High toxocariasis seroprevalence in a tri-border indigenous community (Brazil, Paraguay and Argentina): A One Health perspective

Abstract

Toxocariasis, a neglected parasitic zoonosis, has mostly affected vulnerable populations of subtropical and tropical regions worldwide. In addition to vulnerability, indigenous communities have long existed before bordering areas, particularly in South American countries, leading to cultural isolation, migratory and environmental concerns, lately associated to low human infrastructure and lack of healthcare policies. Accordingly, the study herein has serosurveyed anti-Toxocara spp. antibodies in indigenous persons, and surveyed Toxocara spp. in their dog and soil samples from a Guarani-Mabyá indigenous community located in a tri-border area of Brazil, Paraguay and Argentina. Overall, seropositivity was detected in 246/258 (95.3 %; 95 % CI: 92.1–97.3) indigenous persons, with no statistically associated risk factor to seropositivity, likely due to the highest human toxocariasis seroprevalence reported to date worldwide. Although detected in only 8/124 (6.5 %) dog feces samples, Toxocara spp. eggs were present in 13/42 (30.9 %) soil samples of common areas and 17/32 (53.1 %) of households, molecularly identified as T. canis by DNA amplification. The significant number of infective Toxocara spp. eggs found in the soil samples has reinforced the role of daily environmental exposure in sustaining transmission within this community, which may reflect the pattern of disease status in other nearby indigenous communities. Moreover, migratory behavior of Guarani ethnicity across the tri-border may have spread infection to other border indigenous communities of Brazil, Paraguay and Argentina.

1. Introduction

Parasitic nematodes of the genus Toxocara have been socioeconomically important zoonotic pathogens [1], with toxocariasis economic impact estimated at 2.5 billion USD per year, generally related to costs of medical treatment and lost income [2]. Although underrated, approximately one fifth of the global population may be infected by toxocariasis agents [3], namely Toxocara canis and T. cati, the common roundworms of dogs and cats, respectively [4].

Human infection has been commonly occurred by accidental ingestion of embryonated Toxocara spp. eggs in soil [5] or by consumption of raw or undercooked meat of paratenic hosts, mainly cattle and chickens [6]. After ingestion, L₃ are released from the egg or meat, penetrating the small intestine, accessing the circulation, and then wandering throughout the body, potentially invading all organs [5]. Although rare, two cases of congenital toxocariasis transmission have been reported to date [7,8].

Although human infections have been mostly subclinical, the larvae of Toxocara spp. may trigger local eosinophilia-associated inflammation with increased production of cytokines and specific antibodies [9]. The clinical pathology of toxocariasis has varied following the infection form and intensity, along with the larvae localization and host age [10]. The visceral form of toxocariasis may result in hepatic [11,12], pulmonary [13,14] and cardiac [15] injuries, or rarely resulting in fatal outcome [16]. Ocular toxocariasis may lead to peripheral or posterior granuloma, endophthalmitis, retinal distortions and tractional detachments, resulting in strabismus and blindness [17,18]. Neurotoxocariasis may also occur in a wide spectrum of neurological manifestations such as meningitis, encephalitis, myelitis, cerebral vasculitis, seizures, and headache [19,20].

Toxocariasis, a neglected parasitic zoonosis, has largely impacted impoverished communities [4] of subtropical and tropical regions worldwide [9], which may disproportionately affect indigenous populations living in extreme poverty [21]. Brazilian indigenous populations have historically been conquered, decimated and translocated to remote geographic areas, mostly conservation units, lacking basic sanitation, potable tap water, and veterinary assistance to novel domestic species such as pets and livestock, resulting in exposure to zoonotic agents and socioeconomic disadvantage, when compared to other Brazilian ethnicities [22].

As no domesticated native species was found in Brazil at the time of European invasions in the early 1500s, showing that Brazilian native indigenous communities may have never domesticated native species during 20–30,000 years of existence [23]. Thus, domestic dogs and cats may have brought new pathogens and established new disease cycles, deeply impacting indigenous health, particularly toxocariasis. In addition to inherent vulnerability, indigenous communities located in bordering areas of developing countries may be exposed to even lower human development indices, associated to environmental concerns, migratory flow, and health policies which may present weaknesses from a One Health perspective [24].

