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Article

Haplotypes of Echinococcus granulosus sensu stricto in Chile and Their Comparison Through Sequences of the Mitochondrial cox1 Gene with Haplotypes from South America and Other Continents

by
Nicole Urriola-Urriola
*,
Gabriela Rossi-Vargas
and
Yenny Nilo-Bustios
Laboratorio de Microbiología y Parasitología, Departamento de Ciencias Biomédicas, Facultad de Medicina, Universidad Católica del Norte, Coquimbo 1281, Chile
*
Author to whom correspondence should be addressed.
Parasitologia 2025, 5(3), 40; https://doi.org/10.3390/parasitologia5030040 (registering DOI)
Submission received: 11 April 2025 / Revised: 5 July 2025 / Accepted: 10 July 2025 / Published: 1 August 2025
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Abstract

Cystic echinococcosis is a zoonosis caused by the cestode Echinococcus granulosus sensu stricto. Population genetic studies and phylogeographic patterns are essential to understanding the transmission dynamics of this parasite under varying environmental conditions. In this study, the genetic diversity of E. granulosus s.s. was evaluated using 46 hydatid cyst samples obtained from sheep, goats, cattle, and humans across three regions of Chile: Coquimbo, La Araucanía, and Magallanes. Mitochondrial cox1 gene sequences were analyzed and compared with reference sequences reported from South America, Europe, Africa, Asia, and Oceania. In Chile, the EG01 haplotype was the predominant haplotype. A total of four haplotypes were identified, with low haplotype diversity (Hd = 0.461 ± 0.00637) and low nucleotide diversity (π = 0.00181 ± 0.00036). The haplotype network displayed a star-like configuration, with the EG01 genotype at the center, suggesting a potentially ancestral or widely distributed lineage. In Coquimbo (Tajima’s D = −0.93302, p = 0.061; Fu’s Fs = −0.003, p = 0.502) and Magallanes (Tajima’s D = −0.17406, p = 0.386; Fu’s Fs = −0.121, p = 0.414), both neutrality tests were non-significant, indicating no strong evidence for recent population expansion or selection. Star-like haplotype network patterns were also observed in populations from Europe, the Middle East, Asia, Africa, and Oceania, with the EG01 genotype occupying the central position. The population genetic structure of Echinococcus granulosus s.s. in Chile demonstrates considerable complexity, with EG01 as the predominant haplotype. Further comprehensive studies are required to assess the intraspecific genetic variability of E. granulosus s.s. throughout Chile and to determine whether this variability influences the key biological traits of the parasite. This structure may prove even more complex when longer fragments are analyzed, which could allow for the detection of finer-scale microdiversity among isolates from different hosts. We recommended that future cystic echinococcosis control programs take into account the genetic variability of E. granulosus s.s. strains circulating in each endemic region, to better understand their epidemiological, immunological, and possibly pathological differences.

1. Introduction

The genus Echinococcus is a monophyletic group of small cestodes with morphological, developmental, and genetic similarities that exhibit asexual reproduction and constitute important zoonotic pathogens [1,2]. The definitive hosts are typically canids and felids, which acquire the infection through the ingestion of cysts containing the metacestode in its larval stage. This larval form parasitizes intermediate hosts, usually herbivorous or omnivorous mammals. Humans serve as accidental intermediate hosts, developing a disease known as echinococcosis or hydatid disease [1]. In both humans and animals, metacestode cysts are located primarily in the liver and lungs, and less frequently in other organs, causing a serious zoonotic disease of global public health importance. This zoonosis affects human health, livestock production, and local economic development [1].
The classification and nomenclature of species within the genus Echinococcus have long been subjects of controversy. Phylogenetic analyses have been employed to clarify the taxonomic uncertainties, leading to the identification of species complexes and diverse genotypes [1,2]. In this way, Echinococcus granulosus sensu lato (s.l.) comprises a complex of cryptic species that have been extensively studied over the years, resulting in the description of five different species: E. granulosus sensu stricto (G1–G3; sheep strain and buffalo strain), E. equinus (G4; horse strain), E. ortleppi (G5; cattle strain), E. intermedius (G6–G7), and E. canadensis (G8–G10) [3,4,5,6,7,8]. The G1 genotype is the most widespread and accounts for the majority of human hydatidosis cases worldwide [9,10].
To date, relatively few studies have investigated the genetic structure of Echinococcus granulosus s.s. Phylogenetic analyses based on mitochondrial DNA (mtDNA) sequences have identified geographically distinct clades in Europe, Asia, and North America [1,11,12,13,14,15]. However, the available evidence suggests that the majority of genetic variation occurs within geographically restricted populations [1,16].
In South America, Kamenetzky et al. [17] reported the presence of five strains of E. granulosus in Argentina, including G1 (common sheep strain) and G2 (Tasmanian sheep strain), identified in sheep, cattle, and pigs. The G1 strain exhibited a widespread distribution across the country and presented five genetic variants (G1A–B-C-D-E), based on a cox1 gene sequence analysis. Haag et al. [18] also identified the G1 as the predominant strain infecting various hosts and regions throughout Argentina. The haplotypes defined by Kemenetzky et al. [17] were used to estimate genetic differentiation, with an overall FST value of 0.1474. This value increased to FST 0.4081 when considering only the E. granulosus sheep population, suggesting that the observed geographic patterns and population substructuring are not primarily due to a restricted gene flow among the regions; rather, they reflect the historical context, timing, and origin of their introduction in Argentina.
In Chile, E. granulosus is capable of surviving in a wide range of environmental conditions, from an arid climate in the north to a subpolar oceanic climate in the far south. The parasite is highly endemic in four regions—Coquimbo, La Araucanía, Aysén, and Magallanes—where goat, sheep, and cattle farming are prevalent [19,20,21]. E. granulosus has also been detected in the feces of the South American gray fox (Lycalopex griseus) in the Coquimbo region, potentially influencing the geographical distribution of the different haplotypes [22]. Additionally, the presence of E. granulosus G3 in cattle, E. granulosus s.l., E. intermedius (G6), and E. ortleppi (G5) has been reported in the country [23,24,25].
In Chile, Álvarez Rojas et al. [19] reported the presence of E. granulosus s.s. in various host populations across the country. Negative neutrality indexes (Tajima’s D = −201.645 and Fu’s FS = −19.444) were observed, suggesting signatures of negative selection or population expansion. These findings were accompanied by a high haplotype diversity index (0.875 ± 0.032). Hidalgo et al. [21] identified up to five distinct haplotypes of E. granulosus s.s. within individual hosts, distributed across different organs. Additionally, they reported the occurrence of a single haplotype present in multiple organs of the same host. These patterns may be explained by successive infections of the intermediate host or a single infection involving multiple haplotypes.
However, further studies on the population genetic structure and phylogeographic patterns of this parasite are essential to better understand its transmission dynamics. The aim of the present study was to investigate the population genetic structure of Echinococcus granulosus sensu stricto in Chile. Additionally, the results were compared with data from Latin America, Europe, Asia, Australia, and Africa to characterize the genetic variability of this parasite across different geographical regions.

2. Materials and Methods

2.1. Origin of Hydatid Cysts

The germ membranes of Echinococcus granulosus were extracted from the individual hydatid cysts of livestock: 1 goat, 21 cattle, and 24 sheep; the goat and sheep were 5–12 months old, and the cattle were 20–24 months old. The cattle from Coquimbo City were slaughtered at the Danke slaughterhouse in Coquimbo City. The cattle slaughtered at the Nueva Imperial slaughterhouse in Temuco City came from eight locations. The sheep slaughtered at the Patagonia slaughterhouse in the Magallanes region came from locations around Punta Arenas, and the goats and sheep slaughtered at home in Coquimbo came from the Las Cardas sector (Table 1, Figure 1). The cysts were extracted from different organs: 27 from lungs, 15 from livers, and 3 from other tissues (mesentery, kidney, and heart). The cysts were frozen to extract the germ membrane, which was stored and frozen at −80 °C in 70% ethanol for later use. In addition, a human sample of a hydatid cyst isolated from a Chilean patient with cystic echinococcosis from San Pablo Hospital in Coquimbo, Chile, was used (Table 1, Figure 1).

