Production in ascites fluid of biosynthetically labelled monoclonal antibody to Theileria parva sporozoites

A hybridoma cell line has been previously produced which secretes monoclonal antibodies able to neutralize sporozoites of Theileria parva, the causative agent of East Coast fever of cattle. Cells from this line were injected intra-peritoneally into pristane-treated BALB/c mice. During the last 4 days of hybridoma cell growth, mice were given 4 daily intraperitoneal injections of a mixture of tritiated amino acids in order to biosynthetically radiolabel the monoclonal antibody being produced in ascites fluid. The specific activity of the antibody obtained was 100 mCi/mmol. The labelled antibody was used to detect, by autoradiography, a surface coat antigen of T. parva sporozoites in cryostat sections of Theileria-infected tick salivary glands. The method allows the preparation of large quantities of biosynthetically radiolabelled immunological probes for the detection of immunoreactive sites in biological specimens.     

Protective immunity induced by daily bites from irradiated mosquitoes infected with Plasmodium yoelii

Individuals in malaria endemic regions do not develop fully protective immune responses against Plasmodium liver stage infections. In high transmission areas, individuals can be exposed to more than two infective mosquito bites daily. Their exposure to Plasmodium sporozoites, therefore, is in the form of small and frequent doses. This is very different from individuals studied in controlled immunization trials where the delivery of large numbers of radiation-attenuated sporozoites in a limited number of doses can induce sterile protective immunity. Using irradiated mosquitoes infected with Plasmodium yoelii 17XNL, we tested whether daily bites from a few mosquitoes can induce a protective immune response in mice. This immunization strategy successfully induced a protective response, preventing the development of liver stages when mice were challenged with nonirradiated sporozoites. These results provide further support for the development of liver stage vaccines. They are also a call for further study into why fully protective responses against the liver stage are not seen in individuals from endemic regions.     

Monoclonal antibody neutralizes the sporozoite stage of different Theileria parva stocks

Summary Monoclonal antibodies were raised against sporozoites of Theileria parva. One of these antibodies (MAbDi) neutralized the infectivity of sporozoites for lymphocytes in vitro and for cattle in vivo. Neutralization seemed to occur by blocking sporozoite entry into the cell. MAbDi neutralized sporozoites of four unrelated stocks of T. parva , indicating the presence of a common antigenic determinant which may be important in initiating protective immunity.     

Humoral immune responses in Gambians to Pfs16, an immunodominant, Plasmodium falciparum integral membrane protein

Naturally acquired humoral immune responses to Pfs16, an integral membrane protein expressed in Plasmodium falciparum gametocytes and sporozoites, were investigated in The Gambia. A high prevalence of antibodies to this molecule was detected by peptide ELISA. Ninety-three per cent of the people taking part in a survey at the end of the rainy season (November) had serum antibodies to one or more synthetic peptides spanning the sequence; 88% reacted with one particular peptide sequence (IMLIILSGIVGFKVK) whereas only one out of ten non-Gambians (taking anti-malarial prophylaxis with no history of infection) reacted with the peptide. Epitope mapping with mouse MoAbs has shown that this peptide is located on a part of the molecule differing from immunodominant regions of the molecule identified in a previous study in Papua New Guinea.     

微小隐孢子虫cDNA文库的构建及P23、CP15/60基因的克隆

目的　微小隐孢子虫地方株cDNA文库构建及P2 3、CP15 60基因的克隆。　方法　提取微小隐孢子虫总RNA、mRNA ,逆转录合成cDNA。将cDNA与 pUC18DNA连接 ,导入DH5α宿主细胞中生成cDNA文库。根据文献分别设计并合成两对PCR引物 ,从上述文库中筛选保护性基因 ,对PCR产物克隆、测序。　结果　文库容量为 1.9× 10 6个重组子 ,文库中cDNA插入片段大小介于 0 .4× 10 3～ 6.5× 10 3bp。从该文库中克隆出编码 2 3kDa、15 60kDa子孢子表面蛋白的核苷酸序列。　结论　成功地用 pUC18质粒载体构建了C .parvumcDNA文库。     

基于子孢子阳性率和混合样本对疟疾进行早期预警时的临界感染率检验

目的:给出基于混合样本的最小检测次数法和序贯检验法,以检验子孢子阳性率是否超过导致疟疾流行的临界水平.方法:在控制两类错误概率的前提下,推导两种方法的期望检测次数和检验功效的数学表达式.对最大期望检测次数进行数学优化,求解最优混合样本的大小,并通过计算机模拟对有关结果进行验证.结果:给出了最小检测次数法和序贯检验法的最优混合样本计算方法、MATLAB程序和具体操作步骤.前者可用于日常监测,后者可用于早期预警.结论:利用本文给出的最优混合样本和检测步骤,在获得比较满意的检验功效(两类错误概率均低于5%)的同时,可以有效降低检测次数.     

