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.     

An attempt to improve the immunization of sheep against heart-water by using different combinations of 3 stocks of Cowdria ruminantium

Neither sheep immune to the Ball 3, the Kwanyanga or the Mara stocks of Cowdria ruminantium nor those immunized with combinations of these 3 stocks were protected against challenge with the Mali stock. Against challenge with the Welgevonden stock, however, immunization with each of the 3 combinations and with single stocks effected a protective immunity.     

In vitro isolation of Ehrlichia ruminantium from ovine blood into Ixodes scapularis (IDE8) cell cultures

Four stocks of Ehrlichia ruminantium (Welgevonden, Ball3, Nonile and Blaauwkrans), the causative agent of heartwater in domestic ruminants, were isolated into Ixodes scapularis (IDE8) tick cells using the leukocyte fraction of the blood of infected sheep. Organisms of two of the E. ruminantium stocks (Welgevonden and Blaauwkrans) propagated in IDE8 cells were also successfully used to infect bovine endothelial cells. All stocks were successfully propagated in IDE8 cells using Dulbecco's modified Eagle's medium nutrient mixture Ham F-12 containing 10% foetal bovine serum (FBS). The technique should be included in any attempt to isolate uncharacterized E. ruminantium stocks.     

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.     

Identification of the antigenic proteins of Cowdria ruminantium.

Immunoblotting of Cowdria ruminantium proteins with sheep or bovine antiserum identified 2 antigenically conserved proteins, one being an immunodominant 31 kDa and the other a minor 27 kDa protein. These proteins are present in the electrophoretic profiles of the Welgevonden, Ball 3 and Kwanyanga stocks and are recognized by sheep antiserum to the Welgevonden, Ball 3, Kwanyanga, Mali, Comoro, Breed, Germishuys, Kümm and Mara stocks and by bovine antiserum to the Welgevonden stock of C. ruminantium. The stocks did not reveal identical or unique antigenic properties which could explain differences in pathogenicity and cross-immunity observed amongst the various stocks of C. ruminantium.     

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.     

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.     

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.     

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.     

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.     

Ehrlichia ruminantium variants which do not cause heartwater found in South Africa

In 1994 a batch of apparently healthy goats was selected for intended export to the USA from a heartwater-free and vector tick-free region of South Africa. The animals were tested serologically for heartwater, using either or both an IFA and an ELISA test, and 52% were found to be serologically positive. A PCR assay based on Ehrlichia ruminantium 16S gene sequences gave positive results for 54% of the animals, suggesting that apparently non-pathogenic E. ruminantium variants existed in this heartwater-free area. To identify and characterise the agents responsible for the positive serological and PCR results, ticks and animal blood samples were collected from two of the three farms involved in the original survey during two successive seasons of expected peak tick activity. Ticks were kept alive for a minimum of 3 weeks to allow digestion of any blood meal before being processed. Over the two seasons, 28% of the livestock and 15% of the ticks sampled were found to be carrying E. ruminantium. E. ruminantium 16S and pCS20 sequences were detected in all of the four tick species collected from the livestock (Rhipicephalus evertsi evertsi, Rhipicephalus evertsi mimeticus, Hyalomma truncatum, Hyalomma marginatum rufipes), suggesting that some of the species may act as vectors. Animals generally carried multiple E. ruminantium 16S genotypes, whereas ticks rarely carried more than one. Infection levels in both animals and ticks were too low to generate a marked response when a blood stabilate was sub-passaged in a clean sheep, preventing the subsequent establishment of any of the organisms in culture.     

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.     

Some observations on the sero-prevalence of heartwater and tick infestation in Zambian goats

A survey was carried out to define the distribution of heartwater in goats that originated from six districts in communal grazing semi-arid areas of Zambia. A total of 181 samples (40.1%) out of 451 serum samples from adult goats were positive for Ehrlichia ruminantium antibodies after screening using indirect MAP-1B antigen ELISA technique with statistically significant differences (P < 0.01) between the six districts. Out of 1 036 adult goats examined for tick infestation, 105 were infested by ticks, with Amblyomma species being the most dominant tick encountered. Amblyomma variegatum, which is the vector for heartwater transmission in Zambia constituted 42.4% of the tick species, identified. The overall tick infestation rate was 10% while the tick:goat ratio was 2.1:1. Amblyomma variegatum appears to be widespread throughout the study area, as are antibodies to E. ruminantium.     

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.     

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 use of a microagglutination assay for the detection of antibodies to Corynebacterium pseudotuberculosis in naturally infected sheep and goat flocks

Two goat flocks comprising 326 animals and four sheep flocks comprising 343 animals, all with a previously recognized problem of abscesses due to Corynebacterium pseudotuberculosis, were examined for the presence of abscesses and antibody titers to C. pseudotuberculosis as detected by direct microagglutination assay. In sheep there was a strong positive relationship between age and titer (p less than 0.0001). However, the relationship in goats between age and titer could not be determined due to a strong interaction between flock and age. When the relationship between abscesses and titer was examined, it was found that goats with abscesses had higher titers than those that did not (p less than 0.05), whereas there was no difference in titer between sheep with abscesses and those without (p = 0.5753). The sensitivity of the microagglutination test was poor to good for both species (52.3% for goats and 89.7% for sheep). The specificity of the test was fair to poor (64.9% for goats and 21.7% for sheep). Given a disease prevalence of 13.5% for goats and 8.5% for sheep the predictive value of the positive test was very poor (18.9% for goats and 9.6% for sheep) but the predictive value of the negative test was good to excellent (89.7% for goats and 95.8% for sheep). The poor specificity of the test and therefore the positive predictive value may be due in part to the criterion of classification of presence of disease, i.e. presence of an abscess at the time of sampling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of infection rates of Oestrus ovis between sheep and goats kept in mixed flocks

Oestrosis is a nasal myiasis of sheep and goats caused by larvae of the fly Oestrus ovis and can lead to severe clinical signs, which together with the disturbance caused by the adult fly may result into serious economic losses. Infection rates and larval burdens are always higher in sheep than in goats after either natural or artificial infestation. The aim of this study was to compare the host preference of the adult fly O. ovis between sheep and goats in mixed flocks, where they are kept together under the same husbandry conditions and hence, are very similarly exposed to the fly preference. Blood sera samples were collected from a total of 397 sheep and 335 goats, from 43 mixed flocks located at different regions of Greece. Antibodies specific to O. ovis IgG were measured by ELISA. A flock was considered positive when at least one individual was positive, i.e. showed a seropositivity of >or=20% in relation to positive control sera. A total of 193 (48.6%) sheep and 58 (17.9%) goats were found to be seropositive against O. ovis. Thirty-eight (88.4%) out of 43 flocks had at least one seropositive animal. The mean seroconversion against O. ovis in animals from the different flocks was 38.6% and 13.6% for sheep and goats, respectively, whereas the variance of infection within each flock was 0-100%. The mean seropositivity between sheep that were found to be positive or negative was 60.6% and 5.4%, respectively, whereas the corresponding values between goats were 35.2% and 5.2%, respectively. No significant difference in the seroconversion values was noted between flocks from the different areas (P=0.817), whereas a very significant difference was observed between animal species (P=0.001). However, there was no significant difference when seroconversion comparisons were made within samples of the same animals species, sheep or goats from different flocks of all the regions included in the study (P=0.695). The results of this study clearly demonstrate that O. ovis has a widespread distribution in Greece, and the seroprevalence is significantly higher in sheep than goats (P=0.001).     

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.     

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.     

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.