Field collections of Holochilus sciureus
Holochilus sciureus was collected as part of an experimental study coordinated by the State University of Maranhão (UEMA) about the helminth fauna of this wild rodent captured in the municipalities of São Bento (02º41'45 "S 44º49'17 "O) and Peri Mirim (02º34'40" S 44º51'14"W), State of Maranhão, Brazil, between 2017 and 2018 (Fig. 1). Rodent capture and manipulation were authorized by the Biodiversity Authorization and Information System (n°67253-1) and the project was registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (registration number AB9E2EC).
These municipalities have a humid tropical climate with two distinct seasons: a rainy period from February to July and a dry period from August to January. Human schistosomiasis is endemic in both municipalities, but the infection rates are low, with an estimated prevalence of 5% among residents from São Bento and 2.3% among those from Peri Mirim (Brazilian Ministry of Health 2018).
As shown in Fig. 1, capture was performed in 14 collection spots located in the naturally flooded fields of both municipalities. For each collection spot, 10 Tomahawk traps baited with a mixture of banana and peanut butter were placed 10 m apart and kept overnight, as previously described by Do Carmo-Silva et al. (2019). Each trap was checked in the morning, and rodents with the morphological characteristics of H. sciureus, the most common species found in this region (Brandão and Nascimento 2015; Miranda et al. 2017), were taken to the laboratory for species confirmation and necropsies. The captured animals were immediately returned to their original environments. Each collection site was inspected for two days.
Recovery of miracidia
The rodents captured in the field were euthanized with an anesthetic overdose (300 mg/kg of ketamine and 30 mg/kg of xylazine) and the livers were immediately dissected (Council of Ethics and Animal Experimentation at UEMA; protocol nº03/2017). After dissection, specific identification of H. sciureus was confirmed based on skull and mandibular features (Rocha et al. 2011; Brandão and Nascimento, 2015).
The livers with macroscopic changes suggestive of schistosome infection were homogenized in cooled concentrated saline solution (2% NaCl), decanted at 4°C and the pellet containing parasite eggs was resuspended in chlorine-free water at room temperature and exposed to the artificial light for 30 min to stimulate the miracidia hatching (Standen, 1952). Miracidia were concentrated at the top of a sealed volumetric flask exposed to artificial lighting as described by Chaia (1956). The water suspension containing the miracidia was recovered, miracidia were counted under a stereomicroscope (Zeiss Stemi Dv4, Jena, Germany), and used for experimental infection of snails. The Schistosoma strain isolated from H. sciureus in the present study was designated HS.
Isolation process in snails
Biomphalaria glabrata snails descending from specimens collected in the municipality of São Bento (SB strain; sympatric strain) and from specimens collected in Belo Horizonte (BH strain; allopatric strain) were used for the experiments. The snail strains were bred and maintained separately at the molluscarium of the Laboratory of Schistosomiasis and Immunohelminthology (Instituto de Ciências Biológicas/Universidade Federal de Minas Gerais, acronym ICB/UFMG), in aquariums containing chlorine-free water, fed with lettuce (Lectuca sativa) previously cleaned in acetic acid solution (0.01%), and supplied with standardized powdered chow for snails (Rosa et al. 2013). Snails, 5-6 mm in diameter, were used for experimental infection with the S. mansoni HS strain.
For individual infection, each snail was placed in one well of a 6-well culture plate (Plate Flat Bottom, Sarstedt, Massachusetts, USA) containing approximately 6 ml of chlorine-free water and a defined number of miracidia obtained from infected H. sciureus liver homogenates (5–8 miracidium/well). During parasite isolation, some H. sciureus-infected livers yielded a small number of miracidia and did not allow for individual infections. In this situation, a mass infection was performed, in which a whole clean supernatant of liver homogenate from infected rodents was placed in a container with 8–15 B. glabrata of the SB strain. Plates or containers with snails and miracidia were kept for 12 h under direct light.
Snails from individual or mass infections were evaluated between 28 and 80 days post-exposure to miracidia to identify and quantify cercarial shedding. Briefly, once a week, the snails were individually placed into 6-well culture plates 6 ml of chlorine-free water and exposed to incandescent light for 4 h (Brazilian Ministry of Health, 2008). The presence of cercariae was verified, and the number of larvae was counted using a stereomicroscope (Zeiss Stemi Dv4, Oberkochen, Germany). After light exposure, snails that shed cercariae (positives) were separated from negative ones, in a dark environment at 23°C and used as a source of infective larvae for the next experimental infection.
