Chapter one

Introduction and Literature Review

1.1 Introduction:

Schistosomiasis is known as bilharzia is after Theodor bilharz, who first identified the parasite in Cairo in 1851. Infection is widespread with A relatively low mortality rate, but a high morbidity rate, causing severe Debilitating illness in millions of people. The disease is often associated with water resource developmental projects, such as dams and irrigation schemes, where the snail intermediate hosts of the parasite breed (WHO, 2010).

Schistosomiasis is a chronic, parasitic disease caused by blood flukes (Trematode worms) of genus Schistosoma. An estimated 700 million people at risk in 74 endemic countries; as their agricultural, domestic and recreational activities expose them to infested water, more than 207 million people are infected worldwide, most of them live in poor communities without access to safe drinking water and adequate sanitation, and hygiene; play habits make children vulnerable to infection, and in many areas a large proportion of school-age children are infected. The WHO strategy for Schistosomiasis control focuses on reducing disease through periodic, targeted treatment with praziquantel (WHO, 2010). Many plants are known to have molluscicidal activity; such plants may provide cheap, safely produced, biodegradable and effective control agents in rural areas of developing countries (Marston, 1993). Schistosomiasis is second to malaria in terms of socio-economic and public health importance (Doumenge et al., 1987). Two clinical forms of the disease are known: urinary schistosomiasis caused by Schist soma haematobium, and intestinal schistosomiasis caused by Schistosoma mansoni, Schistosoma japonicum, Schistosoma intercalatum and Schistosoma mekonge. Intestinal schistosomiasis caused by Schistosoma mansoni affects millions of people in many tropical and subtropical countries (Eddleston and Pierini, 1999). In chemical laboratories worldwide, compounds, more and more toxic to the vector snails, are being discovered. Copper sulphate (CuSO4), Sodium pentachlorophenate (NaPCP), triphenmorph, and niclosamide are among the synthetic compounds. Niclosamide is the molluscicide of choice, being highly active against different stages of the snail life cycle, and it is not toxic to humans, domestic animals and crops, but is toxic to fishes (Sturrock et al., 1994). However, the application of niclosamide does not prevent recolonization

1 by the remaining snails which may lead to selection of molluscicide-resistant populations. The mollusicidal potency of the present synthesized organophosphorus derivatives of niclosamide was studied. Preparation of such derivatives was encouraged due to their potential rapid metabolism, decomposition in the soil and water and low chronic toxicity (Gruzdyev, 1983). Several other compounds such as nicolinanilides, organotin and lead compounds, and “Endod” an extract from the plant ( phytolacca dodecndra), It is generally agreed that snail control is one of the most rapid and effective means of reducing transmission of schistosomiasis (Mc Cullough et al,. 1980). The potential of using plants for control of the intermediate hosts of human schistosomiasis and other snail –transmitted parasitic infections has received considerable attention (Perret and Whitfield,. 1996). The importance of plants as sources of natural product bioactive molecules to medicine lies not only in their pharmacological or chemotherapeutic effects but also in their role as template molecules for the production of new drug substances (Phillipson, 1994). Many plants are known to have molluscicidal activity; such plants may provide cheap, safe produced, biodegradable and effective control agents in rural areas of developing countries (Marston, 1993).

In the Sudan, a new trend has developed to control vectors of human disease, using natural products including plant extracts as larvicides and molluscicides and schistosomicide. Interest in plants as pesticides has started in the Sudan as early as 1930 s when Archibald (1933) advocated planting the tree, Balanites aegyptica along the water courses of the River Nile. Laboratory trails of this scientist showed that one fruit soaked in 100 litter of water for 24 hours killed cercaria, tadpoles and mosquito larvae, while his field observations showed that fallen fruits of Balanites species inhibited the increase of snail population. One of the alternatives in the integrated control strategies is the use of natural products; the literature contains many examples of plant products that showed considerable activities against mollusks. The dried berries of the Ethiopian herb phytolacca dodecandra “Endod” showed high molluscicidal potency (Lemma, 1965 and 1970) with saponin as an active constituent (Lemma, 1970; Baallway, 1972; Lemma et al,. 1979). The relatively high molluscicidal potency of the seeds of Croton species has been reported by some workers (Amin et al., 1972, Daffalla, 1973 and Daffalla and Amin, 1976). Their detailed laboratory and field evaluation of the molluscicidal potency of Croton macrostachys seeds (locally known as Habat –El-Molok) showed that a concentration of 0.1 ppm of the aqueous extract of these seeds kill 90 percent of B.truncatus, on 24 hours

2 exposure , while 20 ppm of the same solution was required to kill 90 percent of B.pfeifferi. In the field a concentration of 5 ppm of this extracts required to kill 100 percent of the two species. Daffalla (1973) found that B.aegyptiaca and Scilia lihacina have molluscicidal activities against Bulinus truncates and Biomphalaria pfeiffei . Elkheir and El Tohami (1979) screened 51 Sudanese medicinal plant species. Eight plant species possessed molluscicidal activity against B.truncatus. In the Sudan the disease is prevalent in all regions of the country, and it increased in distribution and prevalence as a result of expansion in water resources development projects (Blue Nile Health Project, 1981) (WHO, 1985). Schistosomiasis has been recognized as a common parasitic infection in domestic stock and wild game in Africa, but there has been little recognition of its veterinary significance. Schistosomiasis in cattle and sheep may, however favor intensive disease transmission and cause significance losses. Schistosomiasis control can be achieved through proper sanitation, snail eradication and Chemotherapy (Jordan et al., 1980). Chemotherapy of Schistosomiasis by anti-schistosomal drugs aims at eradication of the worms and stopping the production of eggs in the greatest percentage of patients treated in the shortest time, and with theleast cost.

Praziquantel (PZQ) is a drug of choice for Schistosomiasis control on a population wide basis in endemic areas, because it is effective against all species of Human Schistosomiasis. Substantial reduction in its price with market competition was recorded. Also it is a safe drug with tolerable side effects (Abramowicz, 1992; Cioli et al., 1995; and Doenhoff et al., 2002). Yet in the recent years, a number of reports indicated the apparent failure of the recommended doses of PZQ to yield the expected cure rates in human population in Kenya (Coles et al., 1987), and Egypt ( Ismail et al., 1996 ) This evidence about resistance raised alarm to search for new alternative drugs either synthetic or from natural resources to overcome this problem.

The use of natural resources (e.g. plants, fungi, bacteria.etc) for treatment of many diseases is associated with folk medicine from different parts of world. Natural products from some of these natural resources continue to be used in pharmaceutical preparation either as crude extract, fraction, pure compounds or analogous compounds. Plants have provided a number of useful clinical agents that prove to have considerable potentials as sources of new drugs ( Pillipson, 1994). So, the use of medicinal plants which grow abundantly in areas where Schistosomiasis is indemic may become a useful complement either as

3 molluscicides or chemotherabutic for the control of this disease. However, few studies have addressed the use of medicinal plants with antischstosomal activity as treatment for this disease (Liu and Weller, 1996 and Schulz et al., 1997). In the Sudan, medicinal and aromatic plants and their derivatives are locally sold at special shops called Atareen. vendors of traditional medicine within Khartoum area are of local inhabitants who have well established retail or whole sale outlets. The most outstanding and nationally recognized house of expertise in sudan is known as Timan. They are usually providing counseling to the patients in addition to dispensing these herbal preparations.

1.2 Schistosomes and Schistosomiasis:

1.2.1 Schistosomiasis in Africa:

Schistosomiasis is a chronic, debilitating parasitic caused by blood flukes of the genus Schistosoma, and is also known as “bilharzias”. It is indemic in 74 countries in Africa, South America and Asia. Worldwide, an estimated 200 million people are infected, of which 20 million are assumed to suffer from or less a sever form of the disease creating 4.5 million DALYs1 lost (WHO Expert Committee 2002). Schistosomiasis is endemic in 46 out of the 54 counties in the Africa continent. The disease may cause damage to various tissues (the bladder, liver or the intestines) depending on the species, and lowers the resistance of the infected person to other diseases. There are 16 different known species of Schistosoma (S), of which 5 are infective to man, S.mansoni, S.haematobium, S.intercalatum, S. japonicum and S.mekongi. the species differ according to their snail intermediate host, egg morphology, final location of the adult worms in the human body, resulting symptoms, and their geographical distribution ( Doumenge et al., 1987). In sub-Saharan Africa, approximately 393 million people are at risk of infection with S.mansoni, of which 54 millions are infected. These numbers for S.haematobium are estimated to be as high as 436 millions at risk and 112 millions are infected.

