Dataset Information

Comparative Study of Promastigote- and Amastigote-Initiated Infection of Leishmania infantum (Kinetoplastida: Trypanosomatidae) in Phlebotomus perniciosus (Diptera: Psychodidae) Conducted in Different Biosafety Level Laboratories.

ABSTRACT: Sand flies (Diptera: Psychodidae) are natural vectors of Leishmania. For the initiation of sand fly experimental infections either Leishmania amastigotes or promastigotes can be used. In order to obtain comparable results, it is necessary to adjust and standardize procedures. During this study, we conducted promastigote- and amastigote-initiated infections of Leishmania infantum Nicolle, 1908 parasites in Phlebotomus (Larroussius) perniciosus Newstead, 1911 in two laboratories with different levels of biosafety protection. Protocol originally designed for a biosafety level 2 facility was modified for biosafety level 3 facility and infection parameters were compared. Particularly, specially designed plastic containers were used for blood feeding; feeders were placed outside the sand fly cage, on the top of the mesh; feeding was performed inside the climatic chamber; separation of engorged females was done in Petri dishes kept on ice; engorged females were kept in the cardboard containers until dissection. All experiments, conducted in both laboratories, resulted in fully developed late stage infections with high number of parasites and colonization of the stomodeal valve. We demonstrated that protocol originally designed for biosafety level 2 facilities can be successfully modified for other biosafety facilities, depending on the special requirements of the individual institution/laboratory.

SUBMITTER: Vaselek S

PROVIDER: S-EPMC7044723 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Comparative Study of Promastigote- and Amastigote-Initiated Infection of Leishmania infantum (Kinetoplastida: Trypanosomatidae) in Phlebotomus perniciosus (Diptera: Psychodidae) Conducted in Different Biosafety Level Laboratories.

Vaselek Slavica S Prudhomme Jorian J Myskova Jitka J Lestinova Tereza T Spitzova Tatiana T Bañuls Anne-Laure AL Volf Petr P

Journal of medical entomology 20200201 2

Sand flies (Diptera: Psychodidae) are natural vectors of Leishmania. For the initiation of sand fly experimental infections either Leishmania amastigotes or promastigotes can be used. In order to obtain comparable results, it is necessary to adjust and standardize procedures. During this study, we conducted promastigote- and amastigote-initiated infections of Leishmania infantum Nicolle, 1908 parasites in Phlebotomus (Larroussius) perniciosus Newstead, 1911 in two laboratories with different lev ...[more]

PMID: 31702779

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Project description:BackgroundTrypanosomatids are among the most critical parasites for public health due to their impact on human, animal, and plant health. Diseases associated with these pathogens manifest mainly in poor and vulnerable populations, where social, environmental, and biological factors modulate the case incidence and geographical distribution.MethodsWe used Sanger and amplicon-based next-generation sequencing (NGS) in samples from different mammals to identify trypanosomatid infections in several departments in Colombia. A total of 174 DNA samples (18 humans, 83 dogs, and 73 wild mammals) were analyzed by conventional PCR using a fragment of the heat shock protein 70 (Hsp70) gene and Sanger sequenced the positive samples. Twenty-seven samples were sent for amplicon-based NGS using the same gene fragment. Data obtained were used to perform diversity analyses.ResultsOne hundred and thirteen samples were positive for PCR by Hsp70 fragment; these corresponded to 22.1% Leishmania spp., 18.6% L. amazonensis, 9.7% L. braziliensis, 14.2% L. infantum, 8% L. panamensis, and 27.4% Trypanosoma cruzi. Comparison of the identified species by the two sequencing technologies used resulted in 97% concordance. Alpha and beta diversity indices were significant, mainly for dogs; there was an interesting index of coinfection events in the analyzed samples: different Leishmania species and the simultaneous presence of T. cruzi and even T. rangeli in one of the samples analyzed. Moreover, a low presence of L. braziliensis was observed in samples from wild mammals. Interestingly, to our knowledge, this is the first report of Leishmania detection in Hydrochaeris hydrochaeris (capybara) in Colombia.ConclusionsThe Hsp70 fragment used in this study is an optimal molecular marker for trypanosomatid identification in many hosts and allows the identification of different species in the same sample when amplicon-based sequencing is used. However, the use of this fragment for molecular diagnosis through conventional PCR should be carefully interpreted because of this same capacity to identify several parasites. This point is of pivotal importance in highly endemic countries across South America because of the co-circulation of different genera from the Trypanosomatidae family. The findings show an interesting starting point for One Health approaches in which coevolution and vector-host interactions can be studied.

Project description:Phlebotomine sand flies (Diptera: Psychodidae) are insects of medical importance due to the role that some species play in the transmission of leishmaniasis. This work aimed to study some ecological aspects among sand flies fauna inhabiting two different environments: the várzea (lowland Amazonian forest) and terra firme (upland Amazonian forest), both located in Tefé Municipality, Amazonas State, Braziland to detect Leishmania infection in those phlebotomine populations.Sand flies were collected using HP light traps. Collection took place over the course of six months: January, February, April, August, September, and October of 2013. To detect natural infection by Leishmania, DNA samples were extracted from female sand flies and submitted to Polymerase Chain Reaction (PCR) targeting the kDNA gene; Leishmania species were identified by PCR-RFLP targeting the hsp70 gene and genetic sequencing.In all, 5,716 individuals were collected, and 46 species were identified. Trichophoromyia ubiquitalis (3,330 - 58.26%) and Nyssomyia antunesi (661 - 11.26%) were the most abundant species. Species richness was greater in terra firme environments (42 species) than in the várzea environments (22 species), and forests ecotopes (43 species) were richer than peridomiciles (28 species). DNA of Leishmania was found in Th. ubiquitalis and Psychodopygus davisi, both of which inhabit the terra firme environment and sequencing analysis confirmed the presence of Leishmania (Viannia) lainsoni DNA in Th. ubiquitalis in Tefé Municipality.The high abundance of Th. ubiquitalis and Ps. davisi and detection of DNA of Leishmania sp. may indicate that both species could be putative vectors for American Cutaneous Leishmaniasis (ACL) in the terra firme environment of Tefé. The sand fly fauna found in várzea is rich and diverse, exhibiting several species, nevertheless the seasonal hydric stress during part of the year that could influence the local diversity, if compared with other studies. This is the first report in Amazonas State of Th. ubiquitalis with presence of L. (V.) lainsoni DNA.

Dataset Information

Comparative Study of Promastigote- and Amastigote-Initiated Infection of Leishmania infantum (Kinetoplastida: Trypanosomatidae) in Phlebotomus perniciosus (Diptera: Psychodidae) Conducted in Different Biosafety Level Laboratories.

Publications

Comparative Study of Promastigote- and Amastigote-Initiated Infection of Leishmania infantum (Kinetoplastida: Trypanosomatidae) in Phlebotomus perniciosus (Diptera: Psychodidae) Conducted in Different Biosafety Level Laboratories.

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