A host-restricted viral vector for antigen-specific immunization against Lyme disease pathogen

Newcastle disease virus (NDV) is an avian virus that is attenuated in primates and is a potential vaccine vector for human use. We evaluated NDV as a vector for expressing selected antigens of the Lyme disease pathogen Borrelia burgdorferi. A series of recombinant NDVs were generated that expressed intracellular or extracellular forms of two B. burgdorferi antigens: namely, the basic membrane protein A (BmpA) and the outer surface protein C (OspC). Expression of the intracellular and extracellular forms of these antigens was confirmed in cultured chicken cells. C3H or Balb/C mice that were immunized intranasally with the NDV vectors mounted vigorous serum antibody responses against the NDV vector, but failed to mount a robust response against either the intracellular or extracellular forms of BmpA or OspC. By contrast, a single immunization of hamsters with the NDV vectors via the intranasal, intramuscular, or intraperitoneal route resulted in rapid and rigorous antibody responses against the intracellular or extracellular forms of BmpA and OspC. When groups of hamsters were separately inoculated with various NDV vectors and challenged with B. burgdorferi (10⁸ cells/animal), immunization with vector expressing either intracellular or extracellular BmpA was associated with a significant reduction of the pathogen load in the joints. Taken together, our studies highlighted the importance of NDV as vaccine vector that can be used for simple yet effective immunization of hosts against bacterial infections including Lyme disease.     

Identification of putative vaccine candidates against Helicobacter pylori exploiting exoproteome and secretome: A reverse vaccinology based approach

Helicobacter pylori (H. pylori) is an important pathogen associated with diverse gastric disorders ranging from peptic ulcer to malignancy. It has also been recognized by the World Health Organization (WHO) as class I carcinogen. Conventional treatment regimens for H. pylori seem to be ineffective, possibly due to antibiotic resistance mechanisms acquired by the pathogen. In this study we have successfully employed a reverse vaccinology approach to predict the potential vaccine candidates against H. pylori. The predicted potential vaccine candidates include vacA, babA, sabA, fecA and omp16. Host–pathogen interactions analysis elaborated their direct or indirect role in the specific signaling pathways including epithelial cell polarity, metabolism, secretion system and transport. Furthermore, surface-exposed antigenic epitopes were predicted and analyzed for conservation among 39 complete genomes of H. pylori (Genbank) for all the candidate proteins. These epitopes may serve as a base for the development of broad spectrum peptide or multi-component vaccines against H. pylori. We also believe that the proposed pipeline can be extended to other pathogens and for the identification of novel candidates for the development of effective vaccines.     

Borrelia miyamotoi sensu lato Seroreactivity and Seroprevalence in the Northeastern United States

Borrelia miyamotoi sensu lato, a relapsing fever Borrelia sp., is transmitted by the same ticks that transmit B. burgdorferi (the Lyme disease pathogen) and occurs in all Lyme disease�?"endemic areas of the United States. To determine the seroprevalence of IgG against B. miyamotoi sensu lato in the northeastern United States and assess whether serum from B. miyamotoi sensu lato�?"infected persons is reactive to B. burgdorferi antigens, we tested archived serum samples from area residents during 1991�?"2012. Of 639 samples from healthy persons, 25 were positive for B. miyamotoi sensu lato and 60 for B. burgdorferi. Samples from �%^10% of B. miyamotoi sensu lato�?"seropositive persons without a recent history of Lyme disease were seropositive for B. burgdorferi. Our resultsA suggest thatA human B. miyamotoiA sensu latoA infection may be common in southern New England and that B. burgdorferi antibody testing is not an effective surrogate for detecting B. miyamotoi sensu lato infection.     

Borrelia burgdorferi BBA52 is a potential target for transmission blocking Lyme disease vaccine

The surface-exposed antigens of Borrelia burgdorferi represent important targets for induction of protective host immune responses. BBA52 is preferentially expressed by B. burgdorferi in the feeding tick, and a targeted deletion of bba52 interferes with vector-host transitions in vivo. In this study, we demonstrate that BBA52 is an outer membrane surface-exposed protein and that disulfide bridges take part in the homo-oligomeric assembly of native protein. BBA52 antibodies lack detectable borreliacidal activities in vitro. However, active immunization studies demonstrated that BBA52 vaccinated mice were significantly less susceptible to subsequent tick-borne challenge infection. Similarly, passive transfer of BBA52 antibodies in ticks completely blocked B. burgdorferi transmission from feeding ticks to naïve mice. Taken together, these studies highlight the role of BBA52 in spirochete dissemination from ticks to mice and demonstrate the potential of BBA52 antibody-mediated strategy to complement the ongoing efforts to develop vaccines for blocking the transmission of B. burgdorferi.     

