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Am. J. Trop. Med. Hyg., 78(3), 2008, pp. 472-478
Copyright © 2008 by The American Society of Tropical Medicine and Hygiene

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Two Triatoma dimidiata Clades (Chagas Disease Vector) Associated with Different Habitats in Southern Mexico and Central America

Paulino Tamay-Segovia, Ricardo Alejandre-Aguilar, Fernando Martínez, Guiehdani Villalobos, Francisco J. Zavala-Díaz de la Serna, Patricia de la Torre, Juan Pedro Laclette, Selene Blum-Domínguez, AND Bertha Espinoza*
Centro de Investigaciones en Enfermedades Tropicales, Universidad Autónoma de Campeche, Campeche, México; Departamento de Parasitología, y Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Distrito Federal, México; Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Distrito Federal, México


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Triatoma dimidiata is the only reported Chagas disease vector in Campeche, Mexico. The purpose of this study was to determine the genetic variability of vectors from Campeche coastal and rain forest areas and establish a phylogenetic relationship with other T. dimidiata populations by analyzing the internal transcribed spacer-2 (ITS-2) region. The sequence length of samples from Campeche ranged from 469 to 478 basepairs. The ITS-2 variability among the populations enabled us to classify them into two clades with an 18–22 nucleotide difference. The genetic distance (0.042) between them confirms this divergence. Phylogenetic analysis of gene genealogies confirmed these two clades. Furthermore, the population genetic analyses showed two groups with little genetic similarity or migration between them. One group was associated with the tropical forest area and the other group was associated with a mainly coastal distribution. This correlation was also observed when T. dimidiata from other regions of Mexico and Central America were analyzed.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chagas disease continues to be a severe public health problem in Latin America. According to the World Health Organization, approximately 18 million people are infected with the causal agent, Trypanosoma cruzi, and 100 million are at risk of contracting the infection.1 More recently, some studies seem to indicate a reduction of these figures (10 million persons infected).2 This disease is mainly vector-transmitted (≥ 80% of the infections), and is the main cause of heart disorders in Latin America.3 There is no vaccine or adequate pharmacological treatment for Chagas disease. The most effective form of control is the interruption of vector and blood transmission.1,2

The major Chagas disease vectors are Triatoma infestans in South America and Rhodnius prolixus and Triatoma dimidiata in Central America. In contrast, in Mexico there are several important vectors: Triatoma dimidiata, T. barberi, and several species of the Phyllosoma complex.4,5 Triatoma dimidiata is considered one of the most important Chagas disease vectors in Central America and Mexico because of its wide distribution and ability to colonize domestic and peri-domestic habitats. In South America T. dimidiata is found in Colombia, Ecuador, Venezuela, and northern Peru. In Central America, it is found in Panama, Costa Rica, Nicaragua, El Salvador, Honduras, Guatemala, and Belize.

In Mexico, it is found in the Yucatan peninsula (Campeche, Yucatan, and Quintana Roo); Chiapas; in the states of Veracruz and Tabasco on the Gulf of Mexico; in Oaxaca, Guerrero, Colima, Jalisco, and Nayarit on the Pacific coast; and in Puebla, San Luis Potosi, Guanajuato, and Hidalgo in central Mexico.6–8 In Mexico, a study that included the states of Hidalgo, Oaxaca, San Luis Potosi, and Veracruz showed that T. dimidiata was the most represented species, with a Trypanosoma cruzi infection rate between 4.1% and 14%.9 In the state of Yucatan, T. dimidiata was found in 61% of all houses examined, and 16% of the captured insects were infected.10

Campeche, in southeastern Mexico, has the largest protected tropical forest area in the country. The only studies conducted in this area are those concerning triatomine distribution. These studies have reported only T. dimidiata that was infected with Trypanosoma cruzi. In 1967, Gonzalez-Angulo and Ryckman,11 collected T. dimidiata in Campeche and reported it as T. dimidiata maculipennis, according to the earlier subspecies classification by Usinger,12 based on markings on the corium. Lent and Wygodzinsky13 suggested that these chromatic variations did not establish a subspecies; therefore, these two organisms were the same species.

