Comparison of efficacy of American and African Amblyomma ticks as vectors of heartwater (Cowdria ruminantium) infection by molecular analyses and transmission trials

The ability of Amblyomma americanum, Amblyomma cajennense, Amblyomma maculatum, and Amblyomma variegatum to acquire and transmit Cowdria ruminantium infection was investigated. Uninfected nymphs were fed on clinically reacting C. ruminantium-infected sheep and then analyzed for infection by specific DNA detection assays and by tick transmission trials. By polymerase chain reaction (PCR), the mean infection prevalence of A. maculatum ticks (50.7%) was similar to that of A. variegatum, Elevage strain (43.5%; P = 0.83) and Petit Bourg strain (45.9%; P = 0.26) ticks. Though Amblyomma hebraeum were not tested by PCR, by DNA probe their infection prevalence was 94%. In contrast, A. americanum and A. cajennense ticks demonstrated very low susceptibility to C. ruminantium, and the prevalence of infection by PCR was approximately 1%. The higher susceptibility of A. maculatum and A. variegatum to C. ruminantium correlated with superior vector efficiency, depicted by similar prepatent periods and severity of disease transmissions to sheep. Amblyomma americanum and A. cajennense failed to transmit infection, confirming that low susceptibility to C. ruminantium correlates with the poor vector status of these species. These results highlight the importance of A. maculatum as a potential vector that is likely to play a major role in the establishment and maintenance of heartwater, if the disease were to be introduced to the U.S.A., Central, and South America.     

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.     

Experimental transmission ofCowdria ruminantium (Rickettsiales) by the American reptile tickAmblyomma dissimile Koch, 1844

A Senegalese isolate of the rickettsiaCowdria ruminantium was transmitted transstadially by nymphs of the American reptile tickAmblyomma dissimile. Only eight nymphs, fed as larvae on a Saanen goat reacting to heartwater, were required to transmit fatal heartwater to another susceptible goat.SinceA. dissimile usually feeds on snakes, iguanas and lizards in central America, the tick is not considered to play a significant role in the transmission of heartwater between ruminants. However, the tick could play a role in maintaining a rickettsial reservoir in reptile populations, since it has been shown that an African reptile can be a subclinical carrier ofC. ruminantium, infective to vector ticks.     

Evidence of Cowdria ruminantium infection (heartwater) in Amblyomma sparsum ticks found on tortoises imported into Florida

Amblyomma marmoreum and A. sparsum ticks were collected from tortoises imported into Florida from Africa and were tested for Cowdria ruminantium infection using a C. ruminantium-specific pCS20 polymerase chain reaction assay. In I shipment imported from Zambia, 15 of the 38 A. sparsum male ticks collected from the leopard tortoises (Geochelone pardalis) were found to be positive for infection with C. ruminantium. In contrast, all 148 A. marmoreum tested were negative for C. ruminantium infection. This is the first reported evidence of the introduction of heartwater-infected ticks into the United States, but there were no opportunities to confirm isolation of C. ruminantium from the ticks by either culture or transmission studies.     

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.     

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 19751980 and 19881991 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.Exp Appl Acarol 22: 725740 © 1998 Kluwer Academic Publishers     

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.     

Susceptibility of white-tailed deer to experimental heartwater infections

Nine white-tailed deer (Odocoileus virginianus) were experimentally infected with Cowdria ruminantium, the causal agent of heartwater. All deer developed clinical signs; one was killed, one was treated, and seven died within 2 wk postinoculation. Diagnosis of heartwater was based on clinical signs, postmortem lesions and by microscopic observation of C. ruminantium in endothelial cells of brain capillaries of dead animals. Cowdria ruminantium was passaged by collecting blood from deer at the height of the febrile response and intravenous inoculation of susceptible deer and goats. Tetracycline was effective in the treatment of heartwater in a deer.     

