Recently, we have shown that glycerol induces early fibrosis in rat muscles which persists up to two weeks after injury. The current study aims to determine the possible factor associated with fibrosis of rat muscle following glycerol injury. Eight-week-old male Wistar rats received either glycerol only (as a control) or a co-treatment of neutralizing antibody to transforming growth factor (TGF)-β1 (5 and 12.5 µg). Both antibody doses significantly decreased fibrosis and improved muscle regeneration suggesting that anti-TGF-β1 antibody has both anti-fibrotic and myogenic effects. In conclusion, fibrosis developed in glycerol-injured rat muscles, might be mediated, in part, by the upregulation of TGF-β1 expression. Targeting TGF-β1 could be a promising approach for inhibiting fibrosis and enhancing muscle regeneration.
Trueperella pyogenes is an opportunistic pathogen that causes a wide variety of purulent infections. We recently isolated a T. pyogenes strain unable to be identified by the previously reported T. pyogenes pyolysin gene (plo)-specific PCR from the lung of a sheep with astasia. Sequence comparison of plo among representative strains revealed several nucleotide substitutions in the primer-annealing regions. As such substitutions were considered to be a reason for the low PCR specificity, we designed novel primers in conserved regions of plo. Under optimized conditions, the novel primers precisely identified all T. pyogenes strains tested, and no products were generated from any other bacterial strains, suggesting the usefulness of the novel PCR assay for the diagnosis of T. pyogenes infections.
Streptococcus spp. cause a wide range of diseases in animals and humans. A Streptococcus strain (FMD1) was isolated from forest musk deer lung. To identify the bacterium at the species level and investigate its pathogenicity, whole genome sequencing and experimental infections of mice were performed. The genome had 97.63% average nucleotide identity with the S. equinus strain. Through virulence gene analysis, a beta-hemolysin/cytolysin genome island was found in the FMD1 genome, which contained 12 beta-hemolysin/cytolysin-related genes. Hemolytic reaction and histopathological analysis established the strain’s pathogenicity in mice. This is the first report of a beta-hemolytic S. equinus strain in forest musk deer identified based on phenotypic and genotypic analyzes; this strategy could be useful for analyzing pathogens affecting rare animals.
High-producing dairy cows are easily affected by left displacement of the abomasum (LDA) within 4 weeks postpartum. Although LDA is highly associated with metabolic disturbances, the related information on comprehensive metabolic changes, with the exception of some blood biochemical parameters, remains limited. In this study, the changes in plasma metabolites and in the metabolic profile of postpartum dairy cows with LDA were investigated through liquid chromatography coupled with quadrupole time of flight mass spectrometry (LC-Q/TOF-MS)-based metabolomics, and the metabolic networks related to LDA were constructed through metabolomics pathway analysis (MetPA). An obvious change in the metabolic profile was reflected by significant variations in 68 plasma metabolites in postpartum dairy cows with LDA, and these variations consequently altered 13 metabolic pathways (histidine metabolism, tyrosine metabolism, valine, leucine and isoleucine biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, arginine and proline metabolism, tryptophan metabolism, synthesis and degradation of ketone bodies, linoleic acid metabolism, arachidonic acid metabolism, citrate cycle, butanoate metabolism, vitamin B6 metabolism and pyrimidine metabolism). This study shows that the more detailed information obtained by LC-Q/TOF-MS-based metabolomics and MetPA might contribute to a better understanding of the disordered metabolic networks in postpartum dairy cows with LDA.
