We evaluated the genetic variation of
Echinococcus G7 strain in larval and adult stages using a fragment of the mitochondrial
cox1 gen. Viscera of pigs, bovines, and sheep and fecal samples of dogs were inspected for cystic and canine echinococcosis, respectively; only pigs had hydatid cysts. Bayesian inferences grouped the sequences in an
E. canadensis G7 cluster, suggesting that, in Mexico, this strain might be mainly present. Additionally, the population genetic and network analysis showed that
E. canadensis in Mexico is very diverse and has probably been introduced several times from different sources. Finally, a scarce genetic differentiation between G6 (camel strain) and G7 (pig strain) populations was identified.
Echinococcus granulosus sensu lato (s.l.) includes species that cause cystic echinococcosis (CE), one of the most important and widespread parasitic zoonoses. Recent phylogenetic studies based on both mitochondrial and nuclear DNA genes show that
E. granulosus s.l. consists of at least four valid species:
E. granulosus sensu stricto (s.s.; genotypes G1–G3),
E. equinus (G4),
E. ortleppi (G5), and
E. canadensis (G6–G10). Genotypes G6/G7 are closely related and referred to as camel and pig strains, respectively.
1–3 The pig–dog cycle is mainly present in Mexico and maintains the G7 strain.
4,5 Although there are isolated reports of
E. oligarthrus in a wild cat,
6
E. ortleppi (
E. granulosus s.l.; G5) in a patient,
7 and
E. granulosus s.s. (G1) in a rural pig, there is no evidence that these species are maintained in Mexico.
8 No data of CE caused by G7 have been documented in Mexican patients, although there is a high number of
E. canadensis G7-infected patients in central Europe, pointing to the importance of this strain as a cause of human CE.
9,10 There are only two genetic studies performed in samples from Mexico. Cruz-Reyes and others
5 documented that G7 parasites of Mexican and Polish pig isolates showed similar patterns by restriction fragment length polymorphism (RFLP) of ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1) and random amplified polymorphic DNA (RAPD) techniques, and although polymerase chain reaction (PCR) -sequencing analysis of mitochondrial
cox1 gen fragment was performed, no polymorphism data were reported. Sharma and others
11 identified two variants (A and B) inside of the G6/G7 group consisting of samples from Mexico and Argentina using five nuclear markers (elongation factor 1α, transforming growth factor-β receptor kinase, thioredoxin peroxidase, calreticulin, and ezrin-radixin-moesin-like protein). Because some local slaughter records from northern Mexico indicate the presence of
Echinococcus spp. in livestock animals,
5 the objective of this study was to investigate if parasites in pigs and dogs correspond to G7 and if so, describe its genetic variation.Infected animals were identified in the municipal slaughterhouse of Calera, Zacatecas (north central Mexico), where farm and backyard livestock animals coming from the whole state and other surrounding states were included. For this purpose, viscera from 387 pigs, 243 bovines, and 32 sheep were inspected for the larval stage of
Echinococcus. Nine pigs (six pigs from Zacatecas, two pigs from Aguascalientes, and one pig from Morelos) were found infected, and hydatid cysts were obtained under aseptic conditions. After cyst contents were aspirated and centrifuged, aliquots were examined under microscopy to confirm the presence of protoscolices, and pellets were kept in 70% ethanol at −20°C until DNA extraction. Each cyst from each animal was considered as an isolate.Based on the presence of the parasites previously identified in Calera''s slaughterhouse, a rural community located in the central area of Zacatecas at 22°55′ N, 102°48′ W was selected to look for the adult stage of this parasite. For this search, all dogs (60) present in the community were sampled one time for feces after obtaining verbal consent from the owner; samples were used to identify taeniid eggs by the Faust technique, antigens in stool samples (copro-antigens) by enzyme-linked immunosorbent assay (ELISA; CpAg ELISA), and DNA by Copro-PCR. The CpAg ELISA was performed as described by Allan and others
12 and Moro and others.
13 For Copro-PCR, only positive samples by CpAg ELISA were analyzed using JB3 and JB4 primers to amplify a
cox1 gen fragment.
14 Coprological analysis of dogs showed that 11 samples were positive by CpAg ELISA (18.3%); only 2 of these samples had taeniid tapeworms (3.4%), and 3 of 11 samples yielded products of approximately 450 bp. All amplicons obtained of hydatid cysts and fecal samples were purified, sequenced on both strands, submitted to GenBank (accession numbers
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cox1. Dogs positive for taeniid eggs or antigens were purged and treated with praziquantel at 30 mg/kg and arecoline bromide at 2 mg/kg. The protocol was previously approved by the Ethics and Research Committees of the General Hospital “Dr. Manuel Gea Gonzalez”; government and health authorities of the municipality and community also authorized our study.All sequences were subjected to the Basic Local Alignment Search Tool (BLAST) search in the GenBank database; multiple alignments were performed with the CLUSTAL W and MUSCLE programs,
15,16 with manual adjusted in MEGA program v5
17 to determine the appropriate model of molecular evolution in the Modeltest 3.7 program.
18 The phylogenetic reconstruction using Bayesian inference was performed with Mr Bayes 3.2.1 program.
19 Unrooted haplotype networks were created using NETWORK 4.611 software and nested according to the rules in median-joining networks.
20 An analysis of genetic diversity within and between populations was performed using DnaSPv4
21 and included nucleotide diversity (π), haplotype polymorphism (θ), genetic differentiation index (F
ST), and Tajima''s D test. Analysis of molecular variance (AMOVA) was used to examine the population genetic structure between populations by Φ
ST as the genetic fixation index (analogous to F
ST) obtained by ARLEQUIN software.
22After multiple alignments, all sequences of larval and adult stages showed 98% or higher identity with
E. canadensis, whereas the Bayesian phylogenetic tree and the haplotype network inference grouped these sequences in the
E. canadensis G7 cluster. Sequences for
cox1 of
E. canadensis from Africa, Asia, Europe, Latin America, and North America deposited in the GenBank databases (
N = 58) as well as our sequences (accession numbers
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P < 0.01). Genetic differentiation indexes between different paired sequences of
E. canadensis genotypes are shown in