Cultured cells are generally observed through the bottom of dishes or flasks using an inverted microscope. Two-dimensional and horizontal observation is insufficient for histological analysis of several cell lines, such as embryonic stem cells or cancer cells, because they form three-dimensional colonies. In the present study, we aimed to establish a more informative method for analysis of such stereoscopic cultured cells. We cultured mouse embryonic stem cells using a temperature-sensitive culture dish, embedded these cells in paraffin, and successfully observed vertical sections of embryonic stem cells. This vertical analysis of the stereoscopic colony emphasized structural features such as the dome shape of naïve pluripotent stem cells. This method could have the potential for analysis of three-dimensional structures and histological preservation in cultured cells.
Stable reference genes are important for gene expression analyses such as quantitative PCR. The stability of 15 candidate reference genes that can be used to developing mouse gonads was thoroughly verified using combinations of multiple algorithms. The expression of these genes fluctuated greatly depending on the analysis period and/or gender. Peptidylprolyl isomerase A (Ppia) and polymerase (RNA) II (DNA directed) polypeptide A (Polr2a) were the reference genes that were used stably for a wide analysis period in developing mouse gonads. Furthermore, the stable reference genes corresponding to the analysis period and/or gender were ranked. These results are useful for the selection of the optimal reference gene required for high-precision measurements.
The bactericidal efficacies of 0.2% food additive grade calcium hydroxide (FdCa(OH)2) solution, a quaternary ammonium compound (QAC) diluted at 1:500 (QACx500) and their mixture−Mix500 (FdCa(OH)2 powder added at final concentration 0.2% to QACx500)−were investigated at two different temperatures (room temperature (RT) (25 ± 2°C) and 2°C), using varying contact time, with or without presence of organic materials (5% fetal bovine serum: FBS), either in suspension or on abiotic carrier (steel, rubber and plastic). In the suspension test, QACx500 could inactivate Salmonella Infantis at effective level (≥3 log reductions), within 30 sec and 5 sec, respectively, with or without 5% FBS at RT; however, at 2°C it required 30 min and 1 min, respectively. Mix500 revealed the same efficacy as QACx500 at RT, but, at 2°C it required 1 min and 30 sec, respectively with or without FBS. Whereas, 0.2% FdCa(OH)2 solution alone could inactivate S. Infantis within 1 min and 3 min, respectively at RT and 2°C, even with 5% FBS. In the carrier test, single disinfectant required bit more (3 or 5 min) contact time to reduce bacterial load (S. Infantis or Escherichia coli) down to the effective level on rubber surface than that on steel and plastic surface. However, Mix500 could inactivate both bacteria on carrier surfaces within 1 min, even at 2°C. Thus, synergistic effects were observed in the suspension test and the carrier test at both temperatures toward both bacteria.
This study aimed to characterize two third-generation cephalosporins- and quinolone-resistant Escherichia coli (TGCs- and Q-R-Ec) isolates recovered from the ovaries of a broiler breeder flock and the internal contents of hatching eggs produced by the broiler breeder flock. Clonal relatedness was determined by multilocus sequence typing (MLST). The isolates displayed the same multidrug resistance profile, with resistance to ampicillin, ticarcillin, piperacillin, cefazollin, cephalothin, cefotaxime, nalidixic acid, tetracycline and sulfonamides. Double disk synergy test demonstrated that the two isolates presented an ESBL phenotype. PCR and sequencing results showed that both the isolates harbored the blaCTX-M-1 and qnrS1 genes. MLST revealed a novel allele combination, designated as ST461, in these isolates. This study would contribute to the molecular epidemiological understanding of TGCs- and/or Q-R-Ec.
With the increasing number of cats kept as pets, opportunities to treat cats with lower urinary tract disease (LUTD) have recently increased in the clinical veterinary field. Urine samples collected from 50 cats with bacterial cystitis brought to Maeda Veterinary Hospital between August 10, 2015 and March 31, 2017 were used in the study. Sample preparation of the urine was performed using a MALDI Sepsityper kit and rapid BACpro. To identify the isolates, MALDI-TOF MS was performed on an AutoFlex TOF/TOF mass spectrometer. MALDI-TOF MS using rapid BACpro for pretreatment was found to be a quick and reliable method for identification of bacteria from infected urine, with a shortened analysis time enabling earlier and more accurate selection of antibiotics for treatment of feline LUTD.
