https://orcid.org/0000-0001-6314-8668
Figures
Abstract
Background
Ascaris lumbricoides and Ascaris suum are nematode parasites that infect millions of people and pigs worldwide, respectively. Reports of cross-infection and hybridization between the two species has stimulated molecular epidemiological studies of the Ascaris genus. In this study, we evaluated the dynamics of Ascaris transmission between Guarani indigenous schoolchildren, pigs, and the environment of a village in the state of Paraná, southern Brazil.
Methodology/principal findings
Parasitological and molecular analyses of fecal samples from humans and pigs, and soil samples from the village were carried out. Eggs of Ascaris spp. were observed in 8.4% (7/83) of human samples, 44.4% (8/18) of pig samples, and 8.9% (6/68) of soil samples. PCR amplification of the ITS-1 locus of the rDNA gene in samples that were positive in the parasitological examination revealed mixed infection and/or hybrids of the two species, A. lumbricoides and A. suum, in human and swine hosts. The soil, which was contaminated by both human and swine feces, also contained eggs of the two Ascaris species and hybrids, thus constituting a source of Ascaris infection for both hosts. DNA from A. lumbricoides and A. suum, individually, was detected in samples from humans and pigs, respectively, and the soil, while DNA from hybrid and/or Ascaris spp. was detected in samples from both hosts and the soil.
Author summary
Despite the control measures implemented in Brazilian Indigenous Lands, the prevalence of intestinal parasites continues to vary from moderate (> 20% and < 50%) to high (≥ 50%) in its inhabitants. The number of indigenous people residing in Brazil has been increasing over the last few decades, including in the South region of the country, where a much smaller proportion of indigenous people reside compared to the North region. Ascaris lumbricoides and Ascaris suum are the most prevalent helminth parasites in humans and pigs, respectively. The occurrence of cross-infection and hybridization between human and pig Ascaris has been investigated in order to propose more effective control measures. In this study, we used parasitological and molecular methods to verify the presence of these parasites in fecal samples from schoolchildren and pigs, in addition to soil samples, from a Guarani indigenous village in the state of Paraná, southern Brazil. Genetic material from A. lumbricoides and A. suum, individually, was detected in humans and pigs respectively, and the soil, and hybrid and/or Ascaris spp. was found in both hosts and soil samples, indicating the occurrence of transmission of hybrids and/or both species in these populations. Control measures aimed at the correct disposal of human and animal feces must be implemented to minimize damage to health and prevent new infections.
Citation: Boeira VL, Bezagio RC, Carvalho MSd, Gandra RF, Abreu APd, Massara CL, et al. (2025) Hybrid and/or mixed infection by pig and human Ascaris in a Guarani indigenous village in southern Brazil. PLoS Negl Trop Dis 19(2): e0012271. https://doi.org/10.1371/journal.pntd.0012271
Editor: María Victoria Periago, Consejo Nacional de Investigaciones Cientificas y Tecnicas, Fundación Mundo Sano, ARGENTINA
Received: June 10, 2024; Accepted: December 6, 2024; Published: February 6, 2025
Copyright: © 2025 Boeira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data are in the manuscript and/or supporting information files.
Funding: This work was supported by Conselho Nacional de Desenvolvimento Tecnológico e Científico (CNPq) (https://www.gov.br/cnpq/pt-br) through a productivity grant (granted to MJOT), grant numbers 314927/2020-4 and 315180/2023-4. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Ascaris lumbricoides is the most prevalent species of soil-transmitted helminths. This nematode parasite is a cosmopolitan with wide geographic distribution; around 500 million individuals are infected worldwide and infection rates are above 20% in certain regions of Asia, sub-Saharan Africa, and Latin America [1–3]. In these regions, the high prevalence is closely related to the lack of basic sanitation and poor hygiene practices, conditions directly associated with poverty, in addition to climatic factors that favor the biological cycle of the parasite [4]. Ascariasis is therefore considered a neglected tropical disease and, although the majority of cases do not lead to death, this disease causes approximately 604 thousand years of life lived with disability, representing a serious economic and health problem in the affected countries [5,2].
The estimated percentage of pig herds infected by intestinal parasites worldwide is high (~50%), particularly among pigs raised in extensive and organic animal production systems when compared to those in industrialized systems [6–7]. One of the most reported species of parasite in pig herds is the helminth Ascaris suum, which was first described by Goeze in 1782 [6–9].
