Ticks associated with wild mammals in Ghana.

The host ranges of a collection of 21 tick species found on wild mammals in the savanna, forests and coastal zone of Ghana suggested that most species were adapted to feeding mainly on host species within a single mammalian order, i.e. on artiodactyls (bovids/suids), carnivores, rodents or pholidotes (pangolins). Only a few species were dispersed evenly across a range of orders. Seven out of ten of the most common ticks on forest mammals were significantly associated with a particular host species or a group of closely related host species, which could be viewed as their major host or hosts, but they were also recorded much less frequently on a wide range of host species. Two other species were confined to their major hosts. Only one species appeared to be widely dispersed on forest mammals and to lack a particular major host. The majority of tick species therefore occurred on hosts with very distinctive biological, behavioural and ecological characteristics. The study provided no evidence to support the view that host specificity is an artefact of sampling. Finding that the tick species on Ghanaian wild mammals occurred on particular hosts, as well as in distinct habitats, indicated that tick-host associations are important for tick survival and confirmed the importance of climate and vegetation in tick distribution.

An updated list of the ticks of Ghana and an assessment of the distribution of the ticks of Ghanaian wild mammals in different vegetation zones.

Twenty one species of ticks belonging to five genera of the family Ixodidae (Order Acari, sub-order Ixodida) - Amblyomma, Haemaphysalis, Hyalomma, Ixodes and Rhipicephalus (including the sub-genus Rhipicephalus (Boophilus)) - were collected from 1260 mammals, representing 29 species, 14 families and 6 orders, in four vegetation zones in Ghana during the period 1971-1978. Four other species were collected from humans in 1977. In all, eight species appeared to be new records for Ghana: Amblyomma tholloni Neumann; Dermacentor circumguttatus Neumann; Haemaphysalis houyi Nuttall & Warburton; Ixodes loveridgei Arthur; Ixodes oldi Nuttall; Ixodes vanidicus Schultze; Rhipicephalus complanatus Neumann; Rhipicephalus cuspidatus Neumann. The updated list of tick species in Ghana given here includes 41 species of ixodid ticks and four species of argasid ticks. Most species have been found in neighbouring regions of West Africa but 56 of the 121 different combinations of ixodid tick species and host species found in the collection described here have not apparently been reported before. The new combinations recorded here bring the total number of different combinations of ixodid tick species and mammalian host species now reported in Ghana to 151. The tick species found on wild mammals in Ghana mostly differed from those reported from domestic stock by other authors. The data showed that different tick species occurred in different vegetation zones and that most species displayed a pronounced preference for certain groups of related host species. Some tick species were found in the savanna feeding mainly on large bovids and/or suids; others were found in forests feeding mainly on small bovids, large rodents or small carnivores.

Theileria annulata: attenuation of a schizont-infected cell line by prolonged in vitro culture is not caused by the preferential growth of particular host cell types.

Flow cytometry and monoclonal antibodies to bovine leucocyte surface antigens were used to identify the types of host cells that the sporozoites of Theileria annulata infect in cattle, to determine whether virulent schizont-infected cell lines (lines) differed phenotypically from avirulent lines, and to establish whether attenuation in vitro was accompanied by the preferential growth of particular host cell types. The surface antigens of four pairs of T. annulata (Ta) (Hisar) lines derived ex vivo and in vitro, including the virulent ex vivo-derived Ta Hisar S45 line, were consistent with a myeloid origin for all lines, irrespective of their derivation. The profiles of lines derived from cattle inoculated with a virulent line showed that the schizonts liberated from inoculated cells had transferred to myeloid cells. A number of other lines infected with different stocks of T. annulata expressed myeloid markers; a single line expressed CD21, a B cell marker. During prolonged in vitro culture, the parasites in the ex vivo (virulent)- and in vitro (avirulent)-derived Ta Hisar S45 myeloid lines became clonal, as defined by glucose phosphate isomerase (GPI) polymorphism, and the virulent line became attenuated. The two lines retained phenotypic profiles indicative of a myeloid origin but coexpressed some lymphoid antigens (CD2, CD4, CD8), although not CD3. Cloned schizont-infected lines, representing the three parasite GPI isotypes which constituted the virulent line, expressed similar patterns of myeloid and lymphoid markers to the virulent parent line. Some schizont-infected clones failed to establish as lines during the early weeks of culture because the cells died as the parasites differentiated into merozoites at 37 degrees C, the temperature at which schizont-infected cells normally grow exponentially. These results provided no evidence that prolonged culture induces preferential growth or loss of particular host cell types. However, a number of the alterations in host cell surface antigens induced by prolonged culture were shown to be linked to permanent changes in the parasite genome.

