DEVELOPMENT OF USER FRIENDLY DIAGNOSTICS AND CELL CULTURE ADAPTED VACCINE CANDIDATE FOR DUCK PLAGUE
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Date
2016-05
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Assam Agricultural University, Khanapara, Guwahati
Abstract
Duck plague or duck viral enteritis is an acute and contagious viral disease of ducks, geese swan and other species of the order Anseriformes. The disease is responsible for significant economic losses in duck husbandry due to heavy mortality, condemnation and decrease in egg production in duck. Besides clinical and postmortem findings, laboratory diagnosis is essential to confirm the disease in cases of outbreaks. Conventional diagnostic methods are labour intensive, time consuming and less sensitive. There is an urgent need for development of rapid, sensitive and cost effective in house as well as user friendly diagnostic test so as to confirm the disease at clinical phase in the field itself. Again, vaccination is the only available option for prevention and control of the disease.
The present study was undertaken to develop user friendly diagnostics and potent cell culture adapted vaccine to control duck plague virus (DPV) infection. During the study period a total of 29 outbreaks of duck plague were attended. Various clinical and post mortem samples were processed for detection of viral DNA by PCR and viral antigen by S-ELISA. Serum samples collected from different districts were tested for presence of antibody by I-ELISA and Dot-ELISA. Duck plague virus was isolated in duckling, duck egg and DEF primary cell culture from the field tissue sample. Sequence and phylogenetic analysis of local DPV isolate and a vaccine strain was done to see the circulating virus in Assam. A cell culture adapted vaccine was developed, and safety and potency test was conducted to see the efficacy of the vaccine.
In sero-epidemiological study, among the 445 serum samples tested by I-ELISA 348 (78.20%) were found positive for DPV antibody, however in Dot-ELISA 149 (33.48%) were found to be positive. A total of 380 samples were collected from clinically affected (107) and dead ducks (273). S-ELISA showing positive results in 25 (23.36%) in clinical samples and 188 (68.86%) post mortem samples, however in PCR a total of 231 (84.61%) post mortem samples and 68 (63.55%) clinical samples showed positive for duck plague virus specific nucleic acid. The present study showed that PCR is the suitable and reliable test for detection of duck plague virus. Among different tissue samples collected from dead birds, liver and spleen were found to be most suitable. In cases of clinical samples ducks whole blood was found to be preferred sample than the cloacal swabs and tracheal swab. However, due to simplicity of collection, cloacal swab may be the choice of sample from large flock.
Reviving of field isolate in primary host followed by isolation in duck embryo and duck embryo fibroblast (DEF) cell culture made 100% recovery of virus. However, the DEF cell culture was found to be more suitable than embryonated duck egg for isolation. Sequence and phylogenetic analysis of the local isolates and a vaccine strain showed a close relationship among the local isolates with the vaccine strain. Local isolates also showed a significantly high degree of sequence identity with other DPV isolates from China, Vietnam, Korea and Germany.
A highly virulent local strain was selected as vaccine candidate and adapted in CEF primary cell culture, whereas standard vaccines strain was adapted in CEF primary cell culture as well as in vero cell line. In safety and potency test of the CEF cell culture adapted DPV vaccine strain, ducklings were vaccinated with 0.5 ml of 103, 104 and 105 TCID50/ml dose of vaccine virus. All doses of vaccine were found to be safe and
optimum for eliciting protective immunity in the vaccinated ducklings, and conferred 100% protection of ducklings challenged with 1 ml of 100 DID50 of virulent DPV. Thereby, the minimum dose containing 1 ml of 103 TCID50/ml of vaccine virus can be considered as optimum vaccine dose for providing protection, which can be further used for protection of ducks from duck plague.
The present study clearly showed that duck plague is endemic in Assam causing high mortality in ducklings as well as in growing and adult ducks. Diagnostic tests I-ELISA, S-ELISA and Dot-ELISA along with molecular technique PCR could be companion diagnostic tools for confirmation of DPV as well as assessment of virus antibody. Significantly development of cell culture adapted vaccine and conferring of 100% protection can be an achievement of the study.
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