Ascaris lumbricoides and Ascaris suum are considered cryptic because they have great biological and morphological similarity; the species can only be differentiated by differences in the labial denticles of adult helminths using scanning electron microscopy. Species-specific identification through visualization of eggs by optical microscopy is impossible, since they are morphologically identical [10–12].
The real scenario of human and swine ascariasis in endemic and non-endemic regions is still unknown and it is difficult to address by conventional coproparasitological methods, such as microscopy. Comparative analyses of ribosomal DNA (rDNA) comprising the internal transcribed spacer 1 (ITS-1) locus revealed deletions in the A. lumbricoides sequence and polymorphisms in the alignment between the two species, totaling six divergent nucleotide positions. The sum of these factors results in a genetic divergence of 1.3% between the ITS-1 sequences of A. lumbricoides and A. suum, with a variation of up to 0.2% within the taxon, enabling the species to be differentiated by molecular techniques [13–15]. Comparative phylogenomic analyses of worms from human hosts in African villages, where pig husbandry is rare, showed that the majority of samples had mitochondrial genomes that cluster closer to the pig parasite A. suum than to A. lumbricoides [16]. Nuclear genomes had extensive heterozygosity, and all samples existed as genetic mosaics with inheritance patterns similar to one or the other species, supporting a highly interbreeding Ascaris species genetic complex.
According to 2022 census data, the number of indigenous people residing in Brazil was 1,693,535, which represented 0.83% of the country’s total population; this was an 88.82% increase from the 2010 census number [17]. More than half of Brazilian indigenous people live in the Legal Amazon while 44.48% (753,284 people) are concentrated in the North region of the country [17]. Only 5.20% of this population (88,097 people) reside in the South region of Brazil.
The prevalence of enteroparasitosis in the Brazilian indigenous population continues to be moderate (> 20% and < 50%) to high (≥ 50%) [18], despite the implementation of control activities, such as housing improvements, water pipe installation and water treatment, and mass antiparasitic treatment of the population [19,20]. Therefore, reducing the morbidity and mortality caused by enteroparasite infection in this population still remains a challenge for health authorities.
In principle, indigenous people have more frequent contact with domestic animals and, to a certain extent, with wild animals, than the rest of society. The link between pathogens, the environment, animal species, and humans is inseparable, and thus, developing strategies to avoid or minimize current health problems requires collaboration from different fields of research. This approach is called “One Health” [21] and, following the COVID-19 pandemic, recognition of its relevance has grown [22].
Comparative phylogenomic analyses of a new Ascaris spp. genome proposed by Easton et al. [16] against existing draft genomes of A. lumbricoides and A. suum suggest that A. suum and A. lumbricoides form a genetic complex that is capable of interbreeding, which has apparently undergone a recent worldwide expansion of the multi-species Ascaris population.
In this study, using a One Health approach, we sought to analyze the dynamics of transmission of Ascaris spp. in a Guarani indigenous village in Paraná, southern Brazil, through the identification and molecular characterization of isolates obtained from samples of human and swine feces and soil.
Materials and methods
Ethics statement
This study was approved by the National Research Ethics Commission through Opinion 1.756.060/2016, with participation in the study conditional on the signing of the free and informed consent form by the parents/guardians of the schoolchildren that provided fecal samples. Following the recommendation of the ethics committee, three types of the free and informed consent form were prepared. The first was signed by parents or guardians of younger children (from 1st to 4th year, elementary school 1), the second for those of students in intermediate age groups (from 5th to 9th grade, elementary school 2), and the third for high school students. The three informed consent were offered in writing. However, verbal consent was obtained from most parents or guardians, and written consent was signed by the students in the older age groups themselves, as they had the autonomy to decide about their participation in the study. The research was authorized by local indigenous leaders and the Special Indigenous Health District—South Coast.
Study area
In the state of Paraná, located in the South region of Brazil, 23 indigenous lands/villages exist, corresponding to an area of 85,264.30 hectares [23]. The Tekoha Ocoy Indigenous Land, called Santa Rosa do Ocoy village, is located in the municipality of São Miguel do Iguaçu, west of Paraná (25° 20′ 50″ S; 54° 14′ 6″ W). The village is located 14 km away from the urban center of São Miguel do Iguaçu and covers a territory of around 250 hectares (Fig 1). The village receives assistance from the National Indigenous People Foundation, the National Health Foundation, the Itaipu Binacional Hydroelectric Plant, and the São Miguel do Iguaçu City Hall. The people of the village use Portuguese and Guarani as a form of communication.
