Molecular diagnosis of trypanosomatids in Didelphis marsupialis from Los Montes de María: a first report of Trypanosoma rangeli from Colombian Caribbean region.

Didelphis marsupialis is a primary reservoir of Trypanosoma cruzi, etiologic agent of American Trypanosomiasis-AT or Chagas Disease-CD, in America. Some findings of Trypanosoma rangeli have been recorded in this mammal, in sympatry with T. cruzi. In Los Montes de María, Bolívar, Colombian Caribbean, triatomine insects and potential parasite host has been registered, but little is known about the relationship between these parasites and D. marsupialis. We investigated the natural trypanosomatids infection rate in D. marsupialis, applying a parasitological and molecular diagnosis. Twenty D. marsupialis was investigated between 2018 and 2019 using 21 Tomahawk® traps placed on the sylvatic/domestic corridors. Blood was drawn by cardiopuncture after sedation. An aliquot of blood samples was cultured in Novy, Nicolle, McNeal/Roswell Park Memorial Institute medium at 24 °C/60 days for the detection of motile trypomastigotes. Parasite DNA was obtained by salting out methods from positive blood cultures. Trypanosomatids diagnosis was done by Polymerase Chain Reaction-sequencing of V7V8 region of 18S ribosomal RNA (18S-rRNA) gene. Amplicons were sequenced, and consensus sequences were aligned with reference sequences from GenBank. Four isolates corresponded to T. rangeli (20%) and one to T. cruzi (5%). The natural infection of D. marsupialis by T. rangeli and T. cruzi constitutes the first record of these parasites in didelphids in Los Montes de María and the first record of T. rangeli in this marsupial, in the Colombian Caribbean.

Infections and Coinfections by Trypanosomatid Parasites in a Rural Community of Venezuela.

INTRODUCTION: Trypanosoma cruzi, Trypanosoma rangeli and Leishmania spp. are parasites that coexist in several endemic areas. The identification of these parasites in hosts is important for the control programs. METHODS: 216 samples from human blood (101), blood of other mammals (45) and triatomine intestinal content and hemolymph (70), from an endemic area of Venezuela, were analysed. The samples were evaluated by; serology (only humans) and PCR for T. cruzi in human, other mammals and triatomines, PCR for T. rangeli in mammals-including human and triatomines and PCR for Leishmania in mammals-including human. RESULTS: The 9.9% of the human samples were positive for T. cruzi by serology, 11.9% by PCR, 4% for T. rangeli PCR and none for Leishmania spp. PCR. 60% of the samples of other mammals showed DNA amplification for T. cruzi, 42.2% for T. rangeli and 4.4% for Leishmania spp. 61.4% of the triatomine samples showed DNA amplification for T. cruzi and 10% for T. rangeli. CONCLUSIONS: High T. cruzi infection was detected in mammals and triatomines compared with T. rangeli. Low leishmanial infection was detected in other mammals. It is the first time that T. cruzi/T. rangeli coinfection, in humans, Canis familiaris (dog), and Bos Taurus (cow), were reported world-wide, and that this coinfection was described in Tamandua tetradactyla (anteater) from Venezuela. The coinfection T. cruzi/T. rangeli in mammals-including humans and triatomines, and coinfection T. cruzi/Leishmania spp. in non-human mammals, show the risk for trypanosomic zoonoses in this endemic area.

Ecopathogenic complexes of American trypanosomiasis in endemic areas of Venezuela: Diagnosis and variability of Trypanosoma cruzi.

BACKGROUND & OBJECTIVES: Trypanosoma cruzi, the causative agent of American trypanosomiasis, has been reported in 180 mammalian species and 154 triatomines species of Neotropic. This is a clonal parasite with variable biological behaviour, associated with the genetics of the parasite and its hosts. To know the eco-pathogenic complex of this zoonosis, it was proposed to characterize T. cruzi isolates obtained from triatomines and domestic, peridomestic and wild mammals of the eastern and central-western regions of Venezuela. METHODS: The positivity to T. cruzi was established and the isolates were genetically characterized by PCR amplification of the mini-exon gene, the DNA coding for 24Sa and 18S rRNA, and polymorphic sequences-RFLPs. The sampling sites were georeferenced using the MapSource Software and ArcGis 9.3 programs to generate distribution maps of the isolates. RESULTS: Of the 460 hosts (205 triatomines and 255 mammals), 49% were positive for the parasite. On the other hand, 38 isolates obtained from the triatomines and 23 isolates obtained from mammals were evaluated. The TcI genotype predominated in most of the isolates; however, in those obtained from triatomines the presence of the TcIII genotype in single infections and TcI + TcIII or TcI + TcIV in mixed infections was also evidenced. INTERPRETATION & CONCLUSION: There is a possibility that the triatomines act as biological syringes for these genotypes associated exclusively to them. The heterogeneity in T. cruzi isolates demonstrated the complexity of parasitosis in these regions, presenting its control and prevention as a challenge.

