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. 2020 Apr 1;147(9):949–956. doi: 10.1017/S0031182020000529

Echinococcus granulosus sensu stricto, Echinococcus ortleppi; and E. intermedius (G7) are present in Bolivia

V Ali 1, E Martinez 1, P Duran 1, M A Seláez 2, M Barragan 2, P Nogales 3, A Peña y Lillo 4, M Castañares 5, Y Claros 6, P Deplazes 7, C A Alvarez Rojas 7,✉
PMCID: PMC10317627  PMID: 32234095

Abstract

Cystic echinococcosis (CE) is a zoonotic disease caused by a complex of species known as Echinococcus granulosus sensu lato. CE is endemic in Argentina, Chile, Peru, Uruguay and the South part of Brazil. In contrast, little is known regarding the presence of CE in Bolivia. In this study, 35 cysts isolated from livestock (mostly from the Department of La Paz) and 3 from humans (La Paz, Oruro and Potosi) were genetically characterized analysing the sequence of the cox1 gene (1609 bp). In total, 30 cysts (from La Paz, Cochabamba and Beni) were characterized as E. granulosus sensu stricto (3 fertile and 4 non-fertile cysts from sheep, 8 fertile and 12 non-fertile cysts from cattle and 3 fertile cysts from humans). A detailed analysis of the cox1 haplotypes of E. granulosus s.s. is included. Echinococcus ortleppi (G5) was found in 5 fertile cysts from cattle (from La Paz and Cochabamba). Echinococcus intermedius (G7) was identified in 3 fertile cysts from pigs (from Santa Cruz). Additionally, E. granulosus s.s. was detected in 4 dog faecal samples, while E. ortleppi was present in other two dog faecal samples. The implications of these preliminary results in the future implementation of control measures are discussed.

Key words: Bolivia, cystic echinococcosis, Echinococcus granulosus, molecular epidemiology, haplotypes, genotypes

Introduction

Echinococcus granulosus sensu lato (s.l.) comprises a number of species which are responsible for the zoonotic disease called cystic echinococcosis (CE) (Eckert and Deplazes, 2004). From these species, E. granulosus sensu stricto (s.s.) (including the genotypes G1, G3 and their micro variants) is the most commonly found species infecting humans (Alvarez Rojas et al., 2014) and occurs in a wide spectrum of intermediate hosts worldwide (Cardona and Carmena, 2013). Echinococcus ortleppi (G5 genotype) circulates between cattle and dogs (Romig et al., 2017), although is also able to infect humans is far less relevant than other species for human infection. The genotypes G6/7/8/10 have been conventionally clustered as E. canadensis (Nakao et al., 2006). However, this nomenclature remains controversial (Nakao et al., 2015) and some authors subdivide this species grouping the genotypes G6/G7 as E. intermedius, the genotype G8 as E. borealis and G10 as E. canadensis (Lymbery et al., 2015). In this paper, we use the term E. intermedius to refer to the G6/7 group. The cluster (G6/7) is the second most common cause for CE in humans worldwide. Echinococcus intermedius (G6) infects mostly camels and goats with human CE, caused by this species, occurring mainly in Argentina and Africa. Echinococcus intermedius (G7) uses primarily pigs as intermediate host with high incidences in humans especially in the Baltic countries (Romig et al., 2017).

It is well documented that CE is highly endemic in South American countries including Argentina, Brazil (south), Chile, Peru and Uruguay causing serious public health issues (Moro and Schantz, 2006; Pavletic et al., 2017). In contrast, the evidence regarding the situation of CE in Bolivia is scarce and limited to the understanding that the parasite is present and causing an unknown number of cases (Schantz, 1972; Moro and Schantz, 2006). The distribution of species comprising the E. granulosus s.l. complex in the endemic countries in South America has been investigated mostly with samples from the intermediate hosts. It is known that E. granulosus s.s. is present in all the endemic South American countries; E. ortleppi has been described in Argentina, Brazil, Chile and Uruguay; E. intermedius (G6) has been found in Argentina, Chile and Peru. And E. intermedius (G7) has been isolated in Argentina, Brazil and Peru (Rosenzvit et al., 1999; Kamenetzky et al., 2000; Kamenetzky et al., 2002; Guarnera et al., 2004; Badaraco et al., 2008; Manterola et al., 2008; Santivañez et al., 2008; Moro et al., 2009; Sánchez et al., 2010; Schneider et al., 2010; Soriano et al., 2010; de la Rue et al., 2011; Balbinotti et al., 2012; Sánchez et al., 2012; Armua-Fernandez et al., 2014; Espinoza et al., 2014; Monteiro et al., 2014; Cucher et al., 2016; Alvarez Rojas et al., 2017; Deplazes et al., 2017; Corrêa et al., 2018) and more recently, for the first time in Ecuador (Ramos-Sarmiento and Chiluisa-Utreras, 2020). In the case of Bolivia, this data is limited to two human cases reported as E. granulosus s.s. (Kamenetzky et al., 2002; Jarovsky et al., 2020).

Bolivia shares borders with all endemic countries in South America except for Uruguay. From all Bolivian borders, the ones with Argentina (773 km) and Peru (1000 km) are the most relevant for the proximity with areas where CE is endemic, like Salta and Jujuy in Argentina (Casas et al., 2013; Salud, 2019) and Puno in Peru (Cabrera, 2007). For this study, we have collected cyst parasite material isolated from cattle, sheep, pigs and humans. We also analysed dog faecal samples infected with E. granulosus s.l. from Bolivia. Most of the samples were originated in La Paz Department. We present here the genetic characterization of such samples as a first step to produce data which can contribute to a better understanding of the distribution of the species comprising E. granulosus s.l. present in Bolivia.

