For the purpose of investigation of working mechanisms in endocrine disruptors, we evaluated the dose-related effects of fetal and/or neonatal exposure to an estrogenic compound on the male reproductive organs in adult mice, particularly with respect to gene expression of steroidogenic acute regulatory protein (StAR). The pregnant ICR mice were given subcutaneous injections of 10 μg/day/animal of diethylstilbestrol (DES) to subject the fetal mice to in utero exposure (IUE). Subsequently, the newborn male mice were subjected to neonatal exposure (NE) by treatment with vehicle or 0.1-10 μg/day/animal of DES. Fertility rates of each group were as follows: control, 100%; IUE only, 60%; IUE+NE 0.1 μg, 25%; IUE+NE 1 μg, 0%; IUE+NE 10 μg, 0%. In general histology, germ cell layers in the seminiferous tubules were thinned in the group of IUE+NE 10 μg. Hypoplasia of the Leydig cells, in which the staining intensity of eosin was diminished, was also observed in the groups of IUE+NE 0.1-10 μg. The androgen receptor (AR) and estrogen receptor alpha (ERα) immunoexpression in the Leydig cells of IUE+NE 1-10 μg was slightly lower than that in the controls. Long-term dysfunction of the hypothalamo-pituitary-testicular axis, including sustained hypoproduction of gonadotropin and testosterone, and altered expressions of steroid hormone receptors and StAR genes were observed. The hypothalamo-pituitary control of gonadotropin secretion may be affected by the smaller doses of estrogenic agents than the reproductive organs. Furthermore, the fertility rate in the male mice exposed to this estrogenic agent was closely correlated with the testosterone levels, and even more so with the rate-limiting factor of steroidogenesis, StAR. This finding suggests that endocrine disruptors have an important pronounced effect on StAR gene expression.
The possibility of persorption of bovine serum albumin (BSA) molecules from mucous epithelial cells and its mechanism were investigated in rats orally pre-immunized by BSA for 14 consecutive days. In the small and large intestines, both the BSA antigen (BSA-Ag) and its specific antibody (SpAb) were absorbed by the epithelial cells at the late apoptotic stage (ApoEp), and were subsequently transcytosed by membranes of the small vesicles. The basal cytoplasms containing highly-concentrated BSA-Ag and SpAb were occasionally fragmented into small cytoplasmic droplets that were secreted into the lamina propria. In Peyer's patches, both BSA-Ag and SpAb were more actively absorbed and transcytosed toward the dome area by the ApoEp of the dome apex than by the M cells. BSA-Ag and SpAb were finally persorbed into the portal blood and lymph, but were never secreted into the bile. They were also engulfed by macrophage-like cells in the villous lamina propria, mesenteric lymph node and spleen, and by hepatocytes in the liver. These findings suggest that sensitized soluble luminal antigens are taken up by ApoEp in the small intestine and are finally persorbed into the peripheral blood. The uptake of luminal antigen might be mediated by its luminal SpAb.
The immunolocalization of the efferent duct and the epididymis in canine was firstly examined using an the immunohistochemical method with the canine carbonic anhydrase (CA) -I, CA-II and CA-III antisera. The efferent duct was immunonegative for all present canine CA antisera. However, some slender shaped epithelial cells in the head and body segments of the epididymal duct were intensely reacted to the CA-II antiserum. These results suggested that the CA-II might be controlled in the luminal environment in the head and body segments of the canine epididymis by the proton and bicarbonate balance for the maintenance of the spermatozoal stability and movement.
Reticuloendotheliosis virus (REV) is widespread in the world. No related data has been reported in Taiwan. To determine the REV infection status, antibody determination and virus isolation were performed on chickens in Taiwan. The results revealed that serological flock prevalence for the REV antibody reached 92.8% (39/42) amongst breeders (> 16 weeks old). Two different REV isolates were identified by reverse transcriptase-polymerase chain reaction, electron microscopic, immunofluorescent, and western blot assays after isolation. One of these viruses was isolated from a broiler breeder farm and the other was isolated from a Taiwan Country Chicken farm. Despite their different origins, the percent identity of the nucleotide sequences of the env gene of these two isolates was 99.7%. These two strains were similar to the FPV-UI-REV strain, featuring 99.7% and 99.8% percent identity. Indeed, REV infection would appear to be quite common amongst chickens.
