Longitudinal transcriptome analysis of cattle infected with Theileria parva.

The apicomplexan cattle parasite Theileria parva is a major barrier to improving the livelihoods of smallholder farmers in Africa, killing over one million cattle on the continent each year. Although exotic breeds not native to Africa are highly susceptible to the disease, previous studies have illustrated that such breeds often show innate tolerance to infection by the parasite. The mechanisms underlying this tolerance remain largely unclear. To better understand the host response to T. parva infection we characterised the transcriptional response over 15 days in tolerant and susceptible cattle (n = 29) naturally exposed to the parasite. We identify key genes and pathways activated in response to infection as well as, importantly, several genes differentially expressed between the animals that ultimately survived or succumbed to infection. These include genes linked to key cell proliferation and infection pathways. Furthermore, we identify response expression quantitative trait loci containing genetic variants whose impact on the expression level of nearby genes changes in response to the infection. These therefore provide an indication of the genetic basis of differential host responses. Together these results provide a comprehensive analysis of the host transcriptional response to this under-studied pathogen, providing clues as to the mechanisms underlying natural tolerance to the disease.

Optical mapping compendium of structural variants across global cattle breeds.

Structural variants (SV) have been linked to important bovine disease phenotypes, but due to the difficulty of their accurate detection with standard sequencing approaches, their role in shaping important traits across cattle breeds is largely unexplored. Optical mapping is an alternative approach for mapping SVs that has been shown to have higher sensitivity than DNA sequencing approaches. The aim of this project was to use optical mapping to develop a high-quality database of structural variation across cattle breeds from different geographical regions, to enable further study of SVs in cattle. To do this we generated 100X Bionano optical mapping data for 18 cattle of nine different ancestries, three continents and both cattle sub-species. In total we identified 13,457 SVs, of which 1,200 putatively overlap coding regions. This resource provides a high-quality set of optical mapping-based SV calls that can be used across studies, from validating DNA sequencing-based SV calls to prioritising candidate functional variants in genetic association studies and expanding our understanding of the role of SVs in cattle evolution.

A cattle graph genome incorporating global breed diversity.

Despite only 8% of cattle being found in Europe, European breeds dominate current genetic resources. This adversely impacts cattle research in other important global cattle breeds, especially those from Africa for which genomic resources are particularly limited, despite their disproportionate importance to the continent's economies. To mitigate this issue, we have generated assemblies of African breeds, which have been integrated with genomic data for 294 diverse cattle into a graph genome that incorporates global cattle diversity. We illustrate how this more representative reference assembly contains an extra 116.1 Mb (4.2%) of sequence absent from the current Hereford sequence and consequently inaccessible to current studies. We further demonstrate how using this graph genome increases read mapping rates, reduces allelic biases and improves the agreement of structural variant calling with independent optical mapping data. Consequently, we present an improved, more representative, reference assembly that will improve global cattle research.

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.

Seroprevalence of respiratory viral pathogens of indigenous calves in Western Kenya.

Most studies of infectious diseases in East African cattle have concentrated on gastro-intestinal parasites and vector-borne diseases. As a result, relatively little is known about viral diseases, except for those that are clinically symptomatic or which affect international trade such as foot and mouth disease, bluetongue and epizootic haemorrhagic disease. Here, we investigate the seroprevalence, distribution and relationship between the viruses involved in respiratory disease, infectious bovine rhinotracheitis virus (IBR), bovine parainfluenza virus Type 3 (PIV3) and bovine viral diarrhoea virus (BVDV) in East African Shorthorn Zebu calves. These viruses contribute to the bovine respiratory disease complex (BRD) which is responsible for major economic losses in cattle from intensive farming systems as a result of pneumonia. We found that calves experience similar risks of infection for IBR, PIV3, and BVDV with a seroprevalence of 20.9%, 20.1% and 19.8% respectively. We confirm that positive associations exist between IBR, PIV3 and BVDV; being seropositive for any one of these three viruses means that an individual is more likely to be seropositive for the other two viruses than expected by chance.

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.

Variation and covariation in strongyle infection in East African shorthorn zebu calves.

Parasite burden varies widely between individuals within a population, and can covary with multiple aspects of individual phenotype. Here we investigate the sources of variation in faecal strongyle eggs counts, and its association with body weight and a suite of haematological measures, in a cohort of indigenous zebu calves in Western Kenya, using relatedness matrices reconstructed from single nucleotide polymorphism (SNP) genotypes. Strongyle egg count was heritable (h(2) = 23.9%, s.e. = 11.8%) and we also found heritability of white blood cell counts (WBC) (h(2) = 27.6%, s.e. = 10.6%). All the traits investigated showed negative phenotypic covariances with strongyle egg count throughout the first year: high worm counts were associated with low values of WBC, red blood cell count, total serum protein and absolute eosinophil count. Furthermore, calf body weight at 1 week old was a significant predictor of strongyle EPG at 16-51 weeks, with smaller calves having a higher strongyle egg count later in life. Our results indicate a genetic basis to strongyle EPG in this population, and also reveal consistently strong negative associations between strongyle infection and other important aspects of the multivariate phenotype.

