Heartwater--Ehrlichia ruminantium infection.

Summary Heartwater is a notifiable disease that is listed by the World Organisation for Animal Health. It is caused by Ehrlichia ruminantium, an obligately intracellular Gram-negative bacterium in the order Rickettsiales and the family Anaplasmataceae. The disease is borne byticks 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.

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.

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.

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.

Theileria parva: genomic DNA studies reveal intra-specific sequence diversity.

Theileria parva parva piroplasm DNA was purified from 11 different infections of cattle with 6 different East African isolates of the parasite. Total DNA was also prepared from bovine lymphoblastoid cells infected with schizonts of one of the isolates. Two of the infections were with cloned parasites. The DNA was of high molecular weight and free from protein and RNA, but some of the samples contained a proportion of bovine DNA. The 6 samples least contaminated with bovine DNA had a mean 'melting' temperature (Tm) of 84 degrees C and a mean GC content of 31.3%. Reassociation kinetics gave an estimate of 1.2 x 10(7) base pairs for the genome of T. parva. Repetitive restriction fragments were cloned from two samples, separated from the recombinant vectors and used as probes to demonstrate RFLPs between isolates. Discrimination into five groups was achieved. Schizont and piroplasm DNAs from the same isolate gave identical RFLPs, and one of the cloned parasites appeared to be a sub-population selected from a mixed-infection field isolate. Comparison of RFLPs with monoclonal antibody profiles suggested that neither technique yet provides discrimination between all the isolates which may comprise a strain. The importance of DNA probes for studying the epidemiology of theileriosis and for control programs is discussed.

Characterisation of a glutamine- and proline-rich protein (QP protein) from Theileria parva.

We have isolated a clone from a Theileria parva infected lymphocyte cDNA library which has the potential to encode a protein of 480 amino acids. This protein is particularly rich in glutamine and proline and has some short repeated amino acid motifs based on the sequences QPXP and QPXQ. We have called it the 'QP protein'. Southern blotting suggests that the QP protein gene is present as a single copy in the T. parva Muguga genome. Northern blotting revealed that the gene is transcribed in both schizonts and piroplasms. We have expressed part of the QP protein as a fusion with glutathione S-transferase in Escherichia coli and used this product to raise an anti-QP protein serum. Western blots of T. parva lysates using this serum showed a major polypeptide of approximately 100 kDa and two further polypeptides of approximately 67 and 72 kDa. Indirect immunofluorescence assays using the anti-QP protein serum on infected cells showed that the protein is associated with the schizont. The pattern of staining in the indirect immunofluorescence assays and the structure of the protein suggest that it is a component of the schizont membrane.

An unusual repetitive gene family in Theileria parva which is stage-specifically transcribed.

Tpr1 is a repetitive DNA element from Theileria parva which has previously been shown to be of value in strain characterisation. Further characterisation, described here, has shown that Tpr1 is present in long tandem arrays. The sequence of 8.1 kb from one end of an array has been determined. The sequence showed that Tpr1 is a 1.44-kb element which contains an ORF extending from its 5' end to the 3' end. The sequenced region contains 4 large ORFs; 2 of these consisted only of Tpr1 whilst the third consisted of Tpr1 and a 0.55-kb element (Tpr2) located 5' of Tpr1. The largest ORF consisted of Tpr1 plus Tpr2 as well as an additional 420-bp element (Tpr3) 5' of Tpr2, thus a continuous ORF arranged 5'-Tpr3-Tpr2-Tpr1-3' was formed. This ORF potentially encodes a 795 amino acid polypeptide commencing at an ATG close to the 5' end. In contrast the first in frame ATGs in the other 3 ORFs are at least 417 bp from the 5' end. Southern analysis confirmed that the sequenced region was typical of the rest of the Tpr array(s). Transcripts containing both Tpr3 and Tpr1 were detected in the piroplasm but not the schizont stages of the life cycle.

Evidence for two single copy units in Theileria parva ribosomal RNA genes.