Hydrolyzed proteins are often prescribed for dogs with food hypersensitivity in food elimination programs. However, the potential of these diets to stimulate lymphocyte-mediated hypersensitivity is currently unknown. In this study, two commercially available hydrolyzed diets for dogs, D-1 (Aminopeptide Formula Dry, Royal Canin Japon, Tokyo, Japan), and D-2 (Canine z/d Ultra Dry, Hill’s-Colgate (Japan) Ltd., Tokyo, Japan), were analyzed to identify residual proteins or peptides, as well as activated helper T-lymphocyte reactions in dogs with suspected food hypersensitivity. Proteins and peptides with molecular weights >1 kDa (majority 1.5–3.5 kDa) were detected in both diet extracts with sodium dodecyl sulfate polyacrylamide gel electrophoresis, and size exclusion chromatography. When peripheral blood mononuclear cells (PBMC’s) from 316 dogs with suspected food allergies were cultured with hydrolyzed diet extracts, flow cytometry analysis revealed detectable levels of CD25low helper T-lymphocytes stimulated by D-1 and D-2 in 91 of 316, (28.8%), and 75 of 316 (23.7%) samples, respectively. These data indicated that the extracts contained proteins or peptides large enough to activate the lymphocytes. The percentages of CD25low helper T-lymphocytes stimulated by D-1 and D-2 extracts increased to 38.7% and 29.6%, respectively, in 186 of the original 316 samples (186/316, 58.9%), also reactive to poultry-related antigens. Thus, both poultry-related antigens, and D-1 and D-2 diet extracts may activate helper T-lymphocytes. These results demonstrate that hydrolyzed diets may contain proteins that stimulate helper T-lymphocytes, and may not be effective for treating all dogs with food hypersensitivity.
Toxoplasma gondii can cause severe encephalitis in immunocompromised patients. Although pyrimethamine and sulphadiazine have been standard therapeutic agents for the treatment of acute toxoplasmosis, they have toxic side effects. Therefore, there is a need to identify new drugs that are less toxic. Some traditional Chinese medicines (TCMs) have shown good efficacy in controlling T. gondii replication in mouse models. Here, we screened a natural product library comprising TCMs with the aim of identifying compounds and extracts with anti-toxoplasmosis activities. We found several hit compounds and extracts that could be candidates for new drugs against T. gondii infection.
East Coast fever is caused by Theileria parva, and poses serious concerns for dairy farmers owing to massive economic losses. In the current study, we compared three methods (DNA extraction kits, FTA-NaOH and FTA-TENT) of DNA extraction to identify the most economical and reliable method. A survey for T. parva prevalence was conducted in dairy cattle in Mbarara, Uganda. Cytochrome C oxidase subunit I (COI) and T. parva-p104 genes were amplified to compare the methods. FTA-NaOH-based polymerase chain reaction (PCR) yielded the best detection rate for both COI gene and p104 gene. Prevalence of T. parva was 45.0% and 83.3% at animal and farm-level, respectively. FTA-NaOH based-PCR is simple, highly sensitive and cost-effective tool for T. parva diagnosis in resource constrained settings.
We performed gross and histological examinations of the livers of sika deer (Cervus nippon yesoensis) in Hokkaido, Japan. Out of 1,381 deer slaughtered for venison production, thickening and dilation of the large intrahepatic bile ducts and Fasciola flukes in the duct lumens were detected in 621 deer (45.0%). Furthermore, 107 non-bile lesions (75 intrahepatic and 32 capsular lesions) were detected during gross examinations. Histologically, the bile duct lesions included chronic proliferative cholangitis, papillary hyperplasia, goblet cell and pyloric gland metaplasia, and periductal fibrosis. Many of the intrahepatic non-bile duct lesions (53/75, 71%) were considered to be Fasciola fluke migration-associated lesions, including two lesion types: necrosis, hemorrhage, and eosinophilic granuloma formation (29 lesions), and lymphoid tissue formation (24 lesions). Lymphoid tissue formation was considered to result from the persistent immune responses against dead Fasciola flukes. An epidermoid liver cyst was found incidentally, which has not been reported in the veterinary literature. In summary, this study demonstrated the predominance of fascioliasis-associated lesions in sika deer livers. The gross and histological lesions caused by Fasciola flukes in sika deer were similar to fascioliasis in other animals. Moreover, we described lymphoid tissue formation as a fascioliasis-associated lesion for the first time. The fact that bile duct lesions (45.0%) had a markedly higher prevalence than fascioliasis-associated parenchymal lesions (53/1,381, 3.8%) indicated that sika deer are a permissive host for fascioliasis. Our results provide information that will aid pathological examinations of sika deer.
