Thesis Ref. No. _____ PATHOLOGICAL AND SEROPREVALENCE STUDIES ON INFECTIOUS BURSAL DISEASE IN CHICKENS IN AND AROUND BAHIR DAR, NORTH WEST, ETHIOPIA. MSc Thesis By Natnael Teshager Addis Ababa University, College of Veterinary Medicine and Agriculture, Department of Pathology and Parasitology June, 2015 Bishoftu, Ethiopia PATHOLOGICAL AND SEROPREVALENCE STUDIES ON INFECTIOUS BURSAL DISEASE IN CHICKENS IN AND AROUND BAHIR DAR, NORTH WEST, ETHIOPIA A Thesis submitted to the College of Veterinary Medicine and Agriculture of Addis Ababa University in partial fulfillment of the requirements for the degree of Master of Science in Tropical veterinary Pathology By Natnael Teshager June, 2015 Bishoftu, Ethiopia Addis Ababa University College of Veterinary Medicine and Agriculture Department of Parasitology and pathology As members of the Examining Board of the final MSc open defense, we certify that we have read and evaluated the thesis prepared by: Natnael Teshager entitled: Pathological and Seroprevalence Studies on Infectious Bursal Disease in Chickens in and Around Bahir Dar, North West, Ethiopia and recommend that it be accepted as fulfilling the thesis requirement for the degree of: Masters of Tropical veterinary pathology ________________________ ___________________ ______________ Chairman Signature Date ________________________ _______________ ______________ External Examiner Signature Date ________________________ _______________ ______________ Internal Examiner Signature Date 1. Dr. Hagos Ashenafi _______________ ______________ Major Advisor Signature Date 2. Dr. Asegedech ______________ ______________ Co- Advisors Signature Date 3. Dr. Wieland Barbara _______________ ______________ Co- Advisors Signature Date 4. Dr. Hagos Ashenafi _______________ ______________ Department chair person Signature Date DEDICATION This MSc thesis work is dedicated to memory of my absent father “Teshager Desta “whom I lost at grade 8. He nurtures me with lifelong hopes up to he was alived on: That help me to struggle even in uncomfortable circumstance of life to maintain self steem. STATEMENT OF AUTHOR I declare that the thesis hereby submitted for the MSc degree at the Addis Ababa University, College of Veterinary Medicine and Agriculture is my own work and has not been previously submitted to any other University or institution for the award of any degree. I concede copyright of the thesis in favor of the Addis Ababa University, Collage of Veterinary Medicine and Agriculture. Name: Natnael Teshager Signature: ___________ Addis Ababa University, Collage of Veterinary Medicine and Agriculture, Bishoftu Date of Submission: June, 2015 i TABLE OF CONTENTS ACKNOWLEDGMENTS ............................................................................................... iii LIST OF TABLES ........................................................................................................... iv LIST OF FIGURES .......................................................................................................... v LIST OF ABBREVIATIONS ......................................................................................... vi LIST OF ANNEXES ....................................................................................................... vii ABSTRACTS ................................................................................................................. viii 1. INTRODUCTION ..................................................................................................... 1 2. LITERATURE REVIEW ......................................................................................... 4 2.1. Infectious bursal disease .................................................................................................. 4 2.2. Etiology ............................................................................................................................ 5 2.3. Epidemiology ................................................................................................................... 6 2.3.1. Status of IBD in Ethiopia .............................................................................. 7 2.3.2. Morbidity and mortality ................................................................................ 8 2.4. Clinical signs .................................................................................................................... 8 2.5. Pathology and lesions ...................................................................................................... 9 2.6. Immunosuppression ....................................................................................................... 11 2.7. Diagnosis ....................................................................................................................... 11 2.7.1. Histological diagnosis ................................................................................ 12 2.7.2. Virological diagnosis .................................................................................. 12 2.7.3. Serological diagnosis .................................................................................. 13 2.7.4. Molecular identification ............................................................................. 13 3. MATERIALS AND METHODS ............................................................................ 15 3.1. Study area....................................................................................................................... 15 3.2. Study animals ................................................................................................................. 15 3.3. Study design ................................................................................................................... 15 3.4. Study methodology and data collection ......................................................................... 16 3.4.1. Data collected ............................................................................................. 16 3.4.2. Blood sample collection, serum separation and IELISA test...................... 16 ii 3.4.3. Clinical examination and Postmortem findings.......................................... 17 3.4.4. Histopathology procedures ......................................................................... 17 3.5. Data management and analysis ...................................................................................... 18 4. RESULTS ................................................................................................................. 19 4.1. Questionnaire survey ..................................................................................................... 19 4.2. Clinical manifestation .................................................................................................... 21 4.3. Characterization of gross pathological lesions ............................................................... 21 4.4. Histopathological changes ............................................................................................. 24 4.5. Seroprevalence of IBD Virus ......................................................................................... 29 5. DISCUSSION ........................................................................................................... 32 6. CONCLUSION AND RECOMMENDATIONS .................................................. 39 7. REFERENCES ........................................................................................................ 40 8. ANNEXES ................................................................................................................ 50 iii ACKNOWLEDGMENTS I convey my sincere gratitude and heartfelt thanks to my advisor Dr. Hagos for his advice and guiding through developing this MSc research thesis. Great thanks also given to Dr. Asegedech Sirak who did the histopathology reading accurately and timely. My special thanks given to International livestock research institute in which funding this research thesis. iv LIST OF TABLES Pages Table 1: Reported prevalence of IBD in Ethiopia ............................................................................ 7 Table 2: Distribution of chicken breed in the study population in and around Bahir Dar ............. 19 Table 3: IBD vaccine utilization per farm system in & around Bahir Dar. ................................... 20 Table 4: Lesion score in IBD infected Bursa in vaccinated and unvaccinated chickens .............. 26 Table 5: The overall seroprevalence of IBDV by IELISA in and around Bahir Dar. .................... 29 Table 6: Seroprevalence of IBD in different study sites, management systems, sex and breed in around Bahir Dar............................................................................................................................ 30 Table 7 : Association of risk factor with IBD by using multivariate logistic regression in and around Bahir Dar............................................................................................................................ 31 v LIST OF FIGURES Pages Figure 1: The death rate of the different breeds ranked by the owners in & around Bahir Dar......................................................................................................................... 20 Figure 2: The occurrence of death in different breeds at different seasons of the year as responded by the owners in and around Bahir Dar. .............................................. 21 Figure 3: Clinical signs observed during active case of IBD in 28 – 52 day old chickens in and around Bahir Dar. ................................................................................................... 22 Figure 4: Gross pathological changes in different organ of 21-54 day old chicken infected by IBDV in and around Bahir Dar. ............................................................... 23 Figure 5: Moderate lymphocyte depletion in follicles , formation of cortical rim (double arrow) in IBD infected bursa of chicken. ................................................................. 24 Figure 6: Bursa with intrafollicular edema, interfollicular hemorrhage, congestion, depleted lymphoid cell with pyknotic nuclei, and interfollicular fibrosis . ............. 25 Figure 7: Bursa with marked lymphoid depletion and necrosis, interfollicular hemorrhage (single arrow) and follicular cyst . ......................................................................... 26 Figure 8: The tubules of kidney became congested with purulent exudates; hemorrhage within and between tubules ................................................................................. 27 Figure 9: The tubules of kidney became congested with fibrinous exudates that contain necrotized cuboidal cell; hemorrhage within and between tubules. . ..................... 27 Figure 10: The depletion of lymphocyte in the germinal center of the spleen (arrow), infiltration of heterophils and hemorrhage in red pulp area . ............................... 28 Figure 11: Hemorrhage, congestion (double head arrow, A), infiltration of heterophils on the junction of proventriculus and gizzard . ...................................................... 