Disruption of VirB6 Paralogs in Anaplasma phagocytophilum Attenuates Its Growth.

Many pathogenic bacteria translocate virulence factors into their eukaryotic hosts by means of type IV secretion systems (T4SS) spanning the inner and outer membranes. Genes encoding components of these systems have been identified within the order Rickettsiales based upon their sequence similarities to other prototypical systems. Anaplasma phagocytophilum strains are obligate intracellular, tick-borne bacteria that are members of this order. The organization of these components at the genomic level was determined in several Anaplasma phagocytophilum strains, showing overall conservation, with the exceptions of the virB2 and virB6 genes. The virB6 loci are characterized by the presence of four virB6 copies (virB6-1 through virB6-4) arranged in tandem within a gene cluster known as the sodB-virB operon. Interestingly, the virB6-4 gene varies significantly in length among different strains due to extensive tandem repeats at the 3' end. To gain an understanding of how these enigmatic virB6 genes function in A. phagocytophilum, we investigated their expression in infected human and tick cells. Our results show that these genes are expressed by A. phagocytophilum replicating in both cell types and that VirB6-3 and VirB6-4 proteins are surface exposed. Analysis of an A. phagocytophilum mutant carrying the Himar1 transposon within the virB6-4 gene demonstrated that the insertion not only disrupted its expression but also exerted a polar effect on the sodB-virB operon. Moreover, the altered expression of genes within this operon was associated with the attenuated in vitro growth of A. phagocytophilum in human and tick cells, indicating the importance of these genes in the physiology of this obligate intracellular bacterium in such different environments.IMPORTANCE Knowledge of the T4SS is derived from model systems, such as Agrobacterium tumefaciens The structure of the T4SS in Rickettsiales differs from the classical arrangement. These differences include missing and duplicated components with structural alterations. Particularly, two sequenced virB6-4 genes encode unusual C-terminal structural extensions resulting in proteins of 4,322 (GenBank accession number AGR79286.1) and 9,935 (GenBank accession number ANC34101.1) amino acids. To understand how the T4SS is used in A. phagocytophilum, we describe the expression of the virB6 paralogs and explore their role as the bacteria replicate within its host cell. Conclusions about the importance of these paralogs for colonization of human and tick cells are supported by the deficient phenotype of an A. phagocytophilum mutant isolated from a sequence-defined transposon insertion library.

VirB10 vaccination for protection against Anaplasma phagocytophilum.

BACKGROUND: Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the etiologic agent Anaplasma phagocytophilum. HGA was designated a nationally notifiable disease in the United States in 1998. Currently there are no vaccines available against HGA. Conserved membrane proteins that are subdominant in Anaplasma species, such as VirB9 and VirB10, may represent better vaccine targets than the variable immunodominant surface proteins. VirB9 and VirB10 are constituents of the Type 4 secretion system (T4SS) that is conserved amongst many intracellular bacteria and performs essential functions for invasion and survival in host cells. RESULTS: Immunogenicity and contribution to protection, provided after intramuscular vaccination of plasmid DNA encoding VirB9-1, VirB9-2, and VirB10 followed by inoculation of homologous recombinant proteins, in a prime-boost immunization strategy was evaluated in a murine model of HGA. Recombinant VirB9-1-, VirB9-2-, and VirB10-vaccinated mice developed antibody responses that specifically reacted with A. phagocytophilum organisms. However, only the mice vaccinated with VirB10 developed a significant increase in IFN-γ CD4+ T cells and partial protection against challenge with A. phagocytophilum. CONCLUSIONS: This work provides evidence that A. phagocytophilum T4SS VirB10 is partially protective in a murine model against infection in an IFN-γ-dependent fashion and suggests that this protein may be a potential vaccine candidate against this and possibly other pathogenic bacteria with a T4SS.

Reduced Infectivity in cattle for an outer membrane protein mutant of Anaplasma marginale.

