Bovine babesiosis: Partial purification and characterization of blood culture-derived Babesia bovis

Morphologic, biologic and immunologic properties of corpuscular and soluble fractions of Babesia bovis purified from in vitro blood cultures were studied. Supernatant fluids obtained during routine medium exchange were studied. Supernatant fluids obtained during routine medium exchange were submitted to differential centrifugation to separate soluble and corpuscular babesial antigens from erythrocyte stroma. Extracellular babesiae were sedimented with infected and uninfected erythrocyte ghosts. The majority of babesiae were found in erythrocyte ghosts. Clumps of extracellular parasites were sometimes formed in vitro and generally could not be separated from uninfected erythrocytes. Centrifugation over a discontinuous Ficoll density gradient did not improve separations. Parasites remained viable throughout the purification procedure but were killed by freezing and rapid thawing. Both corpuscular and soluble antigen fractions elicited the production of specific anti-babesial antibodies when injected into calves. Electron microscopy of corpuscular antigen revealed the presence of intra- and extraerythrocytic babesial merozoites. A surface coat was visible loosely adhering to the plasma membrane of the parasites. Parasite suspensions and cell-free supernatant fluids obtained from in vitro cultures of B. bovis should provide a variety of unique antigens for further in vitro and in vivo studies.     

Comparison of the effects of irradiation and splenectomy on Babesia rodhaini infection in mice

Comparison of the effects of irradiation and splenectomy on Babesia rodhaini infection in mice. International journal for Parasitology 3: 773–781. Babesia rodhaini infection was compared in irradiated, splenectomized and control mice. Although irradiation reduced the weight of the spleen by as much as 95 per cent, this reduction in size did not result in parasitaemia levels comparable to those seen in splenectomized mice, which were consistently higher. Parasitaemias were similar in irradiated and control mice, but the mean survival time in control mice was longer than that of irradiated or splenectomized mice, which were comparable. Splenectomy generally resulted in higher parasitaemias than those seen in non-splenectomized mice.

Since B. rodhaini has a predeliction for invading reticulocytes, the apparent failure of irradiated mice to develop parasitaemias comparable to those of splenectomized mice, may have been due to the selective destruction of these immature red cells by irradiation.     

Babesia microti: Molecular and antigenic characterizations of a novel 94-kDa protein (BmP94)

A novel gene, BmP94, encoding 94-kDa protein of Babesia microti was identified by immunoscreening of the cDNA expression library. The full-length of BmP94 was expressed in Escherichia coli (rBmP94), which resulted in insoluble form with low yield, and the truncated hydrophilic C-terminus region of the gene was expressed as a soluble protein (rBmP94/CT) with improved productivity. Antiserum raised against rBmP94/CT recognized the 94-kDa native protein in the parasite extract by Western blot analysis. Next, an ELISA using rBmP94/CT was evaluated for diagnostic use, and it demonstrated high sensitivity and specificity when tested with the sera from mice experimentally infected with B. microti and closely related parasites. Moreover, the immunoprotective property of rBmP94/CT as a subunit vaccine was evaluated in BALB/c mice against a B. microti challenge, but no significant protection was observed. Our data suggest that the immunodominant antigen BmP94 could be a promising candidate for diagnostic use for human babesiosis.     

Babesia bigemina: In Vitro and in VivoEffects of Curdlan Sulfate on Growth of Parasites

Igarashi, I., Njonge, F. K., Kaneko, Y., and Nakamura, Y. 1998.Babesia bigemina:In vitroandin vivoeffects of curdlan sulfate on growth of parasites.Experimental Parasitology90, 290–293.     

Comparison of injectable iron complexes in their ability to iron load tissues and to induce oxidative stress

