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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

Time course analysis of FTA extractions for sand flies fed on human and chicken blood at 24, 48 and 72 h after the blood feed. FTA extractions were followed by multiplex PCR reactions using primer sequences for human, dog, chicken, universal forward and reverse for the cytochrome b gene. (A) Sand flies fed on human blood. (B) Sand flies fed on chicken blood. External lanes correspond to 100 bp DNA ladder. C− and C+ are negative (no DNA) and positive control reactions, respectively.
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fig2: Time course analysis of FTA extractions for sand flies fed on human and chicken blood at 24, 48 and 72 h after the blood feed. FTA extractions were followed by multiplex PCR reactions using primer sequences for human, dog, chicken, universal forward and reverse for the cytochrome b gene. (A) Sand flies fed on human blood. (B) Sand flies fed on chicken blood. External lanes correspond to 100 bp DNA ladder. C− and C+ are negative (no DNA) and positive control reactions, respectively.

Mentions: To optimise PCR conditions initial experiments were performed on DNA isolated from sand flies using a genomic DNA extraction kit. Time-course analysis on chicken and human-fed sand flies in the laboratory showed that host DNA could be detected for up to 48 h after the blood meal (Fig. 1). Prominent non-specific low-molecular weight products were observed in all lanes, including control reactions lacking DNA, and are often seen in multiplex PCR reactions. However, with human DNA two specific bands were detected, corresponding to the predicted human-specific 334 bp and universal mammalian 623 bp bands (Fig. 1A). The lack of products in samples taken after 48 h is presumably due to digestion of the DNA by sand fly gut enzymes. Using flies fed on chicken blood the predicted 210 bp band was observed (Fig. 1B). Next the same procedure was repeated, except that sand fly samples were homogenised and spotted onto FTA cards, then discs punched out and used in the multiplex PCR (Fig. 2). As before, specific bands were detected in sand flies up to 48 h after feeding on either human or chicken blood. Although the results were broadly similar, in comparison to directly extracted genomic DNA (Fig. 1), the human-specific bands were consistently weaker, whereas the chicken-specific bands were consistently stronger. In both sets of experiments the absence of specific bands after 48 h corresponds to the end of blood meal digestion, indicating likely template degradation by digestive enzymes.


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)

Time course analysis of FTA extractions for sand flies fed on human and chicken blood at 24, 48 and 72 h after the blood feed. FTA extractions were followed by multiplex PCR reactions using primer sequences for human, dog, chicken, universal forward and reverse for the cytochrome b gene. (A) Sand flies fed on human blood. (B) Sand flies fed on chicken blood. External lanes correspond to 100 bp DNA ladder. C− and C+ are negative (no DNA) and positive control reactions, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2571954&req=5

fig2: Time course analysis of FTA extractions for sand flies fed on human and chicken blood at 24, 48 and 72 h after the blood feed. FTA extractions were followed by multiplex PCR reactions using primer sequences for human, dog, chicken, universal forward and reverse for the cytochrome b gene. (A) Sand flies fed on human blood. (B) Sand flies fed on chicken blood. External lanes correspond to 100 bp DNA ladder. C− and C+ are negative (no DNA) and positive control reactions, respectively.
Mentions: To optimise PCR conditions initial experiments were performed on DNA isolated from sand flies using a genomic DNA extraction kit. Time-course analysis on chicken and human-fed sand flies in the laboratory showed that host DNA could be detected for up to 48 h after the blood meal (Fig. 1). Prominent non-specific low-molecular weight products were observed in all lanes, including control reactions lacking DNA, and are often seen in multiplex PCR reactions. However, with human DNA two specific bands were detected, corresponding to the predicted human-specific 334 bp and universal mammalian 623 bp bands (Fig. 1A). The lack of products in samples taken after 48 h is presumably due to digestion of the DNA by sand fly gut enzymes. Using flies fed on chicken blood the predicted 210 bp band was observed (Fig. 1B). Next the same procedure was repeated, except that sand fly samples were homogenised and spotted onto FTA cards, then discs punched out and used in the multiplex PCR (Fig. 2). As before, specific bands were detected in sand flies up to 48 h after feeding on either human or chicken blood. Although the results were broadly similar, in comparison to directly extracted genomic DNA (Fig. 1), the human-specific bands were consistently weaker, whereas the chicken-specific bands were consistently stronger. In both sets of experiments the absence of specific bands after 48 h corresponds to the end of blood meal digestion, indicating likely template degradation by digestive enzymes.

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