Anaplasma marginale is the causative agent of anaplasmosis in cattle. Transposon mutagenesis of this pathogen using the Himar1 system resulted in the isolation of an omp10 operon insertional mutant referred to as the omp10::himar1 mutant. The work presented here evaluated if this mutant had morphological and/or growth rate defects compared to wild-type A. marginale. Results showed that the morphology, developmental cycle, and growth in tick and mammalian cell cultures are similar for the mutant and the wild type. Tick transmission experiments established that tick infection levels with the mutant were similar to those with wild-type A. marginale and that infected ticks successfully infected cattle. However, this mutant exhibited reduced infectivity and growth in cattle. The possibility of transforming A. marginale by transposon mutagenesis coupled with in vitro and in vivo assessment of altered phenotypes can aid in the identification of genes associated with virulence. The isolation of deliberately attenuated organisms that can be evaluated in their natural biological system is an important advance for the rational design of vaccines against this species.

Knockout of an outer membrane protein operon of Anaplasma marginale by transposon mutagenesis.

BACKGROUND: The large amounts of data generated by genomics, transcriptomics and proteomics have increased our understanding of the biology of Anaplasma marginale. However, these data have also led to new assumptions that require testing, ideally through classical genetic mutation. One example is the definition of genes associated with virulence. Here we describe the molecular characterization of a red fluorescent and spectinomycin and streptomycin resistant A. marginale mutant generated by Himar1 transposon mutagenesis. RESULTS: High throughput genome sequencing to determine the Himar1-A. marginale genome junctions established that the transposon sequences were integrated within the coding region of the omp10 gene. This gene is arranged within an operon with AM1225 at the 5' end and with omp9, omp8, omp7 and omp6 arranged in tandem at the 3' end. RNA analysis to determine the effects of the transposon insertion on the expression of omp10 and downstream genes revealed that the Himar1 insertion not only reduced the expression of omp10 but also that of downstream genes. Transcript expression from omp9, and omp8 dropped by more than 90% in comparison with their counterparts in wild-type A. marginale. Immunoblot analysis showed a reduction in the production of Omp9 protein in these mutants compared to wild-type A. marginale. CONCLUSIONS: These results demonstrate that transposon mutagenesis in A. marginale is possible and that this technology can be used for the creation of insertional gene knockouts that can be evaluated in natural host-vector systems.

Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum.

BACKGROUND: Anaplasma phagocytophilum is an intracellular organism in the Order Rickettsiales that infects diverse animal species and is causing an emerging disease in humans, dogs and horses. Different strains have very different cell tropisms and virulence. For example, in the U.S., strains have been described that infect ruminants but not dogs or rodents. An intriguing question is how the strains of A. phagocytophilum differ and what different genome loci are involved in cell tropisms and/or virulence. Type IV secretion systems (T4SS) are responsible for translocation of substrates across the cell membrane by mechanisms that require contact with the recipient cell. They are especially important in organisms such as the Rickettsiales which require T4SS to aid colonization and survival within both mammalian and tick vector cells. We determined the structure of the T4SS in 7 strains from the U.S. and Europe and revised the sequence of the repetitive virB6 locus of the human HZ strain. RESULTS: Although in all strains the T4SS conforms to the previously described split loci for vir genes, there is great diversity within these loci among strains. This is particularly evident in the virB2 and virB6 which are postulated to encode the secretion channel and proteins exposed on the bacterial surface. VirB6-4 has an unusual highly repetitive structure and can have a molecular weight greater than 500,000. For many of the virs, phylogenetic trees position A. phagocytophilum strains infecting ruminants in the U.S. and Europe distant from strains infecting humans and dogs in the U.S. CONCLUSIONS: Our study reveals evidence of gene duplication and considerable diversity of T4SS components in strains infecting different animals. The diversity in virB2 is in both the total number of copies, which varied from 8 to 15 in the herein characterized strains, and in the sequence of each copy. The diversity in virB6 is in the sequence of each of the 4 copies in the single locus and the presence of varying numbers of repetitive units in virB6-3 and virB6-4. These data suggest that the T4SS should be investigated further for a potential role in strain virulence of A. phagocytophilum.

Comparative Whole Genome Analysis of an Anaplasma phagocytophilum Strain Isolated from Norwegian Sheep.

