Reflections on IDEAL: What we have learnt from a unique calf cohort study.

The year 2020 marks a decade since the final visit was made in the 'Infectious Diseases of East African Livestock' (IDEAL) project. However, data generation from samples obtained during this ambitious longitudinal study still continues. As the project launches its extensive open-access database and biobank to the scientific community, we reflect on the challenges overcome, the knowledge gained, and the advantages of such a project. We discuss the legacy of the IDEAL project and how it continues to generate evidence since being adopted by the Centre for Tropical Livestock Genetics and Health (CTLGH). We also examine the impact of the IDEAL project, from the authors perspective, for each of the stakeholders (the animal, the farmer, the consumer, the policy maker, the funding body, and the researcher and their institution) involved in the project and provide recommendations for future researchers who are interested in running longitudinal field studies.

The African buffalo parasite Theileria. sp. (buffalo) can infect and immortalize cattle leukocytes and encodes divergent orthologues of Theileria parva antigen genes.

African Cape buffalo (Syncerus caffer) is the wildlife reservoir of multiple species within the apicomplexan protozoan genus Theileria, including Theileria parva which causes East coast fever in cattle. A parasite, which has not yet been formally named, known as Theileria sp. (buffalo) has been recognized as a potentially distinct species based on rDNA sequence, since 1993. We demonstrate using reverse line blot (RLB) and sequencing of 18S rDNA genes, that in an area where buffalo and cattle co-graze and there is a heavy tick challenge, T. sp. (buffalo) can frequently be isolated in culture from cattle leukocytes. We also show that T. sp. (buffalo), which is genetically very closely related to T. parva, according to 18s rDNA sequence, has a conserved orthologue of the polymorphic immunodominant molecule (PIM) that forms the basis of the diagnostic ELISA used for T. parva serological detection. Closely related orthologues of several CD8 T cell target antigen genes are also shared with T. parva. By contrast, orthologues of the T. parva p104 and the p67 sporozoite surface antigens could not be amplified by PCR from T. sp. (buffalo), using conserved primers designed from the corresponding T. parva sequences. Collectively the data re-emphasise doubts regarding the value of rDNA sequence data alone for defining apicomplexan species in the absence of additional data. 'Deep 454 pyrosequencing' of DNA from two Theileria sporozoite stabilates prepared from Rhipicephalus appendiculatus ticks fed on buffalo failed to detect T. sp. (buffalo). This strongly suggests that R. appendiculatus may not be a vector for T. sp. (buffalo). Collectively, the data provides further evidence that T. sp. (buffalo). is a distinct species from T. parva.

A longitudinal assessment of the serological response to Theileria parva and other tick-borne parasites from birth to one year in a cohort of indigenous calves in western Kenya.

Tick-borne diseases are a major impediment to improved productivity of livestock in sub-Saharan Africa. Improved control of these diseases would be assisted by detailed epidemiological data. Here we used longitudinal, serological data to determine the patterns of exposure to Theileria parva, Theileria mutans, Babesia bigemina and Anaplasma marginale from 548 indigenous calves in western Kenya. The percentage of calves seropositive for the first three parasites declined from initial high levels due to maternal antibody until week 16, after which the percentage increased until the end of the study. In contrast, the percentage of calves seropositive for T. mutans increased from week 6 and reached a maximal level at week 16. Overall 423 (77%) calves seroconverted to T. parva, 451 (82%) to T. mutans, 195 (36%) to B. bigemina and 275 (50%) to A. marginale. Theileria parva antibody levels were sustained following infection, in contrast to those of the other three haemoparasites. Three times as many calves seroconverted to T. mutans before seroconverting to T. parva. No T. parva antibody response was detected in 25 calves that died of T. parva infection, suggesting that most deaths due to T. parva are the result of acute disease from primary exposure.

Mortality in East African shorthorn zebu cattle under one year: predictors of infectious-disease mortality.

