莫氏巴贝斯虫滑动体组件蛋白基因gap45、gap50、myo A 和mlc的克隆表达与多克隆抗体制备

【目的】克隆和表达莫氏巴贝斯虫滑动体组件蛋白GAP45(gliding associated protein 45, GAP45)、GAP50(gliding associated protein 50,GAP50)、肌球蛋白A（Myosin A, Myo A）和肌球蛋白轻链（myosin light chain, MLC）基因及制备兔源性多克隆抗体。【方法】利用末端快速扩增（rapid-amplification of cDNA ends, RACE）技术从莫氏巴贝斯虫裂殖子cDNA中扩增gap45、gap50、myo A及mlc基因,将获得的全长或基因的部分序列构建pGEX-4T-1原核表达载体,在大肠杆菌BL21（DE3）pLys中用IPTG诱导表达;融合蛋白经纯化后免疫家兔,制备多克隆抗体,并用ELISA测定抗体的效价及反应性。【结果】获得了gap45、gap50、myo A和mlc基因的全长分别为664、1391、773和2 538 bp,开放阅读框长度分别为567、1194、606和2 514 bp,制备的多克隆抗体具有较好的特异性,效价高于1:25 600。【结论】克隆了滑动体组件蛋白gap45、gap50、myo A和mlc基因的全长,并制备了多克隆抗体,为筛选巴贝斯虫疫苗和诊断用抗原和药物靶位、研究其生物学入侵机制奠定了基础。     

Review of Equine Piroplasmosis

Equine piroplasmosis is caused by one of 2 erythrocytic parasites Babesia caballi or Theileria equi. Although the genus of the latter remains controversial, the most recent designation, Theileria, is utilized in this review. Shared pathogenesis includes tick‐borne transmission and erythrolysis leading to anemia as the primary clinical outcome. Although both parasites are able to persist indefinitely in their equid hosts, thus far, only B. caballi transmits across tick generations. Pathogenesis further diverges after transmission to equids in that B. caballi immediately infects erythrocytes, whereas T. equi infects peripheral blood mononuclear cells. The recent re‐emergence of T. equi in the United States has increased awareness of these tick‐borne pathogens, especially in terms of diagnosis and control. This review focuses in part on factors leading to the re‐emergence of infection and disease of these globally important pathogens.     

Investigation of seroprevalence of <Emphasis Type="Italic">Theileria equi</Emphasis> and <Emphasis Type="Italic">Babesia caballi</Emphasis> in horses in Nigde province,Turkey

The prevalence of equine piroplasmosis caused by Theileria equi and Babesia caballi in Nigde, in central Anatolia, Turkey has remained unknown. Serum samples were obtained from a total of 125 horses and were tested for antibodies to T. equi and B. caballi using the Indirect Fluorescence Antibody Test (IFAT). Twenty-three (18.4%) horses were seropositive for equine piroplasmosis. Anti-T. equi was observed in 16 horses (12.8%) while anti-B. caballi was detected in 12 horses (9.6%). In addition, 5 serum samples were positive for both parasites. The prevalence rates of antibodies to T. equi and B. caballi for female and male horses were statistically indifferent (p = 0.19 and 0.90). The difference between the seropositivity rates to T. equi among age groups was statistically insignificant (p = 0.44) while the difference to B. caballi among age groups is statistically significant (p = 0.01). Seropositivity rates ranged from 2.9% to 25.7% for T. equi and 2.9% to 14.3% for B. caballi from the selected districts in Nigde. A statistically significant difference on seropositivity rates for the study sites was observed for only T.equi (p = 0.03). This study indicates that T. equi is higher than B. caballi in Nigde. This study was supported by the Scientific Research Projects Unit of Nigde University (FEB 2007/08).     

