Nitric oxide (NO) is a free radical and produced from L-arginine by nitric oxide synthase (NOS). Since NO is recently suggested to be involved in olfactory perception, the expression of eNOS, an isoform of NOS, was examined in the rat olfactory epithelium. The activity of NADPH-diaphorase was also examined as a marker of NOS. In the dorsomedial region of the nasal cavity, intensely positive reactions for NADPH-diaphorase were observed in the entire cytoplasm of sensory cells (olfactory cells). By immunohistochemistry, intensely positive reactions for eNOS were also found in the dorsomedial region of the nasal cavity. These reactions were observed on the free border of the olfactory epithelium. By immunoelectron microscopy, positive reactions for eNOS were found in the cilia of olfactory cells. In addition, in situ hybridization analysis of the olfactory epithelium revealed the expression of eNOS mRNA in the olfactory cells. These results indicate the presence of eNOS in the olfactory cells of the rat, and differential expression of eNOS in the olfactory epithelium depending on the regions of the nasal cavity. In addition, NO produced by eNOS may be involved in olfactory perception in the cilia of olfactory cells.
The aim of this study was to determine the developmental changes of small leucine-rich proteoglycans (PGs), decorin, biglycan and fibromodulin, in ICR mouse retinas and to elucidate their role in the adult retina using kainic acid (KA)-induced retinal degeneration model. Retinas of prenatal, postnatal and adult mice were collected for histological and immunohistochemical staining to investigate the changes in distribution of these PGs. Decorin-and fibromodulin-immunostainings were diffusely distributed at prenatal and early postnatal stages and were stronger in the adult retina. However, biglycan was moderately distributed in the prenatal and early postnatal stages and was faint in the adult retina. Retinas were collected at 1, 3 and 7 days after intravitreal injection of KA. Retinas of KA injected eyes underwent shrinkage accompanied by serious damage in the inner layers. Decorin and fibromodulin were upregulated in the inner retinal layers of KA-injected eyes compared to the normal ones. Our results suggest that decorin and fibromodulin play key roles in retinal differentiation, and contribute to the retinal damage and repair process. However, biglycan may have no or only a limited role in the mouse retinal development or repair process.
Spina bifida aperta (SBA) is a congenital malformation of the spinal cord with complications such as spinal ataxia and bowel and bladder dysfunction. We have developed a chick model with surgery-induced SBA that shows spinal ataxia after hatching. In the present study, motor neurons in the early stages in chicks with and without SBA were observed by immunohistochemical staining with a monoclonal antibody against Islet-1, a motor neuron marker. Delay in migration and maturation of motor neurons was observed in SBA. Although the final numbers of Islet-1-positive neurons in these two groups were not different, a defect in the production and elimination of excess motor neurons in the early developmental stages in the SBA group may be involved in the pathological mechanism of the motor complications of this disease.
We examined the number of ribosomal gene (rDNA) loci in the metaphase spreads of 54 dogs by FISH method. We found that in 16 dogs (30%) one or two loci were missing. The total number of rDNA loci was varied from 5 to 7 in males and from 4 to 6 in females. As the male dog consistently bears the rDNA on the Y chromosome, the polymorphism of the rDNA locus was ascribed to the absence of autosomal rDNA loci. Indeed, the frequency of polymorphism is almost equivalent in both sexes in the beagle. In one female beagle dog, remarkable intense fluorescence signals were observed at the four autosomal loci, indicating the in situ amplification of rDNA.
An 8-year-old, intact female Maltese dog was presented with decreased tear production and unilateral loss of eye blinking. Neuro-ophthalmic examination and brain magnetic resonance imaging were performed to determine the origin of facial paresis. A cystic lesion in the left pontomedullary region which displayed equal intensity to cerebrospinal fluid was revealed. Hyposignality was noted on fluid attenuated inversion recovery sequences, and the lesion was suggestive of an arachnoid cyst. This report described unilateral facial nerve dysfunction that resulted from a suspected brainstem arachnoid cyst in an unusual anatomic location.
