Poultry red mite (PRM, Dermanyssus gallinae) is a blood-sucking ectoparasite as well as a possible vector of several avian pathogens. In this study, to define the role of PRM in the prevalence of avian infectious agents, we used polymerase chain reaction (PCR) to check for the presence of seven pathogens: Avipox virus (APV), Fowl Adenovirus (FAdV), Marek’s disease virus (MDV), Erysipelothrix rhusiopathiae (ER), Salmonella enterica (SE), Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG). A total of 159 PRM samples collected between 2004 and 2012 from 142 chicken farms in 38 prefectures in Japan were examined. APV DNA was detected in 22 samples (13.8%), 19 of which were wild-type APV. 16S ribosomal RNA (16S rRNA) of MS was detected in 15 samples (9.4%), and the mgc2 gene of MG was detected in 2 samples (1.3%). Eight of 15 MS 16S rRNA sequences differed from the vaccine sequence, indicating they were wild-type strains, while both of the MG mgc2 gene sequences detected were identical to the vaccine sequences. Of these avian pathogen-positive mite samples, three were positive for both wild-types of APV and MS. On the other hand, the DNAs of ER, SE, FAdV and MDV were not detected in any samples. These findings indicated that PRM can harbor the wild-type pathogens and might play a role as a vector in spreading these diseases in farms.
The prevalence of resistant genes against β-lactams in 119 Aeromonas strains was determined. A large number (99.2%) of the present fish strains were resistant to one or more β- lactams including ceftiofur, amoxicillin-clavulanic acid, ampicillin, piperacillin and cefpodoxime. Among antibiotic resistance phenotypes, the simultaneous resistance to all β-lactams occurred in 25.2% (n=30) of all strains, which consisted of 18 strains of A. dhakensis, 8 strains of A. caviae, 2 strains of A. hydrophila and only one strain of A. veronii. For exploring genetic background of the antibiotic resistances, multiple PCR assays were subjected to detect β-lactamase-encoding genes, blaTEM, blaOXA-B and blaCTX-M. In the results, the blaTEM-1 gene was harbored in all strains, whereas only 3 strains harbored blaOXA gene. In the case of blaCTX-M gene, the gene was detected in 21.0% (25 out of 119) of all strains, which countered with 80% (20 out of 25) of A. dhakensis, 8% (2 out of 25) of A. caviae and 12% (3 out of 25) of A. hydrophila. In addition, most of the blaCTX-M positive strains showed simultaneous resistance to all β-lactams (18 out of 30 strains). In sequence analysis for blaCTX-M genes detected, they were CTX-M group 1-encoding genes including blaCTX-M-33 from 3 eel strains of A. dhakensis. Therefore, A. dhakensis obtained from cultured fish could represent a reservoir for spreading genes encoding CTX-M group 1 enzymes and hence should be carefully monitored, especially for its potential risk to public health.
A real-time PCR for detection and quantification of M. ovipneumoniae was developed using 9 recently sequenced M. ovipneumoniae genomes and primers targeting a putative adhesin gene p113. The assay proved to be specific and sensitive (with a detection limit of 22 genomic DNA) and could quantify M. ovipneumoniae DNA over a wide linear range, from 2.2 × 102 to 2.2 × 107 genomes.
The minimum inhibitory concentrations (MICs), mutation prevention concentrations (MPCs) and contribution of quinolone resistance-determining region (QRDR) mutations to fluoroquinolone (ciprofloxacin, enrofloxacin and orbifloxacin) susceptibility in 23 Pasteurella multocida (Pm) isolates were investigated. Fluoroquinolone-susceptible isolates (MICs ≤0.25 µg/ml, 9 isolates) had no QRDR mutations, and their respective MPCs were low. Fluoroquinolone-intermediate isolates (MICs=0.5 µg/ml, 14 isolates) had QRDR mutations (Asp87 to Asn or Ala84 to Pro in gyrA), and their respective MPCs were high (4–32 µg/ml). First-step mutants (n=5) and laboratory-derived highly resistant fluoroquinolone mutants (n=5) also had QRDR mutations. The MICs of fluoroquinolones for mutant-derived strains were decreased in the presence of efflux inhibitors. The results indicated that the fluoroquinolone resistance of Pm is mainly due to multiple target gene mutations in gyrA and parC and the overexpression of efflux pump genes.
