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Revista Brasileira de Parasitologia Veterinária

Print version ISSN 0103-846XOn-line version ISSN 1984-2961

Rev. Bras. Parasitol. Vet. vol.31 no.1 Jaboticabal  2022  Epub Apr 01, 2022

https://doi.org/10.1590/s1984-29612022016 

Original Article

Cytochrome c oxidase subunit 1 gene reveals species composition and phylogenetic relationships of Oesophagostomum spp. infecting pigs in northeastern Brazil

Inferências filogenéticas e caracterização de Oesophagostomum spp. parasitos de suínos no estado do Piauí, Brasil, por sequenciamento parcial de DNA mitocondrial

Polyanna Araújo Alves Bacelar1  2 

Kerla Joeline Lima Monteiro1  2  * 
http://orcid.org/0000-0002-5024-2628

Deiviane Aparecida Calegar1 

Jéssica Pereira dos Santos1  2 

Beatriz Coronato-Nunes3 

Elis Regina Chaves dos Reis4 

Márcio Neves Bóia5 

Lauren Hubert Jaeger6 

Filipe Anibal Carvalho-Costa1 

1Laboratório de Epidemiologia e Sistemática Molecular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz – Fiocruz, Rio de Janeiro, RJ, Brasil

2Escritório Técnico Regional, Fundação Oswaldo Cruz – Fiocruz, Teresina, PI, Brasil

3Faculdade de Medicina de Petrópolis – FMP, Centro Universitário Arthur Sá Earp Neto – UNIFASE, Petrópolis, RJ, Brasil

4Secretaria Municipal de Saúde de Nossa Senhora de Nazaré, Nossa Senhora de Nazaré, PI, Brasil

5Laboratório de Biologia e Parasitologia de Mamíferos Silvestres Reservatórios, Instituto Oswaldo Cruz – Fiocruz, Fundação Oswaldo Cruz – Fiocruz, Rio de Janeiro, RJ, Brasil

6Departamento de Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal de Juiz de Fora – UFJF, Juiz de Fora, MG, Brasil


Abstract

Helminths of the genus Oesophagostomum cause enteric diseases and affect domestic animals such as pigs. The aim of this study was to explore the species composition and genetic diversity of Oesophagostomum spp. infecting pigs in close contact with humans in the state of Piauí, Brazil. Eighty-seven fecal samples were collected for parasitological tests and molecular analysis. Through microscopy, the overall positivity rate for strongyliform eggs was 81.6% among the pigs studied. Forty-two strongyliform egg samples were subjected to PCR and six cox1 sequences (637 bp) were identified for the genus Oesophagostomum. The sequences were identified as Oesophagostomum dentatum, O. quadrispinulatum and O. columbianum. In the phylogenetic tree and haplotype network, 89 sequences were separated into seven clusters, which also included reference sequences from GenBank. Oesophagostomum dentatum and O. quadrispinulatum were seen to be closely related species and formed a monophyletic group related to O. aculeatum. Oesophagostomum columbianum showed similarity with sequences from parasites infecting small ruminants and the clade was positioned closer to O. bifurcum. High interspecific diversity was found and intraspecific diversity varied according to the species. This was the first study to characterize Oesophagostomum DNA sequences obtained from pigs in Brazil.

Keywords:  DNA barcode; Oesophagostomum; molecular taxonomy; pigs

Resumo

Parasitos do gênero Oesophagostomum causam doenças entéricas e podem afetar a criação de animais, como os suínos. O objetivo deste estudo foi identificar as espécies e explorar a diversidade genética de Oesophagostomum spp. infectando suínos em contato próximo com humanos, no estado do Piauí, Brasil. Oitenta e sete amostras fecais foram coletadas para testes parasitológicos, análise morfométrica dos ovos e análises moleculares. A taxa geral de positividade para ovos estrongiliformes foi de 81,6%. Quarenta e duas amostras de ovos estrongiliformes foram submetidas à PCR e seis sequências cox1 (637 bp) foram identificadas para o gênero Oesophagostomum. As sequências foram identificadas como Oesophagostomum dentatum, O. quadrispinulatum e O. columbianum. Na árvore filogenética e na rede haplotípica, 89 sequências foram separadas em sete clusters, incluindo sequências de referência do GenBank. Oesophagostomum dentatum e O. quadrispinulatum são espécies estreitamente relacionadas e formaram um grupo monofilético com O. aculeatum. Oesophagostomum columbianum apresentou semelhança com sequências de parasitas obtidos de pequenos ruminantes e o clado foi posicionado mais próximo de O. bifurcum. Alta diversidade interespecífica foi encontrada e a diversidade intraespecífica variou de acordo com as espécies. Esse foi o primeiro estudo a caracterizar sequências de DNA de Oesophagostomum isoladas de suínos no Brasil.

Palavras-chave:  Código de barras de DNA; Oesophagostomum; taxonomia molecular; suíno

Introduction

The genus Oesophagostomum (family Chabertiidae, order Strongylida) includes worms ranging in length from 6.5 to 24 mm. The anterior end of the body presents a shallow oral cavity, a dilated cuticle forming a cephalic vesicle and a radiated crown present with varied structure (Taylor et al., 2010). Species within this genus are hosted by a wide diversity of mammals.

