A locus conferring tolerance to Theileria infection in African cattle.

East Coast fever, a tick-borne cattle disease caused by the Theileria parva parasite, is among the biggest natural killers of cattle in East Africa, leading to over 1 million deaths annually. Here we report on the genetic analysis of a cohort of Bos indicus (Boran) cattle demonstrating heritable tolerance to infection with T. parva (h2 = 0.65, s.e. 0.57). Through a linkage analysis we identify a 6 Mb genomic region on bovine chromosome 15 that is significantly associated with survival outcome following T. parva exposure. Testing this locus in an independent cohort of animals replicates this association with survival following T. parva infection. A stop gained variant in a paralogue of the FAF1 gene in this region was found to be highly associated with survival across both related and unrelated animals, with only one of the 20 homozygote carriers (T/T) of this change succumbing to the disease in contrast to 44 out of 97 animals homozygote for the reference allele (C/C). Consequently, we present a genetic locus linked to tolerance of one of Africa's most important cattle diseases, raising the promise of marker-assisted selection for cattle that are less susceptible to infection by T. parva.

IDEAL, the Infectious Diseases of East African Livestock project open access database and biobank.

The Infectious Diseases of East African Livestock (IDEAL) project was a longitudinal cohort study of calf health which was conducted in Western Kenya between 2007-2010. A total of 548 East African shorthorn zebu calves were recruited at birth and followed at least every 5 weeks during the first year of life. Comprehensive clinical and epidemiological data, blood and tissue samples were collected at every visit. These samples were screened for over 100 different pathogens or infectious exposures, using a range of diagnostic methods. This manuscript describes this comprehensive dataset and bio-repository, and how to access it through a single online site ( http://data.ctlgh.org/ideal/ ). This provides extensive filtering and searching capabilities. These data are useful to illustrate outcomes of multiple infections on health, investigate patterns of morbidity and mortality due to parasite infections, and to study genotypic determinants of immunity and disease.

Theileria parva: a parasite of African buffalo, which has adapted to infect and undergo transmission in cattle.

The tick-borne protozoan parasite Theileria parva causes an acute, often fatal disease in cattle throughout a large part of eastern and southern Africa. Infection of African buffalo (Syncerus caffer) is also widespread in this region but does not cause clinical disease in this species. This difference most likely reflects the evolutionary history of the parasites in these species, in that cattle were only introduced into Africa within the last 8000 years. In both hosts, T. parva establishes a carrier state, involving persistence of small numbers of parasites for many months following the acute phase of infection. This persistence is considered important for maintaining the parasite populations. Although cattle and buffalo parasites both produce severe disease when transmitted to cattle, the buffalo-derived parasites are usually not transmissible from infected cattle. Recent studies of the molecular and antigenic composition of T. parva, in addition to demonstrating heterogeneity in the populations in both host species, have revealed that infections in individual animals are genotypically mixed. The results of these studies have also shown that buffalo T. parva exhibit much greater genotypic diversity than the cattle population and indicate that cattle parasites represent a subpopulation of T. parva that has adapted to maintenance in cattle. The parasites in cattle and buffalo appear to be maintained largely as separate populations. This insight into the genotypic composition of T. parva populations has raised important questions on how host adaptation of the parasite has evolved and whether there is scope for further adaptation of buffalo-maintained populations to cattle.

Ancient diversity and geographical sub-structuring in African buffalo Theileria parva populations revealed through metagenetic analysis of antigen-encoding loci.

An infection and treatment protocol involving infection with a mixture of three parasite isolates and simultaneous treatment with oxytetracycline is currently used to vaccinate cattle against Theileria parva. While vaccination results in high levels of protection in some regions, little or no protection is observed in areas where animals are challenged predominantly by parasites of buffalo origin. A previous study involving sequencing of two antigen-encoding genes from a series of parasite isolates indicated that this is associated with greater antigenic diversity in buffalo-derived T. parva. The current study set out to extend these analyses by applying high-throughput sequencing to ex vivo samples from naturally infected buffalo to determine the extent of diversity in a set of antigen-encoding genes. Samples from two populations of buffalo, one in Kenya and the other in South Africa, were examined to investigate the effect of geographical distance on the nature of sequence diversity. The results revealed a number of significant findings. First, there was a variable degree of nucleotide sequence diversity in all gene segments examined, with the percentage of polymorphic nucleotides ranging from 10% to 69%. Second, large numbers of allelic variants of each gene were found in individual animals, indicating multiple infection events. Third, despite the observed diversity in nucleotide sequences, several of the gene products had highly conserved amino acid sequences, and thus represent potential candidates for vaccine development. Fourth, although compelling evidence for population differentiation between the Kenyan and South African T. parva parasites was identified, analysis of molecular variance for each gene revealed that the majority of the underlying nucleotide sequence polymorphism was common to both areas, indicating that much of this aspect of genetic variation in the parasite population arose prior to geographic separation.

