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.     

Characterisation of Escherichia coli O157 isolates from Danish cattle and human patients by genotyping and presence and variants of virulence genes

An important objective in vaccination strategies is to activate lymphocytes with particular effector functions. Cellular immunity and the type I cytokine IFN-gamma have been implicated in protective immunity to heartwater. Furthermore, low molecular weight proteins of Cowdria ruminantium have been shown to induce peripheral blood mononuclear cells to proliferate. To determine which lymphocyte subset responds when stimulated with fractionated C. ruminantium proteins, specific short-term lymphocyte cultures were established from cattle immunized with the Welgevonden isolate. Four cattle were immunized, two by infection and treatment and two with inactivated organisms. Cell surface phenotypic analysis of the cultures indicated that CD4+ lymphocytes were enriched over time. This coincided with increased antigen-specific proliferation and IFN-gamma production. Proteins of molecular weights 13-18kDa induced the CD4+-enriched T-cell cultures, derived from each of the animals, to proliferate and produce IFN-gamma. Although the two groups of cattle were immunized differently, their lymphocytes responded similarly. These results extend previous findings by identifying the responder cells as being predominantly IFN-gamma producing CD4+ lymphocytes. This cytokine has been implicated in immunity to the parasite. The low molecular weight proteins that induced CD4+ lymphocytes to proliferate and produce IFN-gamma are therefore likely to be important in protection against heartwater and may have a role in vaccine development.     

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.     

Problems encountered in the control of heartwater in Angora goats

This preliminary investigation confirmed that when Angora goats were immunized against heartwater, either when kids or young goats, they still had an immunity to heartwater a year later when they were challenged with a vaccine containing Cowdria ruminantium. An overwhelming majority of the uninoculated animals which were challenged at the same time were susceptible to clinical heartwater. Strategic immunization to obtain enzootic stability to heartwater is suggested. In these experiments the immunization of Angora goats was not accompanied by severe losses.     

Protective killed Ehrlichia ruminantium vaccine elicits IFN-gamma responses by CD4+ and CD8+ T lymphocytes in goats

Interferon gamma (IFN-gamma) is considered as a key mediator of protective cell-mediated immunity against intracellular pathogens in general, and against Ehrlichia ruminantium, the causative agent of tick-borne heartwater disease of ruminants, in particular. However, the source of this important cytokine in animals immunized against E. ruminantium remains largely unknown. We have analyzed in goats protected by vaccination with a killed E. ruminantium vaccine, the potential of individual, genuine (i.e., non-cloned), T cell subsets to produce IFN-gamma after antigenic recall in vitro. In all vaccinated but none control animals, E. ruminantium-induced IFN-gamma secretion was observed in 24 h stimulated blood. Flow cytometric analysis of stimulated peripheral blood mononuclear cells (PBMCs) collected after each vaccine inoculation indicated that immune CD4+ and CD8+ T cells contribute to the same extent to the production of IFN-gamma, while WC1+ T cells are less important. This was confirmed by blocking the secretion of IFN-gamma with anti-classes I and II major histocompatibility complex antibodies. Blocking experiments also suggest that CD8+ need the help of CD4+ T cells in order to produce IFN-gamma. Thus, this work underlines the key role of CD4+ T cells in the production of IFN-gamma by immune goat PBMC. It also describes, for the first time in ruminants, E. ruminantium-specific CD8+ effector T cells. Since CD4+ and CD8+ T cells collectively contribute to the production of IFN-gamma in most vaccinated animals, and since these responses are associated with protection, it may be that a recombinant vaccine will need to incorporate E. ruminantium antigens capable of driving both responses.     

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.     