Considering only the species with the greatest impact on public health and veterinary medicine, pigs can be infected with the species O. dentatum and O. quadrispinulatum (Lin et al., 2014). Oesophagostomum bifurcum, O. stephanostomum, O. brumpti and O. aculeatum have previously been described in human and non-human primates (Glen & Brooks, 1985); and O. columbianum, O. venulosum and O. asperum in sheep and goats (Gaddam et al., 2017). Despite the first human case of ‘O. stephanostomum’ infection was recorded from Brazil, the actual systematic position of this worm has not been settled (Railliet & Henry, 1910; Thomas, 1910).

Oesophagostomiasis causes economic losses in pig farming, affecting both large and small producers (Li et al., 2017). Despite differences in size, Strongylida eggs are similar in morphology, which makes it difficult to identify species with parasitological examinations. Although obtaining larvae through stool cultures helps in making genus-specific diagnoses, species identification remains limited (Benavides et al., 2007).

To overcome these limitations, molecular taxonomy through DNA sequencing is widely applied to diagnoses, to enable phylogenetic reconstructions and evolutionary deductions of infectious agents (De & Bandyopadhyay, 2008). The objective of the present study is to explore the species composition and genetic diversity of Oesophagostomum infecting pigs in rural communities in northeastern Brazil.

Material and Methods

The fieldwork was performed from 2014 to 2017 in two regions in the state of Piauí, northeastern Brazil: Nossa Senhora de Nazaré (NSN) and Teresina (TER), in periurban and rural low-resource communities (Figure 1). Fecal samples from pigs (n = 78 in NSN and 9 in TER) were collected after spontaneous defecation, on the ground. The pigs were in rudimentary shelter or in peridomestic or domestic areas during the field visits. These samples were individually stored in properly identified plastic bags, placed in a container with ice and sent to the field laboratory for parasitological examinations. The feces were processed using the Ritchie method (centrifugal sedimentation with ethyl acetate) and sucrose flotation. Samples that contained helminth eggs were then frozen until DNA extraction.

Figure 1 Geographical location of the study in Piauí state, northeastern Brazil. 

Genomic DNA was extracted from 42 fecal samples positive for strongyliform eggs using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany), in accordance with the manufacturer's instructions. The partial cytochrome c oxidase subunit 1 (cox1) gene was amplified using the Platinum Taq DNA polymerase (Invitrogen, Waltham, MA, USA) with a final volume of 50 µL. A cocktail of three primer pairs that recover cox1 barcodes from diverse nematode lineages parasitic on vertebrates, including members of three orders and eight families was used (Prosser et al., 2013). The PCR conditions were as follows: initial denaturation at 94 ºC for 5 min, followed by 35 cycles of 94 ºC for 40 s, 55 ºC for 40 s, 72 ºC for 1 min and a final extension at 72 ºC for 5 min. The PCR products were purified using the DNA Illustra GFX PCR and gel band purification kit (GE HealthCare, Pittsburgh, USA) and were subjected to sequencing of both strands of DNA using the BigDye Terminator v. 3.1 cycle sequencing kit (Thermo Fisher Scientific, Foster City, USA) using the following primers: M13F 5'- TGT AAA ACG ACG GCC AGT – 3' (forward) and M13R 5'- CAG GAA ACA GCT ATG AC – 3' (reverse) (Messing, 1993). Capillary electrophoresis was performed using an ABI 3730 automated DNA sequencer (Applied Biosystems). Sequences showing overlapping peaks on electropherograms were cloned using the pGEM®-T Easy vector system (Promega, Madison, WI, USA) with Escherichia coli DH5-alpha cells in brain heart infusion broth (Sigma-Aldrich, St. Louis, MO, USA) on disposable plates. After cloning, the PCR and sequencing conditions were performed using the universal primers T7 5'-TAA TAC GAC TCA CTA TAG G - 3' (forward) and SP6 5'-GAT TTA GGT GAC ACT ATA G - 3' (reverse).

The Bioedit software v.7.0.4 (Hall, 1999) was used to edit the nucleotide sequences (401 bp). The Basic Local Alignment Search Tool (BLAST) (NCBI, 2022) was used to verify the similarity of the nucleotides with sequences of nematodes from GenBank. Orthologous sequences (n = 83) were retrieved from GenBank (Table 1) and sequences with degenerate bases were not included. The sequences obtained in the study (n = 6) were deposited in GenBank under accession numbers MK282837 to 42.

Table 1 Oesophagostomum spp. cox1 reference sequences used in this study (n = 83).  

