engleski

Detection of antigens of Leptospira spp. reactive with sera from mares that aborted

Abortion in mares may result from a variety of causes ; infectious and noninfectious . One of the infectious causes is bacterial infection with pathogenic Leptospira spp. According to the state annual order of the Ministry of Agriculture in Croatia, all aborted mares must be serologically tested to leptospirosis. A total of 40 aborted mare sera were chosen among those routinely examinated on leptospirosis during the year 2010 and 2011. Sera were tested by microscopic agglutination test (MAT) using 12 Leptospira serovars. Initial dilution was 1:50. Out of 40 sera, 21 showed a positive MAT reaction. Ten (48 %) reacted with the highest titer against serovar Bratislava, six (28.6 %) against serovar Pomona, three (14.3 %) against serovar Saxkoebing and two (9.52%) against serovar Icterohaemorrhagiae. 19 aborted mare sera were considered serologically negative with the titer < 50. Since the MAT is dependent on agglutinating antibody, predominantly IgM against LPS, this work was done to screen MAT positive and negative aborted mare sera for the detection of IgG antibodies to different Leptospira antigens by one and two-dimensional SDS PAGE immunoblot (1D and 2D IB). Proteins of an in vitro cultivated isolate of L. interrogans serovar Copenhageni, strain RJ 16441 were separated by 1D and 2D electrophoresis, visualised by SyproRuby staining (Sigma-Aldrich St. Louis, USA) and transfered to PVDF mambrane. Membranes were probed with mare sera diluted on 1:200, followed by incubation with goat Anti- Equine IgG diluted 1:800. Sera with different MAT results showed different patterns of reactivity with Leptospira antigens. Antigens corresponding to the molecular mass of LipL32, Lip L41 and Lip L21 were detected by 1D IB. In addition, reactive Leptospira proteins with molecular masses of approximately 50, 60, 80, 100 and 120 kDa also were detected. Two-dimensional (2D) immunoblots showed better resolution of reactive Leptospira proteins. Negative sera also showed reactivity with Leptospira antigens, indicating that mares were previously exposed to Leptospira spp. and that 1D and 2D immunoblot can detect antibodies against Leptospira proteins in sera which were negative by MAT.

leptospira; 1D 2D immunoblott; mare abortion

nije evidentirano