Infectious bursal disease (IBD) is characterized by immunosuppression due to the depletion of lymphocytes in the atrophied bursa of Fabricius (BF). We have sometimes encountered contradictory findings: chickens infected with the vaccine IBD virus (IBDV) strain have sometimes exhibited a highly atrophied BF, but not immunosuppression. In this study, chickens administered vaccine or wild-type strains of IBDV were later vaccinated with the B1 strain of the Newcastle disease virus (NDV). Bursal changes were examined histologically with a focus on the bursal follicle. The immunoreactivity to NDV was also evaluated with the hemagglutination inhibition test. In gross examination, we observed a few chickens with a severely atrophied BF in vaccine strain-administered groups (vaccine groups), and the level of severity was the same as that in the wild-type strain-administered group (wild-type group). However, these chickens retained humoral antibody responses to NDV and were revealed to possess a higher number of bursal follicles than those of the wild-type group. These results indicated that macroscopic evaluation dose not accurately reflect the immunoreactivity and degree of bursal damage in IBDV-administered chickens. We also found non-immunosuppressed chickens in the wild-type group. These non-immunosuppressed chickens retained a significantly higher number of normal follicles and total follicles according to our statistical analysis. Furthermore, a high correlation coefficient between the NDV-HI titer and the number of normal follicles was found in the wild-type group. These results implied that the retained number of normal follicles is important for the immunoreactivity of chickens infected with IBDV.
To understand the epidemiology of Avian paramyxovirus serotype-1 (APMV-1) in pigeons in Japan, phylogenetic analysis was comprehensively conducted based on partial fusion protein gene using isolate from the surveillance of this virus with previously known Japanese pigeon strains. This surveillance was conducted using feces obtained from domestic pigeons collected in 40 prefectures throughout Japan from June 2011 to March 2013. From a total of 1,021 samples, a single virus (APMV1/pigeon/Japan/Kanagawa/2013: JP/Kanagawa-pg/2013) was isolated. All Japanese pigeon APMV-1 strains were clustered into a single genetic lineage, which was termed VIb/1 by phylogenetic analysis based on the F gene including the sequence of the cleavage site. These APMV-1 strains were further subdivided into four subgroups identified over 4 separate timeframes: 1984–1995 (group 1), 1995–2000 (group 2), 2001–2007 (group 3) and the novel subgroup isolated in 2013 (group 4). Each subgroup has specific amino acid motifs at a cleavage site of the F protein, namely, 112GRQKR-F117(except for one strain), 112RRKKR-F117, 112RRQKR-F117 and 112RRQKR-F117, respectively. Our data suggest that Japanese APMV-1 strains from pigeons were diverse and reinforced the possibility that there were multiple introduction routes from foreign countries into Japan.
Biofilm-forming ability is increasingly being recognized as an important virulence factor in several Staphylococcus species. This study evaluated the biofilm-forming ability of sixty canine derived clinical isolates of S. pseudintermedius, using three phenotypic methods, microtiter plate test (MtP), Congo red agar method (CRA) and tube adherence test, and the presence and impact of biofilm-associated genes (icaA and icaD). The results showed that icaA and icaD genes were detected concomitantly in 55 (91.7%) of 60 isolates. A majority (88.3%) of the strains screened had matching results by the tube adherence test, MtP and PCR analysis. Better agreement (95%) was found between the PCR-based analysis and the CRA. Results of the icaA and icaD gene PCRs showed good agreement with CRA results, with a kappa of 0.7. Comparing the phenotypic methods, the statistical analysis showed that the agreement among the phenotypical tests using categorical data was generally good. Considering two classes (biofilm producer and biofilm non-producer), the percentage of matching results between the CRA method and the tube adherence test and between the CRA method and the MtP was 93.3%. A concordance of 100% was revealed between the MtP and the tube adherence test. The results indicate a high prevalence of the ica genes within S. pseudintermedius isolates, and their presence is associated with in vitro formation of a biofilm. A combination of phenotypic and genotypic tests is recommended for investigating biofilm formation in S. pseudintermedius.
