Integrative taxonomy of Metastrongylus spp. in wild boars from Brazil – Parasites & Vectors

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Study areas

The samples were collected from wild boars hunted in rural properties from the municipalities of São Simão, Monte Azul, Paraíso, Colina, Matão, Bebedouro e Monte Alto (São Paulo), Ipiranga (Paraná), and Santo Antônio das Missões (Rio Grande do Sul) (Fig. 1).

Fig. 1

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

The study areas in São Paulo state are located in the transition zone between the Cerrado and the Atlantic Forest biomes. According to the modified Köppen climate classification, the climate is humid subtropical, with an average annual temperature above 18 °C and average annual rainfall between 1200 and 1500 mm. They have 4–5 months of drought in winter, between May and September, and are located about 600 m above sea level [9,10,11]. Agriculture is the main economic activity, with emphasis on sugarcane, soy, corn, peanut, and tomato crops, in addition to beef cattle and poultry [12].

In Paraná State, the Campos Gerais region, where Ipiranga is located, has an average annual rainfall between 1400 and 1800 mm and an average annual temperature between 16 ºC and 20 ºC. The wettest season is from September to March, but frequent precipitation occurs during the winter. The prevailing climate is humid subtropical, according to the Köppen classification, and is located at about 800 m above sea level. The characteristic vegetation is the mixed rainforest. The region is characterized by high-tech crops such as soy, corn, wheat, potatoes, and beans, in addition to dairy cattle [13].

The municipality of Santo Antônio das Missões belongs to the Missões region, northwest of Rio Grande do Sul state, located in the Pampa biome. According to the Köppen classification, the climate is humid subtropical with an average annual temperature of about 17 ºC. January is the hottest month (average 32.7 ºC), and July is the coldest (average 10.5 ºC). Rainfall is about 1900 mm/year, with uneven distribution during the period. The economy is based on agricultural products, with emphasis on soy, rice, wheat, corn, sheep, and beef and dairy cattle, in addition to diversified subsistence production [14].

Biological samples

The sampling was carried out without biostatistical criteria due to the lack of data regarding the wild boar population in the region. Instead, it relied on the hunting success of our partner hunters. We examined the lungs of 58 wild boars, comprising 33 males and 25 females. The age of the animals was estimated according to dental eruption [15], categorizing them as either juveniles (less than 6 months old), or adults (more than 6 months old). The classification of the two age groups was established based on our fieldwork observations of pregnant wild boars around 6 months old (EGL Hoppe, personal communication, July 17, 2023) probably due to the random crosses between wild boars and domestic pigs. The organs were removed from the thoracic cavity, packed in individually labeled plastic bags, stored in isothermal boxes with ice, and immediately sent to the Laboratory of Parasitic Diseases (LabEPar) at the Department of Pathology, Reproduction and One Health (DPRSU), within the School of Agricultural and Veterinary Studies (FCAV), at the São Paulo State University (Unesp), Jaboticabal, São Paulo, Brazil.

Morphological identification

The trachea and the lungs were slit opened following the airways, from the trachea and main bronchi to the terminal bronchioles. All obtained nematodes were fixed in 70% ethanol and stored in identified flasks. The parasites were clarified with 80% acetic acid and mounted on temporary slides for taxonomic identification, according to Vicente et al. [16] and Gassó et al. [6]. Images and measurements (in millimeters, expressed as mean ± standard deviation, lower and upper values in brackets) were obtained with an Olympus BX-51 microscope attached to a Q-Color 3 camera (Olympus, Tokyo, Japan) and processed using Image-Pro Plus 4 image analyzer software (Media Cybernetics, Rockville, MD, USA). Vouchers were deposited in the collection of the Oswaldo Cruz Institute (CHIOC/Fiocruz, Rio de Janeiro, Brazil), and additional specimens were kept in LabEPar’s helminthological collection.