To investigate potency of oral disulfiram (DSF) compared with that of dexamethasone (Dexa), on endotoxin-induced uveitis (EIU) in rats. The oral administration with 750 mg/kg DSF suppressed the number of inflammatory cells, protein concentration, and levels of tumor necrosis factor (TNF)-α, Nitric oxide (NO) and prostaglandin (PG) E2 in the aqueous humor and improved the histiologic status of the ocular tissue at 24 hr after lipopolysaccharide (LPS) injection. The anti-inflammatory potency of DSF oral administration was as strong as that observed with 0.5 mg/kg Dexa in the present study. The results suggest that DSF might pave the way for a novel therapeutic agent for the management of uveitis.
A 3-year-old, spayed female miniature dachshund was presented for vomiting and anorexia. Thoracic radiographs and CT scan revealed abnormal pulmonary opacities at bilateral caudal lobe. Cytological analysis of the pulmonary mass revealed the presence of large lymphohistiocytic cells and small lymphocytes with occasional neutrophils and plasma cells. An open lung biopsy was performed and a diagnosis of pulmonary lymphomatoid granulomatosis (LYG) was made. The dog was administered CHOP based therapy (modified UW-25), and it survived for 1,022 days after admission. Immunohistochemistry revealed pulmonary lesions consisted of many CD79a positive B cells aggregation and proliferation with prominent angiocentric pattern. This was the first case of canine pulmonary LYG managed by CHOP chemotherapy.
Blood pressure in female SDT-fa/fa rats was periodically investigated at ages 8, 16, and 24 weeks. Furthermore, an insulin therapy was performed for 5 weeks in the female rats at age 11 weeks, and the change of blood pressure was examined. In addition to obesity, hyperglycemia, hyperinsulinemia, and hyperlipidemia, hyperleptinemia and increased urinary angiotensinogen level were observed during the experimental period. Blood pressure was elevated at ages 8 and 16 weeks, but that at 24 weeks was comparable to that in Sprague-Dawley (SD) rats. Heart rate was decreased from age 8 to 24 weeks. Insulin therapy induced good glycemic control and improvement of hyperlipidemia, but the blood pressure was not reduced. Blood pressure in female SDT-fa/fa rats was elevated temporarily. The blood pressure was not decreased by insulin treatment. SDT-fa/fa rat is a useful model to investigate the relation between diabetes mellitus and hypertension.
This study aims to understand Th2 immune responses and alternative macrophage activation against nematode parasites in aged mice. Eighteen-month (18 M) and three-month (3 M) old C3H/HeN mice were inoculated with Heligmosomoides polygyrus (Hp) larvae. Real-time PCR analysis indicated that interleukin (IL)-4 and IL-13 gene expression was elevated in both groups after infection, but the expression level was significantly low in 18 M mice. Macrophage phenotype was monitored by measuring arginase-1 gene expression and immunofluorescence staining in small intestine, showing a decrease in the number of alternatively activated macrophages (AAMacs) around worm cysts in 18 M mice. These results suggest that the Th2 immune response in aged mice against a nematode parasite was not sufficiently induced to promote AAMacs.
A diagnosis of acute myeloid leukemia was made in a 10-month-old Holstein female calf. The leukemia was macroscopically characterized by great enlargement of the spleen and moderate enlargement of some lymph nodes. Histochemical and immunohistochemical examination disclosed the presence of neoplastic cells either containing metachromatic and tryptase-positive granules or expressing factor VIII-related antigen. The granules, which were positive for naphthol AS-D chloroacetate esterase and did not have particulate contents, were distinct from those of basophilic leukemia cells. This leukemia was thought to be derived from a common myeloid progenitor capable of giving rise to megakaryocyte-erythrocyte progenitors and granulocyte-monocyte progenitors with the ability to differentiate into mast cells.