Evaluation of Salmonella live vaccines with chromosomal expression cassettes for translocated fusion proteins

Salmonella enterica is a versatile live carrier for the presentation of recombinant vaccine antigens. Fusion proteins of a type III secretion system effector and heterologous vaccine antigens can be translocated by live attenuated Salmonella strains and mediate protective immunity against infections. Here we investigated the use expression cassettes for translocated fusion protein consisting of effector SseF and antigens of Listeria monocytogenes after stable integration into the Salmonella chromosome. The efficacy of chromosomal expression cassettes was compared to plasmid-based expression cassettes. Our data indicate that live Salmonella vaccines with chromosomal expression cassettes for translocated fusion proteins, although only present in single copy, efficiently stimulate immune responses.     

Non-pathogenic Borrelia burgdorferi expressing Treponema pallidum TprK and Tp0435 antigens as a novel approach to evaluate syphilis vaccine candidates

Background

Syphilis is resurgent in many developed countries and still prevalent in developing nations. Current and future control campaigns would benefit from the development of a vaccine, but although promising vaccine candidates were identified among the putative surface-exposed integral outer membrane proteins of the syphilis spirochete, immunization experiments in the rabbit model using recombinant antigens have failed to fully protect animals upon infectious challenge. We speculated that such recombinant immunogens, purified under denaturing conditions from Escherichia coli prior to immunization might not necessarily harbor their original structure, and hypothesized that enhanced protection would result from performing similar immunization/challenge experiments with native antigens.

Vector competence studies with hard ticks and Borrelia burgdorferi sensu lato spirochetes: A review

Use of emerging technology allowing for identification of genetic material from pathogens and endosymbionts in ticks collected from humans, domestic animals, wildlife, or the environment has resulted in an avalanche of new data on tick-microorganism associations. This rapidly growing stream of new information is a tremendous resource but also presents challenges, including how detection of pathogen genetic material in ticks should best be interpreted. There is a tendency in the more recent published literature to incorrectly use the term “vector” based on detection of pathogen genetic material from tick species not experimentally confirmed to serve as vectors of the pathogen in question. To serve as a vector of a horizontally maintained pathogen, such as a Borrelia burgdorferi sensu lato (s.l.) Lyme borreliosis spirochete, the tick species in question must be capable of acquiring the pathogen while feeding in the larval or nymphal stage on an infectious host, maintaining it transstadially through the molt, and then transmitting the pathogen to a naïve host while feeding in the subsequent nymphal or adult stage. This review examines the experimental evidence for and against species of hard (ixodid) ticks from different genera to serve as vectors of B. burgdorferi s.l. spirochetes. Of the 18 Ixodes species ticks evaluated to date, 13 were experimentally confirmed as vectors of B. burgdorferi s.l. spirochetes. These studies focused primarily on the three major Lyme borreliosis agents: Borrelia burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii. In striking contrast, none of 8 tick species from other genera (1 Amblyomma species, 5 Dermacentor species, and 2 Haemaphysalis species) evaluated to date were unequivocally experimentally confirmed as vectors of B. burgdorferi s.l. spirochetes. The strength of the evidence for or against each tick species to serve as a vector of B. burgdorferi s.l. spirochetes is discussed together with key knowledge gaps and research challenges.     

Genetic structure and divergence in populations of Lutzomyia cruciata,a phlebotomine sand fly (Diptera: Psychodidae) vector of Leishmania mexicana in southeastern Mexico

