This study was performed to anatomically illustrate the living canine hippocampal formation in three-dimensions (3D), and to evaluate its relationship to surrounding brain structures. Three normal beagle dogs were scanned on a MR scanner with inversion recovery segmented 3D gradient echo sequence (known as MP-RAGE: Magnetization Prepared Rapid Gradient Echo). The MRI data was manually segmented and reconstructed into a 3D model using the 3D slicer software tool. From the 3D model, the spatial relationships between hippocampal formation and surrounding structures were evaluated. With the increased spatial resolution and contrast of the MPRAGE, the canine hippocampal formation was easily depicted. The reconstructed 3D image allows easy understanding of the hippocampal contour and demonstrates the structural relationship of the hippocampal formation to surrounding structures in vivo.
Nitric oxide (NO) has been reported as a key mediator in enhancing hepatocyte proliferation during liver regeneration. Juvenile hepatocytes have a strong ability to proliferate while still in their undifferentiated state but the mechanism of NO production and its contribution to hepatocyte proliferation are not yet fully understood. The present study was designed to investigate NO production in the normal liver and its contribution to hepatocyte proliferation in juvenile rats. Endogenous NO production was evaluated quantitatively using a spin trap followed by electron paramagnetic resonance spectroscopy with the Fe-N, N-diethyldithiocarbamate complex as an NO-trapping reagent in the rat liver. NO production in the liver significantly peaked at 3 weeks after birth, but NO synthase (NOS) 3 expression did not change between 2 to 5 weeks after birth, while NOS 1 and NOS 2 mRNA were not detected. Hepatocyte proliferation, measured by the incorporation of 5-bromo-2'-deoxyuridine into the DNA, was found to decline significantly when endogenous NO production was inhibited by the administration of the NOS inhibitor NG-nitro- L-arginine methyl ester. These findings indicate that endogenous NO production peaked at 3 weeks after birth and hepatocyte proliferation declined significantly when NO production was inhibited. Thus, this study provides a novel insight into the contribution of NO to hepatic growth and liver maturation in juveniles.
Avian liver is structurally different from that of mammal. There are only a few reports about the ultrastructure of the hepatic vessels and bile duct in birds. Casts of the hepatic vessels and bile ducts in chicken and duck were examined by a scanning electron microscope (SEM) to provide further detail. The sinusoidal vessels formed regular three-dimensional networks but the replicated bile ducts formed irregular ones. There was a dense plexiform network around each tubular cavity in biliary casts. This biliary plexus formed around a small branch of the portal vein. It was the transition between the bile capillaries and ductules. The present study shows that the liver of chicken and duck had the characteristic structure at the peripheral part of biliary system, i.e., biliary plexus.
Vitamin E is thought to affect bone formation and bone remodeling. In this study, we investigated the effects of vitamin E (α-tocopherol and δ-tocopherol) on the osteoblasts isolated from rat calvariae. At 4 and 7 days (Day 4 and 7) after induction of osteoblastic differentiation, treatment of α-tocopherol (100 and 200 μM) and δ-tocopherol (2 and 20 μM) for 3 days significantly decreased alkaline phophatase activity of the cultured osteoblasts. At Day 14, however, no significant change was detected in ALP activity and expression of bone sialoprotein mRNA in the osteoblasts treated with α-tocopherol or δ-tocopherol for 3 days. Expression of osteocalcin mRNA was decreased by treatment of α-tocopherol (100 and 200 μM) and δ-tocopherol (2 and 20 μM) at Day 4 and 7. At Day 14, expression of osteocalcin mRNA was decreased only with treatment of 200 μM α-tocopherol. In addition, the noncalcified nodules were decreased by treatment of α-tocopherol (200 μM) and δ-tocopherol (20 μM) at Day 7. However, treatment of α-tocopherol and δ-tocopherol showed no significant change of formation of calcified nodules at Day 14. These results indicate that vitamin E inhibits differentiation of osteoblasts especially from early stage to osteoid-producing stage.
From November 2002 to February 2008, 2,251 crane feces were collected at the Izumi Plain in Kagoshima Prefecture. Salmonella enterica was isolated from 359 feces (15.9%), of which 332 (92.5%) were Salmonella Typhimurium (ST), 9 were S. Hvittingfoss/II, 4 were S. Abaetetuba, 3 were S. Enteritidis, 2 were S. Konstanz, 1 was S. Pakistan and 8 were untyped isolates, respectively. Against 12 antimicrobial agents, no resistant strains were found in 154 isolates examined, but one was found to be resistant to ampicillin. By pulsed-field gel electrophoresis (PFGE), all but one of the 68 ST isolates tested showed indistinguishable banding patterns; one had a different pattern. The results suggest that ST strains from the same origin would spread in crane flocks during their stay at Izumi Plain every winter.
