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At least 19 recordsLinked to original sources
PubMed2004

Characterization of a major outer membrane protein multigene family in Ehrlichia ruminantium.

Ehrlichia ruminantium is a tick-transmitted rickettsial pathogen, which causes heartwater or cowdriosis in wild and domestic ruminants. A dominant antibody response of animals infected with E. ruminantium is directed against the outer membrane protein MAP1 (major antigenic protein 1). Part of the locus containing map1 has been characterized and consists of four map1 paralogs, designated map1-2, map1-1, map1 and map1+1, indicating that map1 is encoded by a multigene family. The purpose of this study was to determine the total number of map1 paralogs and their transcriptional activities. Using genome walking and data from an ongoing E. ruminantium genome sequencing project at the Onderstepoort Veterinary Institute, we found 16 paralogs of the map1 gene tandemly arranged in a 25 kb region of the E. ruminantium genome. The map1 multigene family is downstream of a hypothetical transcriptional regulator gene and upstream of the secA gene. Thirteen paralogs at the 5' end of the 25-kb locus were connected by short intergenic spaces (ranging from 0 to 42 bp) and the remaining three paralogs at the 3' end were connected by longer intergenic spaces (ranging from 375 to 1612 bp). All 16 map1 paralogs were transcriptionally active in E. ruminantium grown in endothelial cells and paralogs with short intergenic spaces were co-transcribed with their adjacent genes.

Antigens, Bacterial↗
PubMed2003

Phylogenetic relationships among Ehrlichia ruminantium isolates.

Ehrlichia ruminantium, the causative agent of heartwater, is a tick-borne pathogen infecting ruminants throughout sub-Saharan Africa and on some Caribbean islands. The most reliable test for E. ruminantium is PCR-based, but this gives positive results in some areas free of clinical heartwater and of the known Amblyomma spp. tick vectors. To investigate the molecular basis for this finding we have sequenced and carried out phylogenetic analysis of a range of genes from a number of E. ruminantium isolates. The genes include ribonuclease III and cytochrome c oxidase assembly protein genes (the pCS20 region), groESL, citrate synthase (gltA), and 16S ribosomal RNA. Relationships among major antigenic protein (map1) genes have been exhaustively investigated in a previous study that showed that the genes are variable in length, have non-synonymous mutations, and show no geographical specificity among isolates. The 16S sequences are highly conserved, except in the V1 loop region. The pCS20, groESL, and gltA genes show only single nucleotide polymorphisms (SNPs) dispersed throughout the sequenced regions. Phylogenetic analysis using pCS20 data differentiates the western African isolates into a single clade, which also includes a southern African isolate. All other southern African isolates and a Caribbean isolate fall into a further clade, which is subdivided into two groups. Sequence variation within this clade is greater than that within the western African clade, suggesting that E. ruminantium originated in southern Africa.

Africa South of the Sahara↗
PubMed2005

Transcription analysis of the major antigenic protein 1 multigene family of three in vitro-cultured Ehrlichia ruminantium isolates.

Ehrlichia ruminantium, an obligate intracellular bacterium transmitted by ticks of the genus Amblyomma, causes heartwater disease in ruminants. The gene coding for the major antigenic protein MAP1 is part of a multigene family consisting of a cluster containing 16 paralogs. In the search for differentially regulated genes between E. ruminantium grown in endothelial and tick cell lines that could be used in vaccine development and to determine if differences in the map1 gene cluster exist between different isolates of E. ruminantium, we analyzed the map1 gene cluster of the Senegal and Gardel isolates of E. ruminantium. Both isolates contained the same number of genes, and the same organization as found in the genome sequence of the Welgevonden isolate (H. Van Heerden, N. E. Collins, K. A. Brayton, C. Rademeyer, and B. A. Allsopp, Gene 330:159-168, 2004). However, comparison of two subpopulations of the Gardel isolate maintained in different laboratories demonstrated that recombination between map1-3 and map1-2 had occurred in one subpopulation with deletion of one entire gene. Reverse transcription-PCR on E. ruminantium derived mRNA from infected cells using gene-specific primers revealed that all 16 map1 paralogs were transcribed in endothelial cells. In one vector (Amblyomma variegatum) and several nonvector tick cell lines infected with E. ruminantium, transcripts were found for between 4 and 11 paralogs. In all these cases the transcript for the map1-1 gene was detected and was predominant. Our results indicate that the map1 gene cluster is relatively conserved but can be subject to recombination, and differences in the transcription of map1 multigenes in host and vector cell environments exist.

Antigens, Bacterial↗
PubMed2006

Determination and quantification of the in vitro activity of Aloe marlothii (A. Berger) subsp. marlothii and Elephantorrhiza elephantina (Burch.) skeels acetone extracts against Ehrlichia ruminantium.

