We investigated the histological changes of extra-intestinal organs, such as the liver, kidney, lung and pancreas in SAMP1/Yit mice, a human Crohn's disease model, using immunohistochemical techniques. The perivascular cellular infiltration was detected around the small vessels after 30 weeks. These infiltrating cells consisted of many CD4-positive T-lymphocytes, and small numbers of CD8- positive T-lymphocytes and IgG-positive B-lymphocytes. MAdCAM-1 and VCAM-1 were detected in vascular endothelial cells in non-affected regions of 13 and 20 week-old, as well as in the affected regions showing perivascular cellular infiltration after 30 weeks. In addition, integrin α4β7 was detected on these infiltrating cells in the perivascular regions after 30 week-old. LT-β and IL-12, cytokines of the Th-1-type immune response, were not observed in these affected regions. However, IL-4, one of the cytokines of the Th-2-type immune response, was detected on the perivascular infiltrating cells after 30 week-old. These results revealed that the changes in extra-intestinal organs were mainly caused by infiltration of CD4-positive T-lymphocytes into the perivascular regions in SAMP1/Yit mice. These cellular infiltrations were thought to be initiated by adhesion of CD4-positive T-lymphocytes to the endothelial cells mediated by MAdCAM-1 and integrin β7. Immunohistochemistry for Th related cytokines indicated that the perivascular cellular infiltration was developed by the Th-2-type immune response in the extra-intestinal organs of SAMP1/Yit mouse.
Development of cerebral perivascular nerves immunoreactive for vasoactive intestinal polypeptide (VIP) was investigated in the Japanese quails, using immunohistochemistry and quantitative analysis. VIP-immunoreactive (VIP-IR) nerves supplying the anterior circulation appeared on the cerebral carotid artery (CCA) at embryonic day 10 and on the cerebroethmoidal artery (CEA) after hatching. Nerves from the CCA increased greatly in number and spread progressively during successive embryonic stages, while those from the CEA were sparse all through the post-hatching stages, mostly remained limited to this vessel wall. The distribution of VIP-IR nerves to the respective major arteries of the anterior circulation from the two vascular routes was basically similar among post-hatching day (P) 15, P20, P30 and P50. Likewise, no clear statistical difference was observed with regard to the nerve density of the corresponding arteries in the four age groups. These findings suggest that VIP-IR innervation of the quail anterior circulation usually attains its mature pattern at the third week after hatching.
Colibacillosis associated with salpingitis occurred in a layer chicken flock on a commercial egg-producing farm in Japan. An increase in mortality was observed when the birds were at 62 weeks of age and reached 0.89% at 68 weeks of age. Postmortem examinations revealed pericarditis, perihepatitis, airsacculitis, and reproductive tract lesions in 4 affected birds at 69 weeks of age. Analysis of pulse-field gel electrophoresis (PFGE) patterns and putative virulence genes of 22 E. coli isolates obtained from the affected birds demonstrated that isolates from liver, heart, and the surface of the reproductive tract of one bird were genetically unrelated with those recovered from the lumen of the oviduct. In the other birds, isolates from liver, heart, and reproductive tract lesions were closely related to each other. These findings suggest that salpingitis in the former bird may be caused by ascending infection of the oviduct from the cloaca and salpingitis in the remaining birds may occur as part of systemic infection.
Nineteen blood samples collected from free-living Japanese serows, Capricornis crispus, between 2006 and 2008 in Iwate prefecture were examined for the anaplasma infection by PCR amplification of a part of the 16S rRNA gene. Ten (52.6%) out of the 19 samples produced a visible band in electrophoresed agarose gels. Positive PCR products were subjected to DNA sequencing. We found the nucleotide sequences were identical. Almost entire length of the 16S rRNA gene for a representative stain was then sequenced. We found the nucleotide sequence of the 16S rRNA gene distinct from previously established Anaplasma species in phylogenetic analysis. Our data first indicated that anaplasma infection occurred continuously among the free-living Japanese serow populations in northern Japan.
The importance of CD4+CD25+Foxp3 + regulatory T cells (Treg cells) in immune response is increasingly being recognized. However, to date, only a few studies on these cells have been conducted in canine species, partly because of the unavailability of appropriate antibodies to detect this cell population. In this study, the crossreactivities of anti-human CD25 antibody (clone ACT-1) and anti-mouse Foxp3 antibody (clone FJK16s) to canine CD25 and Foxp3, respectively, were confirmed using cell lines overexpressing either of these genes. By using these antibodies, we determined if CD4+, CD25+, or Foxp3+ cells were present in unstimulated canine peripheral blood mononuclear cells (PBMCs) and in concanavalin A (ConA)-stimulated PBMCs. CD25+ cells were not detected in unstimulated canine PBMCs, unlike in human or mouse PBMCs. ConA stimulation of canine PBMCs induced Foxp3 and CD25 expression in CD4+ and CD4- cells. These data indicate that the activation of canine lymphocytes might induce the production of adaptive Treg cells, as observed in humans.
