Isoenzyme Profiles of Trypanosoma Cruzi Stocks From

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Mem lnst Oswaldo Cruz, Rio de janeiro, Vol. 96(4):527-533, May 2001 527 Isoenzyme Profiles of Trypanosoma cruz; Stocks from Different Areas of Paraguay

Nidia Acosta, Margarita Samudio, flsa López, Fernando Vargas", Nina Yaksic*, Simone Frédériquebrenière**, Antonieta Rojas de Arias/+ I Dq"ento de Medicina Tropical, Instituto de Investigaciones en Ciencias de la Salud (TJNA), Casilla de l Correo 251 Asuncih, Paraguay *Universidad Mayor de San Andrès, Instituto Boliviano de Biologia de 1, I Altura, La Paz, Bolivia **UMR CNRS/ORSTOM No9926, Mission ORSTOM Mexique, Los Morales, Mèxico DF Twenty one Trypanosoma cruzi stoch from humans, domiciliary triatomines and one sylvatic ani- mal of different areas of Paraguay were subjected to isoenzyme analysis. Thirteen enzyme systems (15 loci in total) were shldied. MN c12 (clonets 39) and SO34 c14 (clonets 20) were used as references. Relationships between stocks were depicted by an UPGMA dendrogram constructed using the Jaccard's distances matrix. Among the Paraguayan stoch I4 zymodemes were identified (Par1 to Parl4), Par 5 being the mostfrequent. Polymorphism rate and clonal diversity were O. 73 and 0.93, respectively. Aver- age number of alleles per polymorphic locus was 2.5 {range 2-4). These measurements show a high diversity, which is confirmed by the dendrogram topology. All stoch belong to the same lineage, as MN c12 reference strain (T. cruzi I.. Moreover three distinct subgroups were ident$ed and two of them correspond to Brazilian and Bolivian zymodemes, respectively. The third subgroup, the most common in Paraguay, is related to Tulahuen stock. The large geographical distribution of some zymodemes agrees with the hypothesis of clonalityfor T. cruzipopulations. However sample size was not adequate to detect " .. genetic recombination in any single locality. :i /I

1 Key words: Ttypanosoma cruzi - zymodemes - genetic polymorphism - clonality - Paraguay 1. . .- .. .." I. ., .. I.

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Chagas disease is widely distributed in Latin The seroprevalence rates in endemic areas of ;j America from the north of Mexico to the south of Paraguay range from 14.3% in communities in the ,I Argentina (Zeledón 1974). In Paraguay, like in a department of San Pedro (Canese 1973), 21.6% in !I many Latin American countries, the disease is an Paraguarí, 24.9% in Cordillera (Rojas de Arias important public health problem, because there is 1996) and 72.9% in an indigenous group of the a high number of Trypanosoma cruzi infected Chaco region, the highest value recorded (Canese people and high triatomine infestation rates in & Brice 1977). dwellings (WHO 1986, Rojasde Arias 1996). Para- Limited studies have analyzed the T. cruzi guay is located in the central part of South America stocks collected in different areas in Paraguay. two and it is divided by the Paraguay River into Chapman et al. (1 984) related T. cruzi stocks iso- main regions: the Western or Chaco and the East- lated in the Presidente Kayes department of the em regions. Most of the population live in mal Paraguayan Chaco to the Bolivian zymodeme 2. areas where living conditions make T. cruzi trans- Yamasaki et al. (1990) described similar mission easy. Triatoma infestans is the main vec- schizodeme profiles of three isolates from acute tor and vector transmission is the most common Chagas patients fiom distant geographical areas and way of acquiring the disease (eojas de Arias 1996). related .to a Brazilian strain. Mimori et al. (1992) foxnd four distinct schizodeme groups among eleven stocks and matched two of them with the Brazilian zymodeme 2. In aprevious study, Acosta et al. j1995) carried out metacyclogenesis, experi- mental infection in mice, lectin agglutination, This study received financial support from the United parasitemic curves and morphometry with T. cruzi Nations Development ProgramlWorld BankMrHO Spe- cial Program for Research and Training in Tropical Dis- stocks from acute cases in humans, and found a ease (grant 940209). great variability, especially in the biological be- *Corresponding author Fax: i-595-21-480-185. Email: havior. Analysis of the geographical distribution sarias@conexion. com.py of T. cruzi is often difficult because of lack of stan- Received 15 May 2000 dardization of techniques, studies with reduced loci, 2000 Accepted 7 December . absence of phylogenetic studies and lack of the cumenhire I