Isolation process in mice and hamsters
Heterogenetic hamsters (Mesocricetus auratus) and isogenic BALB/c mice aged 6–8 weeks were acquired from an established colony at the mouse facility of UFMG and used as a vertebrate model for experimental infections using the S. mansoni HS strain. Experimental animals were kept at the animal facility for infected animals at the Parasitology Department (UFMG), fed with standard chow (Presence, Primor, Brazil), and provided tap water ad libitum. Animal experiments were approved by the UFMG Animal Ethics and Experimentation Council (nº46/2019)
Cercariae used for hamster and mouse infections were obtained from B. glabrata infected with S. mansoni miracidia of the HS strain. Positive snails were pooled in a glass beaker containing 100 ml of chlorine-free water and exposed to light and heat for 4 h. The recovered solution containing cercariae was filtered (40 mesh/inch), concentrated in a Buchner funnel (20–30 µm porosity), and the number of cercariae was counted using a stereomicroscope (Pellegrino and Macedo 1955). When the number of cercariae was very low (less than 20 larvae/500 µL), the mass percutaneous infection was performed. Briefly, these larvae were diluted in 500 mL chlorine-free and the mice were maintained individually in contact with this solution in a bath for 1 h with artificial light exposure, as described by Brener (1956, 1959). When the infected snails shed a large number of cercariae, allowing for concentration and quantification, a defined number (from 20 to 120 cercariae/animal, as defined for each experiment) was diluted in 500µL of saline (0.9% NaCl), and the solution was subcutaneously inoculated in each experimental vertebrate model (Pellegrino and Macedo, 1956).
For qualitative confirmation of infection of vertebrates (mice or hamsters), the feces of cercarial-exposed animals were collected weekly from 35 to 62 days post-infection (dpi) and examined using the sedimentation technique (Hoffman et al. 1934). Eight weeks post-infection (wpi), cercarial-exposed animals were euthanized with an anesthetic overdose (300 mg/kg of ketamine and 30 mg/kg of xylazine) and subjected to circulatory perfusion for recovering the adult worms, which were collected individually in microtubes at -20ºC, for molecular analysis.
DNA isolation, PCR, and sequencing
Genomic DNA from individual adult male worms was isolated from mice experimentally infected with the S. mansoni HS strain using the QIAamp® DNA mini kit (Qiagen, California, USA), according to the manufacturer's guidelines. Polymerase chain reaction (PCR) assays and sequencing were performed for the 16S-12S region of the mtDNA (referred to as 16S herein), using the primers 16SF2 (F) 5’ GTG CTA AGG TAG CAT AAT AT 3’ and 16SR3 (R) 5’ AGA AGC AGT TTA ACC GCG AC 3, and for the cytochrome c oxidase subunit I (cox1) region, using the primers CO1F (F) 5-' GGC ATA TCT GTA TGA GTC TA 3’ and CO1R3 (R) 5' GCA TTT AAA TAR TCA ACA TG 3’ (Morgan et al. 2005). Both primer sets amplify a 730 bp fragment. PCR reactions were performed in a final volume of 25µL, consisting of 1.25 µL of each primer (final concentration, 0.5 µM), 12.5 µL of GoTaq® Colorless Master Mix 2x (Promega, USA), 1 µL of DNA template (about 20 ng/µL) and 9 µL of ultrapure water. Cycling conditions were as follows: 1 min at 95°C, 45 s at 50°C and 90 s at 70°C, followed by 29 cycles of 30 s at 95°C, 30 s at 50°C and 90 s at 72°C, and a final extension at 72°C for 7 min (Morgan et al. 2003). PCR products were subjected to agarose gel electrophoresis to confirm amplification. Positive PCR products were purified by enzymatic treatment with ExoSAP-IT Express (Thermo Fisher, Massachusetts, USA). Purified products were sent for sequencing at the CT-Vacinas facility (BHtec, Belo Horizonte, Brazil) using PCR primer sets (forward and reverse) according to Sanger et al. (1977).
Contiguous sequences were assembled in Geneious (Geneious Prime ver. 2022 created by Biomatters, available at http://www.geneious.com/), and their consensus was extracted and deposited in GenBank. A preliminary basic local alignment search tool (BLAST) search of the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) was performed to confirm the genetic proximity between the present sequences and those from representatives of Schistosomatidae.
Sequences of all species of Schistosoma covering the same genetic region sequenced in the present study, were used for phylogenetic reconstructions (see Online Resources 1 and 2). Sequences from the same isolates (clones or others with 100% genetic similarity), those not identified to the species level, and those too short in length were not included. Sequences were aligned using M-Coffee (Notredame et al. 2000), then evaluated by the transitive consistency score to verify the reliability of aligned positions, and based on score values ambiguous aligned positions were trimmed (Chang et al. 2014). Datasets (i.e., alignments according to each genetic marker) were subjected to maximum likelihood (ML) and Bayesian inference (BI) analyses using PHYML and MrBayes, respectively (Huelsenbeck and Ronquist 2001; Guindon and Gascuel 2003). The model of evolution and its fixed parameters for each model were chosen and estimated under the Akaike information criterion using jModelTest 2 (Guindon and Gascuel 2003; Darriba et al. 2012) and are detailed in Online Resource 3. The nodal support of ML was based on 1,000 bootstrap non-parametric replications. Nodal supports for Bayesian posterior probability values were determined after running the Markov chain Monte Carlo (2 runs 4 chains) for 1 × 106 generations, with sampling frequency every 1 × 103 generations, and discarding the initial ¼ of sampled trees (1 × 106) as burn-in. The outgroup chosen was Trichobilharzia regenti Horak, Dvorak, and Kolarova, 1998, based on previous phylogenies of Schistosoma (Webster et al. 2006).
