应用DNA探针检测蚊体内丝虫幼虫的研究

本文用人工合成的寡核苷酸探针，经斑点杂交法检测蚊体内马来丝虫幼虫。可测到丝虫和微丝蚴２ｎｇ的ＤＮＡ量，不与其它动物丝虫标本发生交叉反应。将单个蚊直接压于硝酸纤维素膜上进行检测，感染蚊中含有１条感染期幼虫不可出现了性反应。将一组蚊虫在裂液中研磨集体检测时，在２０只蚊虫中有１只感染蚊即可检出。显示该探针可用于马来丝虫地区的蚊媒监测。     

马来丝虫非放射性寡核苷酸探针的制备及现场应用研究

目的 寻找一种高度特异，敏感的马来丝虫的检测方法，并将其用于现场检测。方法 根据马来丝虫HhaⅠ重复序列，合成马来丝虫特异性引物1对和寡核苷酸探针1条，采用地高辛加尾标记试剂盒对人工合成寡核苷酸探针进行非放射性标记，并对标记后的探针进行标记效率检测，将血液标本和蚊虫标本分别用蛋白酶K等处理液和Chelex-100处理后，结合PCR技术，对该探针的特异性和敏感展出一进行检测；并将此探针用于平远县马来丝虫病的监测。结果 经测定，标记后探针的有效浓度为1.5-2fmol/μl；结合PCR技术，该探针可检出100μl阳性血样中1条微丝蚴；50只蚊中含有1只只感染1条幼丝虫阳性蚊亦能准确检出，班氏丝虫等其它寄生虫标本的检测结果均为阴性；采自平远县的240份血液标本和1008只中华按蚊标本均为阴性。结论 该探针杂交检测方法特异性强，敏感性高，可作为一种新的马来丝虫病监测方法在现场中使用。     

马来丝虫非放射性寡核苷酸探针的制备及现场应用研究

目的　寻找一种高度特异、敏感的马来丝虫的检测方法 ,并将其用于现场检测。　方法　根据马来丝虫 Hha 重复序列 ,合成马来丝虫特异性引物 1对和寡核苷酸探针 1条 ,采用地高辛加尾标记试剂盒对人工合成寡核苷酸探针进行非放射性标记 ,并对标记后的探针进行标记效率检测 ;将血液标本和蚊虫标本分别用蛋白酶 K等处理液和 Chelex- 10 0处理后 ,结合 PCR技术 ,对该探针的特异性和敏感性进行检测 ;并将此探针用于平远县马来丝虫病的监测。　结果　经测定 ,标记后探针的有效浓度为 1.5～ 2 fmol/μl;结合 PCR技术 ,该探针可检出 10 0 μl阳性血样中 1条微丝蚴 ;5 0只蚊中含有 1只只感染 1条幼丝虫阳性蚊亦能准确检出。班氏丝虫等其它寄生虫标本的检测结果均为阴性 ;采自平远县的 2 4 0份血液标本和 10 0 8只中华按蚊标本均为阴性。　结论　该探针杂交检测方法特异性强 ,敏感性高 ,可作为一种新的马来丝虫病监测方法在现场中使用。     

PCR及PCR-ELISA法检测蚊体内马来丝虫幼虫

目的：建立一种检测蚊体内马来丝虫幼虫的灵敏、快速、特异的方法。方法：选择应用PCR及PCR－ELISA法检测马来丝虫幼虫DNA的最佳反应条件，并在该条件下分别以马来丝虫Ⅰ期、Ⅱ期、Ⅲ期幼虫各1条作模板，测定检测的灵敏度及检测实验室人工感染中华按蚊体内的马来丝虫幼虫。结果：PCR及PCR－ELISA法均能检测出1条Ⅰ期幼虫（L1），而PCR的检测下限为1／10条L1，PCR－ELISA检测下限为1／100条L1；将分离的感染期幼虫加阴性蚊媒进行粗提、扩增及电泳，结果未见明显的扩增条带，扩增产物作ELISA检测，全部为阴 性；个体解剖人工感染的中华按蚊120只，分别收集113只阳性蚊体内的幼丝虫，用两种方法检测，结果均为阳性。结论：初步建立了PCR及PCR－ELISA法检测蚊体内马来丝虫幼虫的方法。     

