ABSTRACT
The aim of this study was to determine the immune response of broiler chickens to Eimeria tenella developmental stages Four hundred broilers divided into six groups (n=40) were used for the study. Each group was subdivided into two (n=20) as treated and non-treated and infected with different developmental stges of Eimeria tenella (local isolate). The molecular identification of the local Eimeria tenella isolate identity was done through polymerase chain reaction (PCR) amplification of the genomic deoxyribonucleic acid (DNA). Clinical signs, gross caecal lesions, humoral and cellular-mediated immune responses were determined in the infected broiler chickens with Eimeria tenella developmental stages. The faeces were processed using simple floatation technique and observed at x 10 and x 40 objectives of the Neiss microscope. Oocysts isolated from the caeca of birds naturally infected in Jos, Nigeria with the local strain were used to obtain the different developmental stages either in vitro or in vivo using bovine monocytes (schizonts), embryonated chicken eggs (gamatocytes) and two weeks old broilers (merozoites). To study the immune response elicited during the primary and secondary infection, each developmental stage was used to infect a group of two, three and half weeks old broilers, twenty of which were treated with the recommended dose of amprolium (250 mg/l (0.025%) for 5 days at the appearance of clinical signs. At the tertiary infection, all the experimental birds except the control group of forty birds were orally infecteded with 105 sporulated oocysts of known characterized virulent Eimeria tenella strain. The mean oocysts output or count was 37.07 x 106 in the infected birds non-treated than 25.65 x 106 in the treated groups, although there was a gradual reduction (groups II – 8.36 x 106 – 7.84 x 106 – 5.10 x 106; III – – 6.58 x 106 – 4.83 x 106; IV – 7.18 x 106 – 7.00 x 106 – 3.83 x 106; V – 6.59 x 106 – 5.87 x 106 – 4.20 x 106) in oocyst count from primary-secondary-tertiary infections except group I (control). There was a significant difference in oocyst output between the groups (II and IV) ( p<0.05). Antibodies (IgG or IgY) titre values were higher in broilers sera infected with sporulated oocyst (0.265 ± 0.010, 0.282 ± 0.005;
0.305 ± 0.002, 0.316 ± 0.010 and 0.252 ± 0.002, 0.281 ± 0.010) and merozoites (0.177 ±
0.001, 0.186 ± 0.003; 0.135± 0.010, 0.141 ± 0.002 and 0.069 ± 0.004, 0.139 ± 0.005 ) reaching a peak on day 10 of post primary and secondary infections and day 5 post tertiary infection in sera of broilers (treated and non- treated). At tertiary infection, antibodies increases at day 5, 7, 11 and 14 indicating that antibodies increases in broilers infected with the invasive or zoite stages, (sporozoite and merozoite) of the parasite.. There was a significant difference in the antibody output between the sera of the broiler groups (p<0.05). The study reveals the proliferation of cytokines in treated and non- treated broilers consisting of IFN- γ, IL-1, IL-2, IL-4, IL-6, TNF and TGF. The CD4 lymphocyte count in the treated and non- treated broilers orally administered with various developmental stages of the parasite reached a peak at day 10 ((groups I – 198.0 x 103 µl,
165.3 x 103 µl; 200.0 x 103 µl, 156 x 103 µl and 196.7 x 103 µl, 173.3 x 103 µl ; II – 199.0 x 103 µl, 186.0 x 103 µl ; 197.0 x 103 µl, 192.7 x 103 µl and 200.0 x 103 µl, 194 x 103 µl;
- – 198 x 103 µl, 153.3 x 103 µl ; 200.0 x 103 µ,l 160.0 x 103 µl and 188.7 x 103 µl, 166.7 x 103 µl ; IV – 193.3 x 103 µl, 183 x 103 µl; 198.7 x 103 µl, 183.3 x 103 µl and 190 x 103
µl , 188.0 x 103 µl ; V – 200.0 x 1 03 µl, 198.0 x 103 µl ; 187.3 x 103 µl , 174 x 103 µl and 188.7 x 103 µl, 175.3 x 103 µl respectively) at primary and secondary infections and day 24 at tertiary infection. There was significant difference in the CD4 cell count between groups of the infected broiler chickens (p<0.05). Caecal lesions were observed to gradually reduce from primary-secondary-tertiary infections. The lesions were significantly different between in groups (II and IV) (p<0.05). Oocyst output and caecal lesions were absent group VI (control). The current study observed a relationship between the different developmental stages of the parasite and immune responses (humoral and lymphocytes responses). The study also observed that broilers with high oocyst output had high antibody production, CD4 lymphocytes count and high levels of cytokines. Thus, the sporozoites and merozoites are the invasive stages that initiate infection of host cells and probably stimulation of the immune response and may be possible vaccine candidate against avian coccidiosis.