Pairwise (patristic) distance (P distances) matrixes were generated, according to each genetic marker, using the software MEGA 7.0 (Kumar et al. 2016), to evaluate intra and interspecific divergences among samples. The Kimura two-parameter (K2P) (Kimura 1980) was used as distance metric, with other parameters set to default.
Parasitological profile of S. mansoni HS strain under laboratory conditions
After isolating an S. mansoni HS strain and confirming its specific identification by genetic analysis, the next step was to describe the parasitological profile of this strain better. For this purpose, we performed standardized experimental infections of B. glabrata snails and BALB/c mice.
The course of infection by S. mansoni of HS strain in B. glabrata host was evaluated in two different snail strains: the BH strain (allopatric) and the SB strain (sympatric). At the time of infection, the snails, 5–6 mm in diameter, were individually exposed to three different quantities of infective larvae: 10, 15, or 20 miracidia of the S. mansoni HS strain. For the experiments, 20 snails of each strain were infected with each miracidia load and 20 snails of each strain were kept uninfected. Each experimental group, infected and control of both snail strains, was evaluated weekly for up to 84 days (or 12 weeks) and dead animals were counted, and the data were used to build a mortality curve. During this period, the number of cercariae shed from each infected snail was also monitored weekly from 28 to 84 dpi after exposure of the infected snails to artificial light. The number of cercaria shedding by each infected snail was estimated under an optical microscope (Nikon, New York, USA) in two 500 µL aliquots collected from a solution containing one infected snail after light exposure.
To assess the infection of mice with S. mansoni HS, 20 male BALB/c mice, 6-8 weeks old, were subcutaneously infected with 20 cercariae/mouse as previously described (Pellegrino and Macedo 1956), and five male BALB/c mice of the same age were kept uninfected. The experimental procedures were evaluated and approved by the Animal Ethics and Experimentation Council of UFMG (protocol nº46/2019).
Animals were monitored weekly for 12 weeks (84 days) to evaluate the mortality rate and number of eggs present in feces (infected group). The mortality rate was determined by counting the number of dead mice in each experimental and control group. Animals showing extreme cachexia, dyspnea, piloerection, and signs of apathy were euthanized and included in the mortality group.
The parasite burden was estimated by the number of adult worms recovered from portal circulation, eggs present in the feces and retained in the tissues. Fecal samples, collected from each infected mouse between the 6th and 12th weeks, were weighed and processed according to Negrão-Corrêa et al. (2004). To evaluate the number of eggs per gram of feces, two aliquots of 200 μl from each sample (final volume of 5 mL of formalin 10%) were analyzed under an optical microscope (Nikon), and the average number of eggs per aliquot of stool was regarded as the total number of eggs per gram of feces.
In addition, 8–10 infected mice were euthanized by anesthetic overdose (300 mg/kg ketamine and 30 mg/kg xylazine) and necropsied after 8 (acute schistosomiasis) and 12 weeks of infection (chronic schistosomiasis) to estimate the number of adult worms in circulation and the number of parasite eggs retained in the lungs, liver, spleen, and intestines. Adult worms of the S. mansoni HS strain were recovered from the portal/mesenteric circulation of each infected mouse after circulatory perfusion, as described by Pellegrino and Siqueira (1956). The recovered worms were counted separately as male and female using a stereomicroscope (Zeiss). After blood perfusion in the infected animals, the lungs, liver, spleen, and intestine (small and large) were isolated and digested in a 5% KOH solution (37ºC/4 h), as described by Cheever (1968). For egg counting, two 200 μl aliquots of each sample (5 ml final volume) were analyzed under an optical microscope, and the data were expressed as eggs/organ (Maggi et al. 2021).
The normal distribution of the measurements was evaluated using the Kolmogorov-Smirnov test. Continuous data with a normal distribution were expressed as mean and standard error, and data without a normal distribution were expressed as median and interquartile range. Categorical data frequencies were compared using Fisher's exact test. Data related to the survival curves were analyzed using log-rank tests. Statistical significance was set at P < 0.05. These analyses along with graphic construction were performed using GraphPad Prism software version 8 (Prism Software, Irvine, CA, USA) and STATA version 11 (Stata Corp., College Station, TX, USA).