Traditionally, species classification is based on morphologic features, including coloration pattern.12,13 Thus, some investigators have included T. dimidiata in the Phyllosoma complex (T. mazzottii, T. longipennis, T. pallidipennis, T. phyllosoma, T. picturata, T. mexicana, and T. bassolsae), which is a controversial taxonomic classification for this species.14–16 Taxonomic classification of these closely related species, as well as that of cryptic species, using only morphologic features is difficult. Nonetheless, recent studies using molecular markers have clarified this situation.15 For example, Marcilla and others,14 using the internal transcribed spacer-2 (ITS-2) of ribosomal DNA as the molecular marker, found numerous nucleotide differences between the T. dimidiata populations of central Mexico (Morelos and San Luis Potosi) and southeastern Mexico (Yucatan). More recently, analysis using ITS-2 and cytochrome B sequences showed that T. dimidiata was outside the Phyllosoma complex.16

The controversial taxonomic classification of T. dimidiata based on morphologic features and the lack of knowledge about its taxonomic status from the molecular point of view motivated the present study in the state of Campeche. The purpose of this study was to determine the genetic variability of T. dimidiata and the phylogenetic relationship of this species with other populations within and between the species, using ITS-2 as a molecular marker. The relationship between the clades and the habitat of T. dimidiata in Mexico and Central America were also studied.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study area. The study was conducted in seven localities in Campeche, Mexico. This state is located at 20°51'N, 17°49'S, 89°9'E, and 92°28'W. Campeche is bordered to the north by the Gulf of Mexico and Yucatan, to the east by Quintana Roo and Belize, to the south by Guatemala and the state of Tabasco, and to the west by the state of Tabasco and the Gulf of Mexico (Figure 1Go). The average annual temperature is 26°C throughout the state and 28°C on the coast.17 The localities selected for this study were the cities of Campeche, Hampolol, Seybaplaya (all at sea level), the protected natural reserve of Calakmul (125 meters above sea level), Bolonchen (120 meters above sea level), Dzibalchen (110 meters above sea level), and Becal (20 meters above sea level).17 The last four localities are situated in the tropical rain and deciduous forest areas (Figure 1Go).


Figure 1
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  		    FIGURE 1. Map of Mexico. The inset shows the state of Campeche and the geographic location of the Triatoma dimidiata capture sites. Collection sites are marked with white stars. Main vegetation types are also shown.

 
Campeche is the state capital. It is located on the coast at sea level. Hampolol is a rural community also located on the coast, at sea level, approximately 17.1 km from Campeche. It is separated from the capital city by a group of small hills. Seybaplaya is a rural area located on the coastline, approximately 27.9 km from Campeche at sea level. All these localities have dry tropical forest vegetation.17 Calakmul is a natural rain forest reserve, located approximately 179.5 km from the capital city, and Dzibalchen is situated approximately 94.14 km from Campeche. These regions have tropical rain forest vegetation. Becal and Bolonchen are rural areas approximately 88.5 km and 83 km, respectively, from the capital city. Their vegetation is tropical deciduous forest, a less dense tropical forest.17

Insects collected. Triatomines were collected manually during the day and at night with a lamp, according to the method described by Schofield.18 Captured insects were placed in labeled flasks containing folded cardboard sheets and transported to the laboratory. At the laboratory, insects were recorded, identified with the morphologic key of Lent and Wygodzinsky,13 and frozen at –70°C until used.

DNA extraction. Triatoma dimidiata specimens kept at –70°C were used for DNA extraction. The legs of the triatomines were selected to extract DNA because they are free from DNA contamination by other hosts. The legs were macerated with a plastic stick in a 1.5 mL microcentrifuge vial, placed in liquid nitrogen, and pulverized. The powder was then resuspended in 1 mL of lysis buffer (50 mM NaCl, 50 mM EDTA, 1% sodium dodecyl sulfate, 50 mM Tris, pH 8.0) and 100 µL of proteinase K (20 mg/mL). The suspension was incubated overnight at 37°C. Total DNA was then extracted using a phenol-chloroform technique previously described.19 After extraction, the supernatant was adjusted with a 10% volume of a 3 M sodium acetate solution, 1 mL of absolute ethanol was added, and the mixture was shaken and incubated at –70°C for one hour. The mixture was centrifuged, the supernatant was decanted, and the pellet was washed with 75% ethanol. The DNA pellet was resuspended in 25 µL of sterile water, and its integrity was determined by electrophoresis on a 1% agarose gel and staining with ethidium bromide. The concentration was determined by spectrophotometry. DNA was kept at –20°C until further use.