Expression of brain-specific phenotypes increased in all trans retinoic acid (ATRA)-induced neural differentiation of mouse P19 embryonal carcinoma cells. Among these phenotypes, expression of class IVa β-tubulin isotype (TUBB4a) was particularly enhanced in neural differentiation. Transient transfection assays employing a reporter construct found that ATRA-mediated regulatory region of the TUBB4a gene lay in the region from −83 nt to +137 nt relative to the +1 transcription start site. Site-directed mutagenesis in the AP-1 binding site at −29/−17 suggested that the AP-1 binding site was a critical region for ATRA-mediated TUBB4a expression. Chromatin immunoprecipitation experiments suggested participation of JunD and activating transcription factor-2 (ATF2) in TUBB4a expression. Additionally, exogenous induction of the dominant-negative (dn) type of JunD canceled ATRA-induced upregulation of TUBB4a, and the dn type of ATF2 suppressed even the basal activity. Further immunoblot study revealed an ATRA-mediated increase in JunD protein, while a significant amount of ATF2 protein was constantly produced. These results suggest that differentiation-mediated activation of JunD results in enhanced TUBB4a expression.
Loperamide is a peripheral opiate agonist that can cause apoptosis and G2/M arrest in human cancer cell lines and may sensitize cells to chemotherapy. The objectives of this study were to investigate the effects of loperamide on viability, apoptosis and cell cycle kinetics in canine cancer cells and to establish whether the drug sensitizes cells to doxorubicin. Cell viability was assessed using Alamar Blue. Cell death and cell cycle were studied using flow cytometry with 7-Aminoactinomycin-D (7-AAD) and propidium iodide (PI), respectively. Loperamide decreased cell viability in a dose-dependent fashion and was most effective against canine osteosarcoma cells. In all cell lines, it induced a dose and time dependent apoptosis and resulted in accumulation in G0/G1. When co-incubated with doxorubicin, loperamide induced a synergistic cell kill in canine carcinoma cells. Investigation is warranted into the role of loperamide in the treatment of canine cancer.
We designed a new method of measuring the length of the ulnar nerve and determining standard values for F-wave parameters of the ulnar nerve in clinically normal beagles. Nerve length must be precisely measured to determine F-wave latency and conduction velocity. The length of the forelimb has served as the length of the ulnar nerve for F-wave assessments, but report indicates that F-wave latency is proportional to the length of the pathway traveled by nerve impulses. Therefore, we measured the surface distance from a stimulus point to the spinous process of the first thoracic vertebra (nerve length 1) and the anterior horn of the scapula (nerve length 2) as landmarks through the olecranon and the shoulder blade acromion. The correlation coefficients between the shortest F-wave latency and the length of nerves 1, 2 or the forelimb were 0.61, 0.7 and 0.58. Nerve length 2 generated the highest value. Furthermore, the anterior horn of the scapula was easily palpated in any dog regardless of well-fed body. We concluded that nerve length 2 was optimal for measuring the length of the ulnar nerve.
Apoptosis inhibitor of macrophage (AIM) plays roles in survival of macrophages. In this study, we cloned canine AIM cDNA and observed its transcriptional expression levels in various tissues. The coding sequence of canine AIM was 1,023 bp encoding 340 amino acid residues, which had around 65% homology with those of the human, mouse and rat. Transcriptional expression of AIM was observed in the spleen, lung, liver and lymph node, which confirmed the expression of canine AIM in tissue macrophages. Moreover, AIM was highly expressed in one of the canine histiocytic sarcoma cell lines. CD36, the receptor of AIM, was also expressed in various tissues and these cell lines. These findings are useful to reveal the actual functions of canine AIM.
An 8-year-old spayed female Netherland Dwarf rabbit presented with a two-month history of dyspnea and snoring. A computed tomography (CT) scan of the head revealed mass lesions in the right nasal cavity. Surgical exenteration of the lesions was performed, and the histopathological diagnosis was an intranasal adenocarcinoma. On the basis of this diagnosis, radiotherapy was planned and consisted of eight fractions of 6 Gy administered once a week. After the completion of radiation therapy, the soft tissue density in the right nasal cavity, as detected by CT, significantly decreased. The prognosis has remained good for over 3 years after treatment. This paper is the first to describe the clinical and pathological features of an intranasal tumor in a rabbit.
