Swiss white mice used in this study, were maintained at the Rodent House, Institute of Primate Research (IPR). They were fed with nutrient pellets (Laboratory Cho from Unga Feeds® CO.) and supplemented with carrots and kale leaves. Water was supplied ad libitum. S. mansoni intermediate host, Biomphalaria pfeifferi were obtained from Kakuyuni, Kangundo and maintained at the Snail room at IPR, at 22-25°C, 12 h of light and 12 h of darkness, and fed on dried lettuce.

The digestive gland of colony bred snails was dissected out from the rest of the snail body. The two tissues were homogenized separately in a glass mortar and pestle. The homogenates were centrifuged for 1 h at 10,000 g at 4°C to obtain the soluble proteins. The concentration of the proteins was assayed using Bradford method (1976). Two groups of mice, DG receiving soluble protein from the snail digestive gland, and RT receiving soluble protein from the rest of the body, were immunized with 50 µg of the specified soluble protein in 100 µl of complete Freund’s adjuvant intra-peritoneally. Each of the groups received 2 boosters, at weeks 2 and 4 of 25 µg of specified protein in 100 µl of incomplete Freund’s adjuvant intraperitoneally. One week after the final vaccination, the two groups of immunized mice, DG & RT, and a control IC, were challenged abdominally with 150 S. mansoni cercariae, using the method of Smithers and Terry (1965).

0-3 hour release protein was prepared by artificially transforming cercariae (Ramalho-Pinto et al. 1974) and separating the heads from tails on a discontinuous Percoll gradient (Ladzins et al. 1982). The heads were cultured in Complete medium [RPMI 1640 (Sigma), containing 10% foetal calf serum (Gibco BRL, Germany), Gentamycine 2mM (Sigma) and 1% β-mercaptoethanol] for 3 h. The schistosomules were pelleted, and the supernatant containing the proteins released during penetration obtained. Protein concentration was determined using Bradford (1976) method. Schistosoma mansoni Soluble worm antigen (SWAP) was prepared as described by Yole et al. 1996.

At weeks 2, 4 and 6 post-challenge, blood was obtained from anaesthetized (mixture of Rompun and Ketamine in ratio of 20:1) DG, RT, IC and naïve mice by heart puncture. Serum was prepared from the blood and stored at -20°C before use in IgG Enzyme linked immunosorbent assay (ELISA).

The inguinal and auxillary lymph nodes, which drain the abdominal area and the spleen, were obtained from the mice which had been bled. Lymph node cells and spleen cells were prepared as described by Yole et al. 2006. Flat-bottomed 48-well culture plates (costar®, Corning Incorporated, NY, USA) were used for culture and 6 x 105 cells were dispensed in each well. Duplicate wells were set for each regime. Negative control had only complete medium and cells. Positive control had 1μg/ml of Concanavalin A, while test wells had 10μg/ml of either SWAP or 0-3 hr release protein. The total volume of culture medium per well was 400μl. The plates were incubated at 37ºC, 5% CO2 for 48 h for Con A and 72 h for the other set-ups. The supernatants were collected from each well and were stored at -20°C before use in Cytokine ELISA.

MABTECH Inteferon gamma and IL-5 kits were used for the assay. Nunc-Immulon ^TM (Maxisorp^TM surface ) ELISA plates were coated with 50 µl of 5 µg/ml solution of monoclonal anti-IFN/anti-IL-5 antibody and incubated overnight at 4°C. They were blocked by1 h incubation with 100 µl/well of 0.1% Bovine Serum Albumin (BSA). In specified wells, 50 µl/well of samples and mouse IFN/IL-5 standards were added in duplicate and plates incubated for 2 h. In each well, 50 µl of 3 µg/ml of rabbit anti-mouse IFN [Detection mAb (R4-6A2-Biotin)] or IL-5 [(Detection mAb (TRFK4-Biotin)] was added and incubated for1 h. Binding was then detected by adding 50 µl/well of Streptavidin-Horseradish peroxidase and incubating for 1 h. The incubations were at 37°C, and there were appropriate washes in between the steps. After the final wash, 50 µg/well of the substrate (Sure Blue ^TM TMB) was added. The plates were incubated in the dark, at 37°C, for 30 minutes. Optical density was read at 630 nm in an ELISA microplate reader (Dynatech MRX).

Nunc-Immuno^TM plates (MaxiSorp ^TM Surface) ELISA plates were coated overnight at 4°C with 50 μl of 10 µg/ml SWAP/ 0-3 hr release antigens. Non-specific binding sites were blocked by 1 h incubation with 100 μl 3% BSA. Diluted (1:200) 50 μl serum samples were dispensed into specified wells in duplicates and incubated for 1 h. IgG binding was detected by incubating for 1 h with 50 μl of 1:2000 peroxidase conjugated goat anti-mouse IgG (SIGMA Goat anti-mouse IgG peroxidase). The incubations were at 37°C, and there were appropriate washes in between the steps. In each well, 50 μl of substrate (Sure Blue ^TM TMB) was added. The plates were incubated at 37°C in the dark for 30 minutes. Optical density was read at 630 nm in an ELISA microplate reader (Dynatech MRX).

At week 6 the abdominal cavity of DG, RT and IC was opened. The gross pathology of the liver was observed in terms of: inflammation, adhesions and presence of granulomas. Granulomas appear as raised pinheads sized foci distributed over the surface of the liver lobes. Severity of the granulomas was categorized as follows: 1-3 granulomas per lobe were considered few, 4-10, moderate and ≥ 10 severe.

