The vasculature of the hemal node (HN) from the bovine cervical region was investigated using a combination of vascular corrosion casting and scanning electron microscopy. A dense vessel network of capsule was found surrounding the cast of HN parenchyma and had no connection with the subcapsular sinus; these vessels converged and exited the HN via the hilar vein. Within the HN, many anastomoses were found between the capillary networks and the surrounding sinuses in the follicular zone and deep cortex. The sinusoid pathway in the HN was characterized by subcapsular sinuses, which were continuous with the trabecular sinuses and tubular sinuses over the parenchyma, and these sinuses finally entered the medullary veins. In our study, direct communications between cortical capillaries and subcapsular sinuses were identified. This may explain the origin of numerous erythrocytes in the HN sinusoids and help to understand lymphocyte migration of the HN.
In rodents, Gαi2-expressing sensory neurons (SNs) that co-express vomeronasal receptor type 1 (V1R) are specifically found in the vomeronasal organ (VNO) and project their axons to the accessory olfactory bulb (AOB). In goats, however, Gαi2/V1R-expressing SNs exist in both the VNO and the olfactory epithelium. Thus, we examined whether the Gαi2-expressing axons functionally project to the main olfactory bulb (MOB). We analyzed the expression of Gαi2 in the olfactory bulb and found small Gαi2-immunoreactive clusters in the MOB. The Gαi2-immunoreactive axons in these clusters made synaptic contacts with second-order neurons in the MOB. These results suggest that some Gαi2-expressing SNs functionally project their axons to the MOB in goats.
The Red-crowned Crane, Grus japonensis, is an endangered species of crane that has two separate breeding populations, one in the Amur River basin (continental population) and the other in eastern or northern Hokkaido, Japan (island population). So far, only two haplotypes (Gj1 and Gj2) have been identified in the mitochondrial D-loop in island population, whereas seven haplotypes have been found in continental population (Gj3–Gj9). We developed a rapid and inexpensive method of extensive genotyping of D-loop haplotypes in Red-crowned Cranes, based on amplification refractory mutation system (ARMS) PCR assay. Two hundred and three cranes in eastern Hokkaido were studied with this method and supplemental DNA sequencing. Only two haplotypes, Gj1 and Gj2, were confirmed in eastern Hokkaido with Gj2 as a major haplotype. Additionally, only Gj2 was identified in twelve feathers from both sexes found in northern Hokkaido. These results suggest scarce genetic diversity in island population of Red-crowned Cranes in Hokkaido, Japan.
Guide dogs help visually impaired persons both physically and psychologically. More than half of all candidate dogs do not qualify, mainly for behavioral reasons. Improved training efficacy is desirable, and earlier prediction of qualification-related traits would be beneficial. In a previous study, we identified ‘Distraction’, assessed during the training period, as an important behavioral trait for judging the qualification of guide dogs at the Japan Guide Dog Association. As a second step, we aimed to develop an index that can predict during the puppy period. In this study, candidate guide dogs, 5-month-old Labrador retrievers, were assessed by puppy raisers using a newly developed questionnaire that consisted of 20 items. The same dogs were assessed later, at 15 months, by trainers to determine ‘Distraction’. In principal components analysis, nine items, including excitability toward strangers, initiative while out for a walk, and exploration, composed the first principal component (PC1). When we compared PC1 points with ‘Distraction’ points, the two categories were positively correlated (n=110, rs=0.31, P=0.0009). Although the accuracy of the questionnaire should be increased, the results of the present study suggest that it may be possible to assess and predict ‘Distraction’, which is associated with disqualification for guide dogs, early in the puppy-raising period.
