Sero-Prevalence and Risk Factors for Infectious Bursal Disease in Local Chicken on Backyard Production System in Selected Districts of Ilubabor Zone, South Western Ethiopia

Moti Wakgari, DVM, MSc

Survey and Surveillance of Endemic Animal Disease Expert, Department of Veterinary Epidemiology, Bedelle Regional Veterinary Laboratory Center, P. O. Box 15, Bedelle, Ethiopia; Tel. +251474450019; E-mail: motiwakgari@gmail.com

INTRODUCTION

Livestock production offers concerning forty-seven per cent of the agricultural gross domestic product and 18% of the national gross domestic product of Federal Democratic Republic of Ethiopia.¹ Chicken production is an important and essential part of most Ethiopian households in rural, urban, and peri-urban areas. Poultry will play a key role in managing financial crisis and food security. Moreover, as chicken farming is often done by ladies and youngsters, this will play a key role in unit labor productivity and gender authorization.² Ethiopian Ministry of Agriculture has recognized chicken production as a key sector to upset food security problems. The intention is to lift the number of meat and eggs created annually by increasing the amount of poultry farms and introduction of improved breeds.³

The backyard chicken production system that accounts for 96% of Ethiopia’s fifty million chicken populations.⁴ It is extremely poor, as scavenging chickens live along side individuals and different species of farm animals. In the backyard chicken production system, no way that of dominant movement and dropping of chickens, since chickens freely rove within the unit compound. Isolation of sick chickens from the flocks and dead chicken disposal has not been practiced.² Around 40-60% of the chicks hatched die at the 1^st eight weeks of life^5,6 that predominantly because of disease and predation. Moreover, Alamargot⁷ recorded a death rate of 20-50% in indigenous chickens due to disease. Since village backyard chickens habitually exposed to overwhelming numbers of microorganisms, infectious bursal disease virus is one amongst the diseases that cause chick mortality.⁸ Infectious diseases like Newcastle disease and infectious bursal disease are reported to be the key health and production constraints of chickens.⁹

The infectious bursal disease is also known as Gumboro disease, an acute, highly contagious, and immunosuppressive^10,11 viral disease affecting mostly young chicks. The causal virus belongs to the genus Avibirnavirus of the family Birnaviridae. Two serotypes of the virus are identified. Serotype one virus is infective to chickens. Sero type two virus is nonpathogenic to chickens and has been isolated from each chickens and Turkeys.¹² It constrains poultry industries worldwide.¹³ It causes appreciable economic losses ensuing from mortality^14,15,16 and an immunological disorder that ends up in vaccine failure against different infectious diseases.¹⁷ Additionally, the immunological disorder will increase the status of chickens to different infectious diseases.¹¹ Maternal antibodies to infectious bursal disease (IBD) in susceptible chickens act chicks up to twenty-one-days.¹⁸

Large losses are a result of opportunist infections encountered by poultry farmers and particularly the crisis in developing countries like Ethiopia.¹⁹ IBD is an extremely communicable disease of young chickens (<17-weeks of age) during which the tissues of the system, and particularly the bursa of  Fabricius, are targeted leading to immunological disorders and prone for different infections, such as E. coli, Salmonella, Mycoplasma, coccidia, Marek’s disease and others.²⁰ The disease is unfolded through contaminated feed and water.²¹ In chickens, severe acute disease, typically in three to six-week-old birds, is related to high mortality, however less acute or subclinical infections are common earlier in life.²²

In Ethiopia, IBD incidence was occur in 2002 for the 1^st time at a personal business poultry farm with a death rate of 45- 50% and therefore the incidence of recent strains of IBD became a challenge to the juvenile poultry business in Ethiopia.²³ The first study on the incidence of IBD in Ethiopian village poultry was in 2 areas within the Amhara region that had received “improved” chicks from an advertisement farm,²⁴ and it has been advised that this was the explanation for the introduction of the disease to village poultry.

The seroprevalence of IBD in backyard chickens was studied in numerous components of Ethiopia. Among these: thirty-nine in East Shoa Zone, Oromia,²⁵ 38.4% in 2 districts of Amhara region, Northwest Ethiopia²⁶; 76.64% in Waliso, Ambo, and Welmera²⁷, 29.4% in Bahir dar²⁴, 83.1% in selected sites of Ethiopia²⁸, 72.7% prevalence in Gondor²⁹, 45.05% around Mekele town North Ethiopia,³⁰ 51.56% in and around Bahir Dar North West Ethiopia,³¹ 84.2% in North Shoa Zone of Oromia and Amhara region^32,33 20.7% and 51.7% In Jigjiga and Harrar, East Ethiopia.³⁴

Due to happening reports of chickens, disease mortality and morbidity in the Iluababora zone in 2020, questionnaire survey was done by Bedelle Regional Veterinary Laboratory Center to assess chickens’ disease and issues. Chick mortality and morbidity were the foremost outstanding issues and IBD was one amongst the diseases assessed throughout the farm survey. However, there was no study on seroprevalence and associated risk factors of infectious bursal in native grounds chicken production during this zone. Therefore, this study was required to be a remedy to chick mortality and morbidity within the study areas with the following objectives.

