Journal of the American Mosquito Control Association, ll(2):162-166, 1995Copyright @ 1995 by the American Mosquito Control Association, Inc.

IDENTIFICATION OF MALARIA-INFECTED DRIEDMOSQUITOES BY BIOTINI-YLATED DNA PROBE

LIU KE-YI,' CHEN XI-XIN, HUANG BING-CHENG AND CHENG YI-LIANG

Shandong Institute ofParasitic Diseases, Jining 272133, Shandong, People's Republic ofChina

ABSTRACT. Dot-blot hybridization with cloned genomic DNA labeled with photobiotin as a probeis used to detect Plasmodium falciparum sporozoites in dried mosquitoes. The assay is sensitive enoughto detect 2 pg Plasmodium parasite DNA, or one infected mosquito in a pool of 40 insects using DNAextraction samples, or one infected mosquito in a pool of 25 insects without DNA extraction. A singlemosquito squashed directly on a nitrocellulose filter for the determination of parasites provides a simplemethod for the detection of sporozoites. The mosquitoes triturated in reduction buffer were efficient forthe screening of malaria infection of mosquitoes in a large number of samples. The species specificity,sensitivity, and ease of performance of this assay as well as the stability of the reagents may make it auseful epidemiological rool.

INTRODUCTION

Malaria transmission to humans occurs whenan Anopheles mosquito infected with sporozoitesof one of the 4 species of human Plasmodium(P. vivax, P.falciparum, P. malariae, and P. ovale)takes a blood meal from a human. Determina-tion of sporozoites is an important componentof epidemiological investigations of malaria.However, the traditional method of dissectionof the salivary glands of individual Anophelesmosquitoes to search for sporozoites using a lightmicroscope is labor intensive and time consum-ing because in many endemic areas, the vectorshave very low rates ofsporozoite infection. Thus,a great number of mosquitoes must be examined.Also, the dissection method cannot determinewhich species of malaria parasites is present inthe mosquitoes because P/a s modium sporozoitesofboth human malaria species and other animalparasite species are difficult to morphologicallydistinguish from each other. Enzyme immuno-assays have been developed to circumvent theseproblems (Wirtz et al. 1985, 1992; Beach et al.1992; Somboon et al. 1993). However, these as-says do have some limitations; for example, mi-crotiter plate assays may require special equip-ment to perform and read the data, the samplesneed special treatment to reduce the background,and false negatives are difficult to avoid due tothe variabilities of the antigens of Plasmodiumparasites.

Because the genetic material DNA has thecharacteristic of species specificity, and DNA hy-bridization is not affected by whether the nucle-otide state is active or inactive, DNA hybridiza-tion technology has been used extensively for thedetection of malarial parasites from human blood

I Present address: Instituto de Biotecnologia, Univ-ersidad Nacional Autonoma de Mexico, Apdo. Postal5 I 0-3. Cuernavaca 6227 l, Morelos. Mexico.

(Franzen et al. 1984, Relf et al. I 990). We reporthere a method using dot-blot hybridization withcloned genomic DNA labeled with photobiotinas a probe to detect P. falciparum sporozoitesfrom dried mosquitoes. The results indicate thatthis method may be of value in monitoring ma-laria control programs and in investigating trans-mission of Plasmodium.

MATERIALS AND METHODS

Preparation of DNA probe: Plasmodium fal-ciparum (Fcc,/HN strain) parasites were ob-tained through in vitro culture and purified bylysis with saponin. Genomic DNA of the para-sites was isolated, subjected to quantitative di-gestion with the restriction endonuclease IlindIII,and ligated to HindIlI-digested plasmid pBR322to construct a genomic DNA library. Chimericplasmids were used to transform Escherichia colistrain HBl0l (Maniatis et al. 1982, Liu et al.I 993). The library was screened for colonies con-taining highly repeated sequences of P. falcipa-rum DNA. This was performed by in situ colonyhybridization using a probe oftotal genomic DNAof P. falcip arum radiolabeled with ( a-32P)dATPby nick translation as described by Rigby et al.(1977). Colonies with strong hybridization sig-nals were selected. The recombinant DNA wasisolated from selected clones by the alkalineminiprep method (Maniatis et al. 1982), and dis-solved in distilled water.

Recombinant plasmid DNA solution (10 pgDNA) was mixed in subdued light with 2 vol-umes of photobiotin acetate (Yap et al. 1988)(product of Company of Chinese Academy ofMilitary Medicine and Science, Beijing), and ir-radiated for 20 min on ice (tungsten filamentreflector lamp LYQI2-100, at a distance of l0cm). The labeled DNA was extracted twice withequal volumes ofbutanol after adding TE buffer(10 mM Tris-HCl, pH 9.0 and I mM EDTA) to

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500 pl, and recovered by precipitation with 2.5volumes of ethanol and resuspended in steriledistilled water. The sensitivity ofthe labeled DNAwas evaluated by direct incubation ofthe probewith avidin-alkaline phosphatase conjugate, andsubstrates of 5-bromo-4-chloro-3-indolyl phos-phate (BCIP) and nitro blue tetrazolium (NBT).

