Molecular identification and phylogenetic characterization of Leptospira spp. in domestic animals from Jordan

Abstract

Background: Leptospirosis is an emerging zoonotic disease-causing substantial morbidity, mortality, and economic losses worldwide, with over 60,000 human deaths annually. Early diagnosis and molecular identification of Leptospira spp. are essential for effective control and prevention. In Jordan, while serological evidence exists, there is a critical lack of molecular data regarding the species circulating in animal hosts. Objectives: This study aimed to detect Leptospira infection using nested polymerase chain reaction (PCR) and to characterize the isolates molecularly through sequencing and phylogenetic analysis of the 16S rRNA (rrs) gene. Materials and Methods: DNA was extracted from serum and urine samples collected between January 2020 and May 2021 from 82 animals suspected of leptospirosis, including 80 cattle, one goat, and one dog. Pooled urine from imported feedlot calves and urine from the goat and dog were analyzed. Positive Leptospira samples were identified using nested PCR targeting the rrs gene, followed by Sanger sequencing. Phylogenetic analysis was performed using MEGA software, and sequences were compared with reference serovars from GenBank. Results: Three samples (3.5%) were positive for the Leptospira rrs gene, producing the expected 299 bp amplicon. Sequencing confirmed Leptospira DNA in two samples—one from pooled urine of imported calves and one from a dog serum sample. The sequences were submitted to GenBank as “Uncultured Leptospira spp. Canine Jordan 2020” (OK394051) and “Uncultured Leptospira spp. Cattle Jordan 2020” (OK394045). Conclusions: This is the first molecular characterization of Leptospira spp. in domestic animals in Jordan. The findings underscore the need to include leptospirosis in differential diagnoses of febrile diseases and to enhance surveillance and preventive programs.