Hemoplasma is a tribal name for epierythrocytic mycoplasmas including Mycoplasma suis and M. parvum which are currently recognized in pigs as causative of porcine hemoplasmosis. Here, we report a real-time PCR assay for differential detection of these swine hemoplasma species by using allelic primers in the16S rRNA gene, and its application to survey for hemoplasma infections in pigs. Universal primers and species-specific primers were designed and evaluated by using swine blood samples positive in hemoplasmas. Mycoplasma suis and M. parvum infections were both confirmed by universal primers, and mixed infections were clearly distinguished by species-specific primers. Further, we applied this real-time PCR assay to 120 swine blood specimens from clinically healthy pigs in eleven farms in Japan, and found six (5.0%) were positive for M. suis and 18 (15.0%) were positive for M. parvum, and three (2.5%) were mixed infection by both hemoplasma species.
Hematological and serum biochemical values in five captive finless porpoises (Neophocaena phocaenoides) were examined, and abnormalities of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase and γ-glutamyltransferase were determined in a male finless porpoise. The numbers of white and red blood cells in pregnant female porpoises were significantly lower than those in nonpregnant ones (P<0.05). Significantly higher values of serum creatinine were observed in summer when compared with the values in winter (P<0.01). Abnormalities of hepatic enzymes could improve with administration of ursodeoxycholic acid and a hepatic hydrolysate drug.
The purpose of this study was to assess the normal distribution of 18F-fluoro-2-deoxy-D-glucose (FDG) uptake of canine brain structures using a high-resolution research tomography-positron emission tomography (HRRT-PET) and 7 T-magnetic resonance imaging (MRI) fusion system. FDG-PET and T2-weighted MRI of the brain were performed on 4 healthy laboratory beagle dogs. On MRI, regions of interests (ROIs) were manually drawn over 51 intracranial structures, including nine gross structures and 42 detailed structures. Relative standard uptake value ratio (rSUV=SUV of ROI/SUV of whole brain) was calculated for each ROI. The HRRT-PET and 7 T-MRI fusion imaging system demonstrated significant differences in glucose metabolism among various intracranial structures. Among gross structures, the midbrain and the pons and medulla oblongata had the highest uptake (rSUV: 1.12 ± 0.03) and lowest uptake (rSUV: 0.90 ± 0.06) of FDG, respectively. When rSUVs were calculated on detailed regions, the caudal colliculus and the longitudinal fibers of pons had the highest (rSUV: 1.62 ± 0.05) and the lowest (rSUV: 0.63 ± 0.03) glucose metabolism, respectively. Because the high resolution of PET-MRI fusion images provided clearly identifiable metabolic activities of canine brain, the HRRT-PET and 7 T-MRI fusion imaging might be a good tool for evaluation of intracranial diseases in canines.
We compared wedged hepatic venous pressure (WHVP), splenic pulp pressure (SPP) and trans-splenic portal vein pressure (TSPVP) in healthy dogs. We found that portal blood pressure could be measured in dogs using any of these techniques. The WHVP, SPP and TSPVP were 7.8 ± 1.0, 6.2 ± 0.8 and 6.8 ± 1.2 mmHg, respectively. Measuring SPP using ultrasound is most simple and minimally invasive, and it might be useful for evaluating portal hypertension in dogs with liver diseases.
In this study, we conducted a survey of the cytochrome b (cytb) gene of Babesia gibsoni (B. gibsoni) isolated from clinical cases to determine the prevalence of potential atovaquone (ATV)-resistant variants. Ninety-two blood samples were collected from naturally B. gibsoni infected dogs. The cytb nucleotide sequence was determined by direct sequencing. Twelve non-synonymous amino acid substitutions were identified in cytb. The principal ATV-resistant substitution, M121I, was detected in three cases. This survey determined that potentially ATV-resistant B. gibsoni strains are present in dogs in Japan.
Simple ectopic kidney was diagnosed in three dogs by means of radiography and ultrasonography. A 2-year-old castrated male Schnauzer, a 13-year-old female Schnauzer and a 9-year-old male Jindo were referred with vomiting, hematuria and ocular discharge, respectively. In all three dogs, oval-shaped masses with soft tissue density were observed in the mid to caudal abdomen bilaterally or unilaterally, and kidney silhouettes were not identified at the proper anatomic places on abdominal radiographs. Ultrasonography confirmed the masses were malpositioned kidney. The ectopic kidneys had relatively small size, irregular shape and short ureter but showed normal function on excretory urography.
