Verification of post-chemotherapeutic clearance of Theileria equi through concordance of nested PCR and immunoblot.

The researchers in this study have validated a method to accurately determine whether horses have been completely rid of Theileria equi, a disease-causing parasite, after treatment with imidocarb diproprionate. This verification process combines two testing methods, nested polymerase chain reaction and immunoblotting.

Context and Objective

• Equine piroplasmosis, triggered by the parasite Theileria equi, is a disease that is not endemic in certain countries such as the U.S., thanks to stringent import regulations and surveillance.
• While countries where the disease is endemic often use exposure to the disease as a control measure (premunition), non-endemic countries rely on chemosterilization and stringent tests to confirm the eradication of the parasite.
• The researchers in this study wanted to verify the effectiveness of a chemosterilant drug, imidocarb diproprionate (ID), via a combination of two methodologies: nested polymerase chain reaction (nPCR) and immunoblotting (IB).

Identification of Challenges

• While the ID has been proven to effectively eradicate T. equi from the horses’ bodies, the process faced two significant challenges:
• Due to the extended persistence of anti-T. equi antibodies, certain tests such as cELISA or IFA tend to show positive results for an extended period post treatment.
• This persistent positive testing poses serious challenges for regulatory veterinary medicine and international trade.

Study Methodology

• The study involved 179 horses treated with ID.
• The researchers adopted nPCR targeting the ema1 gene and immunoblotting (IB) to measure the declination in anti-EMA1 and anti-EMA2 antibodies in order to establish the elimination of T. equi.

Study Findings

• The results supported the use of immunoblot, which demonstrated a consistent decline in anti-EMA1 and EMA2 titers in infected horses after the successful treatment with the ID.
• Concurrent evidence of these results with a negative nPCR thus led to a more effective determination of successful ID clearance of T. equi.
• Study results showed that while nPCR displayed uniformly negative results 14 days post-treatment and cELISA often stayed positive even after a year, immunoblot was, on average, negative after 4 months and 100% harmonious with nPCR results.