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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The examination of mosquito vectors for ingested MF, developing larvae, and infective third-stage larvae (L3) offers the opportunity to monitor the impact of control programs without disturbing the human population. Traditionally, mosquitoes have been examined by dissection and microscopic identification of filarial larvae. In areas with low infection rates, high-throughput screening is required and dissection of individual mosquitoes is not cost-effective; therefore, a number of polymerase chain reaction (PCR) assays have been developed for the screening of mosquito pools.2–4 These methods hold great promise to be widely used for monitoring effects of MDA or other interventions. During a five-year MDA campaign in Egypt, decreases in mosquito infection rates determined by PCR tests of filarial DNA from pools of blood-fed mosquitoes correlated well with decreases in other infection parameters in humans (mf, circulating filarial antigen, and antibody prevalence rates).5 However, the significance of mosquito pool screening PCR results for guiding lymphatic filariasis elimination programs is still not established.
Filarial vector capacity is limited by the number of mf ingested by a mosquito, and less than 100% of them develop into infective L3 within competent vectors.6–9 In contrast, no ingested MF develop into L3s in non-competent vectors. Dead filarial larvae or their remnants may remain free or encapsulated in the hemocoel or in different mosquito tissues (i.e., midgut or thorax). This can provide a source of worm DNA for detection by PCR. It has been suggested that recently blood-fed mosquitoes may have higher detection rates for filarial DNA.2,3 However, there is no experimental evidence to support this hypothesis and it is not known how long DNA from non-developing or dead filarial larvae remain detectable by PCR. Therefore, the aim of the present study was to determine the fate of mf and their DNA in vector competent and non-competent mosquito strains to better define the role of mosquito screening as a monitoring tool for lymphatic filariasis control. We infected three different strains of mosquitoes with B. malayi and examined them by dissection and quantitative real-time PCR for developing larvae and parasite DNA, respectively.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mosquito exposure to infective blood. Brugia malayi (TRS strain) infected cat blood was obtained from the National Institute of Allergy and Infectious Diseases/National Institute of Health Filariasis Research Reagent Repository Center (http://www.filariasiscenter.org) with mf densities of 28–54 mf/20 µL. Mosquitoes were fed using water-jacketed membrane feeders fitted with fresh chicken skin membranes.12 Control mosquitoes were allowed to feed on uninfected dog blood from similar membrane feeders. Fully engorged mosquitoes were separated, placed in 0.5-liter paper cartons, and provided 0.3 M sucrose solution ad libitum. Mosquitoes were sampled for dissection or DNA extraction on days 0 (two hours post-blood feeding), 1, 2, 3, 4, 7, 10, 14, and 21 post-ingestion of mf-positive blood (pi).
Mosquito dissection. Individual mosquitoes were dissected, and the head, thorax, midgut, and abdomen (with midgut removed) were examined separately for parasites. Each body part was placed in a separate drop of Aedes saline13 and teased apart with 0.15-mm insect pin probes. Mosquito tissues were examined for filarial worms with a compound microscope using phase contrast optics. Approximately 15 minutes were spent examining tissues from each dissected mosquito.
Extraction of DNA. Four mosquitoes were pooled (only three on day 0 pi) and stored at –20°C. For extraction of DNA, mosquitoes were homogenized in a parafilm-sealed, 2-mL, round-bottom reaction tube containing 180 µL of phosphate-buffered saline and one zinc-plated 4.5-mm steel BB (Daisy Outdoor Products, Rogers, AR) by vortexing for 20 minutes using a 24-sample vortex adapter (Laney S, unpublished data). Samples were briefly centrifuged and extraction was performed with the DNeasy kit (Qiagen, Valencia, CA) as described previously.3 DNA was eluted in 100 µL of AE buffer (Qiagen) (75 µL for samples from day 0 with a pool size of three mosquitoes) and 1 µL of template was used in a 25-µL real-time PCR.
Quantitative real-time PCR.
Real-time PCR targeting the Hha I repeat of B. malayi was performed as described previously.14 Two different assays were used that amplify either the entire 320-basepair repeat (Taqman assay; Applied Bio-systems, Foster City, CA) or a 120-basepair fragment of the repeat (MGB Eclipse assay; Nanogen, Bothell, WA). All PCRs were carried out in duplicate. The samples were retested if only one of the duplicates was found positive or if the cycle threshold value (Ct) was 
39. The sample Ct value was calculated as the arithmetic mean of duplicates. In all real-time PCRs, water was used as a no-template negative control and 100 pg of B. malayi DNA was used as a positive control.