Material and methods

Parasite material

A total of 35 cysts, identified as the larval stage of E. granulosus s.l., were collected from livestock including 7 cysts from sheep, 3 cysts from pigs and 25 cysts from cattle. Parasite material was collected from established abattoirs with veterinary inspection, except for sheep samples which were collected directly in an ‘open-air’ indigenous abattoir with no veterinary inspection. Additionally, three cysts from CE human patients were also included in the study. Microscopic analysis of cyst fluid was performed for discrimination of fertile and infertile cysts based on the presence or absence of protoscoleces. Furthermore, six dog faecal samples positive for E. granulosus s.l. were also included in this study. These dog faecal samples were identified from a parallel study from which 14 out of 131 samples were found to contain Taenia spp. eggs at microscopic examination (samples were fixed with formalin 10%). The information of the species, organ and fertility (for cysts) and collection place of all the samples used in this study can be seen in Table 1. A more detailed geographic origin of the samples isolated within La Paz Department including cysts isolated from 2 sheep, 18 cattle and all dog faecal samples can be seen in the map in Supplementary Fig. 1. Samples from two other cattle were originated in Punata, in the Cochabamba Department and 3 cysts from 2 pigs were originated in Santa Cruz Department (Table 1). Human cysts were collected after surgery from hospitals in Oruro, La Paz and Potosi and sent to the laboratory in La Paz. Ethics clearance for the use of human samples was provided by the Universidad Mayor de San Andres, La Paz, Bolivia.

Table 1.

Origin, host species and fertility of Echinococcus cysts and faecal dog samples from Bolivia used for the genetic analysis in this study with the corresponding genotype and cox1 haplotype

Sample number Host species/Organ Fertility Community/Municipality/Department Species/Genotype (G)/Haplotype
1 Human/liver Yes ND/ND/Oruro E. granulosus s.s./G1/Eg01
2 Human/abdominal cavity Yes ND/ND/La Paz E. granulosus s.s./G1/Eg01
3 Human/lungs Yes ND/ND/Potosi E. granulosus s.s./G1/Eg01
4 Sheep/lungs Yes Batallas/Batallas/La Paz E. granulosus s.s./G1/Eg01
5 No E. granulosus s.s./G1/Eg01
6 Yes E. granulosus s.s./G1/Eg01
7 No E. granulosus s.s./G1/Eg01
8 No E. granulosus s.s./G1/Eg01
9 Yes E. granulosus s.s./G1/Eg01
10 Sheep/lungs No Batallas/Batallas/La Paz E. granulosus s.s./G1/Eg01
11 Cattle/lungs No Illimani/Palca/La Paz E. granulosus s.s./G1/Eg01
12 Cattle/lungs Yes ND/Mecapaca//La Paz E. granulosus s.s./G1/Eg01
13 Cattle/lungs Yes ND/Viacha/La Paz E. granulosus s.s./G1/Eg01
14 Cattle/lungs No Batallas/Batallas/La Paz E. granulosus s.s./G1/Eg01
15 Cattle/lungs No Pocoata/Viacha/La Paz E. granulosus s.s./G1/Eg01
16 Cattle/lungs No ND/Patacamaya/La Paz E. granulosus s.s./G1/Eg01
17 Cattle/lungs Yes Lahuachaca/Sica Sica/La Paz E. granulosus s.s./G1/Eg01
18 Cattle/lungs No ND/Sica Sica/La Paz E. granulosus s.s./G1/Eg01
19 Cattle/lungs No ND/Mecapaca/La Paz E. granulosus s.s./G1/Eg01
20 Cattle/lungs No Chicani/La Paz/La Paz
 E. granulosus s.s./G1/Eg01
21 Cattle/liver No E. granulosus s.s./G1/EgBol1
22 Cattle/lungs Yes ND/ND/La Paz E. granulosus s.s./G1/EgBol2
23 Yes E. granulosus s.s./G1/EgBol2
24 Yes E. granulosus s.s./G1/EgBol3
25 Cattle/liver No ND/Punata/Cochabamba E. granulosus s.s./G1/EgBol4
26 Cattle/lungs No E. granulosus s.s./G1/EgBol4
27 Cattle/lungs No ND/Batallas/La Paz E. granulosus s.s./G1/EgBol5
28 Cattle/lungs Yes ND/ND/Beni E. granulosus s.s./not G1 G3/EgBol6
29 Cattle/lungs Yes Villa Remedios/Viacha/La Paz E. granulosus s.s./G1/EgBol7
30 Cattle/lungs Yes Villa Remedios/Viacha/La Paz E. granulosus s.s./not G1 G3/EgCL03
31 Cattle/lungs Yes ND/Punata/Cochabamba E. ortleppi/G5/Eo12
32 Cattle/lungs Yes ND/ND/La Paz E. ortleppi/G5/Eo12
33 Cattle/lungs Yes ND/Punata/Cochabamba E. ortleppi/G5/Eo12
34 Yes E. ortleppi/G5/Eo12
35 Cattle/lungs Yes Pocoata/Viacha/La Paz E. ortleppi/G5/Eo12
36 Pig/liver Yes ND/ND/Santa Cruz E. intermedius/G7/EcPer1
37 Pig/lungs Yes E. intermedius/G7/EcPer1
38 Pig/kidney No ND/ND/Santa Cruz E. intermedius/G7/EcPer1
39 Feces – Batallas/Batallas/La Paz E. granulosus s.s.
40 Feces – Batallas/Batallas/La Paz E. ortleppi
41 Feces – Pucarani/Pucarani/La Paz E. granulosus s.s.
42 Feces – Lacaya Baja/Pucarani/La Paz E. granulosus s.s.
43 Feces – Tiahuanacu/Tiahuanacu/La Paz E. granulosus s.s.
44 Feces – Tiahuanacu/Tiahuanacu/La Paz E. ortleppi
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ND, no data.