28 vi LIST OF ABBREVIATIONS BoAAO Bureau of the Amhara Agriculture Office CSA Central Statistics Agency IBD Infectious bursal disease IBDV Infectious bursal virus BB Bovans brown BW Bovans white LB Local breed HB Hybrid breeds vii LIST OF ANNEXES Page Annex I: Questionnaire survey…………………………………………………………..50 Annex II: Serological and pathological sample data collection sheet…………………...51 Annex III: Gross and histopathological changes in different tissue of IBD infected chickens.........................................................................................................52 viii ABSTRACTS A cross sectional study was conducted from January 2015 to May 2015 to characterize the pathological changes and to determine seroprevalence of IBD and its associated risk factors in chickens in and around Bahir Dar. The clinical signs observed in IBD infected chickens were whitish diarrhea mixed with blood, ruffled feather, and massive death within short period. The necropsy findings were petichial hemorrhage in bursa of fabricius, kidney, thymus, spleen, thigh and pectoral muscles. Bursa of fabricius became edematous in serosa and mucosal part, whitish-creamy and atrophied as the course of the disease progressed. Kidney became pale and ureter was turgid with urate in a numbers of cases. The histopathological changes in this study revealed that edema of bursa of fabricius, formation of cystic follicles, depletion of lymphocytes, fibrosis and follicular architecture lost. Severe, moderate and mild lesion score were observed in bursa of fabricius. Infiltrations of heterophils were noted both in lymphoid and non lymphoid tissues. Depletion of lymphoid cell in germinal center of spleen and total depletion of lymphoid cells were encountered in thymus gland. The kidney tubules were filled by exudates and necrotized cuboidal cells. Sera collected from a total of 320 chickens were subjected to IELISA test and disclosed an overall prevalence of 51.56% (CI: 45.95 – 57.14) in study area. The seroprevalence of IBD among chickens showed a statistically significant difference (P<0.05) among study sites namely Andassa (72.73%), Gombat (50%), Wonjeta (47.69%), Meshenty (44.44%) and Bahir Dar (42.42%). The susceptibility of chickens to IBDV revealed a statistically significant difference (P<0.05) among different breeds where Koekoek was found more prone to IBD infection with seroprevalence of 67.11% followed by Bovans brown (57.69%), local breeds (48.31%), and Bovans white (40.28%). The seroprevalence of IBD was also significantly associated (P<0.05) with the age of chickens and among farm systems. These studies clearly indicated that IBD infection is a common and wide spread problem affecting a number of chicken breeds under different management systems and hence an urgent control intervention should be in place. Keywords: Age, Bahir Dar, Breed, Chickens, Histopathology, Lesions, Seroprevalence, sex 1 1. INTRODUCTION Ethiopia has the largest livestock population in Africa, including more than 53.99 million cattle, 49.5 million small ruminants, approximately 0.92 million camels, 9 million equines and 50.38 million chickens. In Ethiopia about 96.9% of the chicken population consists of indigenous chickens, while the remaining 2.56% and 0 .54% consists of exotic and hybrid breeds (CSA, 2013). This indigenous poultry production in Ethiopia contributed to 98.5 and 99.2% of the national egg and poultry meat production, respectively (Alemu and Taddele, 1997). This flock Provide with yearly output of 72,300 metric tons of meat and 78,000 metric tons of eggs (Hailemariam et al., 2006). Chickens are wide spread in the Ethiopia and are important to subsistence, economic and social livelihoods of a large human population. Chickens are especially important to women, children, and aged individuals, who are the most vulnerable member of the society in terms of under-nutrition and poverty. There has been a gradual decline in the Ethiopian poultry population. According to Burley (1957) and the Central Statistical Authority (2004-2005), the Ethiopian poultry population was estimated at 85 and 31 million in 1954 and in 2005 respectively. The Sub- Sector Review (1984) estimated the average number of chickens per household at 6.5 in 1984 whereas the average number of chickens per household is estimated at 4.1 in 2003 (CSA, 2013). These figures show that the country’s poultry population has turned down by 64% in the last 50 years, while the average number of chickens per farmer has reduced by 37% over the last 20 years. This problem attracts the attention of researchers in Ethiopia to improve health management and breeding aspect of chickens (Mazengia, 2012). Despite, Ethiopia owned huge chicken flock; there are different constraints like poor nutrition, poor management and prevalent diseases that hinder the productivity of the chickens in most area of the country (Alemu, 1995; Dessie and Ogle, 1996). Among the above obstacles, the poultry diseases are the main constraints incriminated for reduction of total numbers and compromised productivity (Tadesse, 2000; Ashenafi, 2000). 2 According to the work done by (Tadesse, 2000; Ashenafi, 2000) some farmers have stopped rearing chickens due to disease problems. Infectious bursal disease, Newcastle, Coccidiosis, Salmonellosis and nutritional deficiency have been considered the major diseases inflicting heavy losses in Ethiopia. Beside the above problem, imported breeds and cross-breeds are multiplied in Ethiopia poultry hatchery center and day old chickens are imported from abroad. This day old chicken distributed to farmers by the Bureau of Agriculture to be maintained and produced under the backyard management system. This is aim to advance the livelihood and nutrition of poor farmers and in addition to increase the country economy (Tadelle and Ogle, 2001). Accordingly, the Bureau of the Amhara Agriculture Office schemed poultry production strategy that started in 2003.The goal of the strategy was to enable farmers to generate income through rearing day-old chickens of breeds of Bovans brown, Bovans white and Koekoek breeds which are hatched and distributed from poultry multiplication centers of Andassa, Kombolcha and Ethio - chickens. However, it is becoming a growing issue with introduction of diseases of various etiologies into chickens of backyard and intensive poultry farms. The distribution of these none indigenous breeds to farmers is creating a great treat to the indigenous backyard chickens (Alamargot, 1987; Zeleke et al., 2005). Among these distributed day old chickens to small holder farmers, about 31% of them died within 3-6 weeks and 2% of total died within 7 weeks of age (BOAAO, 2013). Infectious bursal disease is among those diseases introduced with exotic breed that result in death in young chickens and reduction of egg in pullet that impose great economical loss in productivity of chickens in Ethiopia (Taddele and Ogle, 2001; Zeleke et al., 2005). Infectious Bursa Disease (IBD) is an acute and highly contagious viral disease of growing chickens. The virus infect primarily lymphoid cells, especially B-cells in which, Lymphoid tissues of the bursa fabricius are severely affected (Chou and Calnek, 1997). There are two serotypes of IBDV (serotypes 1 and 2). Strains of serotype 1 IBDV are pathogenic only in chickens, and are further classified as classical virulent IBDV (cvIBDV), vvIBDV, antigenic variant IBDV (avIBDV) and attenuated IBDV (atIBDV) (van den Berg et al., 2004). 3 In the world, the poultry industry has encountered heavy economic losses associated with very virulent (vv) IBDV strains in the last several years. These strains may cause high mortality in affected chicken flock and severe immunosuppression that involves both innate and adaptive immune responses (Negash, 2013). Although prevalence and incidence of infectious bursal disease (IBD) have been reported in major field outbreaks of intensive farm and backyards in northwest and central Ethiopia in recent years (Zeleke et al., 2005; Mazengia et al., 2009; Zeryehun and Fekadu, 2012), the pathological changes and susceptibility variation across breed, age, sex and location of IBD remain untouched and with its scanty current information on seroprevalence in backyard and intensive chickens farms in and around Bahir Dar. The present study was designed and proposed to undertake an investigation on the occurrence of IBD in and around Bahir Dar area and further characterize the involved pathological changes. Thus, the objectives of this study include characterization of the major clinical signs, appreciation of the gross and histopathological changes in different tissues of naturally infected chickens as well as determination of the seroprevalence of IBD virus among chickens in different farm systems. Moreover, the association of IBD with different risk factors was assessed. 4 2. LITERATURE REVIEW 2.1. Infectious bursal disease Infectious bursal disease (Gumboro disease) is infectious disease of global economic importance (Pitcovski, 2003). Infectious bursal disease virus (IBDV) is cause of IBD, currently responsible for huge economic impact on the worldwide poultry industry (Vandersluis, 1999). Two serotypes of the virus have been described; these are Serotype 1 IBDV strains, pathogenic to chickens (Muller et al. 2003; Van Den Berg et al., 2004), whereas serotype 2 strains are non-pathogenic (Meferran et al., 1980). Serotype 1 IBDV isolates comprise the variant, classical virulent and vvIBDV strains, which greatly differ in their pathogenecity in chickens. Variant IBDVs do not cause mortality, whereas the classical strains cause up to 20% mortality (Muller et al., 2003). VvIBDV causes mortality exceeding 50% in susceptible chickens (Chettle et al. 1989; Berg et al., 1991; Muller et al., 2003). Currently, this disease reported throughout the globe and an economical important disease causing 100% morbidity. Mortality reaches up to 90% in susceptible flock. The loss is attributing to high mortality, immunosuppression and condemnation of carcasses (Kibeng, 1988). The clinical disease of IBD, first described by Cosgrove in (1962), affects chicks between 3 and 6 weeks of age. It is characterized by ruffled feathers, whitish or watery diarrhea, anorexia, depression, trembling, severe prostration and finally death. The target organ of the virus is the lymphoid tissue, specially the bursa of Fabricius that has a gelatinous yellowish transudate covering the serosal surface. Longitudinal striations on the surface become prominent, and the normal white color turns to cream. The transudate disappears as the bursa returns to its normal size and becomes gray during the following period of atrophy. The infected bursa often shows necrotic foci and at times ecchymotic hemorrhages on the mucosal surface. Occasionally, extensive hemorrhage throughout the entire bursa has been observed (Sellers et al., 1999). 5 2.2.Etiology Infectious bursal disease virus (IBDV) is classified as a member of the Birnaviridae family. The family includes 3 genera: Aquabirnavirus whose type species is infectious pancreatic necrosis virus (IPNV), which infects fish, mollusks and crustaceans; Avibirnavirus whose type species is infectious bursal disease virus (IBDV), which infects birds; and Entomobirnavirus whose type species is Drosophila X virus (DXV), which infects insects (Viruses in this family possess bi-segmented, double-stranded RNA (dsRNA) genomes, which are packaged into single shelled, non-enveloped virions. The capsid shell exhibits icosahedral symmetry composed of 32 capsomeres and a diameter ranging from 55 to 65 nm (Brown et al., 1994). Two serotypes of the virus have been described; these are Serotype 1 IBDV strains, pathogenic to chickens (Muller et al. 2003; Van Den Berg et al. 2004), whereas serotype 2 strains are non-pathogenic (Meferran et al. 1980). Serotype 1 IBDV isolates comprise the variant, classical virulent and vvIBDV strains, which wide differ in their pathogenicity to chickens. Variant IBDVs do not cause mortality, whereas the classical strains cause up to 20% mortality (Muller et al., 2003). VvIBDV causes mortality exceeding 50% in susceptible chickens (Chettle et al. 1989; Berg et al., 1991; Muller et al., 2003). Infectious bursal disease virus is highly resistant to adverse environmental conditions. It is more resistant to heat and ultraviolet light than reovirus (Petek et al., 1973) and is resistant to ether and chloroform. Once infected with IBDV, chickens are capable of shedding the virus in feces for as long as 16 days (Winterfield et al., 1972). Benton et al., (1967) reported that poultry houses which previously harbored infected flocks remained infective for at least 122 days and that fomites (water, feed, droppings) contaminated with IBDV contribute to viral dissemination (Benton et al., 1967). Therefore, the control of this disease depends mainly on vaccination (Al-Natour et al., 2004), but in some cases vaccinations have been ineffective in protecting birds (Islam et al., 2003). 