Cowdria ruminantium DNA is unstable in a SuperCos1 library.

A Cowdria ruminantium genomic library was constructed in a cosmid vector to serve as a source of easily accessible and pure C. ruminantium DNA for molecular genetic studies. The cosmid library contained 846 clones which were arrayed into microtitre plates. Restriction enzyme digestion patterns indicated that these clones had an average insert size of 35 kb. Probing of the arrays did not detect any bovine clones and only one of the known C. ruminantium genes, pCS20, was detected. Due to the high AT content and the fact that C. ruminantium genes are active in the Escherichia coli host, the C. ruminantium clones were unstable in the SuperCos1 vector and most clones did not grow reproducibly. The library was contaminated with E. coli clones and these clones were maintained with greater fidelity than the C. ruminantium clones, resulting in a skewed representation over time. We have isolated seven C. ruminantium clones which we were able to serially culture reproducibly; two of these clones overlap. These clones constitute the first large regions of C. ruminantium DNA to be cloned and represent almost 10% of the C. ruminantium genome.     

A differential lysis method for the isolation of Cowdria ruminantium DNA

In order to isolate pure Cowdria ruminantium DNA an enzymatic lysis procedure was used to lyse Cowdria-infected bovine endothelial cell cultures differentially. Infected host cells were treated with trypsin followed by DNase digestion and centrifugation. This method resulted in the isolation of intact Cowdria organisms and removed bovine DNA effectively.     

The occurrence of Theileria and Cowdria parasites in African buffalo (Syncerus caffer) and their associated Amblyomma hebraeum ticks

The polymerase chain reaction and oligonucleotide probing were used to detect Theileria and Cowdria species in DNA extracted from blood and ticks recovered from 24 African buffalo during a gamecapture operation in the Kruger National Park, South Africa. Species-specific probing indicated that all but one of the buffalo were carrying at least one Theileria species. Indirect fluorescent antibody (IFA) serology indicated that all animals had been exposed to Theileria parva infection but only 33% were positive for T. parva by probing. Twelve (50%) of the animals but only six of the 214 adult Amblyomma hebraeum ticks examined (2.8%) were probe-positive for Cowdria. Only one Cowdria 16S genotype was detected in the animals and ticks.     

The tick-borne rickettsia Cowdria ruminantium has a Chlamydia-like developmental cycle.

The development of the tick-borne rickettsial pathogen Cowdria ruminantium (S stock) was studied in bovine umbilical endothelial (BUE) cell cultures and in goat choroid plexus, by light- and electron microscopy. Cowdria divided by binary fission within intracytoplasmic vacuoles resulting in large colonies of reticulate bodies. After three to four days in culture, reticulate bodies developed into smaller intermediate bodies characterized by an electron-dense core. Shortly before disruption of the host cells, intermediate bodies condensed further into electron-dense elementary bodies, which were released into the culture medium. Elementary bodies invade other endothelial cells thus initiating a new infectious cycle which lasts between 5 and 6 days. In the infected goat choroid plexus similar reticulate and intermediate bodies were identified within vacuoles of capillary endothelial cells. However, extracellular elementary bodies were not detected. Another stock of Cowdria (W) showed an identical developmental cycle as that of the S stock. The W isolate was also pathogenic for mice, making it possible to test the infectivity of reticulate and elementary bodies in these animals. Reticulate bodies appeared to be less infective than elementary bodies. The developmental cycle of Cowdria resembles the cycle known to occur in Chlamydia. Moreover, Cowdria has other similarities with Chlamydia. It has a Gram-negative envelope, it does not store iodine-stainable carbohydrates and may lack peptidoglycan as does Chlamydia. It is concluded, that Cowdria and Chlamydia are to a certain extent related, confirming a recent report that both organisms have certain antigenic determinants in common. Since Cowdria is also related to Ehrlichia it may well be that Cowdria takes an intermediate position between Chlamydia and Ehrlichia. The phylogenetic relationship between Cowdria and Chlamydia and also with Ehrlichia should be further elucidated by molecular analysis using 16S ribosomal DNA sequences.     

