Characterization of the pCS20 region of different Ehrlichia ruminantium isolates

Heartwater is a serious tick-borne disease of ruminants caused by the rickettsial organism Ehrlichia (Cowdria) ruminantium. A diagnostic test, targeting the pCS20 genomic region and using PCR amplification and probe hybridization, detects E. ruminantium infection in ticks and animals. However, only the pCS20 sequence of the Crystal Springs E. ruminantium isolate is available and the existence of sequence variation amongst different E. ruminantium isolates has not been determined. Primers were designed from the published pCS20 sequence to obtain sequences of the pCS20 region of various E. ruminantium isolates. These primers were unable to amplify the pCS20 region from genomic Welgevonden DNA and genome walking was used to characterize the pCS20 region. This technique showed that the published pCS20 sequence is from a chimeric clone. Sequences of the pCS20 region of 14 different E. ruminantium isolates were determined after amplification with newly designed primers. Sequencing data indicated that West African E. ruminantium isolates are highly conserved, whereas more variation occurs amongst the southern African isolates. These results facilitated the design of a short pCS20 probe and a large PCR target that improved the sensitivity of the E. ruminantium detection assay.     

Natural history of Ehrlichia ruminantium

Ehrlichia ruminantium is an obligately intracellular 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.     

In vitro isolation of Ehrlichia ruminantium from ovine blood into Ixodes scapularis (IDE8) cell cultures

Four stocks of Ehrlichia ruminantium (Welgevonden, Ball3, Nonile and Blaauwkrans), the causative agent of heartwater in domestic ruminants, were isolated into Ixodes scapularis (IDE8) tick cells using the leukocyte fraction of the blood of infected sheep. Organisms of two of the E. ruminantium stocks (Welgevonden and Blaauwkrans) propagated in IDE8 cells were also successfully used to infect bovine endothelial cells. All stocks were successfully propagated in IDE8 cells using Dulbecco's modified Eagle's medium nutrient mixture Ham F-12 containing 10% foetal bovine serum (FBS). The technique should be included in any attempt to isolate uncharacterized E. ruminantium stocks.     

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.     

Host cell-specific protein expression in vitro in Ehrlichia ruminantium

Ehrlichia ruminantium, a tick-transmitted pathogen, is the causative agent of heartwater in ruminants. In this study, a proteomic approach was used to identify host cell-specific E. ruminantium proteins encoded by the map1 multigene family, expressed in vitro in bovine endothelial and tick cell cultures. Two-dimensional gel electrophoresis combined with mass spectrometry analysis was used to establish the identities of immunodominant proteins. Proteins extracted from E. ruminantium-infected endothelial cells were shown to be products of the map1 gene, whereas tick cell-derived E. ruminantium proteins were products of a different gene, map1-1. The expressed proteins were found to be glycosylated. Differential expression of MAP1 family proteins in vitro in mammalian and tick cell cultures indicates that the map1 multigene family might be involved in the adaptation of E. ruminantium to the mammalian host and vector tick.     

Lack of cross-protection between Cowdria ruminantium and Ehrlichia phagocytophila.

Antigenically distinct stocks of Cowdria ruminatium from Senegal and South Africa were compared with a Dutch isolate of Ehrlichia phagocytophila in cross-immunity trials in goats. There was a complete absence of cross-immunity between E. phagocytophila and C. ruminantium, despite previous observations that both rickettsial organisms have certain antigenic determinants in common.     

反刍动物埃立克体荧光定量PCR检测技术的建立和应用

为快速、准确地检测反刍动物埃立克体,本研究以反刍动物埃立克体pCS20为靶基因设计特异性引物和探针,建立了 TaqMan和Eva Green荧光定量PCR方法,对其反应的特异性、敏感性和重复性进行了分析,并与OIE推荐的套式PCR方法一起对临床样品进行检测.结果显示,本方法特异性强,与牛巴贝斯虫、牛双芽巴贝斯虫、环形泰...     

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.     

蜱传反刍动物艾立希体巢式PCR检测方法的建立及应用

为建立一种快速、敏感检测反刍动物艾立希体的方法,本研究根据GenBank中登录的反刍动物艾立希体pCS20基因保守区设计2对特异性引物,经各反应条件的优化,建立了反刍动物艾立希体巢式PCR检测方法。结果显示,该方法可以特异性检测反刍动物艾立希体DNA,而对牛巴贝斯虫、双芽巴贝斯虫、牛环形泰勒虫和弓形虫的检测均为阴性,具有良好的特异性;该方法灵敏度可达1.04×101拷贝/μL,是反刍动物艾立希体实时荧光PCR检测试剂盒的10倍,是常规PCR的1 000倍。对50只血蜱、花蜱和微小牛蜱DNA进行检测,巢式PCR、反刍动物艾立希体实时荧光PCR检测试剂盒和常规PCR的阳性检出率分别为26.0%,14.0%和0.0%。本试验建立的巢式PCR检测方法适用于反刍动物艾立希体病的早期诊断和分子流行病学调查,为蜱传反刍动物艾立希体病的防控提供技术支持。     