..- 528 Isoenzymes of T: cruzi from Paraguay Nidia Acosta et al. same reference strains (Barnabé & Breniere 1999). Isoenzynze analysis - Parasite pellet was har- The present study analyzes the isoenzyme profiles vested in the growth peak day and washed twice of 21 T. cmzi stocks mainly from the domestic with PBS at 3,000 rpm, 4°C for 1O min. The pellet cycle of different localities in Paraguay. Identical was kept at -80°C until used. At the time of elec- i loci and similar electrophoretic conditions were trophoresis it was diluted in an equal volume of E- ...... ,..-). used as previously to analyze large samples of T. protein stabilizer solution containing 2 mM cruzi from diverse origins. This allows easier iden- amino caproic acid, EDTA and dithiothreitol. tification of the taxonomic position of the Para- Electrophoresis was carried out in cellulose guayan stocks. acetate plates (Helena, Beaumont, TX) as previ- MATERIALS AND METHODS ously described (Tibayrenc & Le Ray 1984). Thir- teen enzymatic systems were studied: glucose Parasite stocks - Twenty one stocks from dif- phosphate dehydrogenase (GGPD, EC. 1.1.1.49), ferent regions of Paraguay were studied (Fig. 1). glucose phosphate isomerase (GPI, EC.5.3.1.9), Twelve stocks belonged to human cases, eight glutamate dehydrogenase NAD (GDH 1, stocks to domestic T. infestans and one stock to a EC. 1.4.1.2., glutamate dehydrogenase NADP+ domesticated yellow armadillo. The origin of each (GDH 2, EC. 1.4.1,4), isocitrate dehydrogenase stock is shown in Table I. SO34 c14 (clonets 20) (IDH, EC. 1.1.1.42), malate dehydrogenase (MDH, and MN ci2 (clonets 39), previously typed EC. 1.1.1.37), malic enzyme (ME, EC. 1.1.1.40.), (Breniere et al. 1998), were used as reference peptidase (PEP 1, EC.3.4.11.1 I), peptidase (PEP strains. These clones are genetically well separated 14-2, EC.3.4.11.13), phosphoglucomutase (PGM, and they belong to T. cmzi I and T. cruzi II groups, EC.2.7.5.1), 6-phosphogluconate dehydrogenase, .44), respectively (Anon 1999). They correspond to (6PGD, EC. 1.1. I diaphorase (DIA, EC.I.6.-.-), major clones frequently isolated in South America aspartate aminotransferase (GOT, EC.2.6.1.1). and they are the main clones found in the domestic The genotypes were assigned according to the Bolivian cycle (Tibayrenc & Breniere 1988, profiles in the cellulose acetate plates with the‘al- Brenière et al. 1998) ready mentioned criteria of genotype numbers of Isolation and maintename the stocks - The (Tibayrenc & Ayala 1988): band ’1 corresponded stocks were obtained bv-, hemoculture,-- xenocufture to the smallest distance from the anode. . and mice inòculation, maintained and ampliiici ai.....:.- The observed heterozykosiw was estilnated by -I I 28°C in LIT medium (liver infusion tryptose) the following formula: Ho = ho/(N x r), where ho (Camargo 1964) with 50 pgll of hemin, supple- = total number of heterozygous observed, N = mented with 20% fetal bovine 100 U1 of serum, number of stocks and r = number of loci studied. penicillin and 100 pg of streptomycin per ml of medium. The expected heterozygysity was estimatyd by the following relation: H= F hlr, where h = F 1- qi2, and r..= number of loci and qì = frequency of the BOLIVIA allele “Y in locus ‘Y’. The polymorphism rate was determined by the relation: no. of polymorphic loci/no. of studied loci. The clonal diversity was measured by Whittam’s (Whittam 1989) index which is equal Z to: d = n ( 1- Xi2 )/( n- I), where Xi = frequency of each zymodeme and n = no. of stocks. This index measures the probability of obtaining stocks from different zymodemes when performing sampling the study population. The average of alleles per locus was calculated by the relation: I: a / r, where a = no. of alleles per locus and, r = no. of studied loci. In order to estimate the genetic divergence between zymodemes, Jaccard‘s phenetic distance .. . .I was (Jaccard 1908): d = 1 - ( C/2N - C), where C = two ARG E NTlNA no. of common bands between zymodemes and N = no. of total bands of both zymodemes. Fig. I: geographical location of the different Paraguayan The UPGMA (unweightedpair-group method with stocks. I, 2 and 3 correspond to the subgroups of zymodemes arithmetic averages) (Sneath & Sokal 1973) was found. used to cluster the zymodemes according to 96(4), Mem lnst Oswaldo Cruz, Rio de janeiro, Vol. May 2001 529 Jaccard's distances matrix. The dendrogram was 1 to 6 for Par 5 and the probability of sampling built using Mac Dendro (Thioulouse 1989). two different zymodemes in the population mea- RESULTS sured by the Whittam's index was 0.93. Phenetic relationships - The dendrogram (Fig. Genotype variability - Among the 13 enzyme 2) based on the Jaccard's distance matrix showed two systems tested, (ME and DIA) made it pos- two upper branches which included a reference sible to infer the activity of two different loci, con- stock each. They correspond to the two principal sequent1y;the analysis of 15 putative loci in total groups of T. cmzi previoüsly described (Tibayrenc was performed. Tables I and II show the multilocus 1995, Brenière et al. 1998). All the Paraguayan genotypes of the 23 studied stocks deduced from stocks belong to the T. cmzi II as MN c12 refer- the isoenzyme profiles obtained; stocks that showed ence stock. Moreover, T. cruzi II, presented three identica1 isoenzymatic pattems for all the enzyme separated branches (subgroups 1 to 3) with dis- systems belong to the same zymodeme. Fifteen tances ranging from 0.35 to 0.65 between them distinct zymodemes were identified including the (Fig. 2). Four stocks were closely related to Mn two reference stocks MN c12 and SO34 c14 which c12 reference stocks (subgroup 1) which belong to belong to Par1 and Parl5, respectively. the most common zymodeme (clonets 39) found Among the Paraguayan stocks, four loci were in the domestic Bolivian cycle. Except the Arma I monomorphic (Mdh, Pep 2, Got, Dia I) and the 134 (the only stock isolated from a sylvatic mam- I polymorphism rate was 0.73. Regarding the 11 mal) all the stocks of subgroups 1 and 2 presented i variable loci, the average number of alleles per variable heterozygote pattems for the Gpi locus locus was 2.5 (range 2-4). Several pattems ob- and those of subgroups 3 had the same homozy- served for Gpi, Idh, Pgni and 6pgd loci were char- gous profile (genotype 2/2, Gpi locus). The distri- acteristic of heterozygotes, and the observed het- bution of zymodemes according to their hosts and erozygosity was O. 17. The average value of ex- geographical origin is shown in Figs 1 and 2. An pected heterozygosity was 0.34; a deficiency of absence of specific geographical distribution of the heterozygotes was hence apparent. The number of zymodeme groups was observed, since for example , stocks belonging to a single zymodeme range from zymodemes belonging to subgroup 2 were found