In such a scenario, our research group has recently detected a high exposure to Toxocara spp. (73.9 %; 95 %CI:70.0–77.7) in indigenous populations living in southern and southeastern Brazil, along with 9/194 (4.6 %) dog fecal and 4/204 (2.0 %) dog hair samples, with detection of T. canis recovered in 36/90 (40.0 %) soil samples of common areas [25]. However, no study to date has assessed indigenous communities living in far west areas of Brazilian bordering areas. Accordingly, the present study has assessed a major indigenous community located nearby the tri-border of Brazil, Argentina and Paraguay, surveying human toxocariasis, dog infection, soil contamination and potential associated risk factors, in a One Health perspective.

2. Material and methods

2.1. Ethics

This study was approved by the Committee on Ethics in Human Health of the Brazilian Ministry of Health (protocol 52039021.9.0000.0102) and by the Committee on Ethics in the Use of Animals (protocol number 033/2021) of the Federal University of Paraná, Brazil.

2.2. Study design

This was a cross-sectional seroepidemiological study based on a One Health (human, dog and soil samples) approach for toxocariasis in a clinically healthy (asymptomatic) indigenous community located in a three-border area (Brazil, Argentina, and Paraguay). The Ocoy indigenous community was chosen based on its high vulnerability due to highly populated (trading, smuggling and trafficking) three-country boarding area, forced translocation from its original landing after dam flooding, poor general hygiene habits, insalubrious environment (non-potable lakeshore dam water) and mostly inhabited by Guarani-spoken only individuals (translator required). Selection was made by the Special Indigenous Health District (DSEI) - South Coast, Special Secretariat of Indigenous Health (SESAI), Brazilian Ministry of Health, out of 150 indigenous communities with around 20,000 indigenous persons living in the states of Parana, São Paulo and Rio de Janeiro. In addition, as part of the official duties of the Brazilian Unified Health System, this indigenous community was considered priority for One Health approach due to the high presence of abandoned, unneutered, non-dewormed and unvaccinated dogs and cats.

2.3. Study area

The present study was conducted in the indigenous community Avá Guarani Tekoha Ocoy of Guarani ethnicity Mbyá subgroup, located in the city of São Miguel do Iguaçu, far west of Paraná State, Brazil, bordering Argentina at south, far 10 km at west from Paraguay and 40 km to the three-border point of Brazil, Paraguay and Argentina.

2.4. Population characteristics

Historically, the Guarani ethnic groups have been marked by migratory waves across Brazil, Paraguay and Argentina between 1800 and 1900, related to European invasions, religious aspects, and subsistence exploring. As result of such diaspora, the Guarani reached the Atlantic rainforest areas of southern and southeastern Brazilian coast in early 1900 [26]. Three major groups of the Guarani ethnicity named Mbyá, Kaiowá and Nhandeva have been settled, differentiating among themselves by customs, dialect, rituals and land occupation [27].

The Mbyá Guarani community has been traditionally maintained themselves through subsistence on natural resources. The high population mobility among Guarani communities has been known as tekoa (“good place to live” or “place of the Guarani way of being”), historically conducted from forty to one hundred individuals at the time for religious and ritual purposes, besides exchange of raw materials, handcrafts and agricultural goods [27,28].

The Ava Mbyá Guarani nation surveyed herein was removed from their ancestral lands in the Brazilian - Paraguayan border by the military government during the early 1970s, which was submerged due to the binational Itaipu hydroelectric construction, along with the biggest artificial powerplant dam worldwide (and currently third) at the time [29]. The indigenous community was partially relocated to a lakeshore dam area of 251 ha, struggling with their growing population (currently 800 inhabitants) living on an insufficient territory and lack of potable water, as the local lake has been unsuitable for drinking, bathing and fishing, due to pollution by solid waste and pesticides [29].

