title: Molecular characterization and classification of Trypanosoma spp. Venezuelan isolates based on microsatellite markers and kinetoplast maxicircle genes.
abstract: BACKGROUND
  Livestock trypanosomoses, caused by three species of the Trypanozoon subgenus, Trypanosoma brucei brucei, T. evansi and T. equiperdum is widely distributed throughout the world and constitutes an important limitation for the production of animal protein. T. evansi and T. equiperdum are morphologically indistinguishable parasites that evolved from a common ancestor but acquired important biological differences, including host range, mode of transmission, distribution, clinical symptoms and pathogenicity. At a molecular level, T. evansi is characterized by the complete loss of the maxicircles of the kinetoplastic DNA, while T. equiperdum has retained maxicircle fragments similar to those present in T. brucei. T. evansi causes the disease known as Surra, Derrengadera or "mal de cadeiras", while T. equiperdum is the etiological agent of dourine or "mal du coit", characterized by venereal transmission and white patches in the genitalia.
  
  
  METHODS
  Nine Venezuelan Trypanosoma spp. isolates, from horse, donkey or capybara were genotyped and classified using microsatellite analyses and maxicircle genes. The variables from the microsatellite data and the Procyclin PE repeats matrices were combined using the Hill-Smith method and compared to a group of T. evansi, T. equiperdum and T. brucei reference strains from South America, Asia and Africa using Coinertia analysis. Four maxicircle genes (cytb, cox1, a6 and nd8) were amplified by PCRfrom TeAp-N/D1 and TeGu-N/D1, the two Venezuelan isolates that grouped with the T. equiperdum STIB841/OVI strain. These maxicircle sequences were analyzed by nucleotide BLAST and aligned toorthologous genes from the Trypanozoon subgenus by MUSCLE tools. Phylogenetic trees were constructed using Maximum Parsimony (MP) and Maximum Likelihood (ML) with the MEGA5.1® software.
  
  
  RESULTS
  We characterized microsatellite markers and Procyclin PE repeats of nine Venezuelan Trypanosoma spp. isolates with various degrees of virulence in a mouse model, and compared them to a panel of T. evansi and T. equiperdum reference strains. Coinertia analysis of the combined repeats and previously reported T. brucei brucei microsatellite genotypes revealed three distinct groups. Seven of the Venezuelan isolates grouped with globally distributed T. evansi strains, while TeAp-N/D1 and TeGu-N/D1 strains clustered in a separate group with the T. equiperdum STIB841/OVI strain isolated in South Africa. A third group included T. brucei brucei, two strains previously classified as T. evansi (GX and TC) and one as T. equiperdum (BoTat-1.1). Four maxicircle genes, Cytochrome b, Cythocrome Oxidase subunit 1, ATP synthase subunit 6 and NADH dehydrogenase subunit 8, were identified in the two Venezuelan strains clustering with the T. equiperdum STIB841/OVI strain. Phylogenetic analysis of the cox1 gene sequences further separated these two Venezuelan T. equiperdum strains: TeAp-N/D1 grouped with T. equiperdum strain STIB818 and T. brucei brucei, and TeGu-N/D1 with the T. equiperdum STIB841/OVI strain.
  
  
  CONCLUSIONS
  Based on the Coinertia analysis and maxicircle gene sequence phylogeny, TeAp-N/D1 and TeGu-N/D1 constitute the first confirmed T. equiperdum strains described from Latin America.
authors.affiliation: Laboratorio de Fisiología de Parásitos. Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela. evsanchu@ivic.gob.ve.
authors.forename: E
authors.initials: E
authors.lastname: Sánchez
authors.affiliation: Laboratorio de Fisiología de Parásitos. Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela. mgonzat@gmail.com.
authors.forename: T
authors.initials: T
authors.lastname: Perrone
authors.affiliation: Grupo de Bioquímica e Inmunología de Hemoparásitos. Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, 1080, Venezuela. aninnaig.87@gmail.com.
authors.forename: G
authors.initials: G
authors.lastname: Recchimuzzi
authors.affiliation: Laboratorio de Fisiología de Parásitos. Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela. cardozo.iralis@gmail.com.
authors.forename: I
authors.initials: I
authors.lastname: Cardozo
authors.affiliation: Laboratoire de Microbiologie Fondamentale et Pathogénicité, Université Bordeaux. UMR-CNRS 5234, 146, Rue Léo Saignat, 33076, Bordeaux, Cedex, France. nicolas.biteau@u-bordeaux.fr.
authors.forename: N
authors.initials: N
authors.lastname: Biteau
authors.affiliation: Grupo de Bioquímica e Inmunología de Hemoparásitos. Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, 1080, Venezuela. pedrom@usb.ve.
authors.forename: P M
authors.initials: PM
authors.lastname: Aso
authors.affiliation: Laboratorio de Fisiología de Parásitos. Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela. amijares@ivic.gob.ve.
authors.forename: A
authors.initials: A
authors.lastname: Mijares
authors.affiliation: Laboratoire de Microbiologie Fondamentale et Pathogénicité, Université Bordeaux. UMR-CNRS 5234, 146, Rue Léo Saignat, 33076, Bordeaux, Cedex, France. theo.baltz@u-bordeaux.fr.
authors.forename: T
authors.initials: T
authors.lastname: Baltz
authors.affiliation: CIRAD, UMR InterTryp, F-34398, Montpellier, France. david.berthier@cirad.fr.
authors.forename: D
authors.initials: D
authors.lastname: Berthier
authors.affiliation: Laboratorio de Biometría y Estadística, Área de Agricultura y Soberanía Alimentaria, Instituto de Estudios Avanzados, Caracas, 1015A, Venezuela. leobalzano@gmail.com.
authors.forename: L
authors.initials: L
authors.lastname: Balzano-Nogueira
authors.affiliation: Grupo de Bioquímica e Inmunología de Hemoparásitos. Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, 1080, Venezuela. mgonzat@usb.ve.
authors.forename: M I
authors.initials: MI
authors.lastname: Gonzatti
mesh_terms: D000818:Animals; D016000:Cluster Analysis; D018105:DNA, Kinetoplast; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D018397:Equidae / Q000469:parasitology; D017125:Genes, Protozoan; D014644:Genetic Variation; D005838:Genotype; D006736:Horses / Q000469:parasitology; D018895:Microsatellite Repeats; D008969:Molecular Sequence Data; D010802:Phylogeny; D012377:Rodentia / Q000469:parasitology; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D014345:Trypanosoma / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D014687:Venezuela
chemical_list: D018105:DNA, Kinetoplast; D016054:DNA, Protozoan