Competitive enzyme-linked immunosorbent assay for heartwater using monoclonal antibodies to a Cowdria ruminantium-specific 32-kilodalton protein

Hybridomas producing monoclonal antibodies (mAb) to Cowdria ruminantium were raised. Four mAbs of the IgG isotype reacted in western blots with a 32-kilodalton Cowdria protein (Cr32), which had previously been shown to be conserved and immunodominant. A fifth mAb of the IgM isotype recognized a 40-kDa Cowdria protein. The latter mAb was negative in an indirect fluorescent antibody test (IFA), whereas the other four were positive. mAb No. 4F10B4 showed the strongest signal in western blots using three different stocks of Cowdria. Immuno-gold labeling of Cowdria organisms in vitro using 4F10B4 showed that Cr32 has surface-exposed antigenic determinants. Using mAb 4F10B4, a competitive ELISA was developed which detected specific Cowdria antibodies in goat, sheep and cattle sera. Antibodies in animal sera competed with binding of mAb 4F10B4 to a crude sonicated Cowdria antigen obtained from infected endothelial cell cultures. The competition ELISA (CELISA) detected antibodies in 55 out of 70 (79%) goats experimentally infected with one of eight different Cowdria stocks. Fourteen out of the 15 sera which were shown negative in the CELISA were also negative in the IFA. Nevertheless, all 15 sera recognized some epitopes of the immunodominant Cowdria-specific 32 kDa protein as judged from their reaction with this protein in western blots. Overall, there was 89% agreement between CELISA and IFA considering all 70 goat sera. Moreover, antibodies were detected in nine out of nine sheep infected with one of three different stocks of Cowdria and in sera from calves experimentally infected by two different strains of heartwater. There were no cross-reactions with Ehrlichia phagocytophila antibodies in goat sera, nor with Anaplasma marginale antibodies in bovine sera. Lack of cross-reactivity and detection of antibodies to eight geographically widely distributed stocks of Cowdria, makes the competition ELISA a promising test for use in heartwater endemic areas.     

Cowdria ruminantium is recognized by a monoclonal antibody directed against the major outer membrane protein of Chlamydia trachomatis.

The relationship between Cowdria ruminantium and Chlamydia trachomatis was studied by immunofluorescence. A monoclonal antibody directed against the major outer membrane protein of C. trachomatis recognized rickettsial colonies of C. ruminantium in infected goat brain. No specific fluorescence was observed in non-infected brain. Two commercial Chlamydia-specific monoclonal antibodies as well as polyvalent anti-Chlamydia rabbit serum recognized C. trachomatis, but did not recognize Cowdria. Moreover, polyvalent Cowdria antiserum failed to recognize C. trachomatis cultivated in HeLa cells. It is concluded that Cowdria and Chlamydia are to a certain extent related, confirming similarities in ultrastructure and developmental cycle.     

Serotypes in Cowdria ruminantium and their relationship with Ehrlichia phagocytophila determined by immunofluorescence

Two tick-borne rickettsial pathogens of ruminants, Cowdria ruminantium (causative agent of heartwater disease) and Ehrlichia phagocytophila (causative agent of tick-borne fever), were successfully cultivated in caprine or ovine neutrophilic granulocytes. Infected cultures were subsequently used as antigens in the indirect fluorescent antibody test. Low-level bilateral serological cross-reactions could be detected between Cowdria and Ehrlichia. In addition, comparison of five Cowdria stocks using immunofluorescence demonstrated the existence of distinct serotypes within the genus of Cowdria. It is concluded that the occurrence of these serotypes will considerably complicate the current serodiagnosis of heartwater.     

Ultrastructural morphology of Cowdria ruminantium in midgut epithelial cells of adult Amblyomma hebraeum female ticks

Amblyomma hebraeum male and female ticks, experimentally infected as larvae with the Ball 3 stock of Cowdria ruminantium, were fed on a heartwater susceptible sheep. The initial attachment of the males was required as a pre-requisite for female attachment. Reticulate bodies were the predominant morphologic form of Cowdria observed in gut epithelial cells after 1-3 days of feeding. Single intermediate bodies and no elementary bodies were observed. Organisms were found within a membrane-bound vacuole and each organism had a double-unit membrane. Infrequently colonies contained homogeneous electron-dense inclusions. Groups of Cowdria organisms within a haemocyte suggested a possible dissemination of organisms from the gut to various other tissues by haemocytes.     

Asymptomatic carrier state in Creole goats and cattle after recovery from Cowdria infection in Guadeloupe]

Creole goats and cattle in Guadeloupe can be carriers of cowdriosis (heartwater: Cowdria ruminantium) after recovery for a period as long as 11 months in goats and 2 months in cattle. The carrier status was demonstrated by feeding Amblyomma variegatum nymphs on recovered animals and the resulting adult ticks on susceptible goats. Cowdria ruminantium was not detected permanently during the carrier status.     