Species Host GenBank Countries Reference
Species accession number
O. aculeatum Macaca fuscata LC063900 Japan Ota et al. (2015)
O. aculeatum Macaca fascicularis LC428762 Malaysia Frias et al. (2019)
O. aculeatum Macaca fascicularis LC428765 Malaysia Frias et al. (2019)
O. aculeatum Macaca fascicularis LC428773 Malaysia Frias et al. (2019)
O. aculeatum Macaca fascicularis LC428776 Malaysia Frias et al. (2019)
O. aculeatum Macaca fascicularis LC428777 Malaysia Frias et al. (2019)
O. aculeatum Macaca fascicularis LC428778 Malaysia Frias et al. (2019)
O. aculeatum Macaca fascicularis LC428779 Malaysia Frias et al. (2019)
O. aculeatum Macaca fascicularis LC428781 Malaysia Frias et al. (2019)
O. aculeatum Macaca fascicularis LC428782 Malaysia Frias et al. (2019)
O. aculeatum Macaca fascicularis LC428783 Malaysia Frias et al. (2019)
O. aculeatum Nasalis larvatus LC428786 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428789 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428791 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428792 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428793 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428794 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428796 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428797 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428798 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428800 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428801 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428803 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428807 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428809 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428811 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428812 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428813 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428815 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428816 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428817 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428818 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428819 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428820 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428821 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428822 Malaysia Frias et al. (2019)
O. aculeatum Pongo pygmaeus LC428823 Malaysia Frias et al. (2019)
O. stephanostomum Pan troglodytes schweinfurthii LC063867 Uganda Ota et al. (2015)
O. stephanostomum Pan troglodytes schweinfurthii LC063868 Uganda Ota et al. (2015)
O. stephanostomum Pan troglodytes schweinfurthii LC063871 Uganda Ota et al. (2015)
O. stephanostomum Pan troglodytes schweinfurthii LC063876 Uganda Ota et al. (2015)
O. stephanostomum Pan troglodytes schweinfurthii LC063877 Uganda Ota et al. (2015)
O. stephanostomum Pan troglodytes schweinfurthii LC063879 Uganda Ota et al. (2015)
O. stephanostomum Pan troglodytes schweinfurthii LC063881 Uganda Ota et al. (2015)
O. stephanostomum Pan troglodytes schweinfurthii LC063882 Uganda Ota et al. (2015)
O. stephanostomum Pan troglodytes schweinfurthii LC063883 Uganda Ota et al. (2015)
O. stephanostomum Pan troglodytes schweinfurthii LC063886 Uganda Ota et al. (2015)
O. stephanostomum Pan troglodytes schweinfurthii LC063887 Uganda Ota et al. (2015)
O. stephanostomum Pan troglodytes schweinfurthii LC063888 Uganda Ota et al. (2015)
O. stephanostomum Gorilla gorilla gorilla AB821032 Gabon Makouloutou et al. (2014)
O. stephanostomum Gorilla gorilla gorilla AB821033 Gabon Makouloutou et al. (2014)
O. stephanostomum Gorilla gorilla gorilla AB821034 Gabon Makouloutou et al. (2014)
O. stephanostomum Gorilla gorilla gorilla AB821036 Gabon Makouloutou et al. (2014)
O. stephanostomum Gorilla gorilla gorilla AB821037 Gabon Makouloutou et al. (2014)
O. stephanostomum Gorilla gorilla gorilla AB821038 Gabon Makouloutou et al. (2014)
O. stephanostomum Gorilla gorilla gorilla AB821039 Gabon Makouloutou et al. (2014)
O. stephanostomum Gorilla gorilla gorilla AB821040 Gabon Makouloutou et al. (2014)
O. stephanostomum Gorilla gorilla gorilla AB821041 Gabon Makouloutou et al. (2014)
O. stephanostomum Gorilla gorilla gorilla AB821042 Gabon Makouloutou et al. (2014)
O. stephanostomum Pan troglodytes AB821044 Gabon Makouloutou et al. (2014)
O. columbianum Sheep KC715827 China Zhao et al. (2013)
O. columbianum Sheep NC 023933 China Zhao et al. (2013)
Oesophagostomum sp. Ovis aries MK282868 Brazil Monteiro et al. (unpublished data)
Oesophagostomum sp. Ovis aries MK282869 Brazil Monteiro et al. (unpublished data)
Oesophagostomum sp. Capra hircus MK282872 Brazil Monteiro et al. (unpublished data)
O. muntiacum Muntiacus reevesi LC415114 Japan Setsuda et al. (2020)
O. asperum Goat NC_023932 China Zhao et al. (2013)
O. dentatum Pig FM161882 China Lin et al. (2012b)
O. quadrispinulatum Pig FM161883 China Lin et al. (2012b)
O. bifurcum Pan troglodytes schweinfurthii LC063862 Uganda Ota et al. (2015)
O. bifurcum Pan troglodytes schweinfurthii LC063863 Uganda Ota et al. (2015)
O. bifurcum Pan troglodytes schweinfurthii LC063864 Uganda Ota et al. (2015)
O. bifurcum Pan troglodytes schweinfurthii LC063865 Uganda Ota et al. (2015)
O. bifurcum Papio ursinus LC063889 South Africa Ota et al. (2015)
O. bifurcum Papio ursinus LC063890 South Africa Ota et al. (2015)
O. bifurcum Papio ursinus LC063891 South Africa Ota et al. (2015)
O. bifurcum Papio cynocephalus LC063892 Tanzania Ota et al. (2015)
O. bifurcum Papio cynocephalus LC063893 Tanzania Ota et al. (2015)
O. bifurcum Papio cynocephalus LC063894 Tanzania Ota et al. (2015)
O. bifurcum Papio cynocephalus LC063895 Tanzania Ota et al. (2015)
O. bifurcum Papio cynocephalus LC063896 Tanzania Ota et al. (2015)
O. bifurcum Papio cynocephalus LC063897 Tanzania Ota et al. (2015)
O. bifurcum Papio cynocephalus LC063898 Tanzania Ota et al. (2015)

