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. 2020 Mar 26;14(3):e0008077.
doi: 10.1371/journal.pntd.0008077. eCollection 2020 Mar.

Molecular detection and identification of Leishmania DNA and blood meal analysis in Phlebotomus (Larroussius) species

Affiliations

Molecular detection and identification of Leishmania DNA and blood meal analysis in Phlebotomus (Larroussius) species

Latifa Remadi et al. PLoS Negl Trop Dis. .

Abstract

Background: Phlebotomus (Larroussius) perniciosus and Canis familiaris are respectively the only confirmed vector and reservoir for the transmission of Leishmania (L.) infantum MON-1 in Tunisia. However, the vector and reservoir hosts of the two other zymodemes, MON-24 and MON-80, are still unknown. The aim of this study was to analyze the L. infantum life cycle in a Tunisian leishmaniasis focus. For this purpose, we have focused on: i) the detection, quantification and identification of Leishmania among this sand fly population, and ii) the analysis of the blood meal preferences of Larroussius (Lar.) subgenus sand flies to identify the potential reservoirs.

Methodology and findings: A total of 3,831 sand flies were collected in seven locations from the center of Tunisia affected by human visceral leishmaniasis. The collected sand flies belonged to two genus Phlebotomus (Ph.) (five species) and Sergentomyia (four species). From the collected 1,029 Lar. subgenus female sand flies, 8.26% was positive to Leishmania by ITS1 nested PCR. Three Leishmania spp. were identified: L. infantum 28% (24/85), L. killicki 13% (11/85), and L. major 22% (19/85). To identify the blood meal sources in Ph. Lar. subgenus sand flies, engorged females were analyzed by PCR-sequencing targeting the vertebrate cytochrome b gene. Among the 177 analyzed blood-fed females, 169 samples were positive. Sequencing results showed seven blood sources: cattle, human, sheep, chicken, goat, donkey, and turkey. In addition, mixed blood meals were detected in twelve cases. Leishmania DNA was found in 21 engorged females, with a wide range of blood meal sources: cattle, chicken, goat, chicken/cattle, chicken/sheep, chicken/turkey and human/cattle. The parasite load was quantified in fed and unfed infected sand flies using a real time PCR targeting kinetoplast DNA. The average parasite load was 1,174 parasites/reaction and 90 parasites/reaction in unfed and fed flies, respectively.

Conclusion: Our results support the role of Ph. longicuspis, Ph. perfiliewi, and Ph. perniciosus in L. infantum transmission. Furthermore, these species could be involved in L. major and L. killicki life cycles. The combination of the parasite detection and the blood meal analysis in this study highlights the incrimination of the identified vertebrate in Leishmania transmission. In addition, we quantify for the first time the parasite load in naturally infected sand flies caught in Tunisia. These findings are relevant for a better understanding of L. infantum transmission cycle in the country. Further investigations and control measures are needed to manage L. infantum transmission and its spreading.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. ITS1 nested PCR-RFLP results.
The Leishmania species typing using RFLP was done for the 54 PCR-ITS1 positive samples. The specific molecular band sizes are: 127, 55, 52, and 20 bp for L. killicki; 126, 66, and 55 bp for L. infantum; 145 and 126 bp for L. major. Lanes 1, 2, and 3: L. infantum identified from infected Ph. perfiliewi, Lane 4: L. infantum identified from infected Ph. perniciosus, Lk: L. killicki control, Li: L. infantum control, and Lm: L. major control. MW: Molecular Weight marker (25-bp DNA ladder).
Fig 2
Fig 2. Phylogenetic analysis by Maximum Likelihood method based on Leishmania ITS1 sequences.
The numbers above the branches indicate the bootstrap sampling percentages.
Fig 3
Fig 3. Blood meal identification results.
A: electropherogram of part of the sequence S 9 showing superposed peaks, B: part of the alignment of four cytochrome b sequences showing superposed peaks for S9, S 10: cytochrome b sequence of Bos taurus, S 3: cytochrome b sequence of Homo sapiens, S 9: cytochrome b sequence of mixed blood meal (Bos taurus/Homo sapiens), two peaks were detected peak 1 (S9P1 corresponding to Bos taurus sequence) and peak 2 (S9P2 corresponding to Homo sapiens sequence).
Fig 4
Fig 4. Real time PCR results.
A: Representative fluorescence acquisition graph showed comparison of each sand fly according to standards. NTC (non template control), NC (negative control corresponding to 30 ng of DNA from reared sand fly), and standards (106 and 103 promastigotes/ml). B: Graphic showing concentration results of each sample according to standards. Slope = -3.37, efficiency = 0.98. S1: Ph. perfiliewi infected with L. infantum, S2: Ph. perniciosus infected with L. major, S3: Ph. perniciosus infected with L. killicki, S4: Ph. perfiliewi infected with L. major, S5: Ph. (Lar.) spp. infected with L. major, S6: Ph. perniciosus infected with L. infantum, and S7: Ph. perniciosus infected with L. killicki.

References

    1. Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, et al. Leishmaniasis worldwide and global estimates of its incidence. PloS one. 2012;7(5):e35671 Epub 2012年06月14日. 10.1371/journal.pone.0035671 - DOI - PMC - PubMed
    1. Chaara D, Ravel C, Banuls A, Haouas N, Lami P, Talignani L, et al. Evolutionary history of Leishmania killicki (synonymous Leishmania tropica) and taxonomic implications. Parasites & vectors. 2015;8:198 10.1186/s13071-015-0821-6 - DOI - PMC - PubMed
    1. Haouas N, Chaker E, Chargui N, Gorcii M, Belhadj S, Kallel K, et al. Geographical distribution updating of Tunisian leishmaniasis foci: about the isoenzymatic analysis of 694 strains. Acta tropica. 2012;124(3):221–8. 10.1016/j.actatropica.2012年08月01日2 . - DOI - PubMed
    1. Ben Ismail R, Gramiccia M, Gradoni L, Helal H, Ben Rachid MS. Isolation of Leishmania major from Phlebotomus papatasi in Tunisia. Transactions of the Royal Society of Tropical Medicine and Hygiene. 1987;81(5):749 Epub 1987年01月01日. 10.1016/0035-9203(87)90018-6 . - DOI - PubMed
    1. Ghawar W, Snoussi MA, Hamida NB, Boukthir A, Yazidi R, Chaabane S, et al. First report of natural infection of least weasel (Mustela nivalis Linnaeus, 1776) with Leishmania major in Tunisia. Vector borne and zoonotic diseases (Larchmont, NY). 2011;11(11):1507–9. Epub 2011年08月27日. 10.1089/vbz.2011.0673 . - DOI - PubMed

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