TABLE 1 Origin of the Typanosoma cruzi stocks examined Stocks Department Host ZYm Sub-group HV Caaguaz6 Human (acute) Par 9 2 ROFl Central Human (acute transfusional) Par 13 3 JM Central Human (acute transfusional) Par 14 3 RN Lezcano Central (mother from San Pedro) Human (congenital) Par 1 1 JAG Cordillera Human (acute) Par 13 3 DG Paraguari Human (indeterminate) Par 10 3 Xc 116 Paraguari TriatQmg infestons Par 2 1 FALE Paraguari Human (indeterminate) Par 3 1 GS Presidente Hayes Human (acute) Par 11 3 Xc ILSE Presidente Hayes Human {acute) Par 5 2 LAURA Presidente Hayes Human (acute) Par 5 2 Corai 6 Presidente Hayes Triatama infatans Par 5 2 Coraí 14 Presidente Hayes Triatoma infestans Par 5 2 Lengua 15 Presidente Hayes Triatama dnfeTstans Par 5 2 2 Coraí 7 Presidente Hayes Triatoma infestuns Par I6 Corai sin Presidente Hayes Triatoma infestans Par 2 Lengua 16 Presidente Hayes Triatoma infestans Par 7 2 Coraí 18 Presidente Hayes Triatoma infestans Par 8 2 EV San Pedro Human (chronic) Par12 3 ARMA 134 San Pedro Euphractus sexcintus Par 4 1 (yellow armadillo) EM1 San Pedro Human (acute) Par 5 2 SO34 c14' Potosi (Bolivia) Triatoma infestans Par 15 MN c12a IV Region (Chile) Human (chronic) Par 1 1 a: laboratory clones 20 (SO34 c14) and 39 (MN c12) used as references (Tibayrenc & Ayala 1988).

I Paraguay 530 Isoenzymes of 7: cruzi from 9 Nidia Acosta et al.

