Continent-wide genomic analysis of the African buffalo (Syncerus caffer).

The African buffalo (Syncerus caffer) is a wild bovid with a historical distribution across much of sub-Saharan Africa. Genomic analysis can provide insights into the evolutionary history of the species, and the key selective pressures shaping populations, including assessment of population level differentiation, population fragmentation, and population genetic structure. In this study we generated the highest quality de novo genome assembly (2.65 Gb, scaffold N50 69.17 Mb) of African buffalo to date, and sequenced a further 195 genomes from across the species distribution. Principal component and admixture analyses provided little support for the currently described four subspecies. Estimating Effective Migration Surfaces analysis suggested that geographical barriers have played a significant role in shaping gene flow and the population structure. Estimated effective population sizes indicated a substantial drop occurring in all populations 5-10,000 years ago, coinciding with the increase in human populations. Finally, signatures of selection were enriched for key genes associated with the immune response, suggesting infectious disease exert a substantial selective pressure upon the African buffalo. These findings have important implications for understanding bovid evolution, buffalo conservation and population management.

In vitro infection of bovine erythrocytes with Theileria annulata merozoites as a key step in completing the T. annulata life cycle in vitro.

Theileria annulata is a protozoan parasite with a complex life cycle involving a bovine host and a tick vector. It is transmitted by Hyalomma ticks and is the causative agent of tropical theileriosis, a debilitating and often fatal disease in southern Europe, northern Africa and large parts of Asia. Understanding the biology of different life cycle stages is critical for the control of tropical theileriosis and requires the use of experimental animals which poses an ethical concern. We present for the first time the in vitro infection of red blood cells (RBCs) with T. annulata differentiated schizonts. The Ankara cell line of T. annulata was cultured at 41 °C for nine days to induce merogony and subsequently incubated with purified RBCs for one to three days. Percentage of parasitized erythrocyte (PPE) over the short culture period was estimated by Giemsa staining (0.007-0.01%), Flow cytometry activated sorting (FACS) (0.02-1.1%) and observation of FACS sorted cells by confocal microscopy (0.05-0.4%). There was a significant difference in the PPE between FACS and the two other techniques (one-way ANOVA followed by Tukey test, P = 0.004) but no significant difference was observed between the confocal imaging and Giemsa staining methods (ANOVA one-way followed by Tukey test, P = 0.06). Importantly, all three complementary methods confirmed the invasion of RBCs by T. annulata merozoites in vitro. Although the experimental conditions will require further optimization to increase the PPE, the in vitro infection of RBCs by T. annulata merozoites is pivotal in paving the way for the eventual completion of the T. annulata life cycle in vitro when combined with artificial tick feeding.

Dual RNA-seq to catalogue host and parasite gene expression changes associated with virulence of T. annulata-transformed bovine leukocytes: towards identification of attenuation biomarkers.

The apicomplexan parasite Theileria annulata is transmitted by Hyalomma ticks and causes an acute lymphoproliferative disease that is invariably lethal in exotic cattle breeds. The unique ability of the schizont stage of T. annulata to transform infected leukocytes to a cancer-like phenotype and the simplicity of culturing and passaging T. annulata-transformed cells in vitro have been explored for live vaccine development by attenuating the transformed cells using lengthy serial propagation in vitro. The empirical in vivo evaluation of attenuation required for each batch of long-term cultured cells is a major constraint since it is resource intensive and raises ethical issues regarding animal welfare. As yet, the molecular mechanisms underlying attenuation are not well understood. Characteristic changes in gene expression brought about by attenuation are likely to aid in the identification of novel biomarkers for attenuation. We set out to undertake a comparative transcriptome analysis of attenuated (passage 296) and virulent (passage 26) bovine leukocytes infected with a Tunisian strain of T. annulata termed Beja. RNA-seq was used to analyse gene expression profiles and the relative expression levels of selected genes were verified by real-time quantitative PCR (RT-qPCR) analysis. Among the 3538 T. annulata genes analysed, 214 were significantly differentially expressed, of which 149 genes were up-regulated and 65 down-regulated. Functional annotation of differentially expressed T. annulata genes revealed four broad categories of metabolic pathways: carbon metabolism, oxidative phosphorylation, protein processing in the endoplasmic reticulum and biosynthesis of secondary metabolites. It is interesting to note that of the top 40 genes that showed altered expression, 13 were predicted to contain a signal peptide and/or at least one transmembrane domain, suggesting possible involvement in host-parasite interaction. Of the 16,514 bovine transcripts, 284 and 277 showed up-regulated and down-regulated expression, respectively. These were assigned to functional categories relevant to cell surface, tissue morphogenesis and regulation of cell adhesion, regulation of leucocyte, lymphocyte and cell activation. The genetic alterations acquired during attenuation that we have catalogued herein, as well as the accompanying in silico functional characterization, do not only improve understanding of the attenuation process, but can also be exploited by studies aimed at identifying attenuation biomarkers across different cell lines focusing on some host and parasite genes that have been highlighted in this study, such as bovine genes (CD69, ZNF618, LPAR3, and APOL3) and parasite genes such as TA03875.

