The freeze-drying of Cowdria ruminantium

Lyophilized tissues of mice and blood of sheep, infected with either the Kümm, the Welgevonden or the Ball 3 stock of Cowdria ruminantium, remain infective to mice and sheep after storage at 4 degree C for 90 days. Freeze-dried tissues stored at -18 degrees C and -28 degrees C are still infective after 6 months and 2 years, respectively.     

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.     

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.     

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.     

Comparison of diluents for maintaining the viability of Cowdria ruminantium

Thirteen solutions were compared to determine the optimal diluent for preservation of the viability of the Kwanyanga isolate of Cowdria ruminantium. They included the 2 diluents commonly used with C ruminantium and diluents proved effective with other rickettsiae. The capability of each diluent to maintain the viability of C ruminantium over a 3-hour period at room temperature was assessed by comparing the survival distributions of groups of outbred albino mice after they were inoculated IV with infected liver homogenates. The results indicated that the Snyder I diluent was significantly better at maintaining the viability of C ruminantium than were the other diluents studied.     

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.     

Purification of Cowdria ruminantium by density gradient centrifugation

The isolation of Cowdria ruminantium by differential and isopycnic density gradient centrifugation is reviewed with special reference to the suitability of Percoll as density gradient medium. Infected sheep brain, Amblyomma hebraeum nymphae and various mouse organs were used as starting material. By these methods, partially purified viable populations of the organism with distinctly different densities were obtained. The conclusions are based upon results of analyses of density fractions by inoculation into sheep or mice, protein determination, electron microscopy and enzyme-linked immunosorbent assay. Morphological differences were observed in the density fractions obtained from infected brain tissue and A. hebraeum.     

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.     

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.     

The transmission of Cowdria ruminantium by Amblyomma sparsum

Transmission of Cowdria ruminantium (heartwater) by nymphs of Amblyomma sparsum has been demonstrated. Adults of the same tick species failed to transmit the disease after feeding on infected sheep in either the larval or the nymphal stage, and there was no transovarian transmission. A. sparsum seldom parasitizes domestic livestock but could be of importance in maintaining reservoirs of heartwater infection in wildlife populations.     

The isolation of nucleic acid sequences specific for Cowdria ruminantium

Screening of a genomic library of Cowdria ruminantium has yielded twelve clones hybridizing to Cowdria DNA. These clones should be suitable for development into DNA probes specific for this organism.     

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.     

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.     

Preservation of Cowdria ruminantium at low termperatures.

A Nigerian isolate of Cowdria ruminantium was rapidly frozen with or without 10 per cent dimethyl sulphoxide at -85 degrees C and -196 degrees C. All animals inoculated with the frozen stabilates died of heartwater fever.     

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.     

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.     

Purification of Cowdria ruminantium by lectin cellular affinity chromatography

This review covers the isolation of Cowdria ruminantium by lectin cellular affinity chromatography from different Amblyomma hebraeum sources. Cellular affinity chromatography has been reviewed with special attention being given to the application of this technique in the isolation of rickettsiae.     

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.     

Proof of transovarial transmission of Cowdria ruminantium by Amblyomma herbraeum

Transovarial transmission of Cowdria ruminantium by Amblyomma hebraeum does occur in certain instances. Both the transovarial and the filial infection rates appear to be very low. The infection may reappear only in the adults or nymphae, or in all 3 stages of the tick's life cycle.