Featured area of the map of Brazil represents the state of Paraná. The Indigenous Land is indicated within this area. Source: the author, from the U.S. Geological Survey (http://www.usgs.gov).
The indigenous community is located on the edge of Itaipu lake, with the residences surrounding the lake, divided into groups according to the kinship between the families. The houses are made of stone and wood and have straw-thatched roofs. In addition, the village has community spaces, including a prayer house, football fields, a warehouse for meetings and gatherings, a school, and a basic health unit [24].
Population
According to the 2022 census, approximately 30,460 indigenous people were living in Paraná [17], belonging to three ethnicities: Guarani, Kaingáng, and Xetá. The village of Santa Rosa do Ocoy is inhabited by approximately 500 people belonging to the Guarani ethnic group, who are distributed among 106 households. Around 50% of the population is under 15 years of age, which is a common characteristic of the indigenous peoples of Brazil [17].
The study population consisted of schoolchildren, aged between 5 and 19 years, following the Toledo et al. [25] distribution. All students enrolled at the local indigenous school, Colégio Indígena Teko Nemoingo, from nursery to high school age, were invited to participate in the study (around 270 students).
Sample collection
Collection bottles (polypropylene bottles with a screw cap) for fecal samples, duly labeled with the participants’ names, were delivered, along with verbal and written instructions on how to perform the sample collection, to all children at the school, with the help of indigenous community health agents. The sample collection was carried out by parents, guardians, or by the children themselves (if they were able to understand and correctly carry out the collection on their own) between November 2019 and August 2022. The bottles containing the samples were collected by the village health team and placed in thermal boxes with reusable artificial ice, which were sent to the Clinical Parasitology Laboratory of the University of Western Paraná (UNIOESTE), located in the Teaching, Research and Extension Laboratory of the University Hospital of Western Paraná, within 24 h of collection.
Samples of pig feces were collected randomly from fresh evacuations observed by the research team in the closed pigsties. The samples were placed in duly identified collection bottles, which were then placed in thermal boxes with artificial ice and sent to the same laboratory for analysis.
For the soil samples, collection points were chosen close to the school, residences with animals raised in the home, and residences in different locations within the village area, respecting a perimeter of 10 m around each collection location. Approximately 50 g of soil were collected from each point at a depth of 5 cm. Different climatic and humidity conditions that could influence the results were taken into account, and thus, collection was repeated at the same points in each of the four seasons of the years 2019 and 2021. The samples were placed in collection bottles, labelled with the date and location of collection, and then sent for analysis in the same way as the fecal samples. No preservatives were used for any type of sample collected.
Soil sample treatment
The soil of the village was classified as clayey, making it difficult to extract compounds as it is more compacted than other types of soil [26]. To reduce dirt and interference in the analyses, the concentration and purification protocol of the Ministry of Health was performed [27], where 10 g of sample was mixed with 1 M glycine to a volume of 40 mL, homogenized for 30 min at 20 rpm, then completed to 50 mL with 1 M glycine and left to rest for 5 min. The supernatant was then transferred to another tube, centrifuged at 2,100 × g for 10 min, and the sediment was subjected to parasitological analysis.
Parasitological analyses
Analysis of the samples was carried out at the UNIOESTE Clinical Parasitology Laboratory. When immediate analysis was not possible, the samples were kept refrigerated for a maximum of 2 days.
Fecal samples from humans (n = 86), together with those from pigs (n = 18) and soil (n = 68) were processed by spontaneous sedimentation in water [28]. For better use of the material and exclusion of fecal debris, the sediments (3 mL) were then subjected to the Ritchie method [29] adapted by Bezagio et al. [30].
The samples were placed on slides and stained with Lugol, which were then examined by optical microscopy with a 10× objective; structures were confirmed at 400× magnification. The presence of parasite eggs, larvae, or cysts was considered a positive result, regardless of the quantity.
Molecular analyses
Molecular analyses for the detection of Ascaris genetic material and identification of the species was carried out for all fecal and soil samples, regardless of the microscopy results. Positive and negative controls were also used.
