An immunoblotting diagnostic assay for heartwater based on the immunodominant 32-kilodalton protein of Cowdria ruminantium detects false positives in field sera

Abstract

Heartwater, a major constraint to improved livestock production in Zimbabwe, threatens to invade areas which have been previously unaffected. To monitor its spread in Zimbabwe, an immunoblotting diagnostic assay based on the responses of animals to the immunodominant, conserved 32-kDa protein of Cowdria ruminantium was evaluated. In this assay, no false reactions were detected with sera known to be positive and negative, but sera from some cattle, sheep, and goats from heartwater-free areas of Zimbabwe reacted strongly with the 32-kDa protein, suggesting that either these animals had previous exposure to heartwater or they were false positives. To investigate the possibility of previous exposure to heartwater, 11 immunoblot-positive and 6 immunoblot-negative sheep from heartwater-free areas of Zimbabwe were compared regarding their susceptibilities to challenge with C. ruminantium. Prior to challenge, C. ruminantium could not be detected in any sheep by transmission to Amblyomma hebraeum ticks or by the polymerase chain reaction (PCR) conducted with plasma samples. All sheep were equally susceptible to the challenge, and infection was confirmed by brain biopsy, necropsy, PCR, and transmission of C. ruminantium to ticks. Our data suggest that the immunoblotpositive reactions of sera from heartwater-free areas were due not to previous C. ruminantium infection but rather to antigenic cross-reactivity between C. ruminantium and another agent(s), such as Ehrlichia species. In conclusion, the immunodominant 32-kDa protein is not antigenically specific to C. ruminantium and its use in serological diagnosis of heartwater requires reevaluation.

Detection of the Agent of Heartwater, Cowdria ruminantium , in Amblyomma Ticks by PCR: Validation and Application of the Assay to Field Ticks

“…Furthermore, xenodiagnosis in mice has recently been shown to have low sensitivity and is unreliable for the detection of infection (38). More-expedient DNA probe and PCR assays based on the pCS20 and MAP 1 DNA sequences of C. ruminantium have been developed recently and have been used to detect infections in ticks and animals (1,31,37,38,42,43,52,60,62). PCR assays, which have greater sensitivity than DNA probe assays (52), may be useful tests for laboratory and field epidemiological investigations which are needed for improved understanding of heartwater epidemiology and the impact of new control measures (49).…”

Development of a Quantitative PCR Assay for Differentiating the Agent of Heartwater Disease, Ehrlichia ruminantium , from the Panola Mountain Ehrlichia

“…For example, on the Caribbean islands of St. Kitts and Nevis, heartwater has never been diagnosed, but PME has been detected in A. variegatum , which may be the cause of false positives. As further demonstrated here, false‐positive reactions for E. rumanintium on the MAP‐IB ELISA in experimentally inoculated PME‐positive goats indicate that serology appears to be of limited use for diagnosing either heartwater or PME, in HW endemic and non‐endemic areas where PME may be circulating (Mahan et al., ; Simbi et al., ; Kelly et al., ). This underscores the need for a highly sensitive, rapid and specific assay that can differentiate between E. ruminantium and PME.…”

Validation of the Indirect MAP1-B Enzyme-Linked Immunosorbent Assay for Diagnosis of Experimental Cowdria ruminantium Infection in Small Ruminants

“…Antemortem tests for detecting C. ruminantium include animal subinoculation, cell culture isolation, serodiagnostic tests, DNA hybridization, and PCR. Serodiagnostic methods, such as the indirect fluorescent antibody test, immunoblotting, and enzyme-linked immunosorbent assays (ELISA), have been hampered by cross-reactions with Ehrlichia species (18,22,24). However, the use of recombinant major antigenic protein 1 (MAP1) of C. ruminantium has been recently introduced, and an indirect ELISA based on a specific fragment of this protein (fragment B, referred to herein as MAP1-B) has been developed (32).…”