We aimed to document macroscopic variations in the cellular tapetum in the dog, to provide a histologic description of the macroscopic results and to evaluate the correlation between the macroscopic appearance and aging. Fifty three dogs including 5 beagles, 1 Chihuahua and 47 mixed breeds of each gender were used. For a macroscopic study, the fresh tapetal fundi were photographed using digital camera. For a histological study, the glutaraldehyde-formalin fixed eyes were embedded in nitrocellulose and stained with hematoxylin-eosin or thionine. The normal tapetum was triangular with the rounded angles and the smooth contour. The atypical tapetum was smaller and more variable in shape, contour and color than the normal one. In severe cases, the fundus was devoid of the tapetum. The atypical tapetum tended to increase in frequency with aging. Retinal pigment epithelial cells on the normal tapetum were unpigmented. In the eye with the atypical tapetum, regardless of tapetal size and shape, unpigmented retinal pigment epithelial cells showed a similar distribution to that on the normal tapetum, even in a dog without a tapetum. Although there is a congenitally hypoplastic tapetum, the atypical tapetum tends to increase in incidence and severity with aging.

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Three Klebsiella pneumoniae isolates producing extended-spectrum beta-lactamase (ESBL) were obtained from three dairy cows with clinical mastitis in two farms in western Japan. Two of the 3 isolates from cows in different farms were able to transfer plasmids carrying the bla_CTX-M-2 gene to Escherichia coli recipient. Pulsed-field gel electrophoresis (PFGE) patterns of the 2 isolates were different from each other, although restricted-fragment patterns of the two conjugative plasmids were similar to each other. Additionally, PCR-based replicon typing revealed that both the plasmids belonged to type Inc.T. These results suggest that ESBL-encoding genes can be distributed in bacteria on dairy farms through the plasmids.

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We investigated the phenotypic level of albumin in peripheral blood mononuclear cells (PBMC) of streptozotocin (STZ)-induced diabetic rats. A specific reduction of albumin was identified by 2-dimensional electrophoresis and mass spectrometry. Decreased albumin content was also confirmed by immunoblotting and quantitative real-time PCR. Since albumin is a major and predominant antioxidant in plasma, the PBMC albumin may also contribute to their antioxidant activity. By measuring the amount of H₂O₂, lipid peroxidation and the redox form of glutathione, it was found that the production of the oxidative stress was elevated in STZ-diabetic rats compared to that of normal control. We suggest, therefore, that decreased albumin content may lead to the decreased antioxidant activity in the PBMC of type 1 diabetic rats.

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Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine, sharing a common beta subunit (CDw131) with interleukins 3 and 5. GM-CSF is important for its direct and indirect involvement in host defense. In veterinary medicine, human (h) GM-CSF has been used as a substitute for canine GM-CSF to stimulate canine granulocytes and macrophages. In this study, we compared the effects of three distinct hGM-CSFs produced by bacteria, yeasts and Chinese hamster ovary (CHO) cells with those of Escherichia (E) coli-produced canine GM-CSF on the cluster of differentiation 11b (CD11b) expression in canine granulocytes. The median effective dose (ED₅₀) of hGM-CSFs from bacteria, yeasts and CHO cells was 3.09, 4.09 and 4.27 ng/ml, respectively, with no significant difference among three. In contrast, a significant difference was observed between ED₅₀ of canine GM-CSF (0.56 ng/ml) and three hGM-CSFs according to the paired t-test (P<0.05). We conclude that hGM-CSF can activate canine granulocytes, but the average activity of the three rhGM-CSFs was approximately 15% of that of canine GM-CSF.