Iron and copper homeostasis have been studied in various tissues after iron-loading with the polynuclear ferric hydroxide carbohydrate complexes, iron dextran, iron polymaltose, iron sucrose and iron gluconate for four weeks. There were significant increases in the iron content of the different rat tissues compared to controls, with the exception of the brain, which showed no change in its iron content following iron loading. However, the level of iron loading in the different tissues varied according to the preparation administered and only iron dextran was able to significantly increase the iron content of both broncho-alveolar macrophages and heart. The hepatic copper content decreased with iron loading, although this did not reach significance. However the copper content did not alter in the iron loaded broncho-alveolar macrophages. Despite such increases in hepatic iron content, there was little evidence of changes in oxidative stress, the activities of cytosolic (apart from iron dextran) or mitochondrial hepatic superoxide dismutase, SOD, were similar to that of the control rats, confirming the fact that the low reduction potential of these compounds prevents the reduction of the ferric moiety. It was not necessary for macrophages to significantly increase their iron content to initiate changes in NO^. release. Iron gluconate and iron sucrose increased NO^. release, while iron polymaltose and iron dextran decreased NO^. release although only the latter iron preparation significantly increased their iron content. It may be that the speciation of iron within the macrophage is an important determinant in changes in NO^. release after ex vivo stimulation. We conclude that tissues loaded with iron by such polynuclear iron complexes have variable loading despite the comparable iron dose. However, there was little evidence for participation of the accumulated iron in free radical reactions although there was some evidence for alteration in immune function of broncho-alveolar macrophages.     

风车草和香根草在人工湿地中迁移养分能力的比较研究

为研究风车草(Cyperus alternifolius)和香根草(Vetiveria zizanioides)迁移养分的能力,建立17.0m²风车草潜流式人工湿地和13.3 m²香根草潜流式人工湿地处理猪场废水,在四个季节末测定植物生物量和组织氮、磷、铜、锌含量.结果表明,香根草地下部生物量大于风车草,地上部生物量则是风车草大于香根草.风车草年地上部收获量为3406.47 g·m^-2,比香根草的1483.88 g·m^-2高2.3倍;风车草的氮含量为22.69 mg·g^-1,比香根草的15.44 mg·g^-1高7.25 mg·g^-1;风车草的磷含量为6.09 mg·g^-1,比香根草的5.47 mg·g^-1高0.62 mg·g^-1.植株含铜、锌量风车草略比香根草高.风车草每年迁移N 68.72 g·m^-2和P18.49 g·m^-2,香根草迁移N 8.93 g·m^-2和P 3.69 g·m^-2.风车草人工湿地每年由植物迁移的氮、磷、铜、锌比香根草高4～7倍.     

意大利苍耳与乌拉尔甘草种间竞争能力比较

意大利苍耳已经开始入侵乌拉尔甘草农田,然而对其入侵后果目前知之甚少。通过研究意大利苍耳与乌拉尔甘草的种间竞争关系,以期为意大利苍耳对乌拉尔甘草农田生态系统的入侵能力及入侵后果的评估提供试验依据。模拟了甘草农田的土壤水肥条件,采用取代试验法,设置意大利苍耳与乌拉尔甘草的单种种植和混种种植2种种植模式,待意大利苍耳生育期结束后进行收获,分别比较了该两种植物的个体生长及生物量积累在单种种植与混种种植两种处理间的差异,并比较单种和混种模式下乌拉尔甘草的地下器官中甘草酸含量的差别,通过计算相对产量、相对竞争强度和竞争攻击系数来比较该两种植物种间竞争能力的相对强弱。结果表明：与单种模式相比,混种模式下意大利苍耳个体生长的形态学性状和有性繁殖能力均有显著增加,其株高、冠幅和种子数量较单种处理分别增加了13%、27%和56%。而乌拉尔甘草的个体生长及克隆繁殖能力均显著降低,其根瘤的形成也受到显著的抑制,混种处理的乌拉尔甘草的株高、冠幅、根总长度、根总表面积、根平均直径以及根瘤数量比单种处理分别下降了35%、45%、55%、63%、19%和76%。单种处理下每株甘草根状茎的平均条数为3条,而与意大利苍耳混种后,其根状茎的发育被完全抑制。与单种处理相比,混种处理中的意大利苍耳生物量积累均显著的增加了,其中根、茎、叶、果实及总生物量与单种模式相比分别增加了84%、73%、84%、73%和77%,而混种模式却极显著降低了乌拉尔甘草生物量和地下器官甘草酸含量的积累,使其根、茎、叶、总生物量以及甘草酸含量与单种模式相比分别下降了72%、80%、65%、71%和63%。混种模式中的意大利苍耳相对产量（RY_a）大于1,而乌拉尔甘草相对产量（RY_b）小于1,表明意大利苍耳受到邻株同种其他个体的种内竞争压力大于来自邻株乌拉尔甘草的种间竞争压力,而乌拉尔甘草受到邻株同种其他个体的种内竞争压力则小于来自邻株意大利苍耳的种间竞争压力。混种模式中的意大利苍耳相对竞争强度（RCI_a）小于0,其竞争攻击系数（A_q）大于0,而乌拉尔甘草的相对竞争强度（RCI_b）则在0-1之间,其竞争攻击系数（A_b）小于0,表明在该两种植物的混生群落中,乌拉尔甘草的竞争能力弱于意大利苍耳。总体来看,在二者混生的群落中,意大利苍耳在竞争中占据明显的优势地位,对乌拉尔甘草的产量和品质均造成强烈的负面影响。     

