Development Of In-House Indirect Elisa Kit Method For The Serological Detection Of Antibodies Against Fowl Cholera In Chickens

Abstract

Fowl Cholera Is An Infectious Disease Of Birds Caused By Gram Negative Pasteurella Multocida.It Is Endemic To Ethiopia And Has Been Causing Major Economic Losses Due To Mortality And Loss Of Production. Owing To The Lack Of Appropriate Diagnostic Methods, The Exact Circulation Of Fowl Cholera In Ethiopia Has Been Not Well Studied And Documented. Thus, The Present Study Is Aimed To Develop An In-House Indirect Enzyme Linked Immuno-Sorbent Assay (Elisa) Kit Method That Can Detect Antibodies Produced Against Fowl Cholera. For This Purpose A Freeze Dried Fowl Cholera Seed Obtained From National Veterinary Institute?��?S (Nvi) Stock Was Allowed To Grow On Tryptose Media. The Purity And Identity Of The Pasteurella Multocida Wasconfirmed After Being Subjected To Gram Stain And Polymerase Chain Reaction Respectively.The Bacterial Cells Were Lysed By High Speed Centrifugation Through Washing By Phosphate Buffer Saline And The Antigen Was Purified And The Protein Content Was Estimated Using The Bicinconic Acid Assay Method. In This Study All Data Were Analyzed By Microsoft Excel Sheet Of Window 10. The Working Antigen Concentration, Serum Dilution And Secondary Antibody Dilutions Were Optimized At 1.5mg/Ml, 1:600 And 1:2000, Respectively. The Incubation Time And Temperature Were Optimized At 250c (Room Temperature) For 30 Minutes. On The Other Hand, The Cut-Off Value Of The In-House Elisa Test Was Determined To Be 0.28. The Test Sensitivity And Diagnostic Specificity Of This Assay Was Found To Be 89.9% And 95%, Respectively. The Intra And Inte