Theileria parva: significance of leukocytes for infecting cattle

Using an artificial feeding technique, infective particles of Theileria parva were harvested in bovine blood in capillary tubes from prefed female Rhipicephalus appendiculatus over a 2-hr period. Inoculations of this blood feed pool invariably resulted in the establishment of patent East Coast fever in autogeneic or syngeneic cattle, i.e., the blood donors or their monozygotic twins, but not in unrelated animals. Mechanical passage of 4 × 10⁶ macroschizont-infected lymphoid cells, harvested from a heifer with East Coast fever, successfully induced patent theileriosis in the donor's monozygotic twin but not in a susceptible allogeneic bovid. The significance of parasite-cell association and histocompatibility of infected cells is discussed in relation to natural and mechanical transmission of Theileria parva.     

Cryopreservation of infective particles of Theileria parva

Cryopreservation of infective particles of Theileria parva. International Journal for Parasitology3: 583–587. Infective particles of Theileria parva, the causative organism of East Coast fever of cattle, were obtained from infected Rhipicephalus appendiculatus ticks, either by using an in vitro feeding technique or by grinding the ticks in a suitable medium. If foetal calf serum containing 15% glycerol (v/v) was added to the infective material and it was then distributed either to glass capillary tubes (in vitro tick feed) or glass tubes (ground tick supernate) it could be slowly frozen to either ?80°C or ?196°C without loss of viability. Stabilates, tested by rapid thawing and inoculation into ECF-susceptible cattle, remained viable for up to a year at these temperatures.     

Absolute Quantification of the Host-To-Parasite DNA Ratio in Theileria parva-Infected Lymphocyte Cell Lines

Theileria parva is a tick-transmitted intracellular apicomplexan pathogen of cattle in sub-Saharan Africa that causes East Coast fever (ECF). ECF is an acute fatal disease that kills over one million cattle annually, imposing a tremendous burden on African small-holder cattle farmers. The pathology and level of T. parva infections in its wildlife host, African buffalo (Syncerus caffer), and in cattle are distinct. We have developed an absolute quantification method based on quantitative PCR (qPCR) in which recombinant plasmids containing single copy genes specific to the parasite (apical membrane antigen 1 gene, ama1) or the host (hypoxanthine phosphoribosyltransferase 1, hprt1) are used as the quantification reference standards. Our study shows that T. parva and bovine cells are present in similar numbers in T. parva-infected lymphocyte cell lines and that consequently, due to its much smaller genome size, T. parva DNA comprises between 0.9% and 3% of the total DNA samples extracted from these lines. This absolute quantification assay of parasite and host genome copy number in a sample provides a simple and reliable method of assessing T. parva load in infected bovine lymphocytes, and is accurate over a wide range of host-to-parasite DNA ratios. Knowledge of the proportion of target DNA in a sample, as enabled by this method, is essential for efficient high-throughput genome sequencing applications for a variety of intracellular pathogens. This assay will also be very useful in future studies of interactions of distinct host-T. parva stocks and to fully characterize the dynamics of ECF infection in the field.     

Construction of a genetic map for Theileria parva: identification of hotspots of recombination

The tick-borne protozoan parasite Theileria parva is the causal agent of East Coast Fever (ECF), a severe lymphoproliferative disease of cattle in eastern, central and southern Africa. The life cycle of T. parva is predominantly haploid, with a brief diploid stage occurring in the tick vector that involves meiotic recombination. Resolved genetic studies of T. parva are currently constrained by the lack of a genome-wide high-definition genetic map of the parasite. We undertook a genetic cross of two cloned isolates of T. parva to construct such a map from 35 recombinant progeny, using a genome-wide panel of 79 variable number of tandem repeat markers. Progeny were established by in vitro cloning of cattle lymphocytes after infection with sporozoites prepared from Rhipicephalus appendiculatus ticks fed on a calf undergoing a dual infection with the two clonal parental stocks. The genetic map was determined by assigning individual markers to the four chromosome genome, whose physical length is approximately 8309 kilobasepairs (Kb). Segregation analysis of the markers among the progeny revealed a total genetic size of 1683.8 centiMorgans (cM), covering a physical distance of 7737.62 Kb (∼93% of the genome). The average genome-wide recombination rate observed for T. parva was relatively high, at 0.22 cM Kb⁻¹ per meiotic generation. Recombination hot-spots and cold-spots were identified for each of the chromosomes. A panel of 27 loci encoding determinants previously identified as immunorelevant or likely to be under selection were positioned on the linkage map. We believe this to be the first genetic linkage map for T. parva. This resource, with the availability of the genome sequence of T. parva, will promote improved understanding of the pathogen by facilitating the use of genetic analysis for identification of loci responsible for variable phenotypic traits exhibited by individual parasite stocks.     

