用随机扩增多态DNA(RAPD)技术研究不同地理株埃及伊蚊的分化

目的 用RAPD技术对实验室饲养的广东、海南、台湾和印尼Baro等4个不同地理株的8只雌蚊进行随机扩增多态DNA分析。方法 随机扩增多态性DNA(RAPD)技术。结果 选用20个随机引物进行扩增，有8个引物表现清晰的RAPD谱带并呈显著多态性。UPGMA法构建的分子系统树表明埃及伊蚊4个地理株之间存在着一定程度的遗传分化。结论 用RAPD方法可以区分不同地理株埃及伊蚊。     

Evaluation of surveillance devices for monitoring Aedes aegypti in an urban area of northeastern Peru

In this study, we assessed the efficacy of the American Biophysics Corporation Standard Professional (ABC-PRO) light trap, the Omni-Directional Fay-Prince trap (with and without CO2), and the Centers for Disease Control and Prevention Wilton trap as a means of evaluating populations of adult Aedes aegypti in an urban area of northeastern Peru. Efficacies of collections from each of the trap types were compared to backpack-aspirator collections and human-landing collections. Collections were conducted twice daily, 3 days per week, for 27 wk from July 2001 to July 2002. Backpack-aspirator collections yielded significantly more mosquitoes (1,764) than any of the other collecting methods with a mean of 21.80 mosquitoes collected per sampling period. This method was less specific for Ae. aegypti than were human-landing collections because only 28.3% of mosquitoes collected with backpack aspirators were Ae. aegypti. Human-landing collections yielded only 23% (554/2,411) of the total mosquitoes collected. However, more than 80% (445/554) of the mosquitoes collected by this method were Ae. aegypti. None of the trapping devices evaluated collected mosquitoes, specifically Ae. aegypti, as effectively as backpack-aspirator or human-landing collections. The ABC-PRO trap, which was the most effective device in collecting mosquitoes, particularly Ae. aegypti, collected less than 2% of the total mosquitoes (mean of 0.12 mosquitoes/sampling period), and less than 3% of total Ae. aegypti (mean of 0.11 Ae. aegypti/sampling period). We conclude that none of the trap devices evaluated in this study is an acceptable alternative to backpack-aspirator or human-landing collections for monitoring populations of adult Ae. aegypti in Peru.     

Genetic differentiation of Aedes aegypti mainland and island populations from southern Thailand

Isozymes of 5 wild-caught collections of Aedes aegypti were compared by using starch gel electrophoresis. All collections were collected from Surat-Thanee Province, southern Thailand, an area considered to be a hyperendemic zone for dengue virus. One collection was from Donsak Harbor, whereas the other 4 collections were from 4 districts of Samui Island. The percent polymorphic loci (24.2-33%) in the 4 island collections was lower than in the mainland collection (36.4%). This study revealed a large effective migration rate among all 5 collections. No fixed differences were detected. No significant differentiation was found among the 5 collections from Surat-Thanee Province.     

Molecular characterization of Streptococcus pyogenes isolates to investigate an outbreak of puerperal sepsis.

OBJECTIVE: To describe microbiological characteristics and epidemiologic features of an outbreak of postpartum endometritis. METHODS: Various markers were investigated in five patients and three throat carriage isolates of Streptococcus pyogenes obtained during an outbreak of endometritis occurring in a 13-week period. Molecular characterization included biotyping, T-serotyping, emm gene sequence and restriction, pulsed-field gel electrophoresis (PFGE), and random amplified polymorphic DNA (RAPD) analysis. RESULTS: Biotype, T-serotype, and genotypic data (emm analysis, PFGE, and RAPD analysis) revealed a close relationship among the isolates from three patients, suggesting that cross-contamination had occurred. These isolates were biotype 1, T type 28, and emm type 28. The isolates from one patient and one carrier differed from those of the index patient by minor variations of the emm amplicon restriction pattern, PFGE pattern, or RAPD pattern. The remaining isolates were phenotypically and genetically different. CONCLUSION: Identification of different isolates demonstrated that different strains may circulate simultaneously during a true outbreak and that the predominant strain might persist for several months.     