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Matrix metalloproteinases (MMPs) 2 and 9 are zinc-dependent endopeptidases that contribute to the control of breakdown and reconstitution of extracellular matrix under both normal and pathological conditions. The main objective of this study was to identify the presence of MMP-2 and -9 in the mucosa of the small and large intestines of clinically healthy beagle dogs using gelatin zymography technique. Intestinal mucosa samples from four different parts of the intestine (duodenum, jejunum, ileum and colon) were taken from 12 healthy laboratory beagle dogs and examined histologically. Based on WSAVA histology standards, recorded findings of all samples were considered insignificant. Pro-MMP-2 and -9 activities were found in 17/48 (35%) and 25/48 (52%) of the samples, respectively. Among four different parts of the intestine of 12 dogs, the ileum had the highest positivity rates of 7/12 (58.3%) and 8/12 (66.7%) for pro-MMP-2 and -9 activities, respectively. However, statistical analysis showed no significant difference of pro-MMP-2 and -9 activities between the separate parts of the intestine (P>0.05). None of the intestinal samples showed gelatinolytic activity corresponding to the control bands of active MMP-2 and MMP-9. This study showed that pro-MMP-2 and -9 could be detected in the intestinal mucosa of healthy dogs using zymography, which seems to be a useful tool to evaluate the role of MMP-2 and -9 in the pathogenesis of canine chronic enteropathies, including inflammatory bowel diseases.

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Serine proteases elicit cellular responses via protease-activated receptor-2 (PAR-2) which is known to regulate inflammation and the immune response. Although the gastrointestinal tract is exposed to large amounts of proteolytic enzymes, the role of PAR-2 in canine inflammatory bowel disease (IBD) remains unclear. The objective of this study was to investigate the effects of PAR-2 activation on inflammatory cytokine/chemokine gene expression in canine intestine and the expression of intestinal PAR-2 and fecal serine protease activity in dogs with IBD. Duodenal biopsies from healthy dogs were cultured and treated ex vivo with trypsin or PAR-2 agonist peptide, and inflammatory cytokine/chemokine gene expression in the tissues was then quantified by real-time PCR. PAR-2 mRNA and protein expression levels in the duodenal mucosa were examined by real-time PCR and immunohistochemistry, respectively. Fecal serine protease activity was determined by azocasein assay. In ex vivo-cultured duodenum, trypsin and PAR-2 agonist peptide induced significant up-regulation of mRNA expression levels of interleukin-1 β (IL-1β), IL-8, mucosae-associated epithelial chemokine (MEC) and fractalkine, and this up-regulation was inhibited by a serine protease inhibitor. Duodenal PAR-2 mRNA and protein expression levels were higher in dogs with IBD than in healthy control dogs. Fecal serine protease activity was significantly elevated in dogs with IBD, and the level of activity correlated positively with the clinical severity score. These results suggest that PAR-2 may contribute to the pathogenesis of canine IBD by inducing expression of inflammatory mediators in response to luminal serine proteases.

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A 9-month-old intact female Yorkshire terrier dog was presented with episodic partial seizure-like cramping of the limbs. The patient’s episodes began six months previously; the interval between episodes became shorter, and the duration of the episodes increased. Various tests including neurologic examination, blood examination, abdominal radiography, ultrasonographic examination, angiographic computed tomography (CT) and brain magnetic resonance imaging (MRI) detected no remarkable changes. After these tests were conducted, the patient’s condition was suspected to be canine epileptoid cramping syndrome (CECS), which could be a form of paroxysmal dyskinesia (PD), and as a trial therapy, Science Diet k/d (Hill’s Pet Nutrition, Topeka, KS, U.S.A.) was prescribed. The clinical signs were dramatically reduced after diet therapy, and we diagnosed the patient with CECS. This is the first case report of CECS in a Yorkshire terrier dog.

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To clarify the pathophysiology of left displaced abomasum (LDA), beef cattle fed high-starch diets were examined. The abomasal pH in beef cattle with LDA was lower than that in non-LDA reference animals (data from beef cattle at an abattoir), suggesting that it facilitated acidity. Bacteriological examinations of the abomasal fluid in cattle with LDA revealed the presence of Pseudomonas spp., Clostridium spp. and Candida spp., presumably reflecting the accelerated influx of ruminal fluid into the abomasum. Biochemical analyses of serum revealed that LDA cattle had higher lactic acid and lower vitamin A and E levels than non-LDA reference animals. These results indicate that beef cattle with LDA may suffer from vitamin A and E deficiencies due to maldigestion of starch and the high acidity of abomasal fluid.