*Unpublished

Phylogenetic inferences were made using the Molecular Evolutionary Genetics Analysis (MEGA) v.7.0.20 software (Kumar et al., 2016). The maximum likelihood (ML) method was applied and the Hasegawa-Kishino-Yano model (HKY) with gamma distribution (G) (Hasegawa et al., 1985) was selected using the Bayesian information criterion (BIC) in MEGA v.7.0.20 (Kumar et al., 2016). The clade stability of the cox1 sequence tag topologies was evaluated using 1,000 bootstrap replicates.

The relationships in the haplotype network were inferred through median joining using the Network v.10.2.0 and DnaSP v.6 software (Rozas et al., 2017). Free vector images were used in the figures of the phylogenetic tree and haplotype network, to represent the hosts (Flaticon, 2022; SVG SILH, 2022). The genetic diversity indexes of Oesophagostomum populations were calculated using the Arlequin v.5.2.2 software (Excofer & Lischer, 2010). The Fst fixation index was determined on all populations, using the Arlequin v.5.2.2 software to estimate the genetic differentiation among populations, with a significance of 1,000 permutations (Excofer & Lischer, 2010). This study was approved by the Ethics Committee for the Use of Animals (LW-21/13 [P-4/13.3]) of Instituto Oswaldo Cruz (Fiocruz).

Results

Eighty-seven fecal samples from pigs were analyzed and the positivity rate for strongyliform eggs through microscopy was 81.6% (71/87). Six sequences (637 bp) were identified as belonging to the genus Oesophagostomum. From NSN, four samples were characterized as O. quadrispinulatum and one as O. columbianum. In TER, one sample was characterized as O. dentatum. Three cox1 sequences showed overlapping peaks, thus indicating the presence of more than one species or genotype. Cloning of the fragments enabled identification of the species O. quadrispinulatum and O. dentatum. Fecal samples positive for strongyliform eggs which were negative for Oesophagostomum in the molecular analysis allowed the identification of Trichostrongylus sp. (n=1) and Metastrongylus spp. (n=10) through cox1 sequencing. Therefore, no Oesophagostomum species previously characterized in humans was found in the studied swine populations.

Alignment of the sequences of the present study in relation to 83 Oesophagostomum spp. reference sequences (401 bp cox1 sequences) from GenBank (Table 1) was performed. The ML phylogenetic tree (Figure 2) showed that the Oesophagostomum sequences were organized into three main groups. These groups included seven clades: i) clade A containing 37 sequences of O. aculeatum from Japan and Malaysia, identified in non-human primates (Macaca fuscata, Macaca fascicularis, Nasalis larvatus and Pongo pygmaeus); ii) clade B containing two sequences from O. dentatum from China and Brazil (TER) and five from O. quadrispinulatum from China and Brazil (NSN), all from pig hosts; iii) clade C containing 23 sequences of O. stephanostomum from Uganda and Gabon, detected in non-human primate hosts (Pan troglodytes, Pan troglodytes schweinfurthii and Gorilla gorilla gorilla); iv) clade D containing three sequences of O. columbianum from Asia and Brazil (NSN) identified in goats and one pig, and three of Oesophagostomum sp. from sheep and goats in Brazil; v) clade E containing a sequence of O. asperum from China, detected in a goat; vi) clade F containing a sequence of O. muntiacum from Japan, observed in a deer (Muntiacus reevesi); and vii) clade G containing 13 sequences of O. bifurcum from Uganda, Tanzania and South Africa, from non-human primate hosts (Pan troglodytes schweinfurthii, Papio ursinus and Papio cynocephalus).

Figure 2 Maximum likelihood (ML) tree constructed using 401 bp cox1 sequences of Oesophagostomum spp.. Support for the branching order was determined by means of 1,000 bootstrap replicates, and only values > 70% were reported.  

In the study area, O. quadrispinulatum was the predominant species. The sequences of O. quadrispinulatum and O. dentatum were grouped in the same clade, in which there were only sequences from pigs (showing 99% similarity with the reference sequences), and were more closely related to O. aculeatum (cluster A). The O. columbianum sequence in the present study was grouped into the same clade as parasites obtained from small ruminants. Interestingly, three sequences that clustered close to O. columbianum (Figure 2) were located in another arm of the tree (showing 99% similarity with the reference sequence).

