The aim of the present study has been to provide primary evidence of Trypanosoma cruzi landscape genetics in the Mexican Neotropics. T. cruzi and DTU prevalence were analyzed in landscape communities of vectors, wildlife, livestock, pets, and sympatric human populations using endpoint PCR and sequencing of all relevant amplicons from mitochondrial (kDNA) and nuclear (ME, 18S, 24Sα) gene markers.  Although 98% of the infected sample set (N=2963) contained single or mixed infections of DTUI (TcI, 96.2%) and TcVI (22.6%), TcIV and TcII were identified. The sensitivity of individual markers varied and was dependent on the host taxon; kDNA, ME, and 18S combined identified 95% of infections. ME genotyped 90% of vector infections, but 60% of mammals (36% wildlife), while neither 18S nor 24Sα typed more than 20% of mammal infections. Available gene fragments to identify or genotype T. cruzi are not universally sensitive for all landscape parasite populations, highlighting important T. cruzi heterogeneity among mammal reservoir taxa and triatomine species.

Vectors were collected either by inhabitants in and around houses or technical personnel in landscapes which included domestic fragment (housing community) or using various collection methods (light traps, animal bait) in ecotone (agricultural and livestock grazing areas) and sylvatic (conserved) habitats. Specimens of Triatoma dimidiata haplogroup 1 (Hg1), T. dimidiata haplogroup 2 (Hg2), T. dimidiata haplogroup 3 (Hg3), Triatoma pallidipennis, Triatoma phyllosoma, and Panstrongylus rufotuberculatus were taxonomically identified and preserved in 70% EtOH, as previously described. Wildlife specimens were collected as previously described across landscape habitats, taxonomically identified, euthanized, and tissues (exclusively from heart tissue are reported herein) were preserved immediately in 95% molecular grade EtOH or DNazol (Invitrogen, San Diego, California, USA). Blood samples from livestock (5ml), pets (3ml), and one protected bat species (Myotis velifer; 0.3 ml) were drawn in guanidine buffer. Following informed consent, including permission for subsequent use of remaining aliquots, a blood sample (5ml) was drawn in EDTA for standard serology (2-5 tests only for humans), and an additional sample (5ml) preserved in guanidine buffer for molecular T. cruzi detection. The conserved region of the kDNA (S34/S67primers) was chosen as the primary multiple copy mitochondrial fragment for T. cruzi infection in all taxa as previously described. All samples were analyzed herein using the kDNA and the conserved repeat of the spliced leader mini-exon (ME), and all samples amplified using either marker, 100% of kDNA negative vector, bat and human samples, and a random selection of 25% of all other kDNA negative samples were also amplified using primers for the small subunit rDNA 18S gene using the primers SSU561F/SSU561R (N=2103). A random selection of 70% of kDNA-positive samples and all equine and livestock samples were also analyzed using the 24Sα gene using the D71/ D72 primers.  All amplicons of expected band size of the kDNA (120bp), those between 300 and 350 bp of the ME, all amplification products between 520-720 bp of the 18S, and all 24S amplicons within the range of 110 to 125 bp were sequenced.

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