Laboratory and field assessment of some kairomone blends for host-seeking Aedes aegypti

Using laboratory Y-tube olfactometers, the attractiveness of lactic acid and 2 kairomone blends from the United States Department of Agriculture (USDA) and BioGents GmbH (BG) was assessed for attractiveness to Aedes aegypti. Four geographically disparate populations were assessed: North Queensland Australia (NQA), Florida USA, Minas Gerais Brazil (MGB), and Singapore. In descending order, populations were attracted to USDA, BG blends, and lactic acid. MGB was poorly attracted to lactic acid alone. The blends were less attractive than human odor. Proprietary blends were modified, and their attractiveness was assessed to find the optimum attractive mixture for NQA. Adding acetone to BG, and ammonia and caproic acid to USDA, improved attractiveness in the laboratory. Field attractiveness was assessed by coupling the blends with a newly developed BG-Sentinel Ae. aegypti trap. Trials were carried out using the BG blend, BG blend plus acetone, USDA blend, USDA blend plus ammonia and caproic acid, and a control trap with no kairomones. The traps were highly effective, with mean 24-h collections up to 11.15 Ae. aegypti per trap, and this species made up 91.7% of collections. However, the effectiveness of the unbaited control trap indicated that the BG-Sentinel has visual attractive properties for Ae. aegypti and that the kairomone lures added little to trap performance in NQA.     

Population genetic structure of Anopheles maculatus in Thailand

Anopheles (Cellia) maculatus Theobald is a major malaria vector in southern Thailand and peninsular Malaysia, and previous population genetic studies suggested that mountain ranges act as barriers to gene flow. In this study, we examine the genetic variance among 12 collections of natural populations in southern Thailand by analyzing 7 microsatellite loci. Based on analysis of molecular variance (AMOVA), three geographic populations of An. maculatus are suggested. The southern population exists in western Thailand north of 12 degrees north latitude. Mosquitoes to the south fall into two genetic populations: 1) the middle southern collections located on the west side of the Phuket mountain range between 8 degrees and 10 degrees north latitude, and 2) the southern collections located on the east of the Phuket mountain range located between approximately 6.5 degrees and 11.5 degrees north latitude. AMOVA revealed significant genetic differentiation between northern and middle southern and southern populations. The middle southern population was moderately differentiated from the southern population. Furthermore, gene flow was restricted between proximal collections located on different sides of the Phuket mountain range. Collections separated by 50 km exhibited restriction of gene flow when separated by geographic barriers, whereas greater gene flow was evident among collections 650 km apart but without geographic barriers.     

A method for genotoxicity detection using random amplified polymorphism DNA with Danio rerio

The increasing presence of genotoxic pollutants in the aquatic environment has led to the development of quick monitoring methods. We have applied the random amplified polymorphism DNA (RAPD) method to evaluate the toxic effects of genotoxic chemicals. In all 22 normal wide-type Danio rerio (zebrafish), 78 amplified bands were obtained after PCR using the primer set, in which the frequency of emergence above 60% is 21. There do exist four stable bands in the amplified product, which appear in all normal test samples. The zebrafish RAPD fingerprinting database is set up on this base. The RAPD pattern from zebrafish exposed to genotoxic chemicals such as cyclophosphamide in dilution series ranging from 0.1 to 10 mg/L and dimethoate in dilution series ranging from 1.0 to 100 mg/L displayed some changes in polymorphism band patterns including the lost of stable bands. There also exists a distinct distance between the band patterns of exposed zebrafish and the control samples via the cluster method. In addition to that, the result derived from numerical analysis is more sensitive to the result obtained from stable band analysis because it can reveal the distance between the band pattern of the zebrafish exposed to 0.05 mg/L cyclophosphamide and the control sample. These results proved the accuracy and the sensitivity of the two analyses of genotoxicity effect based on RAPD fingerprinting research.     

