Expanding beyond zoonoses: the benefits of a national One Health coordination mechanism to address antimicrobial resistance and other shared health threats at the human-animal-environment interface in Kenya.

In order to manage global and transnational health threats at the human- animal-environment interface, a multisectoral One Health approach is required. Threats of this nature that require a One Health approach include, but are not limited to, emerging, endemic and re-emerging zoonotic diseases, food safety, antimicrobial resistance (AMR), vector-borne and neglected infectious diseases, toxicosis and pesticides. Relevant Kenyan authorities formally institutionalised One Health in 2011 through the establishment of the Zoonotic Disease Unit (ZDU) and its advisory group, the Zoonoses Technical Group. At that time, the One Health agenda focused on zoonotic diseases. As the issue of AMR began to gain traction globally, a One Health approach to its management was advocated in Kenya in 2015. This paper summarises a series of interviews (with respondents and key informants) that describe how AMR institutionalisation evolved in Kenya. It also examines how responses to other health threats at the human-animal- environment interface were coordinated and used to identify gaps and make recommendations to improve One Health coordination at the national level in Kenya. Results showed that the road to the institutionalisation of AMR through the National Action Plan on Prevention and Containment of Antimicrobial Resistance, 2017-2022 and a formally launched One Health coordination mechanism, the National Antimicrobial Stewardship Interagency Committee (NASIC), took ten years. Moreover, supplementary actions are still needed to further strengthen AMR coordination. In addition to the ZDU and NASIC, Kenya has established two other formal multisectoral and multidisciplinary coordination structures, one for aflatoxicosis and the other for health threats associated with pesticide use. The country has four distinct and separate One Health coordination mechanisms: for zoonoses, for AMR, for aflatoxicosis and for the health threats associated with pesticide use. The main gap lies in the lack of overall coordination between these topic-specific structures. An overall coordination mechanism for all One Health issues is therefore needed to improve synergy and complementarity. None of the topic-specific mechanisms plays a critical role in the policy development process, institutionalisation or implementation of activities related to the other topic areas. The authors recommend renaming the ZDU as the One Health Office, and expanding it to include AMR and food safety teams, and their associated technical working groups. Through this restructuring, the One Health Office would become an umbrella organisation dealing with all four issues mentioned above. Based on Kenya's experience, the authors recommend that other countries also consider expanding the scope of multisectoral One Health coordination mechanisms to include other shared health threats.

La gestion des menaces sanitaires mondiales et transnationales à l'interface homme­animal­environnement nécessite de faire appel à une approche Une seule santé multisectorielle. Les menaces de cette nature appelant une approche Une seule santé sont notamment (mais ne s'y limitent pas) les maladies zoonotiques émergentes, endémiques et réémergentes, la sécurité sanitaire des aliments, la résistance aux agents antimicrobiens, les maladies à transmission vectorielle, les maladies infectieuses négligées, les toxicoses et les pesticides. Les autorités kényanes ont institutionnalisé formellement l'approche Une seule santé en 2011 en mettant en place l'Unité Maladies zoonotiques (ZDU : Zoonotic Disease Unit) et son groupe consultatif, le Groupe technique Zoonoses. Le programme d'activités Une seule santé était alors centré sur les maladies zoonotiques. La problématique de l'antibiorésistance ayant gagné du terrain à l'échelle mondiale, en 2015 il a été préconisé de recourir à l'approche Une seule santé pour y faire face au Kenya. Les auteurs résument une série d'entretiens conduits auprès d'interlocuteurs et d'acteurs clés concernant l'évolution de l'institutionnalisation de la lutte contre la résistance aux agents antimicrobiens au Kenya. Ils mettent également en lumière le déroulement de la coordination des réponses mises en place pour contrer d'autres menaces sanitaires à l'interface homme­animal­environnement et l'éclairage que ces réponses ont permis d'apporter afin d'identifier les lacunes et de formuler des recommandations pour améliorer la coordination Une seule santé à l'échelle nationale. Il ressort de cette analyse qu'il a fallu dix ans pour que le Kenya institutionnalise le domaine de l'antibiorésistance à travers le Plan d'action national pour la prévention et la maîtrise de l'antibiorésistance (2017­2022) et pour qu'il mette en place un mécanisme officiel de coordination Une seule santé, le Comité national inter-agences de gestion concertée des agents antimicrobiens (NASIC : National Antimicrobial Stewardship Interagency Committee). Il est également apparu que des mesures complémentaires devaient être prises pour renforcer la coordination en matière d'antibiorésistance. Outre le ZDU et le NASIC, deux autres structures officielles de coordination multidisciplinaires et multisectorielles ont été créées au Kenya, chargées respectivement de l'aflatoxicose et des menaces sanitaires en lien avec l'utilisation de pesticides. Le pays dispose donc de quatre mécanismes de coordination distincts portant respectivement sur les zoonoses, l'antibiorésistance, l'aflatoxicose et les menaces sanitaires liées à l'utilisation de pesticides. La faille centrale est l'absence de coordination d'ensemble entre ces structures thématiques. Il faut donc instituer un mécanisme de coordination général pour toutes les questions relevant de l'approche Une seule santé, afin d'améliorer les synergies et la complémentarité. Aucun des mécanismes thématiques ne joue de rôle déterminant dans le processus d'élaboration des politiques, l'institutionnalisation ou la mise en œuvre de mesures relevant des autres thématiques. Les auteurs recommandent de modifier le nom du ZDU en Bureau Une seule santé et d'en élargir les compétences pour intégrer les équipes chargées de l'antibiorésistance et de la sécurité sanitaire des aliments ainsi que leurs groupes de travail techniques respectifs. Suite à cette restructuration, le Bureau Une seule santé pourrait devenir l'organisation transversale traitant des quatre thèmes précités. En se basant sur l'expérience du Kenya, les auteurs recommandent que d'autres pays s'engagent à leur tour sur la voie d'un élargissement de la portée des mécanismes de coordination multisectoriels Une seule santé afin d'inclure d'autres menaces sanitaires communes.

