Malacological and parasitological surveys

Malacological surveys were focused on the snail intermediate hosts of human schistosomiasis (Bulinus spp.). Two waterbodies of the Estuaire province were visited: Melen, situated in Libreville city (09°30′59.55″ East; 00°23′46.22″ North; 16 m altitude) and Ekouk (10°21′00.20″ East; 00°04′48.64″ South; 65 m altitude). Freshwater snails were collected for 1 h by one person using a stainless-steel strainer (18 cm wide, 8 cm deep, 20 cm handle, and < 1 mm wide holes) attached to a handle. Submerged and emergent vegetation, as well as floating plants, were screened. For each waterbody, collected snails were carefully stored in plastic boxes (70 mm high and 35 mm in diameter), and taken to the laboratory in Libreville, Gabon. The snails were kept in glass vials with 150 ml of well water, and exposed to daylight. The water was examined under a binocular microscope for cercarial emergence 24 h later, and then every 3 days for 40 days. The snails were fed ad libitum with dried lettuce.

The parasitological surveys were carried out in the same sites as the malacological surveys: Melen and Ekouk. Informed consent was obtained from families and the participation was voluntary. Each volunteer was given two pots (50 ml each), one for urine and one for stools. Samples were collected between 08.00 and 10.00 hours and taken to the laboratory for their examination. From each urine sample, an aliquot of 5 ml was centrifuged in a conical plastic tube for 3 min at 1500 rpm. Pellets were examined microscopically for schistosome egg presence. Stool samples were examined by the merthiolate-iodine-formaldehyde concentration (MIF-C) method. Prevalences (number of individuals infected by schistosomes divided by the total number of individuals examined) were calculated.

Egg morphometry

Eggs were extracted from urine samples in Melen and Ekouk and from faeces in Ekouk. The positive urine samples of each site were filtered together through a 45 μm sieve in saline solution (NaCl 0.85%). The positive stool samples were filtered through a series of four sieves 425, 180, 106 and 45 μm pore size in saline solution (NaCl 0.85%). The eggs were collected on the 45 μm sieve and maintained in saline solution (NaCl 0.85%). All eggs were randomly sampled, and measured under the microscope. For each egg, we measured the total length (including the terminal spine) and the maximum width.

Adult worm recovery

Miracidia were obtained only from eggs extracted from urine samples at Melen and at Ekouk. Bulinus globosus was used as the intermediate host since it is more compatible with S. haematobium compared to B. forskalii and since it is compatible with the hybrids of S. haematobium and S. guineensis (Southgate et al., Reference Southgate, Van Wijk and Wright1976; Mutani et al., Reference Mutani, Christensen and Frandsen1985).

For Melen, positive urine samples of nine patients were filtered together through a 45 μm sieve in saline solution (NaCl 0.85%). The eggs were collected on the 45 μm sieve and maintained in well water for miracidial hatching. Twelve positive B. globosus (exposed to five miracidia) were used to infect Meriones unguiculatus as definitive host.

For Ekouk, positive urine samples of 14 patients (four of them also had eggs in the faeces) were filtered together as explained above. Among the collected eggs, we preferentially selected the S. guineensis, and intermediate morphotypes (see fig. 1) in order to get potential hybrid specimens. Nine positive B. globosus (exposed to five miracidia) were used to infect M. unguiculatus as definitive host.

Fig. 1

Schistosoma haematobium, S. guineensis and intermediate morphotypes of schistosome eggs from human urine and faeces in Estuaire province, Gabon.

The rodents were anaesthetized with a solution of 2.5 ml of Rompun (20 mg/ml), 5 ml of Imalgène (100 mg/ml) and 42.5 ml NaCl by intraperitoneal injection of 0.1 ml/10 g of body weight. The abdomens were shaved and exposed to 300 cercariae per rodent. Five months post-exposure, the rodents were killed by lethal injection (0.5 ml) of Doléthal (Vétoquinol, France) and instantly perfused to collect adult worms. The perfusion was followed by supplementary examination to collect the worms trapped in the liver, in the superior and inferior mesenteric system, including rectum, and in the lungs. Worms were preserved in 95% alcohol for DNA analysis.

DNA extraction, ITS2 amplification

DNA was extracted from 80 adult worms from Gabon: 30 males from Melen (18, 2 and 10 from the mesentery–liver, rectum and lungs, respectively) and 50 adult worms from Ekouk (11, 2 and 13 males from the mesentery-liver, rectum and lungs, respectively, and 11, 2 and 11 females from the mesentery–liver, rectum and lungs, respectively). One male adult worm of each of S. haematobium (Benin and Niger), S. intercalatum (Democratic Republic of the Congo) and S. guineensis (Cameroon) were also used for comparison. A manufacture's protocol from the QIAamp DNA Mini Kit (Qiagen) was used.

Internal transcribed spacer-2 (ITS2) amplification was performed by polymerase chain reaction (PCR) using primers ITS2 single-stranded conformational polymorphism (SSCP) forward (5′-GCATATCAACGCGGG6-3′) and ITS2 SSCP reverse (5′-ACAAACCGTAGACCGAACC-3′), according to Kane et al. (Reference Kane, Bartley, Stothard, Vercruysse, Rollinson and Southgate2002). The cycling conditions included an initial denaturing step at 94°C for 5 min and then 40 cycles at 94°C for 30 s (denaturing), 66°C for 30 s (annealing) and 72°C for 1 min (elongation). There was a final elongation period at 72°C for 10 min. Each PCR product was checked by agarose gel electrophoresis of 5 μl of each product (Webster et al., Reference Webster, Tchuem Tchuenté and Southgate2007). PCR products were purified using a modified manufacture's protocol from Wizard SV Gel and PCR Clean-Up System kit (Promega, Madison, Wisconsin, USA).

Single-stranded conformation polymorphism ITS2 analysis

SSCP ITS2 analysis was carried out on the 80 adult worms on Bio-Rad PROTEAN^® II xi Cell (Bio-Rad, Hercules, California, USA), adapting a protocol used elsewhere by Kane et al. (Reference Kane, Bartley, Stothard, Vercruysse, Rollinson and Southgate2002) with specific modifications and run conditions. Acrylamide 12% gel consisting of a mixture of: 21 ml of acrylamide/bis-acrylamide (37.5:1), 42 ml of 1 × Tris-borate-EDTA (TBE) buffer; 470 μl of 10% ammonium persulphate (APS), 117 μl of tetramethylethylenediamine (TEMED) and 7 ml of miliQ H₂O was used. The resultant solution was poured into the assembled gel plates and stored at 4°C for 1 h, as well as 2 litres of 1 × TBE electrophoresis buffer. Sample preparation was as follows. Denaturing of PCR products was carried out by adding 10 μl of each PCR product to 22.5 μl of formamide and 2.5 μl NaOH (100 mm). This mixture was heated for 15 min in a thermocycler at 95°C and placed immediately on ice. The whole mixture of each sample added to 5 μl of charge buffer was loaded into the gel. Run conditions were as follows: system cooled 2 h before migration at 6°C, voltage 110 V and run time 17 h.

Statistical analyses

Yates's chi-square test was used to compare the percentages of eggs found in urine or faeces according to their morphotype. The Mann–Whitney, Kruskal–Wallis (KW) and Dunn's tests were used to compare egg sizes. All data were analysed using Microsoft Excel 2003 and GraphPad InStat, Version 3.05 (GraphPad Software, La Jolla, California, USA). Statistical significance was assessed at the P < 0.05 level.