Data analysis. Group differences were assessed with the chi-square test. The Poolscreen program version 2.0 was used to estimate mosquito infection rates.15
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Among 44 dissected AeR, B. malayi mf were only found in 10 mosquitoes (23%), with most being recovered during the first two days pi. A single, non-moving, and completely melanized mf was recovered from the thorax of one mosquito at day 4, and another single motile mf with little melanin deposition was recovered from the abdomen of another AeR mosquito on day 10 (Figure 1C). In 44 examined Cu mosquitoes, only a single non-moving mf was detected in the midgut two hours pi (Figure 1E).
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). Using the Poolscreen algorithm, this would correspond to an estimated B. malayi DNA detection rate in single mosquitoes of 65% (95% confidence interval [CI] = 50–85%). Examination of the same pools by the Eclipse MGB assay showed B. malayi DNA in all pools, and a DNA detection rate for mosquitoes could not be estimated by Poolscreen.
Screening of AeR pools showed similar results. All pools were positive for filarial DNA using the Taqman and the MGB assays with exception of a single pool for each method on day 14 pi and day 10 pi, respectively (Figure 1D). This corresponds to an estimated B. malayi DNA detection rate in single mosquitoes of 65% (95% CI = 48–85%).
Culex mosquito pools had somewhat different results. Eighty percent of the pools were Taqman positive for B. malayi DNA up to day 3 pi, although only 26% from later collection times were positive (Figure 1F). With the more sensitive MGB Eclipse assay, 82% of the pools through 10 days pi were positive, and on day 14 pi and 21 days pi only 35% were positive. Poolscreen estimates for DNA detection rates in Cu mosquitoes were 14% (95% CI = 10–20%) with the Taqman assay and 28% (95% CI = 21–36%) with the Eclipse MGB assay.
Quantitation of B. malayi DNA in pools of mosquitoes by real-time PCR.
Cycle threshold (Ct) values (with fluorescence signals that exceed background noise levels to indicate a positive real-time PCR) are inversely correlated with the amount of target DNA present in the sample. In general, the Taqman assay had significantly higher Ct values compared with the MGB Eclipse assay (Figure 2; P < 0.05). Cycle threshold values for Cu mosquitoes on days 0 and 1 were equivalent to or lower than those for AeL and AeR. Therefore, the lower B. malayi DNA detection rates and DNA concentrations in Cu at later time points cannot be explained by a reduced uptake of mf during the blood meal.
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). Although some mosquitoes cleared filarial DNA, there was no significant difference in DNA detection rates between the two groups of mosquito pools (P > 0.05, by chi-square test). Thus, filarial DNA was detectable in some Cu mosquitoes after a second blood meal taken one week after mf ingestion.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The natural vectors of B. malayi are mainly Anopheles (nocturnally periodic strains) and Mansonia (subperiodic strains) species.16 AeL and Ae. togoi are efficient laboratory vectors whereas Culex species are not considered to be vectors for B. malayi either in nature or in the laboratory. Dissection of mosquitoes showed that in AeL filarial larvae of different developmental stages were detectable in 85% of the mosquitoes. In contrast, filarial larvae were found mainly during the first two days pi in AeR and only two partly melanized mf were detected at later time points in this strain. A pronounced defense mechanism, such as complete melanization and encapsulation of filarial larvae, does not occur in this strain of Ae. aegypti. This is in agreement with earlier studies of AeR infected with the closely related B. pahangi.17 Few filarial larvae were detected in Cu, and mf were not found outside the midgut. This confirmed earlier observations that mf are unable to penetrate the midgut wall of this mosquito strain (Christensen BM, unpublished data). Staining of filarial larvae using hemalum may enhance the sensitivity of mosquito dissection to detect larvae or parasite fragments. However, it is unlikely that a significant number of larvae were overlooked by our careful dissection procedure because larvae were unambiguously identified by dissection in most of the AeL mosquitoes. Therefore, we postulate that remnants of filarial larvae remain free or encapsulated in the refractory mosquito strains AeR and Cu and that parasite DNA in these remnants is detected by PCR.
We used a relatively small pool size of only four mosquitoes to study the fate of B. malayi DNA in different mosquito strains. With the exception of the negative controls, all mosquitoes were allowed to feed on mf-positive blood. This experimental design ensured that immediately after blood feeding, all pools would contain at least one mf-positive mosquito that could be detected by real-time PCR. Previous evaluation of these real-time PCR assays indicated their high sensitivity.14 Because of the high copy number of the Hha I repeat and the short target sequence of the MGB Eclipse assay, we have estimated that a single cell of B. malayi can be detected in 100 µL of blood. We have no evidence that this assay is less sensitive for the detection of B. malayi DNA in pooled mosquitoes. It can be assumed that all positive pools contain at least one B. malayi cell (or an equivalent amount of DNA). Increasing the pool size to 20 mosquitoes using uninfected mosquitoes should not influence the sensitivity. Therefore, we conclude that the MGB Eclipse assay should be capable of detecting a single B. malayi cell in a pool of up to 20 mosquitoes.
