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.

SfiI and NotI polymorphisms in Theileria stocks detected by pulsed field gel electrophoresis.

DNAs of Theileria parva parva, T. p. lawrencei, T. p. bovis and Theileria mutans stocks, from Kenya, Uganda, Zanzibar and Zimbabwe were digested with either SfiI or NotI and analysed using contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE). The SfiI-digested T. parva genomic DNA resolved into approximately 30 fragments while the NotI digestion produced between 4-7 bands. The summation of the sizes of SfiI fragments gave an estimate of 9-10 X 10(6) base pairs for the size of the T. parva genome. Heterogeneity within T. p. parva Muguga, Pemba/Mnarani and Mariakani stocks was detected. All the T. parva stocks analysed showed SfiI and NotI restriction fragment length polymorphisms (RFLP). Hybridisation of 5 SfiI-digested T. parva DNAs with a Plasmodium berghei telomeric repeat probe suggested that most of the polymorphisms and heterogeneity occurred in the telomeric or sub-telomeric regions of the genome. The recognition by the Plasmodium telomeric probe of 7-8 strongly hybridising SfiI bands indicates that the T. parva genome may possess at least 4 chromosomes. The T. mutans genome was cut frequently with the above enzymes resulting in large numbers of fragments predominantly below 50 kb, thus suggesting either a much higher G + C content than T. parva or the presence of highly reiterated G + C-rich regions.

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.

Ultrastructure of the intra-erythrocytic stage of Theileria species from cattle and waterbuck.

A transmission electron microscopic study of the intra-erythrocytic stages of a pathogenic Theileria parva from cattle and a previously uncharacterized Theileria sp. from waterbuck (Kobus defassa) in Kenya revealed several novel ultrastructural features, associated with feeding and multiplication, in these parasites. In trophozoites a connecting channel was observed between the parasite's cytostome and its intracytoplasmic food vacuole. In some cases the limiting membrane of the food vacuole was seen to be continuous with a close-meshed network of membrane-bounded, anastomosing tubules. This labyrinthine structure, which has not been described previously, may function as a digestive organelle in theilerial trophozoites. Electron micrographs also revealed the mode of intra-erythrocytic multiplication of these parasites in vivo. Prior to division, electron-dense cisternae and rhoptries appeared beneath the parasite's plasmalemmal membrane, marking the sites of merozoite formation. From a single parasite, a maximum of four merozoites were formed by schizogonous division and subsequently separated from a residual body by constriction at the base of each merozoite. In addition, observations on two double-membraned organelles seen in trophozoites and the intra-erythrocytic crystalline structures associated with Theileria sp. in waterbuck are reported.

Transmission of a Trypanosoma sp. to cattle by the tick Hyalomma anatolicum anatolicum.

Hyalomma anatolicum anatolicum engorged nymphs and flat adults were collected from two areas in northern Sudan. Various developing stages of Trypanosoma theileri-like flagellates were observed in the engorged nymphs, freshly moulted adults and mature adults partially engorged on rabbits. When these ticks were applied to two calves, one calf became infected with the trypanosome. The parasites were observed for one day in the enlarged lymph node nearest to the tick-feeding site 5 days after the tick application. Subsequently the trypanosomes were re-isolated in vitro from the infected calf. Inoculation of a ground-up tick supernatant suspension from the infected batch of ticks containing 10(4) trypanosomes into a calf did not produce a patent infection.

Infection rates of Theileria annulata in the salivary glands of the tick Hyalomma marginatum rufipes.

Theileria annulata was experimentally transmitted to cattle on two occasions by the two-host tick Hyalomma marginatum rufipes. Transmission was transstadial; engorged nymphs fed on Theileria annulata-infected calves transmitted the disease as adults. Salivary glands of all partially fed and incubated adult ticks were heavily infected with Theileria parasites. Immatures attached rapidly and fed successfully on cattle. However, since the immature stages of this species normally feed on birds, this tick is unlikely to be an important vector in the field.

Effect of timing and intensity of challenge following immunization against East Coast fever.

When groups of Theileria parva parva Muguga-immunized cattle were given a homologous lethal challenge at different times after immunization, it was found that 4/6, 5/6, 6/6 and 6/6 animals survived when challenged on Days 5, 10, 20 and 30, respectively, post-immunization. With a heterologous challenge (T.p.parva Marikebuni), 2/6, 5/6, 4/6, 4/6 and 5/6 cattle survived when challenged on Days 5, 10, 20 and 30, respectively, after immunization. All controls, except one, died of East Coast fever (ECF). The survivor underwent severe ECF and recovered after a prolonged convalescence. When two T.p.parva Muguga-immunized animals were each given homologous challenge by application of 1000 infected ticks (infection rate of 20 infected acini (i.a.) per tick), both survived a mild ECF reaction. When groups of T.p.parva Muguga- or T.p.parva Muguga/Marikebuni-immunized cattle were challenged with different doses of T.p.parva Muguga sporozoites (equivalent of 140, 1400 and 14,000 i.a. per animal), 28/29 cattle survived. All controls died of ECF. It was concluded that cattle could be safely exposed to tick challenge 1 week after immunization by infection and treatment using appropriate immunizing stock(s). Massive homologous challenge did not break through the immunity induced by the immunization procedure.

