Introduction

Poultry production is an important component of livestock subsector in Nigeria involving thousands of birds that provide employment to both rural and urban dwellers among other advantages (Obi et al., 2008). However, one of the major impediments to this industry are infectious diseases among which egg drop syndrome ’76 is the one posing a serious threat to the layer industry worldwide (Van Eck et al., 1976). The disease affects many species of birds. Although disease outbreaks have been recorded only in laying hens, it is reported that natural hosts for the EDS ‘76 virus are ducks and geese (Bidin et al., 2007). It is caused by Duck adenovirus A, a member of the genus Atadenovirus and the family Adenoviridae (Hess et al., 1997, Benko et al., 2005). The virus replicates to high titres in duck kidney, duck embryo liver, duck embryo fibroblast and chick liver cell cultures (Adair et al., 1986, Yamaguchi et al., 1981). Duck adenovirus A can be transmitted vertically in eggs (Dhama et al., 2011) and by horizontal and mechanical routes of transmission (Abdul-Aziz and Hassan, 1995, McFerran and Adair, 2003; Balamurugan and Kataria, 2004). Iatrogenic transmission is possible by needles (McFerran, 2003, McFerran and Adair, 2003). The disease is characterized by production of thin and soft-shelled eggs and loss of shell colour in pigmented eggs (McFerran et al., 1978).

This disease is of primary economic importance as the affected birds do not appear sick (Van Eck, 1980, CFSPH, 2006). Consequently, diagnosis to detect the viral antigen or antibody is critical to an effective control measure. Serological evidence of EDS ‘76 virus have been demonstrated in some avian species in Nigeria (Oyeduntan and Durojaiye, 1999, Abubakar et al., 2008, Ezeibe et al., 2008, Ezema et al., 2008, Salihu et al., 2010). However, there is paucity of information on the status of the disease in both commercial and local chickens in the study area. Therefore, this research is designed to serologically screen for antibodies against the virus and subsequently know the viral titre- information vital for the disease prevention and control.

Materials and Method

Study Area

This study was carried out in Maiduguri Metropolis, the capital city of Borno State in the North-eastern Nigeria. Borno State lies between latitude 10⁰N and 13⁰N and longitude 12⁰E and 15⁰E. The State has an area of about 69,436 KM² (Encyclopedia Britannica, 1998).

Sampling

A nonprobability sampling technique was used to collect a total of 154 samples from poultry comprising of 32 samples from exotic chickens in university of Maiduguri farm (Animal Science Research Farm); 122 from live bird market (LBM) Maiduguri consisting of 119 from local chickens and 3 samples from guinea fowl.

Blood Collection

About 2ml of blood was collected from each bird in commercial farm through the brachial vein using 23G sterile hypodermic needles and 2ml syringes; while samples from live bird market (LBM) slaughter slabs were collected into vacutainer tubes at the point of slaughter. The samples were transported in a cold park from the commercial farm and LBM to the virus laboratory research unit of the Department of Veterinary Microbiology, Faculty of Veterinary Medicine, University of Maiduguri. The blood was allowed to clot at room temperature from which sera were obtained through centrifuging at 2000rpm for 5minutes. The harvested sera were stored at -20^oC until further processing.

Source of Egg Drop Syndrome 1976 Antigens

Standard EDS ‘76 antigen and antiserum were obtained from National Veterinary Research Institute (NVRI), Vom, Plateau state, Nigeria and used for the detection of EDS ‘76 antibodies. The validity and titre of the EDS ‘76 antigen was determined using haemagglutination (HA) test as described by OIE (2009). The sera were assayed by haemagglutination inhibition (HAI) test as described by OIE (2009).

Preparation of 1% chicken Red Blood Cells

Four mililitres of blood was collected from an apparently healthy local cock and was pooled in an equal volume of Alsever’s anticoagulant solution. The red blood cells (RBC) were washed three times in phosphate buffered saline (PBS) (pH 7.2) by centrifuging at 2000rpm for 5 minutes. One percent RBC suspension was made by adding 1ml of washed RBC to 99ml of PBS.

Determination of Titre of Egg Drop Syndrome 1976 Antigens

Haemagglutination test was carried out according to the method described by OIE (2009). Briefly, twenty five microlitres of PBS was dispensed into each well of “V” bottom microtitre plates except the first well. Twenty five microlitres of antigen suspension in PBS was placed into the first well. A two-fold dilution was done by taking 25ul from the first well into the second well and mixed, the 25ul from second well transferred to the third well and continued till the eleventh well. A 25ul of 1%chicken RBC was dispensed into each well. These were mixed by gently tapping the plate and then allowing the RBC to settle for about 30-40 minutes at room temperature. The HA was determined by tilting the plate and observing for the presence or absence of tear-shaped streaming of the RBC. The titration was read to the highest dilution giving complete HA (no streaming); this represents 1 HAU.

