Development of recombinant sialidase (NanH) protein-based Indirect-ELISA for epidemiological survey of anti-Pasteurella multocida antibodies in bovines

Recombinant NanH protein based Indirect-ELISA for HS in bovine

Abstract

Haemorrhagic septicemia (HS) is a highly contagious and fatal disease of cattle and buffaloes and causes major economic losses to farmers. The indirect hemagglutination (IHA) test has been used to detect a specific antibody against P. multocida, but the test has low specificity for sero-diagnosis of HS. Therefore, development of a rapid, highly sensitive and specific serological test is a prerequisite for the detection of antibodies against HS. This study was conducted in order to develop and evaluate an in-house ELISA method using recombinant antigens for detection of antibodies against P. multocida in bovines. In this study, the NanH gene from P. multocida B:2 strain P52 was cloned and the recombinant mature protein with a C- and N-terminal truncation, was produced as a fusion protein (∼63 kDa) in Escherichia coli. The immunogenic potential of purified rNanH-Tr was assessed by the western blot method using specific anti-rNanH-Tr antibody responses in sera collected from immunized rabbits. An indirect-ELISA based on rNanH-Tr was developed and optimized. Furthermore, the rNanH-Tr ELISA was applied to screen bovine serum samples (n=250). The receiver operating characteristic curve analysis for the detection of anti P. multocida specific antibodies indicated a diagnostic specificity of 86.2 (CI- 73.26% to 96.80%) and specificity of 80.0 (63.06% to 91.56%). No cross reactivity was noted with antibodies against other bovine diseases (e.g., foot-and-mouth disease and brucellosis). Screening of random bovine serum samples showed a 22% sero-positivity for anti P. multocida specific antibodies.