Susceptibility and carrier status of impala, sable, and tsessebe for Cowdria ruminantium infection (heartwater).

Three species of wild African ruminants, impala (Aepyceros melampus), sable (Hippotragus equinus), and tsessebe (Damaliscus lunatus), were experimentally inoculated with in vitro culture-derived Cowdria ruminantium organisms, the tick-borne causative agent of heartwater in domestic ruminants, to determine their susceptibility to infection. No clinical disease was observed in any of the ruminants. However, C. ruminantium was detected in the sable by the transmission of heartwater to susceptible sheep, through the tick vector Amblyomma hebraeum, at 10 and 37 days postinfection (PI). Attempts to detect infection in the impala and tsessebe by tick transmission at 54 days PI failed. The impala and tsessebe were reinoculated with C. ruminantium organisms at 146 days after the first inoculation; however, a tick transmission attempt at 66 days after the reinoculation also failed. Seroconversion, as detected by immunoblotting, was demonstrated in the sable and the tsessebe but not in the impala. The results demonstrate that sable can be carriers of C. ruminantium. The susceptibility of tsessebe and impala, however, remains undetermined.     

Phylogenetic position of Cowdria ruminantium (Rickettsiales) determined by analysis of amplified 16S ribosomal DNA sequences.

The 16S ribosomal DNA sequence of Cowdria ruminantium, the causative agent of heartwater disease in ruminants, was determined. An analysis of this sequence showed that C. ruminantium forms a tight phylogenetic cluster with the canine pathogen Ehrlichia canis and the human pathogen Ehrlichia chaffeensis. Although a close relationship between the genus Cowdria and several members of the tribe Ehrlichieae has been suspected previously, the tight phylogenetic cluster with E. canis and E. chaffeensis is surprising in view of known differences in host preference and target cells.     

Competence of the African tortoise tick, Amblyomma marmoreum (Acari: Ixodidae), as a vector of the agent of heartwater (Cowdria ruminantium)

The ability of the African tortoise tick, Amblyomma marmoreum, to acquire and transmit Cowdria ruminantium infection was investigated experimentally with transmission trials and with a C. ruminantium-specific polymerase chain reaction (PCR) detection assay. Laboratory-reared A. marmoreum larvae and nymphs were fed on small ruminants with clinical heartwater. After molting, the resultant nymphs were fed on Cowdria ruminantium-naive sheep (n = 3), and the adults were ground and inoculated intravenously into sheep (n = 5). Fatal heartwater developed in the 5 recipient animals, demonstrating larvae-nymph transmission and nymph-adult acquisition of infection. Cowdria ruminantium infection was also detected in adult A. marmoreum by PCR analysis, although at lower frequency (10%) than in Amblyomma hebraeum ticks (43%), the major vector of C. ruminantium in southern Africa, which had been fed simultaneously on the infected animals (P<0.0001). Amblyomma marmoreum, therefore, can be an effective vector of C. ruminantium. The potential role of this species in heartwater epidemiology and in the spread of the disease to new areas is highlighted by these results and by the fact that immature stages of this tick feed readily on domestic and wild animals susceptible to C. ruminantium.     

Introduction of the exotic tick Amblyomma hebraeum into Florida on a human host

A resident of Florida returned from a short visit to southern Africa to find a male Amblyomma hebraeum tick attached to the skin behind her knee. Amblyomma hebraeum is a major vector of 2 pathogens that cause important diseases in southern Africa, heartwater of ruminants and African tick-bite fever of humans. The tick was tested by polymerase chain reaction assay for evidence of infection with Cowdria ruminantium and Rickettsia africae (the causative agents of heart-water and African tick-bite fever, respectively) and was found to be negative for both agents. This is the second record of the exotic tick, A. hebraeum, being introduced into the United States on a human host.     

Immune responses to Cowdria ruminantium infections.

Understanding the basis of protective immunity to Cowdria ruminantium will facilitate the development of an effective subunit vaccine against heartwater in ruminants and contribute to a better definition of protective immune mechanisms to obligate intracellular pathogens in general. Until recently, immunological studies of heartwater in ruminants concentrated solely on antibody responses. Since 1995, the mechanisms underlying cell-mediated immunity of heartwater have been analysed. Progress achieved in these areas is discussed here by Philippe Totté and colleagues, with special emphasis on ruminants, the natural hosts of C. ruminantium.     

