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.     

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.     

Distinctive staining of colonies of Cowdria ruminantium in midguts of Amblyomma hebraeum

Mallory's phloxine-methylene blue stain was used to differentiate colonies of Cowdria ruminantium in midgut epithelial cells of nymphal Amblyomma hebraeum that had been infected as larvae. Gut tissues were collected from nymphs that had fed on a susceptible sheep and were fixed in formol-saline on the day of repletion. Paraffin sections, 3-4 micron thick, were then stained and this rendered colonies and cell nuclei densely blue against a uniformly pink background of tick tissues. Colonies were easily distinguished from nuclei by their specific morphology. This method of parasite visualization may be adapted to field-collected ticks for rapid detection of C. ruminantium or to assays of susceptibility of tick populations to various strains of the organism.     

Biochemical studies on the salivary glands and haemolymph of Amblyomma hebraeum

The functional significance of some components of salivary glands and of their secretion and of haemolymph of Amblyomma hebraeum and other tick species is reviewed with respect to host responses at the attachment site, the survival of specific pathogens in the vector, the transmission of pathogens and immunological responses of the host to tick infestation.     

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.     

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.     

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 presence of Cowdria ruminantium antigen in various tissues of Amblyomma hebraeum imagines as detected by an enzyme-linked immunosorbent assay

Investigation into the presence of C. ruminantium antigen, using an enzyme-linked immunosorbent assay (ELISA) in various tick tissues and haemolymph of adult Amblyomma hebraeum ticks revealed that the organism invades a number of body parts and can be demonstrated in A. hebraeum. In females, the gut, salivary glands, hypodermis and synganglion and in males, the salivary glands and gut showed the highest concentration.     

Infection rates with Cowdria ruminantium of nymphs and adults of the bont tick Amblyomma hebraeum collected in the field in Zimbabwe

Cowdria ruminantium (heatwater) infection rates of field populations of the bont tick, Amblyomma hebraeum, were determined at two locations in the southern lowveld of Zimbabwe. At Mbizi Quarantine Station, unfed adult males and females, and nymphs were collected at intervals over a 2-year period using traps. At Lemco Ranch, engorged nymphs were collected on three occasions from weaner calves and allowed to moult to adults. The unfed ticks were fed in small pools on heartwater-susceptible sheep, some of which became infected. The infection rates of the ticks were then estimated statistically. Depending on the date of collection and locality, these rates were in the range 0.0-44.9% for males, 20.0-36.1% for females and 0.0-13.4% for nymphs. Most of these rates are considerably higher than those previously believed to occur.     

A method for determining the Cowdria ruminantium infection rate of Amblyomma hebraeum: effects in mice injected with tick homogenates

Amblyomma hebraeum ticks, collected in the field and individually homogenized, were injected into mice. Thirteen out of 240 ticks were shown to be infected with the heartwater agent. Antibodies against Cowdria ruminantium were detected in the sera of the mice by means of the indirect fluorescent antibody test. Giemsa-stained smears, prepared from the haemocytes of the ticks, revealed morphologically different forms of the heartwater agent. A strain of C. ruminantium, designated the Welgevonden strain, was isolated in mice from one of the infected ticks and passaged in mice for 8 generations. When inoculated intravenously, it was highly infective to mice, sheep and cattle. The murinotropism of the Welgevonden strain is compared with that of other strains previously described.     

The role of males of the bont tick (Amblyomma hebraeum) in the transmission of Cowdria ruminantium (heartwater)

The role of males of the bont tick (Amblyomma hebraeum) in the transmission of Cowdria ruminantium (heartwater) was investigated. Transstadial (nymph to adult) and intrastadial transmission were demonstrated. Males transferred from live or dead hosts to live hosts were shown to transmit C. ruminantium repeatedly. It was concluded that male transmission is of importance in the epidemiology of heartwater.     

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.     

The prevalence of Cowdria ruminantium in free-living adult Amblyomma hebraeum collected at a communal grazing area and in 2 wildlife reserves in South Africa

In order to detect the prevalence of Cowdria ruminantium in the vector tick, Amblyomma hebraeum, free-living, unfed adult ticks were collected with the aid of pheromone/CO2 traps. Ticks were collected at the Rietgat communal grazing area, as well as in the southwestern Kruger National Park and in the Songimvelo Game Reserve, all located in heartwater-endemic areas of South Africa. The presence of C. ruminantium in these ticks was determined by polymerase chain reaction (PCR) analysis. Ticks from the Rietgat communal grazing area were assayed in 2 batches and 4.7% of the one and 11.3% of the other were positive for infection, while 5.7% of the ticks collected in the Kruger National Park and 25% in the Songimvelo Game Reserve were positive. These results support the contention that a vector-wildlife cycle of transmission of C. ruminantium, the cause of heartwater in domestic ruminants, can be maintained in the absence of the latter animals.     

The tick vectors of Cowdria ruminantium (Ixodoidea, Ixodidae, genus Amblyomma) and their distribution

Twelve species of Amblyomma are presently known to be capable of transmitting Cowdria ruminantium. Of these A. variegatum is the most important and widely distributed vector in Africa. It has also extended its range considerably outside this continent, eastward to the Yemen Arab Republic and to the islands of Madagascar, Réunion and Mauritius, and westward to the Cape Verde islands and to some of the West Indian islands. A. hebraeum is probably the only field vector in most parts of southern Africa. A 3rd species, A. lepidum, is known to have been involved in field outbreaks of heartwater in the Sudan. Two other species are also currently regarded as field vectors of Cowdria: A. astrion on the islands of S?o Tomé and Principe, and A. pomposum in Angola. Another 5 African species (A. cohaerens, A. gemma, A. tholloni, A. sparsum and A. marmoreum) have proved to be capable of transmitting heartwater in the laboratory, as have 2 American species (A. maculatum and A. cajennense), but none of these ticks have been implicated in field outbreaks of the disease.     

The occurrence of Theileria and Cowdria parasites in African buffalo (Syncerus caffer) and their associated Amblyomma hebraeum ticks

The polymerase chain reaction and oligonucleotide probing were used to detect Theileria and Cowdria species in DNA extracted from blood and ticks recovered from 24 African buffalo during a gamecapture operation in the Kruger National Park, South Africa. Species-specific probing indicated that all but one of the buffalo were carrying at least one Theileria species. Indirect fluorescent antibody (IFA) serology indicated that all animals had been exposed to Theileria parva infection but only 33% were positive for T. parva by probing. Twelve (50%) of the animals but only six of the 214 adult Amblyomma hebraeum ticks examined (2.8%) were probe-positive for Cowdria. Only one Cowdria 16S genotype was detected in the animals and ticks.     

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.     

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.     

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.     

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.