The intermediate hosts of schistosomes are freshwater pulmonate snails, that belong to the “Planorbidae” family. The species belong to genus, Bulinus; the host for S.haematobium and S.intercalatum (Sturrock, 1993). The Bulinus species prefer water velocities below 0.3 m/s, gradual changes in the water level, a slope of less than 20 m/km, firm mud substrate, little turbidity, some organic pollution, partial shade and optimal water tempreture between 18 C and 28 C(Birley, 1991). Under these conditions, aquatic snails, flourish creating

4 favorable habitats for intermediate snail host. Transmission can take place in almost any type of habitat e.g., from large lakes and rivers to small seasonal ponds and streams. Although transmission may be intensive in both natural and man-made water bodies, the latter seem particularly important as human population density is often high. Within the irrigation schemes transmission is focal and is primarily due to much localized contamination of habitats with human excreta or urine containing schistosoma eggs and, also because of the high incidence of human water contact at a few points. Schistosome transmission usually is seasonal, primarily due to the variation in temperature and the irrigation cycle. Local circumstances influence both time and place of transmission, which should be taken into consideration when designing ecological measures to control the snail population (Birley, 1991).

1.2.2 Schistosomiasis in the Sudan:

The first mention of the existence of Schistosomiasis in the Sudan during the last century was reported by Balfour (1904), when he drew attention to a focus of “endemic heamaturia” in Khartoum primary school where he found 17% of children to be suffering from urinary Schistosomiasis. Archibald (1914) reported four cases of intestinal Schistosomiasis in Khartoum, three of which were from Egyptian soldiers. Before 1918, few cases occurred in the Northern States and in other areas of the country where considerable contact with Egyptian persons had taken place. The infection is thought to have been introduced by the Egyptian laborers and West African pilgrims, who used to cross Sudan in the way to the holy land Mecca ( Rahma, 2010).

The manifestation of the disease began to capture medical attention within a period characterized by the construction of agricultural developmental schemes. In (1925), an agricultural scheme was constructed in Gezira area, after that the prevalence of the disease increased rapidly to reach 15% in 1952 and 80% in 1978 (Omer et at., 1976). The disease is prevalent in all regions of the country, and in recent years, the disease increased in distribution and prevalence as a result of expansion in water –resources and developmental projects. In northern Sudan, Schistosomiasis haematobium was reported along the Nile, North of Khartoum and Schistosoma mansoni in small localized areas (Buchana, 1937). Omer (1978) found the prevalence of Schistosoma haematobium among fisher men in Lake Nassir to be over 38%. In Southern Sudan, both S. haematobium and S.minsoni were reported (Omer et al 1972). (Brown et al., 1984),during the assessment of Jongoly Canal, reported the presence of schistosome intermediate

5 host in the area and emphasized the possibility that the canal might increase snail population and raise the prevalence of parasitic disease. In Western Sudan, S. haematobium prevalence of 14% was reported among children (Eltom, 1976) but also the prevalence of S. haematobium was found to be 8.5%, 29.5% and 37.7% among children in Kadogle, Deleng and Rahad respectively (Dafalla and Suliman, 1988). According to Doumenge et al., (1987); hospital records showed a prevalence of S. haematobium of 8.7% in Kassala and 3.4% in Gadarif. In the White Nile area S. haematobium and S.minsoni have been reported (Doumenge et al., 1987). In Khartoum State types of schistosomiasis have been reported (Omer. 1978). The expansion in agricultural projects has resulted in an increase of schistosomiasis in the Sudan (WHO ., 1985) schistosomiasis is now endemic in Kenana Sugar scheme (Amin, 1978), Guneid Sugar scheme(Collins, 1976), Rahad scheme (BNHP, 1984), New Halfa scheme (Abd Elgalil, 1980) as well as Gazira- Managil scheme. Both types of schistosomiasis were more prevalent in the Managil compared to the Gazira area, where the Blue Nile Health project has adopted comprehensive control strategy consisting of chemotherapy, focused mollusciciding, health education and improvement of water supply and sanitation during the period 1980 – 1990. In April 1999, Gezira Administration Board adopted a new system for imporoving irrigation, consisting of drying and cleaning canals before the irrigation season; this highlights the effectiveness and long – lasting impacts of integrated control programs in high endemic areas (Hilali et al., 1995).

There are least 19 species of Schistosoma of which five were seriously affecting people. Schistosoma japonicum and Schistosoma mekongi, causing intestinal Schistosomiasis, are found in Asia; S.mansoni and S.intercalatum cause intestinal Schistosomiasis in Africa and S.haematobium causes urinary Schistosomiasis in Africa and the Middle East. S.mansoni, S.haematobium and Schistosoma japonicum are the most widespread and important species (Rahma. 2010). Schistosomes vary in their choice of snails as intermediate hosts; the genus Biomphalaria for S.mansoni , Bulinus for S.haematobium and Oncomelania for S.japonicum ( Warren, 1987).

1.2.3 Pathogenesis of Schistosomiasis:

In Schistosomiasis, the pathological change starts with granuloma formation in response to the schistosomal egga and their secretions which are more potent than the schistosomal worm itself (Warren et al., 1974). The primary cause of

6 liver pathology in schistosome infection is the immobilization of the schistosome eggs into the radiculi of the portal system leading to granuloma formation which destroys the involved radical (Von Lichtenberg, 1955). Destruction of the vascular muscle layer of the portal vein follows with subsequent inflammation and fibrosis of it (Andrade, 1965).Such inflammatory reaction with consequent fibrosis usually result in presinusoidal obstruction of the portal blood flow leading to portal hypertension which causes splenomegaly, hepatomegaly, ascites, esophageal and gastric varices as a result of portasystemic shunting (Raia et al., 1985). The liver becomes hard and nodular. Bleeding due to eaeophageal varices is a major cause of death (mahmoud., 1984). The liver is considered to be the main organ responsible for the biosynthesis, uptake and degradation of a number of biological materials in blood including protein and enzymes. Almost all the enzymes are intracellular. Following an injury, or death of physiological active cells, the enzymes are released into the circulation and can be used as an indicator of cell damage rather than cell function (Eastham, 1975). Schistosomal infection causes liver necrosis characterized by fibrosis and absence of regeneration leading to liberation of enzymes contanined within the cell (Salah, 1962 and EL-Haieg et al., 1978) Serum enzymatic activity of alanine transaminase (ALT) is considered a sensitive parameter for functional changes in hepatosplenic bilharziasis (EL-Haieg et al., 1978 and Taha et al., 1992). Increase of these enzymes denotes an active hepatic cell damage and/or an increase in permeability of the cell membrane to this enzyme (Ghanem et al., 1970 and Ebeid et al., 1987).

1.2.4 Chemotherapy of schistosomiasis:

Schistosomiasis is a major health problem in tropical countries involving an estimate of 200 million people (Nash et al., 1982). Schistosomiasis control can be achieved through proper sanitation, snail eradication, immunization and chemotherapy (Macdonald et al., 1969; Jordan et al., 1980). Chemotherapy of Schistosomiasis by antischistosomal drugs aims at stopping the production of eggs in the greatest percentage of patients treated in the shortest time, and with the least cost. It also aims to relief symptoms, heals existing pathological lesions and prevents further damage to tissues and organs of host (Mousa and Zien EL- Abdin, 1971). By the early 1970s, metrifonate was established for use against S.haematobium, although the need for three fortnightly treatments caused logistic problems for community use. Fears for its safety prevented the

7 widespread acceptance of hycanthone, the first single-dose drug effective against S.mansoni and, possibly, S.haematobium. But a similar drug, oxaminiquine, was released for community use against S.mansoni in the 1970s. the real turning point was the arrival in the early 1980s of praziquantel, a safe, effective, single-dose drug active for all schistosomes and many other human and veterinary helminthes, besides (WHO, 1985). It promised to revolutionise community chemotherapy against schistosomiasis, although it was initially too expensive for widespread use. Its price has at last dropped radically and it is now the major weapon for community control of schistosomiasis.

