Analysis of Stage-Specific Protein Expression during Babesia Bovis Development within Female Rhipicephalus Microplus.

Arthropod-borne protozoan pathogens have a complex life cycle that includes asexual reproduction of haploid stages in mammalian hosts and the development of diploid stages in invertebrate hosts. The ability of pathogens to invade, survive, and replicate within distinct cell types is required to maintain their life cycle. In this study, we describe a comparative proteomic analysis of a cattle pathogen, Babesia bovis, during its development within the mammalian and tick hosts with the goal of identifying cell-surface proteins expressed by B. bovis kinetes as potential targets for the development of a transmission blocking vaccine. To determine parasite tick-stage-specific cell-surface proteins, CyDye labeling was performed with B. bovis blood stages from the bovine host and kinetes from the tick vector. Cell-surface kinete-stage-specific proteins were identified using 2D difference in gel electrophoresis and analyzed by mass spectrometry. Ten proteins were identified as kinete-stage-specific, with orthologs found in closely related Apicomplexan pathogens. Transcriptional analysis revealed two genes were highly expressed by kinetes as compared with blood stages. Immunofluorescence using antibodies against the two proteins confirmed kinete-stage-specific expression. The identified cell-surface kinete proteins are potential candidates for the development of a B. bovis transmission blocking vaccine.

Superinfection Exclusion of the Ruminant Pathogen Anaplasma marginale in Its Tick Vector Is Dependent on the Time between Exposures to the Strains.

UNLABELLED: The remarkable genetic diversity of vector-borne pathogens allows for the establishment of superinfection in the mammalian host. To have a long-term impact on population strain structure, the introduced strains must also be transmitted by a vector population that has been exposed to the existing primary strain. The sequential exposure of the vector to multiple strains frequently prevents establishment of the second strain, a phenomenon termed superinfection exclusion. As a consequence, superinfection exclusion may greatly limit genetic diversity in the host population, which is difficult to reconcile with the high degree of genetic diversity maintained among vector-borne pathogens. Using Anaplasma marginale, a tick-borne bacterial pathogen of ruminants, we hypothesized that superinfection exclusion is temporally dependent and that longer intervals between strain exposures allow successful acquisition and transmission of a superinfecting strain. To test this hypothesis, we sequentially exposed Dermacentor andersoni ticks to two readily tick-transmissible strains of A. marginale The tick feedings were either immediately sequential or 28 days apart. Ticks were allowed to transmission feed and were individually assessed to determine if they were infected with one or both strains. The second strain was excluded from the tick when the exposure interval was brief but not when it was prolonged. Midguts and salivary glands of individual ticks were superinfected and transmission of both strains occurred only when the exposure interval was prolonged. These findings indicate that superinfection exclusion is temporally dependent, which helps to account for the differences in pathogen strain structure in tropical compared to temperate regions. IMPORTANCE: Many vector-borne pathogens have marked genetic diversity, which influences pathogen traits such as transmissibility and virulence. The most successful strains are those that are preferentially transmitted by the vector. However, the factors that determine successful transmission of a particular strain are unknown. In the case of intracellular, bacterial, tick-borne pathogens, one potential factor is superinfection exclusion, in which colonization of ticks by the first strain of a pathogen it encounters prevents the transmission of a second strain. Using A. marginale, the most prevalent tick-borne pathogen of cattle worldwide, and its natural tick vector, we determined that superinfection exclusion occurs when the time between exposures to two strains is brief but not when it is prolonged. These findings suggest that superinfection exclusion may influence strain transmission in temperate regions, where tick activity is limited by season, but not in tropical regions, where ticks are active for long periods.

Nilgai antelope display no signs of infection upon experimental challenge with a virulent Babesia bovis strain.

