Characterisation of infection associated microRNA and protein cargo in extracellular vesicles of Theileria annulata infected leukocytes.

The protozoan parasites Theileria annulata and Theileria parva are unique amongst intracellular eukaryotic pathogens as they induce a transformation-like phenotype in their bovine host cell. T. annulata causes tropical theileriosis, which is frequently fatal, with infected leukocytes becoming metastatic and forming foci in multiple organs resulting in destruction of the lymphoid system. Exosomes, a subset of extracellular vesicles (EV), are critical in metastatic progression in many cancers. Here, we characterised the cargo of EV from a control bovine lymphosarcoma cell line (BL20) and BL20 infected with T. annulata (TBL20) by comparative mass spectrometry and microRNA (miRNA) profiling (data available via ProteomeXchange, identifier PXD010713 and NCBI GEO, accession number GSE118456, respectively). Ingenuity pathway analysis that many infection-associated proteins essential to migration and extracellular matrix digestion were upregulated in EV from TBL20 cells compared with BL20 controls. An altered repertoire of host miRNA, many with known roles in tumour and/or infection biology, was also observed. Focusing on the tumour suppressor miRNA, bta-miR-181a and bta-miR-181b, we identified putative messenger RNA targets and confirmed the interaction of bta-miR181a with ICAM-1. We propose that EV and their miRNA cargo play an important role in the manipulation of the host cell phenotype and the pathobiology of Theileria infection.

Sheep as Host Species for Zoonotic Babesia venatorum, United Kingdom.

Babesia venatorum is an increasingly prominent zoonotic parasite that predominantly infects wild deer. Our molecular examination of Babesia infecting mammals in the United Kingdom identified 18S sequences in domestic sheep isolates identical to zoonotic B. venatorum. Identification of this parasite in livestock raises concerns for public health and farming policy in Europe.

Characterization of HSP90 isoforms in transformed bovine leukocytes infected with Theileria annulata.

HSP90 chaperones are essential regulators of cellular function, as they ensure the appropriate conformation of multiple key client proteins. Four HSP90 isoforms were identified in the protozoan parasite Theileria annulata. Partial characterization was undertaken for three and localization confirmed for cytoplasmic (TA12105), endoplasmic reticulum (TA06470), and apicoplast (TA10720) forms. ATPase activity and binding to the HSP90 inhibitor geldanamycin were demonstrated for recombinant TA12105, and all three native forms could be isolated to varying extents by binding to geldanamycin beads. Because it is essential, HSP90 is considered a potential therapeutic drug target. Resistance to the only specific Theileriacidal drug is increasing, and one challenge for design of drugs that target the parasite is to limit the effect on the host. An in vitro cell culture system that allows comparison between uninfected bovine cells and the T. annulata-infected counterpart was utilized to test the effects of geldanamycin and the derivative 17-AAG. T. annulata-infected cells had greater tolerance to geldanamycin than uninfected cells yet exhibited significantly more sensitivity to 17-AAG. These findings suggest that parasite HSP90 isoform(s) can alter the drug sensitivity of infected host cells and that members of the Theileria HSP90 family are potential targets worthy of further investigation.

Identification of candidate transmission-blocking antigen genes in Theileria annulata and related vector-borne apicomplexan parasites.

BACKGROUND: Vector-borne apicomplexan parasites are a major cause of mortality and morbidity to humans and livestock globally. The most important disease syndromes caused by these parasites are malaria, babesiosis and theileriosis. Strategies for control often target parasite stages in the mammalian host that cause disease, but this can result in reservoir infections that promote pathogen transmission and generate economic loss. Optimal control strategies should protect against clinical disease, block transmission and be applicable across related genera of parasites. We have used bioinformatics and transcriptomics to screen for transmission-blocking candidate antigens in the tick-borne apicomplexan parasite, Theileria annulata. RESULTS: A number of candidate antigen genes were identified which encoded amino acid domains that are conserved across vector-borne Apicomplexa (Babesia, Plasmodium and Theileria), including the Pfs48/45 6-cys domain and a novel cysteine-rich domain. Expression profiling confirmed that selected candidate genes are expressed by life cycle stages within infected ticks. Additionally, putative B cell epitopes were identified in the T. annulata gene sequences encoding the 6-cys and cysteine rich domains, in a gene encoding a putative papain-family cysteine peptidase, with similarity to the Plasmodium SERA family, and the gene encoding the T. annulata major merozoite/piroplasm surface antigen, Tams1. CONCLUSIONS: Candidate genes were identified that encode proteins with similarity to known transmission blocking candidates in related parasites, while one is a novel candidate conserved across vector-borne apicomplexans and has a potential role in the sexual phase of the life cycle. The results indicate that a 'One Health' approach could be utilised to develop a transmission-blocking strategy effective against vector-borne apicomplexan parasites of animals and humans.