• To estimate the seroprevalence of infectious bursal disease virus (IBDV) among native chickens in backyard production systems of Hurumu, Metu and Bilo Nopa districts of Ilubabor Zone.
• To assess risk factors for the incidence of IBDV in local chickens of backyard chicken production systems of Hurumu, Metu and Bilo Nopa districts of Ilubabor Zone.

MATERIALS AND METHODS

Description of Study Area

This study was conducted in three districts (Hurumu, Metu, and Bilo Nopa) of Ilubabor Zone of Oromia Regional State, Ethiopia. Ilubabor Zone is located in the South-Western part of Ethiopia, 600 kms away from Addis Ababa (Figure 1). Hurumu district is located at a latitude of 08º21’42” to 08º31’17” North and 35º05’18” to 35º68’30” East. It comprises 48,395 cattle, 20,938 shoots, 4686 equine, 50,559 local chickens, and 13,834 exotic Sasso T-44 and Bovans Brown chicken breeds. There were 6 small-scale poultry farm microenterprises and 2 private small-scale poultry farms in Hurumu district (Hurumu District Livestock Resource and Development Office, 2021).

Figure 1. Map of Study Areas (Ilubabor Zone Livestock Resource Development Office, 2021)

Metu district is located at longitude 035°.32’ to 040°.29’ East and latitude 08°.28’ to 010°.54’ North of the equator. The livestock population of the Mettu district comprises of 146,635 cattle, 93,012 shoats, 24,372 equines, 134,132 local, and 57,260 exotic Sasso T44 and Bovans Brown chicken breeds. There were 17 small-scale poultry farm microenterprises and 8 private small-scale poultry farms in Metu district (Metu District Livestock Resource and Development Office, 2021).

Bilo Nopa district is located at latitude of the area ranging from 07°05.33’ to 08°45.33’ to North while the longitude of the area ranges from 033°47.57’ to 036°52.33’ East. This district comprises a livestock population of 17,289 cattle, 12,614 shoats, 850 equines, 24,550 local chickens, and 7,440 exotic SASO T-44 and Bovans Brown chicken breeds. There were 7 small-scale poultry farm microenterprises and 3 private small-scale poultry farms in Bilo Nopa district (Bilo Nopa District Livestock Resource and Development Office, 2021).

Study Methods

The study was conducted in local chickens raised under the backyard production system of three districts. Most of them were scavenging chickens living together with people and other species of livestock. The chicken movement was unlimited and free-roaming in the household compound. There was no practice of isolating sick chickens from the flocks. The total population of chickens from 160 households were 822, including none sampled chicks less than 3-weeks, and the average flock size was 5.14(822/160) chickens per household, while 6.23(467/75), 4.49(206/46), and 3.82(149/39) average flock density in Metu, Bilo Nopa, and Hurumu districts, respectively.

The breeds of chickens in the study area were Horo local breed and exotic Sasso T-44 and Bovans Brown breeds.³⁵ More than half of the households bought exotic chicken breeds and they scavenge with local breeds of grain, common maize supplement. Most farmers buy exotic chickens from multiplication centers through the livestock sector while few of them buy from smallscale poultry farm microenterprises and open markets. Exotic breeds had been believed as they were vaccinated against IBD before distribution to farmers, thus that was why samples were only collected from the local chicken breed. Chickens were categorized by age young (<17-weeks) and adult ≥17-weeks) based on the clinical characteristics of IBD disease.²⁰ Chicken flocks were categorized based on chicken numbers greater than five (>5) and less than or equal to five (≤5), which was based on the average number of chickens per flock (5.14) per household in the study area.

Study Design

The study was cross-sectional from March 2021 to August 2021. Data to assess risk factors related to IBDV were collected using a questionnaire survey and serum sample collection format. In all studies kebele’s, selected poultry owners were interviewed face to face during blood sample collection. Variables included in the survey were study area, age (young, adult), sex, cleaning activity of the housing area (regular or daily, irregular or not fixed), flocks mixed with exotic chicken breed (yes, no), number of chickens per flock, source of chickens (home breed, purchased) and flocks housing system (separated house, roost in family dwelling or kitchen) were emphasized as risk factors.

Sample Size Determination and Sampling Technique

The sample size was determined according to sequential multiple assignment randomized trial (SMART) methodology³⁶ using cluster random sampling using an expected animal level prevalence of 50% and a desired absolute precision of 5% with 95% CI, since there was no previously expected prevalence in the study area.

t²×p×q

n=———————×DEFF

d²

       (2.045)² ×0.5×0.5

                  n=————————————×1=418

          (0.05)²

Where, n=sample size, t=linked to 95% confidence interval for cluster sampling (2.045), p=expected prevalence (fraction of 1), q=1-p(expected prevalence), d=relative desired precision, DEFF=Design Effect=1.