BACKGROUND: Bovine babesiosis is caused by infection with the protozoal parasite Babesia bovis, which is transmitted by Rhipicephalus (Boophilus) spp. It can cause mortality rates up to 90% in immunologically naive Bos taurus cattle. In south Texas, R. (B.) microplus is known to infest nilgai antelope (Boselaphus tragocamelus); however, their susceptibility to infection with B. bovis and their role in the transmission of the parasite remain unknown. In this study, we challenged nilgai antelope with B. bovis and evaluated their susceptibility to infection. METHODS: Nilgai were needle inoculated with ≈108 B. bovis-parasitized erythrocytes (merozoites) or a homogenate of B. bovis-infected larval ticks (sporozoite) delivered intravenously. Bos taurus beef calves were inoculated in parallel, as this strain of B. bovis is lethal to cattle. Temperature and hematocrit were monitored daily over the course of each study, and whole blood was collected for molecular [polymerase chain reaction (PCR)] and serological [indirect enzyme-linked immunosorbent assay (ELISA)] diagnostic evaluation. Histological sections of nilgai cerebral tissue were examined for evidence of infection. Recipient bovine calves were sub-inoculated with blood from nilgai challenged with either stage of the parasite, and they were monitored for clinical signs of infection and evaluated by a PCR diagnostic assay. Red blood cells (RBCs) from prechallenged nilgai and B. taurus beef cattle were cultured with an in vitro B. bovis merozoite culture to examine colonization of the RBCs by the parasite. RESULTS: Nilgai did not display clinical signs of infection upon inoculation with either the merozoite or sporozoite stage of B. bovis. All nilgai were PCR-negative for the parasite, and they did not develop antibodies to B. bovis. No evidence of infection was detected in histological sections of nilgai tissues, and in vitro culture analysis indicated that the nilgai RBCs were not colonized by B. bovis merozoites. Cattle subinoculated with blood from challenged nilgai did not display clinical signs of infection, and they were PCR-negative up to 45 days after transfer. CONCLUSIONS: Nilgai do not appear to be susceptible to infection with a strain of B. bovis that is lethal to cattle. Tick control on these alternative hosts remains a critical priority, especially given their potential to disseminate ticks over long distances.

Comparative genomic analysis and phylogenetic position of Theileria equi.

BACKGROUND: Transmission of arthropod-borne apicomplexan parasites that cause disease and result in death or persistent infection represents a major challenge to global human and animal health. First described in 1901 as Piroplasma equi, this re-emergent apicomplexan parasite was renamed Babesia equi and subsequently Theileria equi, reflecting an uncertain taxonomy. Understanding mechanisms by which apicomplexan parasites evade immune or chemotherapeutic elimination is required for development of effective vaccines or chemotherapeutics. The continued risk of transmission of T. equi from clinically silent, persistently infected equids impedes the goal of returning the U. S. to non-endemic status. Therefore comparative genomic analysis of T. equi was undertaken to: 1) identify genes contributing to immune evasion and persistence in equid hosts, 2) identify genes involved in PBMC infection biology and 3) define the phylogenetic position of T. equi relative to sequenced apicomplexan parasites. RESULTS: The known immunodominant proteins, EMA1, 2 and 3 were discovered to belong to a ten member gene family with a mean amino acid identity, in pairwise comparisons, of 39%. Importantly, the amino acid diversity of EMAs is distributed throughout the length of the proteins. Eight of the EMA genes were simultaneously transcribed. As the agents that cause bovine theileriosis infect and transform host cell PBMCs, we confirmed that T. equi infects equine PBMCs, however, there is no evidence of host cell transformation. Indeed, a number of genes identified as potential manipulators of the host cell phenotype are absent from the T. equi genome. Comparative genomic analysis of T. equi revealed the phylogenetic positioning relative to seven apicomplexan parasites using deduced amino acid sequences from 150 genes placed it as a sister taxon to Theileria spp. CONCLUSIONS: The EMA family does not fit the paradigm for classical antigenic variation, and we propose a novel model describing the role of the EMA family in persistence. T. equi has lost the putative genes for host cell transformation, or the genes were acquired by T. parva and T. annulata after divergence from T. equi. Our analysis identified 50 genes that will be useful for definitive phylogenetic classification of T. equi and closely related organisms.

Comparative analysis of gene expression between Babesia bovis blood stages and kinetes allowed by improved genome annotation.

