Subolesin: A candidate vaccine antigen for the control of cattle tick infestations in Indian situation

Identification of cross-protective tick vaccine antigens is a challenging area of veterinary research. To address this challenge, a recently identified candidate tick protective antigen, Subolesin (SUB), was targeted in this research. The conservation of subolesin ortholog of Hyalomma anatolicum and Rhipicephalus (Boophilus) microplus across different Indian strains was 98.1–99.4% (within species), while at the amino acid level SUB sequence homology was ≥53.2% (between tick species). Recombinant R. (B.) microplus SUB (rBmSu) was produced in Escherichia coli and characterized. Cross-bred cattle male calves (N = 10) were immunized with three doses of 100 μg each of the rBmSu emulsified in 10% Montanide 888 at monthly intervals on days 0, 30 and 60. The control group was injected with PBS in 10% Montanide 888. For the first tick challenge, calves were infested with larvae of R. (B.) microplus generated from 100 mg eggs 2 weeks after last immunization (day 75). The immunization resulted in 16.3%, 8.0%, 9.4%, and 26.1% reduction in female tick numbers (DT), weight (DW), oviposition (DO) and egg fertility (DF), respectively, when compared to controls. In the subsequent challenge on day 105, DT, DW, DO and DF were reduced by 9.0%, 4.1%, 8.6%, and 24.2%, respectively, when compared to controls. The vaccine efficacy (E) was equal to 44.0% and 37.2% after the first and second challenges, respectively. The results showed a positive correlation between antibody titers for both total IgG and IgG1 and E in the second but not in the first tick challenge. These results suggested the possibility of developing a SUB-based vaccine for control of cattle tick infestations under Indian conditions.     

Cloning and expression of ecto 5'-nucleotidase from the cattle tick Boophilus microplus

Although 5′-nucleotidases are ubiquitous in higher vertebrates, the arthropod enzymes have been little studied. The cDNA sequence of the mature 5′-nucleotidase from the tick Boophilus microplus was therefore determined (Gen Bank accession number: U80634). The enzyme has 39–41% sequence identity with the vertebrate 5′-nucleotidases and contains binuclear metal ion binding sites. There are no significant introns within the coding region of the genomic sequence. Southern blot analysis indicates the presence of multiple related genes encoding 5′-nucleotidases. Recombinant tick 5′-nucleotidase was expressed in both Escherichia coli and in baculovirus-infected insect cells. The E. coli recombinant protein was truncated, inactive and produced in abundance. The enzyme was expressed in baculovirus-infected insect cells as a secreted, soluble, glycosylated and enzymatically active protein. This represents the first successful expression and characterization of enzymatically active recombinant 5′-nucleotidase from any organism. Supplementation of the culture medium with 25 μm zinc resulted in a twofold increase in the activity of the expressed protein. The enzyme was purified to homogeneity. It exists under non-denaturing conditions as a homodimer, with an apparent molecular mass of 135 kDa. The K_m for the hydrolysis of AMP was 0.37 μm and the k_cat= 11.5/s, in agreement with data for the native enzyme.     

Larval membrane antigens protect Hereford cattle against infestation with Boophilus microplus

Hereford cattle (Bos taurus) were immunized with antigens solubilized with Triton X-100 from larval membranes of the cattle tick (Boophilus microplus). Based on tick egg production compared to control cattle, vaccinated cattle were protected (78%) against challenge with 2 x 20,000 tick larvae. The soluble Triton X-100 extract of tick larval membranes was further purified by immunoaffinity chromatography, using immunoglobulin ligands (IgG1 and IgG2) from three immune steers, previously vaccinated with membrane antigens from the midgut of partly engorged adult female ticks. Cattle vaccinated with these purified antigens were protected in two separate experiments (80 and 89% respectively), against challenge with 2 x 20,000 larval ticks compared to control cattle. Whole larval membranes used as vaccines in cattle reduced the amount of eggs produced from ticks by 47% compared to control cattle, but this difference was not significant.     

Immunoglobulin G binding proteins in male Rhipicephalus appendiculatus ticks

Three abundant immunoglobulin G binding proteins (IGBPs) of29kDa (IGBP-MA), 25kDa (IGBP-MB), and 21 kDa (IGBP-MC) were isolated from partly fed male Rhipicephalus appendiculatus ticks using an agar-ose-IgG column. After separating the proteins by SDS-PA GE, each individual IGBP band was cutou tfrom the gel and used to raise antiserum in guinea pigs. Using immuno-blotting, each of the three IGBPs was shown to be antigenically distinct, and specific for feeding male R. appendiculatus ticks. They were detected in the salivary glands of male ticks that had been feeding in either the presence of absence of female ticks, and appeared to be glycoproteins. The smallest protein, IGBP-MC, bound to guinea pig IgG (on which species the ticks were fed) and also bound to human and bovine IgG, whereas IGBP-MA and possibly IGBP-MB only bound to guinea pig IgG. The function of IGBPs in tick salivary glands and their significance in male ticks are discussed.     

