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

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.

Trypanosoma cruzi is the etiological agent of Chagas disease, a condition that affects approximately seven million people, mainly in Latin America.In contrast with T. cruzi, Trypanosoma rangeli causes only temporary manifestations in humans and in other mammals, but is pathogenic for its triatomine vector.T. cruzi and T. rangeli share vectors and reservoirs, making the specific diagnosis of Chagas disease difficult (1) .In vitro cultivation of T. cruzi is used for detection of the parasite in vertebrate hosts and vectors.T. cruzi cultivation can be performed in liquid, solid, or semisolid media (2) (3) .Liver infusion tryptose (LIT) is a liquid medium (4) used for large-scale cultivation of trypanosomatids for genetic characterization and manipulation of parasites.Due to poor growth of T. rangeli in LIT (5) , other supplements must be added to the culture media.The addition of human urine (HU) to LIT medium stimulates the in vitro growth of T. cruzi and T. rangeli, allowing similar maximum parasite densities for these parasites (5) .
Many studies use limiting dilution in cases where cellular cloning is necessary.However, this is a time-consuming technique, and the clonality of the isolated cells often cannot be guaranteed.Other methodologies for the guaranteed isolation of a single cell include micromanipulation or the use of a fluorescence-activated cell sorter (FACS) (6) .The most efficient method for isolating clonal parasite lineages, however, is plating the forms on solid or semisolid media.In spite of the widespread use of liquid media (primarily LIT) for T. cruzi cultivation, there have been few previous attempts to cultivate T. cruzi on semisolid media.In addition, growth differences have been observed between T. cruzi and T. rangeli on solid media (3) .One study showed that BLAB (BHI-LIT-blood agar), a solid medium (2) , resulted in the growth of T. cruzi colonies with reasonable plating efficiencies.However, growth of T. rangeli has not been assessed on BLAB medium.Different types of solid media (3) , each made up of a double layer, were recently assessed for growth of T. cruzi and T. rangeli isolates from triatomine bugs and mice and resulted in high plating efficiencies for T. cruzi.Four colonies of T. rangeli (strain not specified) were also isolated from one of the plates.
Considering the necessity of a simple and inexpensive method for obtaining clonal lineages for genetic manipulation experiments, in this study, we aimed to determine the growth and plating efficiency of T. cruzi and T. rangeli on semisolid LIT (SSL) medium with 3% (v/v) HU added.
Epimastigote forms of T. cruzi (strains Y, JG, and RN1) and T. rangeli (strains P07 and SO29) (7) were maintained by passaging in LIT medium (4) supplemented with 10% (v/v) fetal bovine serum.These were incubated at 28ºC in a biochemical oxygen demand (BOD) incubator.SSL medium was prepared by mixing equal volumes of 2X LIT medium and 2% (w/v) noble agar (Sigma, Missouri, USA).SSL-HU medium was prepared by supplementing SSL with 3% (v/v) sterile HU (5) .Noble agar was replaced with 1% (w/v) bacteriological agar (BA) to produce SSL-BA medium with or without HU.Aliquots of 20mL SSL were poured into Petri dishes with a 9-cm diameter.After drying, the plates were refrigerated at 4ºC until use.
Plates were placed at approximately 25 o C for approximately 15 min before use.Parasites were counted in a hemocytometer and diluted in LIT medium to obtain 1.0 × 10 3 epimastigote forms in a volume of 30-50µL.This volume was transferred to the surface of each plate, and parasites were spread using a disposable plastic Drigalsky spatula.Plates containing T. cruzi and T. rangeli strains were prepared using a single strain or by mixing different parasite strains (Table 1).In this last case, equal quantities of parasites (5.0 ×10 2 forms of each strain) were mixed and then spread on the plate using just one disposable plastic Drigalsky spatula.The plates were sealed and incubated at 28ºC.Plates were checked for the appearance of colonies twice a week for four weeks.Plating efficiency was determined as the ratio of the total number of colonies obtained per plate to the total number of parasites plated (1.0 × 10 3 epimastigote forms).Isolated colonies were picked from plates 30 days after incubation, and each colony was inoculated into 2mL of LIT medium.After 48 hours, aliquots of 2mL of each culture were inoculated into the same volume of a solution containing 6M guanidine hydrochloride and 0.2M ethylenediaminetetraacetic acid (EDTA) for preservation, then DNA extraction was performed as previously described (8) .Twenty colonies from each plate were selected for further pulsed-field gel electrophoresis (PFGE) analysis.
Colonies of T. cruzi and mixed strains became macroscopically visible beginning ten days after plating.There were no differences observed in the time before colony appearance among T. cruzi strains or mixtures of strains (10-12 days).T. rangeli colonies, however, were only observed 21 days after incubation.The plating efficiency of various strains on SSL-HU plates varied from 5.1 to 9.6% (Table 1).Colonies were generally transparent or whitish and differed in size.When observed by microscopy, the colonies revealed epimastigotes  with normal structures and active flagellar movement.In addition to SSL-HU plates, epimastigotes were also plated with 1.0% (w/v) bacteriological agar instead of noble agar and in SSL with no HU.No colonies were detected in plates prepared without HU, using either noble or bacteriological agar (Table 1).When HU was added to plates with 1.0% BA, the plating efficiency was very low (0.3-1.2%) compared to that of plates made with noble agar and HU (Table 1).