1.3 Snail:

Snails which belong to the family planorbidae act as intermediate hosts for the parasitic blood trematodes of the genus Schistosoma which cause the debilitating disease known as Schistosomiasis. The snail control is one of the most rapid and effective means of reducing transmission of Schistosomiasis. The potential of using plants for the control of the intermediate hosts of human Schistosomiasis and other snail-transmitted parasitic infections has received considerable attention (Perrett and Whitefield, 1996).

The intermediate hosts of the Schistosomes in Africa are freshwater pulmonate snails that belong to the planorbidae family. The species belong to two genera, namely Biomphalaria; the host for S.mansoni, and Bulinus; the host for S.haematobium and S.intercalatum (Sturrock, 1993). Both Biomphalaria and Bulinus species prefer water velocities below 0.3 m/s, gradual changes in the water level, a slope of less than 20 m/km, firm mud substrate, little turbidity, some organic pollution, partial shade and optimal water temperature between 18 C and 28 C)65 – 82 f) (Birley, 1991). Under this condition, aquatic vegetation and algae, which are the main sources of nutrition for the aquatic snails, flourish creating favorable habitats for intermediate snail hosts. Transmission can take place in almost any type of habitat e.g., from large lakes and rivers to small seasonal ponds and streams.

1.3.1 Scientific classification

Kingdo: Animalia, Phylum: Mollusca, Class: Gastropoda, Superfamily: Planorboidea, Family: Planorbidae, Subfamily: Bulininae, Genus: Bulinus

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Species: tropicus complex - 14-15 species

Bulinus angolensis, Bulinus depressus , Bulinus hexaploidus, Bulinus liratus, Bulinus mutandensis Bulinus natalensis , Bulinus nyassanus, Bulinus octoploidus , Bulinus permembranaceus , Bulinus succinoides Bulinus transversalis , Bulinus trigonus , Bulinus tropicus , Bulinus truncatus , Bulinus yemenensis (Jorgensen, et al., 2007).

1.3.2 Shell description:

The shell of species in the genus Bulinus is sinistral. It has a very large body whorl and a small spire.

1.3.3 Distribution:

These snails are widespread in Africa including Madagascar and the Middle East. This genus has not yet become established in the USA, but it is considered to represent a potentially serious threat as a pest, an invasive species which could negatively affect agriculture, natural ecosystems, human health or commerce. Therefore it has been suggested that this species be given top national quarantine significance in the USA (Cowie, et al., 2009).

1.3.4 Snail control:

The first fruits of the WHO programs were the development of molluscicides which were available in the middle of 1950s. One of them, niclosamide, is still in use and effective. Careful biological studies suggested an alternative approach to the control of oriental Schistosomiasis: Oncomelania snail populations are less resilient than Biomphalaria and Bulinus spp. Habitat modification was recommended as potential control measures because it also improved rice production and this could potentially, fund the elimination of snail habitats (Rahma, 2010).Another method for achieving a long term but low cost vector control program is by the use of biological control agents. Recently a number of predators of snails have been tested in the laboratory and field. The snails control is an important preventive strategy associated with the treatment of infected people together with environmental and socio-economic improvements and health education with community participation. The method of environmental control depends on the modification of snail environment, rendering the habitat unsuitable for snail breeding. However, environmental control can be most effective in water developmental schemes (WHO, 1980). Although chemical molluscicides are the most used approach of snails control

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(WHO, 1994), yet chemicals have their hazards in addition to their high costs. They may be toxic to aquatic fauna and source of pollution. All factors make it imperative, to consider using molluscicides of plant origin either naturally growing or locally cultivated. Generally, chemicals derived from plants have advantages over synthetics, in that they always have the least side effects, beside their easy application with simple technology and their cheap production especially when the are abundant in the area which is always the case (Sharma and Dwivedi, 1980).

1.3.5. strategic control of schistosome intermediate host:

1.3.5.1 Control through the main host:

1.3.5.1.1 Health education:

Key to long term control of schistosomiasis are improvements in hygiene and sanitation. By eliminating human waste in fresh bodies, part of the complex life cycle of the schistosome can be eliminated. High rates of reinfection demonstrated the need for health education through programs established by the government (Manal , 2010).

1.3.5.1.2 Immunological control:

Generation immunity through use of vaccines iscomplexs. In the presence of high prevalence, vaccine would not be given to naïve patients. Rather, those receiving the vaccine can are expected to have already been exposed and to experience repeated exposure to schistosomiasis after getting the vaccine. It is precisely the host immune response that gives rise to granulomas responsible for the morbidity of schistosomiasis. Potentially, by triggering the production of immunity to various schistosomiasis antigens, the vaccine could promote the production of granuloma formation. In fact, however, progress is being made in phase 1 and 2 clinical trails of different vaccine. Even without eradication of schistosomes from the environment, the vaccine appears to reduce susceptibility to re-infection. It is postulated that the vaccines artificially-induced immunity is boosted by re-exposure to the not-yet-eradicated schistosomes. This suggests that immunogenicity may need to be assessed if and when schistosomes are eliminated (World Health Organization, 2002).many trials of vaccination are based on homologous or heterologous antigens. Bashtar et al., ( 2006) Found

10 that schistosomal worm and egg antigen had a potency role in protection against schistosomiasis.

1.3.5.1.3 Chemotherapy:

Praziquantel (PZQ), a pyrazinoisoquinoline derivative, is mainstay of treatment and a critical part of community-based schistosomiasis control Programs. Recent reports on praziquantel elucidate its fail to stop reinfection as a result of development of drug resistant schistosoma strain, beside it induces hemorrhages in the lung tissue of the host as will as abdominal pain diarrhea by long term application of the drug. Mirazid, the oleo-resin extract from Myrrh of Commiphora molmol tree, was established in Egypt as a new drug against schistosomiasis and other parasitic diseases (Manal, 2010).

1.3.5.2 Control through the intermediate host:

Snail control could be regarded as a rapid and efficient of reducing or eliminating transmission and remains among the methods of choice for schistosomiasis control. The importance of snail control should be overlooked, for despite greatly improved chemotherapy by single dose oral treatment with PZQ , there are logistical problems in mass treatment that threat of reinfection and absence a completely and safe schistosomiasis vaccine support the use of snail control as an important means in control programs (Manal, 2010).Snail control can be divided into three categories; environment, molluscicides and biological control.

1.3.5.2.1 Environment Control:

Environment factors which influence snail distribution and which might be manipulated to be achieved were suggested by Thomas and Tait (1984) these factors are classified as follws:

1.3.5.2.1.1 Water Chemistry:

Water chemistry includes calcium concentration, total dissolved chemical content and oxygen. The densities of all snail species were very low in salt water, high in medium water and somewhat lower in hard water, where low calcium concentration appears to be the direct or indirect cause of the poor snail fauna.the effect of NaCl concentration other aspects of salinity on freshwater pulmonate snails were reviewe by Madsen (1990).

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The level of oxygenation may be an important influence on distribution within habitats. There is a zone of high concentration of oxygen available to snails immediately beneath the floating leaves of, however dense floating vegetation preventing snail from reaching the surface where there is a shortage of dissolved oxygen (manal, 2010).

1.3.5.2.1.2 Temperature:

The power of temperature to limit distribution was simply demonstrated by Pitchford (1981) who observed that biomphalaria snail were during winter when night temperature fell gradually below freezing.

1.3.5.2.1.3 Current Speed:

Some snails are adapted to fast-flowing water and have the shell modified to resist dislodgement, while in small rivers and streams that are slowly flowing or stagnant for most of the year the sudden spates following heavy rainfall sweep away many snails and cause major fluctuations in population density. So, the water current be an important means for the dispersal of snail. The adverse effect of strong current on snail population was attributed also to sweeping away of food and stress caused to the snails (Manal, 2010).

1.3.5.2.1.4 Light and Shade, Circadian Rhythms:

Snail host for schistosomes showed a regular pattern of activity in relation to the 24 h cycle of day and night (Circadian Rhythms). In assessing the effect of solar radiation on snails in the field is difficult to separate the effects of light and temperature. Shaded sites are unfavorable for some species snails and shading by trees is suggested as a mean for controlling the snail. Dense shade beneath mate of floating vegetation is generally unsuitable for snails. The adverse effect of shade is thought to be indirect due to depression of the growth of sub-aquatic vegetation that provides snails with food and oxygenates the water. Yet some species such as Lymnaea libycus and physa waterloti may found most frequently in high shaded places (Manal, 2010).

