First detection of Allobilharzia visceralis (Schistosomatidae, Trematoda) from Cygnus cygnus in Japan.

Adult schistosomes were detected in the veins or capillaries of the large intestine, mesentery, liver, and adrenal glands in eight of 13 whooper swans (Cygnus cygnus) examined in Iwate Prefecture, Japan. However, neither eggs nor severe tissue injuries were observed in any of the swans. The schistosomes were definitively identified as Allobilharzia visceralis based on the nucleotide sequences of the ribosomal internal transcribed spacer (ITS) region. Allobilharzia visceralis infections have been reported in whooper swan in Iceland and tundra swan (Cygnus columbianus) in North America. These detections suggest that A. visceralis is distributed extensively along the swan flyways because the swans are migratory birds. To the best of our knowledge, this is the first report of A. visceralis infection in Asia.

Molecular detection of Mycoplasma haemomuris subspecies using dnaK-targeted real-time PCR with SYBR Green I and melting curve analysis.

Hemoplasmas cause severe infections in mammals, but these pathogens are difficult to detect and identify at the species and subspecies level because of the need for time-consuming sequence based methods. Here, we used real-time PCR with SYBR Green I targeting of the dnaK gene followed by standard melting curve analysis to achieve rapid detection and differentiation of the Mycoplasma haemomuris subspecies 'Candidatus Mycoplasma haemomuris subsp. musculi' and 'Candidatus M. haemomuris subsp. ratti'. The melting temperatures of the PCR products, 84.63 ± 0.14 °C for 'Candidatus M. haemomuris subsp. musculi', and 80.72 ± 0.16 °C for 'Candidatus M. haemomuris subsp. ratti', provided clear differentiation between them. Murine hemoplasma DNA samples, which were used as references, were confirmed for species by an analysis of 16S rRNA sequences. The protocol described herein provides a new rapid detection and identification method suitable for use with two recognized subspecies of M. haemomuris.

A method for estimating the glomerular filtration rate in conscious monkeys.

To establish a method for estimating the glomerular filtration rate (GFR) in conscious monkeys, the radiographic contrast medium iodixanol and the standard agent inulin were coadministered as tracers to male cynomolgus monkeys (Macaca fascicularis) as a bolus injection; blood was collected after 60, 90 and 120 min. An equation based on a single-blood-sample method derived from Jacobsson's formula was prepared using the data from healthy and saline- and gentamicin-treated monkeys by a multisample strategy with iodixanol. The GFR using the equation with iodixanol was in agreement with that from the multisample method with inulin or iodixanol. When the GFR decreased to more than 60% of the basal reference level, serum creatinine concentrations tended to increase, whereas serum blood urea nitrogen concentrations fluctuated. The results suggest that the single-sample-blood method with iodixanol is a practical tool for estimating the monkey GFR in a toxicological research setting therefore minimizing animal sufferings.

Localization of connexin 32 in spontaneous liver lesions of mice.

We examined the localization of connexin 32 (Cx32), a component of gap junctions, in 24-month-old male B6C3F1 mice with spontaneously occurring hepatocellular altered foci or tumors. Immunohistochemically, Cx32-staining intensity in cell-to-cell membranes of altered hepatocytes was decreased in eosinophilic foci and increased in basophilic foci as compared to those in intact hepatocytes. These alterations were enhanced in adenomas and carcinomas with both eosinophilic and basophilic cytoplasm. In cell membranes facing on the sinusoidal portions, the intensities increased in all lesions. Image analyses confirmed that the spot areas of Cx32 were decreased in eosinophilic foci, but increased in basophilic foci, adenomas and carcinomas. These results demonstrate that Cx32 shows different expression in different types of hepatic lesions.

Vertical transmission of Mycoplasma wenyonii in cattle, supported by analysis of the ribonuclease P RNA gene - Short communication.

The vertical transmission of Mycoplasma (M.) wenyonii was investigated in beef cattle raised on a farm in Japan by analysing the ribonuclease P RNA (rnpB) gene sequence using PCR. Peripheral blood samples from 17 dams infected with M. wenyonii and from their neonatal calves were collected and colostrum samples were taken from cows immediately after parturition, and subsequently the blood samples of calves were monitored continuously for three months. At birth on day 0, although no rnpB gene was detected in the colostrum of any of the dams, four (23.5%) of the 17 calves born were positive. At three months after delivery, the number of positive calves decreased to three. Although horizontal transmission by blood-feeding arthropod vectors has been basically accepted as the most common route of haemoplasma infection, these findings suggest that vertical transmission is, at least in part, another most likely route of M. wenyonii infection in cattle.