Accordingly, 418 local backyard chickens were sampled. First, districts were purposively selected based on chicken disease outbreak reports and a questionnaire survey done on backyard chicken mortality and morbidity. Metu, Bilo, Napa, and Hurumu districts have 29, 15, and 14 kebele’s, respectively. Based on the chicken’s population and the number of kebele lists from each district; 5 kebeles from Metu district, 3 kebeles from Hurumu, and 3 kebeles from Bilo Nopa district were randomly selected by lottery method. At the 2^nd stage, the household of each kebele was randomly selected. Finally, except chicks below 3-weeks, all chickens were sampled.

Sample Collection and Transportation

Blood samples were collected aseptically from the wing vein victimization 3 ml disposable syringe. The syringe was placed in an exceedingly slope position for long at room temperature to empty the sera samples. The separated serum was transferred into a sterile cryovials tube, labeled, and transported to Bedelle Regional Veterinary Laboratory Center underneath a cold chain for laboratory analysis. The sera were kept at -20 °C until the check were performed. Indirect enzyme-linked immunosorbent serologic assay was sent to discover IBD virus antibodies employing a commercially ready IBDV enzyme-linked immunosorbent serologic assay check kit. Individual-level connected risk factors: sex, age, and supply of chickens.

Serological Test and Laboratory Analysis

Innovative diagnostic indirect enzyme-linked immunosorbent serologic assay kit (Louis Pasteure Grabels, France) discovered the presence of anti-IBD antibodies within the chicken serum following the kit manufacturers’ suggested protocol. The test sera were pre-diluted by dilution buffer 14 in a pre-dilution plate according to the established protocol or kit instructions, and each was dispensed into the requested number of micro wells. In the ELISA plate pre-diluted samples and dilution buffer 14 were added and incubated for 30 min±3 min at 21 °C. After incubation, the sera were discarded from the plates, and each well was washed 3 times by 300 μl of washing solution. About 100 μl anti-chicken immunoglobulins peroxidase conjugate was dispensed into the wells and the plates were incubated for 30±3 min at 21 °C. After incubation, again the sera were discarded from the plates, and each well was washed 3 times by 300 μl of washing solution. About 100 μl substrate solutions were dispensed into each test well and again incubated for 15±2 min at 21 °C in the dark place. After a final incubation, the substrate chromogen reaction was stopped by adding about 100 μl stop solution and the color reactions were quantified by measuring the optical density of each well at 450 nm.
To ascertain the validity of IBD enzyme-linked immunosorbent serologic assay results, a validity check was done. In the valid IBD enzyme-linked immunosorbent serologic assay results, the mean optical density (OD) price of positive management humor is bigger than (0.25), and the quantitative relation of the mean of the positive and negative management (ODPC and ODNC) is bigger than three. For the interpretation of the results, a humor sample positive (SP) management quantitative relation was needed. Consequently, the sample positive quantitative relation was calculated as follows. If S/P price was >(0.3), the IBD protein standing was thought of to be positive, however, <(0.3) was taken as negative.

    S                  OD_sample– OD_NC
———%=—————————
P                   OD_PC– OD_NC

Data Management and Analysis

All data obtained from the field was recorded in the record sheet format and later entered into Microsoft Excel worksheet and binary logistic regression for flock level data and multilevel mixed effects model (Generalized Leaner Model logit) for chicken level data statistics was used to summarize the data by using stata software version 13. The overall prevalence was calculated by dividing positive samples by the total number of examined samples and multiplied by a hundred. Seroprevalence was categorized into chicken level (sex, age, source, study area) and flock level (study areas, cleaning activity, presence of exotic breeds within the flock, number of chickens per flock and housing system of chickens).

Multivariate logistic regression analysis was used to examine the relationship between the outcome variable (seroprevalence) and the different explanatory variables controlling the possible effect of confounders. The odds ratio (OR) was used to assess the association between the dependent and independent variables. p-value of less than 0.05 (p<0.05) was set for the significance of statistical associations.³⁷

RESULTS

Overall Seroprevalence of IBDV Antibody

Among the 418 chicken serum samples tested for IBDV antibodies to know chicken level IBD infection, 180 samples were positive for IBDV antibody with an overall seroprevalence of 43.10% (95%CI: 38.40, 47.85) in the study area.

District and Village Level Chicken Seroprevalence of IBDV
Antibody

The highest chicken level seroprevalence of IBDV was observed in the Metu district (100/194, 51.55%) followed by the Bilo Nopa district (45/125, 36%) and Hurumu district (35/99, 35.35%). The seroprevalence of IBDV was higher in purchased chickens (64.23%) than home breed chickens (32.74%), in females (48%) than males (31.45%), and in adults (54%) than in young chickens (31.71%) as illustrated in (Table 1).

