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

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 ۲۰۲۰ to September ۲۰۲۱, ۲۰۰ positive nasopharyngeal samples were collected, and ۱۷ 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 (RdRp۱, RdRp۲, RdRp۳), two for the N gene (N۱, N۲), and one for the E gene. Results: In total, out of ۴۰۰ milk samples collected from cow, buffalo, sheep, and goats based on the IS۱۱۱۱ gene, ۶۲ (۱۵.۵%), (۹۵% CI: ۱۲.۳%–۱۹.۴%) samples were positive for C. burnetii. Out of ۶۲ positive samples, ۱۶ (۲۵.۸%), (۹۵% CI: ۱۶.۶%–۳۷.۹%) samples contained QpH۱ plasmid gene and ۵ (۸%), (۹۵% CI: ۳.۵%–۱۷.۵%) samples contained QpRS plasmid gene. Also, there were ۷ (۱۱.۳%), (۹۵% CI: ۵.۶%–۲۱.۵%) positive samples for QpDG and ۵ (۱۱.۳%), (۹۵% CI: ۳.۵%–۱۷.۵%) positive to QpDV gene. The Phylogenetic analysis of plasmid sequences showed that all obtained sequences have ۱۰۰% similarity. A phylogenetic tree constructed based on neighbor-joining analysis of partial genes revealed that ۲۰ sequenced isolates were closely clustered together showing ۹۹.۹% similarity which can be considered identical and also revealed the ۱۰۰% 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.

کلیدواژه ها:

Milk ، Q fever ، Coxiella burnetii ، Plasmid ، Transposon genes ، کوکسیلا بورنتی ، پلاسمید ، درختچه فیلوژنی ، تب کیو ، شیر