Heartwater Research Articles

PCR Detection of Cowdria ruminantium Infection in Ticks and Animals from Heartwater‐Endemic Regions of Zimbabwe

The development of a PCR assay for the detection of Cowdria ruminantium infection in ticks has been previously described. Here we report a further evaluation of this assay by comparison with a DNA probe and with the mouse inoculation assay (MIA). Application of the PCR assay in determining the prevalence of infection in ruminants and ticks from heartwater-endemic areas of Zimbabwe is also described. One hundred uninfected and 120 infected Amblyomma hebraeum ticks were analyzed by PCR, DNA probe and the MIA. These tests detected infection in 92%, 77% and 8% of the infected ticks, respectively, showing PCR to be the most sensitive assay. None of the uninfected ticks were positive by any of the 3 tests. The PCR assay detected infection rates of 10.5%, 12.5% and 3.2% in 200 male, 241 female and 95 nymphal A. hebraeum ticks, respectively, which were collected from a heartwater-endemic farm. The PCR test was also applied to cattle of different age groups and goats from the same heartwater-endemic farm. In a cross-sectional survey, the assay detected infection in 3.3% to 26.7% of the cattle and in 23.3% of the goats. The implications of these findings and the potential for the application of PCR in heartwater diagnosis is discussed.

Validation and Comparison of Three Enzyme‐linked Immunosorbent Assays for the Detection of Antibodies to Cowdria ruminantium Infection a

Serological tests for Cowdria ruminantium infection have been hampered by low specificity. Here, an indirect ELISA based on purified antigen, a competitive ELISA using a recombinant major antigenic protein (MAP-1) and an indirect ELISA based on the MAP-1B region of the recombinant MAP-1 were compared. The tests were validated using 3000 sera of ruminants from 14 islands of the Lesser Antilles as well as sequential serum samples from 10 cattle, 17 goats and 10 sheep vaccinated with inactivated C. ruminantium in ISA 50 adjuvant and from 14 goats infected with a virulent culture supernatant. All tests detected significantly higher percentages of positives on Antigua, Guadeloupe and Marie-Galante, where C. ruminantium had been isolated before. Overall specificity calculated with sera from the other 11 heartwater-free islands was 98.1%, 98.5%, and 99.4% for the ELISA based on crude antigen, recombinant MAP-1 and MAP-1B, respectively. Sensitivities observed with sequential serum samples were similar for all tests. Tests based on recombinant antigens, especially the MAP-1B, showed improved specificity, suggesting their use for epidemiological studies in regions where the distribution of cowdriosis is unknown. In addition, the competitive ELISA is useful for studies in wildlife for which species-specific conjugates do not exist.

Inhibitory effect of Cowdria ruminantium on the expression of MHC class I and class II molecules on bovine endothelial cells.

Bovine endothelial cells constitutively express MHC class I molecules, whereas MHC class II molecules can be induced by interferon gamma (IFN gamma) treatment. Endothelial cells may play a role as antigen presenting cells (APC), but their role in the immune response against Cowdria ruminantium is unknown. We have studied the effect of C. ruminantium infection on the expression of MHC class I and class II molecules on the surface of bovine endothelial cells. Bovine brain endothelial cells (BBEC) from the microvasculature and bovine umbilical endothelial cells (BUEC) from the macrovasculature were cultured in vitro, infected with different concentration of Cowdria, and treated or not with IFN gamma. We observed that Cowdria induced a reduction of MHC molecules expression on the surface of BUEC and BBEC. This inhibitory effect of Cowdria on MHC molecules could affect the capacity of endothelial cells to act as efficient antigen-presenting cells.

Immunization of cattle by infection with Cowdria ruminantium elicits T lymphocytes that recognize autologous, infected endothelial cells and monocytes.

