Molecular Epidemiology of Chagas Disease in the Wild Transmission Cycle: the Evaluation in the Sylvatic Vector Mepraia Spinolai from an Endemic Area of Chile

Molecular Epidemiology of Chagas Disease in the Wild Transmission Cycle: The Evaluation in the Sylvatic Vector Mepraia spinolai from an Endemic Area of Chile

Ximena Coronado , Marlene Rozas , Carezza Botto-Mahan , Sylvia Ortíz , Pedro E. Cattan , and Aldo Solari * Programa de Biología Celular y Molecular, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile; Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile; Departamento de Ciencias Biológicas Animales, Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Santiago, Chile

Abstract. The sylvatic transmission cycle of Chagas disease in Chile is composed of wild mammals and insects of the genus Mepraia. We determined infection rates and Trypanosoma cruzi genotypes in Mepraia spinolai. We collected 227 insects from two ecologically contrasting areas to assess T. cruzi infection. Polymerase chain reaction (PCR)-amplified minicircle DNAs were characterized by Southern blot and hybridization tests with genotype-specific probes. Infection in insects from the more fertile area was almost 2-fold higher than in the poorer area. The genotype TCI was the most prevalent and other genotypes such as TCIIb, TCIId, and TCIIe were found at lower rates. The areas differed in their genotype distribution but not in their genotype diversity. We suggest that the difference in abundance and richness of mammals between the areas may be producing the detected infection levels in vectors. Our results are compared with those reported for mammals from the same area.

