Serological Detection of Antibodies Against Gamma Coronavirus Infection in Scavenging Village Chickens in Ada’a District, Ethiopia

Infectious bronchitis virus (IBV) is a highly contagious disease of birds causing significant economic losses globally. Unvaccinated scavenging chicken may serve as a source of infection for the commercial poultry farms and hence controlling the disease relying only on vaccinating commercial flocks resulted in poor achievement. The objective of this study was to determine the prevalence of IB among village chickens scavenging in Ada’a district, Ethiopia. The study area was selected for the fact that most of the commercial farms are present and thus characterized by a high density of poultry breeding. Eleven peasant associations (PA) from a total of 22 Ada’a districts were selected purposefully considering their close proximity to the large commercial poultry farms. The study was conducted on a total of 426 adult unvaccinated scavenging village chickens and sera were analyzed by an indirect ELISA test. The overall seroprevalence found in this particular study was 64.79%. Among the villages, the highest seroprevalence of IBV was detected at Yatu and Kality villages reaching up to 93.9% and 92.3% and mean antibody titer of 7097.32 and 8942.95 respectively. Given the high prevalence observed in the study area suggests an urgent need for the development of preventive and control strategies against IB not only for commercial farms but also it should consider the scavenging chicken for better achievement.


Introduction
The total chicken population in Ethiopia is estimated to be 56.5 million with native chicken representing 96.9%, hybrid chicken 0.54% and exotic breeds 2.56% [1]. The most dominant chicken types reared in Ethiopia are local ecotypes, which show a large variation in body position, plumage color, comb type and productivity [2]. However, the economic contribution of the sector is not still proportional to the huge chicken numbers, attributed to the presence of many productions, reproduction and infrastructural constraints [3]. The indigenous flocks are said to be disease resistant and adapted to their environment. However, the survival rates of the Ethiopian indigenous chicks kept under natural brooding conditions considered low. Disease and predators are known to be the major causes of mortality in the country [4,5]. According to Negussie, et al. [6], Newcastle disease accounted for the largest DOI: 10.26717/BJSTR.2021.33.005448 proportion of overall flock mortality to be 57.3% followed by fowl pox 31.6%, coccidiosis, 9.4% and predator loss 1.7%. Another study conducted in all zones found in Southern Ethiopia by Aberra, et al. [7] indicated that the major problems of poultry production in the study were Fowl cholera (28.8%), followed by Newcastle disease (26%), coccidiosis 921.6%), Fowl influenza (15.4%), fowl pox (3.4%), fowl typhoid (3.4%), and salmonellosis (1.4%) [8].
Infectious bronchitis (IB) is a highly contagious disease of birds, caused by Infectious bronchitis virus enveloped, positive-sense, and single-stranded RNA virus, belonging to the genus: Gamma coronavirus, family coronaviridae [9,10]. The genome of the virus is approximately 27.6kb in size encoding 4 structure proteins namely phosphorylated nucleocapsid (N) protein, small envelope protein (E), integral membrane glycoprotein (M), and spike glycoprotein (S) in the order of 5′-Pol-S-3a-3b-E-M-5a-5b-N-UTR3′ [11]. The hypervariable regions have the characteristic of virus neutralization and serotype-specific antigenic determinants responsible for binding to the host cell, neutralization, and immune response [12] and responsible for the pathogenicity of the virus to the host cell [13]. The virus is highly contagious and causes upper respiratory disease in chickens [14] with significant economic losses globally [15]. In hens, it also results in reduction of egg production and poor egg quality. Interstitial nephritis and mortality is also often observed in some strains of the virus [16]. All ages groups can be affected by the disease [17] among which extremely susceptible are chicks of 2 to 3 weeks of age. The overall mortality rates reaches as high as 40%-90% in affected chicks [18]. The disease is transmitted through the air-borne, mechanical transmission between birds, houses, and farms [19] and control of IB is essentially based on the use of live attenuated and killed vaccines. However, the low level of cross protection between vaccines of different serotypes is a major obstacle to IB control (Cavanagh and Naqi, 2003). Moreover, scavenging chicken may serve as a source of infection for the commercial poultry farms and hence controlling the disease only by vaccination makes it more difficult. Therefore, the effects of this disease have been a problem to the poultry sector in Ethiopia in particular and worldwide in general. The objective of the study was to determine the prevalence of infectious bronchitis virus among scavenging village chickens in central Ethiopia using an ELISA assay. The study focused on Ada'a district, areas surrounding Bishoftu, a city where most of the commercial farms present and thus characterized by a high density of poultry breeding.