Molecular analysis

Genomic DNA was isolated from the germinal layer of each cyst using the DNA mini kit (Qiagen). DNA was used as a template for polymerase chain reaction (PCR) to amplify and sequence 1609 bp of the cox1 gene of E. granulosus s.l. using the primers previously described (Hüttner et al., 2008). If no PCR product was amplified, then two overlapping fragments were produced by PCR (5′ and 3′ fragments) and sequenced (Alvarez Rojas et al., 2016). PCR products were visualized in a 2% agarose gel stained with GelRed and purified using the MinElute PCR purification kit (Qiagen) for sequencing in both directions using Sanger technology (Microsynth, Switzerland). The final sequence of the cox1 gene (1609 bp) from each cyst was built with the software Geneious (Kearse et al., 2012) using the cox1 haplotype EG01 (accession number: JQ250806) as reference. Sequencing chromatograms were thoroughly checked, only sequences with single peaks were used for subsequent analysis. The identification of genotypes (G-system) was performed comparing the cox1 gene from each isolate with the original reference sequences (366 bp) for E. granulosus s.s. G3 (M84663), E. equinus G4 (M84664), E. ortleppi G5 (M84665), E. intermedius G6 (M84666) and E. intermedius G7 (M84667) from Bowles et al. (1992). The original reference sequence uploaded to GenBank for E. granulosus s.s. G1 (M84661) is incorrect as this is identical to G3 (M84663). The sequence M84661 was manually corrected following Fig. 1 from the original publication and used as a reference for E. granulosus s.s. G1 (Bowles et al., 1992). The identification of new cox1 haplotypes (1609 bp) and haplotype network design was accomplished comparing data produced in this study with all same length cox1 sequences of E. granulosus s.l. deposited in GenBank using TCS and PopArt (Clement et al., 2000; Leigh and Bryant, 2015). For the 14 dog faecal samples microscopically positive for taeniid eggs, DNA was isolated using the alkaline lysis method as previously described (Štefanić et al., 2004) and further used as a template for multiplex PCR and sequencing to discriminate between E. granulosus s.l. (band of 117 bp) and other cestodes comprising Taenia spp. (267 bp) (Trachsel et al., 2007).

Fig. 1.

Fig. 1.

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Distribution of E. granulosus s.s., E. ortleppi and E. intermedius (G7) and host species infected in different departments in Bolivia according to the data presented in this study.

Results

Echinococcus granulosus s.s. was identified in all 7 cysts from sheep (from La Paz), also in 20 out of 25 cysts from cattle (17 from La Paz, 2 from Cochabamba and 1 from Beni) and in all 3 cysts from humans (from Oruro, La Paz and Potosi) (Table 1). When assigning genotypes (G-system) to these sequences, based on 366 bp of the cox1 gene, 28 out of the 30 samples identified as E. granulosus s.s. show 100% identity with the 366 bp of the original reference sequence for G1 and two other differ by a single nucleotide with either the genotypes G1 or G3 (Table 1). Echinococcus ortleppi (G5) was identified in the remainder 5 cysts from cattle (3 cysts were collected in La Paz and 2 others in Cochabamba) (Table 1). Echinococcus intermedius (G7) was identified in the three cysts available from pigs collected in Santa Cruz (Table 1). The presence of protoscoleces in samples identified as E. granulosus s.s. was reported in 3 out of 3 human samples, in 3 out 7 cysts (43%) from sheep and in 8 out of 20 cysts (40%) from cattle. Protoscoleces were observed in all five samples identified as E. ortleppi and in all the three samples identified as E. intermedius (G7). From the 6 dog faecal samples used in this study, E. granulosus s.s. was identified in 4 samples (1 from Batallas, 2 from Pucarani and 1 from Tiahuanacu) and E. ortleppi in 2 dogs (1 from Batallas and 1 from Tiahuanacu) (Supplementary Fig. 1). It was not possible to amplify the full length of the cox1 gene of E. granulosus s.l. in the dog faecal samples due to the use of formalin as a fixative. Taenia hydatigena was found in five dog faecal samples and Taenia pisiformis in two cases (not shown). One dog faecal sample showed a mixed infection with E. granulosus s.s. and T. hydatigena while another faecal sample showed a co-infection with E. ortleppi and T. hydatigena (not shown). A schematic representation of the species of E. granulosus s.l. found in different departments of Bolivia in this study is shown in Fig. 1.

From the 30 isolates identified as E. granulosus s.s. in the cyst samples, 20 of them were recognized as the cox1 haplotype Eg01 (Accession number JQ250806) (Table 1). The cox1 haplotype EgCL03 (KX227118) (Alvarez Rojas et al., 2017) was identified in a single sequence in this study (Table 1). The remainder 9 sequences identified as E. granulosus s.s. do not share 100% homology with any sequence deposited in GenBank and they are named here as EgBol1-EgBol7 (Accession numbers: MT072973-MT072979) (Table 1). Two cattle presented mixed infection with two different cox1 haplotypes of E. granulosus s.s (Table 1). The isolates identified as E. ortleppi in 5 cysts from cattle had an identical sequence, this sequence is 100% homologous with the cox1 haplotype Eo12 (KU743926) originally identified in Namibia (Addy et al., 2017). The isolates identified as E. intermedius (G7) have the same sequence sharing 100% homology with the cox1 haplotype EcPer1 (AB777924) identified in Peru and also in the sequence of the complete mitochondrial genomes of E. intermedius (G7) from Poland, France, Spain, Romania and Mexico (Laurimäe et al., 2018).

The haplotype network for E. granulosus s.s. built with data from Bolivia from the present study and also cox1 sequences from Chile, Peru, Argentina and Brazil is shown in Fig. 2. The network has the typical star-like shape with the cox1 haplotype Eg01 in the centre. This haplotype is present in 20 sequences from Bolivia (this study), 33 from Chile, 16 from Peru, 10 from Argentina and 6 from Brazil. Other cox1 haplotypes found in more than one country in South America included EgCL03 (in Chile, Argentina and Bolivia), EgCL04 (in Chile and Argentina) and the haplotype ARG12 (in Argentina and Brazil). A total of 27 cox1 haplotypes in Fig. 2, including the ‘new’ haplotypes from Bolivia, differ only in a single nucleotide with the sequence of Eg01.

Fig. 2.

Fig. 2.

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Haplotype network built with 1609 bp of the cox1 gene of E. granulosus s.s. isolated in Bolivia in this study (red circles) (MT072973-MT072979) together with similar data from Chile: haplotypes EgCL1-21 (Alvarez Rojas et al., 2017), EgCL22-27 (Hidalgo et al., 2019). Haplotypes found in Chile (Alvarez Rojas et al., 2017) but first described in Australia: EgAus02 (KT968703) and EgAus03 (KT968704) (Alvarez Rojas et al., 2016); Russia: EgRUS7 (AB777904) (Konyaev et al., 2013) and Nepal EgP1 (AB522646). From Peru: haplotypes Eg43 (AB688620) and Eg44 (AB688621) (Yanagida et al., 2012). Also included in the network is the sequence of the cox1 gene from isolates from Argentina: ARG1-ARG17 (KX039937- KX039953), Chile: CHI1-CHI4 (KX039961-KX039964), Brazil: BRA1-BRA6 (KX039955- KX039960) and haplotype AB1 (KX039954) (Laurimäe et al., 2016). Eg01 includes the cox1 sequence from ARG1, ARG3-10, ARG17, BRA1 and CHI2 (Laurimäe et al., 2016). EGCL04 is 100% homologous to ARG13, ARG12 = BRA3, EgCL01 = CHI1, EgCL02 = CHI3. The size of the circles is proportional to the number of sequences in which each haplotype has been identified, the Eg01 haplotype includes 30 sequences from Bolivia (this study), 33 from Chile (Alvarez Rojas et al., 2017), 16 from Peru (Yanagida et al., 2012), 10 from Argentina and 1 from Brazil (Laurimäe et al., 2016).