DNA extraction
First, the efficiency of two commercial DNA extraction kits was tested using samples from the laboratory that were known to be positive for Ascaris spp. The PureLink PCR Purification Kit (Invitrogen, Carlsbad, CA, USA) was used for laboratory samples that had or had not undergone prior sonication treatment for disruption of the Ascaris spp. egg membrane: 50 Hz for 30 s at 4°C, repeated 4 times, with an interval of 1 min between cycles. The QIAmp DNA Stool Mini Kit (Qiagen, Hilden, Germany), which is widely used for DNA extraction from fecal samples, was used only for laboratory samples that had not undergone the sonication procedure. The intensity of the bands observed in an electrophoresis gel after PCR with the extracted samples informed which DNA extraction method was subsequently used for the collected samples.
Amplification of the ITS-1 fragment
The approximately 580 base pair (bp) fragment of the ITS-1 locus was amplified by nested PCR with the primers F2662 5′-GGCAAAAGTCGTAACAAGGT-3′ and R3214 5′-CTGCAATTCGCACTATTTATCG-3′, according to Ishiwata et al. [31]. Each amplification reaction was performed in a final volume of 10 μL, containing 1× reaction buffer (200 mmol/L Tris-HCl pH 8.4, 500 mmol/L KCl), 2.5 mmol/L MgCl2, 1 U of Platinum Taq DNA Polymerase (Invitrogen, Lithuania), 200 μmol/L deoxyribonucleotide triphosphate, 2 pmol of each primer, sterile Milli-Q water, and 2 μL of DNA. Amplification conditions were used according to Sadaow et al. [14]: pre-incubation at 95°C for 5 min, followed by 35 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and then a final extension at 72°C for 10 min.
Genotyping of Ascaris spp
Three primers were used in the same reaction; two of which were species-specific forward primers, F. Al specific for A. lumbricoides (5′-GCGGTTTCTTTT TTTTTTCGCG-3′) and F. As specific for A. suum (5′-GAGAAAGCTCCTCGT TGCGG-3′), and a reverse primer (5′-CCACGAACCGAGTGATCCAC-3′), which anneals to a region common to both species (R. both). Amplification of genetic material from A. lumbricoides results in a fragment of approximately 384 bp, with the combination of primers F. Al + R. both, while for A. suum, the combination of primers F. As + R. both results in a fragment of approximately 176 bp. This technique allows either or both species (or hybrid) to be detected in the same reaction (Fig 2). Conventional PCRs were performed, in a final volume of 10 μL, containing 1× reaction buffer (200 mmol/L Tris-HCl pH 8.4, 500 mmol/L KCl), 2.5 mmol/L MgCl2, 1 U of Platinum Taq DNA polymerase (Invitrogen, Lithuania), 200 μmol/L deoxyribonucleotide triphosphate, 2 pmol of each primer, sterile Milli-Q water, and 5 μL of DNA from the first reaction. Amplifications were performed using the following program: pre-incubation at 95°C for 5 min, 30 cycles at 95°C for 30 s, 60°C for 45 s, and 72°C for 45 s, and a final extension step at 72°C for 8 min [32].
The first PCR used primers F2662 and R3214 for the amplification of the ITS-1 locus (~580 bp), and thus, the identification of Ascaris spp. The second PCR used the fragment resulting from the first PCR, together with two species-specific forward primers (F Al and F As) and a common reverse primer (R both), for the identification of A. lumbricoides (~384 bp) and A. suum (~176 bp). Source: the author.
In all reactions, a positive control for A. lumbricoides (DNA provided by Helminthology Laboratory of the René Rachou Institute), a positive control for A. suum (DNA extracted from samples of adult worms from swine), and a negative control with Milli- Q water in place of DNA were used.
The products of these reactions were visualized on 5% polyacrylamide gels, stained with silver, and digitally photographed.
Results
All students (approximately 270), aged between 5 and 19, enrolled in the indigenous school of the village were invited to participate in the study; however, only 86 bottles (approximately 30.5%) containing fecal samples were returned for analysis. Three samples were excluded because they had an insufficient amount of material to carry out the analyses. The total prevalence of intestinal parasites in the schoolchildren was 81.9% (68/83); 47.0% (39/83) had polyparasitism while the specific prevalence of Ascaris spp. was 8.4% (7/83; Table 1).