Para lidiar con las amenazas sanitarias mundiales o transnacionales en la interfaz de personas, animales y medio ambiente es preciso trabajar desde la óptica multisectorial de Una sola salud. Este tipo de amenazas que apelan al concepto de Una sola salud son, entre otras, las enfermedades zoonóticas emergentes, endémicas o reemergentes, los factores que afectan a la inocuidad de los alimentos, las resistencias a los antimicrobianos, las enfermedades infecciosas de transmisión vectorial o desatendidas, las toxicosis y los efectos del uso de plaguicidas. En 2011, con la creación de la ZDU (Zoonotic Disease Unit: unidad de enfermedades zoonóticas) y de un grupo técnico sobre zoonosis encargado de asesorarla, las autoridades competentes kenianas pusieron en práctica oficialmente la noción de Una sola salud. En aquel momento los programas de Una sola salud se centraban sobre todo en las enfermedades zoonóticas. A partir de 2015, cuando las resistencias a los antimicrobianos empezaron a ganar terreno en todo el mundo, en Kenia se apostó por combatirlas desde la óptica de Una sola salud. Los autores, sintetizando la información obtenida con una serie de encuestas y entrevistas con informadores clave, describen la progresiva institucionalización en Kenia de la lucha contra esas resistencias. También explican cómo se coordinaron las actividades de respuesta a otras amenazas sanitarias surgidas en la interfaz de personas, animales y medio ambiente y cómo ello sirvió para detectar deficiencias y formular recomendaciones encaminadas a mejorar la coordinación en clave de Una sola salud en todo el territorio nacional. Los resultados demuestran que hicieron falta diez años para institucionalizar la lucha contra la resistencia a los antimicrobianos, materializada en un plan nacional de acción sobre prevención y contención de antibiorresistencias para 2017­2022 y en la creación oficial de un mecanismo de coordinación de Una sola salud, el NASIC (National Antimicrobial Stewardship Interagency Committee: comité nacional interinstitucional de gestión de antimicrobianos). No obstante, aún hacen falta más medidas para mejorar la coordinación en todo lo relativo a las antibiorresistencias. Además de la ZDU y el NASIC, Kenia ha creado otras dos estructuras oficiales de coordinación multisectorial y multidisciplinar, una para la aflatoxicosis y otra para las amenazas sanitarias derivadas del uso de plaguicidas. El país cuenta así con cuatro mecanismos distintos e independientes de coordinación en clave de Una sola salud, centrados en las zoonosis, las antibiorresistencias, la aflatoxicosis y los riesgos sanitarios ligados a los plaguicidas. La principal deficiencia estriba en la falta de coordinación global entre estas estructuras de carácter temático. Para lograr mayores cotas de sinergia y complementariedad, por lo tanto, se requiere un mecanismo de coordinación general de todos los ámbitos de trabajo que tocan a la noción de Una sola salud. Ninguno de los mecanismos temáticos cumple una función decisiva en el proceso de formulación de políticas o de institucionalización y ejecución de actividades relacionadas con los demás ámbitos temáticos. Los autores recomiendan que la ZDU pase a denominarse Oficina de Una sola salud y que sea ampliada para integrar en ella a los equipos encargados de las antibiorresistencias y la inocuidad de los alimentos y a los correspondientes grupos de trabajo técnicos. Con semejante reestructuración, la Oficina de Una sola salud pasaría a ser una supraentidad que abarcaría los cuatro temas ya mencionados. Teniendo en cuenta la experiencia de Kenia, los autores recomiendan que otros países se planteen también la posibilidad de ampliar la cobertura de los mecanismos de coordinación multisectorial de Una sola salud para que incluyan otras amenazas sanitarias que tengan elementos en común.