Discussion

Data from the present study show that at least three species of the E. granulosus s.l. complex are circulating in Bolivia. The cosmopolitan species E. granulosus s.s. was found in the majority of the isolates examined, including the human samples. Interestingly, a similar fertility rate was observed in E. granulosus s.s. cysts isolated from sheep and cattle (around 40%). It is usually accepted that fertility of E. granulosus s.s. cysts is much higher in sheep than in cattle (Thompson, 2017). The relatively high fertility of cysts from cattle in this study suggests that this species could play an important role in the transmission of the parasite in Bolivia. In this study cysts from only 2 sheep were included. The sheep population in Bolivia reaches 7 416 185 animals (2017) from which a high proportion is slaughtered at home compared to cattle. In the last decades, the sheep population in the Altiplano has decreased due to high infection with Fasciola hepatica (Plata, 2005). The finding of E. ortleppi and E. intermedius (G7) in Bolivia extends the knowledge of the distribution of these parasites in South America (Fig. 2). It is known that both species can also cause infection in humans (Alvarez Rojas et al., 2014). Echinococcus ortleppi was found in cattle from two different areas of Bolivia (La Paz and Cochabamba), which are located approximately 380 km from each other. This suggests that E. ortleppi could, potentially, be widely spread in Bolivia. It is well known that E. ortleppi produce fertile cysts in cattle unlike the low fertility observed when E. granulosus s.s infects cattle (Thompson et al., 1984; Balbinotti et al., 2012), all the cysts identified as E. ortleppi in this study were fertile (Table 1). The total number of cattle in Bolivia is estimated to be 9 304 572 animals, from which 4 010 258 are located in Santa Cruz. Cattle population in La Paz reaches 548 841 animals, most of them in the Altiplano, north of La Paz (MDR&T, 2017). Interestingly, E. intermedius (G7) was found in all three fertile cysts available from pigs in this study. The isolates were collected in Santa Cruz and all three cysts produced the same cox1 sequence which shares 100% homology with G7 isolates previously found in Peru and also in different European countries. The total number of pigs in Bolivia (in 2014) has been estimated to be 2 941 827 from which 892 232 are in Santa Cruz (MDR&T, 2014). An important number of pigs in Bolivia are raised outdoors with no veterinary inspection (Callisaya, 2018). Most likely these pigs are slaughtered at home continue facilitating the transmission of the parasite. Further investigations of population-based prevalences have to be established to estimate the relative contribution of the different intermediate hosts to the transmission.

Echinococcus granulosus s.s. was also found in 4 faecal dog samples (Batallas, Pucarani and Tiahuanacu) while E. ortleppi was present in 2 samples (Batallas and Tiahuanacu) demonstrating active transmission of both species in La Paz Department. The presence of T. hydatigena in 5 dog faecal samples (together with the finding of E. granulosus s.l.) described in this study shows that some dogs are actively been fed or have access to livestock offal in La Paz Department. Dog samples included in this study were taken from a parallel study investigating intestinal parasites from 131 samples in the La Paz department. Although this is not intended to be a prevalence study, it is possible to say that at least 4.5% (6 out of 131) of the dogs sampled were infected with E. granulosus s.l. This number could have been potentially higher if a more sensitive method for isolation/concentration of taeniid eggs would have been used, for example, the flotation and sieving method widely used in previous studies (Mathis et al., 1996; Trachsel et al., 2007). Previous reports of the prevalence of E. granulosus s.l. in dogs from Bolivia are rather limited. These include a vague report of 50% of infection in dogs near La Paz after arecoline purgation in the 1970's (Moro and Schantz, 2006); a report of 23.9% of positive dogs using copro-ELISA in Tupiza (Potosi) in 2011 (Villena, 2011); and the most recent report of prevalence indicating values of between 3.4 and 30% using copro-ELISA and copro-Western blot, in border towns with Argentina (Casas et al., 2013). Future studies of the prevalence of E. granulosus s.l. in dogs from Bolivia are urgently needed to have a better understanding of the epidemiology of the parasite in this country.

The analysis of 1609 bp of the cox1 gene from the cysts from this study identified a vast majority as the haplotype Eg01. Two animals showed infection with more than one haplotype as it has been previously described (Hidalgo et al., 2019). It is important to mention that the biological significance of the different haplotypes of E. granulosus s.s. remains unknown and no data is available concerning differences in virulence or host/parasite interaction or antigenicity which could affect the response to vaccines or diagnostic methods. In the meantime the description of new cox1 haplotypes of E. granulosus s.s. worldwide contributes to the general knowledge of how the parasite spread worldwide. The haplotype EG01 was originally described by Yanagida et al. (2012) as the likely founder population of E. granulosus s.s. which was disseminated from the Middle East with the export of livestock after their domestication. It is usually located at the centre of haplotype networks in different countries where similar studies have been performed including Iran, Jordan, Peru, Russia, Chile and Australia (Yanagida et al., 2012; Konyaev et al., 2013; Romig et al., 2015; Alvarez Rojas et al., 2016; Alvarez Rojas et al., 2017). The haplotype network built with the cox1 sequences from E. granulosus s.s. from this study and the ones available in GenBank from South American countries shows that 27 haplotypes differ by a single nucleotide with the central haplotype Eg01 (Fig. 2). Although, there is no evidence that the population of E. granulosus s.s. differ significantly between South American countries, it is obvious that most of the cox1 haplotypes are uniquely identified with particular countries. Since the Eg01 cox1 haplotype remains as the most commonly found in South America, we speculate here that it could have been the most common variant of the parasite infecting livestock and also dogs imported into the Americas with the Spanish colonization more than 500 years ago. The parasite and especially the one belonging to the Eg01 haplotype adapted to a variety of climates in South America and random mutations accumulated over time (in the cox1 and other genes) which now we identify as different haplotypes of the parasite. Livestock (including cattle, sheep and pigs) first arrived in La Española Island (currently in the Dominican Republic) 1 year after the arrival of Columbus. Later, livestock also arrived in Brazil with the Portuguese colonization (Primo, 2004) and also directly to Uruguay and Argentina in subsequent trips from Spain (Beteta Ortiz, 1999). After the colonization, most livestock was concentrated in what is currently known as Panama, from where it was distributed south to Venezuela, Colombia, Ecuador and Peru (Primo, 1992; Villalobos Cortés et al., 2009). After conquering the Inca Empire, livestock was mostly concentrated in Peru from where it was distributed to the territories known now as Bolivia, Chile and Argentina. Since the discovery of silver in Potosi (Bolivia) this city was known to be a hub for livestock some of which was brought to the Americas from Argentina and Uruguay (Escobari de Querejazu, 1985). Also, La Paz was a connecting point between the commercial routes that led from Potosí and Oruro to Lima. These events show that the Bolivian territory was an important hub for livestock distribution suggesting that the parasite has been present in Bolivia for centuries and it has been mostly neglected in this country.

