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Blood meal identification and parasite detection in laboratory-fed and field-captured Lutzomyia longipalpis by PCR using FTA databasing paper.

Sant'Anna MR, Jones NG, Hindley JA, Mendes-Sousa AF, Dillon RJ, Cavalcante RR, Alexander B, Bates PA - Acta Trop. (2008)

Bottom Line: The phlebotomine sand fly Lutzomyia longipalpis takes blood from a variety of wild and domestic animals and transmits Leishmania (Leishmania) infantum chagasi, etiological agent of American visceral leishmaniasis.Blood meal identification in sand flies has depended largely on serological methods but a new protocol described here uses filter-based technology to stabilise and store blood meal DNA, allowing subsequent PCR identification of blood meal sources, as well as parasite detection, in blood-fed sand flies.This technique revealed that 53.6% of field-collected sand flies captured in the back yards of houses in Teresina (Brazil) had fed on chickens.

View Article: PubMed Central - PubMed

Affiliation: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK.

ABSTRACT
The phlebotomine sand fly Lutzomyia longipalpis takes blood from a variety of wild and domestic animals and transmits Leishmania (Leishmania) infantum chagasi, etiological agent of American visceral leishmaniasis. Blood meal identification in sand flies has depended largely on serological methods but a new protocol described here uses filter-based technology to stabilise and store blood meal DNA, allowing subsequent PCR identification of blood meal sources, as well as parasite detection, in blood-fed sand flies. This technique revealed that 53.6% of field-collected sand flies captured in the back yards of houses in Teresina (Brazil) had fed on chickens. The potential applications of this technique in epidemiological studies and strategic planning for leishmaniasis control programmes are discussed.

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Related in: MedlinePlus

Detection of Leishmania infantum DNA using the FTA method. (A) Serial dilutions of cultured L. infantum were spotted onto FTA paper, then extraction and PCR performed using primers specific for the parasite small subunit ribosomal RNA. Numbers above bands represent the number of parasites spotted onto the FTA paper. (B) Detection of L. infantum in lab-infected Lu. longipalpis submitted to whole fly FTA extractions from 24 h to 120 h post-infection. Negative control (C−) represents FTA extraction from an uninfected sand fly, and positive control (C+) represents genomic Leishmania DNA extracted from an infected sand fly amplified using the same set of primers.
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fig3: Detection of Leishmania infantum DNA using the FTA method. (A) Serial dilutions of cultured L. infantum were spotted onto FTA paper, then extraction and PCR performed using primers specific for the parasite small subunit ribosomal RNA. Numbers above bands represent the number of parasites spotted onto the FTA paper. (B) Detection of L. infantum in lab-infected Lu. longipalpis submitted to whole fly FTA extractions from 24 h to 120 h post-infection. Negative control (C−) represents FTA extraction from an uninfected sand fly, and positive control (C+) represents genomic Leishmania DNA extracted from an infected sand fly amplified using the same set of primers.

Mentions: In addition to blood meal identification, it would also be useful to detect the presence of Leishmania parasites in sand fly homogenates. Therefore, a sensitivity test was performed by preparing parasite serial dilutions, which were then spotted onto FTA paper and processed for PCR using primers targeting the parasite small subunit ribosomal RNA (Lachaud et al., 2002) (Fig. 3A). The predicted 603 bp product was detected, and this showed that the equivalent of as few as 49 parasites/original 50 μl sample could be detected. Given that the assay uses a 2 mm disc from a spot of approximately 15 mm diameter this represents a theoretical sensitivity of approximately a single parasite (∼77 fg DNA). To assess whether Leishmania infection could be detected in samples of lab-reared Lu. longipalpis, flies were experimentally infected with Le. infantum, homogenised individually in 0.15 M saline and spotted onto FTA filter paper. A time course analysis of individual sand flies homogenised at various time points post-infection was able to detect the presence of parasite DNA as early as 24 h (Fig. 3B). As expected, the band intensity increased with time as the infection matured. Nevertheless, the results show that sand flies sampled 24–48 h after feeding could be analysed for both blood meal identification and the presence of parasites.


