Adult female filarial worms, Setaria digitata were collected from the peritoneum of cattle at a nearby abattoir in sterile alpha – MEM containing 1% glucose, Penicillin-100 units/ml, Streptomycin-100 μg/ml Gentamycin-50 μg/ml and Amphotericin-B 2.5 μg/ml and transported to the laboratory and used on the same day. Individual worms taken in a petridish were washed three times (x3) in sterile medium and dissected into small pieces in about 5 ml of medium and incubated at 37°C for 30 mins. The large pieces were removed and the medium containing intra-uterine stages were harvested, washed in MEM and the final pellet of cells suspended in 1 ml of sheath fluid and analyzed using a flowcytometer (FACSCalibur, Becton Dickinson, USA) using Cell Quest Pro software. The data (5000 events) were acquired for forward and side scatter using the following settings: FSC, voltage E00 and SSC, voltage 340. The three populations were gated and sorted using a FACS sorter under moderate fidelity settings. The sorted suspensions were centrifuged on to microscopic slides using a cytospin and observed under a phase contrast microscope for identification of organisms in the gated populations.

Blood for sera were collected from patients with chronic filariasis (elephantiasis/hydrocoele) and microfilariae carriers from filariasis endemic areas near Bhubaneswar as described by us earlier 6. Sera stored at -20°C were diluted in PBS with 1% BSA and used for binding to intra-uterine stages as described above.

Five Mastomys coucha were immunized with three doses (15 days apart) of intra-uterine stages (50,000 cells per dose) in complete Freund's adjuvant and blood for sera was collected between days 40–45 and tested for antibody reactivity to intrauterine stages as described above.

Microfilariae (Mf) from human blood were purified using 5 μM pore polycarbonate membranes as described by us earlier 6. Nocturnal blood samples collected from W.bancrofti infected Mf carriers or day time blood samples from S.digitata infected cattle were used for purification – Mf washed in Tris-EDTA buffer were taken in sheath fluid and analyzed by flow cytometry for forward and side scatter as described above.

A suspension (500 μl) containing about 10,000 intra uterine stages in PBS 7.2 with 1% BSA were mixed with 500 μl of 4 μg/ml or 1 μg/ml of biotinylated WGA (L-5142, Sigma Chemical Co, USA). Simlarly two dilutions, 500 or 2000 fold diluted biotinylated Con-A (BA-16, Bangalore Genie, India) were used and the suspensions were incubated at RT for 45 min. The cells were then washed x3 in PBS and taken in 0.5 ml of PBS with 1% BSA to which 0.5 ml of 250 fold diluted Avidin – FITC (S-3762, Sigma Chemical Co, USA) was added and incubated for 30 min at RT. The suspension was washed again thrice in PBS and samples taken in 1 ml of sheath fluid and 5000 events were acquired. The three populations were gated and fluorescence intensity was read using a 488 nm laser. The percentage reactivity of lectins in comparison to Avidin-FITC controls was calculated using CellQuest Pro software.

A suspension (500 μl) containing about 10,000 IU stages in PBS-BSA were mixed with 500 μl of 50 fold diluted human or Mastomys sera (normal as well as immunized animals) and incubated at RT for 45 min. The cells were then washed x3 and taken in 0.5 ml of PBS-BSA to which 0.5 ml of 250 fold diluted FITC labeled anti-human Immunoglobulin (F-6506, Sigma Chemical Co.USA) or 50 fold diluted FITC labeled anti-mouse IgG was added and incubated for 30 min at RT. The suspension was washed x3 in PBS and organisms were taken in 1 ml of sheath fluid for acquiring 5000 events in FACS Calibur. The three populations were gated and fluorescence intensity was read using a 488 nm laser. The percentage reactivity as well as mean fluorescence intensity were calculated for antibody binding and compared with FITC labeled second antibody conjugate controls using CellQuest Pro software.

Figs 1a &1c show the dot plots of intrauterine stages in forward and side scatter for two representative adult worms; respective contour maps are shown in Fig 1b &1d. Three distinct populations of organisms, R1, R2, and R3 could be identified. The three populations were consistently found in several worms although the relative numbers in each scatter varied between worms as shown in Fig 1. Purified mf of S.digitata and W.bancrofti (from blood) by using nucleopore membrane filtration scattered exclusively in the R1 region indicating that organisms in this region are microfilariae (Fig 2a and 2b respectively). The organisms in the three scatter groups were purified by sorting them in a FACS Calibur sorter and examined under a phase contrast microscope: Figs 10 &11 show organisms in the unsorted population which contain a mixture of early and late developmental stages of eggs as well as fully stretched Mf, while Fig 12 and 13 reveal pure Mf of S.digitata in the sorted R1 population confirming the data presented in fig 2a and 2b above. Organisms in R2 gate were found to be early developmental stages of eggs (Fig 8) while late developmental stages were found in the R3 population (Fig 9).

Two lectins, Wheat Germ Agglutinin (WGA) and Conconavalin-A (Con-A) that specifically react primarily with N-Acetyl-D-Glucosamine and D-mannose residues respectively (which are present ubiquitously in several parasites including filarial parasites), were chosen as markers in this study to evaluate the flow cytometry based assay procedure. The intrauterine stages incubated with biotinylated WGA or Con-A were analysed for single colour fluorescence using 488 nm laser in the three gated population. The background reactivity and mean fluorescence reactivity of avidin-FITC (controls) for the three gated populations was minimal. The specific binding of WGA to the three populations is shown in Fig 7a–f. There was a dose dependant binding of WGA to intra-uterine stages – lower concentrations Fig 7d,e &7f) bound proportionately less than higher concentrations as shown in histograms in Fig (7a,b &7c). Similarly a dose dependent binding of Con-A to intrauterine stages could also be demonstrated (Fig 3a–f). Both the lectins bound significantly (> 95%) to early and late developmental stages of eggs (R2 and R3 respectively) – their reactivity to intra-uterine Mf (R1) was, however, not high.

The binding of antibodies in human Bancroftian filariasis and in S.digitata immune Mastomys sera to intrauterine developmental stages could also be studied. The results of single colour fluorescence using 488 nm laser in the three-gated populations in two Mastomys sera are shown in Fig 4. The background binding of anti-mouse IgG-FITC conjugate to the three gated populations R1, R2, and R3 was very minimal. The binding profile of pre-immune sera were similar to conjugate controls while significant binding of antibodies to intrauterine eggs (R2 and R3 populations) could be demonstrated in both the immune sera (Fig 4a–f), in addition, similar reactivity was shown in three other immune Mastomys sera also (data not shown). Binding of antibodies in human filariasis sera to intrauterine stages could also be shown by the assay. The assay for two sera of elephantiasis cases is shown in Fig 5a–f. Significant binding of antibodies to intrauterine stages, particularly to egg stages could be demonstrated.

Although antibodies in human sera did not bind well to intra-uterine Mf, very significant binding of antibodies to a small select population of Mf was demonstrable by the assay (Fig 5a &5d). The mean antibody levels in nine patients with chronic filariasis were quantified (Fig 6). The levels were expressed as percentage reactivity and mean geometric fluorescence intensity as shown in Fig 6a &6b respectively. The antibody and lectin binding reported in this study are primarily restricted to reactivity of these molecules to the surface of intra-uterine stages since fresh live stages collected from adult female worms were used for the assays. Fixation with formaldehyde and permeabilising the cells resulted in antibody binding to intracellular components also (data not shown).