| Mosquito-borne diseases are transmitted by mosquitoes, which include malaria, lymphatic filariasis, yellow fever, Dengue fever, Japanese encephalitis, West Nile fever. etc. The epidemics of mosquito-borne diseases could raise important public health problem. Owing to the global climate warming, accelerating of urbanization, rapid development of tourism and international trading, and the deterioration of enviromnent, the incidence of mosquito-borne disease rises and the affected regions expand.With the changing pattern of the vector-borne diseases, it is pressed that much sensitive, specific and high throughput detecting methods been developed to fit the need of Disease Surveillance . Detecting of pathogens-carring mosquito are an earlier detection and surveillance approach in forecasting the epidemics of vector-borne diseases, which is crucial for early warning and effective controlling of mosquito-borne diseases. Most studies in surveillance of mosquito-borne diseases are targeted to detect single specific pathogen, and some of methods are not satisfactory due to a lower sensitivity. It is imperative to develop multiple-pathogens detecting methods in surveillance the mosquito-borne diseases.This study is designed to develop high throughput, multiplex detection hemi-nested PCR methods for screening the field caught mosquitoes. The method includes divide the mosquitoes in pools, and processed the samples in two paralleled vials. One was retro-transcripted for detecting flavivirus. The other one was used for PCR amplifying to detect Plasmodium spp. and filarial worms.For detecting the flavivirus, the nonstructural proteins gene, NS5 gene was selected as the amplification target. The primers were designed in reference of more than 30 viruses in Japanese Encephalitis Virus Complex found in GenBank , including Japanese encephalitis virus(JEV), Dengue virus( DenV), Yellow fever virus,( YFV), West Nile viruss (WNV), etc. which are endemic in China, or is regarded as the potential imported pathogens.To optimize the reaction conditions, we used cDNA of JEV, DenV I～IV types as PCR templates. The RT-hemi-nested PCR method was established for the detection of Japanese encephalitis virus Complex. The sensitivity of the method was then further evaluated with attenuated vaccine strain of Japanese encephalitis virus (SA14-14-2 strains). The threshold concentration for detecting the virus was 5.0×10-4 PFU/ml, ten-fold sensitive then the similar methods.A multiplex hemi-nested PCR method was established to amplify the SSU rRNA gene of Plasmodium spp.,( including P. falciparum, P. vivax, P. malaria, P. knowlesi, etc.), and the mitochondria genes sequence of lymphatic filarial worms reported in GenBank (including Brugia malayi, Dirofilariasis immitis, etc.). We designed 6 universal primers for multiplex hemi-nested PCR. And the genomic DNA from P. falciparum, P. vivax, B. malayi were used for optimizing the reaction conditions. The method was evaluated by detecting P. yoelii sporozoites containing Anopheles dirus. The sensitivity of the method was also evaluated with the genomic DNA from P. falciparum and B. malayi. The minimum amout of DNA from P. falciparum that can be detected by the method was equivalent to be 0.53 parasite. And for B. malayi, the amount was equivalent to be 0.01 parasite. The method could clearly detect P. yoelii in An. dirus.The two methods we established were further evaluated in detection field-collected mosquites. The mosquitoes were trapped from several collecting spots surrounding the Puer cities of Yunnan province, All samples were species identified and allocated into pools, Each mosquito pool contained 10 or less mosquitoes depends on the numbers of each sampling. We used the RT-hemi-nested PCR method to detect 54 pools of Culex tritaeniorhynchus. 14 mosquito pools had positive amplification. Sequencing of the amplicons revealed 9 pools of JEV, 3 pools of DenV type II, 1 pool of DenV type I, and 1 pool of unknown virus (the closest match was Quang Binh virus with 82% similarity). The minimum infection rates for the above viruses are 16.7‰, 5.55‰, 1.85‰, 1.85‰, respectively. 148 pools of mosquitoes was detected for Plasmodium spp., and filarial worms, no positive amplification was found.The two hemi-nested PCR methods we established are well meeting the need in surveillance of mosquito-borne diseases. The methods show superior in detecting mosquito-borne pathogens simultaneously and high throughputly. Monitoring pathogens-infected mosquitoes is an earlier detection and surveillance approach in forecast the transmitting potential of mosquito-borne diseases. The methods are simple, effective, relative rapid for detection, and is superior over virus culture method in its high-throughput detection. On detecting the field caught mosquitoes, we found JEV, DenV type II and I, unknown virus in mosquitoes collected from Puer, Yunnan province. Health workers in each CDC level should pay more attention to the results, and proceed further surveillance in this region and other similar regions. Meanwhile, the data obtained with the utilizing the methods could also provide experimental documents for mosquitoes surveillance, for diagnosis and monitoring of emerging imported mosquitoes-borne diseases. With the incorporation of the GIS in surveillance and forecasting of mosquito-borne diseases, it is no doubts that the methods we established could provide more reasonable and more effective data. |
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