Anaplasma phagocytophilum is a Gram-negative obligate intracellular tick-borne alphaproteobacteria (family Anaplasmatacea, order Rickettsiales) with a worldwide distribution. In Norway, tick borne fever (TBF), caused by A. phagocytophilum, presents a major challenge in sheep farming. Despite the abundance of its tick vector, Ixodes ricinus, and A. phagocytophilum infections in wild and domestic animals, reports of infections in humans are low compared with cases in the U.S. Although A. phagocytophilum is genetically diverse and complex infections (co-infection and superinfection) in ruminants and other animals are common, the underlying genetic basis of intra-species interactions and host-specificity remains unexplored. Here, we performed whole genome comparative analysis of a newly cultured Norwegian A. phagocytophilum isolate from sheep (ApSheep_NorV1) with 27 other A. phagocytophilum genome sequences derived from human and animal infections worldwide. Although the compared strains are syntenic, there is remarkable genetic diversity between different genomic loci including the pfam01617 superfamily that encodes the major, neutralization-sensitive, surface antigen Msp2/p44. Blast comparisons between the msp2/p44 pseudogene repertoires from all the strains showed high divergence between U. S. and European strains and even between two Norwegian strains. Based on these comparisons, we concluded that in ruminants, complex infections can be attributed to infection with strains that differ in their msp2/p44 repertoires, which has important implications for pathogen evolution and vaccine development. We also present evidence for integration of rickettsial DNA into the genome of ISE6 tick cells.

Molecular characterization of "Candidatus Anaplasma testudinis": An emerging pathogen in the threatened Florida gopher tortoise (Gopherus polyphemus).

Members of the family Anaplasmataceae are obligate intracellular bacteria that replicate within membrane bound vacuoles in the cytoplasm of cells in vertebrate and invertebrate hosts. This study reports a putative new Anaplasma species in gopher tortoises in Florida. Two Florida gopher tortoises (Gopherus polyphemus) presented at the University of Florida Veterinary Hospital with anemia and intracytoplasmic vacuoles filled with bacteria within erythrocytes. The bacteria within these parasitophorous vacuoles were morphologically similar to Anaplasma marginale. We inoculated ISE6 cells with blood from one tortoise and isolated bacterial colonies consistent with A. marginale. Molecular characterization targeting Anaplasmataceae 16S rRNA sequences indicated that the clinical isolate, named here provisionally as "Candidatus Anaplasma testudinis", grouped within the genus Anaplasma on a separate clade, most closely related to the A. marginale, Anaplasma ovis and Anaplasma centrale group. We next screened archived red blood cells from 38 wild gopher tortoises with documented clinical anemia. Fourteen of the 38 wild tortoises, representing 5 of 11 geographical locations were PCR-positive for Anaplasmataceae spp. Sequencing analysis revealed 16S rRNA sequence identical to "Ca. A. testudinis". The clinical presentation of significant anemia associated with "Ca. A. testudinis" in a threatened species could have conservation implications. Importantly, the availability of a clinical isolate will aid further studies to develop diagnostic tests and to investigate potential tick vectors and infectivity for other wildlife and domestic animal species.

Biostatistical prediction of genes essential for growth of Anaplasma phagocytophilum in a human promyelocytic cell line using a random transposon mutant library.

Anaplasma phagocytophilum (Ap), agent of human anaplasmosis, is an intracellular bacterium that causes the second most common tick-borne illness in North America. To address the lack of a genetic system for these pathogens, we used random Himar1 transposon mutagenesis to generate a library of Ap mutants capable of replicating in human promyelocytes (HL-60 cells). Illumina sequencing identified 1195 non-randomly distributed insertions. As the density of mutants was non-saturating, genes without insertions were either essential for Ap, or spared randomly. To resolve this question, we applied a biostatistical method for prediction of essential genes. Since the chances that a transposon was inserted into genomic TA dinucleotide sites should be the same for all loci, we used a Markov chain Monte Carlo model to estimate the probability that a non-mutated gene was essential for Ap. Predicted essential genes included those coding for structural ribosomal proteins, enzymes involved in metabolism, components of the type IV secretion system, antioxidant defense molecules and hypothetical proteins. We have used an in silico post-genomic approach to predict genes with high probability of being essential for replication of Ap in HL-60 cells. These results will help target genes to investigate their role in the pathogenesis of human anaplasmosis.

Diversity of Ehrlichia ruminantium major antigenic protein 1-2 in field isolates and infected sheep.

