Genetic Diversity Based on LipL32, 16S rRNA and ColA Expression in Leptospira spp. Isolated From Rodent Hosts in Samarahan and Gedong Districts in Sarawak

Mohamed Nur Azim, Mazlan (2022) Genetic Diversity Based on LipL32, 16S rRNA and ColA Expression in Leptospira spp. Isolated From Rodent Hosts in Samarahan and Gedong Districts in Sarawak. PhD thesis, Universiti Malaysia Sarawak.

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Abstract

Leptospirosis is a major zoonotic disease in the world that is caused by Leptospira spp., that may cause acute febrile illness in mammals with varied pathogenicity and clinical disease. Among factors that have been associated with pathogenicity of Leptopsira are LipL32 and ColA gene. LipL32 has been identified to play an important role in TLR-dependent pathway and inducing erythrocytes. ColA (LA0872), a gene coding for bacterial collagenase, a common invasive enzyme which facilitates the leptospires to quickly invade and spread into internal organs. Gene or protein that had been expressed by leptospires during infection or within specific host may provide glimpses into leptospiral pathogenic strategies. The study was conducted in Samarahan Division, Sarawak involving two locations; Kota Samarahan and Gedong. Traps were set at the study sites and 31 rodents were caught with 96.7% of them are from Rattus rattus species. The kidneys and blood samples were cultured into Ellinghausen-McCullough-Johnson-Harris (EMJH) media added with 5-fluorouracil. DNA and protein isolates from positive cultures were extracted and analysed. From the 31 kidney samples of rodents caught, 28 kidney tissue samples (90.3%) showed presence of leptospire-like organism in cultures. PCR analyses utilizing 16S gene primers confirmed the presence of leptospires, in 90.3% of rodents caught in the study area. Almost half of the leptospires detected were saprophytic (45.2%) and pathogenic Leptospira spp. (38.7%). The highest number of species detected was pathogenic Leptospira, Leptospira kmetyi strain SS-S7 with 38.7%, from the whole isolates sequenced when compared to GenBank, followed by, intermediate Leptospira, Leptospira wolfii strain SS-S17 detected (12.9%), and saprophytic Leptospira, Leptospira biflexa strain Patoc_DB58 (19.4%), Leptospira wolbachii serovar Codice strain and Leptospira idonii strain Eri-1 16S ribosomal RNA (6.5% respectively) and Leptospira terpestrae serovar Hualin strain LT 11-33 (16.1%). There were no related DNA sequences retrieved from LipL32 primers after multiple passages of cultures. The DNA sequence degree of homology was varied from 80% to 99% which were clustered into 3 clusters (A1, A2 and A3). Most of the isolates were delineated into cluster A2 with 15 isolates (48.4%) which is close to NR.043043.1 sequence retrieved from NCBI database, followed by A1 cluster with 11 isolates (35.5%) (KY411405.1), and A3 cluster with 4 isolates (12.9%) (KY411411.1). All samples that shown presence of collagenase was belong to cluster A2. Interestingly, ColA protein was not detected in all intermediate and saphrophytic isolates and only detected in eight of the samples (25.8%) which only infected with pathogenic strain, Leptospira kmetyi. The findings indicated that a high proportion of rodents in the study area were infected with Leptospira spp. and about a third of them were harboured with pathogenic strain, Leptospira kmetyi. It is found that ColA gene was expressed only in pathogenic leptospires, and might indicate its importance in the pathogenesis of pathogenic Leptospira spp. in rodent hosts.

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