Infection, Genetics and Evolution

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1 Infection, Genetics and Evolution 11 (2011) Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: Identification and lineage genotyping of South American trypanosomes using fluorescent fragment length barcoding P.B. Hamilton a, *, M.D. Lewis b, C. Cruickshank a, M.W. Gaunt b, M. Yeo b, M.S. Llewellyn b, S.A. Valente c, F. Maia da Silva d, J.R. Stevens a, M.A. Miles b, M.M.G. Teixeira d a Biosciences, College of Life and Environmental Sciences, University of Exeter, Prince of Wales Road, Exeter EX4 4PS, United Kingdom b Department of Pathogen Molecular Biology Unit, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom c Instituto Evandro Chagas, Belém, PA , Brazil d Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP , Brazil ARTICLE INFO ABSTRACT Article history: Received 20 August 2010 Received in revised form 14 October 2010 Accepted 15 October 2010 Available online 26 October 2010 Keywords: Co-infection Genetic diversity Vector Chagas disease Protozoa Kinetoplastid Trypanosoma cruzi and Trypanosoma rangeli are human-infective blood parasites, largely restricted to Central and South America. They also infect a wide range of wild and domestic mammals and are transmitted by a numerous species of triatomine bugs. There are significant overlaps in the host and geographical ranges of both species. The two species consist of a number of distinct phylogenetic lineages. A range of PCR-based techniques have been developed to differentiate between these species and to assign their isolates into lineages. However, the existence of at least six and five lineages within T. cruzi and T. rangeli, respectively, makes identification of the full range of isolates difficult and time consuming. Here we have applied fluorescent fragment length barcoding (FFLB) to the problem of identifying and genotyping T. cruzi, T. rangeli and other South American trypanosomes. This technique discriminates species on the basis of length polymorphism of regions of the rdna locus. FFLB was able to differentiate many trypanosome species known from South American mammals: T. cruzi cruzi, T. cruzi marinkellei, T. dionisii-like, T. evansi, T. lewisi, T. rangeli, T. theileri and T. vivax. Furthermore, all five T. rangeli lineages and many T. cruzi lineages could be identified, except the hybrid lineages TcV and TcVI that could not be distinguished from lineages III and II respectively. This method also allowed identification of mixed infections of T. cruzi and T. rangeli lineages in naturally infected triatomine bugs. The ability of FFLB to genotype multiple lineages of T. cruzi and T. rangeli together with other trypanosome species, using the same primer sets is an advantage over other currently available techniques. Overall, these results demonstrate that FFLB is a useful method for species diagnosis, genotyping and understanding the epidemiology of American trypanosomes. ß 2010 Elsevier B.V. All rights reserved. 1. Introduction Trypanosoma cruzi and Trypanosoma rangeli are the two species of human-infective trypanosomes occurring in overlapping areas of South and Central America. T. cruzi causes Chagas disease, a condition that affects at least 8 million people, with 100 million at risk and 14,000 deaths annually (Jannin and Salvatella, 2006). Despite recent advances in disrupting vector transmission in Southern Cone countries, this disease remains a major public health problem in Latin America (Schofield et al., 2006; Miles et al., 2009). In regions endemic for Chagas disease, T. cruzi circulates between humans and domestic animals and is transmitted by domiciliated triatomine bugs. However, infection by T. cruzi is a * Corresponding author. Tel.: ; fax: address: p.b.hamilton@exeter.ac.uk (P.B. Hamilton). widespread zoonosis, ranging from the southern half of the USA to the southernmost countries of South America (Marcili et al., 2009c). T. rangeli is not believed to cause disease in humans. A high prevalence of T. rangeli in humans has been reported in Central America and northwestern South America, where concomitant infections and serological cross-reactivity with