1.3.5.2.1.5 Snail-Plant Association:

The greatest species diversities of freshwater snails are usually associated with aquatic or sub-aquatic (emergent) leafy plants (Macrophytes). There may be a symbiotic relationship between snails and aquatic macrophytes, evolved a

12 long period. Plants provide snails with shelter from solar radiation and the water current, sources of food and egg-laying sites. Most aquatic habitats contain rich microflora which, together with decaying vegetable matter, provide the principal food of snail, although there may be no preference for any particular species of microflora (Manal, 2010).

1.3.5.2.2 Control by Molluscicides:

1.3.5.2.2.1 Chemical Molluscicides:

The role that molluscicides can play in the effective control of schistosomiasis had been well established since the discovery of the life cycle of Schistosoma. The chemical control of the snails entails the application of molluscicides to snail habitats in sufficient quantities to destroy the snail population in the most economical way with the least interference to the rest of the biota; Chemical control remains the best method for the destruction of snail host. Many control projects during the seventies decade in many endemic area like Brazil, Congo, Philippines, Zimbabwe, Egypt and Sudan Chemical control is used usually in combination with other methods can reduce or eliminate transmission. In the Sudan over 95% of the infected snails are found in those sites near human dwelling in the absence of any control measure. Molluscicides are either inorganic (synthetic) or organic, naturally occurring compounds that show some degree of selective toxicity to the snails. Synthetic Molluscicides: The Japanese recommend the use of calcium cyanide as combined cercaricidemolluscicides against S. Japonicum. In 1920 copper sulphate used in Egypt and then was introduced in several parts of world including Sudan. In the Gezira scheme the use of copper sulphate was barred in 1969 due to disadvantage in killing fish imposing severe effect on aquatic vegetation and invertebrate fauna. Sodium pentachlorophenate (NaPCP) was tested in many endemic areas. The compound is irritant to handlers and is quickly decomposed by sunlight. New and much more effective molluscicides such as Frescon and Bayluscide (Niclosamide) have gained access into the market of chemical molluscicides. Frescon has advantage of being effective at extremely low concentration, but it has limited biocidal action and decomposes rabidly in water, soil and plant. These disadvantages have resulted in its withdrawn from the market. Although the toxicity of Bayluscide was low, the amphibian and fish are very sensitive to niclosamide and the chemical has strong irritant effect on mucosal membranes. Bayluscide now is the molluscicide of choice in many areas because it seems to fulfill most of the characteristics of the ideal molluscicide, as stated by the World Health Organization, Sahar (2005).

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1.3.5.2.2.2. plants molluscicides:

The high cost of synthetic molluscicides used in the control of the intermediate snail host of schistosomiasis, along with increasing concern over the possible built up of snail resistance of these molluscicide and their toxicity to non-target organisms, has drawn much attention during recent years in renewed interest in the use of plant molluscicides may provide cheap, locally produced, biodegradable and effective control agents in rural areas of developing countries where schistosomiasis is endemic. The same authors suggested that plant molluscicides could be used in low doses at transmission foci to reduce schistosome in infected snails. Molluscicidal potence of many plant as Ambosia maritime, Solanum nigrum, Thymelaea hirsute, Callistemon lanceolatus and Peganum harmala. They attributed the mulloscicidal effect of these plants to the disturbance occurs in glycolytic pathways. They reported that reduction of snail compatibility for the developing parasite was due to the disturbance of hexokinase. Glucose phosphate isomerase and pyruvate kinase as three glycolytic enzymes.Moreover, thy declared that glycolysis is the most important metabolic pathway for infected snails which should be targeted by synthetic or plant molluscicides (manal, 2010). Sahar, (2005) recorded that sublethal concentrations of selected plant molluscicides were effective in reducing the compatibility of Bulinus truncates snails to S.haematobium infection as seen through reducing in cercarial shedding and elongation of the prepatent periods as Balanites aegyptica plant.

1.3.5.2.3 Biological Control:

Biological methods for the control of fresh water snail were reviewed, more emphasis should be put on searching for pathogens or microparasites as agents for control that can affect directly or indirectly on the intermediate snail hosts. Fishes as Trematocranus placodon are employed in the biological control of schistosome intermediate host, where snails are its preferred food. In addition, the possible use of tilapia fish for biological control of snails as intermediate hosts(Manal, 2010).

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1.4 Plants:

1.4.1 Balanites aegyptiaca:

Balanites aegyptiaca Del., also known as ‘Desert date’ in English, a member of the family Zygophyllaceae, is one of the most common but neglected wild plant species of the dry land areas of Africa and South Asia. This tree is native to much of Africa and parts of the Middle East. In India, it is particularly found in Rajasthan, Gujarat, Madhya Pradesh, and Deccan. This is one of the most common trees in Senegal. It can be found in many kinds of habitat, tolerating a wide variety of soil types, from sand to heavy clay, and climatic moisture levels ( Daya and Vaghasiya, 2011).

1.4.1.1 Taxonomy and nomenclature:

Kingdom: Plantae , Class: Magnoliophyta , Order: Zygophyllales ,

Family: Zygophyllaceae , Subfamily:Tribuloideae, Genera: Balanites, And Species: B.aegyptiaca.

Scientific name: Balanites aegyptiaca Delile

Synonyms: Ximenia aegyptiaca L.

Vernacular/common names:

Unani : Hingan, Hanguul. English: Desert date, Soapberry tree, Thorn tree, Egyptian balsam. Arabic: Heglig. French: Dattier du desert, Hagueleg, Balanite. Spanish: corona di Jesus.

1.4.1.2. Botanical description:

It is multibranched, spiny shrub or tree up to l0 m tall. Crown spherical, in one or several distinct masses. Trunk short and often branching from near the base. Bark dark brown to grey, deeply fissured. Branches armed with stout yellow or green thorns up to 8 cm long. Leaves with two separate leaflets; leaflets obovate, asymmetric, 2.5 to 6 cm long, bright green, leathery, with fine hairs when young. Flowers in fascicles in the leaf axils, and are fragrant, yellowish-green.

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1.4.1.3 Fruit and seed description:

Fruit is a rather long, narrow drupe, 2.5 to 7 cm long, 1.5 to 4 cm in diameter. Young fruits are green and tormentose, turning yellow and glabrous when mature. Pulp is bitter-sweet and edible. Seed is the pyrene (stone), 1.5 to 3 cm long, light brown, fibrous, and extremely hard. It makes up 50 to 60% of the fruit.

1.4.1.4 Flowering and fruiting habit:

Flowers are small, inconspicuous, hermaphroditic, and pollinated by insects. Seeds are dispersed by ingestion by birds and animals. The tree begins to flower and fruit at 5 to 7 years of age and maximum seed production is when the trees are 15 to 25 years old.

1.4.1.5 Distribution and habitat: Natural distribution is obscured by cultivation and naturalization. It is believed indigenous to all dry lands south of the Sahara, extending southward to Malawi in the Rift Valley, and to the Arabian Peninsula, introduced into cultivation in Latin America and India. It has wide ecological distribution, but is mainly found on level alluvial sites with deep sandy loam and free access to water. After the seedling stage, its intolerant to shade and prefers open woodland or savannah for natural regeneration. It is a lowland species, growing up to 1000 m altitude in areas with mean annual temperature of 20 to 30°C and mean annual rainfall of 250 to 400 mm.

1.4.1.5 Uses:

Aqueous extract of fruits showed spermicidal activity without local vaginal irritation in human being, up to 4% sperms becoming sluggish on contact with the plant extract and then immobile within 30 s; the effect was concentration- related. Protracted administration of the fruit pulp extract produced hyperglycemia-induced testicular dysfunction in dogs. Seed is used as expectorant, antibacterial, and antifungal. Fruit is used in whooping cough, also in leucoderma and other skin diseases. Bark is used as spasmolytic.The seed is used as a febrifuge.Root extracts have proved ‘slightly effective’ against experimental malaria. In Egyptian folk medicine, the fruits are used as an oral hypoglycemic. and an antidiabetic; an aqueous extract of the fruit mesocarp is used in Sudanese folk medicine in the treatment of jaundice. Fruits are used to treat dysentery and constipation. A fruit is used to treat liver disease.

16

The bark is used in the treatment of syphilis, round worm infections, and as a fish poison (Daya and Vaghasiya, 2011).

