Demonstration of immune responses and biomarkers:
This may be done either on the basis of sero-conversion study on paired sera samples or by challenge (protection) test. In sero-conversion, the first sera sample is collected at the time of disease and the second after 21 to 30 days of the first collection. A rise of 4-fold or more in antibody titer in neutralization, HI and CF tests are taken as a positive evidence of the involvement of the particular infectious agent in the causation of the disease under study. Challenge tests of recovered and control animals with the known virus strain of the disease being suspected, is a sure method to diagnose disease. However, it requires more time and inputs in terms of animals, sheds, attendants, etc.