6 2.3.Epidemiology Cosgrove (1962) reported a specific disease, (IBD) that affecting the bursa of Fabricius in chickens .The first cases were seen in area of Gumboro, United States of America (USA), which is the name derived, even if the terms 'IBD' / 'infectious bursitis' are more accurate descriptions. In the year of 1960 and 1964, the disease observed in most part of the USA (Lasher and Davis., 1997), and become devastating disease in Europe in the years of 1962 to 1971 (Faragher, 1972). With its pandemic movement from the year 1966 to 1974, the disease was reported in the southern and western Africa, Far East, Middle East, India and Australia (Faragher, 1972; Provost et al., 1972; Firth, 1974; Jones, 1986; Lasher and Shane, 1994; Van den Berg, 2000; Van der Sluis, 1999). Infectious bursal disease currently become an international issue, 95 % of the 65 countries that responded to a survey conducted by the (OIE, 1995) announced presence of infection (Eterradossi, 1995), including New Zealand which had been free of disease until 1993 (Jones, 1986). Only chickens develop IBD after infection by serotype 1 viruses. Turkeys may be asymptomatic carriers of serotype 2 (McFerran et al., 1980; Jackwood et al, 1982; Ismail et al., 1988), and at times, of serotype 1 viruses whose pathogenicity for turkeys is ill- defined (Reddy and Silim, 1991;Owoade and Durojaiye, 1995). Anti-IBDV antibodies have been detected in guinea-fowl, common pheasants and ostriches, which have also been found to carry, serotype 2 viruses (Guittet et al., 1992). Neutralizing or precipitating antibodies have been detected, inter alia, in various species of wild duck, goose, tern, puffin, crow and penguin, which may mean that wild birds act as reservoirs or vectors (Gardner et al., 1997; Ogawa et al., 1998; Wilcox et al., 1983). The age of maximum susceptibility to IBDV is between 3 and 6 weeks, which is the period of maximum bursa development, during which the acute clinical signs are observed. Infections occurring before the age of three weeks are generally subclinical and immunosuppressive. Clinical cases may be observed up to the age of fifteen to twenty weeks (Ley et al., 1979; Okoye and Uzoukwu, 1981). Light strains of laying stock are more susceptible to disease than the heavy broiler strains (Van den Berg and Meulemans, 1991; Bumstead et al., 1993; Hassan and Saif, 1996). 7 The IBD transmit with horizontal way only, with healthy subjects being infected by the oral or respiratory pathway. Infected subjects excrete the virus in faces as early as 48 hours after infection, and may transmit the disease by contact over a sixteen-day period (Vindevogel et al., 1976). The possibility of persistent infection in recovered animals has not been researched. The disease is transmitted by direct contact with excreting subjects, or by indirect contact with any inanimate or animate contaminated vectors. Some researchers have suggested that insects may also act as vectors (Howie and Thorsen, 1981). The extreme resistance of the virus to the outside environment enhances the potential for indirect transmission. The virus can survive for four months in contaminated bedding and premises (Benton et al., 1967) , and up to fifty-six days in lesser mealworms taken from a contaminated building (Lucio and Hitchner,1980). In the absence of effective cleaning, disinfection and insect control, the resistance of the virus leads to perennial contamination of infected farm buildings. 2.3.1. Status of IBD in Ethiopia Table 1: Reported prevalence of IBD in Ethiopia Study area Prevalence Authors Central Ethiopia 82.2% Zeryehun and Fekadu, 2012 Selected sites of Ethiopia 83.1% Jebberie et al., 2012 Gondar and west Gojjam 73.5% Kassa and Molla, 2012 Bahir Dar 29.4% Mazengia et al., 2010 Farta 21.7% Mazengia et al., 2010 Andassa poultry farm 100% Woldemariam and Wossene, 2007 Debre Zeit 93.3% Zeleke et al., 2005 8 2.3.2. Morbidity and mortality Infectious bursal disease is extremely contagious and in infected flocks, morbidity is high, with up to 100 % serological conversion, after infection, whilst mortality is variable. Until 1987, the field strains isolated was of low virulence and caused only 1% to 2 % of specific mortality. However, since 1987 an increase in specific mortality has been reported in different parts of the world. In the USA, new strains responsible for up to 5% of specific mortality were described (Rosenberger and Cloud, 1986). At the same time, in Europe, Africa and subsequently in Japan, high mortality rates of 50 % to 60 % in laying hens and 25 % to 30 % in broilers were observed. These hypervirulent field strains caused up to 100 % mortality in specific pathogen free (SPF) chickens (Van den Berg et al., 1991; Nunoya et al., 1992). 2.4. Clinical signs The incubation period is very short which range from 2 to 3 days. In acute cases, the chickens tired, prostrated, dehydrated, suffer from watery diarrhea, and feathers are ruffled. Mortality commences on the third day of infection, reaches a peak by day four, then drops rapidly, and the surviving chickens recover a state of apparent health after five to seven days. Disease severity depends on the age and breed sensitivity of the infected birds, the virulence of the strain, and the degree of passive immunity. Initial infection on a given farm is generally very acute, with very high mortality rates if a very virulent strain is involved. If the virus persists on the farm and is transmitted to successive flocks, the clinical forms of the disease appear earlier and are gradually replaced by subclinical forms. Nonetheless, acute episodes may still occur. Moreover, a primary infection may also be in apparent when the viral strain is of low pathogenicity or if maternal antibodies are present (Faragher, 1972). The clinical signs of IBD vary considerably from one farm, region, country or even continent to another. Schematically, the global situation can be divided into three principal clinical forms, these are; 1) the classical form, caused by the classical virulent 9 strains of IBDV. Specific mortality is relatively low, and the disease is most often subclinical, occurring after a decline in the level of passive antibodies (Faragher, 1972). The second is immunosuppressive form, principally described in the USA, is caused by low pathogenicity strains of IBDV, as well as by variant strains, such as the Delaware variant E or GLS strains, which partially resist neutralization by antibodies against the ‘classical’ viruses (Jackwood and Saif, 1987; Snyder, 1990). The acute form, first described in Europe, Africa and then in Asia, is caused by 'hypervirulent' strains of IBDV, and is characterized by an acute progressive clinical disease, leading to high mortality rates on affected farms (Chettle et al., 1989; Stuart, 1989; Van den Berg et al., 1991). 2.5.Pathology and lesions Even although IBD affected different lymphoid organs (Sharma et al., 1993; Tanimura et al., 1995; Tanimura and Sharma., 1997), the principal target of the virus is the bursa of Fabricius (Kaufer and Weiss, 1980), which is the reservoir of B lymphocytes in birds. Indeed, the target cell is the B lymphocyte in active division, for which the infection is cytolytic (Burkhardt and Müller, 1987). Cell sorting studies have demonstrated that the B lymphocyte is susceptible in the immature stage, during which immunoglobulin M is carried on the surface of the lymphocyte (Hirai et al, 1981; Nakai and Hirai, 1981). This accounts for the paradoxical immune response to IBDV, in which immunosuppression co-exists with high anti-IBDV antibody titres. The mature and competent lymphocytes will expand as a result of stimulation by the virus whereas the immature lymphocytes will be destroyed. A kinetic study using immunofluorescence (Mülle et al., 1979) has shown that, 4 hours after oral inoculation, the virus is found in the lymphoid tissues associated with the digestive tract, where the first cycle of viral replication occurs. The virus subsequently enters the general circulation via the hepatic portal vein. A phase of primary viraemia ensues, during which the virus reaches the bursa, 11 hours after infection, and a major 10 secondary replication cycle occurs. A phase of secondary viraemia then occurs, and the other lymphoid organs become massively infected. Macroscopic lesions are observed principally in the bursa which presents all stages of inflammation following acute infection (Vindevogel et al, 1974; McFerran, 1993). Autopsies performed on birds that died during the acute phase (three to four days following infection) reveal hypertrophic, hyperemic and oedematous bursas. The most severe cases are characterized by a major infection of the mucous membrane and a serous transudate, giving the bursal surface a yellowish colour. This appearance is often accompanied by petechiae and haemorrhages. By the fifth day, the bursa reverts to normal size and by the eighth day becomes atrophied to less than a third of the normal size. The affected animals are severely dehydrated, and many birds have hypertrophic and whitish kidneys containing deposits of urate crystals and cell debris. Haemorrhages in the pectoral muscles and thighs are frequently observed, probably due to a coagulation disorder (Skeeles et al., 1980). Certain variants from the USA are reported that causes rapid atrophy of the bursa without a previous inflammatory phase (Lukert and Saif, 1997). Moreover, in the acute form of the disease caused by hypervirulent strains, macroscopic lesions may also be observed in other lymphoid organs (thymus, spleen, caecal tonsils, Harderian glands, Peyer's patches and bone marrow) (Hiraga et al., 1994; Inoue et al., 1994; Tsukamoto et al., 1995; Inoue et al., 1999) . Pathological study in Bangladesh on natural infected chickens by (Islam and Samad, 2004) observed gross lesions in bursa which was swollen, oedematus and streaks of haemorrhagic on outer and inner surface of bursa. Cut surface of the bursa revealed slimy and gelatinous material. Thigh muscle also revealed petechial hemorrhage and in addition spleen was hemorrhagic and swollen. This investigation also indicated that hemorrhages at the junction of proventriculus and gizzard. Henry et al., (1980) have developed a system for evaluating microscopic lesions of the affected organs, with a score ranging from one to five according to lesion severity. The B lymphocytes are destroyed in the follicles of the bursa as well as in the germinal centres and the perivascular cuff of the spleen. The bursa is infiltrated by heterophils and undergoes hyperplasia of the reticulo- endothelial cells and of the intermolecular tissue. As the disease evolves, the surface 11 epithelium disappears and cystic cavities develop in the follicles. Severe panleukopenia is also observed and these microscopic lesions are exacerbated in the acute forms of the disease. 2.6. Immunosuppression The destruction of immature B lymphocytes IBDV in the bursa creates an immunosuppression, which will be more severe in younger birds (Giambrone et al., 1976). In addition to the impact on production and role in the development of secondary infections, this will affect the immune response of the chicken to subsequent vaccinations which are essential in all types of intensive chicken production (Giambrone et al., 1976). The most severe and longest duration immunosuppression occurs when day-old chicks are infected by IBDV (Allan et al., 1972; Faragher et al., 1974; Sharma et al., 1989; Sharma et al., 1994). In field conditions, this rarely occurs since chickens tend to become infected at approximately two to three weeks, when maternal antibodies decline. Evidence suggests that the virus has an immunosuppressive effect at least up to the age of six weeks (Wyeth, 1975; Giambrone, 1979; Lucio and Hitchner, 1980). 2.7. Diagnosis The clinical diagnosis of the acute forms of IBD is based on disease evolution of a mortality peak followed by recovery in five to seven days and relies on the observation of the symptoms and post-mortem examination of the pathognomonic lesions, in particular of the bursa of Fabricius. The diseases like avian coccidiosis, Newcastle disease in some visceral forms, stunting syndrome, mycotoxicoses, chicken infectious anaemia and nephropathogenic forms of infectious bronchitis are the differential diagnosis for IBD. In all acute cases, the presence of bursal lesions allows for a diagnosis of IBD. In subclinical cases, an atrophy of the bursa may be confused with other diseases such as Marek's disease or infectious anaemia. A histological examination of the bursa will allow differentiation between these diseases (Lukert and Saif, 1997). 