1.4.2 Moringa oleifera lam:

Small, fast growing deciduous tree or shrub 7 – 8 m high, with light bark, and soft wood. Leaves imparipinnate, rachis 12 – 25 cm, pubescent, with 2 – 6 pairs of pinnules, 3 – 6 long, 3 – 5 pairs of leaflets, 1 – 2cm. long, and a slightly larger terminal leaflet. The basal pair of the leaflets may be tri-pinnate. Leaflets obovate, pale green.Flowers during the dry season, in panicles, sweet-scented, cream-coloured; 5 petals unequal and slightly larger than sepals. Fruits in pods of triangular cross section, approximately 30 – 50 cm long or more, containing round, black, oily seeds (Gamal, et al., 2000).

Botanical Name: Moringa oleifera Lam. Family Name : Moringaceae.

Common Name: Moringa, Ma-room (Thai Name). Part Used: Leaves

Synonym: Moringa pterygosperma Gaertn.

1.4.2.1`Distribution:

Originating from Arabia and India, occurring to day all over the tropics of the old world; from South Asia to West Africa.Very common inparts of East and South Africa.

1.4.2.2 uses :

One of the very valuable multipurpose trees for semiarid areas. Young fruit, flowers and leaves eaten as vegetable. Moringa provides wind-protection, shade and support for climbing garden plants. It’s also used to make foul water potable. In medicine Moringa is used as a diuretic and to treat bladder and prostate ailments .the water purifying effect is mainly attributed to bounded seeds. It is very rich in protein and makes an excellent fertilizer. Flowers provide good honey. The bark is used for seasoning and to cure diarrhea.

Numerous medicinal application of different plant parts are recorded, e.g. for treating ascites, anasarca, hydropsy, boils, as a diuretic, for diarrhoea, gonorrhoea, syphilis (Gamal.E.B, et al., 2000).

17

Objectives:

General objective:

The objective of the present study is to evaluate the molluscicidal activities of the medicinal plants Moringa oleifera leaves and Balanites aegyptiaca mesocarp .together with their effect in reducing the degree of the infection to the final host (man and animals).

Specific objectives:

1- Assessment of molluscicidal and cercaricidal activity of B. aegyptiaca mesocarp and M. oleifera leaves. 2- Assessment and measurement of LC50 and LC90.

18

Chapter two

2- Materials and methods:

2.1 Study Area:

The study was carried out in Khartoum locality of Khartoum State. This study is conducted in an snails host the irrigation canals of Seilate Agricultural Schemes.

Plant material:

(a) Balanites aegyptiaca (Mesocarp),(b) Moringa oleifera (leaves). The plant material (Balanites aegyptiaca mesocarp) was collected from Om Ruaba locality , Kordovan North State, Sudan, and (Moringa oleifera leaves) was collected from Omdurman locality, Khartoum State, Sudan , identified at the Medicinal and Aromatic Plant Research Institute , Khartoum, Sudan.

2.2 Preparation of plant extracts:

Balanites aegyptiaca mesocarp were manully removed from their original trees. The plants material were dried anthe shade, and then finely powdered (Rahma, 2010).

2.3 Aqueous exreaction:

50 gm of each of the powder and dried fruits of Balanites aegyptiaca mesocarp And Moringa oleifera leaves were soacked and extracted with 1000 ml of dechlorinated water for 24 hours at room temperature, then, the suspension was filtered. The stock solution of Balanites aegyptiaca mesocarp and Moringa oleifera leaves were placed in glass vials and stored in a deep freezer at – 20 Ċ till beign used (Changbunjong T et al., 2010).

2.4 Snail Collection and Exposure:

Bulinus truncatus snail are the intermediate hosts of S.haematobium they were collected from the from Fetah Elagaliin locality in Khartoum state, using a wire scoop and brought to the laboratory in moistened cotton wool. They were examined in the laboratory for patent Trematode infections by placed in glass beakers with clean water and then maintain in the laboratory for up to four weeks. Only those snails free from any infection were used in the laboratory

19 experiments. In the laboratory the snails were kept in groups of 50 in plastic bowls, 30 cm diameter and 15 cm deep containing dechlorinated tape water. The snails were fed daily on dry previously boiled lettuce. Water in bowls was changed twice week to eliminate faecal dropped and to prevent fouling. The snails were kept for a period of two weeks to acclimatize to laboratory conditions before use in the experiment. Dead as well as unhealthy, algal infected snails were removed as soon as discovered. Ten snails were placed in every dish for tested with medicinal plant.

2.5 Collection of Cercariae:

Urine samples from infected children from Fetah Elagaliin locality in Khartoum were examined under a light microscope at amagnification of 40X in a laboratory, the eggs were caught with pipettes and transferred to petri dishes containg 10 ml of dechlorinated water and placed under lamplight for 30 min. The emerging meracidiae were caught and placed in petri dish containing two snails. The snails became infected, all the number of snails infected were 80 snails. They were kept in plastic bowls, 30 cm diameter and 15 cm deep, contained dechlorinated tape water. They were fed daily on dry previously boiled lettuce. After 5-7weeks Cercariae shedding were induced by exposed snails to light for 1 to 2 hrs, on a daily basis. Like wise 100 cercarias were placed in every dish for being tested. The extracts of the two plants were tested to determine their efficacy against the motilities of cercariae (as infective stages) of the parasite Schistosoma haematobium.

2.6 Molluscicidal activity tests:

Water extracts of B. Aegyptiaca mesocarp and Moringa oleifera leaves, were tested against adult Bulinus truncatus snail according to the method recommended by ( WHO,1983). To each concentration of plant parts used, 10 adult snails of B. truncatus were immersed. Tests were carried out at room temperature. After 24 h of exposure, the suspension was decanted. Ten snails were immersed in separate aged water with the same volumes of the solvent that would serve as control. Three replicates for each test were used. All groups were observed carefully after 24 h, the number and the percentages of death in each group were calculated. Snails were considered dead if they could not move or retracted well into or hanging out of the shell, with the body and shell discolored (Vijay P, 2010).To prepare each plants parts stock solution, 50 grams were added to 1000 milliliters of dechlorinated water to give a

20 concentration of 5% (0.05). Ten snails were transferred by clips to small disposable test cup 400 ml. Small, unhealthy or damaged snails were replaced. The depth of the water in the cups was kept between six and eight centimeters. The Bulinus truncatus snails were exposed to five different concentrations for Balanites aegyptiaca mesocarp (750, 500, 250, 125, 25) respectively and five different concentrations for Moringa oleifera leaves aqueous extract (7500, 6250, 5000, 3750, 2500) respectively and the control was dechlorinated water. Three replicates were set up for each concentrations and an equal number of controls were set up with dechlorinated water. The test containers were kept at 25 to 30 , Laboratory temperatures were measured using a mercury thermometer. After 24 hours exposure, snail mortality in each concentration was recorded.

If the control mortality was between 5% and 20%, the mortalities of treated groups were corrected according to Abbott’s formula:

X - y

Mortality (%) = ———— × 100

X Where:

X = percentage survival in the untreated control and Y = percentage survival in the treated sample. The percentage of death counting was as follow:

Snails death 100 total number of snails

The percentage of death was transformed to the probit units. Concentrations used in each experiment were set as part per million (ppm) logarithms of concentration for (ppm) were calculated. After determining the mortality of snails the results were using to determine LC50 and LC90 values. All the experiments were performed according to WHO standard techniques.

2.7 Data Analysis:

The data were analyzed using Excel 2007 program and other computer soft ware. The computer and the formula of the regression line were used to calculate the LC50 and LC90.

21

Table1: concentrations of Balanites aegyptiaca mesocarp aqueous extract in parts per million in dechlorinated water.

Milliliter Serial Stock Dechlorinated Whole volume Concentration solution Water ppm

1 3 197 200 750 2 2 198 200 500 3 1 199 200 250 4 o.5 199.5 200 125 5 0.1 199.9 200 25 Control 0.00 200 200 0.00

22

Table 2: concentrations of Moringa oleifera leaves. aqueous extract in parts per million in dechlorinated water .