Congenital Chagas disease involves Trypanosoma cruzi sub-lineage IId in the northwestern province of Salta,Argentina

Trypanosoma cruzi is genetically classified into six discrete phylogenetic lineages on the basis of different genetic markers. Identifying lineages circulating among humans in different areas is essential to understand the molecular epidemiology of Chagas disease. In the present study, 18 T. cruzi isolates from congenitally infected newborns in the northwestern province of Salta-Argentina were studied by multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA (RAPD). All isolates were typed by MLEE and RAPD as belonging to T. cruzi IId. Analysis of minor variants of TcIId using probes hybridizing with hypervariable domains of kDNA minicircles, detected three variants with a similar distribution among the isolates. Our findings confirm the presence of T. cruzi IId among congenitally infected newborns in northwestern Argentina and support the assumption that human infection by T. cruzi in the Southern Cone countries of Latin America is due principally to T. cruzi II.     

人源和猪源猪链球菌的同源性研究

目的 进一步鉴定菌种;评价人源株与猪源株猪链球菌的同源性。方法 对分离自猪无菌部位、患者血液和脑脊液的７株猪链球菌Ⅱ型和标准猪链球菌Ⅱ型,用菌体脂肪酸分析和随机引物基因扩增技术进行菌种的表现型和基因型分类。对其结果进行聚类分析和主成分分析。结果 随机引物基因扩增技术分析提示,被检的７株猪链球菌Ⅱ型与标准株一致,均为猪链球菌Ⅱ型;人源株与猪源株同源;分离自病人血液和脑脊液的菌株同源。这些结果在菌体脂     

Genetic structure and phylogeography of Aedes aegypti, the dengue and yellow-fever mosquito vector in Bolivia

Between the 16th and 18th centuries, Aedes aegypti (Diptera: Culicidae), a mosquito native to Africa, invaded the Americas, where it was successively responsible for the emergence of yellow fever (YF) and dengue (DEN). The species was eradicated from numerous American countries in the mid-20th century, but re-invaded them in the 1970s and 1980s. Little is known about the precise identities of Ae. aegypti populations which successively thrived in South America, or their relation with the epidemiological changes in patterns of YF and DEN. We examined these questions in Bolivia, where Ae. aegypti, eradicated in 1943, re-appeared in the 1980s. We assessed the genetic variability and population genetics of Ae. aegypti samples in order to deduce their genetic structure and likely geographic origin. Using a 21-population set covering Bolivia, we analyzed the polymorphism at nine microsatellite loci and in two mitochondrial DNA regions (COI and ND4). Microsatellite markers revealed a significant genetic structure among geographic populations (F(ST)=0.0627, P<0.0001) in relation with the recent re-expansion of Ae. aegypti in Bolivia. Analysis of mtDNA sequences revealed the existence of two genetic lineages, one dominant lineage recovered throughout Bolivia, and the second restricted to rural localities in South Bolivia. Phylogenic analysis indicated that this minority lineage was related to West African Ae. aegypti specimens. In conclusion, our results suggested a temporal succession of Ae. aegypti populations in Bolivia, that potentially impacted the epidemiology of dengue and yellow fever.     

海南岛4种常见蜚蠊遗传多态性的初步研究

目的探讨海南岛常见蜚蠊遗传多态性，寻找蜚蠊分类的分子标记。方法采用随机扩增DNA多态性（RAPD）技术对美洲大蠊、澳洲大蠊、黑胸大蠊和褐斑大蠊进行随机扩增，分析DNA图谱、计算遗传距离并绘出亲缘关系聚类图。结果筛选出4条随机引物，扩增出122个条带，其中多态性条带占71．31％。根据RAPD图谱计算出4种蜚蠊间的遗传距离，以澳洲大蠊和黑胸大蠊间遗传距离最短，褐斑大蠊和美洲大蠊间距离最长。根据4种蜚蠊遗传距离构建出的系统聚类图中，黑胸大蠊先与澳洲大蠊聚成一簇，再与褐斑大蠊、美洲大蠊相聚类。结论蜚蠊基因组DNA遗传多态性丰富，RAPD技术能够反映出不蚓蜚蠊间的亲缘关系，并可准确进行蜚蠊分类。     

Vector competence of Aedes aegypti populations from Senegal for sylvatic and epidemic dengue 2 virus isolated in West Africa