T. cruzi make diagnosis of Chagas disease difficult (Vallejo et al., 2009). Both T. cruzi and T. rangeli have a wide mammalian host range and are transmitted by a large diversity of triatomine bugs, although only species of the genus Rhodnius transmits T. rangeli (Maia da Silva et al., 2007; Vallejo et al., 2009). Molecular studies have revealed high genetic diversity in T. cruzi and T. rangeli, with isolates of both species distributed into several lineages, also called discrete taxonomic units (DTU) within T. cruzi (Stevens et al., 1999; Maia da Silva et al., 2007; Miles et al., 2009; Vallejo et al., 2009). At least six lineages of T. cruzi cruzi have been described using molecular markers including RAPDs, SSU /$ see front matter ß 2010 Elsevier B.V. All rights reserved. doi: /j.meegid

2 P.B. Hamilton et al. / Infection, Genetics and Evolution 11 (2011) rrna gene sequences, microsatellites and mitochondrial genes (e.g. Brisse et al., 2001; Machado and Ayala, 2001; Freitas et al., 2005; Westenberger et al., 2005; Miles et al., 2009). These lineages differ in their host range, ecotope and geographical distribution (Miles et al., 2009) and are potentially associated with variable forms of Chagas disease (Anez et al., 2004). T. cruzi lineages until recently designed as TcI and TcIIa e (Brisse et al., 2001; Miles et al., 2009) were recently redesigned as follows: TcI, TcII (former TcIIb), TcIII (TcIIc), TcIV (TcIIa), TcV (TcIId) and TcVI (TcIIe) (Zingales et al., 2009). Accurate identification of T. cruzi and T. rangeli, and their respective lineages is important for diagnosis and understanding their epidemiology. In addition, other species of mammalian trypanosome can be found in the vertebrate hosts of these species, and there are potentially species that are yet to be discovered (Stevens et al., 1999; Maia da Silva et al., 2008; Marcili et al., 2009a,c; Cavazzana et al., 2010). Distinguishing between T. cruzi and T. rangeli lineages is still problematic, especially in regions where man, wild reservoirs and triatomines can be found infected with different combinations of isolates from different lineages of both T. cruzi and T. rangeli (Yeo et al., 2005, 2007; Vallejo et al., 2009). Morphology is insufficient for species identification, particularly in mixed infections in vectors. In mixed cultures T. cruzi prevails over T. rangeli and, after successive passages, typically only one lineage of T. cruzi is selected (Yeo et al., 2007; Maia da Silva et al., 2008). PCR assays have increased sensitivity and accuracy of diagnosis of T. cruzi, allowing identification directly from tissue samples, and triatomine guts and faeces (Hamano et al., 2001; Virreira et al., 2003). Several PCR-based methods are able to differentiate T. cruzi from T. rangeli, including PCR with species-specific primers developed from several genomic regions: kdna minicircles (Avila et al., 1991); telomeric repeats (Chiurillo et al., 2003); repetitive DNA (Vargas et al., 2000); randomly amplified polymorphic DNA (RAPD)-derived markers (Maia da Silva et al., 2004a), spliced leader gene (Maia da Silva et al., 2007) and Cathepsin L-like gene (Ortiz et al., 2009). Length differences in a region of the 24S rrna gene permitted the identification of the three common trypanosomatid species in triatomines: T. cruzi, T. rangeli and Blastocrithidia triatoma (Schijman et al., 2006). The most widely used methods for differentiating between T. cruzi lineages are based on polymorphism of 24S alpha rdna and spliced leader DNA, although these methods are unable to distinguish all lineages (Souto et al., 1996; Fernandes et al., 2001). Indeed, some studies have shown that use of a single molecular marker can lead to misclassification of T. cruzi isolates (Brisse et al., 2001; Burgos et al., 2007; Marcili et al., 2009a,b,c). The five lineages of T. rangeli can be distinguished through length and sequence polymorphisms of the internal transcribed spacer (ITS) rdna regions, cathepsin L-like and spliced leader (SL) genes (Maia da Silva et al., 2004b, 2007, 2009; Ortiz et al., 2009). Fluorescent fragment length barcoding (FFLB) is a method that discriminates species by size polymorphisms in