Peripheral blood mononuclear cells (PBMC) from immune cattle proliferate in the presence of autologous Cowdria ruminantium-infected endothelial cells and monocytes. Endothelial cells required treatment with T-cell growth factors to induce class II major histocompatibility complex expression prior to infection and use as stimulators. Proliferative responses to both infected autologous endothelial cells and monocytes were characterized by expansion of a mixture of CD4+, CD8+, and gammadelta T cells. However, gammadelta T cells dominated following several restimulations. Reverse transcription-PCR analysis of cytokine expression by C. ruminantium-specific T-cell lines and immune PBMC revealed weak interleukin-2 (IL-2), IL-4, and gamma interferon (IFN-gamma) transcripts at 3 to 24 h after stimulation. Strong expression of IFN-gamma, tumor necrosis factor alpha (TNF-alpha), TNF-beta, and IL-2 receptor alpha-chain mRNA was detected in T-cell lines 48 h after antigen stimulation. Supernatants from these T-cell cultures contained IFN-gamma protein. Our findings suggest that in immune cattle a C. ruminantium-specific T-cell response is induced and that infected endothelial cells and monocytes may present C. ruminantium antigens to specific T lymphocytes in vivo during infection and thereby play a role in induction of protective immune responses to the pathogen.

Nitric oxide is produced by Cowdria ruminantium-infected bovine pulmonary endothelial cells in vitro and is stimulated by gamma interferon.

Nitric oxide (NO) is a labile inorganic free radical produced by NO synthase from the substrate L-arginine in various cells and tissues including endothelial cells. A substantial elevation of nitrite levels indicative of NO production occurred in cultures of Cowdria ruminantium-infected bovine pulmonary endothelial cells (BPEC) incubated in medium alone. Exposure of the infected cultures to recombinant bovine gamma interferon (BorIFN-gamma) resulted in more rapid production of NO, reduced viability of C. ruminantium, and induction of endothelial cell death. Significant inhibition of NO production was noted after addition of the NO synthase inhibitor N-monomethyl-L-arginine (L-NMMA), indicating that the increase in production occurred via the inducible NO synthase pathway. Reduction in the infectivity of C. ruminantium elementary bodies (EBs) occurred in a dose-dependent manner after incubation with the NO donor molecule S-nitroso-N-acetyl-DL-penicillamine (SNAP) prior to infection of endothelial cells. The level of infection in cultures maintained in SNAP was reduced in a dose-dependent manner with significant negative correlation between the final level of infection on day 7 and the level of SNAP (r = -0.96). It was established that pretreatment and cultivation of C. ruminantium EBs with the NO donor molecule SNAP reduced infectivity to cultures and viability of EBs with the implication that release of NO in vivo following infection of endothelial cells may have an effect upon the multiplication of the agent in the host animal and may be involved in the pathogenesis of heartwater through the effect of this molecule upon circulation.

Inhibition of MHC class I and class II cell surface expression on bovine endothelial cells upon infection with Cowdria ruminantium

Endothelial cells constitute a main target for Cowdria ruminantium (CR) and can potentially play a role as antigen presenting cells (APC). Therefore, we measured, in vitro, the effect of CR infections on the expression of MHC class I and class II molecules on bovine umbilical endothelial cells (BUEC) and on bovine brain endothelial cells (BBEC). A dramatic inhibition of the expression of IFN γ induced MHC class II molecules was observed on BUEC and to a lesser extent on BBEC upon CR infection. This inhibitory effect was also observed on constitutively expressed MHC class I molecules. Part of the reduction of cell surface MHC molecules could be ascribed to their accumulation in intracellular compartments pinpointing a disruption in the transit of these molecules to the surface of the cells. The exact mechanisms of inhibition are not yet known but, as opposed to what is described in other models, the involvement of prostaglandin E2 can be excluded. The results obtained in this study show that endothelial cells have a decreased capacity to express both MHC class I and class II molecules on their surface upon CR infection, thus favouring the escape of this pathogen from the host immune system.

Letter to the Editor Regarding a Bool Review on Heartwater

Letter to the Editor Regarding a Bool Review on Heartwater

Evidence to show that an agent that cross-reacts serologically with Cowdria ruminantium in Zimbabwe is transmitted by ticks.