----““““w-- N W W WW- l#l----NN...... N N in Presidente Hayes, San Pedro and Caagmzú de- partments, and zymodemes of the three subgroups were obsemed in the San Pedro department. Re- garding the host, all the studied stocks (except one) were from human cases and T. iizfestans which is the principal vector in Paraguay. Therefore all these stocks can be considered as part of the do- I mestic cycle. As expected similar zymodemes and closely related zymodemes could be found in T. infestans and human (eg. Par 5) but zymodemes of subgroups 3 were only isolated from humans. Nevertheless, such a host specificity must be veri- fied with larger samples of stocks. DISCUSSION The isoenzyme technique has been widely used to characterize T. cruzì populations of different countries belonging to domestic and sylvatic cycles (Miles et al. 1980, Tibayrenc & Miles 1983, Carneiro et al. 1991, De Luca D’oro et al. 1993, Lewicka et al. 1995, Brenibre et al. 1997) but limited data are available from Paraguayan stocks. The comparison of isoenzyme data with other genetic rl markers gave congruent results and showed the division of T. cruzì taxon into two principal groups (Tibayrenc 1995, Souto et al. 1996, Brisse etaal. 1998, Breniére et al. 1998). According to the analy- “’ *

sis of MLEE data (22 loci) and WD(10-mer ..$ a primers) together T. cmzi I and T. cmiII present . i high bootstrap values (97% and 93% respectively), . , a strong support of the T. cruzì division in two lin- % r. eages; the SO34 c14 stock belongs to T. cruzi I and MN c12 to T. cruzi II. In the present study, the isoenzyme analysis of 15 loci allowed the classification . of the 21 Paraguayan stocks into 14 zymodemes and the estimation of the degree of genetic differentiation among them. All the stocks were clustered in an upper branch with the MN c12 stock used as control and none of them were genetically ...... “, r.*,.,...... Ni.*.cn.h¿ li related to the SO34 c14 stock. Moreover, the comparison of the distances with previous data show that the Paraguayan stocks can be considered as belonging to T. cruzi II. Nevertheless, among the stocks, genetic diversity is still important, and three subgroups were observed. Previous analysis also showed that group T. cruzi II presents a high genetic diversity and Tibayrenc (1998) proposed five subgroups. The relationship between these subgroups and the three Paraguayan subgroups is more difficult to interprete. Nevertheless, it is worth not-

” ... ”...... - .. ing that the stocks belonging to subgroups 1 and 2 .... have heterozygous profiles for Gpi,Pgm and 6Pgd loci, hence they should correspond to two differ- 1,;: ent zymodemes named 239 and 243 by Tibayrenc ... .-..-,. .. (1998): 239 include MN cl2 stock and 243 include the laboratory reference strain Tulahuen. The re- maining Paraguayan stocks are clustered apart and Mem lnst Oswaldo Cruz, Rio de Janeiro, Vol. 96(4), May 2001 537 Jaccard's distance O 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 I l l l l I I I

A pari 1

A Par3 fl c Par4 a AB Par5 A B Par6 A B Par7 A 3- B Par8 A 2 A Par9 O

A ParIOU

A Par11 A A Par12. - 3 A Par13.A

A .Par 14 T. cruzi I SO34 *

Fig. 2: dendrogram constructed by the hierarchical ascending method UPGMA from Jaccard's distanceA: matrix (15 loci), for 21 Trypanosoma cmzi zymodemes. Host and geographical origins are indicated for each zymodeme. human,o: B: Trialoma infesfans; C: Euphractus sexcintus (armadillo); A: Presidente Hayes; O: San Pedro; A: Cordillera; M: Central; Caaguazu; O:Paraguan. 1, 2 and 3 correspond to the subgroups ;*: reference stock (S034) did not present heterozygous patterns (subgroup pertaining to one of the most frequent clone circu- 3) but gave the same profile as Esmeraldo c13 stock lating in the Bolivian domestic cycle (clonets 39) (Brazilian 22 reference) at Gpi and 6Pgd loci which (Brenière et al. 1995, 1998) (subgroup 1); and (iii) was previously characterized by Brenière et al. Tulahuen reference stock (subgroup 2). The stocks (1998). Previous analysis of Paraguayan T. cruzi belonging to the latter were.the most abundant in stocks by isoenzyme (Chapman et al. 1984) and Paraguay (50% of the studied sample). restriction enzyme analysis of kinetoplast DNA High genetic variability in the studied popula- (Yamashi et al. 1991, Mimori et al. 1992) showed tion was confirmed by the index of polymorphism that the stocks shared most features of the Boliv- and Whittam's which are similar to others found ian 22 and Brazilian 22 T. cmzi stocks. More re- in other countries (Tibayrenc &Ayala 1988, De cent works showed that these zymodemes belong Luca D'Oro et al. 1993, Brenière et al.1997). to T. cruzi I1 (Brenière et al. 1998), but are clus- The criteria of clonality for T. cmzi populations tered apart in distinct subgroups (Tibayrenc & has been widely described in severa1 studies Miles 1983, Bamabé & Brenière 1999): Bolivian (Tibayrenc & Desjeux 1983, Tibayrenc et al. 1984, 22 and Brazilian 22 belong to 239 and 232, re- Tibayrenc & Ayala 1988). The results obtained.in spectively, according to Tibayrenc (1998). The this study agree with this hypothesis. A stock iso- present and previous results agree with the pres- lated from a human case of San Pedro department ence in Paraguay of stocks genetically related to: presented the same genotype as stocks from hu- (i) the Brazilian 22 (subgroup 3) (ii) Bolivian stocks man and triatomines of Presidente Hayes depart- 532 Isoenzymes of T. cruzi from Paraguay Nidia Acosta et al.