2.2. DNA Extraction and Cox1 Gene Amplification

The DNA extraction was performed from the germ membranes of the hydatid cysts of the different hosts. First, the hydatid cyst was punctured with a 10 mL syringe; next, the hydatid fluid was extracted; last, the germ membrane was extracted and stored in a 15 mL falcon tube in PBS pH 7.0 solution at −80 °C. The total genomic DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions.
The amplification of the mitochondrial cox1 gene was performed using the primers described by Bowles et al. [3]: JB3F 5′–TTTTTTGGGCATCCTGAGGTTTAT-3′; JB3R5′-TAAAGAAAGAACATAATGAAAATG-3′. The PCR mixture was prepared with a final volume of 50 μL, containing 2 μL of DNA template (50 ng/μL), 1 μL of each dNTP (10 mM), 1 μL of each primer (10 μM), 1 μL of PFU polymerase enzyme (Agilent technologies, Santa Clara, CA, USA), 5 μL of 10x buffer, and 39μL of water. Thermal cycling was performed for 50 cycles as follows: initial denaturation at 95 °C for 3 min; denaturation at 95 °C for 35 s; annealing at 50 °C for 35 s; extension at 72 °C for 35 s; and a final extension at 72 °C for 10 min. The PCR products were subjected to electrophoresis in 0.8% agarose gels (w/v), stained with ethidium bromide in Tris-Borate-EDTA 1X buffer at 110 V, and visualized using UV illumination. The amplified products were sent for sequencing in both directions to Macrogen (Seoul, Republic of Korea).

2.3. Sequence Analysis and Construction of Phylogenetic Trees

A total of 45 PCR products (product = 450 bp) from the different hosts were sequenced using the forward and reverse primers by Macrogen (Seoul, Republic of Korea). The sequences were identified and compared with the GenBank database using the Blast (http://www.ncbi.nlm.nih.gov/BLAST/, accessed on 8 March 2025) analysis (8 March 2025). The sequences were then edited and aligned using the Bioedit program (https://bioedit.software.informer.com, accessed on 8 March 2025) and Clustal W (https://www.ebi.ac.uk, accessed on 8 March 2025). These multiple alignments were performed with the reference sequences of E. granulosus (G1, G2, G3, G4, G5, G6, and G7) and variants of the G1 genotype (G1A, G1B, G1C, G1D, and G1E) deposited in the GenBank as references M84661/62/63/64/65/66/67 [3]; AF458871/72/73/74/75 [17]; EF393619, EF595654, EU178103, EU178104 [26]; and JQ250806 [27]; Taenia hydatigena and Taenia crassiceps.

2.4. Cox1 Sequence Comparisons with GenBank Deposited Sequences

The term “genotype” was used to describe the genetic variants described within the E. granulosus s.l. complex (G1–G10) [3,27,28], and the term “haplotype” was used to describe the genetic microvariants observed in E. granulosus s.s. [3,17,26].
The sequences obtained in this study were aligned to obtain a comparison with the 450 bp E. granulosus cox1 sequences, which are a reference for the G1, G2, and G3 genotypes originally described by Bowles et al. [3] and Yanagida et al. [27]. For the same purpose, the sequences of this study were aligned with 101 sequences described for Latin America: Chile (39), Peru (27), Bolivia (9), and Argentina (26) [19,21,24,27,28,29,30,31]. In the Middle East, 17 sequences were also used: Palestine (5), Iran (11), and Saudi Arabia (1) [27,32,33,34,35,36,37]. A total of 61 cox1 sequences were described in Europe: Austria (10), Bulgaria (9), Hungary (6), Italy (10), Portugal (10), Romania (13), Greece (1), and England (2) [12,29,38,39,40,41,42,43]. Similarly, 95 described sequences of the cox1 gene were used in Asia, corresponding to the following: Turkey (21), China (50), Iran-Kurdistan (21), and Kyrgyzstan (3) [5,12,14,27,38,41,44,45,46,47], GenBank: EU929083.1, GenBank: MN990735.1, and GenBank: MT537165.1. A total of 19 sequences of E. granulosus cox1 were described in Australia and Oceania [29,40], and 39 sequences were described for Africa: Libya (4), Tunisia (34), and Kenya (1) [29,48], which are deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 8 March 2025) (Table A1).

2.5. Diversity and Genetic Structuring of the Population

The nucleotide and haplotypic diversity were calculated for the total and for each population—Coquimbo, La Araucanía, and Magallanes (Figure 3)—in the Arlequin 3.5 program [49]. The demographic history for the total sequences and by population was estimated using the neutrality tests of Fu and Li [50] and Tajima [51] using the DnaSP 5.10 program [52]. To estimate the genetic differentiation between pairs of populations, the ΦST (an analogue of the FST that takes into account haplotypic frequency and divergence) with 10,000 permutations in the Arlequin 3.5 program [49]. Additionally, the nearest-neighbor statistic (Snn) [53], which measures how often the closest sequence neighbors are found within the same locality, was measured in the DnaSP 5.10 program [52]. If populations are strongly structured, it is expected to find the nearest neighbor of a sequence in the same locality. Therefore, the Snn is expected to be close to 1.0 when two populations are highly differentiated and about half when two populations are part of a panmictic population [53]. The Molecular Analysis of Variance (AMOVA) was performed to test the hypothesis that the genetic diversity within the sampled populations is not significantly different from that which could result from the union of the populations. The analysis was performed in the Arlequin 3.5 program [49]. The PopART 1.7 software was used, maintaining statistical parsimony, to draw a network of haplotypes (https://popart.maths.otago.ac.nz, accessed on 8 March 2025).
The phylogenetic tree and the relationships between the haplotypes of the present study and the reference sequences [3] and other sequences described in various locations were evaluated by calculating the means of a maximum likelihood tree based on the Kimura 2 Parameters (K2P) model using the MEGA 12 software (https://www.megasoftware.net, accessed on 8 March 2025).

3. Results

The mitochondrial gene cox1 was successfully amplified in 46 of the isolated cysts (amplifying in 100% of them), obtaining sequences of 450 bp. Echinococcus granulosus s.s. was identified in 21 hydatid cysts from the cattle, 23 from the sheep, 1 from the goat, and 1 from the human (Table 1 and Table A1). Most of the samples (N = 34; 73.91%) belong to the EG01 genotype with an accession number of JQ250806 (Table A2).
The presence of the EG1A haplotype (AF458871) was observed in six isolates (13.04%): one goat isolate from the Coquimbo region (EgCLCo03) and five cattle isolates from La Araucanía (EgCLBA03-04-07-10-12), which presented a T → C transition in residue 96. In six (13.04%) sheep isolates from the Magallanes region, the presence of the EG1D haplotype (AF458874) (EgCLOM01-03-05-08-10-21) was observed, presenting a C → T transition in residue 197 (Table A2). It can also be observed that the sheep isolate EgCLOM16 presented an A → G transition at site 253 regarding the EG01 haplotype (JQ250806) (Table A2). The phylogenetic tree clearly separated the haplotypes, showing the distinct clades with the haplotypes EG1A and EG1D (Figure 2).
The parsimony lattice of E. granulosus s.s. shows a star-shaped topology with the haplotypes EG1A (AF548871), EG1D (AF458875), and EG01 (JQ250806, AB491414, JF513058), and the haplotype EgCLOM16 (Figure 3A). The population analysis only shows four haplotypes with a low diversity of haplotypes (Hd) among the three populations analyzed (Hd = 0.461 ± 0.00637), a low presence of only four polymorphic sites (K), and a low diversity of nucleotides, which fluctuated between 0.00084 and 0.00128 (Table 2).
The haplotype diversity in the cattle populations by region was moderate to low (Table 2). The nucleotide diversity (π) was low in all populations, with the lowest value obtained in the Coquimbo population. The neutrality indexes, Tajima’s D and Fu’s Fs, were negative in Coquimbo and Magallanes, with the lowest value found in Coquimbo. However, in the Magallanes population, Tajima’s D and Fu’s Fs were both positive (Tajima’s D = 0.8695, Fu’s Fs = 1.039), although not statistically significant (p > 0.05) in any of the three populations.
To estimate the degree of gene flow among the three populations, the pairwise FST was computed. The FST values among the populations ranged from −0.0876 to 0.1073 (Table 3). A moderate and statistically significant genetic differentiation was observed between the La Araucanía and Magallanes populations (FST = 0.10703, p = 0.00098), which suggests limited gene flow and possible ecological or historical isolation between these regions.
The AMOVA results presented in Table 4 show that 79.66% of the genetic variation is found within the populations, while 20.34% occurs among the three analyzed populations of E. granulosus s.s.
The haplotype network in Figure 4A, constructed with the sequences of the present study and with other sequences from Chile, Peru, Bolivia, and Argentina, shows a star shape with the EG01 haplotype (JQ250806) in the center, present in 46 of the sequences of this study, 20 from other studies in Chile, 16 from Peru, 7 from Bolivia, and 9 from Argentina. Six (6) of the haplotypes described for Chile differ by a single nucleotide from the EG01 sequence (JQ250806) (Figure 4A, Table A3). The analysis of the population genetic structure determined by a paired comparison between populations indicated a small population differentiation (FST = 0.00743) and a negative Tajima’s D index (Figure 4B).
The EG01 haplotype (JQ250806) is common across all geographic populations and constitutes part of the central structure of haplotype networks on all continents (Figure 4A–D,F). In all cases, the haplotype networks exhibited a star-like topology. The networks from Latin America, Europe, and Asia displayed the most complex structures.
In Chile, the EG01 haplotype was detected in cattle and sheep across all three sampled localities: Coquimbo, La Araucanía, and Magallanes.
All regions except the Middle East exhibited negative and significant values for both Tajima’s D and Fu’s Fs. Consistent with the signals of recent population expansion, Latin America showed a Tajima’s D = –2.39 (p = 0.000) and Fu’s Fs = –13.85 (p = 0.000); Africa demonstrated a Tajima’s D = –2.06 (p = 0.000) and Fu’s Fs = –16.62 (p = 0.000); and Australia showed a Tajima’s D = –1.83 (p = 0.001) and Fu’s Fs = –6.63 (p = 0.001). Europe and Asia also showed significant negative values, suggesting a population expansion in these regions. In contrast, the Middle East displayed a non-significant Tajima’s D = –1.29 (p = 0.083) and a significant Fu’s Fs = –3.60 (p = 0.015), which may indicate a mild or localized expansion (Figure 4G, Table 5).
When examining Latin American countries, Chile exhibits a Tajima’s D of −2.36563 (p = 0.000), Fu’s Fs of −2.083 (p = 0.201), and haplotype diversity (hd) of 0.542 ± 0.059, suggesting a moderate or uncertain signal of population expansion and moderate haplotypic diversity. In contrast, Argentina shows a Tajima’s D of −1.71486 (p = 0.010), Fu’s Fs of −8.639 (p = 0.000), and hd of 0.869 ± 0.042, while Peru presents a Tajima’s D of −1.99558 (p = 0.002), Fu’s Fs of −6.421 (p = 0.000), and hd of 0.561 ± 0.114. These results indicate evidence of population expansion in both countries, with Argentina exhibiting high haplotypic diversity (Table 5).

