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PROJECT TOPIC AND MATERIAL ON NEMATOCIDAL ACTIVITY OF METHANOLIC EXTRACTS OF LEAF, STEM BARK AND ROOT OF AZADIRACHTA INDICA A. JUSS. (NEEM) AGAINST HAEMONCHUS CONTORTUS
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- Name: NEMATOCIDAL ACTIVITY OF METHANOLIC EXTRACTS OF LEAF, STEM BARK AND ROOT OF AZADIRACHTA INDICA A. JUSS. (NEEM) AGAINST HAEMONCHUS CONTORTUS
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ABSTRACT
The antihelminthic activity of methanolic extracts of leaf, stembark and root of Azadirachtaindica were evaluated in vitro againstHaemonchuscontortus larvae. Phytochemical screening conducted on the extracts of all the plant parts revealed the presence of carbohydrates, cardiac glycoside, saponins, flavonoids, tannins and alkaloids, leaf had steroids, while stembark and root had triterpenes. The quantitative estimation of the phytochemical constituents revealed a high percentage in flavonoids followed by alkaloids and tannins with least quantitative percentages insaponins: 20.21, 27.50, 20.95; 5.25, 5.48, 5.48; 4.95, 4.93, 4.15 and 0.75, 0.68, 0.67respectively. Eggs of H.contortus were cultured in culture plates maintained at 27°C in an incubator. The infective third stage larvae (L3) were recovered from 7-9 day old sterilefaecal cultures. The larvae harvested were concentrated at 1000 rpm for 15 minutes, 0.1 ml of the larvae containing 120 L3 were put into wells of microtitre plate and concentrations of each of the extracts (leaf, stembark and root) at 0.1mg/ml, 1.0mg/ml, 10.0mg/ml, 100mg/ml, negative control (water) and positive control (Levamisole) were added to each of the wells with six replication. After addition of the treatments, it was viewed under the microscope once in 6 hours for a period of 48 hours and the number of mortality recorded. The lethal concentration (LC50/ EC) value for the leafis 12.30mgml-1, 12.58mgml-1 for the stem bark and 15.84mgml-1 for the root extract. Mortalities of the parasites increased with increase in the concentration and with the time of exposure. At the peak time of exposure (48 hours) and at the highest concentration of 100mgml-l, mortalities were higher than those of the least time of exposure (6 hours) and with the lowest concentration of 0.1mgml-1. The data showed highly significant differences between the plant parts, time of exposure, concentration of the extracts, the time of exposure and concentration and between the plant parts and concentration (p=0.001). Although, mortality of the parasites increased with increase in time, there were no significant differences between the plant parts and the time of exposure (p=0.92) and between the plant parts, time of exposure and concentration (p=0.99).Mortalities recorded were high in the positive control wells with increase in mortality as the time of exposure increased but in the negative control wells, mortalities were not recorded. The study concludes that mortalities recorded were due to the effects of the extracts on the parasites. Validation of the efficacy of the extracts of this plant is suggested to determine the effects of natural or experimental haemonchosis in ruminants.
CHAPTER ONE
Introduction
Azadirachta indica,commonly known as neem in English and Dogonyaro in Hausa,is an evergreentree in the Mahogany family Meliaceae. It is native to India, Pakistan and Burma, growing in tropical and semi-tropical regions (Balakrishnan et al., 2007).
It is the most versatile, multifarious tree of the tropics with immense potential growing to about 25m in height with semi-straight to straight trunk, 3m in girth and spreading branches forming a broad crown (Kumar and Gupta,2002).
It possesses useful non-wood products (leaves, bark, flowers, fruits, seed, gum, oil and neem cake) than any other tree species. These non-wood products are known to have antidermatic, antifeedent, antifungal, anti-inflammatory antipyorrhoeic, antiscabic, cardiac, diuretic, insecticidal, larvicidal, nematocidal, spermicidal and other biological activities (Brahmachari,2004). The wider application and activities of neem have made it a green treasure (Khanna, 1992,Suri and Mehrotra 1994).
The tree has adaptability to a wide range of climatic, topographic and edaphic factors. It thrives well in dry, stony shallow soils and even on hard calcareous soils or clay pan at a shallow depth (Koul et al.,1990,Schmutterer, 1990).
Neem tree requires little water and plenty of sunlight (Anonymous,2006, Sateesh,1998). The tree grows naturally in areas where the rainfall is in the range of 450 to 1200 mm.
However, it has been introduced successfully even in areas where the rainfall is as low as 150 to 250 mm. It grows on altitudes up to 1500 m (Chari, 1996, Jattan et al., 1995, Tewari, 1992).
It can grow well in wide temperature range of 00C and 490C (Hegde,1995). It cannot withstand water-logged areas and poorly drained soils. The pH range for the growth of neem tree lies in between 4 to 10. It grows on almost all types of soil including clayey, saline and alkaline soil but it does well on black cotton soils and deep well drained soils with good sub-soil water. Neem tree has the ability to neutralize acidic soils by a unique property of calcium mining (Hegde, 1995).
