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and domestic ruminants transmitted by ixodid ticks ... schizont stages of certain Theileria species induce a unique, cancer-like, phenotype in infected host leukocytes. Theileria ..... clonal progeny were isolated and in all three progeny isolated ...
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Theileria: intracellular protozoan parasites of wild and domestic ruminants transmitted by ixodid ticks R. BISHOP1*, A. MUSOKE 1, S. MORZARIA 1, M. GARDNER 2 and V. NENE 2 1 2

The International Livestock Research Institute (ILRI), P.O. Box 30709, Nairobi, Kenya The Institute for Genomic Research (TIGR), 9712 Medical Center Drive, Rockville, MD 20850, USA

SUMMARY

Theileria are economically important, intra-cellular protozoa, transmitted by ixodid ticks, which infect wild and domestic ruminants. In the mammalian host, parasites infect leukocytes and erythrocytes. In the arthropod vector they develop in gut epithelial cells and salivary glands. All four intra-cellular stages of Theileria survive free in the cytoplasm. The schizont stages of certain Theileria species induce a unique, cancer-like, phenotype in infected host leukocytes. Theileria undergoes an obligate sexual cycle, involving fusion of gametes in the tick gut, to produce a transiently diploid zygote. The existence of sexual recombination in T. parva has been confirmed in the laboratory, and is presumed to contribute to the extensive polymorphism observed in field isolates. Key parameters in T. parva population dynamics are the relative importance of asymptomatic carrier cattle and animals undergoing severe disease, in transmission of the parasite to ticks, and the extent of transmission by nymphs as compared to adult ticks. Tick populations differ in vector competence for specific T. parva stocks. Recombinant forms of T. parva and T. annulata sporozoite surface antigens induce protection against parasite challenge in cattle. In future, vaccines might be improved by inclusion of tick peptides in multivalent vaccines. Key words: Theileria, intracellular protozoa, sporozoite, ixodid tick, transmission dynamics, recombinant vaccine.

INTRODUCTION

The genus Theileria comprises tick-transmitted sporozoan protozoa that are the causative agents of a variety of disease syndromes in domestic and wild ruminants, and are collectively responsible for economic losses amounting to hundreds of millions of dollars annually in Sub-Saharan Africa and Asia. Theileria are unique among protozoa in that certain species are capable of immortalizing either mammalian lymphocytes or cells of the monocyte/ macrophage lineage that they infect. Theileria are currently classified in the class Sporozoa together with human pathogens, including Plasmodium and Toxoplasma. Along with other Sporozoa, Theileria has been included within a sub-phylum designated the Apicomplexa, based on the common possession of an apical complex containing secretory organelles involved in invasion, or establishment, in the cells of their mammalian and invertebrate hosts. However the evolutionary and functional equivalence of the apical complex between different genera and hence the taxonomic validity of the Apicomplexa remains unclear. Analysis of 18s ribosomal RNA gene sequences demonstrates that the genus Theileria is phylogenetically most closely related to Babesia, a genus of tick-borne protozoans infective to the red

* Corresponding author : Dr Richard Bishop, ILRI, P.O. Box 30709, Nairobi, Kenya. Tel: 254 2 630 743. Fax : 254 2 631 499. E-mail : [email protected]

cells of mammals including domestic livestock, and more distantly to the genus Plasmodium which causes malaria in humans and other species of vertebrates (Allsopp et al. 1994). There are similarities, but also significant differences between features of life cycle, genome organization and mammalian host immune responses to infection when Theileria and Plasmodium are compared. Economically important Theileria species that infect cattle and small ruminants are transmitted by ixodid ticks of the genera Rhipicephalus, Amblyomma, Hyalomma and Haemaphysalis. Theileria species infective to domestic ruminants are summarized in Table 1. Globally the most important species are Theileria parva, transmitted by Rhipicephalus ticks, that causes a rapidly fatal lympho-proliferative disease known as East Coast fever (ECF). The disease was estimated to have been responsible for 170 million US dollars worth of economic loss in 1989 alone (Mukhebi, Perry & Kruska, 1992) and limits introduction of more productive exotic (Bos taurus) cattle in much of eastern, central and southern Africa. The primary host of T. parva is the African cape buffalo (Syncerus caffer) in which the parasite typically does not cause any disease. Theileria annulata, originating from Asian water buffalo (Bulbulus bubulis), and transmitted by several Hyalomma tick species, is responsible for tropical theileriosis from Southern Europe to China, a vast region in which an estimated 250 million cattle are at risk. Both T. parva and T. annulata induce a transformation-like