4. Discussion

In this study, the genetic diversity and population structure of Echinococcus granulosus s.s. in Chile were assessed by analyzing the cox1 gene sequences obtained from various intermediate hosts. This mitochondrial marker has been demonstrated to be effective for detecting intraspecific variability and has been widely used to investigate population structures in other parts of the world [3,5,12,19,21,27,29,44].
All samples in this study were confirmed as belonging to the G1 strain (sheep strain), which is the most globally prevalent genotype (88.44%) and is highly infectious to humans (Figure 3A, Table A2) [54]. This strain is recognized as the most common and dominant strain across most regions worldwide (Figure 3A) [9,12,14,19,27,55].
The most frequently observed haplotype was EG01 (JQ250806), found in 33 of the 46 samples analyzed. This haplotype has been previously reported as predominant in several regions, including Jordan, China, and Peru (AB491414) [27], Europe (JF513058) [12], Australia [9], Russia [56], and Chile [19,21].
The EG01 haplotype (JQ250806) was identified by Yanagida et al. [27] as an ancestral lineage potentially originating from the Middle East. This haplotype is globally distributed and commonly associated with domestic livestock, as evidenced by haplotype networks derived from our samples and previously reported sequences from Europe, Asia, Africa, Australia, and Latin America (Figure 4). It has been hypothesized that EG01 may have been introduced in South America during the Spanish colonization over 500 years ago through the movement of domesticated animals [19,57]. The available data indicate that this haplotype is widespread across geographically distant regions, suggesting its continued presence in domestic animals and livestock populations.
The network of haplotypes constructed from the data in this study (Figure 3) reveals a star-shaped pattern of expansion, with the EG01 haplotype in the center. Most of the remaining haplotypes differ from EG01 by only a single nucleotide substitution, indicating a recent origin from a common ancestral lineage. Neutrality tests (Tajima’s D and Fu’s Fs) did not detect significant departures from neutrality in any of the regions analyzed, suggesting an absence of strong selective pressures or recent demographic events. The slightly negative values observed in Chile and Coquimbo may reflect a historical demographic expansion, while the positive values detected in La Araucanía could indicate genetic stability or the presence of a population substructure. Overall, the haplotype diversity was moderate across all regions, suggesting a relatively stable level of genetic variation. These findings are consistent with a parasite population that has undergone limited recent demographic change, potentially reflecting a stable host–parasite association shaped by historical rather than ongoing transmission dynamics.
The neutrality indexes revealed contrasting patterns among the studied countries. In Chile, Tajima’s D was significantly negative, while Fu’s Fs was also negative but not statistically significant, providing weaker support for demographic expansion. The haplotype diversity was moderate, which is consistent with the possibility that it may have undergone recent growth following a historical reduction in size.
Overall, these results suggest that parasite populations in Argentina and Peru have undergone recent demographic expansion, with similar, though weaker, signals observed in Chile. These phylogeographic patterns and population structures do not appear to result from a lack of flow, as previously suggested by Haag et al. [18], although additional data from other regions are required to better assess the dispersion of various E. granulosus s.s. strains and haplotypes.
Likewise, significantly negative values of Tajima’s D and Fu’s Fs were observed in the pooled samples from Latin America, Europe, Asia, Africa, and Australia, further supporting the hypothesis of a recent global population expansion of E. granulosus s.s. [5,29].
The FST fixation index calculated for the samples in this study (0.19742) indicates moderate genetic differentiation among the Echinococcus granulosus sensu stricto population across the various regions of Chile. This pattern is also reflected in the haplotypes’ network and their geographic distribution. In the Coquimbo region, two of the four haplotypes identified, EG01 and EG1A, were detected in cattle and goats, respectively. Similarly, in La Araucaía, the same haplotypes were found in cattle. In contrast, in the Magallanes region, three haplotypes—EG01, EG1D, and EgCLOM16—were identified in sheep.
The low haplotype diversity observed may be partially attributed to the limited sequence length analyzed, as some researchers have proposed that longer sequences could provide greater resolution for assessing the genetic structure of parasite populations [19,27,58]. Additionally, the low variability may also reflect the sampling of hosts from geographically restricted areas.
The EG1A haplotype was also described by Alvarez Rojas et al. [19] in goats, cattle, and humans from La Araucanía and the Metropolitan region (GenBnk: KX227118). In contrast, the EG1D haplotype has been reported in cattle and sheep in the Maule and Los Lagos regions (KX227120, Kr968703). These findings expand the known distribution of E. granulosus s.s. haplotypes in Chile, underscoring the need for further molecular analyses to better characterize the population structure and phylogeography of the species in the country.
These haplotypes have also been reported from other regions in South America. The EG1A haplotype was described in Bolivia (KX227118) and Tucuman, Argentina (AF458871) [17,57]. The EG1D haplotype has been described in humans, sheep, and dogs in Bulgaria and Hungary (JF513065) [12,17], in human cases in Turkey (GenBank: KX874719.1), and in dogs from Iran (JN604099) [35].
Our results indicate that the population genetic structure of E. granulosus s.s. is complex, with multiple haplotypes circulating in the different regions. The EG01 haplotype appears to represent a potential ancestral lineage, possibly introduced to Chile through the historical introduction of livestock from Europe. This complexity is likely to increase with the analyses of longer sequences, which could facilitate the study of microdiversity within E. granulosus s.s. populations in different hosts.
Therefore, when designing future control programs for cystic echinococcosis (CE), it is essential to consider the strain variability in each endemic region. Further research is needed to determine whether the different haplotypes exhibit biological or antigenic differences, organ tropism, variation in tissue survival or infection rates, host immune response profiles, and potential correlations between the immunodiagnostic test performance and the specific haplotype or strain infecting the host.

