Feline embryo development was examined for 7 days after fertilization using commercially available human media supplemented with 0.3% bovine serum albumin (BSA) or 5% fetal bovine serum (FBS). Cumulus-oocyte complexes were categorized as Grades 1, 2, and 3 according to morphology. Only-One Medium (OM) was used for in vitro culture (IVC) in OM + BSA, OM + FBS, and OM + BSA/FBS, with BSA supplementation for the first 2 days and FBS for the subsequent 5 days. Embryos cultured in Early Culture Medium (1–2 days) and Blastocyst Medium (3–7 days) were defined as EB + BSA and EB + BSA/FBS. The developmental rate until the blastocyst stage of Grade 1 and 2 oocytes cultured in OM + BSA/FBS was higher than for the other groups and was significantly higher than for the OM + BSA and EB + BSA groups (P<0.01). Grade 3 oocytes cultured in OM + BSA/FBS also showed the greatest proportion of blastocyst formation. However, FBS supplementation throughout the IVC period reduced blastocyst number. The percentage of 2 pronuclei after fertilization as well as blastocyst cell number were significantly higher in Grade 1 and 2 than Grade 3 oocytes when cultured in OM + BSA/FBS (P<0.05). These results indicate that commercially available OM supplemented with BSA for the first 2 days of culture and FBS for the subsequent 5 days is suitable for feline embryo development until the blastocyst stage.
The aim of this study is to identify the combined effect of multiple chemicals to the development of allergy. In this study, the effect of prenatal exposure to an organochlorine agent methoxychlor (MXC) and/or an organophosphate agent parathion (PARA) on trimellitic anhydride-induced allergic airway inflammation was examined in mice. Eosinophil infiltration in the bronchoalveolar lavage fluid (BALF) was significantly enhanced by MXC+PARA exposure compared to that of the control, MXC, and PARA groups. In the hilar lymph node, only slight increases in B-cell infiltration, as well as IL-6 and IL-9 secretions were observed in MXC+PARA group, and no effect was observed in the individual treatment groups. Our findings imply that prenatal exposure to some combinations of multiple chemicals may exacerbate the allergic inflammatory responses including eosinophils and cytokine production.
Four-toed hedgehogs presented bloody stool and loss of appetite. Integumental masses were observed in two of the four cases. Intraabdominal masses were observed on radiographs and ultrasonography in the remaining two cases. The masses were surgically removed from all four cases. All samples were histologically and immunohistochemically consistent with histiocytic sarcoma (HS). At the time of surgery, 3 of 4 cases had grossly recognized metastatic/disseminated lesions. Survival time was 48 days, 64 days, and 113 days, respectively, and the remaining case is currently alive (at Day 207). The present report describes the clinical management and outcome of hedgehogs that were diagnosed with HS.
We investigated the clinical characteristics of healthy cats in accordance with the target organ damage (TOD) risk category, on the basis of systolic blood pressure (SBP). This prospective multi-center study included 137 healthy cats. Indirect blood pressure was measured using an oscillometric technique. The median SBP in all cats was 147 mmHg (interquartile range: 134‒158). On the basis of the TOD risk category, 57.7, 19.7, 21.9, and 0.7% of the cats were classified into categories I–IV, respectively. Age, sex, and body weight did not affect the SBP. This study provides basic information on the distribution of TOD risk categories in clinically healthy cats.
Patellar luxation (PL) is one of the most common orthopedic disorders in dogs and a genetic factor is considered to play an important role in the development of PL. Genomic analysis has attempted to identify the genetic markers associated with the development of PL but only suggestive markers have been identified. Carefully selecting breeds with higher incidence rates of congenital PL as well as affected dogs with more severe symptoms are required, but such information remains limited to date. This study aimed to assess the genetic contribution to the development of PL in puppies. Using data on PL from 2,048 puppies of the nine common breeds in Japan, the association of PL grades between the limbs, breed, and sex as well as the concordance of PL between littermates were examined. A significant correlation was found between right and left limbs in PL grades in all the puppies (Spearman rank correlation coefficient (rs)=0.91, P<0.001) and for each breed (rs=0.81–0.93, P<0.001). In total, 20.3% of the puppies were affected. The inter-breed difference in PL prevalence was 2.1–38.1%, and Toy Poodles showed the highest prevalence rates. Littermates of the affected puppies with PL grade ≥2 had a 16.2-fold higher risk (P<0.001). Thus, these results suggest that PL in puppies is primarily influenced by genetics, especially in Toy Poodles. These data highlight the necessity of using a breeding scheme to decrease the prevalence of PL.
Since few studies have been published investigating plasma amino acid abnormalities in calves with illnesses, the aim of this study was to examine plasma amino acid abnormalities in calves with diarrhea. Forty-three Holstein calves aged 10.9 ± 5.6 days old were used for this study. Thirty-one of the 43 calves exhibited clinical signs of diarrhea without severe acidemia. The other 12 healthy calves were used as the control. Concentrations of plasma essential amino acids, non-essential amino acids, branched-chain amino acids, glucogenic amino acids, and ketogenic amino acids in diarrheic calves with hypoaminoacidemia were significantly lower than those in healthy calves. No significant differences were observed between diarrheic calves with normoaminoacidemia and healthy calves when looking at these parameters.
Medetomidine, an α2-adrenoceptor agonist, was reported to decrease tear flow in some species. However, there are no reports about the effect of medetomidine on tear flow in pigs. The purpose of this study was to elucidate it. The study was performed in 10 clinically normal female Landrace pigs aged 3 months. Tear flow was measured by the Schirmer tear test (STT) I before (baseline) and 15 and 30 min after intramuscular administration of 80 µg/kg medetomidine. Compared to the STT I value at baseline, the value decreased significantly at 30 min after administration in both the left and right eyes. In pigs treated with medetomidine, an artificial tear solution or ophthalmic gel should be applied to protect the ocular surface.
