Expression and characterization of recombinant leptospiral outer membrane protein LipL32 from Leptospira interrogans serovar autumnalis.

Leptospira interrogans serovar autumnalis, a causative agent of leptospirosis in Thailand, was isolated from a patient for DNA extraction and amplification of LipL32 gene by polymerase chain reaction (PCR). The 782 bp PCR product was obtained, which was inserted into pAE plasmid with polyhistidine (His6 tag) to construct pAE-LipL32. This recombinant plasmid was transfected into E. coli BL21 (DE3). His6-LipL32 was purified by Ni-NTA affinity chromatography. The recombinant protein was used as antigen for testing with sera from leptospirosis and syphilis patients by dot-ELISA technique. It reacted positively with leptospirosis patient sera and negatively with syphilis and healthy sera.

Identification of clinical isolates of Leptospira spp by pulsed field gel-electrophoresis and microscopic agglutination test.

Twenty-five clinical isolates of Leptospira spp were characterized by microscopic agglutination test (MAT) and pulsed field gel-electrophoresis (PFGE) in comparison with 23 reference Leptospira serovars and with the saprophytic L. biflexa serovar Patoc. PFGE DNA profiling was more specific and reliable than MAT.

Survey of leptospirosis of small mammals in Thailand.

During 1999-2000, kidney tissues of approximately 15% of 1310 rodents trapped from northeastern provinces of Thailand were tested for the presence of leptospires. Our direct immunofluorescent assay (DFA) for detection of leptospires showed 100% sensitivity and 94% specificity with the culture data. Both methods identified R. norvegicus as the highest source of infection. Among isolated Leptospira, 137 were serotyped by cross agglutinin absorption and/or a microscopic agglutination, and gave some variations and similarities at the serovar level to the DFA results. DFA data demonstrated over half of the positive animals were infected with several serovars of Leptospira interrogans. A subsequent DFA study in Bangkok in 2002 revealed leptospiral infection in 33% of 42 rats and shrews. The most common infecting serovars were Autumnalis and Canicola identified in rural and urban animals, respectively. This finding suggests that wild small mammals may act as important sources of pathogenic leptospires and warrant active surveillance to understand the epidemiology of transmission and control of carrier animals.

Comparison of leptospiral serovars identification by serology and cultivation in northeastern region, Thailand.

Blood from patients suspected of leptospirosis 148 specimens were cultured for leptospira. Twenty two specimens were positive (15%). The isolated leptospira were tested against the 24 serovars of standard antisera by Microscopic Agglutination Test (MAT). It was found that all 22 leptospira isolates reacted strongly against L. autumnalis, except 1 isolate that also reacted against serovar djasiman. The patient's sera were collected from only 14 cases. When the sera of the 14 patients were tested with the 24 reference serovars by MAT it was found that sera reacted the most against L. australis and in decreasing order against L. bratislava, L. autumnalis, L. rachmati, L. copenhageni, L. javanica. There had some cross reactions against several serovars in a single patient. The present study showed inconsistency between culture results and serum assays. Since sera showed cross reactivities against several serovars, it was not possible to determine which serovar was etiologic. Therefore, the isolation of leptospira though time consuming is specific in the identification of the serovar.

Prevalence of antibodies to Leptospira serovars in rodents and shrews trapped in low and high endemic areas in Thailand.

OBJECTIVE: To investigate the prevalence of antibodies to Leptospira serovars in rodents and shrews trapped in urban and rural areas in low and high endemic areas in Thailand. MATERIAL AND METHOD: A total of 1,664 serum samples were collected from rodents and shrews in areas of low and high endemicity for leptospirosis. Four areas classified by case rates (CR) per 100,000 population of leptospirosis were urban Area I Bangkok (CR = 0.07), rural Area II (CR = 0.24), rural Area III (CR = 1.97) and rural Area IV (CR = 48.20). All serum samples were investigated for antibodies to leptospires by microscopic agglutination test (MAT) using antigens from each of the 22 pathogenic serovars of Leptospira interrrogans: australis, autumnalis, ballum, bangkok, bataviae, bratislava, canicola, celledoni, copenhageni, djasiman, grippotyphosa, hardjo, hebdomadis, icterohaemorrhagiae, javanica, pomona, pyrogenes, rachmati, saigon, sejroe, tarassovi and wolffi and one non-pathogenic strain of L. biflexa serovar patoc. RESULTS: Ninety-four (5.6%) serum samples were positive for Leptospira antibodies. The most commonly detected antibodies were to serovars pyrogenes (39.1%), sejroe (19.1%), bataviae (10.0%), pomona (6.4%), autumnalis (5.5%), copenhageni (3.6%) and javanica (3.6%). The positive rates in Area I, II, III and IV were 7.6 per cent, 2.9 per cent, 4.6 per cent and 7.1 per cent, respectively. The seroprevalence in rural areas tended to increase significantly with high endemicity for leptospirosis (Chi-square for trend, p = 0.04). The seropositive rates by animal species were 39/496 (7.9%), 22/322 (6.8%), 23/492 (4.7%), 6/170 (3.5%), 4/175 (2.3%), 0/4 (0%) and 0/5 (0%) in Rattus norvegicus, Rattus exulans, Rattus rattus, Bandicota indica, Bandicota savilei, Mus musculus and Suncus murinus, respectively. There was a statistical trend between seropositive rates in R. exulans and endemicity for leptospirosis (Chi-square for trend, p = 0.04). CONCLUSION: The 5.6 per cent of rodents and shrews trapped in urban and rural areas in Thailand were reservoirs of leptospires. The results of high seroprevalence in rats also indicate the high endemicity for leptospirosis.