An Ehrlichia ruminantium culture system was utilized for the anti-rickettsial evaluation of two ethnoveterinary plants, Elephantorrhiza elephantina and Aloe marlothii. Well-established E. ruminantium cultures were incubated with the plant leaf acetone extracts and compared to oxytetracycline and untreated controls. Effectivity was established by comparing the percentage parasitised cells and the calculation of both EC50 and extrapolated EC90 in microg/ml. The plant extracts were also screened for antibacterial activity using bioautography. Elephantorrhiza elephantina and A. marlothii demonstrated anti-ehrlichial activity with an EC50 of 111.4 and 64.5 microg/ml and EC90 of 228.9 and 129.9 microg/ml, respectively. The corresponding EC50 and EC90 for oxytetracycline was 0.29 and 0.08 microg/ml. Both plants appeared to produce their inhibitory activity by a similar mechanism, unrelated to that of the tetracyclines. Both the plant acetone extracts demonstrated antibacterial activity against Escherichia coli and Staphylococcus aureus (ATCC strains).

Acetone↗
PubMed2004

Possible death of a buffalo calf (Syncercus caffer) due to suspected heartwater (Ehrlichia ruminantium).

Rickettsial organisms resembling Ehrlichia ruminantium (the causative organism of heartwater) were demonstrated in brain smears and formalin-fixed brain sections derived from a buffalo calf that died on a private game reserve in northern KwaZulu-Natal. The possibility that the tick-free environment of a quarantine boma may have affected the calf's immunity, is discussed. These findings suggest that monitoring heartwater in wild ruminants and making brain smears as a routine during post mortem evaluations of wild ruminants, should be encouraged.

Animals↗
PubMed2003

In vitro infection of nonendothelial cells by Ehrlichia ruminantium.

The Welgevonden stock of Ehrlichia ruminantium was propagated in eight nonendothelial cell cultures derived from different animal species, both ruminants and nonruminants. The origins of the cells were: bovine fetal testis (BFT), cat ovary (COC), donkey fibroblasts (DFC), sheep fibroblasts (E(2)), horse testis (HTC), lamb fetal testis (LFT), mouse connective tissue (L), and African green monkey kidney (Vero). Four cell culture types (BFT, E(2), LFT and Vero) required supplementation of the medium with cycloheximide for suitable growth of E. ruminantium, whereas the other four (COC, DFC, HTC, and L) did not. Three other stocks of E. ruminantium, Senegal, Ball 3, and Gardel, were also propagated, either in LFT cultures only or in both E(2) and LFT cell cultures. The Welgevonden stock was successfully initiated using E(2) and LFT cell cultures.

Animals↗
PubMed2002

Genetic immunization with Ehrlichia ruminantium GroEL and GroES homologues.

Ehrlichia ruminantium GroEL and GroES genes were amplified from E. ruminantium Welgevonden genomic DNA and were cloned into genetic vaccine and Salmonella expression vectors. These constructs were used to inoculate Balb/c and C57BL/6J mice. Both GroEL and GroES induced low levels of protection in Balb/c and C57BL/6J mice immunized with the Salmonella expression vectors. None of the mice inoculated with the genetic vaccine survived. Immunological memory was also tested in these mice and a correlation between splenocyte proliferation and the survival rate was observed.

Animals↗
PubMed2005

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.

Africa South of the Sahara↗
PubMed2004

Nested PCR for detection and genotyping of Ehrlichia ruminantium: use in genetic diversity analysis.

Ehrlichia ruminantium, the agent of cowdriosis transmitted by Amblyomma ticks, presents an extensive genetic and antigenic diversity of key importance for vaccine formulation. Two means of nested polymerase chain reaction (PCR) targeting were developed to conduct molecular epidemiology studies in the Caribbean and Africa. The first used a conserved DNA fragment for detection of the pathogen in animals and vectors, and the second relied on the polymorphic map1 gene for genotyping. As compared to a PCR, the nested PCR showed a 2-Log10 improvement of sensitivity and allowed amplification from ticks, blood, brain, and lungs from infected animals, providing a more accurate picture of the tick infection rate. In Guadeloupe, this rate reached 36% (N = 212) instead of 1.7% (N = 224), as previously estimated. Genetic typing was done by restriction fragment length polymorphism or sequencing of map1 amplification products. Molecular epidemiology studies conducted in field sites selected for vaccination trials with inactivated vaccine, revealed the circulation of genetically divergent strains in limited geographical areas. It is known, then, that genetic clustering based on map1 has no predictive value regarding the protective value of a given strain against a new strain. However, tracing the strains by this technique revealed the extent of E. ruminantium diversity that one can expect in a given region, and the method allows differentiation between an inadequate immune response and the challenge by a breakthrough strain on animals dying despite vaccination. Up to now, genetic typing does not avoid cross-protection studies, which were conducted in parallel, although on a more limited scale. The importance of pathogen diversity studies for optimization of vaccine design is discussed as well as the research for new polymorphic genes. These genes may allow better predictions on cross-protection, given the recent completion of the sequence of the full genome of two E. ruminantium strains.

