Isolation of infectious Theileria parva sporozoites secreted by infected Rhipicephalus appendiculatus ticks into an in vitro tick feeding system

Background Vector-borne diseases pose an increasing threat to global food security. Vaccines, diagnostic tests, and therapeutics are urgently needed for tick-borne diseases that affect livestock. However, the inability to obtain significant quantities of pathogen stages derived from ticks has hindered research. In vitro methods to isolate pathogens from infected tick vectors are paramount to advance transcriptomic, proteomic, and biochemical characterizations of tick-borne pathogens. Methods Nymphs of Rhipicephalus appendiculatus were infected with Theileria parva by feeding on a calf during an acute infection. Isolation of sporozoites was accomplished by feeding infected adult ticks on an in vitro tick feeding system. Sporozoite viability was tested using in vitro bovine lymphocytes. Results We isolated infectious T. parva sporozoites secreted into an in vitro tick feeding system. Infected adult R. appendiculatus ticks attached to and successfully fed on silicone membranes in the in vitro tick feeding system. Bovine blood in the receptacle was replaced with cell-free medium and the ticks were allowed to feed for 3 h to collect secreted T. parva sporozoites. Secreted sporozoites infected in vitro bovine lymphocytes, demonstrating that isolated sporozoites remained viable and infectious. Conclusions This work is the first to report the isolation of mature infectious T. parva sporozoites using an in vitro tick feeding system, which represents a significant step towards the development of a more efficient control strategy for T. parva. Isolation of infectious tick-stage parasites will facilitate the examination of the vector-pathogen interface, thereby accelerating the development of next-generation vaccines and treatment interventions for tick-borne pathogens. Graphical Abstract

Inherited Tolerance in Cattle to the Apicomplexan Protozoan Theileria parva is Associated with Decreased Proliferation of Parasite-Infected Lymphocytes

Theileria parva is the causative agent of East Coast fever and Corridor disease, which are fatal, economically important diseases of cattle in eastern, central and southern Africa. Improved methods of control of the diseases are urgently required. The parasite transforms host lymphocytes, resulting in a rapid, clonal expansion of infected cells. Resistance to the disease has long been reported in cattle from T. parva-endemic areas. We reveal here that first- and second-generation descendants of a single Bos indicus bull survived severe challenge with T. parva, (overall survival rate 57.3% compared to 8.7% for unrelated animals) in a series of five field studies. Tolerant cattle displayed a delayed and less severe parasitosis and febrile response than unrelated animals. The in vitro proliferation of cells from surviving cattle was much reduced compared to those from animals that succumbed to infection. Additionally, some pro-inflammatory cytokines such as IL1β, IL6, TNFα or TGFβ which are usually strongly expressed in susceptible animals and are known to regulate cell growth or motility, remain low in tolerant animals. This correlates with the reduced proliferation and less severe clinical reactions observed in tolerant cattle. The results show for the first time that the inherited tolerance to T. parva is associated with decreased proliferation of infected lymphocytes. The results are discussed in terms of whether the reduced proliferation is the result of a perturbation of the transformation mechanism induced in infected cells or is due to an innate immune response present in the tolerant cattle.

Clinical Evaluation of Corridor Disease in Bos indicus (Boran) Cattle Naturally Infected With Buffalo-Derived Theileria parva

