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.     

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.     

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.     

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.     

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.     

Observations on mouse-infective stocks of Cowdria ruminantium: attempts to demonstrate persistence of the organism in mice immune to the Kwanyanga stock

Mice immunised against the Kwanyanga stock of Cowdria ruminantium by infection and treated with oxytetracycline proved immune to challenge on day 40 and also to a second challenge on day 125 after infection. Treatment with the experimental dithiosemicarbazone gloxazone on days 59 and 73 did not abolish immunity to challenge on day 125. No persistence of the organism in immune mice that had been challenged on day 40 could be demonstrated by subinoculating blood and liver homogenate on day 126. These results are different from findings reported elsewhere with the mouse-infective Kumm stock.     

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.     

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.     

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.     

An attempt to establish an inbred line of mice genetically resistant to Cowdria ruminantium

An attempt to establish an inbred line of mice resistant to Cowdria ruminantium failed. First generation couples were constituted from those mice out of 100 males and 100 females that had survived infection with the Kümm stock of C. ruminantium and that were serologically negative to the indirect fluorescent antibody test. An attempt to establish 10 separate inbred lines by constituting next generation brother and sister matings from predominantly seronegative survivor mice from the preceding generation, was unsuccessful because too few mice survived the challenge. The percentage seronegative survivors increased to 94% over the first 6 generations, but then declined sharply during the next.     

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.     

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.     

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.     

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.     

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.     

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.     

T cell-mediated immunity to Cowdria ruminantium in mice: the protective role of Lyt-2+ T cells

The inability of athymic nude mice to make a drug-aided recovery from infection with either the Kümm or the Welgevonden stocks of Cowdria ruminantium and their inability to mount an immune response, suggest that immunity in heartwater is cell-mediated. The adoptive transfer of immunity with the spleen cells of mice immune to the Welgevonden stock is supportive evidence. Immune spleen cells depleted of Lyt-2+ T cells are unable to confer resistance to challenge to recipient mice, whereas the depletion of L3T4+ T cells had no effect on the protection conferred by immune spleen cells. This is conclusive evidence that immunity in heartwater is largely cell-mediated. Immune serum, C. ruminantium and complement incubated in the presence of mouse peritoneal macrophages, inhibits the infectivity of the heartwater agent, but not in the absence of macrophages. The decreased resistance to challenge of immune mice treated with gloxazone adds further support to the concept that in heartwater persistence of C. ruminantium in the host is associated with immunity.     

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 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.     

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.