Hemi-Nested PCR and RFLP Methodologies for Identifying Blood Meals of the Chagas Disease Vector, Triatoma infestans

Abstract

Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted by hematophagous reduviid bugs within the subfamily Triatominae. These vectors take blood meals from a wide range of hosts, and their feeding behaviors have been used to investigate the ecology and epidemiology of T. cruzi. In this study we describe two PCR-based methodologies that amplify a fragment of the 16S mitochondrial rDNA, aimed to improve the identification of blood meal sources for Triatoma infestans: a.- Sequence analyses of two heminested PCRs that allow the identification of mammalian and avian species, and b.- restriction fragment length polymorphism (RFLP) analysis from the mammalian PCR to identify and differentiate multi-host blood meals. Findings from both methodologies indicate that host DNA could be detected and the host species identified in samples from laboratory reared and field collected triatomines. The implications of this study are two-fold. First, these methods can be used in areas where the fauna diversity and feeding behavior of the triatomines are unknown. Secondly, the RFLP method led to the identification of multi-host DNA from T. infestans gut contents, enhancing the information provided by this assay. These tools are important contributions for ecological and epidemiological studies of vector-borne diseases.

Modeling Disease Vector Occurrence When Detection Is Imperfect II: Drivers of Site-Occupancy by Synanthropic Triatoma brasiliensis in the Brazilian Northeast

“…Opportunistic exploitation of alternative hosts in wild habitats has also been suggested by bloodmeal analyses [7] , [8] , albeit the methods used to identify blood sources all have their drawbacks (see refs. [6] , [38] , [39] ). Perhaps one further sign of an ancestral association with terrestrial rodents is that wild T. brasiliensis do not seem to occupy arboreal Caatinga habitats such as hardwood trees, where T. pseudomaculata occurs, or Copernicia palms, often infested by Rhodnius nasutus [12] , [17] , [40] .…”

Surveillance of Trypanosoma cruzi infection in Triatomine vectors, feral dogs and cats, and wild animals in and around El Paso county, Texas, and New Mexico

“…cruzi- positive triatomine samples were analyzed to determine the mammalian species on which they fed. Amplification of the 16S rDNA was accomplished by hemi-nested PCR, as previously described [ 36 ] with minor modifications. The first reaction contained 3 μL of DNA template from triatomine, a universal forward primer (VF) (ACC CNT CYM TGT NGC AAA AKR GTG) and a mammalian group-specific forward primer (MF) (CCT GTT TAC CAA AAA CAT CAC) at 0.25 μM final concentration, 12.5 μL 2X GoTaq Green Master mix, and nuclease-free water to a final volume of 25 μL.…”

Supersuppression: Reservoir Competency and Timing of Mosquito Host Shifts Combine to Reduce Spillover of West Nile Virus

“…In brief, DNA was extracted from homogenized individual mosquitoes with a Sella score between 2 and 5 using the QIAamp DNA Mini Kit (Qiagen Inc.), following the manufacturer's protocol. Identification of bloodmeal sources was accomplished using a heminested PCR protocol amplifying a polymorphic region of the 16S ribosomal DNA, described in detail in Roellig and others, 26 which allows for the detection of small amounts of host DNA from naturally degraded blood meals. The primary PCR reaction used a universal vertebrate forward primer and a class-specific reverse primer, either Mammalia or Aves, whereas the secondary reaction used the class-specific primer in both directions.…”