Localization of Toll-like receptors (TLRs) in the exocrine glands associated with the rat alimentary tract was immunohistochemically studied using anti-TLR antibodies. TLR-2, -4 and -9 were detected in the secretory granules of acinar cells or the luminal substances of the gustatory gland, extraorbital lacrimal gland, Harderian gland, proper gastric gland and pancreas. TLR-2 and -9 were also detected in the mucous acinar cells of the sublingual gland. Positivity for all TLRs was found in the striated borders of columnar epithelial cells and the luminal substances of the intestinal crypts throughout the small intestine, and also in the goblet cells throughout the large intestine. Only TLR-4 was detected in the secretory granules of Paneth cells. A reduction of TLR-4-positive secretory granules and the formation of TLR-4-positive vacuoles were found in the ileal Paneth cells under the hyper-proliferation of indigenous bacteria. In the apical to middle intervillous portions of the ileum, Gram-positive bacterial colonies were significantly more abundant than Gram-negative bacterial colonies, whereas this difference disappeared in the basal intervillous portions. These findings suggest that there are distribution differences in the secretory sources of soluble TLRs that possibly neutralize their luminal ligands, in the rat alimentary tract. Therefore, the bacterial ligand-recognition system composed of the membranous TLRs of villous columnar epithelial cells and soluble TLRs from crypt epithelial cells might contribute to host defense mechanisms for the selective elimination of Gram-positive bacteria rather than Gram-negative bacteria in the rat small intestine.
Previously, the specific antibody-mediated persorption of antigenic molecules and particulates from the small-intestinal lumen into the peripheral blood was clarified in rats, but the intermediation of the receptor for the specific antibodies was not. In this study, the existence of receptor for the specific antibody was experimentally examined in the rat small intestine. Glutaraldehyde-fixed sheep erythrocytes (SEs) coated by Fc-fragments of IgG (IgG-Fc), (Fab′)2-fragments of IgG (IgG-Fab) or bovine serum albumin (BSA), were injected into 3 jejunal loops each 2 cm in length in non-orally pre-immunized rats, respectively. Thirty minutes after the injection, IgG-Fc-coated SEs were significantly more engulfed by villous columnar epithelial cells than Fab- or BSA-coated SEs. The most frequent absorption sites were the intestinal villous apices. The IgG-Fc-coated SEs were adhered to the striated borders and were engulfed by villous columnar epithelial cells. IgG-Fc-coated SEs passing through the epithelial cells were also detected in the subepithelial blood capillaries just beneath the villous epithelium, but not in the connective tissue and the lymph vessels. These findings suggest that the absorption of luminal antigenic particulates is probably mediated by the Fc-receptor, and that the absorbed antigenic particulates are directly transferred to the hepatic portal blood by passing through the endothelium of the subepithelial blood capillaries.
A serological survey on bovine brucellosis was carried out 3 times between 2007 and 2009 in 3 districts (Kiboga, Mpigi and Kiruhura) in western Uganda and 2 (Kumi and Mbale) in the east employing the rose bengal test (RBT) for infected-herd screening and an indirect ELISA (iELISA) for testing the serostatus of individual animals. The animal prevalence was significantly higher in the 3 districts of the west (mean 21.5% in 2009) compared with the 2 districts (mean 3.4% in 2008) in the east (P<0.0001), though a significant difference was not observed between Kumi and Mpigi in 2008. In the west, it was the lowest in Mpigi, but a significant increase was observed between 2008 (5.3%) and 2009 (30.0%), as in Kiruhura, in which the prevalence increased from 8.1% in 2007 to 16.8% in 2009. A similar trend was also observed in Kumi, namely, the seropositivity significantly increased from 2.3% in 2007 to 6.2% in 2008 and became remarkably higher than in Mbale (0.64%). As a result, the farm prevalence was also higher in the west, especially in Kiboga in 2007 (77.8%) and 2008 (65.6%), and Mpigi in 2009 (70.8%). The linear predictor of the fitted generalized linear model proved that the logit of RBT positivity increased linearly over the increase in percent positivity values. This study demonstrated an example of an unaided self-help survey as one of the control measures in Uganda.