The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host-parasite interaction.

Babesia spp. are tick-borne, intraerythrocytic hemoparasites that use antigenic variation to resist host immunity, through sequential modification of the parasite-derived variant erythrocyte surface antigen (VESA) expressed on the infected red blood cell surface. We identified the genomic processes driving antigenic diversity in genes encoding VESA (ves1) through comparative analysis within and between three Babesia species, (B. bigemina, B. divergens and B. bovis). Ves1 structure diverges rapidly after speciation, notably through the evolution of shortened forms (ves2) from 5' ends of canonical ves1 genes. Phylogenetic analyses show that ves1 genes are transposed between loci routinely, whereas ves2 genes are not. Similarly, analysis of sequence mosaicism shows that recombination drives variation in ves1 sequences, but less so for ves2, indicating the adoption of different mechanisms for variation of the two families. Proteomic analysis of the B. bigemina PR isolate shows that two dominant VESA1 proteins are expressed in the population, whereas numerous VESA2 proteins are co-expressed, consistent with differential transcriptional regulation of each family. Hence, VESA2 proteins are abundant and previously unrecognized elements of Babesia biology, with evolutionary dynamics consistently different to those of VESA1, suggesting that their functions are distinct.

Modulation of activation-associated host cell gene expression by the apicomplexan parasite Theileria annulata.

Infection of bovine leucocytes by Theileria annulata results in establishment of transformed, infected cells. Infection of the host cell is known to promote constitutive activation of pro-inflammatory transcription factors that have the potential to be beneficial or detrimental. In this study we have compared the effect of LPS activation on uninfected bovine leucocytes (BL20 cells) and their Theileria-infected counterpart (TBL20). Gene expression profiles representing activated uninfected BL20 relative to TBL20 cells were also compared. The results show that while prolonged stimulation with LPS induces cell death and activation of NF-κB in BL20 cells, the viability of Theileria-infected cells was unaffected. Analysis of gene expression networks provided evidence that the parasite establishes tight control over pathways associated with cellular activation by modulating reception of extrinsic stimuli and by significantly altering the expression outcome of genes targeted by infection-activated transcription factors. Pathway analysis of the data set identified novel candidate genes involved in manipulation of cellular functions associated with the infected transformed cell. The data indicate that the T. annulata parasite can irreversibly reconfigure host cell gene expression networks associated with development of inflammatory disease and cancer to generate an outcome that is beneficial to survival and propagation of the infected leucocyte.

Development of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle.

