Increased generation of superoxide in erythrocytes infected with Babesia gibsoni.

The present study was conducted to clarify the mechanism underlying the oxidative process in erythrocytes infected with Babesia gibsoni. The parasite B. gibsoni was cultured together with erythrocytes from normal dogs for 7 days. When parasitemia reached 12.0-13.4% at Day 7. the production of superoxide in erythrocytes was significantly higher in the parasitized culture than in the control culture (p<0.005). The concentration of thiobarbituric acid reactive substances (TBARS) in erythrocytes in parasitized culture was also significantly increased compared with the control culture (p<0.005), indicating that lipid peroxidation was greater in infected erythrocytes than in non-infected cells. In addition, the rates of superoxide generation in the blood of B. gibsoni-infected dogs were also significantly higher than in non-infected dogs (p<0.001). These results indicate that superoxide anions are increased in erythrocytes parasitized with B. gibsoni. and suggest that oxidative damage, due to lipid peroxidation, might be caused in host erythrocytes by the parasite.     

Pathological features of salivary gland cysts in a Shiba dog with GM1 gangliosidosis: a possible misdiagnosis as malignancy

Salivary gland cysts are often concurrent with GM1 gangliosidosis in Shiba dogs. Although the etiology is unknown, these cysts may be misdiagnosed as malignant due to the accumulation of foamy cells. The present study investigated the cytological, histopathological, immunohistochemical and electron microscopic characteristics of salivary gland cysts in a Shiba dog affected with GM1 gangliosidosis. The salivary gland masses were surgically enucleated and examined clinicopathologically and pathologically in a 7-month-old Shiba dog with GM1 gangliosidosis. Many large cells with rich cytoplasm including vacuoles of various sizes, i.e., foamy cells, were observed in stamp smears from the cut-surface of the masses and histopathologically in major parts of the cyst wall. Some of these foamy cells presented features similar to a spider-web appearance. The foamy cells were confirmed to have originated from macrophages based on marked immunohistochemical expression of vimentin, HLA-DR, lysozyme and Iba1. An ultrastructural study demonstrated electron-dense vesicular structures in the vacuolated cells. Therefore, the masses were diagnosed pathologically as benign salivary gland cysts with accumulation of foamy cells. In conclusion, the histopathological features of the salivary gland cysts in this Shiba dog were similar to those of lipoma and/or liposarcoma. In such cases, immunohistochemical and ultrastructural examinations were useful in the differential diagnosis. Practitioners, clinical pathologists and pathologists should take GM1 gangliosidosis into consideration when they encounter salivary gland cysts in Shiba dogs.     

Effects of propofol on the electrocardiogram and systolic blood pressure of healthy cats pre-medicated with acepromazine

OBJECTIVE: To obtain and analyze the electrocardiogram and systolic blood pressure of cats before, during, and after a continuous infusion of propofol. STUDY DESIGN: Prospective, uncontrolled experimental trial. ANIMALS: Twenty healthy adult crossbred male and female cats aged between 3 and 5 years, weighing 2.8-5.0 kg (mean 3.9 kg). METHODS: Cats were pre-medicated with acepromazine 0.1 mg kg(-1) subcutaneously and anesthesia was induced with intravenous (IV) propofol 6 mg kg(-1) and maintained with a continuous infusion of propofol at 0.5 mg kg(-1) minute(-1) for 60 minutes. Electrocardiographic parameters and systolic blood pressure obtained by Doppler ultrasound were recorded before pre-medication (T0), 30 (T30), and 60 (T60) minutes after beginning the continuous infusion, and 30 minutes after its cessation (T90). Repeated measures anova was used to perform statistical analysis. RESULTS: A significant decrease in heart rate was observed at all time points when compared with T0 values. The PR interval increased significantly at T60 and T90. Systolic blood pressures during anesthesia were significantly lower than at T0 and T90. CONCLUSION AND CLINICAL RELEVANCE: The changes seen were not clinically important in normal cats but given the reduction in heart rate and systolic blood pressure, careful consideration should be given before using this technique in patients in which hypotension or a reduction in heart rate would be poorly tolerated.     

Antibabesial activity of protoberberine alkaloids and 20-hydroxyecdysone from Arcangelisia flava against Babesia gibsoni in culture

Bioassay-guided fractionation of the boiled extract from the stems of Arcangelisia flava led to the isolation of palmatine (1), berberine (2), jatrorrhizine (3), dihydroberberine (4) and 20-hydroxyecdysone (5). The chemical structures of these compounds were elucidated on the basis of their chemical and spectral evidence. The isolated compounds were evaluated for their growth inhibiting effects on Babesia gibsoni in culture for a week. Compounds (1-4) showed significant inhibitions at concentrations from 100 to 1.0 microg/ml, while compound 5 at a concentration of 100 microg/ml, only.     

Relation between erythrocyte reduced glutathione and glutamate concentrations in Korean Jindo dogs with erythrocytes possessing hereditary high activity of Na-K-ATPase and a high concentration of potassium

The concentrations of sodium, potassium, reduced glutathione (GSH) and free amino acids and Na-K-ATPase activity in erythrocytes were examined in 35 purebred Jindo dogs in Korea. The incidence of Jindo dogs with a high potassium concentration and high activity of Na-K-ATPase in erythrocytes (HK phenotype) was 25.7%. The erythrocyte GSH concentration in HK Jindo dogs varied widely, from 2.45 to 12.38 mmol/l of RBCs, and was positively correlated with the erythrocyte glutamate concentration. These results indicate that HK Jindo dogs have normal to very high levels of erythrocyte GSH, which might result from the varying quantity of Na-dependent glutamate influx in the erythrocytes.     