The present study was designed to explore the inhibitory mechanism by nitric oxide (NO) of the tachykininergic neuro-muscular transmissions in the hamster ileum. In the presence of guanethidine (1 μM), atropine (0.5 μM), nifedipine (0.1 μM) and apamin (100 nM), electrical field stimuli (EFS; 0.5 ms duration, 15 V) evoked non-adrenergic, non-cholinergic excitatory junction potentials (EJPs) in circular smooth muscle cells. The EJPs were markedly inhibited by the tachykinin NK1 receptor antagonists [D-Pro4, D-Trp7,9]-SP(4-11) (3 μM). Both the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 200 μM) and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 μM), did not affect on the resting membrane potentials, but enhanced the tachykininergic EJPs. In the presence of L-NAME (200 μM), exogenously applied NO (10 μM) and the membrane permeable analogue of guanosine 3',5'-cyclic monophosphate (cGMP), 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP, 3 mM), significantly inhibited the tachykininergic EJPs. Application of EFS (0.5 msec duration, 15 V) with trains of 20 pulses at 20 Hz increased amount of released substance P (SP). The release of SP was further increased by the treatment of L-NAME or ODQ, but markedly reduced by exogenously applied NO and 8-Br-cGMP. These results suggest that the endogenous NO may inhibit the tachykininergic neuro-muscular transmissions by the decrease of SP release from the tachykininergic neurons, possibly through a guanylate cyclase-cGMP-dependent mechanism in the hamster ileum.
A pharmacokinetic study of a commercial florfenicol-tylosin (2:1) combination product was conducted in six beagle dogs after intravenous (IV) and intramuscular (IM) administration at doses of 10 mg/kg (florfenicol) and 5 mg/kg (tylosin). Serum drug concentrations were determined by a validated high performance liquid chromatography (HPLC) using UV detection. A rapid and nearly complete absorption of both drugs with a mean IM bioavailability of 103.9% (florfenicol) and 92.6% (tylosin), prolonged elimination half-life, and high tissue penetration with steady state volume of distribution of 2.63 l/kg (florfenicol) and 1.98 l/kg (tylosin) were observed. Additional studies, including pharmacodynamic and toxicological evaluation are required before recommendations can be made regarding the clinical application of the product in dogs.
Cytochrome P450 (CYP) is important for metabolism of not only xenobiotics such as drugs, but also endogenous compounds including arachidonic acids. CYP4A11, CYP4F3v2, CYP4F11, and CYP4F45 have been identified in cynomolgus macaque, an animal species widely used for investigation of drug metabolism due to its evolutionary closeness to human. However, their metabolic functions have not been investigated. In this study, proteins were heterologously expressed in Escherichia coli and characterized by metabolic assays using arachidonic acids as substrates that are metabolized by CYP4 isoforms in human. The results showed that all four CYPs metabolized arachidonic acids. Therefore, cynomolgus macaque CYP4A11, CYP4F3v2, CYP4F11, and CYP4F45 are functional enzymes.
We previously demonstrated that intracerebroventricular (ICV) injection of orexin-A induces arousal and increased metabolic turnover rates of norepinephrine, dopamine, and serotonin in layer (egg-type) chicks. Because monoamine oxidase-A (MAO-A) is a potent degrading enzyme of these monoamines, we hypothesized that orexin-A may mediate its arousal-inducing effects through MAO-A. Therefore, we simultaneously injected clorgyline, a specific inhibitor of MAO-A, with orexin-A and examined behavior of chicks. Behaviors associated with arousal were attenuated in the group of chicks that received clorgyline and orexin-A compared with those that received orexin-A alone. For the monoamine turnover rate, enhancement of the turnover rate of serotonin by orexin-A was attenuated by clorgyline. Therefore, we conclude that orexin-A-induced arousal is dependent upon monoamine neural activities stimulated by MAO-A in chicks.
