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Home / Equine Research Bank / Parasit Vectors / Identification of phlebotomine sand fly blood meals by real-...
Parasites & vectors2015; 8; 230; doi: 10.1186/s13071-015-0840-3

Identification of phlebotomine sand fly blood meals by real-time PCR.

Abstract: Phlebotomine sand flies are blood-feeding insects of great medical and veterinary significance acting as vectors of Leishmania parasites. Studying the blood-feeding pattern of these insects may help in the understanding of their interactions with potential reservoir hosts of Leishmania parasites. In this study, we developed real time PCR assays for the identification of sand fly blood meal. Methods: Six pairs of primers were designed based on cytochrome b gene sequences available in GenBank of the following potential hosts: dog, cat, horse, chicken, black rat, and human. Firstly, SYBR Green-based real time PCR assays were conducted using a standard curve with eight different concentrations (i.e., 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg and 1 fg per 2 μl) of DNA samples extracted from EDTA blood samples from each target animal. Then, DNA samples extracted from field-collected engorged female sand flies belonging to three species (i.e., Lutzomyia longipalpis, L. migonei and L. lenti) were tested by the protocols standardized herein. Additionally, female sand flies were experimentally fed on a black rat (Rattus rattus) and used for evaluating the time course of the detection of the protocol targeting this species. Results: The protocols performed well with detection limits of 10 pg to 100 fg. Field-collected female sand flies were fed on blood from humans (73%), chickens (23%), dogs (22%), horses (15%), black rats (11%) and cats (2%). Interestingly, 76.1% of the L. longipalpis females were positive for human blood. In total, 48% of the tested females were fed on single sources, 31% on two and 12% on three. The analysis of the time course showed that the real time PCR protocol targeting the black rat DNA was able to detect small amounts of the host DNA up to 5 days after the blood meal. Conclusions: The real time PCR assays standardized herein successfully detected small amounts of host DNA in female sand flies fed on different vertebrate species and, specifically for the black rats, up to 5 days after the blood meal. These assays represent promising tools for the identification of blood meal in field-collected female sand flies.
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Publication Date: 2015-04-16 PubMed ID: 25889289PubMed Central: PMC4410465DOI: 10.1186/s13071-015-0840-3Google Scholar: Lookup
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  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research focuses on identifying the animal sources of blood meals for sand flies, which transmit Leishmania parasites, using real-time PCR. The researchers used DNA testing to successfully identify the blood meals drawn from a wide range of animals and humans.

Overview of Research Process and Methodology

  • The study centered on developing real-time Polymerase Chain Reaction (PCR) assays in order to identify the source of sand fly blood meals.
  • For identification, the team designed six pairs of primers based on cytochrome b gene sequences. The selected sequences were from a dog, cat, horse, chicken, black rat, and human.
  • The team first conducted SYBR Green-based real-time PCR assays using a standard curve with eight diverse concentrations of DNA samples. These samples were derived from blood taken from each of the target animals.
  • The researchers then applied the standardized protocols to the DNA samples drawn from field-collected, blood-engorged female sand flies of three species (Lutzomyia longipalpis, L. migonei, L. lenti).
  • As an extra measure, female sand flies were also fed on a black rat experimentally. The duration of detection for this specific protocol was also evaluated.

Key Findings of the Research

  • The protocols developed in the study showed good performance in the detection limits between 10 picograms to 100 femtograms.
  • The research found that the female sand flies got their blood meals from humans (73%), chickens (23%), dogs (22%), horses (15%), black rats (11%), and cats (2%).
  • For L. longipalpis females, 76.1% were found to have consumed human blood.
  • Of all the tested female sand flies, 48% were found to have only one source of blood meal, 31% had two, and 12% three.
  • When examining the protocol targeting black rat DNA, the team found that they could detect small amounts of the host DNA up to five days after the blood meal.

Conclusion

  • The real-time PCR method was successful in detecting small amounts of host DNA in female sand flies, specifically for blood meals from different vertebrate species.
  • Importantly, the protocol for the black rat DNA demonstrated it could detect small amounts of DNA up to five days after the sand fly had a blood meal.
  • This research is significant because the tools developed represent promising aids for identifying blood meals in field-collected female sand flies.

Cite This Article

APA
Sales KG, Costa PL, de Morais RC, Otranto D, Brandão-Filho SP, Cavalcanti Mde P, Dantas-Torres F. (2015). Identification of phlebotomine sand fly blood meals by real-time PCR. Parasit Vectors, 8, 230. https://doi.org/10.1186/s13071-015-0840-3

Publication

Parasites & vectors
ISSN: 1756-3305
NlmUniqueID: 101462774
Country: England
Language: English
Volume: 8
Pages: 230
PII: 230

Researcher Affiliations

Sales, Kamila Gaudêncio da Silva
  • Department of Immunology, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, Pernambuco, 50740-465, Brazil. kamila@cpqam.fiocruz.br.
Costa, Pietra Lemos
  • Department of Immunology, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, Pernambuco, 50740-465, Brazil. pietra@cpqam.fiocruz.br.
de Morais, Rayana Carla Silva
  • Department of Immunology, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, Pernambuco, 50740-465, Brazil. rayanacarla_m@hotmail.com.
Otranto, Domenico
  • Department of Veterinary Medicine, Università degli Studi di Bari, Valenzano, Bari, 70010, Italy. domenico.otranto@uniba.it.
Brandão-Filho, Sinval Pinto
  • Department of Immunology, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, Pernambuco, 50740-465, Brazil. sinval@cpqam.fiocruz.br.
Cavalcanti, Milena de Paiva
  • Department of Immunology, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, Pernambuco, 50740-465, Brazil. mp@cpqam.fiocruz.br.
Dantas-Torres, Filipe
  • Department of Immunology, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, Pernambuco, 50740-465, Brazil. filipe.dantas@cpqam.fiocruz.br.
  • Department of Veterinary Medicine, Università degli Studi di Bari, Valenzano, Bari, 70010, Italy. filipe.dantas@cpqam.fiocruz.br.

MeSH Terms

  • Animals
  • Cats
  • DNA / genetics
  • DNA / isolation & purification
  • Dogs
  • Feeding Behavior
  • Female
  • Humans
  • Psychodidae / physiology
  • Rats
  • Real-Time Polymerase Chain Reaction / methods
  • Sensitivity and Specificity
  • Species Specificity

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This article has been cited 21 times.
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