2.5. Samplings and testing

2.5.1. Human

Human blood samplings were performed by convenience in indigenous individuals after signed consent and completion of an epidemiological questionnaire. Blood samples (10 mL) were collected by venipuncture using vacuum tubes without anti-coagulant, performed by a certified nurse. The tubes were kept at room temperature (25 °C) until visible clots formation and then centrifuged (800g for 5 min). The obtained sera were stored (−20 °C) until screening for anti-Toxocara spp. IgG antibodies by Enzyme Linked Immunosorbent Assay (ELISA).

Associated risk factors with Toxocara spp. seropositivity were assessed by a questionnaire applied to all indigenous individuals, gathering epidemiological information along with human blood samplings. The survey included questions regarding gender, age, educational level, water source, raw or undercooked meat consumption, game meat consumption, nail-biting behavior, contact with soil, and dog and cat ownership.

Prior to serological analysis, serum samples were pre-adsorbed with Ascaris suum adult worm extracts following a previously established protocol [30], improving the assay specificity and avoiding cross-reactivity with other ascarid antigens [31]. This assay has exhibited an overall 78.3 % sensitivity and 92.3 % specificity [32,33].

Excretory-secretory (TES) antigen preparation for ELISA test was made using a concentrate of proteins from larvae hatched from T. canis eggs collected from adult female parasites, as previously described (Lowry et al. 1951; Fan et al. 2004; Elefant et al. 2006). ELISA was performed in polystyrene 96-well microplates (Corning, Costar, New York, USA) covered with TES antigens (1.9 μg/μL per well) in carbonate-bicarbonate buffer (0.06 M) at pH 9.6 for 1 h at 37 °C and 18 h at 4 °C. The plates were washed (PBS-Tween 5 %) and blocked with 3 % commercial skimmed milk PBS-Tween 5 % solution for 1 h at 37 °C. After a triple 5-minute-washing, pre-adsorbed serum samples were added in duplicate and the plates incubated for 1 h at 37 °C. Anti-Human IgG (Fc-specific) peroxidase antibody produced in goat (Sigma A6029) at a 1:10.000 dilution was added (1 h at 37 °C), followed by another triple 5-minute washing. The reaction was revealed using o-phenylenediamine substrate (0.4 mg/mL, Sigma), and stopped by adding 2 N H2SO4.

Positive and negative controls were included on each plate. The absorbance was read at 492 nm, and the cut-off defined as the mean absorbance of 96 negative control sera plus three standard deviations. The antibody levels were calculated as the ratio between the absorbance values of each sample and the cut-off value, set at 0.314 and expressed as reactivity indices (RI).

2.5.2. Dogs

Fecal and hair samples were collected by convenience from dogs of the indigenous community. Feces were collected from the rectum and stored into graduated tubes (50 mL) containing 10 % formalin solution, followed by refrigeration (4 °C) until microscopic examination. After feces sampling, dog hair samples were collected from perineal and lower back regions using scalpel blades and placed in individual graduated tubes (50 mL) kept under refrigeration (4 °C) until processing.

Feces were processed by a flotation method (Bowman, 2014) with a highly concentrated sodium chloride (NaCl) solution (d = 1.20 g/cm³). The samples were mixed with NaCl solution, filtered and transferred to clean containers covered with a slide. After a 5-minute-flotation, slides were analyzed under optical microscopy at 10× magnification to Toxocara spp. eggs detection.

Hair samples were processed following a previously described protocol with adaptations [34]. Briefly, samples were kept overnight with distilled water (20 mL) and anionic detergent Tween 80 5 % (0.2 mL), then thoroughly homogenized after addition of the same amounts of distilled water and detergent. The mixture was sieved (metal meshes 300, 212, and 38 μm) under running water for 5 min. The material retained onto the last sieve (38 μm) was microscopically (magnifications: 10× and 40×) examined for detection of Toxocara spp. eggs.

2.5.3. Soil

2.5.3.1. Samplings and processing

Soil samples were randomly collected from common areas (school, school playground, praying house, health center, trails and playing fields) and household locations of the indigenous community. The number of collection sets in each location was ruled by the surrounding area characteristics, avoiding sets with the presence of grass or feces.

