gastric Helicobacter spp. infection in dogs.
A four-month-old female Labrador retriever was brought to the Tokyo University of Agriculture and Technology Animal Medical Center for examination of its main symptoms of cough, tachypnea and exercise intolerance. Upon examination, the dog was found to have cyanosis and inadequate growth. Echocardiography revealed tetralogy of Fallot. Cardiac catheterization confirmed that the main pulmonary artery was completely occluded and that blood flowed from the aorta to the pulmonary artery. Accordingly, the animal was diagnosed with extreme tetralogy of Fallot.
A newly developed nested polymerase chain reaction (PCR) assay targeting the internal transcribed spacer (ITS) region of the ribosomal DNA was applied to detect and characterize Encephalitozoon cuniculi DNA from pet rabbits in Japan. The analysis was carried out using 257 urinary samples and 314 fecal samples collected from 307 pet rabbits in the age group of 1 month to 12 years from 30 different prefectures of Japan and 107 fecal samples and 3 urinary samples collected from 1-month-old rabbits from 3 breeding facilities in Japan. We detected 840-bp amplicons in 20 urinary samples (7.78%) from the pet rabbits of the 13 prefectures and in 1 urinary (33.3%) and 6 fecal (5.6%) samples from the rabbits of the 2 breeding facilities. The sequences (803 bp) of the 27 amplicons had no variations and completely coincided with the sequence of E. cuniculi genotype I. To the best of our knowledge, this is the first report on the detection and genotype characterization of E. cuniculi DNA from pet rabbits in Japan.
This report describes an atypical mammary adenoma with a rare histological feature characterized by proliferating single-layered cystic ducts composed of basaloid cells with frequent myoepithelial differentiation. A 9-year-old, intact female Miniature Pinscher dog had mammary tumors on the thorax. Histologically, one of tumors comprised the proliferation of two types of tubular structures; the single-layered cystic ducts lined by flattened cells and double-layered tubules with luminal cells and outer spindle cells. The former ducts were predominant in the tumor and contained pale basophilic mucus, which was Alcian blue (pH 2.5)-positive, but periodic acid Schiff-negative. Immunohistochemical staining indicated that the cells lining single-layered cystic ducts were negative for the luminal epithelial marker, cytokeratin (CK) CAM5.2, but were constantly positive for basal cell markers CK14 and p63 and frequently positive for SMA. Electron microscopy revealed fine, parallel myofilaments within these single-layered neoplastic cells. These histological and immunohistological examinations suggested that the origin of the tumor was bipotential mammary progenitor cells with predominant differentiation into the myoepithelial progenitor linage.
A new-born (8-day-old) male marmoset (Callithrix jacchus) was found dead in a zoo. The littermate and parents had no clinical abnormalities. By gross observations at necropsy, there were moderate to severe multiple necrotic foci in the liver and heart. Histopathological examinations also revealed mild focal necrosis with neutrophilic infiltration in the cerebral cortex. By Giemsa stained sections, intracytoplasmic bundles of large bacilli were observed in the hepatocytes, intestinal epithelial cells, cardiac myocytes and neuronal cells around the necrotic lesions. Immunohistochemically, these bacilli were intensely positive for rabbit sera against Clostridium piliforme, RT and MSK strains. Although Tyzzer’s disease has been rarely reported in primates, the central nervous system (CNS) lesions by Clostridium piliforme infections are very unusual.
Omentin is a recently identified adipocytokine, and we previously demonstrated that omentin played anti-inflammatory roles in vascular endothelial and smooth muscle cells. We also demonstrated that omentin induced vasodilation in rat isolated blood vessels. However, effects of omentin on blood pressure (BP) are not determined. Here, we examined whether intravenously injected omentin acutely alters BP of Wistar rats. Omentin (0.06–18 μg/kg) alone did not alter BP of Wistar rats. On the other hand, omentin (18 μg/kg) significantly inhibited noradrenaline (NA; 2 μg/kg)-induced increases in systolic BP and mean BP. Omentin (18 μg/kg) significantly inhibited angiotensin II (1 μg/kg)-induced increases in diastolic BP. Omentin (18 μg/kg) significantly inhibited dimorpholamine (3 mg/kg)-induced increases in diastolic BP. Omentin (18 μg/kg) failed to inhibit the NA (0.02–2 μg/kg)-induced increases of systolic BP in the nitric oxide (NO) synthase inhibitor, NG-nitro-l-arginine methyl ester (80 mg/kg, 1 day)-treated Wistar rats. In summary, we for the first time demonstrated that omentin inhibited agonists-induced increases in BP. The effect of omentin was suggested to be mediated likely via NO-dependent mechanism.
