Am. J. Trop. Med. Hyg., 74(2), 2006, pp. 303–305 Copyright © 2006 by The American Society of Tropical Medicine and Hygiene

SHORT REPORT: IDENTIFICATION IN TRIATOMINE VECTORS OF FEEDING SOURCES AND TRYPANOSOMA CRUZI VARIANTS BY HETERODUPLEX ASSAY AND A MULTIPLEX MINIEXON POLYMERASE CHAIN REACTION MARIE-FRANCE BOSSENO, LUIS SANTOS GARCÍA, FRANÇOISE BAUNAURE, EZEQUIEL MAGALLÓN GASTELÚM, MARGARITA SOTO GUTIERREZ, FELIPE LOZANO KASTEN, ERIC DUMONTEIL, AND SIMONE FRÉDÉRIQUE BRENIÈRE* Unite de Recherche 008 Pathogénie et Epidémiologie des Trypanosomatidés, Institut de Recherche pour le Développement, Montpellier, France; Departamento de Salud Pública, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara, Jalisco, Mexico; Laboratorio de Parasitología, Centro de Investigaciones Regionales, Universidad Autónoma de Yucatán, Yucatán, Mexico

Abstract. Feeding sources of triatomine vectors (Triatoma longipennis) collected in peridomiciles in Mexico were identified by a heteroduplex assay developed with triatomine blood meals. Trypanosoma cruzi parasites were also characterized in the same blood meal samples by multiplex-polymerase chain reaction assay of mini-exon gene intergenic regions. The main blood meal source was from rats, but the bugs were able to feed on a wide variety of hosts, and human blood meals were identified. Trypanosoma cruzi was the only flagellate species identified in the blood meals. All populations belong to the T. cruzi I lineage, a result that is consistent with the previously assumed predominance of this lineage in Mexico. This combination of blood meal and T. cruzi lineage identification provides a powerful tool for understanding T. cruzi transmission cycles. sis in on 10% acrylamide (29 acrylamide: 1 bisacrylamide) gels in Tris-borate-EDTA buffer and compared with HDA standards obtained with different DNAs from known vertebrate species and potential feeding hosts. To test the blood meal HDA system, five adult Triatoma dimidiata were experimentally blood engorged with pigeons and processed at various times after feeding. Cytochrome b detection was still possible up to 30 days later. Blood meals from 249 T. longipennis specimens collected in 2003 in peridomiciles in the village of Los Guerrero in Jalisco State, Mexico were analyzed. An adequate quantity of PCR products required for the HDA were obtained from 171 samples (68.7%) collected from 52 adults of both sexes and 119 nymphs (second to fifth instars). Comparison of the HDA patterns between them and with those formed with DNAs of known vertebrate species showed that 142 (83%) of 171 samples matched with the patterns used as standards and could thus be directly identified. Eleven (6.4%) samples that did not matched with any standard were analyzed by direct sequencing of the PCR products. BLASTN searches in Genbank nucleic acid sequence database identified two duck species that were not included in our standards (best sequence similarity with Cairina moschata, gi number ⳱ 3088745, 9 cases and with Anas versicolor, gi number ⳱ 3088737, 2 cases). Eighteen samples remained undetermined (10.5%) and 11 (6.4%) showed a complex pattern that could be explained by multiple blood sources and sequencing reactions were unreadable. Figure 1 shows the different HDA patterns observed among T. longipennis blood meal samples and summarizes the overall host feeding determinations. Rattus rattus (rat) was the most frequently detected host (40.5%), followed by the opossum D. virginiana (20.3%), which was determined by PCR product sequencing (best sequence similarity, gi number ⳱ 5835037). Bird feedings (chicken and ducks) represented 18.3%. Two human blood meals were identified from second and fourth instar nymphs found between bricks in two different peridomiciles. Among the current samples, 87 blood meal DNAs of 38 nymphs and 49 adults with flagellate-positive feces were also processed with the mini-exon multiplex PCR-