The persistence of Theileria parva infection in cattle immunized using two stocks which differ in their ability to induce a carrier state: analysis using a novel blood spot PCR assay.

An improved Theileria parva DNA detection assay based on the polymerase chain reaction (PCR) using primers derived from the 104 kDa antigen (p104) gene was developed to detect parasite DNA in blood spots on filter paper. The specificity of the assay was validated using DNA from a wide range of cattle-derived and buffalo-derived stocks of T. parva. DNA of T. annulata, T. buffeli, T. lestoquardi, T. mutans and T. taurotragi was not amplified using the p104 primers. The detection threshold of the assay was approximately 1-2 parasites/microl of infected blood. PCR amplification using the p104 primers was applied to sequential samples from groups of cattle experimentally infected with either the T. parva Marikebuni stock that induces a long-term carrier state or the Muguga stock, which does not induce a carrier state. The study extended for up to 487 days post-infection and PCR data from defined time points were compared with parasitological microscopy and serological data, together with xenodiagnosis by experimental application of ticks. Microscopy first detected piroplasms between days 13 and 16 after infection whereas all cattle became PCR +ve between days 9 and 13. Animals infected with the Muguga stock of T. parva had parasite DNA in the peripheral blood, which could be detected by PCR, for between 33 and 129 days post-infection in different animals. By contrast parasite DNA in the blood of cattle infected with the Marikebuni stock could be detected consistently from day 9 up to 487 days, when the study terminated. The data suggest that the nature and persistence of the carrier state may differ markedly between different T. parva parasite stocks.

Cloning, characterization, and expression of a 200-kilodalton diagnostic antigen of Babesia bigemina.

Current serological tests for Babesia bigemina use semipurified merozoite antigens derived from infected erythrocytes. One of the major drawbacks of these tests is that antigen quality can vary from batch to batch. Since the quality of the antigen contributes to the sensitivity and specificity of serological tests, the use of standardized recombinant antigens should ensure consistency in assay quality. Previously, a 200-kDa merozoite antigen (p200) was identified as a candidate diagnostic antigen for use in a serological assay for the detection of B. bigemina antibodies in infected cattle. In this study, we have cloned, characterized, and expressed p200. A 3.5-kbp cDNA clone encoding p200 was isolated and shown to be almost full length, lacking approximately 300 bp at the 5' end. The predicted amino acid sequence shows that p200 consists of a long, highly charged central repeat region of an uninterrupted alpha helix, indicative of a fibrous protein. Immunoelectron microscopy localized p200 to the merozoite cytoplasm, suggesting that the antigen may be a structural protein involved in forming filament structures within the cytoskeleton. The 3.5-kbp cDNA was expressed in bacteria as a fusion protein with glutathione S-transferase (GST), but the yield was poor. To improve the yield, cDNA fragments encoding antigenic domains of p200 were expressed as fusions with GST. One of these fusion proteins, C1A-GST, is composed of a 7-kDa fragment of the p200 repeat region and contains epitopes that react strongly with sera from cattle experimentally infected with B. bigemina. Recombinant C1A-GST should permit the development of an improved enzyme-linked immunosorbent assay for the detection of antibodies against B. bigemina.