Mechanism(s) of attenuation of Theileria annulata vaccine cell lines.

Attenuated vaccines are an important means of controlling Theileria annulata infection of cattle. Production is by prolonged cultivation of macroschizont-infected cells. The mechanism of attenuation remains unclear. There are three general nonmutually exclusive possibilities: Selection of avirulent subpopulations, genome rearrangements and alterations in gene expression. Several groups, including ours, have provided evidence that the population structure usually tends to simplify during attenuation. Our data on the T. annulata (Ta) Ankara cell line show that attenuation is not necessarily accompanied by the population becoming clonal. We have been unable to detect large DNA rearrangements. Evidence for alterations in host and parasite gene expression during attenuation is available. With respect to the host we have shown that attenuation is accompanied by loss of expression of parasite induced matrix metalloproteinases (MMPs). However, in different lines different protease activities are involved. In the T. annulata Ode line we have shown that 8 activities (including MMP9) are downregulated and that this correlates with a loss of metastatic behaviour. This has previously been shown in vitro using reconstituted basement membrane (Matrigel) and is demonstrated in vivo using scid mice in this study. Thus part of the pathology, namely the ability to disseminate, mediated by host MMPs, is lost upon attenuation. Re-isolation experiments have shown that the reduction/loss of MMP is a stable transferable trait. A logical extension is that loss of MMP activity (and virulence in general) must be at the most fundamental level a genetic trait of the parasite. Evidence for loss of parasite gene expression is implied by the loss of the ability to differentiate into merozoites on attenuation. Specific evidence for loss of parasite gene expression has been obtained using differential RNA display. We view virulence as a multifactorial phenomenon involving interacting subpopulations of cells and attenuation is a threshold effect whereby the number of virulence factors is reduced below a critical level. On this basis there will be many different ways to achieve attenuation.

Protective immune mechanisms to ticks and tick-borne diseases of ruminants.

A workshop of the European Union (EU) Concerted Action Project on the Integrated Control of Ticks and Tick-borne diseases (CA-ICTTD) recently assessed protective immune mechanisms to ticks and tick-borne diseases. The consensus achieved there is summarized here by Patricia Preston and Frans Jongejan. The current understanding of this field is expanded upon by the accompanying articles, and Poster, in this special issue of Parasitology Today.

Innate and adaptive immune responses co-operate to protect cattle against Theileria annulata.

For many years it was assumed that Theileria annulata resembled T. parva, parasitizing lymphocytes and causing lymphoproliferative disease, with the two species being controlled by similar protective immune responses. Patricia Preston et al. here review the evidence that has led to a different view of T. annulata. It is now thought that the schizonts of T. annulata inhabit macrophages and B cells, and that tropical theileriosis is not a lymphoproliferative disease. Both innate and adaptive responses contribute to recovery from infection and resistance to challenge and cytokines produced by infected and uninfected cells influence the outcome of infection. Partial protection has been stimulated recently by defined recombinant antigens; efficacy depended upon the delivery system.

Tissue damage in cattle infected with Theileria annulata accompanied by metastasis of cytokine-producing, schizont-infected mononuclear phagocytes.