Immunization of cattle with a Theileria parva bovis stock from Zimbabwe protects against challenge with virulent T.p. parva and T.p. lawrencei stocks from Kenya.

Following inoculation of 34 Bos indicus (Boran) cattle with a Theileria parva bovis (Boleni) stock from Zimbabwe, 18 animals underwent mild theilerial reactions, 12 underwent moderate reactions, three suffered severe reactions and one died. When these animals were subsequently challenged with different virulent stocks of either T.p. parva (Muguga, Marikebuni or Mariakani) or T.p. lawrencei (Ngong 1 or Nanyuki) from Kenya, all except two animals resisted challenge. The two reactors were part of the group challenged with the T.p. parva (Mariakani) stock. All 12 susceptible control animals underwent severe reactions and 11 died. The results of these experiments suggest that T.p. bovis (Boleni) may be used in some situations to immunize cattle against East Coast fever without the need to provide concomitant chemotherapy as in the infection and treatment method of immunization.

Effect of chronic trypanosomiasis on immunization against East Coast fever.

Two experiments were carried out in which uninfected cattle, or cattle chronically infected with Trypanosoma congolense, were immunized by the infection and treatment method against East Coast fever (ECF; Theileria parva infection). Chronic trypanosomiasis did not prevent cattle mounting an effective immunological response to ECF immunization and resisting subsequent lethal challenge. There appeared to be no difference in the level or quality of immunity between uninfected cattle and trypanosome-infected cattle. Thus, T. congolense infection on its own does not appear to provide a constraint to ECF immunization in the field.

Immunization against East Coast fever: the use of selected stocks of Theileria parva for immunization of cattle exposed to field challenge.

Two antigenically different stocks of Theileria parva parva (Kilifi and Marikebuni), previously characterized as belonging to groups A and C respectively on monoclonal antibody (MAb) profiles, were selected for immunization of different breeds of cattle against East Coast fever (ECF) by the infection and treatment method. A total of 52 immunized cattle and 33 susceptible controls of different group sizes were exposed to field challenge by ticks for periods of 42-90 days at three field sites where ECF is endemic on the Kenyan coast. All immunized cattle survived ECF challenge, but 87% of the controls died of the disease. The cattle exposed at one site had been immunized 1 year earlier and maintained tick-free in the intervening period. The level of immunity in these cattle was similar to that of cattle which had been immunized 1 or 2 months prior to exposure. Thus, immunity had not waned over the 1-year period. A study at another site showed that acaricidal treatment of immunized cattle could be safely extended from twice a week to once every three weeks, whereas in susceptible cattle even twice weekly spraying did not control ECF. The isolates made from infected controls during the trials indicated the presence of three T. p. parva stocks as defined by MAb profiles. Of the two stocks used for immunization, T. p. parva Marikebuni induced broader protection. In view of the apparent limited antigenic diversity of T. p. parva strains within the Coast Province it is suggested that the Marikebuni stock might represent a key stock for vaccination in this area.

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.

Identification of Babesia bigemina in the tick Boophilus decoloratus by the indirect fluorescent antibody technique.

Developing stages of Babesia bigemina were detected in the Giemsa-stained haemolymph smears of replete Boophilus decoloratus females engorged on infected animals. Replicate smears of these were prepared for staining by the indirect fluorescent antibody (IFA) technique. With specific antisera to B bigemina in dilutions up to 1/160 and rabbit antibovine globulin conjugated with fluorescein isothiocyanate (conjugate) the B bigemina stages were seen to fluoresce under the fluorescent microscope. When antisera against cattle Theileria spp or negative control sera were used, fluorescence was not detected in dilution above 1/5 and there was a complete absence of fluorescence when the conjugate alone was used. Thus the developing stages of B bigemina from the haemolymph could be identified using the IFA technique. Both spherical and elongated developing stages were seen to fluoresce specifically. The apical and the perinuclear regions and the posterior end of the vermicules appeared to fluoresce more intensely than the rest of the cytoplasm.

Ultrastructure of Babesia major in the tick Haemaphysalis punctata.