Fecal samples from pigs were collected in March 2020, which coincided with the beginning of the COVID-19 pandemic, and in July 2022, as access to the village was prohibited during the pandemic. The pigs were located outside the residences, being kept in closed pigsties with restricted access to areas close to the houses. Of the 18 samples collected, 44.4% were positive for Ascaris spp. (Table 2).
Samples collected in March 2020 and July 2022.
Of the 68 soil samples collected, 52 (76.5%) were positive for at least one parasite species, the majority with zoonotic potential for infection in humans. Six samples (8.9%) were positive for Ascaris spp. (Table 3).
Samples collected throughout 2019 and 2021.
Sediments of fecal and soil samples, obtained using the Ritchie method adapted by Bezagio et al. [30], were sonicated to rupture the Ascaris spp. egg membrane (Fig 3), thus favoring DNA extraction, as described below.
ruptured (arrows) after sonication at 50 Hz for 30 s at 4°C, repeated 4 times, with a 1 min interval between cycles. 100× magnification. Source: the author.
Three DNA extraction/purification methods were evaluated using a known A. lumbricoides-positive sample and all methods appeared to be efficient. Subtle differences were observed in the intensities of the ITS-1 locus band of the extracted samples following PCR (Fig 4). Extraction with the PureLink PCR Purification Kit (Invitrogen, Carlsbad, CA, USA) preceded by sample sonication resulted in a slightly more intense band than the other two protocols tested, and thus, this method was chosen for the DNA extraction of samples collected in this study (Fig 4).
1. DNA ladder; 2. Ascaris lumbricoides-positive laboratory sample extracted with the PureLink PCR Purification Kit after sonication (50 Hz for 30 sec at 4°C repeated 4 times with 1 min interval between cycles); 3. Ascaris lumbricoides-positive laboratory sample extracted with the PureLink PCR Purification Kit without sonication; 4. Ascaris lumbricoides-positive laboratory sample extracted with the QIAmp DNA Stool Mini Kit without sonication; 5. positive control (A. lumbricoides DNA); and 6. negative control (PCR reaction without DNA).
The sediments of the collected samples (human and pig feces and soil) were submitted to DNA extraction followed by PCR amplification of the ITS-1 locus. Genotyping was then carried out using the PCR products from the ITS-1 amplification reaction to verify the presence of A. lumbricoides, A. suum, and hybrids. Table 4 shows the results of microscopy analysis and the ITS-1 genotyping in the three sample types. The DNA of Ascaris spp. was amplified from all samples that were positive in the microscopy analysis. Twice as many samples that were negative in the microscopy were randomly chosen to undergo the amplification reaction for validation, and all were negative.
Of the human fecal samples that were positive in the parasitological examination and exhibited amplification of the ITS-1 locus (7/83), 57.1% (4/7) presented a band of ~396 bp, characteristic of A. lumbricoides, and 42.9% (3/7) showed a ~396 bp band together with a band of ~176 bp, characteristic of A. suum. The presence of both bands indicated an infection by hybrid and/or mixed infection in humans. Among the positive pig fecal samples (8/18), 87.5% (7/8) presented the ~178 bp band and 1/8 (12.5%) showed both bands, suggesting hybrid and/or mixed infection in these animals. Of the Ascaris-positive soil samples (6/68), 33.3% (2/6) showed the A. lumbricoides characteristic band, 33.3% (2/6) showed the A. suum characteristic band, and 33.3% (2/6) showed both bands. Some representative samples of these results are presented in Fig 5.
The band for A. lumbricoides is ~384 bp and for A. suum is ~176 bp. 1: DNA ladder; 2: positive control for A. lumbricoides; 3: positive control for A. suum; 4 and 5: human feces samples that were positive in the parasitological examination; 6: human feces samples that were negative in the parasitological examination; 7 and 8: pig feces samples that were positive in the parasitological examination; 9: pig feces samples that were negative in the parasitological examination; 10, 11, and 12: soil samples that were positive in the parasitological examination; 13: soil sample that was negative in the parasitological examination; 14: negative control.
Discussion
This is the first molecular epidemiology study to determine the prevalence of Ascaris spp. in an indigenous community of Brazil, genetically and culturally distinct from the surrounding society, and to evaluate the circulation of this pathogen between humans, pigs, and the environment (soil).
