Solanum incanum roots and Carica papaya seeds were collected and placed in plastic bags. The plants were dried at room temperature for 2 months and crushed into tiny particles using Mekon Micromealer Single Phase and passed through a 0.5 mm mesh to standardize the particles. The ground plant material for each plant was divided into two equal portions and separately placed in two clean large bottles. Distilled water was added to one bottle holding each plant type until the sample was slightly submerged, after which it was left to soak for 72 h. The content was filtered; the process of soaking and filtering was repeated 3 times. The three filtrates were mixed in a container and freeze-dried using a freeze-drying machine (model FD-A made in Japan) for a month after which the aqueous extract was obtained in powder form.

Methanol was added to the other samples in the bottles until the samples were slightly submerged, soaked for 36 h and the content was filtered. The filtrate was placed in a round bottomed flask and fixed on a clean rotary vacuum evaporator (RE-100 Bibby, made in Japan). Temperature was set at 70^○C, the mouth of the machine closed and power switched on. The machine was switched off when methanol stopped running from the distiller. The sample was then removed from the machine and further dried on a water bath until there was no evaporation of methanol (methanol extract).

Schistosoma mansoni isolate used in this study originated from infected baboons at the Institute of Primate Research (IPR), Karen, Nairobi and maintained in Biomphalaria pfeifferi snails collected from Kakuyuni in Kangundo. The snails were placed individually in each well of a 24-culture plate. Each snail was infected with 3-6 miracidia. The miracidia were left for 30 minutes to penetrate. The infected snails were placed in plastic tanks containing un-chlorinated water (snail water), sterilized sand and pebbles. Daphnea were included for aeration. The snails were fed on lettuce and maintained at the IPR Malacology laboratory. Four weeks post infection the snails were covered with a dark cloth to prevent shedding of cercariae. Five weeks after infection, snails were placed under strong light to induce shedding of cercariae for mice infection and cercaricidal assay.

BALB/c mice acquired from IPR Animal Resources Department were housed in cages, in groups of five per cage. They were maintained on a commercial diet and water ad libitum. The animals were under light/dark cycle of approximately 12 h/12 h at ambient temperature (20^○C) and 50-60% relative humidity.

Mice were divided into six categories of 15 mice each, representing treatment and infected control groups infected with S. mansoni and an un-infected control group having 5 mice. The mice to be infected with

S. mansoni were anaesthetized using 0.02 ml Ketamine/xylazine mixture (ratio of 3:1). Each mouse received approximately 200 cercariae of S. mansoni through intact skin penetration by abdominal skin exposure using the ring method (Smithers and Terry, 1965). Treatment was done at week 4 post-infection with two doses two days apart. Each dose was 150 mg/kg body weight of the plant extracts. There were two groups for each plant extracts; aqueous and methanol. There were two control groups; one was treated with 450mg/kg body weight of Praziquantel (Farah et al., 2000), and the other was infected-untreated group.

Blood was obtained via heart puncture as follows; 5 naive mice (uninfected control) were sampled at wk 0; and 5 mice from each infected/ treated groups at wk 6. The blood was used to prepare serum for IgG ELISA. Five mice from all experimental groups and controls (PZQ and infected control) were perfused for worm recovery at week 6 and their livers were observed for gross pathology.

Blood obtained from heart puncture was allowed to stand at room temperature for 3 h and incubated overnight at 4 ^○C. The clotted blood was centrifuged in a Microfuge (Sorvall RT 6000D made in Japan) at 2000 rpm for 20 minutes and sera retrieved. Nunc-Immuno^TM plates (MaxiSorp ^TM Surface) ELISA plates were coated overnight with 50 μl of 10 μg/ml of soluble worm antigen preparation (SWAP) or 18 hr schistosomule soluble antigen (SSP) diluted in bicarbonate buffer, pH 9.6 and incubated overnight at 4 ^o C. The antigen was dispensed off on a blotting paper. The plates were washed six times using the washing buffer (0.05 % Tween 20 in PBS). This was followed by blocking of the non-specific binding sites with 100 μl 3% BSA in PBS and incubating at 37 ^o C for 1 h. The plates were washed off unbound BSA six times with washing buffer. Diluted (1:200) serum samples (50 μl) was dispensed into each well in duplicates and incubated overnight at 4 ^o C, and then washed as above. After washing the unbound serum, 50 μl of 1:2000 peroxidase conjugated rabbit anti-mouse IgG was dispensed into the wells and incubated for 1 h at 37^oC. The unbound conjugate was washed off as before and

50 μl TMB micro well peroxidase substrate (Sure Blue ^TM TMB) was added. The plates were incubated at 37 ^o C in the dark for 30 minutes and optical density was read at 630 nm in an ELISA micro plate reader.