INTRODUCTION into T. cruzi I (TCI) and T. cruzi II (TCII). The latter is sub- divided into five subgroups, IIa–IIe. Trypanosoma cruzi kine- Chagas disease vectors are hematophagous reduviid toplast DNA (kDNA) contains two components: minicircles (Hemiptera: Heteroptera) insects belonging to the subfam- and maxicircles. The minicircles are abundant (10.000–20.000/ ily Triatominae and capable of transmitting Trypanosoma cell) and have two kinds of sequence elements: a conserved 1–3 cruzi . Two Triatominae genus occur in Chile: Triatoma and region repeated four times per molecule and four variable Mepraia . Triatoma infestans has been the main domestic vector and divergent regions intercalated among the conserved of T. cruzi , now controlled through widespread interventions repeats.19 Oligonucleotides containing conserved minicircle 4,5 of the Southern Cone Initiative. Meanwhile, Mepraia spino- DNA sequences have been used for the detection of the para- lai is almost entirely sylvatic, and only occasionally reported site by polymerase chain reaction (PCR) assays in blood and/ 6 at homes or in peridomestic habitats. It is the only species or vector samples. Moreover, probes from the highly variable of Triatominae with wing polymorphism, and an aggressive regions of minicircles are directed toward groups of T. cruzi 7 behavior to feed on any potential host. This species presents populations allowing characterization and studying host-para- a longer generation time compared with T. infestans and dif- site relationships by hybridization tests. 8–11 ferent feeding and defecation behavior. Mepraia spinolai is Current information suggests that the agriculture has often found in rocky sylvatic ecotopes associated with small played multiple roles in the evolution of animal pathogens rodents and rabbits and also in peridomestic ecotopes, such as into human pathogens.20 Those roles included both genera- 12 henhouses and goat corrals. Mepraia spinolai is endemic to tion of the large human populations necessary for the evolu- 13 Chile and distributed between the 22° and 33°S. Unlike other tion and persistence of human crowd diseases, and generation triatomines, this species is usually a diurnal bug, but it is also of large populations of domestic animals, with which farmers 14 active at night. came into much closer and more frequent contact than hunter/ Even though the human blood index for M. spinolai indi- gatherers have had with wild animals.21 Moreover, these domes- cates that this species is not an important vector of T. cruzi , tic animals served as efficient conduits for pathogen transfers the insect reaches high population densities in stone walls or from wild animals to human. 22 Wildlife represents a source of dry rivers near human dwellings, suggesting an increasing risk zoonotic diseases; therefore, as human settlements encroach 15 of Chagas disease transmission in these localities. A previous on more rural areas with high numbers of infected vectors and 16 study reported by Botto-Mahan and others in the same geo- reservoirs, the contact between humans and infected vectors graphic area showed 46.2% of T. cruzi infection in M. spino- will be expected to increase. In addition, poor human housing lai using molecular evidence, which is much higher than that conditions provide potential access to the insect vectors. reported for the same area using microscopic observation. In In this study, we examined whether T. cruzi infection and addition, their results indicate that younger nymphs are as genotype distribution in M. spinolai depend on biotic fea- infected as the older ones, therefore, equally important in par- tures of the habitat (i.e., vegetation cover and native mammal asite transmission. richness) and anthropogenic influence (i.e., human activity Trypanosoma cruzi possesses a heterogeneous structure and cattle traffic) in a protected area of north-central Chile. and is composed of different genotypes, which circulate in Results from this study are compared with T. cruzi infection domestic and wild cycles, involving human, mammal res- and genotype distribution for sylvatic mammals.23 ervoirs and vectors. 17 Different genetic variants have been detected in several hosts and geographic regions.18 Phenotypic MATERIAL AND METHODS and genotypic analyses have allowed the division of T. cruzi Population under study. Mepraia spinolai insects were cap- * Address correspondence to Aldo Solari, Program of Cellular and tured from two areas inside Las Chinchillas National Reserve, Molecular Biology, Faculty of Medicine, University of Chile, Casilla Coquimbo Region, Chile (31°30′S, 71°06′W). Indivi duals were 70086, Santiago 7, Chile. E-mail: [email protected] manually collected from December 2001 to March 2002 by 656 MOLECULAR EPIDEMIOLOGY OF CHAGAS DISEASE IN THE WILD CYCLE 657 two trained people in 3 consecutive days for 2 hours within probes. Construction of minicircle probes was performed as the time of maximum insect activity (11:00 to 14:00 hours). previously described, and radio-labeled as described for total Captured insects were classified by nymphal stage and indivi- kDNA. 23,24 dually kept to avoid cross-contamination. In the laboratory, Statistical analyses. Infection level comparison between all insects were maintained under optimal growing conditions populations was performed using χ 2 analysis. Genotype (27°C; 75% relative humidity [RH]) until samples of intestinal distribution within each population was compared by the same content were obtained. statistical test. A replicated G-test of goodness of fit was used Areas of study. The two collecting areas (Population 1 to compare genotype distributions between populations. 25 and Population 2, hereafter) are approximately 8 km apart; A genotype diversity index (H : Shannon-Wiener function) vegetation is characterized by thorny scrub composed mainly of was calculated for each population.26 spiny dicots, bromeliads, and cacti, depending on sun exposure. Population 1 is located in a hill nearby a seasonal sandy stream RESULTS with caprine, equine, and bovine cattle, free-ranging rabbits ( Oryctolagus cuniculus), wild rodents (Phyllotis darwini , A total of 227 nymphs of M. spinolai were captured at Octodon degus , Abrothrix sp., Oligoryzomis longicaudatus , the Reserve (both populations combined). We captured 113 and Abrocoma bennetti ), and a native marsupial ( Thylamys in Population 1 (number of individuals per stage: 28 I, 27 II, elegans). This area presents abundant vegetation cover, 23 III, 22 IV, 13 V) and 114 in Population 2 (number of individ- moderate human activity, and regular traffic of domestic uals per stage: 16 I, 27 II, 24 III, 24 IV, 23 V). Adult individuals animals. On the other hand, Population 2 corresponds to an are not represented because of extremely low abundance dur- abandoned stony quarry with extremely low vegetation cover ing the collecting periods. and native mammal abundance/richness (only the rodent Results from PCR assays indicated that 76.1% (N = 86) Abrothrix sp.), null human activity, and traffic of domestic and 40.4% ( N = 46) of the nymphs captured in Population 1 animals. and Population 2, respectively, were infected with T. cruzi . Triatomine samples. The intestinal content of each insect The infection level in Population 1 was significantly higher was removed through abdominal extrusion and mixed with than in Population 2 (χ 2 = 29.81, degrees of freedom [df] = 1, 200 μL of phosphate buffered saline (PBS) buffer, boiled for P < 0.0001), probably showing an association between ecologic 10 minutes, centrifuged at 10,000g , and individually frozen features and infection rate in M. spinolai . at −20°C for PCR assay. A sample of 1 to 5 μL of this crude Positive samples were subjected to genotyping with the four pre-boiled extract was used as DNA template in 50 μL of specific probes. Figure 1A shows a representative experiment final volume. Intestinal contents were free of fresh blood; with PCR results from a selected group of infected M. spino- therefore, no DNA extraction was required. Samples failing lai . Figure 1B –D show Southern blot analyses with the four to amplify were used on a positive control with kDNA to specific probes and mixed infections with up to two genotypes. ensure that the lack of amplicons was not caused by PCR Probes showed all possible hybridization patterns: samples inhibitors. that hybridized with zero (unknown genotype), one, two, three, PCR reaction. The PCR was performed as previously and four probes ( Table 1 ). Hybridization with more than one reported using primers 121 (5′-AAA TAA TGT ACG G probe is indicative of mixed infection. Within the positive indi- (T/G) GAG ATG CAT GA-3′) and 122 (5′ GGG TTC GAT viduals for Population 1, the genotype TCI was the most abun- TGG GGT TGG TGT-3′) to amplify the variable region of dant (59.8%), followed by TCIIb (20.9%), TCIId (16.3%), and minicircle DNA.24 Each experiment included positive and TCIIe (10.5%) (single and mixed genotypes combined). The negative controls. Samples were tested in triplicate, and a same tendency was detected for Population 2 (TCI = 52.1%, reaction was considered positive when at least two of the three TCIIb = 10.9%, TCIId = 8.7%, TCIIe = 8.7%). In both popu- assays showed positive results. False negatives were evaluated lations, at least one unknown genotype is circulating because by incubation of the DNA sample on a positive control. The PCR products were analyzed by electrophoresis on 2% agarose gels and visualized by staining with ethidium bromide. A 330-basepair product indicated a positive result. Southern analysis. These analyses were performed using 10 μL of each PCR assay. The PCR products were electro- phoresed, transferred onto Hybond N+ nylon membranes (Amersham, Little Chalfont, UK ), and cross-linked by UV light for DNA fixation. After transferring PCR products, membranes were pre-hybridized for at least 2 hours at 55°C, and finally hybridized overnight with total kDNA labeled by random priming with 32 P (1 × 10 6 cpm/membrane) as probe for diagnosis confirmation. After hybridization, membranes were washed three times for 30 minutes each with 2 × SSC, 0.1% sodium dodecyl sulfate (SDS) at 55°C, and later exposed in the Molecular Imager FX (Bio-Rad Laboratories, Hercules, CA). Trypanosoma cruzi genotyping. For T. cruzi genotyping, Figure 1. Hybridization patterns from different Mepraia spinolai four different T. cruzi clones isolated in Chile (sp 104cl1, samples. ( A) Ethidium bromide staining of polymerase chain reac- CBBcl3, NRcl3, and v195cl1) corresponding to TcI, TcIIb, tion (PCR) assays, (B – E ) Patterns of hybridizations with probes TCI, TcIId, and TcIIe, respectively, were used to generate specific TCIIb, TCIId, and TCIIe, respectively. 658 CORONADO AND OTHERS