The soil was collected from each set at 5 to 15 cm depth, placed in individual plastic bags and refrigerated (4 °C) until processing [35]. Samples were submitted to Toxocara spp. eggs retrieving, combining some previously described protocols [35,36]. Each soil sample was weighed (20 g) and kept overnight with anionic detergent Tween 80 5 % (100 mL). Then, the supernatant was disposed, and the soil was mixed thoroughly with more detergent (100 mL). Next, soil was sieved (300, 212, 90, and 38 μm) in running water and the material retained into last sieve (38 μm) collected and submitted to a centrifugal-flotation method using zinc sulfate solution (d = 1.35 g/cm³). The flotation material was transferred to 15 mL tubes, filled with distilled water, and centrifuged at 873g for 5 min. The supernatant was discarded and the washing process repeated twice to remove the zinc sulfate solution. Finally, the entire pellet was screened under optic microscopy (magnifications: 10× and 40×). Toxocara spp. eggs found in soil were classified (non-viable, viable, embryonating egg or embryonated) [37], collected with a 25 μL micropipette and stored (−20 °C) in microtubes with distilled water and stored for subsequent DNA extraction.

2.5.3.2. Molecular characterization

The molecular characterization of Toxocara spp. eggs retrieved from soil was performed according to a protocol previously described [25,38]. Pools of Toxocara spp. eggs retrieved from soil samples were grouped according to collection locations, disrupted using a commercial homogenizer (Scilogex D160, Rocky Hill, CT, USA) and incubated at 65 °C for 16 h with 20 μL of proteinase K solution. Next, genomic DNA was extracted following the manufacturer's instructions of a commercial kit (PureLink™ Microbiome DNA purification kit, Invitrogen, Waltham, MA, USA). For positive control samples, genomic DNA was extracted from eggs collected from female T. canis and T. cati parasites. The DNA concentration was measured using spectrophotometer by absorbance at 260/280 nm (NanoDrop 2000, Thermo Scientific, Waltham, MA, USA).

The polymerase chain reaction (PCR) was performed targeting the sequences of ITS1 and ITS2 of rDNA region, using the pairs of primers previously described [39] for T. canis (Forward: 5′-CTCGAGTCGACGAAGTATGTAC-3′; Reverse: 5′-AATTGGGCCGCCCATCATAC-3′), and T. cati (Forward: 5′-GTAAGATCGTGGCACGCGTACGTA-3′; Reverse: 5′-TCTTTGATGTCAAGACTTCAGCGC-3′).

Each PCR reaction was prepared with 10 μM of primers forward and reverse, 0.02 mM of deoxynucleotide, 30 mM of MgCl2, 2 μL of buffer (10× PCR Buffer), 1 U of Taq DNA polymerase (Invitrogen), and approximately 50 ng of DNA template. RNAse free water was added into reactions to complete 25 μl of volume. PCR was performed in a thermal cycler (Multigene, Labnet International, Edison, NJ, USA) under the following conditions: initial denaturation at 94 °C for 60 s, followed by 35 cycles at 94 °C for 60 s, 55 °C for 45 s and 72 °C for 30 s, and a final cycle at 72 °C for 5 min. Amplicons were visualized on an ethidium bromide stained 1.5 % agarose gel under ultraviolet light after electrophoresis run (80 V for 60 min).

2.6. Statistical analysis

Gathered socioepidemiological information were tabulated and categorized using the Excel commercial software (Excel, version 2401, Microsoft Co., Redmond, WA, USA) and statistical analyses conducted in R software version 4.2.2 [40]. The univariate analysis was performed using Chi-square test or Fisher's exact test to evaluate potential risk factors and seropositivity for Toxocara spp. in indigenous people. Odds ratios (OR) with 95 % confidence intervals were calculated, considering a p-value lower than 0.05 as statistically significant. Fisher's and Mann-Whitney tests were applied for the comparison of soil samples and the number of Toxocara spp. eggs recovered from these samples.

3. Results

3.1. Human

A total of 246/258 indigenous individuals (95.3 %; 95 % CI: 92.1–97.3) tested seropositive for Toxocara spp., consisting of 160/258 (62.0 %) females and 98/258 (38.0 %) males, with ages ranging from 2 to 101 years (median: 18 years). Participants aged ≤18 years represented 133/258 (51.6 %), while 117/258 (45.3 %) were >18 years old. Some participants (8/258; 3.1 %) were unable or refused to report their age or date of birth.

