Development of in vitro atovaquone-resistant Babesia gibsoni with a single-nucleotide polymorphism in cytb

An atovaquone (ATV)-resistant Babesia gibsoni was developed by in vitro exposure of uncloned wild type (WT) B. gibsoni to 800 nM ATV for 6 days. Sequence analysis of mitochondrial genes showed a single-nucleotide polymorphism (SNP) at cytb nt363 (G to T) that resulted in the substitution of methionine with isoleucine (M121I), which is one of the SNPs reported in a previous in vivo study. 363T or 363G allele-specific real-time polymerase chain reaction (PCR) revealed that an M121I variant was present in over 99% of the ATV-resistant population. As neither ATV resistance nor gene polymorphisms appeared in the B. gibsoni WT sibling clones, the expression of ATV resistance in this study was suspected to be because of selective multiplication of the B. gibsoni M121I variant. This ATV-resistant B. gibsoni displayed the same sensitivity as the WT B. gibsoni against 5 other drugs, including diminazene aceturate, azithromycin, doxycycline, clindamycin, and proguanil. This is the first report on the in vitro establishment of an ATV-resistant B. gibsoni with gene polymorphisms.     

Antagonistic effects of atipamezole and yohimbine on medetomidine-induced diuresis in healthy dogs

This study aimed to investigate and compare the antagonistic effects of atipamezole and yohimbine on medetomidine-induced diuresis in healthy dogs. Five dogs were used repeatedly in each of 8 groups. One group was not medicated. Dogs in the other groups received 20 μg/kg of medetomidine intramuscularly and, 0.5 h later, saline (as the control injection), 50, 100, or 300 μg/kg of atipamezole, or 50, 100, or 300 μg/kg of yohimbine intramuscularly. Urine and blood samples were taken 11 times over 24 h for measurement of the following: urine volume, specific gravity, and creatinine concentration; urine and plasma osmolality; urine and plasma concentrations of electrolytes and arginine vasopressin (AVP); and the plasma concentration of atrial natriuretic peptide (ANP). Both atipamezole and yohimbine antagonized the diuretic effect of medetomidine, inhibiting medetomidine-induced decreases in urine specific gravity, osmolality, and concentrations of creatinine, sodium, potassium, chloride, and AVP and reversing both the medetomidine-induced increase in plasma concentrations of sodium, potassium, and chloride and the medetomidine-induced decrease in the plasma AVP concentration. Atipamezole significantly stimulated ANP release. The antidiuretic action of yohimbine was more potent than that of atipamezole but was not dose-dependent, in contrast to the action of atipamezole. The effects of these drugs may not be due only to actions mediated by α₂-adrenoceptors.     

Quantitative structure-activity studies of insect growth regulators: XIX. Effects of substituents on the aromatic moiety of dibenzoylhydrazines on larvicidal activity against the beet armyworm Spodoptera exigua

Larvicidal activity against the beet armyworm, Spodoptera exigua (Hübner), was measured after topical treatment for a series of N-tert-butyl-dibenzoylhydrazines having various substituents in the benzoyl (A-ring) moiety closer to the tert-butyl group, the other benzoyl (B-ring) moiety being unsubstituted. The effects of substituents on the larvicidal activity were analyzed using the classical quantitative structure-activity relationship (QSAR) procedure. Introduction of hydrophobic substituents with a small volume into any position was favourable to activity. The existence of electron-withdrawing substituents at ortho positions was also favourable to activity. For multi-substituted compounds, physico-chemically unknown unfavourable factors were suggested to remain after separating common substituent effects derived from QSAR for mono-substituted analogues. With the exception of the unsubstituted compound RH-5849, the effect of substituents in the A-ring moiety on the larvicidal activity was similar to those found with the lepidopteran rice stem borer, Chilo suppressalis (Walker). The larvicidal activity of RH-5849 against S exigua was significantly lower than the value predicted from the correlation between activities against S exigua and C suppressalis. Topical treatment with piperonyl butoxide, a synergist inhibiting oxidative metabolism, slightly enhanced the larvicidal activity of RH-5849 against S exigua.     