应用单克隆抗体检测蚊体内幼丝虫的实验观察

以马来丝虫感染期幼虫为靶抗原制备多株单克隆抗体（ＭｃＡｂ）从中筛选出１Ｇ１、１Ｈ１单克隆抗体，检测人工感染马来丝虫的中华按蚊。当中华按蚊吸食含马来微丝蚴的兔血后，分别解剖，同时以ＥＬＩＳＡ及Ｄｏｔ－ＥＬＩＳＡ检测饲养不同时间的中华按蚊。结果显示，饲养９ｄ的按蚊马来丝虫幼虫阳性率，人工解剖为８６．６％，ＥＬＩＳＡ和Ｄｏｔ－ＥＬＩＳＡ法分别为８２．２％和７７．８％。经统计学处理，人工解剖与单抗检测无显著性差异，其灵敏度为每只蚊０．５条幼虫。实验证明，此ＭｃＡｂ与牛腹腔指状丝虫、猪肺丝虫抗原无交叉反应，显示了一定的特异性。     

聚合酶链反应检测蚊体内马来丝虫幼虫的实验

目的　建立一种特异、灵敏和简捷的聚合酶链反应 (PCR)检测蚊体内马来丝虫幼虫的方法。方法　在应用一种新的模板纯化处理技术 (Microcon 10 0 )基础上 ,采用适应于检测我国马来丝虫的两套DNA扩增引物(P1、P2与P3、P4) ,对实验感染马来丝虫的中华按蚊进行扩增检测。结果　两套引物均可检出蚊体内不同发育期幼丝虫 (L1、L2 和L3) ,其灵敏度达 1只蚊体内 1/ 6 4条L1和 2 0 0只群体蚊中含有 1只感染蚊 (体内有 1条L3)的水平 ,而对犬恶丝虫及未感染蚊却不能扩增出特异条带。结论　初步建立特异、灵敏和简捷的PCR检测蚊体内马来丝虫幼虫的方法。     

应用单克隆抗体检测蚊体内幼丝虫的实验观察

以马来丝虫感染期幼虫为靶抗原制备多株单克隆抗体从中筛选出１Ｇ１，１Ｈ１单克隆抗体，检测人工感染马来丝虫的中华按蚊。当中华按蚊吸食马来微丝蚴的兔血后，分别解剖同时以ＥＬＩＳＡ及Ｄｏｔ－ＥＬＩＳＡ检测饲养不同时间的中华按蚊。结果显示，饲养９ｄ的按蚊马来丝虫幼虫阳性率，人工解剖为８６．６％，ＥＬＩＳＡ和Ｄｏｔ－ＥＬＳＩＡ法分别为８２．２％和７７．８％。     

嗜人按蚊自然感染班氏丝虫的调查

安徽省舒城县芦镇乡为班氏、马来丝虫病混合流行区，班氏丝虫病占９８．９０％。自然界捕获并解剖嗜人按蚊５５９只，幼丝虫感染率为６．４４％，密度为１～２９条／蚊；感染期幼丝虫率为１．０７％，密度为１～５条／蚊。均为班氏丝虫幼虫。因而认为嗜人按蚊是安徽省班氏丝虫病传播媒介之一。     

人房内中华按蚊,嗜人按蚊自然感染牛腹腔丝虫的纵向观察

中华接蚊、嗜人按蚊均可感染牛腹腔丝虫和马来丝虫。在马来丝虫病流行区进行蚊媒调查，特别是在马来丝虫病防治后期的蚊媒监测中，蚊体内人、畜丝虫幼虫的鉴别尤为重要[1]。为了排除牛腹腔丝虫幼虫对马来丝虫病防治后期蚊媒监测的干扰，1987～1992年，1995～1996年，我们选定已无微丝蚴血症者的蔡金镇两个原马来丝虫病流行村，总人口1963人为观察区、先后共8年观察人房内中华接蚊和嗜人按蚊自然感染牛腹腔丝虫的情况。材料与方法1．牛腹腔丝虫自然传染原调查：上午抽采观察区村民饲养的水牛耳背静脉血各6大滴（约120μl），涂制成1．5×3．0…     