At week 6 post-infection (2 weeks post-treatment), five mice from each group were perfused according to a modified method of Smithers and Terry (1965). Adult worm recovery was done according to the method described by Yole et al. (1996). Worm maturation was calculated using the following formula:

Figure 1

At week 6, gross pathology of the liver was observed in all the groups. The indices of comparison were; inflammation, adhesions and presence or absence of granulomas on the liver. The granulomas were categorized into none (no granuloma), few (1-3 granulomas), moderate (4-10 granulomas) and severe (>10 granulomas) per lobe.

Two millilitre of each of plant extract (5 ug/ml, 15 ug/ml and 30/ml) was dispensed in a well of 24 cell culture plate containing an aliquote of 20 cercariae. Two replicates for each concentration was made. Each preparation was observed under a dissecting microscope for cercariae motility at the following time points: 5, 10, 20, 30, 45 and 60 minutes. Immobile cercariae were noted at every point; when all cercariae were immobile before 1 h, the experiment was terminated. At the end of each experiment, iodine was added for clarity in counting of the total number of cercariae as a confirmation of accuracy of the counting procedure.

Analysis of worm recovery and immunological data was performed using Student’s t-test using computer excel programme and significance difference was defined as p<0.05. Gross pathology was noted by visual observation of liver tissues and cercaricidal assay was by enumerating the larvae under a microscope in percentages.

The maturation level of the Kibwezi isolate of S. mansoni in BALB/c, maintained in snails from Kakuyuni. was 28.5%.

The mean number of S. mansoni worms recovered in the infected-untreated control, Praziquantel, C. papaya aqueous, C. papaya methanol, S. incanum aqueous and S. incanum methanol groups at week 6, was 57±1.3, 25±2.4, 37±1.8, 33±1.4, 31±2.1 and 33±3.4, respectively. Praziquantel had the lowest mean number of worm recovered while infected-untreated control had the highest. The percentage worm recovery for Praziquantel, C. papaya aqueous, C. papaya methanol, S. incanum aqueous and S. incanum methanol groups were as follows; 43.9%, 64.9%, 57.9%, 54.4%and 57.9% respectively (Fig 1).

Worms recovered from different groups were subjected to Student’s t test to determine their significant difference in comparison with each other. Infected-untreated control was significantly different from PZQ control at p<0.001. All the four treatment groups were significantly different from infected-untreated control; C. papaya methanol and S. incanum aqueous at p<0.001, S. incanum methanol at p<0.01 and C. papaya aqueous at p<0.05.

There was a significant difference between three treatment groups and PZQ: C. papaya methanol and S. incanum aqueous, p<0.0l and C. papaya aqueous p<0.05. However, there was no significant difference between S. incanum methanol and PZQ, p>0.05. Statistically this shows that S. incanum methanol and PZQ had similar worm recoveries.

Fig 1: Percentage Worm Recovery in Treatments

Figure 2

In all mice in the six groups, all liver were inflamed and had adhesions. In infected-untreated control group, most of the mice had moderate granuloma while two mice had severe granuloma. Praziquantel group had only one mouse with few granulomas and the rest had no granuloma. In C. papaya aqueous treatment group, 4 out of 5 mice had few granulomas and only one mouse had no granuloma. In C. papaya methanol 3 mice had a few granulomas and 2 mice did not have any granuloma. In S. incanum aqueous and methanol groups, four of the mice had few granuloma and only one mouse had moderate granuloma. Generally the lowest granuloma levels were observed in Praziquantel group, while the infected control had the highest granuloma levels. The extract treatment groups’ granuloma levels lay in between the controls, C. papaya being second best to PZQ as S. incanum followed.

IgG responses were analyzed in seru collected from naive group, infected-untreated control and from treatment groups week-2 post-treatment (6 weeks post-infection). The results are shown in Feig 2.