The mice were perfused using the modified method of Smithers and Terry, 1965 (Yole et al. 2006). The perfusate was collected in a 20 cm glass Petri-dish. The perfusate containing the recovered worms were transferred into urine jar and topped with phosphate buffered saline (PBS). After the worms had settled, the supernatant was sucked out, and the settling procedure repeated three times. When the supernatant was clear, the worms were then placed on a Petri dish containing PBS and then counted. Any worms still left in the mesenteries/ liver were recovered by soaking the mouse in a Petri dish containing PBS for at least 30 minutes, to allow the worms to crawl out. Percentage worm recovery and reduction for each group was calculated as shown in the formulae below.

Figure 1

Figure 2

The percentage worm recovery for RT group was 39.5% while that for DG group was 56.7% when perfused 6 weeks post infection. Both RT and DG had significantly lower worm burden than IC (t-test; p< 0.05). The difference between RT and DG was also significant (p<0.05) with the worm burden being lower in RT mice than DG. Worm reduction in RT was 60.5% while that of DG was 43.3%. This exceeded the World Health Organization goal of 40% protection for both proteins (WHO/TDR, 1996).

Inteferon gamma is a THI cytokine involved in development of inflammatory foci, while Interleukin-5, a TH2 cytokine, is one of the cytokine which stimulate B cells to produce antibodies.

RT group of mice had significantly higher (p< 0.05) IFN gamma responses than both DG and IC at all time points for both lymph node and spleen cells and also for both antigens, SWAP and 0-3 hr release protein (Fig. 1). In most of the times, DG had higher responses than IC, for both cells and both antigens, but the difference was not always significant. This shows that RT vaccination had the stronger interferon gamma production, implying better Th1 protection against the parasite, produced by RT, compared to DG (Pearce & Sher., 1991).

Lymph node cells had a peak production of IFN gamma at week two post-challenge, which is in agreement with Pemberton et al., (1991). Spleen cells had peak responses at week four. This is expected since antigens take longer time to reach the spleen compared with the draining lymph nodes, which encounter parasite antigens presented by dendritic cells soon after the infection. The 0-3 hr release protein gave better responses than SWAP antigens. This is expected since sampling was done at early part of migration of parasites, before most worms had matured to adult worms, implying more shared antigens between 0-3 hr release protein and early stages of development of S. mansoni before maturation.

Figure 3

Fig. 1: Inteferon gamma responses in spleen cells stimulated 0-3 hr release protein

Key: RT - Mice immunized with soluble proteins derived from the rest of the body tissues of the intermediate host and then challenged; DG - Mice immunized with soluble proteins derived from the digestive gland of the Intermediate host then challenged; IC - Infected control

RT mice had a significantly higher IL-5 (p<0.05) responses compared to DG and IC (Fig. 2) except for just one time point were the difference was not significant (LN cells, SWAP week 4). Except for responses to SWAP for the spleen cells, all other responses for RT were higher at week 2 when compared to week 4. DG and IC had similar IL-5 responses throughout the sampling period, for both organs and antigens. These results show that, in addition to IFN gamma, RT was able to stimulate production of IL-5, and hence invoke Th2 protection in Swiss mice. This phenomenon is reported in man, primates and rats (Pearce & Sher., 1991). Again, like in the case of IFN gamma, cells stimulated with 0-3 hr had higher responses than those stimulated with SWAP.

Figure 4

Fig 2: Interleukin-5 responses in mice spleen cells stimulated with 0-3 hr release protein

Key: RT - Mice immunized with soluble proteins derived from the rest of the body tissues of the intermediate host and then challenged; DG - Mice immunized with soluble proteins derived from the digestive gland of the Intermediate host then challenged; IC - Infected control

RT had very strong IgG responses at week 2, which declined slightly at week 4, before rising again at week 6 (Fig. 3). The strong response at week 2 correlates with higher IL-5 responses at the same time point for this group. IL-5 is involved in the Th2 responses, which stimulates B cells to produce antibodies. RT had significantly higher IgG responses than both DG and IC at all sampling points (p<0.05). IgG responses for DG and IC showed an increasing trend from week 2 to 6. There was no significant difference for IgG responses between DG and IC although DG responses for 0-3hr release protein were consistently slightly higher than those of IC. RT, DG and IC had significantly higher IgG responses than naïve control, which is expected. This shows that all the groups produced IgG which is involved in antibody dependent cell–mediated cytotoxicity (ADCC; Hagan et al, 1991). However, the strongest response was in RT, and this correlates very well with the greatest worm reduction recorded in this group.

Figure 5

Fig 3: 0-3 hr Schistosome specific IgG responses

Key: RT - Mice immunized with soluble proteins derived from the rest of the body tissues of the intermediate host and then challenged; DG - Mice immunized with soluble proteins derived from the digestive gland of the Intermediate host then challenged; IC - Infected control

NAIVE: Not infected

Gross pathology was done by physical observation of the liver surface to detect inflammation; adhesions and presence of granulomas. None of the mice had granuloma. This could be attributed to a slower development of the parasite in the Swiss mice model compared to, for example, BALB/c mouse where granulomas were present at week 6 (Kanyugo et al., 2009).

Inflammation in the liver was categorized as slightly inflamed or inflamed. All the mice in RT group had slightly inflamed liver tissues. DG had 5 mice with slightly inflamed liver tissues and one mouse with inflamed liver. All the mice in IC had inflammed liver tissues. This shows that RT had the least pathology, while IC had the worst, with DG lying in between.