ABSTRACT
A study to determine the in vitro and in vivo anticoccidial activities of Khaya senegalensis leaf extracts on Eimeria tenella infection in broilers was conducted. A preliminary investigation was carried out using three plants namely; Khaya senegalensis, Guiera senegalensis and Tamarindus indica to select the one with the most anticoccidial activity. The leaves were extracted by maceration method for 72 hours using 70% methanol and the partial purification of the dried crude methanol extracts done. The separated portions of the extracts were then evaluated by Thin Layer Chromatography (TLC). The chromatography result showed that Khaya senegalensis has phenols and flavonoids. The n-butanol fractions were tested for in vitro anticoccidial activities on the sporulation time of the Eimeria tenella oocysts. Khaya senegalensis n-butanol fraction had the most anticoccidial activity in which there was total sporulation inhibition (100%) while Guiera senegalensis had the least anti coccidial activity with 49% sporulation inhibition. The maximum tolerated dose (MTD) was determined using graded doses of the n-butanol extracts of Khaya senegalensis at 11 mg/kg, 33 mg/kg, 99 mg/kg, 297 mg/kg and 891 mg/kg administered orally to 4 weeks old birds at the same period which were divided into groups A- E respectively while group F birds were given 0.2 ml/bird of distilled water as control. The high survival rate of 100% in the five treated groups of the maximum tolerated dose experiment indicated the high safety margin of Khaya senegalensis even at a dose of 891mg/kg. The progressive weight increase in both the treated (11mg/kg) and un treated group shows the ability of the extract to improve weight gain. The histopathological lesions had some degree of nephro-toxicity seen. A chronic toxicity study was also conducted using the n-butanol portion of the extracts of Khaya senegalensis. Two groups of 7 birds each (1 and 2) of 4 weeks of age broilers were used with group 1 chickens orally dosed with 500 mg/kg of the n-butanol fraction of the extract daily fo two weeks. Group 2 served as the control group and each bird was given distilled water at 0.2ml. The gross lesions showed that 500 mg/kg given over a two weeks period had effects on the internal organs of the birds as revealed by the hypertrophied, congested kidneys. Evaluation of the modulatory effect of the n-butanol fraction of the crude methanol extracts of Khaya senegalensis against E tenella in broilers was carried out using one hundred and twenty, 4 week-old birds which were obtained from the National Animal Production and Research Institute (NAPRI), Shika, Zaria. The higher mean oocyts count in the infected untreated group (48%) than the treated group two weeks post infection indicated that the extract had some inhibitory effects on oocyst count. Among the extract treated birds, the high oocyst count by the 11 mg/kg group (17%) suggests the dose was not adequate to prevent oocyst production while the 99 mg/kg had lower oocyst counts (12%) which was comparable to the group treated with normal dose of Amprolium (10%) and thus was the effective dose 2 weeks post treatment. Also the phenols at a dose of 99mg/kg used to treat the infected birds three weeks post treatment, reduced oocysts count by 2%. The presence of schizonts and merozoites in caecal scrappings showed that reproductive activities of the parasites were not completely halted even after treatment. It also shows that the endogenous stages were not susceptible to the extracts at all doses although the population in the treated infected groups were lower than those seen in the infected un treated group. The survival percentage was higher in the treated groups compared to the infected un treated group (55%). Among the treated groups, the survival rate was numerically higher in the 33 mg/kg (85%) treated group while the least was in the group treated with 99 mg/kg (70%). The infected groups had slightly lower Packed Cell volume (PCV) even after treatment with the extracts than the uninfected or Amprolium treated group. In this experiment the lowest PCV was 24.66% at the third week post infection in the 33mg/kg treated group. The histopathological lesions observed included very severe lesions including severe villous atrophy and fusion of the villi. In conclusion, the in vitro and in vivo modesl were found to be useful tools for rapid screening of anticoccidial activity of the plant preparations against Eimeria tenella parasites. It is recommended that recommended that extract of Khaya senegalensis can be given at a dose of 11 mg/kg as a natural growth promoter. Also anticoccidial in vitro activity of K senegalensis as it relates to in vivo anticoccidial activity is an interesting area for more research. Detailed studies are needed to isolate, characterize and evaluate the active components and the mechanism of action of the identified active principles in Khaya senegalensis leaves as well as other parts of the plant.