Tropical theileriosis, bovine babesiosis and anaplasmosis are tick-borne protozoan diseases that impose serious constraints on the health and productivity of domestic cattle in tropical and sub-tropical regions of the world. A common feature of these diseases is that, following recovery from primary infection, animals become persistent carriers of the pathogen and continue to play a critical role in disease epidemiology, acting as reservoirs of infection. This study describes development and evaluation of multiplex and single PCR assays for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle. Following in silico screening for candidate target genes representing each of the pathogens, an optimised multiplex PCR assay was established using three primer sets, cytob1, MAR1bB2 and bovar2A, for amplification of genomic DNA of T. annulata, A. marginale and B. bovis respectively. The designed primer sets were found to be species-specific, generating amplicons of 312, 265 and 166 base pairs, respectively and were deemed suitable for the development of a multiplex assay. The sensitivity of each primer pair was evaluated using serial dilutions of parasite DNA, while specificity was confirmed by testing for amplification from DNA of different stocks of each pathogen and other Theileria, Babesia and Anaplasma species. Additionally, DNA preparations derived from field samples were used to evaluate the utility of the single and multiplex PCRs for determination of infection status. The multiplex PCR was found to detect each pathogen species with the same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA representing the other pathogens. Moreover, single and multiplex PCRs were able to detect each species with equal sensitivity in serially diluted DNA representing mixtures of T. annulata, B. bovis and A. marginale, and no evidence of non-specific amplification from non-target species was observed. Validation that the multiplex PCR efficiently detects single and mixed infections from field samples was demonstrated. The developed assay represents a simple and efficient diagnostic for co-detection of tropical theileriosis, bovine babesiosis and anaplasmosis, and may be a valuable tool for epidemiological studies aimed at assessing the burden of multiple infection with tick-borne pathogens and improving control of the associated diseases in endemic regions.

Selection of genotypes harbouring mutations in the cytochrome b gene of Theileria annulata is associated with resistance to buparvaquone.

Buparvaquone remains the only effective therapeutic agent for the treatment of tropical theileriosis caused by Theileria annulata. However, an increase in the rate of buparvaquone treatment failures has been observed in recent years, raising the possibility that resistance to this drug is associated with the selection of T. annulata genotypes bearing mutation(s) in the cytochrome b gene (Cyto b). The aim of the present study was: (1) to demonstrate whether there is an association between mutations in the T. annulata Cyto b gene and selection of parasite-infected cells resistant to buparvaquone and (2) to determine the frequency of these mutations in parasites derived from infected cattle in the Aydin region of Türkiye. Susceptibility to buparvaquone was assessed by comparing the proliferative index of schizont-infected cells obtained from cattle with theileriosis before and/or after treatment with various doses of buparvaquone, using the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colourimetric assay. The DNA sequence of the parasite Cyto b gene from cell lines identified as resistant or susceptible was determined. A total of six nonsynonymous and six synonymous mutations were identified. Two of the nonsynonymous mutations resulted in the substitutions V135A and P253S which are located at the putative buparvaquone binding regions of cytochrome b. Allele-specific PCR (AS-PCR) analyses detected the V135A and P253S mutations at a frequency of 3.90% and 3.57% respectively in a regional study population and revealed an increase in the frequency of both mutations over the years. The A53P mutation of TaPIN1 of T. annulata, previously suggested as being involved in buparvaquone resistance, was not detected in any of the clonal cell lines examined in the present study. The observed data strongly suggested that the genetic mutations resulting in V135A and P253S detected at the putative binding sites of buparvaquone in cytochrome b play a significant role in conferring, and promoting selection of, T. annulata genotypes resistant to buparvaquone, whereas the role of mutations in TaPIN1 is more equivocal.

A parasite DNA binding protein with potential to influence disease susceptibility acts as an analogue of mammalian HMGA transcription factors.

Intracellular pathogens construct their environmental niche, and influence disease susceptibility, by deploying factors that manipulate infected host cell gene expression. Theileria annulata is an important tick-borne parasite of cattle that causes tropical theileriosis. Excellent candidates for modulating host cell gene expression are DNA binding proteins bearing AT-hook motifs encoded within the TashAT gene cluster of the parasite genome. In this study, TashAT2 was transfected into bovine BoMac cells to generate three expressing and three non-expressing (opposite orientation) cell lines. RNA-Seq was conducted and differentially expressed (DE) genes identified. The resulting dataset was compared with genes differentially expressed between infected cells and non-infected cells, and DE genes between infected cell lines from susceptible Holstein vs tolerant Sahiwal cattle. Over 800 bovine genes displayed differential expression associated with TashAT2, 209 of which were also modulated by parasite infection. Network analysis showed enrichment of DE genes in pathways associated with cellular adhesion, oncogenesis and developmental regulation by mammalian AT-hook bearing high mobility group A (HMGA) proteins. Overlap of TashAT2 DE genes with Sahiwal vs Holstein DE genes revealed that a significant number of shared genes were associated with disease susceptibility. Altered protein levels encoded by one of these genes (GULP1) was strongly linked to expression of TashAT2 in BoMac cells and was demonstrated to be higher in infected Holstein leucocytes compared to Sahiwal. We conclude that TashAT2 operates as an HMGA analogue to differentially mould the epigenome of the infected cell and influence disease susceptibility.