Effect of vaccination with a recombinant Bm86 antigen preparation on natural infestations of Boophilus microplus in grazing dairy and beef pure and cross-bred cattle in Brazil

Current methods for the control of the cattle tick Boophilus microplus infestations are not effective and the parasite remains a serious problem for the cattle industry in tropical and sub-tropical areas. Recent advances have introduced the possibility for the immunological control of the parasite through the use of recombinant vaccines. Recently, it was shown that the recombinant vaccine Gavac™ (Heber Biotec S.A.) is able to control B. microplus populations in artificially infected grazing dairy cattle in Cuba. To assay the effect of the vaccine on a different B. microplus strain and under different ecological conditions, we conducted a trial in Brazil on grazing dairy and beef pure and cross-bred cattle under natural infestation conditions. A farm in the northeast of the state of Sao Paulo was selected and two groups of animals per breed were included in the experiment and were maintained grazing on separate but similar pastures. For each breed, one group was vaccinated with the vaccine Gavac and the second group was not vaccinated and was employed as a control. In vaccinated cattle, during 36 weeks of experiment, the average infestation rate was maintained below 78 ticks per animal while average infestation peaks (mean ± S.E.) of 144 ± 44 ticks per animal (for dairy cross-bred cattle) and 195 ± 42 ticks per animal (for beef cross-bred cattle) were recorded in the control groups. Tick infestation rates showed statistical significant differences (p=0.04) between both experimental groups throughout the experiment. These results clearly showed, as in the Cuban study, that the vaccine controlled tick numbers in successive generations in the field.     

中华硬蜱和微小牛蜱不同发育阶段蛋白质、糖、脂的比较研究

本文应用盘状电泳方法对中华硬蜱和微小牛蜱不同发育阶段的蛋白质、糖、脂进行了比较研究。结果表明,两种不同属种硬蜱的成虫、幼虫和虫卵的蛋白质、糖、脂电泳图谱都有明显差别;两种硬蜱各自的不同发育阶段的蛋白质、糖、脂电泳图谱也具有明显差异。     

微小牛蜱Bm86基因的原核表达与表达条件的优化

根据微小牛蜱Bm86基因序列设计引物,在重组质粒pMD18-T-Bm86中克隆,并将其定向亚克隆入原核表达载体pGEX-4T-1,转化至大肠埃希菌BL21（DE3）株,用不同浓度异丙基-β-D-硫代半乳糖苷（IPTG）在不同时间进行诱导表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）检测结果表明,37 ℃条件下经1 mmol/L IPTG诱导8 h后,目的重组蛋白表达量最大,表达相对分子质量（Mr）约为94 000的包涵体蛋白,与预期大小一致,目的蛋白约占蛋白总量的29%。蛋白质印迹（Western blotting）分析表明,该重组蛋白可被兔抗微小牛蜱全蜱阳性血清所识别。     

室内饲养条件下三种硬蜱产卵和孵化特性的比较研究

目的:为了认识微小牛蜱和扇头蜱之间的关系。方法:在室内饲养条件下对微小牛蜱(Boophilusmi-croplus)、镰形扇头蜱指名亚种(Rhipicephalushaemaphysaloideshaemaphysaloides)和猛突血蜱(Haemaphysalismontgomeryi)的产卵和孵化特性进行了比较研究。结果:三种硬蜱在产卵期天数上差异不明显(p>0.05),而在孵化期上有极显著差异(p<0.01);微小牛蜱和镰形扇头蜱间除了吸血后体重(p<0.05)、产卵前期(p<0.01)、孵化期(p<0.01)外,其余参数均差异不明显(p>0.05);猛突血蜱与微小牛蜱间仅在产卵前期和产卵期天数上差异不显著(p>0.05),与镰形扇头蜱间仅在产卵期天数上差异不明显(p>0.05),其余各项参数间都有显著或极显著差异;猛突血蜱的生殖效率和孵化率均明显高于微小牛蜱和镰形扇头蜱(p<0.05)。此外,三种硬蜱不仅日产卵量曲线相似,而且产卵量与吸血后体重之间均存在着极显著的正相关关系(r>0.93,p<0.01)。结论:微小牛蜱和扇头蜱之间有较高的相似度。     

Effect of salivary gland extracts from the tick,Boophilus microplus,on leucocytes from Brahman and Hereford cattle

The effect of salivary gland extract (SGE) from Boophilus microplus on peripheral blood lymphocytes, neutrophils and monocytes from Brahman (Bos indicus) and Hereford (Bos taurus) cattle was investigated. SGE (8 micro g) significantly inhibited the proliferation response of lymphocytes to concanavalin A from both Brahman and Hereford cattle by 89% and 41%, respectively. The difference in inhibition between the two breeds was highly significant (P < 0.01), whilst at 1 micro g of SGE, significant inhibition of lymphocytes occurred only in Hereford cattle (34%). Flow cytometric analysis of monocytes and neutrophils showed that SGE (40 micro g) significantly reduced both the proportion of cells actively phagocytosing Escherichia coli labelled with fluorescein isothiocyanate (E. coli-FITC) and the uptake of E. coli-FITC in Brahman cattle. However, in Hereford cattle, a significant depression in uptake was only observed in neutrophils. The proportion of monocytes and neutrophils with oxidative activity was significantly suppressed in the presence of SGE in both breeds of cattle. These results indicate that peripheral blood leucocytes from different breeds of cattle respond differently to SGE.     

豫西地区莱姆病血清学及病原学研究

目的　了解豫西地区有关人群血清中是否存在抗莱姆病螺旋体抗体及可能存在的生物媒介。方法　采用间接或直接免疫荧光技术 ,对人血清相关抗体及蜱中肠带菌情况进行研究。结果　豫西地区人群中存在莱姆病自然感染 ,人群感染率为 13.5 3% ;蜱类调查显示 ,长角血蜱和微小牛蜱的中肠带菌阳性率为 2 6 .0 0 % ,这两种蜱可能为传播媒介 ;并从褐家鼠和黄胸鼠的血清中也检测到莱姆病抗体。结论　初步显示豫西地区存在莱姆病疫源地。