Hindawi Publishing CorporationISRN ParasitologyVolume 2013 Article ID 328794 7 pageshttpdxdoiorg1054022013328794

Research ArticleFirst Report of Trypanosoma sp in Spectacled Caiman(Caiman crocodilus) Morphological and PhylogeneticRelationships

Arlei Marcili Andrea P da Costa Herbert S Soares Igor C L Acosta Julia T R de LimaAntonio H H Minervino and Solange M Gennari

Faculdade de Medicina Veterinaria e Zootecnia Universidade de Sao Paulo Avenida Prof Orlando Marques de Paiva 87Cidade Universitaria 05508-270 Sao Paulo SP Brazil

Correspondence should be addressed to Arlei Marcili amarciliuspbr

Received 5 June 2013 Accepted 17 July 2013

Academic Editors D Cone A Jabbar G Mkoji and P Somboon

Copyright copy 2013 Arlei Marcili et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

In Crocodylidae family three trypanosomes species were described T grayi in African crocodilian and T cecili and Trypanosomasp in Caimans species from Brazil T grayi was transmitted by tsetse flies and the vector of Brazilian caimans trypanosomes isunknown We characterized first Brazilian trypanosome isolated in spectacled caiman (Caiman crocodilus) from Mato GrossoState in Brazil Morphological findings in epimastigotes forms from axenic culture showed high similarity with Trypanosomasp described in Caiman yacare from Brazilian Pantanal Phylogenetic studies performed with SSU rDNA and gGAPDH(glyceraldehydes-3-phosphato dehydrogenase glycosomal) clustering in T grayi Clade and together to genotype Cay 01 fromTrypanosoma unnamed species isolated in C yacare This is the first isolate of Trypanosoma sp from C crocodilus and thephylogenetic position with isolates in C yacare from Pantanal region and demonstrates the low host specificity of caymantrypanosomes in Brazil

1 Introduction

The order Crocodylia includes 23 living species and threefamilies are recognized Crocodylidae Gavialidae and Alli-gatoridae [1] Most species of families Crocodylidae andGavialidae occur in Africa and Asia Only Alligatoridaeoccurs in South America and is composed of six speciesPaleosuchus palpebrosus P trigonatus Melanosuchus nigerCaiman yacare C latirostris and C crocodilus [2]

The spectacled caiman (Caiman crocodilus) has thewidestdistribution of the New World crocodilians with geographicrange from southern Mexico to Peru and Brazil This geo-graphic variability enabled a segregation of this species intofour subspecies [3 4] A single subspecies occur in North andCentral Brazil Caiman crocodilus crocodilus [5]

The crocodilians are host to a wide variety of parasiteslike intestinal parasites (nematodes and trematodes) [6ndash10]

and hemoparasites (haemogegarines and trypanosomes) [10ndash13]

The species of genus Trypanosoma are parasites of allvertebrate classes (fish amphibians reptiles birds andmammals) with life cycles alternating between vertebratesand invertebrates hosts Most species develop in arthropodvectors which may belong to different orders and familieswhile fish amphibian and reptiles parasites are transmittedby leeches or insects Other species are only mechanicallytransmittedThis genus has several stages present in differentcombinations in blood andor tissues in the vertebrate andinvertebrate hosts [14ndash19]

In reptiles are described about 80 species of Trypanosomaparasites including 42 in lizards 14 in turtles 21 in snakesand 3 in crocodilians The species descriptions are based onmorphology of blood forms host and geographic origin [20]In Africa T grayi has been described inOsteolaemus tetraspis

2 ISRN Parasitology

and Crocodilus niloticus both in family Crocodylidae [21]In Brazil Trypanosoma unnamed species was described inCaiman yacare [22] and T cecili in Caiman crocodilus [13]

Phylogenetic studies performed with a large numberof isolates from Africa (T grayi) and Brazil (Trypanosomasp) positioned all sequences from whole SSU rDNA andgGAPDH in a unique monophyletic branch named T grayiClade [19 23 24]The Brazilian isolates are segregated in twogenotypes named Cay01 and Cay02 and are different from Tgrayi isolates [19]The host of Brazilian trypanosomes isolatesis Caiman yacare captured in Pantanal region

In the present study we described a first record ofTrypanosoma sp in spectacled caiman (Caiman crocodilus)from Mato Grosso State and addressed the phylogeneticrelationships with other crocodilian trypanosomes

2 Materials and Methods

21 Study Areas and Capture of Spectacled Caiman Thesample was collected from a single animal captured by ethnicIndians Tapirape within the area of their Indian reservein Mato Grosso State county of Confresa (10∘3810158402210158401015840 S51∘3410158400810158401015840W) composed of a mosaic of Amazonian RainForest and Cerrado biomes The animal included in thisstudy was obtained from legal hunting activity authorized byIBAMA held by the indigenous populationThree specimensof C crocodilus after being handed and immobilized wereanaesthetized and the blood samples and tissue samples(spleen heart kidney lung and liver) were collected

22 Isolation in Culture of Trypanosoma sp from SpectacledCaiman For Trypanosoma isolation blood samples fromspectacled caiman (Caiman crocodilus) were inoculated invacutainer tubes with a biphasic medium containing 15of sheep red blood cells with 4 Blood Agar Base andoverlaid with liquid LIT medium supplemented with 20of FBS as before [19] The culture was incubated at 28∘Cand expanded in LIT (Liver Infusion Tryptose) medium forDNA preparation The isolate (CBT 02) was cryopreservedin liquid nitrogen in the Department of Preventive Veteri-nary Medicine and Animal Health Faculty of VeterinaryMedicine University of Sao Paulo Samples from cultureswere smeared on glass slides and fixed with methanol andstained with Giemsa and photographed

23 Molecular Study DNA sample was extracted fromparasite mass of trypanosome culture using the phenol-chloroform method and primary samples were purifiedusing the Wizard DNA Clean-Up System (Promega Fitch-burg Wisconsin) Extracted DNA samples were subjectedto conventional polymerase chain reaction (PCR) targetinga fragment of approximately 900 base pairs (bp) of theV7V8 SSU rDNA [18 19] and approximately 800 bp ofthe gGAPDH (glyceraldehydes-3-phosphate dehydrogenaseglycosomal) as previously described [23] PCR products ofthe expected sizes were purified and sequenced in an auto-matic sequencer (Applied BiosystemsPerkinElmer modelABI Prism 310 Genetic Foster City CA) according to the

manufacturerrsquos recommendations The nucleotide sequencesgenerated were deposited in GenBank under the accessionnumbers JQ768791 and JQ768792 respectively for V7V8 SSUrDNA and gGAPDH genes These sequences were concate-nated and aligned using ClustalX [25] and adjusted manuallyusing GeneDoc software [26] with sequences previouslydetermined from other trypanosomatids species availablein Genbank (Table 1) The V7V8 region of SSU rDNA ofcrocodilian isolates was used to construct a dendogram forintraspecific analysis The phylogenetic tree was inferredby the maximum parsimony (MP) method using PAUPversion 40b10 [27] with 500 replicates of random additiontaxa and TBR branch swapping Bayesian analysis (B) wasperformed by Mrs Bayes v312 [28] and 1000000 genera-tions were employed as using GTR substitution model andfour categories range proportion of invariant sites Posterioriprobabilities were used to support branches

3 Results

The positive hemoculture obtained from a single spectacledcaiman captured generated one isolate maintained in culture(33) Cultures were maintained in axenic medium LIT andused for morphological analysis Unfortunately no smearswere obtained from the blood of the spectacled caimanAll tissues including the positive animal were negative totrypanosome DNA barcoding

The morphology consisted of epimastigotes with a largekinetoplast positioned near the nucleus and small and narrowundulating membrane (Figure 1) similar to the morphotype 1in C yacare trypanosomes previously described [19]

Wedetermined sequences fromgGAPDHand SSU rDNA(V7-V8 region) and these sequences were aligned withsequences from different trypanosomatids species retrievedfrom GenBank (Table 1)

Phylogenetic relationships based on gGAPDH and SSUrDNA sequences inferred by parsimony and Bayesian clus-tered this new isolate together to the major branch whichcorresponds to crocodilian trypanosomes isolates (T grayiClade) (Figures 2(a) and 2(b)) The T grayi Clade includedboth African crocodilians trypanosomes and American cay-mans trypanosomes The isolates from Brazilian caimanswere segregated into genotypes assigned Cay01 and Cay02previously described [19]

Despite distinct host and different geographic originsthe isolate from C crocodilus and Cay01 genotype fromC yacare shared high similarity (sim01 for gGAPDH andidentical SSU rDNA V7-V8 region) Phylogenetic analysisusing the concatenated data set of gGAPDH and SSU rDNAgenes generated very similar phylogenetic topologies (datanot show)

4 Discussion

In Brazil only two trypanosomes have been reported inCaiman species based on morphological analysis of bloodtrypomastigote unnamed species in C yacare from Pantanal[22] and Trypanosoma cecili in C crocodilus from Amazonia

ISRN Parasitology 3

Table 1 Trypanosomatids isolates host geographic origin and sequences of SSU rDNA and gGAPDH used for phylogenetic analysis

Trypanosomatids species Isolate code Host Geographic origin Accession numbera

gGAPDH SSU rDNAFish

Trypanosoma sp CLAR Clarias angolensis Africa AJ620251Trypanosoma sp MARV Cyprinus carpio Czech Republic AJ620248T granulosum Portugal Anguilla anguilla Portugal AJ620247T granulosum UK Anguilla anguilla United Kingdom AJ620246

ReptileTrypanosoma sp Gecko Tarentola annularis Senegal AJ620259T varani V54 Varanus exanthematicus Senegal AJ620261Trypanosoma sp T610 Caiman yacare Brazil EU596256 EU596252Trypanosoma sp T624 Caiman yacare Brazil EU596257 EU596253Trypanosoma sp T625 Caiman yacare Brazil EU596259Trypanosoma sp T1092 Caiman yacare Brazil EU596258 EU596254Trypanosoma sp T1100 Caiman yacare Brazil EU596260Trypanosoma sp T1101 Caiman yacare Brazil EU596261Trypanosoma sp T1102 Caiman yacare Brazil EU596262Trypanosoma sp T1119 Caiman yacare Brazil EU596263Trypanosoma sp T1120 Caiman yacare Brazil EU596255Trypanosoma sp CBT02 Caiman crocodilus Brazil JQ768791 JQ768792

BirdT avium Chaffinch Fringilla coelebs Czech Republic AJ620263T avium Rook Corvus frugilegus Czech Republic AJ620262

MammalsT conorhini USP Rattus rattus Brazil AJ620267T cruzi marinkellei B7 Phyllostomus hastatus Brazil AJ620270T cruzi X52898T lewisi L32 Rattus rattus AJ620272T microti TRL132 Microtus agrestis England AJ620273Trypanosoma sp ABF Wallabia bicolor Australia AJ620276Trypanosoma sp R5 Oryctolagus cuniculus Australia AJ620276T theileri K127 Bos taurus Germany AJ620282Trypanosoma sp D30 Cervus dama Germany AJ620279T dionisii P3 Pipistrellus pipistrellus United Kingdom AJ620271T vespertilionis P14 Pipistrellus pipistrellus England AJ620283T brucei rhodesiense 058 Homo sapiens Zambia AJ620284T vivax Desowitz Ovis aries Nigeria AJ620295T vivax AF053744T congolense Forest Cam22 Capra capra Cameroon AJ620289T congolense Forest TSW103 Sus scrofa Liberia AJ620286T evansi AF053743T cyclops LV492 Macaca sp Malaysia AJ620265T minasense LSTM Saimiri boliviensis South America AJ620274T rangeli AF053742

InvertebrateCrithidia fasciculata AF047493T congolense Forest ANR3 Glossina palpalis Gambia AJ620285T congolense Tsavo 114 Glossina pallidipes Tanzania AJ620291T cruzi C8 cl2 Triatoma infestans Bolivia AJ620268T cruzi VINCH 89 Triatoma infestans Chile AJ620269

4 ISRN Parasitology

Table 1 Continued

Trypanosomatids species Isolate code Host Geographic origin Accession numbera

gGAPDH SSU rDNAT grayi ANR4 Glossina palpalis Gambia AJ620257 AJ005278T grayi BAN1 Glossina palpalis Gambia AJ620258 AJ620546T simiae Ken 2 Glossina morsitans Gambia AJ620293T godfreyi Ken 7 Glossina morsitans Gambia AJ620292T simiae Tsavo Ketri 1864 Glossina pallidipes Kenya AJ620294Trypanosoma sp TLAQ22 Philaemon sp Australia AJ620280aSequences determined in this study and deposited in GenBank are underlined and bold

F

F

F

F

N

N

N

N N

K

K

K

K

K

(a)

(b)

(d)

(e)(c)10120583m

Figure 1 Microphotographs of Giemsa-stained trypanosome formsfromCayman crocodilus (CBT 02) obtained by in vitro culture (LIT)Epimastigotes forms (a b c) and trypomastigotes forms (d e) Kkinetoplast N nucleus F flagellum

[13] Recently trypanosomes isolates were obtained fromC yacare in Pantanal from Mato Grosso do Sul State andshowed similar morphology of blood trypomastigote withTrypanosoma sp described previously [19 22] In this presentstudy we obtained the first trypanosome isolate from Ccrocodilus

The prevalence of Trypanosoma sp in C yacare variesfrom 35 to 46 in different studies performed in Pantanalregion from Mato Grosso do Sul state [19 22] Only onespectacled caiman captured in Mato Grosso was positive inhemoculture (33) but the prevalence in this species has notbeen established and a further study involving a large numberof individuals (C crocodilus) is necessary to determine theprevalence of Trypanosoma sp and T cecili

