Epidemiology and Control of Bovine Theileriasis

News Box 1. Submission of Minicircle Sequence to GenBank or EMBL We recommend the following standard format for minicircle sequences: • The sequence of whole minicircles must begin with the Universal Minicircle Sequence (GGGGTTGGTGTAA, boxed below). • The sequence of partial minicircles must be orientated in this way such that they can be directly aligned with whole minicircles. • All entries must state whether or not the minicircle sequence is complete. • Once minicircle sequences have been submitted to GenBank or EMBL, please inform Susan Brewster ([email protected]) so that the database can be kept up-to-date. GGGGTTGGTG TAATATAGTG GGCCGCGCAC TCTCGCTGAG GTTAGAACTC GATAAACTAG

60

GCCTCTTAGA GGCTTGATTT TATTATTTCT AAATTTTAAA ATATTATTTT GATTTAAATG

120

ATAAATTAAC TTTAATTTTT ACTTATTAGC TTTGTTAGTG CTAGTTAAAT AATTAAATTT

180

TAATAATTAG TAATAATTAG AATGTTATTT GAATATAATT AATTAGTTTT ATAAATTTAC

240

TATCTTATAC TATCTCTGCG CTACATAGTT AATGGTTGGT AATGATACGT GATAATGAGC

300

ATAATAATAC TAATATAACT AATTAATATA TTATATTGTA GCCATACTAT TACTATGATA

360

TTACTAACAC TACACTGATA ATAGCGCTGA GATAGAACTA TGATATAGAG TTATGATAGT

420

ATTAATATTA TACATCTTAA TTATATATTT AACATCTTTT ATCTTCTCTC TATACGATCT

480

CTGTTCCGTC TCTAGTTTAT GCAGTTCAAT TATACTTCAA TTCATAGTTC AATTCATTAT

540

AATAAATTTA TTTTTAATAA AAATTATTTT TAAATACACG ATGTTACACC GCATTACCTC

600

TCACTATGAT TCATAAAAAT GGGGGAAAAT CGTACTCCCC GACATGCCTC TGGGTAGGGG

660

CGTTCTGCGA AAACCGAAAA ATGGCATACA GAAACCCCGT TCAAAAATCC CCCAAAATTC

720

GCGTTTTTTG GCCTCCCCGT GCACAATTA

740

Minicircle sequence of a complete L. (V.) braziliensis minicircle is shown in the Fig. above; accession number LVMINIB, orientated in the suggested format for new minicircle sequence submissions.

(only 210 sequences) in comparison to the estimated 10000 minicircles in each kinetoplast6. Of these 210 entries, only around one quarter are whole minicircle sequences, many of which have been submitted by our laboratory. Sequences have been added to the database in a number of different orientations, and many do not specify whether or not they are complete sequences, even if they are of whole minicircle size. This makes it difficult to orient sequences in the same way (to use for alignments, for example) since the ends

of linear sequences can only be joined if it is certain that the minicircle is complete. The database was constructed as a starting point towards standardizing the submission of minicircle sequences into GenBank and EMBL (see Box 1). In total, the database contains sequences from 35 species in seven genera within the Kinetoplastida. Other genera are not represented because there are no minicircle sequence data available. Four species stand out as having a large number of sequence entries: Crithidia fasciculata, Leishmania tarentolae, Trypano-

soma cruzi and Trypanosoma brucei. These sequences are almost all guide RNA genes from minicircles, and are not complete minicircle sequences. The minicircle database lists sequences organized by genera, species and, finally, by accession number, which is linked directly to the EMBL entry. The database also contains a brief description and illustrations of the kinetoplast and its DNA, together with links to the pages listed in Table 1, and a list of useful references. We hope the database will be useful for researchers interested in minicircle sequences, and those who want to find out a little more about the kinetoplast and the organization of its DNA. Acknowledgements We thank the Wellcome Trust and the Medical Research Council for financial support. References 1 Vickerman, K. (1976) in Biology of the Kinetoplastida (Lumsden, W.H.R. and Evans, D.A., eds), pp 1–34, Academic Press 2 Kable, M.L., Heidmann, S. and Stuart, K.D. (1997) Trends Biochem. Sci. 22, 162–166 3 Souza, A.E. and Goringer, H.U. (1998) Nucleic Acids Res. 26, 168–169 4 Simpson, L. et al. (1998) Nucleic Acids Res. 26, 170–176 5 de Bruijn, M.H.L. and Barker, D.C. (1992) Acta Trop. 52, 45–58 6 Barker, D.C. (1980) Micron 11, 21–62