The serological diagnosis of heartwater based on reactions to the immunodominant Cowdria ruminantium major antigen protein-1 (MAP-1) is impaired by the detection of false-positive reactions. In this study, the prevalence of false-positive reactions on seven heartwater-free farms in Zimbabwe was determined to be 8-94% by immunoblotting against C. ruminantium antigens. The highest prevalence of false-positives on Spring Valley Farm correlated with the presence of Rhipicephalus evertsi evertsi ticks. The other tick species found on these seven farms were Hyalomma truncatum and Hyalomma marginatum rufipes. Rhipicephalus evertsi evertsi ticks collected from Spring Valley Farm and fed on seronegative sheep caused seroconversion in one of two sheep. This sheep developed a mild febrile reaction and C. ruminantium MAP-1 antigen reactive antibodies 3 weeks after the ticks started feeding. Polymerase chain reactions (PCRs), conducted using C. ruminantium-specific primers on ticks collected from the seven farms and on some of the R. e. evertsi ticks that had caused seroconversion in one sheep, were negative. However, some of these ticks gave positive PCRs with DNA primers which amplify a 350 bp DNA fragment of the 16s rRNA gene from all ehrlichial agents indicating the presence of infection with one or more Ehrlichia species. Although attempts to isolate the cross-reacting agent from the sheep were unsuccessful, this study demonstrates that false-positive reactions with the MAP-1 C. ruminantium antigen are associated with agents transmitted by ticks.

Immunodominant major outer membrane proteins of Ehrlichia chaffeensis are encoded by a polymorphic multigene family.

Several immunodominant major proteins ranging from 23 to 30 kDa were identified in the outer membrane fractions of Ehrlichia chaffeensis and Ehrlichia canis. The N-terminal amino acid sequence of a 28-kDa protein of E. chaffeensis (one of the major proteins) was determined. The gene (p28), almost full length, encoding the 28-kDa protein was cloned by PCR with primers designed based on the N-terminal sequence of the E. chaffeensis 28-kDa protein and the consensus sequence between the C termini of the Cowdria ruminantium MAP-1 and Anaplasma marginale MSP-4 proteins. The p28 gene was overexpressed, and antibody to the recombinant protein was raised in a rabbit. The antibody and serum from a patient infected with E. chaffeensis reacted with the recombinant protein, three proteins (29, 28, and 25 kDa) of E. chaffeensis, and a 30-kDa protein of E. canis. Immunoelectron microscopy with the rabbit antibody revealed that the antigenic epitope of the 28-kDa protein was exposed on the surface of E. chaffeensis. Southern blot analysis with a 32P-labeled p28 gene probe revealed multiple copies of genes homologous to p28 in the E. chaffeensis genome. Six copies of the p28 gene were cloned and sequenced from the genomic DNA by using the same probe. The open reading frames of these gene copies were tandemly arranged with intergenic spaces. They were nonidentical genes and contained a semivariable region and three hypervariable regions in the predicted protein molecules. One of the gene copies encoded a protein with an internal amino acid sequence identical to the chemically determined N-terminal amino acid sequence of a 23-kDa protein of E. chaffeensis. Immunization with the recombinant P28 protein protected mice from infection with E. chaffeensis. These findings suggest that the 30-kDa-range proteins of E. chaffeensis represent a family of antigenically related homologous proteins encoded by a single gene family.

Distributions of the vectors of heartwater, Amblyomma hebraeum and Amblyomma variegatum (Acari: Ixodidae), in Zimbabwe.