Obesity is associated with the chronic inflammation and senescence of adipose tissues. Macrophage is a key mediator of chronic inflammation that infiltrates obese adipose tissue and stimulates metabolic disorders. However, the fat depot-specific differences of macrophage infiltration and senescence, especially the influence on intramuscular adipose tissue, have remained unclear. We investigated the fat depot-specific differences of macrophage infiltration and senescence in obese bovine adipose tissue from three different anatomical sites (subcutaneous, intramuscular and visceral). Macrophage infiltrations and crown-like structures were observed in visceral adipose tissue, although there were few macrophages in subcutaneous and intramuscular adipose tissues. The positive reaction of senescence marker SA-βgal activity was observed in visceral adipose tissue. In contrast, the activity of SA-βgal in subcutaneous and intramuscular adipose tissues were low. The expression of p53 gene, the master regulator of cellular senescence, in visceral adipose tissue was higher than that of subcutaneous and intramuscular adipose tissue. At the cellular level, p53 gene expression was negatively correlated with the size of subcutaneous adipocytes. In contrast, p53 gene expressions were positively correlated with the size of intramuscular and visceral adipocytes. These results indicate that anatomical sites of obese adipose tissue affect macrophage infiltration and the senescent state in a fat depot-specific manner.
An 8-month-old male Japanese Black calf was referred for the evaluation of a slow-growing conjunctival mass in the right eye. A superficial keratectomy was performed followed by recurrence on two occasions. No metastases were found in surrounding tissues. Histological, immunohistochemical, and ultrastructural investigation revealed that both the primary and the recurrent lesions were benign, conjunctival, myofibroblastomas. Interestingly, bovine papillomavirus type 2 (BPV-2) DNA was detected in both myofibroblastoma lesions. Archival bovine myofibroblastomas from the vulva and neck were also analyzed for papillomaviral genomes. BPV-2 DNA was also amplified from these lesions. To the best of our knowledge, this is the first report describing a potential causal relationship between BPV-2 infection and conjunctival myofibroblastoma.
Pneumatosis coli is a rare intestinal disorder in dogs that is characterized by submucosal or subserosal emphysema of the colon. An 8-year-old castrated male Shih Tzu developed anorexia and hematochezia after undergoing surgery for a congenital splenophrenic shunt. Abdominal radiographic examination revealed linear radiolucency throughout the wall of the large intestine. Results of abdominal computed tomography revealed intramural gas tracking along the colon and rectum. Based on these findings, a diagnosis of pneumatosis coli was made. The dog was treated with antibiotics, but the general condition gradually deteriorated, and the dog died 6 days after the day of diagnosis. It was suspected that portal hypertension following partial ligation of congenital portosystemic shunt may have been associated with pneumatosis coli in this case.
Pancreatitis is an inflammatory disorder of pancreas which leads to varying degrees of pancreatic endocrine and exocrine dysfunction and manifests in either acute or chronic forms. Spontaneous pancreatitis in experimental animals has rarely been reported. Here, we found acute to chronic courses of spontaneous pancreatitis in spontaneously hypertensive rats (SHRs), showing the formation of tubular complexes (TCs) and enhanced islet regeneration. We investigated the expression pattern of clusterin in the pancreas of SHRs based on immunohistochemistry (IHC). IHC analysis revealed the strong expression of clusterin in dedifferentiated duct-like cells and regenerative islets of TCs. These results imply that clusterin might be involved in the formation of TCs and parenchymal regeneration during rat pancreatitis.