Salmonella enterica subspecies enterica serovar Gallinarum biovar Gallinarum is the causative agent of fowl typhoid in chickens, outbreaks of which have devastated poultry populations in Korea since 1992. In order to identify genetic differences among S. Gallinarum isolates, bacteria were examined using the random amplified polymorphic DNA (RAPD) method. Of 13 arbitrary primers screened initially, the primer designated as universal rice primer-6 (URP-6) was selected for subsequent typing assays because it produced a distinctive and reproducible DNA fingerprint for a S. Gallinarum reference strain. URP-6-based RAPD analysis assigned 30 S. Gallinarum isolates into 6 types, with 26 isolates (86.6%) belonging to 2 major RAPD types. The distribution of virulence genes in S. Gallinarum isolates was examined by Southern hybridization. All tested isolates had the invasion gene, invA, the virulence plasmid gene, spvB, and the S. Enteritidis fimbrial gene, sefC. The distribution of virulence genes among S. Gallinarum isolates did not correlate with any specific RAPD type.
Twenty-six out of 46 representative lactic acid bacteria (LAB) that we isolated from 36 dogs in a previous study were agglutinated by concanavalin A (ConA) at a concentration of 0.1563 mg/ml, while isolates did not agglutinate without the addition of ConA. Amongst the isolates, L. reuteri, L. mucosae, and E. canintestini agglutinated strongly, while L. gallinarum, L. kitasatonis, L. acidophilus, L. saerimneri, B. animalis ssp. animalis, P. acidilactici, and E. hirae did not agglutinate. ConA-agglutination of LAB was specifically inhibited by D-glucose, D-galactose, and D-mannose at a concentration of 1.563 mg/ml. Among the sugars, ConA-agglutination was strongly inhibited by D-mannose, while the inhibition level by D-glucose and D-galactose were lower than that of D-mannose. ConA- agglutination of all the LAB isolates was inhibited by D-mannose, except for L. reuteri (one species) and L. mucosae (two species). ConA-agglutination of Bifidobacterium spp. was inhibited by only D-mannose. Based on our results, ConA-agglutination of LAB seems to be strain-specific, but not species-specific.
This study was performed to examine the bacteriological findings in 58 mammary secretions from 15 heifers at 4-5 weeks before parturition, and to evaluate whether a high prevalence of S. aureus infection in lactating cows affects the occurrence of S. aureus infection in prepartum heifers in the dairy herd. A total of 86.7%(13/15) of the heifers and 60.3%(35/58) of quarter milk samples from the heifers were bacteriologically positive. No S. aureus isolate was detected in mammary secretions from the heifers. Coagulase-negative staphylococcus (CNS) species were predominantly detected in 54.3%(19/35) and Streptococci other than Streptococcus agalactiae were isolated from 22.9%(8/35) of the quarters. A high S. aureus prevalence in the herd may not necessarily be a decisive factor for S. aureus infection in heifers.
In total, 582 sera from 116 black goat herds were analyzed by a commercially available ELISA kit to monitor the seroprevalence of Mycobacterium avium subspecies paratuberculosis (Mpt) in Korean black goats (Capra hircus aegagrus). The mean number of goats sampled per herd was 5.11, 4.66, and 5.38 for the northern, central, and southern regions of Korea, respectively. The apparent regional prevalence of Mpt was estimated at 18.2-38.2% and 4.6-15.3% for herds and goats, respectively. The Mpt-positive goats were predominantly detected in the south (n=28), compared to either the northern (n=9) or central (n=11) regions (χ=14.459, P<0.05). Our findings indicate that Mpt is prevalent among the goat population, but regional variation exists.
