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.
BarnettJ. K.,
BarnettD.,
BolinC. A.,
SummersT. A.,
WagarE. A.,
ChevilleN. F.,
HartskeerlR. A.,
HaakeD. A.1999; Expression and distribution of leptospiral outer membrane components during renal infection of hamsters. Infect Immun 67:853–861
BhartiA. R.,
NallyJ. E.,
RicaldiJ. N.,
MatthiasM. A.,
DiazM. M.,
LovettM. A.,
LevettP. N.,
GilmanR. H.,
WilligM. R. & other authors; 2003; Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis 3:757–771[CrossRef]
ChapmanA. J.,
AdlerB.,
FaineS.1988; Antigens recognised by the human immune response to infection with Leptospira interrogans serovar Hardjo. J Med Microbiol 25:269–278[CrossRef]
ChoS. N.,
UhmJ. R.,
KimJ. D.1992; Comparative analysis of lipopolysaccharide and lipid antigens of Leptospira interrogans serovars. Yonsei Med J 33:24–31[CrossRef]
CullenP. A.,
CordwellS. J.,
BulachD. M.,
HaakeD. A.,
AdlerB.2002; Global analysis of outer membrane proteins from Leptospira interrogans serovar Lai. Infect Immun 70:2311–2318[CrossRef]
DikkenH.,
KmetyE.1978; Serological typing methods of leptospires. In Methods in Microbiologyvol. 11 pp 259–307 Edited by
BerganT.,
NorrisJ. R.
London: Academic Press;
DohertyJ. P.,
AdlerB.,
RoodJ. I.,
BillingtonS. J.,
FaineS.1989; Expression of two conserved leptospiral antigens in Escherichia coli
. J Med Microbiol 28:143–149[CrossRef]
DoungchaweeG.,
PhulsuksombatD.,
NaigowitP.,
KhoaprasertY.,
SangjunN.,
KongtimS.,
SmytheL.2005; Survey of leptospirosis of small mammals in Thailand. Southeast Asian J Trop Med Public Health 36:1516–1522
FarrellyH. E.,
AdlerB.,
FaineS.1987; Opsonic monoclonal antibodies against lipopolysaccharide antigens of Leptospira interrogans serovar Hardjo. J Med Microbiol 23:1–7v[CrossRef]
GittonX.,
Andre-FontaineG.,
AndreF.,
GaniereJ. P.1992; Immunoblotting study of the antigenic relationships among eight serogroups of Leptospira
. Vet Microbiol 32:293–303[CrossRef]
GravekampC.,
van de KempH.,
FranzenM.,
CarringtonD.,
SchooneG. J.,
van EysG. J. J. M.,
EverardC. O. R.,
HartskeerlR. A.,
TerpstraW. J.1993; Detection of seven species of pathogenic leptospires by PCR using two sets of primers. J Gen Microbiol 139:1691–1700[CrossRef]
HaakeD. A.,
MatsunagaJ.2002; Characterization of the leptospiral outer membrane and description of three novel leptospiral membrane proteins. Infect Immun 70:4936–4945[CrossRef]
JohnsonR. C.,
FaineS.1984; Leptospira
. In Bergey's Manual of Systematic Bacteriology vol 1 pp 62–67 Edited by
KriegN. R.,
HoltJ. G.
Baltimore, MD: Williams & Wilkins;
JostB. H.,
AdlerB.,
VinhT.,
FaineS.1986; A monoclonal antibody reacting with a determinant on leptospiral lipopolysaccharide protects guinea pigs against leptospirosis. J Med Microbiol 22:269–275[CrossRef]
KelsonJ. S.,
AdlerB.,
ChapmanA. J.,
FaineS.1988; Identification of leptospiral flagellar antigens by gel electrophoresis and immunoblotting. J Med Microbiol 26:47–53[CrossRef]
MidwinterA.,
VinhT.,
FaineS.,
AdlerB.1994; Characterization of an antigenic oligosaccharide from Leptospira interrogans serovar Pomona and its role in immunity. Infect Immun 62:5477–5482
SitprijaV.,
PipatanagulV.,
MertowidjojoK.,
BoonpucknavigV.,
BoonpucknavigS.1980; Pathogenesis of renal disease in leptospirosis: clinical and experimental studies. Kidney Int 17:827–836[CrossRef]