The distribution of schizont-infected cells in six calves undergoing acute, lethal sporozoite-induced infections with Theileria annulata was examined, the calves being killed in the early, middle or late stages of disease. A combination of histological and immunocytochemical techniques showed that schizont-infected cells became disseminated rapidly through the lymphoid tissues from the prescapular lymph node draining the site of inoculation to distant lymph nodes (e.g., precrural, mesenteric and mediastinal) and to the spleen and thymus. The parasitized cells also spread rapidly into non-lymphoid organs, being found in the liver, kidney, lung, abomasum, adrenal glands and pituitary gland by day 7, in the brain by day 12 and in the heart by day 14 after infection. As infection progressed, the schizonts differentiated into merozoites. By the late stages of disease, the cells containing merozoites greatly out-numbered schizont-infected cells. The parasitized mononuclear cells were labelled by antibodies to bovine interferon-alpha1 and tumour necrosis factor-alpha and, during the later stages of the disease, contained erythrocytes parasitized by piroplasms. The results suggested that the parasitized mononuclear cells themselves played a role in the development of clinical disease and in tissue damage. These findings provide new evidence that tropical theileriosis can no longer be viewed as a lymphoproliferative disease resulting from the uncontrolled multiplication and metastasis of lymphoid cells infected with T. annulata schizonts, but is caused by a parasite that lives in, and is disseminated by, cytokine-secreting, proliferating mononuclear phagocytes.

Theileria annulata: the expression of two novel macroschizont antigens on the surface of infected mononuclear cells differs during in vitro attenuation of a virulent cell line.

The first part of this study of the biological mechanisms underlying attenuation of virulent Theileria annulata macroschizont-infected cell lines screened four pairs of T. annulata (Hisar) in vivo- and in vitro-derived macroschizont-infected cell lines (lines) and identified a single in vivo-derived line, which induced lethal tropical theileriosis. The other seven lines were relatively avirulent. Analysis of the clinical, hematological, and parasitological responses of cattle immunized with different passages of the virulent line after in vitro culture showed that it was partly attenuated by passage (p) 50 and avirulent by p130. Clones representing the three glucose phosphate isomerase (GPI) isotypes, which constituted the newly isolated virulent culture, were obtained from p3 by limiting dilution; p50 and p130 consisted of one isotype. The second part of the study raised monoclonal antibodies (MAbs) against macroschizont-infected cells, as reagents for detecting antigenic differences between virulent and avirulent parasites, and identified two MAbs that recognized the surface of infected cells as well as macroschizonts. MAb EU1 recognized an antigen expressed by all the lines tested, whether in vitro- or in vivo-derived, whether uncloned or cloned, and irrespective of extent of subpassage in culture. MAb EU106 recognized an antigen whose expression by the virulent line and its clones disappeared on passage in culture. This antigen was not expressed at all by the avirulent in vitro-derived line prepared with cells from the same calf. Both antigens were expressed by lines infected with other stocks of T. annulata, including two lines known to induce lethal disease. The different profiles of expression of the two novel antigens, recognized by MAbs EU1 and EU106, by the line undergoing attenuation suggest (1) that the two antigens interact differently with the bovine immune system; and (2) that there are two, very different, potential roles for these antibodies in the development of vaccines against T. annulata infections.

Nitric oxide is produced by Cowdria ruminantium-infected bovine pulmonary endothelial cells in vitro and is stimulated by gamma interferon.

Nitric oxide (NO) is a labile inorganic free radical produced by NO synthase from the substrate L-arginine in various cells and tissues including endothelial cells. A substantial elevation of nitrite levels indicative of NO production occurred in cultures of Cowdria ruminantium-infected bovine pulmonary endothelial cells (BPEC) incubated in medium alone. Exposure of the infected cultures to recombinant bovine gamma interferon (BorIFN-gamma) resulted in more rapid production of NO, reduced viability of C. ruminantium, and induction of endothelial cell death. Significant inhibition of NO production was noted after addition of the NO synthase inhibitor N-monomethyl-L-arginine (L-NMMA), indicating that the increase in production occurred via the inducible NO synthase pathway. Reduction in the infectivity of C. ruminantium elementary bodies (EBs) occurred in a dose-dependent manner after incubation with the NO donor molecule S-nitroso-N-acetyl-DL-penicillamine (SNAP) prior to infection of endothelial cells. The level of infection in cultures maintained in SNAP was reduced in a dose-dependent manner with significant negative correlation between the final level of infection on day 7 and the level of SNAP (r = -0.96). It was established that pretreatment and cultivation of C. ruminantium EBs with the NO donor molecule SNAP reduced infectivity to cultures and viability of EBs with the implication that release of NO in vivo following infection of endothelial cells may have an effect upon the multiplication of the agent in the host animal and may be involved in the pathogenesis of heartwater through the effect of this molecule upon circulation.

