Cloning and expression of two genes from Babesia equi merozoites and evaluation of their diagnostic potential.

The research article discusses the cloning and expression of two genes derived from Babesia equi merozoites. These genes were evaluated for their potential in diagnosing Babesia equi, a parasitic disease in horses. The team developed a test, with 99% specificity and 88% sensitivity, that can recognize the disease in horses infected with a specific strain but failed to recognize it in other strains.

Research Methodology

• The team used high-titre equine immune sera to screen a lambda gt 11 expression library of Babesia equi cDNA fragments.
• Out of the screened clones, two cDNA clones that did not cross-hybridize to each other were studied further.
• These clones were specifically hybridized to DNA from Babesia equi, but not to DNA from Babesia caballi, Babesia divergens or Babesia ovis – different species of Babesia.
• Recombinant proteins from these clones were expressed as glutathione S-transferase (GST) fusion proteins. These proteins were of apparent molecular weights of 40 kDa and 75 kDa.

Observations and Findings

• Antibodies developed against the 40 kDa and 75 kDa recombinant proteins detected native antigens of 55 kDa and 50 kDa, respectively in Babesia equi merozoite lysates. This showed that neither cDNA clone was full-length, taking into account that GST itself is 26 kDa.
• During Western blotting experiments, it was found that the 75 kDa protein showed cross-reactivity with sera from horses infected with Babesia caballi. Thus, this protein was not further investigated for specificity to Babesia equi.
• The 40 kDa protein was additionally tested in an enzyme-linked immunosorbent assay (ELISA) to develop a diagnostic test.

Development of Diagnostic Test and its Efficacy

• A diagnostic test was developed using the 40 kDa protein in ELISA. The test was calculated to have a specificity of 99% and a sensitivity of 88% with sera from horses infected with the corresponding strain of Babesia equi.
• However, the ELISA test failed to recognize sera from horses infected with Babesia equi strains from Brazil and Morocco, indicating that the 40 kDa protein may not universally recognize all strains of the disease.