Effects of low protein intake on the development of the remaining kidney in subtotally (5/6) nephrectomized immature rats were examined. Three week-old weaning rats were kept on a diet containing either 12% (Lp rats) or 18% (Np rats) protein for 4 or 8 weeks after subtotal nephrectomy. Blood urea nitrogen (BUN) concentration was determined at 2, 4, 6, and 8 weeks after 5/6 nephrectomy. At 4 or 8 weeks after the operation, glomerular sclerosis and tubulointerstitial damage were assessed by a standard semiquantitative analysis and were expressed as the glomerular sclerosis index (GSI) and interstitial fibrosis score (IFS), respectively. The localization of DNA fragmented cells in the kidney was examined by the terminal deoxynucleotidyl transferase (TdT) -mediated d-UTP-biotin nick end labeling (TUNEL) method and the localization of the epidermal growth factor (EGF) by immunohistochemical methods. BUN concentration was significantly lower in the Lp rats compared with that in the Np rats. Both 4 and 8 weeks after subtotal nephrectomy, GSI and incidence of TUNEL positive cells in the distal tubules were significantly lower in the Lp rats than in the Np rats. Four weeks after the operation, IFS was significantly lower in the Lp rats than in the Np rats. Four and 8 weeks after the operation, EGF positive cells in the distal tubules were more observed in the Lp rats than in the Np rats. These findings reveal that protein restriction is effective in preventing renal tubular scarring in immature rats and that EGF is involved in the process of this prevention.

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We examined time-dependent histological changes of the calcified fibrocartilage area in a tibial cranial cruciate ligament (CCL) insertion after ligament resection in rabbits. The animals were divided into two groups: those undergoing CCL substance resection in the right stifle (resected group) and those receiving the same operation without CCL resection in the left stifle (sham operated group). Five animals were euthanized with deep anaesthesia at four time periods (1, 2, 4 and 6 weeks), and Haematoxylin-eosin and Safranin-O stainings and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining were performed. The average percentage of TUNEL-positive chondrocytes and the average thickness of the glycosaminoglycan (GAG)-stained area in the calcified fibrocartilage area were measured. Two and 4 weeks after the surgery, the average percentages of TUNEL-positive chondrocytes in the resected group (23.8 ± 10.3% and 15.9 ± 6.7%, respectively) were significantly higher than those in the sham operated group (8.9 ± 3.8% and 7.4 ± 1.6%, P<0.05, respectively). Six weeks after the surgery, the average thickness of the GAG-stained area in the resected group (7.7 ± 13. 5μm) was significantly smaller than that in the sham operated group (69.4 ± 39.9 μm, P<0.05). Our results suggest that the average percentage of TUNEL-positive chondrocytes became a peak in 2 weeks and that histological changes occurred in 6 weeks. The chondrocyte apoptosis can induce decrease of GAG-stained area after resection of CCL. Therefore, chondrocyte apoptosis in the calcified cartilage area in the CCL tibial insertion might lead to histological changes.

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Estradiol is known to exert neuroprotective effect against glutamate toxicity in hippocampal-derived cell line (HT22). This study investigated whether estradiol modulates the anti-apoptotic signal through the phosphorylation of Akt and its downstream targets, including Bad, forkhead transcription factors FKHR and FKHRL1. Pretreatment with 17β-estradiol decreased glutamate toxicity-induced cell death in HT22 cells. Also, pretreatment with 17β-estradiol significantly decreased the positive cells of TUNEL stain, compared to that of only glutamate-treated cells. Potential activation was measured by phosphorylation of Akt at Ser⁴⁷³, Bad at Ser¹³⁶, FKHR at Ser²⁵⁶, and FKHRL1 at Thr³² using Western blot analysis. 17β-estradiol pretreatment prevented the glutamate-induced decrease of pAkt, pBad, pFKHR, and pFKHRL1. These findings clearly confirm that 17β-estradiol plays a potent neuroprotective role against glutamate-induced toxicity and suggest that phosphorylation of Akt and its downstream targets by 17β-estradiol mediated these protective effects.