Serum amyloid A (SAA) is used as a biomarker for infections and inflammation in humans and veterinary medicine. We cloned ferret cDNA encoding SAA from the liver of a ferret via reverse transcription PCR (RT-PCR). The sequence of the cDNA clone revealed that ferret SAA has an open reading frame of 387 bp that encodes 129 amino acids. The deduced amino acid sequence of ferret SAA has 96.1, 89.9, 86.0, 83.8, 83.0, 73.8 and 65.3% similarity to the mink, dog, cat, cattle, horse, human and mouse SAA genes, respectively. Compared to human SAA, the deduced ferret SAA amino acid sequence had an insertion of an 8-amino acid fragment between amino acids 88 and 95. Recombinant ferret SAA (rfrSAA) was expressed using an Escherichia coli (E. coli) strain, BL21 Star. Using Western blot analysis, anti-SAA mAb provided with the multispecies SAA ELISA kit reacted with purified rfrSAA. A significant dose-response relationship was observed between the rfrSAA protein and a commercial multispecies SAA ELISA kit. In contrast, rfrSAA was not recognized with the antibodies included in a commercial human SAA ELISA kit. These results suggest that the structure of ferret SAA is antigenically similar to other domestic animal SAAs, and the multispecies ELISA kit allows for the detection and quantification of ferret SAA in vivo.
Aspergillus udagawae and A. viridinutans are members of the section Fumigati; both cause invasive aspergillosis in humans. These two Aspergillus species are discriminated from A. fumigatus by molecular methods. Herein, we report two cases of feline orbital aspergillosis, one caused by A. udagawae and the other by A. viridinutans. To our knowledge, Case 1 represents the first reported case of treatment of A. udagawae with a high dosage of itraconazole, and Case 2 represents the first reported case of A. viridinutans infection associated with sarcoma. Identification of the etiologic agents of these cases was confirmed by comparative analyses of the sequences of β-tubulin-encoding genes. With the spread of non-fumigatus aspergillosis, increasing emphasis should be placed on molecular identification of the infecting Aspergillus species and the use of in vitro drug susceptibility tests to ensure the selection of appropriate antibiotics.
A total of 87 Thoroughbred horses and 10 ixodid ticks from a ranch in Hidaka district, Hokkaido were tested for tick-borne diseases. Using the indirect fluorescent antibody (IFA) method, 3.4, 92.0 and 97.7% of the horses showed antibody titers of ≥80 against Anaplasma phagocytophilum, Rickettsia helvetica, and Borrelia garinii, respectively. This is the first report of infection with the 3 pathogens in horses in Japan. Using PCR, DNAs from the peripheral blood of all horses were found negative with any Anaplasma, Rickettsia and Borrelia spp., while those from Haemaphysalis megaspinosa ticks were found positive for Anaplasma sp. closely related to A. phagocytophilum in Japan, and A. bovis. B. japonica was also detected in an H. flava tick for the first time.
Japanese Black cattle occasionally demonstrate growth retardation despite sufficient nutrient intake. To clarify hormonal and transcriptional characteristics, we investigated differences in blood components, including hormones, and differences in exhaustive gene expressions in the liver and peripheral lymphocytes of six cattle with growth retardation (GR cattle) and eight control cattle of the same age and pedigree with normal growth. Hematocrit values and concentrations of hemoglobin, serum albumin, total cholesterol, insulin-like growth factor 1 (IGF-1), thyroxine and insulin in GR cattle were significantly lower than those in controls. GR cattle also excreted higher levels of GH. We used three GR and three control cattle for a microarray analysis in the liver and found that 279 gene expressions were significantly different. However, gene expressions related to the GH-IGF-1 axis, such as the GH receptor and IGF-1, were not significantly different from those of controls. Immune-related gene expressions were significantly lower. To clarify these gene expression levels, peripheral lymphocytes were used for real-time RT-PCR. The expression rates of genes that were significantly lower in the liver, such as chemokine ligand 8, interferon gamma receptor 1 and immunoglobulin light chain VJ region were also significantly lower in three GR cattle than those in the three control cattle. These results suggest that the cause of growth retardation in the present study was due to other factors, not abnormal gene expressions of factors related to the GH-IGF-1 axis in the liver, and that GR cattle were susceptible to infectious disease.