Flock Level Seroprevalence of IBDV Antibody

Out of 160 flocks tested for IBDV, 73 flocks were found positive for IBDV antibody and flock level seroprevalence of IBDV was 45.63% (95%CI: 37.91, 53.34%). On average, 3 serum samples (480/160=3) were collected per flock and 2.8 chickens per flock (180/73=2.5) were positive for the IBDV antibody, which indicated almost all positive flock chickens were seropositive. The highest flock level seroprevalence of IBDV was observed in Metu district (38/75, 50.66%), followed by Bilo Nopa district (20/46, 43.48%) and Hurumu district (15/39, 38.46%). The seroprevalence of IBDV was higher in flocks mixed with exotic chickens (66%) than those not mixed (20%), in flocks greater than five (>5) 60% than in flocks less than or equal to five (≤5) 31%, in an irregularly cleaned house of flocks 49% than a regularly cleaned house of flocks 38% and roost in family dwelling 46% than separated housing 44% chickens as illustrated in (Table 2).

Chicken level Risk Factors Associated with Inflammatory Bowel Disease

In multivariate logistic regression analysis, sources of chicken (p=0.000), study area (Metu p=0.000, Bilo Nopa p=0.023), sex (p=0.001) and age (p=0.017) were independent predictors of IBD infection. The odds of IBD seroprevalence was more likely higher in females than males, in adult than young, in purchased than home breed, in Metu and Bilo Nopa districts compared to Hurumu district (Table 3).

Flock level Risk Factors Associated with IBD

In multivariate logistic regression analysis, the presence of an exotic breeds within the flock (p=0.000), the number of chickens per flock (p=0.004) were independent predictors of IBD infection. The odds of IBD seroprevalence were more likely higher in flocks mixed with exotic chickens than flocks who did not mix with exotic breeds and in larger flock sizes (greater than five chickens) than smaller flock sizes as shown in (Table 4).

DISCUSSION

There has been increasing interest to estimate the prevalence of IBD in the local backyard chicken production systems since 40- 60% of the chicks hatched die during the first 8-weeks of life^5,6 mainly due to disease and predation. The current finding has a role in the reduction of chicken mortality and morbidity, ensuring improved chicken production and productivity by generating realtime epidemiological information to the poultry sector.

The overall seroprevalence of IBDV in the local chickens of backyard production system in the present study was 43.13% (CI: 38.69, 47.56). The overall seroprevalence of IBDV in this study is in line with the study done in India, 46.2% by Singh et al³⁸, 45% in Taiwan³⁹, and around Mekelle town, Northern Ethiopia 45.05% by Zegeye et al.³⁰ In contrast, the overall seroprevalence of IBDV in local chickens of backyard production system in this study was higher than the reports of 33.9% in Cameroon^40,41 30.7% in Sudan,⁴² 30% in Bostwana,²⁵ 39.2% in East Shoa zone, Oromia region Ethiopia,²⁶ 38.4% in two districts of Amhara region, Northwest Ethiopia,²⁴ 29.4% in two districts of Amhara region Ethiopia,⁴³ 7.26% in Nigeria at Zuria,³³ 20.7% in Eastern Shewa Zone Oromia region Ethiopia⁴⁴ and 33.4% in Nigeria.

However, the current prevalence is lower than the previous studies^{27,28,29,31,32,34,45,46} reported elsewhere with the prevalence of 76.64%, 83.1%, 73.5%, 63.5%, 82.2%, 51.56%, 84.2%, 51.7%, respectively in different parts of Ethiopia in backyard chickens production system. Moreover, in other African countries, the overall seroprevalence of IBD in the backyard chicken production system in this study was lower than and who reported an overall prevalence of 55% from Zimbabwe^40,47 and 60% from Nigeria, respectively. The current seroprevalence difference with different studies done before in different parts of Ethiopia may be due to less distribution of exotic chicken breeds among backyard chickens than in comparison to other zones, the test kit we used was with 100% sensitivity and specificity, study area far (600 km) away from the central part of Ethiopia where the chicken intensification more practiced that in agreement with Zeleke et al⁹,⁴⁸ who investigated IBDV being introduced and disseminated into Ethiopia through exotic chicken breeds, the lower human population in comparison to other study areas, there have been lower chicken product demand consumption and lower purchase of live chickens in the open market which was one of the risk factors responsible for the spread of IBDV.⁴⁹

The odds of IBDV seroprevalence was 2.52 (95% CI: 1.58-4.04, p=0.000) and 1.81 (95% CI: 1.08-3.05, p=0.023) times higher more likely in chickens in Metu and Bilo Nopa districts, respectively in compare to Hurumu district. This finding was in agreement with the findings of different researchers^{27,30,32,34,46,50} that showed a significant association of IBDV seroprevalence between different study areas. In contrast, studies conducted by researchers⁵¹ and reported that no variation was found in the seroprevalence of infectious bursal disease in different study areas.²⁹ The higher seroprevalence of IBDV in Metu and Bilo Nopa districts as compared to Hurumu district in the present study could be due to larger average flock density per household, larger exotic chicken breeds distributed to farmers, and larger small-scale poultry farm enterprises in Metu and Bilo Nopa in compare to Hurumu that agree with previous reports of Farooq et al⁵² who showed overcrowding increase transmission of disease and previous reports of Zeleke et al⁹ , Zeleke et al⁴⁸ who showed introduction and dissemination of IBDV in Ethiopia through exotic chicken breed importation and distribution to farmers.

