Elaboration of diagnostic means for detection antibodies to Newcastle disease virus

ObjectiveA test kit for the detection of antibodies to Newcastle disease virus(NDV) based on haemagglutination inhibition (HI) assay has beendeveloped and introduced into practice for the first time in Ukraine.IntroductionNewcastle disease (ND) is the most important infectious viraldisease of poultry. The world-wide economic loss from it is 2-3billion USD per year. ND is reportable to the World Organizationfor Animal Health (OIE). ND is caused by virulent strains of avianParamyxoviruses belonging to type 1. Industrial poultry farmingis rapidly developing in Ukraine. Ornithological fauna of Ukraineincludes about four hundred species of birds, 207 of which nestwithin its borders. The territory of Ukraine transits 3 out of 14transcontinental global migration flows. The wild birds are themain natural reservoir of ND agents. It is necessary to control theintensity of post-vaccination immunity in poultry and the timing ofrevaccinations. OIE recommends enzyme linked immunosorbentassays (ELISA) and HI test for these purposes [1]. However, it shouldbe noted that HI test, possessing high specificity and sensitivity, ismuch cheaper. Therefore, it is the excellent means for ND timelysurveillance.MethodsDuring the development of a new diagnostic kit, we used thereference strain “La-Sota”, which was obtained from the NationalCenter of Microorganism Strains of Ukraine. We have producedhaemagglutinating antigen using embryonated SPF fowl eggs and10-11 day incubation. A dilution of the virus was inoculated in0.1 ml volumes into the allantoic cavity and incubated at 35-37° Cfor 80-96 hours. For the purpose of NDV inactivation, we usedaminoethyleneimine at the final concentration of 0.1%. Positive serumwas prepared by immunizing 60-day-old chickens with live virus onceand by inactivated virus twice with an interval of 2 weeks. Negativeserum was obtained from healthy birds that did not contain antibodiesto NDV. The investigated blood sera were inactivated by heating(56 C/30 minutes). Samples of 1% suspension of chicken erythrocytesin phosphate buffered saline (pH 7.0-7.1) were used in HI tests.ResultsThe specific haemagglutination activity of the obtained antigenamounted to 10-11 log2.The test was performed using the 4HA unitsof the antigen. Positive control serum activity was in the range of 7-9log2. Negative control serum did not give results of more than 2 log2.The estimation of the quality indexes of the components of thediagnostic test-kit was performed using harmonized methods. In orderto examine sensitivity and specificity of HI test kits, antigens and serafrom commercial diagnostic kits were used. Also, certified negativecontrol serum and samples of International Standard sera were used,which were obtained from reference laboratories, namely againstthe following pathogens: Avian Influenza A (H5), Avian InfluenzaA (H7), Egg Drop Syndrome'76 Virus, Paramyxoviruses of 2 and3 serotypes, Reovirus, Avian Infectious Laryngotracheitis, AvianInfectious Bronchitis Virus, Mycoplasma gallisepticum, and NDV.In order to ensure a high degree of specificity for the antigen,special attention was given to the selection of a stabilizer for freeze-drying (the subject of a patent).Comparison between the national diagnostic test kit for HI andcommercial ELISA kit (IDEXX) in the evaluation of humoralimmune response to ND in vaccinated chickens was investigatedby examining of serum samples (n=152). Statistical analysis of datashowed that the correlation coefficient for the results of both tests was0.92. The relative sensitivity of HI test kit was 93.5% and the relativespecificity - 91.5%.The developed test kit was successfully used for the examinationof field samples. We developed regulatory documents, completed theprocedure of validation and registration in Ukraine of the commercialHI test kit for the detection of antibodies to NDV.ConclusionsThe use of the national standardized diagnostic test kit based onHI for detection of antibodies to NDV allows assessing the post-vaccination antibodies level that helps to maintain the disease-freestatus of the Ukrainian poultry industry with regard to ND