The northern snakehead Channa argus, native to China, Russia and Korea, is currently found widespread throughout Japan following its original introduction during the 1920s. A parasitological study of 10 snakeheads fished from the Fushinogawa River running through Yamaguchi City, Japan, detected 2–101 (average, 23.7) metacercariae per 100 g of trunk muscle from each fish. The trematode was identified as metacercariae of Posthodiplostomum sp. (Strigeidida: Diplostomidae) morphologically and characterized genetically based on the ribosomal RNA gene (rDNA). Phylogenetic trees were constructed on the basis of either the 18S, ITS or 28S region of rDNA to assess the relationship among members of the family Diplostomidae. The addition of genetic data from more diplostomid taxa, particularly Posthodiplostomum cuticola recorded from a variety of freshwater fish in Eurasia, would facilitate the precise identification of the present species.
Multiple, pigmented, verrucous, cutaneous lesions in a 2-year-old female cat were pathologically examined. The lesions were linearly arranged on the right side of the body, and had developed along with moderate pruritus since infancy. Histologically, prominent exophytic, papillomatous outgrowths of the epidermis and acanthosis with intense ortho and parakeratotic hyperkeratosis were characteristic of the lesions. Dermal inflammation with mononuclear cells, neutrophils, and eosinophils was also noted. Inclusion bodies, cellular degeneration, and intranuclear viral particles suggesting papillomavirus infection in the keratinocytes were not observed. Papillomavirus antigen and DNA were not detected in the lesions by immunohistochemistry and polymerase chain reaction, respectively. In accordance with these clinical and histopathological features, the cutaneous lesions of the present cat were diagnosed as epidermal nevi, which were consistent with human inflammatory linear verrucous epidermal nevi.
In clinical settings, cellular resistance to chemotherapy and radiotherapy is a significant component of tumor treatment failure. The mechanisms underlying the control of localization of DNA repair proteins play a key role in the regulation of DNA repair activity. The DNA repair protein XRCC4, which is a regulator of DNA ligase IV activity, might be a key contributor to not only chemoresistance to anticancer agents, e.g., etoposide, but also radioresistance. However, it remains unclear whether XRCC4, which is a key player in nonhomologous DNA-end-joining (NHEJ), plays a role in low-dose radioresistance. In this study, we confirmed that human XRCC4 tagged with the enhanced green fluorescent protein (EGFP-XRCC4), as well as the DNA damage sensor Ku80 tagged with EGFP, mainly localized in the nuclei and its accumulation at DNA damaged sites began immediately after microirradiation. Moreover, we generated and characterized cell lines expressing EGFP-XRCC4 in XRCC4-deficient cells, i.e., XR-1 cells derived from the Chinese hamster ovary. Our findings showed that XR-1 cells were more sensitive than controls (CHO-K1) to low-dose X-irradiation (<0.5 Gy), whereas the radiosensitive phenotype of XR-1 cells was rescued by the expression of EGFP-XRCC4. We also confirmed that EGFP-XRCC4 expressed stably in XR-1 cells stabilizes DNA ligase IV. Altogether, these cell lines might be useful for the study of not only the dynamics and function of XRCC4, but also the molecular mechanism underlying the cellular resistance via the NHEJ pathway to low-dose radiation in mammalian cells.
To elucidate the role of glycogen in the contraction of tracheal smooth muscle, we investigated the changes in the glycogen contents of the bovine trachea during contractions induced by high K+ and hypoxia (achieved by bubbling N2 instead of O2), either in a glucose-free condition or in the presence of iodoacetic acid (IAA), an inhibitor of glycolysis. Hyperosmotic addition of 65 mM KCl (H-65 K+) induced a sustained contraction. A glucose-free condition did not affect H-65 K+-induced contraction. However, hypoxia slightly inhibited the contraction, and glucose-free PSS with hypoxia or IAA remarkably inhibited the H-65 K+-induced contraction. H-65 K+ induced a sustained increase in reduced pyridine nucleotide (PNred) fluorescence, representing glycolysis activity. Hypoxia alone slightly enhanced PNred fluorescence, and when combined with a glucose-free condition, it remarkably enhanced the H-65 K+-induced PNred fluorescence. IAA inhibited PNred fluorescence. In the presence of H-65 K+, a glucose-free condition, hypoxia and the combination of glucose-free PSS and hypoxia decreased the glycogen contents. However, IAA had no effect on glycogen contents. Although hypoxia or glucose-free PSS did not affect PCr and ATP contents, the combination of hypoxia and glucose-free PSS or IAA induced a gradual decrease of PCr content. In conclusion, we suggest that endogenous glycogen was utilized to increase the activity of glycolysis for maintaining high K+-induced contraction of the bovine trachea in the glucose -free and/or hypoxic condition.