Bacteriophage clones containing ribosomal RNA genes of Theileria parva were isolated from genomic DNA libraries. Physical mapping studies revealed 2 ribosomal DNA units, which were distinguishable by restriction enzyme site polymorphisms in flanking sequences. The cloned ribosomal DNA units were mapped to 2 separate T. parva chromosomes. Analysis of sequences contained in lambda EMBL3 recombinants, together with Southern blot analysis of genomic DNA and data on the copy number of the rRNA genes, suggested that the rDNA units were not tandemly repeated. This organisation of ribosomal transcription units is similar to that described for other genera of apicomplexan protozoa, but 2 rDNA units, each containing single copies of the rRNA coding genes, would be the lowest copy number described for any eukaryote in which amplification of rRNA genes is not known to occur. EcoRI restriction fragment length polymorphisms, which were revealed using rRNA gene probes, separated T. parva stocks into 2 categories. Nucleotide sequence analysis of polymerase chain reaction-amplified internal transcribed spacer DNA revealed 2 different ITS sequences derived from rDNA transcription units within the genome of a cloned T. parva parasite. Polymorphism was also observed between ITS sequences amplified from the DNA of different T. parva stocks. A synthetic oligonucleotide derived from T. parva Uganda ribosomal ITS DNA sequences hybridised to DNA from the T. parva Uganda stock, but not to the DNA of the T. parva Muguga stock. This oligonucleotide is potentially useful as a marker for the T. parva Uganda stock.

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.

Major outer membrane proteins of Ehrlichia ruminantium encoded by a multigene family.

Immune responses of infected animals and humans have been reported to be directed against variable outer membrane proteins of Ehrlichia species that are encoded by polymorphic multigene families. In Ehrlichia (= Cowdria) ruminantium, two immunodominant proteins have been identified, namely major antigenic protein 1 (MAP1) and open reading frame 2 (ORF2). The aim of the present study was to identify additional map1-like genes in the E. ruminantium genome. A 12 kb clone that hybridized with the map1 probe was amplified using long template PCR. The PCR product was partially digested, cloned, and sequenced. Four map1-like genes are located in tandem, namely map1-1 (orf2) and map1-2 upstream of map1 as well as map1+1 downstream of map1. A large ORF (2.4 kb) at the 3' end is homologous to secA genes of other organisms. The sequence data in this study support other findings that outer membrane proteins are located in tandem and are encoded by a polymorphic multigene family.

Sequencing of a 15-kb Ehrlichia ruminantium clone and evaluation of the cpg1 open reading frame for protection against heartwater.

A 1.2 kb polymorphic fragment from the Gardel isolate of Ehrlichia (formerly Cowdria) ruminantium was used to isolate a 15kb clone from the E. ruminantium Welgevonden LambdaGEM-11 library. This clone, WL2EL1, was subcloned and sequenced. Eight open reading frames (ORFs) were identified. The ORF in WL2EL1 which contained the Welgevonden homologue of the 1.2 kb polymorphic fragment was designated Cowdria polymorphic gene 1 (cpg1). The cpg1 ORF was cloned into pCMViUB, a genetic vaccine vector. Mice and sheep were immunized with pCMViUB/cpg1 by intramuscular injection and gene gun inoculation. Although all of the immunized mice died, there was a trend for mice that received larger amounts of pCMViUB/cpg1 DNA to survive longer. Four out of five sheep immunized with the construct survived lethal challenge.

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.

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.

Comparative evaluation of 16S, map1 and pCS20 probes for the detection of Cowdria and Ehrlichia species in ticks.

We have developed a panel of 16S ribosomal RNA gene probes for heartwater epidemiology; five of these detect different Cowdria genotypes (Ball3, Senegal, Omatjenne, Crystal Springs, and Mara 87/7); one detects all five of these genotypes; one detects any Group III Ehrlichia species other than Cowdria; one detects any Group II Ehrlichia species. We have used these probes on PCR-amplified rickettsial 16S rRNA genes from over 200 Amblyomma ticks. Control ticks were laboratory-reared and either uninfected or fed on sheep experimentally infected with different Cowdria isolates, field ticks were harvested from animals in heartwater-endemic and heartwater-free areas. All the samples were also examined by PCR amplification and probing for two other Cowdria genes (map1 and pCS20) which have been used for heartwater epidemiology. This paper describes the first direct comparison of all the currently available DNA probes for heartwater-associated organisms.

A partially protective clone from Cowdria ruminantium identified by using a Salmonella vaccine delivery system.