Currently, no simple assays are available for routine quantitative detection of Escherichia coli-produced Shiga toxin 2e (Stx2e) that causes porcine edema disease. Here, we present a novel quantitative detection method for Stx2e based on the measurement of Stx2e binding to the specific globotetraosylceramide (Gb4) receptor by ELISA (Gb4-ELISA). No cross-reactivity was found with the other Shiga toxins Stx1 and Stx2, indicating high specificity. When the recombinant Stx2e B subunit (Stx2eB) was used, the absorbance measured by Gb4-ELISA increased linearly with Stx2eB concentration in the range of 20–2,500 ng/ml. The Gb4-ELISA method can be easily performed, suggesting that it would be a useful diagnostic tool for porcine edema disease.
A rapid and efficient DNA extraction method was developed for detecting mastitis pathogens in milk. The first critical step involved cell wall disruption by bead-beating, as physical disruption using beads was more effective for DNA extraction from Gram-positive bacteria, such as Staphylococcus aureus, than enzymatic disruption using proteinase K. The second critical step involves the use of acetic acid and ammonium sulfate in the purification process, as these reagents effectively and efficiently remove the lipids and proteins in milk. Using these methods, DNA suitable for loop-mediated isothermal amplification was obtained within 30 min. Also, the rapid and sensitive detection of S. aureus in milk was possible at levels as low as 200 cfu/ml.
Malignant melanoma is one of the most common and aggressive tumors in the oral cavity of dog. The tumor has a poor prognosis, and methods for diagnosis and prediction of prognosis after treatment are required. Here, we examined metabolite profiling using gas chromatography-mass spectrometry (GC-MS) for development of a discriminant model for evaluation of prognosis. Metabolite profiles were evaluated in healthy and melanoma plasma samples using orthogonal projection to latent structure using discriminant analysis (OPLS-DA). Cases that were predicted to be healthy using the OPLS discriminant model had no advanced lesions after radiation therapy. These results indicate that metabolite profiling may be useful in diagnosis and prediction of prognosis of canine malignant melanoma.
Feline renal diseases are increasingly noted in veterinary practice. It is important to diagnose and identify the pathological basis of renal dysfunction accurately at an early stage, but there are only a few reports on this area in clinical veterinary medicine. We investigated the efficacy of measurement of urinary albumin (u-Alb) and urinary transferrin (u-Tf) for early diagnosis using 5-µl urine samples collected noninvasively by catheterization from normal (IRIS stage I) cats and cats with stage I chronic kidney disease (CKD). The u-Alb levels in normal and stage I CKD cats were 6.0 ± 4.5 and 11.2 ± 8.4 mg/dl, respectively, and the u-Tf levels were 0.09 ± 0.42 and 0.52 ± 0.79 mg/dl, respectively. Based on ROC curve analysis, the sensitivity and specificity of u-Alb and u-Tf were higher than those of the currently used biomarker, the plasma creatinine level. The sensitivity of u-Alb was higher than that of u-Tf, whereas the specificity of u-Tf was higher than that of u-Alb. The validity of the threshold albumin level (20 mg/dl) was confirmed by measurements using SDS-PAGE. Since leakage of u-Tf in urine precedes leakage of u-Alb, inclusion of u-Tf in biochemistry tests may be appropriate for IRIS staging as a diagnostic marker of early diagnosis of renal disorder in cats.
The aim of the present study was to compare the ability of the commercially available portable test system (PTSTM) to detect endotoxin activity in bovine serum, with that of the traditional LAL-kinetic turbidimetric (KT) and chromogenic (KC) assays. Prior to testing, serum samples, which were obtained from endotoxin-challenged cattle, were diluted 1:20 in endotoxin-free water and heated to 80°C for 10 min. The performance of the PTSTM was not significantly different from that of the traditional LAL-based assays. The results using PTSTM correlated with those using KT (r2=0.963, P<0.001) or KC assays (r2=0.982, P<0.001). Based on these findings, the PTSTM could be applied as a simplified system to assess endotoxin activity in bovine serum.