This study evaluated the expression of genes involved in the concentration of Ca2+ in precursor osteoblast-like cell, MC3T3-E1 subjected to stretching stimuli. Transient receptor potential vanilloid 4 (Trpv4) gene expression, the factor that is activated by stretch stimulation and enables inflow of Ca2+ from the extracellular space, was not affected as a result of stretch stimulation; conversely, the expression of sodium–calcium exchanger 1 (Ncx1) gene involved in outflow of intracellular Ca2+ increased, depending on stimulation intensity. Localization of Ca2+ correlated with the positioning of the endoplasmic reticulum, and intracellular Ca2+ decreased in inverse proportion to the intensity of the stretching force. These results suggest that stretch stimulation activates intracellular Ca2+ elimination rather than Ca2+ uptake before osteoblast differentiation.
The basement membrane surrounding cardiomyocytes is mainly composed of α1 and α2 chain of type IV collagen. Arresten and canstatin are fragments of non-collagenous C-terminal domain of α1 and α2 chain, respectively. We previously reported that the expression of canstatin was decreased in infarcted area 2 weeks after myocardial infarction in rats. In the present study, we investigated the regulatory mechanism for expression of arresten and canstatin. Myocardial infarction model rats were produced by ligating left anterior descending artery. Western blotting and immunohistochemical staining were performed to determine the protein expression and distribution. Arresten and canstatin were highly expressed in the heart. One day and three days after myocardial infarction, the expression of arresten and canstatin in infarcted area was lower than that in non-infarcted area. The expression of cathepsin S, which is known to degrade arresten and canstatin, was increased in the infarcted area. A knockdown of cathepsin S gene using small interference RNA suppressed the decline of arresten and canstatin in the infarcted area 3 days after myocardial infarction. This study for the first time revealed that arresten and canstatin are immediately degraded by cathepsin S in the infarcted area after myocardial infarction. These findings present a novel fundamental insight into the pathogenesis of myocardial infarction through the turnover of basement membrane-derived endogenous factors.
Postpartum uterine disease due to poor uterine involution continues to be a significant factor that contributes to poor reproductive efficiency in dairy cattle. Therapy that increases the frequency, duration and strength of uterine contractions in the postpartum period might enhance uterine involution, resulting in improved reproductive performance. The objective of this clinical trial was to study the effect of two uterine ecbolic therapies, oxytocin and prostaglandinF2α on uterine involution, postpartum endometritis, and reproductive performance. A randomized double-blinded clinical trial was conducted in 118 dairy cows from two research herds that had normal parturition and expulsion of the fetal membranes. Within 24 hr after calving, cows were randomly assigned to receive intramuscular injections twice a day of 50 IU of oxytocin, or 25 mg of dinoprost (PGF2α) or saline (control) for 7 days. Cows were monitored from day 3 to day 63 ± 3 days postpartum by transrectal palpation of the uterus, vaginoscopy, Metricheck® examination and by endometrial cytology. Blood samples were collected for measurement of progesterone in weeks 3 (21 ± 3 days), 5 (35 ± 3 days), 7 (49 ± 3 days), and 9 (63 ± 3 days) postpartum. Herd breeding records were obtained to determine reproductive performance in all cows. Neither oxytocin nor prostaglandin F2α therapy during the first week postpartum had any significant effect on the rate of uterine involution, prevalence of endometritis or reproductive performance, compared to untreated controls. Ecbolic drugs, as used here, are not recommended for use in clinical practice to improve involution or reproductive tract health in normal cows.
A solitary firm nodule was found in the lung of a sika deer (Cervus nippon yesoensis). Histologically, it was a biphasic lesion composed of epithelial and stromal cell elements and exhibited a leaf-like growth pattern. The epithelial cells were immunohistochemically positive for pancytokeratin, cytokeratin 7, napsin A, and thyroid transcription factor-1, and the stromal cells were positive for vimentin and partially positive for desmin and α-smooth muscle actin. These observations were consistent with pulmonary adenofibroma, which is an extremely rare lesion in humans. To the best of our knowledge, this is the first reported case of pulmonary adenofibroma in an animal.
The inhibitory activities of grapefruit seed extract (GSE) on avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), Salmonella Infantis (SI) and Escherichia coli (EC) were evaluated. Original GSE contained 0.24% benzalkonium chloride (BZC), however, 0.0025% BZC solution could not inactivate bacteria. The activity of diluted GSE (×100, ×500 and ×1,000 with redistilled water) against selected viruses and bacteria was evaluated in this study. The GSE solutions were incubated with the pathogens over a period of time after which the remaining viruses were titrated and the bacterial colonies were counted. In the presence of organic material—5% fetal bovine serum (FBS), the test solutions were sprayed at 1 cm and 30 cm distances to test the efficacy of GSE in a spray form. Furthermore, the efficacy of GSE against bacteria on clothes was tested using non-woven cloth. GSE×100 reduced the viral titer of both AIV and NDV even in 5% FBS condition. IBDV showed high resistance to GSE. GSE×1,000 inactivated both SI and EC within 5 sec, even in the presence of 5% FBS. The disinfectant was able to maintain its efficacy in the spray form at 30 cm distance. GSE was also effective against SI and EC inoculated on fabric. GSE is a potential novel disinfectant against viruses and bacteria, effective even within a short contact time.
Six atypical Actinobacillus pleuropneumoniae serovar 15 strains were isolated from pneumonic lesions of naturally infected dead pigs from the same farm in Japan. Genetic analyses of apx genes revealed that the atypical isolates contained the toxin-associated genes apxIBD,apxIIICA, apxIIIBD, and apxIVA, but not apxIICA. Coinciding with the result of the atypical gene profile, analyses of toxin protein production revealed that these atypical isolates expressed only ApxIII but not ApxII. A mouse pathogenicity test showed that the atypical isolate tested seemed to be less virulent than the typical isolates. This is the first report describing the emergence of atypical A. pleuropneumoniae serovar 15, which does not produce ApxII due to the absence of apxIICA genes, in Japan.
The nutria (Myocastor coypus) was introduced to South Korea in 1987 for breeding of individuals for fur and meat industry, and was accidentally released into the wild. Here, we report the development of microsatellites for the nutria collected from South Korea using Illumina MiSeq genome sequencing to identify the genetic variability and demographic history of these introduced populations. A total of 626,282 microsatellite sequences were identified, and nine polymorphic loci were characterized. We used four novel loci developed and three previously known loci to investigate the genetic diversity of twelve South Korean populations. A low level of diversity was found, and no signature of genetic structuring was revealed among populations, indicating that Korean nutria individuals originated from a single population or a highly inbred reared herd.