Animals↗
PubMed2005

Ehrlichia ruminantium: a promiscuous genome.

The intracellular bacterium, Ehrlichia ruminantium, is the causative agent of heartwater, a tick-borne disease of livestock. Because vaccines need to incorporate components from several virulent isolates, it is essential to have information on the extent of genetic variation among isolates. We therefore amplified and sequenced a panel of seven core function genes.

Antigenic Variation↗
PubMed2002

Ehrlichia ruminantium infection (heartwater) in wild animals.

Several wild animal species have been implicated as hosts of Ehrlichia ruminantium (formerly Cowdria ruminantium), the rickettsial agent causing heartwater, a fatal disease of domestic ruminants in sub-Saharan Africa and eastern Caribbean. However, evidence for infection in most wild species is inconclusive because of inadequate diagnostic techniques. Infection has been proven only in 12 African ruminants, three non-African ruminants and two African rodents. A subclinical carrier state occurs in eight of the African ruminant species. Further studies on E. ruminantium infection in wild animal species are needed in order to determine the host range of this pathogen accurately. The host range of Ehrlichia ruminantium in wildlife is reviewed here and the role played by these species in the epidemiology and spread of heartwater is discussed.

Africa↗
PubMed2006

Comparative genomics of three strains of Ehrlichia ruminantium: a review.

The tick-borne Rickettsiale Ehrlichia ruminantium (E. ruminantium) is the causative agent of heartwater in Africa and the Caribbean. Heartwater, responsible for major losses on livestock in Africa represents also a threat for the American mainland. Three complete genomes corresponding to two different groups of differing phenotypes, Gardel and Welgevonden, have been recently described. One genome (Erga) represents the Gardel group from Guadeloupe Island and two genomes (Erwo and Erwe) belong to the Welgevonden group. Erwo, isolated in South Africa, is the parental strain of Erwe, which was maintained for 18 years in Guadeloupe under different culture conditions than Erwo. The three strains display genomes of differing sizes with 1,499,920 bp, 1,512,977 bp, and 1,516,355 bp for Erga, Erwe, and Erwo, respectively. Gene sequences and order are highly conserved between the three strains, although several gene truncations could be pinpointed, most of them occurring within three regions of accumulated differences (RAD). E. ruminantium displays a strong leading/lagging compositional bias inducing a strand-specific codon usage. Finally, a striking feature of E. ruminantium is the presence of long intergenic regions containing tandem repeats. These repeats are at the origin of an active process, specific to E. ruminantium, of genome expansion/contraction based on the addition or removal of tandem units.

Animals↗
PubMed2006

Understanding the mechanisms of transmission of Ehrlichia ruminantium and its influence on the structure of pathogen populations in the field.

The understanding of the structure of Ehrlichia ruminantium stock population in the field was highlighted by experiments done in controlled conditions on the goat model. The mixture of strains observed in ticks seemed to be due to simultaneous infections rather than successive infections of the carrier. During a dual infection, the timing of Ehrlichia ruminantium circulation of the two stocks in hosts influenced their selection by ticks.

Animals↗
PubMed2006

Comparative genomic analysis of three strains of Ehrlichia ruminantium reveals an active process of genome size plasticity.

Ehrlichia ruminantium is the causative agent of heartwater, a major tick-borne disease of livestock in Africa that has been introduced in the Caribbean and is threatening to emerge and spread on the American mainland. We sequenced the complete genomes of two strains of E. ruminantium of differing phenotypes, strains Gardel (Erga; 1,499,920 bp), from the island of Guadeloupe, and Welgevonden (Erwe; 1,512,977 bp), originating in South Africa and maintained in Guadeloupe in a different cell environment. Comparative genomic analysis of these two strains was performed with the recently published parent strain of Erwe (Erwo) and other Rickettsiales (Anaplasma, Wolbachia, and Rickettsia spp.). Gene order is highly conserved between the E. ruminantium strains and with A. marginale. In contrast, there is very little conservation of gene order with members of the Rickettsiaceae. However, gene order may be locally conserved, as illustrated by the tuf operons. Eighteen truncated protein-encoding sequences (CDSs) differentiate Erga from Erwe/Erwo, whereas four other truncated CDSs differentiate Erwe from Erwo. Moreover, E. ruminantium displays the lowest coding ratio observed among bacteria due to unusually long intergenic regions. This is related to an active process of genome expansion/contraction targeted at tandem repeats in noncoding regions and based on the addition or removal of ca. 150-bp tandem units. This process seems to be specific to E. ruminantium and is not observed in the other Rickettsiales.