Regarding educational background, 107/258 (41.5 %) participants reported having studied up to elementary school, and 150/258 (58.1 %) had at least completed middle school. Behavioral and dietary habits revealed that 103/258 (39.9 %) individuals had the habit of game meat consumption, while 21/258 (8.1 %) reported consuming raw or undercooked meat. Artesian well was reported as water source by 65/258 (25.2 %) individuals, while the majority (184/258; 71.3 %) reported the use of other sources as river and spring water. Additionally, 227/258 (88.0 %) participants declared owning dog and 135/258 (52.3 %) owning cats. Nail-biting behavior was reported by 108/258 (41.9 %) individuals.

Univariate analysis was performed to assess the association between the presence of anti-Toxocara spp. antibodies and variables evaluated. However, no variable was considered statistically significant (Table 1).

Table 1.

Association between the presence of anti-Toxocara spp. antibodies (IgG) and characteristics of indigenous individuals (N = 258) in a community of Paraná state, Brazil, by univariate analysis.

	Positive n (%)	Negative n (%)	Univariate analysis
	246 (95.3)	12 (4.7)	OR (95 % CI)	p-overall
Variables
Gender				0.77
Female	153 (62.2)	7 (58.3)	1.0 [Reference]
Male	93 (37.8)	5 (41.7)	0.84(0.26–3.01)
Age (years old)				0.065
18 under	123 (51.7)	10 (83.3)	1.0 [Reference]
19 above	115 (48.3)	2 (16.7)	4.39(1.11–31.8)
Educational level				0.246
Middle school or above	145 (59.2)	5 (41.7)	1.0 [Reference]
Until elementary	100 (40.8)	7 (58.3)	0.50(0.14–1.64)
Water source				0.194
Artesian well	64 (27.0)	1 (8.33)	1.0 [Reference]
Others/river	173 (73.0)	11 (91.7)	0.28(0.01–1.49)
Game meat consumption				1
No	64 (26.7)	3 (27.3)	1.0 [Reference]
Yes	176 (73.3)	8 (72.7)	1.06(0.22–3.89)
Raw/undercooked meat consumption				1
No	197 (83.8)	10 (83.3)	1.0 [Reference]
Yes	38 (16.2)	2 (16.7)	0.91(0.22–6.70)
Owning dog				1
No	28 (11.5)	1 (8.33)	1.0 [Reference]
Yes	216 (88.5)	11 (91.7)	0.79(0.03–4.36)
Owning cat				0.624
No	114 (46.7)	7 (58.3)	1.0 [Reference]
Yes	130 (53.3)	5 (41.7)	1.58(0.48–5.63)
Nail biting				0.183
No	134 (57.3)	4 (33.3)	1.0 [Reference]
Yes	100 (42.7)	8 (66.7)	0.38 (0.10–1.27)
Contact with soil				1
No	24 (10.1)	1 (8.33)	1.0 [Reference]
Yes	214 (89.9)	11 (91.7)	0.91 (0.04–5.08)

3.2. Dogs

The flotation technique used for processing dog feces revealed the presence Toxocara spp. eggs in 8/124 (6.5 %) samples. No Toxocara spp. egg was retrieved from hair samples.

3.3. Soil

Overall, Toxocara spp. eggs. were found in 30/74 (40.5 %) soil samples, retrieved from 13/42 (30.9 %) samples from common areas and 17/32 (53.1 %) from 16 household locations (2 samples per house). Toxocara spp. eggs were found in soil samples from all six common areas (school, playground, health center, playing field, praying house) and from 11/16 (68.7 %) assessed houses.

A total amount of 388 (average of 5 eggs/20 g of soil) Toxocara spp. eggs were recovered from soil samples, mostly classified as embryonated egg (303/388; 78.0 %) containing larva, followed by viable (53/388; 13.6 %), non-viable (19/388; 4.9 %) and embryonating eggs (13/388; 3.3 %). A higher number of Toxocara spp. eggs were retrieved from household locations (370 eggs in total; average of 11 eggs/20 g of soil) than from common areas (18 eggs in total; average of 0.4 eggs/20 g soil).