Vector competence of Aedes aegypti populations from six locations in different bioclimatic zones and habitats of Senegal was assessed for sylvatic (ArD 140875) and epidemic (ArA 6894) dengue 2 virus (DENV-2) isolates. F1 generation mosquitoes were fed with a blood meal containing 10(6.5) and 1.6 x 10(7) tissue culture infectious dose (TCID50) of the sylvatic DENV-2 isolate or 10(6.5) TCID50 of the epidemic isolate and incubated for 14 days. After this period, legs and wings of each individual were pooled and the corresponding body was collected separately. An immunofluorescence assay was used to test bodies and wings/legs for DENV-2 infection separately. The results showed that: (i) Senegalese Ae. aegypti populations have lower vector competence (infection rate 0-26%, dissemination rate 10-100%) for sylvatic and epidemic dengue viruses than populations from Asia and America; (ii) there was no obvious geographic variation in susceptibility among mosquito populations; (iii) the only apparent factor affecting the susceptibility was the virus titre; and (iv) except for that of Kédougou, all populations were able to disseminate the virus. Overall our findings, while extended to four other populations, confirm previous studies showing low susceptibility of Senegalese Ae. aegypti populations.     

Genetic structure of natural populations of Aedes aegypti at the micro- and macrogeographic levels in Brazil

Genetic variation in 13 populations of Aedes aegypti from 3 regions of Brazil was compared using variation at 10 isozyme loci. Heterozygosities varied from 0.050 +/- 0.027 to 0.280 +/- 0.120, and a large genetic differentiation (F(ST) = 0.144) was observed among all populations. The largest within-regions differences were found between populations from the urban areas of northeast Brazil (F(ST) = 0.152). Ecological conditions are likely having an impact on the population structure of Ae. aegypti in the different regions of Brazil.     

Comparison of an improved RAPD fingerprinting with different typing methods for discriminating clinical isolates of Staphylococcus spp.

Different epidemiological markers were used to characterize 2 Staphylococcus epidermidis and 8 Staphylococcus aureus strains isolated from patients with severe infections. We compared random amplified polymorphic DNA (RAPD) fingerprints, biotypes, antibiotic assays, plasmid profiles and chromosomal DNA restriction endonuclease analysis (REA). Data analysis based on numerical taxonomy methods indicates that RAPD and REA give similar results allowing a good discrimination of the two species and of each isolate. The RAPD method is easier and faster than REA, but the reproducibility of RAPD fingerprints obtained in independent experiments can be problematic. We have found simple technical devices to improve the reproducibility of the RAPD procedure which is therefore a very useful tool in epidemiology for identification and characterization of Staphylococcus spp.     

Integration of genotoxicity and population genetic analyses in kangaroo rats (Dipodomys merriami) exposed to radionuclide contamination at the Nevada Test Site, USA

We examined effects of radionuclide exposure at two atomic blast sites on kangaroo rats (Dipodomys merriami) at the Nevada Test Site, Nevada, USA, using genotoxicity and population genetic analyses. We assessed chromosome damage by micronucleus and flow cytometric assays and genetic variation by randomly amplified polymorphic DNA (RAPD) and mitochondrial DNA (mtDNA) analyses. The RAPD analysis showed no population structure, but mtDNA exhibited differentiation among and within populations. Genotoxicity effects were not observed when all individuals were analyzed. However, individuals with mtDNA haplotypes unique to the contaminated sites had greater chromosomal damage than contaminated-site individuals with haplotypes shared with reference sites. When interpopulation comparisons used individuals with unique haplotypes, one contaminated site had greater levels of chromosome damage than one or both of the reference sites. We hypothesize that shared-haplotype individuals are potential migrants and that unique-haplotye individuals are potential long-term residents. A parsimony approach was used to estimate the minimum number of migration events necessary to explain the haplotype distributions on a phylogenetic tree. The observed predominance of migration events into the contaminated sites supported our migration hypothesis. We conclude the atomic blast sites are ecological sinks and that immigration masks the genotoxic effects of radiation on the resident populations.     