A 32 kDa surface antigen of Theileria parva: characterization and immunization studies.

Previous studies using monoclonal antibody (mAb) 4C9 specific for a 32 kDa antigen (p32) of Theileria parva demonstrated expression of the antigen on the surface of the sporozoite, making it a potential antigen for sporozoite neutralization. A full-length cDNA encoding the major merozoite/piroplasm surface antigen (mMPSA) of T. parva was cloned and expressed in bacteria. The expressed product reacted strongly with mAb 4C9, demonstrating identity between the p32 and mMPSA of T. parva. Using immunoblot analysis and immunoelectron microscopy with mAb 4C9 it was shown that the mMPSA is a major antigen of the merozoite and piroplasm at the cell surface, while lower levels of antigen are expressed in the sporozoite and schizont stages. Upregulation of the mMPSA occurs at merogony and can be induced by culturing schizont-infected lymphocytes at 42 degrees C. Recombinant mMPSA of T. parva induced high titres of specific antibodies in cattle but failed to confer protection against a T. parva sporozoite stabilate challenge. The pre-challenge sera also failed to neutralize infectivity of sporozoites in an in vitro assay. Possible reasons for the lack of parasite neutralization in vivo and in vitro are discussed.

Reciprocal cross-protection induced by sporozoite antigens SPAG-1 from Theileria annulata and p67 from Theileria parva.

Theileria annulata and Theileria parva both possess a major surface antigen on the sporozoite stage of the life-cycle, called SPAG-1 and p67, respectively. In each case, these antigens are vaccine candidates and have been shown to induce a degree of homologous protection in earlier work. These antigens share sequence homology and are serologically cross-reactive. Here, we confirm that these antigens confer protection against homologous species challenge. More importantly, they mutually confer a degree of cross-species protection raising the prospect of a common vaccine in the future.

A statistically derived index for classifying East Coast fever reactions in cattle challenged with Theileria parva under experimental conditions.

A statistically derived disease reaction index based on parasitological, clinical and haematological measurements observed in 309 5 to 8-month-old Boran cattle following laboratory challenge with Theileria parva is described. Principal component analysis was applied to 13 measures including first appearance of schizonts, first appearance of piroplasms and first occurrence of pyrexia, together with the duration and severity of these symptoms, and white blood cell count. The first principal component, which was based on approximately equal contributions of the 13 variables, provided the definition for the disease reaction index, defined on a scale of 0-10. As well as providing a more objective measure of the severity of the reaction, the continuous nature of the index score enables more powerful statistical analysis of the data compared with that which has been previously possible through clinically derived categories of non-, mild, moderate and severe reactions.

Protective immune mechanisms against Theileria parva: evolution of vaccine development strategies.

Theileria parva is an intracellular sporozoan parasite that infects and transforms bovine lymphocytes, causing a severe lymphoproliferative disease known as East Coast fever in eastern, central and southern Africa. In this article, Declan McKeever and colleagues summarize the current understanding of immune mechanisms provoked by the parasite with regard to their role in both pathogenesis and protection. In particular, the influence of genomic polymorphism in parasite and host on the development of immunity is discussed, along with the evolution of current vaccine development strategies as a result of immunological research on the disease.

Development of sero-diagnostic and molecular tools for the control of important tick-borne pathogens of cattle in Africa.

Tick-borne diseases (TBDs) are a major economic constraint to livestock production in sub-Saharan Africa. ILRI is focussing on developing a range of products, such as vaccines, diagnostics and decision support services to underpin improved control programmes against these diseases. We have developed three highly sensitive and specific enzyme linked immuno-assays (ELISAs), which allow precise diagnosis of Theileria parva, Babesia bigemina and Anaplasma marginale. These tests have been standardised and validated using defined experimental and field infection sera. Parasite specific recombinant antigens and monoclonal antibodies against bovine immunoglobulins as secondary antibodies have played an important role in in enhancing the sensitivity and specificity of the assays. They have been further evaluated in on-farm longitudinal sero-epidemiological studies to define infection dynamics and disease risks in various farming systems in Kenya and Uganda. In addition, DNA-based tests for differentiation of Theileria species and characterisation of Theileria parva stocks have been developed. These tests have been derived through physical mapping and sequencing of key elements of the T. parva genome, which include repetitive and telomeric regions, minisatellite sequences, antigen genes and a number of random DNA sequences. These tools are currently being deployed in conjunction with field immunisation programmes to determine the biological impact of introducing live vaccines of T. parva on population dynamics.