Hemorrhagic fever with renal syndrome (HFRS) is a rodent-borne zoonotic disease caused by hantavirus infection. Many HFRS cases have been reported in East Asia and North Europe, while the situation in Southeast Asia remains unclear. In this study, the prevalence of hantavirus infection in rodents and humans in Thousand Islands regency, which is close to the port of Jakarta, one of the largest historic ports in Indonesia, was investigated. A total of 170 rodents were captured in 2005, and 27 (15.9%) of the rodents were antibody-positive against Hantaan virus antigen in an immunofluorescence assay (IFA) and Western blotting. Despite the high prevalence in rodents, human sera collected from 31 patients with fever of unknown origin and 20 healthy volunteers in the islands in 2009 did not show positive reaction to the antigen in IFA. To identify the virus in rodents genetically, a total of 59 rodents were captured in 2009. Sera from the rodents were screened for antibody by ELISA, and lung tissues were subjected to RT-PCR. 20 (33.9%) of the 59 rodents were antibody-positive, and 3 of those 20 rodents were positive for S and M genome segments of hantaviruses. Genetic analysis showed that the viruses belonged to Seoul virus and formed a cluster with those in Vietnam and Singapore. These results suggest that a unique group of Seoul viruses has spread widely in Southeast Asia.
To evaluate the prevalence of extended-spectrum cephalosporin (ESC)-resistant Enterobacteriaceae in broiler chickens, 41 rectal samples taken from 4 commercial farms were examined. Desoxycholate hydrogen sulfide lactose agars, supplemented with either 4 μg/ml cefotaxime or 16 μg/ml ceftazidime, were used to screen ESC-resistant bacteria. ESC-resistant bacteria were isolated from all samples. Of the 164 ESC-resistant bacteria (included 4 isolates per a sample), 163 were Escherichia coli, while 1 isolate was identified as Enterobacter cloacae. Extended-spectrum β-lactamase (ESBL) genes and plasmid-mediated AmpC β-lactamase genes in the isolates were determined by PCR and sequencing. One AmpC β-lactamase gene, blaCMY-2 (66%), and 4 ESBL genes, blaCTX-M-1 (26%), blaCTX-M-55 (10%), blaSHV-5 (4%) and blaCTX-M-2 (3%), were detected in the E. coli isolates. The epidemiological relationship of the CMY-2 and CTX-M β-lactamase-producing isolates among the farms was analyzed by pulsed-field gel electrophoresis using the XbaI restriction enzyme. Forty-one (Y1–Y41) and 14 (X1–X14) clusters were found in the CMY-2 and CTX-M-carrying E. coli isolates, respectively. Some clusters included isolates derived from more than 1 farm, indicating some cross-contamination of clonal strains and spread of CMY-2 AmpC β-lactamase or CTX-M ESBL among the farms.
The aim of this study was to assess in vitro antimicrobial susceptibility of Brucella melitensis isolates isolated from naturally infected sheep cases in an area where human brucellosis is endemic, focusing on rifampin (RIF), streptomycin (SM), ciprofloxacin (CPFX), trimethoprim/sulfamethoxazole (TMP/SMZ), gentamicin (GM) and tetracycline (TC) and on 11 other antimicrobials. The identification and typing of Brucella isolates were carried out using standard classification tests and polymerase chain reaction (PCR) methods. Antimicrobial susceptibility testing was carried out on Mueller-Hilton agar. The resistance to SM, CPFX and GM was determined at the rate of 7.3% and to RIF at the rate of 9.7%. The highest (46.3%) resistance was determined against TMP/SMZ. All strains were found to be sensitive to TC at the rate of 100.0%. In conclusion, ovine origin B. melitensis strains evaluated in this study were resistant to at least one antimicrobial (51.2%) that is commonly used in human clinical medicine against brucellosis.