Adult urodele amphibians such as newts are capable of regenerating lost structures including their limbs. In these species, dedifferentiation of myofiber is essential for the regenerative process. Upon terminal differentiation, nuclei of myofiber (myonuclei) are withdrawn from cell cycle, but prior to dedifferentiation, myonuclei reenter the cell cycle. In contrast with urodele amphibians, it is generally accepted that mammalian myofibers are not able to dedifferentiate in response to muscle injury. A recent study has suggested that electroporation can induce dedifferentiation response of skeletal muscle in newt limbs. In the present study, we examined whether myonuclei of skeletal muscle of mammals are capable of reentering the cell cycle by means of electroporation. Electroporation was applied to tibialis anterior muscle of the rat with or without plasmid DNA. Histological analyses revealed that, while electroporation induces degenerative/regenerative responses in skeletal muscle irrespective of the presence of plasmid DNA, the expression of proliferating cell nuclear antigen (PCNA) in myonuclei was observed only in the presence of plasmid DNA. The present results indicate that myonuclei of skeletal muscle are capable of reentering the cell cycle and suggest that in vivo electroporation can induce dedifferentiation of mammalian skeletal muscle.
The odor preference of female mice for male odor is reported to have cyclical variations in relation to the estrus cycle. Females prefer the odor of genetically dissimilar males to that of genetically similar ones, but the causal relation between this preference and the estrus cycle has scarcely been investigated. The Y-maze test demonstrated that BALB/c females stayed for a longer duration near the urine of C57BL/6 males than that of BALB/c males when they were in metestrus, diestrus and proestrus, but not in estrus. The prolonged stay disappeared after ovariectomy, and administration of estradiol-17β restored the tendency. The present results suggest that the odor preference of BALB/c females for C57BL/6 over BALB/c males temporally changes according to the estrus cycle and that estrogen can be one of endogenous factors regulating this phenomenon.
A gross pathology and histological investigation was carried out on bovine target organs of anabolic substances in the Molise Region (Italy). One hundred forty-four bovines (12–24 months old, 123 males and 21 females) were included in the survey. An antemortem assessment of their behavior and clinical examination were performed. After slaughter, samples of prostate, Cowper’s glands, Bartholin’s glands, mammary gland, ovaries, thymus and thyroid were collected, inspected and processed for histopathology, as suggested in the guidelines of the Italian national program for residue surveillance (PNR). Overall, 15.3% of the examined animals were classified as “suspect,” 44.4% were classified as “uncertain,” and the remaining 40.3% were classified as “negative.” The most frequent lesion was a severe thymus atrophy with fat infiltration (15.4% of males and 14.3% of females), strongly suggesting the illegal use of corticosteroids.
We developed a new system for detection of whole-genome differentiation using DNA-DNA hybridization, and tested its sensitivity with three closely-related Fusarium species. We compared DNA-DNA relatedness to nucleotide sequence homologies of five genetic regions between each of five strains of three Fusarium species. DNA-DNA relatedness by our system was 16.2–86.6%. Sequence homologies of 18S rDNA, rDNA cluster region from ITS1 to 28S rDNA, β-tub, EF-1α and lys2 were 100.0, 99.0–100.0, 96.7–100.0, 95.1–99.4, and 94.7–100.0%, respectively. Our system could clearly detect differentiation between closely-related fungal species which have very similar morphological-characteristics, and exhibit little diagnoses in nucleotide sequences. Our results suggest that this system is a good tool for identification and phylogenetic analysis of closely-related fungal species.
Use of firocoxib in dogs for postoperative pain control has not been published in any of the journals in Japan. A field study was conducted to evaluate the efficacy and safety of firocoxib in dogs in controlling pain associated with soft tissue surgery in Japan. The study followed a negative control, double-blind, multicenter clinical efficacy study using a randomized block design. A total of 131 client-owned dogs presented to the clinical practices for soft tissue surgery were enrolled. Sixty-nine dogs were allocated to the firocoxib-treated group and received 5 mg/kg of firocoxib orally on Day 0 before the surgery and once daily through Day 2, while 62 dogs were allocated to the non-treated group handled in a similar manner only without the firocoxib administration. Pain assessment took place on Day 0 before the surgery through Day 2. The primary efficacy variable was a success/failure variable based on whether the dog needed rescue medication (based on pain assessment after the surgery or Investigator’s judgment) and a significant difference between firocoxib-treated group (16.4%) and non-treated group (50.0%) (P=0.0031) was observed. There was no adverse event during the study that was considered to be related to the administration of firocoxib. This study indicated the clinical efficacy and safety profile of firocoxib administered to control pain associated with soft tissue surgery under field condition.