East Coast fever: the infectivity for cattle of infective particles of Theileria parva harvested in various substrates

Cunningham M.P., Brown C.G.D., Burridge M.J., Joyner L.P. and Purnell R.E. 1973. East Coast fever : the infectivity for cattle of infective particles of theileria parva harvested in various substrates. International Journal for Parasitology3: 335–338. Female Rhipicephalus appendiculatus ticks infected with Theileria parva were pre-fed on rabbits for 4 days before being removed and restrained on ‘plasticine’. They were then fed for 2-h periods on capillary tubes containing various substrates, the contents of which were subsequently inoculated into East Coast fever-susceptible cattle. Using this technique, precolostral calf serum and Eagle's Minimum Essential Medium, with the addition of Bovine Albumin Powder, were selected as substrates suitable for further laboratory investigation on East Coast fever as they were acceptable to the ticks and supported the viability of the parasites.     

Theileria parva: Early events in the development of bovine lymphoblastoid cell lines persistently infected with macroschizonts

The cellular origin and development of bovine lymphoblastoid cell lines persistently infected with macroschizonts of Theileria parva was studied. Cultures of lymphoblastoid cells isolated from cattle with patent East Coast fever were compared with those obtained by infecting normal lymphocytes in vitro with sporozoites. The young lines were contrasted with a continuous line which had been isolated earlier. The mononuclear cells were separated from the blood and the inoculum enriched for lymphoblastoid cells and/or lymphocytes by removing the monocytes. The lines arose directly from lymphoblastoid cells transplanted into culture or from lymphocytes infected by sporozoites. In primary cultures of lymphoblastoid cells from the peripheral blood, there was an increase in the proportion of infected cells without the eclipse of the parasite, the macroschizonts were larger than those observed in the inoculum or the continuous line, and there was concurrent microschizont differentiation. In lymphocyte cultures challenged with sporozoites, small mononucleated trophozoites were observed after 2 days which differentiated into typical macroschizonts but microschizonts were rare. In all cultures, the infected cells had mitotic indices of 4 to 5%. As the young lines were passaged, the parasites came to resemble those of the continuous line. The macroschizont size in the continuous line was stable and most had six to eight nuclei but when cultured at high cell concentrations the number of parasite nuclei increased. Minicultures of lymphocytes were used to quantitate the infectivity of sporozoites obtained from organ cultures of Rhipicephalus appendiculatus savliary glands. Sporozoites from ticks fed on rabbits for 5 days were approximately six times more infective than those from glands of ticks fed for 2 days and then cultured at 32 °C for 3 days. Glands from unfed ticks cultured for 5 days failed to yield infective sporozoites.     

Whole-Genome Sequencing of Theileria parva Strains Provides Insight into Parasite Migration and Diversification in the African Continent

The disease caused by the apicomplexan protozoan parasite Theileria parva, known as East Coast fever or Corridor disease, is one of the most serious cattle diseases in Eastern, Central, and Southern Africa. We performed whole-genome sequencing of nine T. parva strains, including one of the vaccine strains (Kiambu 5), field isolates from Zambia, Uganda, Tanzania, or Rwanda, and two buffalo-derived strains. Comparison with the reference Muguga genome sequence revealed 34 814–121 545 single nucleotide polymorphisms (SNPs) that were more abundant in buffalo-derived strains. High-resolution phylogenetic trees were constructed with selected informative SNPs that allowed the investigation of possible complex recombination events among ancestors of the extant strains. We further analysed the dN/dS ratio (non-synonymous substitutions per non-synonymous site divided by synonymous substitutions per synonymous site) for 4011 coding genes to estimate potential selective pressure. Genes under possible positive selection were identified that may, in turn, assist in the identification of immunogenic proteins or vaccine candidates. This study elucidated the phylogeny of T. parva strains based on genome-wide SNPs analysis with prediction of possible past recombination events, providing insight into the migration, diversification, and evolution of this parasite species in the African continent.     

Cell fusion, using Sendai virus, to effect inter-species transfer of a cell-associated parasite (Theileria parva)

Baby hamster kidney (BHK) cells were fused with Theileria parva-infecled bovine lymphoid cells, using u.v. light-inactivated Sendai virus. The resultant hamster/bovine heterokaryons were shown to be infected with T. parva. In some cases parasites were detected in cells which apparently contained only BHK nuclei.     

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.     