Zinc is one of the essential microelements involved in the regulation of enzyme activity, as well as metabolism of nucleic acid and proteins. There have been few reports on equine serum zinc concentrations during the training period, and little is known about the relationship between zinc levels and diseases in horses. In this study, we measured serum zinc levels in healthy Thoroughbred racehorses, as well as in other horses, under general disease or training conditions. The reference value for serum zinc levels in Thoroughbred horses was 41–79 μg/dl. There were no differences in serum zinc levels due to sex or age. Significant decreases in serum zinc levels were observed after training, but serum zinc levels did not vary with intensity of sweating. Serum zinc levels were lower in horses clinically diagnosed as having shipping fever (36.3 ± 2.7 μg/dl), fever (45.3 ± 3.0 μg/dl) and cellulitis (44.0 ± 3.4 μg/dl), as compared to control values (59.7 ± 9.7 μg/dl). They also tended to decrease in experimentally infected horses one day after inoculation. Changes in serum zinc levels reached nadir one day after surgical invasion, except for a horse that experienced complicating shock. These results suggest that zinc is a serological indicator of inflammatory status in Thoroughbred horses.
Monitoring of blood glucose concentration is important to evaluate the diabetic status of dogs. Continuous glucose monitoring systems (CGMS) have been applied in veterinary medicine for glucose monitoring in diabetic dogs. The purpose of the study was to evaluate the daily glycemic profiles obtained with CGMS and compare glucose fluctuations between day- and night-time in diabetic dogs. Five diabetic dogs were used in this study and were treated with either NPH insulin or insulin detemir. For data analyses, day-time was defined as 9:00 am–9:00 pm and night-time as 9:00 pm–9:00 am. Using glucose profiles, we determined the mean glucose concentrations (1- and 12-hr intervals), and times spent in hyperglycemia >200 mg/dl or hypoglycemia <60 mg/dl. None of the parameters differed significantly between day-time and night-time in dogs treated with NPH insulin or insulin detemir. In conclusion, this study confirmed, using CGMS, that there are no differences in glucose fluctuations between day- and night-time, in diabetic dogs on a similar feeding regimen and insulin administration.
Narrow-band UVB (NB-UVB) is light over a very narrow band of wavelengths (around 311 nm) that is concentrated in the therapeutic range and minimally in the sunburn range. It has therefore become the phototherapy treatment of choice for skin diseases. The minimal erythema dose (MED) on canine skin for standardizing dosage schedules in NB-UVB treatment and histopathological analyses were performed in these dogs. In all 32 dogs tested, the MED ranged from 432 to 864 mJ/cm2. There were no significant differences in MED among breeds, sex and age groups. Histopathology obtained from areas irradiated by MED showed only mild vascular dilatation. These findings might be valuable for the application of NB-UVB phototherapy to canine skin diseases.
Superoxide dismutase (SOD) catalyzes the breakdown of superoxide into hydrogen peroxide and oxygen in the antioxidant defense system. We had reported that the SOD activities in the ceca of germ-free (GF) mice were significantly higher than those in conventional (CV) mice. In this study, we confirmed the location where SOD activity and protein expression increased in the ceca of GF mice. An immunohistochemical analysis and total SOD activity assay were conducted using the mucosa and other remaining tissues in the ceca. In addition to SOD activity in the ceca, 4 sites of intestinal (duodenal, jejunal, ileal and colonic) mucosae in GF mice were compared with those of CV mice. Total SOD activity in the cecal mucosa of GF mice was significantly higher than that in CV mice (P<0.01), and the intensity of CuZnSOD-positive cells in cecal mucosa was increased in all GF mice. Total and CuZnSOD activities in the duodenal, jejunal, ileal, cecal and colonic mucosae of GF mice were significantly higher than those in CV mice (P<0.05, or P<0.01). Furthermore, CuZnSOD mRNA showed similar tendencies with respect to these activities. Our results suggest for the first time that upregulation of SOD activity occurs in the entire intestinal mucosa of GF mice.