The prevalence of serum antibodies to tick-borne infections in cattle in smallholder dairy farms in Murang'a District, Kenya; a cross-sectional study.

The most important tick-borne disease of cattle in eastern, central and southern Africa is East Coast fever (ECF) caused by Theileria parva and transmitted by the tick Rhipicephalus appendiculatus. Other less-important tick-borne diseases in cattle are benign theileriosis caused by Theileria mutans, babesiosis caused by Babesia bigemina, anaplasmosis caused by Anaplasma marginale and cowdriosis caused by Cowdria ruminatum. In Murang's District, Central Province of Kenya, five agroecological zones (AEZs) are defined according to climate, altitude and agricultural activities. A cross-sectional serological study was conducted on 750 smallholder dairy farms in Murang's District, selected in a stratified random sampling method. The farms had a total of 362 calves. One hundred and fifty farms were studied from three administrative sublocations in each of the five AEZs. Prevalence of serum antibodies to three tick-borne parasites, that is T. parva, T. mutans and B. bigemina, were determined using the enzyme-linked immunosorbent assay (ELISA) technique. Antibody prevalence values differed across the AEZs. The ranges of means for the prevalences were: T. parva (18-72%), T. mutans (1.5-28%) and B. bigemina (12-49%). The above results serve as indicators of the possible existence of endemic stability in some AEZs for some parasites.

Factors influencing infections in Rhipicephalus appendiculatus ticks fed on cattle infected with Theileria parva.

A large database on the transmission of a stabilate of the Theileria parva Muguga stock from one breed of cattle using two stocks of Rhipicephalus appendiculatus, Muguga and Ol Pejeta was developed and analysed. Factors associated with the ticks and cattle, and the infections developing in cattle were studied in relation to the infection variables in the tick batches harvested daily from cattle. Generalized Linear Interactive Modelling (GLIM) was used to determine the importance of factors and interactions in influencing the levels of tick infection variables using Type I and Type III sums of squares analyses. Analysis of the 6 variables, prevalence (percentage of ticks infected), abundance (mean number of infected salivary gland acini per tick examined) and intensity (mean number of infected salivary gland acini per infected tick) in batches of 30 male and 30 female ticks showed that 24 covariates, factors or interactions had a significant effect (P < 0.05). Certain covariates and factors were particularly important for all 6 tick infection variables; parasitaemia of animal on the day of tick harvest, stabilate dilution administered to animal, month in which tick batch was harvested, minimum packed cell volume of animal over the sampling period, age of animal, and the minimum leukocyte count of the animal over the sampling period. The GLIM analyses were found to be a useful tool in identifying factors that influence infection levels and in devising methods of producing tick batches with more predictable infections.

Artificial feeding of ixodid ticks.

Ixodid ticks are economically important as they cause direct damage to livestock and are vectors of several pathogens that cause diseases in humans and animals. Some of the important tick-borne pathogens of livestock are Theileria parva, T. annulata, Babesia bigemina, B. bovis, Anaplasma marginale and Cowdria ruminantium. These pathogens are responsible for causing enormous losses in livestock. Identification of factors that influence transmission and development of these pathogens in ticks will greatly facilitate development of rational strategies for control of tick-borne diseases. This research has been hampered by the lack of suitable artificial feeding methods. In this paper, Sam Waladde, Aian Young and Subhash Morzaria review recent developments in the artificial feeding of ixodid ticks and evaluate how this method can potentially be exploited. They use an example the transmission of an important livestock pathogen, T. parva, by Rhipicephalus appendiculatus.

Generation and characterization of cloned Theileria parva parasites.