A white nodule was detected in the liver of a wild female sika deer. The nodule was histologically diagnosed as squamous cell carcinoma (SCC), and it transitioned into a hyperplastic and chronically inflamed intrahepatic bile duct showing Fasciola infection. Therefore, the tumor was demonstrated to have originated from the biliary epithelium of the intrahepatic bile duct. Hyperplastic and chronic inflammatory changes of the biliary epithelium might have contributed the carcinogenesis of the present case, as proposed in human primary intrahepatic SCC cases. To the best of our knowledge, this is the first reported case of primary intrahepatic SCC in an animal.
Leiomyoma is the most common mesenchymal tumor in the gastrointestinal (GI) tract. Leiomyomas usually have a single or multinodular mass of various sizes, and affected animals can develop alimentary symptoms depending on the location and size. A 3-year old female miniature dachshund died after a history of refractory rectal prolapse, esophagectasis and aspiration pneumonia. At necropsy, the GI wall at the gastroesophageal and anorectal junctions was circumferentially thickened. Histologically, both GI lesions were composed of bundles of well-differentiated smooth muscles without mass formation or invasive growth. The neoplastic cells had little cellular atypia and low proliferative activity, and were positive for α-smooth muscle actin. The lesions were diagnosed as diffuse leiomyomatosis with circumferential thickening of the GI wall and has not been described in the veterinary literature.
A 26-year and 6-month-old male sika deer that was kept at the Showa Park, Tokyo, Japan, collapsed and died of severe disease wasting and severe tabefaction. Grossly, numerous masses, 0.3–1.0 cm diameter, were dispersed throughout the liver. The multiple masses were composed of tumor cells, which had hypochromatic nuclei and abundant faintly eosinophilic cytoplasm, arranged in nests of various sizes. Immunohistochemically, tumor cells were positive for cytokeratin, chromogranin A, synaptophysin and gastrin. Ultrastructurally, the cytoplasm of the tumor cells contained abundant membrane-bound electron-dense granules. A metastatic lesion was observed in the renal, hepatic and pancreatic lymph nodes. On the basis of these findings, this tumor was diagnosed as a neuroendocrine carcinoma with metastases to the lymph nodes.
In our previous studies, by simply inducing burn injuries on bullfrog hearts or partially exposing their surface to high-potassium (K+) solution, we could reproduce a ST segment elevation in the electrocardiogram (ECG), which is a characteristic finding in human ischemic heart disease. In the present study, using our burn-induced subepicardial injury model, we could additionally reproduce “reciprocal” ST segment changes for the first time in frog hearts, mimicking those observed in human acute myocardial infarction. Immunohistochemistry demonstrated markedly decreased Na+/K+-ATPase protein expression in the ventricular surface after the burn injury. The loss of this pump expression in injured cardiomyocytes was thought to be responsible for the creation of “currents of injury” and the subsequent ST segment changes observed in acute myocardial infarction.
The aim of this study was to evaluate the normal range of motion of the canine tibiofemoral joint and the movement of the tibia relative to the femur, especially the internal/external rotation under flexion. Right stifle joints were harvested from eight skeletally mature Beagle dogs, which were euthanized for reasons unrelated to this study. All muscle tissue was removed from the limbs prior to testing. Flexion and extension tests were conducted using a robotic manipulator with six degrees-of-freedom. Cranial/caudal and medial/lateral displacement and varus/valgus and internal/external rotation were measured at various degrees of flexion. We observed that the tibia rotated internally at an increasing flexion angle with mean peak internal and external rotations of 20.0 ± 13.8° and 4.5 ± 3.6°, respectively. The tibia also tended to displace cranially at an increasing flexion angle, with a mean peak cranial displacement of 8.9 ± 4.4 mm; there was minimal medial displacement when increasing the flexion angle. Valgus rotation also tended to occur at an increasing flexion angle. During the flexion of the canine stifle joint, approximately 20° of internal tibial rotation occurred around the longitudinal axis, along with a rollback motion involving the cranial displacement of the tibia.