Motivation is one of the most important factors in dog training. To generate motivation, people use various reinforcer mechanisms. In particular, many pet owners use food because it is simple and convenient. The aim of this study was to investigate the association between dogs' level of interest in food and their responsiveness to commands. Thirty-four dogs were divided into three groups based on their feeding patterns (Fast, Slow, and Leftover). The fast group (n=15) had the highest interest in food and showed a high response to commands when food was used as a reinforcer, rather than praise/stroking. The leftover group (n=6) showed the least interest in food and had a low reaction to commands, regardless of the reinforcer. In contrast, the slow group (n=13) had modest interest in food and maintained the same response to commands regardless of the reinforcer. Results of this study indicate that dogs' feeding patterns are indicative of their level of interest in food, and may be useful in determining the optimal training reinforcer. This can help dog owners improve their relationships with their dogs.
For efficacious DNA vaccine development against infectious bronchitis virus (IBV), the immunogenicity of a multivalent DNA vaccine was evaluated. Three expression plasmids each targeting spike protein (S1), nucleocapsid protein (N), and membrane protein (M) of IBV were prepared. Chickens were immunized with either individual plasmids (monovalent) or with a combination of all plasmids (multivalent). Immunization with the multivalent DNA vaccine induced synergistic augmentation of humoral and cellular responses in comparison with the individual vaccines, and provided up to 85% immune protection. Thus the multivalent DNA vaccine represents an innovative approach for enhancing DNA vaccine potency, and has potential clinical application for vaccination against IBV.
This is the first report on the cDNA sequences of bat interleukin (IL)-2, IL-4, IL-6, IL-10, IL-12 p40, and tumor necrosis factor (TNF)-α. The cDNAs of bat IL-2, IL-4, IL-6, IL-10, IL-12 p40, and TNF-α comprise 459, 405, 624, 537, 990, and 699 base pairs respectively. Moreover, each of the cDNAs of bat IL-2, IL-4, IL-6, IL-10, IL-12 p40, and TNF-α contain a single open reading frames encoding 152, 134, 207, 178, 329, and 232 amino acids, respectively. The comparison of bat cytokines with Perrissodactyla (horse), Carnivora (dog and cat), and Cetartiodactyla (cattle and pig) orthologs revealed a high degree of homology. Although the N-terminal amino acids and cysteine residues are highly conserved in each mature cytokine, the deduced N-linked glycosylation sites vary across species.
The concentrations of branched-chain amino acids (BCAA; valine, leucine, isoleucine) were determined in plasma of 7 healthy thoroughbred mares and their foals from birth (0 week) to 24 weeks of age, using automated high-performance liquid chromatography. In foals, the concentrations of plasma valine were significantly high (p<0.05) at 16, 20 and 24 weeks. The concentrations of plasma leucine were significantly high (p<0.05) at 1 and 3 weeks. The concentrations of plasma isoleucine were significantly high (p<0.05) from 1 to 24 weeks. In mares, the concentrations of plasma valine were significantly high (p<0.05) at 16 and 24 weeks. The concentrations of plasma leucine and isoleucine were significantly high (p<0.05) at 16 weeks. It was clear that the concentrations of plasma BCAA in foals and mares were at different levels at various times after birth. Since mares and foals were kept in health during this study, we could get the base data of the concentrations of BCAA in plasma of healthy foals and mares from birth to 24 weeks.
This study determined the appropriate biochemical assay for measuring plasma tartrate-resistant acid phosphatase isoform 5b (TRAP5b) activity; this information is important to clarify the relationship between plasma TRAP5b and known biochemical bone markers in cattle. When plasma TRAP5b was measured using fluorometric and spectrophotometric methods, hemolysis products in plasma did not affect the former method. In plasma from healthy cattle, there was a good correlation (r=0.66) between the 2 methods. In age-related profiles, plasma TRAP5b (r=-0.53), hydroxyproline (HYP, r=-0.56) and bone-specific alkaline phosphatase (BALP, r=-0.44) showed significant negative correlations with age; these three parameters decreased until 4 or 5 years of age and then remained constant. There were significant correlations between TRAP5b and HYP (r=0.83) or BALP (r=0.83). Our results show that the fluorometric assay can be performed with a high degree of precision and reproducibility without interference from hemolysis, and that the age-related changes in plasma TRAP5b, HYP, and BALP constitute additional background values for clinical guidance in bovine medicine.
