Population genetic structure of Taenia solium from Madagascar

and Mexico: implications for clinical profile diversity

and immunological technology

Rodrigo Vegaa,*, Daniel Pinerob, Bienvenue Ramanankandrasanac, Michel Dumasc,Bernard Bouteillec, Agnes Fleuryd, Edda Sciuttoa, Carlos Larraldea,e, Gladis Fragosoa

aDepartamento de Inmunologıa, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico,

Circuito Interior S/N, Ciudad Universitaria, Mexico D. F. 04510, MexicobDepartamento de Ecologıa Evolutiva, Instituto de Ecologıa, Universidad Nacional Autonoma de Mexico, Mexico D. F. 04510, Mexico

cInstitute d’Epidemiologie Neurologique et de Neurologie Tropicale, Faculte de Medecine, Limoges 87025, FrancedInstituto Nacional de Neurologıa y Neurocirugıa, Mexico D. F. 14269, Mexico

eCentro Internacional de Ciencias, Cuernavaca, Morelos 62131, Mexico

Received 19 May 2003; received in revised form 30 June 2003; accepted 14 July 2003

Abstract

Taenia solium is a cestode parasitic of humans and pigs that strongly impacts on public health in developing countries. Its larvae

(cysticercus) lodge in the brain, causing neurocysticercosis, and in other tissues, like skeletal muscle and subcutaneous space, causing

extraneuronal cysticercosis. Prevalences of these two clinical manifestations vary greatly among continents. Also, neurocysticercosis may be

clinically heterogeneous, ranging from asymptomatic forms to severely incapacitating and even fatal presentation. Further, vaccine design

and diagnosis technology have met with difficulties in sensitivity, specificity and reproducibility. Parasite diversity underlying clinical

heterogeneity and technological difficulties is little explored. Here, T. solium genetic population structure and diversity was studied by way of

random amplified polymorphic DNA in individual cysticerci collected from pigs in Madagascar and two regions in Mexico. The

amplification profiles of T. solium were also compared with those of the murine cysticercus Taenia crassiceps (ORF strain). We show

significant genetic differentiation between Madagascar and Mexico and between regions in Mexico, but less so between cysticerci from

different localities in Mexico and none between cysticerci from different tissues from the same pig. We also found restricted genetic

variability within populations and gene flow was estimated to be low between populations. Thus, genetic differentiation of T. solium suggests

that different evolutionary paths have been taken and provides support for its involvement in the differential tissue distribution of cysticerci

and varying degrees of severity of the disease. It may also explain difficulties in the development of vaccines and tools for immunodiagnosis.

q 2003 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Taenia solium; Cysticercosis; Random amplified polymorphic DNA; Genetic variability; Population structure

1. Introduction

Human cysticercosis is a parasitic disease known since

antiquity and prevalent since prehistoric times (Hoberg

et al., 2001). It is caused by the larvae (cysticercus) of

the parasitic cestode Taenia solium. Humans are the only

definitive hosts for the adult stage (tapeworm), which

usually lodges alone in the intestinal tract. There it

reproduces by autofecundation, and sheds its terminal

segments replete with thousands of fertile eggs that reach

the environment upon defecation. Eggs ingested by

humans and pigs (the intermediate hosts) develop onto

cysticerci within their tissues. The life cycle is com-

pleted when humans consume undercooked pork meat

infected with living cysticerci that transform in

new tapeworms. Taenia solium disease is endemic in

0020-7519/$30.00 q 2003 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/S0020-7519(03)00206-6

International Journal for Parasitology 33 (2003) 1479–1485

www.parasitology-online.com

* Corresponding author. Tel.: þ52-55-5622-3152; fax: þ52-55-5622-

3369.

E-mail address: vegabernal@correo.biomedicas.unam.mx (R. Vega).

countries of Latin America, Asia and Africa, and North

America and Western Europe still report imported

human cases of cysticercosis (Schantz et al., 1998;

Sciutto et al., 2000).

Extensive research has come to indicate to the great

heterogeneity of T. solium disease and to the almost

ignored role of parasite diversity in explaining it. The

prevalences of neurocysticercosis and extraneuronal

cysticercosis vary greatly between continents: Asia and

Africa reporting more extraneuronal cysticercosis cases

than Latin America (Ito et al., 2002; Sciutto et al., 2003).

