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.     

The pathology of heartwater. III. A review

The pathological changes in cattle, sheep, goats, mice and various game species infected with Cowdria ruminantium are summarized. Macroscopical lesions in most animals include effusion of body cavities, oedema of the lungs and lymph nodes and splenomegaly. Apart from the presence of heartwater organisms in most organs the histopathological lesions are not striking. The ultrastructural lesions in the lungs of sheep and goats infected with the Ba11 3 strain of heartwater, and mice infected with the Welgevonden strain, are discussed. Damage to capillary endothelial cells of the alveoli is limited and the mild cytopathic changes in parasitized cells indicate that the damage caused by the organisms is most probably not responsible for the increased vascular permeability associated with the disease. Pathological changes in domestic ruminants and game animals are briefly compared.     

Heartwater. The development and life cycle of Cowdria ruminantium in the vertebrate host, ticks and cultured endothelial cells

Various aspects of the development and life cycle of Cowdria ruminantium are discussed. C. ruminantium is transmitted transstadially by certain Amblyomma species. Apparently organisms initially develop in the gut epithelial cells of ticks and subsequent stages of C. ruminantium invade and develop in the salivary gland acini cells of the vector. Stages at which transmission to the final host are attained appear to be coordinated with the feeding cycle of the ticks and the vertebrate host is infected via salivary glands of the tick. In the vertebrate host, ticks and cultured endothelial cells, different morphological forms of C. ruminantium (electron-dense and reticulated forms) are found. Organisms enter cells through a process resembling phagocytosis and reticulated forms of the organisms appear to be the main vegetative stage. In the vertebrate host, organisms proliferate in vascular endothelial cells, neutrophils, macrophages and reticulo-endothelial cells.     

Heartwater. The morphology of Cowdria ruminantium and its staining characteristics in the vertebrate host and in vitro

The morphology of Cowdria ruminantium is described and its staining characteristics in the vertebrate host and in vitro are summarized. Morphologically, the organisms are characterized in the cytoplasm of endothelial cells, macrophages and reticulo-endothelial cells. Based on the morphology of the internal structure of the organisms, elementary (electron-dense), intermediate and reticulate bodies are identified. Each organism is surrounded by a double membrane and a "capsule" is evident around a few organisms in vitro. Usually, only organisms of the same form are found within a particular vacuole, although mixed colonies are described in the in vitro studies.     

Histologic and immunochemical study of the pathogenesis of heartwater (Cowdria ruminantium infection) in goats and mice

Eleven adult goats and 32 adult outbred mice were inoculated IV with Cowdria ruminantium-infected blood (Kwanyanga isolate), monitored clinically, then serially euthanatized. Predominant clinical signs of disease in goats were depression, head tremors, seizures, and dyspnea. In mice, dyspnea and depression were the only clinical signs of disease noticed. Tissues were examined histologically and immunohistochemically for C ruminantium colonies or antigen. In goats, C ruminantium was detected only in endothelial cells of the brain, even though gross and microscopic lesions were confined to the thorax. In mice, C ruminantium was detected only in endothelial cells of the heart and lungs.     

Diagnosis of heartwater at post-mortem in ruminants and the confirmation of Cowdria ruminantium in mice

Criteria that are required to confirm heartwater in ruminants and mice are discussed. In ruminants it entails the macroscopical and microscopical lesions as well as the identification of Cowdria ruminantium in brain smears or histological sections. Macroscopical lesions in the majority of animals that die of the disease include effusion of body cavities, hydropericardium, oedema of the lungs, brain, mediastinum, and its associated lymph nodes, and splenomegaly. The effect of specific chemotherapy on the morphology of heartwater organisms in vivo is outlined. A severe nephrosis in heartwater-infected Angora goats, treated after the first day of the febrile reaction, is described.     

A method for determining the Cowdria ruminantium infection rate of Amblyomma hebraeum: effects in mice injected with tick homogenates

Amblyomma hebraeum ticks, collected in the field and individually homogenized, were injected into mice. Thirteen out of 240 ticks were shown to be infected with the heartwater agent. Antibodies against Cowdria ruminantium were detected in the sera of the mice by means of the indirect fluorescent antibody test. Giemsa-stained smears, prepared from the haemocytes of the ticks, revealed morphologically different forms of the heartwater agent. A strain of C. ruminantium, designated the Welgevonden strain, was isolated in mice from one of the infected ticks and passaged in mice for 8 generations. When inoculated intravenously, it was highly infective to mice, sheep and cattle. The murinotropism of the Welgevonden strain is compared with that of other strains previously described.     