A one-year and six-month-old female Maine Coon cat presented with skin problems and paravertebral induration with a history of seven months. Survey radiographs and computed tomography revealed prominent calcifications in both sides of cervical, thoracic and lumbar vertebrae and soft tissue in femoral regions, below knee regions and in brachial regions. Histopathological findings from muscle biopsy samples showed connective tissue proliferation around adjacent skeletal muscle, cartilage formation and endochondral ossification. On the basis of these findings, this feline patient was diagnosed with fibrodysplasia ossificans progressiva (FOP). The most prominent signs observed in this FOP case were significant calcifications of dorsal muscle and presentation of cutaneous signs at the early stage.
Canine alimentary lymphoma is currently diagnosed on the basis of findings of cytological or histopathological examination. However, it is often difficult to histopathologically distinguish alimentary lymphoma from lymphocytic-plasmacytic enteritis. Recently, the application of polymerase chain reaction for antigen receptor gene rearrangement (PARR) has been reported. In the present study, we assess the sensitivity of PARR analysis in diagnosing canine alimentary lymphoma using endoscopically obtained biopsy specimens from 12 dogs that were histopathologically diagnosed as having lymphoma. The sensitivity of PARR analysis in diagnosing alimentary lymphoma was found to be 66.7%, which was lower than that of other lymphoid malignancies. A combination of histipathological examination and findings of PARR analysis may improve the diagnostic accuracy.
The aim of this study was to investigate the effect of fetal exposure to diethylstilbestrol (DES) on the induction of mammary tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) in female rats. Pregnant rats were fed only normal diet, diet mixed with 0.1 ppm DES throughout pregnancy period or diet mixed with 0.1, 1 or 10 ppm DES from day 13 of pregnancy till the end of pregnancy. Delivered pups were given 10 mg DMBA by gastric intubation at 50 days after birth and observed till 336 days after birth. Some rats exposed to DES throughout pregnancy and those from day 13 till the end of pregnancy showed endocrine disrupting effects such as absence of CL and active lactation in mammary glands at necropsy, while no abnormal estrus cycle such as persistent estrus was seen during the observation period until 88 days after birth. Fetal exposure to 0.1 ppm DES throughout pregnancy period, 0.1 and 1 ppm DES from day 13 of pregnancy increased the incidence and number of mammary carcinomas (MCs) at the earlier period while exposure to 0.1 ppm DES throughout pregnancy period enhanced the incidence and number of benign proliferative lesions (PLs) at the later period. MCs appeared earlier than benign PLs. These results suggest that exposure to DES throughout pregnancy and from day 13 of pregnancy could induce endocrine disrupting conditions and enhance the induction of MCs and that exposure to DES throughout pregnancy enhance PLs.
An adult male hare (Lepus brachyurus angustidens) was discovered in a moribund condition in the bush in the mountains of Aomori prefecture in Japan. Upon gross inspection, many ticks were found on the neck and the external ear regions, and more than half the ticks contained blood in the intestine. The skin around the tick bite wounds was alopecic and mildly thickened. At necropsy, enlargement of the cervical lymph nodes and spleen were observed. Histologically, acute necrotizing splenitis, lymphadenitis, hepatitis, pneumonia, myelitis, adrenalitis, and encephalitis with bacterial organisms were observed. The cutaneous lesions were chronic and cysts had formed in the areas marked by tick bites. Immunohistochemically, the organisms in the skin, liver, spleen, lymph nodes, lungs, adrenal glands, brain, bone marrow, and ticks were positive for F. tularensis antigen. Microbiological and polymerase chain reaction results were consistent with F. tularensis subsp. holarctica. Because the cutaneous lesions were more chronic than those in the visceral organs and F. tularensis was detected in the ticks, we inferred that F. tularensis was transmitted to the hare via tick bites.
A captive male Bolivian squirrel monkey (Saimiri boliviensis) of less than 1 year of age died following diarrhea and debilitation on the day of death. At necropsy, necrotizing enteritis accompanied with enlarged Peyer's patches, solitary lymphatic follicles and mesenteric lymph nodes, and multiple yellowish-white nodules in the spleen and liver were found. Histopathologically, these lesions were necrotizing inflammation containing Gram-negative bacilli. Yersinia pseudotuberculosis serotype 7 was isolated from the spleen and liver. The virF gene, which is an essential virulent plasimid (pYV) in pathogenic Y. pseudotuberculosis isolates, and the ypmA gene, which is a superantigenic toxin, were detected in the isolates. This is the first report of a fatal case of Yersinia pseudotuberculosis 7 infection in the world.