Determination of Titre of Egg Drop Syndrome – 76 Antibodies

The antibody titre was determined by haemagglutination inhibition (HI) test as described by Allan and Gough, (1974). Twenty five microlitres of PBS was dispensed into each well of a plastic “V” bottom microtitre plate. Twenty five microlitre of serum was dispensed into the first well to the eleventh well. Two-fold dilutions of the 25ul volumes of the serum were made across the plate from the first well. Four HAU of the antigen suspension in 25ul was added to each well and left for a minimum of 30 minutes at room temperature. A 25ul of washed chicken RBC was added to each well and after gentle mixing, it was allowed to settle for about 40 minutes at room temperature. The HI titre was the highest dilution of the serum causing complete inhibition of RBC agglutination by the 4HAU antigen. Wells considered positive to HI were wells in which RBC stream at the same rate as positive control wells (wells A12 – H12) and the result were expressed in Log₂ (OIE, 2012).

Data Analyses

The data obtained from serology for EDS ‘76 antibodies were analyzed by cross tabulations and descriptive statistics using GraphPad InStat 3. The frequency, mean standard error, mean antibody titre and confidence interval of the cross tabulations were analyzed. Values of p<0.05 were considered significant.

Result and Discussion

A total of 154 sera samples were tested for EDS ‘76 antibodies and an overall sero-prevalence of 24.03% was obtained. Samples from commercial farm have a percentage sero-prevalence of 12.50% while those from live birds had a percentage sero-prevalence of 27.05%. The overall mean EDS ‘76 antibody titre in the birds sampled in the two different sample units was 17.536±3.211log₂. The highest mean EDS ‘76 antibody titre was 17.508±3464log₂ from live bird market. The lowest mean EDS ‘76 antibody titre was 16.000±7.603log₂ in birds from commercial farm (Table 1). The findings of this work have demonstrated the presence of EDS ‘76 virus antibody in chickens in the study area. The commercial farm tested in this study did not vaccinate their birds against egg drop syndrome 1976; therefore detection of antibodies could be as a result of natural infection. Similarly, local chickens which are mostly kept in rural household areas according to Abubakar et al. (2008) are rarely vaccinated against EDS ‘76 virus; therefore detection of antibodies against EDS ‘76 in this study could also be as a result of natural infection. This is in agreement with findings of similar works carried out by various authors (Nawathe and Abegunde, 1980; Abubakar et al., 2008; Ezema et al., 2008; Sanda et al., 2008; and Salihu et al., 2010) who reported the presence of EDS ‘76 antibodies in different parts of Nigeria.

The higher sero-prevalence of 27.05% (Table 1) obtained from live birds market could be due to multi-species, multi-age, multi-type of birds kept and sold together in the market, consequently, this may ease lateral spread of EDS ‘76 virus from an infected bird to a susceptible ones, although reported to be slow and intermittent, taking weeks to achieve as reported by Raj et al. (2007).

Table 1: Distribution of EDS-‘76 in relation to sampling units

P ˂ 0.000; Key: CI = Confidence interval

In this study, a lower sero-prevalence of 12.50% and a mean antibody titre of 16.000±7.603log₂ (Table 1) is obtained when compared to 38.0% obtained by Ussa, (2015). This might be because the chickens sampled were vaccinated against EDS ‘76 virus. However, samples from this study were collected from farm that lacked history of vaccination against EDS ‘76 virus. Therefore, antibodies detected from this study might be as a result of natural infection.

Based on the type of poultry sampled (Table 2), chickens had the highest number of positive cases with a sero-prevalence of 24.50% and mean antibody titre of 17.536±3.211log₂ compared to guinea fowl which has no positive case. This is of economic significance as a drop in egg production with poor egg quality are the usual clinical signs in such birds and will therefore affect farmers income (CFSPH, 2006). This is higher than the findings of Abubakar et al. (2008) who worked in North Eastern Nigeria and obtained a sero-prevalence of 17.65% in chickens, this could be because most chickens sampled were from live bird market where there could be cross infection between different poultry species.

Table 2: Distribution of EDS-‘76 in relation to species of poultry sampled

P ˂ 0.0001; Key: CI = Confidence interval

No antibody was detected from samples collected from guinea fowl, this does not agree with the finding of Abubakar et al. (2008) who obtained a seroprevalence of 25.23%. The reason for the difference could be attributed to the number of samples collected. Fewer samples were collected in this study (3 samples) while Abubakar et al. (2008) collected 107 samples in their study.

Seroprevalence of 27.73% and 12.50% (Table 3) was obtained in local and exotic chickens respectively. High prevalence was detected in local chickens; this result does not correlate with the finding of Oyeduntan and Durojaiye (1999). Local chickens are known to roam about, may have been infected by field virus. A significant difference (P<0.0001) was observed between local and exotic chickens. This is of great concern as they could serve as source of infection to commercial laying birds.

Table 3: Distribution of EDS-‘76 in relation to type of chickens sampled

P ˂ 0.0001; Key: CI = Confidence interval

Conclusion and Recommendations

This study shows that the overall seroprevalence of EDS ‘76 in chickens in both commercial and LBM is 24.03% with an overall mean antibody titre of 17.536±3.211log₂. Therefore, it is recommended that further studies should be conducted to confirm the virus through isolation and characterization. In addition, EDS ‘76 should be included as a differential to cases with history of drop in egg production. Furthermore, vaccination against EDS ‘76 virus should be included in the routine vaccination schedule of poultry in Maiduguri and environs.

Acknowledgments

Authors are thankful to the staff of the Department of Veterinary Microbiology, University of Maiduguri for their technical assistance. Authors are also grateful to the staff of University of Maiduguri poultry farm for their cooperation and assistance during sampling.

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