Corridor disease (CD) is a fatal condition of cattle caused by buffalo-derived Theileria parva. Unlike the related condition, East Coast fever, which results from infection with cattle-derived T. parva, CD has not been extensively studied. We describe in detail the clinical and laboratory findings in cattle naturally infected with buffalo-derived T. parva. Forty-six cattle were exposed to buffalo-derived T. parva under field conditions at the Ol Pejeta Conservancy, Kenya, between 2013 and 2018. The first signs of disease observed in all animals were nasal discharge (mean day of onset was 9 days post-exposure), enlarged lymph nodes (10 days post-exposure), and pyrexia (13.7 days post-exposure). Coughing and labored breathing were observed in more than 50% of animals (14 days post-exposure). Less commonly observed signs, corneal edema (22%) and diarrhea (11%), were observed later in the disease progression (19 days post-exposure). All infections were considered clinically severe, and 42 animals succumbed to infection. The mean time to death across all studies was 18.4 days. The mean time from onset of clinical signs to death was 9 days and from pyrexia to death was 4.8 days, indicating a relatively short duration of clinical illness. There were significant relationships between days to death and the days to first temperature (chi2 = 4.00, p = 0.046), and days to peak temperature (chi2 = 25.81, p = 0.001), animals with earlier onset pyrexia died sooner. These clinical indicators may be useful for assessing the severity of disease in the future. All infections were confirmed by the presence of macroschizonts in lymph node biopsies (mean time to parasitosis was 11 days). Piroplasms were detected in the blood of two animals (4%) and 20 (43%) animals seroconverted. In this study, we demonstrate the successful approach to an experimental field study for CD in cattle. We also describe the clinical progression of CD in naturally infected cattle, including the onset and severity of clinical signs and pathology. Laboratory diagnoses based on examination of blood samples are unreliable, and alternatives may not be available to cattle keepers. The rapid development of CD requires recognition of the clinical signs, which may be useful for early diagnosis of the disease and effective intervention for affected animals.

Antigenic Diversity in Theileria parva Populations From Sympatric Cattle and African Buffalo Analyzed Using Long Read Sequencing

East Coast fever (ECF) in cattle is caused by the Apicomplexan protozoan parasite Theileria parva, transmitted by the three-host tick Rhipicephalus appendiculatus. The African buffalo (Syncerus caffer) is the natural host for T. parva but does not suffer disease, whereas ECF is often fatal in cattle. The genetic relationship between T. parva populations circulating in cattle and buffalo is poorly understood, and has not been studied in sympatric buffalo and cattle. This study aimed to determine the genetic diversity of T. parva populations in cattle and buffalo, in an area where livestock co-exist with buffalo adjacent to the Serengeti National Park, Tanzania. Three T. parva antigens (Tp1, Tp4, and Tp16), known to be recognized by CD8+ and CD4+ T cells in immunized cattle, were used to characterize genetic diversity of T. parva in cattle (n = 126) and buffalo samples (n = 22). Long read (PacBio) sequencing was used to generate full or near-full length allelic sequences. Patterns of diversity were similar across all three antigens, with allelic diversity being significantly greater in buffalo-derived parasites compared to cattle-derived (e.g., for Tp1 median cattle allele count was 9, and 81.5 for buffalo), with very few alleles shared between species (8 of 651 alleles were shared for Tp1). Most alleles were unique to buffalo with a smaller proportion unique to cattle (412 buffalo unique vs. 231 cattle-unique for Tp1). There were indications of population substructuring, with one allelic cluster of Tp1 representing alleles found in both cattle and buffalo (including the TpM reference genome allele), and another containing predominantly only alleles deriving from buffalo. These data illustrate the complex interplay between T. parva populations in buffalo and cattle, revealing the significant genetic diversity in the buffalo T. parva population, the limited sharing of parasite genotypes between the host species, and highlight that a subpopulation of T. parva is maintained by transmission within cattle. The data indicate that fuller understanding of buffalo T. parva population dynamics is needed, as only a comprehensive appreciation of the population genetics of T. parva populations will enable assessment of buffalo-derived infection risk in cattle, and how this may impact upon control measures such as vaccination.

Development of a Potential Yeast-Based Vaccine Platform for Theileria parva Infection in Cattle

East Coast Fever (ECF), caused by the tick-borne apicomplexan parasite Theileria parva, remains one of the most important livestock diseases in sub-Saharan Africa with more than 1 million cattle dying from infection every year. Disease prevention relies on the so-called “Infection and Treatment Method” (ITM), which is costly, complex, laborious, difficult to standardise on a commercial scale and results in a parasite strain-specific, MHC class I-restricted cytotoxic T cell response. We therefore attempted to develop a safe, affordable, stable, orally applicable and potent subunit vaccine for ECF using five different T. parva schizont antigens (Tp1, Tp2, Tp9, Tp10 and N36) and Saccharomyces cerevisiae as an expression platform. Full-length Tp2 and Tp9 as well as fragments of Tp1 were successfully expressed on the surface of S. cerevisiae. In vitro analyses highlighted that recombinant yeast expressing Tp2 can elicit IFNγ responses using PBMCs from ITM-immunized calves, while Tp2 and Tp9 induced IFNγ responses from enriched bovine CD8+ T cells. A subsequent in vivo study showed that oral administration of heat-inactivated, freeze-dried yeast stably expressing Tp2 increased total murine serum IgG over time, but more importantly, induced Tp2-specific serum IgG antibodies in individual mice compared to the control group. While these results will require subsequent experiments to verify induction of protection in neonatal calves, our data indicates that oral application of yeast expressing Theileria antigens could provide an affordable and easy vaccination platform for sub-Saharan Africa. Evaluation of antigen-specific cellular immune responses, especially cytotoxic CD8+ T cell immunity in cattle will further contribute to the development of a yeast-based vaccine for ECF.