Preparation of mosquito samples: Plasmodi-um falciparun sporozoite-infected mosquitoeswere obtained by membrane feeding (Collins etal. 1986) Anopheles dirus Peyton and Harrisonmosquitoes with blood from gametocyte-carry-ing humans in Hainan Province of China. I-evelsof P. falciparum illrfection of mosquitoes wereassessed by examining the midguts of represen-tative samples of mosquitoes from different lotsfor oocysts. Then, lots of infected mosquitoeswere kept for a time sufficient to allow the par-asites to complete sporogonic development (as-sessed by looking for sporozoites in the salivaryglands). Subsequently, the mosquitoes were col-lected and kept in bottles without any reagents,and stored at room temperature for use. For thecontrol, some of mosquitoes were kept in 75o/oethanol and washed with water before use.

A single dried mosquito was soaked in a so-lution of 10% sodium dodecyl sulfate (SDS) forl0 min, then put on a nitrocellulose filter,squashed with a heat-sealed micropipette tip. Forthe portion test, the mosquito was cut trans-versely between the thorax and the abdomen andthe head portions (head plus thorax) and bodyportions (abdomen) were placed onto the filter.The filters were placed onto puddles of lysis buFer (0.5 M EDTA, pH 8.0, 0.50/o SDS, 100 pelmlproteinase K), incubated at 50.C for t h, andthen air dried.

Detection of hybrids: The filters with mos-quito samples were placed onto puddles of 0.5M NaOH for 5 min to denature the DNA. thenneutralized by 0.5 M Tris-HCl, pH 8.0, 1.5 MNaCl for 5 min after picking off the mosquitodebris. The filters were then baked under vacuumfor 30 min at 75"C after air drying. The prehy-bridization was carried out for 2 h at room tem-perature in prehybridization buffer (5 x Den-hardt's solution, 5x SSC [20x SSC: 3 M NaCl,0.3 M sodium citrate, pH 7 .Ol, IOO 14/ml salmonsperm DNA, 500/o formamide). The filters werehybridized overnight with photobiotinylatedpBF4 DNA probe (100 nglml) at room temper-ature. The filters were washed twice in 2 x SSC.0.lolo SDS, then twice in 0.1x SSC, 0.lolo SDSfor l0 min at room temperature with vigorousagitation.

The washed filters were blocked for 20 min atroom temperature in blocking buffer (100 mMTris-HCl, pH 7.5, 1.0 M NaCl, 2 mM MgClr,0.05o/o Triton X-100, 3olo BSA). The blocking

solution was replaced by I pglml avidin-alkalinephosphatase in the same buffer and incubatedfor l0 min at room temperature. The filters werewashed twice in blocking buffer for l0 min, thenin developing buffer (100 mM Tris-HCl, pH 9.5,1.0 M NaCl, 5 mM MgClr) for l0 min. Theresults were revealed by adding fresh preparedsubstrates of BCIP (final concentration 0.2 mg/ml) and NBT (final concentration 0.3 mglml)after incubation for 20 min in the dark (Yap etal. 1988).

Specificity identification' To examine thespecificity of the probe, various genomic DNAswere extracted by phenoVchloroform extractionand ethanol precipitation (Maniatis et al. 1982)from P. falciparum cultured in vitro, a Plasmo-dium cynomolgi-infected monkey, and, a Plas-modium berghei-infected mouse. An oligonucle-otide specific to P. vivax (Relf et al. 1990) (5'-CGGCCACCCTCCAGGACAG AAGACAGGT.GTCCACC-3') was synthesized automatically.These DNAs were mixed with genomic DNA ofAnopheles mosquitoes, spotted onto nitrocellu-lose filters, and hybridized to 32P-nick-translatedpBF4. The results were demonstrated by auto-radiography.

Examination of sensitivity: Plasmodium fal-ciparum genomic DNA was extracted from cul-tured parasites in vitro and diluted serially withdistilled water. then dot-blotted onto nitrocel-lulose filters for hybridizatiort. One P. falciparumsporozoite-infected mosquito was mixed u.ith 9,19, or 24 uninfected insects. Then the pooledmosquitoes were soaked in 100 pl, 200 pl, or 250pl, respectively, of lysis buffer (20lo SDS, 20oloB-mercaptoethanol, 100 pglml proteinase K), andsubsequently triturated completely with a pestle.Two microliters of each group were spotted ontothe nitrocellulose filter for detection. GenomicDNA was extracted from pools of 50 mosquitoeswith an infective rate of 20o/o, and, resuspendedwith 400 rrl distilled water. Two microliters ofthe DNA solution were spotted onto the nitro-cellulose filter for analysis.

Comparison with dissection method: The samebatch of infected mosquitoes was divided into 2parts; one was used for dissection ofthe salivaryglands for examining sporozoites, and the otherpart was used for DNA hybridization. These weredone by different persons without communica-tion with one another.

RESULTS

Clone containing repeated sequences: 32P-la-beled P. falciparum total genomic DNA was usedas a hybridization probe to screena P.falciparumgenomic library under conditions that alloweddetection of repeated sequences (Meinkoth and

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sulfate solution and then squashed on nitrocel-lulose filters, parasite DNA within the mosqui-toes is easily detected, which simplifies the pro-cessing of samples, and enables the detection ofindividually infected mosquitoes. If a large num-ber of samples needs to be screened in an epi-demiological survey, the triturating methodshould prove to be very efficient and rapid.

ACKNOWLEDGMENTS

We thank Cai Xian-Zhengand Liangftte-Tangfor technical assistance in mosquito infection anddissection. This study was carried out with fi-nancial support from the Ministry of PublicHealth of China.

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