Also, within the human neurocysticercosis cases the

clinical profiles differ widely (Singh, 1997), from

asymptomatic to benign to severe neurological conditions

not infrequently fatal (Sotelo and Del Bruto, 2000;

Fleury et al., 2003; Sciutto et al., 2003). The heterogen-

eity of the immune response of infected humans and pigs

is well known and probably behind the difficulties in

developing reliable technology for diagnosis and vaccine

development (Larralde et al., 1989; Garcia et al., 1994;

Sloan et al., 1995; Sciutto et al., 1998). Host differences

in human leukocyte antigen, gender and immune profiles

are suspected to contribute to the variable clinical and

immunological profiles (Schantz et al., 1998).

Of all the factors used to explain the heterogeneity of

cysticercosis, those intrinsic to the parasite are seldom

considered: the homogeneity among individuals of T. solium

is implied by default in hundreds of studies. Genetic

differentiation and variability of T. solium could be a

significant source of clinical heterogeneity of the disease

and could be involved in the difficulties encountered in the

design of reliable immunodiagnosis and reproducible

vaccine efficiencies.

We explored population genetic structure (genetic

diversity, differentiation and distance) by random ampli-

fied polymorphic DNA (RAPD; Welsh and McClelland,

1990; Williams et al., 1990) in T. solium cysticerci

infecting pigs of Mexico and Madagascar. The cysticerci

of Mexico came from two different geographic areas

(Central and South-eastern Mexico) and different

localities within the Central area (Huatlatlauca, Cuente-

pec and Zacatepec), in order to explore the possibility of

differentiation within Mexico. The cysticerci from

Madagascar were all obtained from skeletal muscle,

whilst those of Mexico were also from skeletal muscle,

but some came from the brain of the same pig to study

differences in cysticerci from different tissues. We also

measured the genetic distance, based on the amplification

profiles, of T. solium with respect to the murine cestode

Taenia crassiceps (ORF strain; Freeman, 1962; Smith

et al., 1972), because the latter has been used as a

laboratory model in the study of cysticercosis and as a

source of cross-reacting antigens for immunodiagnosis of

disease in humans (Larralde et al., 1990) and for

vaccine design in pigs (Gevorkian et al., 1996; Sciutto

et al., 2002).

2. Materials and methods

2.1. Taenia solium cysticerci

Thirty five T. solium cysticerci were obtained from 13

infected pigs bred in the rural Central Mexico localities of

Zacatepec and Cuentepec (Morelos) and Huatlatlauca (Pue-

bla), and 16 cysticerci were obtained from six infected pigs in

the South-eastern Mexico locality of Kochol (Yucatan).

Respectively, three and four cysticerci lodged in brain tissue

were obtained from three pigs from Central Mexico and four

pigs from South-eastern Mexico. Thirty nine cysticerci from

Madagascar were obtained from skeletal muscle of nine pigs

found infected in slaughterhouses of Antananarivo and

Fianarantsoa.

A sample of T. crassiceps-ORF cysticerci was collected

alive from the peritoneal cavity of an experimentally infected

female BALB/CAnN mouse (Padilla et al., 2001). Pig skeletal

muscle free of cysticerci was collected immediately after

slaughter to extract pig DNA for use as a control.

2.2. DNA extraction, quantification and RAPD

Taenia solium, T. crassiceps-ORF and pig tissue DNA

was extracted using the Genomic Prep Cells and Tissue

DNA Isolation Kit following the manufacturer’s protocol

(Amersham Pharmacia Biotech) and quantified by spectro-

photometry. RAPD was conducted using the Ready-To-Go

RAPD Analysis Beads following the manufacturer’s

protocol (Amersham Pharmacia Biotech) with 20 ng of

sample DNA and nine different primers (Primer 01:

50-GGTGCGGGAA-30, Primer 02: 50-GTTTCGCTCC-30,

Primer 03: 50-GTAGACCCGT-30, Primer 04:

50-AAGAGCCCGT-30, Primer 05: 50-AACGCGCAAC-30

and Primer 06: 50-CCCGTCAGCA-30 from Amersham

Pharmacia Biotech, and OPA03: 50-AGTCAGCCAC-30,

OPA07: 50-GAAACGGGTG-30 and OPA13: 50-CAG-

CACCCAC-30 from Operon Technologies). Negative

controls were done using sterile water instead of DNA.

