Molecular diagnosis of Coxiella burnetii in milk based on Plasmid and Transposon Genes

 All Coxiella burnetii isolates carry one of four large, conserved, autonomously replicating plasmids or a plasmid-like chromosomally integrated sequence.

 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.

 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.

 Our results indicated that nested PCR has high sensitivity in detecting plasmids.