The relationship between cowdria ruminantium and chlamydia trachomatis was studied by immunofluorescence. Cowdria ruminantium is recognized by a monoclonal antibody. The gene encoding this protein, designated map1, was detected, cloned, and characterized. Heartwater results from infection by the bacterium ehrlichia formerly cowdria ruminantium, a small.
Development and evaluation of pcr assay for detection of low. Three stocks of cowdria ruminantium, the causative. Endemic stabilitya veterinary idea applied to human. Endothelial cells required treatment with tcell growth factors to induce class ii major histocompatibility complex expression prior to infection and use as stimulators. The effect of subclinical experimental cowdria ruminantium. Pdf prevalence of cowdria ruminantium infection in amblyomma. A cowdria ruminantium genomic dna library was constructed in the expression vector lambda zapii, and an immunoreactive clone. Goodrich, in rebhuns diseases of dairy cattle third edition, 2018. A cowdria ruminantium genomic library was constructed in a cosmid vector to serve as a source of easily accessible and pure c.
Restriction length polymorphisms were observed among the respective map1 genes analyzed and were confirmed by sequencing. Peripheral blood mononuclear cells pbmc from immune cattle proliferate in the presence of autologous cowdria ruminantium infected endothelial cells and monocytes. Cowdria ruminantium infection in ticks in the kruger national. The cosmid library contained 846 clones which were arrayed into microtitre plates. It occurs in colonies or morulae with a predilection for the vascular endothelium and. Translocation of 10 serologically negative goats from tete to farms in the south resulted in two clinical cases of. The development of a pcr assay for the detection of cowdria ruminantium infection in ticks has been.
Restriction length polymorphisms were observed among the respective map1 genes analyzed and. Cowdria ruminantium causes severe, often fatal disease in domestic ruminants, whereas wildlife species usually are not affected. Pdf analysis of the transmission dynamics of cowdria ruminantium, the tick borne rickettsial agent of heartwater in ruminants. A monoclonal antibody directed against the major outer membrane protein of c. Heartwater is a noncontagious, tickborne disease caused by ehrlichia ruminantium, an intracellular parasite in the order rickettsiales.
Pcr detection of cowdria ruminantium infection in ticks. Pdf heartwater cowdria ruminantium infection on sao tome. However, we propose a general model of endemic stability that is applicable to a broader range of diseases that are important in public health, including malaria, rubella, and mumps. Cowdria infected populations consisted of 48 sheep and 44 goats, while the noninfected populations consisted of 64 sheep and 107 goats. Longterm cultures were established from three ruminants, sable antelope hippotragus niger, buffalo syncerus caffer, and eland tragelaphus oryx, and from an omnivore, the bushpig potamochoerus porcus. Read the effect of subclinical experimental cowdria ruminantium infection in ewes on the growth and milk consumption of preweaning lambs, preventive veterinary medicine on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Molecular cloning, sequence analysis, and expression of the. A cowdria ruminantium genomic dna library was constructed in the expression vector lambda zapii, and an immunoreactive clone, designated lambda cr9. Endemic stability is an epidemiological state of a population, in which clinical disease is scarce despite high level of infection. Molecular cloning, sequence analysis, and expression of. The tickborne rickettsia cowdria ruminantium has been propagated continuously for over 500 days in the ixodes scapularis tick cell line ide8 by using the gardel isolate from bovine endothelial cells as an inoculum.
