Semisolid liver infusion tryptose supplemented with human urine allows growth and isolation of Trypanosoma cruzi and Trypanosoma rangeli clonal lineages

Abstract: INTRODUCTION This work shows that 3% (v/v) human urine (HU) in semisolid Liver Infusion Tryptose (SSL) medium favors the growth of Trypanosoma cruzi and T. rangeli. METHODS Parasites were plated as individual or mixed strains on SSL medium and on SSL medium with 3% human urine (SSL-HU). Isolate DNA was analyzed using polymerase chain reaction (PCR) and pulsed-field gel electrophoresis (PFGE). RESULTS SSL-HU medium improved clone isolation. PCR revealed that T. cruzi strains predominate on mixed-strain plates. PFGE confirmed that isolated parasites share the same molecular karyotype as parental cell lines. CONCLUSIONS SSL-HU medium constitutes a novel tool for obtaining T. cruzi and T. rangeli clonal lineages

DNA content analysis allows discrimination between Trypanosoma cruzi and Trypanosoma rangeli.

Trypanosoma cruzi, a human protozoan parasite, is the causative agent of Chagas disease. Currently the species is divided into six taxonomic groups. The genome of the CL Brener clone has been estimated to be 106.4-110.7 Mb, and DNA content analyses revealed that it is a diploid hybrid clone. Trypanosoma rangeli is a hemoflagellate that has the same reservoirs and vectors as T. cruzi; however, it is non-pathogenic to vertebrate hosts. The haploid genome of T. rangeli was previously estimated to be 24 Mb. The parasitic strains of T. rangeli are divided into KP1(+) and KP1(-). Thus, the objective of this study was to investigate the DNA content in different strains of T. cruzi and T. rangeli by flow cytometry. All T. cruzi and T. rangeli strains yielded cell cycle profiles with clearly identifiable G1-0 (2n) and G2-M (4n) peaks. T. cruzi and T. rangeli genome sizes were estimated using the clone CL Brener and the Leishmania major CC1 as reference cell lines because their genome sequences have been previously determined. The DNA content of T. cruzi strains ranged from 87,41 to 108,16 Mb, and the DNA content of T. rangeli strains ranged from 63,25 Mb to 68,66 Mb. No differences in DNA content were observed between KP1(+) and KP1(-) T. rangeli strains. Cultures containing mixtures of the epimastigote forms of T. cruzi and T. rangeli strains resulted in cell cycle profiles with distinct G1 peaks for strains of each species. These results demonstrate that DNA content analysis by flow cytometry is a reliable technique for discrimination between T. cruzi and T. rangeli isolated from different hosts

Genetic identification <i>Trypanosoma cruzi</i> and <i>Trypanosoma rangeli</i> strains.

<p>Duplex PCR to detect specific subtelomeric sequences in <i>T</i>. <i>cruzi</i> and <i>T</i>. <i>rangeli</i>. The amplified products were observed in 2.0% agarose gel stained with ethidium bromide. <i>Trypanosoma rangeli</i> strains: P02; P07; P19; Cas4; SO48; LDG. <i>T</i>. <i>cruzi</i> strains and clone: Dm28c; JG; RN1; Hel; 3663; CLBr (CL Brener). MM: Molecular marker 100bp. NTC: No-template control.</p

Flow cytometric analysis of the relative DNA content in <i>Trypanosoma cruzi</i> and <i>Trypanosoma rangeli</i>.

<p>(A) DNA histogram showing superimposed traces of the clone CL Brener, <i>T</i>. <i>cruzi</i> strains and <i>L</i>. <i>major</i>. (B) DNA histogram showing superimposed traces of the clone CL Brener, <i>T</i>. <i>rangeli</i> strains and <i>L</i>. <i>major</i>.</p

Discrimination between <i>T</i>. <i>cruzi</i> and <i>T</i>. <i>rangeli</i> by DNA content analysis.

<p>(A) DNA histogram of the clone CL Brener of <i>T</i>. <i>cruzi</i>, P07 strain (KP1+) of <i>T</i>. <i>rangeli</i>, and mixed CL Brener + P07. (B) DNA histogram of the clone CL Brener of <i>T</i>. <i>cruzi</i>, SO48 [KP1(−)] strain of <i>T</i>. <i>rangeli</i>, and mixed CL Brener + SO48.</p

Genetic characterization <i>Trypanosoma cruzi</i> and <i>Trypanosoma rangeli</i> strains.

<p>(A) kDNA analysis of <i>T</i>. <i>rangeli</i> strains in a silver stained 6% polyacrylamide gel containing PCR products obtained with primers S35/S36/KP1L. The presence of a 165-bp band indicates the presence of KP1 minicircles. (B) Detection of the 832-bp fragment of TcSC5D gene in <i>T</i>. <i>cruzi</i> strains and clone. (C) PCR–RFLP of TcSC5D products digested with <i>Hpa</i>I and <i>Sph</i>I enzymes. MM: Molecular marker 100bp. NTC: No-template control.</p

Representative flow cytometric gating strategy for DNA content analysis of <i>Trypanosoma cruzi</i> CL-Brener and <i>Leishmania major</i> CC1 strains stained with propidium iodide (PI).

<p>Panels A and D represent relative size (FSC) and complexity (SSC). Panels B and E panels represent PI-A and PI-W parameters indicating single cells stained with PI. Panels C and F show DNA content histogram and G1-0 and G2-M peaks are pointed. Relative PI fluorescence intensity at G1-0 was used for DNA content estimation for all strains analyzed using <i>T</i>.<i>cruzi</i> CL-Brener as reference strain.</p