Interferons (IFNs) are key mediators that activate host defense mechanisms against viruses. The recently identified mammalian Type III IFN has biological effects similar to type I IFN. However, the biological effects of type III IFN have not yet been characterized in birds. We compared the effects of chicken type III IFN (IFN-λ) with type I (IFN-β) and type II (IFN-γ) IFNs on IFN-stimulated genes (ISGs) using recombinant proteins expressed in Escherichia coli. Recombinant chicken IFN-λ inhibited influenza virus replication and induced the mRNA expression of the ISGs, Mx and OAS, in chicken embryonic fibroblasts (CEFs) in a dose-dependent manner. However, the effective dose of IFN-λ was higher than that of IFN-β and IFN-γ. Furthermore, the effect of IFN-λ on induction of Mx and OAS was lesser than that of IFN-β, but comparable to that of IFN-γ. These results indicate that chicken IFN-λ has the potential to induce ISGs and inhibit viral replication in chicken cells.
Four methods were evaluated for isolating exosomes from bovine milk: (1) ExoQuick precipitation, (2) ultracentrifugation with ExoQuick precipitation, (3) ultracentrifugation with density gradient centrifugation, and (4) human milk exosome isolation. Methods 1 and 4 failed due to differences between bovine and human milk. Exosomes were efficiently isolated by ultracentrifugation with either ExoQuick precipitation (method 2) or density gradient centrifugation (method 3). The highest yield of exosomes was achieved using ultracentrifugation with ExoQuick precipitation, whereas higher quality exosome isolation with intact morphological structures was achieved by ultracentrifugation with density gradient centrifugation.
This report describes an enzyme-linked immunosorbent assay (ELISA) for tissue-bound metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) and the application to residue analysis in cultured fish samples. The residue is monitored as a marker for the drug furaltadone. The assay enables the detection of protein bound AMOZ in the form of a 2-nitrophenyl derivative (2-NP-AMOZ) in sample supernatant or extract after acid hydrolysis and derivatization with o-nitrobenzaldehyde. Polyclonal rabbit antibodies were produced with a new immunogen hapten, 2-NP-HXA-AMOZ. The new ELISA had adequate analytical sensitivity (IC50 value 0.325 μg kg-1; limit of detection 0.1 μg kg-1) to determine a trace of AMOZ residue and had a high selectivity. Recoveries of AMOZ fortified at the levels of 0.1, 0.5 and 1.0 μg kg-1 ranged from 89.8 to 112.5% with coefficients of variation of 12.4-16.2% over the range of AMOZ concentrations studied. The results obtained with the ELISA correlated well with those obtained by commercial test kits for 150 tested samples (r=0.984). The results suggest that the developed ELISA is a highly specific, accurate, and sensitive method suitable for high throughput screening for AMOZ residues.
Gastrointestinal stromal tumor (GIST), a mesenchymal neoplasm affecting the gastrointestinal tract, shows a variety of clinical behaviors from inactive benign to aggressive malignant in dogs. In this study, the feasibility of using clinically significant ultrasonographic features to predict the metastatic potential of canine GIST was investigated through comparison with actual metastatic incidence and findings of malignancy obtained by postoperative pathological examination. Ultrasonographic features, including large tumor size, irregular margin and heterogeneous internal echogenicity with large hypoechoic areas, related closely with the presence of metastasis as well as a high-risk ranking by the human classification system according to pathological findings. Based on these ultrasonographic features, the potential of metastasis in canine GIST could be preoperatively predicted.
An 8-month-old Holstein heifer with cervical enlargement was suspected of thymic lymphosarcoma given clinical signs of depression, tendency to lie down, cervical mass, jugular vein distension, conjunctival hyperemia, and ruminal tympany. Unilateral Horner’s syndrome was also observed. Increased serum total lactate dehydrogenase (LDH), LDH isozyme (LDH-2, and LDH-3) and serum thymidine kinase activity were observed. The findings of fine needle aspiration cytology of the cervical mass revealed large lymphoblasts with mitoses present. These findings strongly suggested the diagnosis of lymphosarcoma. Necropsy revealed a large mass in the cervical thymic region, which compressed the esophagus and trachea. Cranial masses in the frontal sinus and multiple extradural sites throughout the cranial vault were also recorded. Histopathological examination confirmed the diagnosis of thymic lymphosarcoma and demonstrated the brain involvement of neoplastic lymphoid cells in the cerebrum. This is a rare clinical case of thymic lymphosarcoma accompanied by brain metastasis in a Holstein heifer.