BACKGROUND: Infectious livestock diseases remain a major threat to attaining food security and are a source of economic and livelihood losses for people dependent on livestock for their livelihood. Knowledge of the vital infectious diseases that account for the majority of deaths is crucial in determining disease control strategies and in the allocation of limited funds available for disease control. Here we have estimated the mortality rates in zebu cattle raised in a smallholder mixed farming system during their first year of life, identified the periods of increased risk of death and the risk factors for calf mortality, and through analysis of post-mortem data, determined the aetiologies of calf mortality in this population. A longitudinal cohort study of 548 zebu cattle was conducted between 2007 and 2010. Each calf was followed during its first year of life or until lost from the study. Calves were randomly selected from 20 sub-locations and recruited within a week of birth from different farms over a 45 km radius area centered on Busia in the Western part of Kenya. The data comprised of 481.1 calf years of observation. Clinical examinations, sample collection and analysis were carried out at 5 week intervals, from birth until one year old. Cox proportional hazard models with frailty terms were used for the statistical analysis of risk factors. A standardized post-mortem examination was conducted on all animals that died during the study and appropriate samples collected. RESULTS: The all-cause mortality rate was estimated at 16.1 (13.0-19.2; 95% CI) per 100 calf years at risk. The Cox models identified high infection intensity with Theileria spp., the most lethal of which causes East Coast Fever disease, infection with Trypanosome spp., and helminth infections as measured by Strongyle spp. eggs per gram of faeces as the three important infections statistically associated with infectious disease mortality in these calves. Analysis of post-mortem data identified East Coast Fever as the main cause of death accounting for 40% of all deaths, haemonchosis 12% and heartwater disease 7%. CONCLUSION: The findings demonstrate the impact of endemic parasitic diseases in indigenous animals expected to be well adapted against disease pressures. Additionally, agreement between results of Cox models using data from simple diagnostic procedures and results from post-mortem analysis underline the potential use such diagnostic data to reduce calf mortality. The control strategies for the identified infectious diseases have been discussed.

Bluetongue and epizootic haemorrhagic disease virus in local breeds of cattle in Kenya.

The presence of bluetongue virus (BTV) and Epizootic Haemorrhagic Disease virus (EHDV) in indigenous calves in western Kenya was investigated. Serum was analysed for BTV and EHDV antibodies. The population seroprevalences for BTV and EHDV for calves at 51 weeks of age were estimated to be 0.942 (95% CI 0.902-0.970) and 0.637 (95% CI 0.562-0.710), respectively, indicating high levels of circulating BTV and EHDV. The odds ratio of being positive for BTV if EHDV positive was estimated to be 2.57 (95% CI 1.37-4.76). When 99 calves were tested for BTV and EHDV RNA by real-time RT-PCR, 88.9% and 63.6% were positive, respectively. Comparison of the serology and real-time RT-PCR results revealed an unexpectedly large number of calves that were negative by serology but positive by real-time RT-PCR for EHDV. Eight samples positive for BTV RNA were serotyped using 24 serotype-specific real-time RT-PCR assays. Nine BTV serotypes were detected, indicating that the cattle were infected with a heterogeneous population of BTVs. The results show that BTV and EHDV are highly prevalent, with cattle being infected from an early age.

Hematological profile of East African short-horn zebu calves from birth to 51 weeks of age.

This paper is the first attempt to accurately describe the hematological parameters for any African breed of cattle, by capturing the changes in these parameters over the first 12 months of an animal's life using a population-based sample of calves reared under field conditions and natural disease challenge. Using a longitudinal study design, a stratified clustered random sample of newborn calves was recruited into the IDEAL study and monitored at 5-weekly intervals until 51 weeks of age. The blood cell analysis performed at each visit included: packed cell volume; red cell count; red cell distribution width; mean corpuscular volume; mean corpuscular hemoglobin concentration; hemoglobin concentration; white cell count; absolute lymphocyte, eosinophil, monocyte, and neutrophil counts; platelet count; mean platelet volume; and total serum protein. The most significant age-related change in the red cell parameters was a rise in red cell count and hemoglobin concentration during the neonatal period. This is in contrast to what is reported for other ruminants, including European cattle breeds where the neonatal period is marked by a fall in the red cell parameters. There is a need to establish breed-specific reference ranges for blood parameters for indigenous cattle breeds. The possible role of the postnatal rise in the red cell parameters in the adaptability to environmental constraints and innate disease resistance warrants further research into the dynamics of blood cell parameters of these breeds.

Characterization of the gene encoding the polymorphic immunodominant molecule, a neutralizing antigen of Theileria parva.