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Hyperadrenocorticism (HAC) is a common endocrine disorder in dogs, in which excess glucocorticoid causes insulin resistance. Disturbance of insulin action may be caused by multiple factors, including transcriptional modulation of insulin signal molecules which lie downstream of insulin binding to insulin receptors. In this study, gene expressions of insulin signal molecules were examined using neutrophils of the HAC dogs (the untreated dogs and the dogs which had been treated with trilostane). Insulin receptor substrate (IRS)-1, IRS-2, phosphatidylinositol 3-kinase (PI3-K), protein kinase B/Akt kinase (Akt)-2 and protein kinase C (PKC)-lambda were analyzed in the HAC dogs and compared with those from normal dogs. The IRS-1 gene expressions decreased by 37% and 35% of the control dogs in the untreated and treated groups, respectively. The IRS-2 gene expressions decreased by 61% and 72%, the PI3-K gene expressions decreased by 47% and 55%, and the Akt-2 gene expressions decreased by 45% and 56% of the control dogs, similarly. Collectively, gene expressions of insulin signal molecules are suppressed in the HAC dogs, which may partially contribute to the induction of insulin resistance.

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Ataxic rolling Nagoya (PROD-rol/rol) mice, which carry a mutation in the α1 subunit of the Ca_v2.1 channel (Cacna1a) gene, were discovered in 1969. They show white spots on agouti coat and have a mutation in the piebald spotting (s) locus. However, mutation analysis of the s locus encoding the endothelin receptor type B (Ednrb) gene in PROD-rol/rol mice had not been performed. Here, we examined the genomic and mRNA sequences of the Ednrb gene in PROD-rol/rol and wild-type rolling Nagoya (PROD-s/s) and studied the expression patterns of Ednrb and Cacna1a genes in these mice in comparison with C57BL/6J mice. Polymerase chain reaction analyses revealed two silent nucleotide substitutions in the coding region and insertion of a retroposon-like element in intron 1 of the Ednrb gene. Expression analyses demonstrated similar localizations and levels of Ednrb and Cacna1a expression in the colon between PROD-rol/rol and PROD-s/s mice, but the expression levels of both genes were diminished compared with C57BL/6J mice. Microsatellite genotyping showed that at least particular regions of chromosome 14 proximal to the Ednrb locus of the PROD strain were derived from Japanese fancy piebald mice. These results indicated that PROD-rol/rol mice have two mutant genes, Ednrb and Cacna1a. As no PROD strain had an intact Ednrb gene, using congenic rolling mice would better serve to examine rolling Nagoya-type Ca_v2.1 channel dysfunctions.

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To examine the effect of Babesia infection on the level of the drug-metabolizing enzyme hepatic cytochrome P450 (CYP) 2D, we intraperitoneally inoculated Babesia microti into male ICR mice. CYP2D protein and CYP2D9 mRNA were significantly decreased at 12 days after infection with B. microti. The activity of bunitrolol 4-hydroxylase, which is catalyzed by CYP2D, was also significantly decreased. The mRNA levels of transcriptional regulators of CYP2D9, hepatocyte nuclear factor 4α and signal transducer and activator of transcription 5b, were markedly suppressed. These results suggest that Babesia infection represses CYP2D expression in the mouse liver. The decline in CYP2D-dependent drug metabolism might be involved in the incidence of adverse drug reactions in patients with babesiosis.