Virulence of two strains ofcowdria ruminantium in mice and their use to predict drug activity against heartwater

A study was made of the infectivity of two mouse-adapted strains of Cowdria ruminantium in mice. The Kwanyanga strain was most virulent in Balb/C mice which died nine days after infection with homogenate of liver from infected mice. CD-1 mice were least susceptible of six strains tested. The du Plessis strain of C. ruminantium was equally virulent in all six mouse strains. The du Plessis strain in CD-1 mice was used as the basis of a drug screen to detect activity against heartwater (C. ruminantium infection) and was highly predictive when active compounds were tested in sheep infected with the Ball 3 strain of C. ruminantium.     

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.     

Morphology and development of Cowdria ruminantium in Amblyomma ticks

The morphology and development of Cowdria ruminantium have been studied in Amblyomma hebraeum and A. variegatum. Colonies of C. ruminantium have so far been demonstrated microscopically in gut, salivary gland cells, haemocytes and malphighian tubules of infected Amblyomma ticks. Colonies in gut cells were seen in both unfed and feeding ticks but colonies in salivary gland acini were observed only in nymphs that had fed for 4 days. Although the predominant type seen in both tick stages was the reticulated form that appeared to divide by binary fission, electron dense forms were also present. The latter are similar to those forms documented in endothelial cells of the vertebrate host as well as in cell culture. The presence of colonies of C. ruminantium in salivary glands of feeding ticks, along with the demonstration of different morphologic forms of the organism, suggests that a developmental cycle of the organism occurs in its invertebrate host. It is thought that organisms first infect and develop within gut cells. From there subsequent stages continue their development in haemolymph and salivary glands and are then transferred to the vertebrate host during tick feeding. Further studies are needed to completely understand the development of C. ruminantium in ticks and its subsequent transmission by these parasites.     

Observations on mouse-infective stocks of Cowdria ruminantium: microscopical demonstration of the Kwanyanga stock in mouse tissue and the carrier-status of the Senegal stock in mice

The behaviour of two different stocks of Cowdria ruminantium was investigated in mice. The mouse-pathogenic Kwanyanga stock of C ruminantium was microscopically demonstrated in mice in capillary endothelial cells of the lung, spleen, kidney, liver and brain. Mice of the ninth passage of the Senegal stock, which is infective but not pathogenic to mice, were kept alive for a year. Their blood and homogenised spleens, inoculated intravenously, caused fatal heartwater in a goat. However, the Senegal stock could not be demonstrated microscopically in mice. These results indicate the possible role of rodents in the epidemiology of heartwater.     

Heartwater in Angora goats. II. A pathological study of artificially infected, treated and untreated goats

Pathological lesions in untreated Angora goats infected with the Ball3 strain of Cowdria ruminantium corresponded with those previously reported. A severe nephrosis was the most prominent pathological lesion in the animals treated after the 1st day of the febrile reaction. Renal ischaemia appears to be central to the pathogenesis of the kidney lesions.     

In vitro isolation of Cowdria ruminantium from plasma of infected ruminants

A new and simple technique for isolation of C. ruminantium in bovine and ovine vascular endothelial cells (aorta, pulmonary artery) is described. Unlike previous studies, no efforts were made to retard cell growth by irradiation or chemicals. Instead, heparin-derived plasma samples obtained from only those animals exhibiting prolonged or extremely high temperatures were used. In this way, C. ruminantium was isolated from 27 of 37 samples (73%) and from 22 of 26 animals (85%). A total of six Zimbabwean stocks of C. ruminantium were isolated in cell culture.     

The Kümm isolate of Ehrlichia ruminantium: in vitro isolation,propagation and characterization

An effective culture system for Ehrlichia (Cowdria) ruminantium comb. nov. was first established in 1985 and many stocks were subsequently isolated and propagated in vitro. A notable exception, however, was the Kümm isolate that resisted all attempts at in vitro culture until the successful experiment described here. In one experiment white blood cells were harvested from heparinized blood derived from a sheep infected with the Kümm isolate. The cells were added to DH 82 cells and incubated at 37 degrees C. The high metabolic activity of the DH 82 cells necessitated that cell growth be retarded by the addition of cycloheximide. Colonies were first detected 19 days after culture initiation and, once the cultures were established, they could be passaged every 3 days. Bovine and sheep endothelial cells were readily infected with culture supernatant obtained from the infected DH 82 cells. In a further experiment another sheep was infected, using a higher dose of the same batch of Kümm stabilate, and we attempted to infect several different cell lines: these were DH 82 cells, bovine aorta (BA 886) cells, sheep brain endothelial (SBE 189) cells and sheep fibroblastoid cells (E2). Ten days after culture initiation only the E2 cells had become positive for E. ruminantium. Culture supernatant from the first cultured isolate (Kümm-1) was less virulent for mice than that of the second cultured isolate (Kümm-2) which killed all mice. Upon molecular characterization with E. ruminantium 16S probes we found that Kümm-1 hybridized with a Senegal 16S genotype probe, whereas Kümm-2 hybridized only with an Omatjenne 16S genotype probe. The original stabilate used to infect the sheep hybridized with both probes. These results clearly indicate that two different stocks had been isolated in culture.     

