Leptospirosis as a zoonotic disease is caused by Leptospira bacteria. Transmission occurs by contact with contaminated biological fluids of the infected animals. Rodents are major sources of infection for humans or other animals. The disease is distributed mainly in tropical regions with rainfall like northern part of Iran. The aim of this study was to find association of prevalent Leptospira species with different rodents of three Northern Provinces in Iran using microscopic agglutination test (MAT). Methods: In this study, 404 rodents were captured alive at 10 different parts of each Mazandaran, Gilan and Golestan Provinces. Identification of the infecting serovars and the antibody titers were done by MAT in the sera samples using a panel of 20 strains of live Leptospira spp. as the source of antigens. RESULTS: Antibodies against one or more serovars were detected in 94 (23.27%) sera at dilution ≥1:200 and 76.73% were detected to be negative. The prevalent Leptospira serovars were detected as L. autumnalis (25.53%), L. serjoehardjo (24.47%) and L. cynopteri (6.38%). The majority of rodents were identified during this study in three provinces included Rattus norvegicus (67.33%), Apodemus sylvaticus (13.86%) and Rattus rattus (13.61%). The common prevalent rodent in three provinces was Rattus norvegicus, which was associated with L. serjoehardjo in Mazandaran (81.8%), L. autumnalis in Gilan (67.2%) and L. canicula in Golestan (50.0%). Conclusion: The dominant srovars of leptospira were L. autumnalis, L. serjoehardjo and L. cynopteri and the most prevalent rodents as reservoir were Rattus norvegicus, Apodemus sylvaticus and Rattus rattus in three Northern provinces of Iran. The results indicated a moderate prevalence of leptospirosis in rodents during this study in north of Iran. This study provided the first epidemiological data about the association between leptospirosis with rodents in Iran.

Outer membrane proteins (OMPs) are the main surface antigens of the pathogenic Leptospires. One of the highly conserved outer membrane proteins expressed only by pathogenic Leptospires is Loa22. This study aims is to obtain the optimum conditions for high expression and purification of Loa22 recombinant (rLoa22) protein. Based on evidence of phylogenetic studies, complete coding sequence of loa22 gene was optimized based on codon usage chart and sub-cloned into a pET32a (+) expression vector. BL21 (pLysS) was used as expression host for transformation. The recombinant clones selected on ampicillin plates and subjected to PCR by using pET T7 primers and expression conditions optimized then by adjusting parameters such as culture media, induction time, temperature, and IPTG concentration. SDS-PAGE Analysis showed that the production of rLoa22 protein was at the highest level when post induction incubation, IPTG concentration, and duration of induction were 37ºC, 0.1 M and 5 h in 2xTY medium respectively. Due to the soluble nature of the protein, the purification of the rLoa22 protein under native conditions using Ni-NTA pull-down was optimum in one hour binding process at 37°C, five times washing process and elution buffer with a pH 7.4 and a 0.3 M imidazole concentration. Based on the results of this study, optimizing the expression and purification process for over production of rLoa22 protein resulted in the large quantity of pure recombinant antigen that forms the basis for future investigation on the design of rapid diagnostic tests and more effective subunit vaccine candidates for leptospirosis.

Background Outer membrane proteins (OMPs) are the main surface antigens of the pathogenic Leptospires. One of the highly conserved outer membrane proteins expressed only by pathogenic Leptospires is Loa22. This study aims is to obtain the optimum conditions for high expression and purification of Loa22 recombinant (rLoa22) protein. Methods Based on evidence of phylogenetic studies, complete coding sequence of loa22 gene was optimized based on codon usage chart and sub-cloned into a pET32a (+) expression vector. BL21 (pLysS) was used as expression host for transformation. The recombinant clones were selected on ampicillin plates and subjected to PCR by using pET T7 primers and expression conditions optimized then by adjusting parameters such as culture media, induction time, temperature, and IPTG concentration. Results SDS-PAGE Analysis showed that the production of rLoa22 protein was at the highest level when post induction incubation, IPTG concentration, and duration of induction were 37ºC, 0.1M and 5h in 2xTY medium respectively. Due to the soluble nature of the protein, the purification of the rLoa22 protein under native conditions using Ni-NTA pull-down was optimum in one hour binding process at 37°C, five times washing process and elution buffer with a pH 7.4 and a 0.3 M imidazole concentration. Conclusion Based on the results of this study, optimizing the expression and purification process for over production of rLoa22 protein resulted in the large quantity of pure recombinant antigen that forms the basis for future investigation on the design of rapid diagnostic tests and more effective subunit vaccine candidates for leptospirosis.