specific regions of the 18S and 28S ribosomal RNA genes (Hamilton et al., 2008). It has been applied to the identification of African trypanosomes, both in tsetse flies and in vertebrates, and its use has led to the discovery of new strains and species (Adams et al., 2008, 2009, 2010; Adams and Hamilton, 2008; Hamilton et al., 2009). It has proved to be quick, accurate, and able to detect mixed infections of up to three different strains (Hamilton et al., 2008; Adams et al., 2009). The high diversity and complexity of T. cruzi and T. rangeli suggest that many genotypes remain to be described, especially from the generally poorly investigated sylvatic vertebrate and invertebrate hosts of unexplored geographical regions and ecotopes. Here we apply this technique to the issue of species and lineage identification of the American trypanosomes, T. cruzi and T. rangeli. Our results provide evidence that FFLB is a useful tool for elucidating the genetic diversity present within these species and for better understanding of the epidemiology of American trypanosomes. 2. Materials and methods 2.1. T. cruzi and T. rangeli isolates The isolates of T. cruzi and T. rangeli were selected for this study to represent the broad genetic diversity found in a range of vertebrate and vector species from their full geographical range (Table 2). They represented the six recognised lineages of T. cruzi cruzi, one new genotype of this species associated with bats (TCbat) and T. c. marinkellei, the subspecies most closely related to T. cruzi cruzi (Stevens et al., 1999) and thought to be restricted to bats from Central and South America (Marcili et al., 2009a; Cavazzana et al., 2010). Isolates of the five currently recognised lineages of T. rangeli (Maia da Silva et al., 2004b, 2007, 2009; Ortiz et al., 2009) were also selected for this study. Identity of species/isolates was as confirmed in previous studies (Maia da Silva et al., 2008; Marcili et al., 2009a,b,c) Fluorescent fragment length barcoding FFLB analysis was carried out using primers and the PCR programme described previously (Hamilton et al., 2008), except REDTaq 1 DNA Polymerase (Sigma) was used. All DNA samples were isolated from cultured trypanosomes. A total of four primer sets were used (two sets within the 18S rrna gene and two within the 28S a rrna gene) to create a barcode for each sample, consisting of the lengths of the four amplified regions. These were then compared to barcodes from other trypanosomes obtained in previous studies (Hamilton et al., 2008, 2009; Adams et al., 2009). 3. Results and discussion 3.1. Identification of American trypanosomes using fluorescent fragment length barcoding In this study, we evaluated the use of fluorescent fragment length barcoding (FFLB) for identification and diagnosis of species of American trypanosomes and genotyping of lineages of T. cruzi and T. rangeli. We compared isolates from all recognised major lineages of T. cruzi and T. rangeli. All isolates examined gave peaks with the four primer sets. Figs. 1 and 2 show example FFLB profiles from a range of species. The method was able to differentiate T. cruzi from T. rangeli independent of lineages of these two species, as the size ranges of 18S1, 18S3 and 28S1 did not overlap between the two species (Tables 1 and 2). The FFLB patterns of T. cruzi and T. rangeli also differed from those of several other species that are known from South American mammals: T. evansi, T. dionisii-like, T. lewisi, T. theileri and T. vivax (Table 1). Additionally, all T. rangeli lineages and the several of T. cruzi lineages could be distinguished, demonstrating that FFLB could be useful for epidemiological studies. Indeed, two loci 18S1 and 28S, used together, were sufficient to discriminate all lineages except the two T. cruzi hybrid lineages, while the other loci often provided additional confidence in the results Identification of T. cruzi lineages Sixty-four T. cruzi cruzi isolates belonging to the six established T. cruzi lineages (TcI TcVI), together with two genotypes that are apparently restricted to bats: TCbat and T. c. marinkellei, were characterised using the FFLB method (Tables 1 and 2 and Fig. 1).