The seroprevalence of IBDV was higher in purchased chickens (64.50%) than home breed chickens (32.60%). The odds of IBDV seroprevalence was 3.58 (95% CI: 2.60-4.93, p=0.000) times higher more likely in chickens purchased than those breeds at home. This result was in agreement with Rashid et al⁴⁹, Swai et al⁵⁰ that showed that the purchase of live poultry from an open market is the main risk factor for IBDV dissemination. In the current study, the higher seroprevalence in purchased chickens than home breed could be due to contact of hundreds of chickens at local open-air markets from different farmers, villages, towns, kebeles, districts, and zones especially during the ceremony which were then taken back to different localities that certainly facilitate the spread of IBDV among backyard chickens.

The seroprevalence of IBDV was higher in females (48%) than males (31.20%). The odds of IBD seroprevalence were 2.25 (95% CI: 1.34-3.67, p=0.001) times higher more likely in females than males. This finding is in agreement with a report from Zegeye et al,³⁰ who reported 54.18% of females and 27.82% of males. The significant association observed between sex and IBD infection might be because sexual maturity in females corresponds with a reduction in T-lymphocyte numbers leading to suppression of cellular immunity, so the reproductive demands placed on females may raise the pathogen load of frequently encountered infections.⁵³ In contrast to current study, reports from^{29,32,33,34,46,50} showed that there was no significant association of seroprevalence between sexes.

The seroprevalence of IBDV was higher in adults (53.70%) than in young chickens (31.10%). The odds of IBDV seroprevalence was 1.95 (95% CI: 1.13-3.36, p=0.017) times higher more likely in adult chickens than young chickens which are similar to the studies^30,32,54 of that reported an increased seroprevalence of IBD infection as the age of chickens increase. Contrary to this finding reported by Lemma et al,³⁴ seroprevalence of IBD was not significantly associated with age. The production of backyard chickens of different age groups together might make the infection within a given flock increase exposure as suggested by Nawathe et al.⁵⁵ The significant association of IBD infection with age in the current study might be because adult chickens need enough time and space for scavenging in their surroundings and ingest more contaminated feed by microorganisms while, chicks below sixweeks of age confined in the house.⁵⁶

In this study, flocks mixed with exotic chickens and those who had not were compared and a higher seroprevalence of (66%) in flock mixed with exotic chickens than those not mixed (20%) with exotic chickens. The odds of IBDV seroprevalence were 5.02 (95% CI: 2.24-11.27, p=0.000) times more likely in flocks mixed with exotic chicken breeds compared to those that did not mixed. The current study agrees with studies^9,48 who investigated IBDV was introduced and disseminated in Ethiopia via exotic chicken breeds. According to the report of this author, Ethiopia has been known to be free from IBD⁵⁷ until its first occurrence in 2002. It also, in agreement with Mazengia et al²⁴ who suggested that the first study on the incidence of IBD in Ethiopian village poultry was due to the distribution of “improved” chicks from a commercial farm to farmers. The significant variation of IBDV seroprevalence noted between flocks mixed with exotic chicken breeds and to none mixed backyard flocks could be related to the dissemination of IBD virus through the distribution of improved breeds of chickens from infected poultry breeding and multiplication centers to backyard village chickens.

Flock size had a significant effect on the seroprevalence of IBD in the study area when multivariable logistic regression analysis was carried out. The highest prevalence of IBD antibody was recorded in flocks with chicken numbers greater than five (>5) 60% than in flocks with chicken numbers less than or equal to five (≤5) 31%. The odds of IBDV seroprevalence were 3.17 (95% CI: 1.43-6.90, p=0.004) times higher more likely in larger flock size than that of smaller flock size less than or equal to 5. This result was in agreement with Jarso, 2016 who reported higher odds of IBD infection in larger flock size than smaller flock size. In the current study, the higher seroprevalence in larger flock size could be due to no supplementary feed in backyard chicken under backyard chicken production systems, and there is higher feed competition in larger flocks as a result chickens need to scavenge for a longer time for survival and routinely exposed to IBD virus.

CONCLUSION

The current study indicates that the seroprevalence of IBDV is high and the IBD virus was found circulating in study sites which may cause economic losses in the poultry sector. Furthermore, the present study demonstrated that the seroprevalence of infectious bursal disease virus in local chickens of the backyard production system was influenced by the study area, source of chicken, sex, age, presence of exotic breeds within the flock, and number of chickens per flock. This seroprevalence might be due to field exposure of chickens to the disease and indicates the importance of further study on the serotype and strains of IBDV that are circulating in the study sites to design appropriate control and prevention measures.