DBA/2J and DBA/2Ha lymphocytes differ in their ability to induce graft-vs-host disease

Graft-vs-host disease (GVHD) is a common occurrence after bone marrow transplantation despite the use of MHC-matched donors and recipients. This indicates that non-MHC loci play an important role in the regulation and development of GVHD. Non-MHC loci have been shown to regulate GVHD in a murine model where acute GVHD results from i.v. injection of C57BL/6J spleen cells into B6D2F1/J [C57BL/6J X DBA/2J)F1) recipients while chronic GVHD results from injection of DBA/2J spleen cells. In contrast to the hyperproduction of Ig and auto-antibodies that is characteristic of the chronic GVHD that occurs after injection of DBA/2J cells, injection of DBA/2Ha cells was found to induce CTL and suppressor cells characteristic of the acute GVHD that results from injection of C57BL/6 cells into B6D2F1/J recipients. Genetic analysis indicated that one autosomal locus is responsible for the different GVHD responses of DBA/2J and DBA/2Ha cells and that the DBA/2Ha allele is dominant. Further studies indicate that the different responses by DBA/2J and DBA/2Ha cells is not due to functional differences between the two sets of cells but by a radiosensitive B6D2F1 recipient immune response which discriminates between the DBA/2J and DBA/2Ha spleen cells.     

Characteristics of a clinical isolate of urease-negative Helicobacter pylori and its ability to induce gastric ulcers in Mongolian gerbils

BACKGROUND: We clinically obtained urease-negative mutant strains of Helicobacter pylori. The goal of this study was to investigate the ability of the urease-negative strain to colonize and subsequently damage the gastric mucosa in Mongolian gerbils. In addition, the genes encoding the urease production in the test strain were analyzed, and other genes encoding the virulence factors, cytotoxin-associated protein and vacuolating-cytotoxin were evaluated. MATERIALS AND METHODS: The character of urease-negative isolates of H. pylori was defined. The identification of H. pylori was confirmed by polymerase chain reaction (PCR). The H. pylori isolate was transfected into Mongolian gerbils as previously described, which were followed up to 42 weeks, and the changes in their gastric mucosa were examined histologically. RESULTS AND CONCLUSION: Fifteen Mongolian gerbils orally infected with 10(7) colony forming units of urease-negative H. pylori were killed at 4, 12, 24, 36 and 42 weeks (n = 3) after infection. Culture medium without urease-negative H. pylori was given to the Mongolian gerbils as control. H. pylori continued to exist in the subject's stomach and gastric ulceration was observed and compared with the control. Clinically obtained urease-negative H. pylori continued to exist for at least 42 weeks in the subject's stomach and it induced gastric ulcers. These data demonstrated that the urease in H. pylori was not a necessary factor in the formation of gastric ulcers in the Mongolian gerbil model.     

蛹虫草线粒体DNA与细胞核DNA进化关系的比较

【目的】分析蛹虫草是否存在核内线粒体DNA片段,比较蛹虫草线粒体DNA与细胞核DNA的碱基变异程度及所反映的菌株间的系统发育关系。【方法】通过本地BLAST或LAST对蛹虫草线粒体基因组和核基因组进行序列相似性搜索;从10个已知线粒体基因组的蛹虫草菌株中分别扩增7个细胞核蛋白编码基因片段,并与其在14个线粒体蛋白编码基因上的碱基变异情况进行比较。【结果】蛹虫草核基因组中存在5处较短的核内线粒体DNA片段,总长只有278bp。蛹虫草核DNA的变异频率整体上高于线粒体DNA。核DNA和线粒体DNA所反映的蛹虫草菌株间的系统发育关系存在显著差异。【结论】蛹虫草线粒体DNA与核DNA间不存在长片段的基因交流,二者变异频率不同,所反映的蛹虫草菌株间的系统发育关系也有差异。本研究增加了对蛹虫草线粒体与细胞核DNA进化关系的认识。     