Estimation of glomerular filtration rate in dogs by a single-blood sample method involving iodixanol.

OBJECTIVE: To establish a simplified single-blood-sample method (SBSM) involving iodixanol to estimate glomerular filtration rate (GFR) in dogs and compare data provided by that procedure with data provided by a conventional multiple-blood-sample method (MBSM) involving inulin. ANIMALS: 26 healthy dogs and 36 dogs with naturally occurring renal disease. PROCEDURES: Dogs were used in various preliminary experiments to establish protocols for the SBSM and the MBSM of GFR estimation. To evaluate the relationship between GFRs obtained by the SBSM and the MBSM each involving iodixanol, iodixanol (40 mg of I/kg) was administered IV to 26 healthy dogs and 36 dogs with renal disease; blood sample collection was performed before and at 60, 90, and 120 minutes after the injection. To evaluate the relationship between GFRs obtained by the SBSM involving iodixanol and the MBSM involving inulin, iodixanol (40 mg of I/kg) and inulin (50 mg/kg) were coadministered IV to 22 healthy dogs and 3 dogs with renal disease, followed by blood sample collection 30, 60, 90, and 120 minutes later. Serum iodixanol and inulin concentrations were separately determined by reverse-phase high-performance liquid chromatography. RESULTS: Findings revealed a correlation (r = 0.99) between GFR estimated by the SBSM and MBSM each involving iodixanol. Likewise, GFR estimated by the SBSM involving iodixanol was correlated (r = 0.89) with that estimated by the MBSM involving inulin. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that the SBSM involving iodixanol can be applied to estimate GFR in dogs, instead of use of an MBSM.

Relationship of glomerular filtration rate based on serum iodixanol clearance to IRIS staging in cats with chronic kidney disease.

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.

Pharmacokinetics of metoclopramide in calves with renal dysfunction.

To clarify the effect of renal dysfunction on pharmacokinetics of the prokinetic agent metoclopramide (MCP), we administered intravenously 0.4 mg/kg MCP to healthy calves and calves subjected to right kidney vessel ligation (ligation) without or with a subsequent left nephrectomy (ligation plus removal). Plasma MCP concentration, glomerular filtration rate (GFR) and plasma prolactin level were measured by liquid chromatography-tandem mass spectrometry, simplified equation using iodixanol and enzyme-linked immunosorbent assay, respectively. Only in calves with ligation plus removal, plasma MCP concentrations were increased significantly 6, 8 and 12 hr after injection, showing that a negative correlation was observed between the plasma MCP concentrations and GFR value. A tendency to increase in plasma PRL concentration was noted also in these calves. In conclusions, plasma MCP concentrations depend on the GFR mode in calves, and its critical GFR value was estimated.

Glomerular filtration rate in cows estimated by a prediction formula.

To testify the relevance of Jacobsson's equation for estimating bovine glomerular filtration rate (GFR), we prepared an integrated formula based on its equation using clinically healthy dairy (n=99) and beef (n=63) cows, and cows with reduced renal function (n=15). The isotonic, nonionic, contrast medium iodixanol was utilized as a test tracer. The GFR values estimated from the integrated formula were well consistent with those from the standard multisample method in each cow strain, and the Holstein equation prepared by a single blood sample in Holstein dairy cows. The basal reference GFR value in healthy dairy cows was significantly higher than that in healthy beef cows, presumably due to a breed difference or physiological state difference. It is concluded that the validity for the application of Jacobsson's equation to estimate bovine GFR is proven and it can be used in bovine practices.

Estimation of glomerular filtration rate in conscious mice using a simplified equation.

To develop an expedient procedure for estimating glomerular filtration rate (GFR) in conscious mice, we first established a simple technique for repeated blood collections from the tail vein using a microcapillary tube attached to a 24-gauge needle without the hub. Then, we devised a definition equation for estimation of the GFR using the contrast medium iodixanol as a test tracer. Iodixanol was administered as a bolus injection at 1500 mg I/kg to ddY mice, and the GFR was determined by the conventional multisample strategy. Based on cumulative data from the multisample method, an equation for the single-blood-sample method including the iodixanol dose, estimated distribution volume (Vd), and plasma iodixanol concentration at 60 min later was sought. The GFR values from the multisample method were in good agreement with those calculated using the equation. In clinically healthy mice, the GFR decreased gradually with ages from 11 weeks old in both sexes, suggesting the necessity of the corresponding control in each protocol. In nephropathy mice induced by cisplatin, the GFR values decreased with rises in serum BUN and creatinine concentrations, and serum creatinine became elevated when the GFR decreased to approximately 70% of the basal value. The results suggest that the simplified equation using iodixanol, allowing for the repeated use of the same mice, is a versatile procedure for research purposes.