The petit rat (pet/pet) is a new semi-lethal dwarf mutant with anomalies in the thymus and testes, defects inherited as a single autosomal recessive trait. At birth, these pet/pet rats show low birth weight and extremely small thymuses; at 140 days of age, their thymuses show abnormal involution. In the present study, we examined early postnatal development of hypoplastic pet/pet thymuses. In addition to being hypoplastic at birth, pet/pet thymus growth was almost completely impaired during the early postnatal period. As shown by cellular incorporation of BrdU, the mitotic activity was lower in pet/pet than in normal thymuses, and terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that apoptosis occurred more often in pet/pet than in normal thymus cells during the first few days after birth. These results indicate that postnatal development of the hypoplastic pet/pet thymus is defective due to the reduced proliferation and increased apoptosis of thymic cells.
Endemic spotted fever group rickettsiosis was reported in Shimane Prefecture, Japan. From an analysis of 14 clinical cases found in the endemic area, the infectious agent of spotted fever group rickettsiosis was identified as Rickettsia japonica. In this study, we also found that Rickettsia japonica was highly infected with the vector tick, Haemaphysalis longicornis, in the endemic area. These findings suggest that the high incidence of rickettsiosis in Shimane Prefecture can be explained by the high prevalence of Rickettsia japonica among Haemaphysalis longicornis ticks.
The purpose of the study was to compare two different electroretinography (ERG) instruments used on the same animal in a laboratory setting. Retinal function in both eyes of 12 healthy miniature schnauzers was evaluated under general anesthesia. Scotopic and photopic ERGs were recorded by the HMsERG and the RETIport using the Dog Standard Protocol recommended by the European College of Veterinary Ophthalmologists (ECVO). The waveforms of the ERGs obtained by the two ERG units were similar to those described in previous studies. The 90% reference ranges using the multiple of medians (MoM) showed that the changes in ERG parameters obtained by the HMsERG unit were very similar to those of the RETIport for most ERG responses, except for a few. The results demonstrate that the two ERG systems are comparable for evaluating retinal function in dogs. Further, the results also show that it is necessary to establish ERG system-specific limits of normality in each laboratory or clinic in order to obtain a correct diagnosis, for example by using a graphical representation of the 90% reference range.
This study examined the prevalence and genetic diversity of the bovine group C rotaviruses (GCRVs) in a total of 127 diarrhea fecal samples of calves from 52 Korean native beef calf herds using RT-PCR and nested PCR. Overall, seven of the 127 fecal samples (5.5%) from seven of the 52 herds (13.5%) tested positive for bovine GCRVs only by nested PCR. Sequence and phylogenetic analyses of a partial VP6 gene showed that Korean bovine GCRVs had marked genetic diversity; two Korean strains belonged to the bovine lineage, whereas five Korean strains belonged to the porcine lineage. These results suggest that the genetically diverse bovine GCRVs cause sporadic infections in diarrheic calves in South Korea.
In 2010, the World Organisation for Animal Health recommended the inclusion of a Florida sublineage clade2 strain of equine influenza virus (H3N8), which is represented by A/equine/Richmond/1/07 (Richmond07), in equine influenza vaccines. Here, we evaluate the antigenic differences between Japanese vaccine strains and Richmond07 by performing hemagglutination inhibition (HI) assays. Ferret antiserum raised to A/equine/La Plata/93 (La Plata93), which is a Japanese vaccine strain, reacted with Richmond07 at a similar titer to La Plata93. Moreover, two hundred racehorses exhibited similar geometric mean HI antibody titers against La Plata93 and Richmond07 (73.1 and 80.8, respectively). Therefore, we can expect the antibody induced by the current Japanese vaccines to provide some protection against Richmond07-like viruses.
Feline parvoviruses were isolated from frozen samples of intestines taken from a snow leopard (Uncia uncia) and a serval (Leptailurus serval) that died successively at Sapporo Maruyama Zoo in Hokkaido, Japan. Isolates possessed an antigenic epitope for both the feline panleukopenia virus (FPLV) and mink enteritis virus, identified with a hemagglutination inhibition test. Sequencing analyses of the VP2 region of the isolates revealed that the two isolates were identical and of the FPLV-type. These results suggested that FPLV was introduced from a feral cat which entered the zoo and transmitted the virus inside the zoo.