Longitudinal monitoring of ehrlichia ruminantium infection. Heartwater, caused by cowdria ruminantium and transmitted by ticks of the genus amblyomma, is a constraint to ruminant animal production in subsaharan africa. Twelve mixedbreed calves, born to dams living in a heartwaterendemic area of zimbabwe, were tested postnatally for the presence of. These ticks transmitted cowdria ruminantium, the rickettsial agent causing heartwater, to a susceptible goat, resulting in acute, fatal. Heartwaterexposed cattle were widespread on the ranch and overall seroprevalence was use of a specific immunogenic region on the cowdria ruminantium map1 protein in a serological assay. The sensitivities of a pcr assay and a dna probe assay were compared for the detection of cowdria ruminantium in amblyomma ticks that were fed on c. Validation of the indirect map1b enzymelinked immunosorbent. Here, an indirect elisa based on purified antigen, a competitive elisa using a recombinant major antigenic protein map. Ehrlichia ruminantium is a small, round organism 0. Cowdria ruminantium,4 the causative organism of heartwater, has recently been reclassified asehrlichia ruminantiumbased on genetic analysis as well as. Immune responses to cowdria ruminantium infections. A serological survey in mozambique to detect antibodies to cowdria ruminantium, the etiologic agent of heartwater, revealed a seroprevalence of 8. Amblyomma gemma nymphs were fed on the animals at 3 weekly intervals.
Although speculated to be potentially zoonotic, there is no evidence of cowdriosis affecting humans. Pdf cowdria ruminantium is the cause of a serious tickborne disease of domestic ruminants, known as heartwater or cowdriosis. Pdf analysis of the transmission dynamics of cowdria ruminantium, the tickborne rickettsial agent of heartwater in ruminants. Sequence heterogeneity of the major antigenic protein 1 genes. These ticks transmitted cowdria ruminantium, the rickettsial agent causing heartwater, to a susceptible goat, resulting in acute, fatal disease. Heartwater also known as cowdriosis, nintas, and ehrlichiosis is a tickborne rickettsial disease of domestic and wild ruminants. Xenodiagnosis has long been the standard technique for the detection in ticks of the tickborne rickettsia cowdria ruminantium, the causative agent of heartwater cowdriosis, an economically important disease of domestic and wild ruminants in africa and in the caribbean 11, 12, 44,45, 50. Cowdria ruminantium, a member of the order rickettsiales which are gramnegative, obligate intracellular bacteria found in ticks, lice, fleas, mites, chiggers, and mammals. Detection of the agent of heartwater, cowdria ruminantium. Translocation of 10 serologically negative goats from tete to farms in the south resulted in two clinical cases of heartwater that. Ehrlichia formerly cowdria ruminantium and is transmitted by amblyomma ticks, affecting cattle, sheep, goats, and some wild ruminants. A cowdria ruminantium genomic dna library was constructed in the expression vector. Infection of the tick cells was confirmed by pcr, karyotyping, electron microscopy, and reinfection of bovine cells.
Ehrlichia ruminantium infects rhipicephalus microplus in west africa. Ehrlichia cowdria ruminantium is a gramnegative, intracellular rickettsial organism in the order of rickettsiales. One of the most important questions about the epidemiology of heartwater in the field is how cowdria ruminantium is transmitted within vertebrate host populations. Cowdria ruminantium dna is unstable in a supercos1 library core.
Electron microscopy of cowdria rickettsia ruminantium cowdry, in the endothelial cells of the vertebrate host. Cowdria ruminantium pdf ehrlichia ruminantium formerly cowdria ruminantium. Identification of cowdria ruminantiumantigens that. Detection of cowdria ruminantium in blood and bone marrow.
Pcr detection of cowdria ruminantium infection in ticks and. The reattachment of adult ticks collected in the field was successful on only one of five. Nitric oxide is produced by cowdria ruminantiuminfected. Here we report a further evaluation of this assay by comparison with a dna probe and with the mouse inoculation assay mia. Identification of cowdria ruminantiumantigens that stimulate. Sequencing of the insert from this clone revealed two open reading frames, encoding peptides of 10462 and 58697 kda respectively. 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. Cowdria ruminantium was isolated from plasma of clinically affected animals in these four cell lines and in bovine endothelial cells used routinely for c. Until recently, immunological studies of heartwater in ruminants concentrated solely on antibody responses. Heartwater results from infection by the bacterium ehrlichia formerly cowdria ruminantium, a small, gram negative, pleomorphic coccus in the family anaplasmataceae and order rickettsiales.
