Background Existing molecular assays for filarial parasite DNA in mosquitoes cannot distinguish between infected mosquitoes that contain any stage of the parasite and infective mosquitoes that harbor third stage larvae (L3) capable of establishing new infections in humans. We now report development of a molecular L3-detection assay for Brugia malayi in vectors based on RT-PCR detection of an L3-activated gene transcript. Methodology/Principal Findings Candidate genes identified by bioinformatics analysis of EST datasets across the B. malayi life cycle were initially screened by PCR using cDNA libraries as templates. Stage-specificity was confirmed using RNA isolated from infected mosquitoes. Mosquitoes were collected daily for 14 days after feeding on microfilaremic cat blood. RT-PCR was performed with primer sets that were specific for individual candidate genes. Many promising candidates with strong expression in the L3 stage were excluded because of low-level transcription in less mature larvae. One transcript (TC8100, which encodes a particular form of collagen) was only detected in mosquitoes that contained L3 larvae. This assay detects a single L3 in a pool of 25 mosquitoes. Conclusions/Significance This L3-activated gene transcript, combined with a control transcript (tph-1, accession # U80971) that is constitutively expressed by all vector-stage filarial larvae, can be used to detect filarial infectivity in pools of mosquito vectors. This general approach (detection of stage-specific gene transcripts from eukaryotic pathogens) may also be useful for detecting infective stages of other vector-borne parasites.
References
[1]
Michael E, Bundy DA (1997) Global mapping of lymphatic filariasis. Parasitol Today 13: 472–476. doi: 10.1016/S0169-4758(97)01151-4
[2]
Ottesen EA (2000) The global programme to eliminate lymphatic filariasis. Trop Med Int Health 5: 591–594. doi: 10.1046/j.1365-3156.2000.00620.x
[3]
Weil GJ, Lammie PJ, Weiss N (1997) The ICT Filariasis Test: A rapid-format antigen test for diagnosis of bancroftian filariasis. Parasitol Today 13: 401–404. doi: 10.1016/S0169-4758(97)01130-7
[4]
Lammie PJ, Weil G, Noordin R, Kaliraj P, Steel C, et al. (2004) Recombinant antigen-based antibody assays for the diagnosis and surveillance of lymphatic filariasis - a multicenter trial. Filaria J 3: 9. doi: 10.1186/1475-2883-3-9
[5]
Abdul Rahman R, Hwen-Yee C, Noordin R (2007) Pan LF-ELISA using BmR1 and BmSXP recombinant antigens for detection of lymphatic filariasis. Filaria J 6: 10. doi: 10.1186/1475-2883-6-10
[6]
Williams SA, Laney SJ, Bierwert LA, Saunders LJ, Boakye DA, et al. (2002) Development and standardization of a rapid, PCR-based method for the detection of Wuchereria bancrofti in mosquitoes, for xenomonitoring the human prevalence of bancroftian filariasis. Ann Trop Med Parasitol 96: Suppl 2S41–S46. doi: 10.1179/000349802125002356
[7]
Rao RU, Atkinson LJ, Ramzy RM, Helmy H, Farid HA, et al. (2006) A real-time PCR-based assay for detection of Wuchereria bancrofti DNA in blood and mosquitoes. Am J Trop Med Hyg 74: 826–832.
[8]
Plichart C, Sechan Y, Davies N, Legrand AM (2006) PCR and dissection as tools to monitor filarial infection of Aedes polynesiensis mosquitoes in French Polynesia. Filaria J 5: 2. doi: 10.1186/1475-2883-5-2
[9]
Plichart C, Laney SJ, Sechan Y, Davies N, Legrand A-M (2007) Correction: PCR and dissection as tools to monitor filarial infections of Aedes polynesiensis mosquitoes in French Polynesia. Filaria J 6: 1. doi: 10.1186/1475-2883-6-1
[10]
Fischer P, Wibowo H, Pischke S, Ruckert P, Liebau E, et al. (2002) PCR-based detection and identification of the filarial parasite Brugia timori from Alor Island, Indonesia. Ann Trop Med Parasitol 96: 809–821. doi: 10.1179/000349802125002239
[11]
Lizotte MR, Supali T, Partono F, Williams SA (1994) A polymerase chain reaction assay for the detection of Brugia malayi in blood. Am J Trop Med Hyg 51: 314–321.
[12]
Hoti SL, Vasuki V, Lizotte MW, Patra KP, Ravi G, et al. (2001) Detection of Brugia malayi in laboratory and wild-caught Mansonioides mosquitoes (Diptera: Culicidae) using Hha I PCR assay. Bull Entomol Res 91: 87–92.
[13]
Bockarie MJ (2007) Molecular xenomonitoring of lymphatic filariasis. Am J Trop Med Hyg 77: 591–592.
[14]
Farid HA, Morsy ZS, Helmy H, Ramzy RM, El Setouhy M, et al. (2007) A critical appraisal of molecular xenomonitoring as a tool for assessing progress toward elimination of Lymphatic Filariasis. Am J Trop Med Hyg 77: 593–600.
[15]
Fischer P, Erickson SM, Fischer K, Fuchs JF, Rao RU, et al. (2007) Persistence of Brugia malayi DNA in vector and non-vector mosquitoes: implications for xenomonitoring and transmission monitoring of lymphatic filariasis. Am J Trop Med Hyg 76: 502–507.
[16]
Gyapong JO, Twum-Danso NA (2006) Editorial: Global elimination of lymphatic filariasis: fact or fantasy? Trop Med Int Health 11: 125–128. doi: 10.1111/j.1365-3156.2005.01542.x
[17]
Williams SA, Lizotte-Waniewski MR, Foster J, Guiliano D, Daub J, et al. (2000) The filarial genome project: analysis of the nuclear, mitochondrial and endosymbiont genomes of Brugia malayi. Int J Parasitol 30: 411–419. doi: 10.1016/S0020-7519(00)00014-X
[18]
Liang F, Holt I, Pertea G, Karamycheva S, Salzberg SL, et al. (2000) An optimized protocol for analysis of EST sequences. Nucleic Acids Res 28: 3657–3665. doi: 10.1093/nar/28.18.3657
[19]
Gregory WF, Atmadja AK, Allen JE, Maizels RM (2000) The abundant larval transcript-1 and -2 genes of Brugia malayi encode stage-specific candidate vaccine antigens for filariasis. Infect Immun 68: 4174–4179. doi: 10.1128/IAI.68.7.4174-4179.2000
[20]
Williams S, Laney S (2002) Filarial Genomics: Gene Discovery and Gene Expression. The Filaria. Springer US. pp. 31–42.
[21]
Ghedin E, Wang S, Spiro D, Caler E, Zhao Q, et al. (2007) Draft genome of the filarial nematode parasite Brugia malayi. Science 317: 1756–1760. doi: 10.1126/science.1145406
[22]
Ramachandran CP (1966) Biological aspects in the transmission of Brugia malayi by Aedes aegypti in the laboratory. J Med Entomol 3: 239–252.
[23]
Mak JW (1985) The identification of infective larvae. In: Mak JW, Yong HS, editors. Kuala Lumpur: World Health Organization. pp. 45–50.
[24]
World Health Organization (2004) Technical Advisory Group on the Global Elimination of Lymphatic Filariasis (TAG-ELF): report of the fifth meeting, Geneva, Switzerland, 3–6 February 2004. Geneva: World Health Organization.