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A dog developed a cervical mass, and computed tomography verified a mass surrounding the trachea with some pulmonary masses. Histopathologically, the cervical mass was composed of malignant neoplastic cells showing follicular appearance which reacted positive for thyroglobulin on immunohistochemistry. A characteristic feature of the tumor was abundant and metaplastic stromal components. Anastomosed collagenous tissues connecting to capsule of the tumor were abundant in the stroma. In parts of the collagenous tissues, mature cartilages and bones were continuously formed. There was no cellular atypia or invasion in the components. We diagnosed this case as follicular thyroid carcinoma with metaplastic stroma. This is the first case report that characterizes stromal components with chondroid and osseous metaplasia in a canine thyroid carcinoma.

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We measured the plasma ghrelin and cortisol concentrations in non-lactating cows under fixed-time feeding conditions followed by an acute or gradual fasting treatment. During the 4 days before fasting, animals in Group 1 were fed a fixed amount of rations at 0800 and 1600 hr, and those in Group 2 were fed a gradually reduced amount. Thereafter, the plasma ghrelin concentrations of each animal were measured for 40 hr. The plasma ghrelin concentrations, which were low at the onset of fasting, increased before and after 0800 during fasting in Group 1, but not in Group 2. There were no significant differences in the plasma cortisol concentration within or between the groups. It was demonstrated that acute fasting induces elevation of the plasma ghrelin concentration, but that gradual fasting does not. This result suggests that fixed-time and fixed-quantity feeding caused a daily ghrelin rhythm in the cow and that this rhythm influenced changes in plasma ghrelin.

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Botulinum neurotoxin type A (BoNT/A) cleaves SNAP-25 and interrupts the release of acetylcholine. We previously reported that BoNT/A subtype 2 (BoNT/A2) ameliorates pathologic behavior more effectively than subtype 1 (BoNT/A1) in a rat Parkinson’s disease model. Here, we further show BoNT/A2 has fewer adverse effects than BoNT/A1. We first confirmed that intrastriatal treatments of both BoNT/As had no-effect on dopaminergic terminals in the striatum. SNAP-25 cleaved by BoNT/A2 was strictly localized to the striatum on the injected side; however, SNAP-25 cleaved by BoNT/A1 diffused contralaterally. Furthermore, treatment with BoNT/A1 caused a significant reduction in body weight, while BoNT/A2 treatment did not. These results suggest that BoNT/A2 is more beneficial for clinical application against Parkinson’s disease than BoNT/A1.

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Contamination of estrogenic compounds disrupts endocrinological and neurological reproductive systems in animals. Neonatal exposure to 17α-ethinyl estradiol (EE) induced an abnormal estrous cycle at postnatal day (PND) 180, but not at PND90. We found that serum level of luteinizing hormone (LH) at the latter half of proestrus in EE-treated rats was lower than in the controls at PND90 when there was no significant difference on estrous cyclicity. Additionally, kiss1 mRNA levels in the anteroventral periventricular nucleus-preoptic area (AVPV/POA) were lower in EE-treated rats than in the controls. The expression of GnRH precursor (GNRH1) mRNA in the AVPV/POA and that of LH beta subunit (LHb) mRNA in the pituitary were similar in the control- and EE-treated groups. Our results indicated that neonatal exposure to EE leads to reduced expression of kiss1 mRNA in AVPV/POA and LH-surge, which is likely related to the delayed reproductive dysfunction seen in adult female rats.

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The serum amyloid A (SAA) concentration is higher in mammary tumors with metastases in both humans and animals. In the present study, the direct effects of recombinant feline SAA (rfSAA) protein on invasiveness of feline mammary carcinoma cells were evaluated. As an indicator of invasiveness, matrix metalloproteinase-9 (MMP-9) expression was investigated in 4 feline mammary carcinoma cell lines of different origins. In 3 of 4 cell lines, MMP-9 expression was significantly increased by rfSAA stimulation. The invasive capacities of feline mammary carcinoma cells were also stimulated by rfSAA. The findings of this study have identified a novel role for SAA in mammary tumorigenesis and suggest that therapeutic strategies targeting SAA may provide new alternatives in treating tumor invasion and metastasis.