DATA AVAILABILITY

All data used during analysis of this research to support the findings of this research are included within the article.

DISCLOSURE

The research was performed at Bedele Regional Veterinary Laboratory center, Ethiopia.

ACKNOWLEDGEMENTS

I would like to express my deepest gratitude for my office Bedelle Regional Veterinary Laboratory Center for granting me an excellent supply of laboratory inputs and covering all the cost that needed for the research.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

A local ethics committee ruled that no official ethics approval was required to carry out this study since it was only blood collection.
Before conducting the study, informed permission was obtained
from the owners of the chickens used in this study.

1. IGAD Centre for Pastoral Areas and Livestock Development, ICPALD. The Contribution of Livestock to the Ethiopian Economy. Ethiopia. 2013. Website. https://igad.int/attachments/714_ ETHIOPIA%20BRIEF%20. Accessed June 4, 2022.

2. Habte T, Amare A, Bettridge J, Collins M, Christley R, Wigley P. Guide to chicken health and management in Ethiopia. 2017. Website. https://core.ac.uk/download/pdf/132697854.pdf. 1-25. Accessed June 4, 2022.

3. Tegegn GG. Ethiopia Livestock Master Plan: Roadmaps for growth and transformation. Paper presented at: Livestock Master Plan Development Project; 2015; Nairobi, Kenya.

4. Gurmu F. Assessment of Farmers’ Criteria for Common Bean Variety Selection: The case of Umbullo Watershed in Sidama Zone of the Southern Region of Ethiopia. Soybean variety development in Ethiopia. 2013; 5(2): 4-13.

5. Dessei T. Studies on Village Poultry Production Systems in the Central Highlands of Ethiopia. [master’s thesis]. Uppsala, Sweden: Swedish University of Agricultural Sciences; 1996; 521-537.

6. Central Statistical Authority (CSA). 2003. Ethiopian agricultural sample enumeration, 2001/02 (1994 EC). Results at country and regional level. statistical report on farm management practices, livestock and farm implements. Part II. (Raw data in softcopy). CSA, Addis Ababa, Ethiopia.

7. Alamargot J. Avian pathology of industrial poultry farms in Ethiopia. Paper presented at: National Livestock Improvement Conference, Addis Ababa University; 1987; Debre Zeit, Ethiopia. 114-117.

8. Olwande PO. Epidemiological Assessment of Productivity Constraints and Appropriate Intervention Measures on Indigenous Chicken Production. [dissertation]. Southern Nyanza, Kenya: University of Nairobi; 2014.

9. Zeleke A, Gelaye E, Sori T, Ayelet G, Sirak A, Zekarias B. Investigation on an infectious bursal disease outbreak in Debre Zeit, Ethiopia. International Journal of Poultry Science. 2005; 4(7): 504-506. doi: 10.3923/ijps.2005.504.506

10. Rauf A. Persistence, Distribution and Immunopathogenesis of Infectious Bursal Disease Virus in Chickens. [dissertation]. Columbus, OH, USA: Ohio State University); 2011.

11. Yao Q, Shijun JZ. Infectious bursal disease virus-host interactions: Multifunctional viral proteins that perform multiple and differing jobs. Int J Mol Sci. 2017; 18: 161. doi: 10.3390/ijms18010161

12. Lim BL, Cao Y, Yu T, Mo CW. Adaptation of virulent infectious bursal disease virus to chicken embryonic fibroblasts by sitedirected mutagenesis of residues 279 and 284 of viral coat protein VP2. J Virol. 1999; 73(4): 2854-2862. doi: 10.1128/JVI.73.4.2854- 2862.1999

13. Toro H, Van Santen VL, Hoerr FJ, Breedlove C. Effects of chicken anemia virus and infectious bursal disease virus in commercial chickens. Avian Dis. 2009; 53(1): 94-102. doi: 10.1637/8408- 071408-Reg.1

14.2 Jackwood DJ, Sommer-Wagner SE, Stoute ST, et al. Characteristics of a very virulent infectious bursal disease virus from California. Avian Dis. 2009; 53(4): 592-600. doi: 10.1637/8957-061109- Reg.1

15. Mahgoub HA. An overview of infectious bursal disease. Arch Virol. 2012; 157: 2047-2057. doi: 10.1007/s00705-012-1377-9

16. Müller H, Mundt E, Eterradossi N, Islam MR. Current status of vaccines against infectious bursal disease. Avian Pathol. 2012; 41(2): 133-139. doi: 10.1080/03079457.2012.661403

17. Jackwood DJ, Sommer-Wagner SE. Detection and characterization of infectious bursal disease viruses in broilers at processing. Prev Vet Med. 2010; 97(1): 45-50. doi: 10.1016/j.prevetmed.2010.07.010

18. Zaheer A, Saeed A. Role of maternal antibodies in protec tion against infectious bursal disease in commercial broilers. International Journal of Poultry Science. 2003; 2: 251-255. doi: 10.3923/ijps.2003.251.255

19. Mohammed N, Fisseha M, Hailu M, Getnet Z. Observation of free chicken disease in selected Districts of North Western Amhara. Advanced Journal of Agricultural Research. 2014; 2(11): 166-172.