Genetic diversity of Hyalomma marginatum in Tunisia is not influenced by the bio-climate.

Ticks are important ectoparasites responsible for the transmission of several pathogens with significant medical, veterinary, and economic impacts. Climate and social changes have generated substantial changes in ticks' distribution, abundance, and activity patterns, including ticks belonging to the Hyalomma marginatum species. Knowledge on the genetic structure and dynamics of H. marginatum populations might contribute to a better understanding of their current and future evolution under the effects of anthropogenic factors and eco-climatic changes. In the present study, we investigated the genetic structure and phylogenetic distribution of H. marginatum across three bioclimatic regions in Tunisia using two mitochondrial markers (16S and 12S rRNA). The molecular investigations were based on 47 adult H. marginatum ticks collected from humid, upper semi-arid, and sub-humid regions of Tunisia. Our results revealed a genetic diversity of 0.278% and 0.809% using the 16S and 12S markers, respectively. The low genetic diversity that we observed raises the hypothesis of a bottleneck event occasioned by a reduction in the size of the tick population under the effects of environmental factors and/or human activities. This hypothesis is supported by the population's demographic history analysis, which revealed a clear deviation from neutrality and supports the occurrence of a bottleneck event followed by a demographic expansion. The fact that most 16S and 12S variability was present in the ticks from the humid bioclimatic zone may suggest that those ticks represent the ancestral population. Overall, the analysis has shown that the phylogenetic clusters do not correspond to the bioclimatic zones.

Conservation and variation in the region of the Theileria parva p104 antigen coding gene used for PCR surveillance of the parasite.

The range of the protozoan parasite Theileria parva, which causes East Coast fever in cattle, has been expanding to countries where it has not previously been detected, as a result of cross-border domestic cattle movement. Countries where T. parva has not previously been observed until recently include Cameroon and South Sudan. This raises the issue of the conservation of the p104 antigen gene, on which the nested PCR assay that is widely used for T. parva surveillance in the blood of infected cattle is based. We sampled 40 isolates from six countries widely distributed across the geographical range of the parasite, including eastern, central and southern Africa, for p104 sequence polymorphism. These included parasites from both domestic cattle and the Cape buffalo (Syncerus caffer) wildlife reservoir. The most frequent allelic variants were present in cattle transmissible isolates from multiple widely separated geographical regions in Zambia, Uganda, Kenya, Tanzania, Rwanda and South Africa. These frequent p104 variants were also present in the three component stocks of the Muguga cocktail used for the infection and treatment live immunisation procedure to control T. parva in the field. Other isolates exhibited unique alleles. This includes some of the p104 sequences from Cameroon, which is outside the known range of the Rhipicephalus tick vector and whose origin is therefore unclear. The nested primer oligonucleotides used to generate the amplicons were universally conserved in cattle-derived parasites and a majority of buffalo-derived isolates across the geographical range of the parasite. However, some rare South African buffalo-derived isolates exhibited one or two mismatches with the primer sequences. It therefore remains possible that some p104 alleles may be so divergent that they do not amplify with the current diagnostic primers and are not detectable in surveys, hence the need for increasing knowledge of genetic heterogeneity of diagnostic targets. There was no evidence for positive selection among those p104 mutations that resulted in residue changes. Importantly, the data indicate that the p104-based PCR detection assay should be effective across the majority of the range of T. parva, and if the one or two mismatches are shown in future to result in the primers annealing less efficiently, then the assay can be further improved by introduction of degenerate bases to enable amplification of the less frequent South African buffalo-derived variant p104 genes.