Throughout their life cycle, Babesia parasites alternate between a mammalian host, where they cause babesiosis, and the tick vector. Transition between hosts results in distinct environmental signals that influence patterns of gene expression, consistent with the morphological and functional changes operating in the parasites during their life stages. In addition, comparing differential patterns of gene expression among mammalian and tick parasite stages can provide clues for developing improved methods of control. Hereby, we upgraded the genome assembly of Babesia bovis, a bovine hemoparasite, closing a 139 kbp gap, and used RNA-Seq datasets derived from mammalian blood and tick kinete stages to update the genome annotation. Of the originally annotated genes, 1,254 required structural changes, and 326 new genes were identified, leading to a different predicted proteome compared to the original annotation. Next, the RNA-Seq data was used to identify B. bovis genes that were differentially expressed in the vertebrate and arthropod hosts. In blood stages, 28% of the genes were upregulated up to 300 fold, whereas 26% of the genes in kinetes, a tick stage, were upregulated up to >19,000 fold. We thus discovered differentially expressed genes that may play key biological roles and serve as suitable targets for the development of vaccines to control bovine babesiosis.

blaCMY-2-positive IncA/C plasmids from Escherichia coli and Salmonella enterica are a distinct component of a larger lineage of plasmids.

Large multidrug resistance plasmids of the A/C incompatibility complex (IncA/C) have been found in a diverse group of Gram-negative commensal and pathogenic bacteria. We present three completed sequences from IncA/C plasmids that originated from Escherichia coli (cattle) and Salmonella enterica serovar Newport (human) and that carry the cephamycinase gene blaCMY-2. These large plasmids (148 to 166 kbp) share extensive sequence identity and synteny. The most divergent plasmid, peH4H, has lost several conjugation-related genes and has gained a kanamycin resistance region. Two of the plasmids (pAM04528 and peH4H) harbor two copies of blaCMY-2, while the third plasmid (pAR060302) harbors a single copy of the gene. The majority of single-nucleotide polymorphisms comprise nonsynonymous mutations in floR. A comparative analysis of these plasmids with five other published IncA/C plasmids showed that the blaCMY-2 plasmids from E. coli and S. enterica are genetically distinct from those originating from Yersinia pestis and Photobacterium damselae and distal to one originating from Yersinia ruckeri. While the overall similarity of these plasmids supports the likelihood of recent movements among E. coli and S. enterica hosts, their greater divergence from Y. pestis or Y. ruckeri suggests less recent plasmid transfer among these pathogen groups.

Transcriptome dataset of Babesia bovis life stages within vertebrate and invertebrate hosts.

Babesia bovis is a hemoprotozoan parasite of cattle that has a complex life cycle within vertebrate and invertebrate hosts. In the mammalian host, B. bovis undergoes asexual reproduction while in the tick midgut, gametes are induced, fuse, and form zygotes. The zygote infects tick gut epithelial cells and transform into kinetes that are released into the hemolymph and invade other tick tissues such as the ovaries, resulting in transovarial transmission to tick offspring. To compare gene regulation between different B. bovis life stages, we collected parasites infecting bovine erythrocytes and tick hemolymph. Total RNA samples were isolated, and multiplexed libraries sequenced using paired-end 100 cycle reads of a HiSeq 2500. The data was normalized using the TMM method and analysed for significant differential expression using the generalized linear model likelihood ratio test (GLM LRT) in edgeR. To validate our datasets, ten genes were selected using NormFinder. Genes that had no significant fold change between the blood and tick stages in the RNA-Seq datasets were tested by quantitative PCR to determine their suitability as "housekeeping" genes. The normalized RNA-Seq data revealed genes upregulated during infection of the mammalian host or tick vector and six upregulated genes were validated by quantitative PCR. These datasets can help identify useful targets for controlling bovine babesiosis.

Equid infective Theileria cluster in distinct 18S rRNA gene clades comprising multiple taxa with unusually broad mammalian host ranges.

Equine theileriosis, a tick-transmitted disease caused by the hemoprotozoan parasites Theileria equi and Theileria haneyi, affects equids throughout tropical and subtropical regions of the world. It is a significant regulatory concern in non-endemic countries, where testing for equine theileriosis is required prior to horse import to prevent parasite entry. Within endemic areas, infection causes significant morbidity and mortality, leading to economic losses. No vaccine for equine theileriosis is available, and current drug treatment protocols are inconsistent and associated with significant side effects. Recent work has revealed substantial genetic variability among equine theileriosis organisms, and analysis of ribosomal DNA from affected animals around the world indicates that the organisms can be grouped into five distinct clades. As these diverse parasites are capable of infecting a wide range of both tick and mammalian hosts, movement of different equine Theileria species between endemic countries, and eventually into non-endemic countries, is a significant concern. Furthermore, the substantial genetic variability of these organisms will likely render currently utilized importation diagnostic tests unable to detect all equine Theileria spp. To this end, more complete characterization of these diverse parasites is critical to the continued global control of equine theileriosis. This review discusses current knowledge of equine Theileria spp. in this context, and highlights new opportunities and challenges for workers in this field.