Trehalose has several novel anti-inflammatory and cell-protective functions. We hypothesized that lyophilized aspirin/trehalose could decrease the severity of aspirin-induced gastropathy. Thirteen dogs were assigned into aspirin, lyophilized aspirin/trehalose, and control groups, and the gastric lesions were assessed on gastroscopy with the modified Lanza scale. Another 6 dogs were used to measure the plasma aspirin concentration by high-performance liquid chromatography after aspirin or lyophilized aspirin/trehalose administration. The results indicated that lyophilized aspirin/trehalose induced less gastric ulceration than aspirin despite maintaining therapeutic concentrations of plasma aspirin in both the groups. Lyophilized aspirin/trehalose might be a solution to decrease aspirin-induced gastropathy.
A 9 year-old, neutered, male French Bulldog showing cluster seizures was diagnosed with a glioma in the right piriform cortex by MRI. Hypofractionated radiation therapy (RT) was performed using a linear accelerator. Although the lesion had involuted significantly at 2 months after RT, recurrence was observed at 4 months after RT. Chemotherapy was started using CCNU (60 mg/m2 every 6–9 weeks) and was continued for one year. Follow-up MRI revealed involution of the lesion and the intervals of CCNU were increased to every 9–14 weeks. Two years after the first presentation, the dog suffered status epilepticus, followed by deficits of left sided postural reaction with cognitive dysfunction. The dog died on day 910, and histopathological diagnosis confirmed anaplastic oligodendroglioma.
Euglycemic-hyperinsulinemic glucose clamp (EHGC) method is a gold standard for assessing insulin resistance in humans. However, this method has yet to be commonly used with dogs, due to the requirement of frequent blood sampling for glucose measurement and adjusting glucose infusion rate (GIR). The purpose of this study was to evaluate insulin resistance, induced either by Cushing Syndrome (CS) or diestrus in dogs, as determined by GIR by EHGC, using an artificial pancreas apparatus. Twenty animals were used in this study with ten (7 females and 3 males) serving as healthy controls, four (3 females, 1 male) diagnosed with CS, and six (all females) undergoing diestrus. A higher GIR value indicates increased insulin sensitivity and lower insulin resistance. GIR of healthy control animals was determined to be within a reference range of [10.6–21.3] with a median of 15.2 mg/kg/min. In comparison, the CS group had a median of 5.4 mg/kg/min; whereas the diestrus group exhibited a median of 8.9 mg/kg/min. Insulin resistant animals suffering from CS and undergoing diestrus demonstrated reductions of 65 and 40% in GIR, respectively; thus indicating differences in degree of insulin insensitivity can be discerned using the EHGC method.
Ferulic acid plays a neuroprotective role in cerebral ischemia. The aim of this study was to identify the proteins that are differentially expressed following ferulic acid treatment during ischemic brain injury using a proteomics technique. Middle cerebral artery occlusion (MCAO) was performed to induce a focal cerebral ischemic injury in adult male rats, and ferulic acid (100 mg/kg) or vehicle was administered immediately after MCAO. Brain tissues were collected 24 hr after MCAO. The proteins in the cerebral cortex were separated using two-dimensional gel electrophoresis and were identified by mass spectrometry. We detected differentially expressed proteins between vehicle- and ferulic acid-treated animals. Adenosylhomocysteinase, isocitrate dehydrogenase [NAD+], mitogen-activated protein kinase kinase 1 and glyceraldehyde-3-phosphate dehydrogenase were decreased in the vehicle-treated group, and ferulic acid prevented the injury-induced decreases in these proteins. However, pyridoxal phosphate phosphatase and heat shock protein 60 were increased in the vehicle-treated group, while ferulic acid prevented the injury-induced increase in these proteins. It is accepted that these enzymes are involved in cellular metabolism and differentiation. Thus, these findings suggest evidence that ferulic acid plays a neuroprotective role against focal cerebral ischemia through the up- and down-modulation of specific enzymes.