A 3-step procedure for cloning Theileria parva parasites was developed. The first step involved the in vitro infection of a fixed number of bovine lymphocytes with titrated sporozoites. The cell lines obtained from infections initiated using sporozoite/lymphocyte ratios below 1:100 were then selected for cloning as these contained schizont-infected cells, each of which was derived from infection with a single sporozoite. In the second step, these cell lines were cloned by limiting dilution. As sporozoites infect lymphocytes and transform to induce clonal multiplication, this step produced infected cell lines containing both cloned parasites and cloned lymphocytes. In the third step, the cloned cell lines were used to infect cattle and isolation of the parasite in ticks was made during piroplasm parasitaemia. Finally, sporozoites were harvested from infected ticks and used for further characterization. Sporozoites derived from cloned cell lines of T. parva Muguga, Marikebuni, Boleni, Uganda and buffalo-derived 7014 were characterized using monoclonal antibody profiles, DNA restriction fragment length polymorphism detected using repetitive and telomeric probes, in vivo infectivity and, in one case, cross-immunity studies. Additionally, several distinct schizont-infected lymphocyte clones were isolated from the Muguga, Mariakani and buffalo-derived 7014 stocks. The combined results of the characterization revealed that the cloning procedure selected clones of T. parva from the parental stocks which were known to contain a mixture of genetically different parasite populations. The cloning method and the clones generated will be of value in studies of the biology of the parasite and in elucidating the strain specificity of immune responses in cattle.

Characterisation of the telomeres at opposite ends of a 3 Mb Theileria parva chromosome.

Bacteriophage lambda clones containing Theileria parva genomic DNA derived from two different telomeres were isolated and the nucleotide sequences of the telomeric repeats and adjacent telomere-associated (TAS) DNA were determined. The T.parva telomeric repeat sequences, a tandem array of TTTTAGGG or TTTAGGG interspersed with a few variant copies, showed a high degree of sequence identity to those of the photosynthetic algae Chlamydomonas reinhardtii (97% identity) and Chlorella vulgaris (87.7% identity) and the angiosperm Arabidopsis thaliana (84.4% identity). Unlike most organisms which have been studied, no significant repetitive sequences were found in the nucleotide sequences of TAS DNA located centromere-proximal to the telomeric repeats. Restriction mapping and hybridisation analysis of lambda EMBL3 clones containing 16 kilobases of TAS DNA derived from one telomere suggested that they did not contain long regions of repetitive DNA. The cloned TAS DNAs were mapped to T.parva Muguga genomic SfiI fragments 8 and 20, which are located at opposite ends of the largest T.parva chromosome. A 126 bp sequence located directly centromere-proximal to the telomeric repeats was 94% identical between the two cloned telomeres. The conserved 126 bp sequence was present on all T.parva Muguga telomeric SfiI fragments.

Parasite strain specificity of precursor cytotoxic T cells in individual animals correlates with cross-protection in cattle challenged with Theileria parva.

Class I major histocompatibility complex-restricted parasite-specific cytotoxic T lymphocytes (CTL) are known to be a major component of the bovine immune response to the protozoan parasite Theileria parva, but formal proof for their role in protection of cattle against infection with T. parva has been lacking. Animals immunized with one stock of T. parva show variations in the degree of protection against heterologous challenge and also in the parasite strain specificity of their CTL responses. The present study investigated the relationship of strain specificity of CTL responses and cross-protection in an effort to verify the role of CTL in protection. The parasite strain specificity of the CTL responses generated in 23 cattle immunized with either of two immunologically distinct parasite populations was examined, and the susceptibility of individual cattle to challenge with the heterologous parasite population was determined. The frequency of stock-specific or cross-reactive CTL precursor cells (CTLp) in individual animals was measured by a limiting-dilution microassay. A proportion of animals immunized with either parasite exhibited cross-reactive CTLp, whereas CTLp detected in the remaining animals were specific for the homologous parasite. On challenge with the heterologous stock, those animals with cross-reactive CTLp were solidly protected while those with strain-specific CTLp showed moderate to severe reactions, although many of them recovered. The finding of a close association between strain specificity of the CTL response and protection against challenge provides strong evidence that CTL are important in mediating immunity.

Theileria parva carrier state in naturally infected and artificially immunised cattle.

