Table of Contents Annual report of the International Laboratory for Research on Animal Diseases Table of Contents ILRAD 1987 The International Laboratory for Research on Animal Diseases (ILRAD) was established in 1973 with a mandate to develop effective control measures for livestock diseases which seriously limit world food production. ILRAD's research program focuses on African animal trypanosomiasis and East Coast fever, a form of theileriosis. ILRAD is one of 13 centres in a worldwide agricultural research network sponsored by the Consultative Group on International Agricultural Research (CGIAR). In 1987, funding for ILRAD's essential research and training activities was provided by the World Bank (International Bank for Reconstruction and Development—IBRD), the United Nations Development Program (UNDP), the African Development Bank (ADB), the Rockefeller Foundation and the Governments of Belgium, Canada, Denmark, France, Germany (Federal Republic), India, Italy, Japan, the Netherlands, Norway, Sweden, Switzerland, the United Kingdom (UK) and the United States of America (USA). Additional research activities were supported by special funding arrangements from the European Economic Community (EEC) and the World Health Organization (WHO) and capital funds were provided by the Government of The Netherlands for construction of a new training and ourteach building. All responsibility for views and information expressed in this report remains with ILRAD. The use of trade names does not imply endorsement of any product. The correct citation for this report is: ILRAD (International Laboratory for Research on Animal Diseases). 1988. ILRAD 1987. Nairobi: ILRAD. ISBN 92-9055-087-2. US Library of Congress Catalogue Card Number 82-981264 ILRAD: 1987 Annual Report of the International Laboratory for Research on Animal Diseases file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/toc.htm[5/19/2016 3:34:59 PM] Table of Contents P O BOX 30709 Nairobi, Kenya Published in 1988 in Nairobi, Kenya Compiled and designed by S.B. Westley Cover photo by D. Elsworth Photos by D. Elsworth, P. Webster, M. Shaw Typeset and printed by Waldman Graphics Pennsauken, New Jersey, USA Table of Contents Foreword Theileriosis Epidemiology and experimental immunization Strain characterization Screening with monoclonal antibodies Analysis of parasite proteins Observation of DNA sequences DNA hybridization probes Efforts to obtain Theileria clones Immunization by infection and treatment Reappearance of parasites following treatment with parvaquone Sporozoite immunization: the search for protective antigens Immunization with irradiated sporozoites Bovine immune responses and schizont-infected cells Proliferation of infected cells Immune responses to schizont-infected cells Strain specificity Antigenic changes on infected cells Characterization of bovine cell types Cells types involved in immunity Cell types infected with Theileria Studies on the bovine MHC file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/toc.htm[5/19/2016 3:34:59 PM] Table of Contents Characterization of cattle according to MHC type Elucidation of MHC gene products Mapping the bovine MHC Non-MHC histocompatibility antigens Role of the MHC in immune responses to Theileria Embryo transfer to produce cattle of specified MHC types Exploring vaccine delivery systems Trypanosomiasis Trypanosome biology and biochemistry Changes associated with the trypanosome life cycle Growth and transformation of bloodstream forms Variable surface glycoproteins T vivax surface antigens Aspects of trypanosome metabolism Trypanosome cultivation in vitro Host responses to trypanosome infection Early events in the skin Parasite-host interaction in the bloodstream Control of parasite growth Host antibody production Pathogenic effects of trypanosomiasis Anaemia Endocrine dysfunction Control of infection in Trypanotolerant and susceptible cattle Control of parasitaemia and anaemia Mechanisms of resistance Epidemiology Trypanosome characterization Serological typing Chromosome profiles Use of DNA probes Identification of species-specific proteins Livestock production under trypanosomiasis risk Trypanocidal drugs: detection and testing in vitro Trypanocidal drugs: detection in treated animals Trypanocidal drugs: evaluation of protection African Trypanotolerant Livestock Network Tsetse challenge and trypanosome infection Evaluation of trypanosomiasis control Indications of heritability file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/toc.htm[5/19/2016 3:34:59 PM] Table of Contents Research on N'Dama productivity in The Gambia and Senegal Epidemiology and socio-economics Training and information services Training Information Services The ILRAD library Research support Tsetse laboratory Tick Laboratory Large animal production Laboratory animal production Clinical and Diagnostic Services Biostatistics and Computing Services 1987 publications Board of Directors Staff Financial statements file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/toc.htm[5/19/2016 3:34:59 PM] Foreword Foreword The International Laboratory for Research on Animal Diseases (ILRAD) was established in 1973 with a mandate to conduct intensive research leading to improved control of livestock diseases. ILRAD now occupies a modern complex of research laboratories and support facilities at Kabete on the outskirts of Nairobi, Kenya, and has a cattle breeding ranch at Kapiti, about 50 km from Nairobi. In 1987, ILRAD's full complement of staff included 59 senior scientific and administrative personnel, 24 specialised technicians, 51 technical support staff and 287 in general support categories. Research and training activities concentrate on immunological and related aspects of two diseases which seriously limit food production in Africa and other developing regions of the world—African animal trypanosomiasis and East Coast fever (ECF), a virulent form of theileriosis. Despite intensive research efforts at ILRAD and in other laboratories, the current situation regarding control of ECF and trypanosomiasis is not yet satisfactory. Control of ECF is still based primarily on control of the tick vector by regular acaricide treatment plus pasture management and exclusion of wildlife. Control of trypanosomiasis is largely based on insecticidal control of the tsetse vectors and the use of trypanocidal drugs, both to prevent infection and to treat infected animals. New techniques have been developed to control some tsetse species, but overall the area of Africa infested by tsetse has increased rather than diminished. At the same time, no new trypanocidal drug has been introduced for general use for nearly 30 years and the potential development of drug resistance remains a serious threat. Thus ILRAD continues to work towards developing a practical means of immunization against ECF and improving control measures for trypanosomiasis. As ILRAD has matured, the need has grown for stronger research management to maintain program focus and efficiency in the use of resources. In line with recommendations made in ILRAD's External Program and Management Reviews and accepted by the Board of Directors, research program management has been strengthened by the appointment of Coordinators for the Theileriosis and Trypanosomiasis Research Programs. The Director of Research and the two Program Coordinators now comprise a Research Review Committee which considers program needs, planning, assessment and direction together with the Program Committee of the Board of Directors. ILRAD's Theileriosis Research Program continues to be organized in three project areas. These deal with aspects of epidemiology and the development of vaccines based on antigenic molecules derived from either Theileria sporozoites or schizonts. The Trypanosomiasis Research Program has been partly restructured to give increasing emphasis to research activities which will lead to benefits in the shorter term. Thus, research on trypanosomiasis epidemiology now includes work on the development of diagnostic tests, improved chemotherapy, the prevention or management of drug resistance and the use of trypanotolerant livestock. ILRAD's other trypanosomiasis research activities, with a longer-term perspective, will continue to include basic research on the biochemistry of trypanosomes and on host responses to infection. The goal is to identify points in the cycle of infection which might be susceptible to chemical or immunological intervention. A new project area has also been added to the program concerned with the pathogenesis of infection, particularly the development of anaemia and reproductive disorders. Developments in 1987 included the construction of a new laboratory, changes in the use of three existing laboratories and some regrouping and relocation of scientific staff to relieve congestion and to meet new program needs. ILRAD is now well placed to move into a further phase of productive research on both theileriosis and trypanosomiasis. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Foreword.htm[5/19/2016 3:34:59 PM] Foreword The results of ILRAD's research and training activities in 1987 comprise the bulk of this report and need not be detailed here. However some notable achievements from the research programs may be mentioned. In the theileriosis program, field trials to test the efficacy of immunization by infection with Theileria sporozoites plus antibiotic treatment have been completed successfully in Kenya and are now in progress in Tanzania and Zimbabwe. A well- characterized strain of Theileria parva has been made available to the Kenya Agricultural Research Institute for field evaluation in infection and treatment immunization in Kenya's Coast Province. Parasite stocks are being characterized using monoclonal antibodies and recombinant DNA (deoxyribonucleic acid) technology and substantial progress has been made in the development of techniques for preparing genetically homogeneous Theileria populations. In the search for T parva antigens which might be used as the basis of a vaccine, several candidate molecules have been identified in the sporozoite form of the parasite and a protein associated with the schizont stage of parasite development has been recognized by monoclonal antibodies. Scientists are now characterizing the genes which encode these molecules. Techniques which allow the transfer of genes from one cell to another have been introduced at ILRAD, primarily to facilitate research on immune mechanisms in theileriosis. Studies on the bovine immune system are a basic requirement for research on both theileriosis and trypanosomiasis and have wider relevance for studies on other diseases. Monoclonal antibodies were prepared in 1987 which recognize subsets of bovine B lymphocytes and macrophages to supplement reagents identified in previous years which characterize subsets of bovine T lymphocytes. The bovine major histocompatability complex (MHC) plays a fundamental role in the generation of immune responses. The emphasis in ILRAD's research on the MHC has moved from definition of class I molecules to class II molecules during the year. Current work on the MHC in ruminants, rodents and man was reviewed at an international workshop held at ILRAD in September. This meeting of specialists from many laboratories has led to new collaborative research projects on the bovine MHC. Research on the biology and biochemistry of trypanosomes emphasizes mechanisms of antigenic variation and development through different life cycle stages which are essential for parasite survival. Particular attention has been given to the ingestion, metabolism and transport of protein molecules and to the cell organelles involved in these processes which could provide targets for immune or chemical attack. Research continues on the nature of trypanotolerance in cattle. Experiments at ILRAD have clearly demonstrated the superior ability of N'Dama cattle to control parasite numbers in the bloodstream and to resist anaemia during repeated trypanosome infections. The role of immunological memory in this superior resistance is now being investigated. Additional N'Dama calves are being produced at ILRAD by embryo transfer. In 1987, 31 embryos recovered from 5 N'Dama heifers were implanted in Boran foster mothers, resulting in 22 pregnancies and 11 births. Scientists are developing better methods for diagnosing trypanosome infection using monoclonal antibodies and DNA probes. Attention focuses on simplifying the technology for more widespread use in the field, particularly to identify species such as Trypanosoma congolense and T simiae which are difficult to distinguish by conventional techniques. To support improvement in trypanosomiasis control by drug treatment, scientists are using trypanosome culture techniques developed earlier to assess the drug sensitivity of different trypanosome stocks. New methods are also being developed to measure the levels of trypanocidal drugs present in treated animals. The trypanosomiasis epidemiology program includes participation in the African file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Foreword.htm[5/19/2016 3:34:59 PM] Foreword Trypanotolerant Livestock Network covering sites in 9 countries. ILRAD is collaborating with the International Livestock Centre for Africa (ILCA) and national livestock ministries to determine how to maximize the productivity of N'Dama and other trypanotolerant livestock under a variety of conditions. One Network project involves typing N'Dama cattle from Zaire and The Gambia according to class I MHC antigens in order to identify genetic markers which may predict inherent resistance to trypanosome infection. An international conference was held at ILRAD in 1987 to review research findings and consider the future direction of Network activities. During 1987, a new Epidemiology and Socio-economics Unit was established at ILRAD. Its goal is to assess the impact of trypanosomiasis and ECF in selected areas of Africa and to evaluate the potential economic, social and environmental effects of improved disease control. Initial emphasis will be on evaluating the impact of infection and treatment immunization against ECF. A total of 13 scientists, veterinarians and senior technicians came to ILRAD in 1987 for training attachments or to broaden their research experience. All were staff members of univerisities or national veterinary research laboratories and most came from African countries. In addition, four courses were held during the year, attracting a total of 44 participants. Fifteen university graduates from African countries worked at ILRAD in 1987 as Research Fellows on projects leading to an M.Sc. or Ph.D. degree and two Senior Research Fellows came to ILRAD from universities in East Africa to undertake 1-year research projects. During 1987, ILRAD prepared proposals for research and training programs for the 5-year period from 1988 to 1992. These medium-term proposals took into account consideration of ILRAD's short-term and long-term research strategies and the recommendations of the External Program and Management Reviews. They were approved by the Technical Advisory Committee of the Consultative Group on International Agricultural Research (CGIAR) and recommended to the donors for funding. The CGIAR's acceptance of research planning on a 5-year basis is a welcome development, since it eases the burden of detailed annual program and budget reviews by the Technical Advisory Committee and recognizes the long-term nature of many aspects of basic research. In June 1987, ILRAD hosted the mid-year meeting of the Technical Advisory Committee and the Directors of the other 12 international agricultural research centres supported by the CGIAR. ILRAD scientists participate actively in a number of collaborative research and training projects with national and international organizations in African countries affected by trypanosomiasis and ECF. Collaborative activities in 1987 included participation in the biennial meeting of the Organization of African Unity's International Scientific Council for Trypanosomiasis Research and Control (OAU/ ISCTRC) held in Togo and in a review of the Food and Agriculture Organization of the United Nations' (FAO) Program for the Control of Tick-borne Diseases, covering activities in Zimbabwe, Malawi, Tanzania, Burundi, Uganda and Ethiopia. In concluding this foreword, I would like to express appreciation to the Board of Directors, the scientific and supporting staff and the donor organizations and nations who collectively support ILRAD's research and training activities. Three members of the Board of Directors retired in 1987. These were Dr K. Warren, Dr C. L'Ecuyer and Dr G. Kazyumba. Three new members joined the Board—Dr P. Englund of the Johns Hopkins University School of Medicine (United States of America—USA), Professor I. Maansson of the Swedish University of Agricultural Sciences and Dr A.S. Sidibe of the OAU's InterAfrican Bureau for Animal Resources (IBAR) in Mali. Professor Hans Jahnke was elected Chairman of the Board of Directors. We acknowledge with gratitude the interest and financial support ILRAD receives from many file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Foreword.htm[5/19/2016 3:34:59 PM] Foreword countries and organizations. In 1987, ILRAD continued to receive core funding from the World Bank (International Bank for Reconstruction and Development—IBRD), the United Nations Development Program (UNDP), the African Development Bank (ADB), the Rockefeller Foundation and the governments of Belgium, Canada, Denmark, France, Germany (FR), Italy, Japan, the Netherlands, Norway, Sweden, Switzerland, the United Kingdom (UK) and the USA. The Government of India added its support during the year. Additional research activities were supported by the European Economic Community (EEC) and the World Health Organization (WHO). In 1987, ILRAD was honoured to receive visits from a number of important guests. These included Ms W. Demeester, the Belgian Secretary of State for Public Health, the Honourable P. Nvue, Minister of Agriculture for the Equatoria Region of Sudan, Dr E. Schuh, Director of Agriculture of the World Bank, Professor J. Di Bioggio, President of Michigan State University (USA), Professor W. Lavery, President of Virginia College of Science and Technology (USA), Dr D. Acker and Dr W. Furtick, Directors of Agriculture of the United States Agency for International Development (USAID), Dr R. Manning, Deputy Director General of the Australian Development Assistance Bureau, Mr A. Bennett, Chief Natural Resources Adviser to the Overseas Development Administration (UK), and Dr W. Dietel, Chairman of Winrock International (USA). Several important visitors from the Government of Kenya came to ILRAD during the year. These included the Honourable W.O. Omamo, Minister for Research, Science and Technology, the Honourable K. Mwendwa, Minister for Livestock Development, the Honourable R. Oyondi, Assistant Minister for Livestock Development and Mr J. Adamba and Mr D. Mbiti, Permanent Secretaries in the Ministry of Livestock Development. ILRAD received visits from Their Excellencies, the Ambassadors of Denmark, Italy, Japan, The Netherlands and the USA, and the High Commissioners of Australia, Canada and the UK. Dr S. Sinding, Director of the USAID Mission in Kenya, also visited ILRAD. Finally, it is always a pleasure to acknowledge the support ILRAD has received through the years from the people and Government of Kenya. We look forward to the continuation of this warm relationship with our Kenyan hosts. A.R. Gray Director General ILRAD file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Foreword.htm[5/19/2016 3:34:59 PM] Theileriosis Theileriosis Epidemiology and experimental immunization Strain characterization Screening with monoclonal antibodies Analysis of parasite proteins Observation of DNA sequences DNA hybridization probes Efforts to obtain Theileria clones Immunization by infection and treatment Reappearance of parasites following treatment with parvaquone Sporozoite immunization: the search for protective antigens Immunization with irradiated sporozoites Bovine immune responses and schizont-infected cells Proliferation of infected cells Immune responses to schizont-infected cells Strain specificity Antigenic changes on infected cells Characterization of bovine cell types Cells types involved in immunity Cell types infected with Theileria Studies on the bovine MHC Characterization of cattle according to MHC type Elucidation of MHC gene products Mapping the bovine MHC Non-MHC histocompatibility antigens Role of the MHC in immune responses to Theileria Embryo transfer to produce cattle of specified MHC types Exploring vaccine delivery systems Theileria is a genus of tick-transmitted protozoan parasites infecting wild and domestic animals in many parts of the world. In East and Central Africa, the most important species—Theileria parva—represents a major constraint on the development of beef and dairy production. Theileria annulata causes disease in cattle over a broad area, extending from the Mediterranean littoral to the Middle East, India, Southern Russia and Asia, while Theileria orientalis affects cattle in the Far East. In the Middle East, Theileria hirci causes disease in sheep and goats. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis Theileria parva causes a virulent form of theileriosis that threatens some 25 million cattle in Kenya, Tanzania, Uganda, southern Sudan, Rwanda, Burundi, Zaire, Malawi, Zambia, Zimbabwe and Mozambique. Three forms of the disease occur in the region. For convenience, these are described by distinguishing three subspecies of the parasite. The term East Coast fever—ECF—is used to describe the classical disease caused by Theileria parva parva, which is transmitted between cattle by the brown ear tick, Rhipicephalus appendiculatus. Corridor disease is caused by T p lawrencei, transmitted by the same tick to cattle from buffalo (Syncerus caffer). A milder form of theileriosis is caused by T p bovis, also transmitted by the tick Rhipicephalus appendiculatus. All these parasites have a complex life cycle in the mammalian host and the arthropod vector. R appendiculatus is a three-host tick, feeding on separate animals as a larva, a nymph and an adult. The parasites are transmitted most commonly when ticks feed on infected animals as nymphs and then on susceptible cattle as adults. Theileria parasites pass through a sexual stage in the gut of the tick and enter the salivary glands where they form an elaborate intracellular sporoblast. During tick feeding, the sporoblast gives rise to 30,000 to 50,000 infective sporozoites. These are injected into cattle in tick saliva. In cattle, they attach to and enter lymphocytes, white blood cells of the immune system. Within the lymphocytes, the sporozoites develop into schizonts and the infected lymphocytes are transformed into enlarged lymphoblasts which multiply together with the schizonts, resulting in a rapidly expanding population of parasitized cells. The final stage of disease is characterized by large-scale cell destruction, often leading to death. During the course of infection, some of the Theileria schizonts differentiate into merozoites which are released from the lymphocytes into the bloodstream. These invade red blood cells and develop into piroplasms. Ticks become infected when they ingest red blood cells containing piroplasms, initiating a new cycle of parasite development. ECF is controlled principally by dipping or spraying cattle with acaricides to kill the tick vectors. However, frequent dipping or spraying is expensive, and cattle remain fully susceptible to disease if there is any interruption of the acaricide regime. Tick resistance to the available acaricides is also a problem and most acaricides are environmentally damaging. Two curative drugs have been developed for the treatment of ECF, but they are expensive and theileriosis must be diagnosed at an early stage for treatment to be most effective. Thus, there is an urgent need for alternative methods of ECF control. At the same time, cattle which recover from ECF show long-lasting immunity, suggesting that there are good prospects for controlling the disease by immunization. However, there is antigenic diversity among theilerial strains and immunity against one strain does not necessarily protect cattle against another. The total number of strains is unknown, but it may be limited, and certain isolates or combinations of isolates appear to provide a broad protection. ILRAD places particular emphasis on improving existing methods of immunization by infection and treatment and on developing new approaches to immunization against ECF. Epidemiology and experimental immunization Experimental procedures for immunization against ECF were initiated in southern Africa and further developed at the former East African Veterinary Research Organization in Kenya. These are based on infection with T parva sporozoites and simultaneous treatment with tetracycline antibiotics. This method offers the most practical and effective form of immunization currently available and it is being tested in extended field trials in several countries. The disadvantages are that infection with live parasites must be carefully controlled and protection may not be achieved in areas where immunologically unrelated strains of the parasite are present. Also, cattle may become carriers of the parasites used for immunization. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis The most urgent requirement for a better understanding of ECF epidemiology and for improving and extending control by infection and treatment is a simple and reliable method for characterizing parasite strains. The primary method available at present is cross-immunity testing in cattle, but this approach is time consuming and expensive and the results are not always free of ambiguity. For this reason, scientists at ILRAD are exploring several in vitro methods of parasite characterization. Field immunization trials were initiated in 1986 in Tanzania and Zimbabwe, using locally isolated parasites. In combination with earlier results from Kenya and elsewhere, this work should provide important information on the effectiveness of infection and treatment immunization in a variety of epidemiological situations. Strain characterization Cattle which are immune to one population of Theileria may not resist infection with another, indicating that different, immunologically distinct, groups of parasites exist in the field. If a vaccine against ECF is to protect cattle against infection, it must stimulate immunity against the different theilerial parasites which the animals are likely to encounter. Thus, in order to develop an effective ECF vaccine, there is an urgent requirement for practicable and reliable laboratory methods to categorize Theileria parasites into antigenic groups and elucidate their cross-protective potential in cattle. Regional organizations and national governments in Eastern and Southern Africa also need improved methods of parasite characterization in order to define the patterns of ECF infection in their countries and to assess current control programs. A formal system of parasite characterization is being developed at ILRAD based on a systematic examination of several different techniques. This work has focused primarily on six Theileria stocks which have already been relatively well characterized—T p parva Muguga, Mariakani and Marikebuni from Kenya, T p parva Uganda, T p bovis Boleni from Zimbabwe and T p lawrencei 7014/Laikipia from Kenya. Bulk stabilates of each stock have been prepared from ground-up infected ticks and from dissected tick salivary glands. These have all been tested for infectivity. A standard battery of bovine cells has been infected with each stock and cell lines established so that all parasites may be tested against a common host-cell background. Cloned infected cell lines have been established for many of these stocks by limiting dilution of schizont-infected cells. These stocks are now being analysed by monoclonal antibody screening, protein analysis and two methods of DNA characterization. Work in this area also focuses on developing techniques to obtain homogeneous populations of Theileria originating from cloned sporozoites. In addition, cells infected with each of the parasite stocks selected for study are being examined for sensitivity to titrated doses of the drugs available to treat ECF—oxytetracycline, parvaquone (Clexon: Wellcome), buparvaquone (Butalex: Wellcome) and halofuginone (Terit: Hoechst). Finally, randomized groups of cattle will each be immunized with a different one of the six selected bulk stocks and challenged with the other five. Results will be analysed to determine any relationships or differences between the stocks. in terms of cross-protection. By the end of 1988, it should be possible to compare the results of all the studies reported here and to identify any correlations among the different methods of characterization. Screening with monoclonal antibodies A great deal of work on the characterization of Theileria stocks in vitro has been based on the recognition of parasite antigens by monoclonal antibodies, detected by the indirect fluorescent file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis antibody (IFA) test. Monoclonal antibodies are produced from hybrid cells formed by the fusion of mouse spleen cells, primed to produce specific antibodies, and mouse tumour cells capable of growing and multiplying in vitro. Whereas sera from infected cattle can be expected to respond to a wide range of parasite antigens, monoclonal antibodies are useful for distinguishing parasite stocks because each one recognizes a single antigenic determinant. The antigens recognized by monoclonal antibodies are not necessarily responsible for stimulating immunity in cattle, and thus patterns of monoclonal antibody response may not correspond with cross-protection patterns in vivo. However, monoclonal antibody responses are consistent when a parasite stock is used to infect cells from different cattle or after passage through ticks, indicating that the antigens recognized by monoclonal antibodies are a fixed character of the parasites. The monoclonal antibodies raised at ILRAD against Theileria sporozoites appear to detect surface antigen(s) common to many different parasite stocks, suggesting that a sporozoite vaccine might confer broad protection. By contrast, some monoclonal antibodies raised against schizont-infected cells detect antigens specific to different parasite stocks, making them useful reagents for detecting antigenic differences that might define Theileria strains. Originally, a panel of 20 anti-schizont monoclonal antibodies was used to screen hundreds of schizont-infected cell lines isolated throughout the ECF endemic region. As additional monoclonal antibodies were raised and more isolates were screened, the patterns of monoclonal antibody response appeared more and more complex. Recent work in this area has focused primarily on T p lawrencei because parasites isolated from buffalo show extensive antigenic diversity and, perhaps for this reason, immunity sometimes breaks down when cattle are exposed to buffalo-derived parasites in the field. These studies have suggested that buffalo may be infected simultaneously with several antigenically distinct Theileria populations. Work in 1987 concentrated on identifying any antigenic characteristics which might be used to distinguish T p lawrencei, T p parva and T p bovis. Seven new monoclonal antibodies were raised against T p lawrencei and used to screen five of ILRAD's reference stocks plus parasites derived from buffalo, but none was specific for T p lawrencei, that is recognizing antigens on all the buffalo-derived parasites and on none of the others. Analysis of parasite proteins The analysis of Theileria proteins by two-dimensional gel electrophoresis is being investigated as a means of distinguishing parasite species, subspecies and strains. Another objective is to identify changes in host-cell proteins which may be associated with Theileria infection. Parasite schizonts were purified from cloned and uncloned cell lines infected with different Theileria stocks. These were radiolabeled with 35S-methionine, which labels the majority of proteins, and the proteins were separated in polyacrylamide gels according to electrical charge and size. Using this technique, minor differences were observed between ILRAD's bulk stabilates of T p parva Muguga, Marikebuni and Mariakani (Figure 1). T p bovis Boleni and the 7014/Laikipia stock of T p lawrencei were indistinguishable from the T p parva stocks, but another stock of T p lawrencei (IC11/E8) showed marked differences. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis Figure 1. Theileria schizonts were purified from infected cells which had been radiolabeled with 35S-methionine. When the purified schizonts were subjected to two-dimensional gel electrophoresis, more than 200 parasite proteins could be identified. A comparison of two T p parva stocks—Muguga and Marikebun I—revealed differences in several proteins, as indicated by the arrowheads. A comparison of proteins from T parva infected and uninfected bovine cells using different types of radiolabeling revealed some interesting differences. Using 35S-methionine, a basic protein of 14,000 daltons was identified which was specific for cells infected with two different stocks. When another radiolabel was used specifically to identify glycoproteins (protein- carbohydrate compounds), marked differences were observed between uninfected and infected cells, with the appearance of some glycoproteins and disappearance of others after infection. Using a third radiolabel which is specific for cell surface proteins, an acidic protein was detected which occurred only on the surface of infected cells. Present work in this area includes biochemical studies of infection-specific proteins plus further analysis of proteins from different Theileria stocks. Observation of DNA sequences Scientists are looking for differences in DNA sequences which might be used to identify specific Theileria strains using gel electrophoresis. Theileria DNA is cleaved into fragments using restriction enzymes and the fragments are applied to one end of an agarose gel. When electrical charges are passed through the gel, the DNA moves towards the other end. Because smaller DNA fragments move through the gel more quickly than larger ones, a pattern emerges, called a genomic profile, with fragments of different sizes at different positions along the gel. Digestion of DNA from T p parva Muguga by the restriction enzyme Sfi 1 resulted in approximately 28 fragments ranging in size from 1 million to 40,000 base pairs. Comparison among different stabilates of T p parva Muguga showed no differences in genomic profiles, suggesting that the parasites had remained genetically stable following passage through cattle file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis and ticks. When genomic profiles were compared using DNA from different T p parva stocks and from an isolate of another Theileria species, T mutans, there were detectable differences in the number and size of fragments in each stock (Figure 2). A second restriction enzyme, Not 1, cleaved T p parva Uganda, Marikebuni and Kibarani DNA into four fragments and T p parva Muguga DNA into five. Cleavage with both restriction enzymes revealed clear differences between T mutans and all the T p parva stocks examined. These differences remained constant whether DNA was prepared from the sporozoite, schizont or piroplasm stage of the parasite life cycle. This technique appears promising for the identification of Theileria stocks and species, but it is not yet clear whether it can be used successfully to identify potentially cross-protective strains. Figure 2. DNA from five T p parva stocks and one T mutans isolate was digested with the restriction enzyme Sfi 1 and analysed using contour-clamped homogeneous electric field gel electrophoresis. The sizes of different fragments were estimated in 1000 base pairs (kb). No differences could be observed among three different stabilates of T p parva Muguga (lanes 1- 3), but differences were detected among different T p parva stocks: Muguga, Pemba/Mnarani (lane 4), Mariakani (lane 5), Uganda (lane 6) and Kibarani (lane 7). There were major differences between all these stocks and the isolate of T mutans (lane 8). DNA hybridization probes Studies were initiated in 1985 to identify DNA sequences which could be used as probes to distinguish T parva subspecies and strains. Three clones were produced 1986 containing segments of T p parva Muguga piroplasm DNA which were present as multiple copies in the parasite genome. In 1987, additional DNA clones were prepared from T p parva Mariakani piroplasms. The T p parva Muguga DNA clones were used successfully as probes to distinguish stocks of T p parva, T p bovis and T p lawrencei in preparations of schizont- infected cells or purified piroplasms. None of the probes hybridized with DNA from T mutans, Theileria taurotragi or Theileria annulata, suggesting that they were species specific. Detailed analysis with these DNA probes has suggested the presence of mixed parasite populations in file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis some of the stocks tested. Experiments were conducted in 1987 to identify any genotypic characteristics which might be used to distinguish T p lawrencei from T p parva. Two of ILRAD's T p parva Muguga DNA probes were used to screen DNA prepared from three infected buffalo and from seven cattle infected with Theileria sporozoites derived from one of the buffalo. The DNA probes revealed considerable variation in the parasites isolated from the cattle (Figure 3) and none of the cattle isolates showed the same DNA hybridization patterns as those obtained from the buffalo, suggesting either that minor parasite populations from the buffalo had been selected and had established infection in the cattle or that a genetic rearrangement had occurred during passage of the parasites from buffalo to cattle. None of the buffalo-derived parasites used in this study showed any specific phenotypic or genotypic characteristics—detectable with ILRAD's existing monoclonal antibodies or DNA probes—which would distinguish them from T p parva. These results confirmed evidence from several other studies suggesting that the classification of Theileria parva parasites into three subspecies, though convenient for workers in the field, may be without a sound genetic basis. Figure 3. Seven cattle were infected with sporozoite stabilates prepared from ticks which fed on a Theileria-infected buffalo. Infected cell lines were isolated from the lymph nodes of the cattle and parasite DNA from these cells was split into fragments with a restriction enzyme. This autoradiogram shows the hybridization of one of ILRAD's T p parva Muguga DNA probes (1gTpM-23) with Theileria DNA derived from the seven cattle. Fragment sizes are indicated in 1000 base pairs (kb). The hybridization patterns reveal considerable genomic diversity. In 1987, ILRAD scientists also tested whether infections with different Theileria stocks could be detected in tick salivary glands using the DNA probes developed to detect parasites in bovine cells. Parasites were detected with two of ILRAD's DNA probes in as few as one infected acinus from the salivary glands of infected ticks. Infections were detected in more than 90% of the ticks which had been shown to be infected by light microscopy, indicating that ILRAD's DNA probes were highly sensitive. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis Future plans include using ILRAD's T p parva DNA probes to examine ticks infected with other species of Theileria. Work is also in progress using DNA probes to investigate the possibility of selection or genetic rearrangement during parasite development in the bovine host, the tick vector or in culture. In addition to their potential use in ECF epidemiological studies, Theileria DNA probes may eventually provide information on evolutionary relationships between different T parva stocks. Efforts to obtain Theileria clones There is an urgent requirement for cloned populations of Theileria derived unequivocally from single parasites. The presence of mixed parasite populations within many of the Theileria stocks currently available complicates the interpretation of cross-protection experiments. If cloned parasites can be produced which stimulate protection, these would provide more standardized material for cross-immunity trials and field immunization programs and would greatly simplify the analysis of breakthrough infections. Cloned parasites are also required to explore the possible role of genetic recombination in the antigenic diversity observed in Theileria parasites. As mentioned earlier, lymphoid cells from cattle or buffalo are readily infected with Theileria sporozoites and the resultant infected cell lines subsequently cloned in vitro. However, the schizonts contained in these cloned cells are not necessarily homogeneous cloned populations because single host cells may have been infected initially by more than one sporozoite. To overcome this problem, lymphocytes were infected in vitro with sporozoites titrated to less than 100 sporozoites per 1 million bovine cells. The resultant infected cell lines were then cloned twice by limiting dilution and these clones were used to infect the cattle from which the cells were originally derived. In most instances, infections with these cloned cells were mild, producing low numbers of Theileria piroplasms. However, parasites were successfully picked up by feeding ticks and prepared as stabilates and their infectivity is now being tested. These stabilates will be used for immunological and characterization studies and to examine for genetic recombination of the parasites in ticks. Immunization by infection and treatment A collaborative project was initiated in 1984 between the Tanzanian Government, the Irish Government, FAO and ILRAD to test ECF immunization by infection and treatment on Zanzibar and Pemba islands in Tanzania. Theileria parasites were isolated from both islands and two stocks—Zanzibar South and Pemba/Mnarani—were characterized which appeared suitable for use in immunization trials. The first field trials were conducted on Zanzibar and Pemba in late 1986. These involved infecting Jersey (Bos taurus) dairy cattle on each island with the locally isolated parasite stock and treating with oxytetracycline. Cattle on Zanzibar were immunized safely by this procedure using two doses of the drug and resisted subsequent ECF challenge, but the majority of cattle immunized on Pemba developed severe ECF reactions following immunization using a single dose of oxytetracycline. The situation on both islands was complicated by the presence of several other tickborne diseases. Two new procedures were tested at ILRAD using Friesian (Bos taurus) cattle and the Pemba/Mnarani stock. These involved two treatments with oxytetracycline—at the time of infection and 4 days later—or one treatment with buparvaquone at the time of infection. Both procedures were effective in controlling the immunizing infection and conferred resistance to subsequent challenge with the same stock. Further experiments will test immunization against the Zanzibar South stock using one dose of file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis buparvaquone at the time of infection. Both Theileria stocks will also be tested to determine whether calves below 1 month of age can be immunized successfully. Based on these results, the Tanzanian Government will be advised on the feasibility of using these stocks for ECF immunization programs on Zanzibar and Pemba islands. Reappearance of parasites following treatment with parvaquone Treatment of bovine cell lines infected with T p parva Muguga with a high dose (10µg/ml) of parvaquone resulted in the disappearance of schizonts and death of the cells within a few days. However, when treated cells were cultured with T-cell growth factor, they continued to multiply and in over half the cultures Theileria schizonts reappeared several days later. Schizonts also reappeared when treated cells were cultured with the growth factor human recombinant interleukin-2 (IL-2) or with supernatant collected from a Theileria-infected bovine cell line which is known to produce IL-2-like growth factors. Cultures in which schizonts reappeared eventually became completely parasitized and grew normally in the absence of growth factors. Before the reappearance of schizonts in the treated cells, inclusion bodies were observed in the cell cytoplasm (Figure 4). These became increasingly dense over time, often appearing as circles or commas within cell vacuoles. The inclusion bodies appeared in all cell cultures, including those in which schizonts did not reappear. They consisted of DNA and were recognized by a Theileria-specific DNA probe but not by a schizont-specific monoclonal antibody. This phenomenon provides a potential explanation for the establishment of carrier infections in cattle treated with theileriacidal drugs. Figure 4. Giemsa-stained cytocentrifuge smears showing the development of intracytoplasmic inclusions and the formation of schizonts in parvaquone-treated, T p parva Muguga-infected file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis cells cultured in T-cell growth factor: (A) untreated cells with normal schizont morphology, (B) small inclusions observed 7 days after treatment, (C) densely staining inclusions 11 days after treatment and (D) reforming schizonts and schizonts with normal morphology 16 days after treatment. Sporozoite immunization: the search for protective antigens Cattle which survive in ECF endemic areas appear to develop a degree of immunity to Theileria infection. Immune responses in these animals include both antibody reponses against the sporozoite form of the parasite and cytolytic (cell-killing) responses against cells infected with Theileria schizonts. Antibodies raised against sporozoites of one T parva stock recognize parasites of other stocks, suggesting that anti-sporozoite antibodies might offer broad protection against ECF infection. Sera from cattle exposed repeatedly to killed sporozoites contain antibodies against 11 major sporozoite antigens. Considerable research at ILRAD focuses on identifying and characterizing these antigens and determining whether they might be used to stimulate protective immune responses in cattle. The objective is to produce sporozoite antigens by expression of the appropriate genes and to test them to determine their potential value in an ECF vaccine. One of these antigens, with an approximate molecular mass of 67,000 daltons, is a glycoprotein, with antibody responses apparently stimulated by the protein moiety. Results from several experiments suggest that it contains at least two sets of amino acids, called epitopes, which stimulate the production of neutralizing antibodies. Out of a panel of 17 monoclonal antibodies raised against the 67,000-dalton protein, two appear to recognize different epitopes. Used in combination, they neutralize sporozoites better than when used singly. The identification of more than one protective epitope will be important in the design of a peptide vaccine. The purified 67,000-dalton protein was used in 1987 to raise monospecific antisera in rats. The antisera recognized the 67,000-dalton protein and also recognized the surface of live sporozoites and neutralized their infectivity. These results confirm earlier findings that the 67,000-dalton protein is located on the surface of the sporozoite and that it is responsible for stimulating neutralizing antibodies. Further studies were conducted in collaboration with Wistar Institute (USA) to determine the total amino acid composition of the 67,000-dalton protein. The enzyme trypsin was used to break the protein down into peptides, these were separated by high-performance liquid chromatography (HPLC) and their amino acid composition and sequences were analysed. Six peptide sequences have now been determined. In order to identify the genes which encode protective sporozoite antigens, both genomic and cDNA (copy DNA) libraries have been constructed and screened with antisera from cattle immunized with killed sporozoites. A cDNA clone was obtained which contained a 207 base pair insert from which an amino acid sequence could be inferred. This sequence was identical to the sequence of 24 amino acids contained in one of the peptides recently obtained in collaboration with the Wistar Institute. The cDNA clone hybridized to DNA from piroplasms and/or schizonts of T p parva Muguga, Marikebuni, Mariakani, Pemba/Mnarani and Uganda, T p lawrencei and T p bovis, but not to DNA from T annulata, T mutans or T taurotragi. This suggests that the gene is specific to the species T parva but not to particular subspecies or stocks. Work now focuses on obtaining the entire sequence of the gene that encodes the 67,000-dalton antigen. The next step will be an attempt to produce this antigen as an expressed product of the gene for vaccination trials. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis Another approach to the identification of genes encoding sporozoite antigens began with total genomic DNA isolated from T p parva Muguga piroplasms. This was broken up by shearing and the fragments inserted in the lambda gt 11 expression vector. When the resulting library of cloned DNA fragments was screened with serum from a sporozoite-immunized cow, three clones were identified that encoded different, unique sets of antigenic determinants. All of the other Theileria-specific antigens identified on Western blots appear to be derived from these three gene products. This suggests that the three cloned sequences may encode three different proteins that exhibit most, if not all, of the major antigenic determinants found on or inside the sporozoite. Determination of the DNA sequence of one of these clones has shown that it contains the entire coding region for a protein with a molecular weight of approximately 100,000 daltons as well as some sequences preceding the gene that may represent a theilerial control or promoter region. The presumed amino-acid sequence exhibits characteristics suggesting that this protein could be a membrane component, though it has not yet been possible to determine the location of the antigen on the surface or inside the sporozoite. The regions of the gene that react with the anti-sporozoite antiserum have been subcloned and expressed and the resulting peptides have been used to immunize cattle. Experiments are now in progress to try to express the entire antigen in sufficient quantities for use in immunization trials. Immunization with irradiated sporozoites A study was initiated in 1986 on the effects of irradiation on T parva sporozoites. Early results, using a sporozoite-binding assay and in vitro infection of lymphocytes, showed that irradiated sporozoites bind and enter lymphocytes as efficiently as non-irradiated sporozoites, but within the infected cells only a small proportion of irradiated sporozoites develop into schizonts. Further experiments in 1987 showed that bovine lymphocytes could be induced to multiply in vitro when infected with irradiated sporozoites, although initial cell growth was slow. When lymphocytes were infected with irradiated sporozoites and examined by electron microscopy 1 week later, 20 to 30% of the cells contained immature or damaged schizonts, whereas lymphocytes which had been infected with non-irradiated sporozoites contained normal schizonts (Figure 5). Both irradiated and non-irradiated sporozoites transformed lymphocytes into dividing lymphoblasts and, once cell lines were established, growth and infection rates were similar. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis Figure 5. Electron micrographs of Theileria parva Muguga sporozoites entering bovine lymphocytes and developing into schizonts 1 week later. Both irradiated (A) and non-irradiated (B) sporozoites (Sp) are capable of entering bovine lymphocytes (L) in similar numbers. However, the development of irradiated sporozoites (C) into schizonts (Sc) during the first week after infection is abnormal, compared to the development of schizonts from non- irradiated sporozoites (d). The early development of irradiated sporozoites after entry in the host cell is now being studied in more detail. Cells infected with irradiated sporozoites are also being cultured with cytolytic T lymphocytes specific for T parva-infected cells. Any cell killing observed in these cultures could shed light on possible host immune responses to infected cells. Bovine immune responses and schizont-infected cells The third major component of ILRAD's theileriosis research program concentrates on the schizont stage of parasite development. When cattle develop immunity to ECF—either following natural infection and recovery or after immunization by infection and treatment— there is evidence that cytolytic (cell-killing) T lymphocytes are stimulated specifically to kill Theileria-infected cells. By contrast, when susceptible cattle become infected, cytolytic T cells are stimulated which are capable of killing both infected and uninfected cells. This non- specific cell killing is probably involved in the destruction of the immune system which is a prominent feature of acute infection. Thus cell-mediated immune responses appear to play an important role both in immunity to ECF and in the pathology of the disease. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis An immune response is initiated when one subpopulation of T lymphocytes recognizes antigens associated with `foreign' disease agents. These helper T lymphocytes do not recognize antigens in isolation, but only when they are presented on the surface of specialized cells, known as antigen-presenting cells. The helper T cells then become activated and produce soluble factors which enable other cells of the immune system to mount a protective immune response. The second, effector, stage of the immune response can be mediated by cytolytic T lymphocytes in the case of cell-mediated immunity, or by B lymphocytes which produce antibodies. Studies continued in 1987 on the complex process of immune responses to schizont-infected cells, from the first recognition of the infected cell as a 'foreign' body to its destruction by cytolytic T cells. Work focuses on identifying the antigenic changes displayed by schizontinfected cells which stimulate cell-mediated immunity. The primary objective is to develop improved immunization procedures based on schizont-associated antigens which will protect cattle against ECF. Another objective is to gain a better understanding of immune responses in cattle which could lead to new methods for enhancing resistance to ECF and other diseases. This work has involved the development of techniques and reagents for identifying bovine cell types involved in immunity. Another aspect is research on the bovine major histocompatibility complex—MHC —a group of genes encoding molecules which play a central role in the presentation of antigens to cells of the immune system. Proliferation of infected cells An understanding of how Theileria parasites regulate the growth of host cells might lead to new methods for treating infected animals or attenuating the parasites and making them more vulnerable to destruction by the host immune system. Earlier research on the proliferation of Theileria-infected T lymphocytes showed that some infected cells secrete a molecule of 25,000 daltons which shares several characteristics with the bovine T-cell growth factor, interleukin 2 (IL-2). Work has concentrated on the possible role of IL-2 in maintaining the rapid cell proliferation which is an important feature of ECF infection. A human IL-2 cDNA hybridization probe was used to screen messenger RNA (mRNA) from infected and uninfected cells originating from cloned T-cell lines. Experiments are now in progress to assess the levels of IL-2-specific mRNA in these cell lines using a bovine genomic DNA probe and a new oligonucleotide probe provided by Johns Hopkins University (USA). Studies in collaboration with the Kernforschungszentrum laboratory in Karlsruhe (FR Germany) are exploring the possible role of transformed cell-membrane molecules in stimulating and maintaining proliferation of Theileria-infected cells. Some of the genes which encode cell membrane receptors for hormones and growth factors have similarities with oncogenes—genes introduced by viral infections and implicated in the transformation and growth of cancer cells. By screening DNA from Theileria-infected and uninfected bovine cells for genes whose transcription is enhanced during infection, scientists identified an oncogene which appears to be associated with Theileria infection. Subsequent screening with a viral oncogene DNA probe revealed consistent differences in DNA from bovine cell lines before and after infection with different stocks of T p parva. When oncogenes stimulate cells to multiply, enzymes called protein kinases trigger the attachment of phosphates to protein substrates through a process called phosphorylation. Characterization of kinase activity during Theileria infection has shown a considerable increase in phospho-proteins in infected cells compared with their uninfected parent clones. Current efforts are directed towards identifying the genes which encode protein kinases and file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis purifying and characterizing the host-cell substrates for kinase activity.One objective is to test the possible role of protein kinases in host-cell transformation and induction of immune responses by transfecting the genes which code for protein kinases into quiescent bovine lymphoid cells. Immune responses to schizont-infected cells There is substantial evidence that the cell-mediated immune responses which play an important role in immunity to ECF are directed specifically against antigenic changes on the surface of parasitized cells. Previous work has also shown that cytolytic T-cell responses are restricted by MHC molecules on the surface of host cells: they kill infected cells from the same ani mal and from animals of matched MHC type, but not infected cells from animals of different MHC types. This indicates that the cytolytic cells recognize parasite-induced antigenic changes on the surface of parasitized cells in conjunction with host MHC molecules. However, it has not yet been possible to identify the target antigens on the cell surface. Strain specificity Studies with T-cell clones in 1986 showed that both helper and cytolytic T lympho cytes participate in the parasite-specific immune response and that in some circumstances both types of T cell exhibit parasite strain specificity. In 1987, scientists continued to analyse the specificity of the cytolytic T cell response, both in vivo and using cloned cell lines in vitro. The principal aims are to determine whether the specificity of T-cell responses to individual parasite strains correlates with the strain specificity of responses observed in immunized cattle and to provide reagents that can be used to help identify the antigens on the surface of parasitized cells which stimulate immunity. Studies on cytolytic T lymphocytes have concentrated on the Muguga and Marikebuni stocks of T p parva. These stocks were selected because cattle immunized with the Marikebuni stock are protected against challenge with both stocks of the parasite, whereas a proportion of cattle immunized with the Muguga stock succumb to challenge with Marikebuni. Cytolytic T cells generated in all cattle immunized against T p parva Marikebuni kill target cells infected with either of the two parasite stocks. Cytolytic T cells derived from some cattle immunized against the Muguga stock only kill target cells infected with Muguga, whereas cells derived from other cattle immunized against Muguga also kill some cells infected with Marikebuni. There is preliminary evidence that these differences may be associated with differences in MHC type among individual animals. These results are consistent with results from cross-immunity testing in cattle and provide evidence for the importance of the cytolytic T-lymphocyte response in immunity. Experiments conducted in 1987 indicated that cellular immune responses in buffalo are similar to those in cattle: both proliferative and cytolytic responses appear to be parasite specific and MHC restricted. To investigate strain specificity, T lymphocytes were derived from a buffalo infected with T p lawrencei and stimulated in vitro with cloned infected cells derived from the same animal. These cells were then assessed for cytolytic activity against cells lines derived from the same animal and infected in vitro with five different T p lawrencei stocks. Screening with ILRAD's panel of monoclonal antibodies had indicated that these cell lines were all antigenically distinct. However, the cytolytic buffalo cells killed all the infected cells lines, suggesting either that the infected buffalo had generated cytolytic cells against a variety of antigens or perhaps that the different parasite stocks shared antigens in common. This work was conducted in collaboration with the Kenya Government's Veterinary Research Laboratory, partially supported by the Government of The Netherlands. It is not possible to obtain definitive results on strain-specific immunity using uncloned T file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis lymphocytes from immunized animals because uncloned lymphocytes may include mixed populations of T cells which differ in their parasite strain specificity. Clear results can only be obtained using cloned cells. Thus, cytolytic T lymphocyte clones have been derived from cattle immunized with either T p parva Muguga or T p parva Marikebuni. The target cell lines used in these experiments were cloned by limiting dilution following infection with Theileria sporozoites and thus probably contained cloned parasites. Clones generated against the Marikebuni stock killed target cells infected with either of the two stocks, whereas clones generated initially against the Muguga stock only killed target cells infected with T p parva Muguga. These findings suggest that cloned T lymphocytes derived from cattle immunized with T p parva Muguga or Marikebuni recognize two different sets of antigenic determinants, one of which is shared between the two parasite stocks. In subsequent studies the Muguga-specific cytolytic T-cell clones also killed some of the target cell lines infected with Marikebuni. This may reflect heterogeneity of parasites within the Marikebuni stock. Further work is now being undertaken to determine whether the responses of helper T lymphocytes are also parasite strain specific. Antigenic changes on infected cells Considerable research is in progress to identify the antigenic determinant(s) on schizont- infected cells which are recognized by Theileria-specific T lymphocytes. Such antigen(s) might provide the basis for a vaccine against this stage of the parasite. Several monoclonal antibodies are available at ILRAD which recognize T parva schizont antigens but none reacts with the surface of infected cells. Interestingly, many of these monoclonal antibodies appear to recognize the same protein in infected cells, suggesting that this molecule is dominant in the induction of antibody responses. Some of the monoclonal antibodies recognize different epitopes on the protein molecule which are specific to particular parasite stocks. The protein also differs in terms of molecular weight between stocks and between different cloned parasitized cell lines derived from the same stock (Figure 6). Preliminary results indicate that this protein is present in purified sporozoites as well as schizonts. Experiments are in progress to purify the protein and test its capacity to stimulate Theileria-specific T lymphocytes. Research in this area also includes efforts to produce monoclonal antibodies against other schizont-associated molecules. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis Figure 6. Western blots derived from Theileria-infected cells incubated with a monoclonal antibody which recognizes a schizont antigen. Clear differences are detected in the molecular weight of the molecule recognized by the monoclonal antibody, both between parasite stocks and between parasite populations derived from the same stock. Lane 1 shows the antigen detected by the monoclonal antibody in a cell line infected with T p parva Muguga. Lanes 2 to 5 show the antigens detected by the same monoclonal antibody in different cell lines, all infected with T p parva Marikebuni. Other experiments involve breaking up infected cells and testing different cell components for their capacity to stimulate the proliferation of parasite-specific helper T lymphocytes. Initial results have shown T-cell stimulation by membrane and soluble fractions derived from T parva-nfected lymphocytes and by schizonts purified from infected cells, but not by similar fractions prepared from uninfected lymphocytes stimulated to proliferate by the mitogen concanavalin A. Both soluble and membrane fractions induce proliferation only in the presence of irradiated accessory or antigen-presenting cells from MHC-matched cattle. This ability of parasitized cells to stimulate both proliferative and cytolytic responses is lost a few days after treatment with the anti-theilerial drug parvaquone, indicating that the stimulation of cell- mediated immune responses requires the active participation of the parasite within the infected cell. Current work concentrates on purifying the antigens present in the soluble fraction and generating helper T-cell clones and antibodies which recognize these antigens. Monospecific antibodies will then be used to screen Theileria DNA libraries in order to identify the genes which encode antigens that stimulate helper T-cell proliferation. Although cytolytic T lymphocytes are readily generated which recognize schizont-infected cells, it has not yet been possible to stimulate cytolytic T cells with crude preparations of antigens from purified schizonts. Studies on T-cell responses in other species have shown that cytolytic T lymphocytes generally do not respond to proteins that have been internalized and processed by antigen-presenting cells.Thus, in order to test whether a specific parasite antigen will trigger a cytolytic T-cell response it may be necessary to identify the gene which encodes the antigen and transfect it into a noninfected cell. Transfection involves the introduction and integration of cloned DNA sequences into a recipient genome and their file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis consequent expression. If cells transfected with parasite DNA are to be recognized by cytolytic T lymphocytes, they must also express the appropriate bovine class I MHC antigens. Unfortunately, no cell of this type is available which is suitable for transfection. For this reason, present work concentrates on transfecting bovine MHC genes into mouse fibroblast cells which are known to be suitable recipients for foreign DNA. Once cells expressing the appropriate bovine class I gene are obtained, these will be used as recipients for parasite genes. Studies carried out in 1987 have shown that biologically active mRNA can be isolated from purified Theileria schizonts. In the coming year, a cDNA library will be prepared from schizont mRNA and screened, along with existing Theileria genomic DNA libraries, with polyclonal antisera and ILRAD's panel of anti-schizont monoclonal antibodies to identify genes which encode schizont antigens and which may prove suitable for transfection. To help anticipate potential problems in transfecting parasite DNA into mammalian cells, a cloned Theileria gene obtained from genomic DNA and encoding a sporozoite antigen will be transfected into mouse cells. Characterization of bovine cell types Studies of both cellular and humoral immune responses in cattle require techniques to identify the different cell types which play a role in immunity. In general terms, humoral immune responses involve the production of antibodies by B lymphocytes produced in the bone marrow, while cell-mediated immune responses are mediated by T lymphocytes which mature in the thymus. However, both types of immune response are highly complex processes which involve more than one cell type. Cells types involved in immunity Monoclonal antibodies have been raised at ILRAD which recognize all T lymphocytes, the subpopulation of T lymphocytes which function as helper cells, stimulating other cells to mount an immune response, and the subpopulation of T lymphocytes with a cytolytic function aimed specifically at cells infected with Theileria schizonts. The target molecule recognized by one of ILRAD's monoclonal antibodies on the surface of all T lymphocytes has been designated BoT2, whereas the target molecule recognized specifically on helper T cells is called BoT4 and the molecule recognized on cytolytic T cells is BoT8. Work in this area in 1987 concentrated on completing the definition of these and other monoclonal antibodies produced at ILRAD and further investigating the functional properties of the cells they recognize. Additional monoclonal antibodies are also being sought which will distinguish subgroups within the two functionally important T-lymphocyte populations displaying the BoT4 or the BoT8 molecule. Several monoclonal antibodies have been raised which react with a different molecule on mature bovine T lymphocytes. a protein of 130,000 daltons. The monoclonal antibodies which recognize this molecule appear to stimulate proliferation of bovine T cells, suggesting that the molecule has an important function in the activation of T lymphocytes in cattle. In collaboration with the Agriculture and Food Research Council's Animal Disease Research Institute at Compton (UK), ILRAD has gained access to two monoclonal antibodies that recognize not only T cells expressing BoT2 and BoT4 or BoT8, but also a small, distinct population of lymphocytes lacking these markers. Monoclonal antibodies have been produced which react specifically with these null lymphocytes. These cells appear to originate in the thymus, but have a distinctly different tissue distribution from conventional T cells (Figure 7). They proliferate strongly in mixed reactions of white blood cells and also proliferate in file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis response to the T-cell mitogen concanavalin A when also in the presence of T-cell growth factors. Several other research groups have produced monoclonal antibodies which appear to recognize the same or similar cells and in all instances the antibodies react with cells from both sheep and cattle. ILRAD scientists are collaborating with colleagues in Australia, Switzerland and the UK to elucidate the functional properties of these cells. Figure 7. Frozen sections of normal bovine lymph nodes stained by the immunoperoxidase technique with monoclonal antibodies that react with (A) all T lymphocytes and (B) a population of non-T, non-B lymphocytes. ILRAD now has five monoclonal antibodies which react with granulocytes and monocytes/macrophages, including the large, frilly macrophages in afferent lymph which appear to be distinct from monocytes and mature macrophages in the blood. Several other monoclonal antibodies are being examined which react with dendritic cells or with dendritic cells plus B lymphocytes. Many of these reagents also react with cells in the bone marrow. Scientists are investigating the functional significance of these different cell types in antigen presentation. There is an increasing body of evidence that the large, frilly macrophages in afferent lymph play a role in transporting antigen to the lymph node and presenting it to helper T lymphocytes. The antigen is not presented to the helper T cells in its native form, but as processed peptide fragments associated with MHC molecules on the cell surface. Cannulation techniques have recently been developed which make it possible to collect afferent lymph from cattle for periods of up to 14 days. Preliminary investigation of the frilly file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis cells in these samples of afferent lymph (Figure 8) indicates that they present soluble antigens, such as the variable surface glycoproteins (VSGs) of trypanosomes, to primed T lymphocytes with great efficiency. Experiments are now in progress to obtain VSG specific T- cell clones which can be used to study the efficacy of antigen presentation by these frilly cells in more detail and determine whether different subpopulations of T lymphocytes are stimulated preferentially by specific populations of antigen-presenting cells. The processing of antigen within these cells will also be investigated, as well as their capacity to transport antigen to specific sites within the lymph node. Figure 8. Electron micrograph of a frilly cell from a sample of afferent lymph collected by cannulation procedures from a normal Boran calf. Of the five monoclonal antibodies raised at ILRAD against monocytes/macrophages and granulocytes, two react with all of the frilly cells, two react with a subpopulation of the frilly cells and one does not react with the frilly cells. Additional monoclonal antibodies are now being raised specifically against these cells. Cell types infected with Theileria Previous studies have shown that both B and T lymphocytes can be infected in vitro with T parva, but in mixed cell populations T cells invariably overgrow B cells so that in a short time the cultures contain virtually all T cells. Among T lymphocytes, both helper cells displaying the BoT4 molecule and cytolytic cells displaying the BoT8 molecule are readily infected and generally retain the antigenic determinants expressed before infection. Null lymphocytes can also be infected and after infection some of these cells acquire T-cell surface markers. Acquisition of different T-cell antigens varies somewhat according to the T parva stock used to initiate infection. By contrast, B cells infected with T parva do not acquire any T-cell markers; a proportion of infected B cells continues to express surface antibody (usually IgM) for periods of several weeks or months after infection Monocytes and neutrophils file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis cannot be infected. Most parasitized cells derived from infected cattle are T lymphocytes. In order to assess the role of different cell types in Theileria infections in cattle, different cell types were derived from cattle, infected with T p parva Muguga in vitro, cloned and inoculated back into the original animals. Only one of the cattle inoculated with infected B lymphocytes developed detectable infection and some of the animals in which parasites were not detected remained fully susceptible to subsequent challenge with the same parasite stock. By contrast, all the cattle inoculated with T lymphocytes or null lymphocytes developed detectable—though mostly mild —infections and all resisted subsequent challenge. These results confirm earlier observations that B lymphocytes probably do not play a major role in the pathogenesis of T parva infections in cattle. The mild reactions observed in these experiments are in marked contrast to the severe infections observed in cattle inoculated with their own cells infected in vitro but not cloned. This suggests either that with continued culture in vitro parasitized cells become less well adapted to growth in cattle or that different parasitized cell types complement each other's cell growth in infected animals. An experiment was conducted in 1987 to determine whether differences in immune responses might relate to the specific cell type infected with Theileria. Peripheral blood cells were sorted into separate populations of helper and cytolytic T lymphocytes and both groups were infected with Theileria sporozoites. However, when the two groups of infected cells were incubated with Theileria-specific cytolytic T lymphocyte clones, no differences in levels of cell killing were observed. Studies on the bovine MHC A group of genes—known as the major histocompatibility complex or MHC—has been found in all mammalian species studied. These genes play an important role in the induction of both humoral and cellular immune responses. Significantly, both helper and cytolytic T lymphocytes can only detect foreign antigens in association with the glycoprotein products of host MHC genes. The MHC gene products fall within two major groups, called class I and class II, which are distinguished on the basis of their molecular nature, function and cellular distribution. These products—the MHC antigens—are integral components of cell membranes. Research on the bovine MHC at ILRAD has concentrated in four areas. These are the development of improved techniques to type individual cattle according to class I and class II MHC genes and their products, studies on the structure of the MHC and the complex relationship of individual genes to the MHC antigens expressed on the surface of bovine cells, research on the role of the bovine MHC in immune responses to Theileria and more recently, in collaboration with ILCA and the International Trypanotolerance Centre in The Gambia, preliminary studies on the role of the bovine MHC in resistance to trypanosomiasis. In September 1987, ILRAD held a workshop to discuss recent advances in research on the class II MHC, primarily in livestock but also in laboratory animals and man. The objectives were to assess the state of knowledge on the class II region in cattle and to identify ways in which work in cattle may be advanced in the light of experience in other species. The participants also discussed ways in which the association between disease resistance and MHC type might be identified. Characterization of cattle according to MHC type Cattle are typed serologically at ILRAD according to MHC antigens, using a panel of over 180 antisera and 20 monoclonal antibodies which react with determinants of MHC gene products. The majority of these reagents react with the products of class I MHC genes. Efforts to file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis improve class I MHC typing has focused primarily on two areas—accurate assessment of typing reaction and the handling and storage of large amounts of typing data. More than 2000 cattle have been typed with the full panel of reagents using a computer-controlled automated typing system developed at ILRAD. A program for the storage and analysis of bovine MHC data has been written by scientists at ILRAD in collaboration with colleagues at the University of Strathclyde (UK). It is called BOLA-PC and is now being used in laboratories in Denmark, Italy, the UK and the USA. Researchers have rapid access to information on the pattern of antisera reactivity for any animal tested, plus other information including parentage, breed, age and disease status. Among the typing reagents, monoclonal antibodies have considerable advantages over conventional typing alloantisera. So far, more than 20 mouse monoclonal antibodies have been produced at ILRAD which recognize different bovine class I MHC antigens. These are now being used for MHC typing in laboratories in France, Germany (FR), Great Britain, Italy, Norway, Switzerland and the USA. They have also proved indispensable in various biochemical and cellular studies of the bovine MHC. Scientists from ILRAD and from the Agriculture and Food Research Council's Institute of Animal Physiology and Genetics Research in Cambridge (UK) have also produced the first bovine monoclonal antibody which reacts with a class I determinant in cattle. Using both alloantisera and monoclonal antibodies, ILRAD scientists have completed a comparative analysis of class I MHC phenotypes for more than 1400 cattle of diverse origin. This work was carried out in collaboration with colleagues at the Institute of Animal Physiology and Genetics Research in Edinburgh (UK). Figure 9 illustrates results for West African N'Dama cattle (Bos taurus) which display resistance to trypanosomiasis, East African Boran (Bos indicus) and European taurine cattle of various breeds. The frequencies of some MHC types were clearly different among the different populations (e.g. KN18), whereas the frequencies of other MHC types were remarkably similar for all the animals tested (e.g. W10). Where differences occur between African and European breeds, these may be the result of natural selection operating in different disease environments. If this is the case, the results of surveys such as the one described suggest which MHC types are particularly advantageous in a specific environment. Such types are promising candidates for further laboratory study and disease-challenge experiments to identify individual genes associated with superior disease resistance. The typing of cattle according to class II MHC antigens is not as straightforward as typing according to class I. However, three methods are being pursued which may prove useful. The first involves attempts to produce monoclonal antibodies specific for bovine class II antigens. The second is based on measuring proliferative responses when lymphocytes from two different animals are mixed together. T lymphocyte clones have been produced at ILRAD which proliferate in response to specific antigens on the surface of lymphocytes from different animals. These are proving to be a discriminating and powerful tool for typing cattle according to class II MHC antigens. The third method is based on the analysis of specific DNA sequences contained in and around MHC genes. This work is being undertaken in collaboration with scientists at the Swedish University of Agricultural Sciences and the Institute of Animal Physiology and Genetics Research in Edinburgh. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis Figure 9. MHC class I gene frequencies in Boran, European and N'Dama cattle. Genes are identified through characterization of their products which are prefixed and numbered. The development of a practicable and reliable system for typing large numbers of cattle according to class I MHC antigens, and selected individuals according to class II antigens, now makes it possible to begin searching for associations between MHC types and resistance to disease in African breeds. Such associations could provide the basis for future breeding programs. Elucidation of MHC gene products In cattle, it is now possible to distinguish about 30 different expression forms (called alleles) of one class I MHC gene using antisera and monoclonal antibodies. These are all located at one site on the chromosome—the A locus. Studies suggested that other class I gene products may also be expressed in cattle. Recently, ILRAD scientists have found evidence for the expression of class I MHC antigens at a second locus, the B locus. This was accomplished using mouse and cattle monoclonal antibodies to study mutant lines of cattle cells lacking expression of certain class I antigens and also using a mouse cell line transfected with a bovine MHC gene. The MHC gene expressed at the B locus also expresses a number of different antigenic forms, apparently independently of the forms expressed at the A locus. Present efforts to overcome many of the difficulties inherent in class II MHC typing concentrate file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis on the production of class IIspecific monoclonal antibodies. Work in this area involves innovative approaches to monoclonal antibody production, including immunizing mouse B lymphocytes in vitro, rather than immunizing mice in vivo, before deriving B cells. Scientists are also trying to produce monoclonal antibodies from fusions of mouse and cattle cells. This work is being conducted in collaboration with the Institute of Animal Physiology and Genetics Research in Cambridge. In order to produce antibodies which respond to class II antigens, mice are being immunized with mutant cell lines which are normal for class II expression but deficient in class I expression. Mapping the bovine MHC The availability of mutant cell lines with some MHC genes deleted has made it possible to begin mapping the MHC region of the bovine genome. DNA from normal and mutant cell lines has been digested into fragments with different restriction enzymes, and the fragments separated by field inversion gel electrophoresis and screened with class I and class II DNA probes. Large fragments of DNA are deleted from some mutant cell lines. By using several restriction enzymes separately and in combination, it is possible to identify the genes associated with these fragments, thus gradually constructing a simple map of the location of MHC genes. Using this technique, it has been estimated that class I and related MHC genes cover approximately 1.6 million base pairs in the bovine genome. Class II genes appear to cover a much shorter region. It is not possible to estimate the total size of the bovine MHC at present because other genes lie between the class I and class II regions. Non-MHC histocompatibility antigens In the course of studies on the bovine MHC, two monoclonal antibodies have been derived which react with antigens on the white blood cells of cattle quite distinct from MHC antigens. This antigenic system is now known to include at least four alleles, all described in various Bos indicus and Bos taurus breeds including Boran and N'Dama. The functional significance of this system is not known, but an awareness of its existence is important for the correct interpretation of MHC typing data. Role of the MHC in immune responses to Theileria Cytolytic T lymphocytes, taken from the blood of a cow following Theileria immunization or challenge, are capable of killing Theileria-infected cells in vitro as long as the cells are derived from the same animal and thus share the same MHC antigens. This phenomenon is known as MHC restriction. Now that it is possible to determine the MHC types of experimental animals at ILRAD, scientists have been able to demonstrate that the parasite-specific cytolytic response generated in vivo is also predominantly restricted by the MHC. The important role of the MHC in cellular immune responses against Theileria parasites has been confirmed by patterns of reactivity in vitro between panels of target and responder (effector) cells which are partially or fully identical in terms of MHC: immune responses occur only when the target and responder cells are matched for an appropriate MHC product. Restriction by either class I or class II MHC has been observed: the cell-killing or proliferative/helper functions of T lymphocytes are inhibited when class I or class II MHC gene products are neutralized by adding the appropriate monoclonal antibodies to in vitro assays. Thus it appears that T lymphocyte responses in cattle undergoing immunization or challenge with T parva are typically MHC restricted, with many similarities to the well characterized T-cell responses to viral antigens in mouse and man. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis ILRAD scientists have obtained evidence that the strain specificity of cytolytic T-cell responses to Theileria-infected cells may be influenced by MHC type. In 1986, T lymphocytes derived from two cattle immunized with T p parva Muguga killed target cells infected with the Muguga stock, but not with T p parva Marikebuni. These T-cell responses were restricted by two class I MHC alleles—w10 and KN18. In 1987, cytolytic T cells derived from two different cattle immunized with T p parva Muguga killed target cells infected with either the Muguga or the Marikebuni stacks at similar levels. T cells from both these animals were restricted by the w6.2 class I MHC allele. These findings suggest that an animal's MHC type may determine which parasite-induced antigen becomes the target for cell-mediated immunity, thus determining the strain specificity of the response. To explore this possibility further, scientists are conducting an immunization and challenge experiment with groups of cattle of defined MHC types. A limiting dilution assay has been developed to determine the frequencies and specificities of cytolytic T lymphocytes in animals immunized with T p parva Muguga prior to challenge with T p parva Marikebuni. Thus it should be possible to compare the strain specificity of cytolytic T cells in vitro with MHC type and with the subsequent strain specificity of the immune response. Embryo transfer to produce cattle of specified MHC types An important aspect of ILRAD's MHC research program has been the application of embryo transfer techniques to provide cattle which are full siblings or identical twins of specified MHC types. Hormonal treatment regimes have been developed for inducing superovulation , and synchronizing oestrus among donor and recipient animals in ILRAD's breeding herd of Boran cattle. Using these techniques, approximately 60% of cows respond to superovulation, yielding an average of 3.7 embryos each. Surgical transfer of these embryos gives a pregnancy rate of 63%. By the end of 1987, seven sets of identical twins had been produced by embryo splitting and transfer of demi-embryos into separate recipients. One set of identical twin calves is now being used to investigate the possibility of conferring immunity against Theileria infection by inoculating a naive animal with Theileria-specific T cells generated from its immunized twin. Using conventional breeding and embryo transfer, ILRAD has obtained 12 cattle, representing five MHC types, which are homozygous over the MHC region. These animals possess identical MHC genes inherited from both parents. They provide an important research resource which considerably simplifies studies on the structure and function of the MHC region. Exploring vaccine delivery systems If Theileria sporozoite or schizont antigens are identified which appear to stimulate protective immunity against ECF, the best methods for eliciting the appropriate immune responses in cattle must be determined in order to develop an effective vaccine. For this reason, experiments were initiated in 1987 to explore different antigen delivery systems for inducing humoral or cell-mediated immune responses in cattle. This work has concentrated on two areas: the characterization of antigen-presenting cells in afferent lymph and the development of transfection techniques. Both have already been discussed in different section of this report. Experiments to establish transfection techniques have started by transfecting bovine class I MHC genes from genomic DNA into mouse fibroblast cells. The approach is to break up bovine genomic DNA by shearing, transfect the fragments into mouse cells and select cells expressing bovine class I MHC molecules using specific monoclonal antibodies with a fluorescence-activated cell sorter (FACS). To date, one transfectant expressing a bovine class file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Theileriosis I molecule has been selected and cloned. Transfected cells will be useful not only for isolating different class I gene products, but also potentially for transfecting Theileria genes. Work in 1988 will concentrate on extending transfection techniques to bovine cells. Studies on recombinant vaccinia viruses will also begin in 1988. The aim of this work will be to explore the potential of recombinant viruses as a means of presenting parasite antigens to cattle for induction of protective immunity. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Theileriosis.htm[5/19/2016 3:35:00 PM] Trypanosomiasis Trypanosomiasis Trypanosome biology and biochemistry Changes associated with the trypanosome life cycle Growth and transformation of bloodstream forms Variable surface glycoproteins T vivax surface antigens Aspects of trypanosome metabolism Trypanosome cultivation in vitro Host responses to trypanosome infection Early events in the skin Parasite-host interaction in the bloodstream Control of parasite growth Host antibody production Pathogenic effects of trypanosomiasis Anaemia Endocrine dysfunction Control of infection in Trypanotolerant and susceptible cattle Control of parasitaemia and anaemia Mechanisms of resistance Epidemiology Trypanosome characterization Serological typing Chromosome profiles Use of DNA probes Identification of species-specific proteins Livestock production under trypanosomiasis risk Trypanocidal drugs: detection and testing in vitro Trypanocidal drugs: detection in treated animals Trypanocidal drugs: evaluation of protection African Trypanotolerant Livestock Network Tsetse challenge and trypanosome infection Evaluation of trypanosomiasis control Indications of heritability Research on N'Dama productivity in The Gambia and Senegal Trypanosomiasis is a disease complex caused by several species of blood-and tissue-dwelling file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis protozoan parasites. The disease occurs throughout the tropical regions of Africa and in large areas of Asia and South America. It affects cattle, sheep, goats, pigs, horses, camels and man. Wild animals can also be infected with the parasites but generally do not suffer from disease. They are the source (reservoir) of infection for domestic animals. The most important trypanosome species affecting domestic livestock in Africa are Trypanosoma congolense, T vivax and T brucei brucei in cattle, sheep and goats, T simiae in pigs and T evansi in camels. T vivax also has a significant impact on cattle production in South America, while T evansi affects camels in Asia and horses, cattle and domestic buffalo in South America, India and Southeast Asia. The human disease is caused by T brucei gambiense and T b rhodesiense. African trypanosomes are transmitted from the reservoir hosts to livestock by several species of tsetse flies and also by other biting insects. Infected animals develop fever, lose weight and progressively become weak and unproductive; breeding animals may abort or become infertile. Left untreated, many animals die of anaemia, heart failure or intercurrent bacterial infections that take advantage of the animal's weakened resistance. Highly productive exotic breeds tend to be especially susceptible. In man, a similar course of events takes place with parasites ultimately invading the brain, resulting in the disease syndrome known as `sleeping sickness'. In Africa, trypanosomiasis occurs in 37 countries, extending over 10 million square kilometres or roughly one-third of the continent. Throughout this region, an estimated 50 million cattle— about 30% of Africa's total cattle population—are exposed to the risk of infection. Largely due to the widespread incidence of trypanosomiasis, Africa produces about 70 times less animal protein per hectare than Europe. Control of trypanosomiasis is based primarily on insecticide spraying to control tsetse populations and on regular treatment of livestock at risk with trypanocidal drugs. Rarely, however, has complete control been achieved using these methods, even after substantial effort. The high cost of regular drug and insecticidal treatment, the limited effectiveness of insecticide application in high-rainfall areas, the possibility of environmental pollution by insecticides, the increasing incidence of parasite resistance to available drugs and the absence of new drugs to replace them—these are some of the problems that make tsetse and trypanosomiasis control difficult and expensive. What's more, the disease problem may be increasing with the expansion of areas infested by tsetse flies. There is an urgent need not only to improve existing control methods, but equally importantly to seek new methods for trypanosomiasis control. This search will require intensive research into several aspects of the basic biology of the parasites, the role of tsetse flies in transmitting disease and the responses to infection of different breeds and species of livestock. The goal is to achieve a fuller understanding of the entire disease process in order to identify promising avenues for practical immunological, chemical or genetic control. Trypanosome biology and biochemistry The three major tsetse-transmitted trypanosome species pass through several different stages during their life cycle in both the insect vectors and the mammalian hosts. When a tsetse fly feeds on an infected animal, it ingests trypanosomes along with blood. The parasites undergo a cycle of development within the tsetse, passing through procyclic and epimastigote stages and finishing as animal-infective metacyclic forms. When the infected fly next feeds, infective metacyclic trypanosomes are injected into the skin of a mammalian host along with tsetse saliva. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis When T congolense or T brucei parasites are transmitted to a susceptible animal by a tsetse fly, a local skin reaction several centimetres in diameter—called a chancre—may develop at the site of the bite. The metacyclic trypanosomes develop further in the chancre, invade the local lymph vessels and move through the regional lymph node into the bloodstream. T vivax parasites also move from the skin through the lymphatic system to the bloodstream, but the local skin reaction, if it occurs, is less severe. Infection in the bloodstream is characterized by successive waves of parasitaemia as trypanosome populations multiply rapidly, then most of the parasites die, but a few survive and begin multiplying again. T brucei parasites may also be found in the connective tissues and, in later stages of infection, all three trypanosome species may invade the central nervous system. Metacyclic trypanosomes and the forms which develop in the mammalian bloodstream have a thick coat which covers the entire surface of the parasite. The surface coat is composed of glycoproteins referred to as variable surface glycoproteins or VSGs (Figure 10). The procyclic and epimastigote forms which develop in the tsetse are not coated with VSGs until they reach the metacyclic stage, ready for transmission to a mammalian host. Figure 10. The molecular structure of a trypanosome VSG. A chain of amino acids is linked at the C-terminal to a carbohydrate which contains an element, called the cross-reacting determinant, that appears to be identical for a wide range of trypanosomes. One or more additional carbohydrave(s) is linked to the amino acid chain. The carbohydrate containing the cross-reacting determinant is part of a glycolipid which also includes two molecules of myristic acid. These are thought to anchor the VSG in the membrane of the cell. When an animal becomes infected, it produces antibodies against the VSGs displayed by the first wave of invading trypanosomes. However, before all the parasites can be eliminated, new trypanosomes emerge, coated with different VSGs, which are not recognized by the animal's initial immune response. These trypanosomes multiply rapidly and the host produces new antibodies, but then parasites appear with yet another VSG, always keeping a step ahead of the host response. This remarkable ability to change the biochemical and antigenic composition of the surface coat—called antigenic variation—is the primary mechanism which prevents most domestic animals from developing effective immunity against trypanosomiasis. The same mechanism would enable the parasite to evade a conventional vaccine based on priming the antibody responses against one or a small number of variable antigens. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis In 1987, several studies at ILRAD focused on genetic and biochemical changes which occur when a trypanosome passes from one developmental stage to another, a process called differentiation. Work also concentrated on various aspects of trypanosome metabolism and on the mechanisms of antigenic variation. The goal of ail these studies is to identify factors which could be manipulated to disrupt trypanosome development or to make the parasites more vulnerable to host defences. Changes associated with the trypanosome life cycle As trypanosomes develop in tsetse and mammals, different genes are expressed at different stages of development. Research is in progress to identify such genes, define the elements in the parasite genome which regulate their expression and determine how changes in gene regulation take place. All the genetic material contained in a trypanosome or other organism is present in the DNA, located primarily in the cell nucleus. However, only a small proportion of genes present in the DNA is expressed at any one time. These expressed genes are transcribed to mRNA and translated into protein. A cDNA library constructed from mRNA obtained at a given stage of parasite development should contain only those genes transcribed at that particular stage. ILRAD scientists have constructed cDNA libraries using as templates mRNA from bloodstream, procyclic and metacyclic forms of T congolense. Libraries from the different stages are being compared to identify genes which are present, or abundant, only at particular stages. Several mRNA transcripts are now being analysed which are specific to bloodstream or metacyclic parasites. The objectives are to identify the nature and function of the proteins encoded by stage-specific genes, the point at which stage-specific genes are switched on and off and the mechanisms for regulating these genes. A study was initiated in 1987 to characterize the respiratory metabolism of metacylic trypanosomes. Trypanosomes use two distinct pathways for respiration in the different environments of the tsetse and the mammalian bloodstream. In the bloodstream, respiration is entirely dependent upon the trypanosome alternative oxidase (TAO), a mitochondria glycerophosphate oxidase which is unique to trypanosomes and might provide a prime target for trypanocidal drugs. Procyclic trypanosomes utilize a mitochondria) cytochrome electron transport system for 70 to 80% of respiration and the TAO for only 20 to 30%. Work in 1987 showed that metacyclic parasites also use both systems, but with the emphasis in reverse—20 to 30% mitochondria cytochrome electron transport and 70 to 80% the TAO—indicating that metacyclic forms are an intermediate differentiation stage between procyclic and bloodstream parasites. Further experiments concentrate on a detailed analysis of the TAO and the mechanisms involved in switching from one system to the other. Growth and transformation of bloodstream forms Most populations of T b brucei in the mammalian bloodstream change from rapidly dividing to nondividing forms. The nondividing trypanosomes are not infective for mammals; they may represent an adaptation for transmission to the tsetse vector. Attention focuses on this transition because parasite populations which switch more quickly to nondividing forms appear to produce lower levels of parasitaemia and may be more readily controlled by the host than parasites which remain in a rapidly dividing state. Studies in mice have indicated that host factors may play a role in regulating the transition process: identical, cloned parasite populations switch to nondividing forms more rapidly in mice which are resistant to trypanosome infection than in susceptible mice. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis Molecular biologists are screening cDNA libraries from populations of the two T b brucei bloodstream forms to identify any genes which are switched on specifically during the nondividing stage of parasite development. Such genes might be involved in the negative regulation of cell division. A comparison of cDNA libraries from dividing and nondividing parasites led to the identification of two DNA sequences which appear to be specific for dividing forms and nine sequences which may be specific for nondividing forms. Experiments are now in progress to characterize the most promising of these sequences and to determine their location within the trypanosome genome. Once scientists have determined the location of stage-specific genes within the trypanosome genome, it may be possible to identify DNA sequences associated with these genes which play a role in controlling gene expression. Because changes in protein structure have been shown to modulate cell differentiation in other organisms, scientists at ILRAD are examining protein phosphorylation in the transition of T b brucei from rapidly dividing to nondividing bloodstream forms. Two phosphorylated proteins have been identified in nondividing trypanosomes which are not present in substantial amounts in rapidly dividing forms. Scientists are now working to characterize these proteins, to find the genes which encode them and to identify the enzymes (protein kinases) involved in the phosphorylation process which may be associated with the transition from rapidly dividing to nondividing trypanosomes. Some T b brucei parasites do not appear to switch from rapidly dividing to nondividing bloodstream forms, but continue dividing until death of the host. Early work suggested that whether or not a parasite population switched to nondividing forms was an intrinsic trait of specific parasite clones, though also influenced by host factors. However, research results obtained in 1987 have challenged this view. When large numbers of one T b brucei clone were used to infect mice, the parasites multiplied without remission until death of the hosts. By contrast, when smaller numbers of the same parasites were injected into mice, populations of bloodstream forms increased and then diminished. Scientists are now maintaining this T b brucei clone in a cell-free culture system in order to investigate its multiplication and transition to nondividing forms in more detail. Variable surface glycoproteins Trypanosomes which develop in the bloodstream of an infected animal are capable of expressing a very large number of different VSGs. Early studies showed that bloodstream forms of T b brucei and T congolense can also express the same VSG a second time during the course of infection. In 1987, ILRAD scientists obtained evidence suggesting that T vivax bloodstream forms may also express the same VSG a second time. Cattle and goats infected intravenously with a cloned population of T vivax bloodstream forms responded with increasing antibody production 9 days after infection and again 16 to 21 days later, presumably to the same VSG which had reappeared among the infecting parasites. To continue these studies, techniques are now being established to measure more precisely the immune responses against specific trypanosome VSGs. Metacyclic trypanosomes are also capable of expressing a number of VSGs. Work is in progress to determine how many different VSG genes are present in metacyclic forms of a genetically identical parasite population (serodeme) and whether metacyclic VSG genes have any peculiarities with respect to their activation or expression which might serve as targets for intervention. Specific antibody against T congolense metacyclic VSG is being used to screen a cDNA expression library prepared from metacyclic mRNA. Several gene sequences have been identified which encode proteins and further experiments are in progress to confirm whether these proteins—which may be VSGs—are specific to T congolense metacyclic forms. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis In collaboration with the Free University of Berlin (FR Germany), scientists are also analysing the carbohydrate structures associated with T congolense metacyclic and bloodstream-form VSGs (see Figure 10). This work focuses on the role of the VSG carbohydrate component in the process of antigenic variation and possibly in blocking host recognition of trypanosome VSG. Preliminary results indicate that VSG becomes more accessible to degradation by digestive enzymes after removal of the carbohydrate component. Previous studies suggested that VSG is attached to the surface of T b brucei bloodstream forms by a lipid anchor. This lipid may be cleaved by a specific enzyme —phospholipase-C— when the trypanosome sheds its surface coat, suggesting that pharmacological stimulation of this enzyme might be exploited as a method for stimulating VSG cleavage and exposing nonvariable surface molecules in bloodstream-form trypanosomes. Experiments conducted in 1987, in collaboration with the European Molecular Biology Laboratory in Heidelberg and the Max-Planck Institute in Tübingen (FR Germany), showed that phospholipase C is present in low concentration within the trypanosome cell. Research at ILRAD has shown that bloodstream forms of T congolense have less phospholipase-C than T b brucei, while T vivax has little or none. Detailed biochemical studies focusing on metacyclic forms of T congolense indicate that other mechanisms may also be involved in conformational changes, and perhaps release, of trypanosome VSG. T vivax surface antigens Research on VSG composition and synthesis in T vivax has been limited because this species tends to be more fragile than other trypanosomes and it is difficult to obtain T vivax parasites in large numbers. Earlier work indicated that the VSG coat is less dense on T vivax than on T b brucei or T congolense, suggesting that it may provide a less effective protective covering than the VSG coats of the other species. VSG purified from a well characterized T vivax clone originating from Nigeria is a relatively small glycoprotein of 46,000 daltons. In 1987, VSG was partially purified from another T vivax clone, from Lugala in Uganda. Preliminary results suggest that this is the smallest VSG so far described in any trypanosome. Antibodies raised against VSGs from these two clones do not crossreact, indicating that they do not recognize a crossreacting determinant on these parasites. Because the T vivax surface coat is less dense that that of other species, other antigens might be exposed on the surface which could trigger a protective immune response in infected animals. Three nanvariable antigen molecules have been identified in bloodstream forms of both T vivax clones which appear to be present on the cell surface and are also present on uncoated procyclic trypanosomes. Two other antigens have been identified which occur exclusively on the surface of procyclic forms. One of these two, with a molecular weight of 30,000 daltons, is of particular interest because it is capable of stimulating a strong antibody response. Scientists are also investigating factors which may have cell-killing functions when added to T vivax parasites. One of these is aerolysin, a bacterial toxin which can make holes in membranes. Aerolysin appears to kill T vivax parasites in vitro but not T congolense or T brucei. Other laboratories have reported that sera from Cotton rats (Sigmodon hispidus) contain an `antivivax' factor, but experiments conducted at ILRAD in 1987 failed to substantiate this. Recently, molecular biologists at ILRAD have shown that mRNA can be isolated from T vivax and translated into proteins. Some of these gene products are recognized by the antisera raised against T vivax variable and nonvariable surface antigens. The synthesis of these proteins by recombinant DNA technology should make it possible to extend studies of T vivax surface antigens despite the constraints posed by the fragility and limited availability of these file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis parasites. Aspects of trypanosome metabolism If trypanosome VSGs are too antigenically diverse to be used as material for a vaccine, research on the cell biology of the parasites may reveal other sites which might be vulnerable to antibody attack. One possible target could be the structures involved in endocytosis, the process trypanosomes use to ingest biologically important molecules—such as essential nutrients and growth and differentiation signals—from their environment. In trypanosomes, endocytosis seems to occur through the specialized membrane of the flagellar pocket. It is clear that various particulate and soluble substances enter the parasite at this site. Bovine transferrin (a serum protein responsible for binding and transporting iron) can be coupled with particles of colloidal gold which serve as markers, making it possible to visualize by electron microscopy the organelles involved in endocytosis within the trypanosome. Work at ILRAD has shown that transferrin/gold from the culture medium enters the fiagellar pocket and is taken up by coated vesicles in the cell cytoplasm. VSG from the surface of the parasite is endocytosed through the same process (Figure 11). The examination of trypanosomes in serial section has revealed that the vesicles involved in endocytosis occur as complex interconnected networks within the cell (Figures 12 and 13). Mathematical techniques have also been introduced, in collaboration with the European Molecular Biology Laboratory, for estimating the volume of these interconnected networks and of other compartments within the parasite. Further work has shown that some parts of these endocytosis networks have molecular sorting functions: T b brucei parasites are capable of separating VSG molecules from transferrin/gold and also of separating the cross-reacting determinant from other parts of the VSG molecule (Figure 14). Figure 11. If trypanosomes are incubated in bovine transferrin coupled to colloidal gold, the transferrin/gold is endocytosed and enters membrane-bound organelles inside the parasite. In this electron micrograph the small gold particles (5nm) in the tubular network are endocytosed transferrin/gold. The larger particles (15nm) are labeling the cross-reacting determinant of trypanosome VSG. The cross-reacting determinant is found on the surface of the trypanosome, in the flagellar pocket and in the same organelles as the transferrin/gold, indicating that the two substances are internalized within the parasite in the same way (magnification 64,800x). file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis Figure 12. Section through a trypanosome incubated in bovine transferrin coupled with small particles (5nm) of colloidal gold. Cross-reacting determinant from trypanosomal VSG was labeled with larger (15nm) gold particles. The cross-reacting determinant is found on the surface of the parasite, in the flagellar pocket and in many intracellular organelles, including the Golgi apparatus and structures that also contain transferrin/gold (magnification 64,800x). file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis Figure 13. Section through a trypanosome incubated in bovine transferrin coupled with small particles (5nm) of colloidal gold. Cross-reacting determinant from trypanosomal VSG is labeled with larger (15nm) gold particles. The transferrin/gold is found in a lysosome-like structure and in cytoplasmic vesicles. The cross-reacting determinant is found on the surface of the parasite, in the lysosome-like structure and in a tubular structure which connects with the transferrin/goldcontaining vesicle, suggesting that the transferrin/gold is being excluded from parts of the endocytosis pathway (magnification 127,575x). file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis Figure 14. Sections through trypanosomes doubled labeled with antibodies directed against the cross-reacting determinant and against another part of the VSG occasionally show tubular structures that either label only with the cross-reacting determinant (labeled here with 15nm particles of colloidal gold) or, as in this example, with the cross-reacting determinant and, at one end only, with low levels of VSG (20nm gold particles). The VSG is associated mostly with the trypanosome suface and the flagellar pocket, whereas