Most causative agents of babesiosis, Babesia parasites, are transmitted transovarially in ixodid ticks. In this study, B. gibsoni, the causative agent of canine babesiosis which has transovarial transmission, was detected in tissues of the vector tick, Haemaphysalis longicornis using a modified quantitative PCR assay. Conventional PCR results showed that the newly designed primer set, which amplifies a 143-bp fragment of rhoptry-associated protein-1 (BgRAP-1) gene in B. gibsoni, was 100 times more sensitive than primers targeting P18 gene encoding 18 kDa protein of B. gibsoni, which was recently renamed as thrombospondin related adhesive protein (BgTRAP) gene, in an artificially generated sample solution containing metagenomic DNA (B. gibsoni DNA extracted from infected dog blood mixed with tick DNA). The TaqMan probe-based quantitative PCR (qPCR) for BgRAP-1 could also detect infected RBCs (iRBCs) at levels of 3.5 × 105 to 3.5 × 101/μl, a range that is broader than that of a past SYBR Green-based qPCR method for P18/BgTRAP, which had a detection limit of 3.5 × 103 iRBCs/μl. Using this qPCR assay, we attempted to quantify the B. gibsoni burden in tick ovaries and embryonated eggs. Levels of infection were normalized to the copy number of tick’s genomic DNA fragment of ribosomal DNA internal transcribed spacer region 2 (ITS2) for the standardization. According to this, low levels of parasite burden were quantified in ovaries and eggs. This detection system is sensitive and is recommended as a tool for elucidating the biological interactions between the vector tick H. longicornis and the parasite, B. gibsoni.
Anaplasma marginale has been detected in the Philippines only by peripheral blood smear examination and serological methods. This study generally aimed to molecularly detect and characterize A. marginale in cattle and ticks in Cebu, Philippines. A total of 12 bovine blood samples and 60 Rhipicephalus (Boophilus) microplus ticks were collected on the Cebu Island in 2011. 16S rRNA-based screening-PCR and DNA sequencing revealed 8 cattle (66.7%) and 8 ticks (13.3%) to be positive for A. marginale, and 1 tick (1.7%) to be positive for A. centrale. Selected positive DNA samples were further characterized based on 16S rRNA (longer sequence), Msp5, Msp1α,gltA and groEL genes for phylogenetic analyses. Sequence identities of partial DNA fragments of A. marginale from the Philippines revealed 99.1–100% (16S rRNA, gltA, groEL and Msp5) and 94.3–97.6% (Msp1α) identities to the closest isolates from other countries. Moreover, sequence analysis of the Msp1α, gene showed 3 variants, including a case of co-infection with 2 variants. Phylogenetic analyses based on Msp1α and Msp5 genes revealed that Philippine A. marginale isolates formed a monophyletic lineage, and were phylogenetically related to Brazilian and Chinese isolates. On the other hand, a highly specific and sensitive nested PCR based on groEL, with a detection limit of 2 copies/PCR, was developed to detect A. marginale in the Philippines. This study reported the first molecular detection and characterization of A. marginale in cattle and R. microplus in Cebu, Philippines.
Although Phlebotomus argentipes as the only known vector of visceral leishmaniasis (VL) is zoophilic in nature, VL is considered to be anthroponotic in the Indian subcontinent. Peripheral blood samples from 85 stray dogs were examined for any molecular evidence of Leishmania infection in VL endemic areas of Bangladesh. Parasite DNA was detected in a blood sample from 1 of 85 (1.2%) stray dogs using ITS1-PCR, and PCR sequencing of the rRNA-ITS and cytochrome b gene confirmed that the parasitic DNA was Leishmania donovani. The results support the assumption that dogs are a probable animal reservoir for the Leishmania parasite in Bangladesh. It will be important to investigate the possible epidemiological role of dogs in domestic foci of VL endemic areas in Bangladesh.
