Growth of Cowdria ruminantium, the causative agent of heartwater, in a tick cell line.

The tick-borne rickettsia Cowdria ruminantium has been propagated continuously for over 500 days in the Ixodes scapularis tick cell line IDE8 by using the Gardel isolate from bovine endothelial cells as an inoculum. Infection of the tick cells was confirmed by PCR, karyotyping, electron microscopy, and reinfection of bovine cells.

Natural prevalence of infection with Ehrlichia (Cytoecetes) phagocytophila of Ixodes ricinus ticks in Scotland.

Ixodes ricinus nymphs and adults were collected from vegetation and from sheep at four sites in Scotland typical of areas endemic for tick-borne fever in sheep caused by infection with Ehrlichia (Cytoecetes) phagocytophila (Rickettsiales). The great majority of ticks examined was from woodland sites adjacent to sheep farms where there was a high probability of them feeding on roe deer (Capreolus capreolus) in a non-domestic focus of infestation and infection. Ticks were examined for infection by five methods. Batches of ticks were examined either by feeding on susceptible sheep or by feeding on rabbits and then prepared as stabilate which was inoculated into susceptible sheep. The sheep were monitored for clinical signs of tick borne fever. Batches of ticks were examined by polymerase chain reaction for Ehrlichia phagocytophila. Salivary glands were dissected out and stained by the Feulgen method to detect Ehrlichia masses, and were examined by indirect fluorescent antibody test. Each of the methods detected infection in ticks and the prevalence of infection in nymphs with the various methods ranged from >0.25% to 2.0%. Small samples of adults examined by Feulgen staining of salivary glands indicated infection prevalences of 2.1% in males and 1.6% in females. It is considered that these low infection prevalences may be typical of natural foci of infection where deer could be a major host of ticks and E. phagocytophila.

The Cowdria ruminantium groE operon.

A Cowdria ruminantium genomic DNA library was constructed in the expression vector lambda ZAPII, and an immunoreactive clone, designated lambda Cr9.4, was isolated by screening with serum from a C. ruminantium-infected goat. Sequencing of the insert from this clone revealed two open reading frames, encoding peptides of 10462 and 58697 kDa respectively. Database searching indicated that the two genes were homologues of groES and groEL, genes encoding a group of heat shock proteins involved in protein processing, export and assembly. Western blotting experiments showed that the recombinant GroEL protein was recognized by sera raised against four isolates of C. ruminantium which originate from South Africa, West Africa and the Caribbean, but not by antisera to the closely related Ehrlichia species (E. ovina, E. [Cytoecetes] ondiri, E. bovis, E. phagocytophila) of African and European ruminants which can be expected to occur in similar geographical areas to C. ruminantium. This suggests that this protein may be useful in development of serodiagnostic tests for C. ruminantium infection which are not subject to cross-reactions with antibodies to Ehrlichia species. The cloning and expression of the GroE operon will also facilitate further study of the roles of the GroE proteins in the immune response to C. ruminantium.

Comparison of two anticoagulants for production of antigens of Cowdria ruminantium in neutrophils.

Ethylene diamine tetra-acetate (EDTA) and lithium heparin were used comparatively as anticoagulants for blood obtained from goats clinically infected with Cowdria ruminantium. Neutrophils were extracted from the blood and cultured for the production of heartwater antigen. EDTA proved superior to heparin in terms of the recovery rate and the better separation of neutrophils from other leucocytes. The antigen produced was tested in the indirect fluorescent antibody test (IFAT) and proved to be of good quality. Production of antigen slides by this method is recommended for moderately well-equipped laboratories in heartwater-endemic areas.

Seroconversion to Cowdria ruminantium of Malawi zebu calves, reared under different tick control strategies.

The seroconversion by indirect ELISA to Cowdria ruminantium over the first year of life of sixty-six Malawi zebu calves born into groups which were dipped 17 times per year was compared to seroconversion of 32 calves born into non-dipped groups. Amblyomma variegatum tick counts and clinical disease in each group of cattle were monitored throughout the study period. No cases of heartwater were seen in either group of calves over the first 22 months of life. Only one case of heartwater was observed, in an 8 year old cow, in the 1,800 intensively monitored cattle over the same period. By 12 months of age almost all undipped calves had seroconverted and 50% of seroconversions were attributed to nymphal challenge. In contrast, only 41% of calves had become seropositive by 12 months of age in the dipped groups. The dipping regime used therefore significantly decreased seroconversion rates to C. ruminantium in these calves. 73% of calves had detectable levels of maternal antibodies to C. ruminantium in the first 4 weeks of life. Antibody levels in each of the calves in dipped groups had waned to below the cut off point for the ELISA by 8-12 weeks. Seroconversion did not occur in the first 8-12 weeks of life in dipped herds. The indirect ELISA test results were not significantly different in the proportion positive in single tests at 12 months of age, or by cumulative test results of the previous 9 months, and therefore the test may be of value as a test of herd immunity.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of antibodies to the agent of tick-borne fever by indirect immunofluorescence.

Cytospin preparations of sheep granulocytes infected with Cytoecetes phagocytophila, the causative agent of tick-borne fever (TBF), enabled the development of an improved and reproducible indirect fluorescent antibody test to detect antibodies in the sera of sheep convalescing from TBF. The test was compared with counter-immunoelectrophoresis.