Serotypes in Cowdria ruminantium and their relationship with Ehrlichia phagocytophila determined by immunofluorescence

Two tick-borne rickettsial pathogens of ruminants, Cowdria ruminantium (causative agent of heartwater disease) and Ehrlichia phagocytophila (causative agent of tick-borne fever), were successfully cultivated in caprine or ovine neutrophilic granulocytes. Infected cultures were subsequently used as antigens in the indirect fluorescent antibody test. Low-level bilateral serological cross-reactions could be detected between Cowdria and Ehrlichia. In addition, comparison of five Cowdria stocks using immunofluorescence demonstrated the existence of distinct serotypes within the genus of Cowdria. It is concluded that the occurrence of these serotypes will considerably complicate the current serodiagnosis of heartwater.     

Mining the genetic diversity of Ehrlichia ruminantium using map genes family

Understanding bacterial genetic diversity is crucial to comprehend pathogenesis. Ehrlichia ruminantium (E. ruminantium), a tick-transmitted intracellular bacterial pathogen, causes heartwater disease in ruminants. This model rickettsia, whose genome has been recently sequenced, is restricted to neutrophils and reticulo-endothelial cells of its mammalian host and to the midgut and salivary glands of its vector tick. E. ruminantium harbors a multigene family encoding for 16 outer membrane proteins including MAP1, a major antigenic protein. All the 16 map paralogs are expressed in bovine endothelial cells and some are specifically translated in the tick or in the mammalian host.In this study, we carried out phylogenetic analyses of E. ruminantium using sequences of 6 MAP proteins, MAP1, MAP1-2, MAP1-6, MAP1-5, MAP1+1 and MAP1-14, localized either in the center or at the borders of the map genes cluster.We show that (i) map1 gene is a good tool to characterize the genetic diversity among Africa, Caribbean islands and Madagascar strains including new emerging isolates of E. ruminantium; (ii) the different map paralogs define different genotypes showing divergent evolution; (iii) there is no correlation between all MAP genotypes and the geographic origins of the strains; (iv) The genetic diversity revealed by MAP proteins is conserved whatever is the scale of strains sampling (village, region, continent) and thus was not related to the different timing of strains introduction, i.e. continuous introduction of strains versus punctual introduction (Africa versus Caribbean islands).These results provide therefore a significant advance towards the management of E. ruminantium diversity. The differential evolution of these paralogs suggests specific roles of these proteins in host–vector–pathogen interactions that could be crucial for developing broad-spectrum vaccines.     

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.     

Preparation of Ehrlichia ruminantium challenge material for quantifiable and reproducible challenge in mice and sheep

The causative agent of heartwater, Ehrlichia ruminantium, is a tick-transmitted pathogen that infects bovine endothelial cells. Due to the obligate intracellular nature of this organism obtaining pure material in sufficient quantities for challenge studies is difficult. A murine model is frequently used to study potential vaccine candidates but giving reproducible challenges in this model for heartwater has always been problematic. We have therefore performed a series of experiments to optimize the parameters governing the reproducibility of challenge material. Two cryoprotectants were compared for the preparation of challenge material, buffered lactose peptone (BLP) and sucrose-potassium-glutamate (SPG). In addition two sources of virulent E. ruminantium were used, infected bovine endothelial cultures and infected mouse spleen homogenates. We also examined practical parameters affecting the reproducibility of challenge experiments: the time it takes to deliver the challenge material, the length of time a mouse remains immune to E. ruminantium challenge, and the effect of a given challenge dose. Finally, we performed a pilot study to determine whether mice could be used to titrate challenge material to be used for experiments in sheep. We found that: (a) E. ruminantium-infected mouse spleen homogenate provides more reproducible challenges than tissue culture material; (b) SPG is a better cryoprotectant than BLP; (c) challenge material should be used within 20min of thawing; (d) it is not essential to use syngeneic material for murine challenge experiments; (e) Balb/c mice are more sensitive to E. ruminantium challenge than C57BL/6J mice; (f) mice immunized by infection and treatment for use as positive immune controls should be challenged within 3 months of immunization; and (g) mice should be challenged with a dose not exceeding 10 LD(50)s.     