Days to obtain colonies
PCR analysis allowed the identification of DNA bands obtained from T. cruzi (100bp) and T. rangeli (170bp) colonies present on the same plates (Figure 1).In mixed platings (T.cruzi/ T. rangeli), only T. cruzi colonies were detected (Figure 1).PFGE allows the determination of the molecular karyotypes of the parasites in each colony, permitting their association with the respective parental strain.On mixed plates with T. cruzi (JG strain) and T. rangeli (SO29/P07), only T. cruzi was rescued (Figure 2A and Figure 2B), confirming the telomeric PCR data.Mixed plates containing the JG and RN1 strains of T. cruzi resulted in clones belonging to both parental strains (Figure 2C), as demonstrated by the karyotype profiles.Those containing the T. cruzi Y and RN1 strains, however, only resulted in Y strain colonies (Figure 2D).Similarly, when the Y strain was cultivated with the JG strain, only JG was rescued (Figure 2E).This may be due to the number of colonies analyzed, since other T. cruzi mixed platings provided colonies from both strains (Figure 2C).
In this study, the use of SSL-HU medium resulted in the growth of a large number of T. cruzi and T. rangeli colonies.In liquid media, the addition of HU leads to similar growth rates of T. cruzi and T. rangeli when they are independently cultivated (5) .HU has also been successfully used for cultivation of several Leishmania spp.isolates (10) .Here, we also obtained similar plating efficiencies for T. cruzi and T. rangeli when they were independently plated in SSL medium supplemented with HU.When plates were prepared by mixing T. cruzi and T. rangeli strains, however, only T. cruzi colonies were observed, as revealed by PCR and PFGE.These results confirm that SSL-HU medium favors T. cruzi growth, as previously observed.In a previous study (3) , when triatomine feces from Rhodnius prolixus were plated, colonies from both species were obtained; however, the number of T. rangeli colonies was lower than that of T. cruzi.In our study, the difference in time required for colony appearance between T. cruzi (approximately 12 days) and T. rangeli (21 days) explains the reason that T. cruzi colonies predominate on mixed plates.Differences in the growth curves of T. cruzi and T. rangeli have been described previously (5) and may explain the presence of only T. cruzi colonies on mixed T. cruzi/T.rangeli plates.
No colonies were detected on plates prepared with SSL without HU, suggesting that the presence of a component of HU promotes T. cruzi and T. rangeli growth (5) .Taken together, these results point to differences in the metabolisms of these parasites that can be assessed with comparative genomics (11) .
Our results confirm that SSL-HU medium allows the isolation of clonal parasite lineages.Even when seeded on the same plate, trypanosomatids formed isolated colonies, each with a specific molecular karyotype.As mentioned previously, other studies have assessed the growth of T. cruzi on BLAB (2) or double-layered (3) media, resulting in plating efficiencies comparable to those in this study.Thus, depending on the aim of the study, SSL-HU can be used as an alternative to these other media.SSL-HU, in particular, is simple to make, and HU is free, easily available, and can be obtained without invasive methods.In studies in which the genetic background of the parasites is not known, techniques with higher discriminatory power, such as microsatellite analysis (12) (13) (14) and multilocus sequence typing (MLST) (15) , may be required to identify all clones present in a parasite population.Future studies should compare the results obtained with SSL-HU with those of other media and investigate the HU factor that supports parasite growth.SSL-HU medium is a new alternative for the isolation of T. cruzi and T. rangeli clonal lines, opening new avenues for genetic studies in these organisms.

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. Number of colonies and plating efficiency (%) a SSL-HU b SSL c SSL-HU-BA d SSL-BA e
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.a Percentage 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.b Semisolid LIT medium prepared with 1.0% (w/v) noble agar and 3% (v/v) HU. c Semisolid LIT medium prepared with 1.0% (w/v) noble agar with no HU added.d Semisolid LIT medium prepared with 1.0% (w/v) bacteriological agar (BA) and 3% (v/v) HU. e Semisolid LIT medium prepared with 1.0% (w/v) bacteriological agar (BA) with no HU added.