This study evaluated the effect of sevoflurane anesthesia on neuromuscular blockade with rocuronium in dogs. Six healthy beagle dogs were anesthetized four times with a minimum 14-day washout period. On each occasion, the dogs were administered 1.25-, 1.5-, 1.75-, or 2.0-fold of the individualized minimum alveolar concentration (MAC) of sevoflurane and received an infusion of rocuronium (0.5 mg/kg followed by 0.2 mg/kg/h) for 120 min. Neuromuscular function was monitored with acceleromyography and train-of-four (TOF) stimulation of the left hind limb. Time to achieve TOF count 0 (onset time), time from the onset of neuromuscular blockade to the reappearance of TOF count 4 (blockade period), and time from the onset of rocuronium infusion to attaining a 70 or 90% TOF ratio (TOFR70 or TOFR90) were recorded. There were no significant differences in the onset time, blockade period, and plasma rocuronium concentration between the sevoflurane MAC multiples. The TOFR70 and TOFR90 were dose-dependently prolonged with the sevoflurane MAC multiples. There were significant differences in the TOFR70 and TOFR90 between the 1.25 sevoflurane MAC (median: 55 and 77.5 min, respectively) and 1.75 sevoflurane MAC (122.0 and 122.6 min; P=0.020 and P=0.020, respectively), 1.25 sevoflurane MAC and 2.0 sevoflurane MAC (126.0 and 131.4 min; P=0.020 and P=0.020), and 1.5 sevoflurane MAC (97.5 and 121.3 min) and 2.0 sevoflurane MAC (P=0.033 and P=0.032). In dogs, sevoflurane anesthesia produced dose-dependent prolongation of recovery from neuromuscular blockade produced by rocuronium.
Elephant endotheliotropic herpesvirus type 1 (EEHV1) is the most important causative agent of an acute fatal hemorrhagic disease in Asian elephants (Elephas maximus). We employed loop-mediated isothermal amplification (LAMP) to develop a rapid and simple detection method for EEHV1 in blood. When used to test 21 clinical samples collected in Japan, the EEHV1 assay correctly identified one positive and 20 negative clinical samples. It was observed that when samples were spiked with synthetic DNA plasmids including EEHV1 polymerase gene, the detection limit of the LAMP assay was 101.2 copies/μl and 100-fold higher than that of conventional PCR. These advantages of the LAMP assay for EEHV1 detection may facilitate better veterinary practices for treating elephants suffering from the acute disease.
This study investigated whether treatment with the mitogen-activated protein kinase kinase inhibitor U0126 during in vitro maturation (IVM), which has previously been reported to improve oocyte developmental competence, is practical for use in calf production using ovum pick up (OPU)-derived oocytes. Two Japanese Black cows were repeatedly and simultaneously treated to stimulate follicular growth and were prepared for OPU. Cumulus-oocyte complexes (COCs) were collected from one cow using a collection medium containing 5 μM U0126 and were cultured in medium supplemented with the same concentration of U0126 for the first 2 hr of IVM; COCs from the other cow were used as controls without U0126 treatment. The cows were exchanged between the two groups at every sequential OPU (n=8). The number of oocytes developing to blastocysts in the U0126-treated group (39.1%, 34/87) was significantly higher than that in the control group (22.1%, 19/86). Eight blastocysts produced with U0126 treatment were transferred to recipients, and four normal calves were obtained. The results indicate that embryos develop efficiently from OPU-derived oocytes treated with U0126, and that these embryos may be of practical use in calf production.
Serial block-face scanning electron microscopy (SBF-SEM) is useful for three-dimensional observation of tissues or cells at high-resolution. In this study, SBF-SEM was used to three-dimensionally analyze the characteristics of fibroblast-like cells (FBLCs) in the rat ileal lamina propria (LP). The results revealed that FBLCs in LP could be divided into four types, tentatively named FBLC type I-IV, based on the external cellular appearance, abundance or shape of each organelle, detailed distribution in the LP and relationship with surrounding cells. FBLC-I and -II localized around the intestinal crypt (InC), FBLC-III localized from the lateral portion of InC to the apical portion of the intestinal villus (InV), and FBLC-IV localized in the InV. FBLC-I, -II and -III, but not FBLC-IV, localized beneath the epithelium. FBLC-II possessed thin lamellar-shaped endoplasmic reticula, whereas FBLC-I possessed expanded endoplasmic reticula that occasionally showed a spherical shape. FBLC-III and -IV possessed a cytoplasmic region with high-electron density and no organelles immediately beneath the cellular membrane; this region was found at the epithelial sides in FBLC-III and scattered in FBLC-IV. FBLC-IV were in constant close proximity to villous myocytes throughout the InV and also in contact with FBLC-III especially in the apical portion of the InV. FBLC-I, -II and -IV, but not -III, were constantly found to be in contact with various immunocompetent cells in the LP. Three-dimensional analysis using SBF-SEM indicates that four types of FBLC localized in the rat ileal LP.
In the present study, we evaluated the antibacterial and anti-quorum sensing qualities of phillyrin. The minimum inhibitory concentration (MIC) of phillyrin with regard to Pseudomonas aeruginosa is 0.5 mg/ml. The production of virulence factors—such as rhamnolipid (>78.69%), pyocyanin (>85.94%), and elastase (>89.95%)—that affect the pathogenicity of the P. aeruginosa strain PAO1 apparently declined in the presence of 0.25 mg/ml phillyrin. Biofilm formation decreased by 84.48%. In a Caenorhabditis elegans–Pseudomonas aeruginosa infection model, diseased worms lived longer (63.33%) in a phillyrin-containing medium than in a drug-free medium, and the drug did not directly kill the pathogen. Therefore, the present work suggests that phillyrin has potential as an antimicrobial agent for the control of infectious pathogens.
The relation between complete or partial ligation of extrahepatic portosystemic shunting and intraoperative mesenteric portovenography (IMP) was evaluated in 72 canines. Of the 72 dogs, 55 had complete ligation and 17 underwent partial ligation of abnormal vessels. IMP allowed evaluation of the number of intrahepatic portal branches and ratio of the diameter of cranial (CrPV) and caudal main portal vein (CaPV) at the shunt location. Nearly all cases in the complete ligation group and nearly half of the cases in the partial ligation group had three or more portal vein branches. CrPV/CaPV was 0.75 ± 0.24 in the complete ligation group and 0.29 ± 0.15 in the partial ligation group. CrPV/CaPV can be an effective new method for assessing IMP.