Serial Milliliter Stock Dechlorinated Whole volume Concentration solution Water ppm

1 30 170 200 7500 2 25 175 200 6250 3 20 180 200 5000 4 15 185 200 3750 5 10 190 200 2500 Control 0.00 200 200 0.00

23

2.8 Cercariaecidal activity tests: To prepare each plants parts stock solution, mentioned above,The Cercariae of Schistosoma haematobium were exposed to five different concentrations for Balanites aegyptiaca mesocarp and five different concentrations for Moringa oleifera leaves aqueous extract and the control was dechlorinated water. Water extracts of B. Aegyptiaca mesocarp and Moringa oleifera leaves, were tested against Cercariae of Schistosoma haematobium according to the method recommended by ( Sahar, 2005). To each concentration of plant parts used, 100 Cercariae were immersed. Tests were carried out at room temperature.

100 Cercariae were immersed in separate dechlorinated water with the same volumes of the solvent that would serve as control. Three replicates for each test were used. All groups were observed carefully after 1 to 4 h, the number and the percentages of mortality in each group were calculated. Cercariae were considered dead if they could not move (Sahar, 2005). Three replicates were set up for each concentrations and an equal number of controls were set up with dechlorinated water, After 1 to 4 hours exposure, Cercariae mortality in each concentration was recorded.

24

Table3. Balanites aegyptiaca mesocarp aqueous extract in parts per million in dechlorinated water.

Milliliter Serial Stock Dechlorinated Whole volume Concentration solution Water ppm

1 0.75 49.25 50 750 2 0.5 49.5 50 500 3 0.25 49.75 50 250 4 0.125 149.875 50 125 5 0.025 49.975 50 25 Control 0.00 50 50 0.00

25

Table4. Moringa oleifera leaves. aqueous extract in parts per million in dechlorinated water.

Serial Milliliter Stock Dechlorinated Whole volume Concentration solution Water ppm

1 7.5 42.5 50 7500 2 6.25 43.75 50 6250 3 5 45 50 5000 4 3.75 46.25 50 3750 5 2.5 47.5 50 2500 Control 0.00 50 50 0.00

26

CHAPTER THREE

3- RESULT

Three of laboratory experiments were conducted to examine the effect as molluscicidal and cercariaecidal activity of Balanites aegyptiaca mesocarp and Moringa oleifera leaves aqueous extract against Bulinus truncatus and cercariae of Schistosoma heamatobium. The responses of snails and Cercariae tested, in terms of probit death, were found to form a linear relationship between percentage concentration (ppm) and probit unit of the extracts, tables ( 8, 12, 16, 20) And Figures (4, 8, 12, 16), show the responses of the snails and cercariae to different concentration of the extracts in terms of percentage death together with their relevant statistical data. In addition, they show the concentration (ppm) of extracts that killed 50 percent of the population and that killed 90 percent.

3-1 effect of aqueous extract of Balanites aegyptiaca mesocarp to Bulinus truncatus and cercariae of S- heamatobium:

Tables (8, 16) and figures (4, 12) illustrated the molluscicidal and cercariaecidal activities of aqueous extract of Balanites aegyptiaca mesocarp against Bulinus truncatus and cercariae of S- heamatobium. The aqueous extract of Balanites aegyptiaca mesocarp was found to possess molluscicidal and cercariaecidal activities, cercariae was found to be more susceptible than B. truncates with LC50 92.8324 ppm (fig12), 119.2614 ppm(fig4) and LC90 672.0475 ppm(fig12), 439.1369 ppm(fig4) respectively. The Average percentage deaths for Bulinus truncates in the three experiments using Balanites aegyptiaca mesocarp ranged between 10% and 100% as has been shown in

Tables 8. The LC50 for Balanites aegyptiaca mesocarp was 119.2614 ppm and the LC90 for the same plant was 439.1369 ppm (Fig. 4). The Average percentage mortality for Cercariae in the three experiments of Balanites aegyptiaca mesocarp aqueous extract ranged between 26% and 96.3% has been shown in

Table 16. The LC50 for Balanites aegyptiaca mesocarp was 92.8324 ppm and the LC90 for the same extract was 672.0475 ppm (Fig. 12).

27

3-2 effect of aqueous extract of Moringa oleifera leaves to Bulinus truncatus and cercariae of S.heamatobium:

Tables (12, 20) and figures (8, 16) illustrated the molluscicidal and cercariaecidal activities of aqueous extract of Moringa oleifera leaves against Bulinus truncatus and cercariae of S.heamatobium. The aqueous extract of Moringa oleifera leaves was found to possess molluscicidal and cercariaecidal activities, cercariae was found to be more susceptible than B. truncatus with

LC50 3788.7853 ppm (fig16), 3929.1625 ppm (fig8) and LC90 6188.7081 ppm (fig16), 5953.8789 ppm (fig8) respectively. The Average percentage deaths for Bulinus truncatus in theThree experiments of Moringa oleifera leaves aqueous extract ranged between 10% and 100% has been shown in Table 12. The LC50 for Moringa oleifera leaves was 3929.1625 ppm and the LC90 for the same extract was 5953.8789 ppm (Fig. 8).

The Average percentage mortality for Cercariae in the Three experiments of Moringa oleifera leaves aqueous extract ranged between 20% and 98.6% as has been shown in Table 20. The LC50 for Moringa oleifera leaves was 3788.7853 ppm and the LC90 for the same extract was 6188.7081 ppm (Fig. 16). Thus, the Moringa oleifera leaves aqueous extract were far less toxic than Balanites aegyptica mesocarp aqueous extract. Higher concentrations of the Moringa oleifera leaves aqueous extract had to be used in these experiments to kill Bulinus truncatus and cercariae of Schistosoma heamatobium compared to Balanites aegyptiaca mesocarp aqueous extract. Other with 7.5 gm of B. aegyptiaca mesocarp dissolve in 10.000 litters to kill 100% of B. truncatus snails compare with 7.5 gm of M. oleifera leaves dissolve in 10.000 litters to kill 10% of B. truncates. Also 7.5 gm of B. aegyptiaca mesocarp dissolve in 10.000 litters to kill 96% of Cercariae compare with 7.5 gm of M. oleifera leaves. dissolve in 10.000 litter to kill 9.8% of Cercariae.

28

Table 5: Percentage death in Bulinus truncatus Treated with different Concentrations of Balanites aegyptiaca mesocarp.

Concentration Logarith-ms of Death Calculate death Probit unit concentr-ation Ppm % %

750 2.875 100 % 100 % 7.33

500 2.698 90 % 90 % 6.28

250 2.397 80 % 80 % 5.84

125 2.096 50 % 50 % 5.00

25 1.397 10 % 10 % 3.72

29

Table 6: Percentage death in Bulinus truncatus Treated with different Concentrations of Balanites aegyptiaca mesocarp.

Concentration Logarith-ms of Death Calculated death Probit unit concentr-ation ppm % %

750 2.875 100 % 100 % 7.33

500 2.698 80 % 80 % 5.84

250 2.397 70 % 70 % 5.52

125 2.096 30 % 30 % 4.48

25 1.397 10 % 10 % 3.72

30

Table 7: Percentage death in Bulinus truncatus Treated with different Concentrations of Balanites aegyptiaca mesocarp.

Concentration Logarith-ms of Death Calculated death Probit unit concentr-ation ppm % %

750 2.875 100 % 100 % 7.33 500 2.698 90 % 90 % 6.28 250 2.397 60 % 60 % 5.25 125 2.096 40 % 40 % 4.75 25 1.397 10 % 10 % 3.72

31

Table 8: The Average Percentage deaths for Bulinus truncatus in the three Experiments when Treated with different Concentrations of Balanites aegyptiaca mesocarp.

Concentration Logarithms of Percentage of death Average Probit concentration percentage unit ppm EX-1 EX-2 EX-3 Of death 750 2.875 100 % 100 % 100 % 100 % 7.33 500 2.698 90 % 80 % 90 % 87% 6.13 250 2.397 80 % 70 % 60 % 70 % 5.52 125 2.096 50 % 30 % 40 % 40 % 4.75 25 1.397 10 % 10 % 10 % 10 % 3.72

32

Fig.1: Average death Percentages for the three Experiments of Bulinus truncatus Treated with different Concentrations of Balanites aegyptiaca mesocarp.

When: y = 5

X = 2.0765

LC50 = 119.2614 ppm

When: y = 6.28

X = 2.6426

LC90 = 439.1369 ppm

33

Table 9: Percentage death in Bulinus truncatus Treated with different Concentrations of Moringa oleifera leaves.