The low dispersal capacity of sand flies could lead to population isolation due to geographic barriers, climate variation, or to population fragmentation associated with specific local habitats due to landscape modification. The phlebotomine sand fly Lutzomyia cruciata has a wide distribution throughout Mexico and is a vector of Leishmania mexicana in the southeast. The aim of this study was to evaluate the genetic diversity, structure, and divergence within and among populations of Lu. cruciata in the state of Chiapas, and to infer the intra-specific phylogeny using the 3′ end of the mitochondrial cytochrome b gene. We analyzed 62 sequences from four Lu. cruciata populations and found 26 haplotypes, high genetic differentiation and restricted gene flow among populations (Fst = 0.416, Nm = 0.701, p < 0.001). The highest diversity values were recorded in populations from Loma Bonita and Guadalupe Miramar. Three lineages (100% bootstrap and 7% overall divergence) were identified using a maximum likelihood phylogenetic analysis which showed high genetic divergence (17.2–22.7%). A minimum spanning haplotype network also supported separation into three lineages. Genetic structure and divergence within and among Lu. cruciata populations are hence affected by geographic heterogeneity and evolutionary background. Data obtained in the present study suggest that Lu. cruciata in the state of Chiapas consists of at least three lineages. Such findings may have implications for vector capacity and hence for vector control strategies.     

Increased diversity of zoonotic pathogens and Borrelia burgdorferi strains in established versus incipient Ixodes scapularis populations across the Midwestern United States

The center of origin theory predicts that genetic diversity will be greatest near a specie’s geographic origin because of the length of time for evolution. By corollary, diversity will decrease with distance from the origin; furthermore, invasion and colonization are frequently associated with founder effects that reduce genetic variation in incipient populations. The blacklegged tick, Ixodes scapularis, harbors a suite of zoonotic pathogens, and the geographic range of the tick is expanding in the upper Midwestern United States. Therefore, we posited that diversity of I. scapularis-borne pathogens across its Midwestern range should correlate with the rate of the range expansion of this tick as well as subsequent disease emergence. Analysis of 1565 adult I. scapularis ticks from 13 sites across five Midwestern states revealed that tick infection prevalence with multiple microbial agents (Borrelia burgdorferi, Borrelia miyamotoi, Babesia odocoilei, Babesia microti, and Anaplasma phagocytophilum), coinfections, and molecular genetic diversity of B. burgdorferi all were positively correlated with the duration of establishment of tick populations, and therefore generally support the center of origin – pathogen diversity hypothesis. The observed differences across the gradient of establishment, however, were not strong and were nuanced by the high frequency of coinfections in tick populations at both established and recently-invaded tick populations. These results suggest that the invasion of ticks and their associated pathogens likely involve multiple means of pathogen introduction, rather than the conventionally presented scenario whereby infected, invading ticks are solely responsible for introducing pathogens to naïve host populations.     

Evolution of Northeastern and Midwestern Borrelia burgdorferi, United States

The per capita incidence of human Lyme disease in the northeastern United States is more than twice that in the Midwest. However, the prevalence of Borrelia burgdorferi, the bacterium that causes Lyme disease, in the tick vector is nearly identical in the 2 regions. The disparity in human Lyme disease incidence may result from a disparity in the human invasiveness of the bacteria in the Northeast and Midwest caused by fundamentally different evolutionary histories. B. burgdorferi populations in the Northeast and Midwest are geographically isolated, enabling evolutionary divergence in human invasiveness. However, we found that B. burgdorferi populations in the Northeast and Midwest shared a recent common ancestor, which suggests that substantial evolutionary divergence in human invasiveness has not occurred. We propose that differences in either animal ecology or human behavior are the root cause of the differences in human incidence between the 2 regions.     

Prevalence of Borrelia burgdorferi and diversity of its outer surface protein C (ospC) alleles in blacklegged ticks (Ixodes scapularis) in Delaware

Characterizing the diversity of genes associated with virulence and transmission of a pathogen across the pathogen's distribution can inform our understanding of host infection risk. Borrelia burgdorferi is a vector-borne bacterium that causes Lyme disease in humans and is common in the United States. The outer surface protein C (ospC) gene of B. burgdorferi exhibits substantial genetic variation across the pathogen's distribution and plays a critical role in virulence and transmission in vertebrate hosts. In fact, B. burgdorferi infections that disseminate across host tissues in humans are associated with only a subset of ospC alleles. Delaware has a high incidence of Lyme disease, but the diversity of ospC in B. burgdorferi in the state has not been evaluated. We used PCR to amplify ospC in B. burgdorferi-infected blacklegged ticks (Ixodes scapularis) in sites statewide and used short-read sequencing to identify ospC alleles. B. burgdorferi prevalence in blacklegged ticks varied across sites, but not significantly so. We identified 15 previously characterized ospC alleles accounting for nearly all of the expected diversity of alleles across the sites as estimated using the Chao1 index. Nearly 40% of sequenced infections (23/58) had more than one ospC allele present suggesting mixed strain infections and the relative frequencies of alleles in single infections were positively correlated with their relative frequencies in mixed infections. Turnover of ospC alleles was positively related to distance between sites with closer sites having more similar allele compositions than more distant sites. This suggests a degree of B. burgdorferi dispersal limitation or habitat specialization. OspC alleles known to cause disseminated infections in humans were found at the highest frequencies across sites, corresponding to Delaware's high incidence of Lyme disease.     

