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.     

Ehrlichia ruminantium seroprevalence in domestic ruminants in Ghana. II. Point prevalence survey

Serum samples collected on a single occasion from cattle, sheep and goats at sites in all 10 regions of Ghana were tested for antibodies to Ehrlichia (previously Cowdria) ruminantium, the causative agent of heartwater, by polyclonal competitive ELISA (PC-ELISA). The survey revealed the presence of heartwater-exposed ruminants throughout the country, with local seroprevalence up to 100%. Seronegative, and therefore presumably susceptible, animals were also present in all regions, in some areas in numbers high enough to indicate local endemic instability. Overall seroprevalences in cattle, sheep and goats were 61, 51 and 28% respectively, and were generally higher in the northern part of the country and lower in the forest zone. Amongst animals over 1 year old, two thirds of cattle and sheep, and around one third of goats throughout the country had been exposed to E. ruminantium. In the north, seroprevalence in sheep sampled with and without cattle was similar, whereas in the south seroconversion rates in sheep were significantly higher in areas where cattle were present.     

The pathology of heartwater. I. A study of mice infected with the Welgevonden strain of Cowdria ruminantium

Gross and microscopical lesions in mice intravenously infected with the Welgevonden strain of Cowdria ruminantium closely resembled the lesions described in cattle, sheep and goats. A high concentration of organisms was present in alveolar endothelial cells. Cytopathic changes in parasitized and non-parasitized endothelial cells and the morphology of the organisms are described and compared with the Ball3 strain of C. ruminantium. Possible mechanisms in the development of the lung oedema are considered and the role of mice as animal model is discussed.     

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.     

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.     

Preliminary observations on the use of the capillary flocculation test for the diagnosis of heartwater (Cowdria ruminantium infection).

A capillary flocculation test was developed to diagnose heartwater disease of ruminants. Antigen was prepared from the brains of cattle and goats highly infected with Cowdria ruminantium. Sera were obtained from experimentally infected ruminants which either recovered naturally or with the aid of oxytetracycline treatment. Antibodies were first detected one to two weeks after clinical recovery or after treatment, and persisted for periods varying between one and four weeks. Control sera collected from cattle (sheep) and goats in the Netherlands where heartwater does not occur, or from animals serologically positive for Anaplasma marginale or Eperythrozoon ovis infections, did not react to the test.     

Observations on mouse-infective stocks of Cowdria ruminantium: attempts to demonstrate antigenic changes of the Kwanyanga stock in mice

Starting from a stabilate of caprine blood infected with the Kwanyanga stock of Cowdria ruminantium, eight serial passages were made in groups of mice, and eight parallel serial passages in goats. Cross-immunity tests in goats and mice failed to demonstrate any difference between stabilates made after the eighth passage. The Kwanyanga stock was of exceptionally low virulence for Dutch goats.     

Cowdria ruminantium: stability and preservation of the organism

Blood collected in either sodium heparin or disodium edetate vacutainers from febrile goats infected with 4 isolates of Cowdria ruminantium and cryopreserved with 10% dimethyl sulphoxide at -70 degrees C and -196 degrees C was an effective stabilate to initiate heartwater infections in goats. A homogenized pool of whole Amblyomma variegatum ticks in Snyder's buffer, maintained at -196 degrees C, was used to infect a goat with C. ruminantium. Liver and spleen collected from Swiss mice infected with the Kwanyanga isolate of C. ruminantium were homogenized in Snyder's buffer, maintained at -196 degrees C and were used to initiate infections in mice. Fresh blood collected from febrile goats and maintained at 4 degrees C for as long as 72 h was infectious to mice. Neutrophils separated from blood of C. ruminantium infected goats and maintained in modified RPMI medium at 37 degrees C for 68 h were infectious for a goat. Similarly neutrophils from a 2nd infected goat maintained for 96 h at 37 degrees C were infectious for mice.     

Problems with the interpretation of epidemiological data in heartwater: a study on 23 farms

In an epidemiological study undertaken on 23 farms where heartwater occurs endemically, it was found that on an overall average, antibodies to Cowdria ruminantium were detected in 64.3% of the cattle, 6 adult Amblyomma hebraeum ticks were counted per animal and 7.0% of ticks were infected with the heartwater agent. It was found that the seropositivity of the animals was determined largely by the tick loads to which they were subjected and that the influence of the tick C. ruminantium infection rate was less evident. There was no parallel between the prevalence of heartwater on the farms and the immune status of the animals. In general, higher tick counts were recorded in herds where strategic tick control is practised than on farms with a total tick control programme. The method of tick control did not, however, appear to influence the immune status of the cattle, the tick infection rate, or the prevalence of heartwater.     