12 2.7.1. Histological diagnosis Histological diagnosis is based on the detection of modifications occurring in the bursa. The ability to cause histological lesions in the non-bursal lymphoid organs, such as the thymus (Inoue et al; 1994), spleen or bone marrow (Inoue et al., 1999) has been reported as a potential characteristic of hypervirulent IBDV strains. The histological diagnostic method has the advantage of allowing for diagnosis of both the acute and chronic or subclinical forms of the disease. Detection of viral antigens: thin sections of the bursa of Fabricius prepared to detect viral antigens specific to IBDV done by direct and indirect immunofluorescence (Allan et al, 1984; Meulemans et al, 1977) or by immunoperoxidase staining (Cho et al, 1987) in the bursal follicles of infected chickens between the fourth and sixth day after inoculation. No viral antigen is detectable from the tenth day. However, the virus can be isolated from bursae sampled from the second to the tenth day, with a maximum infectious titre after four days (Winterfield et al, 1972; Vindevogel et al, 1976). The use of monoclonal antibodies in IHC techniques for detection of the virus enhances the specificity of the test (Cho et al, 1987). 2.7.2. Virological diagnosis Infectious bursal disease virus may be detected in the bursa of Fabricius of chicks in the acute phase of infection, ideally within the first three days following the appearance of clinical signs. Isolation: A filtered homogenate of the bursa of Fabricius is inoculated in nine- to eleven-day-old embryonated eggs originating from hens free of anti- BDV antibodies. The most sensitive route of inoculation is the chorioallantoic membrane (CAM); the yolk sac route is also practicable, and the intra-allantoic route is the least sensitive. The specificity of the lesions observed must be demonstrated by neutralizing the effect of the virus with a monospecific anti-IBDV serum. Isolation in embryonated eggs does not 13 require adaptation of the virus by serial passages, and is suitable for vvIBDVs. In the absence of lesions, the embryos from the first passage should be homogenized in sterile conditions and clarified, and two additional serial passages should be performed (Hitchner, 1970; Rosenberger, 1989; Lukert P and Saif, 1997). 2.7.3. Serological diagnosis In areas contaminated by IBDV, most broiler flocks have anti-IBDV antibodies when leaving the farm. Current serological tests cannot distinguish between the antibodies induced by pathogenic IBDV and those induced by attenuated vaccine viruses, so serological diagnosis is of little interest in endemic zones. Nonetheless, the quantification of IBDV-induced antibodies is important for the medical prophylaxis of the disease in young animals, in order to measure the titre of passive antibodies and determine the appropriate date for vaccination (Dewit, 1999; Kouwenhoven and van den Bos, 1994; Muskett et al, 1979) or in laying hens to verify success of vaccination (Lucio, 1987; Meulemans et al., 1987). Serology is likewise essential to confirm the disease-free status of flocks. Each serological analysis must include a sufficient number (at least twenty) of individual serum samples representative of the flock under study. A kinetic study requires at least two serological analyses separated by an interval of three weeks (paired sera). 2.7.4. Molecular identification Most efforts at molecular identification have focused on the characterization of the larger segment of IBDV (segment A) and especially of the vVP2 encoding region. Several protocols have been published on characterization using restriction endonucleases of RT- PCR products. These approaches are known as RTPCR/RE or RT-PCR-RFLP (restriction fragment length polymorphism) (Jackwood, 1990; Lin et al, 1993; Zierenberg et al, 2001). The usefulness of the information they provide depends on the identification of enzymes that cut in restriction sites that are phenotypically relevant. Some sites involved in antigenicity have already been identified, however, restriction sites reliably related to virulence still need to be defined and validated. Nucleotide sequencing of RT-PCR 14 products, although more expensive than restriction analysis, provides an approach to assessing more precisely the genetic relatedness among IBDV strains. Markers have been demonstrated experimentally, using a reverse genetics approach, for cell culture-adapted strains, which exhibit amino acid pairs 279 N–284 T (Lim et al., 1999) or 253 H–284 T (Mundt , 1999). In most very virulent viruses, four typical amino acids are present (222 A, 256 I, 294 I and 299 S) (Brown et al, 1992; Lin et al, 1993; Eterradossi et al, 1999). However, it is not yet known whether these amino acids play a role in virulence or if they are merely an indication of the clonal origin of most vvIBDV isolates. Several recent studies indicate that although VP2 is an important virulence determinant, it may not be the only one (4). It has been reported that segment A and B of IBDV mostly co-evolve (i.e. most significant IBDV clusters, such as vvIBDV-related strains, may be identified by analysis of both genome segments), however some potentially reassortant viruses have been identified (Lenouen et al., 2006). 15 3. MATERIALS AND METHODS 3.1.Study area The study was conducted in and around Bahir Dar, which include Bahir Dar city, Gombat, Andassa, Meshenty and Wonjeta in Northwest of Ethiopia. The day old chickens were highly distributed by government to the small holder farmers, semi- intensive and intensive farms. Bahir Dar district is located in the Northwest part of Ethiopia at about 578 kms from the capital city, Addis Ababa. Bahir Dar town has an altitude range of 1730 - 2300 meter above sea level. The district has mid-altitude agro- climatic zone, with an annual rainfall of 800 to 1250 mm. (BoAAO, 2003). Except Andassa, Gombat, Wonjeta and Meshenty have the same climatically condition as Bahir Dar. Andassa is located 17 km away from Bahir Dar town in southern direction. It located 110 29' N latitude and 370 29' E longitudes with an elevation of 1730 m.a.s.l. It receives average annual rainfall of 1150mm with the mean annual temperature varying from a maximum of 29.5oC in March to a minimum of 8.8oC in January. 3.2.Study animals Exotic and indigenous breeds of chickens within 3- 9 weeks of age, which were owned by farmers, semi-intensive and intensive farm owners considered and then stratified on the basis of age, sex, and breed. Following, the study samples were drawn by systemic random method from each stratified group. The age of the chickens was categorized in weeks, which ranges between 3- 9week old (Abdu, 1986; Abdu et al., 1987). 3.3. Study design Semi-structured questionnaire was prepared (Annex A) and administered to small chicken holder farmers, semi-intensive and intensive farm owners which were selected every fifth by systematic random sampling method. The selected respondents were 16 interviewed about their chickens, vaccination history, diseases occurrence and mortality proportion in early age of chickens. A cross sectional study was implemented to assess pathological changes and seroprevalence of IBD with its associated risk factors in study flocks in and around Bahir Dar. The sample size was according to the formula described by Thrusfield, 2004. n= 1.962 p (1-p) d2 Where, n = sample size of the study population D = desired precision p = previous/expected prevalence in the study area 29.4% CI = confidence interval (95%) Accordingly, a total of 320 chickens (from backyard, semi-intensive and intensive production systems) were considered. 3.4. Study methodology and data collection 3.4.1. Data collected History of vaccination, clinical signs, pathological changes from affected tissues, serological results and questionnaire survey data from backyard and intensive chickens were recorded. 3.4.2. Blood sample collection, serum separation and IELISA test Approximately 1.5 to 2 ml of blood was collected from the humeral region of the wing vein with a 3-ml syringe. The syringe was laid nearly horizontally until the blood clotted. The sample was kept at 37 °C for 24 hours or left before the serum was separated. After clotting at laboratory, the syringe was returned to a vertical inverted position to facilitate the serum to ooze out. The separated serum was transferred to cryovials, labeled, and 17 stored at –20 °C until the indirect enzyme-linked immunosorbent assay (IELISA) was performed to detect antibodies against the IBD virus (Onunkwo and, Okoye, 1991; Smith, 1992) at Amhara Regional Animal Health and Diagnostic Laboratory Center. IELISA test was valid if the mean of optical density (OD) of the positive control is greater than 0.25(ODpc > 0.025). The ratio of the mean of the OD of positive and negative controls (ODPC and ODNC) is greater than 3, so if the calculated S/P ratio is > 0.3, the serum taken as positive to IBD and ≤ 0.3 considered as free of IBD. 3.4.3. Clinical examination and Postmortem findings The clinical signs observed and histories were recorded, clinically suspected chickens from vaccinated and none vaccinated flocks were opened with standard post mortem procedure. The observed the gross pathological changes in the bursa of fabricius, spleen, kidney, thymus, pectoral and thigh muscle and the junction of proventriculus and gizzard muscle were recorded (annex II). Representative sample (0.5cm-1cm) from each tissue was collected and fixed with the10% neutral buffered formalin for histopathology processing (Philip et al, 2014). 3.4.4. Histopathology procedures For histopathological examination, gross lesions in affected tissues (bursa, thymus, the junction of proventriculus and gizzard, spleen and kidney) were trimmed to the thickness of 5 mm up to 1cm in size and fixed with the 10% buffered formalin. Following, tissues samples were subjected to different steps of fixative, it was dehydrated in a series of alcohol concentrations (70%, 80%, 100%, 100%, 100%), cleared by zylene I and II. Then, it was impregnated in paraffin wax at temperature of 60-62 0C and embedded by molten wax. The embedded tissues were sectioned by rotary microtome with thickness of 5 micrometer (Luna, 1968) at the Pathology unit of the National Animal Health diagnostic and investigation Center (NAHDC). The sectioned ribbon were added to floating bath and picked it up by slide and labeled. The labeled slide was stained with Meyer’s hematoxylin and eosin dye and after stained, it was mounted with Canada 18 balsam. The microscopic changes were examined by 10x 40x objectives and the major histopathological were recorded. Tissues were histopathologically evaluated on the basis of the extent of necrosis and degeneration of follicular lymphocytes, the presence of follicle and muscle wall edema, hyperemia, and heterophilic infiltration (Bennoune, 2011). Cloacal bursa and other tissue microscopically lesions was scored based on the following system: 1 = normal, no IBDV related lesions; 2 = mild, scattered IBDV related cell depletion in a few follicles; 3 = moderate, 1/2 of the follicles with IBDV related atrophy or depletion of follicular lymphocytes; and 4 = acute / sever, totally depletion of lymphoid cells and disappearance of bursa follicle architecture (Hair-Bejo et al., 2004). Spleen was assessed for increased germinal center formation, as well as, lymphoid depletion in the germinal centers and around the periellipsoid and periarteriolar lymphoid sheath (Elankumaran et al., 2002). 3.5.Data management and analysis Data entry and management was done by Microsoft excel window 2007, while statistical analysis was done by SAS university edition version 9.4. Descriptive statistics like frequency, 2 x 2 tables, chi – square (χ2) and fishery exact test were used to compare the distribution and score of lesion in affected tissues, seroprevalence of IBD, across age and breeds. Logistic regression was used to test association of prevalence of IBD with age, breed and management factor in backyard and intensive farm chickens. 