5. Conclusions

The population genetic structure of Echinococcus granulosus sensu stricto in Chile exhibits considerable complexity, with the ancestral haplotype EG01 being predominant. The presence of shared haplotypes between the Coquimbo–La Araucanía (G1A) and La Araucanía–Magallanes (G1D) regions across multiple host species (including goats, cattle, and sheep) suggests the potential translocation of infected livestock among these geographic areas. Further comprehensive studies are needed to assess the intraspecific genetic variability of E. granulosus s.s. throughout Chile and to determine whether this variability affects the key biological traits of the parasite. Such insights are essential for guiding the development of targeted, host-specific control strategies for cystic echinococcosis.

Author Contributions

The work described in this manuscript has not been published previously and is not under consideration for publication elsewhere. All authors have approved this submission. Material preparation and data collection were carried out by N.U.-U., G.R.-V., and Y.N.-B. All authors were involved in data analysis and manuscript write-up. N.U.-U. and Y.N.-B. conceptualized this study and prepared the first draft of this manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financed by the project VRIDT N°10301390 “Genotypic characterization of the Echinococcus granulosus: for the preparation of a future vaccine”. 2014–2016. Universidad Católica del Norte.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Research Committee of the Faculty of Medicine Ethical Committee (Nº 04/12), Universidad Católica del Norte, Coquimbo, Chile.

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study. Written informed consent was also obtained from the patients for the publication of this paper.

Data Availability Statement

The database used to carry out the present study is not publicly available due to internal policy of our departments. However, the database could be available, after a justification of its use, upon request from the corresponding author.

Acknowledgments

Lorena Ortega for her great help in collecting the necessary samples for this research, Giuliano Bernal for providing the laboratory equipment, and Rodrigo Sandoval for helping us with the revision of the manuscript.

Conflicts of Interest

The authors declare no competing interests.

Abbreviations

The following abbreviations are used in this manuscript:
AMOVA Molecular Analysis of Variance
cox1cytochrome oxidase subunit I
FSTFixation index
G1-G3E. granulosus sensu stricto (sheep strain and buffalo strain)
G4E. equinus (horse strain)
G5E. ortleppi (bovine strain)
G6-G7E. intermedius
G8-G10E. canadensis
Hdhaplotypic diversity
Knumber of haplotypes
mtDNAmitochondrial DNA
Nnumber of samples
s.l.sensu lato
s.s.sensu stricto
Snumber of segregation sites
πnucleotide diversity

Appendix A

This section collects any data citations, data availability statements, or Supplementary Materials included in this article.
Table A1. Sequences of mitochondrial cytochrome oxidase gene subunit 1 (cox1) of Echinococcus granulosus s.s. and E. granulosus s.l. used in this study.
Table A1. Sequences of mitochondrial cytochrome oxidase gene subunit 1 (cox1) of Echinococcus granulosus s.s. and E. granulosus s.l. used in this study.
Locality/CountryDog (HF)Sheep (HF)Goats (HF)Bovine (HF)Human (HF)
South America
La Araucanía (IX),
Maule (VII),
Coquimbo (IV),
Chile		KX227122 (EgCLP20) KX227123 (EgCLP21)KX227116 (EgCLO11) KX227120 (EgCLO17) KX227126 (EgCLO22)
MN399400 (EgCLO27)
EgCLOM01-6,8-23*
EgCLOCo07*
KX227131(EgCL36)
KX227125 (EgCL37)
KX227121 (EgCL38)
KX227124 (EgCL39)
KX227119 (EgCL40)		KX227118 (EgCLC14)
EgCLCCo03*		JQ250806.1 (EgCLB28) KT968704.1 (EgCLB05) KT968703 (EgCLB06) AB777904 (EgCLB08) KX227116 (EgCLB10)
KX227117 (EgCLB13) KX227118 (EgCLB15) KX227120 (EgCLB18) KX227130 (EgCLB24)
EgCLBA01-16,19,20* EgCLBCo17,18,21*
AF458872 (EgCLB30)		U50464 (EgCLH01) M84666 (EgCLH02) AB522646 (EgCLH03) JQ250806.1 (EgCLH04) AB777904 (EgCLH07) KX227116 ((EgCLH09) KX227117 (EgCLH12) KX227118 (EgCLH16) KX227127 (EgCLH23) KX227128 (EgCLH25) KX227129 (EgCLH26) EgCLHCo01*
AF458874 (EgCLH19)
AF458875 (EgCLH29)
KX227136 (EgCL31)
KX227135 (EgCL32)
KX227134 (EgCL33)
KX227133 (EgCL34)
KX227132 (EgCL35)
PerúKT001408 (EgPeP09)
KT001398 (EgPeP10)
KT001407 (EgPeP11)
KT001406 (EgPeP12)
KT001405 (EgPeP13)
KT001404 (EgPeP14)
KT001403 (EgPeP15)
KT001402 (EgPeP16)
KT001401 (EgPeP17)
KT001400 (EgPeP18)
KT001399 (EgPeP19)
KT001397 (EgPeP20)
KT001396 (EgPeP21)
KT001395 (EgPeP22)		AB458672 (EgPeO01) AB470527 (EgPeO02) AB688621 (EgPeO03) GU233854 (EgPeO04)
GU23395 (EgPeO06)		 MN732663 (EgPeLL23)
AB458674 (EgPeB24)
AB458673 (EgPeB23)
AB688620 (EgPeB24)
AB688621 (EgPeO25)		GU233952 (EgPeH05) AB458675 (EgPeH07) GU233951 (EgPeH08)
Bolivia JQ250806 (EgBolB01)
KX227118 (EgBolB02)
MT072973 (EgBolB03)
MT072974 (EgBolB04)
MT072975 (EgBolB05)
MT072976 (EgBolB06)
MT072977 (EgBolB07)
MT072978 (EgBolB08)
MT072979 (EgBolB09)
Argentina AF458875 (EgArgO03)
MT796074 (EgArg07)
KC579445 (EgArgO09)
KC579450 (EgArgO10)
KC579447 (EgArgO13)
FN564569 (EgArgO16)
GU980907 (EgArgO27)
GU980906 (EgArgO28)
KC954601 (EgArgO30)
MT796487 (EgArgC24)AF458873 (EgArgB05)
KC579445 (EgArgB08)
KC579449 (EgArgB11)
KC579448 (EgArgB12)
KC579446 (EgArgB14)
KC579447 (EgArgB18)
KC954602 (EgArgB29)		M84661 (EgArgH01)
AF458875 (EgArgH02)
AF458873 (EgArgH04)
AF458872 (EgArgH06)
KT719395 (EgArgH15)
MT800802 (EgArgH17)
MT800801 (EgArgH19)
MT800800 (EgArgH20)
MT800797 (EgArgH21)
MT800796 (EgArgH22)
MT800795 (EgArgH23)
MT796079 (EgArgH26)
Middle East
Palestine KC109640 (EgPaO01), KC109657 (EgPaO02), KC109651 (EgPaO03)
Iran




Saudi Arabia		JN604097 (EgIP08), JN604099 (EgIP09, JN604102 (EgIP10)JQ250806 (EgIO05),
JQ250808 (EgIO06), JQ250811 (EgIO07)