The goal of this study was to prove the possibility of using silicone stents broadly used for human medicine in canine obstructive tracheal disease. A silicone stent anatomically designed for canine trachea was tested on 5 beagle dogs for 8 weeks. The stent was carefully inserted using a newly developed delivery device under fluoroscopic guidance. There were no technical difficulties in placing the stent during the procedure. Previously reported complications of airway stenting such as stent migration or granulation tissue formation did not occur in any of the cases. In addition, removal of the stent was as simple as inserting it, and complications were absent. The stent introduced in this study could possibly be applied to various canine obstructive tracheal diseases.
The objective of the study was to examine changes of intraocular pressure (IOP) undergoing anesthesia in Rhesus Macaques (Macaca mulatta) with Laser-induced Ocular Hypertension. Twenty male rhesus macaques (hypertensive glaucoma eye in OD; normal eye in OS) between 6 and 20 years of age were used for the study. The monkeys were anesthetized with ketamine hydrochloride (10 mg/kg intramuscularly) and 1% isoflurane, and then IOP in both eyes was measured by a single investigator using a calibrated TonopenTM applanation tonometer (Mentor, Norwell, MA, U.S.A.). The mean IOP with ketamine anesthesia was 36.70 ± 12.04 (right eye: OD) and 15.88 ± 2.84 (left eye: OS). The mean IOP with isoflurane anesthesia was 19.98 ± 6.67 (right eye: OD) and 15.32 ± 2.15 (left eye: OS). Undergoing isoflurane anesthesia, the IOP of OD was significantly decreased. Conclusively, careful examination of IOP is required to prevent unexpected contraindication on glaucoma patient with isoflurane anesthesia.
In the present study, the effect of two types of allogeneic-derived feeder cells [porcine ear and tail skin fibroblasts (PESF, PTSF)] and three types of xenogeneic-derived feeder cells [human foreskin fibroblasts (HFK), mouse embryonic fibroblasts (MEF) and immortalized mouse embryonic fibroblasts (STO)] on the isolation and cultivation of putative porcine embryonic stem cells (pESCs) was evaluated. In vivo derived zona pellucida (ZP)-free blastocysts were cultured on different mitotically inactivated feeder layers. The rates of ICM outgrowth and primary colony formation were observed, and further passage onto new feeders was performed. The characteristics of pESCs, including alkaline phosphatase (AP) activity, and pluripotent-related markers (OCT3/4, NANOG, SSEA-4) and genes were examined. Attached blastocysts cultured on HFK and STO feeders showed a higher percentage of ICM outgrowths than those cultured on PESF (76.7, 72.9 and 38.9%, respectively; P<0.05). The rates of primary ES-like colony formation and the number of putative ESC lines were significantly decreased when ICM outgrowths were cultured on PESF, compared with those cultured on HFK (30.6 vs. 76.7%, respectively; P<0.05). Only ES-like colonies from one (25%) and three (50%) cell lines developed on PTSF and STO feeders, respectively, were further maintained in an undifferentiated morphology associated with the presence of all ES characteristics; however, these characteristics disappeared when colonies were continued to the 8th and 6th passages, respectively. The present study indicated that feeder cell types affect the success of pESC establishment and maintenance of their pluripotency.
A Thoroughbred colt with ambiguous external genitalia was presented for clinical and histological examinations. The colt had a short penis that faced backward between his hind limbs. The measurements of luteinizing hormone, follicle stimulating hormone, testosterone and ir-inhibin showed a tendency to increase gradually from April. Both the sex-determining region of the Y chromosome and amelogenin gene fragments were detected by the PCR method. A cytogenetic analysis revealed the 63,XO/64,XY mosaic karyotype (ratio 83:17). In autopsy, immature symmetrical subcutaneous testes were found in the inguinal regions. The testes and other accessory sex organs were histologically normal. These results add to our knowledge of chromosomal abnormality and information concerning disorders of sex development in the horse.
