Demonstration of the humoral immune response of horses to Babesia caballi by western blotting.

This research brief discusses the immune response of horses to Babesia caballi, a malarial-like parasite, as demonstrated by Western blotting, an analytical technique used to detect specific proteins in a sample.

Techniques Used

• The researchers used sodium dodecyl sulfate (SDS) buffer to solubilize Babesia caballi-infected and normal equine erythrocytes (red blood cells).
• These samples were then analyzed with two primary techniques: (1) SDS-polyacrylamide gel electrophoresis (SDS-PAGE) – a method used to separate proteins based on their molecular weight, and (2) Western blotting – a method used to identify specific proteins out of a mixture of proteins.
• The antigens (substances that trigger an immune response) were then exposed to the sera (liquid component of blood) from both horses that were experimentally or field-infected with B. caballi and those from non-infected horses.

Findings

• The major babesial antigens that were recognized by the immune sera showed molecular weights of 141, 112, 70, 50, 48, 34, and 30 kDa.
• The polypeptides (protein fragments) at 50 and 48 kDa were recognized earliest during and throughout infection, but were also weakly acknowledged by some equine sera that tested negative (3/100) and false positive (1/33) by the complement fixation test (CFT) and immunofluorescence antibody test (IFAT).

Implications

• The findings suggest the need for further characterization and purification of B. caballi antigens to identify target antigens which would improve the enzyme immunosorbent assay – an analytical biochemistry assay that uses antibodies and an enzyme-mediated color change to detect the presence of either an antigen (in a sample) or an antibody in a solution.
• Until such an enhanced assay is available, Western blotting can offer a specific tool for diagnosing B. caballi infections, particularly in cases where results from the CFT and IFAT contradict.