TGF-b2 induction regulates invasiveness of Theileria-transformed leukocytes and disease susceptibility.

Theileria parasites invade and transform bovine leukocytes causing either East Coast fever (T. parva), or tropical theileriosis (T. annulata). Susceptible animals usually die within weeks of infection, but indigenous infected cattle show markedly reduced pathology, suggesting that host genetic factors may cause disease susceptibility. Attenuated live vaccines are widely used to control tropical theileriosis and attenuation is associated with reduced invasiveness of infected macrophages in vitro. Disease pathogenesis is therefore linked to aggressive invasiveness, rather than uncontrolled proliferation of Theileria-infected leukocytes. We show that the invasive potential of Theileria-transformed leukocytes involves TGF-b signalling. Attenuated live vaccine lines express reduced TGF-b2 and their invasiveness can be rescued with exogenous TGF-b. Importantly, infected macrophages from disease susceptible Holstein-Friesian (HF) cows express more TGF-b2 and traverse Matrigel with great efficiency compared to those from disease-resistant Sahiwal cattle. Thus, TGF-b2 levels correlate with disease susceptibility. Using fluorescence and time-lapse video microscopy we show that Theileria-infected, disease-susceptible HF macrophages exhibit increased actin dynamics in their lamellipodia and podosomal adhesion structures and develop more membrane blebs. TGF-b2-associated invasiveness in HF macrophages has a transcription-independent element that relies on cytoskeleton remodelling via activation of Rho kinase (ROCK). We propose that a TGF-b autocrine loop confers an amoeboid-like motility on Theileria-infected leukocytes, which combines with MMP-dependent motility to drive invasiveness and virulence.

Longitudinal field study on bovine Babesia spp. and Anaplasma phagocytophilum infections during a grazing season in Belgium.

Anaplasmosis and babesiosis are major tick-borne diseases with a high economic impact but are also a public health concern. Blood samples collected in the spring, summer, and autumn of 2010 from 65 cows in seven different farms in Belgium were monitored with an indirect immunofluorescence antibody test to assess seroprevalence against these pathogens. Seroprevalences to Babesia spp. were measured as 10.7%, 20%, and 12.3% in spring, summer, and autumn, respectively, whereas seroprevalences to Anaplasma phagocytophilum were 30.8%, 77%, and 56.9%, respectively. A total of 805 Ixodes ricinus ticks were collected at the same time from both cattle (feeding ticks) and grazed pastures (questing ticks). The infection level of ticks, assessed by PCR assay, for Babesia spp. DNA was 14.6% and 7.9% in feeding and questing ticks, respectively, whereas 21.7% and 3% of feeding and questing ticks were found be positive for A. phagocytophilum cDNA. Fifty-five PCR-positive samples were identified by sequencing as Babesia sp. EU1, of which five from feeding ticks were positive for both A. phagocytophilum and Babesia sp. EU1. The high density of wild cervids in the study area could explain these observations, as deer are considered to be the main hosts for adults of I. ricinus. However, the absence of Babesia divergens both in feeding and questing ticks is surprising, as the study area is known to be endemic for cattle babesiosis. Increasing cervid populations and comorbidity could play an import role in the epidemiology of these tick-borne diseases.

Novel equi merozoite antigen (ema-1) gene heterogeneity in a geographically isolated Theileria equi population in Croatia.