High linear energy transfer (LET) heavy charged particles have previously been applied clinically to human cancer radiotherapy because of their excellent physical properties of selective dose distribution and higher relative biological effectiveness (RBE) for human; however, such an approach has yet to be applied to cat patients. The present study investigates the biological effectiveness of low-LET γ-rays (0.2 keV/μm) compared to high-LET carbon ions (114 keV/μm) in feline T- lymphocyte FeT-J cells. Clonogenic survival analysis revealed that the RBE value of carbon ions was 2.98 relative to a 10% survival dose (D10) by γ-rays, and that the inactivation cross-section in cells exposed to γ-rays and carbon ions was 0.023 and 38.9 μm2, respectively. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) analysis revealed that TUNEL-positive frequency in carbon-irradiation cells is higher than for γ-irradiated cells against exposure to the same physical doses, but that very little difference in TUNEL-positive frequency is observed between cells exposed to the respective D10 dose of γ-rays. Our data thus indicate that carbon ions are more effective for cell killing than γ-rays at the same physical doses, but kill cells to an extent that is comparable to γ-rays at the same biological doses. Carbon ion radiotherapy is therefore a promising modality for cat patients.
To investigate the clinical utility of C-reactive protein (CRP) in idiopathic polyarthritis (IPA), its concentration was measured in dogs with IPA. The CRP concentration was markedly increased in all the IPA dogs at the time of diagnosis and decreased significantly in response to the initial corticosteroid treatment; this indicated that CRP can be used as an index for therapeutic response in IPA cases. Furthermore, at 6 months after the diagnosis, a significant association was observed between the CRP concentration at follow-up (6-13 days after the treatment was started) and the frequency of medication ("no or seldom-medicated (NSM)" groups or "continuing medication (CM)" groups). These results suggest that the initial response of CRP to corticosteroid treatment may be a prognostic factor of canine IPA.
Interleukin 2 (IL-2) is a T cell proliferation factor released by Th0- and Th1-type helper T cells and is an essential cytokine for immune responses. In the present study, recombinant glutathione S-transferase (GST)-guinea pig IL-2 (GPIL-2) fusion protein was prepared by Escherichia coli (E. coli) and by using this protein as an immunogen, monoclonal antibodies (mAbs) against GPIL-2 were produced to establish a basis for a research on immune responses in guinea pigs. Three stable hybridoma cell lines were established, and specific binding of each mAb to recombinant GPIL-2 produced by E. coli and insect cells infected with recombinant baculovirus was shown by enzyme linked immunosorbent assay (ELISA) and/or immunoblot analysis. Isotype analyses of these mAbs revealed that all three mAbs were IgG1 and had κ chain. Furthermore, assessment of their epitopes by competition binding assay indicated that the mAbs obtained in this study bound to three different epitopes. Thus, a sandwich ELISA based on the two mAbs specific to different GPIL-2 epitopes was developed for detection of GPIL-2, which had a sensitivity threshold of about 0.3 ng/ml of GPIL-2.
A protein chip based on surface plasmon resonance (SPR) was develpoped to measure the antibody (Ab) titers of classical swine fever virus (CSFV) using the recombinant gp55 protein as an antigen. The diagnostic potential of this SPR assay for detecting the Ab titers to CSFV gp55 was compared that of the enzyme-linked immunosorbent assay (ELISA) using 170 serum samples from 14 pig farms. The SPR assay was highly specific and sensitive, and there were no cross-reactions detected. There was a strong positive correlation between the SPR and ELISA titers (n=170, r=0.869, p<0.01). Therefore, the SPR label-free method is a valuable tool in the serodiagnosis of CSFV infection and determining Ab titers after vaccination.
Seven novel cell lines from canine histiocytic sarcoma (HS), three of which were disseminated cutaneous HS and four of which were synovial HS, were established. All of the established cell lines had the same morphological (by light and electron microscopic findings), cytochemical (α-naphthyl butyrate esterase-positive), and immunohistochemical (vimentin- and lysozyme-positive, and cyto-keratin-negative) characteristics as the original HS tumor cells. All of the established cell lines injected into nude mice subcutaneously produced solid tumors. Because the established cell lines also showed phagocytic and processing activities, the HS tumor cells appear to originate from the mononuclear phagocytic system cells, despite their differences in locations or organs.
In this study, a high amount of bioactive recombinant canine interferon-α subtype 4 (CaIFN-α4) was expressed in a baculovirus system. For easy purification, it was expressed as a CaIFN-α4 bearing histidine hexamer at the C-terminal region, designated CaIFN-α4His. CaIFN-α4His was detected in culture supernatants of insect cells infected with the recombinant virus using sodium dodecyl sulfate-polyarcylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue staining. The level of expression was very high, and approximately 1 mg of purified protein, with 5.0 × 107 units/mg, was obtained from 300 ml of culture supernatant. The purified product showed antiviral activity against Vesicular stomatitis virus on canine tumor cell line A72 and chicken embryo fibroblast cells.
