AJTMH Tropical Medicine and Hygiene News
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

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

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by BOSSENO, M.-F.
Right arrow Articles by BRENIÈRE, S. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by BOSSENO, M.-F.
Right arrow Articles by BRENIÈRE, S. F.
Related Collections
Right arrow Triatomine bugs
Right arrow Chagas Disease

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 inter-genic 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.

The presence and colonization of human habitats by tri-atomines, 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 electrophoresis 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 peri-domiciles 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 1Go 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-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.


Figure 1
View larger version (63K):
[in this window]
[in a new window]
  		    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 hetero-duplex 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.

 
Although immunologic methods have been generally used for identification of triatomine blood meal origins, these techniques 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 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.

Received July 12, 2005. Accepted for publication October 5, 2005.

* Address correspondence to Simone Frédérique Brenière, Unite de Recherche 008 Pathogénie des Trypanosomatidés, Institut de Re-cherche pour le Développement, 911 Avenue Agropolis, BP 5045, 34032 Montpellier Cedex 1, France. E-mail: breniere{at}mpl.ird.fr Back

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: mbosseno{at}mpl.ird.fr, fbaunaure{at}montp2.fr, and breniere{at}mpl.ird.fr. 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{at}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: mge28525{at}cercs.udg.mx, msg0059_2001{at}hotmail.com, and f_lozano_k{at}hotmail.com.

 

REFERENCES

  1. Brenière SF, Bosseno MF, Noireau F, Yacsik N, Liegeard P, Aznar C, Hontebeyrie M, 2002. Integrate study of a Bolivian population infected by Trypanosoma cruzi, the agent of Chagas disease. Mem Inst Oswaldo Cruz 97: 289–295.[ISI][Medline]
  2. Momen H, 1999. Taxonomy of Trypanosoma cruzi: a commentary on characterization and nomenclature. Mem Inst Oswaldo Cruz 94: 181–184.
  3. Yeo M, Acosta N, Llewellyn M, Sanchez H, Adamson S, Miles GA, Lopez E, Gonzalez N, Patterson JS, Gaunt MW, de Arias AR, Miles MA, 2005. Origins of Chagas disease: Didelphis species are natural hosts of Trypanosoma cruzi I and armadillos hosts of Trypanosoma cruzi II, including hybrids. Int J Parasitol 35: 225–233.[ISI][Medline]
  4. Diosque P, Padilla AM, Cimino RO, Cardozo RM, Negrette OS, Marco JD, Zacca R, Meza C, Juarez A, Rojo H, Rey R, Corrales RM, Nasser JR, Basombrio MA, 2004. Chagas disease in rural areas of Chaco Province, Argentina: epidemiologic survey in humans, reservoirs, and vectors. Am J Trop Med Hyg 71: 590–593.[Abstract/Free Full Text]
  5. Lee JH, Hassan H, Hill G, Cupp EW, Higazi TB, Mitchell CJ, Godsey MS Jr, Unnasch TR, 2002. Identification of mosquito avian-derived blood meals by polymerase chain reaction-heteroduplex analysis. Am J Trop Med Hyg 66: 599–604.[Abstract]
  6. Fernandes O, Santos SS, Cupolillo E, Mendonca B, Derre R, Junqueira AC, Santos LC, Sturm NR, Naiff RD, Barret TV, Campbell DA, Coura JR, 2001. A mini-exon multiplex polymerase chain reaction to distinguish the major groups of Trypanosoma cruzi and T. rangeli in the Brazilian Amazon. Trans R Soc Trop Med Hyg 95: 97–99.[Medline]
  7. Wisnivesky-Colli C, Ruiz AM, Ledesma O, Gurtler RE, Lauricella M, Salomon DO, Solarz N, Segura EL, 1987. Home ecology of trypanosomiasis americana: feeding profile of Triatoma infestans in a rural area of the Santiago del Estero province, Argentina. Rev Inst Med Trop Sao Paulo 20: 31–39.
  8. Christensen HA, Sousa OE, de Vasquez AM, 1988. Host feeding profiles of Triatoma dimidiata in peridomestic habitats of western Panama. Am J Trop Med Hyg 38: 477–479.
  9. Noireau F, Brenière SF, Ordonez J, Cardozo L, Morochi W, Gutierrez T, Bosseno MF, Garcia S, Vargas F, Yaksic N, Dujardin JP, Peredo C, Wisnivesky-Colli C, 1997. Low probability of transmission of Trypanosoma cruzi to humans by domiciliary Triatoma sordida in Bolivia. Trans R Soc Trop Med Hyg 91: 653–656.[Medline]
  10. Brenière SF, Pietrokovsky S, Gastelum EM, Bosseno MF, Soto MM, Ouaissi A, Kasten FL, Wisnivesky-Colli C, 2004. Feeding patterns of Triatoma longipennis Usinger (Hemiptera, Reduviidae) in peridomestic habitats of a rural community in Jalisco State, Mexico. J Med Entomol 41: 1015–1020.[ISI][Medline]
  11. Boakye DA, Tang J, Truc P, Merriweather A, Unnasch TR, 1999. Identification of bloodmeals in haematophagous Diptera by cytochrome B heteroduplex analysis. Med Vet Entomol 13: 282–287.[Medline]
  12. Njiokou F, Simo G, Mbida Mbida A, Truc P, Cuny G, Herder S, 2004. A study of host preference in tsetse flies using a modified heteroduplex PCR-based method. Acta Trop 91: 117–120.[Medline]
  13. Bosseno MF, Barnabé C, Magallón Gastelúm E, Lozano Kasten F, Ramsey J, Espinoza B, Brenière SF, 2002. Predominance of Trypanosoma cruzi lineage I in Mexico. J Clin Microbiol 40: 627–632.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am J Trop Med HygHome page
A. WALTER, F. LOZANO-KASTEN, M.-F. BOSSENO, E. G. C. RUVALCABA, M. S. GUTIERREZ, C. E. M. LUNA, F. BAUNAURE, P. PHELINAS, E. MAGALLON-GASTELUM, and S. F. BRENIERE
PERIDOMICILARY HABITAT AND RISK FACTORS FOR TRIATOMA INFESTATION IN A RURAL COMMUNITY OF THE MEXICAN OCCIDENT
Am J Trop Med Hyg, March 1, 2007; 76(3): 508 - 515.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by BOSSENO, M.-F.
Right arrow Articles by BRENIÈRE, S. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by BOSSENO, M.-F.
Right arrow Articles by BRENIÈRE, S. F.
Related Collections
Right arrow Triatomine bugs
Right arrow Chagas Disease

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS