Use of immunoblotting as an alternative method for serogrouping Leptospira.
Leptospirosis is a worldwide zoonotic disease caused by a spirochaete bacterium, Leptospira. Serological detection of this micro-organism basically relies on a conventional microscopic agglutination test (MAT), which has some limitations and disadvantages. In the present study, immunoblotting has been applied as an alternative method for differentiating serogroups and serovars of leptospires. Leptospiral whole-cell lysates from a total of 26 serovars were subjected to immunoblotting using rabbit antisera against individual serovars. The findings clearly demonstrated that the pattern of immunoreactive bands could be used to differentiate between leptospires of different serogroups, consistent with MAT results. There was a multi-band pattern that was unique for the pathogenic Leptospira antigens and was not observed in the non-pathogenic Leptospira biflexa and non-leptospiral bacteria (i.e. Escherichia coli, Burkholderia pseudomallei and Helicobacter pylori). For pathogenic Leptospira species, a prominent smear-like band at approximately 19-30 kDa was present when the antigens were probed with the homologous antisera. The molecular size of the prominent band, although it showed a cross-reaction between members within the same serogroup, differed among different serovars. The results obtained from polyclonal antibodies (antisera) were confirmed using mAb. With its simplicity and safety of experimental procedures, it is proposed that immunoblotting may potentially be useful as an alternative method for differentiating between serogroups of leptospires.
Doungchawee
Galayanee
G
Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Sirawaraporn
Worachart
W
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Icksang-Ko
Albert
A
Division of International Medicine and Infectious Disease, Weill Medical College of Cornell University, New York, NY 10021, USA.|Gonçalo Moniz Research Center, Oswaldo Cruz Foundation/Brazilian Ministry of Health, Rua Waldemar Falcão, 12140295-001 Salvador, Bahia, Brazil.
Kongtim
Suraphol
S
Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Naigowit
Pimjai
P
Research Center for Leptospira Laboratory, National Institute of Health, Nonthaburi, Thailand.
Thongboonkerd
Visith
V
Medical Molecular Biology Unit, Office for Research and Development, Department of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
D000907:Antibodies, Bacterial / Q000276:immunology; D000942:Antigens, Bacterial / Q000032:analysis / Q000276:immunology; D016957:Burkholderia pseudomallei / Q000276:immunology; D000939:Epitopes; D004926:Escherichia coli / Q000276:immunology; D016480:Helicobacter pylori / Q000276:immunology; D015151:Immunoblotting; D007919:Leptospira / Q000145:classification* / Q000276:immunology*; D008970:Molecular Weight; D012703:Serotyping / Q000379:methods*
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D000939:Epitopes