BACKGROUND: The apicomplexan haemoparasite Theileria equi, a causative agent of equine piroplasmosis, is an established pathogen of significant welfare and economic concern within the Croatian equine population. A previous large surveillance study of T. equi has identified two distinct parasite populations, one in the north and one in the south, geographically separated by the Dinaric Alps, which traverse the country. This study aimed to further investigate the genetic diversity within these two populations, focussing on allelic variability of the equi merozoite antigen gene, ema-1. METHODS: Following nested PCR of DNA isolates, the generated ema-1 amplicons were subsequently sequenced and compared by phylogenetic analysis to available sequences representing previously described ema-1 genotypes (groups A-C). RESULTS: Isolates from the southern T. equi population clustered with the existing ema-1 groups A and B. Strikingly, isolates from the northern population clustered into two novel ema-1 genotypes, named groups D and E. CONCLUSIONS: This detection of hitherto unreported genotypes suggests that historic geographical isolation has led to a degree of divergent evolution in this northern T. equi population. Additionally, current global regulatory testing of equine piroplasmosis relies heavily on EMA-1 based immunodiagnostics, and the discovery of unique ema-1 genotypes may question the efficacy of current diagnostics in international equine movement, with ramifications for the global equine community.

Theileria annulata histone deacetylase 1 (TaHDAC1) initiates schizont to merozoite stage conversion.

A fungal metabolite, FR235222, specifically inhibits a histone deacetylase of the apicomplexan parasite Toxoplasma gondii and TgHDAC3 has emerged as a key factor regulating developmental stage transition in this species. Here, we exploited FR235222 to ask if changes in histone acetylation regulate developmental stage transition of Theileria annulata, another apicomplexan species. We found that FR235222 treatment of T. annulata-infected transformed leukocytes induced a proliferation arrest. The blockade in proliferation was due to drug-induced conversion of intracellular schizonts to merozoites that lack the ability to maintain host leukocyte cell division. Induction of merogony by FR235222 leads to an increase in expression of merozoite-marker (rhoptry) proteins. RNA-seq of FR235222-treated T. annulata-infected B cells identified deregulated expression of 468 parasite genes including a number encoding parasite ApiAP2 transcription factors. Thus, similar to T. gondii, FR235222 inhibits T. annulata HDAC (TaHDAC1) activity and places parasite histone acetylation as a major regulatory event of the transition from schizonts to merozoites.

Susceptibility to disease (tropical theileriosis) is associated with differential expression of host genes that possess motifs recognised by a pathogen DNA binding protein.

BACKGROUND: Knowledge of factors that influence the outcome of infection are crucial for determining the risk of severe disease and requires the characterisation of pathogen-host interactions that have evolved to confer variable susceptibility to infection. Cattle infected by Theileria annulata show a wide range in disease severity. Native (Bos indicus) Sahiwal cattle are tolerant to infection, whereas exotic (Bos taurus) Holstein cattle are susceptible to acute disease. METHODOLOGY/PRINCIPAL FINDINGS: We used RNA-seq to assess whether Theileria infected cell lines from Sahiwal cattle display a different transcriptome profile compared to Holstein and screened for altered expression of parasite factors that could generate differences in host cell gene expression. Significant differences (<0.1 FDR) in the expression level of a large number (2211) of bovine genes were identified, with enrichment of genes associated with Type I IFN, cholesterol biosynthesis, oncogenesis and parasite infection. A screen for parasite factors found limited evidence for differential expression. However, the number and location of DNA motifs bound by the TashAT2 factor (TA20095) were found to differ between the genomes of B. indicus vs. B. taurus, and divergent motif patterns were identified in infection-associated genes differentially expressed between Sahiwal and Holstein infected cells. CONCLUSIONS/SIGNIFICANCE: We conclude that divergent pathogen-host molecular interactions that influence chromatin architecture of the infected cell are a major determinant in the generation of gene expression differences linked to disease susceptibility.

Extensive polymorphism and evidence of immune selection in a highly dominant antigen recognized by bovine CD8 T cells specific for Theileria annulata.