Reports of dogs with H3N2 canine influenza virus (CIV) have been documented frequently. To better understand the seroprevalence of H3N2 CIV among dogs in northeast China, here we report for the first time a relatively high seroprevalence of H3N2 CIV infection in dogs in northeast China. Forty-five of the 223 canine sera (20.2%) and 166 of the 500 canine sera (33.2%) tested feral dogs and pet dogs were seropositive by NP-ELISA, which is higher than that in southern China. The relative data provided in this report can be useful for small animal practitioners or public health policy makers to carry out relative measures for the prevention of this disease. Meanwhile, similar seroprevalence studies and prospective natural history and incidence studies should also be undertaken in other places.
In this study, eighteen monoclonal antibodies (MAbs) to recent Japanese encephalitis virus (JEV) genotype I were produced and characterized by plaque reduction neutralization test, western blot analysis, indirect immunofluorescence assay and enzyme-linked immunosorbent assay. All MAbs recognized only envelope (E) protein or conformational epitope of E and precursor membrane proteins. Two MAbs (7E5 and 3-3H8) possessed virus-neutralization activity, and their escape mutants possessed a change of glutamine to histidine at the position of 52 of E protein, suggesting that these neutralizing MAbs recognize the domain I–II hinge region of E protein. Five MAbs recognized all examined flaviviruses, and two were specific to JEV. These MAbs may be useful for differentiation and diagnosis of flaviviruses.
For a survey of Coxiella burnetii, the Q fever agent, ticks infesting companion dogs were collected in Aomori, Tochigi, Gifu and Okinawa Prefectures, Japan. A total of 261 ticks were collected, and their species were identified morphologically. Five tick species were identified: Ixodes ovatus, Haemaphysalis concinna, H. flava, H. longicornis and Rhipicephalus sanguineus. Total DNA was extracted from them individually followed by real-time PCR to detect a C. burnetii-specific gene. The results of real-time PCR were all negative, which might suggest a low risk of C. burnetii infection via these ticks and their hosts in urban residential areas in Japan.
It is suggested that topical application of opioids may provide localized analgesia without delay in corneal wound healing. This study was designed to evaluate the effect of topical application of 0.8% nalbuphine on post-operative ocular pain in dogs. Twelve eyes from 11 dogs undergoing phacoemulsification cataract surgery were divided into a nalbuphine group (n=6) and saline group (n=6). Postoperatively, the nalbuphine group received 0.1 ml of topical 0.8% alkalinized nalbuphine (pH 5.6) every 8 hr, and the saline group received 0.1 ml of topical saline (pH 5.9) as a placebo. All dogs received systemic postoperative pain managements with oral tramadol (4 mg/kg) and prednisolone (0.5 mg/kg) every 8 hr. All dogs received pre- and post-ophthalmic examinations. Pain was scored in the dogs using a pain scoring system modified from the University of Melbourne pain scale at 15, 30 and 60 min following the topical treatment on days 1 and 2 (24 and 48 hr after surgery). Eye blink frequency and corneal touch threshold (CTT) were recorded at the same time. There was no statistical difference in the pain score between groups. Significant decreases in CTT, blepharospasm and eye blink frequency were observed after the topical nalbuphine treatment. This indicated that topical application of 0.8% nalbuphine solution can produce a rapid reduction of corneal discomfort in dogs.
Morbilliviruses use signaling lymphocyte activation molecule (SLAM) as a receptor for their entry to cells. In this study, a complete gene encoding SLAM of a domestic cat was identified. The identity of feline SLAM with canine one was 73%, and feline SLAM formed the same cluster with those of carnivores. Furthermore, feline cell expressing feline SLAM supported growth of canine distemper virus (CDV) as well as that expressing canine one. These results indicated that feline SLAM can function as a receptor for morbilliviruses, and our established feline cells that express feline SLAM might be useful for analysis of morbilliviruses originated from felids.