A strategy has been developed to screen the Cowdria genome, using a Salmonella vaccine delivery system, to identify genes that code for protection-stimulating proteins. We have cloned mini-libraries of Cowdria into this Salmonella system, used the recombinant bacteria to immunize outbred mice, and then challenged them after two weeks with a lethal dose of Cowdria. When one of these mini-libraries was tested in a group of 5 mice, one mouse lived much longer than the others. The experiment was repeated with each of the clones from the mini-library being tested individually in 10 mice, and one mouse survived the challenge. This clone has been tested repeatedly in larger groups of mice and is proven to protect 14% of outbred mice against a lethal Cowdria challenge.

Purification of Cowdria ruminantium organisms for use in genome analysis by pulsed-field gel electrophoresis.

Cowdria ruminantium is an obligate intracellular rickettsial pathogen which is responsible for a tick-borne disease of domestic and wild ruminants called heartwater or cowdriosis. Although several genes have been cloned and partially sequenced, the genome size, gross structure, and organization of the C. ruminantium genome is unknown. Genome analysis of the organism has been hindered because it is difficult to obtain C. ruminantium DNA free from contaminating host cell DNA, and this probably accounts for the lack of genome size data for this organism. In this study we investigated several methods for purifying C. ruminantium from bovine cellular contaminants and organisms of a relatively high purity were obtained. These were used to prepare Cowdria DNA which was analyzed by pulsed-field gel electrophoresis (PFGE) and which revealed a genome approximately 1900 kbp in length plus an additional extra-chromosomal fragment migrating with an apparent size of 815 kbp. This is the first time that the genome size of C. ruminantium has been determined and the first demonstration of an extrachromosomal element.

Low molecular weight proteins of Cowdria ruminantium (Welgevonden isolate) induce bovine CD4+-enriched T-cells to proliferate and produce interferon-gamma.

An important objective in vaccination strategies is to activate lymphocytes with particular effector functions. Cellular immunity and the type I cytokine IFN-gamma have been implicated in protective immunity to heartwater. Furthermore, low molecular weight proteins of Cowdria ruminantium have been shown to induce peripheral blood mononuclear cells to proliferate. To determine which lymphocyte subset responds when stimulated with fractionated C. ruminantium proteins, specific short-term lymphocyte cultures were established from cattle immunized with the Welgevonden isolate. Four cattle were immunized, two by infection and treatment and two with inactivated organisms. Cell surface phenotypic analysis of the cultures indicated that CD4+ lymphocytes were enriched over time. This coincided with increased antigen-specific proliferation and IFN-gamma production. Proteins of molecular weights 13-18kDa induced the CD4+-enriched T-cell cultures, derived from each of the animals, to proliferate and produce IFN-gamma. Although the two groups of cattle were immunized differently, their lymphocytes responded similarly. These results extend previous findings by identifying the responder cells as being predominantly IFN-gamma producing CD4+ lymphocytes. This cytokine has been implicated in immunity to the parasite. The low molecular weight proteins that induced CD4+ lymphocytes to proliferate and produce IFN-gamma are therefore likely to be important in protection against heartwater and may have a role in vaccine development.

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.

Isolation of Theileria taurotragi and Theileria mutans from cattle in Botswana.

Two Theileria species demonstrated in peripheral Giemsa-stained blood smears of sick cattle from various parts of Botswana were subsequently identified as Theileria mutans and T. taurotragi using DNA hybridization and the polymerase chain reaction. Initial screening for Theileria species was done using microscopy, the indirect fluorescent antibody technique and the micro Elisa test. The syndrome was characteristically that of high morbidity but low mortality and vague malaise. A low parasitaemia of pleomorphic intra-erythrocytic piroplasms and the absence of schizont stages in circulating lymphocytes and lymph node aspirates were evident. Dual infections were common and often complicated by intercurrent disease conditions.

Construction and initial analysis of a representative lambda ZAPII expression library of the intracellular rickettsia Cowdria ruminantium: cloning of map1 and three other Cowdria genes.

The causative agent of heartwater, the rickettsia Cowdria ruminantium, is very poorly understood at the molecular level owing to a profound lack of suitable tools. We have developed an immunoaffinity chromatographic method to purify C. ruminantium from host cell components and the purified rickettsial cells have been used to prepare substantially pure Cowdria DNA. This DNA has been used to construct what we believe to be the first fully representative C. ruminantium expression library. A clone containing the complete Cowdria map1 gene has been isolated and sequenced. This gene has been expressed in E. coli cells from the native Cowdria promoter, suggesting that the mechanisms for gene transcription and translation are similar between these two organisms. Parts of three other Cowdria genes have also been isolated and sequenced.