Susan Brewster and Douglas C. Barker are at the MRC Outstation of the NIMR, Molteno Laboratory of Parasitology, Department of Pathology, Tennis Court Road, Cambridge, UK CB2 1QP. Martin Aslett is at the European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, UK CB10 1SD. Tel/Fax: +44 1223 333737, e-mail: [email protected]

Epidemiology and Control of Bovine Theileriasis D. Geysen, J. Brandt, S. Geerts and D. Berkvens Antwerp, Belgium December 1997 This meeting focused mainly on Theileria parva and its vector Rhipicephalus appendiculatus, with a major input of interesting data from field projects. Theileria parva An overview of the history of East Coast fever (ECF) was given by T. Dolan (Livestock Services, Nairobi, Kenya). The 438

current status of the disease and its control by immunization through the infection and treatment method was presented by G. Uilenberg (Route du Port, Cargèse, Corsica), with emphasis on the problems encountered in mass immunization campaigns. These campaigns were organized on a large scale in Zambia and on a more limited scale in Zimbabwe and Tanzania. To use available resources more efficiently in the expensive titration experiments of live sporozoite stabilates, a three-stage design

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for titration trials, starting with a wide dose range and few animals per dose followed by a narrow range on more animals, was proposed [L. Duchateau, International Livestock Research Institute (ILRI), Nairobi, Kenya]. Vector Dynamics A population model providing vector distribution, abundance and seasonality, which leads to risk maps for tick-borne diseases, was presented by S. Randolph Parasitology Today, vol. 14, no. 11, 1998

News (University of Oxford, UK). The development of tick survival models is recent, and they show important differences to the known insect models, as a result of three-stage development and overlapping generations, diapausing behaviour and differences in density dependency according to the different stages of the complex life cycle. Disease transmission dynamics and the basic reproductive number, R0, are driven primarily by seasonal tick population variations. R0 is related to vector survival; when the interstadial development period increases from two to seven months, there is a great reduction in R0 (to 1.5% of the original).

In eastern Zambia, which might be considered as a transition zone, R. appendiculatus enters a diapause that is terminated by increasing physiological age of the tick or a weakening of its photoperiodic maintenance. This has not been observed in the vector from equatorial Africa. The humidity requirements of the immature stages are the main limits to the geographical range of the tick. Understanding this diapausing behaviour and the degree of resistance to climatological conditions is the main priority for future research [G. Chaka, Asveza project, Chipata, Zambia; N. Speybroeck, Asveza project, Mazabuka, Zambia; M. Madder, Institute of Tropical Medicine (ITM), Antwerp, Belgium].

Epidemiology Molecular Characterization Epidemiological data from two field studies in different ecological zones in Zambia were discussed by members of the Asveza project, Zambia. In the East, an endemic unstable situation exists mainly as a result of a unimodal rainfall pattern, which restricts the activities of the R. appendiculatus instars, and year-to-year variation in rainfall, which causes fluctuations in tick phenology and T. parva transmission. This determines whether second waves of pronounced activity of adults and nymphs, contributing to more continuous and efficient transmission of T. parva, develop. These second waves do not always occur, but are essential for endemic stability. They also play a key role in the dynamics of prolonged outbreaks in epidemic areas at the edge of the tick–parasite distribution range. Immunizations under these conditions, creating a reservoir of carrier animals, lead to an artificial endemic stability (M. Billiouw, Asveza project, Chipata, Zambia). In Southern Province, data pointed to a disease incidence over a wide age range that was concentrated during the dry season, suggesting the relative importance of larval/nymphal transmission as opposed to the normal nymphal/adult transmission during the rainy season (M. Mulumba, Asveza project, Mazabuka, Zambia). The 5°C difference in the average minimum temperatures between the East and Southern Province strongly influences the ecology of the vector, the secondary generations of larvae occurring one month earlier in the Eastern Province. This fact contributes to an epidemic situation of ECF in Southern Province, but also seems to influence the period of peak incidence. Parasitology Today, vol. 14, no. 11, 1998