The antigen recognition portion of African buffalo class I MHC is highly polymorphic, consistent with a complex pathogen challenge environment, and the 3' region suggests distinct haplotype configurations.

African buffalo (Syncerus caffer) have been distinct from the Auroch lineage leading to domestic cattle for 5 million years, and are reservoirs of multiple pathogens, that affect introduced domestic cattle. To date, there has been no analysis of the class I MHC locus in African buffalo. We present the first data on African buffalo class I MHC, which demonstrates that gene and predicted protein coding sequences are approximately 86-87% similar to that of African domestic cattle in the peptide binding region. The study also shows concordance in the distribution of codons with elevated posterior probabilities of positive selection in the buffalo class I MHC and known antigen binding sites in cattle. Overall, the diversity in buffalo class I sequences appears greater than that in cattle, perhaps related to a more complex pathogen challenge environment in Africa. However, application of NetMHCpan suggested broad clustering of peptide binding specificities between buffalo and cattle. Furthermore, in the case of at least 20 alleles, critical peptide-binding residues appear to be conserved with those of cattle, including at secondary anchor residues. Alleles with six different length transmembrane regions were detected. This preliminary analysis suggests that like cattle, but unlike most other mammals, African buffalo appears to exhibit configuration (haplotype) variation in which the loci are expressed in distinct combinations.

Sequence polymorphisms in a Theileria annulata surface protein (TaSP) known to augment the immunity induced by live attenuated cell line vaccine.

Theileria annulata is a tick-borne protozoan causing tropical theileriosis in cattle. The use of attenuated cell line vaccines in combination with subunit vaccines has been relatively successful as a control method, as exemplified by a recent study in which immunization with a local cell line followed by booster vaccinations with recombinant T. annulata surface protein (TaSP) resulted in 100% protection upon field challenge in Sudan. However, these findings cannot be directly extrapolated to other countries as culture-attenuated live vaccines are generated using local strains and no systematic evaluation of genotype differences between countries has been undertaken. In this study, we sequenced the TaSP gene from T. annulata cell lines and field isolates from Tunisia (n = 28) and compared them to genotypes from Sudan (n = 25) and Morocco (n = 1; AJ316259.1). Our analyses revealed 20 unique TaSP genotypes in the Tunisian samples, which were all novel but similar to genotypes found in Asia. The impact of these polymorphisms on the ability of the TaSP antigen to boost the immunity engendered by live cell line vaccines, especially in Tunisia where studies with TaSP have not been conducted, remains to be examined. Interestingly, phylogenetic analyses of publicly available TaSP sequences resolved the sequences into two clusters with no correlation to the geographical origin of the isolates. The availability of candidate vaccines that were recently attenuated using local strains from Sudan, Tunisia, Egypt and Morocco should be exploited to generate a comprehensive catalogue of genetic variation across this regional collection of attenuated live vaccines.

Phenology and phylogeny of Hyalomma spp. ticks infesting one-humped camels (Camelus dromedarius) in the Tunisian Saharan bioclimatic zone.

In this study, we report the results of a survey of Hyalomma ticks infesting one-humped camels in southern Tunisia. Examinations were conducted every second or third month on 406 camels in Tataouine district from April 2018 to October 2019. A total of 1902 ticks belonging to the genus Hyalomma were collected. The ticks were identified as adult H. impeltatum (41.1%; n = 782), H. dromedarii (32.9%; n = 626), H. excavatum (25.9%; n = 493), and H. marginatum for a single specimen. Although the camels were infested by ticks throughout the year, the highest overall infestation prevalence was observed in April 2018 (p < 0.01). The overall infestation intensity varied between 2.7 and 7.4 ticks/animal. There were no statistically significant differences in tick infestation prevalence based on age categories of the camels, and the overall infestation prevalence was between 82.7% and 97.4%. Female camels were significantly more infested with ticks (88.3%) than males (65.5%) (p < 0.01). The infestation prevalence of camels varied significantly according to the region where sampling took place (p < 0.01), but no correlations were found with abiotic factors. The preferred attachment sites for adult Hyalomma ticks were the sternum (38.3%; n = 729/1902), around the anus (36.2%; n = 689/1902), udder (18.4%; n = 350/1902), and inner thigh (6.9%; n = 132/1902). Morphological classification of ticks was corroborated by sequencing the cytochrome c oxidase I (Cox1) and 16S rDNA genes, and these sequences were also used to infer phylogenetic relationships. A single H. dromedarii seemed to be a natural hybrid with H. rufipes. More attention should be devoted by the veterinary services to the infestation of camels by ticks.