嗜人按蚊自然感洒斑氏丝虫的调查

安徽省舒城县芦镇乡为斑氏，马来丝虫病混合流行区，斑氏丝虫病占９８．９０％。自然界捕获为并解剖嗜人按蚊５５９只，幼丝虫感染率为６．４４％，密度为１－２９条／蚊；感洒期幼丝虫率为１．０７％，密度为１－５条／蚊。均为斑氏丝虫幼虫。因而认为嗜人按蚊是安徽省班氏丝虫病传播媒介之一。     

Persistence of Brugia malayi DNA in vector and non-vector mosquitoes: implications for xenomonitoring and transmission monitoring of lymphatic filariasis

Xenomonitoring (detection of filarial larvae or their DNA in mosquitoes) is a sensitive marker for assessing the endemicity of filariasis and a useful tool for evaluating elimination programs. To examine the fate of microfilariae (mf) and filarial DNA in vector competent and non-competent mosquito strains, we compared the detection of Brugia malayi parasites by dissection and by quantitative real-time polymerase chain reaction (PCR) in three different mosquito strains. We conclude that PCR is much more sensitive than dissection for detecting filarial larvae, especially their remnants in mosquitoes. However, parasite DNA can be detected in both vector and non-vector mosquitoes for two weeks or longer after they ingest mf-positive blood. Thus, although xenomonitoring with vector and non-vector mosquito species may be a sensitive method for indirectly detecting filarial parasites in human populations, positive test results for parasite DNA in mosquitoes do not necessarily prove that transmission is ongoing in the study area.     

Monoclonal antibody to a unique surface epitope of the human filaria Brugia malayi identifies infective larvae in mosquito vectors.

We describe properties of an IgM monoclonal antibody (NEB-D1E5) raised against the human filarial parasite Brugia malayi. The antibody reacts with a stage- and species-specific determinant located on the surface of the infective-stage larva, as determined by indirect immunofluorescence. To use this reagent in epidemiological field studies, we developed an enzyme-linked immunoassay with which B. malayi larvae can be differentiated from other filarial parasites in mosquito vectors, including the morphologically indistinguishable parasite of animals Brugia pahangi. The immunoenzyme assay was 91-94% specific and 90-97% sensitive when performed on infected mosquitoes. In the absence of mosquito tissue, the levels of specificity and sensitivity increased to 100% and 97.5-100%, respectively. Binding of antibody to the surface of living larvae was abrogated by treatment of the worms with the enzymes pronase and proteinase K and with the detergents Triton X-100, octyl beta-D-glucopyranoside, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS). In contrast, treatment with trypsin, endoglycosidase-F, O-Glycanase, N-Glycanase, lipase, various phospholipases, boiling, 2-mercaptoethanol at 37 degrees C, or periodate did not reduce the antigenicity of the larval surface to antibody NEB-D1E5. These results suggest that the species-specific epitope is a peptide domain attached to a hydrophobic anchoring residue.     

A rapid and simplified method of DNA extraction for the detection of Brugia malayi infection in mosquitoes by PCR assay.

Currently used protocols for the extraction of filarial parasite DNA from mosquito samples are tedious and involve extensive use of expensive and hazardous chemicals. Therefore, in order to arrive at a simple procedure, four different methods (A, B, C and D) were tried for the extraction of DNA from mosquitoes infected with filarial parasite, Brugia malayi. Method D was found to be as efficient as the current procedure for the extraction of DNA from a single microfilaria in pools of 25 mosquitoes and the DNA was suitable for polymerase chain reaction (PCR) amplification, yielding a band of 322 base pairs with primers specific for B. malayi. Method D involved drying and crushing the mosquitoes to a powder, which was homogenized in 100 microl TE buffer, vortexed, boiled for 10 min, centrifuged at 14000 r.p.m. for 10 min, and the supernatant used for the PCR assay. Dot-blot hybridization confirmed the specificity of the PCR amplified fragment. The DNA extracted by this method was stable for about 1 year. When comparing with the standard method, the cost of a single PCR reaction, inclusive of DNA extraction, was reduced by 50% and the hands on time was minimized fivefold. Hence, this simple TE-based method is rapid, safe and also cost-effective in assessing the B. malayi infection in pools of vector mosquitoes.     