Possible death of a buffalo calf (Syncercus caffer) due to suspected heartwater (Ehrlichia ruminantium)

Rickettsial organisms resembling Ehrlichia ruminantium (the causative organism of heartwater) were demonstrated in brain smears and formalin-fixed brain sections derived from a buffalo calf that died on a private game reserve in northern KwaZulu-Natal. The possibility that the tick-free environment of a quarantine boma may have affected the calf's immunity, is discussed. These findings suggest that monitoring heartwater in wild ruminants and making brain smears as a routine during post mortem evaluations of wild ruminants, should be encouraged.     

Identification of drugs effective against Cowdria ruminantium using a mouse screening system

Heartwater is a tick-transmitted rickettsial infection of ruminants, caused by Cowdria ruminantium. The tetracyclines are the only compounds available for therapy of the disease. A screen, using mice infected with C. ruminantium, was developed and used to identify new compounds with potential for the control of heartwater. A series of di-4-methyl-thiosemicarbazones was shown to be highly active in the mouse model and their efficacy was confirmed in further trials in sheep infected with C. ruminantium. The mouse screen was shown to be simple to operate and reliably predictive of activity against heartwater. Ways in which the screen may be improved are suggested.     

Specific immunity in farm animals to heartwater

The duration of immunity to heartwater normally varies from 6 months to 4 years. In Angora goats it may be difficult to produce a specific immune response with vaccination. Humoral immunity does not appear to play a role in the development of immunity. It has been suggested that cellular immunity may play a role, but the actual mechanism involved remains obscure. The specific immune response developing in farm animals following infection with Cowdria ruminantium is reviewed.     

The pathology of heartwater. III. A review

The pathological changes in cattle, sheep, goats, mice and various game species infected with Cowdria ruminantium are summarized. Macroscopical lesions in most animals include effusion of body cavities, oedema of the lungs and lymph nodes and splenomegaly. Apart from the presence of heartwater organisms in most organs the histopathological lesions are not striking. The ultrastructural lesions in the lungs of sheep and goats infected with the Ba11 3 strain of heartwater, and mice infected with the Welgevonden strain, are discussed. Damage to capillary endothelial cells of the alveoli is limited and the mild cytopathic changes in parasitized cells indicate that the damage caused by the organisms is most probably not responsible for the increased vascular permeability associated with the disease. Pathological changes in domestic ruminants and game animals are briefly compared.     

Preparation of Ehrlichia ruminantium challenge material for quantifiable and reproducible challenge in mice and sheep

The causative agent of heartwater, Ehrlichia ruminantium, is a tick-transmitted pathogen that infects bovine endothelial cells. Due to the obligate intracellular nature of this organism obtaining pure material in sufficient quantities for challenge studies is difficult. A murine model is frequently used to study potential vaccine candidates but giving reproducible challenges in this model for heartwater has always been problematic. We have therefore performed a series of experiments to optimize the parameters governing the reproducibility of challenge material. Two cryoprotectants were compared for the preparation of challenge material, buffered lactose peptone (BLP) and sucrose-potassium-glutamate (SPG). In addition two sources of virulent E. ruminantium were used, infected bovine endothelial cultures and infected mouse spleen homogenates. We also examined practical parameters affecting the reproducibility of challenge experiments: the time it takes to deliver the challenge material, the length of time a mouse remains immune to E. ruminantium challenge, and the effect of a given challenge dose. Finally, we performed a pilot study to determine whether mice could be used to titrate challenge material to be used for experiments in sheep. We found that: (a) E. ruminantium-infected mouse spleen homogenate provides more reproducible challenges than tissue culture material; (b) SPG is a better cryoprotectant than BLP; (c) challenge material should be used within 20min of thawing; (d) it is not essential to use syngeneic material for murine challenge experiments; (e) Balb/c mice are more sensitive to E. ruminantium challenge than C57BL/6J mice; (f) mice immunized by infection and treatment for use as positive immune controls should be challenged within 3 months of immunization; and (g) mice should be challenged with a dose not exceeding 10 LD(50)s.     