Identification and molecular characterization of a novel protein Saglin as a target of monoclonal antibodies affecting salivary gland infectivity of Plasmodium sporozoites

Molecular mechanisms underlying the interaction between malarial sporozoites and putative receptor(s) on the salivary glands of Anopheles gambiae remain largely unknown. In previous studies, a salivary gland protein of ~100 kDa was identified as a putative target based on recognition of the protein by a monoclonal antibody (mAb) 2A3 that caused a >/= 70% reduction in the average number of sporozoites per infected salivary gland when fed to mosquitoes. Using affinity purification we purified the target of this mAb from extracts of female A. gambiae salivary glands and it was found to be a novel protein by tandem mass spectrometric analysis. Biochemical and molecular characterization of the 100 kDa protein showed that this molecule, designated Saglin, exists as a disulphide-bonded homodimer of 50 kDa subunits. The ability to form homodimers was retained even in the recombinant Saglin expressed in mammalian cells (HEK293). The amino acid sequence of Saglin contains a signal peptide suggesting that Saglin is a secreted protein. If Saglin is indeed involved in the process of invasion of A. gambiae salivary glands by sporozoites of Plasmodium, it could provide a novel target for future investigations aimed at interruption of malaria transmission.     

枯否氏细胞、肝细胞对疟原虫子孢子感染和发育的影响

１次接种９００万约氏疟原虫子孢子悬液，３０ｍｉｎ后的肝脏超微结构显示，枯否氏细胞吞噬复合体，邻近肝细胞线粒体空泡化，嵴突消失；枯否氏细胞内可见形态退变的子孢子；线粒体、内质网、核糖体丰富的肝细胞内子孢子结构清晰；线粒体空泡化、嵴突消失的肝细胞内子孢子退变；子孢子进入枯否氏细胞或肝细胞的时间不一，１个肝细胞内可见２个子孢子。     

环丙沙星对疟原虫子孢子活力的影响

约氏疟原虫子孢子与新型抗生素环丙沙星不同剂量共同孵育３０ｍｉｎ后腹腔接种昆明株小鼠体内．观察环丙沙星对子孢子活力的影响。环丙沙星分为１、２、４、６、μｇ／ｍｌ５种剂量，另外设双抗（青霉素十链霉素）对照组和ＰＢＳ对照组，每组接种５只小鼠，每鼠０．１ｍｌ，内含１０．５×１０４子孢子，３ｄ后尾静脉取血镜检，结果环丙沙星各试验药物组原虫血症出现的平均时间分别为３．８、３．６、３．８、３．８、３．２ｄ，双抗对照组和ＰＢＳ对照组均为３．８ｄ。结果提示环丙沙星剂量达８μｇ／ｍｌ与子孢子孵育３０ｍｉｎ后仍对子孢子活力无影响。     

Seasonal density, sporozoite rates and entomological inoculation rates of Anopheles gambiae and Anopheles funestus in a high-altitude sugarcane growing zone in western kenya

Summary An entomological study was conducted on vectors of malaria and their relative contribution to Plasmodium falciparum transmission in Mumias, a high-altitude site and large-scale sugarcane growing zone in Kakamega district, western Kenya. Anopheles gambiae s. l ., the predominant vector species, represented 84% ( n = 2667) of the total Anopheles mosquitoes collected with An. funestus comprising only 16%. Polymerase chain reaction (PCR) identified all 600 specimens of the An. gambiae complex tested as An. gambiae sensu stricto , an indication that it is the only sibling species represented in the high-altitude sites in western Kenya. Plasmodium falciparum sporozoite rates of 6.3% (133/2118) for An. gambiae s.l . and 9.5% (38/402) for An. funestus by ELISA were obtained in Mumias. None of 1600 mosquitoes tested for P. malariae sporozoites was positive. ELISA tests of mosquito blood meals indicated a high tendency of anthropophagy, a behaviour contributing significantly to malaria transmission by the vector species, with 95.9%, 4.86% and 0.2% having taken at least one blood meal on human, bovine and avian hosts, respectively.  Malaria transmission intensity was low as revealed by the low entomological inoculation rates (EIR) recorded. The EIR values for An. gambiae s.l . were 29.2 infective bites per person per year (ib/p/year) and 17.5 ib/p/year for An. funestus in Mumias. The highest inoculation rate for both vector species was 7.0 ib/p/month in July. Plasmodium falciparum parasite rate among asymptomatic children was 55.4% and 44% in the wet (July–September) and dry (December–February) seasons, respectively. These results indicate that malaria transmission intensity in the high-altitude site is low but perennial, with transmission being maintained by An. gambiae s.s . and An. funestus .