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A total of 328 cloacal swabs and 163 footpads of wild birds were investigated for the presence of salmonellae. All 19 isolates from cloacal swabs were serotyped as Salmonella Typhimurium susceptible to all five conventional antimicrobial agents (ampicillin, chloramphenicol, streptomycin, oxytetracycline and nalidixic acid) tested. In contrast, 15 salmonellae isolated from footpads included S. Muenhen, S. Virchow, S. Bareily and S. Bovismorbificans, including S. Typhimurium; these non-Salmonella Typhimurium isolates showed multiple drug resistance.

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Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL) is a nuclear IκB protein that is also known as interleukin-1-inducible nuclear ankyrin repeat protein and inhibitor of nuclear factor κBζ (IκBζ). We previously observed that MAIL-deficient mice were affected by atopic dermatitis-like skin lesions and demonstrated the importance of MAIL in the skin. In this study, we investigated MAIL expression in mouse keratinocytes. MAIL mRNA was constitutively expressed in the skin epidermis. MAIL expression was also confirmed in primary keratinocytes and the PAM212 keratinocyte cell line. The inhibitors of nuclear factor κB (NF-κB)-Bay11-7082 and the IκBαM supersuppressor-considerably downregulated MAIL expression in the keratinocytes. Immunoreactivity for NF-κB components was localized in the cytoplasm and nucleus of normal unstimulated keratinocytes. The expression level of MAIL in the skin did not change following lipopolysaccharide (LPS) administration to mice. Interestingly, in accordance with the in vivo findings, the MAIL expression level did not change following LPS stimulation even in primary keratinocytes; however, MAIL expression was strongly increased by interleukin-1 stimulation. These results collectively suggest that the constitutive expression of MAIL in keratinocytes is controlled, at least in part, by NF-κB and that there may be LPS-specific repressive mechanisms that inhibit MAIL induction.

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Cerebrospinal fluids (CSFs) from 9 Pug dogs with necrotizing meningoencephalitis (NME: Pug dog encephalitis) were examined to identify the antigens for anti-astrocyte autoantibodies. Each CSF exhibited a positive reaction to the cytoplasm of cultured canine astrocytes by an indirect fluorescent antibody test. In an immunoblotting analysis on normal canine brain proteins, eight of 9 CSFs showed a common band of 52 kDa, corresponding to glial fibrillary acidic protein (GFAP), and all of 9 CSFs reacted with purified bovine GFAP. From these results, GFAP is one of the common autoantigens in Pug dogs with NME. On the other hand, the reactivity of CSFs to chymotrypsin-digested bovine GFAP fragments were variable among dogs, indicating that the antibodies in the CSFs recognized different epitopes on GFAP.

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Leptin secretion by adipose tissue is involved in many physiological control systems, including those that determine growth, development, body composition, milk production, and reproductive function. In the adipocyte of monogastric animals, malonyl CoA (coenzyme A) seems to link the flux of energy substrates to the control of leptin production. In this study, we tested this for ruminants by examining the effect of cerulenin, an inhibitor of de novo fatty acid synthesis at the step from malonyl CoA to palmitate, on leptin production by cultured bovine adipocytes derived from intermuscular fat. Purified preadipocytes were obtained by the ceiling culture method, and adipogenic media were used to induce their differentiation into adipocytes. We found that leptin concentrations increased significantly with time in culture, and with increases in glucose concentration. Addition of 2-deoxy-D-glucose to the medium, a competitive inhibitor of glucose transport and metabolism, suppressed leptin secretion. In media with high glucose concentrations, cerulenin enhanced leptin secretion. We conclude that, as in monogastrics, malonyl CoA may play a key role in the control of leptin secretion in ruminants.