Identification and antigenicity of the Babesia caballi spherical body protein 4 (SBP4).

BACKGROUND: The tick-borne intra-erythrocytic apicomplexan Babesia caballi is one of the etiological agents of equine babesiosis, an economically important disease of equids in most tropical and subtropical areas of the world. Discovering candidate antigens for improved diagnostic tools and vaccines remains needed for controlling equine babesiosis. This study describes the B. caballi sbp4 (Bcsbp4) gene and protein (BcSBP4) and analyzes its antigenicity in infected equids. METHODS: BLAST searches of an uncurated B. caballi assembly genome using the B. bovis SBP4 as a query were carried out, followed by PCR amplification and sequencing of a newly identified BcSBP4. Characterization of this novel gene and protein was performed by bioinformatics analysis, western blots, immunofluorescence (IFA) and an in vitro neutralization test using anti SBP4 peptide antibodies. Antigenicity of recombinant BcSBP4 (rBcSBP4) was tested with sera from field animals (n = 18) using an indirect ELISA (iELISA). RESULTS: Babesia caballi genome searches using B. bovis SBP4 as a query allowed identification of a novel gene termed Bcsbp4. The Bcsbp4 gene encodes for a protein of 30.58 kDa, which is fully conserved among B. caballi isolates from USA and Egypt. Bioinformatics analysis indicates that BcSBP4 contains a signal peptide and lacks additional transmembrane domains. Expression of BcSBP4 in blood stages of B. caballi was confirmed by western blot and IFA using antibodies against synthetic peptides representing putative B-cell epitopes of BcSBP4 predicted by in silico analysis. In vitro neutralization tests using anti-BcSBP4 peptide antibodies showed a marginal, but statistically significant inhibitory effect on the infectivity of B. caballi merozoites in horse red blood cells. Sera from eight B. caballi-infected equids, but none out of ten negative equid control sera, gave a positive signal in an rBcSBP4 based iELISA. CONCLUSIONS: The Bcsbp4 gene is expressed in B. caballi blood stages. The BcSBP4 protein is a potential candidate for developing a novel serological test that could detect B. caballi infection in equids in tropical and subtropical countries worldwide.

Conservation in the face of diversity: multistrain analysis of an intracellular bacterium.

BACKGROUND: With the recent completion of numerous sequenced bacterial genomes, notable advances have been made in understanding the level of conservation between various species. However, relatively little is known about the genomic diversity among strains. We determined the complete genome sequence of the Florida strain of Anaplasma marginale, and near complete (>96%) sequences for an additional three strains, for comparative analysis with the previously fully sequenced St. Maries strain genome. RESULTS: These comparisons revealed that A. marginale has a closed-core genome with few highly plastic regions, which include the msp2 and msp3 genes, as well as the aaap locus. Comparison of the Florida and St. Maries genome sequences found that SNPs comprise 0.8% of the longer Florida genome, with 33.5% of the total SNPs between all five strains present in at least two strains and 3.0% of SNPs present in all strains except Florida. Comparison of genomes from three strains of Mycobacterium tuberculosis, Bacillus anthracis, and Nessieria meningiditis, as well as four Chlamydophila pneumoniae strains found that 98.8%-100% of SNPs are unique to each strain, suggesting A. marginale, with 76.0%, has an intermediate level of strain-specific SNPs. Comparison of genomes from other organisms revealed variation in diversity that did not segregate with the environmental niche the bacterium occupies, ranging from 0.00% to 8.00% of the larger pairwise-compared genome. CONCLUSION: Analysis of multiple A. marginale strains suggests intracellular bacteria have more variable SNP retention rates than previously reported, and may have closed-core genomes in response to the host organism environment and/or reductive evolution.