The roundworm,Haemonchus contortus (Rudolphi 1803, Cobb 1898) which is also known as red stomach worm, wire worm or Barber’s pole worm, is a very common parasite and one of the most pathogenic nematode of ruminants (Burke, 2005). In domestic animals, this parasite is a source of great economic loss (Hickman, 2008).
It is a highly pathogenic parasitic nematode that affects large number of wild and domesticated ruminant species and is the most economically important parasite of sheep and goats worldwide. It is responsible for 80% of worm infections in small ruminants (Balakrishnan et al., 2007). This worm is a worldwide threat, but it is more prevalent in sub-temperate and temperate regions under warm and wet conditions (Fleming, 2006).
Haemonchus contortus is cylindricall shaped, tapered at both ends and has a complete digestive system. Adult worms are blood feeders that reside in the abomasum (stomach) and are approximately 2cm in length when mature(Roberts and Janovy, 2000). They are dioecious with single females typically producing several thousand eggs (5000-10,000) per day which pass out of the host in faeces and develop to infective L3 larvae on the pasture (Merck, 1998; Roberts and Janovy, 2000; White and Newton, 2001).
H. contortus is a member of the superfamily Trichostrongyloidea (Strongylida) which contains most of the economically important parasitic nematodes of grazing livestock. H. contortus cost the global livestock industry billions of dollars per annum in lost production and drug costs. The major problem lies within the agricultural industry. The parasites cause great economic losses in domestic animals, specifically sheep, cattle and goat.Haemonchus contortusbeing a blood sucker, it can induce anaemia and oedema. Also, the haemolytic proteins that the parasite releases can lead to other intestinal disturbances. The host will often die of major disorders such as diarrhea, severe dehydration and severe blood loss.
Haemonchus contortus is known to adapt well to harsh conditions, which makes it more difficult to eliminate (Jacquiet et al., 1998; White and Newton, 2001). Resistance to all the major antihelminthic classes is common worldwide often leading to failure of treatment and control.
1.1 Statement of Research Problem
Control of nematode infections has traditionally been done using antihelminthics drugs (chemotherapy) with best results obtained when this approach is initiated with proper grazing management and resistant animals. However, in the last two to three decades there has been over dependency and even misuse of the chemotherapeutic approach with consequent evolution of antihelminthic resistance (Ngomuo et al., 1990; Prichard, 1994). Apart from antihelminthic resistance, poor availability and affordability of antihelminthics to resource-poor farmers in developing countries have compounded the problem (Hammond et al., 1997). Moreover, there is growing concern over drug residues in the food chain and environment. Search for novel antihelminthics that are more suitable and less toxic is undoubtedly a sensible approach to the control of parasitic infections. One such alternative could be harnessing the available ethno botanical knowledge (Hammond et al., 1997), i.e., the use of medicinal plants with antihelminthic activity. Plantbased antihelminthics have been known and used in many parts of the world for a long time but little research has been done to validate their use, especially in veterinary medicine.
1.2 Justification
Three approaches to the control of nematodiasis are; chemotherapy, chemoprophylaxis and vector control. Attempts to develop a vaccine against nematodes have been dwarfed by the parasite’s ability to avoid total detection by the host’s immune system and in some cases, there are drug resistance problems.
Deficiencies such as lack of efficacy of antihelminthic drugs against all stages of the diseases, the need for parenteral administration and the inability to eliminate all strains of species of nematodes make treatment costly and difficult. Furthermore, the methods used in the control of insect/vectors are either expensive or very tedious.
In view of the problems presented by present nematocides, there is therefore an urgent need to source for suitable alternative drugs in order to combat the disease. Plants used in indigenous medicine are considered to be potential sources for the development of alternative therapeutics (Cox and Blick, 1994). Since herbal treatment for various diseases in Africa is still widespread practice (Anokbongo,1992), an ethno-botanical approach through collaboration with traditional healers may prove to be a rich source of drug discovery. These local knowledge systems are reported to be as effective and cheaper than their orthodox equivalents (Sibanda and Okoh 2008). On the other hand, herbal remedies are the only source of day to day health care for a large number of the world’s population in rural areas. Therefore, it is vital to identify those plants which are suitable for use, focusing on the efficacy and less toxicity. Natural products from plant materials are increasingly being explored for the extraction of bioactive agents for the development of drugs against diseases. Different regions of Ngeria are affected by nematode vectors hence endemic for nematodiasis. In this light, there is the need to create and enrich the pool of information on plants with medicinal value which will serve as a referral in the future for the development of drugs against several pathogen causing diseases.
1.3 Aim
To evaluate the potency of methanolic extracts ofAzadirachta indica(neem) against Haemonchus contortus.
1.4 Hypotheses
i. There is no significant difference between the effects of different concentrations of methanolic extracts of the leaf, stembark and root of Azadirachta indica against Haemonchus contortus/ere is no significant difference between the time of exposure of Haemonchus contortus to the variousplant parts extracts
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