Quantification of Ehrlichia ruminantium by real time PCR.

Ehrlichia ruminantium (ER) is the causative agent of Heartwater, one of the most common tick-borne diseases affecting ruminants in African countries and West Indies. Although ER can be used as an inactivated vaccine for wild and domestic animals, there are currently no easy and reliable methods for the quantification of this obligate intracellular bacterium. This report describes the development of a SYBR Green I based real time PCR protocol for the quantification of ER for vaccine production purposes. The method was validated for four ER strains. The external-standard-based PCR protocol developed has a large dynamic quantitative range allowing accurate ER measurement in samples containing from 10(2) to 10(8) gene copies; the method is also reproducible and precise, with intra- and inter-assay coefficients below 5%. The detection limits were validated for samples collected from bovine aortic endothelial cell culture bulks, which are commonly used to produce the ER vaccine. In contrast to the methods based upon protein content, no interference from the host cells in ER quantification was observed. Furthermore, the extended applicability of the new technique was demonstrated by monitoring ER production in cell culture thus rendering it a valuable tool to ensure consistency between vaccine lots and to evaluate optimal vaccine dosage.

Validation and comparison of three enzyme-linked immunosorbent assays for the detection of antibodies to Cowdria ruminantium infection.

Serological tests for Cowdria ruminantium infection have been hampered by low specificity. Here, an indirect ELISA based on purified antigen, a competitive ELISA using a recombinant major antigenic protein (MAP-1) and an indirect ELISA based on the MAP-1B region of the recombinant MAP-1 were compared. The tests were validated using 3000 sera of ruminants from 14 islands of the Lesser Antilles as well as sequential serum samples from 10 cattle, 17 goats and 10 sheep vaccinated with inactivated C. ruminantium in ISA 50 adjuvant and from 14 goats infected with a virulent culture supernatant. All tests detected significantly higher percentages of positives on Antigua, Guadeloupe and Marie-Galante, where C. ruminantium had been isolated before. Overall specificity calculated with sera from the other 11 heartwater-free islands was 98.1%, 98.5%, and 99.4% for the ELISA based on crude antigen, recombinant MAP-1 and MAP-1B, respectively. Sensitivities observed with sequential serum samples were similar for all tests. Tests based on recombinant antigens, especially the MAP-1B, showed improved specificity, suggesting their use for epidemiological studies in regions where the distribution of cowdriosis is unknown. In addition, the competitive ELISA is useful for studies in wildlife for which species-specific conjugates do not exist.

[Asymptomatic carrier state in Creole goats and cattle after recovery from Cowdria infection in Guadeloupe]. / Le portage asymptomatique de bovins et chèvres Créole guéris de la cowdriose en Guadeloupe.

Creole goats and cattle in Guadeloupe can be carriers of cowdriosis (heartwater: Cowdria ruminantium) after recovery for a period as long as 11 months in goats and 2 months in cattle. The carrier status was demonstrated by feeding Amblyomma variegatum nymphs on recovered animals and the resulting adult ticks on susceptible goats. Cowdria ruminantium was not detected permanently during the carrier status.

[Comparison between 3 antigens for the serodiagnosis of heartwater disease by indirect immunofluorescence]. / Comparaison de trois antigènes pour le sérodiagnostic de la cowdriose par immunofluorescence indirecte.

A cell line of bovine endothelial cells (E5), infected with 3 different stocks of Cowdria ruminantium, was used as antigen in an indirect fluorescent antibody test for the serodiagnosis of heartwater. These antigens were compared to peritoneal macrophages from mice infected with the Kümm stock and to caprine neutrophils in primary cultures from goats infected with 4 different stocks of Cowdria. The use of endothelial cell cultures proved to be superior in all respects. The antigens can be produced in large quantities at a low cost, contrary to the other types. The reaction is easily and quickly read, compared to the laborious reading of neutrophil or macrophage antigens which often contain few and small colonies of Cowdria. Moreover, not all stocks are suitable for the preparation of neutrophil antigens, while macrophage antigen can only be obtained with the Kümm stock. Endothelial cell antigens also distinguish serotypes in C. ruminantium, but these differences seem to be less pronounced than those found with neutrophil antigens. Finally, the specificity of endothelial cell antigens appears to be better than that of Kümm antigen and comparable to that of neutrophil antigens. The use of Kümm antigen may have been responsible to a large extent for past unexplained positive serological results on certain Caribbean islands where it has not been possible to isolate Cowdria and where no clinical evidence of the disease has been found.

Diagnosis of heartwater in the live animal: experiences with goats in Guadeloupe.

A presumptive diagnosis of heartwater in the living animal can be based on clinical and epidemiologic observations. In Guadeloupe, heartwater can be confused with haemonchosis in goats or cerebral babesiosis in cattle. Confirmation of the clinical diagnosis by brain biopsy is useful in experimental infections but is hardly applicable in the field. Positive results were obtained from 92% of animals 16 to 18 days after experimental infection. In febrile animals the best results were obtained between the 3rd and 6th days of the thermal response. Diagnosis can also be supported by serological tests. These are useful for monitoring experimental infections and for checking recovered animals in the field. Nineteen goats out of 27 were negative on Day 1 of the febrile reaction but positive a week later. The remaining 8 goats were positive on Day 1 and had greatly increased antibody titres a week later. Confirmation of a diagnosis can also be achieved by subinoculating into susceptible animals either blood or suspensions of ticks collected from suspect animals and then homogenized. Ticks that have engorged on a suspect animal can be allowed to moult and then fed on a susceptible animal to test their infectivity. These methods are time consuming but useful for heartwater surveys.