3 [()TD$FIG] 46 P.B. Hamilton et al. / Infection, Genetics and Evolution 11 (2011) Table 1 Fluorescent fragment length barcoding profiles of American trypanosomes. Fragment lengths (bp) 18S1 18S3 28S1 28S2 T. cruzi TcI (formerly I) a T. cruzi TcII (formerly IIb) T. cruzi TcIII (formerly IIc) T. cruzi TcIV (formerly IIa) T. cruzi TcV (formerly IId) T. cruzi TcVI (formerly IIe) T. cruzi TcBAT All T. cruzi cruzi T. cruzi marinkellei T. rangeli TrA T. rangeli TrB T. rangeli TrC T. rangeli TrD , 193 b T. rangeli TrE All T. rangeli Other trypanosomes/trypanosomatids found in South America T. dionisii-like T. theileri c T. evansi d T. lewisi T. vivax e f 172 Unidentified trypanosomatid Blastocrithidia triatoma g a either indicates multiple peaks within this range for individual isolates or, different isolates of the strain give different sized peaks within the range. b 191, 193 indicates two peaks for one isolate. c From Hamilton et al. (2009). d From Hamilton et al. (2008). e From Adams et al. (2009). f Dash indicates that no peak was detected consistently for multiple isolates. g predicted from DNA sequence AF153037; no sequences for the 28S rdna are currently available for this species. Fig. 1. Example electropherograms from T. cruzi. x axis, size of fragment in base pairs; y axis, fluorescence intensity. Small peaks without numbers are size standard. Tcm = T. cruzi marinkellei. TcI was clearly distinguished from all other lineages using one locus, 28S2. However, there was significant heterogeneity within this lineage. All four loci varied in size, with the largest size range (15 bp) at 18S1. Most TcI isolates, including three that were cloned (Xe5740, SJM39, M7) gave up to three peaks at two loci, 18S1 and 28S1 (Fig. 1). This could result from the presence of multiple divergent ribosomal copies within the genome of some TcI isolates, although the multiple peaks may result from PCR artifacts. Recent studies have shown high variability and distinct genotypes within TcI on the basis of the polymorphism of the intergenic region of spliced leader gene (Cura et al., in press) and DNA microsatellites (Llewellyn et al., 2009b). However, while in this study isolates originated from throughout the range of TcI, no obvious geographical pattern of FFLB profiles was apparent. TcI is the most common lineage in sylvatic cycles from North (southern USA), Central and South America, transmitted mainly by Rhodnius species. This lineage predominates as an agent of human infection from the Amazon basin northwards, where it is the main cause of Chagas disease in endemic areas of Venezuela, Colombia, Panama and Mexico (Miles et al., 1981; Bosseno et al., 2006; Burgos et al., 2007; Samudio et al., 2007; Anez et al., 2009; Mejia-Jaramillo et al., 2009). In Brazil, this lineage is reported to infect humans in rural endemic areas (Teixeira et al., 2006) and in the Amazonia region (Miles et al., 1981; Marcili et al., 2009c; Valente et al., 2009). TcII (formerly IIb) is common in domestic transmission cycles in Southern Cone countries of South America and is mainly transmitted by T. infestans. TcII could not be clearly distinguished from TcVI using FFLB, although at the loci 28S1 and 28S2 some fragment sizes were found only in TcII. TcIII (IIc) has a widespread distribution, occurring from Venezuela and Brazilian Amazonia to southern Brazil, Argentina, and Paraguay, transmitted by Triatoma and Panstrongylus species mainly in sylvatic and peridomestic cycles (Yeo et al., 2005; Freitas et al., 2006; Llewellyn et al., 2009a; Marcili et al., 2009b; Miles et al., 2009). TcIII showed heterogeneous profiles, and could not be distinguished from TcV, although some 28S2 fragment sizes were restricted to TcIII. The 28S1 loci of three Brazilian isolates, JA2cl2, M6241cl6 and TCC1437 were longer (336 bp, compared to bp) than the other isolates typed, although the