TITLE: Phénologie et phylogénie des tiques Hyalomma spp. infestant les dromadaires (Camelus dromedarius) dans la zone bioclimatique saharienne tunisienne. ABSTRACT: Dans cette étude, les résultats d'une enquête concernant les tiques Hyalomma infestant les dromadaires dans le sud de la Tunisie sont présentés. Des examens ont été menés tous les deux ou trois mois sur 406 dromadaires dans le district de Tataouine entre avril 2018 et octobre 2019. Au total, 1902 tiques appartenant au genre Hyalomma ont été collectées. Les tiques adultes ont été identifiées comme H. impeltatum (41,1 % ; n = 782), H. dromedarii (32,9 % ; n = 626), H. excavatum (25,9 % ; n = 493) et un seul spécimen de H. marginatum. Bien que les dromadaires aient été infestés par les tiques tout au long de l'année, la prévalence globale d'infestation la plus élevée a été observée en avril 2018 (p < 0,01). L'intensité globale d'infestation variait entre 2,7 et 7,4 tiques/animal. Il n'y avait pas de différence statistiquement significative de l'infestation par les tiques en fonction des catégories d'âge des dromadaires, et la prévalence globale d'infestation se situait entre 82,7 et 97,4 %. Les dromadaires femelles étaient significativement plus infestés par les tiques (88,3 %) que les mâles (65,5 %) (p < 0,01). La prévalence d'infestation variait significativement en fonction des régions (p < 0.01) mais il n'y avait pas de corrélation avec les facteurs abiotiques. Les sites de fixation préférés des tiques Hyalomma adultes étaient le sternum (38,3 % ; n = 729/1902), autour de l'anus (36,2 % ; n = 689/1902), la mamelle (18,4 % ; n = 350/1902) et la face interne de la cuisse (6,9 % ; n = 132/1902). La classification morphologique a été corroborée par le séquençage des gènes de l'ADNr du cytochrome c oxydase I (Cox1) et du 16S, et ces séquences ont également été utilisées pour déduire les relations phylogénétiques. Un seul spécimen de H. dromedarii semblait être un hybride naturel avec H. rufipes. Une plus grande attention doit être accordée par les services vétérinaires quant à l'infestation des dromadaires par les tiques.

Unique Mitochondrial Single Nucleotide Polymorphisms Demonstrate Resolution Potential to Discriminate Theileria parva Vaccine and Buffalo-Derived Strains.

Distinct pathogenic and epidemiological features underlie different Theileria parva strains resulting in different clinical manifestations of East Coast Fever and Corridor Disease in susceptible cattle. Unclear delineation of these strains limits the control of these diseases in endemic areas. Hence, an accurate characterization of strains can improve the treatment and prevention approaches as well as investigate their origin. Here, we describe a set of single nucleotide polymorphisms (SNPs) based on 13 near-complete mitogenomes of T. parva strains originating from East and Southern Africa, including the live vaccine stock strains. We identified 11 SNPs that are non-preferentially distributed within the coding and non-coding regions, all of which are synonymous except for two within the cytochrome b gene of buffalo-derived strains. Our analysis ascertains haplotype-specific mutations that segregate the different vaccine and the buffalo-derived strains except T. parva-Muguga and Serengeti-transformed strains suggesting a shared lineage between the latter two vaccine strains. Phylogenetic analyses including the mitogenomes of other Theileria species: T. annulata, T. taurotragi, and T. lestoquardi, with the latter two sequenced in this study for the first time, were congruent with nuclear-encoded genes. Importantly, we describe seven T. parva haplotypes characterized by synonymous SNPs and parsimony-informative characters with the other three transforming species mitogenomes. We anticipate that tracking T. parva mitochondrial haplotypes from this study will provide insight into the parasite's epidemiological dynamics and underpin current control efforts.