Methods currently used for the control of heartwater: their validity and proposals for future control strategies

Major advances made in the last few years in the knowledge of heartwater have not yet resulted in significant improvement of its prevention and control. Acaricide resistance and the high cost of chemical vector control stimulate research on alternative methods of vector control, such as immunization against the tick and the use of pheromones. Recently developed in vitro methods for the culture of Cowdria ruminantium may make it possible to arrive at safe recombinant or molecular vaccines. Specific chemotherapy may be improved by new laboratory screening methods, and studies on the pathogenesis of heartwater could lead to better non-specific treatment as well. Studies on the heritability of natural resistance to heartwater should determine whether or not practical selection and transfer of resistance is feasible. More research is also needed on the immunology and diagnosis of the disease and the factors involved in the resistance of young animals.     

Introduction of potential heartwater vectors and other exotic ticks into Florida on imported reptiles

Following the discovery of establishment of the African tortoise tick Amblyomma marmoreum in Florida, the present study was undertaken to determine the extent of introduction of exotic ticks into Florida on imported reptiles. Exotic ticks were identified on 29 (91%) of 32 reptile premises in 18 counties of Florida. The ticks, found on a variety of imported tortoises, snakes, and monitor lizards, belonged to 4 Amblyomma species (A. marmoreum, Amblyomma nuttalli, Amblyomma sabanerae, and Amblyomma sparsum) and 4 Aponomma species (Aponomma exornatum, Aponomma flavomaculatum, Aponomma latum, and Aponomma varanensis). The most commonly encountered ticks were A. latum and A. marmoreum. The identifications of A. marmoreum on 8 premises in 7 counties, and of A. sparsum on 1 premises, are of great concern because both species are vectors of heartwater, a lethal disease of cattle, sheep, goats, and deer.     

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.     

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.     

Ehrlichia ruminantium infection (heartwater) in wild animals

Several wild animal species have been implicated as hosts of Ehrlichia ruminantium (formerly Cowdria ruminantium), the rickettsial agent causing heartwater, a fatal disease of domestic ruminants in sub-Saharan Africa and eastern Caribbean. However, evidence for infection in most wild species is inconclusive because of inadequate diagnostic techniques. Infection has been proven only in 12 African ruminants, three non-African ruminants and two African rodents. A subclinical carrier state occurs in eight of the African ruminant species. Further studies on E. ruminantium infection in wild animal species are needed in order to determine the host range of this pathogen accurately. The host range of Ehrlichia ruminantium in wildlife is reviewed here and the role played by these species in the epidemiology and spread of heartwater is discussed.     

Amblyomma marmoreum on tortoises of southern province, Zambia

Reptiles from Southern Province, Zambia were captured and inspected for ticks. Seven of 18 tortoises (Geochelone pardalis) were infested by small numbers of adults and nymphs of Amblyomma marmoreum. Under such circumstances, the tick had little apparent effect on the host. None of the 13 specimens representing four species of snakes examined were infested with ticks.     

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.     

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.     

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.     

Comparison of the attachment rates of males of the ticksAmblyomma hebraeum andA. variegatum to cattle,sheep and rabbits in the absence of aggregation-attachment pheromone

Losses in domestic ruminants caused by heartwater (Cowdria ruminantium infection) in Zimbabwe and Mozambique are greater when the vector isAmblyomma hebraeum than when the vector isA. variegatum. It has been suggested that the epidemiology of the disease may be influenced by the rates at which unfed adults of these two tick species attach to uninfested hosts (i.e. in the absence of the male-produced aggregation-attachment pheromone [AAP]). In this study we confined unfed males ofA. hebraeum andA. variegatum on uninfested cattle, sheep and rabbits and recorded their attachment rates. Males of both species attached more rapidly on cattle than on sheep or rabbits. Males ofA. hebraeum attached more rapidly than males ofA. variegatum on all three host species. The differences in the attachment rates between the two species were much greater on sheep and rabbits than on cattle. The findings suggest that in the absence of AAP, pioneer males of both tick species may attach to cattle, and pioneer males ofA. hebraeum may also attach to sheep. The differences in the attachment rates ofA. hebraeum andA. variegatum provide a possible explanation for observed differences in the epidemiology of heartwater associated with these two vector species.     

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.