Nitric oxide causes the macroschizonts of Theileria annulata to disappear and host cells to become apoptotic.

The proliferation of Theileria annulata macroschizont-infected cell lines in vitro was significantly inhibited by nitric oxide (NO) generated by S-nitroso-N-acetyl-DL-penicillamine (SNAP). Incubation with SNAP caused the macroschizonts to disappear and host cells to become apoptotic. SNAP-derived NO also significantly inhibited the incorporation of tritiated thymidine by cultures of cells in which the schizonts had been induced to differentiate into merozoites by maintenance at 41 degrees C instead of 37 degrees C, the temperature used for culturing macroschizont-infected cells. These results point to NO as the mediator of macrophage anti-T. annulata activity and provide new evidence that the protective immune mechanisms which allow cattle to recover from primary infection and resist challenge may be attributed principally to the products of activated macrophages. These findings indicate that effective inactivated vaccines against T. annulata should include antigens able to stimulate the type of CD4+ T cell response which elicits macrophage activation and NO synthesis.

Bovine cells infected in vivo with Theileria annulata express CD11b, the C3bi complement receptor.

Bovine cells from cattle infected with Theileria annulata were phenotyped with monoclonal antibodies recognizing bovine leukocyte antigens. Macroschizont-infected, transformed cell lines prepared from peripheral blood mononuclear cells of cattle, infected with sporozoites, were assessed by flow cytometry; parasitized cells in tissues from infected cattle were examined by immunocytochemical techniques. Co-expression of markers for different cell lineages by the cell lines precluded a definite conclusion as to their phenotypic origins. For, while the pattern of leukocyte antigens expressed by these in vivo-derived schizont-infected cells, which included CD11b, was indicative of a myeloid origin, the possibility that they were NK cells could not be excluded. The monoclonal antibody (MAb) IL-A15, which recognizes CD11b, reacted with a high proportion of parasitized cells in sections of tissues from infected cattle at all stages of acute disease. Mononuclear cells infected with parasites at all stages of differentiation, from macroschizont to microschizont, expressed CD11b. Such parasitized cells occurred throughout the lymphoid tissues, being found in the thymus, spleen and lymph nodes, particularly the prescapular node draining the site of infection, the hepatic, mesenteric and precrural nodes, as well as in the reticulo-endothelial tissue of the liver, kidney, lung, abomasum, adrenal and pituitary glands. These observations provided the first evidence for a myeloid origin for the parasitized T. annulata cells found in infected bovine tissues and blood and suggested a mechanism whereby schizonts could transfer from cell to cell during mechanical infection with schizont-infected cells.

Protective immune responses to Theileria annulata of relevance to vaccine development.

A series of projects on Theileria annulata funded by the European Union (STD1/STD2/STD3) have provided convincing evidence that macrophage and natural killer (NK) cell-dependent immune mechanisms may directly control the proliferation of different stages of T. annulata in cattle. The evidence for this conclusion and the implications for vaccine development are discussed in the following paper.

Theileria annulata: altered gene expression and clonal selection during continuous in vitro culture.