The haplotype network (Figure 3) based on the cox1 locus showed topology similar to the phylogenetic tree. The 89 sequences used in the phylogenetic analyses were distributed into 86 haplotypes (Table 2). Six novel haplotypes of Oesophagostomum were identified in the present study. The species O. aculeatum showed a star shape, with a central haplotype, which has been identified in Asia. In general, the groups had long arms due to the number of polymorphisms identified among the species. Genetic diversity indices revealed high interspecific diversity in the genus Oesophagostomum, with H ± SD = 0.9992 ± 0.0018 and 129 polymorphic sites (Table 2). The intraspecific diversity varied according to the species. Oesophagostomum columbianum showed the lowest intraspecific variability with H ± SD = 0.6667 ± 0.3143 and 4 polymorphic sites. The genetic divergence (Fst) results were similar to the genetic diversity analyses (Table 3). The intraspecific divergence among specimens of O. columbianum was greater than the interspecific divergence of the samples analyzed (O. columbianum Asia Fst = 1; O. bifurcum Fst = 0.55). Furthermore, the Fst value between O. quadrispinulatum and O. dentatum was high: Fst = 1.

Figure 3 Maximum likelihood (ML) network of 401 bp cox1 locus of Oesophagostomum spp. The area of the circle was proportional to the sequence number. 

Table 2 Molecular diversity indexes of Oesophagostomum spp. based on cox1 locus (401 bp, n = 89). 

Species (N) Region (N) Statistics
H ± SD Nº of haplotypes Nº of polymorphic sites Nº of substitutions Nº of transitions Nº of transversions
O. aculeatum (37) Asia (37) 1.0000 ± 0.0063 37 56 56 51 10
O. bifurcum (14) Africa (14) 0.9890 ± 0.0314 13 54 54 47 9
O. stephanostomum (23) Africa (23) 0.9960 ± 0.0142 22 51 54 48 6
Oesophagostomum sp. (3) South America (3) 1.0000 ± 0.2722 3 2 2 2 0
O. columbianum** (3) All (3) 0.6667 ± 0.3143 2 4 4 4 0
Asia (2) 0.0000 ± 0.0000 1 0 0 0 0
O. dentatum** (2) All (2) 1.0000 ± 0.5000 2 7 7 6 1
O. quadrispinulatum** (5) All (5) 1.0000 ± 0.1265 5 10 10 8 2
Brazil (4) 1.0000 ± 0.1768 4 5 5 4 1
All* (89) 0.9992 ± 0.0018 86 129 129 99 63

H ± SD: gene diversity ± standard deviation;

All*: O. aculeatum, O. asperum, O. bifurcum, O. muntiacum, O. stephanostomum, Oesophagostomum sp., O. columbianum, O. dentatum, O. quadrispinulatum. Further details of reference sequences can be found in Table 1. In bold: sequences obtained in this study (Brazil);

**Groups formed only by one sequence have been removed.

Table 3 Population pairwise Fst values based on cox1 Oesophagostomum spp. (401 bp, n = 89). 

Population O. aculeatum_Asia O. asperum_Asia O. bifurcum_Africa O. muntiacum_Asia O. stephanostomum_Africa Oesophagostomum sp._ O. columbianum_ O. columbianum_Asia O. columbianum_Brazil O. dentatum_ O. dentatum_Asia O. dentatum_Brazil O. quadrispinulatum_ Asia and Brazil O. quadrispinulatum_Asia O. quadrispinulatum_Brazil All*
South America Asia and Brazil Asia and Brazil
O. aculeatum_Asia
O. asperum_Asia 0.83 0.00
O. bifurcum_Africa 0.75 0.57 0.00
O. muntiacum_Asia 0.84 1 0.60 0.00
O. stephanostomum_Africa 0.78 0.65 0.64 0.68 0.00
Oesophagostomum sp._South America 0.87 0.97 0.66 0.97 0.74 0.00
O. columbianum_Asia and Brazil 0.86 0.94 0.64 0.94 0.69 0.95 0.00
O. columbianum_Asia 0.86 1 0.63 1 0.68 0.98 -0.20 0.00
O. columbianum_Brazil 0.84 1 0.55 1 0.65 0.96 0.00 1 0.00
O. dentatum_Asia and Brazil 0.83 0.86 0.67 0.87 0.69 0.94 0.92 0.93 0.86 0.00
O. dentatum_Asia 0.82 1 0.61 1 0.65 0.97 0.94 1 1 -1 0.00
O. dentatum_Brazil 0.83 1 0.65 1 0.69 0.97 0.95 1 1 -1 1 0.00
O. quadrispinulatum_ Asia and Brazil 0.85 0.90 0.70 0.90 0.73 0.94 0.92 0.92 0.90 0.88 0.88 0.89 0.00
O. quadrispinulatum_Asia 0.84 1 0.60 1.00 0.69 0.98 0.95 1 1 0.84 1 1 0.20 0.00
O. quadrispinulatum_Brazil 0.85 0.94 0.69 0.94 0.72 0.96 0.94 0.95 0.94 0.91 0.93 0.93 -0.19 0.60 0.00
All* 0.21 0.14 0.26 0.19 0.28 0.40 0.34 0.31 0.14 0.29 0.13 0.20 0.36 0.18 0.35 0.00

*O. aculeatum, O. asperum, O. bifurcum, O. columbianum, O. dentatum, O. muntiacum, O. quadrispinulatum, O. stephanostomum, Oesophagostomum sp.;

In bold: sequences obtained in this study (Brazil).