Limited genetic and antigenic diversity within parasite isolates used in a live vaccine against Theileria parva.

An infection and treatment protocol is used to vaccinate cattle against Theileria parva infection. Due to incomplete cross-protection between different parasite isolates, a mixture of three isolates, termed the Muguga cocktail, is used for vaccination. While vaccination of cattle in some regions provides high levels of protection, some animals are not protected against challenge with buffalo-derived T. parva. Knowledge of the genetic composition of the Muguga cocktail vaccine is required to understand how vaccination is able to protect against field challenge and to identify the potential limitations of the vaccine. The aim of the current study was to determine the extent of genetic and antigenic diversity within the parasite isolates that constitute the Muguga cocktail. High throughput multi-locus sequencing of antigen-encoding loci was performed in parallel with typing using a panel of micro- and mini-satellite loci. The former focused on genes encoding CD8(+) T cell antigens, believed to be relevant to protective immunity. The results demonstrate that each of the three component stocks of the cocktail contains limited parasite genotypic diversity, with single alleles detected at many gene/satellite loci and, moreover, that two of the components show a very high level of similarity. Thus, the vaccine incorporates very little of the genetic and antigenic diversity observed in field populations of T. parva. The presence of alleles at low frequency (<10%) within vaccine component populations also points to the possibility of variability in the content of vaccine doses and the potential for loss of allelic diversity during tick passage. The results demonstrate that there is scope to modify the content of the vaccine in order to enhance its diversity and thus its potential for providing broad protection. The ability to accurately quantify genetic diversity in vaccine component stocks will facilitate improved quality control procedures designed to ensure the long-term efficacy of the vaccine.

Co-infections determine patterns of mortality in a population exposed to parasite infection.

Many individual hosts are infected with multiple parasite species, and this may increase or decrease the pathogenicity of the infections. This phenomenon is termed heterologous reactivity and is potentially an important determinant of both patterns of morbidity and mortality and of the impact of disease control measures at the population level. Using infections with Theileria parva (a tick-borne protozoan, related to Plasmodium) in indigenous African cattle [where it causes East Coast fever (ECF)] as a model system, we obtain the first quantitative estimate of the effects of heterologous reactivity for any parasitic disease. In individual calves, concurrent co-infection with less pathogenic species of Theileria resulted in an 89% reduction in mortality associated with T. parva infection. Across our study population, this corresponds to a net reduction in mortality due to ECF of greater than 40%. Using a mathematical model, we demonstrate that this degree of heterologous protection provides a unifying explanation for apparently disparate epidemiological patterns: variable disease-induced mortality rates, age-mortality profiles, weak correlations between the incidence of infection and disease (known as endemic stability), and poor efficacy of interventions that reduce exposure to multiple parasite species. These findings can be generalized to many other infectious diseases, including human malaria, and illustrate how co-infections can play a key role in determining population-level patterns of morbidity and mortality due to parasite infections.

Parasite co-infections and their impact on survival of indigenous cattle.

In natural populations, individuals may be infected with multiple distinct pathogens at a time. These pathogens may act independently or interact with each other and the host through various mechanisms, with resultant varying outcomes on host health and survival. To study effects of pathogens and their interactions on host survival, we followed 548 zebu cattle during their first year of life, determining their infection and clinical status every 5 weeks. Using a combination of clinical signs observed before death, laboratory diagnostic test results, gross-lesions on post-mortem examination, histo-pathology results and survival analysis statistical techniques, cause-specific aetiology for each death case were determined, and effect of co-infections in observed mortality patterns. East Coast fever (ECF) caused by protozoan parasite Theileria parva and haemonchosis were the most important diseases associated with calf mortality, together accounting for over half (52%) of all deaths due to infectious diseases. Co-infection with Trypanosoma species increased the hazard for ECF death by 6 times (1.4-25; 95% CI). In addition, the hazard for ECF death was increased in the presence of Strongyle eggs, and this was burden dependent. An increase by 1000 Strongyle eggs per gram of faeces count was associated with a 1.5 times (1.4-1.6; 95% CI) increase in the hazard for ECF mortality. Deaths due to haemonchosis were burden dependent, with a 70% increase in hazard for death for every increase in strongyle eggs per gram count of 1000. These findings have important implications for disease control strategies, suggesting a need to consider co-infections in epidemiological studies as opposed to single-pathogen focus, and benefits of an integrated approach to helminths and East Coast fever disease control.