Amplification, cloning, and sequencing. Polymerase chain reaction amplification of the ITS-2 gene was performed with primers forward (5'-CTAAGCGGTGGATCACTCGG-3') and reverse (5'-CACTATCAAGCAACACGACTC-3')14 in a 25 µL reaction mixture containing 1 µL of Taq polymerase (5 U/µL; Invitrogen, Carlsbad, CA) and 1 µL of template DNA (200 ng).16 Reaction conditions were initial denaturalization at 95°C for 10 minutes and 94°C for 5 minutes, followed by 35 cycles at 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute, and a final extension at 72°C for 7 minutes. The amplification fragment was subcloned in vector pCR-2.1 (Invitrogen), extracted with the QIAprep Miniprep kit (Qiagen, Valencia, CA), and sequenced with an ABI Prism 310 sequencer (Applied Biosystems, Foster City, CA) and vector initiators (T7 promoter and M13 reverse).

Sequence aligning and phylogenetic analysis. The ITS-2 sequences of T. dimidiata were aligned with the Clustal X program version 1.8120 with the sequences for T. dimidiata from Yucatan, San Luis Potosi, Oaxaca, Veracruz, Guatemala, El Salvador, Nicaragua, and Honduras and with sequences of the Phyllosoma complex (T. phyllosoma, T. longipennis, and T. pallidipennis), and with T. barberi, Dipetalogaster maxima, T. infestans, and Rhodnius prolixus obtained from GenBank (Table 1Go).


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TABLE 1
Triatoma dimidiata populations used in this study
 
Distance analysis. A DNA evolution model was used considering the transition/transversion percentages and nucleotide frequency, with the MEGA21 and DAMBE22 programs. The distance trees were generated with the neighbor-joining method based on Kimura two-parameter23 distance matrices using the Phylip 3.61 package. Group support was statistically validated with a 1,000 bootstrap analysis according to the Felsenstein24 re-sampling technique. The parsimony trees were constructed with the heuristic tree search using the PAUP 4.0b10 program.25 Both distance and parsimony methods used R. prolixus as the outgroup. Consistency index (CI), homoplasy index (HI), retention index (RI), and rescaled consistency index (RCI) were calculated.26

For the isolation for distance analysis, ordinary linear regressions for the sequences were carried out with in-transformed geographic distances, and the non-parametric Mantel test was used to determine statistical significance27 with the NTSYSpc version 2.02 program.28

Population genetic analysis. Population genetic statistics were used to investigate and describe the genetic structure between the T. dimidiata populations in different regions. Analysis of molecular variance (AMOVA) and Wrights’ F statistics were calculated using ARLEQUIN version 200029 and GENEPOP version 3.1.30 The coancestry coefficient (Fst) (correlation of genes of different individuals in the same population) and migration index (Nm) (interpreted as the absolute number of migrant organisms that come into each subpopulations in each generation), were calculated in populations across each major group to assess genetic variation among populations within each ITS-2 sequence.

Gene genealogies. Unrooted statistical parsimony haplotype networks were created using TCS 1.21.31 This network was nested according to the rules outlined by Templeton and Sing,32 Templeton and others,33 Crandall and others,34 and Castelloe and Templeton.35


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The triatomines captured and used for this study were identified as T. dimidiata according to the dichotomy classification of Lent and Wygodzinsky.13 A total of 18 T. dimidiata ITS-2 clones from the same number of individuals were sequenced from triatomines captured in seven localities in Campeche. Additionally, for 4 individuals, of the 18 vectors, 2 clones were sequenced. In the latter cases, slight differences were observed that did not modify the final topology of the genetic tree and were excluded from the final analysis. The sequences were aligned with the T. dimidiata sequences of triatomines obtained from other localities and reported in GenBank (Table 1Go). The complete alignment showed a length of 527 basepairs. Of these, 276 nucleotide positions were variable and 68 were parsimony-informative sites. The average base composition percentage was 76.2% of A + T. Four sequences were identical (Becal642, Bolon52, Dzib55, and Calak35). Furthermore, there were other similar sequences to this genotype: Calak36 and Calak37, which varied in two insertion/deletion (indel)/ one transversion (Tv), and one transition (Ts)/one Tv, respectively, Becal661 in one Tv and two indel, Bolon63 in one Ts and three indel, and Bolon504 in one Ts and two Tv. This group was designated ITS clade 1 and differed in 18–22 nucleotides with respect to those from Campeche, Hampolol, and Seybaplaya. With respect to the Phyllosoma complex, the difference was 14–19 nucleotides. In addition, a different group of five identical sequences (Cam6, Cam21, Cam23, Cam14, and Dzib62) was found mainly in vectors from the coast (Campeche, Hampolol, and Seybaplaya) (Table 1Go). Sequences similar to the coastal population were observed: Cam4, which varied in one (Tv) and two indel, Seyba43 in one Ts, Seyba44 in one Tv, and Dzib563 in one Ts and one Tv. This group was designated ITS clade 2 and differed by 21–24 nucleotides from those of the Phyllosoma complex.

