Coxiella burnetii, an obligate intracellular coccobacillus, is a major cause of abortion in livestock and a re-emerging zoonotic pathogen responsible for Q fever in humans. Iran, particularly Khorasan-e-Razavi Province, is considered endemic for Q fever. Rapid and accurate detection of C. burnetii in animal shedders, especially asymptomatic ones, is essential to control its spread among animals and from animals to humans. This study provides the first multi-year (2020–2022) quantitative assessment of C. burnetii infection load in ovine abortions in this endemic region. The objectives were to: (i) develop and validate a region-specific real-time PCR assay targeting the IS1111a gene through sequencing of the amplified fragment; (ii) quantify pathogen load using a standard curve derived from a sequenced reference fragment; and (iii) evaluate associations between prevalence and factors such as year, season, geographic location, and confirmed co-infections with other abortifacient agents. Liver samples from 116 aborted sheep fetuses were examined by real-time PCR, revealing a C. burnetii prevalence of 53.4%, while in 54.8% of these positive cases, C. burnetii was identified as the sole infectious pathogen contributing to abortion. A significant association (p < 0.05) between sampling year and prevalence was observed, with a rising trend over time. The high prevalence of C. burnetii and its role in abortion cases underscore the need for enhanced surveillance and control programs to mitigate the spread and impact of this pathogen in endemic regions.

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