It is thought that differences in conception rate between feline epididymal sperm and ejaculate sperm occur because, unlike ejaculated sperm, caudal epididymal sperm have not been sensitized with seminal plasma (SP). In this study, we investigated whether collection of feline epididymal sperm with SP influences sperm qualities after freezing-thawing. Sperm were sensitized with SP for 10 min at room temperature. As a result, the motility of caudal epididymal sperm sensitized with SP immediately after collection was significantly lower than that of ejaculate sperm, and no difference was noted in sperm qualities after freezing-thawing. This shows that the qualities of caudal epididymal sperm cannot be improved to a level higher than those of ejaculate sperm by sensitization with SP.
Intensive agricultural practices are recognized as significant sources of metal pollution in soils and pasture. This study investigated metal contamination in cattle offal from an agricultural area in Zambia, where inorganic fertilizers, agricultural lime, and pesticides are routinely applied. The highest median values (mg/kg, wet weight) of Cu (40.9), Zn (35.2), Cr (1.35) and Ni (0.594) were recorded in the liver, whereas the highest median values of Pb (0.061) and Cd (0.049) were found in kidneys. Maximum levels of Hg, As and Co were under 0.2 mg/kg in both organs. Pb and Cd did not exceed the benchmark values in cattle offal for human consumption and did not pose immediate health risks. Concentrations of Ni and Cr could present a public health concern. Monitoring of metal accumulations in offal of cattle, not only from well-known polluted environments but also agricultural areas, should be done regularly for the health of human consumers.
Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome. Two major PCV2 genotypes, PCV2a and PCV2b, have been identified. To explore the prevalence of different subgroups of PCV2 in Taiwan, 37 PCV2 isolates collected during 2002–2011 were analyzed. The genotypes of the PCV2 isolates collected before 2007 belonged to either PCV2a or PCV2b. However, all of the isolates collected after 2008 were PCV2b. Most of the isolates obtained since 2008 have been classified into a novel genotype within a subgroup of PCV2b based on complete ORF2 sequence analysis. Moreover, analysis of the PCV2 isolates from the same pig farm but from different years revealed that the viruses shifted from a PCV2b genotype to a novel subgroup of the PCV2b genotype. Collectively, PCV2b was the dominant PCV2 genotype in Taiwan currently, and the viruses have shifted into a new emerging subgroup of the PCV2b genotype.
From 2001 to 2010, 17,392 Japanese cats were examined for feline coronavirus (FCoV) antibodies. The seroprevalence of purebreds (66.7%) was higher than that of random breds (31.2%). Seroprevalence increased greatly in purebreds by three months of age, while it did not fluctuate greatly in random breds with aging, indicating that cattery environments can contribute to FCoV epidemics. Purebreds from northern regions of Japan were likely to be seropositive (76.6% in Hokkaido, 80.0% in Tohoku), indicating cattery cats in cold climates might be more closely confined. Among purebreds, the American shorthair, Himalayan, Oriental, Persian, and Siamese showed low seroprevalence, while the American curl, Maine coon, Norwegian forest cat, ragdoll and Scottish fold showed high seroprevalence. There would also be breed-related differences in Japan similar to the previous studies in Australia.
The glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), the prototype arenavirus, is a promising envelope protein of lentiviral pseudotype vectors for gene therapy. The distribution of dystroglycan, a known receptor for LCMV, cannot explain the narrow tropism of LCMV-GP-pseudotypes. Here, we examined whether infection of LCMV-GP-pseudotypes was affected by the expression of four cell surface molecules-Axl and Tyro3 (from the TAM family) and DC-SIGN and LSECtin (from the C-type lectin family)-that are known receptors of Lassa virus, another arenavirus. All four molecules enhanced LCMV-GP-pseudotype infection of cells. These results help explain the tropism of LCMV-GP-pseudotypes and further our understanding of LCMV infection in animals.
Elk prion protein (PrPC) has been confirmed to be capable of rendering rabbit epithelial RK13 cells permissive to temporal infection by chronic wasting disease (CWD) prions. The present study satisfactorily generated persistently CWD prion-affected RK13 cells (RKC1-11) using elk PrPC expressing cells (elkRK13) that were generated via the lentiviral expression system with high efficiency. The elkRK13 cells have been shown to be permissive to accumulation of abnormal isoforms of prion protein (PrPSc) resulting from CWD prions up to 97 serial passages thus far. This novel prion-affected cell line will help facilitate investigation of the molecular basis of CWD prion pathogenesis and confirmation of CWD prion infectivity in vitro.