Discussion

In the present study, the proportion of fecal samples from pigs that were positive for strongyliform eggs was higher than was found in Colombia (12.9%; 36/279) (Pinilla et al., 2020), India (19.9%; 74/371) (Yadav et al., 2021) and other region of Brazil, where it reached 46.6% (41/88) (Barbosa et al., 2015). Considering that the rearing systems reported by these authors were also extensive, our results may be explained by the handling and hygiene conditions of the pigs in the areas studied. Factors such as type of rearing, non-disinfection of drinking fountains and non-deworming are related to high frequencies of enteric helminths in pigs (Nansen & Roepstorff, 1999).

Making diagnoses based only on observation of eggs can lead to erroneous results due to similarities in morphology. Strongyliform eggs may belong to several species of pig parasites, including Oesophagostomum spp., hookworms, Trichostrongylus spp., Hyostrongylus rubidus and Metastrongylus salmi.

The present study used DNA barcoding to access species composition and genetic diversity. Primers for the mitochondrial target cox1 are “eclectic” due to high levels of intraspecific conservation and moderate interspecific variability, thereby providing identification of haplotypes and species in biological material (Hebert et al., 2004). It was possible to identify three distinct Oesophagostomum species in the area studied: O. dentatum, O. quadrispinulatum and O. columbianum. Interestingly, all sequences obtained in the present study were from different and undescribed haplotypes. None of these species are recognized as having zoonotic potential. Despite this, we cannot rule out the possibility of transmission of Strongylida parasites from pigs to human hosts. A previous study by our research group in NSN demonstrated that the strongyliform eggs found in human samples belong to the genus Necator, but not the species N. americanus (Monteiro et al., 2019).

Although O. dentatum and O. quadrispinulatum are parasites normally found in pigs, O. columbianum usually infects small ruminants. In the communities studied, pigs, goats and sheep are raised in close contact with each other, thus indicating the possibility of cross-host transmission. This type of management facilitates ingestion of goat and sheep feces by pigs, given that they are coprophagous, and enables passage of O. columbianum eggs or larvae through the digestive tract and presence of their DNA in fecal samples (pseudoparasitism), or even cross host transmission. In the present study, feces were collected fresh after defecation in the environment and, even though appropriate measures were taken at the time of collection, occurrence of contamination from the environment cannot be ruled out.

The phylogenetic tree and the haplotype network were structured based on mitochondrial DNA sequences and, therefore, were based on matrilineal inheritance. Within this perspective, O. dentatum and O. quadrispinulatum are closely related and formed a monophyletic group with two distinct clades. Similarly, phylogenetic analyses on O. dentatum and O. quadrispinulatum recovered from pigs in different regions of China generated a cluster with two clades that formed a monophyletic group (Lin et al., 2012a). The last authors used ribosomal DNA targets, which resulted in similar formation of a monophyletic group, with different groupings in the species O. quadrispinulatum, thereby indicating the presence of distinct genotypes or subspecies (Lin et al., 2014). Four distinct and novel haplotypes were identified in our O. quadrispinulatum sequences.

Pigs (Sus scrofa domesticus) are not autochthonous species from the Americas, having been introduced during the colonization process by Europeans. More recently, the introduction of the wild boar (Sus scrofa scrofa) in the 1990s led to their conversion into wild animals, as an exotic species, which population growing is uncontrolled in Brazil. Piauí is one of the states where its presence has not yet been registered. Oesophagostomum dentatum and O. quadrispinulatum infect domestic pigs in Brazil, Europe and Asia, with O. dentatum being identified in wild boar in Brazil as well (Silva & Müller, 2013; Li et al., 2017). It can be deduced that the process of introduction of pig farming in Brazil and its expansion also enabled the expansion of these helminth species.

Inclusion of sequences from other Oesophagostomum species in the phylogenetic analysis demonstrated the existence of seven distinct clades for this genus and that the Oesophagostomum species in pigs are closer to the non-human primate species O. aculeatum (which parasitizes monkeys and orangutans in Asia) and O. stephanostomum (which infects chimpanzees and gorillas in Africa). Oesophagostomum columbianum was found in pigs in the present study. A nucleotide BLAST (BLASTn) analysis in GenBank showed a similarity of 99% with O. columbianum in sheep from China (Zhao et al., 2013), and with Oesophagostomum sp. in goats and sheep from Brazil (Monteiro et al., unpublished data).

This is the first study exploring nucleotide sequences of Oesophagostomum in Brazil. These findings highlight the usefulness of molecular tools for investigating the taxonomy of strongyliform eggs observed in parasitological examinations, monitoring the presence of infection in herds ante-mortem, guiding control measures and providing data for studies on resistance to anthelmintics.