MN720282 (EgASO01)		 JQ250810 (EgIH01), JQ250812 (EgIH02),
JQ250815 (EgIH03),
AB677811 (EgIH04)
MT073987 (EgIH11), MN807921 (EgITH12)
Europe
Austria JF513058 (EgAO01) JF513060 (EgAO03) JF513061 ( (EgAO05) JF513058 (EgAH02) JF513060 (EgAH04) JF513061 (EgAH06) AJ508018 (EgAH07) AJ508019 (EgAH08) AJ508021 (EgAH09) AJ508028 (EgAH10)
Bulgaria JF513058 (EgBO01) JF513063 (EgBO02) JF513065 (EgBO03) JF513058 (EgBH07) JF513063 (EgBH08) JF513065 (EgBH09)
Hungry JF513058 (EgHO01) JF513061 (EgHO02) JF513063 (EgHO03) JF513064 (EgHO04) JF513065 (EgHO05) JF513067 (EgHO06)
Italiy JF513058 (EgIO01) F513059 (EgIO02) JF513060 (EgIO03) JF513062 (EgIO04) DQ062857 (EgIO06) DQ062857 (EgIB05)JF513058 (EgIH08) JF513060 (EgIH09) JF513062 (EgIH10)
Portugal JF513058 (EgPO01) JF513060 (EgPO02)
JF513079 (EgPO03) HF947595 (EgPO04) HF947592 (EgPO05) HF947558 (EgPO06) HF947585 (EgPO07) HF947574 (EgPO08)
HF947579 (EgPO09) HF947557 (EgPO10)
HF947566 (EgPO11)
Romania JF513058 (EgRO01) JF513061 (EgRO02) JF513063 (EgRO03)
JF513072 (EgRO04) JF513079 (EgRO05)		 JF513058 (EgRH06) JF513061 (EgRH07) JF513063 (EgRH08) JF513079 (EgRH09)
JF520817 (EgRH010) JF520818 (EgRH011) AY686564 (EgRH012) AY686565 (EgRH013)
Greece DQ856467 (EgGO01)
UKKT001398 (EgUKP01) KT001397 (EgUKP02)
Asia
Türkiye JF513058 (EgTO01) JF513060 (EgTO02) JF513062 (EgTO03) JF513067 (EgTO04) JF513071 (EgTO05) JF513079 (EgTO06) JF775380 (EgTO07) EU929083 (EgTO08) JF513058 (EgTH09) JF513060 (EgTH10) JF513062 (EgTH11) JF513067 (EgTH12) JF513079 (EgTH13) JF775379 (EgTH14) GU951512 (EgTH15) GU951513 (EgTH16) MT537165 (EgTH17) MN990735 (EgTH18) KX874714 (EgTH19) KX874725 (EgTH20) KX874719 (EgTH21)
ChinaDQ356881 (EgCHP01) DQ356882 (EgCHP02)AB491414 (EgCHO03) AB491418 (EgCHO04) AB491421 (EgCHO05) AB491424 (EgCHO06) AB491425 (EgCHO07) AB491432 (EgCHO08) AB491438 (EgCHO09) AB491449 (EgCHO10) AB491454 (EgCHO11) AB688612 (EgCHO12) MG674403 (EgCHO31) MG674404 (EgCHO34) MG674406 (EgCHO40)
MG674412 (EgCHO43) MG674416 (EgCHO46) MG674418 (EgCHO49)		 MG674403 (EgCHY32) MG674404 (EgCHY35) MG674406 (EgCHY41) MG674412 (EgCHY44) MG674416 (EgCHY47) MG674418 (EgCHY50)AB688602 (EgCHH13) AB688611 (EgCHH14) AB688613 (EgCHH15) AB688619 (EgCHH16) AB491419 (EgCHH17) AB491420 (EgCHH18)
AB491422 (EgCHH19) AB491423 (EgCHH20) AB491428 (EgCHH21) AB491431 (EgCHH22)
AB491434 (EgCHH23) AB491437 (EgCHH24) AB491439 (EgCHH25) AB491447 (EgCHH26) AB491451 (EgCHH27)
AB491453 (EgCHH28) AB491455 (EgCHH29) MG674403 (EgCHH30) MG674404 (EgCHH33) MG674406 (EgCHH39) MG674412 (EgCHH42) MG674416 (EgCHH45) MG674418 (EgCHH48)
Iraq, Kurdistan MF004305 (EgKO01)
MF004277 (EgKO04) MF004283 (EgKO07) MF004285 (EgKO10) MF004279 (EgKO13) MF004273 (EgKO14) MF004288 (EgKO15)		MF004305 (EgKC02) MF004277 (EgKC05) MF004283 (EgKC08) MF004285 (EgKC11) MF004278 (EgKO16) MF004287 (EgKO17) MF004293 (EgKO18) MF004306 (EgKO19)MF004305 (EgKB03) MF004277 (EgKB06) MF004283 (EgKB09) MF004285 (EgKC12) MF004280 (EgKB20) MF004291 (EgKB21)
Kyrgyztan MN787554 (EgKirH01)
MN787559 (EgKirH02)
MN787560 (EgKirH03)
Oceania
AustraliaKT001398 (EgAusP05) KT001395 (EgAusP05) KT968706 (EgAusD07)
KT968707 (EgAusM08)
KU697314 (EgAusD14)
JQ250806 (EgAusM16)
JQ250809 (EgAusM17)		AJ508005 (EgAusO01) AJ508006 (EgAusO02) AJ508009 (EgAusO03) AJ508010 (EgAusO04)
KT968705 (EgAusO09)
KT968704 (EgAusO10)
KT968703 (EgAusO11)
KT968708 (EgAusO12)
KT968702 (EgAusO13)
AB522646 (EgAusO15)
AB688591 (EgAusO18)
Africa
LibyaKT001407 (EgLP01)
KT001405 (EgLP02) KT001398 (EgLP03)		 HM636641 (EgLH04)
TunisiaKM014635 (EgTuP03)
KM014636 (EgTuP04)
KM014638 (EgTuP06)
KM014640 (EgTuP08)
KM001399 (EgTuP13)
KT001401 (EgTuP14)
KT001402 (EgTuP15)
KT001403 (EgTuP16)
KT001399 (EgTuP17)
KT0014610 (EgTuP32)		KM014641 (EgTuO09)
KM014644 (EgTuO12)
KT0014628 (EgTuO20)
KT0014627 (EgTuO21)
KT0014614 (EgTuO28) KT0014613 (EgTuO29)		KT0014624 (EgTuC22)
KT0014616 (EgTuC26)		KM014634 (EgTuB01)
KM014633 (EgTuCam02)
KM014639 (EgTuB07)
KM014642 (EgTuB10)
KT0014620 (EgTuB24
KT0014617 (EgTuB25)
KT0014615 (EgTuB27)
KT0014612 (EgTuB30)
KT0014611 (EgTuCe31)
KT0014606 (EgTuCe33)
KT0014607 (EgTuCe34)		KM014637 (EgTuH05)
KM014643 (EgTuH11)
KT0014631 (EgTuH18)
KT0014629 (EgTuH19)
KT0014621 (EgTuH23)
KenyaKM01398 (EgKP01)
Sequence nomenclature = GenBank number (Acronym current study: E: Echinococcus, g: granulosus, X: country, X: animal, X: number); e.g., KX227122 (EgCLP20) = KX227122: GenBank number (E.g.: Echinococcus granulosus, CL: Chile, P: dog, 20: N°20).
Table A2. The segregation sites between the cox1 gene sequences of the E. granulosus s.s. haplotypes identified in this study (EgCLOM01-06, 8-23, EgCLOCo07 haplotypes; EgCLCCo03, EgCLBA01-16, 19, 20; EgCLBCo17, 18, 21; EgCLHCo01) compared to the sequence of haplotypes of E. granulosus s.s. described by Yanagida et al. [3,17,27]. The positions of the nucleotides are numbered from the first nucleotide of the gene.
Table A2. The segregation sites between the cox1 gene sequences of the E. granulosus s.s. haplotypes identified in this study (EgCLOM01-06, 8-23, EgCLOCo07 haplotypes; EgCLCCo03, EgCLBA01-16, 19, 20; EgCLBCo17, 18, 21; EgCLHCo01) compared to the sequence of haplotypes of E. granulosus s.s. described by Yanagida et al. [3,17,27]. The positions of the nucleotides are numbered from the first nucleotide of the gene.
Haplotypes196101134137182197218251253348359368371383400
EGO1-JQ250806CTGCGGCGAAATGAGT
EG1A-AF458871.C..............
EG1B-AF458872..........G.....
EG1C-AF458873.............G..
EG1D-AF458874 ......T.........
EG1E-AF458875.. T............
EgCLCCoO3 .C..............
EgCLBCo17 ................
EgCLBCo18 ................
EgCIBCo21 ................
EgCLH Co01 ................
EgCLBA01 ................
EgCLBA02................
EgCLBA03.C..............
EgCLBA04.C..............
EgCLBA05................
EgCLBA06................
EgCLBA07.C..............
EgCLBA08................
EgCLBA09................
EgCLBA10.C..............
EgCLBA11................
EgCLBA12.C..............
EgCLBA13................
EgCLBA14................
EgCLBA15................
EgCLBA16................
EgCLBA19................
EgCLBA20................
EgCLOM01......T.........
EgCLOM02................
EgCLOM03......T.........
EgCLOM04................
EgCLOM05......T.........
EgCLOM06................
EgCLOCo07................
EgCLOM08......T.........
EgCLOM09................
EgCLOM10......T.........
EgCLOM11................
EgCLOM12................
EgCLOM13................
EgCLOM14................
EgCLOM15................
EgCLOM16.........G......
EgCLOM17................
EgCLOM18................
EgCLOM19................
EgCLOM20................
EgCLOM21......T.........
EgCLOM22................
EgCLOM23................
Table A3. The segregation sites of the cox1 gene sequences of the haplotypes of E. granulosus s.s. described for Chile, Peru, Bolivia, and Argentina in Latin America. The positions of the nucleotides are numbered from the first nucleotide of the gene.
Table A3. The segregation sites of the cox1 gene sequences of the haplotypes of E. granulosus s.s. described for Chile, Peru, Bolivia, and Argentina in Latin America. The positions of the nucleotides are numbered from the first nucleotide of the gene.
Haplotypes1226323638395960667479808996101116128131134137146159167170182197218251260269271280287290299306320332338341344348349371378383400
EG01-JQ250806CTTTAAATAGTCGCTGTGTCGTAGGGCGAGGGTTGAGGTATGATAAGT
EG1A AF458871--............C.................................
EG1B AF458872--........................................G.....
EG1D AF458874--........................T.....................
EG1E AF458875--.................T............................
EG02 M84662--.........T.T..................C..............-
EG03 M84663--...........T..................C..............-
EG6 M84666--..GT.G.TGTTT...A.TTA.AATTTGATAC.TGAT.CCT..GGA-
EU178104...........T....................................
EgCLH01--..............................................
EgCLH02--..GT.G.TGTTT...A.TTA.AATTTGATAC.TGAT.CCT..GGA-
EgCLH04................................................
EgCLB28................................................
EgCLH03.............T..................C...............
EgCLB05..........................T.....................
EgCLB06...................T............................
EgCLH07................................................
EgCLB08................................................
EgCLH09................................................
EgCLB10................................................
EgCLO11................................................
EgCLH12..C.............................................
EgCLB13..C.............................................
EgCLC14..............C.................................
EgCLB15..............C.................................
EgCLH16..............C.................................
EgCLO17..........................T.....................
EgCLB18..........................T.....................
EgCLP20................................................
EgCLP21................................................
EgCLO22................................................
EgCLH23.............T..................C...............
EgCLB24................................................
EgCLH25...........T.T..................C...............
EgCLH26.................................C..............
EgClH19--........................T.....................
