doi:10.5455/vetworld.2013.350-353 Incidence of egg drop syndrome – 1976 in Namakkal district,

Aim: To know the magnitude of influence by Egg Drop Syndrome – 1976 (EDS –'76) virus infection in causing drop in egg production in and around Namakkal. Materials and Methods: A total of 150 cloacal swabs and 15 pouch shell glands (uteri) homogenates from 15 poultry farms in and around Namakkal area were used for virus isolation. Three numbers of 10 –day- old embryonated duck eggs were used for the inoculation of each suspected material for virus isolation. The isolate was identified by HA property, by specific inhibition of HA and by AGPT using hyperimmune serum raised against reference EDS –'76 virus strain 127. Results: Out of samples from 15 farms only one isolate (6.6%) was obtained from poultry farm No.5. Conclusion: The results of the present study revealed that the EDS –'76 virus influence in causing drop in egg production in this area to be minimal.


Introduction
Isolation and identification of the virus from commercial layer farms at Namakkal.Egg drop syndrome -1976 (EDS -'76) is a major cause for loss of egg production upto 40 per cent and

laying of thin shelled and shell less eggs by apparently
Samples: A total of 150 cloacal swabs and 15 pouch healthy birds.The syndrome is caused by an shell glands (uteri) [10] homogenates from 15 poultry adenovirus with transmission occurring vertically and farms were collected in Hank's balanced salt solution horizontally.It has been reported to affect a wide range (HBSS) with antibiotics from apparently healthy birds of birds including turkeys and layers and is a major showing sudden drop in egg production in Namakkal constraint to the profitability of egg production in both area.The samples were transported at 4°C to the commercial and village laying birds [1].It was first laboratory for further work.reported in Netherlands [2] and has since been reported from numerous countries world-wide including Ethical approval : The permission was accorded by the goslings in Hungary [3].Serological evidence of Institutional Animal Ethics Committee to collect the EDS'76 virus in turkeys [4] and free-range chickens is sample from live birds.also observed as a concern in Nigeria [5,6,7].In India Embryonated duck Eggs: Duck eggs were purchased the disease was reported in the year 1984 [8].The drop from the local farmers, cleaned with anti septic lotion in egg production in layer birds has become a major (1% savlon) and incubated at 37°C with proper turning.concern in India [9] and particularly at Namakkal due th Fertile eggs were selected on 10 day and the viral to the enormous economic burden faced by the farmers.
samples were inoculated in ten-day-old embryos The loss incurred by poultry industry due to reduced through allantoic cavity route.productivity, culling and cost of medicine is considered Processing of cloacal swabs: The cloacal swabs [11] to be often greater than loss due to mortality.Earlier were collected in HBSS directly from live birds workers studied the multiple etiology of "Egg drop manifesting the sign of drop in egg production.The syndrome" in this area.But the magnitude of influence collected samples were homogenized and centrifuged by EDS -'76 virus alone has not been studied.Hence at 2500 rpm for 15 minutes at 4°C.The supernatant a comprehensive research study was formulated to was collected and filtered through a disposable syringe assess the incidence of EDS -'76 virus infection by filter of pore size 0.2m.
Inoculation into duck embryo: Three numbers of 10 -day-old embryonated duck eggs were used for the inoculation of each suspected material for virus positive samples).After 20 minutes 25 l of 0.8 per cent isolation.The eggs were inoculated with 0.2 ml of v/v suspension of chicken erythrocytes was added.inoculum via allantoic cavity route, sealed and The plates were left to stand for 45 min at room incubated at 37°C.The eggs were candled twice daily temperature before the results were read.The plates and deaths within 24 h were considered non-specific were observed for inhibition of the haemagglutinating and were discarded.On the fifth day post inoculation, activity by EDS -'76 specific antiserum.Those the eggs were chilled overnight at 4°C and opened to samples which showed specific HI was taken as collect the allantoic fluid.The harvested allantoic fluid positive for EDS -'76 [12].was clarified at 2500 rpm for 20 min and screened for Agar gel precipitation test: Glass slides were cleaned haemagglutination (HA) activity.A minimum of 3 with alcohol and air dried.The slides were overlaid blind passages for each sample were made before with 4 ml of 1 % agar gel and allowed to solidify at declaring it as negative for the presence of viral agent room temperature.After solidification, the slides were [12].
kept at 4°C for 30 min in a humid chamber.The gels were immersed in log2 10 /25 l and Freund's complete adjuvant by intra normal saline for 48 hours with three changes followed muscular route at the dose rate of 0.6 ml / cockerel.The by distilled water for 24 hours to remove the unprecisecond injection of 0.5 ml. of infected allantoic fluid pitated proteins.The gels were covered with two layers alone was administered by intravenous route on the th of wet Whatman No.1 filter paper.Then finally the gels tenth day and the serum was collected on the 28 day were dried in an incubator overnight.The dried gel was after the first injection.The serum samples from the 6 stained with coomassie brilliant blue stain for 30 min cockerels were pooled, inactivated at 56°C for 30 min and then destained with destaining solution until the and stored at -20°C until use.
precipitation lines were clearly visible.Haemagglutination test (HA) : The HA test was performed