To conclude, the finding of three different species of E. granulosus s.l. have important implications in the design and implementation of control measures. For example, it is known that the improved general slaughtering hygiene without any specific control measure per se has reduced the presence of E. ortleppi (G5) in parts of Central and Western Europe (Deplazes et al., 2017). Recently, a pilot control programme in Lithuania, based on the deworming of dogs in specific times before the slaughtering of pigs, has achieved a strong reduction of E. intermedius (G7) (Šarkūnas et al., 2019). However, in areas with extensive sheep production, it is known that further measures have to be applied to control E. granulosus s.s. (e.g. registration and regular deworming of dogs, public health education, construction of elimination ditches for the correct disposal of offal, etc.) (Craig et al., 2017). Also, it remains unclear if the EG95 vaccine which prevents the infection in livestock, based on E. granulosus s.s., is effective to prevent the infection with different species of E. granulosus s.l. For example, it has been suggested that differences in the antigenicity of the EG95 proteins from E. intermedius G6 could make the current vaccine less effective against this species (Alvarez Rojas et al., 2013). Therefore, the use of the EG95 vaccine, based in E. granulosus s.s., in a control programme where multiple species occur, as is the case of Bolivia, would need to be thoroughly monitored. Data reported here represents the first step to understand the epidemiological situation of CE in Bolivia. However, a systematic sample collection of parasite material in different regions from a diverse number of intermediate hosts and humans is needed to understand the real distribution of different species of E. granulosus s.l. in Bolivia.

Acknowledgements

The project ‘Cystic echinococcosis in Bolivia’ is a joint research between the University of Zurich and the Universidad Mayor San Andres in Bolivia. A mobility grant of the Leading House of the Latin American Region ‘Epidemiological investigation of cystic echinococcosis in humans and animals in Bolivia’ is directly linked to this project and has been financed by the Leading House of the Latin American Region/State Secretariat for Education, Research and Innovation (SERI). We acknowledge the authorities of the Faculty of Medicine, Universidad Mayor de San Andrés, for administrative collaboration for this joint initiative; to the Arq Anna Hilda Portugal Peña and the staff of the Centro Municipal de Faeno, Gobierno Autónomo Municipal de La Paz, for their collaboration with collecting of samples from cattle; to Javier Larico and Ever Quispe, from the Servicio Nacional de Sanidad Agropecuaria e Inocuidad Alimentaria (SENASAG) at La Paz, for their collaboration with this project. Our gratefulness to Dr George Garcia for providing parasite material collected from a human. Special acknowledgements to the authorities and inhabitants of the Municipalities of Batallas, Pucarani and Tiahuanacu for their collaboration during the fieldwork.

Financial support

The analyses of the samples were financed by the Institute of Parasitology, University of Zurich. The sample collection, transportation and parasitological analysis were funded by IINSAD and the Research Laboratory of Parasitology, Faculty of Medicine, Universidad Mayor de San Andrés. Cristian A. Alvarez Rojas is a recipient of the mobility grant funded by the Leading House of the Latin American Region/State Secretariat for Education, Research and Innovation (SERI).

Ethical standards

Ethics clearance for the use of human samples was provided by the Universidad Mayor de San Andres, La Paz, Bolivia.

Supplementary material

For supplementary material accompanying this paper visit https://doi.org/10.1017/S0031182020000529.

S0031182020000529sup001.docx (2.5MB, docx)

click here to view supplementary material

Conflict of interest

The authors declare no conflicts of interest.