20. Washington Avian Disease Diagnostic Laboratory, WADDL. Infectious Bursal Disease. Washington State University.ag animal health spotlight. 2014. Website. http://www.vetmed.wsu.edu/ depts_waddl/. Accessed June 4, 2022.

21. Sharma JM, Kim IJ, Rautenschlein S, Yeh HY. Infectious bursal disease virus of chickens: pathogenesis and immunosuppression. Dev Comp Immunol. 2000; 24(2-3): 223-235. doi: 10.1016/s0145305x(99)00074-9

22. Animal Health Australia (AHA). Infectious bursal disease: Fact sheet. 2017. Website. https://www.wildlifehealthaustralia.com.au/Portals/0/Documents/FactSheets/Avian/Infectious%20Bursal%20Disease%20May%202017%20(2.0).pdf. Accessed June 4, 2022.

23. Zeleke A, Yami M, Kebede F, et al. Gumboro: An emerging disease threat to poultry farms in Debre Zeit. Paper presented at: the 17^th Annual Conference of Ethiopian Veterinary Association; 2003; Addis Ababa, Ethiopia.

24. Mazengia H, Tilahun SB, Negash T. Newcastle disease and infectious bursal diseases are threat to village chicken production in two districts of Amhara National Regional State, Northwest Ethiopia. IUP Journal of Life Sciences. 2010; 4(2): 62-72.

25. Reta T. Sero-prevalence Study of Infectious Bursal Disease in Nonvaccinated Backyard Chickens in Agro-ecological Areas of East Shoa Zone. [dissertation]. Deber Zeit, Ethiopia: Addis Ababa University; 2008.

26. Mazengia H, Bekele ST, Negash T. Incidence of infectious bursal disease in village chickens in two districts of Amhara Region, Northwest Ethiopia. Livestock Research for Rural Development. 2009; 21(12): 214.

27. Hailu D, Melese B, Moti Y, Mekedes G. Seroprevalence of infectious bursal disease in backyard chickens of Oromia regional state, Ethiopia. Veterinary Research. 2010; 3(4): 89-93. doi: 10.3923/ vr.2010.89.93

28. Jenbreie S, Ayelet G, Gelaye E, Kebede F, Lynch SE, Negussie H. Infectious bursal disease: Seroprevalence and associated risk factors in major poultry rearing areas of Ethiopia. Trop Anim Health Prod. 2012; 45(1): 75-79. doi: 10.1007/s11250-012-0176-3

29. Kassa SA, Molla W. Seroprevalence of infectious bursal disease in backyard chickens of North West Ethiopia. Scientific Journal of Crop Science. 2012; 1(1): 20-25.

30. Zegeye S, Tsegaye Y, Abreha H, Awol N. Sero-prevalence of infectious bursal disease in backyard chickens around Mekelle, Northern Ethiopia. African Journal of Biotechnology. 2015; 14(5): 434- 437. doi: 10.5897/AJB2014.14349

31. Natnael T. Pathological and Seroprevalence Studies on Infectious Bursal Disease in Chickens in and Around Bahir Dar, North West, Ethiopia. [master’s thesis]. Bishoftu, Ethiopia: Addis Ababa University; 2015.

32. Kebede B, Mitike G, Bekele M. Seroprevalence of Infectious Bursal Disease in Backyard Chickens of Six Districts of North Shewa Zone of Oromia and Amhara Regions, Ethiopia. Journal of Veterinary Science. 2017; 3(2): 1-9. doi: 10.15226/2381- 2907/3/2/00129

33. Jarso D. Incidence of village chicken diseases in Eastern Shewa Zone, Ethiopia: The case of newcastle and infectious bursal disease. J Vet Sci Res. 2016; 1(4): 000121.

34. Lemma F, Zeryehun T, Kebede A. Seroprevalence of infectious bursal disease in non-vaccinated village chicken in Jigjiga and Harar Districts, Eastern Ethiopia. J Vet Sci Technol. 10, 572, 2019.

35. Ayele G, Rich KM. Poultry value chains and HPAI in Ethiopia. 2010. Website. https://cgspace.cgiar.org/bitstream/handle/10568/5418/129495.pdf?sequence=2. Accessed June 4, 2022.

36. Sequential Multiple Assignment Randomized Trial (SMART). Sampling Methods and Sample Size Calculation for the SMART Methodology. 2012. Website. https://www.humanitarianresponse. info/sites/www.humanitarianresponse.info/files/documents/ files/sampling_methods_and_sample_size_calculation_for_ smart_medhodology_june_2012.pdf. Accessed June 4, 2022.

