Ehrlichia ruminantium variants which do not cause heartwater found in South Africa

In 1994 a batch of apparently healthy goats was selected for intended export to the USA from a heartwater-free and vector tick-free region of South Africa. The animals were tested serologically for heartwater, using either or both an IFA and an ELISA test, and 52% were found to be serologically positive. A PCR assay based on Ehrlichia ruminantium 16S gene sequences gave positive results for 54% of the animals, suggesting that apparently non-pathogenic E. ruminantium variants existed in this heartwater-free area. To identify and characterise the agents responsible for the positive serological and PCR results, ticks and animal blood samples were collected from two of the three farms involved in the original survey during two successive seasons of expected peak tick activity. Ticks were kept alive for a minimum of 3 weeks to allow digestion of any blood meal before being processed. Over the two seasons, 28% of the livestock and 15% of the ticks sampled were found to be carrying E. ruminantium. E. ruminantium 16S and pCS20 sequences were detected in all of the four tick species collected from the livestock (Rhipicephalus evertsi evertsi, Rhipicephalus evertsi mimeticus, Hyalomma truncatum, Hyalomma marginatum rufipes), suggesting that some of the species may act as vectors. Animals generally carried multiple E. ruminantium 16S genotypes, whereas ticks rarely carried more than one. Infection levels in both animals and ticks were too low to generate a marked response when a blood stabilate was sub-passaged in a clean sheep, preventing the subsequent establishment of any of the organisms in culture.This research was funded by the Red Meat Research and Development Trust (RMRDT) of South Africa. We thank Prof. Ivan Horak for advice and critical reading of the manuscript

The Kumm isolate of Ehrlichia ruminantium: in vitro isolation, propagation and characterization

An effective culture system for Ehrlichia (Cowdria) ruminantium comb. nov. was first established in 1985 and many stocks were subsequently isolated and propagated in vitro. A notable exception, however, was the Kumm isolate that resisted all attempts at in vitro culture until the successful experiment described here. In one experiment white blood cells were harvested from heparinized blood derived from a sheep infected with the Kumm isolate. The cells were added to DH 82 cells and incubated at 37degreesC. The high metabolic activity of the DH 82 cells necessitated that cell growth be retarded by the addition of cycloheximide. Colonies were first detected 19 days after culture initiation and, once the cultures were established, they could be passaged every 3 days. Bovine and sheep endothelial cells were readily infected with culture supernatant obtained from the infected DH 82 cells. In a further experiment, another sheep was infected, using a higher dose of the same batch of Kumm stabilate, and we attempted to infect several different cell lines: these were DH 82 cells, bovine aorta (BA 886) cells, sheep brain endothelial (SBE 189) cells and sheep fibroblastoid cells (E₂). Ten days after culture initiation, only the E₂ cells had become positive for E. ruminantium. Culture supernatant from the first cultured isolate (Kumm-1) was less virulent for mice than that of the second cultured isolate (Kumm-2) which killed all mice. Upon molecular characterization with E. ruminantium 16S probes, we found that Kumm-1 hybridized with a Senegal 16S genotype probe, whereas Kumm-2 hybridized only with an Omatjenne 16S genotype probe. The original stabilate used to infect the sheep hybridized with both probes. These results clearly indicate that two different stocks had been isolated in culture.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat v.9 was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.Agricultural Research Council of South Africa. European Union (Cowdriosis Network) Grant no. IC18-CT95-0008 (DG12-SNRD)

Identification of Ehrlichia ruminantium proteins that activate cellular immune responses using a reverse vaccinology strategy