Acknowledgements

The authors would like to thank the staff of the Municipal Health Department of Nossa Senhora de Nazaré, Piauí, and the managers of Camp 8 de Março and Settlement 17 de Abril, in Teresina, Piauí. This study used resources from the regular funds of Instituto Oswaldo Cruz / Fiocruz and from Fiocruz Piauí.

These authors contributed equally to the work.

References

Barbosa AS, Bastos OMP, Dib LV, Siqueira MP, Cardozo ML, Ferreira LC, et al. Gastrointestinal parasites of swine raised in different management systems in the State of Rio de Janeiro, Brazil. Pesq Vet Bras 2015; 35(12): 941-946. http://dx.doi.org/10.1590/S0100-736X2015001200001. [ Links ]

Benavides M, Hassum I, Berne M, De Souza CJH, Moraes J. Variação individual de ovos de nematódeos gastrintestinais por grama de fezes (OPG) dentro de um rebanho ovino. Rio Grande do Sul: Embrapa Pecuária Sul; 2007. [ Links ]

De S, Bandyopadhyay S. Molecular taxonomy: an approach based on molecular markers. Sci Cult 2008; 74: 397-496. [ Links ]

Excofer L, Lischer HEL. Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under linux and windows. Mol Ecol Resour 2010; 10(3): 564-567. http://dx.doi.org/10.1111/j.1755-0998.2010.02847.x. PMid:21565059. [ Links ]

Flaticon. Vector icons and stickers [online]. 2022 [cited 2022 Jan 4]. Available from: https://www.flaticon.com/br. [ Links ]

Frias L, Stark DJ, Salgado Lynn M, Nathan S, Goossens B, Okamoto M, et al. Molecular characterization of nodule worm in a community of Bornean primates. Ecol Evol 2019; 9(7): 3937-3945. http://dx.doi.org/10.1002/ece3.5022. PMid:31015978. [ Links ]

Gaddam R, Murthy GSS, Kommu S. Ultrastructural studies of three species of Oesophagostomum (nematoda) by scanning electron microscopy. J Parasit Dis 2017; 41(3): 826-830. http://dx.doi.org/10.1007/s12639-017-0897-3. PMid:28848286. [ Links ]

Glen DR, Brooks DR. Phylogenetic relationships of some strongylate nematodes of primates. Proc Helminthol Soc Wash 1985; 52(2): 227-236. [ Links ]

Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 1999; 41: 95-98. [ Links ]

Hasegawa M, Kishino H, Yano T. Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J Mol Evol 1985; 22(2): 160-174. http://dx.doi.org/10.1007/BF02101694. PMid:3934395. [ Links ]

Hebert PDN, Stoeckle MY, Zemlak TS, Francis CM. Identification of birds through DNA barcodes. PLoS Biol 2004; 2(10): e312. http://dx.doi.org/10.1371/journal.pbio.0020312. PMid:15455034. [ Links ]

Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol 2016; 33(7): 1870-1874. http://dx.doi.org/10.1093/molbev/msw054. PMid:27004904. [ Links ]

Li K, Lan Y, Luo H, Shahzad M, Zhang H, Wang L, et al. Prevalence of three Oesophagostomum spp. from tibetan pigs analyzed by genetic markers of nad1, cox3 and ITS1. Acta Parasitol 2017; 62(1): 90-96. http://dx.doi.org/10.1515/ap-2017-0010. PMid:28030349. [ Links ]

Lin RQ, Liu GH, Hu M, Song HQ, Wu XY, Li MW, et al. Oesophagostomum dentatum and Oesophagostomum quadrispinulatum: characterization of the complete mitochondrial genome sequences of the two pig nodule worms. Exp Parasitol 2012b; 131(1): 1-7. http://dx.doi.org/10.1016/j.exppara.2012.02.015. PMid:22414328. [ Links ]

Lin RQ, Liu GH, Song HQ, Zhang Y, Li MW, Zou FC, et al. Sequence variability in three mitochondrial genes between the two pig nodule worms Oesophagostomum dentatum and O. quadrispinulatum. Mitochondrial DNA 2012a; 23(3): 182-186. http://dx.doi.org/10.3109/19401736.2012.668892. PMid:22651230. [ Links ]

Lin RQ, Shu L, Zhao GH, Cheng T, Zou SS, Zhang Y, et al. Characterization of the intergenic spacer rDNAs of two pig nodule worms, Oesophagostomum dentatum and O. quadrispinulatum. ScientificWorldJournal 2014; 2014: 147963. http://dx.doi.org/10.1155/2014/147963. PMid:25197691. [ Links ]

Makouloutou P, Mbehang Nguema P, Fujita S, Takenoshita Y, Hasegawa H, Yanagida T, et al. Prevalence and genetic diversity of Oesophagostomum stephanostomum in wild lowland gorillas at Moukalaba-Doudou National Park, Gabon. Helminthologia 2014; 51(2): 83-93. http://dx.doi.org/10.2478/s11687-014-0214-y. [ Links ]

