Elsevier

Vaccine

Volume 25, Issue 46, 14 November 2007, Pages 7939-7947
Vaccine

Immunisation of sheep against heartwater in The Gambia using inactivated and attenuated Ehrlichia ruminantium vaccines

https://doi.org/10.1016/j.vaccine.2007.09.002Get rights and content

Abstract

Heartwater (cowdriosis) is a disease of ruminants caused by a rickettsial pathogen Ehrlichia ruminantium and transmitted by ticks of the genus Amblyomma. The purpose of this work was to evaluate the protective efficacies of inactivated and attenuated vaccines to protect sheep against heartwater in The Gambia. An inactivated vaccine, prepared from E. ruminantium (Gardel stock), and a live attenuated vaccine from E. ruminantium (Senegal stock), were evaluated in two independent on-station trials. A local stock of E. ruminantium (Kerr Seringe) was used as challenge material. Inactivated and live attenuated vaccines provided 43% and 100% protection, respectively, against virulent needle challenge. In a subsequent field trial, the attenuated vaccine protected 75% of sheep against virulent tick challenge, which was fatal for all control sheep. Quantification by real-time PCR showed that an immunising dose of approximately 23,000 attenuated E. ruminantium organisms was sufficient. Moreover, restriction fragment length polymorphism (RFLP) analysis indicated that the local Kerr Seringe genotype caused mortality amongst control sheep, whereas fatalities in the vaccinated group could be attributed to a different genotype.

Introduction

Heartwater, caused by Ehrlichia ruminantium and transmitted by ticks of the genus Amblyomma, is a disease of major economic importance affecting cattle and small ruminants, in sub-Saharan Africa and the Caribbean [1], [2], [3]. Recent surveys in West Africa indicated the existence in some areas of high levels of heartwater disease risk for susceptible livestock introduced from non-endemic areas [4], [5]. The disease can be controlled by a combination of acaricides [6], chemotherapy and immunophylaxis using an infection and treatment method [7]. The latter method is based on the virulent Ball 3 stock employed in South Africa, where it does not provide a broad protection against field isolates. The use of chemotherapy is constrained by the acute nature of the disease, which often does not allow timely intervention to prevent a fatal outcome. The application of acaricides by spraying or dipping of livestock can be effective, but carries the risk of contaminating the food chain. This is further complicated by the ability of ticks to develop resistance to the acaricides [8]. Hence, immunisation using a safe, effective and affordable vaccine appears to be the most desirable and sustainable control option.

Several attempts which have been made include the development of inactivated vaccines, attenuated vaccines and also DNA vaccines [9], [10]. Although progress has been made, the antigenic diversity identified amongst different stocks of E. ruminantium resulting in a lack of protection between heterologous stocks, has been identified as a major obstacle in vaccine development. Results of field trials on the efficacy of inactivated vaccines in sub-Saharan Africa vary [10]. For instance, an inactivated vaccine prepared from the Mbizi stock of E. ruminantium provided protection to field challenge at specific locations [11]; trials in West Africa with inactivated E. ruminantium (Gardel) vaccine have been less satisfactory (A. Gueye, personal communication).

Attempts to develop a live attenuated vaccine were first reported using the Senegal stock of E. ruminantium, which became attenuated after 11 passages in vitro[12]. Although this stock provided complete protection against homologous challenge [12], it did not provide efficient cross-protection against virulent heterologous challenge on station and in the field [13], [14]. However, in a recent report, the South African Welgevonden stock of E. ruminantium was successfully attenuated in vitro[9] and was able to protect sheep against challenge with four virulent heterologous stocks [15].

In this study, the protective efficacy of inactivated and live attenuated E. ruminantium vaccines against heartwater was evaluated in Sahalian sheep in The Gambia under a smallholder traditional husbandry system.

Section snippets

In vitro cultivation of E. ruminantium (Kerr Seringe): challenge material

The E. ruminantium (Kerr Seringe) isolate was made from a naturally infected West African dwarf goat (#1946) reared at the International Trypanotolerance Centre (ITC), Kerr Seringe, The Gambia. Whole blood collected into vacutainer tubes containing ethylenediaminetetraacetic acid (EDTA) as anticoagulant (Becton-Dickson®), just before death due to heartwater (confirmed by identification of E. ruminantium in Giemsa-stained brain smears), was cryopreserved at −80 °C in 2 ml aliquots with 10%

In vitro isolation and cultivation of E. ruminantium (Kerr Seringe)

Microscopic examination of Giemsa-stained smears of culture supernatant prepared on day 22 after inoculation revealed the presence of E. ruminantium and confirmed the successful isolation of the organism in cell culture (data not shown). The map1 and pCS20 PCRs also amplified target sequences and gave specific amplicons (data not shown). The map1 coding sequence of the Kerr Seringe stock, named after the location of origin, was cloned, sequenced and submitted to GenBank and assigned accession

Discussion

Current methods available for evaluation to protect small ruminants in West Africa against heartwater are based on inactivated [11], [23], [34], [35], [36] and live attenuated vaccines [12], [15]. In the present study, we evaluated the efficacy of both vaccination methods in laboratory (on-station) and field trials using Sahelian sheep in The Gambia. Prior to the on-station trials, it was necessary to first isolate a local stock of E. ruminantium (Kerr Seringe), which was successfully

Acknowledgements

This work was supported by The European Development Fund, contract no. REG/6061/006, Rothamsted International (UK), The International Foundation for Science (Sweden) in partnership with COMSTECH, Epigenevac project under contract number no. INCO-CT-2005-003713, The Integrated Consortium on Ticks and Tick-borne Diseases (ICTTD-3) financed by the European Union through Coordination Action Project no. 510561, the Utrecht Scholarship Programme (Utrecht University) and The Rural Finance and

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