Serum‐free Media for the <i>in Vitro</i> Cultivation of <i>Cowdria ruminantium</i>

Zweygarth, E.; Josemans, Antoinette I.; Horn, Ernst

doi:10.1111/j.1749-6632.1998.tb11063.x

Cited by 7 publications

(5 citation statements)

References 4 publications

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“…An existing library constructed in Lambda ZAP II (4) was used for initial sequencing. A second small insert library was constructed in pMOSBlue (Amersham Pharmacia) by using E. ruminantium genomic DNA prepared from the culture stock derived from the South African Welgevonden isolate (5) grown in a bovine aorta endothelial cell line (6). Genomic DNA was nebulized for 2 min at 100 kPa in a Medel (San Polo di Torrile, Italy) jet nebulizer reservoir, and fragments in the 600-to 1,500-bp range were cloned into the plasmid.…”

Section: Methodsmentioning

confidence: 99%

The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number

Collins

Liebenberg

Villiers

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

153

144

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Heartwater, a tick-borne disease of domestic and wild ruminants, is caused by the intracellular rickettsia Ehrlichia ruminantium (previously known as Cowdria ruminantium). It is a major constraint to livestock production throughout subSaharan Africa, and it threatens to invade the Americas, yet there is no immediate prospect of an effective vaccine. A shotgun genome sequencing project was undertaken in the expectation that access to the complete protein coding repertoire of the organism will facilitate the search for vaccine candidate genes. We report here the complete 1,516,355-bp sequence of the type strain, the stock derived from the South African Welgevonden isolate. Only 62% of the genome is predicted to be coding sequence, encoding 888 proteins and 41 stable RNA species. The most striking feature is the large number of tandemly repeated and duplicated sequences, some of continuously variable copy number, which contributes to the low proportion of coding sequence. These repeats have mediated numerous translocation and inversion events that have resulted in the duplication and truncation of some genes and have also given rise to new genes. There are 32 predicted pseudogenes, most of which are truncated fragments of genes associated with repeats. Rather then being the result of the reductive evolution seen in other intracellular bacteria, these pseudogenes appear to be the product of ongoing sequence duplication events.gene duplication ͉ bacterial genome ͉ molecular sequence data ͉ intracellular adaptation E hrlichia ruminantium (previously known as Cowdria ruminantium) is an obligate intracellular bacterium in the order Rickettsiales. Species in this order cause serious diseases in man and domestic animals throughout the world. E. ruminantium is transmitted by ticks of the genus Amblyomma and causes heartwater, a fatal and economically important disease of wild and domestic ruminants. The disease occurs throughout subSaharan Africa and on several Caribbean islands, from which it threatens to invade the Americas (1), but the existing immunization procedures are rudimentary and relatively ineffective (2). E. ruminantium is a fragile bacterium with exacting culture requirements in eukaryotic cell lines; genetic manipulation has not been attempted, and little is known about its mechanisms of virulence or pathogenesis. Heartwater affects all domestic ruminants, and 80-95% of naïve animals die within 3 weeks, but those that recover have a T cell-mediated immunity to subsequent homologous challenge (3). In the absence of any directed strategy to identify T cell-stimulatory proteins we sequenced the E. ruminantium genome in the expectation that access to the complete protein-coding repertoire of the organism would facilitate the search for vaccine candidate genes.

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Section: Methodsmentioning

confidence: 99%

The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number

Collins

Liebenberg

Villiers

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

153

144

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“…E. ruminantium (Welgevonden) was grown in bovine endothelial cells (Zweygarth & Josemans 2001 ) and genomic DNA prepared by Percoll density gradient and DNeasy blood and tissue kit (QIAGEN) as described previously (Mahan et al 1995 ). For ease of cloning an adenine (A) and thymine (T) rich gene sequence, Erum2510 was cleaved into six overlapping subfragments, for which amplicon specific primers were designed ( Appendix 1 ).…”

Section: Methodsmentioning

confidence: 99%

Th1 and Th2 epitopes of Cowdria polymorphic gene 1 of Ehrlichia ruminantium

Ngoepe¹,

Pretorius²,

Steyn³

et al. 2023

Onderstepoort j. vet. res.

