Leptospirosis research: fast, easy and reliable enumeration of mobile leptospires

Leptospirosis caused by Leptospira interrogans is the most widespread zoonosis and a major public health problem worldwide. Based on light-scattering and absorption, quantification of leptospires using UV-VIS spectroscopy was used as an indirect counting technique by measuring the optical density and comparing this to automated direct counting using a counting chamber in combination with imaging and analyzing software. Two serovars, Bangkok and Copenhagenii, from log-phase growth were used for the establishment of standard curves. They were found to be linear and slightly different in gradient for each serovar. The ease, rapidity, and reliability of these two adapted and optimized counting techniques may provide a useful alternative enumeration technique for leptospirosis research.

Western immunoblot analysis using a ten leptospira serovars combined antigen for serodiagnosis of leptospirosis.

Leptospirosis is a major zoonotic disease throughout the world. There are unavailable accuracy diagnostic methods for the acute phase of infection. To demonstrate the advantage of Western immunoblot, a mixed leptospira serovars antigen for the serodiagnosis of leptospirosis was employed. SDS-PAGE and Western immunoblot was performed using a 10 mixed leptospira serovars antigen and stained with 16 reference rabbit anti-leptospirosis antibodies. The result showed different immunoreactive band patterns for each reference serum. The bands with molecular weights of 15-20, 23-24, 41 and 45 kDa were commonly found (88% to 100% of the 16 reference sera). Using combined leptospira antigens in a Western immunoblot technique is an alternative and practical strategy for a more sensitive leptospirosis serodiagnosis.

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.

Surveillance of leptospirosis after flooding at Loei Province, Thailand by year 2002.

In this surveillance, suspected leptospirosis patients in Loei Hospital, Loei Province were studied by conventional methods of cultivation and microscopic agglutination test (MAT) during July-October, 2002. It was found that 63% of 64 admitted patients and 35% of 34 outpatients were found positive by leptospire cultivation. Antibodies determined by MAT were positive in 78% of 63 admitted patients. Particularly, the five most common agglutinating antibodies were reactive with serovars bratislava (57%), autumnalis (48%), new (38%), australis (37%) and bangkok (29%). The MAT results of 15 OPD patients were 67% positive with the following five serovars, including bratislava (47%), new (20%), bangkok (7%), ranarum (7%) and australis (7%). Accordingly, preventive strategies against leptospirosis outbreaks after flooding in Thailand should be undertaken, including the prompt treatment of the disease in this endemic area.