Theileria annulata: cross-reactions between a cell culture schizont antigen and antigens of East African species in the indirect fluorescent antibody test

A schizont antigen for the indirect fluorescent antibody test was prepared from an in vitro culture suspension of lymphoid cells infected with Theileria annulata macroschizonts. Two cattle recovered from T. annulata infection showed marked rises in antibody titer to this schizont antigen, with peak titers of 1:40,960 and 1:2560. Using sera from these recovered cattle, T. annulata cell culture schizont antigen was shown to cross-react markedly with Theileria parva and Theileria lawrencei cell culture schizonts and with Theileria mutans piroplasms in the indirect fluorescent antibody test. In contrast, using high-titer antisera to T. parva, T. lawrencei, and T. mutans, serological cross-reactions with T. annulata schizonts were only detected with T. parva and T. lawrencei antisera, and in both instances these were minimal. The value of the indirect fluorescent antibody test in the differential diagnosis of Theileria species pathogenic for cattle is discussed.     

Survival of Rhipicephalus appendiculatus (Acarina: Ixodidae) and persistence of Theileria parva (Apicomplexa: Theileriidae) in the field

Newson R. M., Chiera J. W., Young A. S., Dolan T. T., Cunningham M. P. and Radley D. D. 1984: Survival of Rhipicephalus appendiculatus (Acarina: Ixodidae) and persistence of Theileria parva (Apicomplexa: Theileriidae) in the field. International Journal for Parasitology14; 483–489. Two paddocks with populations of the African brown ear tick (Rhipicephalus appendicuiatus), one of which carried virulent Theileria parva (the causative organism of East Coast fever [ECF] of cattle), were left unstocked for periods of 338 and 354 days. Groups of 1–3, ECF-susceptible cattle were then introduced eight times during the following year to assess the tick and disease challenge, The ticks were also monitored continuously on the ground. The test cattle developed fatal ECF from adult ticks which had fasted for up to 554 days. A non-pathogenic, antigenicaliy distinct, Theileria species was also detected which was still transmitted by adult ticks after 600 days. The mean survival time of unfed larvae was 175 days (max. 240 days); unfed nymphs 270 and 450 (max. 540 days); unfed adults 420 days (max. 730 days), with females in a clear majority after 270 days.     

Some factors controlling the stimulation of sporogony of Theileria parva in its tick vector Rhipicephalus appendiculatus

Young A. S., Leitch B. L. and Mutugi J. J. 1984. Some factors controlling the stimulation of sporogony of Theileria parva in its tick vector, Rhipicephalus appendiculatus. International Journal for Parasitology14: 97–102. The effect of various temperature treatments on the sporogony cycle of Theileria parva in the salivary glands of unfed adult Rhipicephalus appendiculatus ticks of various ages was investigated. It was found that ticks incubated at 28 or 37°C would develop sporozoites infective to cattle but never in such large numbers as in ticks fed on rabbits. Heat stimulation of sporogony was possible for isolates of T. parva with minimal laboratory handling. The age of the ticks incubated at 28 or 37°C was important since sporozoites could only be induced at the earliest on day 27 or 28 p.repl. (post-repletion) and at the latest by day 41 p.repl. The age of ticks fed on rabbits was not as important for the production of sporozoites.     

East coast fever: 60Co-irradiation of Theileria parva in its tick vector, Rhipicephalus appendiculatus

Three experiments were carried out in which Theileria parva was irradiated in its tick vector, Rhipicephalus appendiculatus. In the first experiment, infected unfed adult ticks were irradiated at doubling doses from 4 to 32 krad. Some of the ticks were then fed for 4, 5, 6, 7 and 8 days on rabbits, and the parasites in their salivary glands examined. Five male and 5 female ticks from each irradiation dose were put onto each of a pair of susceptible cattle, whose reactions were recorded. Increasing doses of irradiation resulted in progressive destruction of the parasites. All cattle receiving ticks irradiated at doses up to and including 16 krad died of East Coast fever (ECF), and one of the cattle receiving ticks irradiated at 32 krad died.In the second experiment, recently engorged nymphs were irradiated at 1, 2 or 4 krad, and moulting nymphs at doses of 2, 4, 8, 12, 16 or 32 krad. The salivary glands of the resultant adult ticks were examined after the ticks had fed for 4, 5, 6, 7 or 8 days on rabbits. Engorged nymphs irradiated at 4 krad failed to moult, whilst moulting nymphs irradiated at 32 krad moulted but failed to attach to rabbits. Doses of irradiation survived by the ticks had no apparent morphological effect on the parasites they contained.In the third experiment, infected unfed adult ticks were irradiated at 0, 5, 10, 15, 20 25 30, 35, 40, 45, 50 or 60 krad. The ticks were fed on rabbits for 5, 6 or 7 days. Some of them were then examined morphologically, whilst others were ground in MEM/BPA and aliquots of the supernatant used to inoculate groups of 5 cattle. The reaction of these cattle, together with the morphological examination of the parasites, suggested that increasing doses of irradiation destroyed increasing numbers of parasites.     