The aim of the present study was to investigate the influence of different injection sites, i.e., the neck area and thigh muscle, on the pharmacokinetics of cefquinome in piglets following intramuscular (i.m.) injection. Cross-bred (Landrace × Duroc × Yorkshire) piglets were administered the same dose of cefquinome (2 mg/kg body weight) via intravenous injection and intramuscular injection into the neck area or thigh. The mean maximum concentrations (Cmax) of cefquinome following i.m. injection into neck or thigh area were 4.62 ± 0.31 μg/ml at 0.38 ± 0.14 hr and 4.39 ± 0.53 μg/ml at 0.42 ± 0.13 hr, respectively. The absolute bioavailabilities (F) of cefquinome after i.m. injection into the neck or thigh area were 103.04 ± 13.01 and 97.56 ± 16.14%, respectively (P>0.05). There were no differences noted between the two different injection sites for the pharmacokinetic properties of cefquinome after i.m. injection in piglets. Further studies will be needed to determine the incidence or severity of injection site reactions following repeated administrations of cefquinome into both injection sites.
An outbreak of encephalitozoonosis occurred in a rabbit colony at a zoo in Japan. Throughout the two years after the onset, all 42 rabbits were investigated clinically, pathologically and serologically for prevention and control of the disease. Eleven rabbits (11/42, 26.2%) showed clinical symptoms. Of 38 rabbits examined to detect specific antibodies against Encephalitozoon cuniculi, 71.1% (n=27) were found seropositive; 20 out of 30 clinically healthy rabbits (except for 8 clinical cases) were seropositive. The infection rate was 76.2% (32/42), including 5 pathologically diagnosed cases. The results of serological survey revealed that asymptomatic infection was widespread, even among clinically healthy rabbits. However, encephalitozoonosis was not found by pathological examination in any other species of animals kept in the same area within the zoo. Isolation and elimination of the rabbits with suspected infection based on the results of serological examination were carried out immediately; however, encephalitozoonosis continued to occur sporadically. Therefore, all the remaining rabbits were finally slaughtered. Then, the facility was closed, and all the equipment was disinfected. After a two-month interval, founder rabbits were introduced from encephalitozoonosis-free rabbitries for new colony formation. Since then, encephalitozoonosis has not been seen in any animals at the zoo. In this study, biosecurity countermeasures including staff education, epidemiological surveillance and application of an “all-out and all-in” system for rabbit colony establishment based on serological examination were successfully accomplished with regard to animal hygiene and public health for the eradication of E. cuniculi.
We utilized autoradiography to visualize radioactive contamination in the skeletal muscles of a pig raised within 20 km of the Fukushima Daiichi nuclear power plant following the nuclear accident. The autoradiogram of control muscle showed relatively homogenous exposure. In contrast, the autoradiogram of affected muscle showed a heterogeneous and sporadically dense imaging pattern. Photo luminescence densities of the affected and control muscles were 3.89 ± 0.67 and 2.13 ± 0.43 PSL/mm2, respectively. This difference indicated that radioactive cesium was distributed in the skeletal muscle of the affected pig.
The objective of this study was to evaluate glomerular filtration rate (GFR) and the cardiovascular effects of the combination of tepoxalin (TPX) and medetomidine (MED) in dogs. Six healthy dogs of either sex (5 males and 1 female), aged 2.5 ± 2.2 years and weighing 14.7 ± 4.4 kg, were studied. Each dog received four randomized treatments with a minimum of 1 week between treatments: no medication as the control group (C); MED (750 μg/m2, intravenously [IV]); TPX (10 mg/kg orally for 3 days); and MT (TPX 10 mg/kg orally for 3 days plus MED 750 μg/m2, IV). Iohexol (300 mg iodine/kg, IV) was injected in all dogs in each treatment as an indicator of GFR. Blood samples for serum iohexol clearance analysis were collected before and 1, 2, 5, 10, 15, 20, 60, 120, 240 and 360 min after the iohexol administration. Rectal temperature, heart rate, respiratory rate and direct arterial pressure (AP) were obtained before and 5, 10, 15, 20, 60, 120, 240 and 360 min after the iohexol injection. GFR did not differ between treatments. Heart rate was significantly lower in the MED and MT groups than in C or TPX. Mean AP was significantly higher with MT than TPX, but only at 5 min after the iohexol injection. TPX, MED and the combination of these two drugs do not alter GFR. The combination has minimal effect on cardiovascular variables at these doses in healthy dogs.
