Protective immunity to heartwater (Cowdria ruminantium infection) is acquired after vaccination with in vitro-attenuated rickettsiae

Abstract

A Senegalese (S) stock of Cowdria ruminantium was passaged on bovine umbilical endothelial cells with an average interval of 13.9 days (range, 8 to 34 days) between passages. The virulence of infected bovine umbilical endothelial cultures was tested in susceptible goats and sheep by intravenous inoculation of culture supernatant from passages 2 (51 days in vitro), 3 (69 days), 11 (229 days), 14 (264 days), and 16 (291 days). Both animals inoculated with passages 2 and 3 died of heartwater. However, clinical reactions were completely absent in goats and sheep that were inoculated with C. ruminantium from passages 11, 14, and 16. High antibody titers were detected, with immunofluorescence in all vaccinated animals, and a strong signal was found against a 32-kDa Cowdria protein in Western blots (immunoblots). Moreover, the vaccinated animals proved solidly immune when challenged with virulent Cowdria sp.-infected blood stabilate (S strain), whereas all control goats died. No attenuation of a second Cowdria stock (W) was achieved after 226 days in culture, at which time passage 17 was tested in a recipient goat which died of typical heartwater. This is the first report of vaccination with live attenuated C. ruminantium. These attenuated organisms may replace vaccination with virulent blood currently in use in areas where heartwater is endemic.

Experimental Inoculation with Human Granulocytic Ehrlichia Agent Derived from High- and Low-Passage Cell Culture in Horses

“…Differences in the virulence of E. phagocytophila strains in cattle and sheep have been shown in previous studies (18,21). Loss of virulence after several in vivo or in vitro passages has been shown for a variety of rickettsial agents, such as Anaplasma marginale (20) or Cowdria ruminantium (12). The low-and high-passage cell culture-derived HGE agents were both able to induce a febrile disease in the infected horses; however, differences in the degree and occurrence of clinical signs were found.…”

Molecular cloning, sequence analysis, and expression of the gene encoding the immunodominant 32-kilodalton protein of Cowdria ruminantium

“…Eight stocks of C. ruminantium were used in this study: one each from Senegal (Senegal [23]), Sudan (Um Banein [19]), Zambia (Lutale [23]), and Zimbabwe (Crystal Springs [7]) and four stocks from South Africa (Ball 3 [16], Kumm [13], Kwanyanga [27], and Welgevonden [10]). Four stocks of C. ruminantium (Crystal Springs, Senegal, Um Banein, and Welgevonden) were cultivated in vitro in bovine umbilical endothelial cells (isolate BUE 9) under conditions described previously (17).…”

“…Genomic DNA was obtained from BUE 9 cell cultures infected with C. ruminantium. When the cultures reached an infection score of 3 + for elementary bodies and reticulate bodies (17), infected endothelial cells were scraped from the bottom of the flask and mixed with the rickettsia-containing supernatant. This mixture was centrifuged for 10 min at 15,000 x g and 4°C and resuspended in 1 ml of sucrose-phosphateglutamate buffer (6) per 162-cm2 flask.…”

Validation of the Indirect MAP1-B Enzyme-Linked Immunosorbent Assay for Diagnosis of Experimental Cowdria ruminantium Infection in Small Ruminants

“…Forty-eight adult female Tesselaar sheep, all nonpregnant and 12 to 18 months old, were used as the infected reference sheep population. The animals were challenged with different C. ruminantium isolates by needle infection 1 month after vaccination with an attenuated Cowdria isolate originating from Senegal (17,20). Twenty-four sheep were challenged with the Senegal isolate, four with Welgevonden, and five sheep each with the Umpala, Lutale, Gardel, and Ball 3 isolates.…”