ment. These two regions are geographically sepa- cruzi in Bolivian triatomines. Am J Trop Med Hyg rated by the Paraguay River (see Fig. l). In the 53: 179-184. same way, the stock RN Lezcano showed the same Brenière SF,Bosseno MF, Telleria J, Bastrenta B, Yaksic profiles as MN c12 reference strain isolated in other N, Noireau F, Alcazar JL, Barnabé C, Wincker P, Tibayrenc M 1998. Different behavior of two Try- countries and at different times. the possibil- . .. , Also, panosoma cruzi major clones: transmission and cir- ity of contamination was minimal because the para- culation in Bolivian young patients. Exp Parasitol sites were processed separately by different labo- 89 285-295. ratories. The fact of finding stocks with the same Brenière S, López J, Vargas F, Bamabe C 1997. Ge- profile in geographically distant areas and isolated netic variability and microdistribution of Triatoma in different moments confirms the ubiquitous fea- infestans genotypes and Trypanosoma cruzi clones ture of these zymodemes which is one of the in Arequipa Region (Pení). Mem Inst Oswaldo Cruz clonality criteria (Tibayrenc & Ayala 1988). The 92: 401-408. difference between expected and observed het- Brisse S, BamabC C, Tibayrenc M 1998. Tppanosoma erozygosity values also supports the clonality. cruzi: how many relevant phylogenetic subdivisions are there? Parasitol Today 14: 178-179. Some studies suggest genetic exchanges in this Camargo E 1964. Growth and differentiation in Trypa- organism (Bogliolo et al. 1996, Carrasco et al. nosoma cruzi I. Origen of metacyclic trypanosomes 1996). The clonal model does not exclude the pos- in liquid media. Rev Inst Med Trop São Paulo 6 sibility of the occurrence of genetic exchange. It 93- 1OO. is only that the exchange is not frequent enough Canese A 1973. Epidemiologia de la enfermedad de for the current genotypes or zymodemes to be con- Chagas en el Paraguay. Rev Parag de Microbio1 8: sidered unstable or transient entities. 13-18. The identification of the clonal types of cir- Canese J, Brice E 1977. Diagnbstico de la enfermedad culating T. cruzi and their geographical distribu- de Chagas en aborígenes del Chaco paraguayo. Rev tion are important for the study of Chagas disease. Parag de Microbio1 II: 9-10: .. Carneird M, Romanha AJ, Chiari E 1991. Biological Based on clonal types main and secondary species characterizationof Trypanosoma cruzi strains from of vector can be identified, the relationship between different zymodemes and schizodemes. Mem Inst wild and domestic cycles can be evaluated, reser-. Qswaldo Cr(l.2-86387-393. voirs can be identified and the observed patholo- Cairasco HK, Frame IA, Valente SA, Miles MA 1996. gies' can be compared (Barnabé & Breniere 1999). Genetic exchange as a possible source of genomic ACKNOWLEDGMENTS diversity in sylvatic populations of Tvpanosoma cruzi. Ani J Trop Med Hyg 54: 41 8-424. To Luis Sanabria, Alba Inchaustti, Ma. Elena Ferreira Chapman M, Baggaley R, Godfrey-Fausset P, Malpas for their cooperation in the isolation of the stocks used T, White G, Canese J, Miles M 1984. Tvpanosoma in this work; to Dr Marisel Maldonado for revising the cruzi from the Paraguayan Chaco: isoenzyme pro- English of the manuscript. To the technical staff of the files of strains isolated at Makthlawaiya.JProtozool Department of Tropical Medicine: Pedro Recalde, Elvio 3: 482-486. Benitez, Victoria Bogado and Jazmina Rívarola for their De Luca D' Oro GM, Gardenal CN, Perret B, Crisci JV, valuable cooperation. Montamat E 1993. 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