EgClH29--.................T............................
EgClB30--........................................G.....
EgCL31................................................
EgCL32..........................T.....................
EgCL33.............T..................C...............
EgCL34................................................
EgCL35................................................
EgCL36................................................
EgCL37................................................
EgCL38................................................
EgCL39................................................
EgCL40................................................
EgBolB01................................................
EgBolB02..............C.................................
EgBolB03................................................
EgBolB04................................................
EgBolB05................................................
EgBolB06................................................
EgBolB07................................................
EgBolB08......................................C.........
EgBolB09................................................
EgArgH01.............T..................C...............
EgArgH02..............C.................................
EgArgO03..............C.................................
EgArgH06..........................................G.....
EgArgH04............................................G...
EgArgB05............................................G...
EgArgH07................................................
EgArgB08................................................
EgArgO09........G.......................................
EgArgO10...........T.T..................C...............
EgArgB11.............................................G..
EgArgB12...............A.A..............................
EgArgO13..............C.................................
EgArgB14...................T............................
EgArgH15..............C.................................
EsArgO16........G.......................................
EgArgH17..........................T.....................
EgArgB18..............C.................................
EgArgH19..............C.................................
EgArgH20................................................
EgArgH21................................................
EgArgH22................................................
EgArgH23................................................
EgArgC24...............................................-
EgArgH25.............T.................................-
EgArgH26...................T...........................-
EgArgO27...............................................-
EgArgO28...............................................-
EgArgB29..............C................................-
EgArgO30......................G........................-
EgPeH07...............................................-
EgPeP09.A................CT............................
EgPeP10................................................
EgPeP11...................T............................
EgPeP12...C............................................
EgPeP13..............................T.................
EgPeP14................................................
EgPeP15................................................
EgPeP16......G.........................................
EgPeP17................C...............................
EgPeP18................................................
EgPeP19...............A................................
EgPeP20................................................
EgPeP21...........T....................................
EgPeP22...........T.T..................C...............
EgPeLL23................................................
EgPeO02...............................................-
EgPeB24(2)...........................................G...-
EgPeB23...............................................-
EgPeO01...............................................-
EgPeO03................................................
EgPeB24...........................................G....
EgPeO04................................................
EgPeH08................................................
EgPeH05................................................
EgPeO06................................................
EgPeO25................................................
Table A4. The segregation sites of 61 sequences of the cox1 gene of E. granulosus s.s. described for Austria, Hungary, Bulgaria, Italy, Romania, Portugal, Greece, and England in Europe. The positions of the nucleotides are numbered from the first nucleotide of the gene.
Table A4. The segregation sites of 61 sequences of the cox1 gene of E. granulosus s.s. described for Austria, Hungary, Bulgaria, Italy, Romania, Portugal, Greece, and England in Europe. The positions of the nucleotides are numbered from the first nucleotide of the gene.
Haplotypes1262963667479808996109130134146182197206214224227230280329338371377378379383385386388391398400
EG01-JQ250806CTAAGTCGCTTGCTGCTTTTTTGAATATGATACTT
EG1A AF458871.........C.........................
EG1C AF458873........................G..........
EG1D AF458874...............T...................
EG2 M84662......T.T............C.............
EG3 M84663........T............C.............
EU178103............T......................
EU178104......T............................
EgAO01...................................
EgAH02...................................
EgAO03........T............C.............
EgAH04........T............C.............
EgAO05......T............................
EgAH06......T............................
EgAH07.C.........A.......................
EgAH08......T............................
EgAH09..G...T.T.C..........C.............
EgAH10.................C..C..............
EgBO01...................................
EgBH07...................................
EgBO02.............C.....................
EgBH08.............C.....................
EgBO03...............T...................
EgBH09...............T...................
EgBO04........T.............A............
EgBO05........T....C.......C.............
EgBO06........T......T.....C.............
EgHO01...................................
EgHO02......T............................
EgHO03.............C.....................
EgHO04..................C................
EgHO05...............T...................
EgHO06.C....T............................
EgItO01...................................
EgItH08...................................
EgItO02......T.T............C.............
EgItO03........T............C.............
EgItH09........T............C.............
EgItO04............T......................
EgItH10............T......................
EgItB05...................................
EgItO06...................................
EgItP07...................................
EgPO01...................................
EgPO02........T............C.............
EgPO03........T..........................
EgPO04...................................
EgPO06...................................
EgPO07.........................A.ATTAGAA.
EgPO08...................................
EgPO09...................................
EgPO10...................................
EgPO11...................................
EgRO01...................................
EgRH06...................................
EgRO02......T............................
EgRH07......T............................
EgRO03.............C.....................
EgRH08.............C.....................
EgRO04................C..C...............
EgRO05........T..........................
EgRH09........T..........................
EgRH010...................................
EgRH011...G....T............C.............
EgRH012................C..................
EgRH013......T............................
EgGO01........T............C.G...........
EgUKP01...................................
EgUKP02...................................
Table A5. The segregation sites of 95 sequences of the cox1 gene of E. granulosus s.s. described in countries such as Turkey, Kyrgyzstan, Kurdistan, and China in Asia. The positions of the nucleotides are numbered from the first nucleotide of the gene.
Table A5. The segregation sites of 95 sequences of the cox1 gene of E. granulosus s.s. described in countries such as Turkey, Kyrgyzstan, Kurdistan, and China in Asia. The positions of the nucleotides are numbered from the first nucleotide of the gene.
Haplotypes12911131517192629384244477980899196134146197230249268269280282287291299319329332335344348362365371377400
EG01-JQ250806CTTTTTGTTAAATTCGCATCTCTTCGTATGATGTGGATTATT
EG1A-AF458871..................C.......................
EG1B-AF458872....................................G.....
EG1C-AF458873.......................................G..
EG1D-AF458874.....................T....................
EG2-M84662..............T.T.........C...............
EG3-M84663................T.........C...............
EgTH17..........................................
EgTH18..........................................
EgTH19..........................................
EgTH20.....................T....................
EgTH21..........................................
EgKirH01..........................................
EgKirH02............C.............................