Positive controls for the RAPD beads were done to make

sure they were working properly.

A pilot study was conducted in order to make sure that

the amplification profiles were reproducible. For this, DNA

from several cysticerci was amplified using the nine primers

and reactions were repeated three times. In all cases the

same amplification profile was obtained (data not shown).

2.3. Gel electrophoresis and gel analysis

DNA amplification products were separated by 2%

agarose tris borate EDTA 1X 0.8 mg/ml ethidium bromide

gel electrophoresis ran for 3:30 h at 60 V. Gels were

photographed in UV light and gel photographs were

scanned. The amplification profile (presence and absence

of all bands) for every individual was assessed using the

Gene Profiler software (Scanalytics, Fairfax, VA).

R. Vega et al. / International Journal for Parasitology 33 (2003) 1479–14851480

2.4. Data analysis

The amplification profile of every individual cysticercus

was analysed in the population genetics program

Popgene (Yeh et al., 1997). Gene frequencies were obtained

by q ¼ (C/N)1/2, where q the recessive allele’s frequency, C is

the number of individuals with the recessive allele and N is the

number of individuals in the population. Percentage of

polymorphic loci was obtained as the percentage of all loci

that are polymorphic regardless of allele frequencies. From the

allelic frequencies of T. solium cysticerci banding patterns, the

genetic variability and genetic differentiation were

calculated. Nei’s genetic diversity (h) was obtained by

h ¼ 1 2 ( pi2 þ qi

2), where pi and qi are the allelic

frequencies for each loci of the present band (1) and the

absent band (0), respectively (Nei, 1973). Genetic differen-

tiation (GST) was obtained by GST ¼ (HT 2 HS)/HT, where

GST is the coefficient of genetic differentiation in the context of

multi allelic loci of Nei calculated with the values of HT and

HS, where HT ¼ 1 2 Sppi2 and HS ¼ 1 2 Spsi

2, being HT the

expected heterozygosity over the total population and ppi the

frequency of the i allele averaged over the subpopulations, and

HS the expected heterozygosity in the subpopulation s and psi

the frequency of the i allele in the subpopulation s (Nei, 1973).

Gene flow (Nm) was obtained as an estimate of GST by

Nm ¼ 0.5 (1 2 GST)/GST (Slatkin and Barton, 1989). Popu-

lation genetics parameters were obtained for three hierarchical

levels: countries (Mexico and Madagascar), regions (Central

and South-eastern Mexico and Madagascar) and Mexican

localities (Zacatepec, Cuentepec and Huatlatlauca from the

Central region and Kochol from the South-eastern region). Nei

and Li’s genetic distance (GD) was obtained among

individuals as GDxy ¼ 1 2 (2 Nxy)/(Nx þ Ny), where Nxy

is the number of shared bands in samples X and Y, Nx is the

number of bands in sample X, and Ny is the number of bands in

sample Y (Nei and Li, 1979). These distances where used to

construct phenograms by the unweighted pair-group method

with arithmetic means (UPGMA) algorithm using the Treecon

software (Van de Peer and De Wachter, 1997). The robustness

of the branches was assessed by bootstrap (100 replicates).

3. Results

3.1. Genetic distances and phenogram characteristics

A total of 113 loci were obtained in the T. solium

cysticerci using nine different primers, of which 49

(43.36%) were polymorphic between the samples from

Mexico and Madagascar. Fig. 1 illustrates the banding

patterns and major differences among samples in

geographic contrasts. Nei and Li’s method of genetic

distance among sampled individuals was used to

construct a phenogram by UPGMA algorithm (Fig. 2).

The three regions analysed (Madagascar, Central Mexico

and South-eastern Mexico) formed three separate clusters

Fig. 1. Illustrative electrophoresis gels showing different random amplified

polymorphic DNA profiles between two samples of T. solium cysticerci

from each region, Central Mexico, South-eastern Mexico (SE Mexico) and

Madagascar (lanes 1 and 2), and one sample of T. crassiceps-ORF (Tc-

ORF; lane 1). MW ¼ molecular weight standards (100 bp ladder, Roche).