Ehrlichia ruminantium is an obligate intracellular bacterial pathogen which causes heartwater, a serious tick-borne disease of ruminants throughout sub-Saharan Africa. The development of promising recombinant vaccines has been reported previously, but none has been as effective as immunisation with live organisms. In this study we have used reverse vaccinology to identify proteins that elicit an in vitro cellular immune response similar to that induced by intact E. ruminantium. The experimental strategy involved four successive steps: (i) in silico selection of the most likely vaccine candidate genes from the annotated genome; (ii) cloning and expression of the selected genes; (iii) in vitro screening of the expressed proteins for their ability to induce interferon-gamma (IFN-ᵧ) production in E. ruminantium–immune lymphocytes; and (iv) further examination of the cytokine response profiles of those lymphocytes which tested positive for IFN-ᵧ induction. Based on their overall cytokine induction profiles the recombinant proteins were divided into four distinct groups. Eleven recombinant proteins induced a cytokine profile that was similar to the recall immune response induced by immune peripheral blood mononuclear cells (PBMC) stimulated with intact E. ruminantium. This response comprised the upregulation of cytokines associated with adaptive cellular immune responses as well as innate immunity. A successful vaccine may therefore need to contain a combination of recombinant proteins which induce both immune pathways to ensure protection against heartwater.The South African Department of Agriculture OV9/23/C167 grant and the FP6 EU INCO-DEV EPIGENEVAC FP6-003713 grant.http://www.elsevier.com/locate/vetimmab201

Cowdria ruminantium DNA is unstable in a SuperCos1 library

A Cowdria ruminantium genomic library was constructed in a cosmid vector to serve as a source of easily accessible and pure C. ruminantium DNA for molecular genetic studies. The cosmid library contained 846 clones which were arrayed into microtitre plates. Restriction enzyme digestion patterns indicated that these clones had an average insert size of 35 kb. Probing of the arrays did not detect any bovine clones and only one of the known C. ruminantium genes, pCS20, was detected. Due to the high AT content and the fact that C. ruminantium genes are active in the Escherichia coli host, the C. ruminantium clones were unstable in the SuperCos 1 vector and most clones did not grow reproducibly. The library was contaminated with E. coli clones and these clones were maintained with greater fidelity than the C. ruminantium clones, resulting in a skewed representation over time. We have isolated seven C. ruminantium clones which we were able to serially culture reproducibly; two of these clones overlap. These clones constitute the first large regions of C. ruminantium DNA to be cloned and represent almost 10% of the C. ruminantium genome.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat v.9 was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201

Protection against heartwater by DNA immunisation with four Ehrlichia ruminantium open reading frames

We have reported previously that a recombinant DNA vaccine consisting of four Ehrlichia ruminantium (Welgevonden) open reading frames (ORFs) known as the 1H12 cocktail provided protection against a virulent E. ruminantium (Welgevonden) needle challenge in sheep. In this study, we have investigated the vaccine effectiveness of two other cocktails of E. ruminantium (Welgevonden) ORFs, as well as single ORFs from the 1H12 cocktail, to protect sheep against a virulent needle challenge with the homologous strain. Each individual 1H12 ORF provided protection, but all the animals vaccinated with the other cocktails succumbed to the challenge.The authors thank Stephen Johnston (University of Texas Southwestern Medical Center) for supplying the pCMViUBs vector and Dr. Erich Zweygarth and Antoinette Josemans (ARC-Onderstepoort Veterinary Institute, SA) for providing E. ruminantium tissue culture material for DNA extraction. This work was supported by the South African Department of Science and Technology LEAD biotechnology grant and the EU INCO-ICA4-CT-2000-30026 grant

Extensive genetic recombination occurs in the field between different genotypes of Ehrlichia ruminantium

The intracellular bacterium Ehrlichia ruminantium is the causative agent of heartwater throughout sub-Saharan Africa, Madagascar, and some islands of the Caribbean. The disease is tick-borne and causes substantial livestock losses, threatening food security and productivity in both the commercial and small-scale farming sectors in endemic areas. Immunization by infection and treatment is currently practised in South Africa, and it is known that a variety of immunotypes of the organism occur in the field, and that cross-protection between them varies widely from total to minimal. Future vaccines may therefore need to incorporate components from different genotypes so it is essential to have information on the extent of genetic variation among isolates. To obtain this information we amplified and sequenced a panel of eight core function genes from 12 different cultured stocks originally isolated in different areas of Africa and the Caribbean. Phylogenetic trees inferred from the sequences yielded different branching orders for different genes, and the reason for this inconsistency appears to be that extensive recombination takes place between different genotypes in the field. It is possible that recombination occurs during the period when the organisms are extracellular within the tick, immediately after feeding and before intracellular infection is established, although detection of more than one genotype in DNA from single ticks is encountered infrequently. The results of the analysis show that the phylogenetic variation is greatest among the isolates of southern African origin, suggesting that this is the region where the parasite first evolved. It also appears likely that the Gardel genotype, isolated in the Caribbean, originally came from west central Africa, not from west Africa as had long been assumed.This research was supported by the National Research Foundation of South Africa under Heartwater Vaccine Grant no. FA2004042200063; the European Union under Cowdriosis Network Grant no. IC18-CT95-0008 (DG12-SNRD), and by the Agricultural Research Council of South Afric