Human ascariasis is a serious public health problem in developing countries [19]. The biological cycle of the parasite is favored by poor basic sanitation, poor hygiene practices, and a population living in poverty, which, together with climatic conditions, contribute to high levels of prevalence, especially in school-age children [1,3,4,33,34].
In this study, the highest prevalence of Ascaris spp. was recorded in pigs (44.4%), followed by the soil (8.8%) and, finally, in humans (8.4%). The low prevalence of Ascaris spp. observed in schoolchildren from this Guarani village does not correspond to the reality of many other Indigenous Lands in Brazil; in a study conducted in the state of Minas Gerais, southeastern Brazil, the infection rate was reportedly 34.4% [35], while a 63.5% infection rate was reported for another region of Paraná [25]. This divergence could be the result of periodic antiparasitic treatment, since in the study area there are active deworming programs for schoolchildren and, to a lesser extent, to housing improvements implemented by the government in this region in recent years [20,25,36,37]. However, the deworming status of pigs is unknown. It is believed that they are not routinely treated for parasites, which explains the high prevalence of Ascaris in this host.
Although the prevalence of soil-transmitted helminths, such as Ascaris spp., Trichuris trichiura, and hookworm, and intestinal helminths in the schoolchildren of the village was generally low (32.5% compared to 74.7% for intestinal protozoa), Hymenolepis nana was the most prevalent helminth in this population. Similar prevalence rates of H. nana have been reported for other Indigenous Lands of Paraná, inhabited by Kaingáng indigenous people, where similar prophylactic measures were adopted [25].
The epidemiological picture of human ascariasis around the world has shown some considerable changes, with increasing reports of this parasitic disease in developed countries, where cases of infection in humans were low or non-existent [38,39]. While A. lumbricoides typically affects humans and A. suum infects pigs [10], when human infection during travel or residence in endemic areas is ruled out, contamination with fecal material from pigs has been identified as the main source of infection [40]. Studies have shown that non-endemic regions usually present high levels of cross-infection, with humans being infected by Ascaris spp. derived from pigs, as well as a high percentage of hybrids, and thus, ascariasis has been characterized as a zoonosis in these regions [37, 41–42]. These reports strongly point to the zoonotic potential of A. suum [6,38,43], and the ability of A. suum to develop infection in experimentally infected humans has already been demonstrated [44].
The methods routinely used for the diagnosis of ascariasis do not allow identification at the species level, owing to the indistinguishable morphology of the eggs between species. The diagnosis is therefore inferred based on the host species under analysis and the epidemiology of the region [45–47]. However, given the changing epidemiological picture, molecular techniques that allow the identification of Ascaris species from the DNA of a single egg have been developed. These typically require the use of three primers in the same reaction, in which two primers are species-specific (one for each allele) and one primer is common [33,48,49].
To perform these molecular analyses, DNA must first be extracted from the sample. Three DNA extraction/purification procedures were evaluated in the current study. As Ascaris spp. eggs have three membranes that protect from environmental changes [50], sonication of the samples prior to DNA extraction appeared to favor the extraction process [51] and thus, this was performed prior to extraction with the PureLink PCR Purification Kit.
It is possible to identify and differentiate the species of Ascaris through the molecular analysis of the ITS-1 locus from a single egg. However, when using a pool of eggs in samples from human or swine feces, the presence of both bands (~396 bp and ~176 bp) in a single sample could indicate the occurrence of mixed infection and/or hybrid Ascaris. As demonstrated in Table 4 of this study, although the presence of the ~176 bp band alone, characteristic of A. summ, was not observed in any human fecal sample, and no swine fecal sample presented a single band of ~396 bp, characteristic of A. lumbricoides, we cannot rule out the possibility of cross-infection in these hosts and that the samples with both bands could indicate the presence of both species and/or hybrids. So, based on the methods we used our results could indicate a mixed infection and/or hybrid Ascaris, since the DNA was extracted from samples consisting of a pool of eggs. In other words, the eggs may come from hosts that could harbor both worms and/or hybrids.
In the village investigated in this study, 42.9% of positive human fecal samples presented the bands characteristic of both A. lumbricoides and A. suum, suggesting the occurrence of swine-human transmission of both species and/or hybrid Ascaris in a high proportion of infected individuals. This is different from other studies which have demonstrated that, in endemic regions, cases of human infection are the result of human-to-human transmission [14, 42]. The description of 54 dominant haplotypes and genotypes in Ascaris originating from a given host reveal a host affiliation in sympatric populations of endemic regions [52–53]. Therefore, it is crucial to understand the distribution of haplotypes and frequency of hybrids in the Ascaris population in a given region [41,54–56].