CHAPTER ONE
INTRODUCTION
1.1 Background Information
Avian coccidiosis is caused by intracellular protozoans parasites belonging to seven species of Eimeria. Eimeria tenella is the most virulent causing severe heamorrhagic enteritis by infection of the epithelium and submucosa of the caeca and eventually death in infected chickens (Mehlhorn, 2005). Lawal et al. (2008) showed that the infection can occur in both local and exotic birds with the former serving mainly as reservoir hosts. The parasite development cause diarrhoea, morbidity and mortality, and has serious economic impact in the poultry industry (Takagi et al., 2006; Gyorke et al., 2013). Coocidiosis causes annual losses of US $ 2.4 billion to the poultry industry worldwide (Shirly et al., 2005) in both the layer and broiler industries (Chandrakesan et al., 2009).
Conventional disease control strategies depend on vaccination and proplylactic use of anticoccidial drugs. However, resistance against anticoccidial compounds is widely spread and coccidiostats as feed additives was banned in Europe by the year 2012 (Regulation (EC) No 1831/2003 of the European parliament and of the council of 22 September, 2003 on additives for use in animal nutrition). Eimeria infection promotes antibody and cell-mediated immune responses, and cellular immunity mediated by various cell populations, including lymphocytes, natural killer (NK) cells and macrophages plays a major role in disease resistance (Lillehoj and Choi, 1998). There is increase evidence of CD4+ and intraepithelial lymphocyte (IEL) involvement during a primary infection, while T-cell receptor a -and b -chain-positive CD8+ IEL play a key role in secondary infection
(Lillehoj, 1998). The low level of homology between chicken genes and their mammalian counterparts has made it difficult to discover immunologically relevant chicken genes. However, there have been increasing numbers of chicken gene sequences appearing in the data bases due to the emergence of chicken genome projects. Among the cytokines cloned, one can find gene coding for interleukins (interleukin -1 b (IL-1 b ), (Weining et al.1998), IL-2 (Sundick et al., 1997) and interferons (alpha/beta interferon [IFN- a / b ]
(Sekellick et al., 1994) and IFN- γ (Digby and Lowenthal, 1995) and also for a macrophage growth (Leutz et al., 1989) and three isofornes of transforming growth facto (TGF- b ) (Jakowlew et al; 1990). In addition, several members of the chemokine family, have recently been cloned: C chemokines cc chemokines (macrophage inflammatory protein 1b (MIP-1b ) (Hughes et al;, 1999) and K203 (Sick et al., 2000) and C X C chemokines (k60 (Sick et al., 2000) and IL-8 (Kaiser et al.., 1999). A number of receptors have also been identified including the IL-1 receptor (IL-IR) (Guida et al., 1992) and a putative chemokine receptor (chem.-R) (Gupta et al., 1998). However, the analysis by reverse transcription – polymerase chain reaction (RT-PCR) of the expression of an available panel of genes will provide initial clues about the development of immune response to Eimeria infection. In this study, we intend to analyse the local immune response of broiler chickens to Eimeria tenella commonly found in poultry farms in Jos, Nigeria by PCR, lymphoproliferation assay or non radioactive assay, Enzme linked-immunosorbent assay (ELISA) and flow cytometric analysis.