The African pygmy hedgehog (Atelerix albiventris) is becoming a popular pet in Japan. The aim of this study was to determine the incidence of various diseases in African pygmy hedgehogs. We histologically investigated 105 samples from 100 privately-owned pet African pygmy hedgehogs that were submitted to two laboratories (North Lab and Patho Labo) between 2012 and 2017. Tissues submitted for this study were taken from female reproductive organs (33 cases; 31.43%), skin (20 cases; 19.05%), and the oral mucosa (19 cases; 18.1%). The most common histological diagnoses included endometrial stromal nodules identified as benign uterine neoplasia (14 cases; 13.33%); endometrial polyps identified as non-neoplastic polyps (7 cases; 6.67%), gingival hyperplasia and chronic suppurative inflammation in the oral mucosa (11 cases; 10.48%), fibrosarcomas in the skin (8 cases; 7.62%), and mammary tumors (8 cases; 7.62%). In this study, lymphoma and oral squamous cell carcinoma were less common than in the previous reports. The present study revealed the disease prevalence in captive African pygmy hedghogs that were histopathologically examined.
A hard palate mass was surgically removed from an Australian green tree frog (Litoria caerulea) and examined pathologically. The tumor consisted of sheets of small cells arranged in a tubular structure and cords or rosettes with fibrovascular stroma. Immunohistochemically, neoplastic cells were diffusely positive for cytokeratin and neuron-specific enolase and partially positive for S-100 and doublecortin. These findings indicate that the tumor originated from the neuroectodermal tissue. Based on these findings, the tumor was classified as a neuromastoma (neuroepithelioma). Sensory cells located in the hard palate of the frog were considered to be the origin of the tumor. The frog died after going through 3 surgeries and experiencing difficulties closing its mouth.
A 13-year-old female scimitar-horned oryx (Oryx dammah) died after progressive anorexia, weight loss, and depression. The necropsy showed that the retroperitoneum was compressed by a large white-to-tan uterine mass and on several sections of the mass, the uterine wall was markedly thickened because of ill-defined transmural tumor tissue. Metastatic nodules were detected in the omentum, mesentery, diaphragm, and lung. The genital tract and pulmonary and abdominal nodules exhibited highly pleomorphic sarcoma. The primary and metastatic neoplastic cells showed positive results for vimentin, desmin, and sarcomeric actin, and negative results for smooth muscle actin. Uterine metastatic rhabdomyosarcoma was diagnosed on the basis of the gross, histopathology and immunohistochemistry results.
We surveyed the kidneys of dogs with immune-complex mediated glomerulonephritis (ICGN) by lectin histochemistry using seven lectins—namely WGA, RCA-I, ConA, PNA, SBA, DBA, and UEA-I. Their binding patterns were compared with those from normal dogs. RCA-I signals became weak in the brush borders of the proximal tubules, whereas DBA signals became positive in Bowman’s capsules. Also, varying intensity of the UEA-I signal was noted in the distal tubules, especially in the macula densa. The binding pattern profiles varied among the cases; this diversity in the lectin-binding patterns might be induced as a result of the diverse pathologies seen in canine ICGN.
A 5-month-old crossbred beef steer died after exhibiting astasia. A postmortem examination revealed verrucous endocarditis and numerous renal hemorrhages. Gram-positive bacteria were identified in the necrotic lesions of the verruca and mitral valve via histopathological analysis. Multifocal necrosis and hemorrhage were detected in the renal cortex. Gram-positive cocci isolated from the verruca were identified via biochemical tests and 16S rRNA gene sequence analysis as Streptococcus suis. Serotyping indicated that the S. suis isolates were untypable, following which these isolates were classified as a new sequence type (ST1000) via multi-locus sequence typing. S. suis is an important pathogen of pigs. However, clinical cases in cattle are rare. This report is intended to provide information that may be useful in the diagnosis of streptococcal disease in cattle.
A large abdominal mass was found in a dog. Histopathologically, the surface of the mass was covered with compressed adrenal gland tissue. The neoplastic cells showed typical features of malignant peripheral nerve sheath tumor (MPNST), including Antoni type A and type B pattern, and nuclear palisading. Immunohistochemically, these cells were positive for S100 protein, nerve growth factor receptor, nestin and claudin-1. The dog was euthanized because of the developing multiple metastatic lesions. The metastatic cells showed quite similar histopathological and immunohistochemical features as those in the original tumor. Although MPNST can develop at many body sites, this is the first report of MPNST originating from the adrenal gland in a dog.