the cross-reacting determinant is found mostly within the cell (magnification 127,575x). Experiments conducted in collaboration with the Chester Beatty Research Laboratories (UK) have indicated that transferrin enters trypanosomes after recognition by a receptor molecule on the surface of the parasite. The transferrin is carried into the cell where it releases iron in an acidic compartment. The iron-free transferrin is then recycled to the cell surface where it is released into the medium. Future work will concentrate on characterization of the organelles involved in endocytosis and identification of the transferrin receptor on the surface of trypanosomes. Trypanosomes can be broken up under high pressure and cell fractions separated according to size. Using this technique, ILRAD scientists have isolated membrane-bound structures from bloodstream forms of T b brucei which contain a proteolytic (protein-splitting) enzyme probably involved in the digestion of nutrients. This enzyme has been extensively purified and appears as a single band of activity of approximately 27,000 daltons. The addition of serum from rats, rabbits, cattle or humans to cell fractions containing this enzyme results in new bands of enzymatic activity. The apparent molecular sizes of these bands of activity are different for each mammalian species, suggesting that the host molecules which activate enzymatic digestion in trypanosomes are species specific. Trypanosome cultivation in vitro Culture systems are established which support the development of all three major African trypanosome species throughout their life cycles. In vitro cultivation techniques are being tested and improved on a life cycle stages for biochemical and continuous basis in order to provide adequate numbers of trypanosomes at various life cycle stages for biochemical and immunological research. Scientists also use trypanosomes produced in vitro to study the action of trypanocidal drugs and to investigate drug resistance. In 1987, studies concentrated on increasing the production of T b brucei metacyclic forms and improving techniques for initiating cultures of T vivax. T b brucei was the first trypanosome species to be maintained in culture, but it has proven difficult to produce large numbers of metacyclic forms of this species and to distinguish T b brucei metacyclics from bloodstream forms. A system to produce large numbers of T b brucei metacyclic forms was introduced in 1986 and further developed in 1987, based on passaging parasites alternately between in vitro culture and mice. Cultures were initiated with bloodstream forms obtained from infected mice. These were maintained for 5 days in the presence of bovine fibroblast cells and foetal bovine serum without changing the medium. On the sixth day, the trypanosome population, containing epimastigotes, was transferred to a feeder layer-free system and the medium was changed every 3 days. After a total of 14 days in culture at 27°C, over 95% of the trypanosomes were infective for mice and parasites separated from these populations on DE-52 columns appeared morphologically identical to metacyclic forms observed in the saliva of infected tsetse. Using this system, it was possible to produce nearly 300,000 metacyclic-type parasites from a single culture flask. Three in vitro cultivation systems appear to stimulate the transformation of T b brucei procyclics to epimastigote and metacyclic forms. These systems use embryonic cells from the meadow vole Microtus montanus or cells from different insects as feeder cells, plus whole file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis tissues or organs from tsetse or other flies. Work conducted at the University of Massachusetts at Amherst (USA) and at ILRAD showed that T b brucei procyclics could be cultured in the presence of a cell line from the Anopheles mosquito, the vector for malaria, which is easily maintained in vitro. Procyclic forms previously maintained for periods of up to 96 days in liquid medium without feeder layer cells transformed into epimastigote and metacyclic forms following the introduction of Anopheles cells into the cultures. Infective trypanosomes, coated with VSG, were isolated from these cultures on DE-52 columns or by agglutination of uncoated trypanosomes in bovine plasma. These could be used to initiate cultures of bloodstream forms, thus completing the T b brucei cycle of development. One stock of T congolense (C-49) isolated from the Trans Mara region of Kenya has proven to be the most prolific of the ILRAD's T congolense stocks and clones for the production of metaeyclic forms in vitro. Several new clones were derived from this stock in 1986 and one was identified—IL3000—which appears to possess superior growth characteristics, both in vitro and in experimental animals. Further experiments in 1987 showed that IL3000 is infective for cattle, goats and mice and susceptible to the trypanocidal drug diminazene aceturate (Berenil: Hoechst). Epimastigote forms of T congolense IL3000 were readily established in culture and within 1 month were producing a high percentage of metacyclic trypanosomes. Five months of production yielded 80 billion metacyclic forms, sufficient for a range of biological, molecular biological and biochemical studies. The unusual growth characteristics displayed by T congolense IL3000 may be due to increased expression of genes involved in positive growth regulation. To identify any genomic features peculiar to this clone, genomic DNA from IL3000 was compared with DNA from other isolates of the C-49 stock as well as DNA from an unrelated T congolense clone, IL1180. A repeated nucleotide sequence was identified in all the T congolense parasites examined which has not been detected in T brucei (Figure 15). Copies of this sequence, arranged in tandem arrays, occur in much higher numbers in IL3000 and other isolates from the C-49 stock than in T congolense IL1180. Further studies indicated that this sequence encodes the 35-nucleotide mini-exon repeat sequence found at one end of all trypanosomal cytoplasmic messenger RNA. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis Figure 15. Relative abundance of the miniexon repeat in T congolense IL3000 (track 2) compared with another T congolense clone, IL1180 (track 1), from a different serodeme. DNA (1µ g) from each clone was digested with the restriction enzyme Hind III, separated on a 1 % agarose gel and transferred to a nylon membrane. Hybridization was with a radiolabeled, cloned Hind III fragment of 1,000 base pairs containing the repeat sequence. Culture systems are available which support the development of several stocks of T vivax in vitro throughout all phases of the parasite life cycle, including stocks which are restricted to ruminants and the experimentally more amenable stocks which infect laboratory rodents. For East African stocks of T vivax which only grow in cattle, it is possible to initiate low- temperature cultures using infected tsetse proboscides or trypanosomes isolated from bovine blood. All the vector stages of the parasite develop in these cultures, culminating in the production of metacyclic trypanosomes which are infective for cattle. Host responses to trypanosome infection Early events in the skin The development of a local skin reaction, or chancre, at the site of an infected tsetse bite is the first host response to trypanosome infection. In domestic livestock, the largest and most intensely inflamed chancre lesions often occur in animals that are most resistant to trypanosome infection, suggesting that reactions in the chancre may contribute to host resistance by limiting the rate of parasite growth during this first stage of infection in the skin. Experiments conducted in collaboration with the University of Ibadan (Nigeria) showed that some stocks of T congolense stimulate the development of chancres more readily than others. When cattle or goats were infected with T congolense stocks which induced chancres and then treated with trypanocidal drugs, they developed resistance to subsequent challenge with the same parasite stocks. By contrast, infection with stocks which did not induce chancres file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis failed to confer resistance against homologous challenge. When goats were infected with T congolense, nearly all the trypanosomes derived from the resulting chancres up to 9 days after infection displayed VSGs characteristic of metacyclic forms, even though the parasites resembled bloodstream forms. These findings suggest that trypanosomes which display metacyclic VSGs may multiply in the chancre, allowing the host to mount a comprehensive immune response to all the metacyclic variable antigen types characteristic of a particular trypanosome stock. In earlier experiments conducted in collaboration with the Kenya Goverenment's Veterinary Research Laboratory, four buffalo and four susceptible Boran cattle were infected with T congalense by tsetse. Skin reactions at the sites of infected tsetse bites were much less severe in the buffalo than in the cattle and bloodstream parasites were detected much later in the buffalo. To define more precisely the role of the skin reaction in the delayed appearance of trypanosomes in the bloodstream, buffalo and cattle were infected in 1987 with the same T congolense serodeme by intravenous injection directly into the bloodstream. Trypanosomes were detected in the bloodstream sooner in both species than when infection had been by tsetse transmission, but again bloodstream parasites appeared much later in the buffalo than in the cattle. These results indicated that differences between the two species in control of parasite numbers at the early stages of infection were not, or at least not entirely, due to mechanisms in the skin. Parasite-host interaction in the bloodstream Two responses are involved in host control of T b brucei infection in the bloodstream—control of parasite growth by nonimmunological processes and destruction of parasites by host antibodies. Detailed studies on both of these mechanisms have been in progress for several years, concentrating on trypanoresistant and susceptible strains of mice. The goal is to identify mechanisms which might be enhanced by chemotherapeutic or immunological means. Control of parasite growth In vitro studies have confirmed previous observations that bloodstream forms of T b brucei multiply in response to host-derived growth nutrients present in trace amounts in the sera of infected animals. The growth rate of T b brucei bloodstream forms in a cell-free culture system depended on the concentration of foetal bovine serum in the culture. Serum recovered from cultures at peak trypanosome density had a reduced capacity to support further parasite growth, suggesting that the trypanosomes had depleted the serum of nutrients. The serum molecules which supported parasite growth were large but were otherwise extremely heterogeneous, suggesting that growth promotion was associated with molecules of more than one type. In 1987, the cell-free culture system was improved, making it possible to maintain trypanosomes in cell-free culture for long periods. Recent experiments have concentrated on identifying the serum molecules required to support trypanosome growth. This work showed that foetal bovine serum no longer supported trypanosome growth when lipoproteins were removed. The re-introduction of either high- or low-density lipoprotein restored the serum's growth-supporting capacity. Yet pure lipoprotein without serum did not support parasite growth, nor did the addition of lipoprotein fully restore the growth-supporting capacity of foetal bovine serum which had been depleted of growth nutrients. These results suggested that lipoprotein may be one of several large molecules in foetal bovine serum which are required for the multiplication of T b brucei in vitro. Further experiments are in progress to analyse the role of high-and low-density lipoprotein in supporting trypanosome growth and to identify other growth-supporting molecules. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis Non-immune responses to trypanosome infection were studied in previous years by treating mice with dead Corynebacterium parvum, a bacterium which acts as a nonspecific immunostimulator. C parvum treatment enhanced the ability to control T brucei infection, both in intact mice and in mice whose immune system had been suppressed in different ways. In studies conducted in 1987 using the cell-free culture system, macrophages derived from C parvum-treated mice and macrophages which had been fed C parvum in vitro both released molecules which inhibited multiplication of T brucei. The T brucei growth-inhibitors produced by C parvum-stimulated macrophages could, in turn, be inhibited by increasing the concentration of foetal bovine serum in the cultures. This suggested that the inhibitory molecules might interact with growth-promoting molecules in foetal bovine serum or with a T brucei receptor for the growth-promoting molecules. Present work concentrates on defining the biochemical nature of the growth inhibitors and testing whether they compete with lipoprotein or other molecules in foetal bovine serum. Host antibody production In infected animals, antibodies directed against trypanosomal VSGs attach to the surface of the parasites and mark them for destruction by macrophages or by the complement system. Previous studies of T b brucei infection in mice suggested that host antibody responses do not play a role in stimulating trypanosome transformation in the bloodstream, but only become effective after most of the parasites have already switched to nondividing forms. In vitro experiments were conducted in 1987 to examine the effects of anti-trypanosomal antibodies on rapidly dividing forms of the parasite. Both polyvalent and monoclonal antibodies raised against trypanosome VSG bound to the surface of rapidly dividing bloodstream forms of T b brucei. While the parasites were exposed to moderate levels of antibody they stopped multiplying but when the antibody was removed they began multiplying again, indicating that they had not made an irreversible switch to nondividing forms. The antibodies added to the cultures were rapidly destroyed; most likely, they were endocytosed by the parasites. Work is now in progress to examine whether trypanosomes in this system are in fact endocytosing anti-VSG antibody by the same pathways identified for the uptake of transferrin/gold and VSG. The amount of antibody required to halt trypanosome multiplication in vitro is also being compared with the amount required to neutralize the parasites in an infected animal. Mice which are highly susceptible to trypanosomiasis do not produce detectable antibodies against the parasites, although their B lymphocytes are extensively activated and transformed into plasma cells which contain antibody. In vitro analysis of spleen cells from infected susceptible mice demonstrated that their activated B cells were defective in their capacity both to synthesize and to secrete antibody. When resistant mice were infected with trypanosomes, they produced antibodies which controlled the first parasitaemic wave, but showed similar defective plasma cell function during the second parasitaemic wave. However, in resistant mice antibody synthesis and secretion eventually recovered and parasitaemia was controlled. At the time of parasite remission, vast amounts of antibody were synthesized and secreted although the number of cells containing antibody did not increase. In vitro studies conducted in 1987 indicated that loss of plasma cell function during trypanosome infection was probably not due to the presence of suppressor host cell populations, which could be detected by adoptive transfer, nor was the recovery of plasma cell function due to the presence of contra-suppressor cells. Plasma cell function was restored in vitro by incubation with T-cell growth factor preparations or the growth factor recombinant human IL-2. Research is now in progress to analyse the signals required to induce antibody synthesis and secretion by trypanosome-induced defective plasma cells. This work should help clarify the mechanisms involved in the suppression and restoration of immune responses file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis in infected animals. In order to analyse immune responses to trypanosome infection in cattle, highly sensitive techniques are required to measure the small quantities of antibody produced by bovine B lymphocytes in vitro. A method was developed in 1987 using an inhibition enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies raised against different types of bovine immunoglobulin (Ig). Using monoclonal antibodies already available at ILRAD, it was possible to calculate the total amounts of IgM and IgG present in bovine serum, lymph, nasal secretions and white blood cells. Monoclonal antibodies are now being raised and tested which recognize IgG1, IgG2 and IgA, as well as immunoglobulin light chains. Detailed analysis of three monoclonal antibodies which all recognized bovine IgM revealed that they detected three different epitopes on the IgM molecule. Only one recognized IgM on B lymphocytes of all cattle tested. The proportion of lymphocytes expressing IgM varied widely —from 4 to 31%—among different cattle, but remained the same over time for individual animals and was very similar for identical twins. These findings suggested that the proportion of B cells expressing IgM among the total B cell population may be genetically determined. Work is now in progress to produce monoclonal antibodies which recognize B cells at different stages of maturation. These will be used to study the activation of B-cell immunoglobulin production during trypanosome infection. Research is in progress on T vivax infection in goats to determine the type and level of antibody response produced in animals infected with this species and the degree of protection achieved against rechallenge with the same (homologous) parasites used in an initial infection. In previous years, attempts to immunize goats by tsetse-transmitted infection followed by drug treatment were not fully successful. Possibly the infected tsetse which transmitted the initial infection extruded too few metacyclic parasites to induce an adequate immune response or to represent the full repertoire of antigenic types which the animals encountered later on rechallenge. To overcome this problem, two groups of goats were infected with large numbers of metacyclic trypanosomes, transmitted to one group by 100 infected tsetse feeding for 15 days and to the other group by injection with 100 to 1 million metacyclic forms produced in vitro. All goats were treated with diminazene aceturate 4 weeks after infection and rechallenged 3 weeks later. Only 8 out of 36 animals showed resistance to the challenge infection, though infections were delayed and parasitaemias were lower than in controls. Sera from resistant goats killed 50 to 60% of homologous metacyclic forms in vitro, while sera from the goats which remained susceptible to challenge only killed 8 to 15%. Thus, induction of immunity was erratic and appeared unrelated to the number of trypanosomes used in the original infection. Pathogenic effects of trypanosomiasis Anaemia In most early cases of trypanosomiasis there is an acute onset of anaemia corresponding closely with the detection of parasites in the bloodstream. Later, in chronic infections, levels of anaemia are observed which appear unrelated to the rise and fall of parasite populations. A project was initiated in 1987 to examine these two types of anaemia and to explore how some breeds of cattle, such as the trypanotolerant N'Dama withstand the pathogenic effects of trypanosome infection better than susceptible breeds. Anaemia in trypanosome-infected animals may be due in art to impaired bone marrow function. Undifferentiated stem cells in the bone marrow give rise to both erythrocytes (red blood cells) and white blood cells of different types. Trypanosome infection in susceptible file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis animals could have an inhibitory effect on cell production in the bone marrow and/or result in the premature destruction of developing cells. In order to investigate the effects of trypanosome infection on bone marrow function, a project was initiated in 1987 to establish culture systems to support the growth of bovine bone marrow cells in vitro. Both short- and long-term culture systems are being developed, as well as improved methods for identifying cell types in bone marrow cultures. Bone marrow derived from the sternum of calves was used to establish colonies which gave rise to erythrocytes monocytes/macrophages, neutrophils. eosinophils and mast cells. Once these systems are fully established, they will be used to compare bone marrow function in trypanosome-infected and noninfected cattle. Within the bone marrow, a network of stromal cells appears to influence the survival, proliferation aid development of undifferentiated stem cells probably through the release of a variety of soluble growth factors. In collaboration with Rockefeller University and biotechnology organizations, ILRAD scientists are using DNA hybridization techniques to produce bovine growth factors and investigate their rote in cell development in the bone marrow. Endocrine dysfunction In susceptible livestock growth, fertility and immune responses are all impaired during trypanosome infection. These important functions are influenced by hormones—some of which are small proteins secreted by glands of the endocrine system. Previous studies showed that T b brucei parasites, perhaps when they were dying, released enzymes into the bloodstream of infected mice which were capable of degrading host proteins. Research was initiated in 1987 to assess the effects of trypanosome infection on hormones in the bloodstream of infected cattle and to elucidate the mechanisms involved. Scientists are measuring hormone levels in Boran cattle infected with T congolense and comparing these with levels in noninfected controls. An in vitro assay system is also being developed to measure the level of parasite enzymes in the bloodstream of infected animals. This will be used to investigate whether increased levels of parasite enzymes are associated with severity of disease. Control of infection in trypanotolerant and susceptible cattle Studies in the field and more recently at ILRAD under experimental conditions have established that N'Dama cattle are more capable of controlling trypanosome infection and resisting clinical signs of disease than African Bos indicus cattle or the European taurine breeds. In a study initiated in 1985, eight N'Dama cattle and eight age- and sex-matched Boran were challenged with tsetse-transmitted T congolense on five occasions. The first four T congolense challenges were with clones belonging to four different serodemes of East African origin. The fifth challenge was with the same clone used for the first challenge 2 years earlier. In addition to the 16 cattle used throughout the experiment, groups of 8 naive Boran were included in the second, third, fourth and fifth challenge. Levels of parasitaemia, anaemia as indicated by packed red cell volume (PCV), red and white blood cell counts and weight were measured throughout the series of experiments. Changes in white blood cell populations and reproductive performance were also monitored during part of the experimental period. At the end of each infection period, all the cattle were treated with diminazene aceturate and an additional curative dose was given to any animal which developed severe anaemia during infection, as demonstrated by a drop in PCV to 15% or lower. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis Control of parasitaemia and anaemia None of the eight N'Dama required drug treatment during any of the five challenges. By contrast, among the eight Boran challenged with four different serodemes, more than half required treatment after each challenge. Four Boran required treatment after the second challenge with the first serodeme, indicating that their resistance to trypanosome infection did not improve significantly with repeated exposure. Altogether, the eight Boran required treatment in 30 out of a total of 40 infection periods (75%). Half or more of the naive Boran included in the second, third, fourth and fifth infection period also required treatment. These experiments showed that the N'Dama were significantly more capable than the Boran of withstanding the pathogenic effects of trypanosome infection, as indicated by levels of anaemia, and also that all four serodemes of T congolense used in the experiment were of similar virulence. The responses of the two groups to the first and second challenge with the same T congolense serodeme were considerably different, as shown in Figure 16. Parasitaemia levels were slightly lower in the Boran following the second challenge, but the pattern was similar during the two challenge periods. Only five of the eight N'Dama became parasitaemic during the second challenge. These animals showed sporadic parasitaemia from 22 to 67 days after challenge and thereafter no parasites were detected. During the first challenge, six out of eight Boron required drug treatment; when challenged with the same serodeme 2 years later, four out of eight still required early treatment. Their average PCVs dropped sharply in a similar pattern during both challenge periods. Among the N'Dama, average PCVs dropped during the first 35 days of the first challenge, though less steeply than among the Boron, reached a low of 22.5%, and then gradually recovered. When the N'Damas were challenged a second time with the same serodeme there was no appreciable drop in PCVs. Figure 16. Development of parasitaemia and anaemia in N'Dama and Boran cattle during two infections with the same T congolense serodeme (IL1180), in May 1985 and April 1987. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis Groups of eight 13-month-old N'Dama and Boron were challenged successively with tsetse- transmitted T congolense IL1180, IL2642, IL1587, IL2079 and again with IL1180. All the cattle were treated with diminazene aceturate 4 to 6 months after each challenge. In addition, six of the Boron required treatment during the first infection with IL1180 when their PCVs dropped to 15% and four required treatment during the second infection with IL1180. None of the N'Dama required treatment. Graphs show levels of parasitaemia during the first (A) and second (B) infections with IL1180, expressed as estimated average number of trypanosomes per ml of blood, and levels of anaemia during the first (C) and second (D) infections, expressed as average PCVs. Mechanisms of resistance An analysis of sera collected during the first T congolense challenge period showed that levels of antibody production were similar in the N'Dama and the Boron and antibodies produced by both groups were capable of neutralizing trypanosomes in vitro with equal efficiency. However, in the presence of antibodies, white blood cells from the N'Dama destroyed trypanosomes more efficiently than white blood cells from the Boran. Such information on the mechanisms involved in resistance to trypanosome infection could facilitate the search for genetic markers to identify trypanotolerant animals for selective breeding programs. Epidemiology Trypanosome characterization An important goal of ILRAD's trypanosomiasis research program is to develop accurate, sensitive and simple methods to detect trypanosomes in infected tsetse and livestock and to identify parasite species and serodemes. This work is important for trypanosomiasis diagnosis and treatment and for epidemiological studies, for instance to assess levels of resistance to trypanosome infection in livestock maintained in tsetse-infested areas, to evaluate the efficacy of trypanocidal drug treatment programs or to predict the level of trypanosomiasis risk in an area before introducing susceptible livestock. Serological typing Monoclonal antibodies have been raised against in vitro-propagated procyclic forms of T congolense, T vivax and T brucei (T b brucei and T b rhodesiense). In 1987, these reagents were tested with procyclic and bloodstream forms of 13 T congolense, 6 T vivax, 4 T b rhodesiense, 5 T b gambiense and 3 T simiae isolates from different parts of Africa, using IFA and ELISA. The antibody raised against T brucei reacted specifically with the brucei group of parasites, while the antibody against T congolense reacted with the T congolense and T simiae isolates and the antibody against T vivax reacted only with parasites of that species. Another monoclonal antibody raised against T congolense reacted specifically with T congolense and not with T simiae. These monoclonal antibodies have been used successfully to detect species-specific trypanosomal antigens in sera from experimentally infected cattle. Arrangements are now being made to test them for use in the field. Experiments conducted in 1987 in collaboration with the Vrije Universiteit Brussel (Belgium) indicated that the monoclonal antibody raised against T brucei can be used to diagnose T evansi. This reagent recognized T evansi antigens in sera from goats experimentally infected with isolates from South America and Indonesia and in sera from naturally infected water buffalo and cattle from Indonesia. The T brucei monoclonal antibody also detected circulating trypanosomal antigens in sera from human patients infected with T b rhodesiense. Out of 142 file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis patients known to be infected by parasitological diagnosis, the monoclonal antibody detected trypanosomal antigens in 128 individuals, or 90.1%. The antibody also detected antigen in sera from 8 out of 69 (11.6%) patients suspected of having trypanosomiasis but without a positive parasitological diagnosis. When tested with sera from 425 non-infected individuals, there were no false positive reactions. This test has been offered for further evaluation to the WHO/UNDP/World Bank Special Program for Research and Training in Tropical Diseases. Present work at ILRAD focuses on standardizing diagnostic assays for use in the field. The monoclonal antibody raised against T brucei recognizes a protein epitope on trypanosomal antigens, whereas the monoclonal antibodies against T congolense and T vivax recognize carbohydrate epitopes. Efforts now concentrate on identifying a protein antigen for T congolense and purifying the antigens recognized by ILRAD's monoclonal antibodies for each species. The objective is to construct amino acid sequences and possibly to synthesize the species-specific antigens for improved diagnostic tests. Another project in 1987 concentrated on elucidating the antigenic composition of different stocks of T vivax isolated at various locations in Africa. Preliminary results showed no reactivity between parasites obtained from Nigeria and two different stocks from Kenya. However, late infection sera from animals infected with a T vivax stock from Uganda recognized a percentage of the Nigerian parasites. This confirms earlier evidence that geographically separate populations of T vivax in Africa may possess overlapping antigenic repertoires. Chromosome profiles The development of improved methods for identifying trypanosome serodemes has concentrated initially on T congolense stocks isolated from cattle at Kilifi in Kenya's Coast Province. A technique known as molecular karyotyping makes it possible to compare the size distribution of chromosomes from different groups of parasites. Using this technique, it was possible to divide 117 T congolense clones, derived from 54 stocks isolated at Kilifi, into 18 chromosome-profile groups. Work in 1987 concentrated on identifying any correlation between chromosome profiles and trypanosome serodemes. Seven chromosome-profile groups were selected at random and a representative clone from each group was inoculated into either a cow or a goat. The animals developed chronic infections or became infected and recovered spontaneously. Sera obtained from each animal at monthly intervals were then tested for cross-neutralizing antibody activity against other parasite clones from the same chromosome profile group or from different groups. Chronic infection sera from animals infected with one trypanosome clone neutralized most, if not all, other clones from the same chromosome profile group, but not clones from different groups. These results suggest that clones with the same chromosome profile may express similar VSG repertoires and thus belong to the same serodeme. Use of DNA probes ILRAD scientists have developed six species-specific DNA hybridization probes which recognize T b brucei and T b gambiense, the T congolense parasites isolated at Kilifi, savannah-type T congolense isolated elsewhere in East Africa, T simiae and East and West African populations of T vivax. Work in this area has focused primarily on trypanosomes of the Nannomonas subgenus—savannah-type T congolense, Kilifi-type T congolense and T simiae. In 1987, a collaborative project was carried out with the University of Bristol (UK) to determine whether either of ILRAD's T congolense probes could detect West African forest/riverine T congolese isolated in The Gambia and whether ILRAD's East African T simiae probe could file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis detect West African T simiae. ILRAD's T simiae probe hybridized to the T simiae sample from West Africa, but the savannah-type T congolense probe hybridized only very weakly to the riverine/forest T congolense and the Kilifi-type T congolense probe did not hybridize to this sample at all (Figure 17). Re-examination of the riverine/forest isolates revealed that they comprised mixed populations including both savannah-type and forest-type parasites. A DNA probe for forest- type T congolense was subsequently cloned at Bristol which did not hybridize with savannah- type T congolense or with T congolense from Kilifi. Figure 17. Dot blots were made on filter paper using DNA from six different trypanosome isolates known or suspected to be T congolense or T simiae. T b brucei material was used as a control. Each sample was spotted several times across the filter and the filter was cut into three strips, each containing two spots from each isolate. The strips were hybridized separately with DNA probes specific for East African savannah-type T congolense (A), Kilifi- type T congolense (B) or T simiae (C). Spots at positions 1, 2, 3 and 5 contained DNA from West African T congolense, which hybridized weakly with the savannah-type probe but not with the others. Subsequent analysis revealed that these isolates contained mixed populations of riverine/forest and savannah-type parasites. Spots at position 4 contained DNA from T congolense isolated at Kilifi which hybridized exclusively with the Kilifi-type probe, and spots at position 6 contained DNA from West African T simiae which hybridized exclusively with the East African T simiae probe. The arrow indicates the position of T brucei DNA. This work led to the following conclusions: (1) ILRAD's T simiae DNA probe detects both West and East African T simiae; (2) ILRAD's T congolense probe detects savannah-type T congolense from both East and West Africa but not West African riverine/forest or Kilifi-type T congolense; and (3) West African riverine/ forest T congolense may comprise a genomically distinct group. Several other laboratories have expressed interest in ILRAD's trypanosome DNA probes and more collaborative experiments will be conducted in this area. One objective is to test ILRAD's probes with a larger number and greater diversity of trypanosomes isolated in different parts of Africa. Identification of species-specific proteins In 1987, scientists investigated glycoproteins on the surface of procyclic forms of different file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis trypanosome species. Species specific differences in procyclic surface proteins might be used to identify trypanosome species which are difficult to distinguish by other methods. Glycoproteins were identified on the surface of T congolense, Kilifi-type T congolense, T simiae, T b gambiense, T b rhodesiense and T b brucei procyclic forms. Using several labeling methods, it was possible to distinguish Kilifi-type T congolense, T simiae and T b gambiense from the other species. This project was carried out in collaboration with the University of Nairobi. Livestock production under trypanosomiasis risk In the vast tsetse-infested regions of Africa, livestock production depends largely on trypanocidal drugs, both to prevent trypanosome infection and to treat infected animals. Regular drug treatment will continue to be the most important measure for trypanosomiasis control until the problems associated with initiating and maintaining tsetse control are overcome or an effective trypanosomiasis vaccine becomes available. However, no new trypanocidal compound has been introduced commercially for almost 30 years. At the same time, there have been increasing reports from the field of trypanosome resistance to some of the most widely used trypanocides, including isometamidium chloride (Samorin: May and Baker), most often used to prevent trypanosome infection in livestock, and diminazene aceturate, generally used to cure infection. Trypanocidal drugs: detection and testing in vitro One major constraint on the development of new trypanocidal drugs for human and veterinary use is the expense of drug testing in experimental animals. In vitro systems are urgently needed to assess drug resistance in trypanosome populations and to test the effectiveness of new trypanocidal compounds. Although the activity of a trypanocidal compound in the laboratory may not always correspond precisely with its effectiveness in the field, in vitro drug testing can provide important insights into problems associated with trypanosomiasis control and possibilities for improvement. Initial research in this area was conducted to determine whether in vitro cultivation might alter the drug sensitivity or resistance of trypanosome populations. In collaboration with the Kenya Trypanosomiasis Research Institute and the Free University of Berlin, ILRAD scientists maintained a stock of T b brucei in vitro which was known to be sensitive to one commercial trypanocide and resistant to four others. Parasites maintained for 4 months as bloodstream forms or propagated through different stages of the life cycle all retained their initial range and level of drug sensitivity or resistance. Two simple, inexpensive methods for assessing drug resistance are now being tested. The first is a drug incubation infectivity test. Bloodstream-form trypanosomes are isolated from an infected animal and cultured in vitro with various concentrations of a trypanocidal drug. After a period of incubation, some of the parasites are injected into mice while the remainder are assessed for their ability to multiply in vitro in the presence of the drug. Drug-resistant trypanosomes continue multiplying in vitro and retain their infectivity for mice, while drug- sensitive parasites do not. This method has been tested successfully with stocks of T b brucei and T congolense; it cannot be used with most stocks of T vivax since this species is generally not infective for mice. The second method involves isolating bloodstream-form trypanosomes from an infected animal and allowing the parasites to develop into uncoated procyclic forms in vitro. Different concentrations of trypanocidal drug are incubated with the cultures and parasite growth is assessed using a Coulter counter. This approach may be more suitable for small laboratories in the field because procyclic trypanosomes are easier to maintain in culture than bloodstream file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis forms: no feeder-layer cells or incubator is required. Preliminary experiments showed that both diminazene aceturate and isometamidium chloride inhibited the multiplication of T b brucei and T congolense procyclic forms, and clear distinctions could be observed between drug-resistant and sensitive parasite strains. This approach has the advantage of simplicity, but again it is not suitable for use with T vivax because the procyclic forms of this species do not multiply in vitro, even in the absence of trypanocidal drugs. Scientists are also using in vitro assay systems to assess the trypanocidal activity of several new drugs. One of these, 9-deazainosine, may be useful for treating trypanosome infections which are resistant to currently available compounds. In in vitro tests, 9-deazainosine had a trypanocidal effect on a T brucei stock which was resistant to both diminazene aceturate and isometamidium chloride. This drug is now being tested using other drug-resistant trypanosome stocks. Trypanocidal drugs: detection in treated animals To evaluate the effectiveness of trypanocidal drug treatment and devise more effective treatment regimes, techniques are required to assess the level of trypanocidal activity present in the bloodstream of treated animals. Several methods have been developed and tested to measure the amount of trypanocidal drugs present in mammalian sera. Different techniques for extraction and measurement are required for each different compound. ILRAD scientists began work in 1987 on a new approach using a bioassay to measure the inhibition of parasite growth and infectivity as an indication of trypanocidal activity in serum samples from treated animals. First, T b brucei bloodstream forms were maintained for 6 months in an in vitro culture system which supported a daily growth rate of approximately 500%. Parasites from this population were then incubated with sera taken from goats after treatment with diminazene aceturate. Sera from treated goats inhibited both trypanosome growth and infectivity, with infectivity the more sensitive indicator of drug activity. A difference in the inhibition of infectivity could be observed between drug-resistant and susceptible parasite strains. Further studies involve culturing trypanosomes with sera taken from cattle at different times after drug treatment in order to estimate the period of effective protection. One drawback is that serum from some animals has a trypanocidal effect even in the absence of drugs. Very few trypanocidal drugs can pass through the natural physiological barriers that separate the central nervous system from the bloodstream. For this reason, drug treatment becomes much more difficult in cases where trypanosomes enter the central nervous system. In a project supported by WHO, ILRAD scientists have developed a cannulation system, using goats, to monitor drug levels in cerebrospinal fluid. The bioassay which has been developed to measure trypanocidal activity in sera from treated animals will now be tested using cerebrospinal fluid. Trypanocidal drugs: evaluation of protection Livestock owners in Africa have been using isometamidium chloride for over 25 years to prevent trypanosome infections in cattle. Despite the widespread use of this and other trypanocidal drugs, very little is known about the effectiveness or duration of protection afforded by drug treatment. Reports from the field have indicated that the period of protection provided by isometamidium chloride can vary considerably from one area to another. ILRAD scientists are investigating this variation in drug protection in collaboration with the University of Glasgow (UK). Although earlier studies had shown that cattle treated with isometamidium chloride (0.5 mg/kg) file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis were protected for at least 3 months against monthly tsetse-transmitted challenge with certain stocks of T congolense, drug treatment conferred protection against a Nigerian population of T vivax for only 2 months and protection against a T vivax population isolated in Kilifi, Kenya, lasted less than 4 weeks. However, the same drug was fully effective as a curative against pre-existing infections with either parasite population. In 1987, similar experiments were conducted using two other Kenyan T vivax populations, one originating from Galana and one from Likoni (Figure 18). Cattle treated with isometamidium chloride (0.5 mg/kg) became infected with the Galana parasite population 1 month after drug treatment and with the Likoni parasites 2 months after treatment. As in the previous experiments, both parasite populations were fully sensitive to the therapeutic activity of isometamidium chloride (at 0.5 mg/kg) yet resistant to the drug's prophylactic activity. T vivax Animal —Infection Detected— strain number Month Day After Monthly Challenge IL2969 A1 2 7 (Kilifi) A2 2 9 A3 1 12 A4 1 23 A5 1 24 IL2982 B1 1 9 (Galana) B2 1 9 B3 1 9 B4 1 14 B5 1 9 IL2986 C1 2 12 (Likoni) C2 2 12 C3 2 12 C4 2 12 C5 2 14 Figure 18. Effectiveness of isometamidium chloride for preventing T vivax infection in cattle: results from two experiments using parasites isolated in Kenya. Three groups of five yearling Boran steers were inoculated with isometamidium chloride (0.5 mg/kg) and then challenged with one of three different stocks of T vivax derived from different locations in Kenya's Coast Province. All animals were challenged monthly with 10 infected tsetse, beginning 1 month after drug treatment. Two untreated controls were also challenged monthly with each stock as a check on the infectivity of the parasites. These results are interesting because isometamidium chloride is used primarily as a prophylactic drug. They indicate that tests using isometamidium chloride to treat pre-existing infection do not provide a reliable indication of the effectiveness of the drug as a prophylactic to prevent infection. African trypanotolerant livestock network Small populations of cattle, sheep and goats are found in West and Central Africa which possess some degree of resistance to trypanosomiasis. This resistance, which has been termed trypanotolerance, is generally attributed to the N'Dama and West African Shorthorn breeds of cattle, Djallonké sheep and Dwarf West African goats. Field studies have shown that file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis trypanotolerant cattle can be as productive as other indigenous African breeds in areas of zero or low trypanosomiasis risk; in areas of high risk, only trypanotolerant breeds can survive. Trypanotolerant N'Dama cattle originating in the Fouta Djallon region of Guinea have been established in several tsetse-infested areas of Central Africa—in the Central African Republic, Gabon, Congo and Zaire. N'Dama cattle are now being imported by several other countries in West and Central Africa where they are forming the nucleus of livestock development programs in tsetse-infested areas. ILRAD is collaborating with ILCA and with national livestock ministries and development programs in West and Central Africa in a research network established to study the health and productivity of trypanotolerant and susceptible cattle, sheep and goats under different management conditions and exposed to different levels of tsetse-transmitted trypanosomiasis risk. The goal of this African Trypanotolerant Livestock Network is to provide information on genetic and acquired aspects of trypanotolerance and the factors affecting the susceptibility of livestock to trypanosome infection. This information should lead to improved breeding programs and the optimum use of trypanotolerant livestock in the vast tsetse-infested regions of Africa. The research network will also provide a framework for testing existing and future trypanosomiasis control measures as they become available. Scientists from national programs are collecting information on animal health, animal production and tsetse challenge at 15 sites in West and Central Africa—5 in The Gambia, 4 in Zaire, 2 in Togo, 2 in Ivory Coast, 1 in Gabon and 1 in Senegal. Comparable information is also being collected at three associated sites in East Africa—in Kenya, Tanzania and Ethiopia. ILRAD provides training and supervision for monitoring tsetse populations, trypanosome infection rates in tsetse and livestock and the health status of animals included in the study. ILCA helps collect information on animal production and nutrition and is responsible for processing and analysing health and productivity data from all the network sites. By the end of 1987, participants in the network were collecting data every month on over 10,000 animals. Because of the crucial importance of accurate data collection and standardization among the different sites, field staff working within the network receive intensive training and careful supervision. Two 7-week training courses are conducted at ILRAD every year, in English and in French. By the end of 1987, 63 staff members working in the network had received training at ILRAD. Tsetse challenge and trypanosome infection The tsetse component of the African Trypanotolerant Livestock Network involves collecting data at 12 sites on a monthly basis. Tsetse density is measured for 5 to 10 consecutive days each month, using biconical traps. Relative density, expressed as the average number of flies captured per trap each day, is a function of real tsetse density, behaviour and nutritional status plus position of the traps and weather conditions. Analyses completed in 1987 revealed a statistically significant relationship between monthly estimates of tsetse challenge and trypanosome prevalence in trypanotolerant cattle—both across sites and, in Ivory Coast and Zaire, between locations at individual sites. Intensive research has been initiated at several sites aimed at developing more accurate methods for estimating tsetse challenge. This work includes tsetse mark/release/recapture experiments to improve estimates of actual population density and experiments to determine the nutritional status of tsetse populations by fat/haematin analysis. Network scientists are also sending samples of tsetse flies and mammalian blood to ILRAD for analysis to detect trypanosome infection and determine parasite species. Evaluation of trypanosomiasis control file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis In association with the African Trypanotolerant Livestock Network, the Kenya Ministry of Livestock Development initiated a project in 1982 at Muhaka near the Kenya coast. The goal was to monitor the health and productivity of susceptible East African Zebu cattle maintained under village conditions in a tsetse-infested area with and without regular prophylactic drug treatment. The area was infested with Glossina pallidipes, G brevipalpis and G austeni. T congolense and T vivax infections were detected in cattle, with monthly infection rates averaging from 1 to 8%. Two-thirds of the cattle included in the study were treated on a regular basis with isometamidium chloride, while the others, acting as controls, received only occasional curative treatment. Results reported in 1987 showed that cattle receiving regular drug treatment were 20% more productive than controls (Figure 19). Production With Without Parameter Treatment Treatment Viability Annual cow viability (%) 95.0 95.0 Calf viability to 12 months (%) 96.3 91.3 Young stock viability 12-30 months (%) 98.9 97.5 Body weights Cow weight (kg) 185.0 189.0 Calf weight at 12 months (kg) 63.4 58.5 Productivity Annual calving rate (%) 77.0 76.4 Annual extracted milk yield (kg) 150.6 120.5 Annual productivity index: weight of 12-month-old calf plus liveweight equivalent of milk extracted Productivity per cow (kg) 56.4 47.6 Productivity per 100 kg body weight 30.5 25.2 of cow (kg) Productivity per 100 kg 124.8 103.8 liveweight of cow (kg) Figure 19. Effect of regular prophylactic treatment with isometamidium chloride on the productivity of East African Zebu cattle in Muhaka, Kenya. A tsetse control campaign was initiated in 1987 at a network site in the Boundiali area of Ivory Coast using insecticide-impregnated traps and screens. Estimates of tsetse challenge and trypanosome prevalence in livestock in the experimental area and in a control area will allow a thorough evaluation of the effectiveness of this type of tsetse control operation. Indications of heritability If genetic improvement of trypanotolerant livestock is to be achieved, it is vital to determine whether the components of trypanotolerance are under genetic control. Trypanotolerance appears to be associated with at least three possibly related but genetically independent characteristics—the ability to control parasitaemia, to resist anaemia and to develop an effective immune response. Breeding programs have been introduced at network sites with high natural tsetse challenge in Gabon and Zaire to build up groups of paternal half-siblings file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis which will make it possible to estimate the heritability of these traits. Preliminary results suggest that the ability to maintain PCV levels under high natural challenge might provide the basis for a practical selection approach for anaemia control. In certain circumstances it may also be possible to select for the ability to acquire resistance after repeated infection. An association between disease resistance and MHC type has been demonstrated in some domestic and laboratory animals. The development of a practicable and reliable system for typing large numbers of cattle according to class I MHC antigens—and selected individuals according to class II antigens—has made it possible to begin searching for associations between MHC type and trypanotolerance. Network scientists have collected blood samples from approximately 800 N'Dama cattle in The Gambia and Zaire and 200 of these have been typed using ILRAD's panel of antisera and monoclonal antibodies. An association between MHC type and trypanotolerance would make it possible to develop highly cost-effective programs to select trypanotolerant animals. Preliminary examination of class I antigens has indicated that the N'Dama have a number of characteristics seen in no other breed so far examined. These differences will be analysed in detail once MHC typing of all the animals is complete. Research on N'Dama productivity in The Gambia and Senegal ILCA and ILRAD are participating in a research project on the productivity of N'Dama cattle at five village sites in The Gambia and Senegal. The project was initiated in 1985 under the African Trypanotolerant Livestock Network with funding from the European Economic Community (EEC). The objective is to evaluate the productivity of N'Dama cattle exposed to different levels of tsetse challenge and trypanosomiasis risk under traditional village management conditions. Information collected through this project will allow a critical evaluation of the degree of trypanotolerance exhibited by N'Dama, the possible role of acquired resistance and the influence of various management factors. The research team works in close collaboration with a tsetse research project funded by the British Government's Overseas Development Administration (ODA) and with a large-scale livestock development project funded by the African Development Bank. All three projects are based at the International Trypanotolerance Centre at Kerr Serigne in The Gambia. Study sites in The Gambia (Figure 20) are located at Gunjur and Pirang villages near the coast, with low trypanosomiasis challenge transmitted by G palpalis gambiensis; at Keneba and Nioro Jattaba villages about 150 km inland in an area moderately infested by G morsitans submorsitans; and further inland at Bansang, where both tsetse species are present and trypanosomiasis challenge appears to be higher. Information has been collected on a regular basis since January 1986 covering 1200 cattle in Gunjur and Keneba. In June 1986, the study was extended to cover an additional 700 cattle in Pirang and Nioro Jattaba. In 1987, the project was extended to Bansang in The Gambia and to Kolda in southern Senegal in collaboration with the Centre de Recherches Zootechniques. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis Figure 20. Map of The Gambia and southern Senegal showing the location of six research sites included in the African Trypanonotolerant Livestock Network and the headquarters of the International Trypanotolerance Centre (ITC). The ILCA/ILRAD team is investigating the health and productivity of N'Dama cattle at the study sites by monitoring trypanosome prevalence, anaemia as indicated by PCV levels and productivity as indicated by calving rates, viability, weight changes and milk production. Information is also being gathered on the presence of anti-trypanosomal antibodies and the incidence of other infectious diseases, and N'Dama herds at Kerr Serigne and Keneba are being typed in terms of class I MHC. The ODA-funded team collects data on tsetse distribution, density and trypanosome infection rates and on the movement of livestock in relationship to the location of tsetse at different times of year. Blood samples are taken from all cattle included in the study on a monthly basis and trypanosomiasis prevalence is assessed as the percentage of animals with detectable infections. Figure 21 shows monthly trypanosomiasis prevalence at four sites from June 1986 through May 1987, revealing a pattern of higher infection rates during the rainy season. T congolense was encountered more frequently than T vivax at all four sites, while no T brucei infection was observed. Analysis of data collected at two sites (Keneba and Gunjur) over a 17- month period showed that higher trypanosomiasis prevalence was associated with higher tsetse infestation rates, higher anti-trypanosomal antibody levels, lower PCV levels and lower average body weights. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis Figure 21. Monthly trypanosomiasis prevalence in N'Dama cattle at four study sites in The Gambia from June 1986 ;o May 1987. Trypanosomiasis prevalence differed widely between age groups, with young adult cattle most frequently infected. Trypanosomiasis prevalence was higher and average PCV values were lower for male than for female cattle, perhaps reflecting greater stress among male animals used for traction. Tests for anti-trypanosomal antibodies revealed maternal antibodies in calves up to 3 months of age. A comparison of average monthly body weights from June 1986 through May 1987 revealed a dramatic weight loss at all four sites during the latter part of the dry season, probably related to nutritional factors. Calf growth and daily milk offtake increased during the rains and dropped during the dry season. Milk offtake for an average 10-month lactation period was 300 kg in Gunjur and 333 kg in Keneba. The age at first calving was 4 to 5 years and the average calving interval was 23 months. This late age at first calving and prolonged calving interval prompted an investigation of the length and frequency of normal oestrus cycles in N'Dama cows. However, it has proven difficult to detect oestrus in animals kept under traditional husbandry conditions. Scientists are testing the effects of dry-season supplementary feeding on calving rates and calf growth. The provision of groundnut meal to young calves at Keneba 3 times a week increased liveweight gains by an average of 49% during the 1987 dry season. During the same 6-month period at Nioro Jattaba, milk offtake, calf growth and cow body weight improved among cows supplemented with groundnut meal. By the end of 1987, data collection was well established at all five sites in The Gambia and at Kolda in Senegal. The collection of large data sets on the same animals every month should make it possible to calculate productivity indices for the different sites and to design a model to predict the influence of various factors on production values. Strategic interventions will also be tested in the areas of animal health, nutrition and management. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Trypanosomiasis file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Trypanosomiasis.htm[5/19/2016 3:35:02 PM] Epidemiology Epidemiology and Socio-economics A research program on the epidemiology and socio-economics of animal disease and its control in Africa was initiated in late 1987 with funding provided by the Rockefeller Foundation. The objectives are to identify the factors which govern the successful application of improved disease control measures—particularly the widespread use of immunization—and to assess the likely impact of improved disease control in economic, social and environmental terms. The impact of disease control measures will be considered at the regional, national and farm level. The project will focus initially on the control of theileriosis within the general framework of tick-borne disease control. In the initial stages, a major aspect of this program will be research into methods of assessing the incidence of tick-borne diseases and their impact on productiviy. Work will also focus on the epidemiological and socio-economic consequences of intervention strategies, such as immunization, chemotherapy and strategic tick control. The methodologies developed through this program should eventually be of value for assessing the impact of control options for other diseases in livestock. Much of this research will be carried out in close collaboration with livestock ministries and research organizations in the countries affected by theileriosis and with other international and regional institutions having similar objectives. ILRAD has agreed on a memorandum of understanding with the Kenya Agricultural Research Institute for collaborative research in this area. The program at ILRAD will include: the compilation of extensive data bases on livestock production in the countries most severely affected by theileriosis the characterization and zonation of livestock production systems based on disease epidemiology, management system, agro-ecological zone and socio-economic environment with a view to determining the impact of potential disease control options under a variety of circumstances the development and verification of predictive models which can be used to ensure the optimal application of improved disease control measures for specific locations or production systems the development and testing of protocols for disease-control impact studies at the farm and community level. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Epidemiology.htm[5/19/2016 3:35:04 PM] Training Training and Information Services Training Information Services The ILRAD library Training ILRAD's training program is designed primarily to develop veterinary research and disease control capabilities in African countries where trypanosomiasis and ECF are important animal health problems. The development of scientific and technical resources in these countries receives high priority at ILRAD: in 1987, training activities accounted for nearly 10% of the annual budget. These funds support individualized training programs for scientists and technicians from laboratories and field programs in Africa and other developing areas, postgraduate training for students working towards master's and doctoral degrees and post-doctoral positions to enhance the professional knowledge and experience of young scientists from many parts of the world. In addition to training for individuals, courses, conferences and workshops are held throughout the year on topics directly related to ILRAD's research program. In 1987, 13 scientists and technicians came to ILRAD for specialized technical training for periods lasting from 1 week to 7 months (Figure 22). Their training programs were planned on an individual basis according to the needs of their home institutions. All were staff members of national research laboratories or livestock development programs in developing countries. In addition, ILRAD sponsored three scientists—from Malawi, Kenya and Zaire—who attended a 2-week training seminar on African trypanosomiasis in Togo organized by the OAU, FAO and WHO. Country 1986 1987 Country 1986 1987 Specialized technical training Burundi 2 1 Somalia 2 – Central African 1 – Tanzania 2 1 Republic Ethiopia 2 – Uganda – 2 India – 1 Zimbabwe 2 – Kenya 5 7 Italy 2 – Nigeria 1 1 USA 1 – Rwanda 2 – Post-graduate training Ethiopia – 1 Zaire 1 1 Ghana 1 1 Canada – 1 Kenya 8 9 Italy 2 2 2 Nigeria 2 1 Netherlands 1 1 Sudan 1 1 UK 1 2 file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Training.htm[5/19/2016 3:35:04 PM] Training Uganda 1 1 USA – 1 Post-doctoral training Kenya 3 3 Germany(FR) 1 2 Tanzania – 1 Ireland 1 2 Australia 1 1 UK 2 2 France 1 1 USA 4 2 Figure 22. Individual training at ILRAD in 1986 and 1987: number of participants in different types of training by country of origin. Post-graduate training includes both Research Fellows and Visiting Research Fellows. Post-doctoral training includes both Senior Research Fellows and Post-Doctoral Fellows. Fifteen post-graduate students were working at ILRAD in 1987 on Research Fellowships lasting from 1 to 4 years (Figure 22). The 12 Research Fellows from African countries were all supported by ILRAD; two also received support from the Kenya Government and one from WHO. Three Research Fellows from developed countries were supported by their home governments. All these students worked with ILRAD scientists on projects closely related to the research program. Three Research Fellows received their Ph.D. degrees and one received an M.Sc. degree in 1987, based on research carried out at ILRAD, and one Kenyan staff member from the ILRAD/Veterinary Research Laboratory Wildlife Project received his M.Sc. degree in the USA. Seven Visiting Research Fellows also worked at ILRAD for shorter periods during the year. Two Senior Research Fellows worked at ILRAD during 1987, one from Kenya and one from Tanzania. This program is for young scientists working at universities or research institutes, principally in Africa, who come to ILRAD for several months to enhance their research experience. Post-Doctoral Fellows are selected on an international basis to meet specific requirements of the research program. They normally work at ILRAD for 2 years. Twelve Post-Doctoral Fellows worked at ILRAD in 1987—from Kenya, Ireland, Germany (FR), the UK, the USA, Australia and France. In addition to training activities organized on an individual basis, four training courses were held in 1987, with a total of 44 participants from 24 African countries. The first, from January to March, was a joint course on African animal trypanosomiasis and the development of regions affected by the disease, conducted by ILRAD, FAO and the Centre de Recherche sur les Trypanosomiases animales in Burkina Faso. The course was conducted in French and held in Bobo Dioulasso. Three courses were conducted at ILRAD during the year, one in English on the diagnosis of haemoprotozoan diseases, with emphasis on trypanosomiasis, and two in French on trypanosomiasis and other parasitic diseases, intended primarily for staff members working in the African Trypanotolerant Livestock Network. ILRAD held a major international workshop in September on ruminant class II MHC genes and gene products and their association with disease susceptibility and resistance. Participants in the 4-day meeting included 24 specialists from 9 different countries, 6 participants from the University of Nairobi and research institutes in Kenya and several ILRAD staff members. The proceedings are being published as a special issue of Animal Genetics. In November, ILRAD hosted an international meeting of the African Trypanotolerant Livestock Network. Researchers working at Network sites in nine African countries presented results from the past 2 years, evaluated future research plans and heard scientific reviews from invited specialists. Information services file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Training.htm[5/19/2016 3:35:04 PM] Training The primary responsibility of ILRAD's Information Services Department is publication of the annual report and the quarterly newsletter, ILRAD Reports, in English and French. In 1987, major articles in ILRAD Reports reviewed research on drug treatment for the prevention and cure of trypanosomiasis, collaborative programs with national and regional institutions in Kenya and other African countries, studies on the bovine MHC and work on Theileria strain characterization. The newsletter also described conferences and workshops held at ILRAD and listed articles and other publications by ILRAD staff. For the first time at the beginning of 1987, an annual highlights report was published in English and French, giving a brief description of research and training activities in 1986. The annual report, quarterly newsletter and annual highlights are intended for a wide range of readers, with emphasis on laboratory and field workers in Africa who are responsible for animal health. The Information Services Department also issues a weekly Internal Newsletter in English and Kiswahili. This is distributed to staff members and the Board of Directors. Detailed results from ILRAD's research program are published in international journals and scholarly books in a form suitable for a specialized scientific audience. In 1987, ILRAD staff members published 64 journal articles plus 7 chapters and 1 full-length report. These are listed in a separate section of this annual report. In September 1987, ILRAD participated for the fourth time in the Nairobi International Show, which is sponsored every year by the Agricultural Society of Kenya. ILRAD's exhibit included a poster display and presentation of video productions on ILRAD in English and Kiswahili. Information Services produced-a short brochure—ILRAD: questions and answers—in both languages for distribution during the Show and for visitors to ILRAD. An updated brochure, Welcome to ILRAD, was also produced in 1987 with information about travel, health, accommodation and other areas of concern for visitors from abroad. Considerable effort is devoted to maintaining and verifying ILRAD's distribution list and responding to requests for publications from around the world. The mailing list gradually expanded in 1987 as scientists, government officials, librarians, journalists and others wrote in asking to receive ILRAD publications on a regular basis (Figure 23). Countries Institutions/Individuals Region 1986 1987 1986 1987 Anglophone Africa 25 24 745 883 Francophone Africa 23 23 268 316 Anglophone and other Europe 19 19 235 254 Francophone Europe 2 2 41 47 North and South America 25 26 277 286 Near and Far East 31 3 233 274 Total 125 125 1799 2060 Figure 23. Comparison of ILRAD Reports distribution at the end of 1987 and the end of 1986. In addition to keeping journalists informed about scientific progress at ILRAD, the Information Services Department contacts local newspaper reporters when important visitors come to IL,RAD or meetings take place which would be of interest to the general public. Information Services also distributes press releases and photographs on these occasions. The three daily newspapers in Nairobi published 21 articles in 1987 giving favourable coverage to activities at ILRAD. The ILRAD library file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Training.htm[5/19/2016 3:35:04 PM] Training The ILRAD library serves staff members, visiting scientists, participants in the training program and staff of other research institutes, government departments and universities in Kenya. The specialized collection concentrates on topics related to ILRAD's research program, with emphasis on scientific journals. Subscriptions to 228 international journals and 36 monograph series cover such fields as parasitology, immunology, biochemistry, entomology, cell biology and veterinary medicine, including several indexing and abstracting journals. Journal subscriptions are reviewed every year according to the recommendations of the scientific staff. The collection of scientific reference books is reviewed on a continuous basis. In 1987, the library acquired 230 new books, including several titles requested by the new Epidemiology and Socio-economics Unit. With irrelevant and out-dated material removed, the book collection has been trimmed to about 2,500 volumes. The ILRAD library participates in an interlibrary loan network along with the University of Nairobi, the Kenya Government's Veterinary Research Laboratory, the Veterinary Research Department of the Kenya Agricultural Research Institute, the Ministry of Health and ICIPE. Interlibrary loans increased in 1987, with ILRAD staff members borrowing 824 items from other libraries in the network and the ILRAD library lending 645 items. The ILRAD -library also donated 213 books and 181 journal issues to other libraries in the Nairobi area. The ILRAD library offers current awareness services through weekly announcements of accessions in the Internal Newsletter and through subscriptions to three monthly selective dissemination of information services. These are provided as printouts by Biosciences Information Services (BIOSIS), the International Information System for Agricultural Sciences and Technology (AGRIS) of FAO and the abstracts service of the Commonwealth Agricultural Bureaux International (CABI). Future plans include acquiring CABI Abstracts and other databases in computerized form on CD-ROM as these become available. The library carries out computer-based literature searches for ILRAD staff, using agents in Europe. In 1987, ILRAD carried out 30 literature searches on a wide range of subjects. The library also orders copies of scientific articles which are not available in Kenya as requested by ILRAD staff members. In 1987, photocopies of 612 articles were purchased from the British Library Document Supply Centre. All articles and reference lists acquired in response to requests by individual staff members are retained in the library for other users. Reprints of scientific articles by ILRAD staff members are supplied on request to researchers and field workers, primarily in developing countries. The distribution of reprints was centralized in the library in 1987 and 608 reprints were distributed free of charge. Computerization of the library's catalogue and acquisition system was begun in 1986 and completed in 1987; now the list of journals is being added to the data base. The next step will be to acquire a system which will give library users access to the computerized catalogue. The ILRAD library has supplied information and advice on library computerization to several other organizations, including in 1987 the University of Nairobi, Kenyatta University and the Nairobi office of the International Planned Parenthood Federation. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Training.htm[5/19/2016 3:35:04 PM] Research Research Support Research support Tsetse laboratory Tick Laboratory Large animal production Laboratory animal production Clinical and Diagnostic Services Biostatistics and Computing Services Tsetse laboratory A great deal of the trypanosomiasis research conducted at ILRAD requires trypanosomes which have developed in tsetse. Research on parasite development in the vector and trypanosomiasis transmission is conducted in close collaboration with staff of the Tsetse Laboratory. ILRAD's Tsetse Laboratory maintained breeding colonies of seven tsetse species in 1987. These were Glossina morsitans centralis originating from mainland Tanzania, G austeni from Zanzibar, G palpalis palpalis from Nigeria, G p gambiensis from Burkina Faso, G fuscipes fuscipes from the Central African Republic, G tachinoides from Tchad and G brevipalpis from Kenya. These species represent the three taxonomic groups of tsetse—morsitans, palpalis and fusca. Their breeding performance in 1987 is summarized in Figure 24. Breeding Daily Female Weekly Pupae Puparial Annual Females Mortality per Female Weight Number Tsetse Species (mean) (mean %) (mean) (mean mg) Puparia G m centralis 10,510 0.37 0.62 33.63 340,036 G austeni 1,293 0.32 0.66 27.26 44,086 G p palpalis 1,303 0.84 0.56 32.06 37,890 G p gambiensis 1,414 0.62 0.71 29.34 51,312 G f fuscipes 1,342 0.37 0.53 36.99 36,719 G tachinoides 1,291 0.61 0.66 20.23 44,268 G brevipalpis 1,374 0.92 0.58 75.18 41,693 Figure 24. Performance of seven tsetse breeding colonies maintained at ILRAD in 1987. Two new tsetse breeding colonies were established during the year from flies captured at two different locations in Kenya. These are G pallidipes (morsitans group) from Shimba Hills and G longipennis (fusca group) from Nguruman. By the end of 1987, the G pallidipes colony consisted of 198 breeding females and had produced 1556 pupae with a mean puparial weight of 41.04 mg. The G longipennis colony had 130 breeding females and had produced 557 pupae with a mean weight of 79.48 mg. All breeding colonies are maintained at 25°C and fed 5 days a week on rabbits. G m centralis and G austeni are kept at 70% relative humidity, and the other species at 80%. These breeding colonies provided all the tsetse required for trypanosomiasis research at file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Research.htm[5/19/2016 3:35:04 PM] Research ILRAD in 1987, as well as tsetse used during two training courses conducted under the African Trypanotolerant Livestock Network. Tsetse puparia and adult flies were also supplied to colleagues at the University of Nairobi, the National Council for Scientific Research (Zambia), the Federal Department of Pest Control (Nigeria), the Swiss Tropical Institute, the Prince Leopold Institute of Tropical Medicine (Belgium), the University of Auckland (New Zealand) and the University of Salford (UK). Tick laboratory ILRAD's ECF research program requires a steady supply of the infective sporozoite forms of T parva. The Tick Laboratory provides large numbers of sporozoites, ticks and tick tissues for a variety of research projects. Theileria sporozoites can only be obtained by continuous passage of the parasites between the tick vectors and the mammalian hosts. In ILRAD's Tick Laboratory, ticks are infected with Theileria parasites by feeding on infected cattle. They are then dissected and the sporozoites are isolated from their salivary glands. Colonies of infected and uninfected ixodid (hard) ticks are maintained by feeding on cattle and rabbits. The most important species is Rhipicephalus appendiculatus, the principal vector of T p parva and T p lawrencei in East Africa. Colonies of other tick species maintained at ILRAD include Amblyomma variegatum and A gemma, vectors of Cowdria ruminantium (heartwater) and of two relatively benign Theileria species—T mutans and T velifera; Boophilus decoloratus and B microplus, which transmit Babesia spp and Anaplasma marginale; and three other Rhipicephalus species, R evertsi evertsi, R pulchellus and R zambeziensis. In 1987, facilities and procedures in ILRAD's Tick Laboratory were reviewed with the aim of improving disease security. Future plans include some expansion and remodeling of the facilities and the installation of a computerized system for storing and retrieving information on the tick colonies. Large animal production The large animal facilities at Kabete housed 590 cattle and 725 sheep and goats during 1987, all allocated for research projects at ILRAD. Of these, 70% of the cattle were used for ECF research, 25% for trypanosomiasis research and 5% for other projects, such as antigen production. Most of the small ruminants were used for trypanosomiasis research. ILRAD's ranch, Kapiti Plains Estate Ltd, supplied 217 Boran calves, 53 Bos taurus calves, 7 Boran steers, 6 Boran heifers and 13 Bos taurus heifers to ILRAD in 1987 and sold 617 cattle to other users. Cattle production at Kapiti was adversely affected during the year by low rainfall and by two outbreaks of foot and mouth disease, one on the ranch and one in the neighbouring area. Quarantines were imposed on both occasions, interrupting the sale of livestock from the ranch and leading to high stocking rates. Cattle at Kapiti are vaccinated 3 times a year against foot and mouth disease but the outbreak on the ranch in 1987 was caused by a new, highly virulent strain which was not included in the vaccine. The disease outbreak also interrupted embryo transfer work at Kapiti. Altogether, 967 calves were born on the ranch during the year from a breeding herd of 1118 cows, resulting in a calving rate of 86%. Improvement of facilities at Kapiti included the construction of a workshop and staff housing. Both Boran and N'Dama calves are now produced at ILRAD by embryo transfer on a regular basis. Over a 4-year period, methods have been tested and standardized for synchronizing the ovulation of donor and recipient cows, stimulating superovulation in donors, assessing the suitability of recipients and handling and splitting embryos before implantation. In both breeds, file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Research.htm[5/19/2016 3:35:04 PM] Research an average of just under three embryos is now obtained per embryo-producing donor during each superovulation. The objective of embryo transfer work, in Boran cattle is to produce animals of specific MHC types, both as single calves and as twins. In 1987, 42 Boran calves were produced by embryo transfer from selected, MHC-typed cows and bulls. This included 32 single calves matched for one or both MHC haplotypes, plus 5 sets of identical twins produced by embryo-splitting. Another 43 Boran calves of specified MHC type were produced during the year by natural coatings. ILRAD's five N'Dama heifers were superovulated four times between November 1986 and November 1987. During this period, all the N'Dama were infected with trypanosomiasis. Unlike susceptible cattle, these trypanotolerant N'Dama were capable of retaining reproductive activity in spite of trypanosome infection. A total of 42 N'Dama embryos were recovered and surgically transferred into Boran recipients, 22 pregnancies were established and 4 N'Dama calves were born by the end of 1987. The goal is to increase the number of N'Dama available for ILRAD's trypanosomiasis research program by producing as many N'Dama calves as possible every year. Laboratory animal production The Laboratory Animal Unit supports ILRAD's research and training activities with a regular supply of mice, rats and rabbits. The unit also maintains small colonies of meadow voles (Microtus montanus), Cotton rats (Sigmodon hispidus) and guinea pigs, which are occasionally required for experimental work. All laboratory animals are housed and maintained according to international animal welfare conventions. ILRAD is generally self-sufficient in terms of mouse and rat production. Colonies of three inbred mouse strains—BALB/c, C3H/He and C57/B16—are maintained. The C3H/He and C57B16 mice are used primarily fo r studies on host resistance to trypanosomiasis. The BALB/c mice and a colony of (BALB/c x Swiss) F1 mice are maintained for the production of monoclonal antibodies. Random-bred Swiss mice are used increasingly for all other research work because they are more productive and faster growing than the inbred strains. ILRAD scientists use rats primarily for the production of trypanosomes. All are random-bred, originating from the Sprague Dawley strain. Rabbits are used mainly to produce antisera and to support the tsetse and tick colonies. Expansion of rabbit production has been impeded by outbreaks of Staphylococcus aureus. Although the number of rabbits weaned in 1987 increased by 32% over the 1986 level, ILRAD still relied on outside suppliers for about one- third of the rabbits required during the year. Figure 25 shows the total number of mice, rats and rabbits produced and used at ILRAD in 1987. The demand for mice decreased by 15% from the 1986 level, while the demand for rats increased by 15% and the demand for rabbits increased by 13%. In addition to supplying nearly all the laboratory animals required by ILRAD scientists, the breeding unit provided a total of 2806 mice and 870 rats to other research organizations. In Kenya, these were the Kenya Government's Veterinary Research Laboratory, the Kenya Trypanosomiasis Research Institute (KETRI), the Kenya Medical Research Institute (KEMRI), the Institute for Primate Research, four departments of the University of Nairobi and several secondary schools. Mice were also donated to a heartwater research project in Zimbabwe and to the National Veterinary Research Laboratory in Somalia. Number Number Supplied Species and Strain Weaned to Scientists file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Research.htm[5/19/2016 3:35:04 PM] Research Mice random-bred Swiss 30,362 19,762 inbred BALB/c 10,444 8,828 inbred C3H/He 5,271 3,524 inbred C57B1/6 2,986 1,674 BALB/c × Swiss F1 5,298 3,117 Rats 18,432 15,336 Rabbits 1,081 1,467 Figure 25. Rats, mice and rabbits produced and supplied to ILRAD scientists in 1987. Clinical and diagnostic services The Diagnostic Laboratory carries out several types of routine analysis in support of ILRAD's research program and animal production facilities. In 1987, a total of 37,519 samples were received for analysis. These included 26,783 serological analyses, 3,001 bacteriological analyses, 7,235 haematological analyses and 500 helminthological analyses. Serum samples from experimental livestock were screened throughout the year for antibodies to seven bloodstream parasites—Theileria parva, T mutans, Trypanosoma brucei, T congolenzse, T vivax, Anaplasma marginale and Babesia bigemina. In addition to services provided to ILRAD scientists, the Diagnostic Laboratory continued to provide assistance to. other institutes and projects. In 1987, antigens, conjugates and lymphocyte lysates were provided to the International Trypanotolerance Centre in The Gambia, a German (FR) Gesellschaft fur technische Zusammenarbeit (GTZ) project in Burundi, a Belgian Development Cooperation project in Zanzibar and the National Veterinary Services in Australia. In addition, 30 serum samples were screened for antibodies to bloodstream parasites for the Directorate of Animal Resources and Fisheries in Southern Sudan. Most screening for Theileria and Trpanosoma parasites was by IFA and ELISA. In 1987, veterinarians from the National Health Laboratories in Uganda, the International Atomic Energy Agency (IAEA) in Austria and the Belgian Development Cooperation agency visited ILRAD's Diagnostic Laboratory to learn these techniques. Biostatistics and computing services ILRAD's Biostatistics and Computing Services Unit, in conjunction with the University of Strathclyde (UK), advises research staff on statistical problems and the installation and use of microcomputers. Major projects in 1987 included statistical support for trypanosomiasis experiments in N'Dama and Boran cattle, for ECF infection and treatment field trials at three sites in Burundi and for a study on the frequency of cytolytic T cells in cattle immunized against ECF. The Unit helps maintain databases on bovine MHC types and on all trypanosome and tick-borne disease stabilates available at ILRAD. In 1987, the Unit helped prepare a user's manual on ILRAD's automated MHC typing system. Work continues on the expansion and improvement of ILRAD's telecommunications system. Data communication with other centres in the CGIAR is fully functional through the CGNET system and ILRAD is now also linked with research organizations and universities in many countries through the BITNET international electronic mail service. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Research.htm[5/19/2016 3:35:04 PM] Research file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Research.htm[5/19/2016 3:35:04 PM] 1987_Pubs 1987 Publications Baldwin, C.L., Goddeeris, B.M. and Morrison, W.I. (1987). Bovine helper T-cell clones specific for lymphocytes infected with Theileria parva (Muguga). Parasite Immunology. 9: 499–513 (461). Baldwin, C.L. and Teale, A.J. (1987). Alloreactive T cell clones transformed by Theileria parva retain cytolytic activity and antigen specificity. European Journal of Immunology. 17: 1859–62 (533). Chema, S., Chumo, R.S., Dolan, T.T., Gathuma, J.M., Irvin, A.D., James, A.D. and Young, A.S. (1987). Clinical trial of halofuginone lactate for the treatment of East Coast fever in Kenya. Veterinary Record. 120: 575–77 (531). Conrad, PA., Stagg, D.A., Grootenhuis, J.G., Irvin, A.D., Newson, J., Njamunggeh, R.E.G., Rossiter, P.B. and Young, A.S. (1987). Isolation of Theileria parasites from African buffalo (Syncerus caffer) and characterization with anti-schizont monoclonal antibodies. Parasitology. 94: 413–23 (468). Conrad, P.A., Iams, K., Brown, W.C., Sohanpal, B. and ole MoiYoi, O.K. (1987). DNA probes detect genomic diversity in Theileria parva stocks. Molecular and Biochemical Parasitology. 25: 213–26 (515). Davis, W.C., Grootenhuis, J.G., Magondu, J., Teale, A.J., Ellis, J.A. and Morrison, W.I. (1987). The use of flaw microfluorimetry to identify monoclonal antibodies that cross-react with leucocyte differentiation antigens in domestic and wild ruminants. Animal Genetics. 18, supplement 1: 87 (615). Dolan, T.T. (1987). Immunization to control East Coast fever: Parasitology Today. 3: 4–6 (473). Dwinger, R.H., Lamb, G., Murray, Max and Hirumi, H. (1987). Dose and stage dependency for the development of local skin reactions caused by Trypanosoma congolense in goats. Acta Tropica. 44: 303–14 (447). Dwinger, R.H., Murray, Max and Moloo, S.K. (1987). Potential value of localized skin reactions (chancres) induced by Trypanosoma congolense transmitted by Glossina morsitans centralis for the analysis of metacyclic trypanosome populations. Parasite Immunology. 9: 353–62 (365). Ellis, J.A., Morrison, W.I., Goddeeris, B.M. and Emery, D.L. (1987). Bovine mononuclear phagocytic cells: identification by monoclonal antibodies and analysis of functional properties. Veterinary Immunology and Immunopathology. 17: 125–34 (499). Ellis, J.A., Scott, J.R., MacHugh, N.D., Gettinby, G. and Davis, W.C. (1987). Peripheral blood leucocytes subpopulation dynamics during Trypanosoma congolense infection in Boran and N'Dama cattle: an analysis using monoclonal antibodies and flow cytometry. Parasite Immunology. 9: 363–78 (478). Emery, D.L., MacHugh, N.D. and Ellis, J.R. (1987). The properties and functional activity of non-lymphoid cells from bovine afferent (peripheral) lymph. Immunology. 62: 177–83 (553). file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/1987_Pubs.htm[5/19/2016 3:35:05 PM] 1987_Pubs Emery, D.L., Moloo, S.K. and Murray, Max (1987). Failure of Trypanosoma vivax to generate protective immunity in goats against transmission by Glossina morsitans morsitans. Transactions of the Royal Society of Tropical Medicine and Hygiene. 81: 611 (466). Fawcett, D. W. , Conrad, P.A., Grootenhuis, J.G. and Morzaria, S.P (1987). Ultrastructure of the intra-erythrocytic stage of Theileria species from cattle and waterbuck. Tissue and Cell. 19: 643–55 (537). Fish, WR., Nelson, R.T. and Hirumi, H. (1987). Cell adhesion in Trypanosoma: in vitro studies of the interaction of Trypanosoma vivax with immobilized, organic dyes. Journal of Protozoology. 34: 457–64 (479). Gardiner, P .R. and Wilson, A.J. (1987). Trypanosoma (Duttonella) vivax. Parasitology Today. 3: 49-52 (498). Gardiner, P.R., Pearson, T. W , Clarke, M.W. and Mutharia, L.M. (1987). Identification and isolation of a variant surface glycoprotein from Trypanosoma vivax. Science. 235: 774–77 (472). Gathuo, H.K.W., Nantulya, V.M. and Gardiner, P.R. (1987). Trypanosoma vivax: adaptation of two East African stocks to laboratory rodents. Journal of Protozoology. 34: 48–53 (384). Goddeeris, B.M. and Morrison, W.I. (1987). The bovine autologous Theileria mixed leukocyte reaction: influence of monocytes and phenotype of the parasitized stimulator cell on proliferation and parasite specificity. Immunology 60: 63–69 (483). Goddeeris, B.M., Morrison, W.I., Naessens, J. and Magondu, J.G. (1987). The bovine autologous mixed leukocyte reaction: a proliferative response of non-T cells under the control of monocytes. Immunobiology. 176: 47–62 (467). Grab, D.J., Webster, P, Ito, S., Fish, W.R., Verjee, Y. and Lonsdale-Eccles, J.D. (1987). Subcellular localization of a variable surface glycoprotein phosphatidylinositol-specific phospholipase-C in African trypanosomes. Journal of Cell Biology. 105: 737–46 (493). Grootenhuis, J.G., Young, A.S., Stagg, D.A., Leitch, B.L., Dolan, T.T. and Conrad, PA. (1987). Infection of African buffalo (Syncerus caffer) and cattle with Theileria parva lawrencei after serial passage in cattle. Research in Veterinary Science. 42:326–30 (550). Grootenhuis, J.G., Leitch, B.L., Stagg, D.A., Dolan, T.T. and Young, A.S. (1987). Experimental induction of Theileria parva lawrencei carrier state in an African buffalo (Syncerus caffer). Parasitology. 94: 425–3I (554). International Laboratory for Research on Animal Diseases (1987). Program plans and funding requirements: 1988-1992. Nairobi: ILRAD, 52pp (561). Irvin, A.D. (1987). Characterization of species and strains of Theileria. Advances in Parasitology. 26: 145–97 (559). Irvin, A.D. (1987). Control of tick-borne diseases. International Journal for Parasitology. 17 (special issue): 649–57 (435). Irvin, A.D. (1987). Monitoring patterns of distribution of Rhipicephalus appendiculatus and Theileria parva. In R.W. Sutherst, ed. Ticks and Tick-borne Diseases. Canberra: ACIAR, p. 65 (585) . Irvin, A.D. (1987). Performance and productivity of cattle following immunization against East Coast fever (Theileria parva) infection. In R.W. Sutherst, ed. Ticks and Tick-borne Diseases. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/1987_Pubs.htm[5/19/2016 3:35:05 PM] 1987_Pubs Canberra: ACIAR, p. 121 (586). Irvin, A.D. (1987). The impact of vaccination against tick-borne diseases on future strategies for tick control. In R. W. Sutherst, ed. Ticks and Tick-borne Diseases. Canberra: ACIAR, p. 148 (587). Irvin, A.D. and Morrison, W.I. (1987). Immunopathology, immunology and immunoprophylaxis of Theileria infections. In E.J.L. Soulsby, ed. Immune Responses in Parasitic Infections: Immunology, Immunopathology and Immunoprophylaxis. Volume 3. Boca Ratan, Florida: CRC Press, pp. 223–74 (272). Katende, J.M., Musoke, A.J., Nantulya, V.M. and Goddeeris, B.M. (1987). A new method for fixation and preservation of trypanosomal antigens for use in the indirect immunofluorescence antibody test for diagnosis of bovine trypanosomiasis. Tropical Medicine and Parasitology. 38: 41–44 (465). Katende, J.M., Nantulya, V.M. and Musoke, A.J. (1987). Comparison between bloodstream and procyclic form trypanosomes for serological diagnosis of African human trypanosomiasis. Transactions of the Royal Society of Tropical Medicine and Hygiene. 81: 607–8 (497). Kemp, S.J. and Teale, A.J. (1987). An assay for the fully automated reading of the lymphocytotoxicity test and its application to the detection of bovine class I and class II MHC antigens. Animal Genetics. 18, supplement l: 15–16 (617). Kemp, S.J., Gettinby. G., King, D. and and Teale, A J. (1987). BoL A-PC: a database package for the storage and analysis of animal tissue-typing records on an IBM-PC microcomputer system. Animal Genetics. 18. supplement 1: 16–17 (616). Kimmel, B.E.. ole MoiYoi, O.K. and Young, J.R. (1987). Ingi, a 5.2-kb dispersed sequence element from Trypanosoma brucei that carries half of a smaller mobile element at either end and has homology with mammalian LINEs. Molecular and Cellular Biology. 7: 1465–75 (488). Knowles, G., Black. S.J. and Whitelaw, D.D. (1987). Peptidase in the plasma of mice infected with Trypanasoma brucei brucei. Parasitalogy. 95: 291–300 (512). Kocan, K.M., Morzaria. S.P. Voigt, W.P, Kiarie. J. and Irvin, A.D. (1987). Demonstration of colonies of Cowdria ruminantium in midgut epithelial cells of Amblyomma variegatum. American Journal of Veterinary Research. 48: 356–60(464). Kukla, B.A.,Majiwa. PA.O.. Young, J.R., Moloo. S.K. and ole MoiYoi, O. (1987). Use of species-specific DNA probes for detection and identification of trypanosome infection in tsetse flies. Parasitology. 95: 1–16 (486). Llewelyn, C.A.. Munro.. C.D.. Luckins, A.G., Jordt. T.. Murray, Max and Lorenzini, E. (1987). Behavioural and ovarian changes during the oestrous cycle in the Boran (Bos indicus). British Veterinary Journal. 143: 75–82 (520). Lonsdale-Eccles, J.D. and Grab, D.J. (1987). Lysosomal and non-lysosomal peptidyl hydrolases of the bloodstream forms of Trypanosoma brucei brucei. European Journal of Biochemistry. 169: 467–75 (484). Lonsdale-Eccles, J.D. and Grab, D.J. (1987). Purification of African trypanosomes can cause biochemical changes in the parasites. Journal of Protozoology. 34: 405–8 (494). Majiwa, P.A.O. and Webster, P (1987). A repetitive deoxyribonucleic acid sequence distinguishes Trypanosoma simiae from T congolense. Parasitology. 95: 543–58 (456). file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/1987_Pubs.htm[5/19/2016 3:35:05 PM] 1987_Pubs Masake, R.A., Nantulya, V.M., Musoke, A.J., Moloo, S.K. and Nguli, K. (1987). Characterization of Trypanosoma congolense serodemes in stocks isolated from cattle introduced onto a ranch in Kilifi, Kenya. Parasitology. 94: 349–57 (438). Moloo, S.K. and Kamunya, G.W. (1987). Suppressive action of Samorin on the cyclical development of pathogenic trypanosomes in Glossina morsitans centralis. Medical and Veterinary Entomology. 1: 285–87 (463). Moloo, S.K. and Kutuza, S.B. (1987). Effect of Samorin administered to a bovine host on the survival and reproductive performance of female Glossina morsitans centralis. Annals of Tropical Medicine and Parasitology. 81: 743–44 (469) . Moloo, S.K., Kutuza, S.B. and Desai, J. (1987). Comparative study on the infection rates of different Glossina species for East and West African Trypanosoma vivax stocks. Parasitology. 95: 537–42 (496). Morrison, W.I. (1987). Host effector mechanisms against parasites. Veterinary Parasitology. 25: 163–76 (535). Morrison, W.I., Goddeeris, B.M. and Teale, A.J. (1987). Bovine cytotoxic T cell clones which recognize lymphoblasts infected with two antigenically different stocks of the protozoan parasite Theileria parva. European Journal of Immunology. 17: 1703–9 (541). Morrison, W.I., Goddeeris, B.M., Teale, A.J., Groocock, C.M., Kemp, S.J. and Stagg, D.A. (1987). Cytotoxic T-cells elicited in cattle challenged with Theileria parva (Muguga): evidence for restriction by class 1 MHC determinants and parasite strain specificity. Parasite Immunology. 9: 563–78 (500). Morzaria, S.P., Irvin, A.D., Taracha, E., Spooner, P.R., Voigt, W.P., Fujinaga, T. and Katende, J. (1987). Immunization against East Coast fever: the use of selected stocks of Theileria parva for immunization of cattle exposed to field challenge. Veterinary Parasitology. 23: 23–41 (418). Morzaria, S.P., Irvin, A.D., Voigt, W.P. and Taracha, E. (1987). Effect of timing and intensity of challenge following immunization against East Coast fever. Veterinary Parasitology. 26: 29–41 (458). Morzaria, S.P and Young, A.S. (1987). Maintenance of parasites in ticks. In R.W. Sutherst, ed. Ticks and Tick-borne Diseases. Canberra: ACIAR, p. 93 (588). Mutharia, L.M. and Pearson, T.W. (1987). Surface carbohydrates of procyclic forms of African trypanosomes studied using fluorescence activated cell sorter analysis and agglutination with lectins. Molecular and Biochemical Parasitology. 23: 165–72 (470). Naessens, J. and Hamers, R. (1987). Polymorphism of immunoglobulins—IgM allotypes. In S. Dubriski, ed. The Rabbit in Contemporary Immunological Research. Harlow (UK): Longman Scientific and Technical, pp. 78–84 (607). Nantulya, V.M., Musoke, A.J., Rurangirwa, RR., Saigar, N. and Minja, S.H. (1987). Monoclonal antibodies that distinguish Trypanosoma congolense, T vivax, and T brucei. Parasite Immunology. 9: 421–31 (509). Nwagwu, M. and Hirumi, H. (1987). Trypanosorna (Nannomonas) congolense: properties of hexokinase and phosphofructokinase from cultured procyclic trypomastigotes and bloodstream forms. Acta Tropica. 44: 283–92 (250). ole MoiYoi, O.K. (1987). Trypanosome species-specific DNA probes to detect infection in tsetse flies. Parasitology Today. 3: 371–74 (523). file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/1987_Pubs.htm[5/19/2016 3:35:05 PM] 1987_Pubs Paling, R.W., Moloo, S.K. and Jenni, L. (1987). Trypanosoma congolense: host responses following tsetse transmitted infection of Kilifi isolates in goats. Experimental Parasitology. 63: 279–87 (382). Paling, R.W., Leak, S.G.A., Katende, J., Kamunya, G. and Moloo, S.K. (1987). Epidemiology of animal trypanosomiasis on a cattle ranch in Kilifi, Kenya. Acta Tropica. 44: 67–82 (448). Pearson, T.W., Moloo, S.K. and Jenni, J. (1987). Culture form and tsetse fly midgut form procyclic Trypanosoma brucei express common proteins. Molecular and Biochemical Parasitology. 23: 273–78 (513). Peregrine, A.S., Moloo, S.K. and Whitelaw, D.D. (1987). Therapeutic and prophylactic activity of isometamidium chloride in Boran cattle against Trypanosoma vivax transmitted by Glossina morsitans centralis. Research in Veterinary Science. 43: 268–70 (521). Schönefeld, A., Röttcher, D. and Moloo, S.K. (1987). The sensitivity to trypanocidal drugs of Trypanosoma vivax isolated in Kenya and Somalia. Tropical Medicine and Parasitology. 38: 177–80 (574). Shah, J.S., Young, J.R., Kimmel, B.E., lams, K.P and Williams, R.O. (1987). The 5' flanking sequence of a Trypanosoma brucei variable surface glycoprotein gene. Molecular and Biochemical Parasitology. 24: 163–74 (460). Shapiro, S.Z., Büscher, G. and Dobbelaere, D.A. E. (1987). Acquired resistance to Rhipicephalus appendiculatus (Acari: Ixodidae): identification of an antigen eliciting resistance in rabbits. Journal of Medical Entomology. 24: 147–54 (442). Shapiro, S.Z. and Kimmel, B.E. (1987). Differential protein synthesis during the life cycle of the protozoan parasite Trypanosoma brucei. Journal of Protozoology 34: 58–62 (436). Shapiro, S.Z. and Kimmel, B.E. (1987). A simple method for the production of specific antiserum to protein encoded in cloned genes: immunization with precipitin lines. Journal of Immunological Methods. 97: 275–79 (489). Shapiro, S.Z., Fujisaki, K., Morzaria, S.P., Webster, P., Fujinaga, T., Spooner, P.R. and Irvin, A.D. (1987). A life-cycle stage-specific antigen of Theileria parva recognized by antimacroschizont monoclonal antibodies. Parasitology. 94: 29–37 (450). Stagg, D.A., Conrad, P.A. and Rossiter, PB. (1987). Isolation of Theileria parva lawrencei-infected lymphoid cell lines from free-ranging African buffaloes (Syncerus caffer). Research in Veterinary Science. 43: 124–26 (502). Teale, A.J. and Kemp, S. (1987). A study of BoLA class II antigens with BoT4 + T lymphocyte clones. Animal Genetics. 18: 17–28 (471). Teale, A.J., Baldwin, C.L., Morrison, W.I., Ellis, J. and MacHugh, N.D. (1987). Phenotypic and functional characteristics of bovine T lymphocytes. Veterinary Immunology and Immunopathology. 17: 113–23 (S 14). Voigt, W. P. (1987). Theileria parva infections in Rhipicephalus appendiculatus ticks. In R. W. Sutherst, ed. Ticks and Tick-borne Diseases. Canberra: ACIAR, p. 94 (589). Young, A.S., Leitch, B.L., Morzaria, S.P., Irvin, A.D., Omwoyo, PL. and de Castro, J.J. (1987). Development and survival of Theileria parva parva in Rhipicephalus appendiculatus exposed in the Trans-Mara, Kenya. Parasitology. 94: 433–41 (539). file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/1987_Pubs.htm[5/19/2016 3:35:05 PM] 1987_Pubs file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/1987_Pubs.htm[5/19/2016 3:35:05 PM] Board Board of Directors Dr D.M. Chavunduka Highlands Veterinary Surgery, Harare, Zimbabwe Professor P. Doherty Experimental Pathology Department, John Curtin School of Medical Research, Canberra City, Australia Dr P.T. England Biological Chemistry Department, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Dr A.R. Gray Director General, ILRAD, Nairobi. Kenya Professor H.E. Jahnke (Chairman) International Agricultural Development Faculty, Technical University of Berlin, Berlin, Germany (Federal Republic) Professor L. Jenni Swiss Tropical Institute, Basel, Switzerland Dr W.K. Ngulo Deputy Director of Veterinary Services, Ministry of Livestock Development, Nairobi, Kenya Dr A.R. Njogu Director, Kenya Trypanosomiasis Research Institute, Muguga, Kenya Professor I. Maansson Veterinary Microbiology Department, Swedish University of Agricultural Sciences, Uppsala, Sweden Professor E.N.W. Oppong Animal Health/Husbandry Officer, Food and Agriculture Organization of the United Nations (FAO), Jos, Nigeria Dr W.R. Pritchard School of Veterinary Medicine, University of California at Davis, Davis, California, USA file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Board.htm[5/19/2016 3:35:05 PM] Board Dr A.S. Sidibé Coordinateur, InterAfrican Bureau for Animal Resources, Organization of African Unity (OAU), Bamako, Mali file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Board.htm[5/19/2016 3:35:05 PM] Staff Staff Administration Biochemistry/parasitology of trypanosomiasis A.R. Gray Director General P .R. Gardiner P.R. Rowe Senior Scientist/Laboratory Coordinator Director of Administration D.J. Grab J.J. Doyle Scientist Director of Research G. Knowles B.C. Lloyd Scientist Financial Controller J. Lonsdale-Eccles G. Migwi Scientist Chief Personnel Officer A S .Peregrine M.N. Kanyi Post-Doctoral Fellow Principal Administrator G. Mpimbaza Research Associate M.A. Craig Business Manager R.W Hampton Visiting Scientist M. W. Holt Chief Engineer D.D. Whitelaw Visiting Scientist M.A. Lobo Electronics Engineer L.M. Mutharia Senior Research Fellow S. Kasera Purchasing Officer G. Abebe Research Fellow G. Mzera Stores Superintendent T. Aboagye-Kwarteng Research Fellow A. Mathenge Security Officer J.G. Vos Research Fellow L. Hartley Transport Officer A. Adema Laboratory Technician C. Ndungi Transport Officer R. Thatthi Laboratory Technician Y. Verjee Laboratory Technician Biochemistry/Molecular Biology O. ole MoiYoi Senior Scientist/Laboratory Coordinator K. Iams Scientist file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Staff.htm[5/19/2016 3:35:05 PM] Staff V. Nene Scientist J.R. Young Scientist D. Barnes Visiting Scientist B.A. Kukla Post-Doctoral Fellow M.K. Limo Post-Doctoral Fellow P. Majiwa Post-Doctoral Fellow N. Murphy Post-Doctoral Fellow E. Gobright Research Associate M. Macklin Research Associate W. Endege Research Fellow M. Limo Research Fellow P. Chen Visiting Research Fellow M. Kibe Laboratory Technician A. Nayar Laboratory Technician Cell Biology H. Hirumi Senior Scientist/Laboratory Coordinator W.R. Fish Scientist Y. Eshita Visiting Scientist R. MacSween Visiting Scientist file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Staff.htm[5/19/2016 3:35:05 PM] Staff E. Reinwald Visiting Scientist I. Weissleder Visiting Scientist T. Yanagi Visiting Scientist G. Fritsch Post-Doctoral Fellow R. Kaminsky Post-Doctoral Fellow H. Waithaka Research Fellow I. Gumm Research Associate K. Hirumi Research Associate R.T. Nelson Research Associate E. Omolo Laboratory Technician Immunobiology S.J. Black Senior Scientist/Laboratory Coordinator E.J. Bienen Scientist S. Mahan Post-Doctoral Fellow D. Williams Post-Doctoral Fellow J. Naessens Visiting Scientist W. Ponti Visiting Scientist V. Vandeweerd Visiting Scientist S. Kinuthia Research Fellow file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Staff.htm[5/19/2016 3:35:05 PM] Staff V. Lutje Research Fellow J. Olobo Research Fellow J. Magondu Research Associate J. Newson Research Associate Parasitology/Theileriosis A.J. Musoke Senior Scientist/Laboratory Coordinator T.T. Dolan Senior Scientist S.B. Morzaria Scientist B. Allsop Visiting Scientist E. Flach Visiting Scientist T. Kamio Visiting Scientist C. Sugimoto Visiting Scientist P.A. Conrad Post-Doctoral Fellow E.I.P. Kamanga-Sollo Senior Research Fellow M. Butera Research Fellow A. Kairo Visiting Research Fellow M. Ismail Visiting Research Fellow S.H. Minja Research Associate C.G. Nkonge Research Associate P.R. Spooner file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Staff.htm[5/19/2016 3:35:05 PM] Staff Research Associate L.N. Mburu Laboratory Technician J. Kiarie Laboratory Technician R. Njamunggeh Laboratory Technician Cellular Immunology/Pathology W.I. Morrison Senior Scientist/Program Coordinator C. Baldwin Scientist W.C. Barry Scientist A.J. Teale Scientist S. Alberti Visiting Scientist A. Bensaid Visiting Scientist J. DeMartini Visiting Scientist T. Gliozzi Visiting Scientist B. Goddeeris Visiting Scientist C. Howard Visiting Scientist I. Joosten Visiting Scientist F. Rowell Visiting Scientist B. Tucker Visiting Scientist S. Kemp Post-Doctoral Fellow D. McKeever file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Staff.htm[5/19/2016 3:35:05 PM] Staff Post-Doctoral Fellow P. Toye Post-Doctoral Fellow E. Lorenzini Research Fellow H. Pereira Research Fellow E. Taracha Research Fellow T. Kariuki Visiting Research Fellow C. O'Callaghan Visiting Research Fellow G.M. Lamb Research Associate K.S. Logan Research Associate N.D. MacHugh Research Associate S.A. Khaushal Laboratory Technician J.K. Mburu Laboratory Technician Parasitology/Epidemiology of Trypanosomiasis V.M. Nantulya Scientist/Program Coordinator L.L. Logan Scientist R.A. Masake Scientist R. W. Paling Scientist V. Anosa Visiting Scientist R. Brun Visiting Scientist file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Staff.htm[5/19/2016 3:35:05 PM] Staff K. Lindqvist Visiting Scientist G. Ordner Visiting Scientist J. Makumi Visiting Research Fellow J. Katende Research Associate K. Nguli Research Associate H. Gathuo Laboratory Technician N. Saigar Laboratory Technician Epidemiology and Socio- Economics Unit B. Perry Scientist/Unit Head B. Grandin Scientist A. Mukhebi Scientist Electron Microscopy M. Shaw Scientist/Unit Head M. Marsh Visiting Scientist G. Griffiths Visiting Scientist P. Webster Research Associate Tsetse Laboratory S.K. Moloo Senior Seientist/Unit Head S.B. Kutuza Research Associate file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Staff.htm[5/19/2016 3:35:05 PM] Staff S.G.A. Leak Research Associate G. Kamunya Laboratory Technician E. Ndhine Laboratory Technician Tick Laboratory A. Norval Scientist/Unit Head W.P.Voigt Scientist/Unit Head F.N. Mwakima Research Associate S. Mwaura Laboratory Technician Central Core Unit C. Hinson Research Associate/Radiation Safety Officer Experimental Animal Units T. Jordt Scientist/Unit Head R.C. King Research Associate/Unit Head S.J. Kimani Farm Manager/Kabete L.J. Howard General Manager/Kapiti Plains Estate Biostatistics J. R. Scott Research Associate/Biostatistics G. Gettinby Visiting Scientist Training and Information Services file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Staff.htm[5/19/2016 3:35:05 PM] Staff J.K. Lenahan Training and Outreach Officer S.B. Westley Writer/Editor W. Umbima Librarian D. Elsworth Scientific Photographer/Graphic Arts Supervisor Wildlife Diseases Project J.G. Grootenhuis Senior Scientist N'dama Trypanotolerance Project R.H. Dwinger Scientist P. Jeannin Scientist A.S. Grieve Research Associate file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Staff.htm[5/19/2016 3:35:05 PM] Fin_stat Financial Statements Rattansi Educational Trust Building, Koinange Street , P O Box 41968 Nairobi, Kenya, Telephone 21244, Telex: 22140 CHUNGA, Cables PRICEWATER, Telecopier 335937 Price Waterhouse REPORT TO THE DIRECTORS OF THE INTERNATIONAL LABORATORY FOR RESEARCH ON ANIMAL DISEASES (ILRAD) We have reviewed the abridged financial statements set out in Figures 26 to 29 which contain information extracted from the accounting records of ILRAD for the years ended 31 December 1986 and 1987. We confirm that the information set out in the abridged financial statements is consistent with that contained in the audited financial statements for the years ended 31 December 1986 and 1987, on which we expressed an unqualified opinion. PRICE WATERHOUSE Certified Public Accountants 15 April 1988 1987 1986 Research Parasitology—Trypanosomiasis 502 484 Biochemistry 675 640 Cell Biology 656 490 Immunobiology 653 484 Parasitology—Theileriosis 626 563 Pathology 649 605 Immunoparasitology 342 – Tsetse Laboratory 345 314 Tick Laboratory 121 150 Electron Microscopy 142 131 Epidemiology and Socio-Economics 176 33 International Trypanotolerance 204 - Centre—Gambia Wildlife Project 220 - Total Research 5311 3894 file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Fin_stat.htm[5/19/2016 3:35:07 PM] Fin_stat Research Support Office of Director of Research 470 550 Farm Animal Production 584 597 Laboratory Animal Production 170 209 Clinical and Diagnostic Services 57 88 Radioisotope and Central Core 516 388 Services Total Research Support 1797 1832 Training and Conferences 1022 793 Library and Information Services 448 394 Administration Board of Directors 108 86 Office of the Director General 444 373 Finance 334 342 Personnel 88 80 Purchasing 426 343 Total Administration 1400 1224 General Operations Engineering 738 661 Transport 208 190 Services 285 237 Food and Housing 76 58 Stores 55 53 Total General Operations 1362 1199 Total Operations 11340 9336 Figure 26. Summary costs by program and activity (US$'000). 1987 1986 Unrestricted Funds United States Agency for International 2150 2525 Development (USAID) World Bank (IBRD) 1250 1400 United Kingdom 930 794 Canadian International Development 760 730 Agency (CIDA) Switzerland 644 474 Germany (Federal Republic) 630 510 Netherlands 350 280 file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Fin_stat.htm[5/19/2016 3:35:07 PM] Fin_stat Japan 346 351 Sweden 340 290 Norway 285 250 Italy 232 170 Belgium 223 255 African Development Bank 190 190 France 133 90 Denmark 80 60 India 24 - Australia ___- 230 Total Unrestricted Funds from Donors 8567 8599 Restricted Funds United Nations Development Program 828 766 (UNDP) Italy 570 500 Japan 215 150 Rockefeller Foundation 209 33 Belgium 195 95 Total Restricted Funds from Donors 2017 1544 Total Unrestricted/Restricted Funds from 10584 10143 Donors Figure 27. Summary of core operating funds from donors (US$'000). 1987 1986 SOURCES Core Operating Funds Unrestricted Unrestricted Funds from Donors 8567 8599 Earned Income Applied in Year 727 583 Total Unrestricted Operating Funds 9294 9182 Total Restricted Operating Funds 2017 1544 Total Unrestricted/Restricted Operating 11311 10726 Funds Transfer to Capital Funds (962) (954) Net Unrestricted/Restricted Operating 10349 9772 Funds Capital Funds Transferred from Core Operating Funds 962 954 Unexpended Balance from Previous Year 714 278 Balance of Working Funds 1123 823 Balance of Revolving Fund from Previous 100 100 Year Total Capital Funds 2899 2155 Special Projects Wildlife 220 156 file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Fin_stat.htm[5/19/2016 3:35:07 PM] Fin_stat Trypanotolerance 204 296 Total Special Projects 424 452 TOTAL SOURCES 13672 12379 APPLICATIONS Core Operations 11340 9788 Capital 962 954 Unexpended Balance Unrestricted Core 55 714 Working Funds 1123 823 Revolving Fund 100 100 Capital Development Fund 92 - TOTAL FUNDS 13672 12379 Figure 28. Summary of sources and application of funds (US$'000). 1987 1986 ASSETS Fixed Assets Land and Buildings 11279 10671 Research Equipment 5807 5415 Other Assets 1967 1869 Subsidiary Company Investment 1786 1786 Longterm Loan 20 10 Total Fixed Assets 20859 19751 Revolving Fund 100 100 Capital Development Fund 92 – Net Current Assets 1178 1537 TOTAL ASSETS 22229 21388 EMPLOYED FUND BALANCES Capital Fund 20859 19751 Working Capital 1123 823 Unrestricted Core Surplus 55 714 Revolving Fund 100 100 Capital Development Fund 92 - TOTAL FUNDS 22229 21388 Figure 29. Balance sheet as at December 1987. file:///C|/Users/dhmichael/Desktop/fulldoc_html/ilrad87/Fin_stat.htm[5/19/2016 3:35:07 PM]