Theileria parva, a tick-transmitted protozoan parasite related to Plasmodium spp., causes the disease East Coast fever, an acute and usually fatal lymphoproliferative disorder of cattle in Africa. Previous studies using sera from cattle that have survived infection identified a polymorphic immunodominant molecule (PIM) that is expressed by both the infective sporozoite stage of the parasite and the intracellular schizont. Here we show that mAb specific for the PIM Ag can inhibit sporozoite invasion of lymphocytes in vitro. A cDNA clone encoding the PIM Ag of the T. parva (Muguga) stock was obtained by using these mAb in a novel eukaryotic expression cloning system that allows isolation of cDNA encoding cytoplasmic or surface Ags. To establish the molecular basis of the polymorphism of PIM, the cDNA of the PIM Ag from a buffalo-derived T. parva stock was isolated and its sequence was compared with that of the cattle-derived Muguga PIM. The two cDNAs showed considerable identity in both the 5' and 3' regions, but there was substantial sequence divergence in the central regions. Several types of repeated sequences were identified in the variant regions. In the Muguga form of the molecule, there were five tandem repeats of the tetrapeptide, QPEP, that were shown, by transfection of a deleted version of the PIM gene, not to react with several anti-PIM mAbs. By isolating and sequencing the genomic version of the gene, we identified two small introns in the 3' region of the gene. Finally, we showed that polyclonal rat Abs against recombinant PIM neutralize sporozoite infectivity in vitro, suggesting that the PIM Ag should be evaluated for its capacity to immunize cattle against East Coast Fever.

Reactivity of workshop antibodies on L cell and COS cell transfectants expressing bovine CD antigens.

Mouse L cells and COS cells were transfected with either genomic DNA or cDNAs encoding leukocyte differentiation antigens. Positive transfectants were isolated by FACS and cloned by limiting dilution. Transfectants expressing CD4, CD5, CD8, CD25, CD44, two different WC1 gene products and a transfectant expressing an unknown gene product were isolated using these techniques. Antibodies from the various preliminary clusters were analysed for reactivity on these transfectants. The results confirm the fine specificity of mAbs for two alleles of CD4 and CD5; they also subdivide mAbs recognizing CD8 into two groups based on their specificity for the CD8 alpha chain or a combination of both alpha and beta chains. The data also provides support for the clustering of mAbs recognizing CD25 and CD44 based on transfection of cDNAs encoding these specificities. These results reflect the ability of transfection technology to elucidate the fine specificity of mAbs recognizing bovine CD antigens.

Identification of expressed bovine class I MHC genes at two loci and demonstration of physical linkage.

A cDNA library prepared from lymphocytes of a cow (E98), homozygous at major histocompatibility complex (MHC) loci (BoLA phenotype w10, KN104), was screened with a bovine MHC class I probe. Of the cDNA clones isolated, two, (2.1 and 5.1) were selected and showed divergence at both 5' and 3' termini. E98 DNA was digested with rare-cutter enzymes (Sfi I, Mlu I, Not I, and Cla I) and fragments were size-separated by field inversion gel electrophoresis (FIGE). Hybridization with an entire class I cDNA probe revealed multiple fragments generated by each enzyme. When the 3' untranslated regions (UT) of 2.1 and 5.1 were used as probes, only one fragment was revealed in each digested sample, showing locus specificity of these probes in cattle. Further, DNA of transfected mouse fibroblasts L4 (expressing KN104) and L10 (expressing w10) hybridized to the 3'UT regions of clones 2.1 and 5.1, respectively. Northern blot analysis of the mRNA of the L4 and L10 transfected cells provided further evidence that the cDNA clones 2.1 and 5.1 code for the BoLA-KN104 and BoLA-w10 class I molecules respectively, and thus these represent the products of two different genes. A long range physical mapping of the BoLA-w10 and KN104 genes was performed using FIGE analysis of DNA of an homozygous and an heterozygous animal. This analysis revealed that the BoLA-w10 and KN104 genes are separated by not more than 210 kilobases (kb) and that they are components of a multigene family spanning 1550 kb. As the w10 gene is at the BoLA-A locus we assign the KN104 gene to a B locus.

Characterization of a polymorphic immunodominant molecule in sporozoites and schizonts of Theileria parva.