It has been reported that drugs intended for epidural administration through the lumbosacral junction are accidentally administered into the subarachnoid space frequently in rabbits. Therefore, we evaluated the epidural single-bolus injection technique for the administration of bupivacaine into the coccygeal spinal canal of rabbits. After epidural distribution was confirmed by the injection of iohexol into the coccygeal spinal canal, 0.3 ml/kg 0.5% bupivacaine or 0.3 ml/kg normal saline was injected via the same needle. After the first attempt of iohexol injection, although the contrast was found in the epidural space in all rabbits, the additional contrast was also found in blood vessel in 3 rabbits and in muscular layer in 1 rabbit. Subarachnoid distribution was not observed in any of the rabbits. The time taken to regain normal anal reflex, movement of the hind limbs during walking, conscious proprioception of the hind limbs, and pain sensation of the tail and left hind limb, following coccygeal spinal canal injection, were significantly longer in the bupivacaine group than in the normal saline group. These findings indicated that coccygeal epidural injection of bupivacaine in rabbits may provide anesthesia for the hind limbs, perineum, and tail, but inadvertent vascular entry of the epidural drug may occur.
Two-month-old (Case 1) and one-year-old (Case 2) Japanese Black cattle presented with bilateral rostral mandibular open fractures. At presentation, the proximal edges of each fracture were discolored and had a severe stench, indicating necrosis caused by infection. In both cases, a bilateral rostral mandibulectomy over the symphysis was performed. Although the tongues of both animals prolapsed post-surgery, they had no significant problems with eating or drinking. Case 1 showed excellent growth during fattening, and Case 2 successfully became pregnant and gave birth. Rostral mandibulectomy can be an effective surgical option for the treatment of cattle with difficulty in internal or external fixation due to unfavorable necrotic cranial mandibular open fractures.
Serum anti-Müllerian hormone (AMH), a marker of equine cryptorchidism, is detectable in intact and cryptorchid stallions but not in geldings because it is secreted from Sertoli cells. A 4-year-old uncastrated Thoroughbred racehorse had no visible testes; therefore, the horse was considered a bilateral cryptorchidism. However, the serum AMH was undetectable (<0.08 ng/ml). Human chorionic gonadotrophin (hCG) stimulating test result indicated that the horse was a gelding. The results of sex chromosomal analysis and sequence analysis of SRY gene suggested that the horse was a genetically-intact stallion (X/Y). Only one small degenerative testis was present in the abdominal cavity. The reasons of undetectable serum AMH levels and negative response to hCG stimulation might be low numbers of Sertoli and Leydig cells. This study reports a case of serum AMH-undetectable cryptorchid stallion.
A 3.5-year-old, 2.9 kg, multiparous Chihuahua presented with abdominal distension; pregnancy was diagnosed. On Day 7 before parturition, prenatal sonograms showed anechoic bilateral dilated cerebral lateral ventricles, suggesting fluid-filled regions (ventriculomegaly) in one foetus. A Caesarean section was performed and the male newborn had an abnormally enlarged dome-shaped head and a cleft lip, and died 6 days after birth. According to the family pedigree, the X-linked recessive inheritance of an orofacial cleft from the unaffected mother was suggested. This report clearly demonstrates that canine foetal ventriculomegaly (hydrocephalus) can be diagnosed in utero. For dog breeds predisposed to congenital ventriculomegaly, early detection is important for the prediction of perinatal survival and adequate supportive care can be applied at delivery.
Zearalenone (ZON), produced by Fusarium fungi, exhibits estrogenic activity. Livestock can be exposed to ZON orally through contaminating feeds such as cereals, leading to reproductive disorders such as infertility and miscarriage via endocrine system disruption. However, the details of ZON metabolism remain unclear, and the mechanism of its toxicity has not been fully elucidated. In this study, we investigated the kinetics of ZON absorption and metabolism in rat segmented everted intestines. ZON absorption was confirmed in each intestine segment 60 min after application to the mucosal buffer at 10 µM. Approximately half of the absorbed ZON was metabolized to α-zearalenol, which tended to be mainly glucuronidated in intestinal cells. In the proximal intestine, most of the glucuronide metabolized by intestinal cells was excreted to the mucosal side, suggesting that the intestine plays an important role as a first drug metabolism barrier for ZON. However, in the distal intestine, ZON metabolites tended to be transported to the serosal side. Glucuronide transported to the serosal side could be carried via the systemic circulation to the local tissues, where it could be reactivated by deconjugation. These results are important with regard to the mechanism of endocrine disruption caused by ZON.