Growth promotion and cell binding ability of bovine lactoferrin to Bifidobacterium longum

Lactoferrin, a major whey protein of human milk, is considered as growth promoter for bifidobacteria, the predominant microorganisms of human intestine. In the present study, in vitro growth promotion and cell binding ability of bovine lactoferrin to several strains of Bifidobacterium longum have been demonstrated. A dose-dependent as well as strain-dependent growth promotion effect by lactoferrin was observed. Cell binding ability of lactoferrin was inspected under an inverted confocal laser scanning microscope by incubation bacterial cells with biotinylated bovine lactoferrin and FITC-conjugated avidin. Fluorescence staining showed bovine lactoferrin binding to all tested strains. A lactoferrin-binding protein with a molecular weight of approximately 67 kDa was also detected in the extracted membrane and cytosolic fraction of each B. longum strain by far-Western blot technique using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin. Based on these results, we suggest that existence of lactoferrin-binding protein could be a common characteristic in bifidobacteria. It can also be hypothesized that lactoferrin-binding protein in bifidobacteria is not only involved in growth stimulation mechanism but also could play different roles.     

Microcalorimetric studies of the interactions between cytochromes c and c1 and of their interactions with phospholipids

Thermotropic properties of purified cytochrome c₁ and cytochrome c have been studied by differential scanning calorimetry under various conditions. Both cytochromes exhibit a single endothermodenaturation peak in the differential scanning calorimetric thermogram. Thermodenaturation temperatures are ionic strength, pH, and redox state dependent. The ferrocytochromes are more stable toward thermodenaturation than the ferricytochromes. The enthalpy changes of thermodenaturation of ferro- and ferricytochrome c₁ are markedly dependent on the ionic strength of the solution. The effect of the ionic strength of solution on the enthalpy change of thermodenaturation of cytochrome c is rather insignificant. The formation of a complex between cytochromes c and c₁ at lower ionic strength causes a significant destabilization of the former and a slight stabilization of the latter. The destabilization of cytochrome c upon mixing with cytochrome c₁ was also observed at high ionic strength, under which conditions no stable complex was detected by physical separation. This suggests formation of a transient complex between these two cytochromes. When cytochrome c was complexed with phospholipids, no change in the thermodenaturation temperature was observed, but a great increase in the enthalpy change of thermodenaturation resulted.     

Multiple in vivo passages enhance the ability of a clinical Helicobacter pylori isolate to colonize the stomach of Mongolian gerbils and to induce gastritis

The Mongolian gerbil is an excellent animal model for Helicobacter pylori-induced gastritis in humans. In this study, initially low colonization rates of the H. pylori strains ATCC 43504, SS1, or HP87 inoculated into gerbils caused difficulties in establishing this model. In order to increase the colonization ability and pathogenicity, the clinical HP87 isolate was selected for adaptation to the gerbil stomach by multiple in vivo passages through gerbils. Development of gastritis was examined histologically at 4-52 weeks after infection. The proportion of gerbils which tested positive for H. pylori by culture at four weeks after inoculation gradually increased from 11.1% of gerbils inoculated with HP87 without prior in vivo passage (P0) to 100% of gerbils inoculated with HP87 with seven in vivo passages (P7). In addition, adaptation of HP87 resulted in more severe histopathological changes. Gerbils infected with adapted HP87 (P7) exhibited severe infiltration by monomorphonuclear and polymorphonuclear leukocytes in the mucosa, submucosa, and subserosa of the gastric antrum, as well as epithelial changes consisting of hyperplasia, erosion, and ulceration. Histopathological changes increased in severity from four to 52 weeks after infection. Adaptation of HP87 during its passages through gerbils could be due to genetic changes in bacterial colonization factors. Identification of these changes might be useful to understand the underlying mechanism of gastric adaptation and pathogenesis of H. pylori.     