Most studies of caiman trypanosomes were performedin the Pantanal region The only study in Amazon withC crocodilus and Paleosuchus trigonatus found a very lowprevalence with rare trypomastigote forms in blood or tissueThis species was designated T cecili and isolation has not yetbeen possible [13]

The epimastigotes forms in axenic culture of spectacledcaiman isolate are very similar to Trypanosoma sp from Cyacare from Pantanal (Figure 1) The tissue imprint forms ofT cecili and blood forms of Trypanosoma sp from Pantanalare distinguishable but similar tissue forms are detected inCyacare and C crocodilus [13 19 22]

The variableV7-V8 region of SSU rDNAhas been used forDNAbarcoding of trypanosomatids and is able to distinguishall species polymorphisms and genetic relationships amongclosely related taxa [18 19 29ndash33] In addition the gGAPDHsequences were used to improve the phylogenetic analysis[19 23 24 33] In all phylogenetic analyses inferred by V7-V8SSU rDNA and gGAPDH the isolated CBT 02 was includedin monophyletic branch called T grayi Clade together withTrypanosoma sp from Brazilian caiman The V7-V8 regionsegregated the Brazilian caimans isolates in two branchesCay01 and Cay02 Most isolates were comprised of Cay01genotype probably the most prevalent in Pantanal regionThe new isolate from C crocodilus (CBT 02) nested in Cay01genotype

The phylogenetic positions of CBT02 isolate show thatTrypanosoma sp from Cay01 genotype I is able to infecttwo species of Caiman genus in Brazil These findings clarifythe morphological approaches in tissues imprints whichhave trypomastigote similar morphology in C yacare and Ccrocodilus

The distribution of Cay01 genotype is not restricted toPantanal region of Mato Grosso do Sul state and vertebratehostThe Confresa county is located in north of Mato Grossostate and comprises a mosaic of Amazonia and Cerradobiomes and is irrigated by Tocantins-Araguaia basin The C

ISRN Parasitology 5

T sp T610

T grayi BAN1

T grayi ANR4

T sp T1119

T sp T1101T sp T624

T sp T1102

T sp T1092

T sp T1100

T sp T625

T sp T1120

110

Crithidia fasciculata

Trypanosoma genus

T granulosum UKT granulosumT sp MARVT sp CLAR

T sp T610

T grayi ANR4T grayi BAN1

T sp T1092T sp T624

T congolense ForestT congolense Forest

T congolense Forest

T congolense TsavoT simiae

T godfreyi

T simiae TsavoT brucei rhodesienseT evansi

T varaniT sp Gecko

T avium RookT avium Chaffinch

T sp TLAQ22T sp ABF

T cyclopsT theileri

T sp D30

T microtiT sp Habbit

T lewisi

T cruziT cruzi

T c marinkelleiT dionisii

T cruziT cruzi

T vespertilionisT conorhini

T rangeliT minasense

T vivaxT vivax

T grayi Clade

Cay01

Cay02

(a) (b)

100 100

100 100100 100

100 100

100 100100 100

100 100

100 100

100 100

100 100

100 100

100 100100 100

99 100

100 100

100 100

100 10097 100

88 100

91 8975 69

82 100

75 67

73 75 88 100

76 100

75 68

75 68

67 99

119931 sp CBT 02

119931 sp CBT 02

Figure 2 (a) Phylogenetic trees inferred by maximum parsimony and Bayesian methods based on gGAPDH gene sequences of 43trypanosomes and Crithidia fasciculata as outgroup (515 characters 34 parsimony informative) (b) Dendogram inferred by parsimonyanalyses based on V7-V8 SSU rDNA sequences from 12 crocodilian trypanosomes isolates (734 characters 24 parsimony informative)Numbers at nodes show bootstrap (first) and posterior probabilities (second) values for MP and BI respectively The support values forthe major branches are derived from 500 replicates respectively for MP

yacare occurred in upper Madeira system of the Amazonbasin and Paraguay River and lower Parana system includingBrazil Bolivia Paraguay and Argentina C crocodiles occurin both Amazon and Orinoco drainage [2 5] According toBrazaitis et al [34] sympatric areas occur inMatoGrosso andRondonia states However in Tocantins-Araguaia basin onlyC crocodilus occurs

The presence of the same trypanosome species inC yacare and C crocodilus indicates transmission cyclesbetween these species evidencing sympatry of caiman speciesor exposure to the same vector The T grayi in Africa istransmitted by tse-tse flies [35] In Brazil different speciesof tabanids can bite reptiles like C crocodilus and Eunectesmurinus [36] The low host specificity of tabanids and hostswitch of caiman trypanosomes suggest an important role ofinsects in the transmission cycle of these parasites in Brazil

The addition of new isolates of caiman trypanosomesof different vertebrate hosts is necessary for better under-standing of diversity and phylogenetic relationships of these

parasites and demonstrates the low host specificity of caymantrypanosomes in Brazil

Acknowledgments

The authors are grateful to several people for their invaluablehelp during capture This research was financially supportedby Fundacao de Amparo a Pesquisa do Estado de Sao Paulo(FAPESP) SolangeMGennari andAntonioHHMinervinoare in receipt of a fellowship from CNPq and Arlei MarciliHerbert S Soares Julia T R de Lima ATLM and Igor C LAcosta from FAPESP

References

[1] A Janke A Gullberg S Hughes R K Aggarwal and U Arna-son ldquoMitogenomic analyses place the gharial (Gavialis gangeti-cus) on the crocodile tree and provide pre-KT divergence timesfor most crocodiliansrdquo Journal of Molecular Evolution vol 61no 5 pp 620ndash626 2005

6 ISRN Parasitology

[2] J P Roos Crocodiles Status Survey and Conservation ActionPlan VIII 96 IUCN SS Crocodile Specialist Group GlandSwitzerland 2nd edition 1998

[3] F Medem Los Crocodylia De Sur America 1 ColcienciasBogota Colombia 1981

[4] F W King and R L Burke Crocodilian Tuatara and TurtleSpecies of the World A Taxonomic and Geographic ReferenceAssociation of Systematics Collections Washington DC USA1989

[5] A Velasco and J Ayarzaguena ldquoSpectacled Caiman Caimancrocodilosrdquo in Crocodiles Status Survey and ConservationAction Plan S C Manolis and C Stenvenson Eds 3rd edition2010

[6] K Junker D R Brooks and J Boomker ldquoProctocaecum gairhein sp (Digenea Cryptogonimidae Acanthostominae) fromGavialis gangeticus (Gmelin) in Nepal and a revised cladogramof Proctocaecum Baugh 1957rdquo Systematic Parasitology vol 71no 3 pp 229ndash236 2008

[7] V V Tkach E J Strand and L Froese ldquoMacroderoides texanusn sp (Digenea Macroderoididae) from alligator gar Atractos-teus spatula in Texasrdquo Parasitology Research vol 104 no 1 pp27ndash33 2008

[8] L J La Grange G Marucci and E Pozio ldquoTrichinella zimbab-wensis in wild Nile crocodiles (Crocodylus niloticus) of SouthAfricardquoVeterinary Parasitology vol 161 no 1-2 pp 88ndash91 2009

[9] L J La Grange D Govender and S Mukaratirwa ldquoTheoccurrence of Trichinella zimbabwensis in naturally infectedwild crocodiles (Crocodylus niloticus) from the Kruger NationalPark South Africardquo Journal of Helminthology vol 16 pp 1ndash62012

[10] A R Waddle J M Kinsella J P Ross E Rojas-Flores H FPercival and D J Forrester ldquoNematodes collected by gastriclavage from live American Alligators Alligator mississippiensisin Floridardquo Journal of Parasitology vol 95 no 5 pp 1237ndash12382009

[11] L A Viana and E J Marques ldquoHaemogregarine parasites(Apicomplexa Hepatozoidae) in Caiman crocodilus yacare(Crocodilia Alligatoridae) from Pantanal Corumba MSBrazilrdquo Revista Brasileira de Parasitologia Veterinaria vol 14no 4 pp 173ndash175 2005

[12] L A Viana F Paiva M E Coutinho and R Lourenco-De-Oliveira ldquoHepatozoon caimani (Apicomplexa Hepatozoidae)in Wild Caiman Caiman yacare from the Pantanal RegionBrazilrdquo Journal of Parasitology vol 96 no 1 pp 83ndash88 2010

[13] R Lainson ldquoTrypanosoma cecili n sp a parasite of the SouthAmerican cayman Caiman crocodilus crocodilus (Linnaeus1758) (Crocodilia Alligatoridae)rdquo Protozoology vol 3 pp 87ndash93 1977

[14] C A Hoare The Trypanosomes of Mammals A ZoologicalMonograph Blackwell Scientific Oxford UK 1972

[15] K Vickerman ldquoThe evolutionary expansion of the trypanoso-matid flagellatesrdquo International Journal for Parasitology vol 24no 8 pp 1317ndash1331 1994

[16] J R Stevens H A Noyes C J Schofield and W GibsonldquoThe molecular evolution of trypanosomatidaerdquo Advances inParasitology vol 48 pp 1ndash56 2001

[17] A G B Simpson J R Stevens and J Lukes ldquoThe evolution anddiversity of kinetoplastid flagellatesrdquo Trends in Parasitology vol22 no 4 pp 168ndash174 2006

[18] R C Ferreira A A De Souza R A Freitas et al ldquoA phyloge-netic lineage of closely related trypanosomes (Trypanosomati-dae Kinetoplastida) of anurans and sand flies (Psychodidae

Diptera) sharing the same ecotopes in Brazilian AmazoniardquoJournal of Eukaryotic Microbiology vol 55 no 5 pp 427ndash4352008

[19] L B Viola M Campaner C A Takata et al ldquoPhylogenyof snake trypanosomes inferred by SSU rDNA sequencestheir possible transmission by phlebotomines and taxonomicappraisal bymolecular cross-infection andmorphological anal-ysisrdquo Parasitology vol 135 no 5 pp 595ndash605 2008

[20] S R Telford ldquoThe kinetoplastid hemoflagellates of reptilesrdquo inParasitic Protozoa J P Kreier Ed vol 10 Academic press SanDiego Calif USA 2nd edition 1995

[21] L Lloyd W B Johnson and W A Young ldquoThe trypanosomeinfections of tsetse flies in northern Nigeria and a new methodof estimationrdquo in Bulletin of Entomology Research vol 14 pp265ndash288 1924

[22] V L B Nunes and E T Oshiro ldquoTrypanosoma sp em jacarCaiman crocodilus yacare (Daudin 1802) (Crocodilia Alliga-toridae)rdquo Semina vol 11 pp 62ndash65 1990

[23] P B Hamilton J R Stevens M W Gaunt J Gidley and WC Gibson ldquoTrypanosomes are monophyletic evidence fromgenes for glyceraldehyde phosphate dehydrogenase and smallsubunit ribosomal RNArdquo International Journal for Parasitologyvol 34 no 12 pp 1393ndash1404 2004

[24] P B Hamilton W C Gibson and J R Stevens ldquoPatterns ofco-evolution between trypanosomes and their hosts deducedfrom ribosomal RNA and protein-coding gene phylogeniesrdquoMolecular Phylogenetics and Evolution vol 44 no 1 pp 15ndash252007

[25] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997

[26] K B Nicholas H B Nicholas Jr and D W Deerfield ldquoGene-Doc analysis and visualization of genetic variationrdquo EMBNEWNEWS vol 4 p 14 1997

[27] D L Swofford ldquoPAUPlowast Phylogenetic Analysis Using Parsi-mony (and Other Methods) Beta Version 4 0b10rdquo Sinauer andAssociates Sunderland Mass USA 2002

[28] J P Huelsenbeck and F Ronquist ldquoMRBAYES bayesian infer-ence of phylogenetic treesrdquo Bioinformatics vol 17 no 8 pp754ndash755 2001

[29] F M Da Silva H Noyes M Campaner et al ldquoPhylogenytaxonomy and grouping of Trypanosoma rangeli isolates fromman triatomines and sylvatic mammals from widespread geo-graphical origin based on SSU and ITS ribosomal sequencesrdquoParasitology vol 129 no 5 pp 549ndash561 2004

[30] A P Cortez R M Ventura A C Rodrigues et al ldquoThetaxonomic and phylogenetic relationships of Trypanosomavivax from South America and Africardquo Parasitology vol 133no 2 pp 159ndash169 2006

[31] A C Rodrigues F Paiva M Campaner J R Stevens H ANoyes and M M G Teixeira ldquoPhylogeny of Trypanosoma(Megatrypanum) theileri and related trypanosomes revealslineages of isolates associated with artiodactyl hosts divergingon SSU and ITS ribosomal sequencesrdquo Parasitology vol 132 no2 pp 215ndash224 2006

[32] A Marcili V C Valente S A Valente et al ldquoTrypanosomacruzi in Brazilian Amazonia lineages TCI and TCIIa in wildprimates Rhodnius spp and in humans with Chagas diseaseassociated with oral transmissionrdquo International Journal forParasitology vol 39 no 5 pp 615ndash623 2009

ISRN Parasitology 7

[33] M Cavazzana Jr A Marcili L Lima et al ldquoPhylogeo-graphical ecological and biological patterns shown by nuclear(ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes oftrypanosomes of the subgenus Schizotrypanum parasitic inBrazilian batsrdquo International Journal for Parasitology vol 40 no3 pp 315ndash320 2010

[34] P Brazaitis M EWatanabe and G Amato ldquoThe caiman traderdquoScience American vol 278 pp 52ndash58 1998

