David R. Herndon

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 ∼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.

Association of Mycoplasma ovipneumoniae Infection with Population-Limiting Respiratory Disease in Free-Ranging Rocky Mountain Bighorn Sheep (Ovis canadensis canadensis)

ABSTRACT Bronchopneumonia is a population-limiting disease in bighorn sheep in much of western North America. Previous investigators have isolated diverse bacteria from the lungs of affected sheep, but no single bacterial species is consistently present, even within single epizootics. We obtained high-quality diagnostic specimens from nine pneumonic bighorn sheep in three populations and analyzed the bacterial populations present in bronchoalveolar lavage specimens of seven by using a culture-independent method (16S rRNA gene amplification and clone library analyses). Mycoplasma ovipneumoniae was detected as a predominant member of the pneumonic lung flora in lambs with early lesions of bronchopneumonia. Specific PCR tests then revealed the consistent presence of M. ovipneumoniae in the lungs of pneumonic bighorn sheep in this study, and M. ovipneumoniae was isolated from lung specimens of five of the animals. Retrospective application of M. ovipneumoniae PCR to DNA extracted from archived formalin-fixed, paraffin-embedded lung tissues of historical adult bighorn sheep necropsy specimens supported the association of this agent with bronchopneumonia (16/34 pneumonic versus 0/17 nonpneumonic sheep were PCR positive [P < 0.001]). Similarly, a very strong association was observed between the presence of one or more M. ovipneumoniae antibody-positive animals and the occurrence of current or recent historical bronchopneumonia problems (seropositive animals detected in 9/9 versus 0/9 pneumonic and nonpneumonic populations, respectively [P < 0.001]). M. ovipneumoniae is strongly associated with bronchopneumonia in free-ranging bighorn sheep and is a candidate primary etiologic agent for this disease.

Comparative genomic analysis and phylogenetic position of Theileria equi

BackgroundTransmission 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.ResultsThe 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.ConclusionsThe 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.

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

BackgroundWith 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.ResultsThese 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.ConclusionAnalysis 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.

Complete genome sequence of Anaplasma marginale subsp. centrale.

Anaplasma marginale subsp. centrale is a naturally attenuated subtype that has been used as a vaccine for a century. We sequenced the genome of this organism and compared it to those of virulent senso stricto A. marginale strains. The comparison markedly narrows the number of outer membrane protein candidates for development of a safer inactivated vaccine and provides insight into the diversity among strains of senso lato A. marginale.

Genome Sequences of Mannheimia haemolytica Serotype A2: Ovine and Bovine Isolates

This report describes the genome sequences of Mannheimia haemolytica serotype A2 isolated from pneumonic lungs of two different ruminant species, one from Ovis aries, designated ovine (O), and the other from Bos taurus, designated bovine (B).

Non-maternal transmission is the major mode of ovine lentivirus transmission in a ewe flock: a molecular epidemiology study.

Transmission of ovine progressive pneumonia virus (OPPV), a lentivirus of sheep, occurs through both maternal and non-maternal means. Currently, the contribution of each route to the overall flock OPPV prevalence is poorly understood since previous serological epidemiologic studies lacked the ability to accurately track routes of transmission within an infected flock. In this study, the amount of maternal OPP transmission was assessed in a naturally infected ewe flock by applying molecular analyses to proviral sequences derived from peripheral blood leukocytes of OPP positive dam-daughter pairs (N=40). Both proviral envelope (env) and long terminal repeat (LTR) sequences, separately and combined, were utilized in the following 2 sequence analysis methods: phylogenetic analysis and pairwise distance calculations. True maternal transmission events were defined as agreement in 2 out of the 2 sequence analysis methods. Using this criterion, proviral env sequences resulted in a 14.3% maternal transmission frequency, and proviral LTR sequences resulted in a 10% maternal transmission frequency. Both proportions of maternal transmission varied significantly from equality (P<0.0001). This indicates that the remaining 85.7-90% of daughters are infected via non-maternal transmission. This is also the first study to calculate the OPP proviral rate of change for the env gene and LTR promoter. Accurately defining the routes of OPPV transmission provides critical epidemiological data supporting management intended to reduce flock transmission and viral dose.

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.

Integration of a transfected gene into the genome of Babesia bovis occurs by legitimate homologous recombination mechanisms.

This study examines the patterns of gene integration of gfp-bsd upon stable transfection into the T3Bo strain of Babesia bovis using a plasmid designed to integrate homologous sequences of the parasites two identical ef-1α A and B genes. While the transfected BboTf-149-6 cell line displayed two distinct patterns of gene integration, clonal lines derived from this strain by cell sorting contained only single gfp-bsd insertions. Whole genome sequencing of two selected clonal lines, E9 and C6, indicated two distinct patterns of gfp-bsd insertion occurring by legitimate homologous recombination mechanisms: one into the expected ef-1α orf B, and another into the ef-1α B promoter. The data suggest that expression of the ef-1α orf B is not required for development of B. bovis in cultured erythrocyte stages. Use of legitimate homologous recombination mechanisms in transfected B. bovis supports the future use of transfection methods for developing efficient gene function assignment experiments using gene knockout techniques.

Anaplasma marginale Superinfection Attributable to Pathogen Strains with Distinct Genomic Backgrounds

ABSTRACT Strain superinfection occurs when a second pathogen strain infects a host already infected with a primary strain. The selective pressures that drive strain divergence, which underlies superinfection, and allow penetration of a new strain into a host population are critical knowledge gaps relevant to shifts in infectious disease epidemiology. In regions of endemicity with a high prevalence of infection, broad population immunity develops against Anaplasma marginale, a highly antigenically variant rickettsial pathogen, and creates strong selective pressure for emergence of and superinfection with strains that differ in their Msp2 variant repertoires. The strains may emerge either by msp2 locus duplication and allelic divergence on an existing genomic background or by introduction of a strain with a different msp2 allelic repertoire on a distinct genomic background. To answer this question, we developed a multilocus typing assay based on high-throughput sequencing of non-msp2 target loci to distinguish among strains with different genomic backgrounds. The technical error level was statistically defined based on the percentage of perfect sequence matches of clones of each target locus and validated using experimental single strains and strain pairs. Testing of A. marginale-positive samples from tropical regions where A. marginale infection is endemic identified individual infections that contained unique alleles for all five targeted loci. The data revealed a highly significant difference in the number of strains per animal in the tropical regions compared to infections in temperate regions and strongly supported the hypothesis that transmission of genomically distinct A. marginale strains predominates in high-prevalence areas of endemicity.