Molecular diagnosis of Trypanosoma cruzi/Leishmania spp. coinfection in domestic, peridomestic and wild mammals of Venezuelan co-endemic areas.

American trypanosomiasis and leishmaniases are diseases caused by protozoans of the Trypanosomatidae family. In Venezuela, although several endemic foci of both diseases coincide, there are no reports of coinfection in mammals. The molecular diagnosis of the coinfection T. cruzi-Leishmania spp. was done in 527 blood samples collected on filter paper of several species of mammals (Canis familiaris, Equus asinus, Didelphis marsupialis, Equus mulus, Rattus rattus, Equus caballus, Artibeus fraterculus, Felis catus, Sus scrofa, Bos taurus, Capra hircus and Sciurus granatensis) from the states Cojedes, Aragua, Anzoátegui, Guárico, Miranda and Capital District. The T. cruzi infection was determined through PCR amplification of DNA of kinetoplast minicircles (kDNA) and satellite DNA (sDNA). The Leishmania spp. infection was detected by Leishmania nested PCR (Ln-PCR), and ribosomal DNA internal transcribed spacer 1 PCR (ITS1-PCR). The percentage of infection by T. cruzi was 23.5%, by Leishmania spp. 12.9% and coinfection was 5.7%. D. marsupialis was the species with the highest percentage of infection for each parasitosis (T. cruzi 34.3%, Leishmania spp. 20.0%) and coinfection (14.3%). Anzoátegui was the state with the highest percentage of infection for each parasitosis (T. cruzi 64.9%, Leishmania spp. 64.9%) and coinfection (43.2%). Infections were determined in species not reported as natural reservoirs of T. cruzi (E. asinus and E. mulus) and of Leishmania spp. (E. mulus and S. scrofa). Coinfection was a frequent phenomenon in mammals in several co-endemic zones evaluated.

Triatoma maculata, the Vector of Trypanosoma cruzi, in Venezuela. Phenotypic and Genotypic Variability as Potential Indicator of Vector Displacement into the Domestic Habitat.

Triatoma maculata is a wild vector of Trypanosoma cruzi, the causative agent of Chagas disease; its incursion in the domestic habitat is scant. In order to establish the possible domestic habitat of T. maculata, we evaluated wing variability and polymorphism of genotypic markers in subpopulations of T. maculata that live in different habitats in Venezuela. As markers, we used the mtCyt b gene, previously apply to evaluate population genetic structure in triatomine species, and the ß-tubulin gene region, a marker employed to study genetic variability in Leishmania subgenera. Adults of T. maculata were captured in the period 2012-2013 at domestic, peridomestic (PD), and wild areas of towns in the Venezuelan states of Anzoátegui, Bolívar, Portuguesa, Monagas, Nueva Esparta, and Sucre. The phenotypic analysis was conducted through the determination of the isometric size and conformation of the left wing of each insect (492 individuals), using the MorphoJ program. Results reveal that insects of the domestic habitat showed significant reductions in wing size and variations in anatomical characteristics associated with flying, in relation to the PD and wild habitats. The largest variability was found in Anzoátegui and Monagas. The genotypic variability was assessed by in silico sequence comparison of the molecular markers and PCR-RFLP assays, demonstrating a marked polymorphism for the markers in insects of the domestic habitat in comparison with the other habitats. The highest polymorphism was found for the ß-tubulin marker with enzymes BamHI and KpnI. Additionally, the infection rate by T. cruzi was higher in Monagas and Sucre (26.8 and 37.0%, respectively), while in domestic habitats the infestation rate was highest in Anzoátegui (22.3%). Results suggest domestic habitat colonization by T. maculata that in epidemiological terms, coupled with the presence in this habitat of nymphs of the vector, represents a high risk of transmission of Chagas disease.