濒危植物毛瓣金花茶与其同属广布种茶光合特性的比较

毛瓣金花茶为山茶科山茶属植物,该研究分别对野生种群和栽培种群的毛瓣金花茶及其同属广布种茶的光合特性进行了测定及差异比较。结果表明:在野生和栽培环境下,毛瓣金花茶的光补偿点(LCP)(分别为1.17和3.87μmol·m-2·s-1)和光饱和点(LSP)(分别为395.8和423.6μmol·m-2·s-1)均较低,最大净光合速率(Pmax)(分别为4.25和3.91μmol·m-2·s-1)较小,是典型的阴生植物;而茶的LCP(分别为6.57和9.09μmol·m-2·s-1)较低,LSP(分别为765.0和809.6μmol·m-2·s-1)较高,Pmax(分别为9.37和9.75μmol·m-2·s-1)较大,为耐荫植物。野生和栽培的毛瓣金花茶的Pmax、表观量子效率(AQY)、最大羧化速率(Vcmax)、最大电子传递速率(Jmax)和潜在最大净光合速率(Pmax)均显著低于茶(P0.05),这表明毛瓣金花茶的光合能力和CO2利用能力都比茶要弱。栽培的毛瓣金花茶叶片的Chla、Chlb、Chl(a+b)含量与茶相比无显著差异(P0.05),表明毛瓣金花茶较低的光合能力与其叶绿素含量无关。野生和栽培的毛瓣金花茶的叶面积与茶相比无显著差异(P0.05),而比叶重则显著高于茶(P0.05),与茶相比,毛瓣金花茶对光强的适应范围狭窄,光合能力和CO2利用能力低下,这可能是其分布狭窄的重要生理原因。     

扎龙保护区散养与野生丹顶鹤巢址选择比较

为了探讨扎龙保护区散养与野生丹顶鹤(Grus japonensis)巢址选择的异同,2009年3～5月在扎龙国家级自然保护区内用生境因子测定法对散养丹顶鹤与野生丹顶鹤巢址选择进行比较。独立样本t-检验(independent-samples t-test)结果表明,散养丹顶鹤和野生丹顶鹤在巢址选择中,植被高度、植被密度、巢周围苇丛面积及巢距人为干扰地距离均存在着显著差异。说明野生丹顶鹤对巢址选择具有严格要求,倾向于选择人为活动较少,植被高度较高,植被密度和巢周围苇丛面积较大的生境中筑巢;散养丹顶鹤对生境要求不高。     

Mutants of Agrobacterium tumefaciens with temperature sensitivity in respect to their tumor inducing ability

Summary The isolation of six mutants of Agrobacterium tumefaciens which can induce tumors at low temperatures (22°C) but fail to do so at 28°C is described. At the nonpermissive temperature the following characteristics of the mutants are the same as those of the wild type: growth rates in vitro, growth rates in planta, and sensitivity towards agrocin 84, a marker for the presence of the virulence-plasmid. The tumors induced by the mutants at low temperature grow without addition of hormones at both 22°C and 28°C. The induction of the tumors but not the maintenance of the tumorous phenotypes are affected in the mutants isolated.     

Evidence for recombination and segregation of virulence to pine in a hybrid cross between Gibberella circinata and G. subglutinans

Two species associated with the Gibberella fujikuroi species complex, G. circinata (the cause of pitch canker in pines) and G. subglutinans (avirulent on pine), were found to have limited interfertility in hybrid crosses. MAT idiomorphs, polymorphisms in the histone H3 gene, vegetative compatibility, and virulence phenotypes were used to verify recombination. The MAT idiomorphs appeared to be assorting independently, but the histone H3 haplotype was disproportionately represented by that of the G. subglutinans parent. Ninety-eight percent (45/46) of the progeny tested were vegetatively incompatible with both parents. All F₁ progeny were avirulent to pine, but a wide range of virulence was restored through a backcross to the virulent parent (G. circinata). Attempts at hybrid crosses using other isolate combinations were rarely successful (1/26). This limited interfertility supports retention of G. circinata and G. subglutinans as separate species, but offers opportunities to characterize the inheritance of virulence to pine.     

