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 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.     

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.     

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.     

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.     

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 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 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.     

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.     

Experimental transmission of Cowdria ruminantium by the Gulf coast tick Amblyomma maculatum: danger of introducing heartwater and benign African theileriasis onto the American mainland

The tick Amblyomma maculatum proved to be an experimental vector of Cowdria ruminantium, the cause of heartwater of ruminants. Transstadial transmission from larva to nymph and from larva through nymph to adult was successful; however, an experiment on transovarial transmission gave negative results. Neither A americanum nor A cajennense transmitted the infection. There is a potential danger of the disease gaining access to the American mainland from the Caribbean where it is known to occur and of maintaining itself in areas where A maculatum occurs. Transstadial transmission experiments, using 3 American Amblyomma spp and an African strain of Theileria mutans, which is thought to occur in the Caribbean, were unsuccessful.     

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.     

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 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.     

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.     

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.     

Transmission of Cowdria ruminantium by Amblyomma gemma from Infected African Buffalo (Syncerus caffer) and Eland (Taurotragus oryx) to sheep

Two African buffalo (Syncerus caffer), an eland (Taurotragus oryx) and a waterbuck (Kobus defassa) were intravenously inoculated with Cowdria ruminantium (Kiswani). Amblyomma gemma nymphs were fed on the animals at 3 weekly intervals. Jugular blood was also collected at 3 weekly intervals and inoculated into sheep. Nymphal ticks that fed on one buffalo on days 16 and 37 and on the other buffalo on day 58 after infection transmitted the disease as adults to sheep. Nymphs that were applied to the eland 16 days after infection also transmitted the disease to sheep. No nymphs that had fed on the waterbuck transmitted the disease. This is the first report of transmission of heartwater by Amblyomma gemma from infected wild ruminant species to a susceptible domestic ruminant species.     

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.     

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.     

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.