References

  1. Addy F, Wassermann M, Banda F, Mbaya H, Aschenborn J, Aschenborn O, Koskei P, Umhang G, De La Rue M, Elmahdi IE, Mackenstedt U, Kern P and Romig T (2017) Genetic polymorphism and population structure of Echinococcus ortleppi. Parasitology 144, 450–458. [DOI] [PubMed] [Google Scholar]
  2. Alvarez Rojas CA, Gauci CG and Lightowlers MW (2013) Antigenic differences between the EG95-related proteins from Echinococcus granulosus G1 and G6 genotypes: implications for vaccination. Parasite Immunology 35, 99–102. [DOI] [PubMed] [Google Scholar]
  3. Alvarez Rojas CA, Romig T and Lightowlers MW (2014) Echinococcus granulosus Sensu Lato genotypes infecting humans – review of current knowledge. International Journal for Parasitology 44, 9–18. [DOI] [PubMed] [Google Scholar]
  4. Alvarez Rojas CA, Ebi D, Gauci CG, Scheerlinck JP, Wassermann M, Jenkins DJ, Lightowlers MW and Romig T (2016) Microdiversity of Echinococcus granulosus Sensu Stricto in Australia. Parasitology 143, 1026–1033. [DOI] [PubMed] [Google Scholar]
  5. Alvarez Rojas CA, Ebi D, Paredes R, Acosta-Jamett G, Urriola N, Roa JC, Manterola C, Cortes S, Romig T, Scheerlinck J-P and Lightowlers MW (2017) High intraspecific variability of Echinococcus granulosus Sensu Stricto in Chile. Parasitology International 66, 112–115. [DOI] [PubMed] [Google Scholar]
  6. Armua-Fernandez MT, Castro OF, Crampet A, Bartzabal Á, Hofmann-Lehmann R, Grimm F and Deplazes P (2014) First case of peritoneal cystic echinococcosis in a domestic cat caused by Echinococcus granulosus Sensu Stricto (genotype 1) associated to feline immunodeficiency virus infection. Parasitology International 63, 300–302. [DOI] [PubMed] [Google Scholar]
  7. Badaraco JL, Ayala FJ, Bart JM, Gottstein B and Haag KL (2008) Using mitochondrial and nuclear markers to evaluate the degree of genetic cohesion among Echinococcus Populations. Experimental Parasitology 119, 453–459. [DOI] [PubMed] [Google Scholar]
  8. Balbinotti H, Santos GB, Badaraco J, Arend AC, Graichen DÂS, Haag KL and Zaha A (2012) Echinococcus Ortleppi (G5) and Echinococcus granulosus Sensu Stricto (G1) loads in cattle from Southern Brazil. Veterinary Parasitology 188, 255–260. [DOI] [PubMed] [Google Scholar]
  9. Beteta Ortiz M (1999) Llegada del vacuno español a Suramérica. Vol. 2019 (ed. EspaÑa, R. A. d. C. V. d.).
  10. Bowles J, Blair D and McManus DP (1992) Genetic variants within the genus Echinococcus identified by mitochondrial DNA sequencing. Molecular and Biochemical Parasitology 54, 165–173. [DOI] [PubMed] [Google Scholar]
  11. Cabrera R (2007) Equinococosis quística en el Perú: necesidades de prevención y control de una enfermedad postergada. Bol Epidemiol (Lima) 16, 669. [Google Scholar]
  12. Callisaya (2018) Registro de Granjas Porcinas. (ed. Tierra, M. d. D. R. y.).
  13. Cardona GA and Carmena D (2013) A review of the global prevalence, molecular epidemiology and economics of cystic echinococcosis in production animals. Veterinary Parasitology 192, 10–32. [DOI] [PubMed] [Google Scholar]
  14. Casas N, Otero SC, Céspedes G, Sosa S and Santillán G (2013) Detección de coproantígenos para el diagnóstico de echinococosis canina en la zona fronteriza de La Quiaca-Villazón. Revista Argentina de Microbiología 45, 154–159. [DOI] [PubMed] [Google Scholar]
  15. Clement M, Posada D and Crandall KA (2000) TCS: a computer program to estimate gene genealogies. Molecular Ecology 9, 1657–1659. [DOI] [PubMed] [Google Scholar]
  16. Corrêa F, Stoore C, Horlacher P, Jiménez M, Hidalgo C, Alvarez Rojas CA, Figueiredo Barros G, Bunselmeyer Ferreira H, Hernández M, Cabrera G and Paredes R (2018) First description of Echinococcus Ortleppi and cystic echinococcosis infection status in Chile. PLOS ONE 13, e0197620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Craig PS, Hegglin D, Lightowlers MW, Torgerson PR and Wang Q (2017) Chapter two – Echinococcosis: control and prevention. In Thompson RCA, Deplazes P and Lymbery AJ (eds), Advances in Parasitology, vol. 96. London: Academic Press, pp. 55–158. [DOI] [PubMed] [Google Scholar]
  18. Cucher MA, Macchiaroli N, Baldi G, Camicia F, Prada L, Maldonado L, Avila HG, Fox A, Gutiérrez A, Negro P, López R, Jensen O, Rosenzvit M and Kamenetzky L (2016) Cystic echinococcosis in South America: systematic review of species and genotypes of Echinococcus granulosus Sensu Lato in humans and natural domestic hosts. Tropical Medicine & International Health 21, 166–175. [DOI] [PubMed] [Google Scholar]
  19. de la Rue ML, Takano K, Brochado JF, Costa CV, Soares AG, Yamano K, Yagi K, Katoh Y and Takahashi K (2011) Infection of humans and animals with Echinococcus granulosus (G1 and G3 strains) and E. ortleppi In Southern Brazil. Veterinary Parasitology 177, 97–103. [DOI] [PubMed] [Google Scholar]
  20. Deplazes P, Rinaldi L, Alvarez Rojas CA, Torgerson PR, Harandi MF, Romig T, Antolova D, Schurer JM, Lahmar S, Cringoli G, Magambo J, Thompson RCA and Jenkins EJ (2017) Chapter six – global distribution of alveolar and cystic Echinococcosis. In Thompson RCA, Deplazes P and Lymbery AJ (eds), Advances in Parasitology, vol. 95. London: Academic Press, pp. 315–493. [DOI] [PubMed] [Google Scholar]
  21. Eckert J and Deplazes P (2004) Biological, epidemiological, and clinical aspects of Echinococcosis, a zoonosis of increasing concern. Clinical Microbiology Reviews 17, 107–135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Escobari de Querejazu L (1985) Producción y comercio en el espacio sur andino en el siglo XVII: Cuzco-Potosí, 1650–1700, Embajada de España en Bolivia.