Molecular analysis

DNA extraction

Genomic DNA was extracted from at least two male specimens per municipality studied. Selected specimens were individually washed with sterile phosphate-buffered saline (PBS) pH 7.4 solution and transferred to 1.5 µl microtubes containing 50 µl of tissue lysis buffer (ATL) from the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) and macerated with the aid of sterilized plastic rods. Subsequently, glass beads treated with Triton X-100, 130 µl of ATL buffer, and 20 µl of proteinase K were added to the microtubes. The rest of the extraction proceeded according to the manufacturer’s protocol. The analysis of DNA concentration and quality, whose absorbance ratio between the wavelengths of 260 and 280 nm is desirable between 1.8 and 2.0 ng/dl [17], was performed using the NanoDrop One Spectrophotometer (Thermo Fisher Scientific), and the extraction products were stored at −20 °C until amplification by conventional polymerase chain reaction (PCR).

Amplification

Four genetic regions were amplified: 18S ribosomal DNA (rDNA), internal transcribed spacer (ITS), 28S rDNA, and the cytochrome c oxidase subunit I (cox-1) of the mitochondrial DNA (mtDNA). The primers set are expressed in Table 1. The reactions were composed of 1× buffer (KCl 50 mM, TRIS–HCl 200 mM, pH 8,4); 50 mM of MgCl2; 10 mM dNTPs; 0.5 U Platinum Taq [Thermus aquaticus] DNA Polymerase (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA); 5 pmol of each forward and reverse primer; 60 ng of genomic DNA and ultrapure water to complete a final volume of 20 µl. Amplifications were performed in a Nexus thermal cycler (Eppendorf, Hamburg, Germany) programmed to perform one cycle at 95 ºC for 3 min, and 35 cycles at 94 ºC for 40 s; each primer’s annealing temperature (Table 1) was kept for 30 s, and 72 ºC for 50 s, followed by a final extension cycle at 72 ºC for 10 min.

Table 1 Primer sets and annealing temperature used in polymerase chain reactions with their respective amplicon size

To verify the amplification reaction, the PCR products were submitted to electrophoresis in 1% agarose gel, stained with ethidium bromide, and visualized in a Geldoc XR photodocumenter (Bio-Rad®). In the case of low DNA yield, the reamplification was performed using the same protocol and primers cited previously. The products were purified with the Wizard® SV Gel and PCR Clean-Up System kit (Promega, Madison, WI, USA) according to the manufacturer’s instruction and submitted to PCR sequencing using the BigDye Terminator v3.1 kit (Applied Biosystems, Waltham, MA, USA), according to manufacturer’s instructions. Sequencing was performed by capillary electrophoresis on an ABI 3130 sequencer (Applied Biosystems, Waltham, MA, USA) according to Sanger’s method [18].

Phylogenetic analysis

The electropherograms generated in the sequencing were submitted to the Phred/Phrap/Consed software package [19,20,21] to verify the quality of the bases and trim the sequences considering bases with Phred quality up to 20 or higher. The qualified sequences were compared to others deposited in the National Center for Biotechnology Information (NCBI) database using BLAST (Basic Local Alignment Search Tool) [22]. The sequences from this study and the selected sequences from NCBI’s database were aligned using the ClustalW tool [23] on the software BioEdit v. 7.0.5.3 [24].

Phylogenetic trees were obtained by a maximum likelihood analysis using the W-IQ-Tree software [25]. The best evolutionary model was selected according to the Bayesian information criterion (BIC) using the W-IQ-Tree software [26]. The clade stability was evaluated using 1000 bootstrap replicates. The phylograms were graphically edited and rooted on the TreeGraph 2.15.0–887 beta software [27].

Data analysis

Infection descriptors of prevalence, mean intensity (range of intensity), and mean abundance were based on Bush et al. [28]. Fisher’s exact test was performed to compare parasite prevalence among sex, age group, and states of collection. The Mann–Whitney U test was used to evaluate the differences in mean intensity between the age groups and sex. Differences between the states’ mean intensity were not calculated due to the low parasite burden observed in Paraná state (only one animal was infected). All analyses were performed using the R software version 4.0.4. Values of p < 0.05 were considered statistically significant.

Ethical procedures

All procedures were approved by the Committee on Ethics in the Use of Animals (CEUA) of FCAV/Unesp Jaboticabal protocol no. 3683/20 and Chico Mendes Institute for Biodiversity Conservation (ICMBio), request in the Biodiversity Authorization and Information System (SISBIO) no. 84726–1.

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