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In our previous study, it was demonstrated that the administration of anion salts, which slightly lower the dietary cation-anion difference (DCAD), in the prepartum period is safe and effective for preventing milk fever in multiparous cows. In the present study, several biomarkers, which might show activation of Ca metabolism, were analyzed using stored samples in the previous study to investigate the mechanism of the preventive effect on milk fever by lowering DCAD. Changes in bone-specific alkaline phosphatase activity, osteocalcin and insulin-like growth factor I concentrations in serum were almost the same among the three groups of multiparous cows with or without the oral administration of anion salts, while the levels of these serum biomarkers in the group of primiparous cows (heifer group) were much higher compared with those in the three multiparous groups throughout the experimental period. Urinary deoxypyridinoline excretion was not a useful biomarker for dairy cows because it hardly changed during the peripartum period in all groups. However, serum tartrate-resistant acid phosphatase (TRAP) activity, which is known as a biomarker of osteoclast activity, was well associated with the administration of anion salts lowering DCAD because among the three multiparous groups, only the group of multiparous cows fed the anion salts (anion group) showed an increased level, which rose to the level in the heifer group, and was markedly higher than those in the other control groups of multiparous cows. The increased activity of serum TRAP in the anion group suggested that Ca in the plasma pool was mobilized smoothly from bone-bound Ca via mature osteoclasts at parturition, which might be due to prior activation under mild acidosis induced by slightly lowering DCAD. Therefore, TRAP was the best biomarker to monitor the activation of Ca metabolism in dairy cows fed anion salts.

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Human skin barrier function is evaluated by measuring transepidermal water loss (TEWL). However, this conventional method has not been applied to assess canine skin barrier function because the equipment is not suitable for dogs due to the effects of air turbulence resulting from movement of the subject and vapor from the subject's hair coat. The TEWL analyzer CC-01 was developed as a closed-chamber method device; this means that instead of using the open-chamber method, it has a ventilated chamber that uses dry air. TEWL values measured by CC-01 show less variability than those measured by the conventional method. An ambient temperature of 20-26°C is optimal for measurement with the CC-01, and humidity affects the length of measurement but not the values. The CC-01 may be more reliable for measurement of TEWL than the conventional methods and may give new insights in the evaluation of skin barrier function in dogs.

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A new enzyme immunoassay (EIA) for the measurement of furosemide in horse plasma is described. The lower limit of detection of this EIA method was 7.8 ng/ml. The intra-and inter-assay coefficients of variation ranged from 2.5% to 4.9% and 7.5% to 9.8%, respectively. Cross-reactivity with other compounds was not observed. There was a high correlation (r²=0.987) between the high-performance liquid chromatography and EIA results obtained for furosemide concentrations in horse plasma. These results indicate that the newly developed EIA method is useful for the quantitative analysis of furosemide in horse plasma.

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A 4-months-old calf of Japanese black cattle was diagnosed with orotic aciduria by gas-chromatography/mass-spectromerty (GC/MS). Until now orotic aciduria had not been reported in Japanese black cattle. The animal showed repeated diarrhea. The hematocrit was low, and microcytes and acanthocytes were observed in blood smears. The calf had lower serum total protein concentrations with a higher blood ammonia concentration. Needle-shaped crystals of orotic acid were observed in urinary sediments. Sequence homologous analysis with cattle uridine monophosphate synthase DNA indicated silent mutation in the affected calf.

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Serum amyloid A (SAA) and haptoglobin (Hp) levels were determined in 25 cows suffering from amyloidosis. SAA levels in cows with amyloidosis ranged between < 0.3 and 225.8 μg/ml, with a median level of 105.1 μg/ml, and Hp levels ranged between < 20 and 1860 μg/ml, with a median level of 950 μg/ml. These levels were significantly higher than the levels observed in healthy cows (SAA levels ranged from < 0.3 to 13.5 μg/ml, with median of 1.4 μg/ml, and Hp levels were undetectable in all cases), but were not significantly different from the levels observed in control cows with chronic inflammation. There was a significant correlation between SAA and Hp levels in cows with chronic inflammation , but not in cows with amyloidosis. It was concluded that the serum SAA levels in cows with amyloidosis might be changed by some factor other than inflammation.