Modulation of inter-vaccination interval to avoid antigenic competition in multivalent footrot (Dichelobacter nodosus) vaccines in sheep

Virulent footrot is a significant disease of sheep in most sheep farming countries; a strain/serogroup of the anaerobic bacterium Dichelobacter nodosus is the essential transmitting agent. Commercial multivalent footrot vaccines containing nine fimbrial serogroups (A through I) of D. nodosus produce relatively low and short term antibody responses due to antigenic competition, in contrast to higher and longer responses provided by monovalent or bivalent vaccines. The latter were important components of successful eradication programs for endemic footrot caused by either one or two serogroups of D. nodosus in Nepal, Bhutan, and several flocks in Australia. However, the presence of up to six serogroups in some Australian flocks and the use of an annual bivalent vaccination regime to progressively eradicate serogroups would require a long term program. In this study we report the results of a sequential vaccination trial testing different time intervals between different bivalent vaccinations. Intervals of 12, 9, 6, 3 and 0 months were tested. The 1st vaccination was with recombinant fimbrial antigens for serogroups A and B while the 2nd vaccination was with D and E. There were no significant differences between the antibody responses for time intervals of 3, 6, 9 and 12 months whereas there was a reduced response when sheep were vaccinated with two bivalent vaccines (four antigens) concurrently, indicating antigenic competition. Therefore an inter-vaccination interval of 3 months can be applied between two different bivalent vaccines without detrimental impact on the humoral immune responses to the various fimbrial antigens of D. nodosus. These results could have wider applications in vaccination against diseases caused by multivalent or multistrain microbes.     

Harnessing donor unrestricted T-cells for new vaccines against tuberculosis

Mycobacterium bovis bacille Calmette-Guérin (BCG) prevents extrapulmonary tuberculosis (TB) and death among infants but fails to consistently and sufficiently prevent pulmonary TB in adults. Thus, TB remains the leading infectious cause of death worldwide, and new vaccine approaches are urgently needed. T-cells are important for protective immunity to Mycobacterium tuberculosis (Mtb), but the optimal T-cell antigens to be included in new vaccines are not established. T-cells are often thought of as responding mainly to peptide antigens presented by polymorphic major histocompatibility complex (MHC) I and II molecules. Over the past two decades, the number of non-peptidic Mtb derived antigens for αβ and γδ T-cells has expanded rapidly, creating broader perspectives about the types of molecules that could be targeted by T-cell-based vaccines against TB. Many of these non-peptide responsive T-cell subsets in humans are activated in a manner that is unrestricted by classical MHC-dependent antigen-presenting systems, but instead require essentially nonpolymorphic presentation systems. These systems are Cluster of differentiation 1 (CD1), MHC related protein 1 (MR1), butyrophilin 3A1, as well as the nonclassical MHC class Ib family member HLA-E. Thus, the resulting T-cell responses can be shared among a genetically diverse population, creating the concept of donor-unrestricted T-cells (DURTs). Here, we review evidence that DURTs are an abundant component of the human immune system and recognize many antigens expressed by Mtb, including antigens that are expressed in BCG and other candidate whole cell vaccines. Further, DURTs exhibit functional diversity and demonstrate the ability to control microbial infection in small animal models. Finally, we outline specific knowledge gaps and research priorities that must be addressed to realize the full potential of DURTs as part of new TB vaccines approaches.     

On the use of hemagglutination-inhibition for influenza surveillance: Surveillance data are predictive of influenza vaccine effectiveness