The pathology of heartwater. II. A study of the lung lesions in sheep and goats infected with the Ball3 strain of Cowdria ruminantium

Lung lesions in sheep and goats infected with the Ball3 strain of Cowdria ruminantium corresponded with those reported in mice infected with the Welgevonden strain of Cowdria ruminantium. Ultrastructural changes in the alveolar endothelial and epithelial cells are described and the pathogenesis of the lung oedema is briefly discussed.     

A mouse lethal dose assay for detection and titration of Cowdria ruminantium (Kwanyanga strain) in goats and ticks

A mouse lethal dose assay was used to detect a mouse pathogenic strain (Kwanyanga) of Cowdria ruminantium, the etiological agent of heartwater in goats and ticks. The titer of the rickettsial organisms in goat blood was directly related to the febrile response of the goat and the rickettsia were undetectable after the fever subsided. The maximum rickettsial titer in goat blood was 10(3) mouse LD50 ml-1. Cowdria-infected goat blood was shown to retain infectivity when held on ice for up to 2 h, but when held at room temperature infectivity declined by greater than 50% in 2 h. The mouse assay detected Cowdria in feeding female Amblyomma variegatum only on the eighth day of feeding and in feeding males on the second and eleventh days of feeding. Cowdria was shown to persist in the hemolymph of the soft tick Ornithodoros coriaceus for a period of at least 2 years.     

Comparison between 3 antigens for the serodiagnosis of heartwater disease by indirect immunofluorescence

A cell line of bovine endothelial cells (E5), infected with 3 different stocks of Cowdria ruminantium, was used as antigen in an indirect fluorescent antibody test for the serodiagnosis of heartwater. These antigens were compared to peritoneal macrophages from mice infected with the Kümm stock and to caprine neutrophils in primary cultures from goats infected with 4 different stocks of Cowdria. The use of endothelial cell cultures proved to be superior in all respects. The antigens can be produced in large quantities at a low cost, contrary to the other types. The reaction is easily and quickly read, compared to the laborious reading of neutrophil or macrophage antigens which often contain few and small colonies of Cowdria. Moreover, not all stocks are suitable for the preparation of neutrophil antigens, while macrophage antigen can only be obtained with the Kümm stock. Endothelial cell antigens also distinguish serotypes in C. ruminantium, but these differences seem to be less pronounced than those found with neutrophil antigens. Finally, the specificity of endothelial cell antigens appears to be better than that of Kümm antigen and comparable to that of neutrophil antigens. The use of Kümm antigen may have been responsible to a large extent for past unexplained positive serological results on certain Caribbean islands where it has not been possible to isolate Cowdria and where no clinical evidence of the disease has been found.     

Increased pathogenicity of an Ehrlichia-like agent after passage through Amblyomma hebraeum: a preliminary report

After being passaged through 3 generations of Amblyomma hebraeum, an Ehrlichia-like agent isolated from an adult Hyalomma truncatum female became more pathogenic and elicited a disease in sheep indistinguishable from heartwater. Cross-immunity between this agent and several stocks of Cowdria ruminantium and high levels of antibody elicited by the agent against 2 stocks of C. ruminantium in the indirect fluorescent antibody test, confirmed its close relationship to Cowdria.     

Electron microscopy of Cowdria-infected macrophages suggests that in the absence of binary fission a mosaic of organisms develops from an amorphous electron dense matrix.

Electron microscopy of mouse peritoneal macrophages infected with the Kümm stock of Cowdria ruminantium suggests that in the final stage of intracellular growth, a mosaic of organisms develops from an amorphous matrix of varying electron density by a process in which double unit membranes portion off the Cowdria particles. This stage is preceded by inclusions consisting of a network of aggregated electron dense granules and these in turn by homogeneous dense bodies. The study failed to show how these dense bodies develop from internalized Cowdria particles introduced in the infective inoculum. The replication of the heartwater agent in macrophages differs from that in vascular endothelial cells in two important respects. First, at no stage during the course of development in macrophages is binary fission in evidence and second, in the absence of a limiting membrane the inclusions and colonies of organisms throughout the cycle of development in macrophages are in intimate contact with the host cell cytoplasm.     