Ehrlichia ruminantium: genomic and evolutionary features

Ehrlichia ruminantium is the causative agent of heartwater, an important tick-borne disease of livestock in Africa and the Caribbean that threatens the American mainland. The genome sequences of three strains of E. ruminantium have recently been published, revealing the presence of specific features related to genomic plasticity. E. ruminantium strains have traces of active genomic modifications, such as high substitution rates, truncated genes and the presence of pseudogenes and many tandem repeats. The most specific feature is the presence in all Ehrlichia of independent long-period tandem repeats, which are associated with expansion or contraction of intergenic regions.     

Heartwater infection (cowdriosis) in a sitatunga (Tragelaphus spekei) in Nigeria

Cowdria ruminantium was isolated from a natural infection of heartwater in a captive sitatunga (Tragelaphus spekei) which died after a short illness in Jos Zoo, Nigeria. The isolate killed laboratory mice in a shorter period of time than did isolates of C. ruminantium recovered from natural cases in cattle. This is the first confirmed case of heartwater in a captive wild ruminant in Nigeria, where the disease is endemic.     

A subset of Cowdria ruminantium genes important for immune recognition and protection

Cowdria ruminantium causes the tick-borne rickettsial disease of heartwater, which is devastating to livestock production in sub-Saharan Africa. Current diagnosis and control methods are inadequate. We have identified and sequenced a subset of genes encoding recombinant antigens recognized by antibody and peripheral blood mononuclear cells from immune ruminants. The identified genes include many with significant similarity to those of Rickettsia prowazekii, genes predicted to encode different outer membrane proteins and lipoproteins and a gene containing an unusual tandem repeat structure. Evidence is presented for immune protection by recombinant antigens in a mouse model of C. ruminantium infection. These data identify new recombinant antigens for evaluation in vaccines and diagnostic tests to control heartwater.     

Infection of a goat with a tick-transmitted Ehrlichia from Georgia, U.S.A., that is closely related to Ehrlichia ruminantium.

We detected a novel tick-transmitted Ehrlichia in a goat following exposure to lone star ticks (Amblyomma americanum) from a park in the metropolitan area of Atlanta, GA, U.S.A. Nineteen days after infestation with field-collected adult ticks, the goat developed a fever of two days duration, which coincided with mild clinical pathologic changes and the presence of DNA from a novel Ehrlichia in peripheral blood. The goat transmitted ehrlichiae to uninfected nymphal A. americanum that fed upon the goat, and the ticks maintained the pathogen transstadially. Five months after exposure, immunosuppression of the goat resulted in transient ehrlichemia with transmission of ehrlichiae to feeding ticks. Sequencing and phylogenetic reconstructions of the 16S rRNA, gltA, map1, map2, and ribonuclease III genes suggest the agent might be a divergent strain of Ehrlichia ruminantium, the agent of heartwater, or a new, closely related species. Convalescent serum from the goat reacted with the MAP-1 protein of E. ruminantium and with whole-cell Ehrlichia chaffeensis antigen. DNA from the novel Ehrlichia was detected in 5/302 field-collected adult A. americanum from the park. Our data suggest that A. americanum is a natural vector and reservoir of this Ehrlichia and that domestic goats can be reservoirs. The geographic range of the agent and its pathogenicity to humans and livestock needs to be evaluated.     

Development of multiple-locus variable-number tandem-repeat analysis for rapid genotyping of Ehrlichia ruminantium and its application to infected Amblyomma variegatum collected in heartwater endemic areas in Uganda

The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a serious tick-borne disease in ruminants. The genetic diversity of organisms in the field will have implications for cross-protective capacities of any vaccine developed, and for an effective vaccine design strategy proper genotyping and understanding of existing genetic diversity in the field is necessary. We searched for variable-number tandem-repeat (VNTR) loci for use in a multi-locus VNTR analysis (MLVA). Sequencing analysis of 30 potential VNTRs using a panel of 17 reference strains from geographically diverse origins identified 12 VNTRs with allelic profiles differing between strains. Application of MLVA to 38 E. ruminantium-infected Amblyomma variegatum collected from indigenous cattle in 6 different districts of Uganda identified 21 MLVA types. The discriminatory power of MLVA was greater than that of map1 PCR-restriction fragment length polymorphism analysis, with which only 6 genotypes were obtained. The high discriminatory power as well as cost- effective performance of MLVA provide the potential for this technique to be applied in the future with respect to optimizing vaccine trials by identifying local strain diversity, and also raise the possibility of exploring the association between E. ruminantium genotypes and phenotypes such as pathological outcome in the ruminant host.     