IG response to SWAP antigen in infected-untreated control at week 6 was not very high (O.D 0.319). Praziquantel had a high IgG response in week 6 (O.D 0.352) which was significantly higher than the response in infected-untreated control (p<0.05).

In C. papaya aqueous, the IgG level (O.D 0.452) was higher than that of PZQ (O.D 0.352) and infected-untreated control (O.D 0.319) at week 6. However in C. papaya methanol treatment, IgG response was lower (O.D 0.367) at week 6 in comparison to the aqueous treatment but was slightly higher than that of PZQ and infected-untreated control. The IgG response in both C. papaya methanol and aqueous was significantly higher than PZQ (p<0.05).

Solanum incanum aqueous group had IgG level of O.D 0.331, which was not significantly different from PZQ (p>0.05) while S. incanum methanol group had the lowest IgG response, O.D 0.218, at week 6. The IgG response in the aqueous and methanol treatments was significantly lower than that of PZQ (p<0.05).

In assay for SSP-specific IgG responses, infected-untreated control group had higher IgG responses (O.D 232) than Praziquantel treatment (O.D 0.162). This difference was significant (p<0.05).

In C. papaya aqueous, IgG level (O.D 0.247) was significantly higher than that of C. papaya methanol (O.D 0.213; p<0.05). The responses in S. incanum aqueous group (O.D 0.144) was significantly higher than that of S. incanum methanol group (O.D 0.090; p<0.05). C. papaya had significantly higher IgG response compared to S. incanum extract treatments (p<0.05). C. papaya methanol (p<0.05), S. incanum aqueous and methanol groups (p<0.01) had significantly lower responses than infected-untreated control.

IgG responses to SSP antigen were lower in relation to IgG responses to SWAP antigens in all the treatments and controls

Figure 3

Fig 2: IgG Responses to SWAP and SSP Antigen in Schistosome Infected Mice

KEY

Wk = week

SWAP = soluble worm antigen preparation

SSP =18 hr schistosomule soluble antigen

The cercaricidal effect of the plant extracts is shown in Table 1. The results are the means of duplicate observations of lowest (5 µg/ml), moderate (15 µg/ml) and maximum (30 µg/ml) concentrations of the plant extracts at different time intervals.

In both aqueous and methanol C. papaya treatments, at concentrations of 5 µg/ml and at the 5 ^th minute, about a quarter of the cercariae was dead; at the 10 ^th minute about a half of the cercariae were dead and at the 20 ^th minute all the cercariae were dead. The two treatments had similar strength in killing the larvae worms at the lowest concentrations. However there was great difference in the effects of both methanol and aqueous of C. papaya extracts at the moderate (15 µg/ml) and maximum (30 µg/ml) concentrations. At the 5 ^th minute; 15 µg/ml and 30 µg/ml concentrations of C. papaya methanol group, all the cercariae had lysed and could not be enumerated. In C. papaya aqueous at, 15 µg/ml and 30 µg/ml about three quarters and slightly over three quarters of the cercariae were dead, respectively, at the 5 ^th minute. At the 10 ^th minute all cercariae at the same concentrations were dead. The methanol extracts at increased concentrations (15 and 30 µg/ml) showed a stronger ability in killing the cercariae than the aqueous ones, as it led to lysis of the cercariae.

In S. incanum treatments at concentrations of 5 µg/ml at the 5 ^th minute, about a quarter of cercariae were dead in aqueous treatment, and about a third of the cercariae were dead in methanol group. At the 10 ^th minute in both aqueous and methanol extracts, about a half of the cercariae were dead while at the 20 ^th minute, all the cercariae were dead in both concentrations. The killing strength of aqueous and methanol extracts was almost the same at the lowest concentrations. At 15 µg/ml, 5 ^th minute, in aqueous group, there were over three quarter deaths of cercariae and 100 % death was realised at the 10 ^th minute. However at the same concerntration, all cercariae were dead in the methanol treatment at the 5th minute.

At a concentration of 30 µg/ml of S. incanum aqueous treatment, all the cercariae were dead and not lysed at the 5 ^th minute, while in the methanol treatment; cercariae had been lysed at the same duration and concentration. However, no cercariea was dead after one hour in aqueous control.

Figure 4

Table 1: Effects of Plant Extracts on Cercariae at different concentrations

KEY

*_ cercariae were dead and lysed
_ Observation stopped