Iranian Journal of Medical Microbiology

Background and Aim: 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).
Materials and Methods: 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.
Results: 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).
Conclusion: 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).

Background and Aim: All Coxiella burnetii isolates carry one of four large, conserved, autonomously replicating plasmids or a plasmid-like chromosomally integrated sequence.
Materials and Methods: In Sulaimani City, Iraq, from September 2020 to September 2021, 200 positive nasopharyngeal samples were collected, and 17 known variants with the S gene were randomly selected for whole RdRp, E, and N gene sequencing. To facilitate sequencing, six primer sets were designed for the RdRp gene (RdRp1, RdRp2, RdRp3), two for the N gene (N1, N2), and one for the E gene.
Results: In total, out of 400 milk samples collected from cow, buffalo, sheep, and goats based on the IS1111 gene, 62 (15.5%), (95% CI: 12.3%–19.4%) samples were positive for C. burnetii. Out of 62 positive samples, 16 (25.8%), (95% CI: 16.6%–37.9%) samples contained QpH1 plasmid gene and 5 (8%), (95% CI: 3.5%–17.5%) samples contained QpRS plasmid gene. Also, there were 7 (11.3%), (95% CI: 5.6%–21.5%) positive samples for QpDG and 5 (11.3%), (95% CI: 3.5%–17.5%) positive to QpDV gene. The Phylogenetic analysis of plasmid sequences showed that all obtained sequences have 100% similarity. A phylogenetic tree constructed based on neighbor-joining analysis of partial genes revealed that 20 sequenced isolates were closely clustered together showing 99.9% similarity which can be considered identical and also revealed the 100% similarly of these sequences with more sequences in the gene bank from different sources.
Conclusion: Our results indicated that nested PCR has high sensitivity in detecting plasmids.