Imidocarb dipropionate clears persistent Babesia caballi infection with elimination of transmission potential.

Antimicrobial treatment of persistent infection to eliminate transmission risk represents a specific challenge requiring compelling evidence of complete pathogen clearance. The limited repertoire of antimicrobial agents targeted at protozoal parasites magnifies this challenge. Using Babesia caballi as both a model and a specific apicomplexan pathogen for which evidence of the elimination of transmission risk is required for international animal movement, we tested whether a high-dose regimen of imidocarb dipropionate cleared infection from persistently infected asymptomatic horses and/or eliminated transmission risk. Clearance with elimination of transmission risk was supported by the following four specific lines of evidence: (i) inability to detect parasites by quantitative PCR and nested PCR amplification, (ii) conversion from seropositive to seronegative status, (iii) inability to transmit infection by direct inoculation of blood into susceptible recipient horses, and (iv) inability to transmit infection by ticks acquisition fed on the treated horses and subsequently transmission fed on susceptible horses. In contrast, untreated horses remained infected and capable of transmitting B. caballi using the same criteria. These findings establish that imidocarb dipropionate treatment clears B. caballi infection with confirmation of lack of transmission risk either by direct blood transfer or a high tick burden. Importantly, the treated horses revert to seronegative status according to the international standard for serologic testing and would be permitted to move between countries where the pathogen is endemic and countries that are free of the pathogen.

Genome sequence of Babesia bovis and comparative analysis of apicomplexan hemoprotozoa.

Babesia bovis is an apicomplexan tick-transmitted pathogen of cattle imposing a global risk and severe constraints to livestock health and economic development. The complete genome sequence was undertaken to facilitate vaccine antigen discovery, and to allow for comparative analysis with the related apicomplexan hemoprotozoa Theileria parva and Plasmodium falciparum. At 8.2 Mbp, the B. bovis genome is similar in size to that of Theileria spp. Structural features of the B. bovis and T. parva genomes are remarkably similar, and extensive synteny is present despite several chromosomal rearrangements. In contrast, B. bovis and P. falciparum, which have similar clinical and pathological features, have major differences in genome size, chromosome number, and gene complement. Chromosomal synteny with P. falciparum is limited to microregions. The B. bovis genome sequence has allowed wide scale analyses of the polymorphic variant erythrocyte surface antigen protein (ves1 gene) family that, similar to the P. falciparum var genes, is postulated to play a role in cytoadhesion, sequestration, and immune evasion. The approximately 150 ves1 genes are found in clusters that are distributed throughout each chromosome, with an increased concentration adjacent to a physical gap on chromosome 1 that contains multiple ves1-like sequences. ves1 clusters are frequently linked to a novel family of variant genes termed smorfs that may themselves contribute to immune evasion, may play a role in variant erythrocyte surface antigen protein biology, or both. Initial expression analysis of ves1 and smorf genes indicates coincident transcription of multiple variants. B. bovis displays a limited metabolic potential, with numerous missing pathways, including two pathways previously described for the P. falciparum apicoplast. This reduced metabolic potential is reflected in the B. bovis apicoplast, which appears to have fewer nuclear genes targeted to it than other apicoplast containing organisms. Finally, comparative analyses have identified several novel vaccine candidates including a positional homolog of p67 and SPAG-1, Theileria sporozoite antigens targeted for vaccine development. The genome sequence provides a greater understanding of B. bovis metabolism and potential avenues for drug therapies and vaccine development.

Infection dynamics of Theileria equi and Theileria haneyi, a newly discovered apicomplexan of the horse.