Blood examinations and genotyping of Factor XI (F11) were performed in growth retardation Japanese Black cattle and their dams. Genotyping of F11 revealed that the recessive homozygous and heterozygous genotype frequencies were 5.2% and 50.0% in the Claudin-16 (CL-16) deficiency group (n=58), 0% and 14.2% in the renal dysplasia group (n=7), 0% and 26.1% in the non-CL-16 deficiency nephritis group (n=23), 8.9% and 46.7% in the hypogenesis syndrome group (n=45), 6.2% and 25.0% in the neonatal weak calf syndrome group (n=32), 9.1% and 38.6% in the respective dams group (n=44), 0% and 23.1% in the normal cattle group (n=13), and 5.9% and 38.2% in total (n=222), respectively. These results showed that the carrier rate of F11 deficiency was high in Japanese Black cattle, and that the CL-16 deficiency, hypogenesis syndrome, neonatal weak calf syndrome, and dams groups had a large amount of recessive homozygous genotype than the other groups. No abnormal bleeding was observed clinically in the present study, and 4 of the recessive homozygous dams showed normal growth and parturition.
Right ventricular (RV) Tei index (index of myocardial performance) has been demonstrated to be clinically useful in estimating RV function in various human cardiac diseases. The purposes of this study were to validate the correlation between RV Tei index and RV function obtained by cardiac catheterization in healthy dogs, and to evaluate the RV Tei index in dogs with tricuspid regurgitation (TR). In healthy dogs, the RV Tei index significantly correlated with the RV peak +dP/dt (r=-0.80, p<0.0001) and -dP/dt (r=0.69, p=0.0001). In normal dogs, the RV Tei index was not significantly correlated with heart rate, body weight, and age. The RV Tei index significantly increased in dogs with moderate to severe TR (0.39 ± 0.35, p=0.0015), filariasis (0.46 ± 0.16, p=0.0131), and trivial to mild TR and severe mitral regurgitation (MR; 0.61 ± 0.14, p=0.0017) when compared with the normal dogs (0.17 ± 0.10). In addition, the RV Tei index in dogs with TR significantly increased in association with pulmonary hypertension [PH(-), 0.19 ± 0.09; PH(+), 0.65 ± 0.14; respectively p<0.0001]. Our study has demonstrated that RV Tei index is a feasible approach to estimate RV function in dogs and is not influenced by heart rate, body weight, and aging. Further investigations are required to clarify the clinical significance of RV Tei index in dogs with right-sided cardiac diseases.
A 14-year-old, spayed female, domestic shorthair cat was referred to us with anorexia, pyrexia, and jaundice. Total bilirubin (TBIL) and feline trypsin-like immunoreactivity (fTLI) levels were remarkably high. Based on laparoscopic biopsy of the pancreas, the cat was diagnosed as having pancreatitis. As a result of treatment with a synthetic protease inhibitor and corticosteroid, the TBIL and fTLI values returned to normal and the clinical course was good.
The aim of the present study was to evaluate short term urinary NAG levels in a model of reduced kidney mass. The half and quarter kidney mass were made from ligation of the renal artery. Both groups decreased in the level of excreted NAG on day 1 and 2 after operation. On day 5 after operation, both groups achieved urinary enzyme levels comparable to that of the sham-operated group. The remaining compensated nephrons held normal range of excreted urinary NAG levels, although reduced number of nephrons resulted in a decline in urinary NAG levels.
We report a case of EMND in a heavy horse that was bred and trained in Hokkaido, Japan. Clinical symptoms included severe ataxia of all four limbs, tilted head, lethargy, and flaccid lips. Numerous axonal degenerations and swellings were observed in nuclei, mostly in the cerebellar dentate nucleus and the nucleus of the hypoglossal nerve, and in the ventral horn of the spinal cord. In the ventral horn of the spinal cord, neuronal degeneration, swelling, and/or necrosis were observed sporadically. The case was diagnosed as EMND from the clinical symptoms and pathological findings.