[35] J J McNamara and W F Snow ldquoImproved identification ofNannomonas infections in tsetse flies from The Gambiardquo ActaTropica vol 48 no 2 pp 127ndash136 1990

[36] R L M Ferreira A L Henriques and J A Rafael ldquoActivityof tabanids (Insecta Diptera Tabanidae) attacking the reptilesCaiman crocodilus (Linn) (Alligatoridae) and Eunectesmurinus(Linn) (Boidae) in the Central Amazon Brazilrdquo Memorias doInstituto Oswaldo Cruz vol 97 no 1 pp 133ndash136 2002

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2 ISRN Parasitology

and Crocodilus niloticus both in family Crocodylidae [21]In Brazil Trypanosoma unnamed species was described inCaiman yacare [22] and T cecili in Caiman crocodilus [13]

Phylogenetic studies performed with a large numberof isolates from Africa (T grayi) and Brazil (Trypanosomasp) positioned all sequences from whole SSU rDNA andgGAPDH in a unique monophyletic branch named T grayiClade [19 23 24]The Brazilian isolates are segregated in twogenotypes named Cay01 and Cay02 and are different from Tgrayi isolates [19]The host of Brazilian trypanosomes isolatesis Caiman yacare captured in Pantanal region

In the present study we described a first record ofTrypanosoma sp in spectacled caiman (Caiman crocodilus)from Mato Grosso State and addressed the phylogeneticrelationships with other crocodilian trypanosomes

2 Materials and Methods

21 Study Areas and Capture of Spectacled Caiman Thesample was collected from a single animal captured by ethnicIndians Tapirape within the area of their Indian reservein Mato Grosso State county of Confresa (10∘3810158402210158401015840 S51∘3410158400810158401015840W) composed of a mosaic of Amazonian RainForest and Cerrado biomes The animal included in thisstudy was obtained from legal hunting activity authorized byIBAMA held by the indigenous populationThree specimensof C crocodilus after being handed and immobilized wereanaesthetized and the blood samples and tissue samples(spleen heart kidney lung and liver) were collected

22 Isolation in Culture of Trypanosoma sp from SpectacledCaiman For Trypanosoma isolation blood samples fromspectacled caiman (Caiman crocodilus) were inoculated invacutainer tubes with a biphasic medium containing 15of sheep red blood cells with 4 Blood Agar Base andoverlaid with liquid LIT medium supplemented with 20of FBS as before [19] The culture was incubated at 28∘Cand expanded in LIT (Liver Infusion Tryptose) medium forDNA preparation The isolate (CBT 02) was cryopreservedin liquid nitrogen in the Department of Preventive Veteri-nary Medicine and Animal Health Faculty of VeterinaryMedicine University of Sao Paulo Samples from cultureswere smeared on glass slides and fixed with methanol andstained with Giemsa and photographed

23 Molecular Study DNA sample was extracted fromparasite mass of trypanosome culture using the phenol-chloroform method and primary samples were purifiedusing the Wizard DNA Clean-Up System (Promega Fitch-burg Wisconsin) Extracted DNA samples were subjectedto conventional polymerase chain reaction (PCR) targetinga fragment of approximately 900 base pairs (bp) of theV7V8 SSU rDNA [18 19] and approximately 800 bp ofthe gGAPDH (glyceraldehydes-3-phosphate dehydrogenaseglycosomal) as previously described [23] PCR products ofthe expected sizes were purified and sequenced in an auto-matic sequencer (Applied BiosystemsPerkinElmer modelABI Prism 310 Genetic Foster City CA) according to the

manufacturerrsquos recommendations The nucleotide sequencesgenerated were deposited in GenBank under the accessionnumbers JQ768791 and JQ768792 respectively for V7V8 SSUrDNA and gGAPDH genes These sequences were concate-nated and aligned using ClustalX [25] and adjusted manuallyusing GeneDoc software [26] with sequences previouslydetermined from other trypanosomatids species availablein Genbank (Table 1) The V7V8 region of SSU rDNA ofcrocodilian isolates was used to construct a dendogram forintraspecific analysis The phylogenetic tree was inferredby the maximum parsimony (MP) method using PAUPversion 40b10 [27] with 500 replicates of random additiontaxa and TBR branch swapping Bayesian analysis (B) wasperformed by Mrs Bayes v312 [28] and 1000000 genera-tions were employed as using GTR substitution model andfour categories range proportion of invariant sites Posterioriprobabilities were used to support branches

3 Results

The positive hemoculture obtained from a single spectacledcaiman captured generated one isolate maintained in culture(33) Cultures were maintained in axenic medium LIT andused for morphological analysis Unfortunately no smearswere obtained from the blood of the spectacled caimanAll tissues including the positive animal were negative totrypanosome DNA barcoding

The morphology consisted of epimastigotes with a largekinetoplast positioned near the nucleus and small and narrowundulating membrane (Figure 1) similar to the morphotype 1in C yacare trypanosomes previously described [19]

Wedetermined sequences fromgGAPDHand SSU rDNA(V7-V8 region) and these sequences were aligned withsequences from different trypanosomatids species retrievedfrom GenBank (Table 1)

Phylogenetic relationships based on gGAPDH and SSUrDNA sequences inferred by parsimony and Bayesian clus-tered this new isolate together to the major branch whichcorresponds to crocodilian trypanosomes isolates (T grayiClade) (Figures 2(a) and 2(b)) The T grayi Clade includedboth African crocodilians trypanosomes and American cay-mans trypanosomes The isolates from Brazilian caimanswere segregated into genotypes assigned Cay01 and Cay02previously described [19]

Despite distinct host and different geographic originsthe isolate from C crocodilus and Cay01 genotype fromC yacare shared high similarity (sim01 for gGAPDH andidentical SSU rDNA V7-V8 region) Phylogenetic analysisusing the concatenated data set of gGAPDH and SSU rDNAgenes generated very similar phylogenetic topologies (datanot show)

4 Discussion

In Brazil only two trypanosomes have been reported inCaiman species based on morphological analysis of bloodtrypomastigote unnamed species in C yacare from Pantanal[22] and Trypanosoma cecili in C crocodilus from Amazonia

ISRN Parasitology 3

Table 1 Trypanosomatids isolates host geographic origin and sequences of SSU rDNA and gGAPDH used for phylogenetic analysis

Trypanosomatids species Isolate code Host Geographic origin Accession numbera

gGAPDH SSU rDNAFish

Trypanosoma sp CLAR Clarias angolensis Africa AJ620251Trypanosoma sp MARV Cyprinus carpio Czech Republic AJ620248T granulosum Portugal Anguilla anguilla Portugal AJ620247T granulosum UK Anguilla anguilla United Kingdom AJ620246

ReptileTrypanosoma sp Gecko Tarentola annularis Senegal AJ620259T varani V54 Varanus exanthematicus Senegal AJ620261Trypanosoma sp T610 Caiman yacare Brazil EU596256 EU596252Trypanosoma sp T624 Caiman yacare Brazil EU596257 EU596253Trypanosoma sp T625 Caiman yacare Brazil EU596259Trypanosoma sp T1092 Caiman yacare Brazil EU596258 EU596254Trypanosoma sp T1100 Caiman yacare Brazil EU596260Trypanosoma sp T1101 Caiman yacare Brazil EU596261Trypanosoma sp T1102 Caiman yacare Brazil EU596262Trypanosoma sp T1119 Caiman yacare Brazil EU596263Trypanosoma sp T1120 Caiman yacare Brazil EU596255Trypanosoma sp CBT02 Caiman crocodilus Brazil JQ768791 JQ768792

BirdT avium Chaffinch Fringilla coelebs Czech Republic AJ620263T avium Rook Corvus frugilegus Czech Republic AJ620262

MammalsT conorhini USP Rattus rattus Brazil AJ620267T cruzi marinkellei B7 Phyllostomus hastatus Brazil AJ620270T cruzi X52898T lewisi L32 Rattus rattus AJ620272T microti TRL132 Microtus agrestis England AJ620273Trypanosoma sp ABF Wallabia bicolor Australia AJ620276Trypanosoma sp R5 Oryctolagus cuniculus Australia AJ620276T theileri K127 Bos taurus Germany AJ620282Trypanosoma sp D30 Cervus dama Germany AJ620279T dionisii P3 Pipistrellus pipistrellus United Kingdom AJ620271T vespertilionis P14 Pipistrellus pipistrellus England AJ620283T brucei rhodesiense 058 Homo sapiens Zambia AJ620284T vivax Desowitz Ovis aries Nigeria AJ620295T vivax AF053744T congolense Forest Cam22 Capra capra Cameroon AJ620289T congolense Forest TSW103 Sus scrofa Liberia AJ620286T evansi AF053743T cyclops LV492 Macaca sp Malaysia AJ620265T minasense LSTM Saimiri boliviensis South America AJ620274T rangeli AF053742

InvertebrateCrithidia fasciculata AF047493T congolense Forest ANR3 Glossina palpalis Gambia AJ620285T congolense Tsavo 114 Glossina pallidipes Tanzania AJ620291T cruzi C8 cl2 Triatoma infestans Bolivia AJ620268T cruzi VINCH 89 Triatoma infestans Chile AJ620269

4 ISRN Parasitology

Table 1 Continued

Trypanosomatids species Isolate code Host Geographic origin Accession numbera

gGAPDH SSU rDNAT grayi ANR4 Glossina palpalis Gambia AJ620257 AJ005278T grayi BAN1 Glossina palpalis Gambia AJ620258 AJ620546T simiae Ken 2 Glossina morsitans Gambia AJ620293T godfreyi Ken 7 Glossina morsitans Gambia AJ620292T simiae Tsavo Ketri 1864 Glossina pallidipes Kenya AJ620294Trypanosoma sp TLAQ22 Philaemon sp Australia AJ620280aSequences determined in this study and deposited in GenBank are underlined and bold

F

F

F

F

N

N

N

N N

K

K

K

K

K

(a)

(b)

(d)

(e)(c)10120583m

Figure 1 Microphotographs of Giemsa-stained trypanosome formsfromCayman crocodilus (CBT 02) obtained by in vitro culture (LIT)Epimastigotes forms (a b c) and trypomastigotes forms (d e) Kkinetoplast N nucleus F flagellum

[13] Recently trypanosomes isolates were obtained fromC yacare in Pantanal from Mato Grosso do Sul State andshowed similar morphology of blood trypomastigote withTrypanosoma sp described previously [19 22] In this presentstudy we obtained the first trypanosome isolate from Ccrocodilus

The prevalence of Trypanosoma sp in C yacare variesfrom 35 to 46 in different studies performed in Pantanalregion from Mato Grosso do Sul state [19 22] Only onespectacled caiman captured in Mato Grosso was positive inhemoculture (33) but the prevalence in this species has notbeen established and a further study involving a large numberof individuals (C crocodilus) is necessary to determine theprevalence of Trypanosoma sp and T cecili

Most studies of caiman trypanosomes were performedin the Pantanal region The only study in Amazon withC crocodilus and Paleosuchus trigonatus found a very lowprevalence with rare trypomastigote forms in blood or tissueThis species was designated T cecili and isolation has not yetbeen possible [13]

The epimastigotes forms in axenic culture of spectacledcaiman isolate are very similar to Trypanosoma sp from Cyacare from Pantanal (Figure 1) The tissue imprint forms ofT cecili and blood forms of Trypanosoma sp from Pantanalare distinguishable but similar tissue forms are detected inCyacare and C crocodilus [13 19 22]

The variableV7-V8 region of SSU rDNAhas been used forDNAbarcoding of trypanosomatids and is able to distinguishall species polymorphisms and genetic relationships amongclosely related taxa [18 19 29ndash33] In addition the gGAPDHsequences were used to improve the phylogenetic analysis[19 23 24 33] In all phylogenetic analyses inferred by V7-V8SSU rDNA and gGAPDH the isolated CBT 02 was includedin monophyletic branch called T grayi Clade together withTrypanosoma sp from Brazilian caiman The V7-V8 regionsegregated the Brazilian caimans isolates in two branchesCay01 and Cay02 Most isolates were comprised of Cay01genotype probably the most prevalent in Pantanal regionThe new isolate from C crocodilus (CBT 02) nested in Cay01genotype

The phylogenetic positions of CBT02 isolate show thatTrypanosoma sp from Cay01 genotype I is able to infecttwo species of Caiman genus in Brazil These findings clarifythe morphological approaches in tissues imprints whichhave trypomastigote similar morphology in C yacare and Ccrocodilus

The distribution of Cay01 genotype is not restricted toPantanal region of Mato Grosso do Sul state and vertebratehostThe Confresa county is located in north of Mato Grossostate and comprises a mosaic of Amazonia and Cerradobiomes and is irrigated by Tocantins-Araguaia basin The C

ISRN Parasitology 5

T sp T610

T grayi BAN1

T grayi ANR4

T sp T1119

T sp T1101T sp T624

T sp T1102

T sp T1092

T sp T1100

T sp T625

T sp T1120

110

Crithidia fasciculata

Trypanosoma genus

T granulosum UKT granulosumT sp MARVT sp CLAR

T sp T610

T grayi ANR4T grayi BAN1

T sp T1092T sp T624

T congolense ForestT congolense Forest

T congolense Forest

T congolense TsavoT simiae

T godfreyi

T simiae TsavoT brucei rhodesienseT evansi

T varaniT sp Gecko

T avium RookT avium Chaffinch

T sp TLAQ22T sp ABF

T cyclopsT theileri

T sp D30

T microtiT sp Habbit

T lewisi

T cruziT cruzi

T c marinkelleiT dionisii

T cruziT cruzi

T vespertilionisT conorhini

T rangeliT minasense

T vivaxT vivax

T grayi Clade

Cay01

Cay02

(a) (b)