Differences between Zambian and exotic T. parva stocks were very pronounced at the molecular level, giving different unrelated restricted fragment length polymorphism (RFLP) profiles with four standard probes on EcoRi digested genomic DNA. Similar results were obtained using RFLP techniques on polymerase chain reaction (PCR) amplified products from four polymorphic loci. The Zambian isolates gave related profiles, still accommodating enough differentiation between eastern and southern stocks on all probes and one marker. They showed a high level of homogeneity, in agreement with results from Zimbabwe, but different from the data from Kenya. With the development of sensitive PCR techniques (in collaboration with ILRI, Nairobi, Kenya), molecular epidemiological data can now be generated from dried blood spots on filter paper. Characterization of field samples from Southern Province indicated that one component of an exotic trivalent vaccine, used on a limited scale until 1993 in Southern Province, has become the dominant strain over a wide area in this province and is causing epidemics with marked mortality (D. Geysen, ITM, Antwerp, Belgium).

Theileria annulata An overview of the epidemiology and control of Theileria annulata was presented by C. Brown [Centre for Tropical Veterinary Medicine (CTVM), University of Edinburgh, UK]. Attenuated cell culture vaccines provide very effective control and are used on a wide scale in North Africa and Asia, yet improvements such as selection of cell lines with low nonspecific T-cell activation are being investigated. By cloning low cytokineproducing cell lines for vaccine production, post-vaccinal reactions could be reduced without affecting immunogenicity (J. Campbell, CTVM, University of Edinburgh, UK). In addition, vaccination alternatives such as SPAG-1, a recombinant subunit sporozoite surface antigen, are under evaluation with variable results. The development of a subunit vaccine strategy is hampered by the lack of fundamental knowledge of the immune response in protozoan infections and seems still some way ahead (N. Boulter, University of York, UK). Insight into parasite attenuation has come from experiments by R. Hall (University of York, UK) and his group (in collaboration with CTVM, University of Edinburgh, UK). They showed that alterations in parasite-induced gene expression of host metalloproteinases (MMPs) accompanied attenuation and is a stable feature. The fact that attenuation is correlated with loss of differentiation into merozoites and piroplasms besides the loss of MMP activity implies that virulence seems to be a multifactorial phenomenon and that there may be many ways to achieve attenuation. B. Shiels (University of Glasgow, Glasgow, UK) and his group have shown that differentiation to merozoites in T. annulata is dependent on the protein versus DNA synthesis rate; this controls the progression towards a commitment point starting the differentiation cycle. The modulation of the timing of differentiation indicated that external regulation might be possible, opening the way for new control opportunities. Theileria orientalis/buffeli/sergenti

Economics of Control Economic evaluation of ECF control in Zambia within the framework of total economic cost showed that immunization reduces the overall cost of ECF and is more efficient than chemotherapy or vector control [L. D’Haese, Rijks Universitair Centrum Antwerpen (RUCA), Antwerp, Belgium; K. Penne, Asveza project, Mazabuka, Zambia].

The taxonomy of the species involved in oriental theileriosis remains unclear. Theileria orientalis (Essex stock; UK) is considered to be identical to Theileria buffeli (Warwick stock; TbW Australia). However, genotypic and phenotypic differences exist between T. buffeli and ‘Theileria sergenti’, the latter being considered as an invalid name for the parasites found in Korea and Japan. Japanese 439

News indigenous cattle are thought to be resistant to ‘T. sergenti’. In exotic breeds, however, it causes anaemia and icterus in Japan and Korea. The geographical limitation of oriental theileriosis may be due to differences in Theileria stocks or factors related to both tick and bovine hosts. Phylogenetic studies, based on p32 and small-subunit ribosomal (ssR) DNA, subdivided T. sergenti into two different types (Ikeda and Chitose), underpinned by serological, pathogenic, vectorial and distribution differences. A p32-based sub-

unit vaccine gave good protection against the Chitose type. A Kenyan parasite (Morula) showed good homology with the Chitose type (C. Sugimoto, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Japan). This session was complemented by the report of a serological survey of theileriosis in West Java, where the parasite showed a close relationship with the Ikeda type of T. sergenti [M. Govaerts, Katolieke Universiteit Leuven (KUL), Leuven, Belgium].