The incidence of metabolic syndrome is rapidly increasing worldwide, and adequate animal models are crucial for studies on its pathogenesis and therapy. In the search of an adequate experimental model to simulate human metabolic syndrome, the present study was performed to examine the pharmacological response of WBN/Kob-Leprfa (WBKDF) rats supplemented with a fructose-rich diet (FRD) to liraglutide, a GLP-1 receptor agonist. Male WBKDF rats fed FRD at 7 weeks of age were divided into 3 groups, and administered liraglutide (75, 300 µg/kg subcutaneously) or saline (control group), once daily for 4 weeks. All rats in the control group became overweight, and developed hyperglycemia, hypertension and dyslipidemia as they aged. The rats given liraglutide exhibited a dose-dependent reduction in body weight, visceral fat content and food intake compared with control rats. In addition, liraglutide suppressed the development of hyperglycemia, hypertension and dyslipidemia. An intravenous glucose tolerance test revealed that liraglutide improved glucose tolerance, insulin secretion and insulin resistance. On histological examination, decreased hepatic fatty degeneration was observed in the liraglutide groups. The present study demonstrated that liraglutide protected against obesity, hyperglycemia, hypertension, dyslipidemia, and hepatic steatosis in WBKDF rats fed FRD, suggesting that WBKDF rats fed FRD may be a useful model to investigate the etiology of human metabolic syndrome.
The present retrospective study was conducted from 2003 to 2015 in Egypt to document common surgical affections of the udder and teat in cattle and buffaloes, and determine medical and surgical treatment options that are feasible in a field setting. We diagnosed 19 different surgical affections and classified them into 4 groups according to their location. Teat orifice affections (12.41%) included imperforate teat, contracted teat orifice, enlarged teat orifice, and black spot. Teat cistern affections (23.76%) included teat fistula, dilated teat cistern, teat polypi, and webbed teat. Teat surface affections (50.35%) included sore teat, supernumerary teat, sloughed teat, teat papilloma and fibropapilloma, teat wounds, and teat viral lesions. Udder affections (13.48%) included hypermastia, udder wounds, and suppurative and gangrenous mastitis. In cattle, the number of surgical affections located on the teat surface (20 ± 5.4) was significantly higher compared with other locations as well as compared with buffaloes (P<0.05). No treatment was indicated in 24% of recorded cases. Medical and surgical treatment was indicated in 73.75% of affected animals. Favorable results were achieved with the recommended treatments when applied in the field.
This study investigated the influence of changing recumbency and mode of ventilation over repeated anesthesias on the alveolar to arterial oxygen tension gradient (PA–aO2) and laboratory analytes in eight horses during a year-long imaging study. Anesthesia was induced with xylazine, diazepam or guaifenesin, and ketamine and maintained with isoflurane. Horses were positioned in right or left lateral recumbency for computed tomography. Ventilation was controlled during 47% of the anesthetics. Blood was sampled from an arterial catheter prior to (30 ± 5 min from connection to anesthetic circuit), within 5 min of changing lateral recumbency, and prior to circuit disconnection (24 ± 6 min after second sample) for measurement of pH, partial pressure of arterial oxygen (PaO2) and partial pressure of arterial carbon dioxide, blood glucose and electrolytes. PA–aO2 was calculated. Data from five anesthetic episodes for each horse were summarized as mean ± standard error and analyzed using a mixed-model ANOVA. t tests were used for pairwise comparisons (P<0.05). PaO2 decreased after turning (198 vs. 347 mmHg), then increased to 291 mmHg prior to disconnection. Correspondingly, PA–aO2 was wider (252 vs.120 mmHg), and improved before disconnection (190 mmHg). Body temperature, ionized-Ca2+ and blood glucose were lower, and Na+ was higher at the last time point. In conclusion, turning anesthetized horses decreases PaO2 and results in a widening PA–aO2 suggesting a cautious approach in animals with pre-existing hypoxemia.