A new reassortant H7N3 avian influenza virus (AIV) was isolated from a duck meat product that was illegally taken on board a passenger flight from China to Japan in March 2018. Sequencing analysis revealed that the H7N3 isolate, A/duck/Japan/AQ-HE30-1/2018 (Dk/HE30-1) (H7N3), was a reassortant highly pathogenic avian influenza virus (HPAIV) that contained the haemagglutinin (HA) gene of Chinese H7N9 HPAIV. Dk/HE30-1 (H7N3) possessed a novel polybasic sequence motif PEVPKRRRTAR/GLF at the HA cleavage site that has never previously been reported in H7 HPAIVs. The HA antigenicity of Dk/HE30-1 (H7N3) slightly differed from that of H7N9 HPAIVs previously reported. These findings will help further our knowledge of the circulation and genetic evolution of emerging AIVs in endemic areas.
A 4-year-old male Toy Poodle was presented with a history of status epilepticus. On presentation, neurological examination revealed a delay in postural reactions in the right pelvic limb. Magnetic resonance imaging showed a fluid-containing cystic lesion that compressed the mesencephalon, hippocampus, and amygdala. The cyst was surgically removed via left rostrotentorial craniotomy. The final diagnosis was an intracranial ectopic choroid plexus cyst. The patient has remained free of seizures for 18 months after surgery. This is the first case report of an intracranial ectopic choroid plexus cyst that was surgically removed in a dog.
Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus within the family Flaviviridae, causes fatal encephalitis with severe sequelae in humans. TBEV is prevalent over a wide area of the Eurasian continent including Europe, Russia, Far-Eastern Asia, and Japan. While it was previously thought that TBEV was not endemic in Japan, the first confirmed case of serologically diagnosed TBE was reported in 1993 in the southern area of Hokkaido Prefecture, Japan. In addition, TBEV has been isolated from dogs, wild rodents and ticks in the area. Our epizootiological survey indicated that endemic foci of TBEV were maintained in Hokkaido and other areas of Honshu. TBEV can be divided into three subtypes based on phylogenetic analyses. The Japanese isolates were classified as the Far Eastern subtype, which causes severe neural disorders with a higher mortality rate up to 30%. However, how viral replication and pathogenicity contribute to the neurological manifestations remains unclear. Recent studies have revealed distinctive mechanisms of TBEV pathogenicity and viral genetic factors associated with virulence. This review discusses the recent findings regarding the epidemiology and pathogenesis of TBEV.
Ticks transmit a wide range of viral, bacterial, and protozoal pathogens, which are often zoonotic. Several novel tick-borne viral pathogens have been reported during the past few years. The aim of this study was to investigate a diversity of tick viral populations, which may contain as-yet unidentified viruses, using a combination of high throughput pyrosequencing and Batch Learning Self-Organizing Map (BLSOM) program, which enables phylogenetic estimation based on the similarity of oligonucleotide frequencies. DNA/cDNA prepared from virus-enriched fractions obtained from Ixodes persulcatus ticks was pyrosequenced. After de novo assembly, contigs were cataloged by the BLSOM program. In total 41 different viral families and order including those previously associated with human and animal diseases such as Bunyavirales, Flaviviridae, and Reoviridae, were detected. Therefore, our strategy is applicable for viral population analysis of other arthropods of medical and veterinary importance, such as mosquitos and lice. The results lead to the contribution to the prediction of emerging tick-borne viral diseases. A sufficient understanding of tick viral populations will also empower to analyze and understand tick biology including vector competency and interactions with other pathogens.
Equine Glandular Gastric Disease (EGGD) is a common disease in sport horses. This disease might be associated with usage of nonsteroidal anti-inflammatory drugs (NSAIDs) for treating inflammatory diseases. Although gastroscopy has been an effective method for diagnosis, but a less invasive, and inexpensive method is preferred. This study used proteomic technology to identify candidate serum proteins that might be used as markers of NSAIDs induced EGGD. Five Thoroughbred horses were given high doses of NSAID, phenylbutazone to treat lameness. The experiment was divided into three periods: (i) Pre-EGGD period, (ii) during EGGD period, and (iii) Post-EGGD period. Gastroscopy were used to diagnose EGGD, serum was collected to perform gel electrophoresis (1D SDS-PAGE) and mass spectrometry (LC-MS) in order to identify serum proteins in each group. The candidate serum proteins were computationally predicted for the interaction between phenylbutazone and proteins, tissue specific expression, and association to gastric ulceration. After EGGD induction, all horses showed clinical signs of colic with marked congestion and erosion appearing in the mucosa of the glandular stomach whereas no change was observed in the mucosa of non-glandular stomach. Our proteomic results identified 14 proteins that might be used as EGGD markers. These proteins were highly expressed in the glandular stomach and some proteins were associated with phenylbutazone or ulcer development. However, confirmation of these candidate marker proteins is required with specific antibodies in the larger horse population before they can be considered for application in the field.
We detected parapoxviruses from environmental samples and calves with and without intraoral clinical signs and conducted molecular and serological analyses. Pseudocowpox virus (PCPV) was detected from a calf showing anorexia, frothy salivation, and erosion in the mucosa of the lip and tongue. At the time that PCPV was detected, bovine papular stomatitis viruses (BPSVs) were detected in environmental samples as well as in calves without intraoral clinical signs. BPSV, but not PCPV, was detected in the same calf after 22 days. Phylogenetic analysis revealed that genetically different PCPV strains exist in Japan. This is the first report on the detection of PCPV and BPSV sequentially in the same calf and coexistence of PCPV and BPSV in the same farm in Japan.