Theileria equi infection, exotic to the United States has reemerged through intravenous (iatrogenic) and tick-borne transmission. Surveillance at the US-Mexico border identified a new species, Theileria haneyi, (T. haneyiEP) (EP = Eagle Pass, Texas) which warranted additional investigation due to inability to detect by PCR targeting of T. equi ema-1 and EMA-1-cELISA validated for T. equi. Infection dynamics of T. haneyiEP were evaluated, including ability to superinfect in the presence of T. equi-Texas (T. equiTX), the isolate responsible for the reemergence of T. equi in the U S. Experimental infection with T. equiTX or T. haneyiEP revealed minimal clinical disease however, T. equiTX infection led to significantly greater neutropenia. Comparison of time to antibody detection following inoculation revealed significantly greater time to detectable anti-T. haneyiEP antibody (26.67 days post-inoculation (DPI)) than T. equiTX (11.67 DPI). Regardless of initial infection with either T. equiTX or T. haneyiEP, superinfection was established. Comparative analysis of antibody responses from a splenectomized horse infected with T. haneyiEP to that of a spleen intact horse infected with T. equiFL revealed a different antibody binding profile to T. haneyiEP, T. equiTX and T. equiFL merozoite antigen and limited shared antigen/cross-reactive antibody(s). Affinity purified T. equi EMA-1 and EMA-2 from T. equiFL were shown as targets for horse antibodies against T. haneyi. Data presented here show (1) T. haneyiEP can superinfect in the presence of T. equiTX infection and co-persists for minimally 25 months, (2) intravenous challenge with T. haneyi is subclinical, and (3) limited cross-reactive antibody between T. haneyiEP and T. equi includes reactivity to EMA-1 and EMA-2.

Persistently infected horses are reservoirs for intrastadial tick-borne transmission of the apicomplexan parasite Babesia equi.

Tick-borne pathogens may be transmitted intrastadially and transstadially within a single vector generation as well as vertically between generations. Understanding the mode and relative efficiency of this transmission is required for infection control. In this study, we established that adult male Rhipicephalus microplus ticks efficiently acquire the protozoal pathogen Babesia equi during acute and persistent infections and transmit it intrastadially to naïve horses. Although the level of parasitemia during acquisition feeding affected the efficiency of the initial tick infection, infected ticks developed levels of > or =10(4) organisms/pair of salivary glands independent of the level of parasitemia during acquisition feeding and successfully transmitted them, indicating that replication within the tick compensated for any initial differences in infectious dose and exceeded the threshold for transmission. During the development of B. equi parasites in the salivary gland granular acini, the parasites expressed levels of paralogous surface proteins significantly different from those expressed by intraerythrocytic parasites from the mammalian host. In contrast to the successful intrastadial transmission, adult female R. microplus ticks that fed on horses with high parasitemia passed the parasite vertically into the eggs with low efficiency, and the subsequent generation (larvae, nymphs, and adults) failed to transmit B. equi parasites to naïve horses. The data demonstrated that intrastadial but not transovarial transmission is an efficient mode for B. equi transmission and that persistently infected horses are an important reservoir for transmission. Consequently, R. microplus male ticks and persistently infected horses should be targeted for disease control.

Transmission of Babesia caballi by Dermacentor nitens (Acari: Ixodidae) is restricted to one generation in the absence of alimentary reinfection on a susceptible equine host.

The tropical horse tick, Dermacentor nitens, is a natural vector of Babesia caballi in the Americas. B. caballi, one of the etiologic agents of equine piroplasmosis, occurs widely throughout the world, but the United States and a few other countries are considered to be free of infection. B. caballi is transovarially transmitted by the one-host tick D. nitens; we tested the hypothesis that B. caballi can persist in multiple generations of D. nitens in the absence of opportunity to reacquire infection from a susceptible equine host. Partially engorged female D. nitens were collected from a B. caballi-infected horse in Puerto Rico and allowed to reattach and feed on an uninfected horse, successfully transmitting the infection. Three subsequent generations of ticks were reared on calves (nonsusceptible hosts for B. caballi), testing for B. caballi infection in each generation by feeding a sample of the larvae on naive horses. The first generation ofD. nitens reared on a nonsusceptible host transmitted B. caballi, whereas the second and third failed to transmit to naive horses, showing that D. nitens infection with B. caballi was restricted to one generation in the absence of alimentary reinfection. These results imply that, in the event of the introduction of this pathogen into areas of the continental United States where D. nitens occurs, the tick could become a short-term reservoir of B. caballi, making control of introduced infections more complex.

Discovery of a novel species, Theileria haneyi n. sp., infective to equids, highlights exceptional genomic diversity within the genus Theileria: implications for apicomplexan parasite surveillance.