Germinal center/lymphoid follicle area ratio and CD3/CD20-positive area ratio were calculated for the spleen, submandibular and mesenteric lymph nodes, and Peyer’s patches in cynomolgus monkeys treated orally with cyclosporin A (CsA), an immunosuppressant which blocks Ca2+/NFAT signaling. A difference in hypocellularity between lymphoid organs was observed after CsA administration in a dose-dependent manner. Regarding drug efficacy, the highest susceptibility to CsA tended to be shown in the Peyer’s patches, and susceptibility then descended in the order of the spleen, mesenteric lymph nodes, and submandibular lymph nodes. It was shown in the present study that decreases in germinal center area and CD3-positive area were sensitive indicators of the efficacy of CsA for lymphoid organs and tissue in cynomolgus monkeys.
A full-length cDNA sequence of canine Na-dependent neutral amino acid transporter (ASCT2) and its distribution were determined. The sequence was 2,090 bp long and was predicted to encode 544 amino acid polypeptides. The amino acid sequence deduced from canine ASCT2 showed 90% similarity to that of humans and mice. Northern blot analysis revealed ASCT2 expression in the kidney, heart, lung and muscles, and Western blot analysis using anti-human ASCT2 antiserum detected the bands at 60 and 65 kDa in membrane protein of the lung. RT-PCR analysis revealed ASCT2 expression in all the tissues examined.
We herein examined the sensitivity of Hep G2 human hepatoma cells to Bacillus cereus emetic toxin. Hep G2 cells were treated with the emetic toxin, and the cell shape was observed. The same experiments were performed for comparison purposes, using HEp-2 cells, which are currently used by most laboratories for a bioassay of the emetic toxin. Hep G2 cells showed clearer vacuolation in the cytosol within 2 hr and required a shorter incubation period than HEp-2 cells (10 hr). The number of vacuoles in the Hep G2 cells was greater, and the size of the vacuoles was larger than those observed in HEp-2 cells. The minimal concentration of the emetic toxin required to induce the vacuolation of Hep G2 cells was 0.04 ng/ml. The concentration for the HEp-2 cells was 1 ng/ml. These findings indicate that Hep G2 cells show higher sensitivity to the emetic toxin. Hep G2 cells may be superior to the currently used HEp-2 cells for the bioassay of the emetic toxin.
The cross-reactivity of Japanese encephalitis virus (JEV)-immunized chicken sera and West Nile virus (WNV)-immunized chicken sera in serological tests, such as the IgG indirect ELISA (IgG-ELISA), hemagglutination inhibition test (HI) and plaque reduction neutralization test (PRNT), for JEV and WNV were examined. The mean JEV/WNV ELISA ratio in IgG-ELISA of JEV-immunized sera was significantly higher than that of WNV-immunized sera (P<0.01). JEV-immunized chicken sera did not cross-react in the WNV HI. However, all the WNV-immunized chicken sera cross-reacted in the JEV HI. JEV-immunized chicken sera did not show the WNV neutralization titer at 90% plaque reduction, and WNV-immunized chicken sera did not showed the JEV neutralization titer at 90% plaque reduction. Therefore, it is possible that chicken JEV serum can be distinguished from WNV serum by comparing the titers of IgG-ELISA, HI or PRNT respectively.
The aim of this study was to assess age-related changes and anatomic variation in trabecular bone mineral density (tBMD) using quantitative computed tomography (QCT) in normal cats. Seventeen normal cats were included in this study and divided into the following 3 age groups:<6 months (n=4), 2–5 years (n=10) and >6 years (n=3). A computed tomographic scan of each vertebra from the 12th thoracic to the 7th lumbar spine and the pelvis was performed with a bone-density phantom (50, 100 and 150 mg/cm3, calcium hydroxyapatite, CIRS phantom®). On the central transverse section, the elliptical region of interest (ROI) was drawn to measure the mean Hounsfield unit (HU) value. Those values were converted to equivalent tBMD (mg/cm3) by use of the bone-density phantom and linear regression analysis (r2 >0.95). The mean tBMD value of the thoracic vertebrae (369.4 ± 31.8 mg/cm3) was significantly higher than that of the lumbar vertebrae (285 ± 58.1 mg/cm3). The maximum tBMD occurred at the T12, T13 and L1 levels in all age groups. There was a statistically significant difference in the mean tBMD value among the 3 age groups at the T12 (P<0.001), T13 (P<0.001) and L4 levels (P=0.013), respectively. The present study suggests that age-related changes and anatomic variation in tBMD values should be considered when assessing tBMD using QCT in cats with bone disorders.