Estimation of glomerular filtration rate in cynomolgus monkeys (Macaca fascicularis).

To estimate the glomerular filtration rate (GFR) in cynomolgus monkeys (Macaca fascicularis), a three-blood-sample method using iodixanol was assessed in comparison with the conventional multisample strategy using inulin. Iodixanol and inulin were coadministered intravenously 40 mg I/kg and 50 mg/kg, respectively, to male monkeys, followed by blood collection 60, 90 and 120 min later. A close correlation (r=0.96) was noted between the GFR values estimated by both methods. In clinically healthy monkeys, the basal values were determined to be 3.06 ± 0.50 ml/min/kg. This is the first report, suggesting that serum clearance of iodixanol is a ready-to-use tool for a screening the GFR in monkeys, although it is necessary to perform a more longitudinal study using animals with reduced renal function.

Cisplatin binding and inactivation of mitochondrial glutamate oxaloacetate transaminase in cisplatin-induced rat nephrotoxicity.

Cisplatin is a widely used chemotherapeutic agent, but its use is limited by nephrotoxicity associated with mitochondrial dysfunction. Because its mechanisms are poorly understood, we aimed to identify the mitochondrial proteins targeted by cisplatin. We isolated renal mitochondrial proteins from Sprague-Dawley (SD) rats and performed cisplatin-affinity column chromatography. The proteins eluted were detected on SDS-PAGE and subjected to in-gel tryptic digestion and LC-MS/MS analysis. We identified glutamate oxaloacetate transaminase (GOT) and mitochondrial malate dehydrogenase (MDH). Next, we administered cisplatin intraperitoneally to SD rats to induce nephrotoxicity and assayed the activities of the enzymes. The results indicated that cisplatin caused a severe decrease in mitochondrial GOT activity on day 1 after cisplatin administration. Three d later, we also identified a decrease in mitochondrial MDH activity. Our results indicate that cisplatin binds to mitochondrial GOT and inhibits its activity, causing mitochondrial dysfunction and subsequent nephrotoxicity.

Application of the single blood sample method to estimate feline glomerular filtration rate in a clinically relevant situation.

To compare glomerular filtration rate (GFR) estimated by a single blood sample method, the non-ionic contrast medium iodixanol (40 mg I/kg) and the standard GFR tracer inulin (50 mg/kg) were co-administered as a bolus intravenous injection to 12 cats, followed by blood collection 60 and 90 mins later. Serum iodixanol and inulin concentrations were measured separately by high-performance liquid chromatography and colourimetric assay. A correlation (r = 0.90, P <0.01) was noted between GFR values estimated by the single-blood-sample method using iodixanol and inulin, indicating that this procedure can apply to feline GFR estimates, even if different GFR tracers are used. In a feline kidney transplantation study, the GFR was monitored subsequently by this simplified iodixanol method throughout a 750-day observation period with no adverse reactions. The results demonstrate that the simplified method, including the volume of distribution, can be used as an alternative or expedient tool in a clinically relevant situation.

Detection of hemotropic mycoplasmas in free-living brown sewer rats (Rattus norvegicus).

The prevalence of hemotropic mycoplasmas in wild rodents is largely unknown. Here, we report the presence of hemoplasmas in blood samples collected from brown sewer rats (Rattus norvegicus) trapped during rodent control around an animal hospital in Morioka, Japan. We examined nine rats using real-time PCR and end-point PCR, and found one rat (11.1%) that was positive for a hemoplasma infection. The 16S rRNA gene and 16S to 23S rRNA intergenic spacer region of the hemoplasma detected in a wild-caught rat were amplified using PCR. The nucleotide sequences of the PCR products were further determined and compared to those of other hemoplasmas. Our examinations revealed the presence of a hemoplasma that has not previously been described in rodents. The pathogenic traits of this hemoplasma remain unexplored.

Background lesions during a 24-month observation period in connexin 32-deficient mice.