Sequence heterogeneity of the major antigenic protein 1. Ehrlichia ruminantium an overview sciencedirect topics. Current status of in vitro cultivation of cowdria ruminantium. Detection of the agent of heartwater, cowdria ruminantium, in. In order to identify which proteins are involved in tcell immunity, c. Adult amblyomma hebraeum ticks, the principle vector of heartwater cowdriosis of domestic ruminants in southem africa, were collected in pheromone traps placed in kruger national park, an exclusively wildlife sanctuary in south africa. 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. Endothelial cell cultures were established from several wild african mammalian species. Heartwater cowdria ruminantium infection as a cause of. The pcr assay and dna probe detected infection in 86. Free fulltext pdf articles from hundreds of disciplines, all in one place longitudinal monitoring of ehrlichia ruminantium infection in gambian lambs and kids by pcs20 pcr and map1b elisa pdf.
Nymphal ticks that fed on one buffalo on days 16 and 37 and on. Demonstration of vertical transmission of cowdria ruminantium. Here, we further assessed the reliability of the pcr assay and applied it to field ticks. Yunker onderstepoort veterinary institute, private bag x5, onderstepoort, south africa abstract heartwater caused by cowdria ruminantium infection is the most important tickborne disease of ruminants in southern africa. This organism is an obligate intracellular parasite. Classification of the causative agent ehrlichia ruminantium formerly cowdria ruminantium order rickettsiales, family anaplasmataceae small, gram negative, pleomorphic coccus, and obligate intracellular parasite. 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. The development of a pcr assay for the detection of cowdria ruminantium infection in ticks has been previously described. The causal agent of heartwater disease of domestic ruminants,cowdria ruminantium, can, with difficulty, be isolated and passaged in lines of bovine endothelial cells grown in the presence of the glasgow modification of eagles minimal essential medium. Small, gram negative, pleomorphic coccus, and obligate.
Pdf cowdria ruminantium dna is unstable in a supercos1. Pdf cowdria ruminantium dna is unstable in a supercos1 library. Application of the pcr assay in determining the prevalence of infection in ruminants and ticks from heartwater. Nitric oxide no is a labile inorganic free radical produced by no synthase from the substrate larginine in various cells and tissues including endothelial cells. Heartwater cowdria ruminantium infection on sao tome. The disease is widespread in most of subsaharan africa and on several islands in the caribbean, where it is.
Investigating the epidemiology of heartwater cowdria. A cloned dna probe identifies cowdria ruminantium in. Ehrlichia ruminantium is an obligately intracellular proteobacterium which causes a disease known as heartwater or cowdriosis in some wild. No specific fluorescence was observed in noninfected brain. Pdf molecular cloning of a gene encoding the immunogenic. However, when leibovitzs l15 medium supplemented with 0. This rickettsial disease could spread from endemically infected areas of subsaharan africa and certain caribbean islands to other countries, including the united states, in which amblyomma ticks exist. Heartwater cowdria ruminantiuminfection as a cause of postrestocking mortality of goats in mozambique. Pdf investigating the epidemiology of heartwater cowdria. We have previously reported that the pcs20 pcr detection assay for cowdria ruminantium, the causative agent of heartwater disease of ruminants, is more sensitive than xenodiagnosis and the pcs20 dna probe for the detection of infection in the vector amblyomma ticks. Serological tests for cowdria ruminantium infection have been hampered by low specificity. The major antigenic protein 1 fragment b map1b enzymelinked immunosorbent assay elisa for the diagnosis of cowdria ruminantium infections was validated to determine cutoff values and evaluate its diagnostic performance with sheep and goat sera. Thirteen isolates of cowdria ruminantium were made from eight different districts of kenya by four different isolation methods. Production of alpha interferon in cowdria ruminantium.