Variant analysis of the sporozoite surface antigen gene reveals that asymptomatic cattle from wildlife-livestock interface areas in northern Tanzania harbour buffalo-derived T. parva.

Buffalo-derived Theileria parva can 'break through' the immunity induced by the infection and treatment vaccination method (ITM) in cattle. However, no such 'breakthroughs' have been reported in northern Tanzania where there has been long and widespread ITM use in pastoralist cattle, and the Cape buffalo (Syncerus caffer) is also present. We studied the exposure of vaccinated and unvaccinated cattle in northern Tanzania to buffalo-derived T. parva using p67 gene polymorphisms and compared this to its distribution in vaccinated cattle exposed to buffalo-derived T. parva in central Kenya, where vaccine 'breakthroughs' have been reported. Additionally, we analysed the CD8+ T cell target antigen Tp2 for positive selection. Our results showed that 10% of the p67 sequences from Tanzanian cattle (n = 39) had a buffalo type p67 (allele 4), an allele that is rare among East African isolates studied so far. The percentage of buffalo-derived p67 alleles observed in Kenyan cattle comprised 19% of the parasites (n = 36), with two different p67 alleles (2 and 3) of presumptive buffalo origin. The Tp2 protein was generally conserved with only three Tp2 variants from Tanzania (n = 33) and five from Kenya (n = 40). Two Tanzanian Tp2 variants and two Kenyan Tp2 variants were identical to variants present in the trivalent Muguga vaccine. Tp2 evolutionary analysis did not show evidence for positive selection within previously mapped epitope coding sites. The p67 data indicates that some ITM-vaccinated cattle are protected against disease induced by a buffalo-derived T. parva challenge in northern Tanzania and suggests that the parasite genotype may represent one factor explaining this.

Monitoring vaccinated cattle for induction and longevity of persistent tick-transmissible infection: Implications for wider deployment of live vaccination against East Coast fever in Tanzania.

The infection and treatment (ITM) procedure remains the only available method of immunization against Theileria parva infection. One constraint to deployment is the perception that the carrier state induced by ITM could result in enhanced disease problems. More than one million cattle have been ITM vaccinated in pastoralist systems in Tanzania over the last 2 decades. We present the results of a longitudinal study of six groups of cattle in Maasai villages in northern Tanzania exposed to natural tick challenge for between 2 weeks and 14 years post-vaccination. The p104 nested PCR revealed a higher frequency of T. parva carriers among vaccinates (30%) compared with controls (8%) (OR = 4.89, p = .000), with the highest frequency of carriers found in calves vaccinated 6 months previously, although carrier state was also detected in cattle vaccinated >10 years prior to the study. Variable number tandem repeat genotype analysis revealed 6 MS7 alleles with sizes ranging from 150 bp to 500 bp, but only two alleles were detected in cattle vaccinated >4 years earlier, relative to five alleles detected in recently vaccinated cattle and controls. In terms of heterozygosity, diversity was maximal in calves vaccinated within the last 2 weeks (h = 0.776) but lowest in cattle vaccinated 4 years earlier (h = 0.375). The analysis suggested close genetic relatedness of parasites in vaccinated and unvaccinated groups and up to 96% of variation was within rather than between the groups. These results confirm that ITM leads to a long-term T. parva carrier state in cattle and the detection of vaccine component VNTR in co-grazing unvaccinated cattle suggests potential vaccine transmission by ticks. However, vaccination stocks did not totally replace local genotypes, at least in cattle populations. These findings should mitigate concerns that ITM modifies T. parva field populations in a way that enhances disease in the medium term.

Current status of tropical theileriosis in Northern Africa: A review of recent epidemiological investigations and implications for control.