This study examines several aspects of a polymorphic, immunodominant molecule (PIM) found in the protozoan parasite, Theileria parva. The antigen is present in all T.p. parva stocks examined, and in the related subspecies, T.p.bovis and T.p.lawrencei. It is the predominant antigen recognized by antisera from immune cattle on Western blot analysis of schizont-infected lymphocytes, and is the only antigen which has been shown to react with anti-schizont monoclonal antibodies (MoAbs) on Western blots or in immunoprecipitations. The antigen shows polymorphism in both size and expression of antibody epitopes among the different stocks of T. parva. The antigen is present in sporozoites as well as schizonts.

Transfection into mouse L cells of genes encoding two serologically and functionally distinct bovine class I MHC molecules from a MHC-homozygous animal: evidence for a second class I locus in cattle.

w10 and KN104 are distinct class I major histocompatibility complex (MHC) serological specificities present in Boran (Bos indicus) cattle. Although these specificities are commonly expressed together, they may also be expressed independently. To establish whether w10 and KN104, when expressed together, are on the same or different molecules, and whether a second class I MHC locus exists in cattle, genomic DNA from an animal homozygous for a haplotype encoding the w10 and KN104 specificities was transfected into thymidine kinase-deficient mouse L cells (Ltk- cells), and the transfected cells were screened with monoclonal antibodies (mAb) specific for the w10 or KN104 allospecificities. Two different populations of transfectants were identified: the cells of one population reacted only with w10-specific mAb, whereas those of the other population were recognized only by the KN104-specific mAb. Alloreactive cytotoxic T lymphocytes (CTL) also distinguished between the two populations. Two CTL clones, shown to be restricted by the KN104 specificity, killed only those L cells expressing molecules recognized by the KN104-reactive mAb. Of eight CTL clones which recognized class I molecules associated with the w10 specificity, four killed the L cells expressing the w10 specificity. The remaining four clones did not kill either population of transfectants. Finally, immunoprecipitation studies revealed that both populations express full-length bovine class I MHC molecules. These results demonstrate that the w10 and KN104 specificities are on distinct class I molecules. As the genes encoding these molecules were derived from a MHC-homozygous animal, the findings also provide strong evidence that there are at least two classical class I loci in cattle.

Strain specificity of bovine Theileria parva-specific cytotoxic T cells is determined by the phenotype of the restricting class I MHC.

To determine whether the major histocompatibility complex (MHC) phenotype of cattle could affect the parasite strain specificity of immunity to Theileria parva by influencing the antigenic specificity of Theileria-specific cytotoxic T lymphocytes (CTL), we investigated the parasite strain specificity of Theileria-specific CTL clones derived from cattle of different class I MHC phenotypes. Thirty-one class I-restricted CTL clones were generated from four cattle immunized with the Muguga stock of T. parva. The MHC restriction and parasite strain specificities were determined for each clone utilizing as targets, parasitized cell lines of different MHC phenotypes and cloned cell lines containing different parasite strains. CTL clones restricted by the same MHC determinant had similar parasite strain specificities. On the other hand, clones restricted by different MHC determinants exhibited different parasite strain specificities. This was true whether the clones were generated from the same animal or from different cattle and tested on a target cell line expressing both MHC determinants. These results provide strong evidence that differences in the strain specificities of CTL derived from animals immunized with the same parasite stock, are determined by the class I MHC phenotype of the immunized animal.

Activation of complement by tetrathyridia of Mesocestoides corti: enhancement by antibodies from infected mice and lack of effect on parasite viability.

Tetrathyridia of the cestode Mesocestoides corti were isolated from the peritoneal cavity of infected mice. The parasites activated guinea pig and mouse complement (C) in vitro by both the classical and alternative pathways as shown by quantitative C fixation and crossed immunoelectrophoresis. The ability of tetrathyridia to activate mouse C was enhanced by preincubating the parasites in serum obtained from mice infected with M. corti. Antibodies of the IgG1 class, an immunoglobulin found in profoundly increased amounts in mice infected with M. corti, as well as IgM and IgG2 antibodies, bound to cultured tetrathyridia and facilitated deposition of the third component of C (C3) from dilute mouse serum, presumably via classical pathway activation. The results demonstrate that mouse IgG1 antibodies do not prevent the activation of C by the tetrathyridia or by C-fixing antibodies of other classes which become attached to the tetrathyridia. The activation of C in vitro by tetrathyridia did not affect their ability to grow in mice, even though C3-derived polypeptides could be detected by immunofluorescence on the surface of the parasites.