Parasitological procedures

Male Rattus norvegicus (Wistar) weighing 100–120 g were maintained in the Centro de Experimentação e Utilização de Animais, Universidade Federal de Uberlândia, provisioned with laboratory chow and water ad libitum.

Strongyloides venezuelensis third-stage infective larvae (L3) were obtained from charcoal cultures of infected rat faeces. The cultures were stored at 28°C for 48 h, and the infective larvae were collected and concentrated using the Rugai method (Rugai et al., Reference Rugai, Mattos and Brisola1954). For infection, each rat was inoculated subcutaneously with 1500 S. venezuelensis L3 larvae.

To recover S. venezuelensis parthenogenetic females, rats were anaesthetized (ketamine:xylazine 60:7 mg/kg) and sacrificed at 8 days post-infection (dpi). The small intestines were then removed from the animals, placed in Petri dishes containing saline solution, longitudinally sectioned and incubated at 37°C for 2 h. Parthenogenetic females were then counted following the methods of Sato & Toma (Reference Sato and Toma1990).

To obtain eggs, experimentally infected rats were placed individually on clean, moist, absorbent paper and allowed to defecate over an 18-h period at 5, 8, 13 and 21 dpi. The faeces were collected, diluted in saturated saline and homogenized. The supernatant was placed in a McMaster chamber, and the number of eggs/g of faeces was estimated using the Cornell–McMaster method (Gordon & Whitlock, Reference Gordon and Whitlock1939).

Experimental infections

Rats were divided into four experimental groups: immunocompetent uninfected (n= 6, day 0); immunocompetent infected (n= 24); immunosuppressed uninfected (n= 6, day 0); and immunosuppressed infected (n= 24). Six animals per group were sacrificed at each time point (5, 8, 13 and 21 dpi). Prior to infection, the immunosuppressed rats received 5 μg/ml dexamethasone disodium phosphate in water for 5 days, as described previously by Romand et al. (Reference Romand, Thulliez and Dubey1998). Animals from the infected groups were then inoculated subcutaneously with 1500 L3 S. venezuelensis larvae. At each time point (0, 5, 8, 13 and 21 dpi.), the rats were anaesthetized, and blood samples were collected by cardiac puncture. The blood was centrifuged, and serum aliquots were stored at − 20°C.

Eggs/g of faeces were counted in triplicate on 5, 8, 13 and 21 dpi to verify infection; mean values were used for the analysis.

Immunological protocols

Alkaline extracts were prepared as described previously (Machado et al., Reference Machado, Ueta, Gonçalves-Pires, Oliveira, Faccioli and Costa-Cruz2003). Briefly, 1 ml of 0.15 m NaOH was added to either 500 S. venezuelensis parthenogenetic females or 300,000 eggs, which were gently agitated for 6 h at 4°C. Subsequently, 0.3 m HCl was added until a pH of 7.0 was reached, and the preparations were then centrifuged at 12,400 ×  g for 30 min at 4°C. The supernatants (alkaline extracts) were analysed for protein content according to Lowry et al. (Reference Lowry, Rosebrough, Farr and Randall1951) and were subdivided into aliquots and stored at − 20°C until use. In a previous study, female extraction analysis using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed three major bands, with sizes of 50, 45 and 28 kDa, and one polypeptide group ( < 15 kDa) (Gonçalves et al. Reference Gonçalves, Rocha, Gonzaga, Gonçalves-Pires, Ueta and Costa-Cruz2012b). Egg extracts resulted in somewhat dispersed, faint bands with apparent molecular weights ranging from 27–28 to 32 kDa.

For the production of immune serum, two rabbits were immunized with either S. venezuelensis parthenogenetic females or S. venezuelensis eggs. Briefly, animals were inoculated with 500 μg/ml of both alkaline extract types (parthenogenic females and eggs) emulsified in Freund's complete adjuvant for the first immunization. Inoculations were conducted at intervals of 2 weeks after the first immunization, using Freund's incomplete adjuvant. When ELISA indicated that the antibody count had reached 15,000, 20 ml of blood was collected, and an IgG fraction was prepared using a Protein A Sepharose CL-4B column. After testing IgG activity against the specific antigen, a portion of the IgG fraction was stored at − 20°C for later use for antibody capture by ELISA. The rest of the IgG fraction was conjugated to horseradish peroxidase (Sigma, St. Louis, Missouri, USA) according to Wilson & Nakane (Reference Wilson, Nakane, Knapp, Holuban and Wick1978).

ELISA was performed according to Gonçalves et al. (Reference Gonçalves, Silva, Ueta and Costa-Cruz2012c). Preliminary experiments were performed to determine the optimal conditions for ELISA via the serial dilution of reagents (antigen, control sera and conjugate). Briefly, polystyrene micro-titre plates were coated with 10 μg/ml of S. venezuelensis parthenogenetic female or egg alkaline extracts in 0.06 m carbonate–bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. Then, the plates were washed three times with PBS-T (phosphate-buffered saline plus 0.05% Tween 20), incubated with serum samples (1:10) for 45 min at 37°C and then incubated with the secondary antibody (peroxidase-labelled goat anti-rat IgG (Sigma)) at the ideal dilution of 1:8000 for 45 min at 37°C. The reaction was revealed by adding the enzyme substrate (0.03% H₂O₂ and o-phenylenediamine (OPD) in 0.1 m citrate–phosphate buffer pH 5.0) and incubating the plate for 15 min at room temperature. Then the reaction was stopped by adding 25 μl/well 2 n H₂SO₄. The optical density (OD) was measured at 492 nm in a plate reader (Titertek Multiskan, Flow Laboratories, McLean, Virginia, USA). The cut-off value is the criterion that defines the detection limit, and it was calculated as the mean absorbance of three negative sera plus three standard deviations. OD values above the cut-off were considered positive.

Immune complex detection was performed according to Gonçalves et al. (Reference Gonçalves, Silva, Ueta and Costa-Cruz2012c). Preliminary experiments were carried out to determine the optimal conditions for ELISA via serial dilution of reagents (antibody, control sera and conjugate). Briefly, polystyrene micro-titre plates were coated with 50 μl/well of IgG obtained from rabbits immunized with parthenogenetic female or egg alkaline extracts (40 μg/ml) in 0.06 m carbonate–bicarbonate buffer (pH 9.6) and incubated for 18 h at 4°C. After three washes with PBS-T, 50 μl of serum sample (1:10) was added to each well, and the plates were incubated for 45 min at 37°C. Positive (OD values above the cut-off) and negative (OD values below the cut-off) serum samples were used as controls. After incubation for 45 min at 37°C and three additional washes, peroxidase-labelled goat anti-rat IgG (Sigma) was added (50 μl/well) at the ideal dilution of 1:8000, and the plates were incubated for 45 min at 37°C. The reaction was revealed as described above, and the results were determined using the same cut-off value.

Data analysis

Each experiment was performed in duplicate. The results are expressed as the means + SEM. Data were analysed using ANOVA, followed by a Bonferroni test. The level of statistical significance was set at P <  0.05.