Fig. 2. Genetic differences among pig T. solium cysticerci. Unweighted

pair-group method with arithmetic means phenogram constructed using Nei

and Li’s genetic distance among 90 T. solium cysticerci and a sample of

T. crassiceps-ORF (Tc-ORF). Individuals with identical amplification

profiles (genetic distance ¼ 0.0) were grouped together and depicted with

letters A–P (refer to Table 1 for cysticerci belonging to groups). Central

Mexico, South-eastern Mexico (SE Mexico) and Madagascar populations

are structured in separate clusters, so is T. crassiceps-ORF (Tc-ORF) which

acts as a positive control for random amplified polymorphic DNA capacity

to distinguish between species of Taenia. The bold numbers at the nodes are

the bootstrap confidence values obtained after 100 replicates.

R. Vega et al. / International Journal for Parasitology 33 (2003) 1479–1485 1481

supported by high bootstrap values illustrating the

robustness of the phenogram. Samples from Mexico

and Madagascar were separated by a large genetic

distance close to 0.2, whilst samples from Central and

South-eastern Mexico showed a small genetic distance

close to 0.05. Cysticerci belonging to different localities

in the same region clustered closer in genetic distances

(,0.01), and several were identical in their amplification

profiles having a zero genetic distance between them.

These identical individuals clustered together and are

depicted in the phenogram in 16 different groups labelled

with the letters A–P (for cysticerci belonging to the

different groups, see Table 1).

The amplification profiles of cysticerci collected from

one region were always different from the ones of other

regions. Because of this, in the UPGMA phenogram no

cysticerci belonging to Central Mexico were found in the

South-eastern region or in the Madagascar group and vice

versa, but individuals belonging to different localities in

Central Mexico were somewhat mixed. Cysticerci obtained

from brain tissue did not form a differentiated group and

appeared in companion with cysticerci collected from

skeletal muscle. The phenogram topology shows that there

is a high population structure or differentiation at the level

of the two countries studied and of regions within Mexico,

but not so much at the level of localities belonging to one

region and not at all among cysticerci from different tissues

in the same pig. The sample of T. crassiceps-ORF

shows the highest genetic distance from the T. solium

population (,0.4).

3.2. Significant genetic differentiation

Genetic differentiation, obtained using the values of

expected total heterozygosity (HT) and expected hetero-

zygosity in the subpopulations (HS), was notably high

between Mexico and Madagascar (0.7577), among regions

(0.8767), between paired regions, and between the three

Central Mexico localities paired with the South-eastern

Mexican locality (Table 2). In contrast, a comparatively

lower genetic differentiation was obtained among the three

localities from Central Mexico (0.2112) and between paired

localities from Central Mexico (Table 2).

Taenia solium cysticerci obtained from brain and skeletal

muscle from the same pig or from pigs belonging to the

same locality were identical in their amplification profiles

and showed no genetic differentiation between tissues (data

not shown).

3.3. Gene flow and variability

The gene flow (Nm), estimated from GST, was low between

countries (0.1599), as expected from the low probability of

migration of human carriers or pig commerce between these

two countries. Gene flow was also low between the two

regions of Mexico (0.1294) (Table 2). In contrast, higher gene

Table 1

Groups of T. solium cysticerci with identical amplification profiles

Country Region Group Cysticerci (code number)a

Mexico Central A Z01-01, Z01-02, Z01-03, Z03-08, Z03-09br, Z06-14, Z06-15, Z06-16br,