Design and descriptive epidemiology of the Infectious Diseases of East African Livestock (IDEAL) project, a longitudinal calf cohort study in western Kenya.

BACKGROUND: There is a widely recognised lack of baseline epidemiological data on the dynamics and impacts of infectious cattle diseases in east Africa. The Infectious Diseases of East African Livestock (IDEAL) project is an epidemiological study of cattle health in western Kenya with the aim of providing baseline epidemiological data, investigating the impact of different infections on key responses such as growth, mortality and morbidity, the additive and/or multiplicative effects of co-infections, and the influence of management and genetic factors.A longitudinal cohort study of newborn calves was conducted in western Kenya between 2007-2009. Calves were randomly selected from all those reported in a 2 stage clustered sampling strategy. Calves were recruited between 3 and 7 days old. A team of veterinarians and animal health assistants carried out 5-weekly, clinical and postmortem visits. Blood and tissue samples were collected in association with all visits and screened using a range of laboratory based diagnostic methods for over 100 different pathogens or infectious exposures. RESULTS: The study followed the 548 calves over the first 51 weeks of life or until death and when they were reported clinically ill. The cohort experienced a high all cause mortality rate of 16% with at least 13% of these due to infectious diseases. Only 307 (6%) of routine visits were classified as clinical episodes, with a further 216 reported by farmers. 54% of calves reached one year without a reported clinical episode. Mortality was mainly to east coast fever, haemonchosis, and heartwater. Over 50 pathogens were detected in this population with exposure to a further 6 viruses and bacteria. CONCLUSION: The IDEAL study has demonstrated that it is possible to mount population based longitudinal animal studies. The results quantify for the first time in an animal population the high diversity of pathogens a population may have to deal with and the levels of co-infections with key pathogens such as Theileria parva. This study highlights the need to develop new systems based approaches to study pathogens in their natural settings to understand the impacts of co-infections on clinical outcomes and to develop new evidence based interventions that are relevant.

Two Theileria parva CD8 T cell antigen genes are more variable in buffalo than cattle parasites, but differ in pattern of sequence diversity.

BACKGROUND: Theileria parva causes an acute fatal disease in cattle, but infections are asymptomatic in the African buffalo (Syncerus caffer). Cattle can be immunized against the parasite by infection and treatment, but immunity is partially strain specific. Available data indicate that CD8(+) T lymphocyte responses mediate protection and, recently, several parasite antigens recognised by CD8(+) T cells have been identified. This study set out to determine the nature and extent of polymorphism in two of these antigens, Tp1 and Tp2, which contain defined CD8(+) T-cell epitopes, and to analyse the sequences for evidence of selection. METHODOLOGY/PRINCIPAL FINDINGS: Partial sequencing of the Tp1 gene and the full-length Tp2 gene from 82 T. parva isolates revealed extensive polymorphism in both antigens, including the epitope-containing regions. Single nucleotide polymorphisms were detected at 51 positions (∼12%) in Tp1 and in 320 positions (∼61%) in Tp2. Together with two short indels in Tp1, these resulted in 30 and 42 protein variants of Tp1 and Tp2, respectively. Although evidence of positive selection was found for multiple amino acid residues, there was no preferential involvement of T cell epitope residues. Overall, the extent of diversity was much greater in T. parva isolates originating from buffalo than in isolates known to be transmissible among cattle. CONCLUSIONS/SIGNIFICANCE: The results indicate that T. parva parasites maintained in cattle represent a subset of the overall T. parva population, which has become adapted for tick transmission between cattle. The absence of obvious enrichment for positively selected amino acid residues within defined epitopes indicates either that diversity is not predominantly driven by selection exerted by host T cells, or that such selection is not detectable by the methods employed due to unidentified epitopes elsewhere in the antigens. Further functional studies are required to address this latter point.