Rodent models of focal cerebral ischemia that do not require craniotomy have been developed by intraluminal suture middle cerebral artery occlusion (MCAo). Mouse MCAo models have been widely used and extended to genetic studies of cell death or recovery mechanisms. Therefore, we compared surgery-related parameters and techniques between such rats and mice. In rodent MCAo models, has to be considered body temperature during the operative period, as well as the need for the use of a standardized tip in terms of the outer diameter of probes. Induction of focal cerebral ischemia was measured by neurological dysfunction parameters. Our methods could induce stable moderate-severity ischemic brain injury models and histological alteration at 24 hr after MCAo surgery. Moreover approximately 80% (rats) and 85% (mice) survival ratios were shown indicating with model engineering success. Finally, we described and compared major parameters between rats and mice, including probe size, thread insert length, operation and occlusion periods, and differences in the procedures.
In this study, we examined the prevalence and molecular characteristics of Cryptosporidium in buffalo, dairy cattle and sheep in different farms at Kafr El Sheikh Province, Egypt. Rectal fecal samples, including 466 samples from buffalo, 1697 from cattle and 120 from sheep, were collected from different ages and screened by modified Ziehl-Neelsen acid-fast microscopy for detection of Cryptosporidium oocysts. All studied farms were positives with an overall prevalence of 1.29% in buffalo (4.17% in claves versus 0.48% in adults), 7.07% in cattle (6.90% in calves versus 10.20% and 6.10% in heifers and adults, respectively) and 2.50% in sheep (4.40% in lambs versus 1.30% in adults). PCR-RFLP analyses of small-subunit rRNA genes from positive specimens revealed the occurrence of C. parvum and C. ryanae in buffalo; C. parvum, C. ryanae, C. bovis and C. andersoni in cattle and only C. xiaoi in sheep. Genotypes distribution showed that C. ryanae was the dominant species (60.0%) followed by C. parvum (40.0%) in buffalo calves. Meanwhile, in cattle calves, C. parvum was the commonest species (74.23%) followed by C. ryanae (16.10%) and C. bovis (9.70%). Subtyping of C. parvum based on sequence analysis of the polymorphic 60 kDa glycoprotein gene locus showed the presence of subtypes IIdA20G1 and IIaA15G1R1 in both buffalo and cattle calves, addressing the potential role of calves in zoonotic cryptosporidiosis in Egypt.
Seven laboratory mammal and bird species were orally inoculated with 200–1,000 encysted Metagonimus hakubaensis metacercariae that had been isolated from naturally infected lampreys (Lethenteron reissneri) captured in Aomori Prefecture. At 8 and 15 days post-infection, adult flukes were recovered from all of the laboratory animals tested, and therefore, hamster, rat, mouse, dog, cat, chicken and quail were considered as final hosts of M. hakubaensis. Recovery rates of the fluke were higher in dogs and hamsters than in cats, rats, mice, chickens and quails. The flukes recovered from dogs and hamsters showed increased body length and higher fecundity than those recovered from the other hosts. These results indicate that the suitability of dogs and hamsters for M. hakubaensis infection is higher than that of the other laboratory animals.
The present study investigated the seroprevalence of Toxoplasma gondii (T. gondii) antibodies by ELISA in horses reared in Korea. Serum samples were collected from 2009 through 2013 from 816 horses reared in Korea. Analysis was performed using a commercial toxoplasmosis ELISA kit to detect anti-T. gondii antibodies. Overall, 24 out of 816 horses (2.9%) were seropositive for T. gondii. The result was analyzed by age, gender, breed and region. Significant differences were observed according to breed and region (P<0.05). This is the first nationwide serological investigation of T. gondii in horses reared in Korea. The study results reveal that T. gondii occurs nationwide in Korean horses.
A male Thoroughbred fetus was aborted on day 251 of pregnancy. Gross and histological examinations detected systemic granulomatous lesions in many superficial and visceral lymph nodes and organs including the liver, tonsils, lungs, thymus, spleen, right thyroid gland and gastrointestinal tract, and suppurative placentitis, pyogranulomatous amnionitis and intralesional acid-fast bacilli were also detected. An examination of the DNA base sequence of the β subunit of RNA polymerase demonstrated that Mycobacterium avium strain 104 had infected several organs. To the best of our knowledge, this is the first report of equine fetal mycobacterial infection in Japan.