EgKirH03..........................................
EgTO01..........................................
EgTH09..........................................
EgTO02................T.........C...............
EgTH10................T.........C...............
EgTO03...................T......................
EgTH11...................T......................
EgTO04........C.....T...........................
EgTH12........C.....T...........................
EgTO05................T...............A.........
EgTO06................T.........................
EgTH13................T.........................
EgTO07..............T...........................
EgTO08.......................C..................
EgTH14..............T...........................
EgTH15...CAGTA..................................
EgTH16..........................................
EgCHP01................T.........................
EgCHP02...................T......................
EgCHO03..........................................
EgCHO04.............C............................
EgCHO05..............T...........................
EgCHO06..........................................
EgCHO07..........................................
EgCHO08.C..................C...T.................
EgCHO09..........................................
EgCHO10....................C.....................
EgCHO11.................G........................
EgCHO12....................C.....................
EgCHH13..........................................
EgCHH14..........................................
EgCHH15.........................T................
EgCHH16................T..T......C...............
EgCHH17..........................................
EgCHH18..........G...............................
EgCHH19..........................................
EgCHH20..................................A.......
EgCHH21..........................................
EgCHH22..........G..C............................
EgCHH23..............................G...........
EgCHH24.........G................................
EgCHH25..............T...........................
EgCHH26..............T...........................
EgCHH27...........G..T...........................
EgCHH28................T.........................
EgCHH29.........................T.G..............
EgKO01..............T...........................
EgKC02..............T...........................
EgKB03..............T...........................
EgKO04..........................................
EgKC05..........................................
EgKB06..........................................
EgKO07......................C...................
EgKC08......................C...................
EgKB09......................C...................
EgKO10..........................................
EgKC11..........................................
EgKB12..........................................
EgKO14.C............T...........................
EgKO13..........................................
EgKO15..........................................
EgKO16..........................................
EgKO17......................................G.G.
EgKO18................T.........C...............
EgKO19..............T...........................
EgKB20..........................................
EgKB21...................T......................
EgCHH30....................C.....................
EgCHO31....................C.....................
EgCHY32....................C.....................
EgCHH33............C.............................
EgCHO34............C.............................
EgCHY35............C.............................
EgCHH36.............C.......T....................
EgCHO37.............C.......T....................
EgCHY38.............C.......T....................
EgCHH39..............T...........................
EgCHO40..............T...........................
EgCHY41..............T...........................
EgCHH42...................T.........C.CCC.C.C....
EgCHO43...................T.........C.CCC.C.C....
EgCHY44...................T.........C.CCC.C.C....
EgCHH45.....................T......C.............
EgCHO46.....................T......C.............
EgCHY47.....................T......C.............
EgCHH48..................................A.......
EgCHO49..................................A.......
EgCHY50..................................A.......
Table A6. The segregation sites of sequences of the cox1 gene of E. granulosus s.s. described in countries such as Pakistan, Iran, and Saudi Arabia in the Middle East. The positions of the nucleotides are numbered from the first nucleotide of the gene.
Table A6. The segregation sites of sequences of the cox1 gene of E. granulosus s.s. described in countries such as Pakistan, Iran, and Saudi Arabia in the Middle East. The positions of the nucleotides are numbered from the first nucleotide of the gene.
Haplotypes1798096101128131134137197218239306310311320383400
EG01-JQ250806CCGTGGTCGCGGGCTGGT
EG1A-AF458871...C..............
EG1E-AF458875.......T..........
EU178104.T................
EgPaO02.T................
EgPaO01..................
EgPaO03..................
EgIH01.T................
EgIH02.T................
EgIH03..................
EgIH04.T................
EgIO05..................
EgIO06..................
EgIO07.T................
EgIP08.................-
EgIP09.......T.........-
EgIP10.............G...-
EgIH11..................
EgITH12..................
EgASO01..................
Table A7. The segregation sites of sequences of the cox1 gene of E. granulosus s.s. described in Africa and Australia. The positions of the nucleotides are numbered from the first nucleotide of the gene.
Table A7. The segregation sites of sequences of the cox1 gene of E. granulosus s.s. described in Africa and Australia. The positions of the nucleotides are numbered from the first nucleotide of the gene.
Haplotypes1232394663667479808996101116125128131134146149151175182197227245251263269280287311335347348359368371400
EG01-JQ250806CTTAAAGTCGCTGTTGTCTTATGCTTAGGTTTGTATGAT
EG1-M84661..........T..................C........-
EG1A-AF458871...........C...........................
EG1B-AF458872..................................G....
EG1C-AF458873.....................................G.
EG1D-AF458874.......................T...............
EG1E-AF458875.................T.....................
EG2-M84662........T.T..................C.........
EG3-M84663..........T..................C.........
EF393619...............................C.......
EF595654.........................C.............
EU178103.................T.....................
EU178104........T..............................
EgAusD07(2).......................................
EgAusO01................C..A...................
EgAusO02.......................................
EgAO03..........T.........G...A.....C........
EgAusO04....T.....T..................C.........
EgAusP05.......................................
EgAusP06........T.T..................C.........
EgAusD07.......................................
EgAus08........T.T..................C.........
EgAus09.......................................
EgAus10.......................T...............
EgAus11.................T.....................
EgAus12.......................................
EgAus13.....G....T...C..............C.........
EgAus14..........T..................C.........
EgAus15..........T..................C.........
EgAus16.......................................
EgAus17........T..............................
EgAus18.......................................
EgLP01.................T.....................
EgLP02............................T..........
EgLP03.......................................
EgTuB01.....................C.................
EgTuCam02..................C....................
EgTuP03...G...................................
EgTuP04..C....................................
EgTuH05.......................................
EgTuP06.......................................
EgTuB07..........................G............
EgTuP08.......................................
egTuO09........T..............................
EgTuB10........T..............................
EgTuH11........T..............................
EgTuO12........T........................C.....
EgTuP13............A..........................
EgTuP14.............C.........................
EgTuP15...G...................................
EgTuP16.......................................
EgTuP17............A..........................
EgTuH18.......................................
EgTuH19.......................................
EgTuO20.......................................
EgTuH21.......................T...............
EgTuC22........................G..............
EgTuH23.......................................
EgTuB24.......................................
EgTuB25..........T.....................A......
EgTuC26................................A......
EgTuB27................................A......
EgTuO28.......................................
EgTuO29.......................................
EgTuB30.......................................
EgTuC31.......................................
EgTuP32.............C.........................
EgTuC33.......................................
EgTuC34.C.....................................
EgKP01.......................................