Tropical theileriosis caused by the apicomplexan hemoparasite Theileria annulata is a tick-borne disease that constraints livestock production in parts of Europe, Asia and Africa. Four Hyalomma tick species transmit T. annulata in at least eight Africa countries (Mauritania, Morocco, Algeria, Tunisia, Egypt, Sudan, South Sudan and Ethiopia). The two dominant T. annulata vector ticks present in Africa, H. scupense and H. anatolicum, underlie two different patterns of transmission, which in turn greatly influence the epidemiology of tropical theileriosis. H. dromedarii and H. lusitanicum are also capable of transmitting T. annulata in North Africa, but their roles are associated with specific production systems and agro-ecological contexts. The emergence of resistance to the most widely used theilericidal compound, buparvaquone, continues to limit the effectiveness of chemotherapy. In addition, acaricide use is increasingly becoming unsustainable. Deployable T. annulata attenuated live vaccines established from local strains in Tunisia, Sudan and Egypt are available, and recent work has indicated that these vaccines can be protective under conditions of natural transmission. However, vaccination programmes may vary over space and time due to differences in the prevalence of disease amongst cattle populations, as well seasonal variation in vector activity. We review recent descriptive and analytical surveys on the epidemiology of T. annulata infection with reference to (a) demographic aspects such as breeds and ages of cattle herds previously exposed to distinct T. annulata infection pressures and (b) seasonal dynamics of tick activity and disease transmission. We then discuss how the wider endemic patterns that we delineate can underpin the development and execution of future vaccination programmes. We also outline options for integrated control measures targeting tick vectors and husbandry practices.

The protection afforded to cattle immunized with Theileria annulata infected cell line is enhanced by subunit vaccine candidate TaSP.

Tropical theileriosis constraints the development of the dairy industry in the Sudan and vaccination using live attenuated schizont vaccines is considered a promising measure for its control. The present study was carried out to investigate the ability of recombinant T. annulata surface protein (TaSP) to improve the efficacy of the attenuated Atbara cell line in protecting calves against field challenge. To this end, 23 cross-bred (Friesian × Kenana) calves were divided into four groups. Animals in group 1 (n = 5) were left unvaccinated. Group 2 (n = 6) received the Atbara cell line, animals in group 3 (n = 6) were immunized with three doses of TaSP on days 21, 49 and 77, while animals in group 4 (n = 6) received the cell line vaccine on day 0 and three doses of TaSP in Freund's incomplete adjuvant at days 21, 49 and 77. Twenty-eight days after the last TaSP boost, all groups were challenged by exposing them to natural field tick infestation in a region known to be endemic for tropical theileriosis. No thermal reactions, piroplasms or schizonts were observed in the immunized animals following immunization. Upon challenge, all animals showed a range of symptoms of clinical theileriosis with variable degrees of severity. The application of TaSP alone appeared to have no effect in terms of protection. The efficacy of the cell line alone was lower than the 100% level of protection against mortality observed in the group that received the combined cell line vaccine and TaSP, suggesting a synergistic effect of this combination.

Antigen gene and variable number tandem repeat (VNTR) diversity in Theileria parva parasites from Ankole cattle in south-western Uganda: Evidence for conservation in antigen gene sequences combined with extensive polymorphism at VNTR loci.

Theileria parva is a tick-transmitted apicomplexan protozoan parasite that infects lymphocytes of cattle and African Cape buffalo (Syncerus caffer), causing a frequently fatal disease of cattle in eastern, central and southern Africa. A live vaccination procedure, known as infection and treatment method (ITM), the most frequently used version of which comprises the Muguga, Serengeti-transformed and Kiambu 5 stocks of T. parva, delivered as a trivalent cocktail, is generally effective. However, it does not always induce 100% protection against heterologous parasite challenge. Knowledge of the genetic diversity of T. parva in target cattle populations is therefore important prior to extensive vaccine deployment. This study investigated the extent of genetic diversity within T. parva field isolates derived from Ankole (Bos taurus) cattle in south-western Uganda using 14 variable number tandem repeat (VNTR) satellite loci and the sequences of two antigen-encoding genes that are targets of CD8+T-cell responses induced by ITM, designated Tp1 and Tp2. The findings revealed a T. parva prevalence of 51% confirming endemicity of the parasite in south-western Uganda. Cattle-derived T. parva VNTR genotypes revealed a high degree of polymorphism. However, all of the T. parva Tp1 and Tp2 alleles identified in this study have been reported previously, indicating that they are widespread geographically in East Africa and highly conserved.

