Nested polymerase chain reaction for detection of Ehrlichia equi genomic DNA in horses and ticks (Ixodes pacificus).

This article presents a study which develops a method using nested polymerase chain reaction (PCR) to detect the presence of Ehrlichia equi bacteria in horses and ticks, specifically Ixodes pacificus. The research determines the sensitivity and specificity of the method, and its success at detecting the bacteria in both blood cells of an infected horse and in ticks that fed off of it.

Research Methodology

• The researchers designed a nested polymerase chain reaction (PCR), which is a sensitive technique for amplifying specific DNA sequence, for the detection of Ehrlichia equi. This type of bacteria commonly infects horses and could be transferred to ticks that feed on infected horses.
• A main PCR product of 928 base pairs was produced in the second-round of PCR processing. The identity of the product was verified using a non-radioactive, digoxigenin-based Southern blotting method, where an internal probe was used.
• Furthermore, DNA sequence analysis was performed to provide additional confirmation of the amplified DNA product, ensuring the precise identity and integrity of the DNA sequence.

Sensitivity and Specificity of the PCR

• To assert the sensitivity of the PCR, a dilution study was carried out which indicated that DNA derived from fewer than or equal to 7.6 but more than 3 infected neutrophils was enough to generate a PCR signal. Neutrophils are a type of white blood cell that contains several types of granules and serves as a first line of defense in inflammatory diseases, infections, and other medical issues.
• The researchers also examined the specificity of the PCR, taking care that it only detects Ehrlichia equi amongst a panel of rickettsiae. With this, they ensured that only E. equi and the closely-related human granulocytotropic ehrlichia produced PCR bands, avoiding false positive outcomes.

In Vivo Infection Study

• An in vivo infection study was conducted for verifying the practical validity of the established method. E. equi DNA was found in the blood cells of a horse infected experimentally, particularly from day three to 14 post-inoculation.
• In an extended study, I. pacificus ticks, which as nymphs had been fed on an experimentically infected horse, were observed. The researchers found that three out of six adult ticks were PCR-positive for E. equi, demonstrating the success of the PCR method at detecting the bacteria in ticks as well as horses.

The research essentially aimed to present a new and efficient diagnostic tool using nested PCR for detecting E. equi, and the in vivo tests carried out in this study exhibit a promising potential for such application.