The hemagglutination-inhibition (HI) assay is the main tool used by epidemiologists to quantify antigenic differences between circulating influenza virus strains, with the goal of selecting suitable vaccine strains. However, such quantitative measures of antigenic difference were recently shown to have poor predictive accuracy with respect to influenza vaccine effectiveness (VE) in healthy adults. Here, we re-examine those results using a more rigorous criterion for predictive accuracy – considering only cases when the vaccine (V) and dominant (D) circulating strains are antigenically different – and greater numbers of HI titers. We find that the Archetti–Horsfall measure of antigenic difference, which is based on both the normalized HI titer (NHI) of D relative to antisera raised against V and the NHI of V relative to D, predicts VE very well (R² = 0.62, p = 4.1 × 10⁻³). In contrast, the predictive accuracies of the NHI of D relative to V alone (R² = 0.01), and two other measures of antigenic difference based on the amino acid sequence of influenza virus hemagglutinin (R² = 0.03 for both measures) are relatively poor. Furthermore, while VE in the elderly is generally high in cases when D and V are antigenically identical (VE = 35%, S.E. = 5%), in other cases VE appears to increase with the antigenic difference between D and V (R² = 0.90, p = 2.5 × 10⁻⁵). This paradoxical observation could reflect the confounding effects of prior immunity on estimates of VE in the elderly. Together, our results underscore the need for consistently accurate selection of suitable vaccine strains. We suggest directions for further studies aimed at improving vaccine-strain selection and present a large collection of HI titers that will be useful to such studies.     

Rabies virus isolates of India – Simultaneous existence of two distinct evolutionary lineages

Rabies is a fatal viral disease of serious public health implication. The disease is enzootic in India. In the present study, thirty six rabies virus isolates were obtained from terrestrial mammals of India during 2002–2012. Ecto-domain coding region of the glycoprotein gene from all the isolates were sequenced and the phylogenetic analysis was performed in relation to the global rabies and rabies related virus isolates. The Indian isolates grouped into two distinctly separate lineages with majority of the Indian isolates in Arctic like 1 lineage and the remaining isolates in sub-continental lineage. Isolates of the two distinct lineages were identified simultaneously from the same geographical region. Time scaled phylogenetic tree indicated that the sub-continental lineage of the virus is one of the earliest clade of rabies virus that diverged from bat rabies virus. On the contrary, the Arctic-like 1 lineage of India appeared to be a more recent divergence event. The amino acid sequence comparison revealed that all the major antigenic sites were almost conserved among the Indian isolates whereas few amino acid variations could be identified around site IIa, minor site I and IV. The d_N/d_S study based on G ecto-domain is in support of the earlier reports of strong purifying selection. In conclusion, it is evident that the Indian rabies virus isolates are of two major distinct lineages with distant phylogenetic and evolutionary relationship.     

Identification of immunoreactive linear epitopes of Borrelia miyamotoi

Borrelia miyamotoi is an emerging tick-borne spirochete transmitted by ixodid ticks. Current serologic assays for B. miyamotoi are impacted by genetic similarities to other Borrelia and limited understanding of optimal antigenic targets. In this study, we employed the TBD-Serochip, a peptide array platform, to identify new linear targets for serologic detection of B. miyamotoi. We examined a wide range of suspected B. miyamotoi antigens and identified 352 IgM and 91 IgG reactive peptides, with the majority mapping to variable membrane proteins. These included peptides within conserved fragments of variable membrane proteins that may have greater potential for differential diagnosis. We also identified reactive regions on FlaB, and demonstrate crossreactivity of B. burgdorferi s.l. C6 with a B. miyamotoi C6-like peptide. The panel of linear peptides identified in this study can be used to enhance serodiagnosis of B. miyamotoi.     

Evolutionary genomics of Borrelia burgdorferi sensu lato: Findings,hypotheses, and the rise of hybrids

Borrelia burgdorferi sensu lato (B. burgdorferi s.l.), the group of bacterial species represented by Lyme disease pathogens, has one of the most complex and variable genomic architectures among prokaryotes. Showing frequent recombination within and limited gene flow among geographic populations, the B. burgdorferi s.l. genomes provide an excellent window into the processes of bacterial evolution at both within- and between-population levels. Comparative analyses of B. burgdorferi s.l. genomes revealed a highly dynamic plasmid composition but a conservative gene repertoire. Gene duplication and loss as well as sequence variations at loci encoding surface-localized lipoproteins (e.g., the PF54 genes) are strongly associated with adaptive differences between species. There are a great many conserved intergenic spacer sequences that are candidates for cis-regulatory elements and non-coding RNAs. Recombination among coexisting strains occurs at a rate approximately three times the mutation rate. The coexistence of a large number of genomic groups within local B. burgdorferi s.l. populations may be driven by immune-mediated diversifying selection targeting major antigen loci as well as by adaptation to multiple host species. Questions remain regarding the ecological causes (e.g., climate change, host movements, or new adaptations) of the ongoing range expansion of B. burgdorferi s.l. and on the genomic variations associated with its ecological and clinical variability. Anticipating an explosive growth of the number of B. burgdorferi s.l. genomes sampled from both within and among species, we propose genome-based methods to test adaptive mechanisms and to identify molecular bases of phenotypic variations. Genome sequencing is also necessary for monitoring a likely increase of genetic admixture of previously isolated species and populations in North America and elsewhere.     