A bottlenose dolphin (Tursiops truncatus) housed in the Port of Nagoya Public Aquarium (PNPA) presented with symptomatic pneumonia caused by Aspergillus fumigatus. The dolphin was treated with micafungin. On days 2 and 11 after the first administration of micafungin, results from a physical examination and laboratory test indicated a decline of body temperature (BT) and leukopenia, with lowest BT, white blood cells (WBCs), and segmented neutrophils (SEGs) of 34.2ºC, 600 cells/μl, and 67 cells/μl, respectively. BT, WBCs, and SEGs returned to normal range after administration of granulocyte colony stimulating factor (G-CSF). To the best of our knowledge, this is the first report of micafungin-induced decline of BT and leukopenia that was successfully treated with G-CSF in a bottlenose dolphin.
We previously showed that the promoter region of the human epidermal growth factor receptor (hEGFR) gene elicits high transduction efficiency, with transgene expression restricted to canine breast tumor cells. However, it was unclear whether this promoter induces tumor cell-specific transgene expression in canine urothelial carcinoma cells. Furthermore, compared with studies in human cancer cells, the utility of the telomerase reverse transcriptase (TERT) gene promoter for therapeutic transgene expression in canine cancer cells has not been evaluated thus far. Here, we compared the activity of these promoters in canine mammary tumor and urothelial carcinoma cells. Our results showed that compared with the TERT promoter, the hEGFR promoter was more useful as a tumor-specific promoter to induce efficient transgene expression in canine tumor cells.
RNA interference (RNAi) can inhibit Influenza A virus (IAV) infection in a gene-specific manner. In this study, we constructed a transgene expressing a short hairpin RNA (shRNA) that targets the noncoding region of the IAV RNA gene encoding nucleoprotein (NP). To investigate the antiviral effects of the shRNA, we generated two transgenic mouse lines with this transgene. Unfortunately, there was no apparent difference in IAV resistance between transgenic and non-transgenic littermates. To further investigate the antiviral effects of the shRNA, we prepared mouse embryonic fibroblasts (MEFs) from transgenic and non-transgenic mice. In experimental infections using these MEFs, virus production of mouse-adapted IAV strain A/Puerto Rico/8/1934 (PR8) in the transgenic MEFs was suppressed by means of the down-regulation of the viral RNA gene transcription in the early stages of infection in comparison with non-transgenic MEFs. These results indicated that expression of the shRNA was able to confer antiviral properties against IAVs to MEFs, although the effects were limited. Our findings suggest that the shRNA targeting the noncoding region of the viral RNA (vRNA) of NP might be a supporting tool in developing influenza-resistant poultry.
This study was undertaken to establish a method for measuring mRNA expression by using real-time RT-PCR in the diagnosis of canine meningiomas. When performing real-time RT-PCR, it is essential to include appropriate control tissues and to select appropriate housekeeping genes as an internal standard. Based on the results of our study, RPS18 constitutes a suitable internal standard for the comparison of mRNA expression between normal meninges and meningiomas. The results showed increased mRNA expression of VEGFA and EGFR; however, mRNA expression of KDR was reduced. Measuring mRNA expression by using real-time RT-PCR with appropriate control tissues and internal standards can provide useful information to understanding the pathogenesis of canine meningiomas, which corresponds with immunohistochemical findings.
We compared the temporomandibular joint structure between species of the order Carnivora and investigated its variation among family lineages. We also investigated the effect of the masticatory muscle physiological cross-sectional area (PCSA) on temporomandibular joint structure. The masticatory muscle is composed of multiple muscles, which contract in different directions and exert pressure on the temporomandibular joint. We investigated the effect of the ratio of each muscle’s PCSA—an indicator of muscle force—and muscle size relative to body size on temporomandibular joint structure. The temporalis PCSA relative to body size showed the highest correlation with temporomandibular joint structure. When the temporalis PCSA is large relative to body size, the preglenoid projects caudally, the postglenoid projects rostrally and the pre-postglenoid angle interval is small, indicating that the condyle is locked in the fossa to reinforce the temporomandibular joint. Most Carnivora use blade-like carnassial teeth when slicing food. However, dislocation occurs when the carnassial teeth are used by the temporalis muscle. Our results suggest that the temporomandibular joint is reinforced to prevent dislocation caused by the temporalis muscle. In Mustelidae, the temporomandibular joint with a rostrally projecting postglenoid is suitable for carnassial biting using the temporalis muscle. In Felidae, the force of the masseter onto the carnassial teeth is diverted to the canine by tightening the temporomandibular joint. In Canidae, the masticatory muscle arrangement is well-balanced, enabling combined action. Hence, reinforcement of the temporomandibular joint by bone structure is unnecessary.
Limb-sparing surgery is one of the surgical options for dogs with distal radial osteosarcoma (OSA). This case report highlights the novel application of a three-dimensional (3D)-printed patient-specific polycaprolactone/β-tricalcium phosphate (PCL/β-TCP) scaffold in limb-sparing surgery in a dog with distal radial OSA. The outcomes evaluated included postoperative gait analysis, complications, local recurrence of tumor, metastasis, and survival time. Post-operative gait evaluation showed significant improvement in limb function, including increased weight distribution and decreased asymmetry. The implant remained well in place and increased bone opacity was observed between the host bone and the scaffold. There was no complication due to scaffold or surgery. Significant improvement in limb function and quality of life was noted postoperatively. Local recurrence and pulmonary metastasis were identified at 8 weeks postoperatively. The survival time from diagnosis of OSA to death was 190 days. The PCL/β-TCP scaffold may be an effective alternative to cortical allograft in limb-sparing surgery for bone tumors.
The mutations of TP53 gene are frequently observed in canine histiocytic sarcoma (HS). The objective of this study was to examine the distribution of tumor cells with TP53 gene mutations. Tumor tissues were divided into three or four regions and TP53 gene mutations were examined. TP53 gene mutations were detected only in parts of the HS tissues from six of the eight dogs, and the frequency of the mutant allele varied (0–65%) among the tumor regions. This study suggests that canine HS can exhibit intratumor heterogeneity. Further studies are needed to examine the clinical significance of the intratumor heterogeneity of TP53 gene mutations.