Six 5-week-old porcine circovirus type 2 (PCV2)-free, cesarean-derived, colostrums-deprived (CDCD) pigs were inoculated intranasally with 106 TCID50 of PCV2. Four CDCD pigs were untreated cohabitants. Forty farm-raised pigs from two PCV2-contaminated herds were randomly selected for PCV2 trace investigations. Blood, nasal, oropharyngeal and fecal samples were collected from all tested pigs weekly. The PCV2 DNA shed at 6-11 and 7-12 weeks of age for PCV2-inoculated pigs and cohabitants, respectively. All the CDCD pigs exhibited seroconversion after PCV2 exposure. In the farm-raised animals, PCV2 shed at 9-15 weeks of age and seroconversion started at 11 weeks of age. Collectively, the pigs had a prolonged PCV2 shedding period following viral exposure, and growing pigs were the source of horizontal PCV2 transmission in PCV2-infected herds.
Bovine coronavirus (BCoV) and group A bovine rotavirus (BRV) are two of major causes for neonatal calf diarrhea. In the present study, a one-step duplex RT-PCR was established to detect and differentiate BCoV and group A BRV from fecal samples. The sensitivity of this method for BCoV and group A BRV was 10 PFU/100 μl and 1 PFU/100 μl, respectively. Twenty-eight diarrhea fecal samples were detected with this method, the result showed that 2 samples were identified as co-infected with BCoV and group A BRV, 26 samples were group A BRV positive, and 2 samples were negative. It proved that this method is sensitive for clinical fecal samples and is worth applying to laboratory diagnosis for BCoV and group A BRV.
An immunoglobulin G (IgG) subclass response against equine herpesvirus type 1 (EHV-1) infection was investigated in horses that were naïve to EHV-1/4 and those that had previously been exposed to EHV-4. The IgG subclass response was determined by an ELISA using EHV-1-specific recombinant gG protein as an antigen. In most horses naïve to EHV-1/4, IgGa, IgGb, and IgG(T) were induced after experimental infection with EHV-1. In contrast, a subclass response dominated by IgGa and IgGb, with no apparent increase in IgG(T), was observed after EHV-1 infection in horses previously infected with EHV-4. Horses naturally infected with EHV-1 in the field showed similar responses. These results indicated that pre-infection with EHV-4 induced a Th-1-biased IgG subclass response against subsequent EHV-1 infection.
A sero-epidemiological survey of human and equine H3 influenza A virus infections in dogs and cats using the hemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests was conducted. Serum samples were collected from 582 dogs and 237 cats in Japan during the periods 2002-2008 and 1997-2008, respectively. Although no HI antibodies against equine H3 virus were detected, 9 (3.8%) from cats and 12 (2.1%) from dogs were HI-positive against human H3 virus. Only one serum each from dogs and cats was NI-positive against N2 virus. These findings suggest that although equine H3 influenza virus infections have not been prevalent in companion animals, human H3N2 influenza A virus infections have occurred in dogs and cats in recent years in Japan.
Embryonated chicken eggs (ECEs) are routinely used to isolate equine influenza virus. Propagation of the virus in ECEs results in selection of variants. In the present study, we determined nucleotide sequences of entire coding regions of parent A/equine/Tottori/1/07 (H3N8) and its derivatives that have different passage histories in ECE. After 12 passages, nucleotide sequence analysis predicted 3 amino acid substitutions in hemagglutinin (HA; 2 in HA1 and 1 in HA2). The two amino acid substitutions in HA1 were located in the vicinity of the cell receptor-binding site. Three other amino acid substitutions were predicted in internal proteins, 1 in the M1, 1 in the NP and 1 in the PA. This is the first report showing mutations in the internal protein genes of equine influenza virus associated with adaptation to ECE.