Metabolism and genetics of chlamydias and rickettsias

Chlamydial and rickettsial diseases pose a hazard to man and to domesticated and wild animals. The virulence mechanisms which aid the establishment of these obligate intracellular parasites in the eukaryotic host are still not within our grasp. Recent knowledge of the biochemical stratagem, the metabolic capabilities and the genetic diversity of these microbes illustrate fundamental differences in ecology and evolutionary divergence. The preferred site of intracellular residence determines the strategy for uptake, for nutrient assimilation and also for evasion of the host's immunological defenses. The Chlamydia, Rickettsia, and Coxiella are the most extensively studied of the genera. Whereas the Ehrlichia and Cowdria are poorly understood, they are also the most intriguing of the Rickettsiae. A number of antigenically and genetically distinct species are identified for the genera Chlamydia, Rickettsia, and Ehrlichia, whereas the Coxiella and Cowdria may not represent such a wide diversity. Recent information on the genetic heterogeneity of the chromosomal and plasmid DNAs of the strains of Coxiella suggest the diversity is greater than was originally envisioned. New information regarding the antigenic structure of Cowdria and their cellular tropisms suggests that they are closely related to the Ehrlichia. In this review we compare the metabolic capabilities and the genetic diversity of these different intracellular bacteria.     

Fluorescent antibody technique to detect Cowdria ruminantium in in vitro-cultured macrophages and buffy coats from cattle, sheep, and goats

Fluorescent antibody tests, Giemsa stain, and electron microscopy were used to detect colonies of Cowdria ruminantium in in vitro-cultured macrophages and buffy coats from heartwater-infected cattle, sheep, and goats. Antibodies were obtained from C ruminantium-infected cattle, sheep, and goats treated with a small dose of oxytetracycline HCl. Cowdria ruminantium elementary bodies were small-coccus forms (0.14 micron) and large-coccus forms (0.22 micron to 0.6 micron). The size of inclusion bodies varied from 1.5 micron to 2 micron. Inclusion bodies and elementary bodies were observed in the cytoplasm of macrophages and neutrophils.     

Future prospects and goal setting regarding research on heartwater

This article highlights the most important research goals identified during the workshop on "Heartwater: Past, Present and Future," which was held from 8-11 September 1986 in the Republic of South Africa. An attempt has also been made to identify the most modern technology which is available for this purpose. All 60 papers presented at the workshop, together with other relevant information, are published in this number of the Onderstepoort Journal of Veterinary Research. With regard to the causative organism it is crucial that research should be conducted on pure isolates. Moreover, existing culture methods should be improved in order to obtain better yields of organisms. Research on the hosts and vectors of Cowdria ruminantium should aim to elucidate the ways in which vector ticks become infected in nature. For this purpose especially, it will be necessary to develop rapid tests (e.g. DNA probes) to detect the organism in living animals and ticks. The nature of immunity and young animal resistance are still obscure and call for basic research. Mice and murino-tropic isolates of C. ruminantium should prove useful in this regard. Since cross-reactions with Ehrlichia occur, it is essential to give particular attention to the sero-epidemiology of Ehrlichia in conjunction with similar studies on Cowdria. The development of a tissue culture vaccine offers the greatest chance of immediate success and should be actively pursued. Studies on a recombinant vaccine should, however, be initiated because of the potential long term advantages.     

The reservoir status of goats recovered from heartwater

Experiments were conducted with Creole goats and Amblyomma variegatum ticks in Guadeloupe to investigate whether it is possible to transmit Cowdria ruminantium to susceptible hosts with nymphs fed in the larval stage on recovered goats. Of 88 batches of larvae fed after the return of the goats' temperature to normal, or after challenge or immunosuppression, only the 9 batches of larvae fed during the febrile reaction, 2 batches applied 2 and 3 days respectively after recovery, and a single batch applied 5 days after challenge, became infective. On average, blood appears to be infective for A. variegatum larvae for only an 8-day period associated with the temperature reaction following a primary infection. Recovered goats are not reservoirs of Cowdria, even if--with one exception in our experiment--they are reinfected after recovery.     

Purification of Cowdria ruminantium by immunoadsorbent affinity chromatography

Immunoselective methods with special reference to immunoadsorbent affinity chromatography as a means for the isolation of Cowdria ruminantium are reviewed. Attention is given to the source of the organism, immunization, purification of antibodies, coupling of antibodies to insoluble matrixes and desorption procedures.     

Cowdria ruminantium infection in the mouse: a review

The current knowledge of the pathogenicity, clinical signs and mortality of artificial infections by syringe inoculation of Cowdria ruminantium in laboratory and wild strains of mice is reviewed. It is concluded that a wide spectrum of pathogenicity for mice exists in stocks of the organism.