Neurohormonal and metabolic effects of medetomidine compared with xylazine in healthy cats

The purpose of this study was to investigate and compare the effects of medetomidine and xylazine on some neurohormonal and metabolic variables in healthy cats. Five cats were used repeatedly in each of 11 groups, which were injected intramuscularly with physiological saline solution (control), 20, 40, 80, 160, and 320 microg/kg of medetomidine, and 0.5, 1, 2, 4, and 8 mg/kg of xylazine. Blood samples were taken over 24 h from the jugular vein for determination of plasma glucose, insulin, cortisol, epinephrine, norepinephrine, glucagon, and nonesterified fatty acid concentrations. Both medetomidine and xylazine induced remarkable hyperglycemia that was dose-dependent except for the response to medetomidine from 0 to 3 h. Both agents suppressed epinephrine and norepinephrine release but not in a dose-dependent manner at the tested dosages. Both agents inhibited insulin release and lipolysis, with similar potency, and tended to suppress cortisol release. The glucagon levels did not change significantly in any of the groups. These results suggest that the effects of medetomidine and xylazine on glucose metabolism and catecholamine release may not be due only to the actions mediated by alpha2-adrenoceptors.     

Development of a system for measuring calcitonin in the stingray Dasyatis akajei (a cartilaginous fish): the possible involvement of stingray calcitonin in gonadal development

To elucidate the physiological role of calcitonin (CT) in stingrays (cartilaginous fish), an enzyme-linked immunosorbent assay (ELISA) system using a specific antibody against stingray CT has been developed. Synthetic stingray CT was subcutaneously injected into mice four times—once every 2 weeks—together with an adjuvant. We purified the IgG antibody fraction using the protein A affinity chromatography from collected antiserum. Evaluating the antibody titer, we found the antibody’s optimum dilution ratio to be 600 times. Competitive ELISA has been developed using the antibody diluted 600 times. Our antibody did not cross-react with teleost CTs and muscle extraction, but cross-reacted with stingray plasma and the extract of the ultimobranchial gland, the secretary organ of stingray CT. Using this ELISA, we measured the plasma CT level in stingrays and examined its correlation with several mineral concentrations. Plasma CT did not show significant correlation to calcium, magnesium, inorganic phosphorus, sodium, chlorine, or urea, although there was a correlation among the factors involved in osmoregulation, such as sodium, chlorine, and urea. On the other hand, plasma CT was significantly correlated to body weight and length. Furthermore, there was a significant correlation between plasma CT and gonad weight. Since plasma CT was correlated with the weight of liver, which is involved in the synthesis of egg yolk protein, we examined the influence of 17β-estradiol (E2) on CT secretion. After E2 injection, the plasma CT level increased significantly. This is the first study to demonstrate that E2 induced plasma CT secretion in cartilaginous fish.     

Effect of extracts from <Emphasis Type="Italic">narezushi</Emphasis>, a fermented mackerel product,on cholesterol metabolism in Wistar rats

Narezushi extract was separated into peptide and nonpeptide fractions by ion-exchange column chromatography. The narezushi extract and fractions were administered to rats in a diet enriched with lipid and cholesterol for 30 days. In the narezushi extract and nonpeptide fraction groups, increases in triglyceride, total cholesterol, and low-density lipoprotein-cholesterol levels in the plasma and accumulation of total lipids and triglyceride in the liver were suppressed, while both lipid and cholesterol fecal excretion were increased. In the peptide fraction group, these effects were also observed, except for the suppressing effect on liver lipid accumulation. Narezushi extract administration tended to increase fecal bile acids and promoted the activity of cholesterol 7α-hydroxylase, the rate-limiting enzyme in the synthesis of bile acid from cholesterol in the liver. However, the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the cholesterol synthesis system in the liver, decreased due to regulation by the feedback of lipid transportation from diet to the liver. These results suggest that both the increase in cholesterol and bile acid fecal excretion and the promotion of cholesterol 7α-hydroxylase activities are related to the hypocholesterolemic effects of narezushi extract. Amino acids and organic acids, which are abundantly contained in the nonpeptide fraction, seemed to have more intensive hypocholesterolemic effects than peptides existing in the peptide fraction.     