Molecular screening of canine GM1 gangliosidosis using blood smear specimens after prolonged storage: detection of carriers among shiba dogs in northern Japan

Molecular screening of GM1 gangliosidosis in Shiba dogs was carried out in northern Japan using blood smear specimens after prolonged storage. Of 125 specimens obtained from 3 veterinary teaching hospitals for this screening, 68 specimens (54%) were adequate for direct amplification in a polymerase chain reaction (PCR)-based DNA test, and the percentage of adequacy was different at each hospital (34%, 73%, and 100%), suggesting that the amount of blood on the smear and the storage condition of specimens may affect adequacy. Of the 68 dogs examined, 2 dogs (2.9%) were heterozygous carriers for this disease and the other dogs were all genotypically normal. The results suggest blood smear specimens can be useful for PCR testing after prolonged storage provided specimens contain a generous amount of blood and have been adequately stored. The study also suggests that GM1 gangliosidosis may be widely prevalent in the Shiba dog population in northern Japan.     

Flow cytometry to evaluate the level of Babesia gibsoni parasitemia in vivo and in vitro by using the fluorescent nucleic acid stain SYTO16

In the present study, we employed flow cytometry to evaluate the level of parasitemia of Babesia gibsoni infecting canine erythrocytes in vivo and in vitro by using fluorescent nucleic acid staining. Peripheral blood samples from a B. gibsoni-infected dog and cultured B. gibsoni parasitizing in canine erythrocytes were stained with a membrane-permeable fluorescent nucleic acid stain, SYTO16. In this study, we utilized normal canine erythrocytes (LK erythrocytes) and canine erythrocytes containing high concentrations of potassium, reduced glutathione, and some free amino acids (HK erythrocytes) as host cells for culture. Parasitized cells in vive were discriminated completely from unparasitized cells and a correlation (r = 0.998) between the percentage of SYTO16-positive cells and parasitemia in vivo was observed. On the other hand, erythrocytes in vitro could not be divided clearly into parasitized and unparasitized cells. However, when LK erythrocytes were used as host cells, the percentage of SYTO16-positive cells was almost the same as, and was well correlated (r = 0.932) with, the level of parasitemia. When HK erythrocytes were used as host cells, the percentage of SYTO16-positive cells was almost half of, but was correlated (r = 0.982) with, the level of parasitemia. Therefore, we attempted to observe the changes in the percentage of parasitized cells after treatment with antiprotozoal drug or mitochondria inhibitors by using flow cytometry. The changes in the percentage of SYTO16-positive cells corresponded well with the changes of the level of parasitemia when the parasites in HK erythrocytes were cultured with each compound. The present results suggest that flow cytometric detection using SYTO16 is a rapid and reliable method for monitoring parasitemia both in vive and in vitro.     

Effects of cycloheximide on parthenogenetic development of pig oocytes activated by ultrasound treatment

The present study was carried out to examine the parthenogenetic development of pig oocytes treated with different concentrations of cycloheximide for different durations following activation by ultrasound stimulation. When oocytes were treated with 10 microg/ml cycloheximide for different durations, the blastocyst formation rate of oocytes treated for 5 h was significantly (P<0.05) higher than those of oocytes treated for 0-2 h. The blastocyst formation rate of oocytes treated with 10 microg/ml cycloheximide for 5 h was significantly (P<0.05) higher than those of oocytes treated with 0-5 or 15-20 microg/ml cycloheximide for the same duration. When oocytes were treated with different concentrations of cycloheximide for 2 h, however, the blastocyst formation rate of oocytes treated with 40 microg/ml cycloheximide was significantly (P<0.05) higher than those of oocytes treated with 0-10 or 50 microg/ml cycloheximide. The blastocyst formation rate of oocytes treated with 10 microg/ml cycloheximide for 5 h was not significantly different from that of oocytes treated with 40 microg/ml cycloheximide for 2 h. These treatments did not affect the activation status of oocytes compared with controls that were not treated with cycloheximide. The results of the present study showed that cycloheximide improves the parthenogenetic development of pig oocytes activated by ultrasound stimulation.     

Biomarkers for the activation of calcium metabolism in dairy cows: elevation of tartrate-resistant acid phosphatase activity by lowering dietary cation-anion difference is associated with the prevention of milk fever

In our previous study, it was demonstrated that the administration of anion salts, which slightly lower the dietary cation-anion difference (DCAD), in the prepartum period is safe and effective for preventing milk fever in multiparous cows. In the present study, several biomarkers, which might show activation of Ca metabolism, were analyzed using stored samples in the previous study to investigate the mechanism of the preventive effect on milk fever by lowering DCAD. Changes in bone-specific alkaline phosphatase activity, osteocalcin and insulin-like growth factor I concentrations in serum were almost the same among the three groups of multiparous cows with or without the oral administration of anion salts, while the levels of these serum biomarkers in the group of primiparous cows (heifer group) were much higher compared with those in the three multiparous groups throughout the experimental period. Urinary deoxypyridinoline excretion was not a useful biomarker for dairy cows because it hardly changed during the peripartum period in all groups. However, serum tartrate-resistant acid phosphatase (TRAP) activity, which is known as a biomarker of osteoclast activity, was well associated with the administration of anion salts lowering DCAD because among the three multiparous groups, only the group of multiparous cows fed the anion salts (anion group) showed an increased level, which rose to the level in the heifer group, and was markedly higher than those in the other control groups of multiparous cows. The increased activity of serum TRAP in the anion group suggested that Ca in the plasma pool was mobilized smoothly from bone-bound Ca via mature osteoclasts at parturition, which might be due to prior activation under mild acidosis induced by slightly lowering DCAD. Therefore, TRAP was the best biomarker to monitor the activation of Ca metabolism in dairy cows fed anion salts.