Development and testing of a field diagnostic assay for peste des petits ruminants virus.

We have developed an immunochromatographic test for the diagnosis of peste des petits ruminants (PPR) under field conditions. The diagnostic assay has been tested in the laboratory and also under field conditions in Ivory Coast, Pakistan, Ethiopia and Uganda. The test is carried out on a superficial swab sample (ocular or nasal) and showed a sensitivity of 84% relative to PCR. The specificity was 95% over all nasal and ocular samples. The test detected as little as 10(3) TCID50 (50% tissue culture infectious doses) of cell culture-grown virus, and detected virus isolates representing all four known genetic lineages of peste des petits ruminants virus. Virus could be detected in swabs from animals as early as 4 days post-infection, at a time when clinical signs were minimal. Feedback from field trials was uniformly positive, suggesting that this diagnostic tool may be useful for current efforts to control the spread of PPR.

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.

Genome-wide analysis reveals the ancient and recent admixture history of East African Shorthorn Zebu from Western Kenya.

The Kenyan East African zebu cattle are valuable and widely used genetic resources. Previous studies using microsatellite loci revealed the complex history of these populations with the presence of taurine and zebu genetic backgrounds. Here, we estimate at genome-wide level the genetic composition and population structure of the East African Shorthorn Zebu (EASZ) of western Kenya. A total of 548 EASZ from 20 sub-locations were genotyped using the Illumina BovineSNP50 v. 1 beadchip. STRUCTURE analysis reveals admixture with Asian zebu, African and European taurine cattle. The EASZ were separated into three categories: substantial (⩾12.5%), moderate (1.56%

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.

Parasite co-infections show synergistic and antagonistic interactions on growth performance of East African zebu cattle under one year.

The co-occurrence of different pathogen species and their simultaneous infection of hosts are common, and may affect host health outcomes. Co-infecting pathogens may interact synergistically (harming the host more) or antagonistically (harming the host less) compared with single infections. Here we have tested associations of infections and their co-infections with variation in growth rate using a subset of 455 animals of the Infectious Diseases of East Africa Livestock (IDEAL) cohort study surviving to one year. Data on live body weight, infections with helminth parasites and haemoparasites were collected every 5 weeks during the first year of life. Growth of zebu cattle during the first year of life was best described by a linear growth function. A large variation in daily weight gain with a range of 0·03-0·34 kg, and a mean of 0·135 kg (0·124, 0·146; 95% CI) was observed. After controlling for other significant covariates in mixed effects statistical models, the results revealed synergistic interactions (lower growth rates) with Theileria parva and Anaplasma marginale co-infections, and antagonistic interactions (relatively higher growth rates) with T. parva and Theileria mutans co-infections, compared with infections with T. parva only. Additionally, helminth infections can have a strong negative effect on the growth rates but this is burden-dependent, accounting for up to 30% decrease in growth rate in heavily infected animals. These findings present evidence of pathogen-pathogen interactions affecting host growth, and we discuss possible mechanisms that may explain observed directions of interactions as well as possible modifications to disease control strategies when co-infections are present.

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.

Cytotoxic T lymphocytes from cattle immunized against Theileria parva exhibit pronounced cross-reactivity among different strain-specific epitopes of the Tp1 antigen.

The protozoan parasite Theileria parva causes a usually fatal disease in cattle, known as East Coast fever. Cattle can be vaccinated by injecting live parasites simultaneously with long acting oxytetracycline (the infection and treatment method, ITM). The immunity induced by ITM is believed to be mediated by cytotoxic T lymphocytes (CTL). Although effective, the ITM vaccine has disadvantages such as the need for a liquid nitrogen cold chain and a complex production process, which may be overcome by the development of a subunit vaccine. However, the high level of antigenic polymorphism among different strains of T. parva may hinder the development of a subunit vaccine aimed at induction of a protective CTL response. In this study, the CTL cross-reactivity among T. parva strains was examined. The Tp1(214-224) epitope has previously been shown to be recognized by cattle of the A18 BoLA type. Three different variants of this epitope have been identified from different T. parva strains. Here, bulk CTL and CTL clones were generated from two animals using both the live sporozoite vaccine composed of three different strains and a Muguga strain for immunization. The cross-reactivity of these CTL with the three variant Tp1 epitopes was examined in interferon gamma ELISPOT assays and CTL killing assays. CD8(+) cells from both animals cross-reacted with the three variant CTL epitopes in interferon gamma ELISPOT assays, although the CD8(+) cells from the Muguga-immunized animal showed a more epitope restricted response. Clones from the vaccine immunized animal showed diverse response patterns with clones responding to each variant peptide. Although some variability in the cytotoxic response was observed, overall strong cross-reactivity among the variant Tp1 epitopes was seen in both animals. Such epitope polymorphism does not, in this case, serve as a potential challenge in a putative subunit vaccine as it would be sufficient to only include one of the variant epitopes.