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Parasitologia 05 00040 g001
Figure 1. Geographical origin and number of hosts of Echinococcus granulosus sensu stricto used in this study.
Figure 1. Geographical origin and number of hosts of Echinococcus granulosus sensu stricto used in this study.
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Figure 2. Phylogenetic tree of Echinococcus granulosus s.l., using 450 bp sequences of the cox1 gene, isolated from 46 hydatid cysts from different hosts in three livestock regions of Chile. The tree was constructed with the maximum likelihood (ML) method using the Kimura 2 Parameters (K2P) evolutionary method. Taenia crassiceps and Taenia hydatigena were used as outgroups. (The meaning of each abbreviation in the phylogenetic tree is as follows: Eg: Echinococcus granulosus, CL: Chile, O: sheep, B: cattle, C: goat, M: Magallanes, A: Araucanía, Co: Coquimbo.)
Figure 2. Phylogenetic tree of Echinococcus granulosus s.l., using 450 bp sequences of the cox1 gene, isolated from 46 hydatid cysts from different hosts in three livestock regions of Chile. The tree was constructed with the maximum likelihood (ML) method using the Kimura 2 Parameters (K2P) evolutionary method. Taenia crassiceps and Taenia hydatigena were used as outgroups. (The meaning of each abbreviation in the phylogenetic tree is as follows: Eg: Echinococcus granulosus, CL: Chile, O: sheep, B: cattle, C: goat, M: Magallanes, A: Araucanía, Co: Coquimbo.)
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Figure 3. (A) The haplotype network built with the sequences of the cox1 gene from the 46 samples of E. granulosus s.s. analyzed in this study. (B) The haplotypes, haplotypic diversity indexes, and neutrality indexes for the 46 samples of E. granulosus s.s. analyzed. Tajima’s D and Fst not significant * p > 0.10.
Figure 3. (A) The haplotype network built with the sequences of the cox1 gene from the 46 samples of E. granulosus s.s. analyzed in this study. (B) The haplotypes, haplotypic diversity indexes, and neutrality indexes for the 46 samples of E. granulosus s.s. analyzed. Tajima’s D and Fst not significant * p > 0.10.
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Parasitologia 05 00040 g004
Figure 4. The haplotype network constructed with the cox1 gene sequences of the 46 samples of E. granulosus (s.s.) analyzed in this study. (A) A total of 39 sequences described for Chile and 66 sequences described in Latin America (Peru, Bolivia, Argentina); (B) Europe (Austria, Belgium, Hungary, Italy, Portugal, Romania, Greece, UK); (C) Asia (Turkey, China, Kurdistan, Kyrgyzstan); (D) the Middle East (Pakistan, Iran, Saudi Arabia); (E) Africa (Libya, Tunisia, Kenya); (F) Australia. (G) The haplotypes, haplotype diversity indexes, and neutrality indexes are shown for each continent and for Chile.
Figure 4. The haplotype network constructed with the cox1 gene sequences of the 46 samples of E. granulosus (s.s.) analyzed in this study. (A) A total of 39 sequences described for Chile and 66 sequences described in Latin America (Peru, Bolivia, Argentina); (B) Europe (Austria, Belgium, Hungary, Italy, Portugal, Romania, Greece, UK); (C) Asia (Turkey, China, Kurdistan, Kyrgyzstan); (D) the Middle East (Pakistan, Iran, Saudi Arabia); (E) Africa (Libya, Tunisia, Kenya); (F) Australia. (G) The haplotypes, haplotype diversity indexes, and neutrality indexes are shown for each continent and for Chile.
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Table 1. Origin and number of samples analyzed in this research.
Table 1. Origin and number of samples analyzed in this research.
RegionCity/LocalityNumber of IsolatesHost Speccies (Number of Isolates)
CoquimboOvalle. Peña Blanca. Monte Patria. Illapel6goats (1). sheep (1). cattle (3). humans (1)
La AraucaníaSanta Cruz. Nueva Imperial. San José. Cunco. Los Maitenes. Puyehue. El Carmen. Carahue18cattle (18)
MagallanesMagallanes22sheep (22)
Total 46goats (1). sheep (23). cattle (21). humans (1)
Table 2. Genetic statistics for the analyzed populations of Echinococcus granulosus s.s.
Table 2. Genetic statistics for the analyzed populations of Echinococcus granulosus s.s.
RegionNSKHπTajima’s DpFu’s FSp
Coquimbo6120.333 ± 0.046300.00084−0.93302 *0.061−0.003 *0.502
La Araucanía17120.425 ± 0.4250.001070.8695 *0.7801.0390.801
Magallanes22230.481 ± 0.008750.00128−0.17406 *0.386−0.121 *0.414
Total45340.461 ± 0.006370.00141−0.52134 *0.326−0.856 *0.279
N: number of samples, K: number of haplotypes, S: number os segregating sites, Hd: haplotypic diversity, π: nucleotide diversity, Fu’s Fs: population fixation index, Significance * p > 0.10.
Table 3. The Fisher (lower triangle) and Snn (upper triangle) fixation indexes of Echinococcus granulosus s.s. in the three regions analyzed.
Table 3. The Fisher (lower triangle) and Snn (upper triangle) fixation indexes of Echinococcus granulosus s.s. in the three regions analyzed.
CoquimboLa AraucaníaMagallanes
Coquimbo-----------0.598090.6823
La Araucanía−0.08761----------0.62728 **
Magallanes0.033410.10703 **--------
** p < 0.05.
Table 4. AMOVA analysis of the cox1 gene sequences of the three Echinococcus granulosus s.s. populations analyzed.
Table 4. AMOVA analysis of the cox1 gene sequences of the three Echinococcus granulosus s.s. populations analyzed.
Source of
Variation		D.f.Sum of the SquaresComponents of
Variance		Percentage of Variation
Between
populations		23.2850.06220.340
Within the
populations		4310.2260.24379.659
Total4513.5110.306
p-value 0.00587 ± 0.00219.
Table 5. The diversity and neutrality indexes for the different populations analyzed.
Table 5. The diversity and neutrality indexes for the different populations analyzed.
PopulationNumber of Sequences AnalyzedDiversity Index Neutrality Index
Hn hd ± SD nd ± SD Tajima’s DpFu’s FSp
Chile (this study) 4640.461 ± 0.006370.00181 ± 0.00036−0.541230.326−0.8560.279
Chile8590.542 ± 0.0590.00461 ± 0.00203−2.36563 **0.000−2.0830.201
Argentina30130.869 ± 0.0420.0.00458 ± 0.00063−1.71486 **0.010−8.639 **0.000
Peru2790.561 ± 0.1140.00251 ± 0.00073−1.99558 **0.002−6.421 **0.000
Bolivia930.417 ± 0.1910.00121 ± 0.00060−1.36240 **0.016−1.081 **0.022
Austria1060.889 ± 0.0750.00766 ± 0.00161−0.708430.263−1.1020.205
Bulgaria960.917 ± 0.0730.00607 ± 0.001040.624960.752−2.0340.052
Hungary860.893 ± 0.1110.00411 ± 0.00095−117,5320.111−3.589 **0.000
Italy1040.733 ± 0.1200.00428 ± 0.001020.203500.652−0.0450.482
Portugal1130.345 ± 0.1720.00146 ± 0.00078−0.778150.211−0.6590.126
Romania1370.897 ± 0.0540.00525 ± 0.00100−0.774850.215−2.6400.015
UK210.000000.00000------------------------
Palestine310.000000.00000------------------------
Iran1240.682 ± 0.1020.00224 ± 0.00050−0.578640.285−1.0480.104
Kiyrgyzstan320.667 ± 0.3140.00182 ± 0.00086-----------------------
Türkiye2150.424 ± 0.1310.00221 ± 0.00077−165,3580.176−3.127 **0.004
China50210.935 ± 0.0170.00729 ± 0.00106−1.63535 **0.032−12.892 **0.000
Kurdistan2160.724 ± 0.0780.00304 ± 0.00065−1.38535 **0.040−1.852 **0.017
Australia19100.825 ± 0.0840.00623 ± 0.00112−142,2040.065−4.186 **0.008
Libya331.00 ± 0.2720.00364 ± 0.00121------------------------
Tunisia1450.800 ± 0.0680.00326 ± 0.00049−2.01165 **0.001−12.237 **0.000
Latin America151190.578 ± 0.002060.000409 ± 0.00371−2.39076 **0.000−13.852 **0.000
Europe61190.832 ± 0.0410.00493 ± 0.00357−1.52138 **0.036−13.824 **0.142
Middle East1640.617 ± 0.009270.00204 ± 0.00168−0.627710.268−1.0190.000
Asia95280.880 ± 0.000650.00581 ± 0.00477−1.95889 **0.003−23.448 **0.000
Africa38170.819 ± 0.003820.00307 ± 0.00358−2.19849 **0.002−17.437 **0.000
total380260.36539 ± 0.0310.00321 ± 0.00054−2.42296 **0.000−35.985 **0.000
N: number of sequences analyzed, K: number of haplotypes, Hd: haplotypic diversity, π: nucleotide diversity, Fu’s FS: Population fixation index, ** p < 0.05.
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Urriola-Urriola, N.; Rossi-Vargas, G.; Nilo-Bustios, Y. Haplotypes of Echinococcus granulosus sensu stricto in Chile and Their Comparison Through Sequences of the Mitochondrial cox1 Gene with Haplotypes from South America and Other Continents. Parasitologia 2025, 5, 40. https://doi.org/10.3390/parasitologia5030040

AMA Style

Urriola-Urriola N, Rossi-Vargas G, Nilo-Bustios Y. Haplotypes of Echinococcus granulosus sensu stricto in Chile and Their Comparison Through Sequences of the Mitochondrial cox1 Gene with Haplotypes from South America and Other Continents. Parasitologia. 2025; 5(3):40. https://doi.org/10.3390/parasitologia5030040

Chicago/Turabian Style

Urriola-Urriola, Nicole, Gabriela Rossi-Vargas, and Yenny Nilo-Bustios. 2025. "Haplotypes of Echinococcus granulosus sensu stricto in Chile and Their Comparison Through Sequences of the Mitochondrial cox1 Gene with Haplotypes from South America and Other Continents" Parasitologia 5, no. 3: 40. https://doi.org/10.3390/parasitologia5030040

APA Style

Urriola-Urriola, N., Rossi-Vargas, G., & Nilo-Bustios, Y. (2025). Haplotypes of Echinococcus granulosus sensu stricto in Chile and Their Comparison Through Sequences of the Mitochondrial cox1 Gene with Haplotypes from South America and Other Continents. Parasitologia, 5(3), 40. https://doi.org/10.3390/parasitologia5030040

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