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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.

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.

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.

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.

The sea urchin spicule is composed of CaCO3 associated with an organic matrix containing at least 10 proteins. We have previously shown that one of these proteins, a 50-kDa glycoprotein (SM 50), is encoded by a gene which is only transcribed in cells of the micromere-mesenchyme lineage. In this report we examined RNA from five adult tissues: ovary, coelomocytes, intestine, tube feet, and spine for the presence of SM 50 transcripts and protein. Only RNA from cells of spine and tube feet hybridized to a SM 50-specific probe on Northern blots. These same two tissues undergo biomineralization and contain a protein which is immunologically related to the SM 50 protein. The restricted accumulation of SM 50 transcripts and protein suggests that the differentiation of biomineralizing mesenchyme cells in embryonic and adult tissue may utilize the same genetic program.