A stillborn calf at 259 days of gestation was examined. The dam showed no clinical signs of disease, and the stillbirth was occurred sporadically and were characterized by focal necrosis surrounded by infiltration of the cells such as macrophages and multinucleated giant cells. The lesions were observed systemically. The hyphae were visible by Grocott's stain and they were positive by immunohistochemical stain using serum of a rabbit immunized with the isolated organism. The isolated bacteria were determined to be Nocardia farcinica by bacteriological and molecular analysis and we confirmed that the stillbirth was caused by infection with and proliferation of Nocardia farcinica. This is the first report of a bovine stillbirth caused by this organism.
Cytological, histopathological and immunohistochemical examinations were carried out on a presumed 10-year-old Japanese cat showing vomiting and emaciation. On cytologic examination of the mass of the upper abdominal cavity, many yeast-like organisms were detected in the macrophages. At necropsy, the upper part of colon was markedly dilated with a thickened wall. The lung did not show significant changes. Histologically, severe necrotic and granulomatous lesions were observed in the colon. In the colonic lesion, the cytoplasm of the macrophages contained yeast-like organisms with irregularly shaped dots, and the cell walls of these organisms were stained black by Grocott-Gomori methenamine-silver stain. Immunohistochemically, they were found to be positive for anti-histoplasma yeast antibody. This is the first report of feline histoplasmosis in Japan.
We report herein a feline salivary gland adenocarcinoma with widespread metastases to draining lymph nodes, liver and lung, as well as an unusual metastasis to the spleen. Histologically, the primary salivary gland tumor consisted of low columnar to polygonal epithelial cells forming tubules and trabeculae. The spleen was infiltrated with sheets of poorly differentiated large round cells. Interestingly, morphologic change in epithelial cells was accompanied with the acquisition of vimentin intermediate filaments, a feature particularly evident in the splenic metastasis. This study highlights the role of epithelial cell plasticity during carcinogenesis and metastasis.
In the present study, we investigated the long-term effects of 17β-estradiol on the motility of small intestine in in vitro organ culture and in vivo treatment studies. When rat ileal circular smooth muscle tissues were cultured with 17β-estradiol (0.1 and 1 μM) for 5 days, carbachol-induced contraction was inhibited. In ileal tissue isolated from ovariectomized rat treated with 17β-estradiol (200 μg/kg/day s.c. for 3 days), carbachol-induced contraction was also impaired. Both in vitro and in vivo, 17β-estradiol treatment upregulated heat shock protein 27 (HSP27) expression, indicating the activation of estrogen receptor in the intestinal smooth muscle. 17β-estradiol did not change protein expression levels of RhoA and RhoA-associated coiled coil-forming serine/threonine kinases (ROCKs); however, it upregulated Rnd2 and Rnd3, Rho-family G-proteins that counteract the functions of RhoA, both in vitro and in vivo. In organ culture, treatment of ileal tissue with 17β-estradiol greatly suppressed the carbachol-induced increase in phosphorylation at Thr38 in CPI-17 without altering total CPI-17 protein expression. These results suggest that 17β-estradiol upregulates Rnd expression to inhibit the RhoA-mediated Ca2+ sensitization of contractile mechanisms, which are mediated by CPI-17 phosphorylation in ileal smooth muscle. This mechanism may contribute to the intestinal motility disorder occurring in gender-dependent bowel diseases.
Protease-activated receptor (PAR)-2 plays important roles in intestinal inflammatory responses and also contributes to intestinal digestive motility. In the distal colon of a rat experimental colitis model, expression level of PAR-2 mRNA was decreased, and relaxation through PAR-2 activation was attenuated. This study shows the effects of proinflammatory cytokines on changes to PAR-2 in rat colonic smooth muscle using an organ culture method. Colonic inflammation was induced in rats by administering dextran sodium sulphate in drinking water. Organ culture of distal colonic smooth muscle layer of normal rat was performed for up to 3 days. In the experimental colitis rat, mRNA expression levels of proinflammatory cytokines such as IL-1β and TNF-α increased with inflammation. After the incubation with IL-1β and TNF-α for 3 days, trypsin (PAR-2 agonist)-induced relaxation was attenuated, simultaneous with suppression of PAR-2 mRNA expression. Conversely, in this preparation, mRNA expression levels of iNOS were significantly increased. When l-NMMA was added to the medium with IL-1β and TNF-α, changes to PAR-2 by these cytokine recovered. Moreover, when samples were cultured with NOC-18 (slow-releasing NO donor) for 3 days, relaxation induced by trypsin and expression of PAR-2 mRNA were attenuated. These results suggest that suppression of PAR-2 expression under inflammatory conditions is at least partially induced by NO produced in the colonic muscularis externa by proinflammatory cytokines.