Concentration Logarith-ms of Death Calculated death Probit unit concentr-ation ppm % %

7500 3.875 100 % 100 % 7.33

6250 3.795 90 % 90 % 6.28

5000 3.698 80 % 80 % 5.84

3750 3.574 60 % 60 % 5.25

2500 3.397 20 % 20 % 4.16

34

Table 10: Percentage death in Bulinus truncatus Treated with different Concentrations of Moringa oleifera leaves.

Concentration Logarith-ms of Death Calculated death Probit unit concentr-ation ppm % %

7500 3.875 100 % 100 % 7.33

6250 3.795 90 % 90 % 6.28

5000 3.698 70 % 70 % 5.52

3750 3.574 40 % 40 % 4.75

2500 3.397 0 % 0 % 0.00

35

Table 11: Percentage death in Bulinus truncatus Treated with different Concentrations of Moringa oleifera leaves.

Concentration Logarith-ms of Death Calculated death Probit unit concentr-ation ppm % %

7500 3.875 100 % 100 % 7.33

6250 3.795 80 % 80 % 5.84

5000 3.698 70 % 70 % 5.52

3750 3.574 30 % 30 % 4.48

2500 3.397 10 % 10 % 3.72

36

Table 12: The Average Percentage deaths for Bulinus truncatus in the three Experiments when Treated with different Concentrations of Moringa oleifera leaves.

Concentration Logarithms of Percentage of death Average Probit concentration percentage unit ppm EX-1 EX-2 EX-3 Of death 7500 3.875 100 % 100 % 100 % 100 % 7.33 6250 3.795 90 % 90 % 80% 87% 6.13 5000 3.698 80 % 70 % 70 % 73 % 5.59 3750 3.574 60 % 40 % 30 % 43 % 4.82 2500 3.397 20 % 00 % 10 % 10 % 3.72

37

Fig.2: Average death Percentages for the three Experiments of Bulinus truncatus Treated with different Concentrations of Moringa oleifera leaves

When: y = 5

X = 3.5943

LC50 = 3929.1625 ppm

When: y = 6.28

X = 3.7748

LC90 = 5953.8789 ppm

38

Table 13: percentage mortality in Cercariae treated with different concentration of Balanites aegyptiaca mesocarp.

Con- Log of Percentage mortality % ∑per-mor-% Living cer- Probit ppm con- 1 h 2 h 3 h 4 h (< 4 hours) (< 4 hours) Unite 750 2.875 85 6 2 1 94 6 6.55 500 2.698 70 8 4 2 84 16 5.99 250 2.397 51 12 9 0 72 28 5.58 125 2.096 34 14 5 0 53 47 5.08 25 1.397 19 7 3 0 29 71 4.45 00.00 Invalid 00.0 00.0 00.0 00, 00.0 100 00.0

39

Table 14: percentage mortality in Cercariae treated with different concentration of Balanites aegyptiaca mesocarp.

Con-ppm Log of Percentage mortality % ∑per-mor-% Living cer- Probit con- 1 h 2 h 3 h 4 h (< 4 hours) (< 4 hours) Unite

750 2.875 87 6 3 2 98 2 7.05

500 2.698 69 7 4 1 81 19 5.88

250 2.397 52 10 6 0 68 32 5.47

125 2.096 30 12 2 1 45 55 4.87

25 1.397 15 4 6 0 25 75 4.33

00.00 Invalid 00.0 00.0 00.0 00, 00.0 100 00.0

40

Table 15: percentage mortality in Cercariae treated with different concentration of Balanites aegyptiaca mesocarp.

Con- Log of Percentage mortality % ∑per-mor-% Living cer- Probit ppm con- 1 h 2 h 3 h 4 h (< 4 hours) (< 4 hours) Unite

750 2.875 86 8 3 0 97 3 6.88

500 2.698 65 9 2 1 77 23 5.74

250 2.397 53 7 9 2 71 29 5.55

125 2.096 29 11 3 0 43 57 5.82

25 1.397 16 3 4 1 24 76 4.29

00.00 Invalid 00.0 00.0 00.0 00, 00.0 100 00.0

41

Table 16: The Average of Percentage mortality in Cercariae treated with different concentration of Balanites aegyptiaca mesocarp.

Con- Log of Percentage mortality % ∑per-mor-% Ave-Per-of Probit ppm con- (< 4 hours) ∑per-mor-% Unite

1, 1, 1 2, 2, 2 3, 3, 3 4, 4, 4 Ex- Ex- Ex- (< 4 hours) Hour Hours Hours Hours 1 2 3 750 2.875 85, 87, 86 6, 6, 8 2, 3, 3 1, 2, 0 94 98 97 96.3 6.75 500 2.698 70, 69, 65 8, 7, 9 4, 4, 2 2, 1, 1 84 81 77 80.6 5.88 250 2.397 58, 52, 53 12,10,7 9, 6, 9 0, 0, 2 72 68 71 70.3 5.52 125 2.096 34, 30, 29 14, 12, 11 5, 2, 3 0, 1, 0 53 45 43 47 4.92 25 1.397 19, 15, 16 7, 4, 3 3, 6, 4 0, 0, 1 29 25 24 26 4.36 00.0 Invalid 00.0 00.0 00.0 00.0 00. 00. 00. 00.0 00.0

42

Fig3: The Average of Percentage mortality for the three Experiments of Cercariae Treated with different Concentrations of Balanites aegyptiaca mesocarp.

When: Y = 5

X = 1.9677

LC50 = 92.8324 ppm When: Y = 6.28

X = 2.8274

LC90 = 672.0475 ppm

43

Table 17: percentage mortality in Cercariae treated with different concentration of Moringa oleifera leaves.

Con- Log of Percentage mortality % ∑per-mor-% Living cer- Pro ppm con- bit 1 h 2 h 3 h 4 h (< 4 hours) (< 4 hours) Unit e 7500 3.875 85 5 4 3 97 3 6.88 6250 3.795 72 9 1 0 82 18 5.92 5000 3.698 50 13 2 1 66 34 5.41 3750 3.574 29 15 2 0 53 47 5.08 2500 3.397 13 1 3 2 19 81 4.12 00.00 Invalid 00.0 00.0 00.0 00, 00.0 100 00.0

44

Table 18: percentage mortality in Cercariae treated with different concentration of Moringa oleifera leaves.

Con-ppm Log of Percentage mortality % ∑per-mor-% Living cer- Prob con- it 1 h 2 h 3 h 4 h (< 4 hours) (< 4 hours) Unit e

7500 3.875 81 10 7 1 99 1 7.33

6250 3.795 70 8 3 0 81 19 5.88

5000 3.698 49 12 1 0 62 38 5.31

3750 3.574 28 9 4 2 43 57 4.82

2500 3.397 14 2 1 4 21 79 4.19

00.00 Invalid 00.0 00.0 00.0 00, 00.0 100 00.0

45

Table 19: percentage mortality in Cercariae treated with different concentration of Moringa oleifera leaves.

Con- Log of Percentage mortality % ∑per-mor-% Living cer- Probit ppm con- 1 h 2 h 3 h 4 h (< 4 hours ) (< 4 hours) Unite 7.33 7500 3.875 83 9 6 2 100 00.0

6250 3.795 68 10 4 3 85 15 6.04

5000 3.698 51 11 2 1 65 35 5.39

3750 3.574 25 12 1 0 38 62 4.69

2500 3.397 10 2 4 4 20 80 4.16

00.00 Invalid 00.0 00.0 00.0 00, 00.0 100 00.0

46

Table 02: The Average of Percentage mortality in Cercariae treated with different concentration of Moringa oleifera leaves.

Con- Log of Percentage mortality % ∑per-mor-% Ave-Per-of Probit ppm con- (< 4 hours) ∑per-mor-% Unite

1, 1, 1 2, 2, 2 3, 3, 3 4, 4, 4 Ex- Ex- Ex- (< 4 hours) Hour Hours Hours Hours 1 2 3 7500 3.875 85, 81, 83 5, 11, 9 4, 7, 6 3, 1, 2 97 99 100 98.6 7.33 6250 3.795 72, 71, 68 9, 8, 11 1, 3, 4 1, 1, 3 82 81 85 82.6 5.95 5000 3.698 51, 49, 51 13, 12,11 2, 1, 2 1, 0, 1 66 62 65 64.3 5.36 3750 3.574 29, 28, 25 15, 13, 12 2, 1, 1 0, 2, 0 53 43 38 44.6 4.87 2500 3.397 13, 14, 11 1, 2, 2 3, 1, 4 2, 4, 4 19 21 20 20 4.16 00.00 Invali 00.0 00.0 00.0 00.0 01 00. 00. 00.0 00.0 d

47

Fig4: The Average of Percentage mortality for the three Experiments of Cercariae Treated with different Concentrations of Balanites aegyptiaca mesocarp.