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Growth factors, Platelet Derived Growth Factor (PDGF) and Transforming Growth Factor (TGF)-β, were demonstrated in vertebrate and invertebrate immmunocytes. It is generally known that the growth factors are important in various biological processes, such as the regulation of cell differentiation, development and wound healing. In the present study, the presence of TGF-β1 and PDGF-receptor-α in plasmatocytes and PDGF-AB in granulocytes of a soft tick, Ornithodoros moubata, was confirmed immunohistochemically. The tick midgut might be damaged by intracellular digestion and penetration of protozoa. Therefore, it is considered that PDGF from granulocytes may affect the PDGF-receptor-α in plasmatocytes and TGF-β from plasmatocytes may function to repair the midgut. The results obtained here add to the elucidation of the functions of tick hemocytes.

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The expressions of cytokeratin 8 and 18 (CK8 and CK18) in the normal canine skin (2 cases) and cutaneous adnexal tumors (127 cases) were investigated immunohistochemically. In the normal skin, co-expression of CK8/18 was found in the glandular epithelium of apocrine sweat glands, and single CK8-immunoreactivity was detected occasionally in the external root sheath at the isthmus and suprabulbar regions of the hair follicles. Neoplastic glandular epithelial cells in all apocrine gland tumors (21/21 cases, 100%) had co-expression of CK8/18. In trichoblastomas (27/28 cases, 96%), most neoplastic cells were diffusely positive for CK8, but those were negative for CK18. Single CK8-expression was also observed in basaloid neoplastic cells in several cases of trichoepitheliomas (7/19 cases, 37%) and pilomatricoma (1/7 cases, 14%). In several cases of trichoblastomas (4/28 cases, 14%) and trichoepitheliomas (2/19 cases, 11%), tumor cells forming glandular structures had co-expression of CK8/18. There were no positive reactions for both CK8 and 18 in infundibular keratinizing acanthomas, and sebaceous and hepatoid gland tumors. The present findings indicate that co-expression of CK8/18 is a specific feature of apocrine sweat glands and single CK8-expression represents the natures of external root sheath or pluripotential stem cells. Thus, the combination of CK8- and 18-immunostainings may have the utility to confirm the directions of differentiation in canine cutaneous adnexal tumors providing a reliable hallmark for histopathological diagnoses.

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To investigate whether inactivation of the p53 and retinoblastoma (Rb) protein pathways contributes to the development of canine hemangiosarcoma (HSA), we examined immunohistochemically the expression of p53, Rb, phosphorylated Rb (phospho-Rb), p16, and cyclin D1 in 39 spontaneous canine HSAs and 10 hemangiomas. In addition, mutations in the p53 gene were analyzed by polymerase chain reaction (PCR)-single-stranded conformation polymorphism and PCR direct sequencing; furthermore, we quantified cyclin D1 mRNA by semiquantitative real-time reverse transcription-PCR. Positive immunoreactivity for p53 was observed in 17.9% of HSAs. However, mutations were not detected in these cases. The labeling indices for Rb, phospho-Rb, and cyclin D1 were markedly higher in all HSAs than in hemangiomas. Of the 7 cases with cyclin D1-positive immunoreactivity, 4 overexpressed cyclin D1 mRNA (to a level more than 10-fold higher than that of GAPDH mRNA). The p16 protein was clearly detected in all hemangiomas; however, 82% of the neoplastic cells in HSA showed a loss of or low immunoreactivity. These results suggest that alteration of the p16-cyclin D1-Rb pathway, rather than the p53 pathway, may be associated with the pathogenesis of canine HSA.