A 2.5-year-old male French Bulldog was evaluated for seizures. Cranial magnetic resonance imaging (MRI) suggested a glioma in the left piriform area. Radiation therapy (RT) and continuous chemotherapy were administered. Although the lesion had regressed significantly 2 months after RT, a follow-up MRI revealed meningeal enhancement in the left piriform area, which expanded further, with hydrocephalus, by day 310 (8 months after RT). Because of the poor prognosis, the dog was euthanized on day 356 and necropsy was performed. Histopathological examination confirmed anaplastic oligodendroglioma with leptomeningeal dissemination. This case suggests that the possibility of leptomeningeal dissemination and hydrocephalus should be considered even after RT and chemotherapy for anaplastic oligodendroglioma.
The protective antibody response of Balb/c mice to Bali rabies virus (RABV) in BHK-21 cells was studied. The virus was isolated from a rabid dog and was adapted to replicate in BHK-21 cell culture for seven passages. The BHK-21-adapted Bali RABV (BHK-Bali RABV) was inactivated with binary ethylenimine and 24 mice were immunized twice at 21-days intervals with the inactivated virus and Rabisin® vaccine. Virus replication was detected using indirect immunofluorescence, immunocytochemistry, and western blotting assays. Enzyme-linked immunosorbent assay examination 2 weeks after the first immunization revealed RABV antibody titers that were mostly below the minimum protective level (<0.5 equivalent unit, EU). Antibody titers increased sharply after the second immunization. Antibody titers in serum of mice induced by inactivated BHK-Bali RABV one week after the second immunization were slightly lower (0.8–3.8 EU) than those induced by Rabisin vaccine (0.9–6.3 EU). RABV antibody titers were stable for at least 6 weeks after the second immunization. Both Rabisin vaccine and inactivated BHK-Bali RABV induced neutralizing antibodies with neutralization titers (50% protective dose per ml) of 29.84 for 0.1 ml Rabisin, 211.41 for 0.2 ml Rabisin, 27.41 for 0.1 ml BHK-Bali RABV, and 28.25 for 0.2 ml BHK-Bali RABV. Thus, inactivated BHK-Bali RABV induces a protective immune response in Balb/c mice, but at lower levels compared to induction by Rabisin vaccine.
Bovine foamy virus (BFV) is endemic in many countries, but has not been reported in Japan. A syncytium-forming virus was isolated from peripheral blood leukocytes of clinically healthy cattle on a farm in Kanagawa prefecture during a periodic epidemiological survey of viral diseases. The isolate was propagated in primary fetal bovine muscle cells and subsequently passaged in Madin–Darby bovine kidney cells. Since the isolate appeared to be distinct from the viruses with syncytium-forming ability previously isolated in Japan, we attempted to identify it using genomic analyses and electron microscopy. A phylogenetic analysis revealed that the isolate belongs to the bovine foamy virus cluster and is highly similar to a BFV strain isolated in China. A sero-epidemiological survey was performed using agar gel immunodiffusion test with the isolated virus as the antigen, and five of the 57 cattle tested were found to be seropositive.
Tetrahymena is commonly used as an alternative eukaryotic system for efficiently expressing heterologous genes. In this study, we inserted the non-structural (NS) 1 gene of avian influenza virus (AIV) into the shuttle vector pD5H8 and transformed conjugating T. thermophila with the recombinant plasmid pD5H8-NS1 by particle bombardment. Positive transformants were selected with paromomycin. We demonstrated that the NS1 protein could be expressed steadily following induction with cadmium in this Tetrahymena system. An enzyme-linked immunosorbent assay detection method was preliminary established using the expressed protein as coating antigens for serodiagnosis. This is the first study in which a Tetrahymena expression system was employed for the expression of the AIV NS1 protein, and it provides a good basis for the development of differential diagnostic kits and vaccines for the prevention and control of avian influenza.
Bovine leukemia virus (BLV) is horizontally transmitted among cattle through infected blood. This 3-year field study (2013–2016) aimed to confirm the potential of the blood-sucking stable fly as a risk factor of BLV transmission and to determine the efficacy of vector control on preventing the transmission of BLV. The BLV-positive conversion rate during summer was higher than that during winter in a model dairy farm, where many stable flies were observed during the summer. After fly nets were fixed onto the barn to prevent fly invasion, the BLV-positive conversion rate during the summer was significantly decreased compared with that in the absence of fly nets (P<0.01). These findings suggest that vector control using a fly net may inhibit BLV transmission.