Two Large Yorkshire piglets were diagnosed as persistent hyperplastic primary vitreous (PHPV). In case 1, the white cord-like structure extending from optic disc to lens was observed in the normal-sized right eye. Case 2 showed buphthalmos of the right eye. The internal structure of the right eye was unclear due to bleeding, but a white cord-like structure was slightly observed. In both cases, histological examinations revealed the fibrovascular cord-like structure in hyaloid vitreous. The retina was detached, and dysplastic nervous tissue was observed in anterior vitreous. Immunohistochemistry using various neural markers suggested that dysplastic nervous tissue was derived from the detached neural retina. By the characteristic macroscopic and histopathological features, both cases were diagnosed as PHPV. To our knowledge, this is the first report of swine PHPV.
The aim of this study was to evaluate the efficacy and safety of the intra-articular (IA) injection of botulinum toxin type A (BoNT/A) to the management of chronic pain in dogs. In a randomized, controlled, double-blinded study sixteen dogs with osteoarthritis secondary to hip dysplasia were distributed into two groups: 25 IU BoNT/A (BoNT) or saline solution (Control) was administered IA in each affected joint. All dogs received oral supplements (90 days) and carprofen (15 days). The dogs were assessed by a veterinarian on five occasions and the owner completed an assessment form at the same time (baseline to 90 days). The data were analyzed using unpaired-t test, Fisher’s exact test, analysis of variance and the Tukey’s test (P<0.05). There were no differences between groups in the veterinarian and owner assessments. Lower scores were observed in both groups during 90 days after IA therapy in the owner assessments (P<0.001). Compared with baseline, the Vet score was lower from 15-90 days after IA injection in the BoNT group, and at 15 and 30 days in the Control group (P<0.001). Both treatments were safe and reduced the clinical signs associated with hip osteoarthritis. However, IA BoNT/A (25 IU) did not provide better pain relief than the control treatment.
We investigated the effects of genetic background on the responses to superovulation in Japanese Black cattle. The genotype frequencies of GRIA1 and FSHR relating to ovulation and follicular development in each of the major bloodlines—Tajiri, Fujiyoshi, and Kedaka—were analyzed. The Tajiri line had the lowest frequency of G allele homozygosity of c.710A>G in GRIA1 among the three bloodlines, and deviation from Hardy–Weinberg equilibrium was detected. Genotype frequencies of c.337C>G, c.871A>G, and c.1973C>G in FSHR were in Hardy–Weinberg equilibrium in all bloodlines. The results of generalized linear mixed-model analyses showed that farm, levels of plasma anti-Müllerian hormone (AMH) concentration, age in months, repeated superovulation, c.337C>G in FSHR, and bloodlines had significant effects on the responses to superovulation. The number of transferable embryos in the group heterozygous for c.337C>G in FSHR was significantly higher than that in the group homozygous for the C allele. The Kedaka line showed a significantly higher number of ova/embryos, fertilized embryos, and transferable embryos than the Tajiri and Fujiyoshi lines. The concentration of circulating AMH is a useful endocrine marker for antral follicle counts. This study revealed the effects of genetic background on the responses to superovulation using levels of plasma AMH concentration as a covariate. The prominent effect of genetic background on superovulation in the Kedaka line requires additional studies to confirm the genomic regions and polymorphisms that are involved in the trait.
When rat pups are isolated from their mothers, they emit ultrasonic vocalizations (USVs). Although previous studies have reported that USVs are related to anxiety, others have reported that they are related to simple, nonemotional factors, such as physiological reactions to coldness. In this study, we examined the influence of three maternal separations on rat pups. The number of USVs during 5 min of USV test under maternal separation, latency in the righting reflex as motor function, and body temperature were recorded twice (the first and second tests) before and after the pups were put in various environments for 10 min. The environments were no maternal separation (Control: CON), maternal separation with littermates (LMS), and single maternal separation with a heater (SMS). In the second test, the SMS pups had fewer USVs, a lower body temperature, and a more rapid righting reflex than the CON and LMS pups. In addition, there was no strong correlation between USVs and righting reflex. As a result, pups undergoing 10 min of SMS while being kept warm by the heater showed rapid righting reflex. Thus, by a single maternal separation, the number of USVs decreased but the decrease was unrelated to decrease in motor function.
Megaesophagus (ME) is a common esophageal disease in dogs and the prognosis is generally poor, especially with aspiration pneumonia (AP). We retrospectively investigated the clinical features and prognosis of canine ME in Japan. Twenty-eight dogs were included in this study, with the Miniature Dachshund breed being significantly overrepresented (odds ratio: 4.33). Most cases (21 of 28) were diagnosed as idiopathic ME and Myasthenia gravis (MG) was the most common cause of secondary ME. The overall median survival time (MST) was not reached and the 3-month survival rate was 85.7%. Ten dogs were diagnosed with AP, at least once during the study period, and the MST of ME dogs with AP was 114 days. The survival time overall and even with AP, was notably more prolonged compared to the previous studies. We hypothesized that treatment for canine ME could prolong the survival time, even in those with both ME and AP.
Several manufacturers recommend to feed mixture comprising equal amounts of oral rehydration salt (ORS) solution and milk for diarrheic calves after milk withdrawal. Such a feeding method is expected to supply more nutrients and energy compared to feeding only the ORS solution. However, little is known about the effects of feeding milk diluted with ORS solution on calves’ digestive process. This study examined the abomasal contents, volumes, and emptying rates in calves fed whole milk, milk diluted by 50% with ORS solution (50% ORS–milk), and ORS solution. Ultrasonography identified curds in the milk–fed calves, but not in the 50% ORS–milk–fed or the ORS–fed calves. The abomasal fluid of the 50% ORS–milk–fed calves contained not only β–lactoglobulin but also α–casein (CN), β–CN, and κ–CN, which were used for curd formation and undetectable in the milk–fed calves. Abomasal pH was relatively higher in the 50% ORS–milk–fed than that in the milk–fed calves. Abomasal emptying rates were significantly faster in the ORS–fed than in the 50% ORS–milk–fed and the milk–fed calves. These data indicate that the formation of abomasal curd is inhibited in the 50% ORS–milk–fed calves due to the resultant high abomasal pH and low κ–CN concentration. The 50% ORS–milk may not provide rehydration as quickly as the ORS solution. In conclusion, we do not recommend feeding 50% ORS–milk to calves.