When: Y = 5

X = 3.5785

LC50 = 3788.7853 ppm When: Y = 6.28

X = 3.7916

LC90 = 6188.7081 ppm

48

CHAPTER FOUR

4- Discussion The present study was conducted in Khartoum locality of Khartoum State in 2013- 2014. The main objective of this study was assessment the effects of Balanites aegyptiaca mesocarp aqueous extract and Moringa oleifera leaves on snails and cercariae of schistosoma heamatobium.The study revealed that the Balanites aegyptiaca mesocarp aqueous extract and Moringa oleifera leaves have the potential of controlling Bulinus truncatus and cerceriae of schistosoma heamatobium.

The LC50 and LC90 values for Balanites aegyptiaca mesocarp aqueous extract on Bulinus truncatus were found to be 119.2614 ppm and 439.1369 ppm respectively. Also The LC50 and LC90 values for Balanites aegyptiaca mesocarp aqueous extract on cercariae of schistosoma heamatobium were found to be

92.8324 ppm and 672.0475 ppm respectively. These LC50 and LC90 were calculated from the average percentage of deaths in the experiments carried out during this study. Extract of Ba. aegyptiaca give LC95 12.0 ppm for B. truncates, This mentioned by Anto, F. e t al (2005). The study showed that the effect of Balanites aegyptiaca mesocarp on cercariae more than its effect on B. truncates. This agreed with Sahar (2005), at the highest and least concentration of B. aegyptiaca mesocarp (750 – 25) respectively, the death rate for the snails was found to be 100% and 10% respectively, compared with 96.3% and 26% for cercariae at the same concentration. This result because of the differ in the time of exposure witch was 24 hours for the snails and 4 hours for cercariae, repeated three times. The LC50 and LC90 values for Moringa oleifera leaves aqueous extract on Bulinus truncatus were found to be 3929.1625 ppm and

5953.8789 ppm respectively. Also The LC50 and LC90 values for Moringa oleifera leaves aqueous extract on cercariae of schistosoma heamatobium were found to be 3788.7853 ppm and 6188.7081 ppm respectively. These LC50 and

LC90 were calculated also from the average percentage of deaths in the experiments carried out during this study. The study showed that the effect of Moringa oleifera leaves on cercariae more than its effect on B. truncates. at the highest and least concentration of M. oleifera leaves (7500 – 2500) respectively, the death rate for the snails was found to be 100% and 10% respectively, compared with 98.6% and 20% for cercariae at the same concentration. This result because of the differ in the time of exposure witch was 24 hours for the snails and 4 hours for cercariae, repeated three times. In study conducted by

49

Osman et al (2012). The mortality rates among snails exposed to different concentrations of the aqueous extract of Balanites aegyptiaca 10000, 5000, 1000, 500, 250 and 125ppm. The concentration caused 100% mortality rate among exposed snails was 10000ppm. Compare with 100% death at 750 ppm of Balanites aegyptiaca.

The molluscicidal activity of Balanites aegyptiaca mesocarp against B. pfeifferi give LC50 65.51 mg/L and LC90 86.50mg/L this mentioned by Eshetu Molla et al (2013). The available data, however, indicated that at an average of 0.25 p.p.m. a barrier was created which was lethal to snails which passed through it, but that at an average of 0.125 p.p.m. results were indeterminate, this mentioned by Teesdale, et al (1961). Moringa oleifera leaves aqueous extract was far less toxic than Balanites aegyptiaca mesocarp, High concentrations of the Moringa oleifera leaves aqueous extract were used in this study contrary to Balanites aegyptica mesocarp, which was used in low concentrations. There are many factors, which may have affected the experimental results since there were differences in mortality in the same concentration possibly because the sources of snails were different. There were differences in the results in the various experiments possibly due to external factors since there was death in the controls. All experiments were conducted in a temperature range of 31°C to 32 °C, which may be different from temperatures of snails habitats ( Birley, 1991).mentioned that 18 °C to 28 °C was found to be the optimum temperature for snails development.

Conclusion and Recommendation

Balanites aegyptica mesocarp and Moringa oleifera leaves have a moderate molluscicidal activity and promising cercariaecidal activity IN-VITRO. Further elucidation of the plant species, time of cultivation, and origin of plant and method of extraction as well as extraction with different solvents are needed. Study of the active constituents in plant parts is important before conducting furtherexperiments.

50

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Appendixes:

Fig 5: Balanites aegyptiaca mesocarp Toxicity to Bulinus truncatus (Experiment 1).

When: y = 5. X = 2.0174. LC50 = 104.0878 ppm

When: y = 6.28. X = 2.5754. LC90 = 376.1837 ppm

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Fig.6: Balanites aegyptiaca mesocarp Toxicity to Bulinus truncatus (Experiment 2).

When: y = 5. X = 2.1219. LC50 = 132.4036 ppm

When: y = 6.28. X = 2.7000. LC90 = 501.1872 ppm

.

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Fig.7: Balanites aegyptiaca mesocarp Toxicity to Bulinus truncatus (Experiment 3).

When: y = 5. x = 2.0897. LC50 = 122.9419 ppm

When: y = 6.28. X = 2.6498. LC90 = 446.4779 ppm

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Fig.8: Moringa oleifera leaves Toxicity to Bulinus truncatus (Experiment 1).

When: y = 5. X = 3.5418. LC50 = 3481.7693 ppm

When: y = 6.28. X = 3.7497. LC90 = 5619.5300 ppm

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Fig.9: Moringa oleifera leaves Toxicity to Bulinus truncatus (Experiment 2).

When: y = 5. X = 3.6834. LC50 = 4823.9189 ppm

When: y = 6.28. X = 3.7726. LC90 = 5923.7947 ppm

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Fig.10: Moringa oleifera leaves Toxicity to Bulinus truncatus (Experiment 3).

When: y = 5. X = 3.6141. LC50 = 4112.4440 ppm

When: y = 6.28. X = 3.7959. LC90 = 6250.2875 ppm

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Fig 11 : Balanites aegyptiaca mesocarp toxicity to Cercariae (experiment1).

When: y = 5. x = 1.9042. LC50 = 80.2047 ppm.

When: y = 6.28. X = 2.8468. LC90 = 702.7486 ppm.

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Fig 12: Balanites aegyptiaca mesocarp toxicity to Cercariae (experiment2).

When: Y = 5. X = 1.9762. LC50 = 94.6673 ppm

When: Y = 6.28. X = 2.7557. LC90 = 569.7705 ppm

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Fig 13 : Balanites aegyptiaca mesocarp toxicity to Cercariae (experiment 3).

When: Y = 5. X = 1.8325. LC50 = 67.9986 ppm

When: Y = 6.28. X = 2.7332. LC90 = 541.0034 ppm

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Fig 14: Moringa oleifera leaves Toxicity to Cercariae (Experiment 1).

When: Y = 5. X = 3.5760. LC50 = 3767.0379 ppm

When: Y = 6.28. X = 3.8187. LC90 = 6587.1871 ppm

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Fig 15: Moringa oleifera leaves Toxicity to Cercariae (Experiment 2).

When: Y = 5. X = 3.5814. LC50 = 3814.1695 ppm.

When: Y = 6.28. X = 3.7980. LC90 = 6280.5835 ppm.

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Fig 16: Moringa oleifera leaves Toxicity to Cercariae (Experiment 3).

When: Y = 5. X = 3.5838. LC50 = 3835.3058 ppm.

When: Y = 6.28. X = 3.7898. LC90 = 6163.1111 ppm.

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Fig (17) showing the relationship between Con (ppm) of B. aegyptiaca mesocarp and Death % of B. truncatus

71

Fig (18) showing the relationship between Con (ppm) of B. aegyptiaca mesocarp and death % of Cercariae.

72

Fig (19) showing the relationship between Con (ppm) of M. oleifera leaves. And death % of B. truncatus.

73

Fig (20) showing the relationship between Con (ppm) of M. oleifera leaves. and death % of Cercariae.

74