Patricia M. Preston

The protozoan parasite, Theileria annulata, induces a distinct acute phase protein response in cattle that is associated with pathology.

Acute phase proteins (APP) are synthesised in the liver in response to the systemic presence of high levels of pro-inflammatory cytokines. Bacteria are considered to be strong inducers of APP whereas viruses are weak or non-inducers of APP. Very few reports have been published on APP induction by parasites. Here, we report that the tick-borne protozoan parasite of cattle, Theileria annulata, induced an atypical acute phase response in cattle. Following experimental infection, serum amyloid A (SAA) appeared first, followed by a rise in alpha(1) acid glycoprotein (alpha(1)AGP) in all animals, whereas haptoglobin, which is a major APP in cattle, only appeared in some of the animals, and generally at a low level. All three APP only became elevated around or after the appearance of schizonts in draining lymph nodes and after the first observed temperature rise. Increased alpha(1)AGP levels coincided with the appearance of piroplasms. The production of SAA and alpha(1)AGP correlated strongly with each other, and also with some clinical measures of disease severity including the time to fever, development of leucopaenia, parasitaemia and mortality. These results are consistent with the hypothesis that T. annulata causes severe pathology in susceptible cattle by inducing high levels of pro-inflammatory cytokines.

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.

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‐γ). 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 neurotransmission, 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.

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.

Cytokines inhibit the development of trophozoite-infected cells of Theileria annulata and Theileria parva but enhance the proliferation of macroschizont-infected cell lines

Summary The following bovine (Bo) and human (Hu) cytokines—Bo rTNF‐a, Bo rIFN‐g, Hu IFN‐a, Hu rIL‐1, Hu rIL‐2—significantly inhibited the in vitro development of trophozoite‐infected cells of three stocks of Theileria annulata and of Theileria parva (Muguga). However, none of these cytokines inhibited the proliferation of established T. annulata or T. parva macroschizont‐infected cell lines. Indeed, Bo rTNF‐a and Hu rIL‐2 consistently enhanced the proliferation of macroschizont‐infected cell lines of both species and the blastogenesis of uninfected lymphocytes in trophozoite‐infected cultures. These results suggest that cytokines could help in resistance to challenge infections by preventing the further development of trophozoite‐infected cells but provide no evidence that any of the above cytokines directly help to resolve primary infections by inhibiting the growth of macroschizont‐infected cells. These findings also suggest that both TNF‐a and IL‐2 could play a role in the pathogenesis of Theileria infections by promoting the proliferation of macroschizont‐infected cells and the associated lymphoid hyperplasia.

Bovine Cells Infected in Vivo with Theileria Annulata Express CD11b, the C3bi Complement Receptor

Forsyth, L.M.G., Jackson, L.A., Wilkie, G., Sanderson, A., Brown, C.G.D. and Preston, P.M., 1997. Bovine cells infected in vivo with Theileria annulata express CD11b, the C3bi complement receptor. Veterinary Research Communications, 21 (4), 249-263Bovine 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.

Haemaphysalis qinghaiensis ticks transmit at least two different Theileria species: one is infective to yaks, one is infective to sheep

Adult Haemaphysalis qinghaiensis ticks from Gannan area, where ovine theileriosis is prevalent but bovine theileriosis has not been reported, transmitted Theileria parasites to yaks and sheep. Blood from the infected yak contained Theileria parasites that were infective to yaks, but not sheep. In contrast, blood from the infected sheep did not contain Theileria parasites infective to yaks. These results suggested that the ticks from the Gannan area were infected with two species of Theileria one which specifically parasitises yaks, one which parasitises sheep. Both infections were more aggressive in splenectomised hosts as compared to intact host. Adult H. qinghaiensis ticks from Zhanjiachuan area, where ovine theileriosis is prevalent but where no yaks are raised, transmitted Theileria parasites to ovine, but not yaks. Taken together these results indicate that H. qinghaiensis ticks may be infected with two different Theileria species. The species infective for sheep could be related to the newly recognised, but not yet named, pathogen recently reported in small ruminants in China. The species infective for yaks appears to be benign and is morphologically similar to parasites of the Theileria sergenti/orientalis/buffeli group.

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

Summary 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.

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‐α) was synthesized when peripheral blood mononuclear cells (PBMC) and purified adherent PBMC from naïve and Theileria annulata‐infected cattle were incubated in vitro with concanavalin A (Con‐A) or bovine recombinant interferon gamma (Bo rIFN‐γ). TNF‐α 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‐γ 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.

Establishment of Theileria‐infected bovine cell lines in scid mice

Summary Bovine cells tranformed by infection with the protozoan parasite Theileria annulata were inoculated subcutaneously or intraperitoneally into C.B.‐17 scid mice. Mice injected subcutaneously developed solid tumours at the injection site, whilst those injected intraperitoneally developed ascites. Schizont‐infected cells were found in other tissues: infected cells spread much more easily from the intraperitoneal site. Karyotyping of cells isolated from tumours showed no evidence of transfer of parasites to murine cells. These results show that the scid mouse can be used as a host for Theileria‐infected bovine cells.