For infection with S. ratti, approximately 8 to 10-week-old female C57BL/6 mice were inoculated with indicated numbers of purified iL3 in 30 μL sterile PBS into the hind footpad. Twenty-four hour faeces samples were collected for the detection of L1. For L. major infection, 3 × 106 (high dose) or 3 × 103 (low

dose) L. major stationary phase promastigotes in a final volume of 30 μL PBS were injected subcutaneously into one of the hind footpads of mice. Re-infection with 3 × 106 promastigote parasites was performed at indicated time points after primary infection. The course of disease was monitored daily, and https://www.selleckchem.com/EGFR(HER).html the footpad thickness was measured weekly. The relative increase in footpad thickness in per cent was calculated by employing the following form: (thickness of infected foot × 100: thickness of non-infected foot) − 100. For analysis of parasite-specific serum Ig, blood from mice was collected at indicated time points by puncture of the tail vein. The blood was allowed

to coagulate for 1 h at 4°C. Serum was collected after centrifugation at 12 000 × g for 15 min at RT and stored at −20°C for further analysis. For analysis of cellular responses, mice were killed at the indicated time points and mesenteric (mes) LN as X-396 manufacturer well as popliteal (pop) LN were prepared. In experimental infections with S. ratti, the egg and L1 output was analysed by collecting faeces over 24-h periods. DNA was extracted from 200 mg representative stool samples using the QIAamp DNA stool kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. The DNA was eluted in 200 μL, 6-phosphogluconolactonase and finally 2 μL of a 1 : 10 dilution in water was used as template for the qPCR. The S. ratti 28S ribosomal RNA gene was quantified by qPCR as described (10). Briefly, a 180 bp fragment of the S. ratti 28S RNA gene was amplified by qPCR in duplicates. For each run, a melting curve analysis was performed to guarantee

the specificity in each reaction tube. Comparative quantification (efficiency-corrected Ct method) was used to transform the difference in Ct values between the test samples and the calibrator sample into a copy number ratio. To count the number of adult nematodes in the gut, mice were killed at the indicated time points post-infection (p.i.) The small intestine was sliced open longitudinally and incubated at 37°C for 3 h in a Petri dish with tap water. The released adult females were collected, centrifuged for 5 min at 300 × g at RT and counted. Microscopic analysis of the small intestine revealed that no significant numbers of adults remained in the intestine. To measure the L. major parasite burden, DNA was isolated from footpads at days 10 and 31 p.i. The concentration of mouse ß-actin DNA was quantified by 5′ nuclease PCR (20).