Conserved Sequence↗
PubMed2005

Characterization of Ehrlichia ruminantium replication and release kinetics in endothelial cell cultures.

Ehrlichia ruminantium is the causative agent of Heartwater, a fatal tick-borne disease affecting ruminants in African countries and West Indies and can be used as an inactivated vaccine for wild and domestic animals. In order to improve E. ruminantium production yields we characterize E. ruminantium growth kinetics in terms of duplication time, maximum production yield, and peak of infectivity. After a 24 h period for E. ruminantium attachment/internalization and a lag phase of 12 h, the exponential growth occurred within 36-108 h post-infection (hpi) with a net increase of up to 2.2 orders of magnitude. Maximum E. ruminantium infectivity was observed at 120 hpi and was defined as the best time of harvesting (TOH) for propagation of E. ruminantium cultures. This study showed that considering the quality constraint of the final product (E. ruminantium vaccine), the E. ruminantium suspension should be harvested at 113 hpi. Overall, the characterization of E. ruminantium progression through the average infection cycle, not only can contribute to the maximization of E. ruminantium production yield, with important consequences for the large scale production and utilization of an inactivated Heartwater vaccine, but also to elucidate growth mechanisms of some of the other ehrlichial species, with emerging impact in human and animal health.

Animals↗
PubMed2003

Effect of dimethyl sulfoxide in the treatment of sheep experimentally infected with Ehrlichia ruminantium.

OBJECTIVE: To evaluate the clinical response of sheep experimentally infected with Ehrlichia ruminantium to treatment with dimethyl sulfoxide (DMSO). ANIMALS: 32 Merino crossbred sheep. PROCEDURES: 16 sheep were infected with E ruminantium; 8 of these were treated twice daily with a 10% solution of DMSO (1 g/kg, i.v.) in polyionic fluid for 3 consecutive days. Treatment was initiated 2 days after the onset of clinical disease. Eight uninfected control sheep were similarly treated with DMSO. Placebo treatments (polyionic fluid administrations) were given to 8 infected and 8 uninfected sheep. Arterial and venous blood samples for blood gas and total plasma protein concentration measurements were collected daily (data from 5 days before until 6 days after onset of clinical disease were analyzed); physiologic variables and food consumption were also monitored. Gross pathologic findings and cytologic confirmation of the disease were recorded for the 16 infected sheep. RESULTS: Infected sheep treated with DMSO were able to maintain pulmonary gas exchange and had reduced pleural effusion and plasma protein loss, compared with infected untreated sheep that became hypoxic. Infected treated sheep developed an uncompensated metabolic acidosis. Uninfected treated sheep had reduced appetite, whereas uninfected untreated sheep maintained normal food intake. CONCLUSIONS AND CLINICAL RELEVANCE: Results of DMSO treatment in sheep with experimentally induced heartwater disease indicated that administration of this agent, in combination with specific antimicrobial treatment, may be of some benefit in treatment of naturally occurring disease.

Acidosis↗
PubMed2006

Homologous recombination and the pattern of nucleotide substitution in Ehrlichia ruminantium.

Patterns of nucleotide substitution at orthologous loci were examined between three genomes of Ehrlichia ruminantium, the causative agent of heartwater disease of ruminants. The most recent common ancestor of two genomes (Erwe and Erwo) belonging to the Welgevonden strain was estimated to have occurred 26,500-57,000 years ago, while the most recent common ancestor of these two genomes and the Erga genome (Gardel strain) was estimated to have occurred 2.1-4.7 million years ago. The search for genes showing extremely high values of the number of synonymous substitutions per site was used to identify genes involved in past homologous recombination. The most striking case involved the map1 gene, encoding major antigenic protein-1; evidence for homologous recombination is consistent with previous phylogenetic analysis of map1 alleles. At this and certain other loci, homologous recombination may have contributed to the evolution of host-pathogen interactions. In addition, comparison of the patterns of synonymous and nonsynonymous substitution provided evidence for positive selection favoring a high level of amino acid change between the Welgevonden and Gardel strains at a locus of unknown function (designated Erum4340 in the Erwo genome).

Computational Biology↗
PubMed2002

Sequence analysis of three Ehrlichia ruminantium LambdaGEM-11 clones.

Three Lambda GEM11 clones were isolated from a large-insert Ehrlichia ruminantium Welgevonden library. The inserts were amplified, sequenced, and analyzed. A total of 39 827 bp was obtained, and 18 different open reading frames (ORFs) were identified. Long repeats (100-200 kbp) were found in all three sequences. These repeats may play a role in the induction of antigenic variation. Along with a 20-kbp sequence of a clone from the E. ruminantium cosmid library, these sequences are the first large sequences to be yielded by the E. ruminantium genome sequencing project.

Animals↗