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Metastasis of malignant carotid body tumor to multiple bones was detected in a 13-year-old female Siberian husky dog. Radiographs exhibited an abnormal mass in the retropharyngeal site and osteolytic lesions in the vertebral bodies, spinous process, tibia, and ribs. At necropsy, multiple masses were observed in the bones as well as at the dorsal area of the retropharynx. Histologically, the tumor cells, arranged in sheets and clusters, had eosinophilic finely granular cytoplasm. Immunohistochemistry showed the tumor cells were positive for neuron-specific enolase and synaptophysin. Electron microscopy demonstrated a number of dense membrane-bound granules in the cytoplasm of the tumor cells. Based on these findings, this case was diagnosed as multiple bone metastases of a malignant carotid body tumor. Spinal cord damage induced by the tumor mass was the cause of the hind limb paralysis of the present dog.

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Neonatal weakness of calves is one of the common reproductive-related problems in captive cetaceans; clinical signs can be observed in the first few hours after delivery. Three 3-day-old bottlenose dolphins died with history of weakness since birth. Pathological study demonstrated purulent bronchopneumonia associated with prominent bacterial colonies and foreign substances in alveoli, suggesting aspiration pneumonia as a cause of neonatal weakness and resultant death of the three calves.

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The aim of this study was to investigate the pharmacokinetics of oseltamivir carboxylate (OC) in horses (n=6) after oral administration of its prodrug oseltamivir. The binding rate of OC to horse plasma proteins was negligible (<1%). Oral administration of oseltamivir of 2 mg/kg body weight of oseltamivir to horses provided a plasma concentration of OC (mean maximum concentration: 257.9 ng/ml) above the inhibitory concentrations against equine influenza A viruses determined in vitro. However, because OC is rapidly eliminated from horse plasma (mean elimination half-life: 2.5 hr), administration intervals should be less than 10 hr to retain a suitable concentration when using a single dose of 2 mg/kg oseltamivir.

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For proper management and conservation of the Kuril harbor seal (Phoca vitulina stejnegeri) through disease control, serological analysis was performed for influenza A virus infection in free-ranging seals in Hokkaido, Japan. Serum samples were collected from seals at Nosappu (231 seals), Akkeshi (16) and Erimo (75), between 1998 and 2005, and were analyzed by ELISA. Antibodies to the influenza A virus were detected only in seals from Nosappu. The incidences were 11% (1/9), 3% (2/66), 12% (7/59) and 6% (5/77) in 1998, 2003, 2004 and 2005, respectively. These suggest sporadic infection. Because antibody-positive seals included juvenile seals in each year, the infections were considered to have been circulated since no later than the late 1990s until recent years. ELISA-positive sera were analyzed by hemagglutination inhibition (HI) tests to determine the subtypes. Antibodies to the H3 and H6 subtypes were detected in 10 and 2 sera, respectively. Two of the sera that had antibodies to the H6 subtype also had antibodies to the H3 subtype. These two seals were considered to have been infected with both the H3 and H6 subtypes. This is the first investigation to find antibodies to the H6 subtype in seals. Although the H6 subtype had been isolated only from avians, genetic analysis had suggested that the H6 subtype could become a novel mammalian pathogen. For definitive diagnosis, detection of the virus from the tissue or mucus of seals is required.

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Canine respiratory coronavirus (CRCoV), which is more closely related to the bovine coronavirus (BCoV), has recently been detected in dogs. In this study, we examined whether BCoV was capable of infecting and exhibiting pathogenicity in dogs. Three 1-month-old pups were oronasally given field isolates of BCoV, and were kept together with 2 control animals. As a result, increases in BCoV-neutralizing antibody titers were confirmed in all pups in the challenged and control groups. Moreover, the virus gene was also detected in oral and rectal swabs by RT-PCR. These results indicate that BCoV infects dogs, and easily infects other dogs that are kept together. However, no clinical symptoms such as respiratory symptoms and diarrhea were observed.

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