No statistical difference was observed comparing the proportion of positive soil samples in common areas and household locations (p = 0.061). However, the number of Toxocara spp. eggs recovered from household locations was significantly higher than from the common areas (p = 0.001).

A pool of eggs recovered from soil in each positive area (n = 17) was submitted to DNA extraction and conventional PCR for Toxocara canis and Toxocara cati. Only T. canis DNA was amplified in 13/17 positive locations. In the remaining 4 locations, neither T. canis nor T. cati DNA was detected, despite being considered positive under microscopy analysis. The number of Toxocara spp. eggs found in each location and the conventional PCR result were gathered and presented (Table 2).

Table 2.

Toxocara spp. eggs recovered from soil samples collected from common areas and household locations in an indigenous community of Paraná State (Southern Brazil) according to morphological characteristics and conventional PCR (cPCR) results for T. canis.

	Positive samples/total (%)	Morphological characteristics of Toxocara spp. eggs recovered from soil (n = 388 eggs)				cPCR result
		V	NV	EM	E
Common areas
School	4/6 (66.6)	1	2	0	1	Positive
Playground	2/6 (33.3)	4	0	0	0	Positive
Health center	1/6 (16.6)	0	1	0	0	Negative
Playing fields	1/15 (6.6)	0	1	0	0	Positive
Praying house	2/5 (40.0)	0	2	0	0	Positive
Trail	3/4 (75.0)	2	4	0	0	Negative
Total	13/42 (30.9)	7	10	0	1
Household locations
House 1	0/2 (0.0)	0	0	0	0	–
House 2	2/2 (100.0)	3	1	4	0	Positive
House 3	1/2 (50.0)	0	1	0	1	Positive
House 4	1/2 (50.0)	0	1	0	0	Positive
House 5	0/2 (0.0)	0	0	0	0	–
House 6	0/2 (0.0)	0	0	0	0	–
House 7	1/2 (50.0)	0	1	0	0	Negative
House 8	0/2 (0.0)	0	0	0	0	–
House 9	2/2 (100.0)	20	1	4	59	Positive
House 10	2/2 (100.0)	5	0	3	13	Negative
House 11	1/2 (50.0)	0	0	1	0	Positive
House 12	2/2 (100.0)	6	2	1	217	Positive
House 13	2/2 (100.0)	12	0	0	0	Positive
House 14	0/2 (0.0)	0	0	0	0	–
House 15	1/2 (50.0)	0	2	0	0	Positive
House 16	2/2 (100.0)	0	0	0	12	Positive
Total	17/32 (53.1)	46	9	13	302

V: viable; NV: non-viable; EM: embryonated egg; E: embryonated (containing larvae).

4. Discussion

This study has assessed human toxocariasis, infection of dogs, and the contamination of soil associated risk factors in an indigenous community with high migratory flow, located nearby to the tri-border of Brazil, Paraguay and Argentina, based on a One Health perspective. To the authors knowledge, the 95.3 % human seroprevalence observed herein was the highest reported to date worldwide, surpassing the other highest rates including 359/387 (92.8 %) individuals in Africa [41]; 172/208 (82.7 %) individuals of quilombola communities in Brazil [42]; and 383/483 (79.3 %) individuals of the Wiwa indigenous community in Colombia [43]. The seroprevalence herein was significantly higher than the overall 73.9 % (342/463) seropositivity reported by our research group in nine indigenous communities distributed in southern and southeastern Brazil [38].

The high number of seropositive indigenous individuals in the present study has indicated a high level of exposure to Toxocara spp., which has impaired the assessment and testing of associated risk factors. Although Toxocara spp. exposure has been associated with rural areas [3], rural populations in Brazil have shown wide seropositivity rates, ranging from 14.4 % [44] to 71.8 % [45] and related to poor living standards and lack of basic sanitary conditions, which may predispose toxocariasis transmission [25,46,47]. In addition, Guarani indigenous communities have presented mortality rates mainly caused by preventable causes (51.6 %), including respiratory (40.6 %), infectious and parasitic diseases (18.8 %), indicating higher disease exposure of indigenous peoples when compared to the general population [48]. In this study, health inequalities included lack of potable water, low sanitation and subsistence conditions, and limited access and quality of healthcare assistance [48]. Thus, such distinct pattern of the Guarani indigenous population may have played a role in increasing exposure to Toxocara spp. and potentially to other soil-transmitted helminths.