Preliminary observations on the development of larval filariae in Toxorhynchites species

Brugia malayi and B. Pahangi microfilariae from gerbil intraperitoneal infections were inoculated into the thorax of male and female Toxorhynchites amboinesis and developed into third-stage larvae as early as 11 days. In a comparative study with Aedes togoi fed on microfilaremic gerbils, third-stage larvae were found at 10 days. Some third-stage larvae of B. malayi inoculated into gerbils developed to advanced stages. Third-stage larvae of Wuchereria bancrofti were recovered in low numbers from Tx. amboinesis and Tx. aurifluus inoculated with microfilariae recovered from human blood by membrane filtration. Development of all filarial species was similar in both male and female mosquitoes. Toxorhynchites species are plant feeders and therefore reduce the hazards of laboratory transmission of pathogenic agents. Because of their large size, manipulations with this mosquito species are easy and the size allows for a larger inoculum to be used. This group of mosquitoes should develop into useful laboratory vectors for the transmission of arthropod-borne diseases.     

不同日龄致倦库蚊对马来丝虫易感性的比较研究

不同日龄致倦库蚊分别经胸内接种马来丝虫微丝蚴后,比较观察了蚊虫体内微丝蚴的黑化率、(?)虫率以及感染性蚊虫的比率。接种后第3、4、5、9d解剖接种蚊虫,结果表明,1～2日龄蚊虫体内微丝蚴黑化率显著高于15～16日龄蚊虫,分别为84.1％和57.5％。三期幼虫率则相反,分别为1.5％和3.7％。黑化的微丝蚴主要分布在蚊虫腹部,占72.3％和69.1％。     

马来丝虫微丝蚴定量注射感染中华按蚊和致倦库蚊的...

With an improved technique, quantitative inoculation of Brugia malayi microfilariae (mff) into Anopheles sinensis and Culex quinquefasciatus was carried out in our laboratory in 1988. The development of B. malayi mff in these two species of mosquitoes was observed. In the system of B. malayi and An. sinensis, a well-fitted linear correlation appeared between the number of mff inoculated and the infective larva (L3) positive rate (r = 0.9910; P less than 0.01). The regression equation with the regression coefficient of 8.490 suggested a high susceptibility of this species of mosquito to B. malayi even under the condition of experimental inoculation. The quantitative relationship between the number of mff inoculated and the average filarial maturity rate was also exhibited. At the same dosages of 4 and 10 mff/mosquito, the L3 positive rates and the average filarial maturity rates in An. sinensis were much higher than those in Cx. quinquefasciatus (P less than 0.01), indicating significant difference occurred inside the bodies of these two species of mosquitoes.     

The effect of p-aminobenzoic acid and folic acid on the development of infective larvae of Brugia malayi in Aedes aegypti

Adult Aedes aegypti mosquitoes, infected with the subperiodic Brugia malayi, were found to enhance the development of the filarial parasites to the infective stage when they were exposed to a cotton pad soaked in 10% sucrose solution containing p-aminobenzoic acid (PABA) in 0.001, 0.005, 0.01, 0.05 and 0.1% concentrations. Similarly, larval development increased when the mosquitoes were fed with folic acid at 0.001, 0.01 and 0.1% concentrations. This stimulation was more when PABA or folic acid was given prior to the infected blood meal through the developmental period of the larvae. The data thus suggest that PABA and folic acid are nutrients for the development of B. malayi-microfilariae to the infective stage in A. aegypti.     

马来丝虫微丝蚴定量注射感染中华按蚊和致倦库蚊的实验观察

本文报告采用改进的微量注射技术和较为精细的计数方法,以2—10条微丝蚴/蚊,定量注射感染中华按蚊和致倦库蚊,找出了这两种蚊虫的微丝蚴感染量与感染期幼丝虫阳性率、幼丝虫平均发育成熟率之间的定量关系;比较了在注射感染条件下两种蚊虫对马来丝虫易感性的差异。