We investigated the effect of supplemental vitamin E on the peripheral blood leukocyte population in Japanese Black calves. Twenty-six calves kept at the same farm were studied. They were divided into two groups; thirteen calves received 300 IU/day of vitamin E orally from 1 to 3 months of age (VE group), and the other thirteen calves did not receive the vitamin E supplement (control group). The VE group showed a higher serum vitamin E concentration at 2 and 3 months of age compared with the control group (P<0.01). The numbers of CD3+ cells and CD4+ cells were higher in the VE group than in the control group, and the difference was statistically significant at 3 months of age (P<0.05). The numbers of CD21+ cells were higher in the VE group than in the control group, and the difference was statistically significant at 2 months of age (P<0.05). The numbers of CD335+ cells tended to be higher in the VE group than in the control group. The numbers of CD8+ cells and CD14+ cells tended to be higher in the VE group than in the control group at 3 and 4 months of age. This study demonstrated that the supplementation of suckling Japanese Black calves with vitamin E might affect the numbers of some immune cell types in the peripheral blood.
To obtain blood biochemical basic data of Japanese Black breeding cows in Kagoshima Prefecture, Japan, blood samples were obtained from 857 clinically healthy multiparous cows on 71 farms. Cows were divided into three stages: the prepartum stage (between 20 and 80 days before parturition, n=290), lactation stage (between 20 and 80 days after parturition, n=283) and maintenance stage (between 120 and 200 days after parturition, n=284). The mean concentration of total protein and total cholesterol, and the mean activities of aspartate aminotransferase in the lactation stage tended to be higher than those in the prepartum and maintenance stages. The mean concentration of glucose in the prepartum stage tended to be lower than that in the lactation and the maintenance stages. The mean concentration of nonesterified fatty acids and beta-hydroxybutyrate in the prepartum stage tended to be higher than those in the lactation and maintenance stages. The mean concentration of serum retinol was approximately 30 µg/dl in all stages. These results suggest that the blood biochemical values of multiparous Japanese Black cows vary with breeding stages, and it is considered that the blood parameters obtained in this study are useful as indices for health management of Japanese Black breeding cows.
We examined the correlation between the glomerular filtration rate (GFR) estimated from an equation based on the serum iodixanol clearance technique and International Renal Interest Society (IRIS) stages of chronic kidney disease (CKD) in cats. The equation included the injection dose, sampling time, serum concentration and estimated volume of distribution (Vd) of the isotonic, nonionic, contrast medium iodixanol as a test tracer. The percent changes in the median basal GFR values calculated from the equation in CKD cats resembled those of IRIS stages 1–3. These data validate the association between the GFR derived from the simplified equation and IRIS stages based on the serum creatinine concentration in cats with CKD. They describe the GFR ranges determined using single-sample iodixanol clearance for healthy cats and cats with various IRIS stages of CKD.
Cryptosporidium oocysts were found in 43 out of 77 calves from two farms in Iwate Prefecture and nine farms on Tanegashima Island, Kagoshima Prefecture, Japan. The DNA fragments of 18S ribosomal RNA (18S rRNA) gene were amplified by a nested PCR from 43 oocyst-positive as well as one oocyst-negative samples. All of them were precisely identified as C. parvum by analyzing the nucleotide sequences of the 18S rRNA gene. C. parvum oocyst-positive calves ranged in age from 6 to 13 days old and significantly have watery diarrhea (P<0.05). Sequences of the gene encoding the 60-kDa glycoprotein (GP60) in 43 Cryptosporidium oocyst-positive samples were identical to that of the zoonotic IIaA15G2R1 subtype. We therefore suggest that calves could be potential sources of C. parvum infections in humans.
We examined 11 Prussian carp, Carassius gibelio (Bloch), from the upper Yellow River running through Inner Mongolia (Wuhai City) to record myxosporean species. Between 6 and 15 elongated cysts of Henneguya doneci were located at the basal part of the gill filaments of 3 carp (27.3%); no more myxosporean plasmodia were found in other organs. Although the morphology and morphometric values of the spores (average measurements of 14 spores in µm: 11.4 long by 9.2 wide with 7.5 in thickness; 2 polar capsules, equal, 5.5 long by 3.2 wide; and a bifurcated caudal process, 51.6 long) with an evident intercapsular appendix were basically coincident with the species, the dimensions of the spore bodies were marginally larger, and the length of the caudal processes was distinctly longer than previously reported values for H. doneci (44.2–59.2 µm vs. 26.8–42.6 µm, respectively). Genetic analysis of the 18S ribosomal RNA gene (rDNA) found few nucleotide substitutions when compared with 3 deposited sequences of H. doneci collected around the Yangtze River (Sichuan and Hubei Provinces), China, indicating that the uniqueness of some of the morphological features exhibited by the present Wuhai isolate should be ascribed to intraspecific variation.
