PCR-RFLP of Coxiella burnetii Plasmids Isolated from Raw Milk Samples in Iran

 Several methods have been employed to identify Coxiella burnetii isolates based on the specific Coxiella burnetii QpH1 plasmid to distinguish the acute form from the chronic form of Q fever disease in humans and animals owing to the presence of unique gene sequences in this plasmid. Therefore, the present study aimed to investigate the panel of nucleic acid fragments resulting from the enzymatic cleavage in the QpH1 plasmid isolated from cow and buffalo milk by nested polymerase chain reaction (Nested-PCR).

 A total of 86 isolates of Coxiella burnetii QpH1 plasmid, which were confirmed by the Nested-PCR method in 2018, were used to determine the RFLP panel of the QpH1 plasmid. Plasmids were first extracted with the kit and were then affected by the Hph1 restriction enzyme. Additionally, 4 nucleic acid samples were sent to Pishgam Company for sequencing with the IS1111 gene primer.

Based on the results of the PCR-RFLP test, all plasmid samples showed a similar two-fragment pattern under the influence of Hph1. The results of the nucleic acid sequencing of all 4 samples indicated that they had a Coxiella burnetii type (Nine Mile RSA493 strain).

 RFLP patterns exhibited no difference on the Coxiella burnetii QpH1 plasmid isolated from cow and buffalo milk. Hence, all isolates were genetically identical, and the infection in animals could originate from one Coxiella burnetii strain (Nine Mile RSA493 strain).