Kept in continuous in vitro culture, the protozoan parasite Theileria annulata gradually loses virulence when inoculated into cattle. These attenuated cell lines form the basis of the in vitro live vaccines which have been used successfully to control tropical theileriosis in several endemic regions. In the study reported here, events occurring during in vitro culture of an Indian (Hisar) cell line, which may be associated with the reduction in virulence, have been investigated. Hybridization with two polymorphic DNA probes following Southern blotting showed that selection of particular parasite genotypes occurs very rapidly with culture; a novel hybridization pattern is observed with both probes after 50-100 passages in vitro. In addition to this selection process, immunofluorescence studies using a monoclonal antibody which specifically recognizes virulent T. annulata revealed alterations in antibody reactivity following in vitro culture. This loss of reactivity was observed in three cloned cell lines derived from the early, virulent Hisar line and implies that phenotypic changes resulting from alterations to parasite gene expression are taking place during the attenuation process. When considered with the results from in vivo infections with serial passages of this cell line, it can be proposed that both altered gene expression and selection may be involved in the loss of pathogenicity of T. annulata during continuous in vitro culture.

Nitric oxide inhibits establishment of macroschizont-infected cell lines and is produced by macrophages of calves undergoing bovine tropical theileriosis or East Coast fever.

Nitric oxide (NO) was produced when bovine peripheral blood mononuclear cells (PBMC) or purified, adherent PBMC (macrophages) were incubated in vitro with bovine recombinant interferon gamma (Bo rIFN-gamma). NO was produced by cells from naive, uninfected calves as well as by cells from cattle either infected with or recovered from infection with Theileria annulata or Theileria parva. PBMC of cattle undergoing tropical theileriosis (T. annulata infection) or East Coast fever (T. parva infection) synthesized NO spontaneously in vitro. NO was also induced when PBMC of immune, but not of naive, cattle were cultured with T. annulata macroschizont-infected cell lines. Macrophages alone were not stimulated to produce NO by such infected cells. In vitro establishment of macroschizont-infected cell lines was suppressed either by incubating sporozoites with S-nitroso-N-acetyl-DL-penicillamine (SNAP), a NO releasing molecule, prior to invasion of PBMC or by pulsing developing cultures of trophozoite-infected cells with SNAP. Proliferation of established macroschizont-infected cell lines was not affected by SNAP. Taken together with the well documented roles of NO in neutrotransmission, vasodilatation, cell and tissue damage and immunosuppression, the results presented here indicate that NO may not only protect cattle against T. annulata and T. parva but, if produced in excess, play a prominent role in the pathogenesis of tropical theileriosis and East Coast fever.

Phenotypic analysis of bovine leukocyte cell lines infected with Theileria annulata.

Six cell lines that were infected with Theileria annulata were compared for expression of a range of surface molecules. Immunofluorescence staining with the panel of monoclonal antibodies formulated for the Second Workshop was analysed by flow cytometry. Four lines generated from bovine peripheral blood mononuclear cells by exposure to sporozoites in vitro and one line generated in vivo from an infected calf were phenotypically similar. Phenotypic analysis of those lines did not demonstrate a monocyte or B cell origin, but indicated they were not derived from T cells. An uncloned line generated in vivo from an infected calf was CD3+ and gamma/delta TCR+. The results indicate that a wider range of host cell types may be infected in vivo than suspected hitherto and that some of the cells may express an abnormal pattern of surface molecules. Both could have profound consequences for the pathogenesis of T. annulata infection.

Synthesis of tumour necrosis factor-alpha and interferons by mononuclear cells from Theileria annulata-infected cattle.

Bovine macrophage-derived tumour necrosis factor-alpha/cachectin (TNF-alpha) was synthesized when peripheral blood mononuclear cells (PBMC) and purified adherent PBMC from naive and Theileria annulata-infected cattle were incubated in vitro with concanavalin A (Con-A) or bovine recombinant interferon gamma (Bo rIFN-gamma). TNF-alpha production was also induced when adherent PBMC were cultured with T. annulata macroschizont-infected cells. In contrast, non-adherent PBMC from sublethally infected cattle produced interferon (IFN) when incubated with Hu rIL-2, Con-A, phytohaemagglutinin (PHA) or T. annulata macroschizont-infected cells growing as cell lines in vitro. Whilst PBMC from lethally infected cattle spontaneously produced IFN-gamma during advanced stages of infection, the sera of such animals contained type 1 IFN (alpha/beta). IFN was also produced by T. annulata macroschizont-infected cell lines maintained in vitro. This work suggests that cytokines serve as crucial links between proliferating Theileira-infected cells and the characteristic clinical symptoms of tropical theileriosis.