Aedes aegypti in Ho Chi Minh City (Viet Nam): susceptibility to dengue 2 virus and genetic differentiation

Aedes aegypti is the principal vector of dengue viruses, responsible for a viral infection that has become a major public health concern in Asia. In Viet Nam, dengue haemorrhagic fever was first detected in the 1960s and is now a leading cause of death in childhood. We studied the variability in competence of Ae. aegypti as a vector for dengue 2 virus and genetic differentiation in this mosquito species. Twenty mosquito samples collected in 1998 in Ho Chi Minh City were subjected to oral infection and isoenzyme polymorphism analysis by starch gel electrophoresis. Ae. aegypti populations from the centre of Ho Chi Minh City were genetically differentiated and their infection rates differed from those of populations from the commuter belt. These results have implications for insecticidal control during dengue outbreaks.     

Laboratory evaluation of susceptibility of natural subpopulations of Aedes aegypti larvae to temephos

Aedes aegypti showed the ability to develop resistance to different insecticides, including temephos, the most widely used larvicide. The objectives of this work were to 1) determine the resistance value of 4 natural subpopulations of Ae. aegypti, identified by their different haplotypes, to the insecticide temephos "Abate 1G (1%)"; 2) determine the lethal concentration (LC)50 and LC90 values by using the Rockefeller strain as control; and 3) estimate the resistance ratios. Mosquito samples were collected in Catamarca, Córdoba, and Posadas (Argentina) and in Yacuiba (Bolivia). Six insecticide concentrations were tested. The Rockefeller strain and the Posadas sample showed susceptibility to the diagnostic concentration (0.012 mg/liter), whereas the mortality in Catamarca was 87%. In the Yacuiba and Córdoba collections, mortality was 74% and 75%, respectively, indicating resistance. These results were coincident with those of the Probit analysis from which the highest resistance ratios were estimated for the last 2 subpopulations (5.2 and 4.9, respectively). Before this study, no information was available about the existence of resistance in natural populations of Ae. aegypti in the studied area.     

Molecular characterization of Vibrio cholerae O1 and non-O1 from human and environmental sources in Malaysia

A total of 31 strains of Vibrio cholerae O1 (10 from outbreak cases and 7 from surface water) and non-O1 (4 from clinical and 10 from surface water sources) isolated between 1993 and 1997 were examined with respect to presence of cholera enterotoxin (CT) gene by PCR-based assays, resistance to antibiotics, plasmid profiles and random amplified polymorphic DNA (RAPD) analysis. All were resistant to 9 or more of the 17 antibiotics tested. Identical antibiotic resistance patterns of the isolates may indicate that they share a common mode of developing antibiotic resistance. Furthermore, the multiple antibiotic resistance indexing showed that all strains tested originated from high risk contamination. Plasmid profile analysis by agarose gel electrophoresis showed the presence of small plasmids in 12 (7 non-O1 and 5 O1 serotypes) with sizes ranging 1.3-4.6 MDa. The CT gene was detected in all clinical isolates but was present in only 14 (6 O1 serotype and 8 non-O1 serotype) isolates from environmental waters. The genetic relatedness of the clinical and environmental Vibrio cholerae O1 and non-O1 strains was investigated by RAPD fingerprinting with four primers. The four primers generated polymorphisms in all 31 strains of Vibrio cholerae tested, producing bands ranging from < 250 to 4500 bp. The RAPD profiles revealed a wide variability and no correlation with the source of isolation. This study provides evidence that Vibrio cholerae O1 and non-O1 have significant public health implications.     

肺炎克雷伯菌随机DNA多态性分型研究

目的 建立肺炎克雷伯菌随机扩增DNA多态性分型技术,以应用于临床菌株流行病学调查。方法 对临床分离的29株肺炎克雷柏菌进行Etest检测,并用酚氯仿法提取其DNA后行RAPD分型。结果 29株临床分离菌有7株ESBL阳性,24株有稳定的RAPD带型,其中ICU分离的5株ESBL阳性株的4株RAPD指纹图谱相同。结论 RAPD基因分型技术不需已知核酸序列,分型率高,分辨力强,简便快捷可为肺炎克雷伯菌株提供分型标志,是分子流行病学研究的有效方法。