Genetic basis of antigenic variation in foot-and-mouth disease serotype A viruses from the Middle East

Foot-and-mouth disease viruses (FMDV) from serotype A exhibit high antigenic diversity. Within the Middle East, a strain called A-Iran-05 emerged in 2003, and subsequently replaced the A-Iran-96 and A-Iran-99 strains that were previously circulating in the region. Viruses from this strain did not serologically match with the established A/Iran/96 vaccine, although most early samples matched with the older A22/Iraq vaccine. However, many viruses from this strain collected after 2006 had poor serological match with the A22/Iraq vaccine necessitating the development of a new vaccine strain (A/TUR/2006). More recently, viruses from the region now exhibit lower cross-reactivity with the A/TUR/2006 antisera highlighting the inadequacy of the serotype A vaccines used in the region. In order to understand the genetic basis of these antigenic phenotypes, we have determined the full capsid sequence for 57 Middle Eastern viruses isolated between 1996 and 2011 and analysed these data in context of antigenic relationship (r₁) values that were generated using antisera to A22/Iraq and A/TUR/2006. Comparisons of capsid sequences identified substitutions in neutralising antigenic sites (1, 2 and 4), which either individually or together underpin these observed antigenic phenotypes.     

Evolutionary epidemiology of Streptococcus iniae: Linking mutation rate dynamics with adaptation to novel immunological landscapes

Pathogens continuously adapt to changing host environments where variation in their virulence and antigenicity is critical to their long-term evolutionary success. The emergence of novel variants is accelerated in microbial mutator strains (mutators) deficient in DNA repair genes, most often from mismatch repair and oxidized-guanine repair systems (MMR and OG respectively). Bacterial MMR/OG mutants are abundant in clinical samples and show increased adaptive potential in experimental infection models, yet the role of mutators in the epidemiology and evolution of infectious disease is not well understood. Here we investigated the role of mutation rate dynamics in the evolution of a broad host range pathogen, Streptococcus iniae, using a set of 80 strains isolated globally over 40 years. We have resolved phylogenetic relationships using non-recombinant core genome variants, measured in vivo mutation rates by fluctuation analysis, identified variation in major MMR/OG genes and their regulatory regions, and phenotyped the major traits determining virulence in streptococci. We found that both mutation rate and MMR/OG genotype are remarkably conserved within phylogenetic clades but significantly differ between major phylogenetic lineages. Further, variation in MMR/OG loci correlates with occurrence of atypical virulence-associated phenotypes, infection in atypical hosts (mammals), and atypical (osseous) tissue of a vaccinated primary host. These findings suggest that mutators are likely to facilitate adaptations preceding major diversification events and may promote emergence of variation permitting colonization of a novel host tissue, novel host taxa (host jumps), and immune-escape in the vaccinated host.     

Evolution and diversity of the EMA families of the divergent equid parasites,Theileria equi and T. haneyi

The equine parasite Theilera equi continues to curtail global equine commerce due primarily to its ability to persist indefinitely in the immunocompetent horse. Details regarding the parasite life cycle, pathogenesis and mechanism of persistence remain unclear. The recently discovered T. haneyi is also capable of persistence in the horse, creating a potential reservoir for additional infections. These two divergent parasites share a unique gene family that expresses surface merozoite antigens, or equi merozoite antigens (EMAs). The EMA family was maintained in number and size in both parasites despite a species divergence of over 30 million years ago. This family is unique amongst Theilerias in number, structure and biochemical properties. In silico analysis revealed no evidence of selection for diversity within this family, indicating a role in host adaptation and persistence rather than antigenic variation and immune escape. Biochemical analysis revealed the presence of a conserved domain, homologous to the hemolysin toxin found in cobra venom. This finding combined with data from protein interaction prediction models may indicate interaction with the structural components of the host erythrocyte and a role in merozoite entry or escape. Additional predicted protein interactions focus on disruption of the enzymatic functions of the host cell, potentially resulting in enhanced parasite survival.