Trypanosoma evansi causes wasting disease in many livestock. T. evansi infection gives rise to inflammatory immune responses, which contribute to the development of inflammation-associated tissue injury. We previously reported that regulatory dendritic cells (DCs), which act as potential regulators of inflammation, were activated in infected mice and transfer of regulatory DCs to infected mice prolonged their survival. However, the kinetics of regulatory DCs in cattle, which are natural hosts of T. evansi, remained unclear. In this study, we report that the expressions of CCL8 and IL-10, which promote the development of regulatory DCs, were up-regulated in cattle experimentally infected with T. evansi. This finding is potentially useful for studying the control strategy of T. evansi infection in cattle.
Adiaspiromycosis is a pulmonary infection caused by the soil fungi, Emmonsia crescens and E. parva. It primarily affects small mammals and can range from an asymptomatic condition to fatal disseminated disease. We detected a granuloma containing fungal spherules, which were morphologically consistent with the adiaspores of E. crescens in the lungs of a female Hokkaido sika deer. This is the first reported case of adiaspiromycosis involving a cervid in the world.
This report presents a new case of mucormycosis encountered in penguin characterized by morphological variation of hyphae and presence of sporangia with numerous sporangiospores. A 4.5-year-old Magellanic penguin (Spheniscus magellanicus) died after exhibiting anorexia, poor nutritional condition and dyspnea. Multiple nodular lesions were observed in the thoracic and abdominal regions. Histopathologically, hyphae of various sizes were seen in the lungs, air sac and nodular lesions. Myriad sporangiospores and several sporangia were observed in/around the bronchi or parabronchi. The very narrow and short hyphae in the nodules were not consistent with the characteristics of Mucorales. However, for most hyphae, including those in the nodules, sporangiospores and sporangia, immunohistochemistry revealed Mucorales-positive reactions. In addition, these fungi were identified as Rhizomucor pusillus by gene analysis.
The plasmid is a very well-known mobile genetic element that participates in the acquisition of virulence genes, such as staphylococcal enterotoxins (SEs), via horizontal transfer. SEs are emetic toxins and causative agents in staphylococcal food poisoning (SFP). We herein identified the types of plasmids harbored by seven SFP isolates and examined their production of plasmid-related SE/SEl to determine whether the new types of plasmid-related SE or SE-like (SEl) toxins (i.e. SElJ and SER) were involved in SFP. These isolates harbored pIB485-like plasmids, and all, except for one isolate, produced SElJ and SER. The amount of SER produced by each isolate accounted for the highest or second highest percentage of the total amount of SE/SEl produced. These new types of plasmid-related SE/SEls as well as classical SE may play a role in SFP. The seven isolates were classified into two SED-production types; a high SED-production type (>500 ng/ml) and no SED-production type. A nucleotide sequencing analysis revealed that three plasmids harbored by the SED-non-producing isolates had a single-base deletion in the sed gene with a resulting stop codon (from 233 amino acids of the intact SED to 154 amino acids of the mutant SED (mSED)). A real-time reverse transcription-PCR analysis showed that the mRNA of the msed gene was transcribed in the isolates. If the msed gene was translated as a protein, mSED may act as an emetic toxin instead of intact SED.
Foot-and-mouth disease (FMD) occurred in Miyazaki, Japan, in 2010, and 290,000 animals were culled. This paper describes the mental distress of the volunteers who had been dispatched to Miyazaki for disease control two years after the epidemic. It also assesses risk factors for post-traumatic stress disorder (PTSD). A participatory appraisal and self-administered questionnaire survey were conducted in 2012 for those who were dispatched to Miyazaki in 2010. The Impact of Event Scale-Revised (IES-R) was used as an indicator of PTSD, and univariate and multivariable analyses were performed. Of the 875 respondents, 1.3% had higher IES-R scores than the cut-off point (25), which is suggestive of PTSD. Mental stresses during and soon after FMD control and after two years were described. Four risk factors associated with high IES-R scores were found: transporting culled animals (P<0.01), stress during FMD control (P<0.01) and at the time of the survey (P<0.01), and lack of someone to talk to about FMD-associated stress at the time of the survey (P<0.01). Veterinarians, livestock technicians and clerical officers involved in FMD control still suffer from mental stress two years later. Public services should provide an opportunity for them to consult with mental health specialists. These findings should be used to better prepare workers who deal with infectious diseases of animals, especially when they must be culled. The establishment of a collaborative framework between veterinary and mental health services is recommended.