The presence and colonization of human habitats by triatomines, the obligatory hematophagous vectors of Chagas disease, rely upon accessibility to blood meal sources. Therefore, identification of feeding sources is very helpful in characterizing feeding habits of the different vectors populations (wild, peridomestic, and domestic), identifying the mammalian species that may favor colonization and long-term infestation of human habitats, and assessing the importance of contacts between humans and vectors. Furthermore, several studies have shown that Trypanosoma cruzi clonal populations should be associated with mammalian host species because the different parasite genotypes have their own biologic and genetic properties that may modify host-parasite relationships.1–4 Thus, identification in vectors of both feeding sources and T. cruzi variants should be a powerful epidemiologic approach to assess transmission cycles of the different parasite populations between vectors and mammalian species. In the present study, digestive tract contents of vectors were processed for the identification of blood meal sources by a heteroduplex assay (HDA) of cytochrome b genes, which shows electrophoretic patterns of heteroduplex molecules, and for the characterization of the T. cruzi major lineage by using the intergenic polymorphism of the mini-exon gene. The digestive contents were stored at −20°C until DNA extraction with the QIAamp DNA mini kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s instructions. A 383-basepair cytochrome b gene fragment was amplified from the DNA samples with primers specific for vertebrate cytochrome b. Heteroduplexes were generated with Didelphis sp. (opossum) and Sus scrofa domesticus (pig) cytochrome b polymerase chain reaction (PCR) products as hybridization drivers following the method previously described.5 The HDA patterns were analyzed by electrophore* Address correspondence to Simone Fre´de´rique Brenie`re, Unite de Recherche 008 Pathoge´nie des Trypanosomatide´s, Institut de Recherche pour le De´veloppement, 911 Avenue Agropolis, BP 5045, 34032 Montpellier Cedex 1, France. E-mail: [email protected]

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FIGURE 1. Host feedings of Triatoma longipennis collected in peridomiciliar area in a village of Jalisco State, Mexico. Top, Observed heteroduplex patterns matching with those of domestic animals taken as standards. Didelphis sp. was first used as the driver and when heteroduplex formation was absent the samples were processed again with Sus scrofa domesticus as the driver to confirm Didelphis virginiana feedings. Each species showed a specific pattern of bands between 400 bp and 1,000 bp. MW1 ⳱ small molecular mass ladder (Eurogentec, Seraing, Belgium); MW2 ⳱ 1-kb molecular mass ladder (Promega, Charbonnières, France); bp ⳱ basepairs. Bottom, Number of feeding hosts of Triatoma longipennis among 153 determinations. R. ⳱ rattus.

based typing for direct detection of T. rangeli and T. cruzi.6 Positive PCR results were obtained from 64 samples (sensitivity ⳱ 73.5%). None exhibited PCR pattern matching T. rangeli, whereas all had a major band of 200 basepairs corresponding to T. cruzi I. Although immunologic methods have been generally used for identification of triatomine blood meal origins, these tech-

niques require the preparation of specific antisera for each vertebrate host whose specificity is limited to genus.7–10 Conversely, we demonstrate in this study that HDA is useful because species are differentiated in agreement with previous studies of other hematophagous insects.5,9,11,12 A second advantage of this method is the possible identification of blood meals in the absence of standards by direct sequencing, as

IDENTIFICATION OF FEEDING HOSTS AND T. CRUZI

shown in the present study for duck feeding determinations.5 Moreover, handling of specimens is simple using the PCR method and electrophoresis. Nevertheless, the detection of natural mixed feeding origins is difficult to characterize because the expected patterns are composed of multiple pairwise combinations of the cytochrome b DNA conformations that correspond to the species present in the mixture. In the present study, the frequency of putative mixed feedings was lower (6.4%) than that previously evaluated in a neighboring village (16.8%) using an immunologic method (double diffusion test).10 Another advantage of this approach is that the same blood sample can be used for the identification of feeding sources and T. cruzi typing. The parasite populations belong to the same lineage, a result consistent with the assumption of the predominance of T. cruzi I in Mexico.13 These preliminary analyses of blood meals of T. longipennis species demonstrate that rats play a major role in the development of peridomicile bug colonies and should be the main reservoir. The results are also consistent with the assumption of a successful adaptation of T. longipennis because of its ability to feed on a wide variety of hosts, and undoubtedly confirm the ability of these bugs to feed on humans.

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Received July 12, 2005. Accepted for publication October 5, 2005. Authors’ addresses: Marie-France Bosseno, Françoise Baunaure, and Simone Frédérique Brenière, Unite de Recherche 008 Pathogénie des Trypanosomatidés, Institut de Recherche pour le Développement, 911 Avenue Agropolis, BP 5045, 34032 Montpellier Cedex 1, France, Telephone: 33-4-67-41-62-98, Fax: 33-4-67-41-63-30, E-mails: [email protected], [email protected], and [email protected]. Luis Santos García and Eric Dumonteil, Laboratorio de Parasitología, Centro de Investigaciones Regionales Dr. Hideyo Noguchi, Universidad Autónoma de Yucatán, Avenida Itzaes No. 490 x 59, 97000, Mérida, Yucatán, Mexico, E-mail: oliver@ tunku.wady.mx. Ezequiel Magallón, Gastelúm, Margarita Soto Gutierrez, and Felipe Lozano Kasten, Departamento de Salud Pública, Centro Universitario de Ciencias de la Salud, AP 2-136, Universidad de Guadalajara, Guadalajara, Jalisco, México, E-mails: [email protected], [email protected], and [email protected].

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