19 4. RESULTS 4.1. Questionnaire survey There were five chicken breeds in the study area. These are local; Koekoek, Bovans white, Bovans brown and hybrid (Table 2). The Koekoek, Bovans brown, and Bovans white breeds were introduced to backyard semi-intensive and intensive from Andassa, Gerado and Ethio-chickens multiplication center without immunization against IBD. Among, the interviewed 100 farmers, semi-intensive and intensive owners, 100% of them responded that there was high chicken mortality in their flocks. Most of the respondent (77%) didn’t have the information as to the presence of vaccine for infectious bursal disease and they did not use it at all for their flock. However, only 23 % the flock owners use IBD vaccine for their flock (Table 3). High death rate was observed in Bovans brown, the lowest death occurrence in local breeds (Figure 1). The respondents emphasize that high death of growing chickens occurred in month of September to November and June to August (Figure 2). Table 2: Distribution of chicken breeds used for the study in and around Bahir Dar Breeds Percent (%) Bovans brown 16 Bovans white 15 Koekoek 22 Local 28 Hybrid 19 Total 100% 20 Table 3: IBD vaccine utilization per farming system in & around Bahir Dar. Farm types Use of IBD Vaccine Total No Yes Intensive 28.57% (4/14) 71.43% (10/14) 14 Semi- intensive 46.67% (7/15) 53.33% (8/15) 15 Backyard 92.96%( 66/71) 7.04 % (5/71) 71 Total 77.00 (74/100) 23.00 (26/100) 100 X2 =51.61; P = 0.000 nodeath = number of death. Figure 1: The death rate of the different breeds ranked by the owners in & around Bahir Dar. 21 Seasonmrt = season of mortality; nodeath = number of death Figure 2: The frequency of death in different breeds at different months of the year as responded by the owners in and around Bahir Dar. 4.2.Clinical manifestation In the study flock, prominent clinical sign in IBD infected chicken were recorded. Among which, tired, clumping together, recumbent on the break, whitish diarrhea mixed with blood, emaciated, ruffled feather, dropping of the wing, anorexia, depression and massive death within short period of time (Figure 1: A, B, C and D). 4.3.Characterization of gross pathological lesions Postmortem finding revealed that the affected chicken become dehydrated and emaciated. Congestion of subcutaneous blood vessels was observed in all part of the affected chickens (Annex 3: figure E). Petichial hemorrhage and congestion were observed on the pectoral and thigh muscles (Annex 3: figure D). Among clinically active cases, 76.67% of Bursa of fabricius was found as swollen, edematous and petichial hemorrhage in 22 serosa and mucosal part (Figure E and F). It also became atrophied, whitish-creamy as the course of the disease progressed (6.66%) and 16.67% of it filled with gelatinous exudates on its serosa (Figure 2: E and F). The spleen becomes swollen, dark red in color and petichial hemorrhagic (Figure 2: E). Petichial hemorrhage and swollen was observed on thymus gland (Figure 2: D). A caudal part of kidney became swollen; petichial hemorrhagic and pale, ureter was turgid in a numbers of cases (Figure 2: A and B). Ecchymotic hemorrhage was also observed on the junction of proventriculus and gizzard (Figure 2: C). Figure 3: Clinical signs observed during active case of IBD in 28 – 52 day old chickens in and around Bahir Dar. B D C A 23 Figure 4: Gross pathological changes in different organ of 21-54 day old chicken infected by IBDV in and around Bahir Dar. B A E C D F 24 4.4. Histopathological changes The major microscopic change in the bursa of fabricius was depletion of lymphocye in follicles, accompanied by interfollicular hemorrhage, congestion, infiltration of heterophils, edema, karyorrhetic nucli of lymphocyte, formation of cortical rim, and follicular cyst (Figure 6, 7 and 8). The lesion score in bursa of IBD infected from vaccinated and unvaccinated ranges from mild to severe depletion of lymphoid cells (Table 4). The tubules of kidney became congested with fibrinous exudates and necrotized cuboidal epithelial cells; hemorrhage within and between tubules. Hemorrhages, congestion, depletion of lymphoid cells and infiltration of heterophils have been notice as the prominent changes in the spleen (Figure 8). Hemorrhage, congestion and infiltration of heterophils observed as the main microscopic changes in the proventriculus (Figure 11). A histopathological change in thymus was total depletion of lymphocytes cells in follicles and formation of tissue cords (Annex 3). Figure 5: Moderate lymphocyte depletion in follicles (arrow), formation of cortical rim (double arrow) in IBD infected bursa of chicken. HE x10. 25 Figure 6: Bursa with intrafollicular edema (yellow arrow), interfollicular hemorrhage (red arrow), congestion, depleted lymphoid cell with pyknotic nuclei (black arrow) and interfollicular fibrosis (double arrow). HE X10. 26 Figure 7: Bursa with marked lymphoid depletion and necrosis, interfollicular hemorrhage (single arrow) and follicular cyst (double arrow). HE X40. Table 4: Lesion score in IBD infected Bursa in vaccinated and unvaccinated chickens Vaccine history Lesion score Sever Moderate Mild Total Unvaccinated 10 (62.50%) 2 (12.50%) 4 (25.00%) 16 (100%) Vaccinated 4 (28.57%) 5 (35.71%) 5 (35.71%) 14 (100%) Total 14 (46.67%) 7 (23.33%) 9 (30.00) 30 (100%) Fisher exact test = 0.01820 (p= 0.1856) there was no lesion score difference between the lesion score in vaccinated and unvaccinated IBD infected chicken’s bursa. 27 Figure 8: The tubules of kidney became congested with purulent exudates; hemorrhage within and between tubules (arrows). HE x10. Figure 9: The tubules of kidney became congested with fibrinous exudates that contain necrotized cuboidal cell; hemorrhage within and between tubules. HE x40. 28 Figure 10: The depletion of lymphocyte in the germinal center of the spleen (arrow), infiltration of heterophils and hemorrhage in red pulp area (head arrow). HE x10. Figure 11: congestion (double head arrow: A) and hemorrhage (B), infiltration of heterophils on the junction of proventriculus and gizzard (B). HE x10. A B 29 4.5. Seroprevalence of IBD Virus A total of 320 sera were collected from backyard, semi- intensive and intensive farm and tested by indirect ELISA and results in over all prevalence of 51.56 % (165/320) IBD in the investigation area (Table 5). Andassa has the highest prevalence of 72.73% (48/66) IBD among sub-district while Bahir Dar experienced comparatively with low prevalence of 42.42% (Table 6). The Koekoek breeds have more susceptible to IBD with prevalence 67.11%, (51/76) than the Bovans white, Bovans brown, local breed and Hybrid breeds (Table 6). The prevalence of IBD in sex wise was proportional with 51.63% (127/246) and 51.35 % (38/74) in female and male chickens respectively (Table 6). High IBD was observed in the age range of 3-6 week old chickens, but it was lower prevalence in age group of 7 – 9 weeks old chickens (Table 6). This disease has different prevalence magnitude among farm systems. It was 57.14 % in backyard farm system, 34.69% in semi-intensive chickens farm and 21.05% in intensive farm system (Table 6). The seroprevalence of the IBDV in study area was influenced by a number of risk factors which include age of chicken, farm type, study site and breeds of chickens (Table 7). The odds of IBDV infection was 15.4% lower in intensive farm system than that of backyard production system and odds of it infection was significantly associated with farming type (OR =0.1543; P =0. 0032). The odds of IDBV infection in Bahir Dar was 16.96% lower than that of Andassa and odds of it infection was significantly associated with study site (OR = 0.1696; P = 0.0001). Table 5: The overall seroprevalence of IBDV by IELISA in and around Bahir Dar. Result Serological test / IELISA Test (N=320) Number positive Prevalence (%) Confidence interval (95 %) 165 51.56 45.95 – 57.14 30 Table 6: Seroprevalence of IBD in different study sites, management systems, sex and breed in around Bahir Dar. Variable Seroprevalence (%) X2 (p-value) Farm type Backyard Intensive Semi-intensive 57.14 (144/252) 21.05 (4/19) 34.69 (17/49) 15.8061 (0.0004) Sex Female Male 51.63 (127/246) 51.35 (38/74) 0.0017 (.967) Breed type Koekoek Bovans brown Bovans white Hybrid Local breed 67.11 (51/76) 57.69 (30/52) 40.28 (29/72) 38.71 (12 /31) 48.31 (43/89) 14.23 (0.0066) Study sites Bahir Dar city Andassa Meshenty Wonjeta Gombat 42.42 (28/66) 72.73 (48/66) 44.44 (28/63) 50.00 (34/68) 45.00 (27/60) 20.0589 (.0033) Age (in weeks) 3 4 5 6 7 8 9 66.67 (50/75) 58.21 (39/67) 51.67 (31/60) 41.67 (30/72) 38.89(14/36) 16.67 (1/6) 0.00 (0/4) 20.3578 (.0024) 31 Table 7 : Association of risk factor with IBD by using multivariate logistic regression in and around Bahir Dar. Variables OR 95% CI for OR P Value Age (in weeks) 6 vs. 3 0.422 0.199 - 0.897 0.0497 7 vs. 3 0.333 0.124 - 0.893 0.0290 8 vs. 3 0.041 0.004 - 0.424 0.0504 Breeds HB/BB 0.347 0.120 - 1.001 0.0479 LB/ BB 0.431 0.187 - 0.992 0.0479 Study sites Bahir Dar vs. Andassa 0.169 0.071 - 0.399 0.0001 Gombat vs. Andassa 0.278 0.115 - 0.672 0.0045 Meshenty vs. Andassa 0.300 0.125 - 0.720 0.0070 Wonjeta vs..Andassa 0.274 0.117 - 0.645 0.0030 Farm types 0.28 Intensive vs. backyard 0.154 0.044 - 0.535 0.0032 Semi-intensive vs. backyard 0.409 0.17 3– 0.965 0.0413 32 5. DISCUSSION The present study clearly showed that IBD is one of the major diseases of chickens in and around Bahir Dar. This study was conducted to characterize the pathological changes, determine the seroprevalence of IBD and associated risk factors in and around Bahir Dar in exotic and local breeds of chickens maintained under different productions systems in areas like Bahir Dar city, Andassa, Wonjeta, Gombat, and Meshenty. Clinical signs, gross and histopathological changes as well as seroprevalence of IBD was assessed. Among, the interviewed 100 farmers, semi-intensive and intensive owners, 100% of them responded that there was high chicken mortality in their flocks. Most of the respondent (77%) didn’t have the information as to the presence of vaccine for IBD and they did not use it at all for their flock. However, only 23 % the flock owners use IBD vaccine for their flock. This high mortality of growing chickens might be related with the absence of scheduled vaccination in the study area (Nunoya et al., 1992). The respondents emphasize that high death of growing chickens occurred in month of September to November and June to august. This might be related to time of high number of day old chickens distributed by government without immunization against major chicken diseases. In addition to that, the season of market in investigation area in which chicken transported with life threaten infectious disease like IBD might increase death of young chicken (Rosenberger and Cloud, 1986). The clinical signs like depression, ruffled feather, anorexia, white diarrhea tinged with blood, massive death, emaciation, dropping of the wing were among the major manifestations. These clinical manifestations agreed with findings of (Zeleke et al, 2005; Shekaro and Josiah, 2015) but clumping of the infected chicken was not noticed by these investigators even if this was the prominent manifestation in this study. This may be due to the difference in study breeds of the chickens and season of the investigation period (Kegne and Chanie, 2014). The gross pathological findings observed in this study include hemorrhagic, edematous, enlarged and gelatinous lesions in bursa of fabricius, congestion of subcutaneous blood 33 vessels in all parts of the affected chickens, petichial hemorrhage and congestion on the pectoral and thigh muscles. These changes were similarly reported by previous researchers (Zeleke et al, 2005; Mazengia et al, 2009 and Rozina et al, 2014). However, none of the investigators did not reveal the gross pathological changes in spleen and thymus gland. In our case, swollen and petichial hemorrhagic lesions were common in spleen and thymus. A caudal part of kidney became swollen; petichial hemorrhagic, pale and ureter was turgid with urate in a numbers of cases. Petichial and ecchymotic hemorrhage was also observed on the junction of proventriculus and gizzard which were agreed with the previous finding of (Islam and Samad, 2004; Babiker et al, 2008; Kegne and Chanie, 2014). Depletion of lymphoid cells, formation of cortical rim, intrafollicular and interfollicular hemorrhage, edema, and formation of cystic follicles were the major microscopic findings in IBD infected bursa of chickens (Figures 6, 7 and 8). These histopathological findings in bursa were related with the studies of (Henry et al., 1980; Skeeles et al., 1980; Chowdhury et al., 