Identification and immunoprotective analysis of an in vivo-induced Edwardsiella tarda antigen

Edwardsiella tarda is a severe aquaculture pathogen that can infect many important fish species cultured worldwide. The aim of this study was to evaluate the vaccine potential of an E. tarda antigen, Eta21, which was identified from a pathogenic E. tarda strain via the method of in vivo-induced antigen technology (IVIAT). Eta21 is 510-amino acid in length and shares 58% sequence identity with a putative peptidase of several bacterial species. eta21 was subcloned into Escherichia coli, and recombinant Eta21 was purified as a histidine-tagged protein. When used as a subunit vaccine, purified recombinant Eta21 was effective against lethal E. tarda challenge in a Japanese flounder model. In order to improve the immunoprotective efficacy of Eta21, the chimera AgaV-Eta21 was constructed, which consists of Eta21 fused in-frame to the secretion domain of AgaV, an extracellular β-agarase. E. coli DH5α harboring plasmid pTAET21, which constitutively expresses agaV-eta21, was able to produce and secret AgaV-Eta21 into the extracellular milieu. Vaccination of Japanese flounder with live DH5α/pTAET21 elicited immunoprotection that is significantly higher in level than that induced by vaccination with purified recombinant Eta21. Vaccination with DH5α/pTAET21 and recombinant Eta21 both induced the production of specific serum antibodies at four to eight weeks post-vaccination. Taken together, these results demonstrate that Eta21, especially that delivered by DH5α/pTAET21, is an effective vaccine candidate against E. tarda infection.     

Production and characterization of monoclonal antibodies to Acanthamoeba castellanii and their application for detection of pathogenic Acanthamoeba spp

Fourteen monoclonal antibodies (mAbs) were produced against a strain of Acanthamoeba castellanii isolated from a human cornea. The reactivity of the mAbs to reference strains of Acanthamoeba was examined by an indirect fluorescence antibody test (IFA) and Western immunoblot analysis. Nine mAbs reacted specifically with a known pathogenic reference strain of A. castellanii, but not with a non-pathogenic strain or other Acanthamoeba spp. The antigen recognized by these mAbs had a molecular mass of 17 kDa. The remaining five mAbs reacted with A. castellanii and A. polyphaga, members of group II (Pussard and Pons) but not with A. astronyxis (group I) or A. culbertsoni (group III). Western immunoblot analysis revealed that the latter mAbs stained many protein bands ranging from 30 to 150 kDa. None of the 14 mAbs reacted with Naegleria gruberi, N. fowleri, or Entamoeba histolytica. These observations suggest that an antigen common in group II as well as a pathogenic A. castellanii-specific antigen are present. Slot blot reactivity was comparable to the IFA. Under certain circumstances, therefore, slot blot analysis with a panel of mAbs should be helpful in the detection of keratitis-producing strains of Acanthamoeba.     

永州广聚萤叶甲和豚草卷蛾的种群动态及对豚草的控制效果

【目的】豚草是一种重要的入侵杂草,严重危害农业生产和人类健康。广聚萤叶甲和豚草卷蛾是豚草的专一性天敌。研究这2种天敌在永州的种群动态及其对豚草的控制效果,可以为永州豚草的防控及这2种天敌的有效利用提供依据。【方法】在湖南省永州市江永县豚草大面积发生区域,人工释放广聚萤叶甲和豚草卷蛾,调查这2种天敌在释放区和扩散区的种群动态和对豚草的防治效果,以及这2种天敌在扩散区的越冬情况。【结果】广聚萤叶甲和豚草卷蛾的扩散能力强。释放1个月后,在释放区,广聚萤叶甲各虫态及豚草卷蛾虫瘿均被发现。整体上,随时间延长,广聚萤叶甲各虫态虫口密度先增后减,而豚草卷蛾虫瘿密度呈逐渐降低趋势。释放2个月后,在距释放区边缘10 km的豚草发生区,发现了广聚萤叶甲和豚草卷蛾,且成功建立了种群并顺利越冬。释放区豚草株高几乎没有增加,且叶片最终被取食精光,几乎全部死亡;扩散区豚草株高略有增加,最终近75%叶片被取食。【结论】广聚萤叶甲和豚草卷蛾可在永州成功建立种群并安全越冬,还能自行扩散,可持续控制野外豚草的发生。