A novel apicomplexan parasite was serendipitously discovered in horses at the United States - Mexico border. Phylogenetic analysis based on 18S rDNA showed the erythrocyte-infective parasite to be related to, but distinct from, Theileria spp. in Africa, the most similar taxa being Theileria spp. from waterbuck and mountain zebra. The degree of sequence variability observed at the 18S rDNA locus also suggests the likely existence of additional cryptic species. Among described species, the genome of this novel equid Theileria parasite is most similar to that of Theileria equi, also a pathogen of horses. The estimated divergence time between the new Theileria sp. and T. equi, based on genomic sequence data, is greater than 33 million years. Average protein sequence divergence between them, at 23%, is greater than that of Theileria parva and Theileria annulata proteins, which is 18%. The latter two represent highly virulent Theileria spp. of domestic cattle, as well as of African and Asian wild buffalo, respectively, which differ markedly in pathology, host cell tropism, tick vector and geographical distribution. The extent of genome-wide sequence divergence, as well as significant morphological differences, relative to T. equi justify the classification of Theileria sp. as a new taxon. Despite the overall genomic divergence, the nine member equi merozoite antigen (EMA) superfamily, previously found as a multigene family only in T. equi, is also present in the novel parasite. Practically, significant sequence divergence in antigenic loci resulted in this undescribed Theileria sp. not being detectable using currently available diagnostic tests. Discovery of this novel species infective to equids highlights exceptional diversity within the genus Theileria, a finding with serious implications for apicomplexan parasite surveillance.

Expression of 6-Cys Gene Superfamily Defines Babesia bovis Sexual Stage Development within Rhipicephalus microplus.

Babesia bovis, an intra-erythrocytic tick-borne apicomplexan protozoan, is one of the causative agents of bovine babesiosis. Its life cycle includes sexual reproduction within cattle fever ticks, Rhipicephalus spp. Six B. bovis 6-Cys gene superfamily members were previously identified (A, B, C, D, E, F) where their orthologues in Plasmodium parasite have been shown to encode for proteins required for the development of sexual stages. The current study identified four additional 6-Cys genes (G, H, I, J) in the B. bovis genome. These four genes are described in the context of the complete ten 6-Cys gene superfamily. The proteins expressed by this gene family are predicted to be secreted or surface membrane directed. Genetic analysis comparing the 6-Cys superfamily among five distinct B. bovis strains shows limited sequence variation. Additionally, A, B, E, H, I and J genes were transcribed in B. bovis infected tick midgut while genes A, B and E were also transcribed in the subsequent B. bovis kinete stage. Transcription of gene C was found exclusively in the kinete. In contrast, transcription of genes D, F and G in either B. bovis infected midguts or kinetes was not detected. None of the 6-Cys transcripts were detected in B. bovis blood stages. Subsequent protein analysis of 6-Cys A and B is concordant with their transcript profile. The collective data indicate as in Plasmodium parasite, certain B. bovis 6-Cys family members are uniquely expressed during sexual stages and therefore, they are likely required for parasite reproduction. Within B. bovis specifically, proteins encoded by 6-Cys genes A and B are markers for sexual stages and candidate antigens for developing novel vaccines able to interfere with the development of B. bovis within the tick vector.

Theileria equi isolates vary in susceptibility to imidocarb dipropionate but demonstrate uniform in vitro susceptibility to a bumped kinase inhibitor.

BACKGROUND: The apicomplexan hemoparasite Theileria equi is a causative agent of equine piroplasmosis, eradicated from the United States in 1988. However, recent outbreaks have sparked renewed interest in treatment options for infected horses. Imidocarb dipropionate is the current drug of choice, however variation in clinical response to therapy has been observed. METHODS: We quantified the in vitro susceptibility of two T. equi isolates and a lab generated variant to both imidocarb dipropionate and a bumped kinase inhibitor compound 1294. We also evaluated the capacity of in vitro imidocarb dipropionate exposure to decrease susceptibility to that drug. The efficacy of imidocarb dipropionate for clearing infection in four T. equi infected ponies was also assessed. RESULTS: We observed an almost four-fold difference in imidocarb dipropionate susceptibility between two distinct isolates of T. equi. Four ponies infected with the less susceptible USDA Florida strain failed to clear the parasite despite two rounds of treatment. Importantly, a further 15-fold decrease in susceptibility was produced in this strain by continuous in vitro imidocarb dipropionate exposure. Despite a demonstrated difference in imidocarb dipropionate susceptibility, there was no difference in the susceptibility of two T. equi isolates to bumped kinase inhibitor 1294. CONCLUSIONS: The observed variation in imidocarb dipropionate susceptibility, further reduction in susceptibility caused by drug exposure in vitro, and failure to clear T. equi infection in vivo, raises concern for the emergence of drug resistance in clinical cases undergoing treatment. Bumped kinase inhibitors may be effective as alternative drugs for the treatment of resistant T. equi parasites.