2015). The microscopic evidence of IBD infected chicken’s bursa revealed that different lesion score and have been grouped according to study of (Hair- Bejo et al., 2004) which were taken from outbreaks of vaccinated flock as well as from none vaccinated flock. These lesion scores were classified as sever, moderate and mild. Sever lesion score consists of 62.50% (10/16) from none vaccinated flock and 28.57% (4/14) from vaccinated flock. This lesion score was characterized by total depletion of lymphoid cell in the follicle, formation of cystic follicles and disruption of follicular architecture with fibrosis. The moderate lesion score in microscopic lesion of bursa comprised 25.00% (4/16) from none vaccinated and 35.71% (5/14) have observed in vaccinated flock’s bursa. The moderate lesion score designated by 3 and characterized by depletion of 1/3-1/2 lymphocyte cell in the bursa follicles, interfollicular and intrafollicular hemorrhage, infiltration of heterophils between follicles, prominent edema and formation of cortical rim. The mild lesion score in IBD infected bursa comprised 12.50% (2/16) and 35.71% (5/14) from none vaccinated and vaccinated flock respectively. The microscopic feature in the mild lesion scored bursa was edema and scattered lymphocyte depletion has been observed in the follicles. There was no 34 significant difference among lesion score (P = 0.1856) in bursa of none vaccinated and vaccinated flock which were collected from outbreaks of IBD in both flocks. This magnificent lesion score in IBD infected bursa of chicken both in vaccinated and none vaccinated flock indicated that either the vaccine was not properly given or it may be transported and stored in poor conditions. The other reason is that, virus strain that causes outbreak in the flock may super pass the protective level of the given vaccine (Onunkwo and Okoye, 1991; Gary and Butcher, 2013). The spleen in IBD infected chicken was infiltrated with heterophils, hemorrhage was observed in arteriole, the periarteriolar lymphoid sheath (PALS), and depletion of lymphocytes in germinal centere (Figure 8). This histological finding in IBD infected chickens spleen was related with study of (Hair-Bejo et al., 2004; Gary and Butcher, 2013). While, the histopathological change in thymus was total depletion of lymphocytes cells in follicles and formation of tissue cords. The study conducted by (Mohammad et al, 2012) also revealed that thymus was found devoid of lymphocyte cell in its follicles following naturally IBD infected broiler chickens which support the present study. The tubules of kidney were filled with fibrinous exudates; hemorrhage between and within tubules. The microscopic changes include nephritis, detached and necrotized cuboidal epithelial cells in the tubules and eosinophilic cast in renal ductus, and infiltration of heterophils adjacent to kidney tubules. These microscopic changes in kidney were not specific to IBD infection, but it is a responsive reaction to acute inflammation and dehydration due to diarrhea and hemorrhage. Study conducted in Nigeria by (Oliwayelu et al, 2002) encountered similar histological changes in kidney with the present findings. Microscopic changes in the junction of proventriculus and the gizzard, includes congestion, hemorrhage and infiltration of hetrophils at the junction of two organs. These microscopic changes only observed in early clinical stages of IBD as the disease progressed, it subsides quickly and could not notified in later stage of the disease (Chowdhury et al., 2015). The current seroprevalence findings of IBD in unvaccinated backyard, semi-intensive and intensive chicken flocks result in the overall prevalence of 51.56% in and around Bahir Dar. This result indicated that IBD is epidemic chicken threatening disease in and around 35 Bahir Dar. This prevalence was higher that the study of (Mazengia et al, 2009) in Bahir Dar and Farta district with the prevalence of 29.04% and 21.70%, respectively. The seroprevalence of IBD from this study (51.56%) was higher than the findings of Reta (2008), who reported a prevalence of 39.2% in unvaccinated backyard chickens in East Shoa zone using AGID (Agar gel immuno-diffusion) test. Kassa and Molla (2012) reported the overall prevalence of 73.5% of backyard chickens in northern Gonder and west Gojjam which was higher than the current prevalence report. Study conducted at Andassa poultry multiplication center by Woldemariam and Wossene (2007) reported a seroprevalence of 100% which was much higher than current prevalence in Bahir Dar and its surrounding areas. Zeryehun and Fekadu (2012) investigated the seroprevalence of IBD in chickens managed under backyard production system in central Oromia and reported that the overall prevalence to be 82.2%, which was higher than the present seroprevalence in and around Bahir Dar. This may be related with intensification of poultry farm in central Oromia which may facilitate more contamination to backyard chicken than less intensification in and around Bahir Dar. Retrospective study of IDB in Nigeria at Zuria by (Mbuk et al, 2010) reported the overall prevalence of 7.26% in broiler chickens. This prevalence was much lower than the current study in and around Bahir Dar and the previous IBD seroprevalence reports from Ethiopian. There was no statistically significant difference (x2= 0.0017; P= 0.967) between the seroprevalence of IBD in female 51.63% (127/246) and male 51.35 % (38/74). In Early age, female and male chickens received the same maternal immunity level and thus female and male chickens might have equal resistance level to IBDV (Hassan and Saif, 1996). The other reason could that female and male chickens in the study area share common house, and feed trough both in intensive, semi-intensive and backyard production systems. This facilitates similar exposure to IBDV to male and female chickens, which agree with the prevalence reported by (Zeryehun and Fekadu, 2012) from central Oromia. 36 The seroprevalence of IBD was significantly associated with study area (x2 = 15.77, P= 0.0033). Andassa was highly exposed area with IBDV than Wonjeta, Gombat, Meshenty and Bahir Dar with the prevalence of 72.73% (Table 6). Chicken multiplication and distributor center have been established around Andassa town. This multiplication center, imported different exotic chicken breeds (Koekoek, White leg horn, Bovans white etc) from abroad for multiplication purpose. After multiplication these imported chickens, the center redistribute the day old chickens to farmers and grouped youths without immunizing against IBD. The absence of programmed vaccination against IBDV in exotic breeds of chickens in the study area resulted in high seroprevalence of IBD compared to others. The odds of IBD infection in Andassa was 0.1696% times higher than that of Bahir Dar and the odds of IBD infection was significantly associated with location (OR= 0.1696; P= 0001). The odds of IBD infection was 0.2783% times lower in Gombat than that of Andassa chickens and the odds of IBD infection was significantly associated with location (OR=0.2783; P= 0.0045). Seroprevalence between the sub-districts, 0.2783% times prevalent than Gombat, 0.3005% times prevalent than Meshenty, and 0.2749% times more prevalent than Wonjeta (Table 6). Bahir Dar has the least seroprevalence of 42.42 % among these study areas; since more than half of the intensive farms owners in Bahir Dar vaccinated their flocks against IBDV, which lowers the seroprevalence of IBD compared to Andassa, Meshenty, Gombat, and Wonjeta. This result agrees with study of Zeryehun and Fekadu (2012) from central Oromia with different seroprevalence in their study sub-districts. For instance, Swaia, et al., (2011) reported statistically significant IBD prevalence which varies from 37.5 % to 91 % between districts and from 75 % to 90 % between regions in northern Tanzania free ranging chickens. The seroprevalence of IBD was significantly associated with the age of chickens (x2 = 20.3578; P= 0.0024). As the chickens got older and older, the seroprevalence decreases significantly (Table 6). Higher seroprevalence of IBD in the current study chickens was observed from age ranging 3-6 weeks old. At this age range, bursa of fabricius attains its maximum growth and development with sufficient B lymphocyte for IBDV 37 multiplication which agrees with the findings of Zeryehun and Fekadu (2012) where age was considered as risk factor for IBDV infection in chickens. Bursa of fabricius begins to atrophy starting from seven weeks old and the number of B lymphocyte decrease gradually. As result of this, IBDV infectivity decrease as B lymphocyte decrease with age of the chickens (Rashid et al, 2013). The odds of IBD infection was 33.38% lower in 7 weeks aged chickens than that of 3 weeks aged chickens. The odd of IBD infection was significantly associated with age (7/3) of chicken (OR =0.3338; P = 0.0290). The odds of IBD infection is 4.14% lower in 8 weeks aged chicken than that of 3 weeks aged chickens. The odds of IBD infection was significantly associated with age (8/3) of chicken (OR =0.0414; P =0.0074). This odd implies that as the age of chicken increase beyond 6 weeks old, it become a protective factor for IBDV. The susceptibility of chickens to IBDV was significantly associated with breed (χ2 =14.23; P = 0066). The highest seroprevalence (67.11%) of IBD among the five breeds of chickens from the present study observed in Koekoek chicken might originated from the contaminated poultry multiplication center of Andassa. Bovans brown is the second most susceptible to IBDV with the prevalence of 57.69% as compared to the rest of the breeds. Local breed of chickens was also the third susceptible with the seroprevalence of 48.31% among the five breeds. This indicated that the indigenous breed of chickens was also exposed to IBDV and this agrees with the study of Mazengia (2009) in Bahir Dar and Farta and Zeryehun and Fekadu (2012) in central Oromia. Bovans white and hybrid breeds have almost similar seroprevalence, 40.28% and 38.71%, respectively. Management, bio-security and vaccination play the key role in chickens farming system to control infectious diseases. This study observed high IBD seroprevalence (57.14%) in backyard chickens. In backyard, there is poor bio-security, poor management and no vaccination (Table 3). There is also chance mobility of chickens from villages to market and vice-versa, all these reasons favors IBDV to be endemic problem in backyard production system (Kassa and Molla, 2012).. 38 In semi-intensive chicken production system, there was a relatively higher seroprevalence (34.69%) than that of backyard chicken farm (21.05%) but lower than intensive production system (Table 6). The odds of IBDV infection was 15.43% lower in intensive chicken’s farm system than that of backyard production system and odds of its infection was significantly associated with production system/ type of farming (OR = 0.1543; P = 0.0032: Table 7). The odds of IBD infection in semi-intensive production system was 0.4092 times lower than that of backyard production, so this study indicated that intensification of production system can act as a means of protective factor against IBDV (Negash, 2013). 39 6. CONCLUSION AND RECOMMENDATIONS This study concludes that IBD is epidemic chicken’s disease in and around Bahir Dar as confirmed by pathological and serological findings. Prominent Gross and histopathological changes were observed in bursa of fabricius, spleen, kidney, thymus, thigh and pectoral muscles and at junction of proventriculus and gizzard. High seroprevalence of IBD virus was found to circulate in various study sites in and around Bahir Dar. The IBDV infection in study area was influenced by age of the chicken, breed, and study site and farming system. It was more prevalent in 3 - 6 week old chickens, backyard production system, unimmunized flock, and in the breeds of Koekoek and Bovans brown. This high prevalence of IBD reduces the income of producer from chicken in and around Bahir Dar. In line with the above concluding remarks, the following recommendations are forwarded:  Management factors like, scheduled vaccine program in backyard, proper bio- security in semi-intensive and intensive farm should be implemented to reduce the magnitude of IBDV infection in investigation area.  