100 100

100 100100 100

100 100

100 100100 100

100 100

100 100

100 100

100 100

100 100

100 100100 100

99 100

100 100

100 100

100 10097 100

88 100

91 8975 69

82 100

75 67

73 75 88 100

76 100

75 68

75 68

67 99

119931 sp CBT 02

119931 sp CBT 02

Figure 2 (a) Phylogenetic trees inferred by maximum parsimony and Bayesian methods based on gGAPDH gene sequences of 43trypanosomes and Crithidia fasciculata as outgroup (515 characters 34 parsimony informative) (b) Dendogram inferred by parsimonyanalyses based on V7-V8 SSU rDNA sequences from 12 crocodilian trypanosomes isolates (734 characters 24 parsimony informative)Numbers at nodes show bootstrap (first) and posterior probabilities (second) values for MP and BI respectively The support values forthe major branches are derived from 500 replicates respectively for MP

yacare occurred in upper Madeira system of the Amazonbasin and Paraguay River and lower Parana system includingBrazil Bolivia Paraguay and Argentina C crocodiles occurin both Amazon and Orinoco drainage [2 5] According toBrazaitis et al [34] sympatric areas occur inMatoGrosso andRondonia states However in Tocantins-Araguaia basin onlyC crocodilus occurs

The presence of the same trypanosome species inC yacare and C crocodilus indicates transmission cyclesbetween these species evidencing sympatry of caiman speciesor exposure to the same vector The T grayi in Africa istransmitted by tse-tse flies [35] In Brazil different speciesof tabanids can bite reptiles like C crocodilus and Eunectesmurinus [36] The low host specificity of tabanids and hostswitch of caiman trypanosomes suggest an important role ofinsects in the transmission cycle of these parasites in Brazil

The addition of new isolates of caiman trypanosomesof different vertebrate hosts is necessary for better under-standing of diversity and phylogenetic relationships of these

parasites and demonstrates the low host specificity of caymantrypanosomes in Brazil

Acknowledgments

The authors are grateful to several people for their invaluablehelp during capture This research was financially supportedby Fundacao de Amparo a Pesquisa do Estado de Sao Paulo(FAPESP) SolangeMGennari andAntonioHHMinervinoare in receipt of a fellowship from CNPq and Arlei MarciliHerbert S Soares Julia T R de Lima ATLM and Igor C LAcosta from FAPESP

References

[1] A Janke A Gullberg S Hughes R K Aggarwal and U Arna-son ldquoMitogenomic analyses place the gharial (Gavialis gangeti-cus) on the crocodile tree and provide pre-KT divergence timesfor most crocodiliansrdquo Journal of Molecular Evolution vol 61no 5 pp 620ndash626 2005

6 ISRN Parasitology

[2] J P Roos Crocodiles Status Survey and Conservation ActionPlan VIII 96 IUCN SS Crocodile Specialist Group GlandSwitzerland 2nd edition 1998

[3] F Medem Los Crocodylia De Sur America 1 ColcienciasBogota Colombia 1981

[4] F W King and R L Burke Crocodilian Tuatara and TurtleSpecies of the World A Taxonomic and Geographic ReferenceAssociation of Systematics Collections Washington DC USA1989

[5] A Velasco and J Ayarzaguena ldquoSpectacled Caiman Caimancrocodilosrdquo in Crocodiles Status Survey and ConservationAction Plan S C Manolis and C Stenvenson Eds 3rd edition2010

[6] K Junker D R Brooks and J Boomker ldquoProctocaecum gairhein sp (Digenea Cryptogonimidae Acanthostominae) fromGavialis gangeticus (Gmelin) in Nepal and a revised cladogramof Proctocaecum Baugh 1957rdquo Systematic Parasitology vol 71no 3 pp 229ndash236 2008

[7] V V Tkach E J Strand and L Froese ldquoMacroderoides texanusn sp (Digenea Macroderoididae) from alligator gar Atractos-teus spatula in Texasrdquo Parasitology Research vol 104 no 1 pp27ndash33 2008

[8] L J La Grange G Marucci and E Pozio ldquoTrichinella zimbab-wensis in wild Nile crocodiles (Crocodylus niloticus) of SouthAfricardquoVeterinary Parasitology vol 161 no 1-2 pp 88ndash91 2009

[9] L J La Grange D Govender and S Mukaratirwa ldquoTheoccurrence of Trichinella zimbabwensis in naturally infectedwild crocodiles (Crocodylus niloticus) from the Kruger NationalPark South Africardquo Journal of Helminthology vol 16 pp 1ndash62012

[10] A R Waddle J M Kinsella J P Ross E Rojas-Flores H FPercival and D J Forrester ldquoNematodes collected by gastriclavage from live American Alligators Alligator mississippiensisin Floridardquo Journal of Parasitology vol 95 no 5 pp 1237ndash12382009

[11] L A Viana and E J Marques ldquoHaemogregarine parasites(Apicomplexa Hepatozoidae) in Caiman crocodilus yacare(Crocodilia Alligatoridae) from Pantanal Corumba MSBrazilrdquo Revista Brasileira de Parasitologia Veterinaria vol 14no 4 pp 173ndash175 2005

[12] L A Viana F Paiva M E Coutinho and R Lourenco-De-Oliveira ldquoHepatozoon caimani (Apicomplexa Hepatozoidae)in Wild Caiman Caiman yacare from the Pantanal RegionBrazilrdquo Journal of Parasitology vol 96 no 1 pp 83ndash88 2010

[13] R Lainson ldquoTrypanosoma cecili n sp a parasite of the SouthAmerican cayman Caiman crocodilus crocodilus (Linnaeus1758) (Crocodilia Alligatoridae)rdquo Protozoology vol 3 pp 87ndash93 1977

[14] C A Hoare The Trypanosomes of Mammals A ZoologicalMonograph Blackwell Scientific Oxford UK 1972

[15] K Vickerman ldquoThe evolutionary expansion of the trypanoso-matid flagellatesrdquo International Journal for Parasitology vol 24no 8 pp 1317ndash1331 1994

[16] J R Stevens H A Noyes C J Schofield and W GibsonldquoThe molecular evolution of trypanosomatidaerdquo Advances inParasitology vol 48 pp 1ndash56 2001

[17] A G B Simpson J R Stevens and J Lukes ldquoThe evolution anddiversity of kinetoplastid flagellatesrdquo Trends in Parasitology vol22 no 4 pp 168ndash174 2006

[18] R C Ferreira A A De Souza R A Freitas et al ldquoA phyloge-netic lineage of closely related trypanosomes (Trypanosomati-dae Kinetoplastida) of anurans and sand flies (Psychodidae

Diptera) sharing the same ecotopes in Brazilian AmazoniardquoJournal of Eukaryotic Microbiology vol 55 no 5 pp 427ndash4352008

[19] L B Viola M Campaner C A Takata et al ldquoPhylogenyof snake trypanosomes inferred by SSU rDNA sequencestheir possible transmission by phlebotomines and taxonomicappraisal bymolecular cross-infection andmorphological anal-ysisrdquo Parasitology vol 135 no 5 pp 595ndash605 2008

[20] S R Telford ldquoThe kinetoplastid hemoflagellates of reptilesrdquo inParasitic Protozoa J P Kreier Ed vol 10 Academic press SanDiego Calif USA 2nd edition 1995

[21] L Lloyd W B Johnson and W A Young ldquoThe trypanosomeinfections of tsetse flies in northern Nigeria and a new methodof estimationrdquo in Bulletin of Entomology Research vol 14 pp265ndash288 1924

[22] V L B Nunes and E T Oshiro ldquoTrypanosoma sp em jacarCaiman crocodilus yacare (Daudin 1802) (Crocodilia Alliga-toridae)rdquo Semina vol 11 pp 62ndash65 1990

[23] P B Hamilton J R Stevens M W Gaunt J Gidley and WC Gibson ldquoTrypanosomes are monophyletic evidence fromgenes for glyceraldehyde phosphate dehydrogenase and smallsubunit ribosomal RNArdquo International Journal for Parasitologyvol 34 no 12 pp 1393ndash1404 2004

[24] P B Hamilton W C Gibson and J R Stevens ldquoPatterns ofco-evolution between trypanosomes and their hosts deducedfrom ribosomal RNA and protein-coding gene phylogeniesrdquoMolecular Phylogenetics and Evolution vol 44 no 1 pp 15ndash252007

[25] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997

[26] K B Nicholas H B Nicholas Jr and D W Deerfield ldquoGene-Doc analysis and visualization of genetic variationrdquo EMBNEWNEWS vol 4 p 14 1997

[27] D L Swofford ldquoPAUPlowast Phylogenetic Analysis Using Parsi-mony (and Other Methods) Beta Version 4 0b10rdquo Sinauer andAssociates Sunderland Mass USA 2002

[28] J P Huelsenbeck and F Ronquist ldquoMRBAYES bayesian infer-ence of phylogenetic treesrdquo Bioinformatics vol 17 no 8 pp754ndash755 2001

[29] F M Da Silva H Noyes M Campaner et al ldquoPhylogenytaxonomy and grouping of Trypanosoma rangeli isolates fromman triatomines and sylvatic mammals from widespread geo-graphical origin based on SSU and ITS ribosomal sequencesrdquoParasitology vol 129 no 5 pp 549ndash561 2004

[30] A P Cortez R M Ventura A C Rodrigues et al ldquoThetaxonomic and phylogenetic relationships of Trypanosomavivax from South America and Africardquo Parasitology vol 133no 2 pp 159ndash169 2006

[31] A C Rodrigues F Paiva M Campaner J R Stevens H ANoyes and M M G Teixeira ldquoPhylogeny of Trypanosoma(Megatrypanum) theileri and related trypanosomes revealslineages of isolates associated with artiodactyl hosts divergingon SSU and ITS ribosomal sequencesrdquo Parasitology vol 132 no2 pp 215ndash224 2006

[32] A Marcili V C Valente S A Valente et al ldquoTrypanosomacruzi in Brazilian Amazonia lineages TCI and TCIIa in wildprimates Rhodnius spp and in humans with Chagas diseaseassociated with oral transmissionrdquo International Journal forParasitology vol 39 no 5 pp 615ndash623 2009

ISRN Parasitology 7

[33] M Cavazzana Jr A Marcili L Lima et al ldquoPhylogeo-graphical ecological and biological patterns shown by nuclear(ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes oftrypanosomes of the subgenus Schizotrypanum parasitic inBrazilian batsrdquo International Journal for Parasitology vol 40 no3 pp 315ndash320 2010

[34] P Brazaitis M EWatanabe and G Amato ldquoThe caiman traderdquoScience American vol 278 pp 52ndash58 1998

[35] J J McNamara and W F Snow ldquoImproved identification ofNannomonas infections in tsetse flies from The Gambiardquo ActaTropica vol 48 no 2 pp 127ndash136 1990

[36] R L M Ferreira A L Henriques and J A Rafael ldquoActivityof tabanids (Insecta Diptera Tabanidae) attacking the reptilesCaiman crocodilus (Linn) (Alligatoridae) and Eunectesmurinus(Linn) (Boidae) in the Central Amazon Brazilrdquo Memorias doInstituto Oswaldo Cruz vol 97 no 1 pp 133ndash136 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

ISRN Parasitology 3

Table 1 Trypanosomatids isolates host geographic origin and sequences of SSU rDNA and gGAPDH used for phylogenetic analysis

Trypanosomatids species Isolate code Host Geographic origin Accession numbera

gGAPDH SSU rDNAFish

Trypanosoma sp CLAR Clarias angolensis Africa AJ620251Trypanosoma sp MARV Cyprinus carpio Czech Republic AJ620248T granulosum Portugal Anguilla anguilla Portugal AJ620247T granulosum UK Anguilla anguilla United Kingdom AJ620246

ReptileTrypanosoma sp Gecko Tarentola annularis Senegal AJ620259T varani V54 Varanus exanthematicus Senegal AJ620261Trypanosoma sp T610 Caiman yacare Brazil EU596256 EU596252Trypanosoma sp T624 Caiman yacare Brazil EU596257 EU596253Trypanosoma sp T625 Caiman yacare Brazil EU596259Trypanosoma sp T1092 Caiman yacare Brazil EU596258 EU596254Trypanosoma sp T1100 Caiman yacare Brazil EU596260Trypanosoma sp T1101 Caiman yacare Brazil EU596261Trypanosoma sp T1102 Caiman yacare Brazil EU596262Trypanosoma sp T1119 Caiman yacare Brazil EU596263Trypanosoma sp T1120 Caiman yacare Brazil EU596255Trypanosoma sp CBT02 Caiman crocodilus Brazil JQ768791 JQ768792

BirdT avium Chaffinch Fringilla coelebs Czech Republic AJ620263T avium Rook Corvus frugilegus Czech Republic AJ620262

MammalsT conorhini USP Rattus rattus Brazil AJ620267T cruzi marinkellei B7 Phyllostomus hastatus Brazil AJ620270T cruzi X52898T lewisi L32 Rattus rattus AJ620272T microti TRL132 Microtus agrestis England AJ620273Trypanosoma sp ABF Wallabia bicolor Australia AJ620276Trypanosoma sp R5 Oryctolagus cuniculus Australia AJ620276T theileri K127 Bos taurus Germany AJ620282Trypanosoma sp D30 Cervus dama Germany AJ620279T dionisii P3 Pipistrellus pipistrellus United Kingdom AJ620271T vespertilionis P14 Pipistrellus pipistrellus England AJ620283T brucei rhodesiense 058 Homo sapiens Zambia AJ620284T vivax Desowitz Ovis aries Nigeria AJ620295T vivax AF053744T congolense Forest Cam22 Capra capra Cameroon AJ620289T congolense Forest TSW103 Sus scrofa Liberia AJ620286T evansi AF053743T cyclops LV492 Macaca sp Malaysia AJ620265T minasense LSTM Saimiri boliviensis South America AJ620274T rangeli AF053742