Chronic wasting disease (CWD) is a naturally occurring prion disease in North American deer (Odocoileus species), Rocky mountain elk (Cervus elaphus nelsoni) and moose (Alces alces). The disease was first confirmed in the Republic of Korea in 2001, and subsequent cases were diagnosed in 2004, 2005 and 2010. The experimental host range of CWD includes ferrets, several species of voles, white-footed mice, deer mice and Syrian golden hamsters. In addition, CWD was transmitted to the transgenic mouse over-expressing elk or deer prion protein efficiently, but not to wild type mouse. Here, we report the experimental transmission of elk CWD to conventional VM/Dk mice reaching 100% attack rate after second passage. The CWD-prion-affected wild type mice will be a useful model for future CWD studies.
A nationwide survey of bovine leukemia virus (BLV) infection was conducted among dairy and beef breeding cattle in Japan from 2009–2011 using an enzyme-linked immunosorbent assay. Of a total of 20,835 cattle tested, 35.2% were seropositive for BLV and the animal type-level seroprevalences in dairy and beef breeding cattle were 40.9 and 28.7%, respectively. By the time animals were 1 year old, 21.0% of dairy and 13.7% of beef breeding cattle were considered infected. Our findings indicate that BLV is widespread among dairy and beef breeding cattle in Japan with the BLV seroprevalences approximately 10- and 4-fold higher, respectively, than previously reported for 1980–1982 in Japan.
We report a novel goose parvovirus (MDGPV/PT) isolated from an affected Muscovy duck in Fujian Province, China. In this study, the NS1 sequence analyses indicated a close genetic relationship between MDGPV/PT and Muscovy duck parvovirus (MDPV) strains, although MDGPV/DY, which was isolated from a Muscovy duck in 2006 in Sichuan Province, could be divided into GPV-related groups. Phylogenetic analysis showed that except for differences in the NS1 gene, MDGPV strains PT and DY are closely related to a parvovirus that infects domestic waterfowls. This is the first demonstration of recombination between goose and Muscovy duck parvoviruses in nature, and MDGPV/PT might have led to the generation of a novel waterfowl parvovirus strain circulating in Muscovy duck flocks in China.
Red-crowned (or Japanese) cranes Grus japonensis are native to eastern Hokkaido, Japan-the island population, and mainland Asia-the continental population that migrates from breeding grounds along the Amur River Basin to winter in east China and the Korean Peninsula. The island population was reduced to about 50–60 birds in early part of the 20th century. Since 1950s, the population has increased to more than 1,300 as a consequence of human-provided food in winter, resulted in change of their habitats and food resource. From the carcasses of 284 wild cranes from the island population, collected in Hokkaido since 1976 until 2010, we measured six physical parameters (body weight and lengths of body, wing, tarsus, tail and exposed culmen) and divided into groups by sex and three developmental stages (juvenile, yearling and adult). All parameters of males were larger than those of females at the same stage. Total body length of females tends to grow up earlier than males, in contrast to body weight. Obvious time trends were not observed in these all parameters during 34 years for these six categories measured, except total length of male juveniles, which showed a significant increasing trend. These results provide the first extensive data on body size and mass in the wild red-crowned cranes.
In order to gather basic reproductive information of the water deer and Reeves’ muntjac, the immunolocalization of the cytoskeleton proteins in the testes and epididymides of these two species was investigated. The distribution pattern of cytoskeletal proteins in these two species was similar. The desmin was detected in the peritubular myoid cells of the testes and the sub-epithelial cells of the epididymal ducts. Vimentin was observed in the myoid cells, Leydig cells and perinuclear region of the Sertoli cells. Intense immunoreactions for α-smooth muscle actin were restricted to the smooth vascular muscle cells and the peritubular myoid cells in the testes. From the present results, it appears that these distribution patterns of cytoskeletal proteins may be common in the cervids.