significance of this is not known (Table 2). TcIV (IIa) is common in wild monkeys and Rhodnius in the Brazilian Amazonia, where it has been sporadically reported from human cases of oral infection (Miles et al., 1981; Maia da Silva et al., 2008; Marcili et al., 2009c). This lineage showed heterogeneous profiles: the North American isolate ( r) gave a distinct profile from the eight South American isolates of TcIV at two loci: 18S1 (288, compared to 286 for South American isolates) and 28S2 (207, compared to 204), corroborating that they are closely related, yet distinct (Barnabe et al., 2001; Marcili et al., 2009a; Bosseno et al., 2009). The hybrid lineages TcV (IId) and TcVI (IIe) occur in Bolivia, Paraguay, Chile, Argentina and southern Brazil, and predominate in humans, domestic and synanthropic (animals that live in close association with humans) mammals and triatomines (Brisse et al.,

4 P.B. Hamilton et al. / Infection, Genetics and Evolution 11 (2011) Table 2 Origins of trypanosomes used in the study and their FFLB profiles. Species/ subspecies Type/ lineage Strain/ isolate Location Host/vector FFLB profile 18S1 18S3 28S1 28S2 T. cruzi cruzi TcI (I) 458 Bajo Calima, Colombia Kinkajou Potus flavus 308 (306) a T. cruzi cruzi TcI (I) Cojedes, Venezuela Human Homo sapiens T. cruzi cruzi TcI (I) Dtto Federal, Venezuela Human Homo sapiens 305 (309) T. cruzi cruzi TcI (I) P Georgia, USA Opossum Didelphis marsupialis T. cruzi cruzi TcI (I) b2026 Para, Brazil Opossum Philander opossum 299 (301) T. cruzi cruzi TcI (I) COTMA22 Cotopachi, Bolivia Grass mouse Akodon boliviensis T. cruzi cruzi TcI (I) FLORID Florida, USA Triatomine Triatoma sanguisuga 309 (305) AC1D12 T. cruzi cruzi TcI (I) JR cl4 Anzoátegui, Venezuela Human Homo sapiens 306 (302, 308) 242, , T. cruzi cruzi TcI (I) M16 Barinas, Venezuela Opossum Didelphis marsupialis (244) 334, T. cruzi cruzi TcI (I) M7 b Barinas, Venezuela Opossum Didelphis marsupialis (334) 199 T. cruzi cruzi TcI (I) PALDA21 Chaco, Argentina Opossum Didelphis albiventris T. cruzi cruzi TcI (I) SJM34 b Beni, Bolivia Opossum Didelphis marsupialis T. cruzi cruzi TcI (I) SJM39 b Beni, Bolivia Opossum Didelphis marsupialis 308 (302, 304) (345) 197 T. cruzi cruzi TcI (I) SJM41 b Beni, Bolivia Opossum Philander opossum T. cruzi cruzi TcI (I) TEV55 Chaco, Argentina Triatomine Triatoma infestans T. cruzi cruzi TcI (I) TCC1358 c Amazonas, Brazil Triatomine Rhodnius brethesi 294, (335) T. cruzi cruzi TcI (I) TCC1360 Para, Brazil Triatomine Rhodnius pictipes 294, 298, T. cruzi cruzi TcI (I) TCC1367 Para, Brazil Triatomine Rhodnius robustus 294 (298) T. cruzi cruzi TcI (I) TCC1380 Para, Brazil Triatomine Rhodnius robustus 295 (301) T. cruzi cruzi TcI (I) TCC1397 Para, Brazil Triatomine Rhodnius pictipes 294, , T. cruzi cruzi TcI (I) Para, Brazil Human Homo sapiens 295, , 337, 198 TCC T. cruzi cruzi TcI (I) TCC1010 Rondonia, Brazil Opossum Didelphis marsupialis 302, , T. cruzi cruzi TcI (I) Para, Brazil Human Homo sapiens 297, TCC1612 T. cruzi cruzi TcI (I) Para, Brazil Human Homo sapiens 294, TCC1613 T. cruzi cruzi TcI (I) TCC651 Rondonia, Brazil Triatomine Rhodnius robustus T. cruzi cruzi d TcI (I) TCC758 Amazonas, Brazil Triatomine Rhodnius brethesi 286, 302, 239, , 335, 198, , T. cruzi cruzi TcI (I) USAO Louisiana, USA Opossum Didelphis marsupialis 303 (309) POSSUM 246 T. cruzi cruzi e TcI (I) Xe1313 Para, Brazil Opossum Philander opossum 234, , , 194, T. cruzi cruzi TcI (I) Xe5167 Para, Brazil Opossum Didelphis marsupialis 294 (299) T. cruzi cruzi TcI (I) Xe5740 b Para, Brazil Opossum Didelphis marsupialis 294 (301) T. cruzi cruzi TcII (IIb) Chaco23 Presidente Hayes, Triatomine Triatoma infestans cl4 Paraguay T. cruzi cruzi TcII (IIb) Esm cl3 Bahia, Brazil Human Homo sapiens T. cruzi cruzi TcII (IIb) IVV cl4 Cuncumen, Chile Human Homo sapiens T. cruzi cruzi TcII (IIb) Pot7a cl1 San Martin, Boqueron, Paraguay