Improved diagnostic performance of a commercial Anaplasma antibody competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5-glutathione S-transferase fusion protein as antigen.

The current study tested the hypothesis that removal of maltose binding protein (MBP) from recombinant antigen used for plate coating would improve the specificity of a commercial Anaplasma antibody competitive enzyme-linked immunosorbent assay (cELISA). The number of 358 sera with significant MBP antibody binding (≥30%I) in Anaplasma-negative herds was 139 (38.8%) when tested using the recombinant major surface protein 5 (rMSP5)-MBP cELISA without MBP adsorption. All but 8 of the MBP binders were rendered negative (<30%I) using the commercial rMSP5-MBP cELISA with MBP adsorption, resulting in 97.8% specificity. This specificity was higher than some previous reports, so to improve the specificity of the commercial cELISA, a new recombinant antigen designated rMSP5-glutathione S-transferase (GST) was developed, eliminating MBP from the antigen and obviating the need for MBP adsorption. Using the rMSP5-GST cELISA, only 1 of 358 Anaplasma-negative sera, which included the 139 sera with significant (≥30%I) MBP binding in the rMSP5-MBP cELISA without MBP adsorption, was positive. This resulted in an improved diagnostic specificity of 99.7%. The rMSP5-GST cELISA without MBP adsorption had comparable analytical sensitivity to the rMSP5-MBP cELISA with MBP adsorption and had 100% diagnostic sensitivity when tested with 135 positive sera defined by nested polymerase chain reaction. Further, the rMSP5-GST cELISA resolved 103 false-positive reactions from selected sera with possible false-positive reactions obtained using the rMSP5-MBP cELISA with MBP adsorption and improved the resolution of 29 of 31 other sera. In summary, the rMSP5-GST cELISA was a faster and simpler assay with higher specificity, comparable sensitivity, and improved resolution in comparison with the rMSP5-MBP cELISA with MBP adsorption.

Genetic characterization of Theileria equi infecting horses in North America: evidence for a limited source of U.S. introductions.

BACKGROUND: Theileria equi is a tick-borne apicomplexan hemoparasite that causes equine piroplasmosis. This parasite has a worldwide distribution but the United States was considered to be free of this disease until recently. METHODS: We used samples from 37 horses to determine genetic relationships among North American T. equi using the 18S rRNA gene and microsatellites. We developed a DNA fingerprinting panel of 18 microsatellite markers using the first complete genome sequence of T. equi. RESULTS: A maximum parsimony analysis of 18S rRNA sequences grouped the samples into two major clades. The first clade (n = 36) revealed a high degree of nucleotide similarity in U.S. T. equi, with just 0-2 single nucleotide polymorphisms (SNPs) among samples. The remaining sample fell into a second clade that was genetically divergent (48 SNPs) from the other U.S. samples. This sample was collected at the Texas border, but may have originated in Mexico. We genotyped T. equi from the U.S. using microsatellite markers and found a moderate amount of genetic diversity (2-8 alleles per locus). The field samples were mostly from a 2009 Texas outbreak (n = 22) although samples from five other states were also included in this study. Using Weir and Cockerham's FST estimator (θ) we found strong population differentiation of the Texas and Georgia subpopulations (θ = 0.414), which was supported by a neighbor-joining tree created with predominant single haplotypes. Single-clone infections were found in 27 of the 37 samples (73%), allowing us to identify 15 unique genotypes. CONCLUSIONS: The placement of most T. equi into one monophyletic clade by 18S is suggestive of a limited source of introduction into the U.S. When applied to a broader cross section of worldwide samples, these molecular tools should improve source tracking of T. equi outbreaks and may help prevent the spread of this tick-borne parasite.