The map1 gene of Cowdria ruminantium is a member of a multigene family containing both conserved and variable genes

Heartwater is an economically important disease of ruminants caused by the tick-transmitted rickettsia Cowdria ruminantium. The disease is present in Africa and the Caribbean and there is a risk of spread to the Americas, particularly because of a clinically asymptomatic carrier state in infected livestock and imported wild animals. The causative agent is closely related taxonomically to the human and animal pathogens Ehrlichia chaffeensis and Ehrlichia canis. A dominant immune response of infected animals or people is directed against variable outer membrane proteins of these agents known, in E. chaffeensis and E. canis, to be encoded by polymorphic multigene families. We demonstrate, by sequence analysis, that map1 encoding the major outer membrane protein of C. ruminantium is also encoded by a polymorphic multigene family. Two members of the gene family are located in tandem in the genome. The upstream member, orf2, is conserved, encoding only 2 amino acid substitutions among six different rickettsial strains from diverse locations in Africa and the Caribbean. In contrast, the downstream member, map1, contains variable and conserved regions between strains. Interestingly, orf2 is more closely related in sequence to omp1b of E. chaffeensis than to map1 of C. ruminantium. The regions that differ among orf2, map1, and omp1b correspond to previously identified variable sequences in outer membrane protein genes of E. chaffeensis and E. canis. These data suggest that diversity in these outer membrane proteins may arise by recombination among gene family members and offer a potential mechanism for persistence of infection in carrier animals.     

Vaccine strategies for Cowdria ruminantium infections and their application to other ehrlichial infections.

Suman Mahan and co-authors review the strategies applied to develop improved vaccines for Cowdria ruminantium infections (heartwater). Inactivated vaccines using cell-cultured C. ruminantium organisms combined with an adjuvant are capable of protecting goats, sheep and cattle against lethal C. ruminantium challenge. Immune responses induced with this vaccine, or after recovery from infection, target outer membrane proteins of C. ruminantium, in particular the major antigenic protein 1 (MAP-1). Genetic immunizations with the gene encoding MAP-1 induce protective T helper cell type 1 responses against lethal challenge in a mouse model. Similarly, homologues of MAP-1 in other phylogenetically and antigenically related ehrlichial agents such as Anaplasma marginale and Ehrlichia chaffeensis are also targets of protective responses. Given the antigenic similarities between the related ehrlichial agents, common strategies of vaccine development could be applied against these agents that cause infections of importance in animals and humans.     

Population-based evaluation of the Ehrlichia ruminantium MAP 1B indirect ELISA

The indirect MAP 1B ELISA based on the recombinant MAP 1B fragment of the immunodominant MAP I protein of Ehrlichia ruminantium is considered to be the most sensitive and specific assay for the serodiagnosis of heartwater. In this study, we evaluated its reliability in detecting exposure to E. ruminantium in field populations of domestic ruminants in Zimbabwe. Cattle and goat herds in endemically stable areas with high infection pressure and an expected close to 100% prevalence of E. ruminantium exposure were sampled. Bovine sera (858) and caprine sera (706) collected at seven locations representative of the two main production systems (communal lands and large scale commercial farms) in the two main agroecological zones of Zimbabwe (highveld and lowveld) were analysed. The prevalence of MAP 1B-specific antibodies in goats was similar and high, ranging from 67 to 100%, at all except one site (43%). Age-specific differences in goats (1, 2, 3, 4, 5 years) were not observed. In contrast, MAP 1B seroprevalence in cattle was significantly lower (P < 0.001), ranging from 46 to 61% in the lowveld communal area and from 24 to 33% in the remaining areas (P < 0.001). Age-specific differences in seroprevalence (1, 2, 3, 4, 5-7 + years) were similarly not evident in cattle (P < 0.15). Hence, the indirect MAP 1B ELISA may be an unreliable indicator of past exposure to heartwater in field-infected cattle in Zimbabwe. Although the reasons for this low response in field cattle are not fully understood, this study illustrates the need for field validation of the performance of new diagnostic tests prior to their use for epidemiological purposes.     

