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

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.

Keywords:
Trypanosoma rangeli; Trypanosoma cruzi; Clonal lineages

Authorship

Emanuella Francisco Fajardo

Instituto de Ciências Biológicas e Naturais, Universidade Federal do Triângulo Mineiro, Uberaba, Minas Gerais, Brasil. Universidade Federal do Triângulo MineiroBrazilUberaba, Minas Gerais, BrazilInstituto de Ciências Biológicas e Naturais, Universidade Federal do Triângulo Mineiro, Uberaba, Minas Gerais, Brasil.

Marlene Cabrine-Santos

Instituto de Ciências da Saúde, Universidade Federal do Triângulo Mineiro, Uberaba, Minas Gerais, Brasil.Universidade Federal do Triângulo MineiroBrazilUberaba, Minas Gerais, BrazilInstituto de Ciências da Saúde, Universidade Federal do Triângulo Mineiro, Uberaba, Minas Gerais, Brasil.

Keila Adriana Magalhães Ferreira

Centro de Educação Profissional, Universidade Federal do Triângulo Mineiro, Uberaba, Minas Gerais, Brasil.Universidade Federal do Triângulo MineiroBrazilUberaba, Minas Gerais, BrazilCentro de Educação Profissional, Universidade Federal do Triângulo Mineiro, Uberaba, Minas Gerais, Brasil.

Eliane Lages-Silva

Luis Eduardo Ramírez

André Luiz Pedrosa

Corresponding author: Dr. André Luiz Pedrosa. e-mail: pedrosa@icbn.uftm.edu.br

The authors declare that there is no conflict of interest

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (2)
Tables (1)

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Figure 1
PCR amplification of a 100-bp fragment of the telomeric Trypanosoma cruzi region and a 170-bp fragment of the subtelomeric Trypanosoma rangeli region from clones obtained by plating of individual parasite strains (Y, JG, RN1, P07, and SO29) and from clones obtained by mixed plating (samples 1 and 2: JG/SO29; 3 and 4: JG/P07; 5 and 6: Y/P07; 7 and 8: Y/SO29; 9 and 10: RN1/P07) in semisolid medium. The MM used was a 100-bp DNA ladder (Invitrogen). MM: molecular marker; NC: negative control; bp: base pair; PCR: polymerase chain reaction.

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Figure 2
Ethidium bromide-stained 1% agarose gels showing Trypanosoma cruzi and Trypanosoma rangeli chromosomes separated by PFGE. Lambda DNA concatemers (BioLabs, Ontario, Canada) (panel A) and Saccharomyces cerevisiae chromosomes (BioLabs) (panels B-E) were used as size markers (M). Figures below each gel show the results of the GelCompar II analysis of chromosomal bands shared between clonal lineages and parental strains. Kb: kilo base pairs; PFGE: pulsed-field gel electrophoresis.

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Table 1
Number of Trypanosoma cruzi and Trypanosoma rangeli colonies obtained after incubation in semisolid liver infusion tryptose media with noble agar or bacteriological agar, with or without supplementation with 3% human urine.

Figure 1 PCR amplification of a 100-bp fragment of the telomeric Trypanosoma cruzi region and a 170-bp fragment of the subtelomeric Trypanosoma rangeli region from clones obtained by plating of individual parasite strains (Y, JG, RN1, P07, and SO29) and from clones obtained by mixed plating (samples 1 and 2: JG/SO29; 3 and 4: JG/P07; 5 and 6: Y/P07; 7 and 8: Y/SO29; 9 and 10: RN1/P07) in semisolid medium. The MM used was a 100-bp DNA ladder (Invitrogen). MM: molecular marker; NC: negative control; bp: base pair; PCR: polymerase chain reaction.

Figure 2 Ethidium bromide-stained 1% agarose gels showing Trypanosoma cruzi and Trypanosoma rangeli chromosomes separated by PFGE. Lambda DNA concatemers (BioLabs, Ontario, Canada) (panel A) and Saccharomyces cerevisiae chromosomes (BioLabs) (panels B-E) were used as size markers (M). Figures below each gel show the results of the GelCompar II analysis of chromosomal bands shared between clonal lineages and parental strains. Kb: kilo base pairs; PFGE: pulsed-field gel electrophoresis.

Table 1 Number of Trypanosoma cruzi and Trypanosoma rangeli colonies obtained after incubation in semisolid liver infusion tryptose media with noble agar or bacteriological agar, with or without supplementation with 3% human urine.

SSL: semisolid LIT; HU: human urine; BA: bacteriological agar; Tr: Trypanosoma rangeli; Tc: Trypanosoma cruzi; NP: not performed; LIT: liver infusion tryptose; BOD incubator: biochemical oxygen demand incubator. aPercentage of T. cruzi or T. rangeli colonies obtained. 1,000 epimastigote forms were spread on each plate and incubated at 28°C in a BOD incubator. bSemisolid LIT medium prepared with 1.0% (w/v) noble agar and 3% (v/v) HU. cSemisolid LIT medium prepared with 1.0% (w/v) noble agar with no HU added. dSemisolid LIT medium prepared with 1.0% (w/v) bacteriological agar (BA) and 3% (v/v) HU. eSemisolid LIT medium prepared with 1.0% (w/v) bacteriological agar (BA) with no HU added.

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

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[1] Corresponding author: Dr. André Luiz Pedrosa. e-mail: pedrosa@icbn.uftm.edu.br