The regional government should set immunization schedules in chicken before and after distribute day old chickens to backyard, semi- intensive and intensive producers.  Molecular diagnostic study should be conducted to identify the current circulating strain of IBDV in the chicken population.  The current vaccine efficacy and the circulated IBDV strain should be investigated. 40 7. REFERENCES Abdu PA, Abdullahi US, and Adesiyun AA, Ezeokoli C.D. (1987): A review: Infectious bursal disease. Zariya Veterinarian, 2(1):58-60. Abdu P. A. (1986): Infectious bursal disease immunization failures in chicken in Nigeria Tropical Animal Health and Production, 18:123-125. Alemu .Y, and Tadelle D. (1997): The status of poultry research and development. Research Bulletin No 4. Poultry Commodity program, Debre zeit Agricultural Research Center, Alemaya University of Agriculture. Alamargot J. (1987): Avian pathology of industrial poultry farms in Ethiopia. Proceeding of first National Livestock Improvement Conference (NLIC), Addis Ababa, Ethiopia, pp. 114-117. Allan G.M., McNulty M.S., Connor T.J., McCracken R.M. and McFerran J.B , (1984): Rapid diagnosis of infectious bursal disease infection by immunofluorescence on clinical material. Avian Pathol, 13:419-427. Al-Natour, M.Q., Ward, M.L.A., Saif, Y.M., Stewart-Brown, B., Keck, L.D.(2004); Effect of different levels of maternally derived antibodies on protection against infectious bursal disease virus. Avian Dis. 48, 177–182. Ashenafi H (2000): Survey of Identification of Major Diseases of local chickens in three- selected agro climatic zones in central Ethiopia. DVM thesis, Addis Ababa University, Faculty of Veterinary Medicine, Debre Zeit, Ethiopia, pp. 1 -60. Babiker .M, Yahia.E, Nora.K, (2008): Investigations on Nine Flocks Infected with Infectious Bursal Disease Virus (IBDV) in Khartoum State, Sudan. International Joun poultry sciences, 7:285 – 288. Bennoune, O. (2011): The effect of Bursa of Fabricius on the bacterial activity of heterophils in broiler chicken. Thesis for graduation PhD, University of Batna, Algeria. Benton, W.J., Cover, M.S., Rosenberger, J.K. (1967): Studies on the transmission of the infectious bursal agent (IBA) of chickens. Avian Dis. 11:430–438. 41 Brown, M.D., P. Green and M.A. Skinner. (1994); Sequences of recent European “very virulent” isolates of infectious bursal virus are closely related to each other but are distinct from those of classical” strains. Journal of General Virology 75: 675-68. Bumstead N., Reece R.L. and Cook J.K.A. (1993). – Genetic differences in susceptibility of chicken lines to infection with infectious bursal disease vims. Poult. Sci., 72 (3), 403-410. Bureau of Agriculture and Rural Development of Amhara Regional State (BoARD) Report 2003 Annual Report, Pp. 5-7. Burkhardt E. and Müller H. (1987): Susceptibility of chicken blood lymphoblasts and monocytes to IBDV. Arch. Virol., 94:297-303. Chettle, N., J. C. Stuart H.H U, P. J. Wyeth. (1989): Outbreak of virulent infectious bursal disease in East Anglia.Vet Rec 125: 271-272. Cho B.R., Snyder D.B., Lana D.P. and Marquardt W.W. (1987): Infectious bursal disease: Rapid diagnoses by immunoperoxydase monoclonal antibody stain. In Proc. 36th Western Poultry Disease Conference, 3-5 March, Davis, California. University of California, Davis, 112. Chowdhury. H., Islam R., and Dawan. T. (2015): Acute infectious bursal disease in checken: Pathology observation and virus isolation. Bangladish University, college of Agriculture, 4-5. Chou, K.C. and Calnek, B.W. (1997): Diseases of poultry, Iowa state university press. 10th Ed. Ames, Iowa, 721-733. COSGROVE, A.S. (1962): An apparently new disease of chickens: avian necrosis. Avian Diseases, 6: 385-389. CSA. (2004-2005): report on livestock and livestock characteristics, production constraints survey, Ethiopia; Pp, 12 -16. CSA. (2013): report on livestock and livestock characteristics, agricultural sample survey (3): 13 -20. Dewit J.J. (1999): Gumboro disease: optimizing vaccination. Int. Poult. Prod., 7 (5), 19- 21. 42 Elankumaran, S., R. A. Heckert, and L. Moura. (2002): Pathogenesis and tissue distribution of a variant strain of infectious bursal disease virus in commercial broiler chickens. Avian Diseases 46:169-176. Eterradossi N., Arnauld C., Tekaia F., Toquin D., LE Coq ., Rivallan G., Guitet M., Domenech J., Vanden Berg T.P. and Skinner M.A. (1999): Antigenic and genetic relationships between European very virulent infectious bursal disease viruses and an early West African isolate. Avian Pathol, 28, 36–46. Faragher J.T. (1972): Infectious bursal disease of chicken. Vet. Bull, 42: 361-369. Fauquet, C.M., Mayo, M.A., Manil of f, J.,Desselb erger, U., Ball, L.A. (2005); Virus Taxonomy. Classification and Nomenclature of Viruses, 8th ICTV Report. Academic Press, Elsevier, San Diego, pp. 56 6–567. Firth G.A. (1974): Occurrence of an infectious bursal syndrome within an Australian poultry flock. Aust. vet. J., 50:128-130. Gardner H., Kerry K., Riddle M., Brouwer S. and Gleeson L. (1997). - Poultry virus infection in Antarctic penguins. Nature, 15: 245. Gary D. Butcher .D. (2013): Infectious Bursal Disease (Gumboro) in Commercial Broilers. University of Florida. Giambrone J.J. (1979): Effect of early infectious bursal disease virus in immunity to Newcastle disease in adult chickens. Poult. Sci., 58:794-798. Giambrone J.J. Eidson C.S., Page R.K., Fletcher O.J., Barger B.O. and Kleven S.H. (1976): Effect of early infectious bursal disease agent on the response of chicken to Newcastle disease and Mareks disease vaccination. Avian Dis., 20, 534-544. Guittet M., Picault J.P. and Bennejean G. (1982): Maladie de Gumboro ,immunité maternelle transmise aux poussins issus de reproducteurs vaccinés. Dev. biol. Standard., 51 , 221-233 . Hailemariam Teklewold, Legesse Dadi, Alemu Yami and Negusse Dana. (2006): Adopting poultry breeds in the highlands of Ethiopia. Ethiopian Institute of Agricultural Research. Pp, 26. Hassan M.K. and Saif Y.M. (1996): Influence of the host system on the pathogenicity, immunogenicity, and antigenicity of infectious bursal disease virus. Avian Dis., 40:553-561. 43 Hair - Bejo, M., M.K. Ng and H.Y. Ng, (2004); Day old vaccination against infectious bursal disease in Broiler chickens. Int. J. Poult. Sci. Asian Network for Scientific Information, 3: 124-128. Henry C.W., Brewer R.N., Edgar S.A. and Gray B.W. (1980): Studies on infectious bursal disease of chickens: 2 – scoring microscopic lesions in the bursa of Fabricius, thymus, spleen and kidney in gnotobiotic and battery reared white Leghorns experimentally infected with infectious bursal disease virus. Poult. Sci., 59, 1006-1017. Hiraga M., Nunoya T., Otaki Y., Tajima M., Saito T. and Nakamura T. (1994): Pathogenesis of highly virulent infectious bursal disease virus infection in intact and bursectomized chickens. J.Vet. Med. Sci., 56: 1057-1063. Hirai K„ Funakoshi T., Nakai T. and Shimakura S. (1981): Sequential changes in the number of surface immunoglobulin-bearing B lymphocytes in infectious bursal disease virus-infected chickens. Avian Dis., 25 (2), 484-496. Hitchner S.B. (1970): Infectivity of infectious bursal disease virus for embryonating eggs. Poult. Sci., 49: 511-516. Inoue M., Fujita A. and Maeda K. (1999): Lysis of myelocytes in chickens infected with infectious bursal disease virus. Vet Pathol, 3 6 (2), 146-151. Inoue M., Fukuda M. and Miyano K. (1994): Thymic lesions in chicken infected with infectious bursal disease virus. Avian Dis., 3 8 (4), 839-846. Islam, M.R., Das, B.C., Hossain, K., Lucky, N., Mostafa, M.G. (2003): A study on the occurrence of poultry diseases in Sylhet Region of Bangladesh. Int. J. Poult. Sci. (2): 354–356. Islam and Samad. (2004): Clinic- pathological studies on natural and experimental infectious bursal disease in broiler chickens. Bangl. J. Vet. Med, (2): 31-35. Jackwood D.J. Jackwood R.J. (1997): Molecular identification of infectious bursal disease virus strains.Avian Dis., 41: 97–104. Jackwood D.J. and Saif Y.M. (1987): Antigenic diversity of infectious bursal disease viruses. Avian Dis., 31:766-770. Kegne.T and Chanie. M, (2014): Review on the incidence and pathology of infectious bursal disease. Birtish Joun, of poultry sciences, 3: 5-6. 44 Jones B.A.H. (1986): Infectious bursal disease serology in New Zealand poultry flocks. N.Z. Vet. . , 34, 36. Kassaa S.A and Molla W. (2012): Seroprevalence of infectious bursal disease in backyard chickens of North West Ethiopia. Scientific Journal of Crop Science 1(1) 20-25. Kibeng.F.B, Dhillon.A.S, Russell.R.G. (1988): The growth of serotype I and II and variant strains of IBD virus in vevo cells. Avian Dis, 32: 298-303. Kouwenhoven B. and van den Bos J. (1994): Control of very virulent infectious bursal disease (Gumboro disease) in the Netherlands with more virulent vaccines. In Proc. First International Symposium on infectious bursal disease and chicken infectious anaemia, 21-24 June, Rauischholzhausen (E. Kaleta, Ed.). World Veterinary Poultry Association, Giessen, 262-271. Lasher H.N. and Davis V.S. (1997): History of infectious bursal disease in the USA. The first two decades. Avian Dis., 41: 11-19 Lasher H.N. and Shane S.M. (1994): Infectious bursal disease. World Poult. Sci. J., 50: 133-166. Ley D.H., Storm N., Bickford A.A. and Yumamoto R. (1979): An infectious bursal disease virus outbreak in 14- to 15-week-old chickens. Avian Dis., 23: 235-240. Le nouen C., Rivallan G., Toquin D., Darlu P., Morin Y., Beven V., De Boisseson C., Cazaban C., Gardin Y. and Eterradossi N. (2006): Very virulent infectious bursal disease virus: reduced pathogenicity in a rare natural segment B-reassorted isolate. J. Gen. Virol., 87, 209–216 Lim B.L., Cao Y., YU T. and MO C.W. (1999): Adaptation of very virulent infectious bursal disease virus to chicken embryonic fibroblasts by site-directed mutagenesis of residues 279 and 284. J. Virol., 73, 2854–2862. Lin Z., Kato A., Otaki Y., Nakamurat T., Sasmaz E. and Ueda S. (1993): Sequence comparisons of a highly virulent infectious bursal disease virus prevalent in Japan. Avian Dis., 37, 315–323. Lucio B. and Hitchner S.B. (1980): Immunosuppression and active response induced by infectious bursal disease virus in chickens with passive antibody. Avian Dis., 24 : 189-196. 45 Luna, L., (1968): Manuel of Histology, Staining methods of armed forces, Institute of pathology. 3rd Ed., McGroaw-Hill Book., Co., New York, pp, 43. Lukert P.D. and Saif Y.M. (1997): Infectious bursal disease. In Diseases of poultry, 10th Ed. (B.W. Calnek with H.J. Barnes, C.W. Beard, L.R. McDougald and Y.M. Saif, eds).Iowa State University Press, Ames, 721-738 . McFerran J.B. (1993): Infectious bursal disease. In Vims infections of birds (J.B. McFerran and M.S. McNulty, Eds), Elsevier Science, Amsterdam, 213-228. Mazengia H. (2012): Review on major viral diseases of chickens reported in Ethiopia. Journal of Infectious Diseases and Immunity, 4(1): 1 - 9. Mazengia H. (2010): prevalence study of infectious bursal disease in bahirdar and farta in local chickens. Journ. of poultry, pp 12-13. Mazengia H, Bekele ST, Negash T. (2009): Incidence of infectious bursal disease in village chickens in two districts of Amhara Region, Northwest Ethiopia. J. Livest. Res. Rural Dev., 21: 12. Mbuk.J, Musal.W,Ibrahim. S, and Sa’idu.L. (2010): A Retrospective Analysis of Infectious Bursal Disease Diagnosed at Poultry Unit of Ahmadu Bello University, Nigeria. Int. Jour. Poultry Science 9 (8): 784-790. Meulemans G., Antoine O. and Halen P. (1977): Application del'immunofluorescence au diagnostic de la maladie de Gumboro. Bull. Off. int. Epiz., 88, 225-229. Mohammad. M, Islam. M, Basu. J and and Zahirul. M (2012): Distribution and quantification of lymphocytes in the major lymphoid Organs of naturally Gumboro infected Broilers. Int. J. Morphol., 30 (4):1585-1589. Müller, H., M. R. Islam u. R. Raue. (2003): Research on infectious bursal disease--the past, the present and the future,Pp, 13-17. Müller R., Käufer-Weiss I., Reinacher M. and Weiss E. (1979): Immunofluorescent studies of early virus propagation after oral infection with infectious bursal disease virus (IBDV). Zentralbl. Veterinär med., 26: 345-352. Muskett J . C , Hopkins I.G., Edwards K.R. and Thornton D.H. (1979): Comparison of two infectious bursal disease vaccine strains. Efficacy and potential hazards in susceptible and maternally immune birds. Vet. Rec., 104, 332-334. 