Chapter One
1.0 Introduction
Coccidiosis is one of the most detrimental and lethal management diseases of poultry (Gyorke et al., 2013; Khan et al., 2008). Chickens are susceptible to at least nine (9) species of Coccidia (but only five are pathogenic) (Jordan, 1990). The most common and pathogenic species are Eimeria tenella which causes caecal coccidiosis and Eimeria necatrix (Gyorke et al., 2013), while Eimeria acervulina and Eimeria maxima cause chronic intestinal coccidiosis (Chandrakesan et al., 2009). The disease may be acute or chronic (Takagi et al., 2006) and is associated with haemorrhagic diarrhoea, emaciation, growth retardation and sometimes death (Takagi et al., 2006). Birds of any age may be affected but problems are not common in chicks under two weeks of age (Wright, 2005).
Poultry keeping is the dorminant form of livestock production in developing countries (Numan et al.,2005). In Nigeria, the rising cost of poultry feeds, problems of drug residues and incubial resistance to diseases have become major problems militating against the poultry industry (Ogbe et al., 2008). Porter (1998) estimated that feed for poultry comprise roughly 66 % of the total production costs for layer, broiler chickens or turkeys. In addition, severe enteric damage by bacterial infection will cause overt illness and high mortality in a poultry flock (Elmulsharaf et al., 2006). Coccidiosis has remained the most important poultry disease in Nigeria (Obasi et al., 2006). Lawal et al. (2008) showed that the infection can occur in both local chickens and exotic birds with the former serving mainly as reservoir hosts. Management systems (deep litter, cage and free range) have also been shown to be of no barriers to the occurrence of avian coccidiosis in Zaria (Lawal et al., 2001). The disease causes considerable economic losses in both the layer and broiler industries (Al-Quraishy et al., 2009; Chandrakesan et al., 2009; Yin et al., 2013). Anticoccidial drugs have been in use for more than 50 years and presently almost all commercial broilers are reared with the agents in their feeds (Chapman, 2001; Chandrakesan et al., 2009). The development of drug- resistant field strains and the necessity for withdrawal period prior to slaughter prompted the exploitation of alternative methods for controlling coccidiosis (Zulpo et al., 2007). Consequently, due to the ban on antimicrobial growth promoters (AGP), the use of plant extracts has gained attention in animal feed strategies and formulations (Tipu et al., 2006).
There is an increasing use of medicinal plants and plant products as alternatives to orthodox medicine especially in developing nations (Mikail and Ajabonna, 2007). Plants native to Nigeria have been experimented and shown to have some anti coccidial activities (Anosa and Okoro, 2011) with the bark of Khaya senegalensis used to treat coccidiosis, helminthosis and diarrhoea in poultry (Gefu et al., 2000). Similarly, Tamarindus indica works as a purgative, diaphorectic and an anthelminthic and is also said to have anti trypanosomal activity (McCorkle et al., 1996; Gefu et al., 2000). Gueira senegalensis is widely used in traditional medicine in West Africa (Males et al., 1998) and is known to have antimicrobial effects (Sanogor et al., 1997). Khaya senegalensis, Gueira senegalensis and Tamarindus indica are widely used as medicinal plants in West Africa (Males et al., 1998) and are also known to have antimicrobial effects (Sanogor et al., 1997). This study is being carried out to determine the possible effects of the leaf extracts of the plants named above on the causative agents of avian coccidiosis and to compare them with the standard conventional treatment.
1.1 Statement of Research Problem
Host specific coccidiosis is caused by Eimeria species which belong to the Phylum Apicomplexa, Family Eimeridae and genus Eimeria. It is the major parasitic disease of poultry causing substantial economic losses due to malabsorption, bad feed conversion rate, reduced weight gain and mortality (Fornace et al., 2013). Although some clinical eimerial infections may be unimportant (Gerhold Jr, 2014), they can cause considerable depression in feed efficiency and egg production, which accounts for 70% of the total coccidiosis disease control cost. Coccidiosis is a disease of poor management and mortalities due to the disease are ranked second after viral diseases of poultry (Hadipour et al., 2013). The disease is responsible for 6-10% of all broiler mortalities and incidence of the disease in commercial poultry ranges from 5-70% (Usman et al., 2011). The poultry industry is facing problems which include drug resistance, lack of new anticoccidial products and consumer demanding decrease in the use of drugs in animal feeds (Hafez, 2008). The extensive use of prophylactic anticoccidial drugs has led to the development of drug resistant strains of Eimeria against all products introduced (Chapman, 1997). Live vaccines can restore drug sensitivity and proved to be efficient in controlling the disease but they are expensive and have adverse effects on early chick growth (Williams, 2002). Also the control of coccidiosis in replacement birds namely broiler breeders and egg producing stock is a continuing problem since most anticoccidials cannot be given to birds in egg production , yet most adult birds live in an infected environment (Usman et al., 2011). This suggests that coccidiosis is likely to have a greater impact on the profitability of broiler meat and egg productions in future (Sunhwa et al., 2014). There is therefore an urgent need to seek alternative strategies to control coccidiosis.