Serum-free in vitro cultivation of Theileria annulata and Theileria parva schizont-infected lymphocytes.

Theileriosis is a tick-borne disease caused by intracellular protozoa of the genus Theileria. The most important species in cattle are Theileria annulata and Theileria parva. Both species transform leucocyte host cells, resulting in their uncontrolled proliferation and immortalization. Vaccination with attenuated T. annulata-infected cell lines is currently the only practical means of inducing immunity in cattle. Culture media for Theileria spp. typically contain 10%-20% foetal bovine serum (FBS). The use of FBS is associated with several disadvantages, such as batch-to-batch variation, safety and ethical concerns. In this study, the suitability of serum-free media for the cultivation of Theileria-transformed cell lines was examined. Three commercial serum-free media (HL-1, ISF-1 and Hybridomed DIF 1000) were evaluated for their ability to support growth of the T. annulata A288 cell line. The generation doubling times were recorded for each medium and compared with those obtained with conventional FBS-containing RPMI-1640 medium. ISF-1 gave the shortest generation doubling time, averaging 35.4 ± 2.8 hr, significantly shorter than the 52.2 ± 14.9 hr recorded for the conventional medium (p = .0011). ISF-1 was subsequently tested with additional T. annulata strains. The doubling time of a Moroccan strain was significantly increased (65.4 ± 15.9 hr) compared with the control (47.7 ± 7.5 hr, p = .0004), whereas an Egyptian strain grew significantly faster in ISF-1 medium (43.4 ± 6.5 hr vs. 89.3 ± 24.8 hr, p = .0001). The latter strain also showed an improved generation doubling time of 73.7 ± 21.9 hr in an animal origin-free, serum-free, protein-free medium (PFHM II) compared with the control. Out of four South African T. parva strains and a Theileria strain isolated from roan antelope (Hippotragus equinus), only one T. parva strain could be propagated in ISF-1 medium. The use of serum-free medium may thus be suitable for some Theileria cell cultures and needs to be evaluated on a case-by-case basis. The relevance of Theileria cultivation in serum-free media for applications such as vaccine development requires further examination.

A review of recent research on Theileria parva: Implications for the infection and treatment vaccination method for control of East Coast fever.

The infection and treatment (ITM) live vaccination method for control of Theileria parva infection in cattle is increasingly being adopted, particularly in Maasai pastoralist systems. Several studies indicate positive impacts on human livelihoods. Importantly, the first detailed protocol for live vaccine production at scale has recently been published. However, quality control and delivery issues constrain vaccination sustainability and deployment. There is evidence that the distribution of T. parva is spreading from endemic areas in East Africa, North into Southern Sudan and West into Cameroon, probably as a result of anthropogenic movement of cattle. It has also recently been demonstrated that in Kenya, T. parva derived from cape buffalo can 'breakthrough' the immunity induced by ITM. However, in Tanzania, breakthrough has not been reported in areas where cattle co-graze with buffalo. It has been confirmed that buffalo in northern Uganda national parks are not infected with T. parva and R. appendiculatus appears to be absent, raising issues regarding vector distribution. Recently, there have been multiple field population genetic studies using variable number tandem repeat (VNTR) sequences and sequencing of antigen genes encoding targets of CD8+ T-cell responses. The VNTR markers generally reveal high levels of diversity. The antigen gene sequences present within the trivalent Muguga cocktail are relatively conserved among cattle transmissible T. parva populations. By contrast, greater genetic diversity is present in antigen genes from T. parva of buffalo origin. There is also evidence from several studies for transmission of components of stocks present within the Muguga cocktail, into field ticks and cattle following induction of a carrier state by immunization. In the short term, this may increase live vaccine effectiveness, through a more homogeneous challenge, but the long-term consequences are unknown.

Sequence diversity between class I MHC loci of African native and introduced Bos taurus cattle in Theileria parva endemic regions: in silico peptide binding prediction identifies distinct functional clusters.