The predominant length of the sequences was 473 basepairs for clade 1 and 476 basepairs for clade 2 (Table 1Go). Most sequences contained the microsatellite (AT)5TTT(AT)7 with the exception of the sequences mentioned below. Both groups had an individual that differed at two basepairs: Calak36 with 475 basepairs and Cam4 with 478 basepairs. These differences were the result of the slightly longer microsatellite sequences (AT)5TTT(AT)8 and (AT)6T(AT)8 of Calak36 and Cam4, respectively. The Bolon63 sequence was 476 basepairs, and it had not only a microsatellite identical to that of Calak36, but also an additional indel. The Becal661 sequence was 469 basepairs and had the smaller microsatellite (AT)5TTT(AT)6

The average distance between the groups of T. dimidiata clade 1 and clade 2 obtained from the Kimura distance matrix was 0.042. The average distance between clade 1 and the Phyllosoma complex was 0.033; the distance between this complex and clade 2 was slightly higher (0.048). In contrast, the average genetic distance within the Phyllosoma complex species was 0.007.

Distance consensus and parsimony trees showed two populations of T. dimidiata in a clear process of divergence (Figure 2Go). Values for CI and HI were 0.971 and 0.028, respectively. The CI and HI excluding uninformative characters were 0.870 and 0.129, respectively. The RI and RCI were 0.958 and 0.931, respectively. The T. dimidiata clade 1 sequences are located in a different group from the clade 2 sequences. Both groups are statistically supported by bootstrap values of 93.6% and 99.7% in the case of the distance tree and 99% for the parsimony tree. Interestingly, a subgroup that included T. dimidiata from Nicaragua, Honduras, and El Salvador was observed in the genetic clade 2. The phylogenetic reconstruction analysis showed the same topology of the trees when the identical sequences were excluded. Both groups of T. dimidiata were separated from the Phyllosoma complex.


Figure 2
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  		    FIGURE 2. Phylogenetic trees. A, Triatomine phylogenetic tree derived from the Kimura two-parameter evolution model using the neighbor-joining method. B, Maximum parsimony tree obtained with the heuristic method. Sequences identified in this study are in bold. Numbers on the branch nodes are bootstrap values.

 
Genetic population analysis also showed two clearly separated groups in the analyzed T. dimidiata vectors with high genetic differences and low migration between them. The Fst values were ≥ 0.88 and Nm values ranged from 0.003 to 0.031 between clade 1 and clade 2. Clade 1 included individuals from Becal, Bolonchen, and Calakmul, which are located in the tropical rain forest and tropical deciduous forest. The individuals within this group had Fst values of 0, which indicated low genetic differentiation (the same results were found when AMOVA analysis was used). Clade 2 included individuals from Campeche, Seybaplaya, and Hampolol, was mainly restricted to the coast in dry tropical forest areas, and showed no significant genetic differences between groups.

The results from the TCS analysis that estimates genealogic relationships among the sequences also showed two separated networks (Figure 3Go). This result confirmed the separation between the sequences from individuals of clade 1 and clade 2. In clade 2, T. dimidiata sequences from Oaxaca, Veracruz, Honduras, El Salvador, and Guatemala (Chaoj) were also included. From this analysis, the ancestral haplotype for clade 1 was the most represented (Calak35, Dzib55, Becal 642, Bolon52, and Guatemala Yaxha). For clade 2, the identical sequences of Cam6, Cam21, Cam23, Cam14, and Dzib62 were considered the ancestral haplotype by the program. It was also evident from this analysis that the individuals from Hon-duras and El Salvador within clade 2 had higher sequence changes. This organization also correlated with the original geographic habitat of the individuals.


