Serological evidence of egg drop syndrome 1976 in backyard poultry flocks in Southwestern Nigeria

In Nigeria, egg drop syndrome 1976 (EDS’76), unlike other poultry diseases, has been given little attention as a cause of economic losses due to decreased egg production, particularly in backyard poultry flocks. This study aimed to investigate the presence of EDS’76 virus in backyard poultry flocks in Oyo and Osun states, Southwestern Nigeria. Blood samples were collected in 24 farms from 218 apparently healthy, unvaccinated birds which comprised 30 Japanese quails, 75 turkeys, 30 ducks, 57 indigenous chickens and 26 guinea fowls. Indirect ELISA was used to detect anti-EDS’76 virus antibodies in sera from the birds, and poultry owners were interviewed on their purposes with regard to bird raising. Overall, 139 (63.8%) sera were positive for EDS’76 with 26.7% (8/30), 90.7% (68/75), 33.3% (10/30), 89.5% (51/57) and 7.7% (2/26) from Japanese quails, turkeys, ducks, indigenous chickens and guinea fowls, respectively. Some of the farmers practiced placing eggs from guinea fowls under brooding indigenous hens for natural incubation, or sold eggs from turkeys and ducks to commercial hatcheries. Our findings suggest that these bird species serve as reservoirs of EDS’76 virus with the probable involvement of backyard poultry in its transmission, particularly to commercial poultry and other birds in Southwestern Nigeria. Thus, backyard poultry should be included in anti-EDS’76 vaccination schedules in the country.


Serological evidence of egg drop syndrome 1976 in backyard poultry flocks in Southwestern Nigeria
Adebowale Idris Adebiyi 1* Adekemi Florence Fagbohun 2

Summary
In Nigeria, egg drop syndrome 1976 (EDS'76), unlike other poultry diseases, has been given little attention as a cause of economic losses due to decreased egg production, particularly in backyard poultry flocks. This study aimed to investigate the presence of EDS'76 virus in backyard poultry flocks in Oyo and Osun states, Southwestern Nigeria. Blood samples were collected in 24 farms from 218 apparently healthy, unvaccinated birds which comprised 30 Japanese quails, 75 turkeys, 30 ducks, 57 indigenous chickens and 26 guinea fowls. Indirect ELISA was used to detect anti-EDS'76 virus antibodies in sera from the birds, and poultry owners were interviewed on their purposes with regard to bird raising. Overall, 139 (63.8%) sera were positive for EDS'76 with 26.7% (8/30), 90.7% (68/75), 33.3% (10/30), 89.5% (51/57) and 7.7% (2/26) from Japanese quails, turkeys, ducks, indigenous chickens and guinea fowls, respectively. Some of the farmers practiced placing eggs from guinea fowls under brooding indigenous hens for natural incubation, or sold eggs from turkeys and ducks to commercial hatcheries. Our findings suggest that these bird species serve as reservoirs of EDS'76 virus with the probable involvement of backyard poultry in its transmission, particularly to commercial poultry and other birds in Southwestern Nigeria. Thus, backyard poultry should be included in anti-EDS'76 vaccination schedules in the country. through contaminated eggs, sporadic outbreaks have also been traced to contact of chickens with other birds, contaminated fomites and droppings (Clark, 2019). Commercial hens are routinely vaccinated against EDS'76 whereas most small backyard flocks are not (Clark, 2019).
Several studies show that all ages and breeds of chickens are susceptible to EDS'76 although differences in the response may occur (McFerran and Smyth, 2000;Cha et al., 2013). In addition, antibodies have been detected in other species of birds such as cattle egrets, owls, pheasants, geese and Muscovy ducks (McFerran and Smyth, 2000).
In Nigeria, EDS'76 unlike other poultry diseases is given little attention as a cause of economic losses in poultry industry due to decreased egg production. Emphasis has been on the control of viral diseases such as Newcastle disease, infectious bursal disease, infectious bronchitis and avian influenza (Monne et al., 2015;Adebiyi and Fagbohun, 2017) without any attention given to the prevention and control of EDS'76 virus infection particularly in backyard poultry flocks. This is probably because the affected birds are apparently healthy (Ezema et al., 2008). In addition, there are sparse studies reporting serological evidence of EDS'76 in backyard poultry in Nigeria with the majority of such reports carried out in commercial chicken flocks (Nawathe et al., 1980;Elayo et al., 2010;Igbokwe et al., 2020). Hence, this study aimed to investigate the presence of this virus in backyard poultry flocks in Southwestern Nigeria.

■ MATERIALS AND METHODS
This study was carried out in line with the National Code for Health Research Ethics and was approved by the Oyo State Ministry of Health Research Ethics Committee (AD13/479/346).

Farmers, and animal sampling
A total of 218 adult unvaccinated apparently healthy birds from backyard flocks were used in this cross-sectional study. The birds comprised 30 Japanese quails, 75 turkeys, 30 ducks, 57 indigenous chickens and 26 guinea fowls from 24 backyard poultry flocks in Oyo and Osun states, Southwestern Nigeria. The poultry owners were interviewed on the major purposes of bird raising.
About 2 ml of blood was aseptically collected from the jugular or brachial vein of each bird. The blood was centrifuged at 1500 g for 5 min and separated sera stored at -20°C until tested.

Detection of EDS'76 antibodies
The sera were screened using an indirect enzyme-linked immunosorbent assay (iELISA) kit (Green Spring, Shenzhen Lvshiyuan Biotechnology, China) that detects avian EDS'76 virus immunoglobulin antibodies. The iELISA was carried out according to the manufacturer's instructions and the optical density (OD) was read with an ELISA reader at 450 nm (630 nm as reference).
According to the manufacturer's protocol, the positive control average OD 450 nm value is ≥ 0.6, whereas the negative control is < 0.15.
To determine whether a sample is positive or negative, the OD value of a particular sample must be higher or lower, respectively, than the addition of 0.2 to the OD value of negative control; in addition, where OD of negative control is < 0.05, it is calculated as 0.05

Statistical analysis
Results were analyzed with GraphPad Prism version 7.0 (San Diego, USA). Data were subjected to descriptive statistics and one-way ANOVA at the level of significance p < 0.05.