The ITS-1 locus was used as the molecular target of choice in this study because it is a region with multiple copies in the parasite genome and has well-characterized single nucleotide polymorphisms between the two species. At nucleotide position 133, a guanine is present in the A. lumbricoides sequence and a cytosine in the A. suum sequence; at position 246 a thymine is present in the A. lumbricoides sequence and an adenine in the A. suum sequence; and at position 323 an adenine is present in A. lumbricoides and a guanine in A. suum [14,37,57].
The results of the molecular analyses confirmed the results of the parasitological microscopy analyses and showed that the samples analyzed in this study demonstrated a pattern of transmission between host species. Although it diverges from the results of analyses carried out by other research groups who, using mitochondrial (mt)DNA and microsatellite markers, analyzed the sharing of Ascaris haplotypes and genotypes between Brazilian human and swine populations, and indicated that mixed infection could be occurring in some regions, more than hybrid infections [11,54,58].
In our study, PCR for the ITS-1 region did not detect Ascaris DNA in any of the samples that were negative in the parasitological examination. In other words, the two techniques used in the examination of the biological materials, light microscopy and PCR, showed high sensitivity, that is, the ability to detect the parasite. However, the molecular technique has an advantage over parasitological techniques by enabling the identification of the species/genotype of the parasite present in the sample.
In other Indigenous Lands of Paraná inhabited by other ethnic groups, such as the Kaingáng, where pigs are raised freely, in an extensive system, the soil constitutes an important source of parasitic contamination since, as already demonstrated, the same species of intestinal parasites can be found in human feces and soil [25,41,59]. Differently, in the Guarani indigenous village of the present study, pigs are raised in a non-extensive, confined regime. However, as was observed in situ by researchers, pigs occasionally escape from the pens, being able to defecate in areas where people circulate, and contaminate the soil with species of parasites with zoonotic potential. This makes it possible for both cross-contamination and mixed infection in children and pigs by both species.
Despite the low rate of contamination by Ascaris spp. found in this study when compared to the rates reported for studies of other Indigenous Lands [25,34,41,60], the human-soil-swine transmission link was demonstrated by genotyping and the hybrid Ascaris and/or mixed infection findings in humans and pigs, and the contamination of soil by both species and hybrids.
Our data corroborate the literature suggesting that A. suum and A. lumbricoides form a genetic complex capable of interbreeding, and that it apparently underwent a recent global expansion of the multispecies Ascaris population [16], being recorded for the first time in Amerindians of the South American continent. Therefore, Ascaris from pigs and humans may be important in human diseases, requiring a One Health approach to control the spread of human ascariasis.
Conclusions
In this study the ITS-1 sequences reveals both mixed infection by the two nematode species, A. lumbricoides and A. suum, and the occurrence and transmission of hybrid Ascaris between human and swine hosts from a Guarani indigenous village in southern Brazil. In addition to the hosts, the soil was contaminated with both species of Ascaris, as well as hybrids. Pure and genetically hybrid samples were observed in both hosts and in the soil, confirming that is a source of infection for human and animal populations.
Given the hybrid and/or mixed infection in the village, which confirms the interbreeding and hybridization between Ascaris species, in addition to the zoonotic nature of ascariasis, more effective and targeted control measures must be implemented, such as better containment of pigs to prevent access to human feces, in order to reduce the infection rates of Ascaris spp.
Our results point in the direction of global and African village data suggesting a recent population expansion event in which genetically similar worms are found worldwide, e.g. that A. suum and A. lumbricoides form a genetic complex capable of interbreeding. The molecular analysis of Ascaris in humans and pigs can contribute to the development of control measures, and future studies should be carried out in this and other sympatric areas to further enhance our knowledge about the transmission dynamics of this parasite.
Acknowledgments
The authors would like to express their gratitude to Lilian Maria Rodrigues and Bonifácia Rero Takua Alves for their valuable assistance in recruiting participants, Eloiza Cristina Martelli and Guilherme Martins Boeira in collecting the soil and pigs’ feces for this study.
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