Others variables have also been associated to toxocariasis, such as male gender [49], younger age [3,50], low educational level [[51], [52], [53]], low income [54], dietary habits (e.g., consuming raw/undercooked meat) [55,56] and contact with soil [50,57,58]. Although an individual epidemiological questionnaire was applied to identify such associated risk factors for Toxocara spp. exposure in this studied community, the high 95.3 % seropositivity has statistically compromised the comparison between groups and assessment of the association between seropositivity and epidemiological variables. Nonetheless, the highest seroprevalence worldwide to date observed herein has warned for the severe exposure and potential health risks faced by this vulnerable population. Despite the highest seroprevalence for Toxocara spp. observed herein, all individuals were considered clinically health at the time of sampling.

Although the ELISA test has been widely employed in seroprevalence studies for detecting anti- Toxocara antibodies, with a 78.3 % sensitivity and 92.3 % specificity [32,33], cross-reaction with other ascaridia may lead to over or underestimate results, biasing the comparison among serosurveys studies. The protocol herein was based on a pre-adsorption of sera with A. suum adult worm extract to reduce cross-reactivity with other Ascaridida, including A. lumbricoides [31]. This protocol has also been used to assess seropositivity in different populations and considered reliable, including in surveys of people living in rural settlements [44,46,52], children [42], adult blood donors [59], persons experiencing homelessness [25], indigenous individuals [38], women deprived of liberty [60], quilombola individuals [49], and pregnant women [58,61], with seropositivity ranging from 11.1 % [42] to 82.7 % [60].

Toxocara spp. eggs are commonly found in the feces of infected dogs and cats, contaminating soil of areas where these animals are present [5]. In this context, contact with soil has been considered an important risk factor for toxocariasis [3,41,49,50], as Toxocara spp. eggs can survive in soil for several years under favorable temperature and humidity conditions [62]. This is particularly relevant for individuals living in rural settings, working in agricultural activities and for children playing with soil [41,50,63]. The soil contamination by Toxocara spp. eggs has been reported in rural villages [64], indigenous communities [38] and quilombola communities [49]. Herein, Toxocara spp. eggs. were found in 30/74 (40.5 %) of analyzed soil samples, in a similar rate of 40.0 % (36/90) previously observed in soil samples collected from common areas in nine indigenous communities located in the Paraná and São Paulo states [38]. In addition to the samples collected from common areas, samples from household locations were also evaluated, resulting in a contamination rate of 17/32 (53.1 %), presenting a higher number of infective eggs in household areas than in common settings. Molecular characterization of positive locations revealed only T. canis amplicons, as previously detected in indigenous communities of southern Brazil [38]. Herein, statistical analysis interpretation suggested that despite similar proportions of positive samples, household areas may present higher presence of T. canis eggs than common areas. As no domesticated species were present in Brazilian indigenous communities before European invasions in early 1500s [23], human toxocariasis observed herein may have been direct consequence of dog and cat presence in indigenous communities, which may have unbalanced, along with other multiple causes, the One Health equilibrium of 20–30,000 years.

The use of unfiltered (unpurified) water has been considered a significant risk factor for toxocariasis, as water may be contaminated with infective Toxocara spp. eggs [50]. Brazilian indigenous communities with inadequate sanitary infrastructure were 2.72-fold more likely seropositive, and the use of river water instead artesian wells increased in 11.4-fold the seropositivity risk in these populations [38]. Herein, despite the water source was not identified as a risk factor, most indigenous individuals (71.3 %) reported the use of these risky sources as river and spring water, indicating water source as potential contribution to the high human seropositivity.