46 Nakai T. and Hirai K. (1981): In vitro infection of fractionated chicken lymphocytes by infectious bursal disease virus. Avian Dis., 4, 831-838. Nunoya T., Otaki Y., Tajima M., Hiraga M. and Saito T. (1992): Occurrence of acute infectious bursal disease with high mortality in Japan and pathogenicity of field isolates in SPF chickens. Avian Dis., 36: 597-609. Onunkwo O, Okoye J. O. (1991): First report of an infectious bursal disease outbreak in a vaccinated chicken flock in Anambra State, Nigeria. Revue d’Élévage et de Medecine Veterinaire des Pays Tropicaux 44: 411–414. Ogawa M., Wakuda T., Yamaguchi T., Murata K.,Setiyono A., Fukushi H. and Hirai K. (1998): Seroprevalence of infectious bursal disease virus in free-living wild birds in Japan. J. vet med. Sci., 60 (11), 1277-1279. Okoye J.O.A. and Uzoukwu. M. (1981): An outbreak of infectious bursal disease amongst chickens between 1 6 and 20 weeks old. Avian Dis., 25: 1034-1038. Oluwayelu.D, Emikpe. B, Lkhaloa. J, (2002): The pathogenecity of infectious bursal disease in crossbreeds of Harco cocks and Hens in Nigeria. African Journal of clinical and experimental microbiology, 5:2-3. Owoade A.A. and Durojaiye. O.A. (1995): Infectious bursal disease in 14-week-old turkeys in Nigeria. Trop. anim. Hlth Prod., 27: 47-49. Petek, M., D’Aprile, P.N., Cancelloti, F. (1973): Biological and physic chemical properties of the infectious bursal disease virus (IBDV). Avian Pathol. 2: 135–152. Philip N. Nyaga, Lilly C. Bebora, Paul G. Mbuthia, Lucy W. Njagi and Peter K. Gathumbi. (2014): Diagnostic poultry post- mortem examination in avian medicine, Poultry workshop, 2-8. Pitcovski, J., B. Gutter, G. Gallili, M. Goldway, B.Perelman, G. Gross, S. Krispel, M. Barbakov and A. Michael. (2003): Development and large scale use of recombinant VP2 vaccine for the prevention of infectious bursal disease of chickens vaccine, 21: 36-43. Provost A., Borredon C. and Bocquet. P. (1972): The pathology of infectious bursal disease in growing chickens, Netherland Poultry farm. Rev. Elev. Méd. vét. Pays trop., 25 (3):347-356. 47 Rashid. H, Xue. C, Islam .R, Islam .T, and Cao.Y. (2013): A longitudinal study on the incidence of mortality of infectious diseases of commercial layer birds in Bangladesh. Preventive Veterinary Medicine 109: 354–358. Reddy S.K. and Silim A.N. (1991): Isolation of infectious bursal disease vims from turkeys with arthritic and respiratory symptoms in commercial farms in Quebec. Avian Dis., 35: 3-7. Reta. T. (2008): Seroprevalence study of infectious bursal disease in non vaccinated backyard chickens in agro ecological areas of East Shoa Zone. DVM Thesis, Addis Ababa University, Faculty of Veterinary Medicine, Deber Zeit, Ethiopia. Pp, 11. Rosenberger J.K. (1989): A laboratory manual for the isolation and identification of avian pathogens. American Association of Avian Pathologists, Kendall-Hunt, Dubuque, Iowa, 165-166. Rosenberger J.K. and Cloud S.S. (1986): Isolation and characterization of variant infectious bursal disease viruses. In Abstracts 123rd American veterinary Medical Association (AVMA) Meeting, 20-24 July, Atlanta, Georgia. AVMA, Schaumburg, Illinois, 181: 104. Rozina.M, Islam. N,Sogra. M, and Hossain.A. (2014): Pathogenicity and immunosuppressive properties of GM-97 strain of infectious bursal disease virus in commercial broiler chickens. J. Adv. Vet. Anim. Res., 1(1): 1-7. Sellers, H.S, Villegas, P.N, Seal, B.S, Jackwood. (1999): D.J. Antigenic and molecular characterization of three infectious bursal disease virus fields isolates. Avian Diseases, 43:.198-206. Sharma J.M., Dohms J.E. and Metz A.L. (1989): Comparative pathogenesis of serotype 1 and variant serotype 1 isolates of infectious bursal disease and their effect on humoral and cellular immune competence of SPF chickens. Avian Dis., 33:112-124 Sharma J.M., Karaca K. and Pertile T. (1994): Viruss-induced immunosuppression in chickens. Poult. Sci., 73, 82- 86. Shekaro. A and Josiah.E. (2015): Infectious Bursal Disease Outbreak in Fifteen Weeks Old Pullets in Kaduna, Nigeria. Journal of Animal production Advances, 5(3):636- 644. 48 Skeeles J.K., Slavik M., Beasley J.N., and Brown A.H. Meinecke C.F., Maruca S. and Welch S. (1980): An age-related coagulation disorder associated with experimental infection with infectious bursal disease virus. Am. J. ve£. Res., 4 1 (9), 58-61. Smith A. J. (1992): Integration of poultry production into the agricultural systems. In The tropical agriculturist: poultry. (The Technical Centre for Agricultural and Rural Development (CTA)). Macmillan, London: 179–191. Swaia. E .S. Kessya, M.J,Sankaa. P. N, and Mtui, P. F, (2011): A serological survey for infectious bursal disease virus antibodies in free-range village chickens in northern Tanzania. Tydskr.S.Afr.vet.Ver, 82(1): 32–35. Tsukamoto K., Tanimura N., Mase M. and Imai K. (1995): Comparison of virus replication efficiency in lymphoid tissues among three infectious bursal disease virus strains. Avian Dis., 3 9 (4), 844-852. Stuart J.C. (1989): Acute infectious bursal disease in poultry. Vet. Rec., 125 (10): 281- 284. Snyder D.B. (1990): Changes in the field status of infectious bursal disease virus - Guest Editorial. Avian Pathol, 19:419-423. Tadelle D. (1996); Studies on village poultry production systems in the central highlands of Ethiopia, MSc thesis, Department of animal nutrition and management Swedish, University of Agricultural Sciences. Pp, 49. Tanimura N. and Sharma J.M. (1997): Appearance of T cells in the bursa of Fabricius and cecal tonsils during the acute phase of infectious bursal disease victims infection in chickens. Avian Dis., 4 1 (3): 638-645. Tanimura N., Tsukamoto K., Nakamura K., Narita M. and Maeda M. (1995); Association between pathogenecity of infectious bursal disease vims and viral antigen distribution detected by immunochemistry. Avian Dis., 39, 9-2 Van den Berg, T.P., Morales, D., Eterradossi, N., Rivallan, G., Toquin, D., Raue, R., Zierenberg, K.,Zhang,M.F.,Zhu,Y.P.,Wang,C.Q.,Zheng,H.J.,Wang,X.,Chen,G.C.,Lim,B.L.,Mül ler.H..(2004); Assessment of genetic , antigenic and pathotypic criteria for the characterization of IBDV strains. Avian Pathol. 33: 470–476. 49 Van den Berg T.P. (2000): Acute infectious bursal disease in poultry. A review, Avian Pathol, 29:175-193. Van den Berg T.P., Gonze M. and Meulemans G, (1991): Acute infectious bursal disease in poultry: isolation and characterization of a highly virulent strain. Avian Pathol, 20 (1): 133-143. Vandersluis, W. (1999): World poultry disease update. World Poult., 15: 30-33. Vindevogel H., Gouffaux M., Meulemans G., Duchatel J.P. and Halen P. (1976): Maladie de Gumboro : distribution et persistance du vims chez le poussin inoculé. Études sur la transmission de la maladie. Avian Pathol, 5: 31-38. Wilcox G.E., Flower R.L.P., Baxendale W. and Mackenzie J.S. (1983): Serological survey of wild birds in Australia for the prevalence of antibodies to EDS-76 and infectious bursal disease viruses. Avian Pathol, 12: 135-139. Winterfield, R.W., Fadly, A.M., Bickford, A. (1972): Infectivity and distribution of infectious bursal disease virus in the chicken. Persistence of the virus and lesions. Avian Dis. 16: 622–632. Zeleke A, Gelaye E, Sori S, Ayelet G, Sirak A, Zekarias B. (2005a): Investigation on infectious bursal disease outbreak in Debre Zeit. Asian Network for Scientific Information. Int. J. Poult. Sci., 7: 504-506. Zeryehun T. and Fekadu G. (2012): Seroprevalence of infectious bursal disease in chickens managed under backyard production system in Central Oromia, Ethiopia. African J. of Microbial. Res., 6(38): 6736-6741. Zierenberg K., Raue R., and Muller H. (2001): Rapid identification of very virulent strains of infectious bursal disease virus by reverse transcription-polymerase chain reaction combined with restriction enzyme analysis. Avian Pathol. 30: 55–62. 50 8. ANNEXES Annex II: Questionnaire survey Addis Ababa University College of veterinary medicine and agriculture A questionnaire survey prepared aim to collect information about the presence of infectious bursal diseases (IBD) in chicken flock at purposively selected small holder farmers or semi-intensive farm. Date ......./......./................ Questionnaire No. ............. Breed.......... Small holder farmer................... semi- intensive farm......... Answer the questionnaire by marking ‘X’ on correct answer corresponding to your chicken flock. 1. Is there chicken mortality in your flock? Yes no 2. If your answer in question 1 is yes, at which age range highly mortality of chicken observed? 1-2 weeks 3-6 weeks 7-10 weeks 11- 15 weeks 16-21 weeks. 3. How many of your chickens died per year from flock? 4. At which season high mortality of chicken occur? a) At summer b) winter c) spring d) autumn 5. How many days it takes to die from the observation of the first clinical signs a) 1-2 days b) 3-5 days c) 6- 8 days 6. Which clinical sign observed before the chicken die? a)…………………………………………… b)……………………………………………. c)……………………………………………. 7. Did your chicken get vaccine in this year? Yes no 8. If your answer yes for question 7, for which disease your chicken have vaccinated? a) …………………………….. c) …………………………… b) …………………………….. d) …………………………… 9. Did you sell the diseased / recovered chicken at market? Yes no 51 10. Is there retardation of growth in your chicken flock? Yes no 11. Did your chicken get treatment at time of morbidity? Yes no Annex II: Serological and pathological sample data collection sheet Date ......./......./................. 1. Case no. .................. Age ............. breed.................. semi-intensive farm ................ backyard.................... 1.1. Physical abnormality observed/ antemortem examination and history a)................................................ e)............................................. b)............................................... f)............................................ c).............................................. g)............................................ 1.2. Serum................. Serology test result......................................... 1.3.Postmortem findings in different tissues and its gross interpretation (1) Bursal fabricious (2) spleen (3) thymus a) .................................... a)............................. a).......................... b) .................................... b).............................. b).......................... c) ....................................... c).............................. c).......................... (4) Proventriculus and gizzard (5) pectoral muscle a).......................................... a)..................................... b)........................................ b).................................... c)........................................... c).................................... 1.4. Pathological sample taken for histopathological procedure and microscopic interpretations a. Bursal fabricious result.................................................................. b. Spleen result ................................................................. c. Thymus result ................................................................. d. Proventriculus & gizzard result ............................................... 52 Annex III: Gross and histopathological changes in different tissue of IBD infected chickens. Figure A: M microscopical change (A) and gross changes (B) in chicken thymus infected with IBD was total depletion of lymphocyte cells in follicles and formation of tissue cords with necrotic cells. HEx40. Figure B .Gross pathology (A) and microscopic view (B) of proventriculus and gizzard junction in IBD infected chickens. Hemorrhage was the major pathological changes. A B A B 53 Figure C: Gross (A) and microscopic changes (B) in chicken kidney due to IBD infection. Swollen caudal lobe kidney (arrow), hemorrhage, proteinous edema in tubules and infiltration of heterophils ((B: arrow and double arrow) Figure D: Petichial hemorrhage (arrow) on thigh muscle of IBD infected 52 day old chicken. A B D 54 Figure E: Congested cutaneous blood vessel (arrow) in IBD infected chickens Figure F: Lesion score in bursa of IBD infected chickens (mild A), sever (B) ad moderate (C). A B C