An 8-month-old common marmoset (Callithrix jacchus) was presented with tic-like symptoms, and a 2-year-old pigmy marmoset (Callithrix pygmaea) was presented with dyspnea and hypersalivation. Both monkeys died within a few days, and necropsies were performed. Histopathological examinations revealed ulcerative stomatitis with epithelial cell swelling and eosinophilic intranuclear inclusion bodies in the oral epithelium of both cases. In the central and peripheral nervous systems, neuronal cell degeneration with intranuclear inclusion bodies was observed. Immunohistochemical examination using anti-herpes simplex virus type 1 antibody revealed virus antigens in both cases. Both animals had been kept as pets with limited exposure to the ambient environment except via their owners. Therefore, herpes simplex virus type-1 was probably acquired from close contact with their owners.
This study was conducted to evaluate the pharmacokinetic characteristics of vincristine and their correlation with its clinical effects in dogs with transmissible venereal tumor (TVT). Dogs with TVT were intravenously administered vincristine sulfate at a dose of 0.7 mg/m2 of body surface area. Blood samples were collected starting from 5 min to 48 hr after drug administration. The plasma concentration of vincristine was determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The pharmacokinetic parameters of vincristine were characterized using a two-compartmental pharmacokinetic model. The volume of distribution, distribution half-life, elimination half-life and plasma clearance were 0.660 ± 0.210 l/kg, 21.5 ± 6.90 min, 47.6 ± 14.2 min and 0.010 ± 0.001 l/min/kg, respectively. Tumor regression was determined at weekly interval by a physical examination and histopathological analysis. In our study, three to eight administrations of vincristine at a dose of 0.7 mg/m2 were able to induce a complete tumor regression without any evidence of gross lesion of disease. Therefore, this investigation provides the pharmacokinetic characteristics of vincristine in dogs with TVT, which may be used as an integration tool to gain a better understanding of the disposition properties of the drug and the correlation of these properties with the drug’s clinical effects. In addition, we validated the LC-MS/MS method and found that it is suitable for the pharmacokinetic study of vincristine in dog plasma.
Cytochromes P450 (P450) are important for not only drug metabolism and toxicity, but also biosynthesis and metabolism of cholesterol and bile acids, and steroid synthesis. In cynomolgus macaques, widely used in biomedical research, we have characterized P450 cDNAs, which were isolated as expressed sequence tags of cynomolgus macaque liver. In this study, cynomolgus CYP7A1, CYP17A1, CYP20A1, CYP27A1 and CYP51A1 cDNAs were characterized by sequence analysis, phylogenetic analysis and tissue expression pattern. By sequence analysis, these five cynomolgus P450s had high sequence identities (94–99%) to the human orthologs in amino acids. By phylogenetic analysis, each cynomolgus P450 was more closely related to the human ortholog as compared with the dog or rat ortholog. By quantitative polymerase chain reaction, among the 10 tissue types, CYP7A1 and CYP17A1 mRNAs were preferentially expressed in liver and adrenal gland, respectively. Cynomolgus CYP27A1 and CYP51A1 mRNAs were most abundantly expressed in liver and testis, respectively. Cynomolgus CYP20A1 mRNA was expressed in all the tissues, including brain and liver. Tissue expression patterns of each cynomolgus P450 were generally similar to that of the human ortholog. These results suggest the molecular similarities of CYP7A1, CYP17A1, CYP20A1, CYP27A1 and CYP51A1 between cynomolgus macaques and humans.
Veterinary staff carrying methicillin-resistant Staphylococcus aureus(MRSA) can be a source of MRSA infection in animals. To identify risk factors of MRSA carriage among veterinary staff, MRSA carriage and epidemiological information (sex, career, contact with MRSA-identified animal patients and others) were analyzed from 96 veterinarians and 70 veterinary technicians working at 71 private veterinary clinics in Japan. Univariate analysis determined sex (percentage of MRSA carriage, male (29.2%) vs. female (10%); P=0.002) and career (veterinarians (22.9%) vs. veterinary technicians (10%); P=0.030) as risk factors. Multivariable analysis revealed that sex was independently associated with MRSA carriage (adjusted odds ratio, 3.717; 95% confidence interval, 1.555–8.889; P=0.003). Therefore, male veterinary staff had a higher risk of MRSA carriage than female staff.