Triatomine Triatoma infestans T. cruzi cruzi TcIII (IIc) JA2 cl2 Amazonas, Brazil Opossum Monodelphis sp T. cruzi cruzi TcIII (IIc) M6241 cl6 Para, Brazil Human Homo sapiens T. cruzi cruzi TcIII (IIc) SABP19 cl1 Vitor, Peru Triatomine Triatoma infestans T. cruzi cruzi TcIII (IIc) SJMO18 Beni, Bolivia Armadillo Dasypus novemcinctus T. cruzi cruzi TcIII (IIc) SMA8 Santa Maria de Armadillo Dasypus novemcinctus Apere, Bolivia T. cruzi cruzi TcIII (IIc) SMA9 Santa Maria de Armadillo Dasypus novemcinctus Apere, Bolivia T. cruzi cruzi TcIII (IIc) TCC135 Sao Paulo, Brazil Rodent Proechimys iheringi T. cruzi cruzi TcIII (IIc) TCC1437 Para, Brazil Rodent Proechimys longicaudatus T. cruzi cruzi TcIII (IIc) TCC712 Amazonas, Brazil Marsupial Monodelphis brevicaudata T. cruzi cruzi TcIV (IIa) r Georgia, USA Raccoon Procyon lotor T. cruzi cruzi TcIV (IIa) Saimiri3 cl1 Venezuela Squirrel monkey Saimiri sciureus T. cruzi cruzi TcIV (IIa) Amapa, Brazil Human Homo sapiens , 204 TCC T. cruzi cruzi TcIV (IIa) Para, Brazil Human Homo sapiens TCC1441 T. cruzi cruzi TcIV (IIa) TCC338 Acre, Brazil Monkey Saguinus labiatus T. cruzi cruzi TcIV (IIa) TCC668 Rondonia, Brazil Triatomine Rhodnius robustus T. cruzi cruzi TcIV (IIa) TCC759 Amazonas, Brazil Triatomine Rhodnius brethesi T. cruzi cruzi TcIV (IIa) TCC760 Amazonas, Brazil Triatomine Rhodnius brethesi T. cruzi cruzi TcIV (IIa) X10610 cl5 Guárico, Venezuela Human Homo sapiens T. cruzi cruzi TcV (IId) cl2 Santa Cruz, Bolivia Human Homo sapiens T. cruzi cruzi TcV (IId) Para6 cl4 Paraguari, Paraguay Triatomine Triatoma infestans T. cruzi cruzi TcV (IId) Bertha Santa Cruz, Bolivia Human Homo sapiens T. cruzi cruzi TcV (IId) TCC656 Santa Cruz, Bolivia Human Homo sapiens T. cruzi cruzi TcV (IId) NR cl3 Chile Human Homo sapiens

5 48 P.B. Hamilton et al. / Infection, Genetics and Evolution 11 (2011) Table 2 (Continued ) Species/ subspecies Type/ lineage Strain/ isolate Location Host/vector FFLB profile 18S1 18S3 28S1 28S2 T. cruzi cruzi TcV (IId) Vinch101 cl1 Limari, Chile Triatomine Triatoma infestans T. cruzi cruzi TcVI (IIe) Chaco9 cl15 Presidente Hayes, Triatomine Triatoma infestans Paraguay T. cruzi cruzi TcVI (IIe) CL Brener Rio Grande do Sul, Triatomine Triatoma infestans Brazil T. cruzi cruzi TcVI (IIe) P251 cl7 Cochabamba, Bolivia Human Homo sapiens T. cruzi cruzi TcVI (IIe) Tula cl2 Tulahuen, Chile Human Homo sapiens T. cruzi cruzi Tcbat TCC793 Sao Paulo, Brazil Bat Myotis levis T. cruzi cruzi Tcbat TCC1122 Sao Paulo, Brazil Bat Myotis albescens T cruzi marinkellei TCC344 Rondonia, Brazil Bat Carollia perspicillata T cruzi marinkellei TCC501 Rondonia, Brazil Bat Carollia perspicillata T. dionisii-like TCC211 Sao Paulo, Brazil Bat Eptesicus brasiliensis , T. dionisii-like TCC495 Sao Paulo, Brazil Bat Carolia perspicillata T. rangeli TrA San Augustin Colombia Human Homo sapiens T. rangeli TrA TCC220 Para, Brazil Monkey Saimiri sciureus T. rangeli TrA TCC369 Rondonia, Brazil Opssum Didelphis marsupialis T. rangeli TrA TCC701 Rondonia, Brazil Triatomine Rhodnius robustus T. rangeli TrB TCC010 Para, Brazil Anteater Tamandua tetradactyla T. rangeli TrB AM80 Amazonas, Brazil Human Homo sapiens T. rangeli TrB TCC207 Acre, Brazil Monkey Cebuella pygmaea T. rangeli TrB TCC236 Acre, Brazil Monkey Saguinus f. weddelli T. rangeli f TrC TCC1250 Panama Triatomine Rhodnius pallescens 266, , , , 198 T. rangeli f TrC TCC1252 Panama Triatomine Rhodnius pallescens 266, , , , 198 T. rangeli TrC TCC1254 Panama Triatomine Rhodnius pallescens T. rangeli TrC PG Panama Human Homo sapiens T. rangeli TrD SC58 Santa Catarina, Brazil Rodent Echimys dasithrix , 193 T. rangeli TrE TCC643 Mato Grosso do Bat Platyrrhinus lineatus Sul, Brazil T. rangeli TrE TCC1182 Amazonas, Brazil Triatomine Rhodnius pictipes T. rangeli TrE TCC1224 Amazonas, Brazil Triatomine Rhodnius pictipes a Values in parentheses are <50% the height of main