Parasitology (2004), 129, S271–S283. f 2004 Cambridge University Press DOI: 10.1017/S0031182003004748 Printed in the United Kingdom

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Table 1. Theileria species infective to domestic ruminants, their ixodid tick vectors and geograpical distribution. * Indicates Theileria species infective to small ruminants. Other species are infective to cattle Theileria species T. parva T. annulata

Major ixodid vectors

Known distribution

Rhipicephalus appendiculatus R. zambesiensis R. duttoni Hyalomma anatolicum and other Hyalomma species

Eastern, Central and Southern Africa

Southern Europe Western, Southern and Eastern Asia Northern Africa T. mutans Amblyomma variegatum and Western, Eastern Central four other Amblyomma species and Southern Africa Caribbean islands T. velifera Amblyomma variegatum and Western, Eastern Central and other Amblyomma species Southern Africa Eastern, Central and T. tarurotragi Rhipicephalus appendiculatus Southern Africa R. zambesiensis R. pulchellus T. sergenti Haemaphysalis species Japan and Korea T. buffeli Haemaphysalis species Europe, Asia, Australia, Eastern Africa T. lestoquardi* Hyalomma species Asia and Northern Africa T. ovis* Hyalomma species Asia T. separata* Hyalomma species Asia

phenotype in nucleated mammalian host cells, which is the major cause of pathology. Several other nontransforming Theileria are also responsible for disease in domestic ruminants as a result of anaemia induced by the intra-erythrocytic stage. T. sergenti and T. buffeli cause disease and economic loss in cattle in East Asia. T. mutans may be responsible for disease in cattle in sub-Saharan Africa particularly in mixed infections with other tick-borne pathogens, but the economic impact of this species has yet to be accurately assessed using sensitive and specific diagnostic tests. Theileria lestoquardi is the most pathogenic of several small ruminant Theileria species infective to sheep and goats in Northern Africa and widely across Asia. Most wild bovids in Africa are infected by one or more species of Theileria and the epidemiological situation can be complex with East African cattle potentially being infected with up to five Theileria species at one time (Norval, Perry & Young, 1992). Cattle can be infested with several species of tick and more than one species of Theileria can also be transmitted by the same tick. For example, the morphologically very similar sporozoites of T. parva and the normally non-pathogenic T. taurotragi can both occur together in R. appendiculatus salivary glands, although they can be distinguished by molecular methods based on hybridization to rRNA (Bishop et al. 1994). Additional Theileria species that are infective to livestock, and may possibly be pathogenic in specific circumstances, continue to be discovered. The range of T. buffeli has been extended to Africa (Ngumi et al. 1994), and a hitherto undescribed Theileria that is pathogenic to small ruminants has

been discovered in North Western China (Schnittger et al. 2000). A Theileria species originally described from schizont-infected lymphocyte culture isolates from Cape buffalo (Conrad et al. 1987 ; Allsopp et al. 1993) has recently been found in cattle subjected to challenge by ticks that are presumed to have fed on buffalo (Bishop & Musoke, unpublished data). Theileria infections have also been associated with bovine fatalities in the United States, and the presence of Theileria was confirmed in both Amblyomma americanum and Dermacentor variabilis by PCR amplification using primers derived from small subunit rRNA (SSU rRNA) genes sequences (Chae et al. 1999). The T. parva genome, which is approximately 8.5 Mbp in size, is small for a eukaryote with a complex life cycle involving several distinct intracellular stages in both the arthropod vector and the mammalian host. The genome is divided into 4 chromosomes, containing 33 SfiI fragments (Morzaria & Young, 1992). Both the T. parva (http://www. tigr.org/tdb/e2k1/tpa1/) and T. annulata (http :// www.sanger.ac.uk/Projects/T_annulata/) genome sequences are currently nearing completion. The current status of knowledge relating to the T. parva genome has been reviewed recently (Nene, Morzaria & Bishop, 1998 ; Nene et al. 2000, 2002) and will not be discussed in detail in this article. Most research on Theileria to date has been devoted to the life-cycle stages occurring in the mammalian host, particularly the pathogenic, intra-cellular schizont stages of T. parva and T. annulata, which respectively infect cells of the lymphoid and myeloid lineages and activate