Table 1 other endemic areas, where genetically related T. cruzi clones Distribution of Trypanosoma cruzi genotypes infecting Mepraia spino- exhibit an absence of cross-hybridization between minicir- lai nymphs from two study areas (Population 1 and Population 2) cles DNAs. 28 Probably, these unidentified T. cruzi genotypes located at Las Chinchillas National Reserve, Chile* are TCIIa, TCIIc, or genetically distant TCI, TCIIb, TCIId, T. cruzi genotypes Population 1 Population 2 and TCIIe to the T. cruzi clones used for probes construc- Single tion. 29 Our results indicate that M. spinolai individuals from TCI 35 (40.1%) 18 (39.1%) the two studied populations present similar genotype diver- TCIIb 3 (3.5%) 1 (2.2%) TCIId 8 (9.3%) 0 sity and are infected by similar parasite genotypes (not con- TCIIe 3 (3.5%) 3 (6.5%) sidering the unidentified ones), but differ in their genotype Mixtures distribution. Notwithstanding, characterization of the still TCI ϩTCIIb 10 (11.6%) 2 (4.3%) unidentified genotype(s) is crucial to understand the whole TCI ϩTCIId 1 (1.2%) 2 (4.3%) scenario. ϩ TCI TCIIe 2 (2.3%) 0 We found that the genotype TCI was predominant in this TCI ϩ TCIIb ϩ TCIId 2 (2.3%) 1 (2.2%) TCIIb ϩ TCIIe 1 (1.2%) 0 study and distributed singly and combined among the wild TCIId ϩ TCIIe 1 (1.2%) 0 vectors, a finding that is consistent with the observation that TCI ϩ TCIIb ϩ TCIId ϩ TCIIe 2 (2.3%) 1 (2.2%) the genotype TCI along with TCIIb are the most pure and Unknown† 18 (20.9%) 18 (39.1%) ancestral ones.29 Our results show that TCI is the most abun- * All values in parentheses indicate percentages of hosts infected by identified T. cruzi dant genotype in the vector M. spinolai and, therefore, with a genotypes. † This group could include single or mixed genotypes . higher chance to be transmitted to native mammals and probably to cattle and humans when in contact. Molecular epidemiology studies of Chagas disease of the sylvatic M. spinolai 20.9% (Population 1; N = 18) and 39.1% (Population 2; are useful not only to detect pathogens, but also their hetero- N = 18) of the T. cruzi -positive samples did not hybridize geneity and dynamics of propagation. with any of the tested probes. Statistical analyses indicate Molecular evidence reported for wild mammals belong- that in both populations TCI genotype is significantly more ing to the wild transmission cycle of Chagas disease in central represented than the others (Population 1: χ 2 = 70.73, df = 4, Chile shows high levels of infection (i.e., 61%) and variable P < 0.0001; Population 2: χ2 = 42.06, df = 4, P < 0.0001; single frequencies of genotypes TCI, TCIIb, TCIId, and TCIIe, being and mixed genotypes combined and including unknown gen- the first two of the most represented ones.23 ,30 In peridomestic otypes). Additionally, we detected statistically significant dif- mammals, frequencies for the same genotypes varied slightly ferences in the distribution of genotypes between populations with a preference for TCIId.23,31 In our study, we detected an (G = 9.60, df = 4, P = 0.048; single and mixed genotypes com- infection level for insect vectors similar to those described bined, including positive and negative samples for the anal- in wild mammals, but unlike native mammals; M. spinolai ysis). However, the diversity of genotypes was very similar individuals present much lower frequencies of the ancestral between populations (Population 1: H1 = 0.61; Population 2: genotype TCIIb. Interestingly, TCI was prevalent in the mar-

