Integrative taxonomy of Metastrongylus spp. in wild boars from Brazil

Abstract

Background: Wild boars (Sus scrofa) may cause substantial damage to crops and can spread zoonotic parasites to domestic animals, posing a risk to health and animal production. Metastrongylus spp. can negatively affect the wild boar population, increasing piglet mortality. In addition to that, studies with Metastrongylus genetic characterization are still scarce in Brazil. The present study aims to characterize Metastrongylus spp. from wild boars hunted in the states of São Paulo, Paraná, and Rio Grande do Sul, Brazil, using traditional morphological description and DNA sequences in an integrative taxonomic approach.

Methods: After nematode collection from 58 wild boars, the parasites were morphologically identified and genetically characterized by the amplification of 18S ribosomal DNA (rDNA), 28S rDNA, internal transcribed spacer (ITS) region, and cox-1 mitochondrial DNA (mtDNA). Descriptors of infection were determined and Pearson's Chi-square test was applied to compare the prevalence of infections among the identified parasite species, host age group (juveniles and adults), and sex. The Mann-Whitney U test was performed to compare the mean intensity between the age groups and sex.

Results: Metastrongylus salmi, Metastrongylus apri, and Metastrongylus pudendotectus were identified in 77.6% (45/58) of the necropsied wild boars. Metastrongylus salmi was the most prevalent and abundant species (70.7%, 11.1), followed by M. pudendotectus (18.9%, 4.3) and M. apri (17.2%, 2.2). Metastrongylus pudendotectus showed the highest mean intensity and range (25.2, 1-93), followed by M. salmi (15.7, 1-58) and M. apri (12.6, 3-27). We found a significantly higher prevalence of Metastrongylus spp. and M. salmi in adult wild boars, probably associated with a more prolonged time of exposure to intermediate host species. The phylogenetic analysis revealed that ITS2 region and cox-1 mtDNA are the most suitable genetic markers for Metastrongylus species characterization. Genetic variability between M. apri and M. salmi isolates was verified.

Conclusions: We expand the knowledge about the Metastrongylus community in the non-captive wild boar population from Brazil as well as the importance of this exotic species in the maintenance of Metastrongylus spp. in its areas of occurrence. The novel genetic sequences obtained may help further studies to understand the genetic diversity in other nematode populations from Brazil and other countries.

Keywords: Invasive species; Lungworms; Metastrongylus; Morphology; Neotropical region; Phylogeny.

Fig. 1

Sampling collection sites of wild boars hunted in São Paulo, Paraná, and Rio Grande do Sul states

Fig. 2

Metastrongylus salmi found in wild boars hunted in São Paulo, Paraná, and Rio Grande do Sul states. a Anterior extremity, showing the trilobated lips (black arrow). Magnification: ×200. Bar: 50 µm. b Female posterior extremity; note the short pre-vulvar swelling (orange arrow). Magnification: ×100. Bar: 100 µm. c Male posterior extremity showing the hook-like form at spicule ending (inset). Magnification: ×40. Bar: 2000 µm

Fig. 3

Metastrongylus pudendotectus found in wild boars hunted in Paraná and Rio Grande do Sul states. a Male posterior extremity; note the anchor-like form at the spicule ending (inset). Magnification: ×100. Bar: 100 μm. b Spicule and gubernaculum (black arrow). Magnification: x400. Bar: 25 μm. c Female posterior extremity; note the prominent swelling surrounding the vulvar opening (orange arrow). Magnification: x100. Bar: 100μm

Fig. 4

Metastrongylus apri found in wild boars hunted in Paraná and Rio Grande do Sul states. a Male posterior extremity showing the hook-like form at the spicule ending (inset). Magnification: ×40. Bar: 2000 µm. b Female posterior extremity; note the protruding vulvar swelling (blue arrow). Magnification: ×100. Bar: 100 µm

Fig. 5

Maximum-likelihood tree using internal transcribed spacer (ITS) region encompassing Metastrongyloidea helminths. Cystocaulus ocreatus and Umingmakstrongylus pallikuukensis were rooted as outgroups. Sequences obtained from the study are highlighted in red. Metastrongylus sequences downloaded from the GenBank are indicated with accession number, species name, and country. Bootstrap values are shown at the nodes. The best-fit model was the transversion model with equal base frequencies and the discrete Gamma model with four rate categories (TVMe + G4)

Fig. 6

Maximum-likelihood tree using cytochrome c oxidase subunit 1 mitochondrial DNA (cox-1 mtDNA) region encompassing Metastrongyloidea helminths. Dirofilaria immitis and Onchocerca lupi were rooted as outgroups. Sequences obtained from the study are highlighted in red. Nucleotide sequences downloaded from the GenBank are indicated with accession number, species name, and country. Bootstrap values are shown at the nodes. The best-fit model was the transition model considering the base frequencies, invariable sites, and discrete Gamma model with four rate categories (TIM + F + I + G4)