Results
by the microtitre method in V bottom microplates.Two fold dilutions of the virus were made in 25 l Isolation and Identification: A total of 150 cloacal volumes, starting from 1:2 dilution using normal swabs and 15 pouch shell glands (uteri) homogenates saline.To each dilution of the virus, an equal volume from 15 poultry farms in and around Namakkal area of 0.8 percent washed chicken erythrocytes were added were used for virus isolation.Cloacal swabs and shell and incubated at room temperature for 30 min.The gland homogenates from poultry farm No.5 yielded reciprocal of the highest dilution of the virus showing haemagglutinating virus in embryonated duck eggs.complete HA was taken as titre [12].
The isolate was identified as EDS -'76 virus by specific inhibition of the HA and by AGPT using antiserum harvest.

Haemagglutination-inhibition test: This test was
Hyper immune serum : The hyperimmune serum raised conducted to find out the specificity of freshly collected against reference EDS -'76 virus strain 127 showed a allantoic fluid and supernatant of the processed tissue HI titer of log 9 per 25 l.
2 samples using EDS -'76 antiserum.This test was performed in V bottom microplates.Two fold dilutions Haemagglutination test (HA): Micro HA test were of 25 l of known EDS -'76 antiserum (log 9) were performed for the EDS -'76 isolate and the HA titer for 2 made in sterile physiological saline and incubated with each subsequent passage is tabulated in table I. HA 25 l of virus containing 4 HA units of the virus (HA titer at fifth passage level was log 15 per 25l. Haemagglutination inhibition test : The isolate was specifically inhibited by reference antiserum (strain confirmed as EDS -'76 by specific inhibition of HA 127) but not with hyperimmune serum to NDV, thus using hyper immune serum.The isolate was subjected confirming the isolate as EDS -'76 virus.In AGPT to HI at the level of second passage.
there was one intense precipitation line between the isolate and hyperimmune serum as that of positive Agar gel precipitation test (AGPT) : The isolate showed control indicating the EDS -'76 isolate [13].one intense precipitation line against hyper immune Only one isolate was obtained after screening the serum after 24 h of incubation at 37° C as that of a line samples from 15 poultry farms in and around observed against reference antigen (positive control).
Namakkal.This result is in agreement with the findings Known negative control in the form of uninfected duck of earlier workers [28] who had stated that the New allantoic fluid and suspected samples did not reveal Castle disease (29.71%), aflatoxicosis (3.62%) and any precipitation line.
Infectious Bronchitis Virus (2.17%) as the major cause

Discussion
of drop in egg production rather than EDS -'76 (1.45%) in this area.The syndrome of egg drop is caused by multiple etiologies such as Newcastle disease virus (NDV),
One isolate of EDS -'76 virus was isolated and coli, Pasteurella multocida and aflatoxicosis.Recent identified by its HA property, by specific inhibition of development in disease management, absence of the HA and by AGPT using anti serum raised against specific symptoms associated with these diseases as reference EDS -'76 virus strain 127.The HA titer of the well as protective vaccination of the flocks with isolate initially was log 4 per 25 l and increased rapidly 2 improved vaccines and vaccination schedules ruled out during every successive passage in duck embryo.Agar their occurrence considerably.Several workers later gel precipitation test revealed one intense precipitation found that EDS -'76 virus was a major etiological agent line between the isolate and hyper immune serum.The for reduced egg production during the peak production present study revealed that the EDS -'76 virus period in commercial birds.Hence a study was influence in causing drop in egg production in this area undertaken to investigate the magnitude of egg drop to be minimal.This conclusion is on the basis of syndrome associated with EDS -'76 virus in and materials that are collected in and around Namakkal.around Namakkal.
Attempts were made to isolate the EDS -'76 virus excretion in faeces is maximum during the first 15 days shellless eggs associated with appearance of precipitins to of infection after that the development of antibody adenovirus in flocks of laying fowls.Avian Pathol., 5 : 261renders the isolation difficult [25].In present study the at right time and placed in 1X 3. Ivanics E, Palya V, Glávits R, Dán Á, Pálfi V, Révész T and tissue culture growth medium with antibiotics and Benkö M. (2001) The role of egg drop syndrome virus in acute respiratory disease of goslings.Avian Pathol., 30:201transported to the laboratory at 4° C [20,26].208.The HA titer of the isolate initially was log 4/25 l, 2 4. Bidin, Z., Lojkic, I., Mikec, M. and Pokric, B. (2007) but later after every successive passages in duck Naturally occurring Egg Drop syndrome infection in embryo the HI titer increased rapidly [27].The HA turkeys.Acta Veterinary Brno 76: 415-421. 5. Elayo, S.A., Mamuela, J.T., F. Chukwuemeka, O.F., Okpabi, activity of the local EDS -'76 virus isolate was
2Lesions in duck embryos: There was no mortality