InvertebrateCrithidia fasciculata AF047493T congolense Forest ANR3 Glossina palpalis Gambia AJ620285T congolense Tsavo 114 Glossina pallidipes Tanzania AJ620291T cruzi C8 cl2 Triatoma infestans Bolivia AJ620268T cruzi VINCH 89 Triatoma infestans Chile AJ620269

4 ISRN Parasitology

Table 1 Continued

Trypanosomatids species Isolate code Host Geographic origin Accession numbera

gGAPDH SSU rDNAT grayi ANR4 Glossina palpalis Gambia AJ620257 AJ005278T grayi BAN1 Glossina palpalis Gambia AJ620258 AJ620546T simiae Ken 2 Glossina morsitans Gambia AJ620293T godfreyi Ken 7 Glossina morsitans Gambia AJ620292T simiae Tsavo Ketri 1864 Glossina pallidipes Kenya AJ620294Trypanosoma sp TLAQ22 Philaemon sp Australia AJ620280aSequences determined in this study and deposited in GenBank are underlined and bold

F

F

F

F

N

N

N

N N

K

K

K

K

K

(a)

(b)

(d)

(e)(c)10120583m

Figure 1 Microphotographs of Giemsa-stained trypanosome formsfromCayman crocodilus (CBT 02) obtained by in vitro culture (LIT)Epimastigotes forms (a b c) and trypomastigotes forms (d e) Kkinetoplast N nucleus F flagellum

[13] Recently trypanosomes isolates were obtained fromC yacare in Pantanal from Mato Grosso do Sul State andshowed similar morphology of blood trypomastigote withTrypanosoma sp described previously [19 22] In this presentstudy we obtained the first trypanosome isolate from Ccrocodilus

The prevalence of Trypanosoma sp in C yacare variesfrom 35 to 46 in different studies performed in Pantanalregion from Mato Grosso do Sul state [19 22] Only onespectacled caiman captured in Mato Grosso was positive inhemoculture (33) but the prevalence in this species has notbeen established and a further study involving a large numberof individuals (C crocodilus) is necessary to determine theprevalence of Trypanosoma sp and T cecili

Most studies of caiman trypanosomes were performedin the Pantanal region The only study in Amazon withC crocodilus and Paleosuchus trigonatus found a very lowprevalence with rare trypomastigote forms in blood or tissueThis species was designated T cecili and isolation has not yetbeen possible [13]

The epimastigotes forms in axenic culture of spectacledcaiman isolate are very similar to Trypanosoma sp from Cyacare from Pantanal (Figure 1) The tissue imprint forms ofT cecili and blood forms of Trypanosoma sp from Pantanalare distinguishable but similar tissue forms are detected inCyacare and C crocodilus [13 19 22]

The variableV7-V8 region of SSU rDNAhas been used forDNAbarcoding of trypanosomatids and is able to distinguishall species polymorphisms and genetic relationships amongclosely related taxa [18 19 29ndash33] In addition the gGAPDHsequences were used to improve the phylogenetic analysis[19 23 24 33] In all phylogenetic analyses inferred by V7-V8SSU rDNA and gGAPDH the isolated CBT 02 was includedin monophyletic branch called T grayi Clade together withTrypanosoma sp from Brazilian caiman The V7-V8 regionsegregated the Brazilian caimans isolates in two branchesCay01 and Cay02 Most isolates were comprised of Cay01genotype probably the most prevalent in Pantanal regionThe new isolate from C crocodilus (CBT 02) nested in Cay01genotype

The phylogenetic positions of CBT02 isolate show thatTrypanosoma sp from Cay01 genotype I is able to infecttwo species of Caiman genus in Brazil These findings clarifythe morphological approaches in tissues imprints whichhave trypomastigote similar morphology in C yacare and Ccrocodilus

The distribution of Cay01 genotype is not restricted toPantanal region of Mato Grosso do Sul state and vertebratehostThe Confresa county is located in north of Mato Grossostate and comprises a mosaic of Amazonia and Cerradobiomes and is irrigated by Tocantins-Araguaia basin The C

ISRN Parasitology 5

T sp T610

T grayi BAN1

T grayi ANR4

T sp T1119

T sp T1101T sp T624

T sp T1102

T sp T1092

T sp T1100

T sp T625

T sp T1120

110

Crithidia fasciculata

Trypanosoma genus

T granulosum UKT granulosumT sp MARVT sp CLAR

T sp T610

T grayi ANR4T grayi BAN1

T sp T1092T sp T624

T congolense ForestT congolense Forest

T congolense Forest

T congolense TsavoT simiae

T godfreyi

T simiae TsavoT brucei rhodesienseT evansi

T varaniT sp Gecko

T avium RookT avium Chaffinch

T sp TLAQ22T sp ABF

T cyclopsT theileri

T sp D30

T microtiT sp Habbit

T lewisi

T cruziT cruzi

T c marinkelleiT dionisii

T cruziT cruzi

T vespertilionisT conorhini

T rangeliT minasense

T vivaxT vivax

T grayi Clade

Cay01

Cay02

(a) (b)

100 100

100 100100 100

100 100

100 100100 100

100 100

100 100

100 100

100 100

100 100

100 100100 100

99 100

100 100

100 100

100 10097 100

88 100

91 8975 69

82 100

75 67

73 75 88 100

76 100

75 68

75 68

67 99

119931 sp CBT 02

119931 sp CBT 02

Figure 2 (a) Phylogenetic trees inferred by maximum parsimony and Bayesian methods based on gGAPDH gene sequences of 43trypanosomes and Crithidia fasciculata as outgroup (515 characters 34 parsimony informative) (b) Dendogram inferred by parsimonyanalyses based on V7-V8 SSU rDNA sequences from 12 crocodilian trypanosomes isolates (734 characters 24 parsimony informative)Numbers at nodes show bootstrap (first) and posterior probabilities (second) values for MP and BI respectively The support values forthe major branches are derived from 500 replicates respectively for MP

yacare occurred in upper Madeira system of the Amazonbasin and Paraguay River and lower Parana system includingBrazil Bolivia Paraguay and Argentina C crocodiles occurin both Amazon and Orinoco drainage [2 5] According toBrazaitis et al [34] sympatric areas occur inMatoGrosso andRondonia states However in Tocantins-Araguaia basin onlyC crocodilus occurs

The presence of the same trypanosome species inC yacare and C crocodilus indicates transmission cyclesbetween these species evidencing sympatry of caiman speciesor exposure to the same vector The T grayi in Africa istransmitted by tse-tse flies [35] In Brazil different speciesof tabanids can bite reptiles like C crocodilus and Eunectesmurinus [36] The low host specificity of tabanids and hostswitch of caiman trypanosomes suggest an important role ofinsects in the transmission cycle of these parasites in Brazil

The addition of new isolates of caiman trypanosomesof different vertebrate hosts is necessary for better under-standing of diversity and phylogenetic relationships of these

parasites and demonstrates the low host specificity of caymantrypanosomes in Brazil

Acknowledgments

The authors are grateful to several people for their invaluablehelp during capture This research was financially supportedby Fundacao de Amparo a Pesquisa do Estado de Sao Paulo(FAPESP) SolangeMGennari andAntonioHHMinervinoare in receipt of a fellowship from CNPq and Arlei MarciliHerbert S Soares Julia T R de Lima ATLM and Igor C LAcosta from FAPESP

References

[1] A Janke A Gullberg S Hughes R K Aggarwal and U Arna-son ldquoMitogenomic analyses place the gharial (Gavialis gangeti-cus) on the crocodile tree and provide pre-KT divergence timesfor most crocodiliansrdquo Journal of Molecular Evolution vol 61no 5 pp 620ndash626 2005

6 ISRN Parasitology

[2] J P Roos Crocodiles Status Survey and Conservation ActionPlan VIII 96 IUCN SS Crocodile Specialist Group GlandSwitzerland 2nd edition 1998

[3] F Medem Los Crocodylia De Sur America 1 ColcienciasBogota Colombia 1981

[4] F W King and R L Burke Crocodilian Tuatara and TurtleSpecies of the World A Taxonomic and Geographic ReferenceAssociation of Systematics Collections Washington DC USA1989

[5] A Velasco and J Ayarzaguena ldquoSpectacled Caiman Caimancrocodilosrdquo in Crocodiles Status Survey and ConservationAction Plan S C Manolis and C Stenvenson Eds 3rd edition2010

[6] K Junker D R Brooks and J Boomker ldquoProctocaecum gairhein sp (Digenea Cryptogonimidae Acanthostominae) fromGavialis gangeticus (Gmelin) in Nepal and a revised cladogramof Proctocaecum Baugh 1957rdquo Systematic Parasitology vol 71no 3 pp 229ndash236 2008

[7] V V Tkach E J Strand and L Froese ldquoMacroderoides texanusn sp (Digenea Macroderoididae) from alligator gar Atractos-teus spatula in Texasrdquo Parasitology Research vol 104 no 1 pp27ndash33 2008

[8] L J La Grange G Marucci and E Pozio ldquoTrichinella zimbab-wensis in wild Nile crocodiles (Crocodylus niloticus) of SouthAfricardquoVeterinary Parasitology vol 161 no 1-2 pp 88ndash91 2009

[9] L J La Grange D Govender and S Mukaratirwa ldquoTheoccurrence of Trichinella zimbabwensis in naturally infectedwild crocodiles (Crocodylus niloticus) from the Kruger NationalPark South Africardquo Journal of Helminthology vol 16 pp 1ndash62012

[10] A R Waddle J M Kinsella J P Ross E Rojas-Flores H FPercival and D J Forrester ldquoNematodes collected by gastriclavage from live American Alligators Alligator mississippiensisin Floridardquo Journal of Parasitology vol 95 no 5 pp 1237ndash12382009

[11] L A Viana and E J Marques ldquoHaemogregarine parasites(Apicomplexa Hepatozoidae) in Caiman crocodilus yacare(Crocodilia Alligatoridae) from Pantanal Corumba MSBrazilrdquo Revista Brasileira de Parasitologia Veterinaria vol 14no 4 pp 173ndash175 2005

[12] L A Viana F Paiva M E Coutinho and R Lourenco-De-Oliveira ldquoHepatozoon caimani (Apicomplexa Hepatozoidae)in Wild Caiman Caiman yacare from the Pantanal RegionBrazilrdquo Journal of Parasitology vol 96 no 1 pp 83ndash88 2010

[13] R Lainson ldquoTrypanosoma cecili n sp a parasite of the SouthAmerican cayman Caiman crocodilus crocodilus (Linnaeus1758) (Crocodilia Alligatoridae)rdquo Protozoology vol 3 pp 87ndash93 1977

[14] C A Hoare The Trypanosomes of Mammals A ZoologicalMonograph Blackwell Scientific Oxford UK 1972

[15] K Vickerman ldquoThe evolutionary expansion of the trypanoso-matid flagellatesrdquo International Journal for Parasitology vol 24no 8 pp 1317ndash1331 1994

[16] J R Stevens H A Noyes C J Schofield and W GibsonldquoThe molecular evolution of trypanosomatidaerdquo Advances inParasitology vol 48 pp 1ndash56 2001

[17] A G B Simpson J R Stevens and J Lukes ldquoThe evolution anddiversity of kinetoplastid flagellatesrdquo Trends in Parasitology vol22 no 4 pp 168ndash174 2006

[18] R C Ferreira A A De Souza R A Freitas et al ldquoA phyloge-netic lineage of closely related trypanosomes (Trypanosomati-dae Kinetoplastida) of anurans and sand flies (Psychodidae

Diptera) sharing the same ecotopes in Brazilian AmazoniardquoJournal of Eukaryotic Microbiology vol 55 no 5 pp 427ndash4352008

[19] L B Viola M Campaner C A Takata et al ldquoPhylogenyof snake trypanosomes inferred by SSU rDNA sequencestheir possible transmission by phlebotomines and taxonomicappraisal bymolecular cross-infection andmorphological anal-ysisrdquo Parasitology vol 135 no 5 pp 595ndash605 2008

[20] S R Telford ldquoThe kinetoplastid hemoflagellates of reptilesrdquo inParasitic Protozoa J P Kreier Ed vol 10 Academic press SanDiego Calif USA 2nd edition 1995

[21] L Lloyd W B Johnson and W A Young ldquoThe trypanosomeinfections of tsetse flies in northern Nigeria and a new methodof estimationrdquo in Bulletin of Entomology Research vol 14 pp265ndash288 1924

[22] V L B Nunes and E T Oshiro ldquoTrypanosoma sp em jacarCaiman crocodilus yacare (Daudin 1802) (Crocodilia Alliga-toridae)rdquo Semina vol 11 pp 62ndash65 1990

[23] P B Hamilton J R Stevens M W Gaunt J Gidley and WC Gibson ldquoTrypanosomes are monophyletic evidence fromgenes for glyceraldehyde phosphate dehydrogenase and smallsubunit ribosomal RNArdquo International Journal for Parasitologyvol 34 no 12 pp 1393ndash1404 2004