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Cowdria polymorphic gene 1 (cpg1, Erum2510, ERUM_RS01380) has been shown to induce 30% and 100% protection in sheep immunised by deoxyribonucleic acid (DNA) prime combined with DNA boost and DNA prime combined with protein boost, respectively, against heartwater infection via needle challenge. To localise its antigenic regions for inclusion in a multi-epitope DNA vaccine against heartwater, Erum2510 was cleaved into five overlapping subfragments. These subfragments were expressed individually in an Escherichia coli host expression system and evaluated for their ability to induce proliferative responses, Th1 and Th2 cytokines (interferon gamma [IFN-γ] and interleukin 4 [IL-4]) via enzyme-linked immunospot (ELISpot), quantitative real time polymerase chain reaction (qRT-PCR) and flow cytometry. Recombinant (r)proteins 3 and 4 were shown to induce immunodominant Th1 and Th2 immune responses characterised by the secretion of effector cytokines IFN-γ and IL-4 in addition to differential messenger ribonucleic acid (mRNA) expression of tumour necrosis factor (TNF), IL-2, IL-1, IL-18, IL-10, transforming growth factor (TGF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and inducible nitric oxide synthase (iNOS). Thirty-seven overlapping synthetic peptides (16 mer) spanning the lengths of these immunodominant rproteins were synthesised and assayed. A peptide pool comprising p9 and p10 derived from rprotein 3 induced a Th1-biased immune response. A peptide pool comprising p28 and p29 derived from rprotein 4 induced a mixed Th1 and Th2 immune response characterised by secretion of IFN-γ and differential mRNA expression of IL-1, IL-2, IL-10, IL-12, iNOS, TGF, TNF and GM-CSF. Only one of the peptides (p29) induced secretion of IL-4. Phenotypic analysis showed significant activation of cluster of differentiation 8+ (CD8+), cluster of differentiation 4+ (CD4+) and B+ lymphocyte populations. Findings suggest that Erum2510 rproteins and synthetic peptides can induce both cellular and humoral immune responses, thereby implicating their importance in protection against heartwater.Contribution: This study will facilitate the design of an effective multi-epitope DNA vaccine against heartwater that will contribute to control this economically important disease in sub-Saharan Africa and beyond.

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“…nov 1. The organism was cultured in a serum‐free medium,15 and E. ruminantium elementary bodies were purified and genomic DNA prepared as described elsewhere 16…”

Section: Methodsmentioning

confidence: 99%

Development of Improved Vaccines for Heartwater

Collins

Pretorius

Kleef

et al. 2003

Annals of the New York Academy of Sciences

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Heartwater is a tick-borne disease of ruminants which causes major economic losses for domestic livestock owners throughout sub-Saharan Africa and the Caribbean. It is caused by the intracellular rickettsia Ehrlichia (formerly Cowdria) ruminantium and the only commercially available vaccination procedure is over 50 years old. It involves infecting animals with cryopreserved sheep blood containing virulent E. ruminantium organisms, followed by antibiotic treatment when fever develops. Experimental attenuated, inactivated, and nucleic acid vaccine procedures have been investigated over the last half-century, but none of them has yet been particularly successful. We have developed two new experimental vaccines, a live attenuated vaccine and a nucleic acid vaccine. The attenuated vaccine was developed by continuous passage of E. ruminantium organisms of the virulent Welgevonden isolate in a continuous canine macrophage-monocyte cell line. After more than 50 passages the cultures produced no disease when inoculated into mice or sheep, and the inoculated animals were 100% immune to a subsequent lethal homologous needle challenge. The nucleic acid vaccine is based on four E. ruminantium genes from a genetic locus involved in nutrient transport. A cocktail of all four genes, cloned in a DNA vaccine vector and used to immunize sheep, engendered 100% protection against a subsequent lethal needle challenge with the homologous isolate and with each of five different virulent heterologous isolates. Sheep immunized with this cocktail were also exposed to a field challenge in a heartwater-endemic area and few animals survived. This suggests that the local E. ruminantium genotypes were different from any which were administered by needle challenge, or that needle challenge is not a good model for tick challenge in the field.

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Serum‐free Media for the in Vitro Cultivation of Cowdria ruminantium

Cited by 7 publications

References 4 publications

The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number

The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number

Th1 and Th2 epitopes of Cowdria polymorphic gene 1 of Ehrlichia ruminantium

Development of Improved Vaccines for Heartwater

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