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

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 immunoblot-positive 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 Cowdria ruminantium by means of a DNA probe, pCS20 in infected bont ticks, Amblyomma hebraeum, the major vector of heartwater in southern Africa.

A DNA probe, pCS20, previously described for use in detection of Cowdria ruminantium infections in Amblyomma variegatum (the principal vector of heartwater) hybridized with C. ruminantium DNA in organs of laboratory-infected A. hebraeum adult ticks (the major southern African vector of heartwater). The probe hybridized with C. ruminantium DNA in 46/49 midguts from male ticks and 26/29 from females, thus indicating infection. Corresponding salivary glands were less heavily infected, but infections were more numerous in glands from males. Infection in ticks was confirmed by transmission of the disease to susceptible goats. The probe did not hybridize with DNA from uninfected ticks or with DNA from a spotted fever group rickettsia commonly associated with A. hebraeum in Zimbabwe. The C. ruminantium specific pCS20 DNA probe can be applied to determine accurately the infection rates in the two major vectors of heartwater and the risk of exposure of ruminants in endemic areas.

The immunodominant 32-kilodalton protein of Cowdria ruminantium is conserved within the genus Ehrlichia.

Serological tests for cowdriosis are hampered by cross-reacting antibodies from animals suspected to be infected with Ehrlichia species. We have monitored infections with Ehrlichia bovis, E. ovina, E. canis and E. phagocytophila in experimental animals by competitive ELISA, Western blotting and immunofluorescence using Cowdria-infected endothelial cell culture antigens. Cross-reactions due to Ehrlichia antibodies could be attributed to the recognition of epitopes on the immunodominant Cr32 Cowdria protein. This was especially true for E. canis and E. ovina, much less for E. bovis, but not at all for E. phagocytophila. In addition, strong cross-reactivity between Cowdria and antibodies to E. Chaffeenis were demonstrated. These findings are in agreement with the phylogenetic relationships, recently reported by van Vliet et al. in 1992, between Cowdria and other members of the tribe Ehrlichieae, which showed Cowdria to be closely related to E. canis and also to E. chaffeensis. Although the tests used in this study remain valuable tools under laboratory conditions, their specificity requires improvement. It is suggested to study recombinant Cowdria antigens for the development of second generation serological tests for cowdriosis.

A cloned DNA probe for Cowdria ruminantium hybridizes with eight heartwater strains and detects infected sheep.

The DNA probe pCS20, which was cloned from the DNA of the Crystal Springs heartwater strain from Zimbabwe, cross-reacted with DNAs of heartwater strains from all endemic areas, including four heartwater strains from Zimbabwe, two strains from South Africa, one strain from Nigeria, and the Gardel strain from the Caribbean island of Guadeloupe. By nucleic acid hybridization, the pCS20 DNA probe detected Cowdria ruminantium DNA in all DNA preparations made from plasma samples from infected sheep before and during the febrile reaction. Synthetic oligonucleotides were prepared for amplification of specific C. ruminantium DNA sequences by the polymerase chain reaction (PCR). Amplification of two DNA products (181 and 279 bp) from pCS20 DNA and C. ruminantium genomic DNA of heartwater strains was demonstrated. In contrast, amplification of these products or any other products was not possible from genomic DNAs of Anaplasma marginale, Babesia bigemina, Trypanosoma brucei brucei, Escherichia coli, and bovine endothelial cells. The cross-reactivities of the 32P-labeled PCR products with genomic DNAs from several heartwater strains were similar to those with the pCS20 DNA probe. A nucleic acid-based test that uses hybridization assays and PCR provides a sensitive method for the detection of heartwater in both animals and ticks and has applications in epidemiological studies for the disease, which may allow for improved disease control.

Lack of cross-protection between Cowdria ruminantium and Ehrlichia phagocytophila.

Antigenically distinct stocks of Cowdria ruminatium from Senegal and South Africa were compared with a Dutch isolate of Ehrlichia phagocytophila in cross-immunity trials in goats. There was a complete absence of cross-immunity between E. phagocytophila and C. ruminantium, despite previous observations that both rickettsial organisms have certain antigenic determinants in common.

Observations on mouse-infective stocks of Cowdria ruminantium: microscopical demonstration of the Kwanyanga stock in mouse tissue and the carrier-status of the Senegal stock in mice.

The behaviour of two different stocks of Cowdria ruminantium was investigated in mice. The mouse-pathogenic Kwanyanga stock of C ruminantium was microscopically demonstrated in mice in capillary endothelial cells of the lung, spleen, kidney, liver and brain. Mice of the ninth passage of the Senegal stock, which is infective but not pathogenic to mice, were kept alive for a year. Their blood and homogenised spleens, inoculated intravenously, caused fatal heartwater in a goat. However, the Senegal stock could not be demonstrated microscopically in mice. These results indicate the possible role of rodents in the epidemiology of heartwater.

Serotypes in Cowdria ruminantium and their relationship with Ehrlichia phagocytophila determined by immunofluorescence.

Two tick-borne rickettsial pathogens of ruminants, Cowdria ruminantium (causative agent of heartwater disease) and Ehrlichia phagocytophila (causative agent of tick-borne fever), were successfully cultivated in caprine or ovine neutrophilic granulocytes. Infected cultures were subsequently used as antigens in the indirect fluorescent antibody test. Low-level bilateral serological cross-reactions could be detected between Cowdria and Ehrlichia. In addition, comparison of five Cowdria stocks using immunofluorescence demonstrated the existence of distinct serotypes within the genus of Cowdria. It is concluded that the occurrence of these serotypes will considerably complicate the current serodiagnosis of heartwater.

Observations on mouse-infective stocks of Cowdria ruminantium: attempts to demonstrate persistence of the organism in mice immune to the Kwanyanga stock.

Mice immunised against the Kwanyanga stock of Cowdria ruminantium by infection and treated with oxytetracycline proved immune to challenge on day 40 and also to a second challenge on day 125 after infection. Treatment with the experimental dithiosemicarbazone gloxazone on days 59 and 73 did not abolish immunity to challenge on day 125. No persistence of the organism in immune mice that had been challenged on day 40 could be demonstrated by subinoculating blood and liver homogenate on day 126. These results are different from findings reported elsewhere with the mouse-infective Kumm stock.

Observations on mouse-infective stocks of Cowdria ruminantium: attempts to demonstrate antigenic changes of the Kwanyanga stock in mice.

Starting from a stabilate of caprine blood infected with the Kwanyanga stock of Cowdria ruminantium, eight serial passages were made in groups of mice, and eight parallel serial passages in goats. Cross-immunity tests in goats and mice failed to demonstrate any difference between stabilates made after the eighth passage. The Kwanyanga stock was of exceptionally low virulence for Dutch goats.