Specific immunity in farm animals to heartwater

The duration of immunity to heartwater normally varies from 6 months to 4 years. In Angora goats it may be difficult to produce a specific immune response with vaccination. Humoral immunity does not appear to play a role in the development of immunity. It has been suggested that cellular immunity may play a role, but the actual mechanism involved remains obscure. The specific immune response developing in farm animals following infection with Cowdria ruminantium is reviewed.     

Problems encountered in the control of heartwater in Angora goats

This preliminary investigation confirmed that when Angora goats were immunized against heartwater, either when kids or young goats, they still had an immunity to heartwater a year later when they were challenged with a vaccine containing Cowdria ruminantium. An overwhelming majority of the uninoculated animals which were challenged at the same time were susceptible to clinical heartwater. Strategic immunization to obtain enzootic stability to heartwater is suggested. In these experiments the immunization of Angora goats was not accompanied by severe losses.     

Identification of Babesia bovis and Cowdria ruminantium on the island of Unguja, Zanzibar

Babesia bovis and Cowdria ruminantium were identified for the first time in cattle on Unguja Island, Zanzibar. B bovis is common and widespread, although clinical disease had not been diagnosed previously. The vector of heartwater, Amblyomma variegatum, is found throughout Unguja but C ruminantium appears to be more localised in distribution than B bovis.     

Demonstration of colonies of Cowdria ruminantium in midgut epithelial cells of Amblyomma variegatum

The development of colonies of Cowdria ruminantium was studied in midgut epithelial cells of adult Amblyomma variegatum that had become infected by feeding as nymphs on cattle with experimentally induced heart-water disease. Colonies were not observed in gut tissues obtained from nymphs during the feeding period, but were present in midgut epithelial cells of ticks obtained at 15 days after they were replete through molting to the adult stage. Colonies were small (1 to 10 micron) initially, but as tick development progressed, their diameter increased to as much as 60 micron. With electron microscopy, colonies were observed to be membrane bound and contained pleomorphic organisms that were reticulated. The organisms seemed to be dividing by binary fission. Many colonies contained a large, electron-dense inclusion that was morphologically similar to hemoglobin deposits found in the cytoplasm of midgut epithelial cells of recently fed ticks. Cowdria ruminantium was often observed adhered to these inclusions.     

Antigenic diversity of Cowdria ruminantium isolates determined by cross-immunity

Antigenic diversity in five stocks of the tick-borne rickettsia Cowdria ruminantium, the causal agent of heartwater disease of ruminants, was studied by cross-immunity trials in goats and sheep. Complete absence of cross-protection was found only between the Kümm and Kwanyanga stocks, and in all other combinations there were various degrees of cross-immunity. Immunological strain differences were more pronounced in goats than in sheep.     

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.     

Immunity of tick-exposed seronegative and seropositive small stock challenged with two stocks of Cowdria ruminantium

Nineteen per cent and 32% of serologically positive sheep and goats in heartwater endemic areas of the Republic of South Africa were susceptible to challenge with, respectively, the Ball 3 and Welgevonden stocks of Cowdria ruminantium. There was good correlation between the results of the indirect fluorescent antibody test and immunity, since without exception all the seronegative animals reacted when they were challenged. The fact that only 74% of the seropositive sheep and goats were immune to challenge can probably be ascribed to the poor cross-protection between stocks of C. ruminantium and not to false positive serological reactions.     

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.     

Antigenic differences between stocks of Cowdria ruminantium

Stocks of Cowdria ruminantium from Senegal, Zambia and South Africa were compared in cross immunity tests in goats. The Senegal stock caused fatal heartwater in three of 10 goats immune to the South African reference stock Ball 3, and five others showed significant febrile reactions and recovered spontaneously. Four goats immune to the Senegal stock did not show any reaction on challenge with Ball 3. The stock from Zambia was fully cross-protective with Ball 3 in experiments with three goats, but these three goats, immune to the Zambia stock and to Ball 3, showed severe febrile responses upon further challenge with the Senegal stock. The Senegal stock was highly virulent for Dutch goats and there were exceptionally large numbers of rickettsiae in brain capillaries after death. This stock has been passaged eight times in mice, without causing disease; the presence of the organism in the mice was shown by subinoculating goats. The Senegalese stock of C ruminantium is the first stock outside South Africa against which the reference Ball 3 stock does not fully immunise.     

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.     

Lack of cross-protection between Cowdria ruminantium and Ehrlichia phagocytophila.

Antigenically distinct stocks of Cowdria ruminatium from Senegal and South Africa were compared with a Dutch isolate of Ehrlichia phagocytophila in cross-immunity trials in goats. There was a complete absence of cross-immunity between E. phagocytophila and C. ruminantium, despite previous observations that both rickettsial organisms have certain antigenic determinants in common.