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.     

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.     

Heartwater. The artificial transmission of Cowdria ruminantium in domestic ruminants and mice

The artificial transmission of Cowdria ruminantium with infected blood, organ homogenates, peritoneal macrophages, tick stabilate and tissue culture cells is discussed. Organ homogenates prepared from the myocardium, spleen, kidneys and liver of diseased animals are commonly used to infect mice. The efficacy of organ homogenates as a source of C. ruminantium depends on factors such as the route of inoculation and the heartwater isolate used. Heartwater is artificially transmitted with infected tick stabilate, haemocytes, rectal ampules and hypodermal homogenates. The infectivity of saliva collected from Amblyomma hebraeum female ticks was very low compared to the ground-up suspensions prepared from the same group of ticks.     

Heartwater in the Caribbean: isolation of Cowdria ruminantium from Antigua

Adult Ambylomma variegatum ticks were collected from 184 cattle, 13 sheep and one goat in Antigua, and ground in phosphate buffered saline. The resultant supernates were cryopreserved in liquid nitrogen. Five supernate pools, each derived from approximately 100 ticks collected from different herds, were thawed and each was inoculated intravenously into a separate experimental goat. One goat exhibited a febrile response with Cowdria ruminantium demonstrable in brain biopsies; after recovery, this animal showed no reaction to a lethal challenge with a Guadeloupe isolate 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.     

The development of Cowdria ruminantium in neutrophils

The sequential development of C. ruminantium (Kwanyanga and Kümm isolates) was followed in caprine leukocyte cultures by light microscopy, direct immunofluorescent microscopy (DFA), indirect immunoflourescent microscopy (IFA) and transmission electron microscopy (TEM). During the febrile response, one to several small cocci, large ring forms or rods were observed in neutrophils in blood smears and cytopreparations of neutrophil fractions using Diff Quik stain, Giemsa stain, DFA and TEM. One to several C. ruminantium colonies were seen in up to 35% of neutrophils maintained in vitro for 18 h to 5 days. The organisms were located in neutrophil phagosomes by TEM and were enveloped by two trilamellar unit membranes. Initially, C. ruminantium was tightly enclosed within phagosomes. At 20 h of incubation, organisms were frequently observed undergoing binary fission within enlarged phagosomal vacuoles. At later time periods, neutrophils harboured fully formed colonies (morula) containing numerous organisms. An occasional C. ruminantium-infected macrophage (Kümm isolate), and an occasional infected eosinophil (Kümm and Kwanyanga isolate) were found.     

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.     

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.     

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.     

Heartwater: an in vitro study of the ultrastructure of Cowdria ruminatium

Notwithstanding morphological differences, the ultrastructure of Cowdria ruminatium cultured in vitro concurred to a large extent with that in previous in vivo studies. Two distinct forms of the organism, elementary and reticulate bodies, and a 3rd group of intermediate organisms were identified. Organisms within a particular vacuole were generally a specific form, but in cells containing many colonies different forms were present in the same colony. Most organisms were enveloped by 2 membranes and a few were surrounded by a 'capsule'. C. ruminantium multiplies mainly by binary fission, but it appears that multiplication can also take place by means of budding. The taxonomy of C. ruminantium is briefly discussed.     

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.     

The present state of Cowdria ruminantium cultivation in cell lines

Attempts were made to grow 4 isolates of Cowdria ruminantium in cell lines. Three of these isolates, viz. Ball 3, Welgevonden and Kwanyanga, could be cultivated in a calf endothelial cell line, but experiments with the Kümm isolate have so far failed. The successful in vitro cultivation of 2 isolates (Welgevonden and Kwanyanga), which are also pathogenic for mice, has great potential for future studies and these aspects are discussed in this review.     

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.     

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.     

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.     

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.     

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.     

Colostrum-derived antibodies to Cowdria ruminantium in the serum of calves and lambs

Antibodies to Cowdria ruminantium were detected in the serum of calves born from artificially immunized heifers, by means of the indirect fluorescent antibody test, only for as long as 4 weeks after birth. Lambs born from artificially immunized ewes, however, were still serologically positive at 8-12 weeks of age. Much higher antibody titres were recorded in the sera of ewes and their lambs than in that of heifers and their calves.