1.2 Justification
Viral and protozoan diseases are the most outstanding infectious diseases militating against productive poultry industries in Nigeria (Abdu et al., 2007). Prominent among the protozoan diseases is coccidiosis. Coccidiosis is a protozoan disease commonly occurring under intensive management system (Biu et al., 2006) where pathogenic population of the causative agent Eimeria specie (Obasi et al.,2006) easily builds up causing significant economic losses through reduction in weight, feed consumption and mortality (Gyorke et al.,2013). It currently represents one of the greatest challenges to the broiler industry especially in the production of broiler chickens (Bliss, 2007). The economic losses are significant being estimated at more than 3 billion US dollars annually worldwide (Gyorke et al., 2013). In the United States, well over 600 million dollars are lost annually due to coccidiosis (Bliss, 2007). Although coccidiosis is known to be endemic in Nigeria, there is a dearth of accurate data on the economic impact (Obasi et al., 2006). There are records of losses due to the disease at clinical and sub clinical levels and cost of control measures are appreciable and reprehensible (Obasi et al., 2006; Mikail and Ajabonna, 2007).
In recent times, there has been increasing global interest in ethno- medical and ethno-veterinary practices especially as it relates to the use of medicinal plants in treating various ailments. In the developed world, this move is in response to the production of animals free from industrial chemical inputs and the need to discover new therapeutic substances of natural origin with possibly low toxicity to man and animals. In Africa this is borne out of economics and affordability (Alawa et al., 2003; Tipu et al., 2006). Khaya senegalensis is a well known medicinal plant (Sale et al., 2008; Ndjonka, et al., 2011; Kolawole, et al., 2012; Ibrahim et al., 2013) and the active compounds of Khaya senegalensis have been described by Yaun et al. (2013) and Nakatani et al. (2002). Proximate analysis of K. senegalensis leaves revealed that they are rich in dry matter, organic matter, minerals, gross energy and crude protein (Abdel-Wareth et al., 2014). In Nigeria, the bark of Khaya senegalensis is used to treat coccidiosis, helminthosis, amoebic dysentery and diarrhoea in poultry (Gefu et al., 2000). It has also been reported to have some therapeutic effects against Trypanosoma species (Otu et al., 2009). Anti oxidants and flavonoids are possible candidates in natural products which occur naturally (Taheri et al., 2005) with many medicinal plants been good sources (Emami, 2007) and are stored in different parts of some plants (Bhakuni et al., 2001; Brisibe et al., 2009). The identification of phenolic compounds in Khaya senegalensis which are also antioxidants has been indicated to have anticoccidial activities (Naidoo et al., 2008; Meskerem and Boonkaewwan, 2013). Oxidants can be produced at elevated rates under patho-physiological conditions (Meskerem and Boonkaewwan, 2013). Phenolic compounds could be a major determinant of antioxidant potentials of foods and are therefore a natural source of antioxidants (Aberoumand and Deokule, 2008). However, the effects of leaf extracts of the plants on Coccidia, another important protozoan parasite is not known. The present study is therefore conceived to bridge this gap of knowledge and probably contribute towards finding solutions to effective treatment and control of avian coccidiosis in Nigeria.
1.3 Aim of the study:
The aim of the study is to determine the chemical constituents and anticoccidial activities of extracts of Khaya senegalensis leaves using experimental Eimeria infection in broilers.
1.4 Objectives of the study:
The objectives of the study are to
i: Fractionate the methanol extracts of Khaya senegalensis, Guiera senegalensis and Tamarindus indica
- Evaluate the in vitro anticoccidial effect of the extracts of Khaya senegalensis, Guiera senegalensis and Tamarindus indica.
- Evaluate the in vivo anticoccidial effect of the extracts.
1.5 Research Question
Do aqueous and n-butanol fraction of the crude methanol extracts of Khaya senegalensis, Guiera
senegalensis and Tamarindus indica have anticoccidial effects against Eimeria tenella infection in broilers in vivo and in vitro?