[24] P B Hamilton W C Gibson and J R Stevens ldquoPatterns ofco-evolution between trypanosomes and their hosts deducedfrom ribosomal RNA and protein-coding gene phylogeniesrdquoMolecular Phylogenetics and Evolution vol 44 no 1 pp 15ndash252007

[25] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997

[26] K B Nicholas H B Nicholas Jr and D W Deerfield ldquoGene-Doc analysis and visualization of genetic variationrdquo EMBNEWNEWS vol 4 p 14 1997

[27] D L Swofford ldquoPAUPlowast Phylogenetic Analysis Using Parsi-mony (and Other Methods) Beta Version 4 0b10rdquo Sinauer andAssociates Sunderland Mass USA 2002

[28] J P Huelsenbeck and F Ronquist ldquoMRBAYES bayesian infer-ence of phylogenetic treesrdquo Bioinformatics vol 17 no 8 pp754ndash755 2001

[29] F M Da Silva H Noyes M Campaner et al ldquoPhylogenytaxonomy and grouping of Trypanosoma rangeli isolates fromman triatomines and sylvatic mammals from widespread geo-graphical origin based on SSU and ITS ribosomal sequencesrdquoParasitology vol 129 no 5 pp 549ndash561 2004

[30] A P Cortez R M Ventura A C Rodrigues et al ldquoThetaxonomic and phylogenetic relationships of Trypanosomavivax from South America and Africardquo Parasitology vol 133no 2 pp 159ndash169 2006

[31] A C Rodrigues F Paiva M Campaner J R Stevens H ANoyes and M M G Teixeira ldquoPhylogeny of Trypanosoma(Megatrypanum) theileri and related trypanosomes revealslineages of isolates associated with artiodactyl hosts divergingon SSU and ITS ribosomal sequencesrdquo Parasitology vol 132 no2 pp 215ndash224 2006

[32] A Marcili V C Valente S A Valente et al ldquoTrypanosomacruzi in Brazilian Amazonia lineages TCI and TCIIa in wildprimates Rhodnius spp and in humans with Chagas diseaseassociated with oral transmissionrdquo International Journal forParasitology vol 39 no 5 pp 615ndash623 2009

ISRN Parasitology 7

[33] M Cavazzana Jr A Marcili L Lima et al ldquoPhylogeo-graphical ecological and biological patterns shown by nuclear(ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes oftrypanosomes of the subgenus Schizotrypanum parasitic inBrazilian batsrdquo International Journal for Parasitology vol 40 no3 pp 315ndash320 2010

[34] P Brazaitis M EWatanabe and G Amato ldquoThe caiman traderdquoScience American vol 278 pp 52ndash58 1998

[35] J J McNamara and W F Snow ldquoImproved identification ofNannomonas infections in tsetse flies from The Gambiardquo ActaTropica vol 48 no 2 pp 127ndash136 1990

[36] R L M Ferreira A L Henriques and J A Rafael ldquoActivityof tabanids (Insecta Diptera Tabanidae) attacking the reptilesCaiman crocodilus (Linn) (Alligatoridae) and Eunectesmurinus(Linn) (Boidae) in the Central Amazon Brazilrdquo Memorias doInstituto Oswaldo Cruz vol 97 no 1 pp 133ndash136 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 ISRN Parasitology

Table 1 Continued

Trypanosomatids species Isolate code Host Geographic origin Accession numbera

gGAPDH SSU rDNAT grayi ANR4 Glossina palpalis Gambia AJ620257 AJ005278T grayi BAN1 Glossina palpalis Gambia AJ620258 AJ620546T simiae Ken 2 Glossina morsitans Gambia AJ620293T godfreyi Ken 7 Glossina morsitans Gambia AJ620292T simiae Tsavo Ketri 1864 Glossina pallidipes Kenya AJ620294Trypanosoma sp TLAQ22 Philaemon sp Australia AJ620280aSequences determined in this study and deposited in GenBank are underlined and bold

F

F

F

F

N

N

N

N N

K

K

K

K

K

(a)

(b)

(d)

(e)(c)10120583m

Figure 1 Microphotographs of Giemsa-stained trypanosome formsfromCayman crocodilus (CBT 02) obtained by in vitro culture (LIT)Epimastigotes forms (a b c) and trypomastigotes forms (d e) Kkinetoplast N nucleus F flagellum

[13] Recently trypanosomes isolates were obtained fromC yacare in Pantanal from Mato Grosso do Sul State andshowed similar morphology of blood trypomastigote withTrypanosoma sp described previously [19 22] In this presentstudy we obtained the first trypanosome isolate from Ccrocodilus

The prevalence of Trypanosoma sp in C yacare variesfrom 35 to 46 in different studies performed in Pantanalregion from Mato Grosso do Sul state [19 22] Only onespectacled caiman captured in Mato Grosso was positive inhemoculture (33) but the prevalence in this species has notbeen established and a further study involving a large numberof individuals (C crocodilus) is necessary to determine theprevalence of Trypanosoma sp and T cecili

Most studies of caiman trypanosomes were performedin the Pantanal region The only study in Amazon withC crocodilus and Paleosuchus trigonatus found a very lowprevalence with rare trypomastigote forms in blood or tissueThis species was designated T cecili and isolation has not yetbeen possible [13]

The epimastigotes forms in axenic culture of spectacledcaiman isolate are very similar to Trypanosoma sp from Cyacare from Pantanal (Figure 1) The tissue imprint forms ofT cecili and blood forms of Trypanosoma sp from Pantanalare distinguishable but similar tissue forms are detected inCyacare and C crocodilus [13 19 22]

The variableV7-V8 region of SSU rDNAhas been used forDNAbarcoding of trypanosomatids and is able to distinguishall species polymorphisms and genetic relationships amongclosely related taxa [18 19 29ndash33] In addition the gGAPDHsequences were used to improve the phylogenetic analysis[19 23 24 33] In all phylogenetic analyses inferred by V7-V8SSU rDNA and gGAPDH the isolated CBT 02 was includedin monophyletic branch called T grayi Clade together withTrypanosoma sp from Brazilian caiman The V7-V8 regionsegregated the Brazilian caimans isolates in two branchesCay01 and Cay02 Most isolates were comprised of Cay01genotype probably the most prevalent in Pantanal regionThe new isolate from C crocodilus (CBT 02) nested in Cay01genotype

The phylogenetic positions of CBT02 isolate show thatTrypanosoma sp from Cay01 genotype I is able to infecttwo species of Caiman genus in Brazil These findings clarifythe morphological approaches in tissues imprints whichhave trypomastigote similar morphology in C yacare and Ccrocodilus

The distribution of Cay01 genotype is not restricted toPantanal region of Mato Grosso do Sul state and vertebratehostThe Confresa county is located in north of Mato Grossostate and comprises a mosaic of Amazonia and Cerradobiomes and is irrigated by Tocantins-Araguaia basin The C

ISRN Parasitology 5

T sp T610

T grayi BAN1

T grayi ANR4

T sp T1119

T sp T1101T sp T624

T sp T1102

T sp T1092

T sp T1100

T sp T625

T sp T1120

110

Crithidia fasciculata

Trypanosoma genus

T granulosum UKT granulosumT sp MARVT sp CLAR

T sp T610

T grayi ANR4T grayi BAN1

T sp T1092T sp T624

T congolense ForestT congolense Forest

T congolense Forest

T congolense TsavoT simiae

T godfreyi

T simiae TsavoT brucei rhodesienseT evansi

T varaniT sp Gecko

T avium RookT avium Chaffinch

T sp TLAQ22T sp ABF

T cyclopsT theileri

T sp D30

T microtiT sp Habbit

T lewisi

T cruziT cruzi

T c marinkelleiT dionisii

T cruziT cruzi

T vespertilionisT conorhini

T rangeliT minasense

T vivaxT vivax

T grayi Clade

Cay01

Cay02

(a) (b)

100 100

100 100100 100

100 100

100 100100 100

100 100

100 100

100 100

100 100

100 100

100 100100 100

99 100

100 100

100 100

100 10097 100

88 100

91 8975 69

82 100

75 67

73 75 88 100

76 100

75 68

75 68

67 99

119931 sp CBT 02

119931 sp CBT 02

Figure 2 (a) Phylogenetic trees inferred by maximum parsimony and Bayesian methods based on gGAPDH gene sequences of 43trypanosomes and Crithidia fasciculata as outgroup (515 characters 34 parsimony informative) (b) Dendogram inferred by parsimonyanalyses based on V7-V8 SSU rDNA sequences from 12 crocodilian trypanosomes isolates (734 characters 24 parsimony informative)Numbers at nodes show bootstrap (first) and posterior probabilities (second) values for MP and BI respectively The support values forthe major branches are derived from 500 replicates respectively for MP

yacare occurred in upper Madeira system of the Amazonbasin and Paraguay River and lower Parana system includingBrazil Bolivia Paraguay and Argentina C crocodiles occurin both Amazon and Orinoco drainage [2 5] According toBrazaitis et al [34] sympatric areas occur inMatoGrosso andRondonia states However in Tocantins-Araguaia basin onlyC crocodilus occurs

The presence of the same trypanosome species inC yacare and C crocodilus indicates transmission cyclesbetween these species evidencing sympatry of caiman speciesor exposure to the same vector The T grayi in Africa istransmitted by tse-tse flies [35] In Brazil different speciesof tabanids can bite reptiles like C crocodilus and Eunectesmurinus [36] The low host specificity of tabanids and hostswitch of caiman trypanosomes suggest an important role ofinsects in the transmission cycle of these parasites in Brazil

The addition of new isolates of caiman trypanosomesof different vertebrate hosts is necessary for better under-standing of diversity and phylogenetic relationships of these

parasites and demonstrates the low host specificity of caymantrypanosomes in Brazil

Acknowledgments

The authors are grateful to several people for their invaluablehelp during capture This research was financially supportedby Fundacao de Amparo a Pesquisa do Estado de Sao Paulo(FAPESP) SolangeMGennari andAntonioHHMinervinoare in receipt of a fellowship from CNPq and Arlei MarciliHerbert S Soares Julia T R de Lima ATLM and Igor C LAcosta from FAPESP

References

[1] A Janke A Gullberg S Hughes R K Aggarwal and U Arna-son ldquoMitogenomic analyses place the gharial (Gavialis gangeti-cus) on the crocodile tree and provide pre-KT divergence timesfor most crocodiliansrdquo Journal of Molecular Evolution vol 61no 5 pp 620ndash626 2005

6 ISRN Parasitology

[2] J P Roos Crocodiles Status Survey and Conservation ActionPlan VIII 96 IUCN SS Crocodile Specialist Group GlandSwitzerland 2nd edition 1998

[3] F Medem Los Crocodylia De Sur America 1 ColcienciasBogota Colombia 1981

[4] F W King and R L Burke Crocodilian Tuatara and TurtleSpecies of the World A Taxonomic and Geographic ReferenceAssociation of Systematics Collections Washington DC USA1989

[5] A Velasco and J Ayarzaguena ldquoSpectacled Caiman Caimancrocodilosrdquo in Crocodiles Status Survey and ConservationAction Plan S C Manolis and C Stenvenson Eds 3rd edition2010

[6] K Junker D R Brooks and J Boomker ldquoProctocaecum gairhein sp (Digenea Cryptogonimidae Acanthostominae) fromGavialis gangeticus (Gmelin) in Nepal and a revised cladogramof Proctocaecum Baugh 1957rdquo Systematic Parasitology vol 71no 3 pp 229ndash236 2008

[7] V V Tkach E J Strand and L Froese ldquoMacroderoides texanusn sp (Digenea Macroderoididae) from alligator gar Atractos-teus spatula in Texasrdquo Parasitology Research vol 104 no 1 pp27ndash33 2008

[8] L J La Grange G Marucci and E Pozio ldquoTrichinella zimbab-wensis in wild Nile crocodiles (Crocodylus niloticus) of SouthAfricardquoVeterinary Parasitology vol 161 no 1-2 pp 88ndash91 2009

[9] L J La Grange D Govender and S Mukaratirwa ldquoTheoccurrence of Trichinella zimbabwensis in naturally infectedwild crocodiles (Crocodylus niloticus) from the Kruger NationalPark South Africardquo Journal of Helminthology vol 16 pp 1ndash62012

[10] A R Waddle J M Kinsella J P Ross E Rojas-Flores H FPercival and D J Forrester ldquoNematodes collected by gastriclavage from live American Alligators Alligator mississippiensisin Floridardquo Journal of Parasitology vol 95 no 5 pp 1237ndash12382009

[11] L A Viana and E J Marques ldquoHaemogregarine parasites(Apicomplexa Hepatozoidae) in Caiman crocodilus yacare(Crocodilia Alligatoridae) from Pantanal Corumba MSBrazilrdquo Revista Brasileira de Parasitologia Veterinaria vol 14no 4 pp 173ndash175 2005

[12] L A Viana F Paiva M E Coutinho and R Lourenco-De-Oliveira ldquoHepatozoon caimani (Apicomplexa Hepatozoidae)in Wild Caiman Caiman yacare from the Pantanal RegionBrazilrdquo Journal of Parasitology vol 96 no 1 pp 83ndash88 2010

[13] R Lainson ldquoTrypanosoma cecili n sp a parasite of the SouthAmerican cayman Caiman crocodilus crocodilus (Linnaeus1758) (Crocodilia Alligatoridae)rdquo Protozoology vol 3 pp 87ndash93 1977

[14] C A Hoare The Trypanosomes of Mammals A ZoologicalMonograph Blackwell Scientific Oxford UK 1972

[15] K Vickerman ldquoThe evolutionary expansion of the trypanoso-matid flagellatesrdquo International Journal for Parasitology vol 24no 8 pp 1317ndash1331 1994

