In vitro cultivation of Ehrlichia ruminantium and development of an attenuated culture-derived vaccine

Abstract

Ehrlichia ruminantium, an intracellular gram-negative rickettsia, is the causative agent of heartwater, a tick-borne disease of domestic and wild ruminants. It is a major constraint to livestock production throughout sub-Saharan Africa and the islands of Madagascar, La Réunion, Mauritius and São Tomé. The disease is also present in the Caribbean, on the French Antillean islands of Guadeloupe and Antigua, to which infected Amblyomma variegatum ticks were introduced, possibly as early as the eighteenth century. Research into heartwater was hampered for many years by the impossibility of obtaining viable E. ruminantium organisms of a defined genotype and free from contamination with mammalian cellular material. A major turning point was reached when a suitable culture system became available. However, the first in vitro culture system was exceptionally fragile, and unexplained failures used to occur frequently, causing frustrating delays in heartwater research. The work described in this thesis has provided an important contribution to improved heartwater vaccine development. A chemically completely defined medium for the continuous in vitro propagation of E. ruminantium has been described. Additional in vitro studies revealed that E. ruminantium has a broader repertoire of host cells than previously assumed. Propagation in an unusual host cell line led to the emergence of an attenuated version of the Welgevonden stock of E. ruminantium, an important basis for the development of a culture-derived heartwater vaccine. Preliminary studies on the efficacy of this vaccine indicated that sheep were fully protected against a lethal challenge with the virulent homologous stock or with any of four different heterologous stocks. Future studies aimed at development of a freeze-dried formulation would greatly improve the acceptance of a live, attenuated vaccine by the end user. Effective, reliable control of heartwater using this vaccine to prevent serious disease outbreaks could have a tremendous positive economic impact for small-scale as well as commercial farmers in South Africa.