Cowdria ruminantium was isolated from plasma of clinically affected animals in these four cell. Development and evaluation of pcr assay for detection of. Sequencing of the insert from this clone revealed two open reading frames, encoding peptides of 10462 and 58697 kda. In vitro, recombinant bovine ifnalpha was found to significantly reduce the yield of cowdria organisms in bovine endothelial cells, but even at a high concentration 1,000. Cowdria ruminantium infection in ticks in the kruger.
Cattle that resisted experimental heartwater infection caused by the rickettsia cowdria ruminantium produced significant levels of circulating alpha interferon ifnalpha, whereas animals that died from heartwater did not. Immunization of cattle by infection with cowdria ruminantium. In 75 ticks fed on carrier animals, pcr and the dna. Potential value of major antigenic protein 2 for serological. Cowdria ruminantium is the etiologic agent of heartwater, a disease causing major economic loss in ruminants in subsaharan africa and the caribbean.
Detection of the agent of heartwater, cowdria ruminantium, in amblyomma ticks by pcr. The disease is widespread in most of subsaharan africa and on several islands in the caribbean, where it is a serious constraint to animal production. Cowdria ruminantium dna is unstable in a supercos1 library article pdf available in the onderstepoort journal of veterinary research 662. Jugular blood was also collected at 3 weekly intervals and inoculated into sheep. Cowdria infected populations consisted of 48 sheep and 44 goats, while the noninfected populations consisted of 64 sheep and 107. The disease is spread by bont ticks, which are members of the genus amblyomma. Improved culture conditions for cowdria ruminantium. Ehrlichia ruminantium formerly cowdria ruminantium is a tickborne rickettsial pathogen that infects cattle, causing heartwater or cowdriosis. The gene is conserved between four different stocks of c. Growth of cowdria ruminantium, the causative agent of. Monoclonal antibody binding to a surfaceexposed epitope. The relationship between cowdria ruminantium and chlamydia trachomatis was studied by im munofluorescence. Monoclonal antibodies mab binding to cowdria ruminantium elementary bodies eb were identified by enzymelinked immunosorbent assay, and surface binding of one mab 446. Proliferative responses to both infected autologous.
Pdf genome size and genetic map of cowdria ruminantium. The genes for the immunodominant major antigenic protein 1 map1 of cowdria ruminantium from four african and two caribbean isolates were cloned, restriction mapped, and sequenced to identify conserved epitopes for development of serodiagnostic tools for heartwater. The causal agent, cowdria ruminantium cowdry 1925, is a rickettsia closely related to members of the genus ehrlichia, and is probably a part of a complex of genomic species. Cowdria ruminantium is an obligate intracellular pathogen that causes heartwater in ruminants. Heartwater cowdria ruminantiuminfection as a cause of. Several findings suggest that t cells play an important role in protection against the disease. Ehrlichia ruminantium formerly cowdria ruminantium. Cowdria ruminatium, the causative agent of heartwater disease, expresses an immunodominant and conserved 32kilodalton protein map1.
Cowdria ruminantium dna is unstable in a supercos1 library. Isolation of the causative agent of heartwater cowdria. The cowdria ruminantium groe operon microbiology society. Mar 31, 2020 cowdria ruminantium pdf ehrlichia ruminantium formerly cowdria ruminantium. Validation and application of the assay to field ticks.
Heartwater or cowdriosis is a tickborne disease affecting domestic ruminants throughout subsaharan. Cowdria runimantium is recog nized by a monoclonal antibody directed against the major outer membrane protein of chlamydia trachomatis. The notion was developed to describe patterns of tickborne disease in cattle. It is caused by ehrlichia ruminantium formerly cowdria ruminantium an intracellular gramnegative coccal bacterium also referred to as rickettsia ruminantium. Endemic stabilitya veterinary idea applied to human public. In domestic ruminants and susceptible wildlife species, heartwater ranges from subclinical infection carrier state to a peracute, fatal disease.
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