1.2 Statement of the Problem
The creation of states in Nigeria and increase human population and activities within the states to earn money for living and increase in population has led to establishment of several poultry farms to meet the increasing demands for animal protein in terms of meat and eggs (Adegoye et al., 1988; FAO, 2006). Chickens also fulfill an array of other functions such as pest control, provision of manure and sacrifice in special festivals. However, one of the major constraints to production of chickens is coccidiosis (Sani et al. 1987; Majoro, 1993; Biu et al., 2006). In Nigeria where intensive poultry farming is less developed, the rising cost of poultry feeds, problems of drug residues, lack of new anticoccidial products and resistance to diseases are major problems militating against the poultry industry (Ogbe et al., 2008; Hafez, 2008). Coccidiosis is a serious disease which causes heavy economic loss. It has remained the most important poultry disease in Nigeria (Obasi et al., 2006). The macroscopic lesions in the digestive tract predispose the infected birds to many gastrointestinal bacterial poultry diseases such as clostridiosis, salmonellosis and collibacillosis (Lanckriert et al., 2010). Mua‟zua et al. (2008) reported a prevalence rates of 52.9% and 36.7% coccidial infection among the adult and younger birds in Vom, Plateau State, Nigeria.
Although the exact losses due to coccidiosis in Nigeria are not known due to lack of statistical indices but they could be in the region of millions of naira. The annual worldwide cost is estimated at about US$2.4 Billion (Shirley et al,, 2005). Problems associated with antigenic variation of field strains and the cost of producing multiple-species live vaccines post limits to the current vaccination approaches (Constantinoiu et al., 2008). The cost involved in the production of recombinant vaccines (proteins or DNA), the difficulty in identifying the antigens or genes that can elicit coccidia-specific protective immunity and the devise of the most efficient method of delivering these recombinant vaccines to the bird immune system are additional obstacles (Bedran and Lukesova, 2006). Besides, there are also the increasing incidence of drug-resistant strains and escalating public anxiety over chemical residues in meat and eggs as well as the complexities of the host immune system and parasite life cycle (Bal, 2009).
1.3 Justification of the Study
Coccidiosis is a protozoan disease of poultry commonly occurring under intensive management system (Biu et al., 2006). This makes coccidiosis one of the major health problems of chickens in Nigeria. An outbreak of coccidiosis in a poultry flock has a very high negative and economic impact. The disease is an important component of poultry diseases, and it is responsible for high morbidity, mortality and reduced market value of affected birds, and sometimes leading to culling or delayed slaughter time. Coccidiosis leads to destruction of the integrity of the intestinal mucosae and interference with nutrient absorption, causing diarrhoea and increase in medication costs. All these setbacks lead to huge losses for the producer. Treatment and control of the disease are beset with several problems prominent of which is the poor understanding of the immune response. Another factor is the increasing incidence of drug resistance in field strains of Eimeria. Knowledge of the immune response to the different stages of E. tenella will give an insight on the possibility of control of the disease through vaccine production, which will ultimately lead to increase in productivity. Furthermore, due to health awareness, there is increasing concern regarding drug residues in poultry products and growing pressure from Government and consumer on the production of drug-free poultry products (Williams, 2002). Consequently, the use of vaccines has become more desirable than ever before. Epidemiological studies have established the economic importance of coccidiosis as a major parasitic disease of poultry in Nigeria (Chapman, 2008).
1.4 Aim of the Study
To determine the immune response of broilers to different developmental stages of Eimeria tenella.
1.5 Objectives of the Study
The objectives of the study are to:
- Monitor the clinical signs in the experimental infection (oral) of birds with different Eimeria tenella developmental stages.
- Determine the gross caecal lesions in broiler chickens cause by infection with different stages of Eimeria tenella.
- Determine the immune responses to the various stages of Eimeria tenella in the orally infected broiler chickens by measurement of immune bodies
- Determine the conferment of immunity in the broiler chickens infected with Eimeria tenella developmental stages.
1.6 Research Questions
- Do exogenous (unsporulated and sporulated oocysts) and endogenous stages (schizonts, merozoites and gametocytes) of Eimeria tenella induce clinical signs in the experimentally infected birds?
- Do exogenous (unsporulated and sporulated oocysts) and endogenous stages (schizonts, merozoites and gametocytes) of Eimeria tenella induce gross caecal lesions in the experimentally infected birds?
- Do exogenous (unsporulated and sporulated oocysts) and endogenous stages (schizonts, merozoites and gametocytes) of Eimeria tenella induce immune responses in the experimentally infected birds?