Effect of methisoprinol on virus replication in cell cultures

The effect of Methisoprinol (active substance: isoprinozine) on the replication of two animal viruses, the TK900 strain of Aujeszky's disease virus and the Roakin strain of the Newcastle disease virus was investigated. When the maximal tolerable doses of the drug were added to two cell cultures (CECC and GMK), its effect on the level of infectious titres of theviruses and their adsorption were assayed. Investigations were also performed to assess the direct effect of Methisoprinol on the viral strains used. The final stage of the experiment aimed at analysing of the replication dynamics of the viruses in the presence of Methisoprinol. Methisoprinol showed no direct effect on the viruses used in the study. Nor did it affect their adsorption. The preparation applied to the culture 24 hours before infection did not influence the replication of viruses, but administered simultaneously with the infection significantly lowered the final titres of viruses. The highest inhibitory effect of the drug was observed during the analysis of the replication dynamics of both viruses in CECC and of pseudorabies virus in GMK cell culture upon the application of the maximal tolerable doses of Methisoprinol and low infectious doses of the viruses.     

Simultaneous detection of Anaplasma and Ehrlichia species in ruminants and detection of Ehrlichia ruminantium in Amblyomma variegatum ticks by reverse line blot hybridization

The detection of Anaplasma and Ehrlichia species is usually based on species-specific PCR assays, since no assay is yet available which can detect and identify these species simultaneously. To this end, we developed a reverse line blot (RLB) assay for simultaneous detection and identification of Anaplasma and Ehrlichia species in domestic ruminants and ticks. In a PCR the hypervariable V1 region of the 16S ribosomal RNA (rRNA) gene was amplified with a set of primers unique for members of the genera Anaplasma and Ehrlichia [Int. J. Syst. Evol. Microbiol. 51 (2001) 2145]. Amplified PCR products from blood of domestic ruminants or Amblyomma variegatum tick samples were hybridized onto a membrane to which eight species-specific oligonucleotide probes and one Ehrlichia and Anaplasma catch-all oligonucleotide probe were covalently linked. No DNA was amplified from uninfected blood, nor from other hemoparasites such as Theileria annulata, or Babesia bigemina. The species-specific probes did not cross-react with DNA amplified from other species. E. ruminantium, A. ovis and another Ehrlichia were identified by RLB in blood samples collected from small ruminants in Mozambique. Finally, A. variegatum ticks were tested after feeding on E. ruminantium infected sheep. E. ruminantium could be detected in adult ticks even if feeding of nymphs was carried out 3.5 years post-infection. In conclusion, the developed species-specific oligonucleotide probes used in an RLB assay can simultaneously detect and identify several Ehrlichia and Anaplasma species. However, as no quantitative data for the detection limit are available yet, only positive results are interpretable at this stage.     

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.     

Proteomic analyses of Ehrlichia ruminantium highlight differential expression of MAP1-family proteins

The Rickettsiales Ehrlichia ruminantium (ER) is the causative agent of heartwater, a fatal tick-borne disease of livestock in sub-Saharan Africa and in the Caribbean, posing strong economical constraints to livestock production. In an attempt to identify the most prominent proteins expressed by this bacterium, especially those encoded by the major antigenic protein 1 (map1) multigene family, a proteome map of ER cultivated in endothelial cells was constructed by using two dimensional gel electrophoresis combined with mass spectrometry. Among the sixty-four spots detected, we could identify only four proteins from the MAP1-family; the other proteins detected were mainly related to energy, amino acid and general metabolism (26%), to protein turnover, chaperones and survival (21%) and to information processes (14%) or classified as hypothetical proteins (23%). Additional studies on MAP1-family protein using immunochemical labeling also revealed that these proteins are differentially expressed along the bacterium life cycle, presenting different structural organization. Interestingly, when infectious elementary bodies (EBs) are released from host cells, MAP1 appears to be organized in SDS and heat-resistant dimers and trimers stabilized by disulfide bridges. Overall, the results presented herein not only reveal the first partial proteome map of ER but provide new insights on the expression ER MAP1-family proteins in host endothelial cells.     

Ehrlichia ruminantium seroprevalence in domestic ruminants in Ghana. II. Point prevalence survey

Serum samples collected on a single occasion from cattle, sheep and goats at sites in all 10 regions of Ghana were tested for antibodies to Ehrlichia (previously Cowdria) ruminantium, the causative agent of heartwater, by polyclonal competitive ELISA (PC-ELISA). The survey revealed the presence of heartwater-exposed ruminants throughout the country, with local seroprevalence up to 100%. Seronegative, and therefore presumably susceptible, animals were also present in all regions, in some areas in numbers high enough to indicate local endemic instability. Overall seroprevalences in cattle, sheep and goats were 61, 51 and 28% respectively, and were generally higher in the northern part of the country and lower in the forest zone. Amongst animals over 1 year old, two thirds of cattle and sheep, and around one third of goats throughout the country had been exposed to E. ruminantium. In the north, seroprevalence in sheep sampled with and without cattle was similar, whereas in the south seroconversion rates in sheep were significantly higher in areas where cattle were present.