Proteins expressed from the map1 multigene family of Ehrlichia ruminantium are strongly recognized by immune T and B cells from infected animals or from animals that were infected and have recovered from heartwater disease (although still remaining infected carriers). Analogous multigene clusters also encode the immunodominant outer membrane proteins (OMPs) in other ehrlichial species. Recombinant protein analogs of the expressed genes and DNA vaccines based on the multigene clusters have been shown to induce protective immunity, although this was less effective in heterologous challenge situations, where the challenge strain major antigenic protein 1 (MAP1) sequence differed from the vaccine strain MAP1. Recent data for several ehrlichial species show differential expression of the OMPs in mammalian versus tick cell cultures and dominant expression of individual family members in each type of culture system. However, many genes in the clusters appear to be complete and functional and to generate mRNA transcripts. Recent data also suggest that there may be a low level of protein expression from many members of the multigene family, despite primary high-level expression from an individual member. A continuing puzzle, therefore, is the biological roles of the different members of these OMP multigene families. Complete genome sequences are now available for two geographically divergent strains of E. ruminantium (Caribbean and South Africa strains). Comparison of these sequences revealed amino acid sequence diversity in MAP1 (89% identity), which is known to confer protection in a mouse model and to be the multigene family member primarily expressed in mammalian cells. Surprisingly, however, the greatest sequence diversity (79% identity) was in the less-studied map1-2 gene. We investigated here whether this map1-2 diversity was a general feature of E. ruminantium in different cultured African strains and in organisms from infected sheep. Comparison of MAP1-2s revealed amino acid identities of 75 to 100% (mean of 86%), compared to 84 to 100% (mean of 89%) for MAP1s. Interestingly, MAP1-2s varied independently of MAP1s such that E. ruminantium strains with similar MAP1s had diverse MAP1-2s and vice versa. Different MAP1-2s were found in individual infected sheep. Different regions of a protein may be subjected to different evolutionary forces because of recombination and/or selection, which results in those regions not agreeing with a phylogeny deduced from the whole molecule. This appears to be true for both MAP1 and MAP1-2, where statistical likelihood methods detect heterogeneous evolutionary rates for segments of both molecules. Sera from infected cattle recognized a MAP1-2 variable-region peptide in enzyme-linked immunosorbent assay, but less strongly and consistently than a MAP1 peptide (MAP1B). Heterologous protective immunity may depend on recognition of a complex set of varying OMP epitopes.

Diversity of Anaplasma phagocytophilum strains, USA.

We analyzed the structure of the expression site encoding the immunoprotective protein MSP2/P44 from multiple Anaplasma phagocytophilum strains in the United States. The sequence of p44ESup1 had diverged in Ap-variant 1 strains infecting ruminants. In contrast, no differences were detected between A. phagocytophilum strains infecting humans and domestic dogs.

Assessing the clinical and bacteriological outcomes of vaccination with recombinant Asp14 and OmpA against A. phagocytophilum in sheep.

Anaplasma phagocytophilum is a tick borne bacterium, causing disease in sheep and other mammals, including humans. The bacterium has great economic and animal welfare implications for sheep husbandry in Northern Europe. With the prospect of a warmer and more humid climate, the vector availability will likely increase, resulting in a higher prevalence of A. phagocytophilum. The current preventive measures, as pyrethroids acting on ticks or long acting antibiotics controlling bacterial infection, are suboptimal for prevention of the disease in sheep. Recently, the increased awareness on antibiotic- and pyrethorid resistance, is driving the search for a new prophylactic approach in sheep against A. phagocytophilum. Previous studies have used an attenuated vaccine, which gave insufficient protection from challenge with live bacteria. Other studies have focused on bacterial membrane surface proteins like Asp14 and OmpA. An animal study using homologous proteins to Asp14 and OmpA of A. marginale, showed no protective effect in heifers. In the current study, recombinant proteins of Asp14 (rAsp14) and OmpA (rOmpA) of A. phagocytophilum were produced and prepared as a vaccine for sheep. Ten lambs were vaccinated twice with an adjuvant emulsified with rAsp14 or rOmpA, three weeks apart and challenged with a live strain of A. phagocytophilum (GenBank acc.nr M73220) on day 42. The control group consisted of five lambs injected twice with PBS and adjuvant. Hematology, real time qPCR, immunodiagnostics and flow cytometric analyses of peripheral blood mononuclear cells were performed. Vaccinated lambs responded with clinical signs of A.phagocytophilum infection after challenge and bacterial load in the vaccinated group was not reduced compared to the control group. rAsp14 vaccinated lambs generated an antibody response against the vaccine, but a clear specificity for rAsp14 could not be established. rOmpA-vaccinated lambs developed a strong specific antibody response on days 28 after vaccination and 14 days post-challenge. Immunofluorescent staining and flow cytometric analysis of peripheral blood mononuclear monocytes revealed no difference between the three groups, but the percentage of CD4+, CD8+, γδ TcR+, λ-Light chain+, CD11b+, CD14+ and MHC II+ cells, within the groups changed during the study, most likely due to the adjuvant or challenge with the bacterium. Although an antigen specific antibody response could be detected against rOmpA and possibly rAsp14, the vaccines seemed to be ineffective in reducing clinical signs and bacterial load caused by A. phagocytophilum. This is the first animal study with recombinant Asp14 and OmpA aimed at obtaining clinical protection against A. phagocytophilum in sheep.