[16] J R Stevens H A Noyes C J Schofield and W GibsonldquoThe molecular evolution of trypanosomatidaerdquo Advances inParasitology vol 48 pp 1ndash56 2001

[17] A G B Simpson J R Stevens and J Lukes ldquoThe evolution anddiversity of kinetoplastid flagellatesrdquo Trends in Parasitology vol22 no 4 pp 168ndash174 2006

[18] R C Ferreira A A De Souza R A Freitas et al ldquoA phyloge-netic lineage of closely related trypanosomes (Trypanosomati-dae Kinetoplastida) of anurans and sand flies (Psychodidae

Diptera) sharing the same ecotopes in Brazilian AmazoniardquoJournal of Eukaryotic Microbiology vol 55 no 5 pp 427ndash4352008

[19] L B Viola M Campaner C A Takata et al ldquoPhylogenyof snake trypanosomes inferred by SSU rDNA sequencestheir possible transmission by phlebotomines and taxonomicappraisal bymolecular cross-infection andmorphological anal-ysisrdquo Parasitology vol 135 no 5 pp 595ndash605 2008

[20] S R Telford ldquoThe kinetoplastid hemoflagellates of reptilesrdquo inParasitic Protozoa J P Kreier Ed vol 10 Academic press SanDiego Calif USA 2nd edition 1995

[21] L Lloyd W B Johnson and W A Young ldquoThe trypanosomeinfections of tsetse flies in northern Nigeria and a new methodof estimationrdquo in Bulletin of Entomology Research vol 14 pp265ndash288 1924

[22] V L B Nunes and E T Oshiro ldquoTrypanosoma sp em jacarCaiman crocodilus yacare (Daudin 1802) (Crocodilia Alliga-toridae)rdquo Semina vol 11 pp 62ndash65 1990

[23] P B Hamilton J R Stevens M W Gaunt J Gidley and WC Gibson ldquoTrypanosomes are monophyletic evidence fromgenes for glyceraldehyde phosphate dehydrogenase and smallsubunit ribosomal RNArdquo International Journal for Parasitologyvol 34 no 12 pp 1393ndash1404 2004

[24] P B Hamilton W C Gibson and J R Stevens ldquoPatterns ofco-evolution between trypanosomes and their hosts deducedfrom ribosomal RNA and protein-coding gene phylogeniesrdquoMolecular Phylogenetics and Evolution vol 44 no 1 pp 15ndash252007

[25] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997

[26] K B Nicholas H B Nicholas Jr and D W Deerfield ldquoGene-Doc analysis and visualization of genetic variationrdquo EMBNEWNEWS vol 4 p 14 1997

[27] D L Swofford ldquoPAUPlowast Phylogenetic Analysis Using Parsi-mony (and Other Methods) Beta Version 4 0b10rdquo Sinauer andAssociates Sunderland Mass USA 2002

[28] J P Huelsenbeck and F Ronquist ldquoMRBAYES bayesian infer-ence of phylogenetic treesrdquo Bioinformatics vol 17 no 8 pp754ndash755 2001

[29] F M Da Silva H Noyes M Campaner et al ldquoPhylogenytaxonomy and grouping of Trypanosoma rangeli isolates fromman triatomines and sylvatic mammals from widespread geo-graphical origin based on SSU and ITS ribosomal sequencesrdquoParasitology vol 129 no 5 pp 549ndash561 2004

[30] A P Cortez R M Ventura A C Rodrigues et al ldquoThetaxonomic and phylogenetic relationships of Trypanosomavivax from South America and Africardquo Parasitology vol 133no 2 pp 159ndash169 2006

[31] A C Rodrigues F Paiva M Campaner J R Stevens H ANoyes and M M G Teixeira ldquoPhylogeny of Trypanosoma(Megatrypanum) theileri and related trypanosomes revealslineages of isolates associated with artiodactyl hosts divergingon SSU and ITS ribosomal sequencesrdquo Parasitology vol 132 no2 pp 215ndash224 2006

[32] A Marcili V C Valente S A Valente et al ldquoTrypanosomacruzi in Brazilian Amazonia lineages TCI and TCIIa in wildprimates Rhodnius spp and in humans with Chagas diseaseassociated with oral transmissionrdquo International Journal forParasitology vol 39 no 5 pp 615ndash623 2009

ISRN Parasitology 7

[33] M Cavazzana Jr A Marcili L Lima et al ldquoPhylogeo-graphical ecological and biological patterns shown by nuclear(ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes oftrypanosomes of the subgenus Schizotrypanum parasitic inBrazilian batsrdquo International Journal for Parasitology vol 40 no3 pp 315ndash320 2010

[34] P Brazaitis M EWatanabe and G Amato ldquoThe caiman traderdquoScience American vol 278 pp 52ndash58 1998

[35] J J McNamara and W F Snow ldquoImproved identification ofNannomonas infections in tsetse flies from The Gambiardquo ActaTropica vol 48 no 2 pp 127ndash136 1990

[36] R L M Ferreira A L Henriques and J A Rafael ldquoActivityof tabanids (Insecta Diptera Tabanidae) attacking the reptilesCaiman crocodilus (Linn) (Alligatoridae) and Eunectesmurinus(Linn) (Boidae) in the Central Amazon Brazilrdquo Memorias doInstituto Oswaldo Cruz vol 97 no 1 pp 133ndash136 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

ISRN Parasitology 5

T sp T610

T grayi BAN1

T grayi ANR4

T sp T1119

T sp T1101T sp T624

T sp T1102

T sp T1092

T sp T1100

T sp T625

T sp T1120

110

Crithidia fasciculata

Trypanosoma genus

T granulosum UKT granulosumT sp MARVT sp CLAR

T sp T610

T grayi ANR4T grayi BAN1

T sp T1092T sp T624

T congolense ForestT congolense Forest

T congolense Forest

T congolense TsavoT simiae

T godfreyi

T simiae TsavoT brucei rhodesienseT evansi

T varaniT sp Gecko

T avium RookT avium Chaffinch

T sp TLAQ22T sp ABF

T cyclopsT theileri

T sp D30

T microtiT sp Habbit

T lewisi

T cruziT cruzi

T c marinkelleiT dionisii

T cruziT cruzi

T vespertilionisT conorhini

T rangeliT minasense

T vivaxT vivax

T grayi Clade

Cay01

Cay02

(a) (b)

100 100

100 100100 100

100 100

100 100100 100

100 100

100 100

100 100

100 100

100 100

100 100100 100

99 100

100 100

100 100

100 10097 100

88 100

91 8975 69

82 100

75 67

73 75 88 100

76 100

75 68

75 68

67 99

119931 sp CBT 02

119931 sp CBT 02

Figure 2 (a) Phylogenetic trees inferred by maximum parsimony and Bayesian methods based on gGAPDH gene sequences of 43trypanosomes and Crithidia fasciculata as outgroup (515 characters 34 parsimony informative) (b) Dendogram inferred by parsimonyanalyses based on V7-V8 SSU rDNA sequences from 12 crocodilian trypanosomes isolates (734 characters 24 parsimony informative)Numbers at nodes show bootstrap (first) and posterior probabilities (second) values for MP and BI respectively The support values forthe major branches are derived from 500 replicates respectively for MP

yacare occurred in upper Madeira system of the Amazonbasin and Paraguay River and lower Parana system includingBrazil Bolivia Paraguay and Argentina C crocodiles occurin both Amazon and Orinoco drainage [2 5] According toBrazaitis et al [34] sympatric areas occur inMatoGrosso andRondonia states However in Tocantins-Araguaia basin onlyC crocodilus occurs

The presence of the same trypanosome species inC yacare and C crocodilus indicates transmission cyclesbetween these species evidencing sympatry of caiman speciesor exposure to the same vector The T grayi in Africa istransmitted by tse-tse flies [35] In Brazil different speciesof tabanids can bite reptiles like C crocodilus and Eunectesmurinus [36] The low host specificity of tabanids and hostswitch of caiman trypanosomes suggest an important role ofinsects in the transmission cycle of these parasites in Brazil

The addition of new isolates of caiman trypanosomesof different vertebrate hosts is necessary for better under-standing of diversity and phylogenetic relationships of these

parasites and demonstrates the low host specificity of caymantrypanosomes in Brazil

Acknowledgments

The authors are grateful to several people for their invaluablehelp during capture This research was financially supportedby Fundacao de Amparo a Pesquisa do Estado de Sao Paulo(FAPESP) SolangeMGennari andAntonioHHMinervinoare in receipt of a fellowship from CNPq and Arlei MarciliHerbert S Soares Julia T R de Lima ATLM and Igor C LAcosta from FAPESP

References

[1] A Janke A Gullberg S Hughes R K Aggarwal and U Arna-son ldquoMitogenomic analyses place the gharial (Gavialis gangeti-cus) on the crocodile tree and provide pre-KT divergence timesfor most crocodiliansrdquo Journal of Molecular Evolution vol 61no 5 pp 620ndash626 2005

6 ISRN Parasitology

[2] J P Roos Crocodiles Status Survey and Conservation ActionPlan VIII 96 IUCN SS Crocodile Specialist Group GlandSwitzerland 2nd edition 1998

[3] F Medem Los Crocodylia De Sur America 1 ColcienciasBogota Colombia 1981

[4] F W King and R L Burke Crocodilian Tuatara and TurtleSpecies of the World A Taxonomic and Geographic ReferenceAssociation of Systematics Collections Washington DC USA1989

[5] A Velasco and J Ayarzaguena ldquoSpectacled Caiman Caimancrocodilosrdquo in Crocodiles Status Survey and ConservationAction Plan S C Manolis and C Stenvenson Eds 3rd edition2010

[6] K Junker D R Brooks and J Boomker ldquoProctocaecum gairhein sp (Digenea Cryptogonimidae Acanthostominae) fromGavialis gangeticus (Gmelin) in Nepal and a revised cladogramof Proctocaecum Baugh 1957rdquo Systematic Parasitology vol 71no 3 pp 229ndash236 2008

[7] V V Tkach E J Strand and L Froese ldquoMacroderoides texanusn sp (Digenea Macroderoididae) from alligator gar Atractos-teus spatula in Texasrdquo Parasitology Research vol 104 no 1 pp27ndash33 2008

[8] L J La Grange G Marucci and E Pozio ldquoTrichinella zimbab-wensis in wild Nile crocodiles (Crocodylus niloticus) of SouthAfricardquoVeterinary Parasitology vol 161 no 1-2 pp 88ndash91 2009

[9] L J La Grange D Govender and S Mukaratirwa ldquoTheoccurrence of Trichinella zimbabwensis in naturally infectedwild crocodiles (Crocodylus niloticus) from the Kruger NationalPark South Africardquo Journal of Helminthology vol 16 pp 1ndash62012

[10] A R Waddle J M Kinsella J P Ross E Rojas-Flores H FPercival and D J Forrester ldquoNematodes collected by gastriclavage from live American Alligators Alligator mississippiensisin Floridardquo Journal of Parasitology vol 95 no 5 pp 1237ndash12382009

[11] L A Viana and E J Marques ldquoHaemogregarine parasites(Apicomplexa Hepatozoidae) in Caiman crocodilus yacare(Crocodilia Alligatoridae) from Pantanal Corumba MSBrazilrdquo Revista Brasileira de Parasitologia Veterinaria vol 14no 4 pp 173ndash175 2005

[12] L A Viana F Paiva M E Coutinho and R Lourenco-De-Oliveira ldquoHepatozoon caimani (Apicomplexa Hepatozoidae)in Wild Caiman Caiman yacare from the Pantanal RegionBrazilrdquo Journal of Parasitology vol 96 no 1 pp 83ndash88 2010

[13] R Lainson ldquoTrypanosoma cecili n sp a parasite of the SouthAmerican cayman Caiman crocodilus crocodilus (Linnaeus1758) (Crocodilia Alligatoridae)rdquo Protozoology vol 3 pp 87ndash93 1977

[14] C A Hoare The Trypanosomes of Mammals A ZoologicalMonograph Blackwell Scientific Oxford UK 1972

[15] K Vickerman ldquoThe evolutionary expansion of the trypanoso-matid flagellatesrdquo International Journal for Parasitology vol 24no 8 pp 1317ndash1331 1994

[16] J R Stevens H A Noyes C J Schofield and W GibsonldquoThe molecular evolution of trypanosomatidaerdquo Advances inParasitology vol 48 pp 1ndash56 2001

[17] A G B Simpson J R Stevens and J Lukes ldquoThe evolution anddiversity of kinetoplastid flagellatesrdquo Trends in Parasitology vol22 no 4 pp 168ndash174 2006

[18] R C Ferreira A A De Souza R A Freitas et al ldquoA phyloge-netic lineage of closely related trypanosomes (Trypanosomati-dae Kinetoplastida) of anurans and sand flies (Psychodidae

Diptera) sharing the same ecotopes in Brazilian AmazoniardquoJournal of Eukaryotic Microbiology vol 55 no 5 pp 427ndash4352008

[19] L B Viola M Campaner C A Takata et al ldquoPhylogenyof snake trypanosomes inferred by SSU rDNA sequencestheir possible transmission by phlebotomines and taxonomicappraisal bymolecular cross-infection andmorphological anal-ysisrdquo Parasitology vol 135 no 5 pp 595ndash605 2008

[20] S R Telford ldquoThe kinetoplastid hemoflagellates of reptilesrdquo inParasitic Protozoa J P Kreier Ed vol 10 Academic press SanDiego Calif USA 2nd edition 1995