Figure 3
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  		    FIGURE 3. Haplotype network for 24 Triatoma dimidiata internal transcribed spacer region-2 sequences analyzed with TCS version 1.21 software. Each line represents one mutational change. Ellipse size is proportional to haplotype frequency with small circles representing missing haplotypes and the squares representing the ancestral haplotype as inferred by TCS using outgroup weights. All connections, up to 30 steps, are within the 95% confidence limits of a parsimonious connection. Clades were nested using the rules outlined in the literature.

 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Campeche in southern Mexico is one of the few regions in the country that still has a large extension of tropical forest as a protected natural reserve. Its animal diversity is currently under study. With respect to the one reported Chagas disease vector in the area, no information about the genetic diversity of this vector was available before this study.

The ITS-2 region has been shown to be useful for the study of genetic variability in the triatomine group. In our study, the ITS-2 nucleotide composition of T. dimidiata populations was high in A and T content (76.2%), a tendency that is consistent with other published data for triatomines.16 The ITS-2 of the T. dimidiata populations collected in Campeche showed large nucleotide variations. These variations are similar to those published by Marcilla and others,14 who used ITS-2 as a molecular marker and reported a divergence process between the populations of T. dimidiata in Yucatan and other Mexican states. However, this study did not include populations of T. dimidiata from Campeche. In our study, variation observed in the nucleotide sequence of ITS-2 of these T. dimidiata populations identified two diverging triatomine populations: clade 1, associated mainly with the tropical rain forest, and clade 2 in the coastal area of Campeche, associated with the dry tropical forest habitat. The possibility of these two clades being the result of the isolation by distance phenomenon was excluded because the Mantel test showed a weak correlation value (r = 0.054) and a P value of 0.398.

Genetically diverging populations within the Yucatan peninsula have not been previously found. Campeche is the first place in Mexico where these two clades are present in close proximity, with a distribution of the sylvatic clade 1 preferentially in the tropical rain forest region. In our study, the ITS clade 1 of T. dimidiata showed a similar ITS-2 sequence to previously sequenced specimens from Yucatan and Guatemala (Yaxha). In contrast, clade 2 of T. dimidiata clustered with those of central and southern Mexico (Veracruz, Oaxaca, and San Luis Potosi) and Central America, (Guatemala-Chaoj, Honduras, Nicaragua, and El Salvador).14,36 Each of these diverging groups showed one predominant nucleotide sequence: 473 basepairs for clade 1 populations and 476 base-pairs for clade 2 populations. The length of ITS-2 in T. dimidiata populations is also consistent with a report for other triatomine species.14 However, each group had a specimen with a longer sequence, (475 basepairs and 478 basepairs). Also, clade 2 had a smaller sequence of 469 basepairs. This phenomenon was also observed by Marcilla and others37 in different Panstrongylus populations.

The nucleotide differences in the sequences within each clade were small (1–5 nucleotides). These differences are probably the result of the close relationship and concerted evolution of ITS-2, which tends to homogenize alterations of its own genetic material.38 This homogenization has also been observed in ITS-1 sequences of the domestic R. prolixus and sylvatic R. colombiensis populations, in which no differences in length were observed within each population.39 This finding suggests a marked stability of ITS. Despite these observations, when the two T. dimidiata groups found in Campeche were compared, a marked nucleotide difference in ITS-2 of clade 1 and clade 2 populations of 18–22 nucleotides was observed. This nucleotide difference is similar to that found between vectors that had undergone a divergence process, such as species of Panstrongylus from different South American countries (Colombia, Ecuador, Peru, and Brazil).37 These differences in the Mexican T. dimidiata could indicate the presence of cryptic species, but the interbreeding experiment has not been conducted to demonstrate the possibility of obtaining fertile F1 and F2 progeny. Furthermore, the distance between clades 1 and 2 is even greater than the distance observed within the Phyllosoma complex.16 Our results confirm the separation of T. dimidiata populations from the Phyllosoma complex, a fact that had been observed by isoenzyme pattern, Cyt B, and ITS analysis of these vectors.16,40

The phylogenetic trees show a clear divergence between the two T. dimidiata groups. These results agree with the presence of two T. dimidiata groups suggested by random amplification of polymorphic DNA–polymerase chain reaction by Calderon and others41 in Guatemala. Recently, the presence of three different genetic types of T. dimidiata in Mexico and Central America was reported.36,42 However, our data from ITS2 analysis showed only two strong, statistically supported genetic groups with individuals from Nicaragua, Honduras, and El Salvador located in a subgroup inside clade 2. The latter group probably presents a differentiation process that must be studied with more individuals and with other genetic markers.