South African Buffalo-Derived Theileria parva Is Distinct From Other Buffalo and Cattle-Derived T. parva

Theileria parva is a protozoan parasite transmitted by the brown-eared ticks, Rhipicephalus appendiculatus and Rhipicephalus zambeziensis. Buffaloes are the parasite’s ancestral host, with cattle being the most recent host. The parasite has two transmission modes namely, cattle–cattle and buffalo–cattle transmission. Cattle–cattle T. parva transmission causes East Coast fever (ECF) and January disease syndromes. Buffalo to cattle transmission causes Corridor disease. Knowledge on the genetic diversity of South African T. parva populations will assist in determining its origin, evolution and identify any cattle–cattle transmitted strains. To achieve this, genomic DNA of blood and in vitro culture material infected with South African isolates (8160, 8301, 8200, 9620, 9656, 9679, Johnston, KNP2, HL3, KNP102, 9574, and 9581) were extracted and paired-end whole genome sequencing using Illumina HiSeq 2500 was performed. East and southern African sample data (Chitongo Z2, Katete B2, Kiambu Z464/C12, Mandali Z22H10, Entebbe, Nyakizu, Katumba, Buffalo LAWR, and Buffalo Z5E5) was also added for comparative purposes. Data was analyzed using BWA and SAMtools variant calling with the T. parva Muguga genome sequence used as a reference. Buffalo-derived strains had higher genetic diversity, with twice the number of variants compared to cattle-derived strains, confirming that buffaloes are ancestral reservoir hosts of T. parva. Host specific SNPs, however, could not be identified among the selected 74 gene sequences. Phylogenetically, strains tended to cluster by host with South African buffalo-derived strains clustering with buffalo-derived strains. Among the buffalo-derived strains, South African strains were genetically divergent from other buffalo-derived strains indicating possible geographic sub-structuring. Geographic sub- structuring was also observed within South Africa strains. The knowledge generated from this study indicates that to date, ECF is not circulating in buffalo from South Africa. It also shows that T. parva has historically been present in buffalo from South Africa before the introduction of ECF and was not introduced into buffalo during the ECF epidemic.

Flow Cytometric Analysis of the Cytotoxic T-Cell Recall Response to Theileria parva in Cattle Following Vaccination by the Infection and Treatment Method

The apicomplexan hemoparasite, Theileria parva, causes East Coast fever (ECF), a frequently fatal disease of African cattle. Vaccine development has been impeded by incomplete understanding of protective immunity following natural exposure or the infection and treatment method (ITM) of immunization. This is attributable to a paucity of methods to characterize the memory T-cell repertoire following infection. To overcome this impediment, assays developed to study the immune response to other intracellular pathogens were adapted for use in studies with T. parva to enable definition of the phenotype and function of effector T cells in T. parva-immune cattle, facilitating vaccine development. As reported herein, stimulation of peripheral blood mononuclear cells (PBMC) from ITM-immunized steers with irradiated, autologous, T. parva-infected cell lines elicited a proliferative recall response comprised of CD45R0+/CCR7− CD4+ and CD8+ T cells. Subsequent co-incubation of stimulated cultures with infected cells demonstrated the presence of cytotoxic T cells (CTLs) with the ability to kill infected cells. Comparison of CTL activity in cultures depleted of CD4+ or CD8+ T cells demonstrated CTL activity was primarily attributed to CD8+ T cells. Importantly, stimulation of PBMC from vaccinated steers always elicited proliferation of CD4+ and CD8+ T cells. This was the first important observation obtained from the use of the assay described herein.