The clinical usefulness of antibodies against Babesia gibsoni detected by ELISA with recombinant P50 was examined in dogs in an area where B. gibsoni infection was endemic. Only 8 among 14 dogs with acute type B. gibsoni infection without a previous history of infection were positive. This high percentage of false-negative results is thought to be a weak point of ELISA as a diagnostic tool. However, 14 other anemic dogs with a confirmed history of B. gibsoni infection were all positive, thus confirming the higher sensitivity of ELISA in detecting a history of infection.
Two genes encoding immunodominant antigens, hlim2 and hlim3, were obtained from a salivary gland cDNA library of the hard tick, Haemaphysalis longicornis. The recombinant proteins were expressed in Escherichia coli as the GST fusion protein and used for immunization. We observed that the attachment rate of nymphal ticks fed on mice immunized with GST-hlim3 was significantly lower than that in the control group during the initial days of feeding. However, immunization with GST-hlim3 did not affect the engorgement rate of the ticks. In sharp contrast, GST-hlim2 did not influence the attachment rate and feeding period of ticks but had a significant reduction in the engorgement body weight. These data highlight the suitability of the 2 recombinant cement-like proteins for use in a cocktail vaccine.
Benign Theileria parasites of cattle distributed in the Okinawa prefecture were characterized by allele-specific polymerase chain reaction (PCR) and DNA sequence analysis of the major piroplasm surface protein (MPSP) gene. Using universal or allele-specific primer sets, parasite DNA was amplified in 31 out of 48 blood samples obtained from beef cattle. Among the positive cases, mixed infections involving various combinations of I-, C-, and B-type parasites were detected in 24 (77.4%) samples. Phylogenetic analysis based on the MPSP gene sequences revealed that parasites with the MPSP types 1-5 and 7, exist within the Okinawa prefecture.
A subcutaneous mass at the digit of the left-hind limb of a 12-year-old, male mongrel dog was examined. A white firm mass, approximately 1 × 2 cm in diameter, was excided surgically. Histopathologically, the mass formed multiple nodules consisting of mixed proliferation of round epithelioid cells arranged in cord or sheet-like structures and small spindle cells forming loose irregular bundles. The epithelioid cells often showed proliferation around the blood vessels. A few giant cells scattering in the neoplastic foci were observed. The neoplastic cells were positive for alpha-smooth muscle actin and vimentin, and were negative for cytokeratin (AE1/AE3), desmin, factor-VIII related antigen, S-100 protein, and neuron specific enolase. On the basis of these findings, this tumor was diagnosed as glomus tumor. Since the present neoplasm had neither recurrence nor distal metastasis during the 12 month after surgical resection, the biological natures of the present neoplasm are supposed to be benign.
The risk of infections from zoonotic pathogens of tissues and/or tissue-derived products has been increasing. One preventive approach in reducing infection risk is tissue decontamination, where selection and screening of highly infectious tissues are strictly followed. Therefore, the development of reliable analytical methods for rapid tissue discrimination is essentially important. In the present study, a procedure has been developed for intact tissue discrimination on the basis of multivariate analysis of visible and near-infrared (Vis-NIR) spectra of certain tissues such as brain, liver, kidney and testis of mice without any pretreatment. Transmittance spectra in the 600- to 1000-nm regions were subjected to a principal component analysis (PCA), and leave-out cross-validation was employed to develop multivariate models for tissue discrimination. The plot of PCA scores against Vis-NIR spectra of brains, kidneys, livers and testes from 11 mice portrayed reliable tissue discrimination. This result suggests that Vis-NIR spectroscopy combined with chemometrics analysis may provide a potentially useful approach for rapid non-destructive discrimination of tissues.
A 2-year-old castrated miniature Dachshund dog was presented to the Rakuno Gakuen Veterinary Teaching Hospital for diagnosis of progressive hindlimb paresis and ataxia. There was no thoracolumbar intervertebral disk hernia and magnetic resonance imaging revealed an intramedullary spinal cord lesion at the ninth and tenth thoracic vertebrae. Following surgical excision of the neoplasm, there was minor amelioration of neurological signs, but forelimb function was not recovered. The extracted tumor was histopathlogically diagnosed as spinal nephroblastoma.