Rhipicephalus appendiculatus adult ticks, collected in the field from zebu (Bos indicus) and exotic (Bos tarus) cattle with high antibody titres to Theileria parva schizont antigen, transmitted Theileria parva infection typical of East Coast fever to susceptible cattle. Uninfected R. appendiculatus nymphs applied to naturally recovered zebu and exotic cattle kept under tick-free conditions in the laboratory for 16 and 7 months respectively, transmitted fatal theileriosis to susceptible cattle. Cattle immunised by the infection and treatment method were shown to be carriers of Theileria parva by examination of the salivary glands of ticks applied to them and by tick transmission. Three and 7 months after immunisation, Theileria parva infected lymphocytes were established in vitro from peripheral blood lymphocytes. This was the first demonstration, in vitro, of the existence of schizonts in Theileria parva (East Coast fever) infection carrier status. These studies show that cattle from endemic and epidemic areas of East Coast fever (ECF) become carriers thereby maintaining the T. parva population. The relevance of the findings in this study to the control of ECF by dipping, immunisation and treatment is discussed.

Sequence and expression of a 90-kilodalton heat-shock protein family member of Theileria parva.

A Theileria parva specific full-length cDNA clone, T7, which encodes a protein with more than 60% homology to heat shock protein 90 (hsp90) of other organisms, has been identified. T7 appears to be a single copy gene. The gene is expressed as a protein of 87 kDa in both the sporozoite and schizont stages of T. parva. The protein was not found in the piroplasm stage, although the corresponding transcript was detected, suggesting post-transcriptional regulation of the gene. In the schizont stage the T7 protein is upregulated upon heat shock and localized in the cytoplasm.

Evidence for two single copy units in Theileria parva ribosomal RNA genes.

Bacteriophage clones containing ribosomal RNA genes of Theileria parva were isolated from genomic DNA libraries. Physical mapping studies revealed 2 ribosomal DNA units, which were distinguishable by restriction enzyme site polymorphisms in flanking sequences. The cloned ribosomal DNA units were mapped to 2 separate T. parva chromosomes. Analysis of sequences contained in lambda EMBL3 recombinants, together with Southern blot analysis of genomic DNA and data on the copy number of the rRNA genes, suggested that the rDNA units were not tandemly repeated. This organisation of ribosomal transcription units is similar to that described for other genera of apicomplexan protozoa, but 2 rDNA units, each containing single copies of the rRNA coding genes, would be the lowest copy number described for any eukaryote in which amplification of rRNA genes is not known to occur. EcoRI restriction fragment length polymorphisms, which were revealed using rRNA gene probes, separated T. parva stocks into 2 categories. Nucleotide sequence analysis of polymerase chain reaction-amplified internal transcribed spacer DNA revealed 2 different ITS sequences derived from rDNA transcription units within the genome of a cloned T. parva parasite. Polymorphism was also observed between ITS sequences amplified from the DNA of different T. parva stocks. A synthetic oligonucleotide derived from T. parva Uganda ribosomal ITS DNA sequences hybridised to DNA from the T. parva Uganda stock, but not to the DNA of the T. parva Muguga stock. This oligonucleotide is potentially useful as a marker for the T. parva Uganda stock.

Isolation and preliminary characterisation of a previously unidentified Theileria parasite of cattle in Kenya.

A Theileria parasite was isolated from cattle on a ranch in Kenya where it caused mild theileriosis in approximately one third of the cattle exposed to natural tick challenge. The parasite was isolated by inoculation of blood into two experimental cattle. Blood from one of these cattle was used to infect two splenectomised cattle which developed high piroplasm parasitaemias and severe anaemia. A blood stabilate was prepared from one of the splenectomised cattle and produced high parasitaemias in splenectomised cattle. Immunofluorescence tests showed that the unidentified Theileria species was distinct from other African Theileria and Babesia species of cattle. The Theileria species was shown to be antigenically distinct by means of species-specific monoclonal antibodies. The piroplasma stage was relatively large for a Theileria parasite and erythrocyte infections were usually associated with veils and bars. The salivary glands of Rhipicephalus appendiculatus, R pulchellus, R evertsi and Amblyomma variegatum fed on parasitaemic cattle did not become infected and these ticks failed to transmit the parasite. It was concluded that this parasite represented a new species of cattle Theileria in Kenya.