37. Hosmer DW, Lemeshow S. Applied Logistic Regression. New York, USA: John Wiley & Sons; 2000.

38. Singh KCP, Dhawedkar RG. Prevalence of subclinical infectious bursal disease and its significance in India. Trop Anim Health Prod. 1992; 24(4): 204-206. doi: 10.1007/BF02356745

39. Tsai HJ, Lu YS. Epidemiology of infectious bursal disease in Taiwan. J. Chinese Soc. Vet. Sci. 1993; 19: 249-258.

40. Durojaiye OA, Kwenkam P. A preliminary note on the prevalence of infectious bursal disease of poultry in Cameroon. Rev Elev Med Vet Pays Trop. 1990; 43(4): 439-440.

41. Mahasin EA, Rahaman I. Studies on Infectious Bursal Disease. [dissertation]. Khartoum, Sudan: University of Khartoum; 1998.

42. Mushi EZ, Binta MG, Chabo RG, Ndebele RT. Seroprevalence of infectious bursal disease in non-vaccinated indigenous and exotic chickens on selected farms around Gaborone, Botswana. Onderstepoort J Vet Res. 1999; 66(2): 135-137.

43. Mbuk J. Musal W, Ibrahim S, Sa’idu L. A retrospective analysis of infectious bursal disease diagnosed at poultry unit of Ahmadu Bello University, Nigeria. International Journal of Poultry Science. 2010; 9(8): 784-790. doi: 10.3923/ijps.2010.784.790

44. Shettima YM, El-Yuguda AD, Oluwayelu OD, et al. Seroprevalence of infectious bursal disease virus antibodies in some species of poultry in Maiduguri, Nigeria. Sokoto Journal of Veterinary Sciences. 2018; 16(1): 90-94. doi: 10.4314/sokjvs.v16i1.13

45. Lawal JR, Jajere SM, Bello AM, et al. Prevalence of infectious bursal disease (Gumboro) antibodies in village chickens in Gombe state, northeastern Nigeria. International Journal of Poultry Science. 2014; 13(12): 703-708. doi: 10.3923/ijps.2014.703.708

46. Zeryehun T, Fekadu G. Seroprevalence of infectious bursal disease in chickens managed under a backyard production system in Central Oromia, Ethiopia. African Journal of Microbiology Research. 2012; 6(38): 6736-6741. doi: 10.5897/AJMR12.1344

47. Kelly PJ, Chitauro D, Rohde C, et al. Diseases and management of backyard chicken flocks in Chitungwiza, Zimbabwe. Avian Dis. 1994; 38(3): 626-629.

48. Zeleke A, Sori T, Gelaye E, Ayelet G. Newcastle disease in village chickens in the southern and Rift Valley districts in Ethiopia. International Journal of Poultry Science. 2005; 4(7): 507-510. doi: 10.3923/ijps.2005.507.510

49. Rashid MH, Xue C, Islam MT, Islam MR, Cao Y. Risk factors associated with infectious bursal disease in commercial chickens in Bangladesh. Prev Vet Med. 2013; 111(1-2): 181-185. doi: 10.1016/j.prevetmed.2013.03.013

50. Swai ES, Kessy MJ, Sanka PN, Mtui PF. A serological survey for infectious bursal disease virus antibodies in free-range village chickens in northern Tanzania. J S Afr Vet Assoc. 2011; 82(1): 32- 35. doi: 10.4102/jsava.v82i1.30

51. Nigussie T. Cross Sectional Study of Infectious Bursal (Gumboro) Disease on Backyard Chickens in Addis Ababa and Adam Tulu areas, Ethiopia. [master’s thesis]. Deber Zeit, Ethiopia: Addis Ababa University; 2007.

52. Farooq M, Durrani FR, Imran N, Durrani Z, Chand N. Prevalence and economic losses due to infectious bursal disease in broilers in Mirpur and Kotli districts of Kashmir. International Journal of Poultry Science. 2003; 2: 267-270. doi: 10.3923/ijps.2003.267.270

53. Bettridge JM, Lynch SE, Brena MC, et al. Infection-interactions in Ethiopian village chickens. Prev Vet Med. 2014; 117(2): 358-366. doi: 10.1016/j.prevetmed.2014.07.002

54. Wahome MW, Njagi LW, Nyaga PN, Mbuthia PG, Bebora LC, Bwana MO. Occurrence of antibodies to infectious bursal disease virus in non-vaccinated indigenous chicken, ducks, and turkeys in Kenya. Inter J Vet Sci. 2017; 6(3): 159-162.

55. Nawathe DR, Lamorde AG. Gumboro disease: Problems of control in Nigeria. Rev Sci Tech. 1982; 1(4): 1163-1166. doi: 10.20506/rst.1.4.95

56. Ann M, Laurence O. Indigenous Chicken Farming Training Manual. Agricultural Research Institute National Animal Husbandry Research Centre, Nairobi Kenya. 2006; 1-58.

57. Office International des Epizooties (OIE). Homepage. Web site. https://www.woah.org/en/home/. HandiStatus visited December 3, 2004. Accessed June 4, 2022.