Study Area
The study was conducted from December 2018 to April 2019 in villages found in Ada'a district of Bishoftu city. The Ada'a district has a total of 22 peasant associations (PAs). Bishoftu is also home to the country's main commercial poultry farms. The area is located at 9 °N latitude and 40 °E longitudes at an altitude of 1850 meters above sea level in central high land of Ethiopia. It has an annual rainfall of 866 mm of which 84% is in the long rainy season (June to September). The dry season extends from October to February.
The mean annual maximum and minimum temperatures are 26 °C and 14 °C respectively, with mean relative humidity of 61.3% [20].

Study Design and Sample Size
Cross-sectional study was conducted on 11 PAs' that were purposefully selected from the total of 24 PAs' considering their geographical location and closeness to the large commercial poultry farms. Hence, a total number of 426 backyard-scavenging chickens were randomly sampled. The sample size was determined using the formula given by Thrusfield (2005). As there was no previous estimated prevalence of IB in scavenging village chickens, 50 % was considered as expected prevalence and desired absolute precision of 5 % at 95% confidence level. Thus, the minimum overall sample size required for the study was 384. However, to increase the precision a total of 426 scavenging village chickens were included in this study. Prior to being assayed, a 1:500 dilution of the samples was made with manufacturer's diluent in a 2-step process. 100µl of each diluted sample was then pipetted into the appropriate well on the antigen-coated plate. One hundred microliters of undiluted positive and negative controls was added to their appropriate wells in duplicate. Thenceforth, the plate was incubated for 30 min at room temperature. Plates were then manually washed five times with deionized water and blotted dry on laboratory tissue paper after washing. Hundred microliters of conjugate was added to all wells and the plate was incubated at room temperature for 30±2min.

Blood Sampling and Storage
Washing and blotting were repeated as described above. One hundred microliters of TMB substrate was added to all wells and incubated at room temperature for 15±1min. To stop the reaction, the test according to [21] IBV antibody titer was classified as low (titer level ranging from 397-1000), medium (1001-5000) or high (titers>5000). The overall district and peasant association level of prevalence was calculated using descriptive statistics.

Results
In the present study, the test carried out on a total of 426 adult unvaccinated scavenging chickens sera from different villages in Ada'a district for infectious bronchitis virus (IBV) prevalence.
Hence an overall seroprevalence of 64.79 (Table 1) (Table 2).  However, Hadipour, et al. [28] indicated that an antibody titer of 1427 was considered as high, because their serological study was conducted on endogenous chicken.
To sum up, the unvaccinated status of the backyard-scavenging chickens in this study would indicate a natural infection with a potentially high virulence strain of IBV. Even if Ethiopia's over 95% of the poultry population is reared under extensive production management system (scavenging), vaccination of these chickens against any poultry diseases is not practiced. The country's current poultry disease prevention strategy is fully targeting the commercial farms. However, this practice encourages easy spread of infectious agents and backyard chickens can also serve as a reservoir for infectious agents [29]. Given the high prevalence observed in the study area suggests an urgent need for the development of preventive and control strategies against IB not only targeting for the commercial farms but also in consideration of the scavenging chickens for a better achievement. We recommend isolation and serotyping of IBV from the study area as a future study.