peak from the same locus. b Cloned isolate. c TCC = Trypanosomatid Culture Collection. d Mixed infection with TCIV. e Mixed infection with unidentified trypanosome. f Mixed infection with TCI. 2003; Yeo et al., 2005; Corrales et al., 2009). The hybrid isolates of TcVI and TcV could be distinguished by three loci but shared patterns with TcII and TcIII respectively. This result is perhaps not surprising because these lineages are products of hybridization of TcII and TcIII (Freitas et al., 2006; Miles et al., 2009). There is geographic overlap in the distribution of these hybrid lineages and their parental lineages, especially for TcII and TcV/VI (domestic cycles across the Southern Cone). The overlap is more limited for TcIII, as it is rare in domestic cycles where TcV and TcVI are found, but there are reports of TcIII being sympatric with TcV and TcVI in Paraguay and Argentina (Chapman et al., 1984; Cardinal et al., 2008). Therefore further primers, such as those targeting ITS rdna (Marcili et al., 2009a), or PCR-RFLP assays (Lewis et al., 2009), would be necessary to discriminate these hybrids from the parent lineages. Nevertheless, as the traditional genotyping method (Souto et al., 2006) shows combined TcII/III profiles for the hybrid lineages, FFLB can be used to differentiate mixed infections from hybrids. The newly discovered T. cruzi genotype that is so far apparently restricted to bats, TCbat, showed unique FFLB pattern, in agreement with its placement in a separated cluster in phylogenetic studies (Marcili et al., 2009a). Two other South American bat trypanosomes, T. c marinkellei and T. dionisii-like, also gave unique patterns (Table 1). The two isolates of T. dionisii-like differed in their profiles (Table 2); there is considerable heterogeneity of T. dionisii-like in South America and these two isolates belong to distinct genotypes of this species (Cavazzana et al., 2010) Identification of Trypanosoma rangeli lineages Seventeen T. rangeli isolates belonging to the five lineages (TrA TrE) were genotyped using FFLB (Tables 1 and 2 and Fig. 2). These lineages were previously established by phylogenetic analysis using ITS rdna, spliced leader and CatL-like gene sequences (Maia da Silva et al., 2004b, 2007, 2009; Ortiz et al., 2009). All lineages could be differentiated, with each lineage giving a distinct combination of fragment sizes at the four loci (Table 1). The FFLB patterns of TrB were most divergent, and the sizes of three of the four loci differed from the other T. rangeli lineages, in agreement with phylogenetic studies (Maia da Silva et al., 2007, 2008; Ortiz et al., 2009). The geographical distributions of T. rangeli lineages are related to the ecogeographical structure of the Rhodnius vector species, with lineage divergence associated with sympatric vectors (Maia da Silva et al., 2004b, 2007, 2009; Vallejo et al., 2009): TrA circulates from Brazil to Guatemala and is related to both domestic and sylvatic cycles of species of the R. prolixus complex, and is commonly found infecting man; TrB so far includes only sylvatic isolates from humans and wild mammals from Brazilian Amazonia and is associated with the R. brethesi complex. TrC is related to

6 [()TD$FIG] P.B. Hamilton et al. / Infection, Genetics and Evolution 11 (2011) expected from mixed infections revealed that many of the common mixed infections that occur in natural conditions such as TcI with TcIII could be identified. T. rangeli, lineages TrA and TrC (Panama, Costa Rica and northwest Colombia), and TrA and TrB (Amazonian region) can infect the same vertebrate hosts but not the same vectors, however, all these lineages could be distinguished using FFLB. In studies of African trypanosomes, the application of FFLB has resulted in discovery of novel species and genotypes (Hamilton et al., 2008, 2009; Adams et al., 2009). In the current study, a barcode was obtained from a recent TcI culture from a marsupial (Fig. 2), Philander opossum, which differed from those of other trypanosome species examined in this study, and may represent a previously undescribed trypanosome species. 