Antibody responses to MAP 1B and other Cowdria ruminantium antigens are down regulated in cattle challenged with tick-transmitted heartwater

Abstract

Serological diagnosis of heartwater or Cowdria ruminantium infection has been hampered by severe cross-reactions with antibody responses to related ehrlichial agents. A MAP 1B indirect enzyme-linked immunosorbent assay that has an improved specificity and sensitivity for detection of immunoglobulin G (IgG) antibodies has been developed to overcome this constraint (A. H. M. van Vliet, B. A. M. Van der Zeijst, E. Camus, S. M. Mahan, D. Martinez, and F. Jongejan, J. Clin. Microbiol. 33:2405-2410, 1995). When sera were tested from cattle in areas of endemic heartwater infection in Zimbabwe, only 33% of the samples tested positive in this assay despite a high infection pressure (S. M. Mahan, S. M. Samu, T. F. Peter, and F. Jongejan, Ann. N.Y. Acad. Sci 849:85-87, 1998). To determine underlying causes for this observation, the kinetics of MAP 1B-specific IgG antibodies in cattle after tick-transmitted C. ruminantium infection and following recovery were investigated. Sera collected weekly over a period of 52 weeks from 37 cattle, which were naturally or experimentally infected with C. ruminantium via Amblyomma hebraeum ticks, were analyzed. MAP 1B-specific IgG antibody responses developed with similar kinetics in both field- and laboratory-infected cattle. IgG levels peaked at 4 to 9 weeks after tick infestation and declined to baseline levels between 14 and 33 weeks, despite repeated exposure to infected ticks and the establishment of a carrier state as demonstrated by PCR and xenodiagnosis. Some of the serum samples from laboratory, and field-infected cattle were also analyzed by immunoblotting and an indirect fluorescent-antibody test (IFAT) to determine whether this observed seroreversion was specific to the MAP 1B antigen. Reciprocal IFAT and immunoblot MAP 1-specific antibody titres peaked at 5 to 9 weeks after tick infestation but also declined between 30 and 45 weeks. This suggests that MAP 1B-specific IgG antibody responses and antibody responses to other C. ruminantium antigens are down regulated in cattle despite repeated exposure to C. ruminantium via ticks. Significantly, serological responses to the MAP 1B antigen may not be a reliable indicator of C. ruminantium exposure in cattle in areas of endemic heartwater infection.

FIG. 1

DNA from adult A. hebraeum ticks hybridized with the pCS20 C. ruminantium-specific DNA probe. The ticks were infected as nymphs by feeding on sheep infected with C. ruminantium and were then used for laboratory infections of cattle. DNA from serial dilutions of C. ruminantium organisms (10⁹ to 10² and 100 ng of C. ruminantium DNA) was used for positive control samples. DNA from uninfected adult A. hebraeum ticks was used for negative control samples.

FIG. 2

Kinetics of total IgG antibodies detected by MAP 1B indirect ELISA in cattle exposed to C. ruminantium by field- or laboratory-infected A. hebraeum ticks. (a and b) Group 1 and 2 cattle, respectively, naturally infected on a heartwater-endemic farm; (c) cattle experimentally infected by repeated infestation with laboratory-infected A. hebraeum ticks.

FIG. 3

Immunoblot reactions of titrated sequential serum samples from naturally and experimentally infected cattle against C. ruminantium antigens. Serum dilutions were as follows: strip 1, = 1:100; strip 2, = 1:1,000; strip 3–1:2,000; strip 4, 1:4,000; strip 5, 1:8,000; strip 6, 1:16,000; strip 7, 1:32,000; strip 8, 1:64,000. Week 9 sera from experimentally infected cattle 103 and 108 had eight dilutions tested. The rest of the serum bleeds had only five or six dilutions tested. At week 9 postinfestation, reciprocal end-point titers of MAP 1 antibody responses for both naturally and experimentally infected cattle were 8,000 to 16,000, which then decreased to 100 to 1,000 by weeks 30 to 45.