[21] L Lloyd W B Johnson and W A Young ldquoThe trypanosomeinfections of tsetse flies in northern Nigeria and a new methodof estimationrdquo in Bulletin of Entomology Research vol 14 pp265ndash288 1924

[22] V L B Nunes and E T Oshiro ldquoTrypanosoma sp em jacarCaiman crocodilus yacare (Daudin 1802) (Crocodilia Alliga-toridae)rdquo Semina vol 11 pp 62ndash65 1990

[23] P B Hamilton J R Stevens M W Gaunt J Gidley and WC Gibson ldquoTrypanosomes are monophyletic evidence fromgenes for glyceraldehyde phosphate dehydrogenase and smallsubunit ribosomal RNArdquo International Journal for Parasitologyvol 34 no 12 pp 1393ndash1404 2004

[24] P B Hamilton W C Gibson and J R Stevens ldquoPatterns ofco-evolution between trypanosomes and their hosts deducedfrom ribosomal RNA and protein-coding gene phylogeniesrdquoMolecular Phylogenetics and Evolution vol 44 no 1 pp 15ndash252007

[25] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997

[26] K B Nicholas H B Nicholas Jr and D W Deerfield ldquoGene-Doc analysis and visualization of genetic variationrdquo EMBNEWNEWS vol 4 p 14 1997

[27] D L Swofford ldquoPAUPlowast Phylogenetic Analysis Using Parsi-mony (and Other Methods) Beta Version 4 0b10rdquo Sinauer andAssociates Sunderland Mass USA 2002

[28] J P Huelsenbeck and F Ronquist ldquoMRBAYES bayesian infer-ence of phylogenetic treesrdquo Bioinformatics vol 17 no 8 pp754ndash755 2001

[29] F M Da Silva H Noyes M Campaner et al ldquoPhylogenytaxonomy and grouping of Trypanosoma rangeli isolates fromman triatomines and sylvatic mammals from widespread geo-graphical origin based on SSU and ITS ribosomal sequencesrdquoParasitology vol 129 no 5 pp 549ndash561 2004

[30] A P Cortez R M Ventura A C Rodrigues et al ldquoThetaxonomic and phylogenetic relationships of Trypanosomavivax from South America and Africardquo Parasitology vol 133no 2 pp 159ndash169 2006

[31] A C Rodrigues F Paiva M Campaner J R Stevens H ANoyes and M M G Teixeira ldquoPhylogeny of Trypanosoma(Megatrypanum) theileri and related trypanosomes revealslineages of isolates associated with artiodactyl hosts divergingon SSU and ITS ribosomal sequencesrdquo Parasitology vol 132 no2 pp 215ndash224 2006

[32] A Marcili V C Valente S A Valente et al ldquoTrypanosomacruzi in Brazilian Amazonia lineages TCI and TCIIa in wildprimates Rhodnius spp and in humans with Chagas diseaseassociated with oral transmissionrdquo International Journal forParasitology vol 39 no 5 pp 615ndash623 2009

ISRN Parasitology 7

[33] M Cavazzana Jr A Marcili L Lima et al ldquoPhylogeo-graphical ecological and biological patterns shown by nuclear(ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes oftrypanosomes of the subgenus Schizotrypanum parasitic inBrazilian batsrdquo International Journal for Parasitology vol 40 no3 pp 315ndash320 2010

[34] P Brazaitis M EWatanabe and G Amato ldquoThe caiman traderdquoScience American vol 278 pp 52ndash58 1998

[35] J J McNamara and W F Snow ldquoImproved identification ofNannomonas infections in tsetse flies from The Gambiardquo ActaTropica vol 48 no 2 pp 127ndash136 1990

[36] R L M Ferreira A L Henriques and J A Rafael ldquoActivityof tabanids (Insecta Diptera Tabanidae) attacking the reptilesCaiman crocodilus (Linn) (Alligatoridae) and Eunectesmurinus(Linn) (Boidae) in the Central Amazon Brazilrdquo Memorias doInstituto Oswaldo Cruz vol 97 no 1 pp 133ndash136 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 ISRN Parasitology

[2] J P Roos Crocodiles Status Survey and Conservation ActionPlan VIII 96 IUCN SS Crocodile Specialist Group GlandSwitzerland 2nd edition 1998

[3] F Medem Los Crocodylia De Sur America 1 ColcienciasBogota Colombia 1981

[4] F W King and R L Burke Crocodilian Tuatara and TurtleSpecies of the World A Taxonomic and Geographic ReferenceAssociation of Systematics Collections Washington DC USA1989

[5] A Velasco and J Ayarzaguena ldquoSpectacled Caiman Caimancrocodilosrdquo in Crocodiles Status Survey and ConservationAction Plan S C Manolis and C Stenvenson Eds 3rd edition2010

[6] K Junker D R Brooks and J Boomker ldquoProctocaecum gairhein sp (Digenea Cryptogonimidae Acanthostominae) fromGavialis gangeticus (Gmelin) in Nepal and a revised cladogramof Proctocaecum Baugh 1957rdquo Systematic Parasitology vol 71no 3 pp 229ndash236 2008

[7] V V Tkach E J Strand and L Froese ldquoMacroderoides texanusn sp (Digenea Macroderoididae) from alligator gar Atractos-teus spatula in Texasrdquo Parasitology Research vol 104 no 1 pp27ndash33 2008

[8] L J La Grange G Marucci and E Pozio ldquoTrichinella zimbab-wensis in wild Nile crocodiles (Crocodylus niloticus) of SouthAfricardquoVeterinary Parasitology vol 161 no 1-2 pp 88ndash91 2009

[9] L J La Grange D Govender and S Mukaratirwa ldquoTheoccurrence of Trichinella zimbabwensis in naturally infectedwild crocodiles (Crocodylus niloticus) from the Kruger NationalPark South Africardquo Journal of Helminthology vol 16 pp 1ndash62012

[10] A R Waddle J M Kinsella J P Ross E Rojas-Flores H FPercival and D J Forrester ldquoNematodes collected by gastriclavage from live American Alligators Alligator mississippiensisin Floridardquo Journal of Parasitology vol 95 no 5 pp 1237ndash12382009

[11] L A Viana and E J Marques ldquoHaemogregarine parasites(Apicomplexa Hepatozoidae) in Caiman crocodilus yacare(Crocodilia Alligatoridae) from Pantanal Corumba MSBrazilrdquo Revista Brasileira de Parasitologia Veterinaria vol 14no 4 pp 173ndash175 2005

[12] L A Viana F Paiva M E Coutinho and R Lourenco-De-Oliveira ldquoHepatozoon caimani (Apicomplexa Hepatozoidae)in Wild Caiman Caiman yacare from the Pantanal RegionBrazilrdquo Journal of Parasitology vol 96 no 1 pp 83ndash88 2010

[13] R Lainson ldquoTrypanosoma cecili n sp a parasite of the SouthAmerican cayman Caiman crocodilus crocodilus (Linnaeus1758) (Crocodilia Alligatoridae)rdquo Protozoology vol 3 pp 87ndash93 1977

[14] C A Hoare The Trypanosomes of Mammals A ZoologicalMonograph Blackwell Scientific Oxford UK 1972

[15] K Vickerman ldquoThe evolutionary expansion of the trypanoso-matid flagellatesrdquo International Journal for Parasitology vol 24no 8 pp 1317ndash1331 1994

[16] J R Stevens H A Noyes C J Schofield and W GibsonldquoThe molecular evolution of trypanosomatidaerdquo Advances inParasitology vol 48 pp 1ndash56 2001

[17] A G B Simpson J R Stevens and J Lukes ldquoThe evolution anddiversity of kinetoplastid flagellatesrdquo Trends in Parasitology vol22 no 4 pp 168ndash174 2006

[18] R C Ferreira A A De Souza R A Freitas et al ldquoA phyloge-netic lineage of closely related trypanosomes (Trypanosomati-dae Kinetoplastida) of anurans and sand flies (Psychodidae

Diptera) sharing the same ecotopes in Brazilian AmazoniardquoJournal of Eukaryotic Microbiology vol 55 no 5 pp 427ndash4352008

[19] L B Viola M Campaner C A Takata et al ldquoPhylogenyof snake trypanosomes inferred by SSU rDNA sequencestheir possible transmission by phlebotomines and taxonomicappraisal bymolecular cross-infection andmorphological anal-ysisrdquo Parasitology vol 135 no 5 pp 595ndash605 2008

[20] S R Telford ldquoThe kinetoplastid hemoflagellates of reptilesrdquo inParasitic Protozoa J P Kreier Ed vol 10 Academic press SanDiego Calif USA 2nd edition 1995

[21] L Lloyd W B Johnson and W A Young ldquoThe trypanosomeinfections of tsetse flies in northern Nigeria and a new methodof estimationrdquo in Bulletin of Entomology Research vol 14 pp265ndash288 1924

[22] V L B Nunes and E T Oshiro ldquoTrypanosoma sp em jacarCaiman crocodilus yacare (Daudin 1802) (Crocodilia Alliga-toridae)rdquo Semina vol 11 pp 62ndash65 1990

[23] P B Hamilton J R Stevens M W Gaunt J Gidley and WC Gibson ldquoTrypanosomes are monophyletic evidence fromgenes for glyceraldehyde phosphate dehydrogenase and smallsubunit ribosomal RNArdquo International Journal for Parasitologyvol 34 no 12 pp 1393ndash1404 2004

[24] P B Hamilton W C Gibson and J R Stevens ldquoPatterns ofco-evolution between trypanosomes and their hosts deducedfrom ribosomal RNA and protein-coding gene phylogeniesrdquoMolecular Phylogenetics and Evolution vol 44 no 1 pp 15ndash252007

[25] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997

[26] K B Nicholas H B Nicholas Jr and D W Deerfield ldquoGene-Doc analysis and visualization of genetic variationrdquo EMBNEWNEWS vol 4 p 14 1997

[27] D L Swofford ldquoPAUPlowast Phylogenetic Analysis Using Parsi-mony (and Other Methods) Beta Version 4 0b10rdquo Sinauer andAssociates Sunderland Mass USA 2002

[28] J P Huelsenbeck and F Ronquist ldquoMRBAYES bayesian infer-ence of phylogenetic treesrdquo Bioinformatics vol 17 no 8 pp754ndash755 2001

[29] F M Da Silva H Noyes M Campaner et al ldquoPhylogenytaxonomy and grouping of Trypanosoma rangeli isolates fromman triatomines and sylvatic mammals from widespread geo-graphical origin based on SSU and ITS ribosomal sequencesrdquoParasitology vol 129 no 5 pp 549ndash561 2004

[30] A P Cortez R M Ventura A C Rodrigues et al ldquoThetaxonomic and phylogenetic relationships of Trypanosomavivax from South America and Africardquo Parasitology vol 133no 2 pp 159ndash169 2006

[31] A C Rodrigues F Paiva M Campaner J R Stevens H ANoyes and M M G Teixeira ldquoPhylogeny of Trypanosoma(Megatrypanum) theileri and related trypanosomes revealslineages of isolates associated with artiodactyl hosts divergingon SSU and ITS ribosomal sequencesrdquo Parasitology vol 132 no2 pp 215ndash224 2006

[32] A Marcili V C Valente S A Valente et al ldquoTrypanosomacruzi in Brazilian Amazonia lineages TCI and TCIIa in wildprimates Rhodnius spp and in humans with Chagas diseaseassociated with oral transmissionrdquo International Journal forParasitology vol 39 no 5 pp 615ndash623 2009

ISRN Parasitology 7

[33] M Cavazzana Jr A Marcili L Lima et al ldquoPhylogeo-graphical ecological and biological patterns shown by nuclear(ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes oftrypanosomes of the subgenus Schizotrypanum parasitic inBrazilian batsrdquo International Journal for Parasitology vol 40 no3 pp 315ndash320 2010

[34] P Brazaitis M EWatanabe and G Amato ldquoThe caiman traderdquoScience American vol 278 pp 52ndash58 1998

[35] J J McNamara and W F Snow ldquoImproved identification ofNannomonas infections in tsetse flies from The Gambiardquo ActaTropica vol 48 no 2 pp 127ndash136 1990

[36] R L M Ferreira A L Henriques and J A Rafael ldquoActivityof tabanids (Insecta Diptera Tabanidae) attacking the reptilesCaiman crocodilus (Linn) (Alligatoridae) and Eunectesmurinus(Linn) (Boidae) in the Central Amazon Brazilrdquo Memorias doInstituto Oswaldo Cruz vol 97 no 1 pp 133ndash136 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

ISRN Parasitology 7

[33] M Cavazzana Jr A Marcili L Lima et al ldquoPhylogeo-graphical ecological and biological patterns shown by nuclear(ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes oftrypanosomes of the subgenus Schizotrypanum parasitic inBrazilian batsrdquo International Journal for Parasitology vol 40 no3 pp 315ndash320 2010

[34] P Brazaitis M EWatanabe and G Amato ldquoThe caiman traderdquoScience American vol 278 pp 52ndash58 1998

[35] J J McNamara and W F Snow ldquoImproved identification ofNannomonas infections in tsetse flies from The Gambiardquo ActaTropica vol 48 no 2 pp 127ndash136 1990

[36] R L M Ferreira A L Henriques and J A Rafael ldquoActivityof tabanids (Insecta Diptera Tabanidae) attacking the reptilesCaiman crocodilus (Linn) (Alligatoridae) and Eunectesmurinus(Linn) (Boidae) in the Central Amazon Brazilrdquo Memorias doInstituto Oswaldo Cruz vol 97 no 1 pp 133ndash136 2002

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

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Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Genetics Research International

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Advances in

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Stem CellsInternational

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Enzyme Research

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International Journal of

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