Evaluation of Indigenous Latex Agglutination Assay based on Recombinant Pasteurella Lipoprotein E (rPlpE) As Antigen for Detection of Anti Mannheimia Haemolytica - IgG Antibodies

Abstract

Mannheimia Haemolytica (M. haemolytica) is an organism causing pneumonia in ruminants. M. haemolytica causes severe economic losses to the global livestock industry. Several diagnostic methods have been developed, including bacterial culture, bacterial DNA detection and serological assays. Diagnosis of M. haemolytica is based on the bacteriological methods such as isolation of the microorganisms from clinical samples. Available methods are time consuming and not easy to perform. Serological tests based on recombinant proteins may provide higher sensitivity and specificity than culture tests. There is a need for new diagnostic methods to detect M. haemolytica -specific antibodies. In this study, a latex agglutination test (LAT) was developed based on recombinant outer membrane pasteurella lipoprotein E (rPlpE) for detecting specific antibodies against M. haemolytica. Expressed recombinant PlpE was coated with latex particles for a latex agglutination test. The recombinant PlpE was able to detect anti-M. haemolytica IgG in positive sera, but showed no immunoreaction with Pasteurella multocida and negative samples. These results suggest that the rPlpE can be used to detect the specific anti Mannheimia Haemolytica - IgG Antibodies. Because the recombinant proteins can be produced efficiently and are inexpensively, their use in diagnostic kits such as LATs as reagents can reduce the cost of them. This rapid and specific anti M. haemolytica antibody detection method using recombinant proteins can save cost and be widely applied for efficient and practical detection of. M. haemolytica.

Keywords: Antibody; Latex agglutination test; PlpE; Recombinant protein; Mannheimia Haemolytica.

Figure 1

PCR amplification of the Lkt and HP genes. The 100 bp DNA Ladder (Lane M) was used as a reference for the amplicons of Lkt and HP (206 bp and 90bp) (Lanes 1,3,4,5,6,7). Lanes 2 and 8 were used as negative controls.

Figure 2

SDS-PAGE of rPlpE (A) and Western blotting with anti-histidine antibody (B). Purified rPlpE (Lane 1). Pre-stained protein ladder (Cinnagen PR911654 [SL7012 ]) (Lane M). The samples derived from the same experiment and gel/blot were processed in parallel

Figure 3

The serum antibody titers in the immunized rabbit with the whole-cell antigen of M. haemolytica were measured by ELISA after five injections (1 mg/animal) with one-week intervals.

Figure 4

Slide latex agglutination test with latex beads coated with rPlpE. (A)The results demonstrated a positive agglutination reaction with anti- M. haemolytica IgG-positive serum. (B) and (C) exhibited negative agglutination with negative samples (serum control) and PBS (D) demonstrated the absence of false-positive reactions with anti-Pasteurella multocida -positive serum.