  23. Espinoza S, Salas AM, Vargas A, Freire V, Diaz E, Sánchez G and Venegas J (2014) Detection of the G3 genotype of Echinococcus granulosus from hydatid cysts of Chilean cattle using COX1and ND1mitochondrial markers. Parasitology Research 113, 139–147. [DOI] [PubMed] [Google Scholar]
  24. Guarnera EA, Parra A, Kamenetzky L, García G and Gutiérrez A (2004) Cystic echinococcosis in Argentina: evolution of metacestode and clinical expression in various Echinococcus granulosus strains. Acta Tropica 92, 153–159. [DOI] [PubMed] [Google Scholar]
  25. Hidalgo C, Stoore C, Pereira I, Paredes R and Alvarez Rojas CA (2019) Multiple haplotypes of Echinococcus granulosus Sensu Stricto in single naturally infected intermediate hosts. Parasitology Research 119, 763–770. [DOI] [PubMed] [Google Scholar]
  26. Hüttner M, Nakao M, Wassermann T, Siefert L, Boomker JDF, Dinkel A, Sako Y, Mackenstedt U, Romig T and Ito A (2008) Genetic characterization and phylogenetic position of Echinococcus Felidis Ortlepp, 1937 (Cestoda: Taeniidae) from the African lion. International Journal for Parasitology 38, 861–868. [DOI] [PubMed] [Google Scholar]
  27. Jarovsky D, Brito CRDS, Monteiro DU, Azevedo MID, Botton SDA, Mimica MJ, Arnoni MV, Sáfadi MAP, Berezin EN, Salgado Filho H, Almeida FJ and Rue MLDL (2020) Imported hepatopulmonary echinococcosis: first report of Echinococcus granulosus Sensu Stricto (G1) in Bolivia. Revista da Sociedade Brasileira de Medicina Tropical 53, 1–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kamenetzky L, Canova SG, Guarnera EA and Rosenzvit MC (2000) Echinococcus granulosus: DNA extraction from germinal layers allows strain determination in fertile and nonfertile hydatid cysts. Experimental Parasitology 95, 122–127. [DOI] [PubMed] [Google Scholar]
  29. Kamenetzky L, Gutierrez AM, Canova SG, Haag KL, Guarnera EA, Parra A, García GE and Rosenzvit MC (2002) Several strains of Echinococcus granulosus Infect livestock and humans in Argentina. Infection, Genetics and Evolution 2, 129–136. [DOI] [PubMed] [Google Scholar]
  30. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P and Drummond A (2012) Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics (Oxford, England) 28, 1647–1649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Konyaev SV, Yanagida T, Nakao M, Ingovatova GM, Shoykhet YN, Bondarev AY, Odnokurtsev VA, Loskutova KS, Lukmanova GI, Dokuchaev NE, Spiridonov S, Alshinecky MV, Sivkova TN, Andreyanov ON, Abramov SA, Krivopalov AV, Karpenko SV, Lopatina NV, Dupal TA, Sako Y and Ito A (2013) Genetic diversity of Echinococcus Spp. in Russia. Parasitology 140, 1637–1647. [DOI] [PubMed] [Google Scholar]
  32. Laurimäe T, Kinkar L, Andresiuk V, Haag KL, Ponce-Gordo F, Acosta-Jamett G, Garate T, Gonzàlez LM and Saarma U (2016) Genetic diversity and phylogeography of highly zoonotic Echinococcus granulosus genotype G1 in the Americas (Argentina, Brazil, Chile and Mexico) based on 8279bp of mtDNA. Genetics and Evolution 45, 290–296 [DOI] [PubMed] [Google Scholar]
  33. Laurimäe T, Kinkar L, Romig T, Omer RA, Casulli A, Umhang G, Gasser RB, Jabbar A, Sharbatkhori M, Mirhendi H, Ponce-Gordo F, Lazzarini LE, Soriano SV, Varcasia A, Rostami Nejad M, Andresiuk V, Maravilla P, González LM, Dybicz M, Gawor J, Šarkūnas M, Šnábel V, Kuzmina T and Saarma U (2018) The benefits of analysing complete mitochondrial genomes: deep insights into the phylogeny and population structure of Echinococcus granulosus Sensu Lato genotypes G6 and G7. Infection. Genetics and Evolution 64, 85–94. [DOI] [PubMed] [Google Scholar]
  34. Leigh JW and Bryant D (2015) Popart: full-feature software for haplotype network construction. Methods in Ecology and Evolution 6, 1110–1116. [Google Scholar]
  35. Lymbery AJ, Jenkins EJ, Schurer JM and Thompson RCA (2015) Echinococcus canadensis, E. borealis, and E. intermedius. What's in a name? Trends in Parasitology 31, 23–29. [DOI] [PubMed] [Google Scholar]
  36. Manterola C, Benavente F, Melo A, Vial M and Roa JC (2008) Description of Echinococcus granulosus genotypes in human hydatidosis in a region of southern Chile. Parasitology International 57, 342–346. [DOI] [PubMed] [Google Scholar]
  37. Mathis A, Deplazes P and Eckert J (1996) An improved test system for PCR-based specific detection of Echinococcus multilocularis eggs. Journal of Helminthology 70, 219–222. [DOI] [PubMed] [Google Scholar]
  38. MDR&T (2014) Producción pecuaria de porcinos. (ed. tierras, M. d. d. r. y.).
  39. MDR&T (2017) Producción pecuaria de bovinos. (ed. Tierra, M. d. D. R. y.).
  40. Monteiro DU, Botton SA, Tonin AA, Azevedo MI, Graichen DAS, Noal CB and de la Rue ML (2014) Echinococcus canadensis (G7) and Echinococcus granulosus Sensu Stricto (G1) in swine of southern Brazil. Veterinary Parasitology 202, 335–338. [DOI] [PubMed] [Google Scholar]
  41. Moro P and Schantz PM (2006) Cystic echinococcosis in the Americas. Parasitology International 55, S181–S186. [DOI] [PubMed] [Google Scholar]
  42. Moro PL, Nakao M, Ito A, Schantz PM, Cavero C and Cabrera L (2009) Molecular identification of Echinococcus Isolates from Peru. Parasitology International 58, 184–186. [DOI] [PubMed] [Google Scholar]
  43. Nakao M, McManus DP, Schantz PM, Craig PS and Ito A (2006) A molecular phylogeny of the genus Echinococcus Inferred from complete mitochondrial genomes. Parasitology 134, 713–722. [DOI] [PubMed] [Google Scholar]
  44. Nakao M, Lavikainen A and Hoberg E (2015) Is Echinococcus intermedius a valid species? Trends in Parasitology 31, 342–343. [DOI] [PubMed] [Google Scholar]