Individual heterogeneity in erythrocyte susceptibility to Babesia divergens is a critical factor for the outcome of experimental spleen-intact sheep infections

Susceptibility of sheep erythrocytes to Babesia divergens was investigated in vitro and a high inter-individual variability in their ability to support parasite population development was demonstrated, with some individuals having refractory red blood cells (RBC). As neither changes in growth conditions nor the use of different B. divergens strains influenced the level of susceptibility, the main factor postulated for this variability is the erythrocyte itself. Sheep therefore represent an excellent in vitro model to study the parasite-erythrocyte interaction. In addition, the existence of refractory RBC should help in the identification of the erythrocyte components required for B. divergens development. Experimental infections were carried out on spleen-intact sheep characterized by refractory or fully susceptible erythrocyte types. These differences translated into the successful infection of only those animals with susceptible erythrocytes: infected animals showed no clinical signs, but maintained an asymptomatic persistent infection, as usually observed in the natural bovine host. Sheep therefore represent model organisms that can allow us to study interactions between B. divergens and its vertebrate host at different levels of biological organisation, from the target cell to the intact animal, and represent an experimental infection model of concomitant immunity. Only a low percentage (13%) of the sheep population tested possessed susceptible erythrocytes and the potential role of sheep as a natural host or reservoir of B. divergens is discussed.     

牛巴贝斯虫PCR检测及体外培养试验

本试验通过PCR方法检测出牛巴贝斯虫单一感染的牛全血样本,并探讨了牛巴贝斯虫病患牛全血体外培养方法及培养条件。试验结果表明,PCR检测结果显示检测方法效果较好,单一感染样本的新鲜血液中虫体在含40%牛血清的完全培养液和37 ℃、5% CO₂的条件下能建立起牛巴贝斯虫的体外培养,虫体可以连续培养14 d,传代13次,最高染虫率可以达到12.5%,平均染虫率为4%。     

东方巴贝斯虫的18S rRNA基因的扩增及系统发育研究

利用真核生物18S rRNA基因的PCR通用引物对寄生于中国水牛的巴贝斯虫（已命名为东方巴贝斯虫-BnbPsia orientalis）基因组DNA进行扩增，得到其18S rRNA全基因片段，测序后blast分析表明该虫种属巴贝斯虫无疑。将该基因1700bp长片段序列与GenBank中15种已知巴贝斯虫的相应序列进行比较分析，建立系统发育树。结果表明，东方巴贝斯虫与南非未定种的巴贝斯虫亲缘关系最近，与羊巴贝斯虫亲缘关系较近，与牛巴贝斯虫、双芽巴贝斯虫的亲缘关系较远。这一结果说明水牛东方巴贝斯虫是一独立种。     

吉氏巴贝斯虫单克隆抗体制备的研究

应用淋巴细胞杂交瘤技术制备杂交瘤细胞株 ,经间接萤光抗体法筛选和克隆 ,获得了 5株能稳定分泌吉氏巴贝斯虫特异性抗体的杂交瘤细胞株 ,分别命名为C3B5、M8B7、E9C5、G6 D8、H2 A7。经过鉴定 ,这 5株杂交瘤细胞分泌的单克隆抗体亚类及相对分子质量分别为IgG2b,1 8× 1 0 4 ;IgG2a,1 8× 1 0 4 ;IgG1 ,1 8× 1 0 4 ;IgM ,3 2× 1 0 4 ;IgM ,1 8× 1 0 4 。腹水效价为 1∶1 0 4 ～ 1∶1 0 5。其中E9C5杂交瘤细胞株分泌的单克隆抗体是一种保护性抗体 ,经对实验感染吉氏巴贝斯虫小鼠体内虫体的杀虫试验证明具有较强的杀灭作用。     

一例犬吉氏巴贝斯虫病的诊治

3岁雄性贵宾犬,体重3.20 kg,免疫完全,未绝育,从未进行过体内外驱虫,院内散养。主诉最近2周发现犬的精神状态渐差,进食量逐渐减少。为了对其进行诊治,采用体格检查、血液学检查以及影像学检查的方法进行诊断,并根据诊断结果进行治疗,结果表明,该犬感染吉氏巴贝斯虫且并发肝脏损伤,通过采取输血、驱虫等治疗措施,患犬的临床症状消失,镜检外周血虫体消失,治疗后未见复发。说明临床上可通过血液学检测对犬的吉氏巴贝斯虫感染进行确诊,且输血结合驱虫治疗可有效治疗该病。