尼泊尔Terai地区小规模农场的奶牛和水牛的饲料品质、营养水平及产奶性能

调查尼泊尔Terai地区两个村庄(A、B)中20个小农场的泌乳奶牛和水牛以确定其饲料品质、产奶量和营养水平。在牧草充足期、牧草枯萎期和饲草短缺期三个时期中分别测定饲料的组成成分、干物质、奶牛的体质条件(BCS)、胸围(HG)、体重(BW)、产奶量(MY)及血浆代谢物。305d的产奶量通过平均日产奶量来估计。牧草充足期的稻草和当地牧草供应比牧草枯萎期少而比牧草短缺期多,分别为5.5kg/d∶9.8kg/d和3.2kg/d∶0.4kg/d,(P<0.01),A村中的饲料中粗纤维含量比B村高,奶牛中为5.0∶2.2,水牛中为9.3∶1.8(P<0.01)。两个村中各时期的饲料组成的差异导致粗蛋白(CP)、中性洗涤纤维(NDF)、总可消化营养物质(TDN)供应的差异。奶牛饲料的CP和TDN浓度在牧草充足期比其它两个时期高,分别为9.1%∶7.3%和57.4%∶51.0%(P<0.01)。A村中供应给奶牛和水牛的CP和供应给水牛的TDN比B村的高,分别为7.5%∶8.7%和6.6%∶9.7%(P<0.01)以及53.1%∶56.2%(P<0.05)。A村中奶牛和水牛的BCS、HG、BW比B村中的低,奶牛分别为2.51∶2.86,156cm∶170cm,300kg∶318kg,水牛为2.83∶4.00,186cm∶216cm,429kg∶531kg(P<0.01)。奶牛在牧草充足期比其他两期的产奶量大,7.9L/d∶6.6L/d(P<0.01)。奶牛在饲草短缺期产后305d的产奶量比其它两期低,1900L∶2251L(P<0.01)。A村中的奶牛和水牛的产奶量都比B村的低6.2L/d∶8.1L/d,3.7L/d∶7.7L/d(P<0.01)。A村中奶牛305d的产奶量比B村低,1935L∶2409L(P<0.01),A村中奶牛的血浆清蛋白和尿氮比B村的低,3.2g/dL∶3.4g/dL(P<0.01),7.4mg/dL∶10.2mg/dL(P<0.05)。在不同时期两个村中饲料中的CP、NDF、TDN可能会对奶牛和水牛的产奶量和营养水平有影响。似乎在饲草短缺期A村对奶牛低水平供应的CP和TDN使得奶牛305d的产奶量更低。     

Efficacy of atovaquone against Babesia gibsoni in vivo and in vitro

The therapeutic efficacy of atovaquone against Babesia gibsoni was examined in three dogs experimentally infected with B. gibsoni isolated from naturally infected dogs in Aomori Prefecture, Japan. Once parasitemia reached 10%, atovaquone was administered orally (30 mg/kg twice daily for 7 days). Within 2 days of atovaquone treatment, the parasite disappeared from blood smears without any clinical side effects. Anemia and thrombocytopenia were significantly improved in all the dogs. However, a polymerase chain reaction assay revealed that a B. gibsoni marker gene was intermittently present in peripheral blood after atovaquone therapy, indicating that the organism had not been eliminated, and parasites reappeared in blood smears 33 days after the last treatment. To investigate the change in sensitivity against atovaquone, an in vitro sensitivity test was performed using peripheral blood obtained from an untreated dog that was infected with the original parasite isolate, and from two of the experimentally infected and atovaquone-treated animals (blood was collected at the time of the post-treatment recurrence of the B. gibsoni infection). Atovaquone was added to the culture medium to final concentrations of 0.1, 1, 10, 100, and 1000 nM. For the untreated parasites, complete growth inhibition occurred at 1000 nM of atovaquone, whereas the recurrent parasites were inhibited by only 39.52 +/- 8.34% and 31.31 +/- 8.14% at this concentration after 48 h of incubation. Thus, the recurring parasites were less sensitive to atovaquone than the untreated originally isolated parasites.