Acknowledgements The International Colloquium on Epidemiology and Control of Bovine Theileriosis, held at the Institute of Tropical Medicine (ITM), Antwerp, Belgium, 10–12 December 1997, was organized by the Veterinary Department of ITM. Dirk Geysen, Jef Brandt, Stanny Geerts and Dirk Berkvens are at the Institute of Tropical Medicine (ITM), Nationalestraat 155, B-2000 Antwerp, Belgium. Tel: +32 3 247 6261, Fax: +32 3 247 6264, e-mail: [email protected]

The Liverpool School of Tropical Medicine: 100 Years of Parasitological Achievement D.H. Molyneux This month, November 1998, the Liverpool School of Tropical Medicine celebrates its centenary. Much of its work has been in the field of parasites, parasitic diseases and insect vectors. This article provides a brief account of the achievements, personalities, historic trends and current activities of the School that have made a significant contribution to parasitology. Changing Approaches: from Expeditions to Collaborations Major advances in the early years of the School stemmed from expeditions that left Liverpool between 1899 and 1914 (listed in Fig. 1); these elucidated the epidemiology, aetiology and transmission of key tropical diseases and outlined control measures. Fundamental discoveries resulted from these expeditions, including: trypanosomes as the cause of sleeping sickness; Anopheles as the vector of Wuchereria bancrofti; the cause and transmission of tick-borne relapsing fever; differentiation of acute and chronic sleeping sickness; recognition of the zoonotic nature of Trypanosoma rhodesiense; and early malaria and yellow fever control recommendations1,2. The expeditions were often hastily organized, underfunded or dependent on benefaction or commission. They relied on the easily available subsidized transportation from Liverpool provided by shipowners, and were the basis for the development of what are now regarded as consultancy services to 440

governments or commercial organizations. Some of the scientists involved in the early development of the School’s parasitology are listed in Box 1. After World War I, the need to develop field stations to replace expeditions led to the building of the Sir Alfred Lewis Jones (the School’s founder) Laboratory in Sierra Leone in 1921. This allowed longer-term studies on parasitic diseases, particularly schistosomiasis and onchocerciasis, but drained the School of resources at home. In 1926, Blacklock discovered the involvement of Simulium damnosum as the vector of Onchocerca volvulus3, and later the classic work on schistosome epidemiology was carried out by Gordon and colleagues4 in Sierra Leone. After World War II, the School recommenced field studies, in Kumba, Cameroon with staff support from the MRC (Medical Research Council) Unit there; the epidemiology and transmission of Loa loa and Paragominus were elucidated (W. Crewe, W.N. Beesley and W.E. Kershaw) and studies on Onchocerca initiated4, leading to Duke’s longterm studies. These field links led to longer-term collaborations in Nigeria, Ghana and Thailand. Laboratory work of the School developed in the Runcorn laboratories in the early 1900s, where a field station allowing large-animal experiments paved the way for the School’s continued interest in veterinary parasitology. Yorke’s group (including J.D. Fulton, E.M. Lourie, A.R.D. Adams and F. Hawking) was installed in the current building after World War I; their studies on the chemotherapy

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of malaria, trypanosomes, Leishmania and spirochaetes resulted in the development of the diamidines, suramin, antimonials and antimalarials5. With ICI, the School was the first to publish research on the activity of proguanil (paludrine) and to describe the ability of malaria parasites to develop drug resistance6. More recent laboratory studies are summarized in Box 2. The School now has active links with four Faculties of the University of Liverpool (Medicine, Veterinary Science, Science, and Social and Environmental Studies), enabling a relatively small institution to benefit from extensive collaboration in a major university. Collaborative activities with overseas institutions, universities and Ministries of Health have been a consistent feature of the School’s work. This has involved the development of institutional and professional links in many countries, but particularly with Mahidol University, Bangkok in the 1960s (creating the Faculty of Tropical Medicine), with the University of Ibadan, Nigeria, and more recently with the University of Malawi College of Medicine, Blantyre, the University of Port Harcourt, Nigeria, the Ministry of Health in Ghana, and several institutions in Latin America. Such studies have been funded by research grants, as well as the judicious use of the School’s funds derived from generous donors and specific benefactions. Approximately half of the School’s annual income is derived from external sources that fund specific research. Parasitology Today, vol. 14, no. 11, 1998