■ RESULTS AND DISCUSSION
Results revealed that out of the 218 sampled birds, 26.7% (8/30), 90.7% (68/75), 33.3% (10/30), 89.5% (51/57), and 7.7% (2/26) were positive for EDSV'76 antibodies in Japanese quails, turkeys, ducks, indigenous chickens, and guinea fowls, respectively (Table I). EDSV'76 seroprevalence in all species was 63.8% (139/218). Although the seroprevalence varied between birds, differences were not significant. In addition, most (45.8%) of the poultry owners kept birds as a source of income, whereas 37.5% and 16.7% raised them for sustenance and mixed purposes, respectively. Other findings were that some farmers placed eggs from guinea fowls under brooding indigenous hens for natural incubation; some sold turkey and duck eggs to commercial hatcheries for poult and duckling production, others were either reluctant to allow sample collection from their birds, others yet outrightly refused.
Since its initial description, EDS'76 has been a major cause of loss in egg production as a result of production of eggs with abnormal shell shapes and sometimes shell-less eggs (Van Eck et al., 1976;Salihu et al., 2010). The detection of EDSV'76 antibodies in apparently healthy birds in this study revealed the circulation of the virus in Japanese quails, turkeys, ducks, indigenous chickens and guinea fowls raised in backyard poultry systems in Oyo and Osun states. Since these birds were unvaccinated against EDS'76, the detected antibodies could only have resulted from seroconversion following natural infection with the virus. Thus, the birds could serve as reservoirs shedding the virus in the environment, playing a crucial role in the epidemiology of the disease. In addition, since the birds showed no clinical signs of disease and were apparently healthy, they may have played a carrier role in the transmission of the virus particularly to commercial poultry (Adebiyi and Fagbohun, 2017) and perhaps to other birds. This finding is consistent with previous reports of infection with EDS'76 in poultry elsewhere (Brash et al., 2009;Cha et al., 2013), including the fact that most backyard poultry are not vaccinated against EDS'76 (Clark, 2019), and that affected birds are apparently healthy (Ezema et al., 2008). The backyard poultry system is usually confronted with poor management, lack of veterinary care and poor disease control which contribute to low productivity and high mortality rates (Salihu et al., 2010). Most of the birds in this study were allowed to scavenge which could allow for unrestricted spread of the disease and contribute to the high prevalence of EDSV'76 antibodies in these birds. Similarly, evidence of EDS'76 infection in free-range flocks that had contact with wild fowls, geese or ducks has been reported (Salihu et al., 2010).
Furthermore, backyard poultry production where the extensive management system is usually practiced, i.e. birds are allowed to roam free, or where different poultry species are raised in the same cages because of the limited breeding space, tend to expose birds to infectious diseases (Adebiyi and Fagbohun, 2017). This possibility of virus transmission from these birds to commercial poultry and other birds may be of veterinary importance considering that passage of EDSV'76 through chickens may result in increased pathogenicity (Brugh et al., 1984).
The practice by some small-hold farmers to place eggs from guinea fowls under brooding indigenous hens may ensure endemicity of EDS'76 within backyard poultry in the study area, as the "virus is excreted through the cloaca and originates in the oviduct and will be present in and on eggs for up to three weeks" (Smyth and Adair, 1988). Furthermore, studies show that "following infection in vivo, there was no excretion until onset of lay, when the unmasking of the virus resulted in virus excretion and rapid spread" (Kaleta et al., 2003;Bidin et al., 2007).
Additionally, some farmers incubated eggs of turkeys and ducks in commercial hatcheries. These practices may cause transmission of the virus to egg-producing flocks and wide spreading of EDS'76 in poultry. "This is due to the presence of virus on the exterior of eggs, leading to contamination of trays and trolleys. In many cases, this equipment is not adequately cleaned or disinfected before being returned from the egg-packing plants to other farms at random" (McFerran and Smyth, 2000).

■ CONCLUSION
The findings of this study revealed that egg drop syndrome '76 virus circulates in Japanese quails, turkeys, ducks, indigenous chickens and guinea fowls in Oyo and Osun states, Southwestern Nigeria, indicating that these bird species serve as reservoirs of EDS'76 virus. They underscore the importance of the routine surveillance for EDS'76 in different avian species. Backyard poultry production, being an important economic activity for small-hold farmers in Southwestern Nigeria, should be given adequate attention in disease control and prevention and considered in EDS'76 vaccination schedules in the country. Also, there is a need to include backyard poultry for further studies to determine the genotype of EDSV'76 circulating in the country.

Conflicts of interest
The study was carried out without conflicts of interest.

Author contributions statement
AIA conceived and designed the study. AIA and AFF collected samples. AIA conducted the experiment. AIA and AFF analyzed the results. AIA drafted the first version of the manuscript. Both authors read and approved the manuscript. Algunos granjeros colocaban los huevos de pintada bajo gallinas autóctonas para su incubación natural o vendían los huevos de pavo y pato a incubadoras comerciales. Nuestros resultados sugieren que estas especies de aves sirven de reservorio del virus EDS'76 y que es probable que las aves de corral de las aldeas estén implicadas en su transmisión, especialmente a las aves de corral comerciales y a otras aves del suroeste de Nigeria. Por lo tanto, las aves de corral de las aldeas deberían incluirse en los programas de vacunación contra el EDS'76 en el país.