There is strong evidence that the immunity induced by live vaccination for control of the protozoan parasite Theileria parva is mediated by class I MHC-restricted CD8(+) T cells directed against the schizont stage of the parasite that infects bovine lymphocytes. The functional competency of class I MHC genes is dependent on the presence of codons specifying certain critical amino acid residues that line the peptide binding groove. Compared with European Bos taurus in which class I MHC allelic polymorphisms have been examined extensively, published data on class I MHC transcripts in African taurines in T. parva endemic areas is very limited. We utilized the multiplexing capabilities of 454 pyrosequencing to make an initial assessment of class I MHC allelic diversity in a population of Ankole cattle. We also typed a population of exotic Holstein cattle from an African ranch for class I MHC and investigated the extent, if any, that their peptide-binding motifs overlapped with those of Ankole cattle. We report the identification of 18 novel allelic sequences in Ankole cattle and provide evidence of positive selection for sequence diversity, including in residues that predominantly interact with peptides. In silico functional analysis resulted in peptide binding specificities that were largely distinct between the two breeds. We also demonstrate that CD8(+) T cells derived from Ankole cattle that are seropositive for T. parva do not recognize vaccine candidate antigens originally identified in Holstein and Boran (Bos indicus) cattle breeds.

Molecular evolution of a central region containing B cell epitopes in the gene encoding the p67 sporozoite antigen within a field population of Theileria parva.

Protective immunity induced by the infective sporozoite stage of Theileria parva indicates a potential role for antibodies directed against conserved serologically reactive regions of the major sporozoite surface antigen p67 in vaccination to control the parasite. We have examined the allelic variation and determined the extent of B cell epitope polymorphism of the gene encoding p67 among field isolates originating from cattle exposed to infected ticks in the Marula area of the rift valley in central Kenya where the African cape buffalo (Syncerus caffer) and cattle co-graze. In the first of two closely juxtaposed epitope sequences in the central region of the p67 protein, an in-frame deletion of a seven-amino acid segment results in a truncation that was observed in parasites derived from cattle that co-grazed with buffalo. In contrast, the variation in the second epitope was primarily due to nonsynonymous substitutions, resulting in relatively low overall amino acid conservation in this segment of the protein. We also observed polymorphism in the region of the protein adjacent to the two defined epitopes, but this was not sufficient to provide statistically significant evidence for positive selection. The data indicates that B cell epitopes previously identified within the p67 gene are polymorphic within the Marula field isolates. Given the complete sequence identity of the p67 gene in all previously characterized T. parva isolates that are transmissible between cattle by ticks, the diversity observed in p67 from the Marula isolates in combination with the clinical reaction of the infected cattle is consistent with them originating from ticks that had acquired T. parva from buffalo.

Genetic and expression analysis of cattle identifies candidate genes in pathways responding to Trypanosoma congolense infection.

African bovine trypanosomiasis caused by Trypanosoma sp., is a major constraint on cattle productivity in sub-Saharan Africa. Some African Bos taurus breeds are highly tolerant of infection, but the potentially more productive Bos indicus zebu breeds are much more susceptible. Zebu cattle are well adapted for plowing and haulage, and increasing their tolerance of trypanosomiasis could have a major impact on crop cultivation as well as dairy and beef production. We used three strategies to obtain short lists of candidate genes within QTL that were previously shown to regulate response to infection. We analyzed the transcriptomes of trypanotolerant N'Dama and susceptible Boran cattle after infection with Trypanosoma congolense. We sequenced EST libraries from these two breeds to identify polymorphisms that might underlie previously identified quantitative trait loci (QTL), and we assessed QTL regions and candidate loci for evidence of selective sweeps. The scan of the EST sequences identified a previously undescribed polymorphism in ARHGAP15 in the Bta2 trypanotolerance QTL. The polymorphism affects gene function in vitro and could contribute to the observed differences in expression of the MAPK pathway in vivo. The expression data showed that TLR and MAPK pathways responded to infection, and the former contained TICAM1, which is within a QTL on Bta7. Genetic analyses showed that selective sweeps had occurred at TICAM1 and ARHGAP15 loci in African taurine cattle, making them strong candidates for the genes underlying the QTL. Candidate QTL genes were identified in other QTL by their expression profile and the pathways in which they participate.