The tick vectors of heartwater (Cowdria ruminantium infection) in Zimbabwe, Amblyomma hebraeum and Amblyomma variegatum, historically were believed to be confined to the low-lying regions of the south and north-west of the country. However, country-wide surveys performed in 1975-1980 and 1988-1991 demonstrated that both species were also established in western parts of the highveld plateau and had started to encroach on the predominantly heartwater-free central and eastern highveld regions. To determine the current distributions of both the vectors and evaluate the potential threat of heartwater to animals in the highveld, a survey of ticks infesting cattle was performed in 1996 at 2994 locations in small-holder and large-scale commercial farming areas throughout Zimbabwe. Amblyomma hebraeum was collected at 1329 locations, A. variegatum at 72 locations and both A. hebraeum and A. variegatum at 13 locations. The results demonstrated that A. hebraeum was present, as previously recorded, throughout the southern half of the country and appeared to have undergone further limited spread into the central and eastern highveld regions. Only the northern-most region of the country appeared to be free of this species. Amblyomma variegatum was collected mainly in the north-west, as previously recorded, but was also found at isolated locations across the central highveld region and along the eastern border with Mozambique. This species was, however, still absent from the southern half and the northern-most regions of the country. An overlap of the distributions of the two species existed within a zone along the southern and eastern regions of the distribution of A. variegatum. These results suggest that the vectors of heartwater are spreading and threaten to introduce heartwater into intensive livestock-producing regions of the country.

Construction and initial analysis of a representative λZAPII 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.

Development of an in vitro cloning method for Cowdria ruminantium.

Cowdria ruminantium is a tick-borne rickettsia which causes severe disease in ruminants. All studies with C. ruminantium reported so far were carried out with stocks consisting of infective blood collected from reacting animals or from the same stocks propagated in vitro. Cloned isolates are needed to conduct studies on immune response of the host, on genetic diversity of the parasite, and on mechanisms of attenuation and the development of vaccines. A method of cloning based on the particular chlamydia life cycle of Cowdria was developed. Instead of cloning extracellular elementary bodies, it appeared more convenient to clone endothelial cells infected by one morula resulting from the infection of the cell by one elementary body of Cowdria. Two hundred and sixteen clones were obtained by limiting dilution of infected cells. The method was experimentally validated by comparing randomly amplified polymorphic DNA fingerprints from individual clones obtained from endothelial cell cultures coinfected with two different stocks of C. ruminantium.

Different organisms associated with heartwater as shown by analysis of 16S ribosomal RNA gene sequences

Cowdria ruminantium is a rickettsial parasite which causes heartwater, an economically important disease of domestic and wild ruminants in tropical and subtropical Africa and parts of the Caribbean. Because existing diagnostic methods are unreliable, we investigated the small-sub-unit ribosomal RNA (srRNA) gene from heartwater-infected material to characterise the organisms present and to develop specific oligonucleotide probes for polymerase chain reaction (PCR) based diagnosis. DNA was obtained from ticks and ruminants from heartwater-free and heartwater-endemic areas and from Cowdria in tissue culture. PCR was carried out using primers designed to amplify only rickettsial srRNA genes, the target region being the highly variable V1 loop. Amplicons were cloned and sequenced; 51% were C. ruminantium sequences corresponding to four genotypes, two of which were identical to previously reported C. ruminantium sequences while the other two were new. The four different Cowdria genotypes can be correlated with different phenotypes. Tissue-culture samples yielded only Cowdria genotype sequences, but an extraordinary heterogeneity of 16S sequences was obtained from field samples. In addition to Cowdria genotypes we found sequences from previously unknown Ehrlichia spp., sequences showing homology to other Rickettsiales and a variety of Pseudomonadaceae. One Ehrlichia sequence was phylogenetically closely related to Ehrlichia platys (Group II Ehrlichia) and one to Ehrlichia canis (Group III Ehrlichia). This latter sequence was from in isolate (Germishuys) made from a naturally infected sheep which, from brain smear examination and pathology, appeared to be suffering from heartwater; nevertheless no Cowdria genotype sequences were found in this isolate. In addition no Cowdria sequences were obtained from uninfected ticks. Complete 16S rRNA gene sequences were determined for two C. ruminantium genotypes and for two previously uncharacterised heartwater-associated Ehrlichia spp. Sequence differences within the V1 loop were sufficient for the derivation of four Cowdria genotype-specific oligonucleotide probes. Four further probes were designed: one for the detection of any Cowdria genotype, one for the detection of any Group II Ehrlichia sp., one for any Group III Ehrlichia sp. and one for all Pseudomonadaceae. All the probes were specific except that for the Cowdria (Ball 3) genotype. The high prevalence (96%) in field samples of pseudomonad-like 16S sequences was the result of environmental contamination. The probes were used to screen DNA from goats in an area free of both Amblyomma ticks and clinical heartwater. A substantial proportion (42%) gave positive reactions for the apparently apathogenic Cowdria (Omatjenne), indicating that this genotype is relatively common.