Messing J. M13 cloning vehicles: their contribution to DNA sequencing. In: Griffin HG, Griffin AM, editors. Methods in molecular biology: DNA sequencing protocols. Clifton, NJ: Humana Press; 1993. p. 9-22. http://dx.doi.org/10.1385/0-89603-248-5:9. PMid:8220775. [ Links ]

Monteiro KJL, Jaeger LH, Nunes BC, Calegar DA, Reis ERC, Bacelar PAA, et al. Mitochondrial DNA reveals species composition and phylogenetic relationships of hookworms in northeastern Brazil. Infect Genet Evol 2019; 68: 105-112. http://dx.doi.org/10.1016/j.meegid.2018.11.018. PMid:30508686. [ Links ]

Nansen P, Roepstorff A. Parasitic helminths of the pig: factors influencing transmission and infection levels. Int J Parasitol 1999; 29(6): 877-891. http://dx.doi.org/10.1016/S0020-7519(99)00048-X. PMid:10480725. [ Links ]

National Center for Biotechnology Information – NCBI. Nucleotide [online]. 2022 [cited 2022 Jan 4]. Available from: https://www.ncbi.nlm.nih.gov. [ Links ]

Ota N, Hasegawa H, McLennan MR, Kooriyama T, Sato H, Pebsworth PA, et al. Molecular identification of Oesophagostomum spp. from ‘village’ chimpanzees in Uganda and their phylogenetic relationship with those of other primates. R Soc Open Sci 2015; 2(11): 150471. http://dx.doi.org/10.1098/rsos.150471. PMid:26716002. [ Links ]

Pinilla JC, Morales E, Delgado NU, Florez AA. Prevalence and risk factors of gastrointestinal parasites in backyard pigs reared in the Bucaramanga Metropolitan Area, Colombia. Rev Bras Parasitol Vet 2020; 29(4): e015320. http://dx.doi.org/10.1590/s1984-29612020094. PMid:33237192. [ Links ]

Prosser SWJ, Velarde-Aguilar MG, León-Règagnon V, Hebert PDN. Advancing nematode barcoding: a primer cocktail for the cytochrome c oxidase subunit I gene from vertebrate parasitic nematodes. Mol Ecol Resour 2013; 13(6): 1108-1115. http://dx.doi.org/10.1111/1755-0998.12082. PMid:23433320. [ Links ]

Railliet A, Henry A. Étude zoologique de l’Oesophagostome de Thomas. Ann Trop Med Parasitol 1910; 4(1): 89-94. http://dx.doi.org/10.1080/00034983.1910.11685702. [ Links ]

Rozas J, Ferrer-Mata A, Sánchez-DelBarrio JC, Guirao-Rico S, Librado P, Ramos-Onsins SE, et al. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol Biol Evol 2017; 34(12): 3299-3302. http://dx.doi.org/10.1093/molbev/msx248. PMid:29029172. [ Links ]

Setsuda A, Kato E, Sakaguchi S, Tamemasa S, Ozawa S, Sato H. Chabaudstrongylus ninhae (Trichostrongylidae: Cooperiinae) and Oesophagostomum muntiacum (Chabertiidae: Oesophagostominae) in feral alien Reeves’s muntjacs on Izu-Oshima Island, Tokyo, Japan. J Helminthol 2020; 94: e48. http://dx.doi.org/10.1017/S0022149X19000245. PMid:30973116. [ Links ]

Silva DS, Müller G. Parasitic helminths of the digestive system of wild boars bred in captivity. Rev Bras Parasitol Vet 2013; 22(3): 433-436. http://dx.doi.org/10.1590/S1984-29612013000300020. PMid:24142179. [ Links ]

SVG SILH. Free SVG Image and Icon [online]. 2022 [cited 2022 Jan 4]. Available from: https://svgsilh.com. [ Links ]

Taylor MA, Coop RL, Wall RL. Parasitologia veterinária. Rio de Janeiro: Koogan Guanabara; 2010. [ Links ]

Thomas HW. The pathological report of a case of Oesophagostomiasis in man. Ann Trop Med Parasitol 1910; 4(1): 57-88. http://dx.doi.org/10.1080/00034983.1910.11685701. [ Links ]

Yadav S, Gupta A, Choudhary P, Pilania PK, Joshi SP. Prevalence of gastrointestinal helminths and assessment of associated risk factors in pigs from Rajasthan districts, India. J Entomol Zool Stud 2021; 9(1): 1418-1423. [ Links ]

Zhao GH, Hu B, Cheng WY, Jia YQ, Li HM, Yu SK, et al. The complete mitochondrial genomes of Oesophagostomum asperum and Oesophagostomum columbianum in small ruminants. Infect Genet Evol 2013; 19: 205-211. http://dx.doi.org/10.1016/j.meegid.2013.07.018. PMid:23891666. [ Links ]

Received: October 18, 2021; Accepted: February 17, 2022

*Corresponding author: Kerla Joeline Lima Monteiro. E-mail: kerla.monteiro@gmail.com

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