Natural history of Ehrlichia ruminantium

Ehrlichia ruminantium is an obligately intra-cellular "-proteobacterium which causes a disease known as heartwater or cowdriosis in some wild, and all domestic, ruminants. The organism is transmitted by ticks of the genus Amblyomma, and it is of serious economic importance wherever the natural vectors occur, an area which includes all of sub-Saharan Africa, and several islands in the Caribbean. The disease was first recognized in South Africa in the 19th century, where its tick borne nature was determined in 1900, but the organism itself was not demonstrated until 1925, when it was recognized to be a rickettsia, initially named Rickettsia ruminantium. It was thus the first species of what are now known as Ehrlichia to be discovered, and most of the early work to elucidate the nature of the organisms, and its reservoirs and vectors, was performed in South Africa. The next milestone was the development, in 1945, of an infection and treatment regimen to immunize livestock, and this is still the only commercially available “vaccine” against the disease. Then in 1985, after fruitless attempts over many years, the organism was propagated reliably in tissue culture, opening the way for the first application of the newly developed techniques of molecular genetics. From 1990 onwards the pace of heartwater research accelerated rapidly, with notable advances in phylogeny, diagnosis, epidemiology, immunology, and vaccine development. The complete genome sequence was published in 2005, and during the last two years a new understanding has arisen of the remarkable genetic variability of the organism and new experimental vaccines have been developed. Despite all this the goal of producing an effective vaccine against the disease in the field still remains frustratingly just beyond reach. This article summarises our current understanding of the nature of E. ruminantium, at a time when the prospects for the development of an effective vaccine against the organism seem better than at any time since its discovery 83 years ago