The prevalence of Campylobacter jejuni in wild birds is a potential hazard for human and animal health. The aim of this study was to establish the prevalence of C. jejuni in wild birds in Tokachi area, Hokkaido, Japan and investigate their virulence in vitro. In total, 173 cloacal swabs from individual wild birds were collected for the detection of Campylobacter spp. Thirty four samples (19.7%) were positive for Campylobacter of which 94.1% (32/34 samples) were C. jejuni. Additionally, one C. coli and one C. fetus were isolated. Seven C. jejuni isolates (one from crows and the other from pigeons) had important virulence genes including all three CDT genes (cdtA, cdtB and cdtC) and flaA, flaB, ciaB and cadF, and the other isolates were lacking cdtA gene. Further studies on in vitro virulence-associated phenotypes, such as motility assay on soft agar and invasion assay in Caco-2 cells, were performed. The wild bird C. jejuni isolates adhered and invaded human cells. Although the numbers of viable intracellular bacteria of wild bird isolates were lower than a type strain NCTC11168, they persisted at 48-hr and underwent replication in host cells.
To follow-up anthrax in Zambia since the outbreak in 2011, we have collected samples from the environment and the carcasses of anthrax-suspected animals, and have tried to isolate Bacillus anthracis. In the process of identification of B. anthracis, we collected two isolates, of which colonies were similar to B. anthracis; however, from the results of identification using the molecular-based methods, two isolates were genetically related to the highly pathogenic B. cereus, of which clinical manifestation is severe and fatal (e.g., pneumonia). In this study, we showed the existence of bacteria suspected to be highly pathogenic B. cereus in Zambia, indicating the possibility of an outbreak caused by highly pathogenic B. cereus.
The sedative effects of intramuscular (IM) alfaxalone in 2-hydroxypropyl-beta-cyclodextrin (alfaxalone-HPCD) were evaluated in cats. The cats were treated with alfaxalone-HPCD in five occasions with a minimum 14-day interval between treatments: an IM injection of 1.0 mg/kg (IM1), 2.5 mg/kg (IM2.5), 5 mg/kg (IM5) or 10 mg/kg (IM10), or an intravenous injection of 5 mg/kg (IV5). The sedative effects were evaluated subjectively using a composite measurement scoring system (a maximum score of 16). Cardio-respiratory variables were measured non-invasively. The median sedation scores peaked at 10 min (score 9), 15 min (score 14), 10 min (score 16), 10 to 20 min (score 16) and 2 to 5 min (score 16) after the IM1, IM2.5, IM5, IM10 and IV5 treatments, respectively. The IM5 treatment produced longer lasting sedation, compared to the IV5 treatment. Durations of maintenance of lateral recumbency after the IM10 treatment (115 ± 22 min) were longer than those after the IM2.5 (40 ± 15 min), IM5 (76 ± 21 min) and IV5 treatments (50 ± 5 min). Cardio-respiratory variables remained within clinically acceptable ranges, except for each one cat that showed hypotension (<60 mmHg) after the IM10 and IV5 treatments. Tremors, ataxia and opisthotonus-like posture were observed during the early recovery period after the IM2.5, IM5, IM10 and IV5 treatments. In conclusion, IM alfaxalone-HPCD produced dose-dependent and clinically relevant sedative effect at 2.5 to 10 mg/kg in healthy cats. Hypotension may occur at higher IM doses of alfaxalone-HPCD.