Biological response of endothelial cells and its modulation by cytokines: prospects for therapy and bioprocesses.

Endothelial cells are involved in important pathological situations. They could be the target for infectious processes as for example in Cowdriosis, an important disease in cattle due to the rickettsia Cowdria ruminantium prevalent in the south of the Sahara. They are also connected to angiogenic processes related to tumor invasion.Our results indicate that AIDS related Kaposi sarcoma cells may be of endothelial origin. We conclude from our data the mobility of those cells, related to the expression of the metalloproteinases (especially the 92 kD form of the enzyme), is an important factor in Kaposi saroma dissemination and is the main factor limiting the scale up of Cowdriosis vaccine production in Bovine Umbilical Endothelial Cell line. We showed that PMA and TNF increased the 92 kD Metallaproteinase and that TGFβ, produced in an inactive form in cultures of Human Umbilical Vein Endothelial Cells, is a potential inhibitor of Kaposi sarcoma spreading, and could also be useful in improving our process for Cowdria ruminantium vaccine production, since it reduces the sensitivity of the cells to mechanical stress without affecting significantly the overall infectious process.

Isolation of the causative agent of heartwater (Cowdria ruminantium) from three Amblyomma species in eight Districts of Kenya

Thirteen isolates of Cowdria ruminantium were made from eight different Districts of Kenya by four different isolation methods. Feeding adult Amblyomma species ticks derived from nymphs collected in the field and the inoculation of homogenates prepared from adult field ticks had the highest success rate. The reattachment of adult ticks collected in the field was successful on only one of five attempts, and the subinoculation of blood from suspected heartwater carriers was unsuccessful. Seven of the isolates were derived from A variegatum ticks, four from A gemma, one from A lepidum and one from a mixed pool of the last two species. This is the first report of the isolation of C ruminantium from A gemma ticks, and the first report of its transtadial transmission from nymphal to adult A gemma.

Comparative efficacy of Freund's and Montanide ISA50 adjuvants for the immunisation of goats against heartwater with inactivated Cowdria ruminantium

Two vaccines, based on inactivated elementary bodies of Cowdria ruminantium, one formulated in Montanide ISA50, the other in Freund's adjuvant, were compared in goats. Administered twice subcutaneously with an interval of 81 days, both protected three out of five goats against a very severe challenge, lethal for all 14 control goats, 3.5 months after the second injection. Both vaccines elicited similar antibody levels. The protection afforded by the Montanide ISA50 vaccine was tested 15 and 17 months after the second injection of the vaccine. Three out of six and five out of six goats, respectively, survived a challenge which killed all four control goats used on each occasion. Antibodies were still detectable in the immunised goats. The level of protection appears to be influenced by the dose of virulent C. ruminantium used for the challenge. As any stock of C. ruminantium can be incorporated in order to cover the antigenic repertoire of the organism, this kind of inactivated vaccine can now be tested in the field.

Integrated tick and tick-borne disease control trials in crossbred dairy cattle in Malawi.

Crossbred dairy heifers on a farm in an East Coast fever (ECF) endemic area in Malawi were immunised against Theileria parva, Anaplasma spp., Babesia bigemina, Babesia bovis and Cowdria ruminantium. They were treated at infrequent intervals with chlorfenvinphos to limit infestation with adult ticks, without providing complete tick control. In one trial, which tested a threshold dipping regimen, 20 heifers were dipped only once in 6 months to control a flush of Boophilus microplus. Unimmunised controls showed serological evidence of exposure to T. parva and B. bigemina, and one died of ECF, but there were no incidents of tick-borne disease in the immunised group. In a second trial, which tested a strategic dipping regimen, 107 animals were dipped 9 times over a 6 month period. Despite heavy challenge by B. bovis and moderate challenge by B. bigemina and Anaplasma spp. demonstrated serologically, there was only a single clinical case of babesiosis. The observations provide encouragement for the introduction of integrated tick and tick-borne disease control programmes in improved cattle in ECF endemic areas.