In eastern Indonesia, Anopheles barbirostris is the vector for Brugia timori, a sibling species of B. malayi that can be detected by identical PCR assays.3 In the same area, Culex spp. (especially Cx. tritaeniorhynchus) are the predominant human-biting mosquitoes, but they are never found to be infected with Brugia larvae. However, we have sometimes detected Brugia DNA in Culex pools, and we assumed that this was due to mf ingested by these mosquitoes with blood meals. The results of the present study prove that Brugia DNA can persist in non-vector Culex species for up to three weeks, which is essentially the entire life span of the of the mosquito in nature. These results are consistent with results from blood meal identification studies in mosquitoes and other blood-sucking arthropods, which show that host DNA can be detected for 2–3 days or significantly longer after a blood meal, depending on the species, the molecular marker, and the DNA detection method used.18 Therefore, our laboratory results explain why filarial DNA can be detected in non-vector mosquito species in filariasis-endemic areas.
The main strategy used in the GPELF is MDA, which attempts to interrupt transmission by reducing the source of mf available to vector mosquitoes. Monitoring of vectors for filarial larvae and their DNA is an important diagnostic tool in intervention programs.5,19–21 There are essentially two monitoring strategies: xenomonitoring and transmission monitoring (Table 2). Our study has clearly shown that vector and non-vector mosquitoes may have filarial DNA detectable by PCR for weeks after they have fed on a microfilaremic host. The results indicate that mosquito collection for xenomonitoring using DNA detection assays should be performed after mf in the human population have decreased and at least two weeks after intervention when new generations of mosquitoes are present. Although high DNA detection rates in pools of a particular mosquito species may suggest this species is a competent vector, it does not provide proof of its ability to support parasite development and transmission. Our studies show that a non-vector mosquito species might have a high filarial DNA prevalence rate (similar to AeR in our experimental setting), and the actual vector is another species that may have a wider host range and less frequent contact with MF-positive blood meals. The L3s have to be detected to assess infectivity. Theoretically, this could be done by the detection of stage-specific transcripts using reverse transcriptase–PCR. However, RNA detection is technically more demanding than DNA detection, and it is difficult to identify candidate genes that are not expressed in other larval stages in mosquitoes. Heads and bodies were screened separately for the large-scale assessment of black flies for transmission of the filarial parasite Onchocerca volvulus by poolscreen PCR.22 Infective-stage larvae were assumed to be present only in the heads, and body pools may be judged only as indicators of vector-parasite contact. This strategy is less suitable for lymphatic dwelling filarial worms because infective-stage larvae are often not limited to the head of the mosquito.
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Received September 12, 2006. Accepted for publication November 22, 2006.
Acknowledgments: We thank Dr. John McCall (University of Georgia, Athens, GA) and the National Institute of Allergy and Infectious Diseases/National Institutes of Health Filariasis Research Reagent Repository Center for providing microfilaremic cat blood.
Financial support: This work was supported in part by the National Institutes of Health grant AI-35855 (Gary J. Weil) and AI-19769 (Bruce M. Christensen).
* Address correspondence to Peter Fischer, Department of Internal Medicine, Infectious Diseases Division, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8051, St. Louis, MO 63110. E-mail: pufische{at}im.wustl.edu 
These authors contributed equally to the study.
Authors’ addresses: Peter Fischer, Kerstin Fischer, Ramakrishna U. Rao, and Gary J. Weil, Department of Internal Medicine, Infectious Diseases Division, Washington University School of Medicine, Campus Box 8051, 660 South Euclid Avenue, St. Louis, MO 63110, Fax: 314-454-5293, E-mails: pufische{at}im.wustl.edu, kefische{at}im.wustl.edu, rrao{at}im.wustl.edu, and gweil{at}im.wustl.edu. Sara M. Erickson, Jeremy F. Fuchs, and Bruce M. Christensen, Department of Animal Health and Biomedical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 1656 Linden Drive, Madison, WI 53706, Fax: 608-262-7420, E-mails: smerickson{at}wisc.edu, fuchs{at}svm.vetmed.wisc.edu, and christensen{at}svm.vetmed.wisc.edu.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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