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.

Mammalian expression of functional autologous red cell agglutination reagents for use in diagnostic assays.

The autologous red cell agglutination assay reagent consists of an antibody or antibody fragment of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to an antigen of interest. This bi-functional reagent causes the agglutination of the patient's erythrocytes in the presence of the antigen-specific antibodies in the patient's serum. Previously, such reagents have been produced either by chemical conjugation or recombinant expression in bacteria. These protocols required laborious processes for purification and refolding. The aim of the work reported in this article was to explore the production of the agglutination assay reagent as both a single chain Fv (scFv) antibody fragment and recombinant full-length mAb, expressed in a secreted form in commonly used mammalian cell lines. The DNA encoding the anti-erythrocyte antibodies was linked to that of a diagnostic peptide from West Nile virus, which requires glycosylation for recognition by antibodies present in the sera of infected horses. The expression vectors were designed to allow the rapid directional insertion of DNA encoding other immunogenic peptides to mediate the secretion of agglutinating scFv and full-length mAb reagents from transfected mammalian cells. Stable cell lines were produced for the expression of most, but not all of the constructs. The recombinant reagents could be used directly from the cell culture media after a simple concentration step. The results indicate that further modifications to increase the yield of recombinant protein will enable the direct use of culture supernatant in diagnostic assays without further processing.

An enzyme-linked immunosorbent assay for detection of Theileria parva antibodies in cattle using a recombinant polymorphic immunodominant molecule.

Field and experimental bovine infection sera were used in immunoblots of sporozoite and schizont lysates of Theileria parva to identify candidate diagnostic antigens. Four parasite antigens of Mr 67,000 (p67), 85,000 (the polymorphic immunodominant molecule, PIM), 104,000 (p104), and 150,000 (p150) were selected for a more detailed analysis. The p67 and p104 antigens were present only in the sporozoite lysates, whereas PIM and p150 were found in both sporozoite and schizont lysates. The four antigens were expressed as recombinant fusion proteins and were compared with each other in an enzyme-linked immunosorbent assay (ELISA) and in the whole-schizont-based indirect fluorescent antibody test (IFAT) in terms of their ability to detect antibodies in sera of experimentally infected cattle. The PIM-based ELISA provided a higher degree of sensitivity and specificity than did the ELISA using the other three recombinant antigens or the IFAT. Further evaluation of the PIM-ELISA using experimental sera derived from cattle infected with different hemoparasites and field sera from endemic and nonendemic T. parva areas showed that the assay had a sensitivity of > 99% and a specificity of between 94% and 98%.

Specificity of a novel red blood cell agglutination assay ('SimpliRED') for HIV-1/HIV-2 infection.

The specificity and ease of use of a novel red blood cell assay for detection of HIV-1/HIV-2 antibodies was evaluated on 125 blood donor samples in Nairobi. The specificity was estimated as > 99%. The assay correctly identified five positive samples in the population, and was easy and rapid to perform. The data confirm results obtained for the assay from other regions and suggest that the assay is suitable for detection of HIV-infected individuals by minimally equipped laboratories.

Identification of neutralization and diagnostic epitopes on PIM, the polymorphic immunodominant molecule of Theileria parva.

The polymorphic immunodominant molecule (PIM) of Theileria parva is expressed by the schizont and sporozoite stages of the parasite. We have recently cloned the cDNA encoding the PIM antigen from two stocks of the parasite: the cattle-derived T. parva (Muguga) stock and a buffalo-derived stock. The cDNAs were used in transient-transfection assays to assess the reactivity of the antigen with monoclonal antibodies (MAb) previously raised against schizont-infected cells and used for parasite strain identification. We demonstrate that 19 of the 25 MAb are specific for PIM. Antibody reactivities with deletion mutants of a fusion protein containing PIM and Pepscan analysis of the Muguga version of the molecule with 13 of the MAb indicate that there are at least 10 different epitopes throughout the molecule. None of the MAb react with a tetrapeptide repeat present in the central region of the molecule, probably because of an inability of BALB/c mice to produce antibodies to this repeat. In contrast, sera from infected cattle react strongly with the repeat region, suggesting that this region alone may be useful as a diagnostic reagent. Previous studies showed that MAb to PIM inhibit sporozoite infectivity of bovine lymphocytes in vitro, which suggests that the antigen may be useful in immunizing cattle against T. parva infection. Pepscan analysis revealed that sera from infected cattle reacted with peptides recognized by the neutralizing MAb, as did sera from cattle inoculated with a PIM-containing recombinant protein. The latter sera did not, however, neutralize sporozoite infectivity in vitro. These results will be useful in exploiting the strain identification, diagnostic, and immunizing potentials of this family of antigens.