European Russia is a highly endemic area of hemorrhagic fever with renal syndrome (HFRS), a rodent-borne zoonotic disease, caused by hantaviruses. In total, 145 small mammals of four species (Myodes glareolus, Apodemus flavicollis, A. agrarius, and A. uralensis) were trapped in the Samara region of European Russia in August 2005 and examined for the presence of hantavirus (HV). Anti-HV antibodies were found in six of 68 (8.8%) M. glareolus and in one of 19 (5.3%) A. flavicollis by indirect immunofluorescent antibody assay (IFA). The Puumala virus (PUUV), which is one of the hantavirus species, was detected in the lungs of seven M. glareolus by RT-PCR. The virus S-segment was extremely similar (96.2% to 99.3%) to the sequence found in a fatal case of HFRS in the Samara region. Phylogenetic analyses of S and M segments showed that the Samara PUUVs form a cluster within the Russian Volga lineage and apparently differ from other European PUUVs. Anti-PUUV antibodies were found in blood sera from seven HFRS patients and from one undiagnosed patient from the Samara region, using IFA and an enzyme-linked immunosorbent assay (ELISA). These data suggest that the bank vole M. glareolus is a primary natural reservoir and vector for PUUV, which is the main causative agent of HFRS in humans in the Samara region.
The objectives of the present study were to measure a percentage of pigs born dead in the number of total pigs born (PPBD), and to investigate an association between PPBD and total pigs born by parity groups and three herd groups formed on the basis of the pigs weaned per mated female per 6 years (PWMFY). The studied data included 236,805 parity records of 51,443 sows in 110 herds. The three herd groups, high-, intermediate- and low-performing herds, were formed on the basis of the upper and lower 25th percentiles of PWMFY. Linear mixed-effects models were performed to analyze the data. Mean (± SEM) PPBD was 9.6 (± 0.03) %. Sows in high-performing herds had lower PPBD for all parity groups than those in low-performing herds (P<0.05). Parity 2 sows had the lowest PPBD between parity groups (P<0.05). The PPBD increased from 7.0 to 13.2% as the parity number increased from 2 to ≥ 6. Parity 1 sows had higher PPBD than parity 2 and 3 sows (P<0.05). As the total pigs born increased from 10 to 16 pigs, PPBD increased in all the parity groups (P<0.05). In parity 3 or higher, PPBD in low-performing herds increased more steeply than that in high-performing herds as total pigs born increased form 6 to 10 pigs. We suggest that farrowing management including assisted farrowing improve PPBD for sows in parity 3 or higher.
Cytochrome P450 2B6 (CYP2B6), an important drug-metabolizing enzyme, is involved in the metabolism of prescribed drugs in humans. Despite its importance, cDNA for a CYP2B6 ortholog has not been identified and characterized in cynomolgus macaques, which are frequently used in preclinical studies. In this study, cDNA highly homologous to human CYP2B6 was cloned from the cynomolgus macaque liver. This cDNA contained an open reading frame of 491 amino acids and functional domains characteristic for CYP protein, such as substrate recognition sites and a heme-binding region. Cynomolgus CYP2B6 was expressed predominantly in the liver with some extra-hepatic expression among 10 tissues. Moreover, cynomolgus CYP2B6 revealed activities toward testosterone 16β-hydroxylation and bupropion hydroxylation. These results suggest that cynomolgus CYP2B6 has a functional role in the liver.
Canine distemper virus (CDV) causes a lethal disease among members of the Carnivora. To clarify the distribution of CDV in wild animals, we examined 106 raccoon sera collected from two prefectures in Japan, Hyogo and Osaka, from 2005 to 2007. Among them, 34 raccoons (32.1%) possessed a virus-neutralizing (VN) antibody to KDK-1 strain (genotype Asia-1). There was no significant difference in seroprevalence of CDV regardless of places, gender, and body weights. In Hyogo, a geometric mean of VN titers to KDK-1 was significantly higher than that to Onderstepoort (vaccine strain), indicating that KDK-1-like CDV different from vaccine strain might have spread among raccoon population in Hyogo. In conclusion, CDV is epidemic among feral raccoons in Japan, suggesting that CDV might have been spreading among Japanese wild animals.