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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

Agarose gel showing host-specific cytochrome b PCR products amplified from FTA extractions of blood-engorged wild caught sand flies. Lanes 2–12, sand flies captured in different houses, each lane representing a single sand fly captured in a single house; lanes 13–16, sand flies captured in a chicken house; lane 17, dog positive control; lane 18, human positive control; lane 19, chicken positive control; lanes 20 and 21, negative controls. Outside lanes contain a 100 bp ladder.
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fig4: Agarose gel showing host-specific cytochrome b PCR products amplified from FTA extractions of blood-engorged wild caught sand flies. Lanes 2–12, sand flies captured in different houses, each lane representing a single sand fly captured in a single house; lanes 13–16, sand flies captured in a chicken house; lane 17, dog positive control; lane 18, human positive control; lane 19, chicken positive control; lanes 20 and 21, negative controls. Outside lanes contain a 100 bp ladder.

Mentions: Finally the FTA method was tested on wild-caught sand flies collected in Teresina, Brazil. A total of 2089 sand flies (1701 males and 392 females) were recovered from 65 CDC traps set up in 61 houses from two distinct neighbourhoods during May–June 2007. Of these, 1739 were identified according to Young and Duncan (1994), 1732 as Lu. longipalpis (99.5%), four as Lu. lenti (0.23%) and one each (0.06%) as Lu. whitmani, Lu. termitophyla and Lu. aragaoi. All 58 blood-engorged female sand flies were Lu. longipalpis and positive blood meal identifications were obtained for 43 of these (74%). An example of the results obtained using the FTA extraction diagnostic assay on wild-caught sand flies is shown in Fig. 4. Sand flies that had fed on dogs (e.g. lane 2) and chickens (e.g. lanes 5–11 and 13) were detected. Some flies did not yield any PCR products (e.g. lanes 4 and 16), whereas others amplified the 623 bp universal band (e.g. lanes 3, 12, 14 and 15). In the example shown, two of these (lanes 3 and 15) also included the 210 bp chicken-specific band and were therefore included as chicken-fed sand flies in the overall analysis. Others (e.g. lanes 12 and 14) did not reveal any specific bands and were recorded as unidentified. In total, 41 (70.7%) of the 58 blood-engorged sand flies collected had fed on chickens, 2 (3.4%) had fed on dogs, and 15 (25.9%) could not be identified. These negative results could be due to loss of DNA during blood meal digestion, the presence of a small blood meal or feeding on a host not covered by the multiplex PCR i.e., non-chicken, dog or human blood meals. Fifteen (53.6%) of 28 engorged sand flies collected outside henhouses were positive for chicken blood, compared to 26/30 (86.7%) of specimens collected inside these shelters. None of the blood-fed sand flies collected seemed to have fed on human hosts. The absence of two specific bands in the PCR reactions suggests that none of the engorged sand flies contained blood from more than one host species. Finally, 205/392 field-collected female sand flies (52.3%) were screened for Le. infantum infection using two different set of primers to amplify fragments of the parasite small subunit ribosomal RNA (SSU rRNA) or kDNA (Lachaud et al., 2002). Parasite DNA could not be detected in any of the wild-caught sand flies analysed, indicating that the infection rate in this sample was less than 0.49% (1 in 205).


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)

Agarose gel showing host-specific cytochrome b PCR products amplified from FTA extractions of blood-engorged wild caught sand flies. Lanes 2–12, sand flies captured in different houses, each lane representing a single sand fly captured in a single house; lanes 13–16, sand flies captured in a chicken house; lane 17, dog positive control; lane 18, human positive control; lane 19, chicken positive control; lanes 20 and 21, negative controls. Outside lanes contain a 100 bp ladder.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Agarose gel showing host-specific cytochrome b PCR products amplified from FTA extractions of blood-engorged wild caught sand flies. Lanes 2–12, sand flies captured in different houses, each lane representing a single sand fly captured in a single house; lanes 13–16, sand flies captured in a chicken house; lane 17, dog positive control; lane 18, human positive control; lane 19, chicken positive control; lanes 20 and 21, negative controls. Outside lanes contain a 100 bp ladder.
Mentions: Finally the FTA method was tested on wild-caught sand flies collected in Teresina, Brazil. A total of 2089 sand flies (1701 males and 392 females) were recovered from 65 CDC traps set up in 61 houses from two distinct neighbourhoods during May–June 2007. Of these, 1739 were identified according to Young and Duncan (1994), 1732 as Lu. longipalpis (99.5%), four as Lu. lenti (0.23%) and one each (0.06%) as Lu. whitmani, Lu. termitophyla and Lu. aragaoi. All 58 blood-engorged female sand flies were Lu. longipalpis and positive blood meal identifications were obtained for 43 of these (74%). An example of the results obtained using the FTA extraction diagnostic assay on wild-caught sand flies is shown in Fig. 4. Sand flies that had fed on dogs (e.g. lane 2) and chickens (e.g. lanes 5–11 and 13) were detected. Some flies did not yield any PCR products (e.g. lanes 4 and 16), whereas others amplified the 623 bp universal band (e.g. lanes 3, 12, 14 and 15). In the example shown, two of these (lanes 3 and 15) also included the 210 bp chicken-specific band and were therefore included as chicken-fed sand flies in the overall analysis. Others (e.g. lanes 12 and 14) did not reveal any specific bands and were recorded as unidentified. In total, 41 (70.7%) of the 58 blood-engorged sand flies collected had fed on chickens, 2 (3.4%) had fed on dogs, and 15 (25.9%) could not be identified. These negative results could be due to loss of DNA during blood meal digestion, the presence of a small blood meal or feeding on a host not covered by the multiplex PCR i.e., non-chicken, dog or human blood meals. Fifteen (53.6%) of 28 engorged sand flies collected outside henhouses were positive for chicken blood, compared to 26/30 (86.7%) of specimens collected inside these shelters. None of the blood-fed sand flies collected seemed to have fed on human hosts. The absence of two specific bands in the PCR reactions suggests that none of the engorged sand flies contained blood from more than one host species. Finally, 205/392 field-collected female sand flies (52.3%) were screened for Le. infantum infection using two different set of primers to amplify fragments of the parasite small subunit ribosomal RNA (SSU rRNA) or kDNA (Lachaud et al., 2002). Parasite DNA could not be detected in any of the wild-caught sand flies analysed, indicating that the infection rate in this sample was less than 0.49% (1 in 205).

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