The object of this study was to evaluate the usefulness of measuring the differences in the values of the serum total protein (DVSTP) concentration of foals and the refractometry index (DVRI) of the milk of dams before and after nursing of the colostrum for assessing failure of passive transfer (FPT) in foals. Serum samples from 31 foals were collected before the first nursing and other 1 to 6 times between 4 and 24 hr after birth. Paired colostrum and milk samples were collected from 14 of their dams at the same time. Serum samples were analyzed for IgG concentration using a single radial immunodiffusion (SRID) test (98 samples) and total protein concentration using a temperature-compensating refractometer (98 samples). Colostrum and milk samples were analyzed for refractometry index (RI) using a Brix refractometer (71 samples). DVSTP concentration and DVRI were significantly correlated with serum IgG concentration. The negative predictive values (NPVs) of DVSTP concentration for detecting serum IgG concentrations<400 mg/dl and<800 mg/dl were 98.2% and 91.3% when the cutoff value is set to 0.4 mg/dl and 0.8 mg/dl, respectively. Furthermore, the NPVs of DVRI for detecting serum IgG concentrations<400 mg/dl and<800 mg/dl were 97.3% and 96.3% when the cutoff value is set to 6% and 10%, respectively. The results suggest that measurement of DVRI is useful in assessing FPT as an initial “stall-side” screening test, because it is easy, inexpensive to perform and allows for rapid interpretation.
We investigated the effects of exposure time and concentration of trichostatin A (TSA) on in vitro development and quality of bovine SCNT embryos. At multiple time points, the relative expression of genes related to pluripotency and reprogramming was analyzed in order to assess the quality of preimplantation embryos cultured in media with TSA using real-time PCR. Development into blastocysts was higher in 100 nM TSA than in controls (35.96 vs. 28.30%, P<0.05). Study of 100 nM TSA exposure time showed development into blastocysts was higher during both short-term and long-term exposure than in controls (36.17 and 34.04 vs. 23.45%), but there was no significant difference between TSA groups. Nanog expression in blastocysts after long-term TSA exposure was similar to that in IVF blastocysts and greater than in controls and short-term exposed embryos. The Oct4 levels in the short-term exposure group were similar to those of IVF blastocysts, while Oct4 expression in long-term exposed embryos was significantly higher than in other groups. Measurement of DNMT1 and HDAC1 in blastocysts showed a similar expression profile among IVF and TSA groups regardless of treatment duration. In conclusion, this study suggests that TSA treatment after SCNT in bovine embryos can improve in vitro development of embryos by increasing the blastocysts formation and positive reprogramming of the reconstructed embryo genome caused by downregulation of DNA methylation and up-regulation of pluripotency.
The objectives of the present study were to advance the development of standard operating procedures for sows and piglets during farrowing and lactation in Japanese herds by surveying management procedures and to examine the relationships between the procedures and herd reproductive performance. In 2009, 115 herds using the same recording system were asked to complete a questionnaire about their management procedures. Data from 96 (83.5%) returned questionnaires were coordinated with the respective herd reproductive performance. The participating herds were classified into two groups based on the upper 25th percentile of pigs weaned per mated female per year: high-performing (>23.8 pigs) or ordinary herds. ANOVA was used to compare the procedures between two herd groups. Modeling with backward elimination was performed to establish the most important procedures for herd performance. More high-performing herds practiced farrowing induction and high-performing herds also had a higher percentage of farrowing-induced sows than ordinary herds (P<0.05). Modeling showed that herds feeding lactating sows with dietary fiber had 1.4% lower preweaning mortality risk than those that did not (P<0.05). Herds practicing fostering techniques or using nurse sows had 0.2 kg heavier average pig weaning weight than those not using these procedures (P<0.05). There was no association between pigs born alive and any of the surveyed management procedures. Based on these results, we recommend improving performances in breeding herds by feeding lactating sows with dietary fiber, performing fostering techniques and using nurse sows.