Haemaphysalis punctata ticks were infected with Babesia major by allowing them to feed on an infected splenectomised calf. Heavily infected ticks were dissected and the guts and the ovaries were processed to study the ultrastructure of B major in these organs. The morphology of the parasites in the gut and the ovary was identical. It was shown that the parasite contained a reduced form of an apical complex consisting of characteristic organelles such as the polar ring, rhoptries, micronemes and microtubules. A primitive conoid was situated at the anterior end of the parasite and consisted of delicate helical rings. The occurrence of these organelles confirmed the inclusion of this parasite in the subphylum Apicomplexa.

The serological relationship of a British Theileria with other Theileria species using the indirect fluorescent antibody test.

Antisera and antigens of a Theileria species isolated from British cattle were compared with those of six strains of Theileria mutans from geographically separated areas in East and South Africa, using the indirect fluorescent antibody (IFA) test. It was found that the British Theileria species did not react in the IFA test with these strains of T mutans. The British Theileria species was also compared with a variety of other Theileria species using the IFA test and no reactions were detected. It was concluded that the British Theileria species, which has been designated T mutans Essex, could be distinct from African T mutans.

Experimental transmission of Babesia major by Haemaphysalis punctata.

Using a newly isolated strain of Babesia major and a clean strain of laboratory reared Haemaphysalis punctata it was shown that adult female ticks could be alimentarily infected by feeding on infected calves but that larvae and nymphae could not. All stages were able to transmit the parasite. Two syringe passages of B major resulted in a complete loss of infectivity to ticks.

Natural infection rates of Hyalomma anatolicum anatolicum with theileria in Sudan.

Hyalomma anatolicum anatolicum ticks were collected around cattle pens at two locations close to Khartoum. They were assessed for theileria infection by four methods. Salivary glands were stained whole with methyl green pyronin and examined for parasite masses. Adult ticks were partially fed on rabbits, ground up in medium, and the suspensions were examined in Giemsa stained smears and by inoculation into bovine lymphocyte cultures. Ticks were fully fed on calves which were monitored for developing theileriosis. H a anatolicum were found infected with parasite masses similar to those seen in experimental infections with T annulata. At one site 38 per cent of 102 ticks were infected and the mean number of parasite masses per tick for the whole sample was 37. At the other site 86 per cent of 156 ticks were similarly infected and the mean parasite masses per tick was 19.5. Suspensions of sporozoites contained sporozoites typical of those found in experimental preparations of T annulata. Sporozoites harvested from ticks from both locations infected and transformed normal bovine lymphocyte cultures. H a anatolicum ticks from both locations produced fatal theileria infection in susceptible calves.

Preliminary investigations on isoenzyme variants of lymphoblastoid cell lines infected wih Theileria species.

The electrophoretic mobilities of seven enzymes from eight theileria-infected bovine lymphoblastoid cell lines originating in Kenya and Iran were compared. The isoenzyme patterns of phosphoglucomutase, malate dehydrogenase, malic enzyme and glucose-6-phosphate dehydrogenase were the same for all cell lines infected with any of the three Theileria species. Theileria annulata could be clearly differentiated from T parva and T lawrencei on the basis of three enzymes: glucose phosphate isomerase, glyceraldehyde phosphate dehydrogenase and aldolase. T parva and T lawrencei isoenzyme patterns were alike except for glucose phosphate isomerase, where two sets of isoenzyme mobility were shown which, however, did not separate the two species.

Babesia major in Britain: infectivity for cattle of cryopreserved parasites derived from Haemaphysalis punctata nymphs.

Babesia major-infected Haemaphysalis punctata nymphs, prefed for four days on rabbits, were ground in a modified Eagle's minimum essential medium containing N-2 hydroxyethylpiperazine-N1-ethane-sulphonic acid (HEPES) buffer using sterile sand or ground glass as triturating agents. The resultant supernatant fluids were shown to be infective for splenectomised calves. When attempts were made to cryopreserve the fluids using either dimethyl sulphoxide or glycerol to a final concentration of 10 per cent, only the former successfully maintained the intravenous infectivity of the organisms released by grinding with glass. A cooling rate of approximately 1 degrees C/min resulted in successful cryopreservation but a more rapid cooling rate did not.

Theileria parva: the immune status of calves born of dams immunised against East Coast fever.

Eight Friesian cross cows three months pregnant to a single Friesian bull were immunised against East Coast fever by infection with Theileria parva (Muguga) sporozoite stabilate and treatment with pyrrolidino-methyl tetracycline. They were challenged with the homologous stock four times before calving and a fifth time after calving, and resisted all five challenges which killed all of the five groups of five susceptible controls. Calves born to these hyperimmunised dams were fully susceptible on challenge with the same stabilate, as were susceptible cows from the same farm and their calves. In both instances the calves died three to seven days earlier than the cows which were approximately 10 times heavier. These results show that one- to two-month-old taurine calves from artificially immunised dams are not protected from experimental T parva sporozoite challenge and that there is no inherent calfhood resistance to East Coast fever.