Standardization of the microtitre agglutination test for seromonitoring of haemorrhagic septicaemia vaccination. Journal of Immunology and Immunopathology

Abstract

Microtitre agglutination test (MAT) is a cheaper, easier and faster method to detect agglutinins in sera of immunized animals compared to ELISA and IHA. However, the test has lower sensitivity, due to imperfect visualization of colorless agglutinated mass and autoagglutinating property of whole cell antigen of Pasteurella multocida. To improve visualisation the colored antigen was prepared using crystal violet and brilliant green dye. Further, to reduce autoagglutinability various parameters were examined viz., (a) Media- nutrient broth (NB), tryptic soya broth (TB), brain heart infusion broth (BHI), and fermenter medium (FM) (b) Duration of growth- 12, 18 and 24 h (c) Inactivating agents - heat and formalin. Maximum autoagglutination (AAG) was recorded in BHI followed by, FM and NB while least in TB. Effect of duration on growth was noticed in BHI, TB and FM where minimum AAG activity existed at 24h culture and maximum at 18 h culture (BHI and TB) and 12 h (FM). However, the difference was less significant compared to medium of growth. Heat inactivation was superior as 0–30 % AAG activity (depending upon media and duration) was recorded, compared to formalin treatment where it ranged from 20–60 %. Finally, the colored antigen was prepared by heat inactivation after growth in NB. The MAT titre was estimated on 27 sera, collected from three buffalo calves after vaccination with oil adjuvant vaccine at different time intervals, and correlation was drawn with ELISA and IHA titre. The MAT titre has 90% correlation with ELISA compared to 69% between ELISA and IHA. However, MAT and IHA were only 37% correlated. The result showed that MAT can be used as a routine test to detect antibody level in laboratory as well as in field conditions.