4. Conclusions Fig. 2. Example electropherograms from T. rangeli, T. dionisii-like and mixed infections. See legend to Fig. 1 for further information. domestic and sylvatic cycles of the R. pallescens complex circulating in humans, and domestic and wild mammals in Panama, Costa Rica and Colombia. Lineage TrD is known from rodents from southern Brazil; a presumed Rhodnius sp. vector of this lineage is unknown. TrE has so far only been found in bats and R. pictipes from Central and Amazon regions in Brazil Identification of Blastocrithidia triatomae Many triatomines, particularly Triatoma spp., may also carry Blastocrithidia triatomae, a trypanosomatid parasite of triatomines that apparently does not have a vertebrate host. As no DNA from this species was available, the FFLB profile was estimated from the 18S rdna sequence (AF153037). The 18S1 locus (344 bp) is 27 bp longer than all trypanosome species examined in this study, so this would not be mistaken for T. cruzi or T. rangeli Mixed infections and novel genotypes Mixed infections of different strains of T. rangeli and T. cruzi are common in both mammalian and triatomine hosts. In previous studies, mixed infections of African trypanosomes have been readily detected using FFLB (Hamilton et al., 2008; Adams et al., 2009). In this study, FFLB detected previously unidentified mixed infections in primary cultures from gut contents of triatomine bugs: TcI and TrC in two R. pallescens (Table 2 and Fig. 2); and TcI and TcIV in R. brethesi (Table 2). Examination of FFLB patterns In conclusion, FFLB is a useful tool for the identification of a wide range of American trypanosomes and for lineage identification of T. cruzi and T. rangeli. The technique is quick and sensitive, as it relies on amplification of relatively small regions of DNA and fluorescence detection and is able to differentiate mixed infections. In previous studies, FFLB genotypes have been obtained from DNA isolated directly from blood (Adams et al., 2009) and digestive tracts/proboscides of insects (Hamilton et al., 2008). In the present study, mixed cultures with two species or two lineages were detected using DNA from primary cultures from the guts of triatomines, so it seems likely that American trypanosomes could also be identified without prior use of culturing, thus avoiding selection of species/genotypes. The present study also highlighted the limitations of the FFLB technique, particularly for characterisation of T. cruzi strains. These were length polymorphism within single isolates, hybrid strains and their evolutionary predecessors giving matching profiles and the inability to identify TcI sublineages. However, additional regions, such as the intergenic spacer of spliced-leader genes, which is known to vary in length between some TcI sublineages (Cura et al., in press), could also be included to provide further discrimination. FFLB also requires access to a DNA sequencer and knowledge of the system, so is not suitable for diagnosis in rural settings. Nevertheless, its ability differentiate many known (and potentially unknown) species and several of their lineages, using the same primer sets, is unique and offers an advantage over other established methods for identifying American trypanosomes and should facilitate large scale diagnostics and epidemiological studies. Acknowledgements We thank M. Tibayrenc, C. Barnabe, P. Diosque, Hernan Carrasco, Angela C.V. Junqueira, Vera C. Valente, Arlei Marcili, Luciana Lima and for samples used in this study. Funding from Wellcome Trust, CNPq-Brazil and EC FP7 project ChagasEpiNet. 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