Although parasite strain-restricted CD8 T cell responses have been described for several protozoa, the precise role of antigenic variability in immunity is poorly understood. The tick-borne protozoan parasite Theileria annulata infects leukocytes and causes an acute, often fatal lymphoproliferative disease in cattle. Building on previous evidence of strain-restricted CD8 T cell responses to T. annulata, this study set out to identify and characterize the variability of the target antigens. Three antigens were identified by screening expressed parasite cDNAs with specific CD8 T cell lines. In cattle expressing the A10 class I major histocompatibility complex haplotype, A10-restricted CD8 T cell responses were shown to be focused entirely on a single dominant epitope in one of these antigens (Ta9). Sequencing of the Ta9 gene from field isolates of T. annulata demonstrated extensive sequence divergence, resulting in amino acid polymorphism within the A10-restricted epitope and a second A14-restricted epitope. Statistical analysis of the allelic sequences revealed evidence of positive selection for amino acid substitutions within the region encoding the CD8 T cell epitopes. Sequence differences in the A10-restricted epitope were shown to result in differential recognition by individual CD8 T cell clones, while clones also differed in their ability to recognize different alleles. Moreover, the representation of these clonal specificities within the responding CD8 T cell populations differed between animals. As well as providing an explanation for incomplete protection observed after heterologous parasite challenge of vaccinated cattle, these results have important implications for the choice of antigens for the development of novel subunit vaccines.

Modulation of NF-kappaB activation in Theileria annulata-infected cloned cell lines is associated with detection of parasite-dependent IKK signalosomes and disruption of the actin cytoskeleton.

Apicomplexan parasites within the genus Theileria have the ability to induce continuous proliferation and prevent apoptosis of the infected bovine leukocyte. Protection against apoptosis involves constitutive activation of the bovine transcription factor NF-kappaB in a parasite-dependent manner. Activation of NF-kappaB is thought to involve recruitment of IKK signalosomes at the surface of the macroschizont stage of the parasite, and it has been postulated that additional host proteins with adaptor or scaffolding function may be involved in signalosome formation. In this study two clonal cell lines were identified that show marked differences in the level of activated NF-kappaB. Further characterization of these lines demonstrated that elevated levels of activated NF-kappaB correlated with increased resistance to cell death and detection of parasite-associated IKK signalosomes, supporting results of our previous studies. Evidence was also provided for the existence of host- and parasite-dependent NF-kappaB activation pathways that are influenced by the architecture of the actin cytoskeleton. Despite this influence, it appears that the primary event required for formation of the parasite-dependent IKK signalosome is likely to be an interaction between a signalosome component and a parasite-encoded surface ligand.

Wild deer in the United Kingdom are a potential reservoir for the livestock parasite Babesia divergens.

Redwater fever is an economically important disease of cattle in the United Kingdom caused by the protozoan parasite Babesia divergens. Control efforts are dependent on accurate local historic knowledge of disease occurrence, together with an accurate appreciation of current underlying risk factors. Importantly, the involvement of red deer in the transmission of this pathogen in the UK remains unclear. We employed a polymerase chain reaction approach combined with DNA sequencing to investigate Babesia infections in livestock and red deer at a UK farm with a history of tick-borne disease. This revealed several B. divergens-infected cattle that were not displaying overt clinical signs. Additionally, 11% of red deer on the farmland and surrounding areas were infected with this parasite. We also found that 16% of the red deer were infected with Babesia odocoilei, the first time this parasite has been detected in the UK. The finding of B. divergens in the red deer population updates our knowledge of epidemiology in the UK and has implications for the effective control of redwater fever.

Evolution and diversity of secretome genes in the apicomplexan parasite Theileria annulata.