Effects of intermittent positive pressure ventilation (IPPV) on cardiopulmonary function were evaluated in horses anesthetized with total intravenous anesthesia using constant rate infusions of medetomidine (3.5 µg/kg/hr), lidocaine (3 mg/kg/hr), butorphanol (24 µg/kg/hr) and propofol (0.1 mg/kg/min) (MLBP-TIVA). Five horses were anesthetized twice using MLBP-TIVA with or without IPPV at 4-week interval (crossover study). In each occasion, the horses breathed 100% oxygen with spontaneous ventilation (SB-group, n=5) or with IPPV (CV-group, n=5), and changes in cardiopulmonary parameters were observed for 120 min. In the SB-group, cardiovascular parameters were maintained within acceptable ranges (heart rate: 33–35 beats/min, cardiac output: 27–30 l/min, mean arterial blood pressure [MABP]: 114–123 mmHg, mean pulmonary arterial pressure [MPAP]: 28–29 mmHg and mean right atrial pressure [MRAP]: 19–21 mmHg), but severe hypercapnea and insufficient oxygenation were observed (arterial CO2 pressure [PaCO2]: 84–103 mmHg and arterial O2 pressure [PaO2]: 155–172 mmHg). In the CV-group, normocapnea (PaCO2: 42–50 mmHg) and good oxygenation (PaO2: 395–419 mmHg) were achieved by the IPPV without apparent cardiovascular depression (heart rate: 29–31 beats/min, cardiac output: 17–21 l /min, MABP: 111–123 mmHg, MPAP: 27–30 mmHg and MRAP: 15–16 mmHg). MLBP-TIVA preserved cardiovascular function even in horses artificially ventilated.
The analgesic effect of wound infiltration with bupivacaine was evaluated in cats undergoing bilateral mastectomy. Twenty-one female cats with mammary gland tumors were anesthetized with propofol and oxygen-isoflurane anesthesia following premedication with atropine. In the trial group (Group I; n=11), 30 ml of saline containing 2 mg/kg of bupivacaine was infiltrated topically into the surgical wound right after removal of the mammary glands, whereas only saline solution was infiltrated in the control group (Group II; n=10). At the same time, carprofen (4 mg/kg) was also administered subcutaneously in both groups. Behavioral signs of pain were monitored during the recovery period after general anesthesia. In order to examine the behavioral changes associated with acute pain, a questionnaire was prepared and given to the owners to be completed 4 hr and then 10 hr after the operation. According to the owners’ anwers to the questionnaire, a pain score was specified using a “numerical rating scale” for each cat. Although some cats showed mild to moderate pain, the pain score recorded at 4 hr after the operation was significantly lower in Group I (P<0.001). No significant difference was found at 10 hr after the operation between the groups. The incidence of vocalization, aggression and convulsion within 2 hr after the operation was also lower in Group I. In conclusion, wound infiltration with bupivacaine before incisional closure provided reliable analgesia at least 4 hr after bilateral radical mastectomy in cats.
STX is an agonist for a recently characterized membrane estrogen receptor whose structure has not been identified. We evaluated whether STX suppresses gonadotropin-releasing hormone (GnRH)–induced luteinizing hormone (LH) release from bovine anterior pituitary (AP) cells. We cultured AP cells (n=12) for 3 days in steroid-free conditions, followed by increasing concentrations (0.001, 0.01, 0.1, 1 and 10 nM) of 17β-estradiol or STX for 5 min before GnRH stimulation until the end of the experiment. Estradiol (0.001 to 0.1 nM) significantly suppressed GnRH-stimulated LH secretion, whereas STX did not affect GnRH-stimulated LH secretion at any of the tested concentrations. In conclusion, STX, unlike estradiol, possesses no suppressive effect on GnRH-induced LH release from bovine AP cells.
Field isolates of BVDV which do not show the exaltation of Newcastle disease virus (END) phenomenon (END–) are rarely reported. In this study, 45 BVDV field isolates from cattle in Hokkaido prefecture in Japan were analyzed by the reverse plaque formation method, the END method and observation of cytopathic effects. END– virus was detected in 34 of 45 isolates (75.6%), although 35 of 45 field isolates contained END phenomenon positive virus as the predominant virus population. We propose that END– viruses are widely distributed in the field and that it is possible that the mixture of biologically distinct BVDV correlates with the appearance of disease in infected animals.