Theileria in the ixodid tick vector

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Fig. 1. Life cycle of Theileria parva in cattle and the ixodid tick vector Rhipicephalus appendiculatus.

host signal transduction pathways thereby inducing cancer-like syndromes in infected mammalian lymphocytes, monocytes and macrophages. The only tick-expressed stage of Theileria for which any molecular information is currently available is the sporozoite, which is secreted from the tick salivary glands into the vertebrate host. This article will concentrate on tick vector-associated aspects of Theileria biology and will review the status of vaccine development based on an antigen isolated from the tick-derived

sporozoite life cycle stage. The data presented primarily relates to T. parva, the species for which most information is currently available. OVERVIEW OF THE THEILERIA PARVA LIFE CYCLE

Theileria parva life-cycle stages in the tick vector and bovine host have been comprehensively described previously and are summarized in Fig. 1.

R. Bishop and others

The three-host brown ear tick Rhiphicephalus appendiculatus is the major vector of the parasite. Theileria parva exhibits a very narrow tick and mammalian host range, and there is no laboratory animal model susceptible to infection with the parasite. Rhipicephalus appendiculatus is classified as a three-host tick because the larvae, nymphs and adults feed on different hosts, which are not necessarily cattle. Transmission is trans-stadial, which means that larval or nymphal instars of the tick acquire infections from a blood-meal, which is then transmitted to a new host, after moulting by nymphs or adults, respectively. Transmission of Theileria has not been shown to be transovarian, unlike the related protozoan Babesia transmitted by the one host tick Boophilus (Norval, Perry & Young, 1992). The Hyalomma vectors of T. annulata are, by contrast, two host ticks, with larval and nymphal instars feeding on the same host. Sporogony takes place in the tick salivary glands in response to a combination of increased temperature and components present within the blood meal. Infective sporozoites are released from about day 3 to day 7 of tick feeding. One infected cell may contain 40–50 000 sporozoites, but the mechanics of tick feeding is believed to result in a trickle release of sporozoites from the salivary glands (Shaw, 2002 ; Shaw & Young, 1994). A tick with a single infected acinar cell can potentially cause severe disease and death. However, the severity of ECF is sporozoite dose dependent and individual animals exhibit different thresholds of susceptibility. Some animals undergo a mild disease reaction and recover due to development of protective cellular immune responses. Once introduced into the host, T. parva sporozoites invade a restricted range of cattle and buffalo cells. In vitro they enter purified B cells or T cells which express receptors which are either CD4+, CD8+, or CD4x/CD8x (Baldwin et al. 1988), and also afferent lymph veiled cells and monocytes (Shaw, Tilney & Mckeever, 1993), although the latter cannot subsequently be immortalised. The entry process is probably energy dependent and is likely to involve specific receptors located on the host cells (Shaw, 1997, 2002). T. annulata infects cells of the monocyte/macrophage lineage, although it can also enter B cells. Other species such as T. taurotragi, originally isolated from eland (Taurotragus oryx), appear to have a much wider host range, at least in vitro (Stagg et al. 1983). Like viruses transmitted by R. appendiculatus (see chapter by Nuttall & Labuda in this Supplement) components of tick saliva appear to promote T. parva pathogen transmission by facilitating sporozoite entry into host leukocytes (Shaw, Tilney & McKeever, 1993), although the mechanism may be an indirect one involving nonspecific activation of host lymphocytes by saliva components. Unlike other sporozoans, T. parva sporozoites are not motile. They do not possess all of

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the components of a typical apical complex as described for other Apicomplexa, and also by contrast with Plasmodium entry into the host cells is not orientation specific, and the rhoptries and microspheres are involved in establishment in the host cell subsequent to entry rather than in the entry process itself (Shaw, Tilney & Musoke, 1991). Unlike other Apicomplexa, T. parva sporozoites also appear to lack morphologically distinguishable micronemes (Shaw, Tilney & Musoke, 1991). The newly internalized parasite is surrounded by a host cell membrane which is rapidly (