The detection of antibodies to Cowdria ruminantium in serum and C. ruminantium antigen in Amblyomma hebraeum by an enzyme-linked immunosorbent assay

A sensitive and reliable enzyme-linked immunosorbent assay for the detection of antibodies to Cowdria ruminantium in serum and C. ruminantium antigen in Amblyomma hebraeum nymphae is described. For the screening of antibodies, C. ruminantium from A. hebraeum nymphae, partially purified by wheat-germ lectin affinity chromatography, was used as antigen. To screen nymph populations, sera from either Ball 3 strain-infected sheep or Kumm-strain infected mice were used. By using appropriate controls the assays were rendered specific with respect to C. ruminantium.     

Preparation of Ehrlichia ruminantium challenge material for quantifiable and reproducible challenge in mice and sheep

The causative agent of heartwater, Ehrlichia ruminantium, is a tick-transmitted pathogen that infects bovine endothelial cells. Due to the obligate intracellular nature of this organism obtaining pure material in sufficient quantities for challenge studies is difficult. A murine model is frequently used to study potential vaccine candidates but giving reproducible challenges in this model for heartwater has always been problematic. We have therefore performed a series of experiments to optimize the parameters governing the reproducibility of challenge material. Two cryoprotectants were compared for the preparation of challenge material, buffered lactose peptone (BLP) and sucrose-potassium-glutamate (SPG). In addition two sources of virulent E. ruminantium were used, infected bovine endothelial cultures and infected mouse spleen homogenates. We also examined practical parameters affecting the reproducibility of challenge experiments: the time it takes to deliver the challenge material, the length of time a mouse remains immune to E. ruminantium challenge, and the effect of a given challenge dose. Finally, we performed a pilot study to determine whether mice could be used to titrate challenge material to be used for experiments in sheep. We found that: (a) E. ruminantium-infected mouse spleen homogenate provides more reproducible challenges than tissue culture material; (b) SPG is a better cryoprotectant than BLP; (c) challenge material should be used within 20min of thawing; (d) it is not essential to use syngeneic material for murine challenge experiments; (e) Balb/c mice are more sensitive to E. ruminantium challenge than C57BL/6J mice; (f) mice immunized by infection and treatment for use as positive immune controls should be challenged within 3 months of immunization; and (g) mice should be challenged with a dose not exceeding 10 LD(50)s.     

In vitro cultivation of Cowdria ruminantium

Cowdria ruminantium was cultivated in a calf endothelial cell line after the cells had been irradiated at 45 & 90 GY. Another experiment in which the inoculum and non-irradiated cells were centrifuged together also yielded positive results. In some irradiated cultures, colonies of organisms could be demonstrated microscopically up to 70 days after the cultures had been inoculated with infected tick stabilate. The infectivity of cultures, even after 4 passages and 88 days post-inoculation, was demonstrated by their intravenous injection in sheep.     

Flow cytometric analysis of T cell response in mice infected with Cowdria ruminantium.

A 3-fold increase in the numbers of Lyt-2+ T cells in the circulating blood of mice infected and re-infected with the Welgevonden stock of Cowdria ruminantium, as determined by flow cytometry, is supportive evidence that immunity in heartwater is cell-mediated. The rise in Lyt-2+ cells only after re-infection of the mice is further evidence that the development of immunity in heartwater is dependent on the unhindered and adequate replication of C. ruminantium.     

Cowdria ruminantium infection in ticks in the Kruger National Park.

Adult Amblyomma hebraeum ticks, the principle vector of heartwater (cowdriosis) of domestic ruminants in southern Africa, were collected in pheromone traps placed in Kruger National Park, an exclusively wildlife sanctuary in South Africa. These ticks transmitted Cowdria ruminantium, the rickettsial agent causing heartwater, to a susceptible goat, resulting in acute, fatal disease. C ruminantium was isolated in bovine endothelial cell culture from the plasma of this animal during the febrile stage of the disease and transmitted to susceptible goats, causing fatal heartwater. The prevalence of C ruminantium infection in 292 ticks was determined by polymerase chain reaction (PCR) analysis to be 1.7 per cent (95 per cent confidence interval 0.71 to 4.0 per cent). A DNA probe analysis, which is less sensitive than PCR, detected infection in three of the five PCR-positive ticks. The remaining infections were below the detection limit of the DNA probe, which is approximately 70,000 organisms. This is the first evidence that a vector-wildlife cycle of transmission of C ruminantium can be maintained independently of domestic ruminants.     

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.