This study aimed to improve the staining of frozen-thawed Japanese Black bull sperm acrosomes with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Spermatozoa were washed, fixed with 1–3% paraformaldehyde (PFA) in suspension for 10, 20, and 30 min, permeabilized with 0–2% Triton X-100 for 5 min, stained with FITC-PNA, and mounted with different antifade agents (0.22 M 1,4-diazabicyclo [2,2,2] octane (DABCO), SlowFade®, and ProLong®) in suspension (In-suspension) or on a smear (On-smear). The spermatozoa were categorized into seven pattern types either immediately or after storage for 24 hr. Experiment 1 showed that 1) the In-suspension method was better than the On-smear method; 2) if spermatozoa were stained using the In-suspension method and examined immediately, the best antifade agent was SlowFade®; 3) if samples were to be stored after staining using the On-smear method, DABCO should be avoided; 4) if spermatozoa were stained using the In-suspension method, storage of the stained samples was not recommended; and 5) if samples were to be stored after staining using the In-suspension method, ProLong® might be the best antifade agent. The results of experiment 2 showed that the concentration of Triton X-100 could be reduced to 0.1 from 1%. The results of experiment 3 showed that the paraformaldehyde concentration used for a 30 min fixation could be reduced from 3 to 2%. It is expected that the improved staining protocol will be useful to determine bull sperm acrosomal integrity.
A severely emaciated adult Steller’s sea eagle (Haliaeetus pelagicus ) was found dead with electrocution-induced severe wing laceration, with multiple cutaneous pock nodules at the periocular regions of both sides nearby the medial canthi and rhamphotheca. Histopathological examination of the nodules revealed hyperplasia of the epidermis with vacuolar degeneration and intracytoplasmic inclusion bodies (Bollinger bodies). The proventriculus was severely affected by nematodes and was ulcerated. Nucleotide sequencing of a PCR-amplified product of Avipoxvirus 4b core gene revealed 100% identity to the sequence of Avipoxvirus derived from other eagle species. This report describes the first detection of Avipoxvirus clade A from a Steller’s sea eagle.
Five-day-old neonatal piglets presented with debilitation and ananastasia. At the necropsy of one piglet, the apex of the tongue was found to be discolored dark red, and disseminated white foci were found on the cut surface. Many white foci were also found in the lungs and on the serosa of the liver and spleen. Histopathological findings revealed multifocal necrotic glossitis and pneumonia with Gram-negative bacilli. The bacilli were identified as Actinobacillus suis through immunohistochemical, biochemical, and genetic tests, including 16S rRNA gene sequencing. Although A. suis usually causes inflammation in thoracic and abdominal organs, lesions were also found in the tongue in the present case. This study is the first report of glossitis caused by A. suis.
A 2-year-old castrated male mongrel dog presented with a well-demarcated fluctuant dermal mass, located on the back of the neck. Grossly along with cystic structures filled with a black greasy fluid, when cut open. Microscopically, the mass was multi-lobulated. The lobules consisted of neoplastic basaloid cells and showed central degeneration, forming multiple central cystic structures filled with dark melanin-pigmented materials. Immunohistochemically, the neoplastic cells were strongly positive for CK14 and partially positive for CK19, but negative for CK7, CK8/18, CD34, S-100, Melan-A and α-SMA. Based on the findings, the present case was diagnosed as a feline-type basal cell tumor characterized by cystic structures filled with abundant black fluid.
Cardiac biomarkers are important tools for monitoring disease progress and can monitor progression of therapy. Endothelin-1 (ET-1) has been studied for its use as a cardiac biomarker in human and small animal medicine while in horses with cardiac disease it has not been evaluated yet. The objective of the present study was to determine the concentration of plasma ET-1 in healthy horses and compare it with ET-1 concentration in horses with cardiac disease during rest and after exercise. 54 horses admitted to the Equine Clinic of Free University of Berlin were used in the present study, of which 15 horses were clinically healthy with no evidence of cardiac disease (Group 1), 22 horses suffered from cardiac disease with normal heart dimensions (Group 2) and 17 horses with cardiac disease and enlarged heart diameters (Group 3). Clinical examination, electrocardiography and echocardiography were performed. Endothelin-1 concentration was determined using ET-1 ELISA kit. The concentration of plasma ET-1 was significantly increased in horses with cardiac disease and normal cardiac dimensions (Group 2) and in horses with cardiac disease and enlargement of the left atrium (Group 3) compared to its concentration in clinically healthy horses (Group 1). In addition, the concentration of plasma ET-1 after exercise was significantly increased in diseased horses compared to its concentration at rest. Detection of ET-1 plasma concentration in horses at rest may be useful for detecting horses with changes in left atrial cardiac dimensions.
The small Indian mongoose (Herpestes auropunctatus) was introduced to Japanese islands and has impacted on the island’s biodiversity. Population control has been attempted through capturing but its efficiency has rapidly declined. Therefore, new additional control methods are required. Our focus has been on the immunocontraceptive vaccines, which act in an especially species-specific manner. The amino-acid sequence of the mongoose ovum zona pellucida protein 3 (ZP3) was decoded and two types of synthetic peptides (A and B) were produced. In this study, these peptides were administered to mongooses (each n=3) and the sera were collected to verify immunogenicity using ELISA and IHC. Treated mongoose sera showed an increasing of antibody titer according to immunizations and the antigen-antibody reactions against the endogenous mongoose ZP. In addition, IHC revealed that immune sera absorbed with each peptide showed a marked reduction in reactivity, which indicated the specificity of induced antibodies. These reactions were marked in peptide A treated mongoose sera, and the antibody titer of one of them lasted for at least 21 weeks. These results indicated that peptide A was a potential antigen, inducing autoantibody generation. Moreover, immunized rabbit antibodies recognized mongoose ZP species-specifically. However, the induction of robust immune memory was not observed. Also, the actual sterility effects of peptides remain unknown, it should be verified as a next step. In any case, this study verified synthetic peptides we developed are useful as the antigen candidates for immunocontraception of mongooses.