Blood meal identification and parasite detection in laboratory-fed and field-captured Lutzomyia longipalpis by PCR using FTA databasing paper.

Sant'Anna MR, Jones NG, Hindley JA, Mendes-Sousa AF, Dillon RJ, Cavalcante RR, Alexander B, Bates PA - Acta Trop. (2008)

Detection of Leishmania infantum DNA using the FTA method. (A) Serial dilutions of cultured L. infantum were spotted onto FTA paper, then extraction and PCR performed using primers specific for the parasite small subunit ribosomal RNA. Numbers above bands represent the number of parasites spotted onto the FTA paper. (B) Detection of L. infantum in lab-infected Lu. longipalpis submitted to whole fly FTA extractions from 24 h to 120 h post-infection. Negative control (C−) represents FTA extraction from an uninfected sand fly, and positive control (C+) represents genomic Leishmania DNA extracted from an infected sand fly amplified using the same set of primers.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2571954&req=5

fig3: Detection of Leishmania infantum DNA using the FTA method. (A) Serial dilutions of cultured L. infantum were spotted onto FTA paper, then extraction and PCR performed using primers specific for the parasite small subunit ribosomal RNA. Numbers above bands represent the number of parasites spotted onto the FTA paper. (B) Detection of L. infantum in lab-infected Lu. longipalpis submitted to whole fly FTA extractions from 24 h to 120 h post-infection. Negative control (C−) represents FTA extraction from an uninfected sand fly, and positive control (C+) represents genomic Leishmania DNA extracted from an infected sand fly amplified using the same set of primers.
Mentions: In addition to blood meal identification, it would also be useful to detect the presence of Leishmania parasites in sand fly homogenates. Therefore, a sensitivity test was performed by preparing parasite serial dilutions, which were then spotted onto FTA paper and processed for PCR using primers targeting the parasite small subunit ribosomal RNA (Lachaud et al., 2002) (Fig. 3A). The predicted 603 bp product was detected, and this showed that the equivalent of as few as 49 parasites/original 50 μl sample could be detected. Given that the assay uses a 2 mm disc from a spot of approximately 15 mm diameter this represents a theoretical sensitivity of approximately a single parasite (∼77 fg DNA). To assess whether Leishmania infection could be detected in samples of lab-reared Lu. longipalpis, flies were experimentally infected with Le. infantum, homogenised individually in 0.15 M saline and spotted onto FTA filter paper. A time course analysis of individual sand flies homogenised at various time points post-infection was able to detect the presence of parasite DNA as early as 24 h (Fig. 3B). As expected, the band intensity increased with time as the infection matured. Nevertheless, the results show that sand flies sampled 24–48 h after feeding could be analysed for both blood meal identification and the presence of parasites.

Bottom Line: The phlebotomine sand fly Lutzomyia longipalpis takes blood from a variety of wild and domestic animals and transmits Leishmania (Leishmania) infantum chagasi, etiological agent of American visceral leishmaniasis.Blood meal identification in sand flies has depended largely on serological methods but a new protocol described here uses filter-based technology to stabilise and store blood meal DNA, allowing subsequent PCR identification of blood meal sources, as well as parasite detection, in blood-fed sand flies.This technique revealed that 53.6% of field-collected sand flies captured in the back yards of houses in Teresina (Brazil) had fed on chickens.

View Article: PubMed Central - PubMed

Affiliation: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK.

ABSTRACT
The phlebotomine sand fly Lutzomyia longipalpis takes blood from a variety of wild and domestic animals and transmits Leishmania (Leishmania) infantum chagasi, etiological agent of American visceral leishmaniasis. Blood meal identification in sand flies has depended largely on serological methods but a new protocol described here uses filter-based technology to stabilise and store blood meal DNA, allowing subsequent PCR identification of blood meal sources, as well as parasite detection, in blood-fed sand flies. This technique revealed that 53.6% of field-collected sand flies captured in the back yards of houses in Teresina (Brazil) had fed on chickens. The potential applications of this technique in epidemiological studies and strategic planning for leishmaniasis control programmes are discussed.

Show MeSH
Related in: MedlinePlus