A population genetic analysis was conducted to investigate the relationship between the coastal group and populations of T. dimidiata from the rain forest area. From this analysis, it was clear that two groups exist, which are located in different ecologic habitats with no apparent genetic flow and scarce migration between them. Furthermore, the TCS analysis showed two separate genealogic groups. One group contained individuals from the tropical rain forest and the other included individuals from the dry tropical forest area in Campeche, central Mexico, and Central America. These results suggest that a broad distribution of clade 2 exists, versus the apparently restricted location (only in the rain forest environment) of the individuals belonging to clade 1. A probable explanation, given the suggested origin of T. dimidiata in the Yucatan peninsula,43 is that clade 1 is the original genotype restricted to the rain forest area, and clade 2 has colonized domestic and peridomestic habitats in other climatic areas. Furthermore, the extended distribution of clade 2 could be the result of the constant movement of original Mayan populations to different geographic regions and the adaptation of T. dimidiata clade 2 to the domestic and peridomestic habitats.

In summary, the present work corroborates the divergence process of T. dimidiata and shows the presence of two clades in the state of Campeche in Mexico, one of them clearly associated with the southeastern tropical rain forest area in Mexico and the other with a broad distribution in domestic and peridomestic habitats from central Mexico to Nicaragua in Central America in a possible sub-process of divergence. The existence of two different populations of T. dimidiata could imply, after the use of insecticides, the colonization of the peridomestic and domestic habitats by the sylvatic group. Our results should be considered in the development of future control vector programs.

Received March 14, 2007. Accepted for publication December 6, 2007.

Financial support. This study was supported by the Universidad Autónoma de Campeche, Programa Institucional de Formación de Investigadores, Coordinación General de Posgrado e Investigación–Instituto Politécnico Nacional, y Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica from La Dirección General de Asuntos del Personal Académico–Universidad Nacional Autónoma de México grant no. IN 212806-3. Paulino Tamay-Segovia was supported by a scholarship from the Programa para el Mejoramiento del Profesorado of the Universidad Autónoma de Campeche.

* Address correspondence to Bertha Espinoza, Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Circuito Escolar, Ciudad Universitaria, CP 04510, Distrito Federal, México. E-mail: besgu{at}biomedicas.unam.mx Back

Authors’ addresses: Paulino Tamay-Segovia and Selene Blum-Domínguez, Centro de Investigaciones en Enfermedades Tropicales, Universidad Autónoma de Campeche, Av. Trueba y Regil S/N, CP, Campeche, Campeche, México, Telephone: 981-81-3-01-76, Fax: 981-81-3-01-71, E-mail: pautamay{at}hotmail.com. Ricardo Alejandre-Aguilar and Francisco J. Zavala-Díaz de la Serna, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Prolongación de Carpio y Plan de Ayala S/N, Col. Casco de Sto. Tomas, CP 11340, Distrito Federal, México, Telephone: 525-57-29-63-00 extensions 62400 and 46209, E-mail: rialejandre{at}yahoo.com.mx and javierzavala{at}yahoo.com. Fernando Martínez, Guiehdani Villalobos, Patricia de la Torre, Juan P. Laclette, and Bertha Espinoza, Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Circuito Escolar, Ciudad Universitaria, CP 04510, Distrito Federal, México, Telephone: 525-56-22-89-43, Fax: 525-56-22-33-69, E-mail: fherxyz @yahoo.com, guiehda{at}yahoo.com.mx, and besgu{at}biomedicas.unam.mx.

Reprint requests: Bertha Espinoza, Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Au-tónoma de México, Circuito Escolar, Ciudad Universitaria, CP 04510, Distrito Federal, México, E-mail: besgu{at}biomedicas.unam.mx.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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