C03-22, C03-23, C04-24, C05-26, H02-32, H02-33, H02-35

B Z04-10, Z04-11

C C02-20, C02-21

D Z02-05

E C05-23, H02-34

F H01-28, H01-29, H01-31

G Z05-12, Z05-13, C01-17, C01-18, C01-19br, C04-25, H01-30

H Z02-04

I Z02-06, Z03-07

South East J K03-07, K04-10, K06-15

K K01-01, K01-02, K01-03br, K02-04, K02-05, K03-06, K04-08, K04-09,

K05-11, K05-12, K05-13br, K06-14, K06-16br

Madagascar L Mg03-30

M Mg07-25

N Mg08-08, Mg06-32

O Mg05-03

P Mg02-01, Mg02-02, Mg08-04, Mg05-05, Mg06-06, Mg07-07, Mg09-09,

Mg01-10, Mg02-11, Mg06-12, Mg04-13, Mg05-14, Mg06-15, Mg09-16,

Mg08-17, Mg09-18, Mg01-19, Mg02-20, Mg08-21, Mg04-22, Mg05-23,

Mg06-24, Mg08-26, Mg09-27, Mg01-28, Mg02-29, Mg05-31, Mg07-32,

Mg08-34, Mg09-35, Mg08-36, Mg02-37, Mg04-38, Mg05-39

a Z, Zacatepec; C, Cuentepec; H, Huatlatlauca; K, Kochol; br, cysticerci obtained from brain tissue; Mg, Madagascar. The first number corresponds to the pig

number and the second number corresponds to the number of cysticercus in the study.

R. Vega et al. / International Journal for Parasitology 33 (2003) 1479–14851482

flow estimates were obtained among the three localities from

Central Mexico (1.8670) and between paired Central Mexico

localities (Table 3).

Nei’s genetic variability (h) was low in the samples from

Mexico (0.0412) and Madagascar (0.0380) and in the total

sample (0.1491). The values of genetic variability were also

low for cysticerci from different regions and localities of

Mexico (Table 3).

4. Discussion

The significant genetic differentiation of T. solium

between countries pertaining to different continents may

have evolved after the introduction of pigs to America

from European stock in the late 1400’s (Okamoto et al.,

2001; Nakao et al., 2002), whilst that between regions of

Mexico may have resulted from the great geographic

distance and historical little contact of rural Central

Mexico with the rural areas of South-eastern Yucatan

peninsula. Commerce of infected pigs between rural

areas is an expedient way of exporting and implanting

new genetic versions of the parasite. Migration of human

tapeworm carriers could cause new cases of human

neurocysticercosis in the place of destination (McManus,

1995; Sanchez et al., 1998) but probably not pig

cysticercosis unless the infected migrant establishes in a

rural area with poor faecal disposal and free roaming

pigs. Commerce is more active between geographically

close rural localities than it is between distant ones, and

so is the movement of T. solium carriers from

neighbouring towns. This is congruent with the lower

genetic differentiation and higher gene flow estimates

among Central Mexico localities. Intensification of pig

rearing in the rural unsanitary conditions prevalent in

undeveloped countries would favor the parasite’s spread

and may have accelerated its evolution and progressive

genetic differentiation. The recent adoption of pig rearing

by rural African people, as substitutes for their traditional

cattle herds, represents new opportunities for the parasite

to enlarge its domain (Bao et al., 1995).

There is only one report in the literature on

immunotaxonomy of T. solium in which antigen differences

between cysticerci collected from different pigs are reported

(Yakoleff-Greenhouse et al., 1982). Recent evidence for

heterogeneity in the diagnosis of pig cysticercosis comes

from the evaluation of serology using glycoproteins and

Table 2

Population structure parameters obtained at different hierarchical levels for the 113 loci surveyed in pig T. solium cysticerci populations

HTa GST

b Nmc P (%)d

Between countries

Mexico-Madagascar 0.1634 0.7577 0.1599 49 (43.36)

Among regions

Central Mexico-SE Mexico-Madagascar 0.1541 0.8767 0.0703 49 (43.36)

Between paired regions

Central Mexico-SE Mexico 0.0462 0.7945 0.1294 15 (13.27)

Central Mexico-Madagascar 0.1546 0.8297 0.1026 46 (40.71)

SE Mexico-Madagascar 0.1601 0.8677 0.0762 42 (37.17)

Among localities

Zacatepec-Cuentepec-Huatlatlauca-Kochol 0.0387 0.7907 0.1323 15 (13.27)

Zacatepec-Cuentepec-Huatlatlauca 0.0119 0.2112 1.8670 8 (7.08)

Between paired localities

Zacatepec-Cuentepec 0.0118 0.1234 3.5534 8 (7.08)

Zacatepec-Huatlatlauca 0.0134 0.2526 1.4790 7 (6.19)

Zacatepec-Kochol 0.0448 0.8102 0.1172 14 (12.39)

Cuentepec-Huatlatlauca 0.0085 0.0944 4.7985 3 (2.65)

Cuentepec-Kochol 0.0463 0.8656 0.0776 12 (10.62)

Huatlatlauca-Kochol 0.0464 0.8741 0.0720 11 (9.73)

a HT, total expected heterozygosity averaged over all loci.b GST, genetic differentiation averaged over all loci.c Nm, gene flow between populations estimated from GST.d P, number of polymorphic loci.