Cowdriosis – infeccion por Ehrlichia ruminantium

Heartwater is a notifiable disease that is listed by the World Organisation for Animal Health. It is caused by Ehrlichia ruminantium, an obligately intracellular Gramnegative bacterium in the order Rickettsiales and the family Anaplasmataceae. The disease is borne by ticks in the genus Amblyomma and causes heartwater, or cowdriosis, in wild and domestic ruminants, primarily in Africa, but also in parts of the Caribbean. The disease was recognised in South Africa in the 19th Century and determined to be tick borne in 1900, while the organism was identified in 1925 and first cultured in vitro in 1985. This latter achievement boosted research into the disease at a time when biology was moving into the molecular genetic age. Over the last 20 years, there have been significant improvements in our understanding of E. ruminantium, yielding major advances in diagnosis, epidemiology, genetic characterisation, phylogeny, immunology, and vaccine development. The organism is genetically highly variable; this has important implications for future control measures, and is making it difficult to develop an effective vaccine for protection against tick challenge. Research is continuing into three different types of vaccine, inactivated, attenuated, and recombinant, and the current state of development of each is discussed.La cowdriose est une maladie à déclaration obligatoire inscrite sur la liste de l’Organisation mondiale de la santé animale. L’agent causal est Ehrlichia ruminantium, une bactérie obligatoire intracellulaire à Gram négatif appartenant à l’ordre des Rickettsiales et à la famille des Anaplasmataceae. Cette bactérie transmise par des tiques du genre Amblyomma provoque la cowdriose chez les ruminants sauvages et domestiques, principalement en Afrique mais aussi dans certaines régions des Caraïbes. La maladie a été décrite pour la première fois en Afrique du Sud au cours du xixe siècle ; le rôle de vecteurs des tiques a été élucidé en 1900 ; l’identification du micro-organisme remonte à 1925 mais la première culture in vitro a été réalisée en 1985. Ce succès a donné un nouvel élan à la recherche sur cette maladie à une époque où la biologie entrait dans l’ère de la génétique moléculaire. Au cours des 20 dernières années, les connaissances sur E. ruminantium ont connu des avancées significatives qui se sont traduites par des améliorations majeures dans les domaines du diagnostic, de l’épidémiologie, de la caractérisation génétique, de la phylogénie, de l’immunologie et du développement de vaccins. La grande variabilité génétique du micro-organisme a des répercussions importantes sur les mesures de contrôle qui seront prises à l’avenir et entraîne des difficultés particulières pour la mise au point d’un vaccin conférant une protection efficace contre l’exposition aux tiques. La recherche se poursuit, axée sur trois catégories différentes de vaccin : inactivé, vivant atténué et recombinant. L’auteur fait le point sur l’état actuel du développement de ces trois vaccins.La cowdriosis es una enfermedad de declaración obligatoria que figura en la lista de la Organización Mundial de Sanidad Animal. Su agente etiológico es Ehrlichia ruminantium, bacteria Gram negativa de vida intracelular obligada que pertenece al orden de los Rickettsiales y a la familia Anaplasmataceae. La infección se transmite por garrapatas del género Amblyomma y causa cowdriosis en rumiantes salvajes y domésticos, principalmente en África, pero también en algunas zonas del Caribe. La enfermedad fue descrita en Sudáfrica en el siglo XIX, en 1900 se comprobó que la transmitían garrapatas y en 1925 se pudo identificar al microorganismo, que fue cultivado por primera vez in vitro en 1985. Aquel logro impulsó las investigaciones sobre la enfermedad, en un momento en que la biología empezaba a entrar en la era de la genética molecular. En los últimos 20 años hemos aprendido mucho acerca de E. ruminantium, lo que a su vez ha deparado grandes progresos en los terrenos del diagnóstico, la epidemiología, la caracterización genética, la filogenia, la inmunología y la elaboración de vacunas. El organismo presenta una gran variabilidad genética, cosa que tiene importantes consecuencias de cara a futuras medidas de control y que está dificultando la obtención de una vacuna que proteja eficazmente contra la infección transmitida por la garrapata. Hay investigaciones en curso sobre tres tipos de vacunas: inactivadas, atenuadas y recombinantes, y el autor expone el actual estado de cosas en cada una de esas líneas de trabajo.http://www.oie.int/en/publications-and-documentation/scientific-and-technical-review-free-accessam201

Trends in the control of heartwater

Heartwater is an economically serious tick-borne disease of ruminants caused by the intracellular bacterium Ehrlichia ruminantium. The disease has traditionally been controlled by four different approaches : controlling the tick vector by dipping, establishing endemic stability, performing immunisation by infection and treatment, and preventing the disease by regular administration of prophylactic antibiotics. The first three of these methods are subject to failure for various epidemiological reasons, and serious disease outbreaks can occur. Prophylaxis is effective, but very expensive, and the logistics are daunting when large herds of animals are involved. The development of a safe, cheap and effective vaccine is the only likely way in which heartwater can be economically controlled, and over the past 15 years three new types of experimental vaccine have been developed: inactivated, attenuated, and recombinant vaccines. These new vaccines have shown varying degrees of promise, but none is as yet sufficiently successful to be marketable. We describe the experimental products, and the various technical and biological difficulties which are being encountered, and report on ways in which new technologies are being used to improve vaccine effectiveness

The occurrence of Theileria and Cowdria parasites in African buffalo (Syncerus caffer) and their associated Amblyomma hebraeum ticks

The polymerase chain reaction and oligonucleotide probing were used to detect Theileria and Cowdria species in DNA extracted from blood and ticks recovered from 24 African buffalo during a game capture operation in the Kruger National Park, South Africa. Species-specific probing indicated that all but one of the buffalo were carrying at least one Theileria species. Indirect fluorescent antibody (IFA) serology indicated that all animals had been exposed to Theileria parva infection but only 33 % were positive for T. parva by probing. Twelve (50 %) of the animals but only six of the 214 adult Amblyomma hebraeum ticks examined (2 ,8 %) were probe-positive for Cowdria. Only one Cowdria 16S genotype was detected in the animals and ticks.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat v.9 was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.mn201