A polarized Th1 type immune response to Cowdria ruminantium infection is detected in immune DBA/2 mice

Immune responses to Cowdria ruminantium, an intracellular organism that causes heartwater in domestic ruminants, were characterized in a DBA/2 mouse model. Immunity induced by infection and treatment was adoptively transferable by splenocytes and could be abrogated by in vivo depletion of T cells but not by inhibition of nitric oxide synthase using NG-monomethyl-L-arginine. IgG2a and IgG2b C. ruminantium-specific responses were detected in immune mice. Culture supernatants of splenocytes from immune DBA/2 mice, which were stimulated with crude C. ruminantium antigens or recombinant major antigenic proteins 1 or 2, contained significant levels of interferon (IFN)-gamma and interleukin (IL)-6, but insignificant levels of IL-1alpha, IL-2, IL-4, IL-5, IL-10, IL-12, tumor necrosis factor-alpha (TNF), and nitric oxide. A similar response was detected during primary infection, although IFN-gamma levels decreased significantly during clinical illness and then increased following natural or antibiotic-aided recovery. These data support the conclusion that protective immunity to C. ruminantium in DBA/2 mice is mediated by T cells and is associated with a polarized T helper 1 type of immune response. This murine model could be utilized to screen for protective C. ruminantium antigens that provoke Th1 type immune responses and for evaluation of these antigens in recombinant vaccines against heartwater.     

Influence of diet on the rumen protozoal fauna of indigenous African wild ruminants

A study was carried out to determine if the protozoal fauna of indigenous African wild ruminants was different from that found in their domestic counterparts and if the animal's diet influenced the number and types of protozoa. Samples of rumen contents were collected in 1997 and 2001 from various indigenous African wild ruminants in Kenya. All three ruminant feeding types were sampled: browsers or concentrate selectors (giraffe and Guenther's dik-dik); intermediate or adaptable mixed feeders (impala, Thomson's gazelle, Grant's gazelle and eland); grass or roughage eaters (hartebeest and wildebeest). Total concentration of ciliate protozoa and percentage generic distribution were determined. In general, protozoal concentrations were higher in concentrate selectors, followed by the intermediate or opportunistic mixed feeders and lowest in the grass and roughage eaters. Both Thomson's and Grant's gazelle were protozoa-free in the 2001 samples. Entodinium percentages were considerably higher in concentrate selectors and intermediate mixed feeders, compared to roughage eaters. Two genera of protozoa previously found in only a few African ruminants, Epiplastron and Opisthotrichum, were observed in several additional animal species and represent new host records. A difference was noted in the protozoal species composition of the indigenous wild ruminants from that previously observed in African domestic ruminants.     

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.     

Natural and experimental infection of white-tailed deer (Odocoileus virginianus) from the United States with an Ehrlichia sp. closely related to Ehrlichia ruminantium

An Ehrlichia sp. (Panola Mountain [PM] Ehrlichia sp.) closely related to Ehrlichia ruminantium was recently detected in a domestic goat experimentally infested with lone star ticks (LSTs, Amblyomma americanum) collected from Georgia, USA. The infected goat exhibited pyrexia and mild clinical pathologic abnormalities consistent with ehrlichiosis. At least two other Ehrlichia species (Ehrlichia chaffeensis and Ehrlichia ewingii) are maintained in nature by a cycle involving LSTs as the primary vector and white-tailed deer (Odocoileus virginanus) as a known or suspected reservoir. To investigate the possibility that white-tailed deer are potential hosts of the PM Ehrlichia sp., whole blood samples collected from 87 wild deer from 2000 to 2002 were screened with a species-specific nested PCR assay targeting the citrate synthase gene. In addition, two laboratory-raised white-tailed deer fawns were each infested with 120 wild-caught LST adults from Missouri, USA, and blood samples were periodically collected and tested for the PM Ehrlichia sp. Of 87 deer tested from 20 locations in the southeastern United States, three (3%) deer from Arkansas, North Carolina, and Virginia were positive for the PM Ehrlichia sp. Wild-caught ticks transmitted the PM Ehrlichia sp. to one of two deer fawns, and colony-reared nymphal LSTs acquired the organism from the deer, maintained it transstadially as they molted to adults, and transmitted the PM Ehrlichia sp. to two na?ve fawns. These findings indicate that white-tailed deer are naturally and experimentally susceptible to infection with an Ehrlichia sp. closely related to E. ruminantium and are able to serve as a source of infection to LSTs.     

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.