The etiology of Porcine respiratory disease complex is complicated by infections with multiple pathogens, and multiple infections increase the difficulty in identifying the causal pathogen. In this present study, we developed a detection system of microbes from porcine respiratory by using TaqMan real-time PCR (referred to as Dempo-PCR) to screen a broad range of pathogens associated with porcine respiratory diseases in a single run. We selected 17 porcine respiratory pathogens (Actinobacillus pleuropneumoniae, Boldetella bronchiseptica, Haemophilus parasuis, Pasteurella multocida, Pasteurella multocida toxin, Streptococcus suis, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynovie, porcine circovirus 2, pseudorabies virus, porcine cytomegalovirus, swine influenza A virus, porcine reproductive and respiratory virus US strain, EU strain, porcine respiratory coronavirus and porcine hemagglutinating encephalomyelitis virus) as detection targets and designed novel specific primer-probe sets for seven of them. In sensitivity test by using standard curves from synthesized DNA, all primer-probe sets showed high sensitivity. However, porcine reproductive and respiratory virus is known to have a high frequency of genetic mutations, and the primer and probe sequences will need to be checked at a considerable frequency when performing Dempo-PCR from field samples. A total of 30 lung samples from swine showing respiratory symptoms on six farms were tested by the Dempo-PCR to validate the assay’s clinical performance. As the results, 12 pathogens (5 virus and 7 bacteria) were detected and porcine reproductive and respiratory virus US strain, Mycoplasma hyorhinis, Haemophilus parasuis, and porcine cytomegalovirus were detected at high frequency. These results suggest that Dempo-PCR assay can be applied as a screening system with wide detection targets.
Orthoreoviruses have been indentified in several mammals, however, there is no information about orthoreoviruses in shrews. In this study, we screened wild animals in Zambia, including shrews, rodents, and bats for the detection of orthoreoviruses. Two orthoreovirus RNA genomes were detected from a shrew intestinal-contents (1/24) and a bat colon (1/96) sample by reverse-transcription (RT)-PCR targeting the RNA-dependent RNA polymerase gene of orthoreoviruses. Phylogenetic analyses revealed that each of the identified orthoreoviruses formed a distinct branch among members of the Orthoreovirus genus. This is the first report that shrews are susceptible to orthoreovirus infection. Our results suggest the existence of undiscovered orthoreoviruses in shrews and provide important information about the genetic diversity of orthoreoviruses.
The objective of this study was to compare the efficacy of a porcine reproductive and respiratory syndrome virus (PRRSV)-1 and PRRSV-2 modified-live virus (MLV) vaccines when administered at 1 day of age under field conditions. The piglets elicited anti-PRRSV antibodies at 1 day of age even in the presence of maternally derived antibodies. The number of PRRSV-2 genomic copies in the sera of pigs from the PRRSV-2 MLV-vaccinated pigs was significantly (P<0.05) lower when compared to PRRSV-1 MLV-vaccinated pigs. The average daily gain in PRRSV-2 MLV-vaccinated pigs was significantly (P<0.05) higher when compared to both PRRSV-1 MLV-vaccinated and unvaccinated pigs. This study demonstrated that vaccination as early as 1 day of age was effective against PRRSV infection.
Prevalence of antibodies to Toxoplasm gondii was studied using the latex agglutination (LA) method, followed by sucrose density gradient centrifugation (SDGC) method on the small Indian mongoose (Herpestes auropunctatus), which inhabits Amami-Oshima Island. Of the 362 samples, 38 (10.5%) revealed positive. Single or double peaks in the 7–8 and/or 12–14 fraction to LA titer by SDGC indicated the early stage of T. gondii infection. It is suggested that domestic/feral cats play an important role for spreading this zoonotic pathogen to the mongoose as well as other species that are endemic to this island. Future studies are warranted to prevent the transmission of T. gondii among cats and wild animals in order to maintain the ecosystem health.