Surveillance of avian influenza virus and paramyxovirus in migratory waterfowl and shorebirds was conducted in the San-in district of western Japan from the winter of 2001 to 2008. From 4,335 fecal samples from wild birds, 41 avian influenza viruses of 12 different HA and NA combinations, including two H5N3 strains, and 13 avian paramyxoviruses were isolated. Phylogenetic analysis of HA genes revealed that H5N3 strains clustered in a different branch from the recent highly pathogenic H5N1 isolates in Japan; however, the introduction of new highly pathogenic avian influenza virus by migratory birds cannot be ignored. Therefore, it is necessary to continue surveillance of these potentially serious pathogens in waterfowl and shorebirds.
Antimicrobial resistant Salmonella are becoming more prevalent. Therefore, alternative methods to control swine Salmonella infection must be explored. We examined whether feeding lactic acid to swine is an effective way to control clinical and subclinical Salmonella Typhimurium infection in these animals. In this experiment, swine were inoculated with 5.6 × 107 CFU (hi-ST) or 5.6 × 105 CFU (lo-ST) of S. Typhimurium per swine to reproduce clinical and subclinical infection. The swine were either fed a commercial feed supplemented with 2.8% lactic acid (LA) or the commercial feed without supplementation (C) to examine the effect of feeding lactic acid. Twenty 21 and 22 days old swine were divided into 4 groups, LA-hiST, C-hiST, LA-loST and C-loST, and fed the respective feed. They were inoculated S. Typhimurium at 51 and 52 days old. Clinical symptoms and the number of S. Typhimurium shed in feces were evaluated. The LA-hiST group did not show obvious clinical symptoms, such as diarrhea or febrile response, but the C-hiST group did show clinical symptoms. The number of S. Typhimurium shed in the feces of the LA-hiST group was lower than in that of the C-hiST group, and that of the LA-loST group was lower than that of the C-loST group. Our data suggest that dietary supplementation with 2.8% lactic acid can be an effective way to control clinical and subclinical infections of S. Typhimurium in swine.
Mycoplasma hyorhinis (M. hyorhinis) infection leads cultured cells to various biological alterations in cell metabolism including apoptosis. Apoptosis induced by M. hyorhinis has mainly been considered to be due to mycoplasmal endonucleases. We previously reported that apoptosis in a human carcinoma cell line AZ-521 infected with M. hyorhinis was enhanced by addition of L-ascorbic acid to cell cultures. Since both L-ascorbic acid addition and M. hyorhinis infection activated cellular iNOS, we examined the hypothesis that nitric oxide (NO) exerts an apoptotic effect on M. hyorhinis-infected cells and down-regulates E-cadherin. In this study, we showed that M. hyorhinis infection activates iNOS mRNA synthesis, NO production, and caspase-3 activity and attenuates E-cadherin mRNA synthesis by quantitative real-time PCR, Griess assay and fluorescence caspase-3 detection. L-NAME decreased the numbers of apoptotic cells through inhibition caspase-3 activity. Our results indicate that NO causes anoikis throughout attenuation of E-cadherin and activation of caspase-3 in human gastric carcinoma cell line AZ-521 cells infected with M. hyorhinis.
Dogs can be divided into two genetic groups (a minor HK phenotype and a major LK phenotype) based on erythrocyte monovalent cation concentrations, which are controlled by the putative hk and lk allelic genes. HK dogs retain Na,K-ATPase in their erythrocytes due to the high activity of the enzyme in their precursor cells, whereas total loss of reticulocyte Na,K-ATPase occurs in LK dogs. Here, we report that the levels of the lipid raft-associated membrane protein stomatin decrease in parallel with those of Na,K-ATPase during reticulocyte maturation due to its extrusion in exosomes. The stomatin content of HK reticulocytes is higher than that of LK reticulocytes, and remains in the erythrocytes at levels compatible with that in human erythrocytes. However, it is almost absent from LK erythrocytes with the lk/lk genotype; similar to the deficiency seen in human red cells with overhydrated stomatocytosis. LK erythrocytes from hk/lk genotype dogs show reduced, but not negligible, levels of stomatin. These results indicate that the erythrocyte stomatin level is a suitable genotypic marker for the HK/LK red cell phenotype, and suggests a functional association between stomatin and Na,K-ATPase. The absence of morphological abnormalities in the erythrocytes of stomatin-deficient LK dogs also confirms that stomatin deficiency and stomatocytic shape change are independent from each other.