Susceptibility to heartwater of calves born to non-immune cows

The resistance to artificial infection with Cowdria ruminantium of calves born to cows fully susceptible to heartwater is no different from that of calves bred in heartwater endemic areas where the tick challenge is negligible to considerable. The sub-inoculation into mice of blood collected 14-26 days after infection proved the presence of the heartwater agent in the blood of 8 out of 10 calves with no other clinical signs than mild to moderate fever. The combined use of a mouse model and the indirect fluorescent antibody test revealed considerable variation in the degrees to which calves become infected and react to artificial infection.     

Virulence of two strains ofcowdria ruminantium in mice and their use to predict drug activity against heartwater

A study was made of the infectivity of two mouse-adapted strains of Cowdria ruminantium in mice. The Kwanyanga strain was most virulent in Balb/C mice which died nine days after infection with homogenate of liver from infected mice. CD-1 mice were least susceptible of six strains tested. The du Plessis strain of C. ruminantium was equally virulent in all six mouse strains. The du Plessis strain in CD-1 mice was used as the basis of a drug screen to detect activity against heartwater (C. ruminantium infection) and was highly predictive when active compounds were tested in sheep infected with the Ball 3 strain of C. ruminantium.     

The pathogenesis of heartwater

Hypotheses on the pathogenesis of heartwater, which have been published so far, are briefly reviewed. Attempts were made at counteracting the effects of vaso-active substances released by mast cells by treating mice infected with Cowdria ruminantium with antagonists to histamin and serotonin on one hand, and with mast cell stabilizers on the other, but were not successful. Preliminary findings suggest that a hypersensitive type of reaction, triggered by the release of pharmacologically active substances, may possibly be basic to the pathogenesis of heartwater. Complement, and the products of arachidonic acid metabolism, possibly play a role in the release of the vaso-active substances.     

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.     

Identification of Ehrlichia ruminantium proteins that activate cellular immune responses using a reverse vaccinology strategy

Ehrlichia ruminantium is an obligate intracellular bacterial pathogen which causes heartwater, a serious tick-borne disease of ruminants throughout sub-Saharan Africa. The development of promising recombinant vaccines has been reported previously, but none has been as effective as immunisation with live organisms. In this study we have used reverse vaccinology to identify proteins that elicit an in vitro cellular immune response similar to that induced by intact E. ruminantium. The experimental strategy involved four successive steps: (i) in silico selection of the most likely vaccine candidate genes from the annotated genome; (ii) cloning and expression of the selected genes; (iii) in vitro screening of the expressed proteins for their ability to induce interferon-gamma (IFN-γ) production in E. ruminantium-immune lymphocytes; and (iv) further examination of the cytokine response profiles of those lymphocytes which tested positive for IFN-γ induction. Based on their overall cytokine induction profiles the recombinant proteins were divided into four distinct groups. Eleven recombinant proteins induced a cytokine profile that was similar to the recall immune response induced by immune peripheral blood mononuclear cells (PBMC) stimulated with intact E. ruminantium. This response comprised the upregulation of cytokines associated with adaptive cellular immune responses as well as innate immunity. A successful vaccine may therefore need to contain a combination of recombinant proteins which induce both immune pathways to ensure protection against heartwater.     

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.     

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.     

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.     

In vitro infection by Ehrlichia ruminantium of baby hamster kidney (BHK), Chinese hamster ovary (CHO-K1) and Madin Darby bovine kidney (MDBK) cells

The Welgevonden stock of Ehrlichia ruminantium, aetiological agent of heartwater, was propagated in baby hamster kidney (BHK) cells, Chinese hamster ovary (CHO-K1) cells and Madin Darby bovine kidney (MDBK) cells. The cultures required supplementation of the medium with cycloheximide for reliable growth of E. ruminantium. Growth of the Welgevonden stock in BHK and CHO-K1 cells could lead to the development of suspension cultures suitable for the mass production of E. ruminantium for an inactivated elementary body vaccine.