The epidemiology of heartwater: Establishment and maintenance of endemic stability

Although heartwater ( Cowdria ruminantium infection) is one of the most economically important tick-borne diseases of sub-Saharan Africa, its epidemiology he's remained poorly understood until recently. New data, suggesting that heartwater is present in an endemically stable state in much of sub-Saharan Africa and demonstrating vertical transmission of Cowdria ruminantium in the field, have altered previously accepted views on heartwater epidemiology. In this paper, Sharon Deem and colleagues present an overview of the epidemiology of heartwater based on recent studies, discuss the factors that make endemic stability possible, make recommendations for future directions in research, and provide a foundation for the development of epidemiological models.

Amino acid and protein depletion in medium of cell cultures infected with Cowdria ruminantium.

Cowdria ruminantium (Rickettsiales) causes heartwater in ruminants of Africa, and some islands off Africa and in the Caribbean Sea. The in vitro culture method for the organism devised in 1985, which provided for the first time a means for production of adequate quantities of live organisms and their products, is erratic and requires improvement. We studied depletion of amino acids (AAs) and major proteins in culture medium taken daily from infected and uninfected ovine and bovine vascular endothelial cell cultures. AAs of these samples were analyzed by Pico Tag reversed phase HPLC precolumn derivatization, and major proteins determined by capillary electrophoresis using a 57 cm x 75 microns fused silica tube at high pH. In both ovine and bovine cell cultures, significant depletion of arginine and glutamine occurred over a 5-day observation period regardless of whether they were infected or uninfected. This indicates that supplementation of nutrient media with these AAs might improve conditions for growth of the organism. Both AAs are essential for survival of cultured cells, and probably for the rickettsia (although the metabolism of C. ruminantium is poorly understood). Concentrations of several AAs increased in infected cultures, implying de novo synthesis and/or proteolysis on the part of the organism. In fact, several protein fractions did decrease in culture medium throughout the course of infection, while increasing or remaining unchanged in uninfected control cultures. Proteolytic activity by C. ruminantium may be essential for nitrogen metabolism by the organism. It is suggested that studies such as these will facilitate the development of a specific medium for optimal in vitro growth of the heartwater organism, and may also lead to an understanding of the metabolic stratagem of C. ruminantium. This knowledge, in turn, could reveal the mechanism for pathogenesis of heartwater, with implications for control.

Recombinant expression and use in serology of a specific fragment from the Cowdria ruminantium MAP1 protein.

The major antigenic protein (MAP1) of Cowdria ruminantium was screened for immunogenic regions by expression of overlapping recombinant DNA clones of the gene encoding the MAP1 protein. Two regions, designated MAP1-A and MAP1-B, were recognized by all antisera to 9 different isolates of C. ruminantium. MAP1-A contained one or more epitopes responsible for false-positive reactions with Ehrlichia antisera in several serological tests for cowdriosis. Cross-reactivity with MAP1-B was limited to antisera to Ehrlichia chaffeensis and Ehrlichia canis. Antisera to Ehrlichia species that infect ruminants (E. bovis, E. ovina, and E. phagocytophila) did not recognize MAP1-B. The sensitivity of an indirect ELISA based on MAP1-B was found to be excellent, since all sera from animals experimentally infected with C. ruminantium (64 out of 64) reacted with MAP1-B. Validation of this ELISA was carried out with field sera obtained from sheep raised in heartwater-free areas in Zimbabwe and from several Caribbean islands. Only 9 out of 111 samples from Zimbabwe, and 1 out of 58 samples from the Caribbean islands, which were considered to be false positives by immunoblot or indirect ELISA, reacted with MAP1-B. Thus, the ELISA based on MAP1-B is at present the most specific and sensitive serological test for cowdriosis.