Survivin overexpression has been reported in relation to tumor malignancy, suggesting that it is an unfavorable prognostic marker, and antibody responses to this protein have been confirmed in human cancer patients. In this study, we investigated antibody responses to survivin in canine cancer cases, and examined the prevalence of such responses by enzyme-linked immunosorbent assay (ELISA) using recombinant canine survivin protein as the antigen. The cut-off value for positivity in the anti-survivin ELISA was 0.35, as determined using the mean absorbance +2 S.D. of samples from healthy dogs. Sera from 16 of 59 (27.1%) cancer and 3 of 25 (12%) non-cancer disease dogs were positive on ELISA. The highest positivity rates (>50%) among the cancer cases were seen in dogs with mammary tumor, squamous cell carcinoma and melanoma.
We investigated the differences between vocalizations of mares in estrus and diestrus and determined the spectrographic parameters to discriminate estrus from diestrus. Thoroughbred brood mares (n=89) were exposed to a teasing procedure for 3 min, and we recorded all vocalizations emitted from them. Among the mares, 56.5% of estrus and 78.6% of diestrus mares emitted calls toward an approaching stallion, indicating that there was higher tendency in the occurrence rate of vocal responses in diestrus than estrus mares. We analyzed the spectrographic data of the mares (25 estrus and 22 diestrus mares) emitting calls in the form of a squeal toward an approaching stallion. Based on broad bandwidth spectrographic analysis, the duration and third formant of the call have a significant effect on discriminating estrus from diestrus.
It is required not to increase the ventricular rate and to preserve the ventricular systolic function in treating supraventricular tachyarrhythmia (SVTA). The objective of this study is to investigate whether or not Efonidipine hydrochloride (EH), a T and L dual type Ca2+ channel blocker, suppresses the increasing ventricular rate without reducing the ventricular systolic function using canine SVTA models by rapid atrial pacing (RAP) method. Clinically healthy fourteen beagles were used. The 14 dogs were randomly assigned to the EH-administered group (EH group, n=7) and non-EH-administered group (control group, n=7). The EH group was orally-administered EH at 5 mg/kg SID during RAP. On the other hand, the control group was applied RAP without oral administration of EH. Duration of RAP was for 3 weeks for both groups. The ventricular rate for the EH group was significantly lower than that for the control group. The left ventricular- fractional shortening for the control group declined significantly compared to baseline. Those for the EH group did not show any changes over time and were significantly higher than the control group. The ratio between pre-ejection period and ejection for the EH group were significantly lower than those of the control group. In conclusion, the study demonstrated that EH suppresses the increasing ventricular rate without reducing the ventricular systolic function in canine SVTA model. Therefore, EH is expected to become a new treatment for canine SVTA.
A six-month-old female Galician Blond beef calf presented signs of apathy, anorexia and weight loss. The analysis of a blood sample confirmed renal failure. Bilateral nephrolithiasis was diagnosed at necropsy. Quantitative analysis revealed the nephroliths to be composed of 100 per cent xanthine. In cattle, xanthinuria has only been described in the Japanese Black breed, but never before in other breeds. Clinical history suggested a naturally occurring xanthinuria.
A 4-year-old neutered male Chihuahua was presented with a history of anorexia and vomiting. Continuous elevation of liver enzymes was found on repeated blood examinations and the dog was referred to us for further evaluation. The absence of gallbladder was suspected on ultrasonography. Exploratory laparotomy and retrograde cholangiography confirmed gallbladder agenesis and a possible hypoplasia of the right medial and lateral liver lobes. Histologically, proliferation of bile ductules associated with portal fibrosis and pseudolobular formation were apparent in the liver lobes.
The rat autosomal dominant Rex (Re) mutation on chromosome 7 causes curly hair in Re/+ and hair loss in Re/Re rats. Histopathologically, the Re/+ rat showed dilatation of the hair follicle and hairs with irregularly-coated cuticles, and the Re/Re rat showed more severe effects. We identified Re as a 7-bp deletion at the splicing acceptor site of intron 1 of the keratin 71 (Krt71) gene, which is located within the Re critical chromosomal region and plays an important role in hair formation. The deletion provoked a 6-amino acid in-frame deletion (p.Val149_Gln154del) in the α-helical rod domain of KRT71 protein. Identification of the Re mutation (Krt71Re) enables us to further understand the biological function of KRT71.