Table 3

Number of sampled individuals, genetic variability values and polymorphic

loci within pig T. solium cysticerci populations

Na hb P (%)c

Total sample 90 0.1491 49 (43.36)

Countries

Mexico 51 0.0412 15 (13.27)

Madagascar 39 0.0380 22 (19.47)

Regions (Mexico)

Central Mexico 35 0.0147 8 (7.08)

South-eastern Mexico (Kochol) 16 0.0043 1 (0.88)

Localities (Central Mexico)

Zacatepec (Morelos) 16 0.0126 6 (5.31)

Cuentepec (Morelos) 11 0.0081 3 (2.65)

Huatlatlauca (Puebla) 8 0.0073 2 (1.77)

a N, number of sampled individuals.b h, Nei’s genetic diversity averaged over 113 loci.c P, number of polymorphic loci.

R. Vega et al. / International Journal for Parasitology 33 (2003) 1479–1485 1483

recombinant chimeric antigen of T. solium using blood

samples from pigs infected in Tanzania, where the antibody

response from pigs with positive tongue inspection for

parasitic lesions were not always detected by ELISA

or immunoblot, and from the immunoblot figures presented

(Sato et al., 2003). There is also recent evidence of limited

nucleotide sequence variation in some genes of T. solium

(Hancock et al., 2001; Yamasaki et al., 2002). Okamoto et al.

(2001) and Nakao et al. (2002) have shown by the sequencing

of mitochondrial cytochrome b and cytochrome oxidase

subunit I genes that African and American T. solium isolates

have limited sequence variation and share a common ancestor,

possibly European. Here we show that Madagascar and

Mexican isolates are highly differentiated and possess low

variability, evidence for a population genetic structure that

could not be detected in previous studies.

Should the significant genetic differentiation of T. solium

between Madagascar and Mexico impinge in the parasites

preference of host’s tissues, it could explain the different

prevalence of neurocysticercosis and extraneuronal cysti-

cercosis of humans between the two countries (Zavala and

Bolio, 1966; Sciutto et al., 2003). The possibility of two

drastically distinct disease syndromes arising from two

distinct taenias differing in their preferred target tissue

(neuro-tropic and extraneuro-tropic) affecting Madagascar

and only one of them (the neuro-tropic form) affecting

Mexico is not supported by the almost complete DNA

identity between cysticerci collected from the same pig in

both countries (i.e. in case of two forms of taenias, the

Madagascar samples should show some different cysticerci

within the same pig).

Likewise, the genetic differences found in the cysticerci

from Mexico suggest the possibility that parasites may vary

in infectivity and pathogenicity and thus contribute to the

heterogeneous clinical and immunological expressions of

neurocysticercosis. Further, genetic differentiation

of T. solium must be considered in the design of

immunodiagnostics or vaccines because antigen differences

in the parasites could bring about difficulties in

reproducibility and efficacy, should the antigens come

from pigs living in different regions and countries.

Our work has shown significant genetic differentiation in

T. solium cysticerci affecting pigs from different countries

and between regions in the same country. We also showed

T. solium to have low genetic variability. Both findings in

T. solium – high genetic differentiation and low genetic

variability – are as expected in a usually hermaphroditic

species (Hartl and Clark, 1989), as the tapeworm is typically

found single in its host, honouring its Spanish common

name ‘solitaria’.

Acknowledgements

This work was supported by the Mexican National grants

from DGAPA (IN 220999) and CONACyT (31378-M),

the Program ANUIES CONACyT-ECOS (M99-S03) and

the Howard Hughes Medical Institute. We thank Dr Jose

Luis Dominguez-Alpizar for providing cysticerci from

Kochol, Yucatan.

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