BACKGROUND: Little is known about how apicomplexan parasites have evolved to infect different host species and cell types. Theileria annulata and Theileria parva invade and transform bovine leukocytes but each species favours a different host cell lineage. Parasite-encoded proteins secreted from the intracellular macroschizont stage within the leukocyte represent a critical interface between host and pathogen systems. Genome sequencing has revealed that several Theileria-specific gene families encoding secreted proteins are positively selected at the inter-species level, indicating diversification between the species. We extend this analysis to the intra-species level, focusing on allelic diversity of two major secretome families. These families represent a well-characterised group of genes implicated in control of the host cell phenotype and a gene family of unknown function. To gain further insight into their evolution and function, this study investigates whether representative genes of these two families are diversifying or constrained within the T. annulata population. RESULTS: Strong evidence is provided that the sub-telomerically encoded SVSP family and the host-nucleus targeted TashAT family have evolved under contrasting pressures within natural T. annulata populations. SVSP genes were found to possess atypical codon usage and be evolving neutrally, with high levels of nucleotide substitutions and multiple indels. No evidence of geographical sub-structuring of allelic sequences was found. In contrast, TashAT family genes, implicated in control of host cell gene expression, are strongly conserved at the protein level and geographically sub-structured allelic sequences were identified among Tunisian and Turkish isolates. Although different copy numbers of DNA binding motifs were identified in alleles of TashAT proteins, motif periodicity was strongly maintained, implying conserved functional activity of these sites. CONCLUSIONS: This analysis provides evidence that two distinct secretome genes families have evolved under contrasting selective pressures. The data supports current hypotheses regarding the biological role of TashAT family proteins in the management of host cell phenotype that may have evolved to allow adaptation of T. annulata to a specific host cell lineage. We provide new evidence of extensive allelic diversity in representative members of the enigmatic SVSP gene family, which supports a putative role for the encoded products in subversion of the host immune response.

Investigating the presence of equine piroplasmosis in Ireland.

BACKGROUND: Equine piroplasmosis (EP) is a notifiable disease in Ireland and a significant concern to domestic and international equine industries. Information regarding EP presence in Ireland is currently limited. This retrospective surveillance study describes a serological and molecular analysis of blood samples submitted to the Irish Equine Centre for EP testing between January 2013 and April 2016. METHODS: Following serological testing, seropositive samples were screened using a PCR targeting the 18S ribosomal RNA gene. Amplicon sequences were bioinformatically analysed to identify the parasite species and to assess genetic diversity. RESULTS: From 2099 screened equine blood samples, 2.5 per cent and 1 per cent were seropositive for Theileria equi and Babesia caballi, respectively. T equi DNA was detected in 9 per cent of the seropositive samples while B caballi DNA was not detected in any sample. The T equi DNA sequences displayed no genetic diversity at this locus, in contrast to samples from the UK and from endemic areas. CONCLUSION: Detection of EP-seropositive and parasitaemic horses in Ireland indicates a clear and present health risk to the equine population. It is recommended that owners adopt appropriate biosecurity measures and that clinicians are mindful of this disease as a differential diagnosis.

Phylogenetic analysis and geographical distribution of Theileria equi and Babesia caballi sequences from horses residing in Spain.

The intraerythrocytic protozoans Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis (EP), one of the most important equine tick-borne diseases due to its significant impact on global international horse trade. Although EP is known to be endemic in Spain, previous phylogenetic studies have only been conducted for limited geographical regions. Therefore, the objective of this study was to evaluate the genetic diversity and distribution of these parasite species nationwide. This was performed by amplification of the 18S small subunit (SSU) rRNA gene from 100 EP positive equine blood samples using a nested PCR protocol, and sequencing the obtained amplicons. Seventy-seven T. equi and six B. caballi isolates were successfully sequenced and phylogenetic analysis revealed that the T. equi isolates grouped into the previously described clades A (n = 21/77), D (n = 1/77) and E (n = 55/77), while B. caballi isolates were placed into clades A (n = 5/6) and B (n = 1/6). Isolates from T. equi clade D and B. caballi clade B have not previously been reported in Spain. A greater intra-clade diversity (97.3-98.3 % identity) was observed between T. equi clade E isolates compared to those within clade A (99.7-100 % identity). Additionally, a multivariable logistic regression model was used to analyse associations between the clade of T. equi infection and available epidemiological data. Horses residing in Spanish northern regions were statistically more likely to be infected with T. equi clade E (p = 0.01). We conclude that while extensive sequence variation of equine piroplasms exists in Spanish infected horses, a requirement for increased equine movement controls between Spain and EP-endemic countries should be considered.