Liver contrast X-ray computed tomography (CT) has been used for evaluation of hepatic vessels for liver transplantation, liver lobectomy, interventional radiology and diagnosis of hepatocellular carcinoma in humans. However, there remains scant available anatomical information on normal hepatic vessels in the veterinary field. In this study, visualization of hepatic vessels was evaluated in 32 normal beagle dogs by X-ray contrast CT using triple phase images. The following hepatic vessels were clearly visualized: arterial, portal and hepatic veins. With regards to the running patterns of the portal vein and hepatic vein, there were no significant differences between the dogs. However, the hepatic artery exhibited some differences in each dog. In particular, the hepatic artery of the quadrate lobe and the right lateral lobe had many running patterns. The results of the present study could be useful for veterinary diagnosis, surgery and interventional radiology.
In the present study, we compare a new carbon dioxide (CO2) absorbent, Yabashi lime® with a conventional CO2 absorbent, Sodasorb® as a control CO2 absorbent for Compound A (CA) and Carbon monoxide (CO) productions. Four dogs were anesthetized with sevoflurane. Each dog was anesthetized with four preparations, Yabashi lime® with high or low-flow rate of oxygen and control CO2 absorbent with high or low-flow rate. CA and CO concentrations in the anesthetic circuit, canister temperature and carbooxyhemoglobin (COHb) concentration in the blood were measured. Yabashi lime® did not produce CA. Control CO2 absorbent generated CA, and its concentration was significantly higher in low-flow rate than a high-flow rate. CO was generated only in low-flow rate groups, but there was no significance between Yabashi lime® groups and control CO2 absorbent groups. However, the CO concentration in the circuit could not be detected (≤5ppm), and no change was found in COHb level. Canister temperature was significantly higher in low-flow rate groups than high-flow rate groups. Furthermore, in low-flow rate groups, the lower layer of canister temperature in control CO2 absorbent group was significantly higher than Yabashi lime® group. CA and CO productions are thought to be related to the composition of CO2 absorbent, flow rate and canister temperature. Though CO concentration is equal, it might be safer to use Yabashi lime® with sevoflurane anesthesia in dogs than conventional CO2 absorbent at the point of CA production.
This study aimed to evaluate the analgesic efficacy of gabapentin as an adjuvant for postoperative pain management in dogs. Twenty dogs undergoing mastectomy were randomized to receive perioperative oral placebo or gabapentin (10 mg/kg). All dogs were premedicated with intramuscular acepromazine (0.03 mg/kg) and morphine (0.3 mg/ kg). Anesthesia was induced with propofol (4 mg/kg) intravenously and maintained with isoflurane. Intravenous meloxicam (0.2 mg/kg) was administered preoperatively. Postoperative analgesia was evaluated for 72 hr. Rescue analgesia was provided with intramuscular morphine (0.5 mg/kg). Dogs in the Placebo group received significantly more morphine doses than the Gabapentin group (P=0.021), despite no significant differences in pain scores. Perioperative gabapentin reduced the postoperative morphine requirements in dogs after mastectomy.
We investigated ontogenetic changes in the trunk muscles of the Japanese black salamander (Hynobius nigrescens) before, during and after metamorphosis. Given that amphibians change their locomotive patterns with metamorphosis, we hypothesized that they may also change the structure of their trunk muscles. The trunk muscles were macroscopically observed, and the weight ratios of each trunk muscle group were quantified at six different developmental stages. Immediately after hatching, we found that the lateral hypaxial muscle was composed of one thick M. ventralis, from ventral edge of which M. transversus abdominis arose later, followed by M. obliquus externus and M. rectus abdominis. The weight ratios of the dorsal and abdominal muscles to the trunk muscles increased with growth. We suggest that a single thick and large lateral hypaxial muscle facilitates swimming during early developmental stages. The increase in the weight ratios of the dorsal and abdominal muscles with growth possibly assists with gravity resistance necessary for terrestrial life.
Lobomycosis is a chronic fungal disease caused by the etiologic agent, Lacazia loboi, in the skin and subcutaneous tissues in humans and dolphins in tropical and transitional tropical climates. An Indo-Pacific bottlenose dolphin (Tursiops aduncus) stranded in Kagoshima, Japan, had severe skin lesions characterized by granulomatous reactions and hyperkeratosis that were similar to those of the lobomycosis, but no fungal organism was observed in the skin lesion. In this paper, we report a stranded Indo-Pacific bottlenose dolphin with lobomycosis-like lesions based on pathological examinations in Japan.