Geographical clustering of Trypanosoma cruzi I groups from Colombia revealed by low-stringency single specific primer-PCR of the intergenic regions of spliced-leader genes

Abstract

A low-stringency single-primer polymerase chain reaction (LSSP-PCR) typing procedure targeted to the intergenic regions of spliced-leader genes (SL) was designed to profile Trypanosoma cruzi I stocks from endemic regions of Colombia. Comparison between SL-LSSP-PCR profiles of parasite DNA from vector faeces and cultures isolated from those faeces showed more conservative signatures than profiles using LSSP-PCR targeted to the minicircle variable regions (kDNA). This was also observed by analysing 15 parasite clones from one stock as well as serial samples of a same stock after in vitro culturing or inoculation into mice. Thus, SL-LSSP-PCR appears more appropriate than kDNA-LSSP-PCR for reliable typing of major T. cruzi I groups from in vitro cultured stocks and triatomine faeces. SL-LSSP-PCR grouped 46 of 47 T. cruzi I Colombian stocks according to their geographical procedences in four clusters: Cluster Cas from Casanare Department, Cluster Mg from Northern Magdalena department, Cluster Mom from Momposina Depression in Southern Magdalena and finally Cluster NW from northwestern Colombia, including Sucre, Chocó, Córdoba and Antioquia departments. Sequence analysis identified punctual mutations among amplicons from each cluster. Within Cluster Mg, sequence polymorphism allowed association with different sylvatic vector species. Novel SL sequences and LSSP-PCR profiles are reported from T. cruzi I infecting Eratyrus cuspidatus, Panstrongylus geniculatus and Rhodnius pallescens vectors.

Gene expression study using real-time PCR identifies an NTR gene as a major marker of resistance to benznidazole in Trypanosoma cruzi

“…In this manner, LSSP-PCR and RAPDs analysis have been shown to be good tools in evaluating the genetic variability within the clones belonging to the same strain. These approaches are able to identify multiple or simple variations on the DNA sequence and genetic characterization of T. cruzi [35,60-62]. Furthermore, the high variability of kDNA markers has been extensively used to identify many clones within a strain [63].…”

“…For LSSP-PCR, both minicircle variable regions (kDNA) and intergenic regions of spliced-leader genes (SL) were used as markers as described by Mejia et al (2009) [35]. RAPD analysis was achieved using a total of three primers M13-40 (5'-GTTTTCCCAGTCACGAC-3'), L15996 (5'-CTCCACCATTAGCACCCAAAGC-3') and lgt11 (5'-GACTCCTGGAGCCCG-3') in four different reactions (all primers together and separately) as described previously by Steindel et al (1993) [36].…”

Rhodnius prolixus Life History Outcomes Differ when Infected with Different Trypanosoma cruzi I Strains

“…All T. cruzi strains were originally isolated from Colombia, and have been characterized as sylvatic DTU I [36][37][38][39] (or "Tc1"), as Tc1 is believed to be the predominant DTU in the country, 9,38,40 and is associated with the genus Rhodnius. to simulate the natural conditions of vector-borne T. cruzi transmission.…”

Trypanosoma cruzi I genotypes in different geographical regions and transmission cycles based on a microsatellite motif of the intergenic spacer of spliced-leader genes

“…Tc I strains typically have less DNA content, fewer and smaller chromosomes (Vargas et al, 2004; Lewis et al, 2009). A remarkable intra-DTU variability has been observed, leading to a proposal of associations between internal structuring and transmission cycles (Diosque et al, 2003; Herrera et al, 2007; O´Connor et al, 2007; Brito et al, 2008; Mejía-Jaramillo et al, 2009; Llewellyn et al, 2009). However, knowledge of internal clustering within Tc I is still in its preliminary phase and there is not yet consensus on classification.…”