Fecal samples from Ruddy Shelduck, Tadorna ferruginea, were screened microscopically for Cryptosporidium oocysts. Five samples out of 148 ones (3.38%) were confirmed to be positive. DNA was extracted individually from positive samples and used for PCR amplification of SSU rDNA and HSP70 gene loci. The obtained PCR products were cloned in E. coli (TG1 strain) using pMD18-T vector and sequenced using standard methods. Microscopical and molecular analyses identified the obtained isolates as Cryptosporidiumbaileyi. To our knowledge, this is the first report of detection of C. baileyi from Ruddy Shelduck, Tadorna ferruginea, in China.
Bovine viral diarrhea virus (BVDV) causes fetal brain malformations in ruminants when the fetuses are infected transplacentally in mid-pregnancy. In both cytopathic and non-cytopatic virus infections, viral lytic infection in actively replicating cells and interruption of vascular integrity have been suggested as the pathogenesis, but functional disturbance of infected neural developing cells has been unclear. In this study, we examined the effect of infection with non-cytopathic BVDV2 on the differentiation of neural stem/precursor cells isolated from the bovine fetus. In the process of differentiation to three types of neural cells, neurons, astrocytes and oligodendrocytes, virus infection significantly and selectively inhibited the differentiation of neural stem/precursor cells into the astrocytic lineage. This inhibition is possibly important for the pathogenesis of congenital brain malformations associated with non-cytopathic BVDV infection.
A 22-year-old Thoroughbred stallion had severe left jugular thrombophlebitis. Macroscopic and microscopic examinations revealed extension of the lesions from a penetrating ulcer on the left buccal mucosa to the underlying muscle and local vein, and sequentially to the left jugular vein. This was a rare case of equine jugular thrombophlebitis caused by direct extension of infection from a traumatic oral lesion.
A 1.5-year-old intact male African hedgehog (Atelerix albiventris) was presented with a firm, non-movable subcutaneous mass on ventral chest area. Microscopically, the tumor was un-encapsulated, invasive up to the muscle layer, and composed of highly pleomorphic polygonal cells arranged in variably-sized lobules. The neoplastic cells had abundant cytoplasm with vacuolation and a large pleomorphic nucleus with prominent nucleoli. Mitotic figures were frequently observed with atypical mitoses. Immunohistochemically, the neoplastic cells were strongly positive for cytokeratin, but negative for vimentin. Based on these findings, a diagnosis of sebaceous carcinoma was made. Three months after the surgery, a recurrent mass was found at the surgical site. On necropsy, the mass has penetrated the underlying intercostal musculature, without metastasis to distant organs. This is the first report of a sebaceous carcinoma in an African hedgehog.
Experiments were performed with the aim of investigating the effect of road transportation for 12 hr on erythrocytes of goats during the hot-dry season and the modulatory role of ascorbic acid. Forty 2.5-3-year-old Red Sokoto goats weighing 23-25 kg and belonging to both sexes served as the subjects of the study. Twenty of the goats served as the experimental group and were administered ascorbic acid (AA) per os at a dosage rate of 100 mg/kg body weight; the other 20 served as controls and were given 10 ml each of sterile water. Forty minutes after the administration and loading, the goats were transported for 12 hr. EDTA blood samples collected before loading, after loading, immediately after transportation and subsequently on the 3rd and 7th days of post-transportation were used to determine the red blood cell (RBC) count, packed cell volume (PCV), hemoglobin (Hb), erythrocyte osmotic fragility (EOF), hematimetric (intrinsic) indices and hemoglobin index levels. The obtained results showed that handling, loading and transportation of the control goats induced significant (P<0.05) increases in RBC, Hb, EOF and hypochromic erythrocytes and a decrease (P<0.05) in the volume and average Hb content in RBCs. AA administration ameliorated all these changes. The present results suggest that road transportation for 12 hr during the hot-dry season could induce serious stress, resulting in hemolysis of erythrocytes, which was ameliorated by AA administration. In addition, the results demonstrated that EOF could be used as a diagnostic tool in road transportation stress.
Reproduction of captive elephants in zoos has shown a low fecundity and requires improvement. One of the reasons for low fecundity is ovarian dysfunction in many female elephants. To investigate whether prolactin has a correlation with ovarian function in female elephants, the serum concentrations of prolactin, progesterone and estradiol-17β in four African female elephants (one cycling female and three non-cycling female elephants) were measured. Cyclic patterns of prolactin and estradiol-17β were observed in the cycling female elephant, which tended to be high during the follicular phase and low during the luteal phase. On the other hand, a cyclic pattern of prolactin was not observed in the non-cycling female elephants. One of the three non-cycling females (Mako) had developed breasts and showed significantly higher average levels of prolactin than the other female elephants. These results suggested that high concentrations of circulating estradiol-17β during the follicular phase stimulated prolactin secretion. They also suggested that hyperprolactinemia in Mako was one of the causes of the developed mammary glands and ovarian dysfunction.