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In this study, reverse genetics was applied to produce vaccine candidate strains against highly pathogenic avian influenza viruses (HPAIVs) of the H5N1 subtype. The H5 subtype vaccine strains were generated by a reverse genetics method in a biosafety level 2 facility. The strain contained the HA gene from the H5N1 subtype HPAIV attenuated by genetic modification at the cleavage site, the NA gene derived from the H5N1 subtype HPAI or the H5N3 subtype of avian influenza virus and internal genes from A/Puerto Rico/8/34. Vaccination with an inactivated recombinant virus with oil-emulsion completely protected chickens from a homologous viral challenge with a 640 HAU or 3,200 HAU/vaccination dose. Vaccination with a higher dose of antigen, 3,200 HAU, was effective at increasing survival and efficiently reduced viral shedding even when challenged by a virus of a different HA clade. The feasibility of differentiation of infected from vaccinated animals (DIVA) was demonstrated against a challenge with H5N1 HPAIVs when the recombinant H5N3 subtype viruses were used as the antigens of the vaccine. Our study demonstrated that the use of reverse genetics would be an option to promptly produce an inactivated vaccine with better matching of antigenicity to a circulating strain.

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Astroviruses and kobuviruses are frequently found in mammalian feces, including that of humans. The present study examined fecal samples from 91 Korean dogs suffering from diarrhea. Canine astroviruses (CAstVs) and canine kobuviruses (CKoVs) were identified in 2 (2.1%) and 46 (50.6%) dogs, respectively. Nucleotide sequence analysis coupled with phylogenetic analysis using the neighbor-joining method showed that CAstVs clustered into four genetically diverse groups. Two Korean CAstVs belonged to group 2 alongside strains isolated in Italy and France. Twelve of the Korean CKoVs belonged to a single clade, along with strain UK003 identified in the UK and six CKoVs identified in the USA. Thus, the results suggest that the Korean strain of CAstV is closely related to strains isolated in Europe. Surely, CKoV in South Korea could identify the circulation among dogs population.

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Borna disease virus (BDV) infection causes neurological disease in cats. Here, we report BDV infection in 199 hospitalized domestic cats in the Tokyo area. BDV infection was evaluated by detection of plasma antibodies against BDV-p24 or -p40. BDV-specific antibodies were detected in 54 cats (27.1%). Interestingly, the percentage of seropositive cats was not significantly different among the three clinical groups, i.e., healthy (29.8%), neurologically asymptomatic disease (22.2%) and neurological disease (33.3%). The specific antibodies were present even in cats aged below one year. The seropositive ratio was constant, irrespective of age and sampling season. The present study suggests that additional factors are required for onset of Borna disease in naturally infected cats and that BDV is transmitted through vertical routes in cats.

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Seven reticulated giraffes were hand-reared at Nogeyama Zoological Gardens, because the dam had agalactia. Six of the 7 calves exhibited polyarthritis and/or phlegmon in the lower legs. However, the cause of the disorder was unclear. The present study reviewed the clinical records of the 7 giraffes, including the type and amount of colostrum ingested during the first 72 hr. The disorder involved the fetlocks and carpal and tarsal joints in 6 of the 7 calves within an average of 8 days of birth. The average amount of fed bovine or powdered colostrum was 0–2.4 l in the first 24 hr and 2.0–6.2 l during the first 72 hr. Insufficient colostrum quantity might be a factor in polyarthritis and/or phlegmon.

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Sarcoptes scabiei is one of the important external parasites. Although ivermectin is the recommended treatment, many raccoon dogs (Nyctereutes procyonoides) that were rescued and brought to the Kanazawa Zoological Gardens (Yokohama, Japan) have died because of S. scabiei, even after receiving single ivermectin treatment. Therefore, supportive treatment should be required. The present study revealed the number of animals that survived was greater after the administration of ivermectin along with an antibiotic for all raccoon dogs, as well as following the administration of fluid therapy to the debilitated raccoon dogs infected with S. scabiei, immediately after the rescue. During the initial period, treatment to improve the general clinical condition was required prior to deworming treatment for S. scabiei.

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