H2 = 0.58). supial Thylamis elegans, same as described in other endemic areas. 32 Interactions between T. cruzi and the insect vectors DISCUSSION continue to be an interesting subject to be intensely explored. Competition within-host generating selection of parasites is In this study, we examined T. cruzi infection levels in the a regularly described phenomenon. 33 Furthermore, the intra- endemic vector M. spinolai from two areas (Population 1 and host competition among parasites of different genotypes has Population 2) located inside a Reserve, which differ in their been described as a main factor shaping parasite ecology and ecologic features (i.e., biotic and abiotic aspects). We found evolution.34 No studies on the evolution of mixed infections significantly more infected insects in the bug population are available, although it is to be expected that the develop- located in an area with more vegetation cover, higher abun- ment of the parasites will be profoundly influenced inside the dance and richness of mammal hosts, and more anthropogenic blood-sucking insect. influence in terms of cattle and human traffic (Population 1) than in the less fertile population (Population 2). This hetero- Received January 28, 2009. Accepted for publication June 25, 2009. geneous or patchy distribution of the infection in vector populations had been previously described in other endemic areas Financial support: This study was supported by grants FONDECYT 1040762 and 1085154 to A. Solari, and CONICYT-PBCT/PSD66 to C. of Chile. Unfortunately, the ecologic characteristics were not Botto-Mahan. described to raise conclusions about areas of higher endemic- ity. 27 We propose that the features of the ecologic environment Disclosure: The authors declare that they have no conflict of interests would determine the higher or lower insect infection rate, and in relation to this work. this would ultimately depend on vegetation abundance as a resource for mammal hosts. Authors’ addresses: Ximena Coronado, Marlene Rozas, Sylvia Ortíz, and Aldo Solari, Program of Cellular and Molecular Biology, Faculty In both populations, the genotype TCI was significantly of Medicine, University of Chile, Casilla 70086, Santiago 7, Chile, more abundant, followed by TCIIb, TCIId, and TCIIe at Tel: 56-2-978-6062, Fax: 56-2-735-5580, E-mail: [email protected] much lower rates. However, at least one still unidentified .uchile.cl. Carezza Botto-Mahan, Department of Ecological Sciences, genotype is circulating in the vector M. spinolai . Unknown Faculty of Sciences, University of Chile, Casilla 653, Santiago, Chile, Tel: 56-2-9787392, Fax: 56-2-272-7363, E-mail: [email protected]. Pedro genotypes can be genetically slightly different from those E. Cattan, Department of Biological Sciences, Faculty of Veterinary studied here, because they do not cross-hybridize with any Sciences, University of Chile, Casilla 2 correo 15, Santiago, Chile, Tel: of the probes used. The same situation has been reported in 56-2-978-5629, Fax: 56-2-978-5526, E-mail: [email protected]. MOLECULAR EPIDEMIOLOGY OF CHAGAS DISEASE IN THE WILD CYCLE 659

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