  45. Pavletic CF, Larrieu E, Guarnera EA, Casas N, Irabedra P, Ferreira C, Sayes J, Gavidia CM, Caldas E, Lise MLZ, Maxwell M, Arezo M, Navarro AM, Vigilato MAN, Cosivi O, Espinal M and Del Rio Vilas VJ (2017) Cystic echinococcosis in South America: a call for action. Revista Panamericana de Salud Publica 41, 1–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Plata W (2005) Pampa Belen comunarios, maestros y residentes defienden su tierra. La Paz: Fundación TIERRA. [Google Scholar]
  47. Primo A (1992) El ganado bovino ibérico en las Américas: 500 años después. Archivos de zootecnia, ISSN 0004–0592 41, 41. [Google Scholar]
  48. Primo AT (2004) América : conquista e colonizac¸ão : a fantástica história dos conquistadores ibéricos e seus animais na era dos descobrimentos, Movimento, Porto Alegre.
  49. Ramos-Sarmiento D and Chiluisa-Utreras V (2020) First molecular identification of hydatid tapeworm Echinococcus granulosus Sensu Lato G6/G7 in Ecuador. Journal of Helminthology 94, e100. [DOI] [PubMed] [Google Scholar]
  50. Romig T, Ebi D and Wassermann M (2015) Taxonomy and molecular epidemiology of Echinococcus granulosus sensu lato. Veterinary Parasitology 213, 76–84. [DOI] [PubMed] [Google Scholar]
  51. Romig T, Deplazes P, Jenkins D, Giraudoux P, Massolo A, Craig PS, Wassermann M, Takahashi K and de la Rue M (2017) Chapter five – ecology and life cycle patterns of echinococcus Species. In Thompson RCA, Deplazes P and Lymbery AJ (eds), Advances in Parasitology, vol. 95. London: Academic Press, pp. 213–314. [DOI] [PubMed] [Google Scholar]
  52. Rosenzvit MC, Zhang LH, Kamenetzky L, Canova SG, Guarnera EA and McManus DP (1999) Genetic variation and epidemiology of Echinococcus granulosus in Argentina. Parasitology 118, 523–530. [DOI] [PubMed] [Google Scholar]
  53. Salud MD (2019) Boletin integrado de vigilancia. Vol. 477.
  54. Sánchez E, Cáceres O, Náquira C, Garcia D, Patiño G, Silvia H, Volotão AC and Fernandes O (2010) Molecular characterization of Echinococcus granulosus From Peru by sequencing of the mitochondrial cytochrome C oxidase subunit 1 gene. Memórias do Instituto Oswaldo Cruz 105, 806–810. [DOI] [PubMed] [Google Scholar]
  55. Sánchez E, Cáceres O, Náquira C, Miranda E, Samudio F and Fernandes O (2012) Echinococcus granulosus genotypes circulating in alpacas (Lama pacos) and pigs (Sus scrofa) from an endemic region in Peru. Memórias do Instituto Oswaldo Cruz 107, 275–278. [DOI] [PubMed] [Google Scholar]
  56. Santivañez SJ, Gutierrez AM, Rosenzvit MC, Muzulin PM, Rodriguez ML, Vasquez JC, Rodriguez S, Gonzalez AE, Gilman RH and Garcia HH and Cysticercosis Working Group in P (2008) Human hydatid disease in Peru is basically restricted to Echinococcus granulosus genotype G1. The American Journal of Tropical Medicine and Hygiene 79, 89–92. [PMC free article] [PubMed] [Google Scholar]
  57. Šarkūnas M, Vienažindienė Ž, Alvarez Rojas CA, Radziulis K and Deplazes P (2019) Praziquantel treatment of dogs for four consecutive years decreased the transmission of Echinococcus intermedius G7 to pigs in villages in Lithuania. Food and Waterborne Parasitology 15, e00043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Schantz PM (1972) Hidatidosis: magnituzd del problema y perspectivas de control. Boletin de La Oficina Sanitaria Panamerica 73, 187–197. [PubMed] [Google Scholar]
  59. Schneider R, Gollackner B, Schindl M, Tucek G and Auer H (2010) Echinococcus canadensis G7 (pig strain): an underestimated cause of cystic echinococcosis in Austria. American Journal of Tropical Medicine and Hygiene 82, 871–874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Soriano SV, Pierangeli NB, Pianciola L, Mazzeo M, Lazzarini LE, Saiz MS, Kossman AV, Bergagna HFJ, Chartier K and Basualdo JA (2010) Molecular characterization of Echinococcus Isolates indicates goats as reservoir for Echinococcus canadensis G6 genotype in Neuquén, Patagonia Argentina. Parasitology International 59, 626–628. [DOI] [PubMed] [Google Scholar]
  61. Štefanić S, Shaikenov BS, Deplazes P, Dinkel A, Torgerson PR and Mathis A (2004) Polymerase chain reaction for detection of patent infections of Echinococcus granulosus (“sheep strain”) in naturally infected dogs. Parasitology Research 92, 347–351. [DOI] [PubMed] [Google Scholar]
  62. Thompson RCA (2017). Chapter two – biology and systematics of Echinococcus. In Thompson RCA, Deplazes P and Lymbery AJ (eds), Advances in Parasitology, vol. 95. London: Academic Press, pp. 65–109. [DOI] [PubMed] [Google Scholar]
  63. Thompson RCA, Kumaratilake LM and Eckert J (1984) Observations on Echinococcus granulosus of cattle origin in Switzerland. International Journal for Parasitology 14, 283–291. [DOI] [PubMed] [Google Scholar]
  64. Trachsel D, Deplazes P and Mathis A (2007) Identification of taeniid eggs in the faeces from carnivores based on multiplex PCR using targets in mitochondrial DNA. Parasitology 134, 911–920. [DOI] [PubMed] [Google Scholar]
  65. Villalobos Cortés A, Martinez A and and Delgado JV (2009) Historia de los bovinos en Panama y su relacion con las poblaciones bovinas de Iberoamerica. Archivos de Zootecnia 58, 121. [Google Scholar]
  66. Villena E (2011) Proyecto de Fortalecimiento de la Cooperación Técnica entre Uruguay y Bolivia para mejorar el control de la Hidatidosis en Bolivia. Tupiza: WHO/PAHO. [Google Scholar]
  67. Yanagida T, Mohammadzadeh T, Kamhawi S, Nakao M, Sadjjadi SM, Hijjawi N, Abdel-Hafez SK, Sako Y, Okamoto M and Ito A (2012) Genetic polymorphisms of Echinococcus granulosus Sensu Stricto in the Middle East. Parasitology International 61, 599–603. [DOI] [PubMed] [Google Scholar]

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