Worldwide use of anticoagulant rodenticides (ARs) for rodents control has frequently led to secondary poisoning of non-target animals, especially raptors. In spite of the occurrence of many incidents of primary or secondary AR-exposure and poisoning of non-target animals, these incidents have been reported only for individual countries, and there has been no comprehensive worldwide study or review. Furthermore, the AR exposure pathway in raptors has not yet been clearly identified. The aim of this review is therefore to comprehensively analyze the global incidence of primary and secondary AR-exposure in non-target animals, and to explore the exposure pathways. We reviewed the published literature, which reported AR residues in the non-target animals between 1998 and 2015, indicated that various raptor species had over 60% AR- detection rate and have a risk of AR poisoning. According to several papers studied on diets of raptor species, although rodents are the most common diets of raptors, some raptor species prey mainly on non-rodents. Therefore, preying on targeted rodents does not necessarily explain all causes of secondary AR-exposure of raptors. Since AR residue-detection was also reported in non-target mammals, birds, reptiles and invertebrates, which are the dominant prey of some raptors, AR residues in these animals, as well as in target rodents, could be the exposure source of ARs to raptors.
The influence of different levels of heat exposure on the functions of ovarian and adrenal gland were investigated in pre-puberty female rats. Three-week old female rats were treated with control (26℃) or three higher temperatures (38, 40 and 42℃) for 2hr/day. After 9 days of treatment, blood samples were collected for measurement of luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol-17β, corticosterone, cholesterol and triglyceride. Adrenal glands, ovaries and liver were collected for analyzing gene expressions. Body and liver weight were significantly low in the 42℃ heating group. Circulating LH and triglyceride in the 42℃ heating group were significantly lower, and estradiol-17β, corticosterone and cholesterol were significantly higher than those of the control group. The gene expression of 3β-HSD and P450c21 in the adrenal gland; 3β-HSD, receptors of LH, FSH and estrogen in the ovary were significantly low in heated rats. The liver gene expressions of caspase 3 and NK-kB were significantly high in 42℃ heated rats, suggesting that the ability of liver metabolic function reduced in the 42℃ heated rats. These results demonstrated that the high temperature is responsible for suppression of ovarian function by decreasing the expression of steroidogenic enzymes, estrogen and gonadotropin receptors in the ovary. Increase in circulating estradiol-17β in the heated rats may be due to accumulate this hormone in circulation by potential changes in liver metabolism during the heat stress.
Duck Tembusu virus disease, caused by the duck Tembusu virus (DTMUV), can lead to a severe reduction in egg production and growth retardation in laying ducks and ducklings, respectively. In this study, we engineered a novel recombinant adenovirus expressing the E protein of DTMUV (rAd-E) in AAV-293 cells (analyzed by western blot and indirect immunofluorescence assays). Intramuscular immunization of Cherry Valley ducks with rAd-E was performed to evaluate host cellular and humoral immune responses. Compared to the phosphate-buffered saline administered group and the negative control wild-type adenovirus (wtAd) group, the rAd-E vaccinated group showed increased cellular and humoral responses. The results from the cytokine release and lymphocyte proliferation assays showed that rAd-E induced a stronger cellular immune response than the control group (P<0.01), 4 weeks after primary immunization. The results of enzyme-linked immunosorbent and virus neutralization assays showed that rAd-E induced higher titers of specific neutralizing antibodies, 2 weeks after primary immunization. The DTMUV challenge experiment showed a higher survival rate (80%) of ducks in the rAd-E group, when challenged with 0.5 ml (ELD50 = 10−2.67/0.2 ml) of the DTMUV strain AH-F10. These results indicate that rAd-E effectively protects ducks against DTMUV infection. Therefore, rAd-E could be a vaccine candidate to provide an effective and safe method for prevention and control of DTMUV infection.
The present study was designed to describe the clinical presentation of abdominal hernias and to evaluate the efficacy of polypropylene mesh in repair of such affection in camels. Twenty-six dromedary camels were included in this study on the basis of clinical and ultrasonographic evidence of abdominal hernia. Factors associated with prevalence and clinical findings of hernia were presented and hernioplasty using polypropylene mesh was evaluated as a surgical intervention. Out of 26 studied camels, abdominal hernia was prevalent in Wadeh camels than other breeds (17 vs 9, P<0.01). Camels <6 years of age exhibited more hernias than other age groups (18 vs 8, P<0.01). Moreover, females showed a significantly higher prevalence (19 vs 7, P<0.01) of abdominal hernia compared to males (26.9%, n=7). The sensitivity (96.8%) and specificity (93.1%) of ultrasonography for diagnosing hernia were higher in comparison to clinical examination (88.3%). At 3 weeks postoperatively, the clinical index score of 26 operated camels was significantly reduced in comparison with pretreated (22 vs 4, P<0.005). However, only 2 cases had recurrence of the hernia and 2 camels had slight swelling in situ. By the 6th month post treatment, all treated camels were completely recovered. In conclusion, the polypropylene mesh is a viable and consistent alternative effective treatment for abdominal hernias in camels. In addition, the clinical index scores and ultrasonography provide a precise paradigm for diagnosis and preoperative planning for abdominal hernias in dromedary camels.
Shiga toxin-producing Escherichia coli (STEC), Enteropathogenic E. coli (EPEC), and Enterotoxigenic E. coli (ETEC) make up an important group of pathogens causing major animal and public health concerns worldwide. The aim of this study was to determine the prevalence of different pathotypes of E. coli in captive wildlife. We analyzed 314 fresh fecal samples from captive wildlife, 30 stool swabs from animal caretakers, and 26 feed and water samples collected from various zoological gardens and enclosures in India for the isolation of E. coli, followed by pathotyping by multiplex PCR. The overall occurrence rate of E. coli was 74.05% (274/370). The 274 E. coli isolates were pathotyped by multiplex PCR targeting 6 genes. Of them, 5.83% were pathotyped as EPEC, 4.74% as STEC, and 1.09% as ETEC. The 16S rRNA genes from the selected isolates were amplified, sequenced, and a phylogenetic tree was constructed. The phylogenetic tree exhibited indiscriminate genetic profiling and some isolates from captive wild animals had 100% genetic identity with isolates from caretakers, suggesting that captive wildlife may serve as a reservoir for infection in humans and vice-versa. The present study demonstrates for the first time the prevalence of these E. coli pathotypes in captive wildlife in India. Our study suggests that atypical EPEC strains are more frequent than typical EPEC strains in captive wildlife. Discovering the implications of the prevalence of these pathotypes in wildlife conservation is a challenging topic to be addressed by further investigations.