Mitral valvuloplasty (MVP) is used in dogs with refractory mitral regurgitation (MR); however, it is difficult to tie the artificial chord, i.e., the expanded polytetrafluoroethylene suture, at the planned height of the mitral valve, because of the slippery nature of the knot. The loop technique has resolved these difficulties in humans. Premanufactured loops (length, 8.0–15.0 mm with 1.0-mm increments) were used in the new modified loop technique. In the current study, cardiac murmurs disappeared, and the MR markedly improved or completely disappeared approximately 3 months after surgery in 3 dogs. Therefore, this new technique might be effective in dogs.
In the present study, the quality of frozen-thawed epididymal and testicular sperm recovered from a Siamese Eld’s deer was examined. The epididymal sperm quality was assessed in fresh, cold-stored at 4°C and frozen-thawed samples. Zona binding ability of the frozen-thawed epididymal samples with Burmese Eld’s deer oocytes was also evaluated. Testicular sperm extracted from tissues frozen at -80 or -196°C for one month were examined for membrane and DNA integrity. Epididymal sperm retained their quality for up to 24 hr of cold storage at 4°C. The percentages of sperm motility, intact membrane, intact acrosome and intact DNA were 30, 46.5, 27 and 89.5% in the frozen and thawed epididymal sperm, and the average ability to bind with oocytes was 92.5 ± 64 sperm/oocytes. Around 70% of the sperm extracted from testicular tissues cryopreserved at -196 and -80°C for one month showed an intact membrane. In conclusion, epididymal and testicular sperm survived for more than 13 hr post-mortem. Furthermore, cold storage at 4°C and cryopreservation at -196 and -80°C maintain the quality of epididymal and testicular sperm. This study represents a model for male gamete rescue in endangered Eld’s deer.
Chronic wasting disease (CWD) has been recognized as a naturally occurring prion disease in North American deer (Odocoileus species), Rocky Mountain elk (Cervus elaphus nelsoni) and moose (Alces alces). The disease was confirmed only in elk in the Republic of Korea in 2001, 2004 and 2005. Epidemiological investigations showed that CWD was introduced via importation of infected elk from Canada between 1994 and 1997. In spite of the increasing geographic distribution and host range of CWD, little is known about the prion strain (s) responsible for distinct outbreaks of the disease. We carried out strain characterization, using transgenic mice overexpressing elk prion protein, including clinical assessment, pathological examination and biochemical analyses, in brain tissues derived following primary through tertiary transmissions. The final incubation period was shortened to approximately 130 dpi due to adaptation. Biochemical profiles remained identical between passages. Lesion profiling in recipient mice brains showed similar patterns of vacuolation scores and intensity. It is clear that there were no biochemical or histopathological differences in Korean CWD cases in 2001 and 2004, suggesting a single strain was responsible for the outbreaks.
A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed for the simultaneous detection of canine distemper virus (CDV), canine respiratory coronavirus (CRCoV) and canine influenza virus (CIV). These viral pathogens are all causative agents of canine infectious respiratory disease (CIRD). The sensitivity and specificity of the mRT-PCR were determined by comparing it to a rapid antigen test (RAT) or immuno-chromatography test kit and reverse transcription-polymerase chain reaction (RT-PCR) in the detection of CDV, CRCoV and CIV antigens present in 100 clinical samples (nasal swabs and whole blood samples) from 50 dogs with respiratory disease symptoms. This study revealed that mRT-PCR had almost exactly the same performance or results were almost 100% in agreement with that of RT-PCR and RAT both in terms of the assay sensitivity and specificity which was more highly evident in detecting CIV, CDV and CRCoV antigens present in canine nasal swab samples. Therefore, this assay could be a better alternative for the definitive and simultaneous ante-mortem detection of the three viral pathogens that cause CIRD by using nasal swabs.