Arboviruses, belonging to the Simbu serogroup of the genus Orthobunyavirus, often cause congenital malformations and reproductive loss in cattle. The recent occurrences of such reproductive problems suggest that new arboviruses have emerged in Japan. However, there is no information on the presence of these viruses in South Korea. The aim of this study was to determine the presence of antibodies for Akabane, Aino, Peaton, Sathuperi, and Shamonda viruses in four regions, namely Gyeonggi, Jeollabuk, Jeollanam provinces, and Jeju Island of South Korea by a serum neutralization test. Antibody positivity against Akabane, Aino, Peaton, Sathuperi, and Shamonda viruses was detected in the country, with average seropositive rates of 10.4, 4.5, 1.1, 4.9, and 5.6%, respectively.
When foot-and-mouth disease (FMD) occurs and a “vaccination-to-live” policy is adopted in a country, the country must perform serological surveillance of a nonstructural protein (NSP) of FMD virus. The NCPanaftosa kit is the only kit for detecting antibodies to NSPs that is officially recognized as the reference regent by the World Organization for Animal Health; however, it is only used in South American countries. In this study, the specificity and sensitivity of the NCPanaftosa kit were compared with those of the PrioCHECK kit sold by an international company. Results in this study suggest that the PrioCHECK kit performs similarly to the NCPanaftosa kit in detecting antibodies to the NSP in the cattle population.
The giraffe (Giraffa camelopardalis) is a difficult animal to keep in captivity as it has high mortality due to nutrition-related disorders, perhaps because the giraffe’s condition is difficult to evaluate. Image analysis techniques have recently become popular and may be useful for evaluating the giraffe’s somatotype. The present study aimed to evaluate the giraffe somatotype using a three-dimensional laser measurement device, and to examine the usefulness of this method. First, ten zoo staff members visually evaluated the somatotypes of three giraffes housed at Kanazawa Zoological Gardens, Japan. Next, three-dimensional point cloud datasets were obtained from these giraffes using the device. The point cloud datasets indicated that the cross-sectional area and width of the largest giraffe’s body were large in the abdominal region in the transverse sections. However, by visual examination, the ten zoo staff members deemed a different giraffe to be the largest. These results indicated that the three-dimensional laser measurement device could be used to evaluate giraffe somatotype in detail, and that this method may be an alternative to visual evaluation.
The prevention and control of infectious diseases transmitted by wildlife are gaining importance. To establish effective management strategies, it is essential to understand the population structure of animals. Raccoon dogs (Nyctereutes procyonoides) in South Korea play a key role in the maintenance of food web stability and possess genetic compositions that are unique compared to those in other areas. However, wild raccoon dogs play another role as the main host of various infectious diseases. To establish long-term strategies for disease management, we investigated the genetic structure and possible geographic barriers that influence the raccoon dog population in South Korea by analyzing 16 microsatellite loci. The present study showed that mountains were the major factors responsible for genetic structuring, along with distance. We proposed potential management units (MUs) for raccoon dogs based on the genetic structuring and gene-flow barrier data obtained in this study. Four MUs were suggested for the Korean raccoon dog population (Northern, Central, Southwestern, and Southeastern). The Korean raccoon dog population structure determined in this study and the proposed MUs will be helpful to establish pragmatic strategies for managing Korean raccoon dog population and for preventing the transmission of infectious diseases.
Subulura (Murisubulura) andersoni (Cobbold, 1876) (Nematoda: Subuluridae) is redescribed in detail using light and scanning electron microscopy based on materials collected from Bunomys chrysocomus and Bunomys andrewsi (Rodentia: Muridae) of Sulawesi, Indonesia, to provide basic reference data for identification. Comparison was made among congeners from adjacent areas, especially Subulura (Murisubulura) suzukii Yagi and Kamiya, 1981 of Japan. Because morphological differences distinguishing S. (M.) andersoni from S. (M.) suzukii and S. (M.) sipiroki were only the thickness of female tail and length of male tail, respectively, further study using DNA sequence analysis is necessary to prove their distinctness.