Proliferation of Theileria annulata and Theileria parva macroschizont-infected bovine cells in scid mice.

Theileria annulata and Theileria parva macroschizont-infected bovine cells formed tumours at the inoculation site when injected subcutaneously into C.B.-17 scid mice. T. annulata tumours showed more vigorous growth than T. parva tumours. The tumours did not regress and infected cells spread to other tissues. Intraperitoneal injection of high doses of T. annulata-infected cells resulted in the development of ascites: the infected cells colonized abdominal organs, in particular mesenteric tissue. Low doses of cells did not establish when administered by this route. Evidence for a role for macrophages in controlling proliferation of Theileria-infected cells was provided by finding (i) that uninfected bovine cells did not survive for as long in the peritoneal cavities of scid mice as in Balb/c mice: (ii) peritoneal macrophages both proliferated in vivo in the presence of infected cells and were activated as assessed by production of interleukin-1. Evidence against a role for NK cells was provided by (i) the failure of an in vivo assay for allogeneic lymphocyte cytotoxicity to reveal any activity against bovine cells in the lungs or liver, i.e. the sites usually associated with NK cell cytotoxicity, and (ii) the lack of correlation between tumour regression and NK cell activity in the spleens of mice with chronic T. annulata tumours.

Tropical theileriosis in Bos taurus and Bos taurus cross Bos indicus calves: response to infection with graded doses of sporozoites of Theileria annulata.

This work extends basic knowledge of tropical theileriosis in taurine and crossbred cattle. Infection of Bos taurus and Bos taurus cross Bos indicus (Sahiwal) calves with graded doses of sporozoites of Theileria annulata (Hissar), an Indian stock of the parasite, showed the following to be dose dependent in both cattle types: the time to appearance and population size of macroschizonts, microschizonts and piroplasms, time and severity of pyrexia, anaemia manifested by erythrocyte counts and haematocrit. All infections were accompanied by a prompt and severe panleucopenia. This effect was dose related in both the taurine and the Sahiwal crossbred calves. Lymphocyte counts returned to preinfection levels in the blood of animals which recovered, but death from theileriosis was characteristically accompanied by a persistent and severe lymphocytopenia. Flow cytometry using monoclonal antibodies to bovine mononuclear cells was used to identify the lymphocyte subsets involved in lymphocytopenia. The outcome of infection was dose dependent in the crossbred calves but not in taurine calves. Although the results obtained did not differ qualitatively between the two cattle types, they provided some preliminary evidence for resistance to tropical theileriosis in Sahiwal crossbred calves.

Growth of Theileria annulata and Theileria parva macroschizont-infected bovine cells in immunodeficient mice: effect of irradiation and tumour load on lymphocyte subsets.

Bovine cells infected with macroschizonts of the protozoan parasites Theileria annulata and Theileria parva formed solid tumours when injected into irradiated Balb/c and irradiated Balb/c nude mice. T. annulata tumours grew more vigorously than T. parva tumours, when initiated with similar doses of infected cells in mice exposed to the same doses of gamma-irradiation. In irradiated Balb/c mice, tumours of both species of parasites began to regress 2-3 weeks after injection of cells but grew without regression in irradiated Balb/c nude mice. Haemorrhage and necrosis of tumours, induced by macrophages and neutrophils, were seen in both mouse strains but were insufficient to cause regression in Balb/c nude mice. Theileria-infected bovine cells failed to establish in C57 beige mice, which lack functional natural killer (NK) cells. Flow cytometry, using monoclonal antibodies to murine leukocyte/lymphocyte antigens, showed that the radiation dose required to allow establishment of T. annulata tumours in Balb/c mice caused a severe depletion of splenic lymphocytes. B cells, helper T and cytotoxic T cells showed differing levels of susceptibility to irradiation. The presence of a tumour promoted the recovery of lymphocyte populations: this recovery was accompanied by destruction of the tumour.