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.     

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.     

Whole genome sequencing of Theileria parva using target capture

Protozoan parasite isolation and purification are laborious and time-consuming processes required for high quality genomic DNA used in whole genome sequencing. The objective of this study was to capture whole Theileria parva genomes directly from cell cultures and blood samples using RNA baits. Cell culture material was bait captured or sequenced directly, while blood samples were all captured. Baits had variable success in capturing T. parva genomes from blood samples but were successful in cell cultures. Genome mapping uncovered extensive host contamination in blood samples compared to cell cultures. Captured cell cultures had over 81 fold coverage for the reference genome compared to 0–33 fold for blood samples. Results indicate that baits are specific to T. parva, are a good alternative to conventional methods and thus ideal for genomic studies. This study also reports the first whole genome sequencing of South African T. parva.     

Linkage of two distinct AT-rich minisatellites at multiple loci in the genome of Theileria parva

Minisatellite tandem repeat elements are well known components of vertebrate genomes, but have not yet been extensively characterized in lower eukaryotes. We describe two unusual, AT-rich minisatellites of the protozoan parasite Theileria parva whose sequences are unrelated to the G/C-rich `chi minisatellite superfamily' of vertebrate and plant genomes. The T. parva tandem repeats, one with a conserved sequence T_2-5ACACA (6–17 copies), and the other with a 6-bp core sequence of either ACTATA or TATACT associated with additional variable sequences in repeats of 10–17 bp (3–7 copies), were closely linked at more than 20 sites in the T. parva genome, separated by 390, 510 and 660 bp at three loci analysed in detail. Such linkage is without precedent in minisatellites so far analysed in other organisms. The minisatellite loci were widely dispersed on 13 out of 33 genomic SfiI fragments, on all four T. parva chromosomes and did not exhibit a telomeric bias in their distribution. Analysis of flanking sequences revealed no obvious conserved sequences between the five loci, or other multicopy repeat sequences outside the minisatellite regions. The T_2-5 ACACA minisatellite was highly effective as a multilocus fingerprinting probe for discrimination of T. parva isolates. Analysis of two individual minisatellite loci revealed variation between the genomic DNAs of two T. parva isolates in the copy number of the constituent repeats within the array, similar to that typical of vertebrate minisatellites.     

Characterization of some East African Theileria species isolates by isoenzyme analysis, with particular reference to T. parva

Eight different isolates of Theileria parva and one isolate of T. taurotragi, in the form of intra-lymphocytic schizonts and/or purified piroplasms, were subjected to isoenzyme analysis for 24 enzymes by both isoelectric focusing in agarose and electrophoresis in starch gel. Twelve enzymes distinguished between T. parva and T. taurotragi; five enzymes (HK, GPI, PEP1, LDH and SOD) showed variations within T. parva. The metabolism of the host cell was affected by schizont infection, which masked intraspecific variations. Piroplasms were of more potential value for characterization of T. parva.     

Serum of rabbits infected with Treponema pallidum (Nichols) inhibits in vitro transformation of normal rabbit lymphocytes.

Serum collected from outbred male New Zealand white rabbits infected intratesticularly with Treponema pallidum (Nichols) was assayed for ability to alter transformation of normal rabbit peripheral blood lymphocytes (PBL) in vitro. Sera collected from 25 infected rabbits inhibited [³H]thymidine incorporation by normal rabbit PBL stimulated with concanavalin A (Con A, 16μg/ml), relative to PBL cultured in normal rabbit serum (NRS). Maximal inhibitory activity was detected in serum collected at the time of peak orchitis. The degree of inhibition was related to the concentration of syphilitic serum in PBL cultures. Inhibition of Con A stimulation was reversed by increased mitogen concentration. Sera which depressed Con A stimulation also depressed lymphocyte transformation induced by oxidation with sodium m-periodate (NaIO₄). Cytotoxic activity was detected in occasional sera. All sera were heat inactivated at 56 °C for 30 min prior to testing. Both freshly collected sera and sera stored at ?70 °C significantly inhibited PBL transformation. These results suggested that serum of syphilitic rabbits contains one or more inhibitors of in vitro lymphocyte transformation.