Connexin 32 (Cx32) is a major gap junction protein in the liver. Neoplastic and non-neoplastic lesions were examined in Cx32-deficient (Cx32KO) mice maintained for 24-month, and compared with those in wild-type mice as a corresponding control. In neoplastic lesions, hepatocellular carcinoma increased significantly only in male Cx32KO mice, suggesting that Cx32 deficiency may be related to their pathogenesis. For females, the incidence of pituitary adenoma in the pars distalis of Cx32KO mice was lower than that of wild-type mice. No non-neoplastic lesions related to Cx32-deficiency were observed in the Cx32KO mice. In conclusion, these results demonstrate that the incidence of hepatocellular carcinoma increases only in male Cx32KO mice, presumably due to enhanced tumor promotion and progression signals associated with Cx32 deficiency.

An autoradiogram of skeletal muscle from a pig raised on a farm within 20 km of the Fukushima Daiichi nuclear power plant.

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/mm(2), respectively. This difference indicated that radioactive cesium was distributed in the skeletal muscle of the affected pig.

Simple and sensitive method for measurement of metoclopramide in cattle plasma by LC-MS/MS using a multimode chromatography.

A quantitative assay method using LC/ESI-MS/MS for simultaneous determination of MCP in cattle plasma was developed and validated. Chromatographic separation was carried out using a multimode column (2 × 150 mm, 3 µm) with gradient elution (0.05% formic acid/methanol with 0.05% formic acid). MCP and levosulpiride (internal standard) were analyzed in the precursor/product ion pair of m/z 300.1/226.9 and 342.0/112.0, respectively. Linear calibration curves were obtained in the range of 2.5-500 ng/ml (R(2)>0.999) with a lower limit of quantification of 0.05 ng/ml. Mean recoveries were 96-103%, and the coefficient of variation was less than 6.5%. Plasma MCP concentrations after intravenous administration at 0.4 mg/kg to 12 cattle were determined by the LC-MS/MS method.

Response of Holstein cows with milk fever to first treatment using two calcium regimens: a retrospective clinical study.

The responses of 64 Holstein cows with milk fever to first treatment with 500 ml of either of 2 intravenous calcium (Ca) solutions, one containing Ca alone (group A, n = 32) or 1 containing Ca, phosphate and magnesium (group B, n = 32), were evaluated by selected clinical signs and serum biochemical data. Based on the cow's ability to stand, treatment response was categorized into "immediate response" (stood after single treatment), "delayed response" (stood after repeated treatments) and "non-response" (slaughtered despite repeated treatments). No significant differences among the response categories were found between the two groups, suggesting that the solution containing Ca borogluconate alone was sufficient for the first treatment of milk fever.

Simplified procedure for the estimation of glomerular filtration rate following intravenous administration of iodixanol in cats.

OBJECTIVE: To compare the use of a single-sample method involving IV administration of iodixanol with a multisample method involving inulin for the estimation of glomerular filtration rate (GFR) in cats. ANIMALS: 24 cats, including 15 healthy cats and 9 cats with naturally occurring renal diseases. PROCEDURES: Each cat was coadministered iodixanol (a nonionic contrast medium; dose providing 40 mg of I/kg) and inulin (50 mg/kg), IV, and blood samples were collected 60, 90, and 120 minutes later. Serum iodixanol and inulin concentrations were determined by means of high-performance liquid chromatography and colorimetry, respectively. Serum urea nitrogen and creatinine concentrations were also measured. RESULTS: Analysis of the data from healthy cats and cats with naturally occurring renal diseases revealed an excellent correlation between GFR values estimated by the multisample and single-sample methods with iodixanol. Likewise, GFR values estimated from the single-sample method with iodixanol were closely correlated with those calculated from the multisample method with inulin. CONCLUSIONS AND CLINICAL RELEVANCE: For estimation of GFR in cats, use of a single-sample method with iodixanol, instead of a multisample procedure, may be an expedient tool in both clinical and research settings because of its benefits to patient well-being as a result of reduced stress associated with blood sample collection.

Early-stage bioassay for monitoring radioactive contamination in living livestock.

Soil samples from the ground surface and feces and blood from a mixed-breed male pig were collected on April 10, 2011 at a farm within 20 km of the Fukushima Daiichi nuclear power plant. The radioactivity of each sample was measured using a Ge semiconductor detector. Despite the fact that the pig had been fed non-contaminated imported feed, (131)I, (134)Cs and (137)Cs were detected in the feces, and (134)Cs and (137)Cs were detected in the blood clots. Because it is considerably difficult to measure radioactive contamination in the edible muscle of living livestock, bioassays are an option for the screening of radioactive contamination in living livestock to ensure food safety.