This study was designed to evaluate the nuclear maturation and maturation promoting factor (MPF) level at different maturation times, and the effect of parthenogenetic activation on nuclear maturation in canine oocytes. Cumulus-oocyte complexes (COCs) were matured in TCM-199 supplemented with 10% fetal bovine serum, hormones, 0.57 mM cysteine, and 10 ng/ml epidermal growth factor for 72 hr at 38.5°C. In Experiment 1, COCs at 0, 24, 48 and 72 hr of culture were assessed for nuclear maturation and MPF levels using histone H1 kinase activity assay. A significantly higher rate of oocytes at 72 hr than 0, 24 and 48 hr of culture developed to metaphase I-anaphase I and metaphase II. Relative abundance of histone H1 kinase activity of oocytes matured for 48 hr increased to ~1.5 ×, with a marked increase to ~2.5 × for 72 hr, significantly higher than others. In Experiment 2, oocytes matured for 48 hr were parthenogenetically activated with 5 μm ionomycin for 5 min (Group 1) and followed by 10 μg/ml cycloheximide for 3 hr (Group 2), or no treatment (Control). Oocytes were then cultured for 24 hr and assessed for nuclear maturation. A significantly higher rate of oocytes in Group 1 developed to metaphase II than in Group 2 and the control. These results indicated that ionomycin treatment at 48 hr of in vitro maturation had a positive influence on oocyte progression to the metaphase II stage.
For determining surveillance programs or infectious disease countermeasures, risk evaluation approaches have been recently undertaken in the field of animal health. In the present study, to help establish efficient and effective surveillance and countermeasures for equine infectious diseases, we evaluated the potential risk of equine infectious disease transmission in non-race horses from the viewpoints of horse movements and health management practices by conducting a survey of non-race horse holdings. From the survey, the non-race horse population was classified into the following five sectors based on their purposes: the equestrian sector, private owner sector, exhibition sector, fattening sector and others. Our survey results showed that the equestrian and private owner sectors had the largest population sizes, and movements between and within these sectors occurred quite frequently, while there was little movement in the other sectors. Qualitative evaluation showed that the equestrian and private owner sectors had relatively high risks of equine infectious disease transmission through horse movements. Therefore, it would be effective to concentrate on these two sectors when implementing surveillance or preventative measures. Special priority should be given to the private owner sector because this sector has not implemented inspection and vaccination well compared with the equestrian sector, which possesses a high compliance rate for these practices. This qualitative risk evaluation focused on horse movements and health management practices could provide a basis for further risk evaluation to establish efficient and effective surveillance and countermeasures for equine infectious diseases.
A simple gold-immunochromatographic assay (GICA) based on indirect reaction format was developed for rapid detection of antibodies against avian infectious bronchitis virus (IBV). The detection time of IBV IgG by the GICA was less than 10 min, whereas the HI test and the enzyme-linked immunosorbent assay (ELISA) require 1-2 hr. Reference sera against newcastle disease virus, infectious bursal disease, avian influenza virus H5 and H9 subtypes were all negatives for anti-IBV antibodies using the GICA. Compared with the HI, the sensitivity of the GICA was 92.11% and specificity was 81.71%. The agreement rate between the 2 tests was 85%. Compared with the ELISA, the sensitivity of the GICA was 92.31% and specificity was 97.06%. The agreement rate between the 2 tests was 95%. The GICA test strip is a reliable and useful tool for the on-site surveillance of anti-IBV antibodies.
A total of 2,703 pig sera from 171 farms in six regions in Japan were screened for virus-neutralizing (VN) antibody against transmissible gastroenteritis virus (TGEV). Although none of the farms had clinical signs of transmissible gastroenteritis (TGE) or vaccination against TGEV, VN antibody was detected in 14.4% of sera at 30 farms (17.5%) across all six regions. On testing of 263 VN antibody-positive sera from 27 farms with a commercial blocking ELISA to distinguish TGEV and porcine respiratory coronavirus (PRCV) antibodies, 78.3% were positive for PRCV antibody only, while 12.5% were positive for TGEV antibody only or both TGEV and PRCV antibodies. Seven of the eight TGEV antibody-positive farms were also positive for PRCV antibody. Five months after the antibody examination, a TGE outbreak occurred at one of these seven farms. These results suggest that most of the detected VN antibodies were to PRCV, and that TGEV infection might be present at some PRCV-positive farms in Japan.