Abstract
Egg Drop Syndrome (EDS'76) is one of the most economically important viral diseases of the layer
birds worldwide and including Bangladesh. Objectives of this study were to isolate and
characterize EDS'76 virus from commercial layer farms and preparation of experimental vaccine
using local isolate. To attain the objectives; 74 sera (unvaccinated) and clinical 282 samples (uterine
tissue, egg washing, feces and cloacal swab) were collected from 15 districts. The samples were
processed for virus isolation in embryonated duck egg (EDE); followed by virus identification
through HA, HI, AGDT and PCR (targeting partial Hexon gene; 1901bp). One isolate was selected
for experimental vaccine preparation after the biological and molecular characterization. The oil
based vaccine was evaluated through three successive trial and potency test. Twenty six percent of
the unvaccinated birds (n=74) were found positive for EDS'76 antibody. Six field isolates of
EDS'76v were obtained. Uterine tissue served as the best sample for virus isolation. The isolates
were able to grow in embryonated duck egg (EDE), embryonated chicken egg (ECE); DEF and
CEF cell culture. The isolates failed to adapt in mammalian cell lines (BHK21, Vero, MDCK and
DH82). The EDE was most suitable for EDS'76v cultivation; HA titer was high and consistent. The
isolates were named as BAU EDS'76 isolate I to VI. Isolate III was chosen for downstream
applications, because isolate III showed a high HA tier (14log2) and EID50 of 1010.67/mL in EDE,
while the oral ID50 in susceptible chicken was 106.706/mL. The pathogenicity test proved the
virus to be highly pathogenic and more than 50% egg production drop (p<0.05) was recorded. In
RFLP the BAU EDS'76 isolates produced similar fingerprints (with TaqI andBstY1), while the strain
BC14 produced a distinct additional band (with 1901bp PCRp) when treated with Taql; indicating
the BAU EDS'76 isolates were similar at molecular level and differed from BC14. Full Hexon
sequence showed 100% similarity among the isolates (GenBank acc. no. KU0532 76-81); and the
isolates showed close evolutionary relationship with majority EDS'76v of the world. It is
interesting to note that BAU EDS'76 isolates were almost identical with EDS'76v strain 127 (one
earliest isolate) at the amino acid level (full Hexon); except one amino acid at Hexon protein amino
acid position 810, although there were 3 nucleotide differences in the Hexon gene (2733bp). The
isolates were 96% to 100% similar (Hexon gene) with other EDS'76v of the world. The highest HA
titer obtained in ECE was 6.5log2, in DEF was 9log2 and CEF was 6log2, while in the EDE the HA
titer was 14log2. Our attempts to inactivate EDS'76 virus failed with the given concentrations of
BEI (0.001-0.004mol/L), but formalin (0.12%) completely inactivated the virus. The inactivation
was proved by passaging the (killed) virus suspension in EDE for 4-5 passages and examining the
inactivation by HA and PCR. The sterility, safety and stability were found satisfactory. To evaluate
the potency of the experimental vaccine three successive vaccine trials with different adjuvants
were carried out in seronegative layer birds. One group of birds was vaccinated with a commercial
vaccine and another group of unvaccinated control was kept for comparison. HI antibody titers
were always significantly (p<0.01) higher compared to the non-vaccinated control birds at all
expected points of sera collection and were always much higher (lowest titer was 112.8) than the
protective level (HIU232). When the ELISA titers were compared, the commercial vaccine induced
slightly higher antibody compared to the experimental vaccines; however, the variation was not
significant, while the titers were significantly higher (p<0.05) and protective (≥650), when
compared to the unvaccinated control birds. In the vaccine trial 3; the BAU EDS'76 2M (25%
Montanide®) vaccine was less satisfactory. The HI and ELISA antibody titers from the vaccinated
birds were almost always ranked at number one; except the BAU EDS'76 2M, that failed to
perform adequately at some points. Vaccine trials also proved the BAU EDS'76 5M to be the
number one compared to the other experimental vaccines produced (different adjuvants). So,
isolation of Bangladesh origin EDS'76v along with the production of an inactivated vaccine was
successful.