Accuracy of an indirect fluorescent-antibody test and of a complement-fixation test for the diagnosis of Babesia caballi in field samples from horses.

We evaluated the indirect fluorescent-antibody (IFA) test and complement-fixation (CF) test for diagnosis of equine piroplasmosis in the absence of a gold standard. Using Evan's blue, we estimated the specificity of the IFA test on a parasite-free, field horse population to be 98% (95% confidence interval=97, 99). We observed an excellent test agreement (kappa=0.83) between two collaborating laboratories when the IFA test was performed on identical samples from an endemic area. Using Bayesian analysis with informative prior probability distributions, we estimated the sensitivity of the IFA test to be 92% (95% probability interval, PI=81, 98), and specificity to be 95% (95% PI=88, 99). The CF test sensitivity and specificity estimates were 28% (95% PI=15, 47) and 99% (95% PI=96, 100), respectively. We found the IFA to be superior to the CF test, and the inclusion of Evan's blue in test protocol improved the performance of the IFA test. We conclude that the IFA test for Babesia caballi is a sensitive and specific test for the diagnosis of equine piroplasmosis.

Validation of the indirect fluorescent antibody and the complement fixation tests for the diagnosis of Theileria equi.

The indirect fluorescent antibody (IFA) test for Theileria equi was evaluated to assess test's suitability for the serological diagnosis of equine piroplasmosis, to provide performance parameters for the purpose of test validation, and to compare it with the complement fixation (CF) test. Using a protocol that included Evan's blue, the specificity of the IFA test was estimated at 99.0% for T. equi by the classical method of analysis, and 96.6% by the Bayesian method. The use of Evan's blue in the test protocol increased test specificity and contributed to an excellent test agreement between two collaborating laboratories (kappa = 0.96). Using Bayesian analysis, the sensitivity estimate for the IFA test was 89.2%. The CF test sensitivity and specificity estimates for T. equi were 63.1 and 96.4%, respectively, as determined by Bayesian analysis. The IFA test was more sensitive than the CF test but the specificity estimates were similar.

An attenuated Ehrlichia ruminantium (Welgevonden stock) vaccine protects small ruminants against virulent heartwater challenge.

Heartwater is a tick-borne disease of ruminants caused by the intracellular rickettsia Ehrlichia ruminantium. The only commercially available immunization procedure involves infecting animals with cryopreserved sheep blood containing virulent E. ruminantium organisms, followed by treatment with tetracyclines when fever develops. The virulent Welgevonden stock of E. ruminantium was attenuated by continuous propagation of the organisms in a canine macrophage-monocyte cell line (DH82), followed by re-adaptation to grow in a bovine endothelial cell line (BA 886). The material used for the present experiments consisted of the attenuated stock between passages 43 and 64 after re-adaptation. When inoculated into sheep or goats the attenuated organisms did not produce disease, and the only symptom observed was a rise in body temperature in most, but not all, animals. All sheep injected with 2 ml of culture suspension were subsequently found to be fully protected against a lethal needle challenge with the virulent homologous stock or with one of four different heterologous stocks (Ball 3, Gardel, Mara 87/7, Blaauwkrans). Titrations of elementary body suspensions showed that 2ml of a 1:10,000 dilution of culture suspension injected into sheep or goats was still sufficient to trigger an immune response which resisted a lethal needle challenge with the virulent Welgevonden stock. Adult Amblyomma hebraeum ticks, fed as nymphs on sheep immunized with DH82-derived organisms of passage 111, were able to transmit the attenuated stock to a naive sheep, which was found to be protected against a subsequent lethal homologous needle challenge.

In vitro isolation of equine piroplasms derived from Cape Mountain zebra (Equus zebra zebra) in South Africa.

Twenty blood samples of zebras (Equus zebra zebra) from the Karoo National Park and the Bontebok National Park in South Africa, all seropositive for Theileria equi, were subjected to in vitro culture to identify carrier animals and to isolate the parasites. Sixteen animals had a detectable parasitaemia in Giemsa-stained blood smears examined before culture initiation, the remaining four animals were identified as T. equi carriers by in vitro culture. Cultures were initiated either in an oxygen-reduced gas mixture or in a 5% CO2-in-air atmosphere. Out of the 20 blood samples, 12 cultures of T. equi and two cultures of T. equi mixed with Babesia caballi were established. None of the four animals seropositive for B. caballi could be identified as carrier animals, whereas two seronegative samples became culture-positive for B. caballi.

Culture, isolation and propagation of Babesia caballi from naturally infected horses.

Thirteen blood samples of horses from South Africa, five of which were seropositive for Babesia caballi and eight for both B. caballi and Theileria equi, were subjected to in vitro culture to identify carrier animals. None of the animals had a detectable parasitaemia on Giemsa-stained blood smears before culture initiation. Cultures were initiated in L-cysteine-enriched medium, either in an oxygen-reduced gas mixture or in a 5% CO2-in-air atmosphere. All five animals seropositive for B. caballi were identified as carrier animals using an oxygen-reduced atmosphere, whereas only four samples became culture positive under normal atmospheric conditions. Among the eight samples seropositive for both B. caballi and T. equi, two were identified as carriers for both. The remaining six samples were identified as carrying only T. equi.