This study was undertaken to develop a simple and practical method to control the time of ovulation in cynomolgus monkeys. Diets containing a synthetic gestagen, levonorgestrel (LNG) were given daily to normally cycling female monkeys for 2 weeks, and plasma concentrations of estradiol-17β and progesterone were determined by EIA in order to estimate the time of ovulation. Doses of LNG (0, 3.2, 8, 20, 50, or 125 μg) were given from Day 2 (Day 0 =the first day of menstruation) through Day 15. The numbers of days from the last administration of LNG to the estimated ovulation in the groups treated with LNG at 20 μg and above were significantly greater than those in the controls, and the values in the group treated with LNG at 50 μg were within a narrow range. In a second experiment, LNG was administered at 50 μg in different phases of the menstrual cycle (Days 9–22, 16–29, and 23–36), and the results indicated that ovulation occurred more than 12 days after the last administration in all monkeys, and the number of days from the last administration of LNG to the estimated ovulation in the group treated on Days 16–29 (luteal phase) was significantly greater than that in the group treated on Days 23–36. These results indicate that daily provision of a diet containing 50 μg LNG could be applicable for delaying ovulation, and suggest that the total level of (exogenous and endogenous) progestins is critical for determining the length of ovulation delay in cynomolgus monkeys.
The reproductive performance of postpartum cows is affected by factors such as suckling and nutrition. We investigated the effect of a restricted suckling period on the superovulatory response and the fertility after flushing in postpartum Japanese Black cows. Forty-seven postpartum cows were used in this study. At 7 days postpartum, the cows were divided into 2 groups: (1) continuous access to calves from birth to weaning at 3 months postpartum (ad libitum suckling group; n=20); and (2) twice daily suckling to the calves penned adjacent to them (restricted suckling group; n=27). All cows were initiated a superstimulatory treatment with a controlled internal drug releasing device and follicle stimulating hormone at 40 days postpartum. Embryos were nonsurgically collected at 7 or 8 days after estrus. After uterine flushing, the cows were again used for reproduction. There were no significant differences between the ad libitum and restricted suckling groups in terms of the numbers of transferable (6.7 ± 5.4 versus 7.9 ± 7.0) and freezable embryos (5.5 ± 4.9 versus 6.2 ± 7.0). In contrast, the interval to the first estrus after flushing in the restricted suckling group was lower (P<0.05) than that in the ad libitum suckling group (8.9 ± 5.7 days versus 27.9 ± 24.2 days). These results suggest that restricted suckling in postpartum Japanese Black cows does not affect the superovulatory response and embryo quality; however, it improves their fertility after flushing.
The present study focused on the in vitro infection of Madin-Darby bovine kidney (MDBK) cells and bovine peripheral blood mononuclear cells (PBMCs) from naÏve animals with non-cytopathic (ncp, BVDV-1b NY-1) and cytopathic (cp, BVDV-1a NADL) strains. Infections with 0.1 and 1 multiplicity of infections (MOI) and incubation times of 18 and 36 hr were compared. Twelve BVDV naÏve heifers were enrolled to collect PBMCs. The viral loads in MDBK cells and in PBMCs after in vitro infections were measured by real-time polymerase chain reaction (PCR) assays. The highest viral loads were measured at 1 MOI and 36 hr post infection in both cell systems and the lowest at 0.1 MOI and 18 hr with the exception of the cp strain NADL in PBMCs, for which the highest viral load was observed at 0.1 MOI and 36 hr. Viral load mean values were higher for the cp strain than the ncp strain irrespective of the extent of the infection period and MOI. The models of infection studied uncovered different replication activities respectively according to the biotype of virus, the cell substrate and the duration of infection. Replication tends to be higher in PBMCs, particularly at low MOIs and for the ncp strain.
As a tool to understand the role of mucins in the infection of respiratory viruses, we established cell lines stably expressing inactive mutants of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3), which initiates O-glycosylation of mucins. We introduced single amino acid mutations into the regions essential for the enzyme activity of GALNT3 using the expression plasmid of human GALNT3 and transfected the mutant constructs into a human bronchial epithelial cell line, BEAS-2B. We showed that although the mutants of GALNT3 exhibit an authentic localization at the Golgi apparatus, the glycosylation pattern of the expressing cell lines appeared to be different from that of the cells expressing wild-type GALNT3. These results suggested that the established cell lines express inactive forms of GALNT3 and might be useful in investigation of the significance of O-glycosylation of mucins in respiratory virus infections.