Direct contact with dogs and cats, particularly not regularly dewormed or cleaned, has increased the risk of human toxocariasis as Toxocara eggs into feces and on the fur may be sources of infection [34,37,65]. Herein, no Toxocara spp. egg was retrieved from dog hair samples, indicating that direct dog fur contact may not be a major transmission route in this indigenous community. Results of the present study corroborated to quilombola communities in Brazil, which found few or no positive dog hair samples [49]. However, 22/104 (21.15 %) dog hair samples presented eggs of Toxocara spp. in traditional fisherman communities of southern Brazil [66].

Only 8/124 (6.45 %) dog feces samples herein were positive for Toxocara canis, similar to 9/194 (4.6 %) found in other indigenous communities [38]; 5/96 (5.2 %) in quilombola communities [49]; and 12/115 (10.43 %) in seashore areas in Brazil [66]. In addition to the low frequency of Toxocara spp. generally found in adult dog feces, result herein may be underestimated since puppies were not sampled due to sanitary and animal welfare reasons. Puppies have a significantly higher percentage of adult Toxocara canis worms in their intestines (80 %) compared to adults (22.5 %) [37], mostly due to transplacental transmission, where the infection may vertically pass from the mother to the puppies before birth [67].

As limitation, no cat feces were sampled in the present study. Cats from the studied community herein were mostly free-range and feral cats. Thus, due to stress and unknown health conditions of feral cats, catching and restrain were not conducted. In the present study, no T. cati was identified in the soil samples. Previous studies have shown that T. cati recovery in soil samples of urban areas, specially parks, was higher than in rural areas [35,[68], [69], [70]]. Cats have the habit of burying their feces in soil and sand, which may explain the lack of T. cati DNA amplification in such rural area herein [71]. Also, as DNA was amplified from a pool of recovered eggs, T. cati may not be excluded. In addition, the land-use type and precipitation may affect the distribution of T. canis and T. cati eggs in soil samples [72]. For example, T. cati eggs buried along with the feces by cats may be protected from desiccation and adverse weather conditions, prolonging the longevity and the timeframe for human and cat transmission [73]. Thus, further studies should also consider soil collection during all year-long seasons and throughout the different landscapes and soil uses.

In summary, the present study has revealed the highest seroprevalence of toxocariasis reported worldwide to date, found in an indigenous community from a tri-border area in southern Brazil. Despite the absence of risk factors due to impaired assessment of statistical analysis, the significant number of infective eggs found in soil has reinforced the role of environmental exposure in sustaining transmission in this community. The high seropositivity herein may be also consequence of Guarani migratory culture and additional exposure to Toxocara sp. during tekoa movements in the tri-border area of Brazil, Paraguay and Argentina. Since the treatment of soil contaminated by Toxocara spp. eggs has not been well stablished, the prevention of soil contamination and hygienic practices remains the better available choice. In this way, interventions targeting environmental sanitation, regular deworming of dogs, and health education on toxocariasis and other zoonotic diseases may be crucial for a better human, animal and environmental health (One Health) of these indigenous communities.

In conclusion, the One Health approach applied herein has emphasized the interconnection of human, animal, and environmental health, highlighting the need for comprehensive strategies to address the toxocariasis transmission and health impact, particularly within vulnerable populations as indigenous communities. Further studies involving indigenous populations should always consider the ethnicity, its culture and habits to assess pathogen transmission routes and apply effective actions along community strategies to successfully monitor, control and prevent toxocariasis and other pet-related zoonotic diseases.

CRediT authorship contribution statement

Isabella Braghin Ferreira: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Roberto Teixeira de Souza Filho: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis. Susana Angélica Zevallos Lescano: Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis. Rogério Giuffrida: Writing – review & editing, Writing – original draft, Validation, Software, Methodology, Investigation, Formal analysis, Conceptualization. Daniele Rodrigues: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Data curation, Conceptualization. Suelen Teixeira de Faria Resende: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Data curation, Conceptualization. Fabiano Borges Figueiredo: Writing – review & editing, Writing – original draft, Resources, Methodology, Investigation, Funding acquisition, Conceptualization. Louise Bach Kmetiuk: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Data curation, Conceptualization. Andrea Pires dos Santos: Writing – review & editing, Writing – original draft, Supervision, Methodology, Investigation, Conceptualization. Alexander Welker Biondo: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization. Vamilton Alvares Santarém: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Funding

No funding.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.