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

Attenuated vaccines are an important means of controlling Theileria annulat a infection of cattle. Production is by prolonged cultivation of macroschiz ont-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. S everal groups, including ours, have provided evidence that the population s tructure usually tends to simplify during attenuation. Our data on the T. a nnulata (Ta) Ankara cell line show that attenuation is not necessarily acco mpanied by the population becoming clonal. We have been unable to detect la rge DNA rearrangements. Evidence for alterations in host and parasite gene expression during attenuation is available. With respect to the host we hav e shown that attenuation is accompanied by loss of expression of parasite i nduced matrix metalloproteinases (MMPs). However, in different lines differ ent protease activities are involved. In the T. annulata Ode line we have s hown that 8 activities (including MMP9) are downregulated and that this cor relates with a loss of metastatic behaviour. This has previously been shown in vitro using reconstituted basement membrane (Matrigel(TM)) and is demon strated 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 atte nuation. Re-isolation experiments have shown that the reduction/loss of MMP is a stable transferable trait. A logical extension is that loss of MMP ac tivity land virulence in general) must be at the most fundamental level a g enetic 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 bee n obtained using differential RNA display We view virulence as a multifacto rial phenomenon involving interacting subpopulations of cells and attenuati on 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.