Outer membrane protein sequence variation in lambs experimentally infected with Anaplasma phagocytophilum.

Anaplasma phagocytophilum has long been known to cause tick-borne fever in ruminants and has been identified more recently as the causative agent of the emerging disease human granulocytic anaplasmosis. The related organism Anaplasma marginale uses gene conversion of the expression site for two major outer membrane proteins (OMPs) to generate extensive sequence and antigenic variation in these OMPs. This is thought to present a continuously varying repertoire of epitopes to the mammalian host and allow disease persistence. Recent genomic and structural data on human strains of A. phagocytophilum, together with animal studies in model systems, have implicated an orthologous OMP of A. phagocytophilum in a similar mechanism of variation. However, to date there has been little investigation of the mechanisms of antigenic variation or disease persistence in hosts naturally infected with field strains of A. phagocytophilum. Approximately 300,000 lambs in Norway suffer severe disease caused by A. phagocytophilum annually. We show here the persistent and cyclic nature of infection in these animals that is accompanied by loosely programmed sequence variation of the major OMP expression site in each rickettsemic peak. These data will allow analysis of interactions between A. phagocytophilum and the host immune system in naturally occurring persistent infections and provide an important comparison with enduring infections of cattle caused by A. marginale.

In situ detection of Anaplasma spp. by DNA target-primed rolling-circle amplification of a padlock probe and intracellular colocalization with immunofluorescently labeled host cell von Willebrand factor.

Endothelial cell culture and preliminary immunofluorescent staining of Anaplasma-infected tissues suggest that endothelial cells may be an in vivo nidus of mammalian infection. To investigate endothelial cells and other potentially cryptic sites of Anaplasma sp. infection in mammalian tissues, a sensitive and specific isothermal in situ technique to detect localized Anaplasma gene sequences by using rolling-circle amplification of circularizable, linear, oligonucleotide probes (padlock probes) was developed. Cytospin preparations of uninfected or Anaplasma-infected cell cultures were examined using this technique. Via fluorescence microscopy, the technique described here, and a combination of differential interference contrast microscopy and von Willebrand factor immunofluorescence, Anaplasma phagocytophilum and Anaplasma marginale were successfully localized in situ within intact cultured mammalian cells. This work represents the first application of this in situ method for the detection of a microorganism and forms the foundation for future applications of this technique to detect, localize, and analyze Anaplasma nucleotide sequences in the tissues of infected mammalian and arthropod hosts and in cell cultures.

Viral Enrichment Methods Affect the Detection but Not Sequence Variation of West Nile Virus in Equine Brain Tissue.

West Nile virus (WNV), a small, positive sense, single stranded RNA virus continues to encroach into new locales with emergence of new viral variants. Neurological disease in the equine can be moderate to severe in the face of low to undetectable virus loads. Physical methods of virus enrichment may increase sensitivity of virus detection and enhance analysis of viral diversity, especially for deep sequencing studies. However, the use of these techniques is limited mainly to non-neural tissues. We investigated the hypothesis that elimination of equine brain RNA enhances viral detection without limiting viral variation. Eight different WNV viral RNA enrichment and host RNA separation methods were evaluated to determine if elimination of host RNA enhanced detection of WNV and increase the repertoire of virus variants for sequencing. Archived brain tissue from 21 different horses was inoculated with WNV, homogenized, before enrichment and separation. The protocols utilized combinations of low-speed centrifugation, syringe filtration, and nuclease treatment. Viral and host RNA were analyzed using real-time PCR targeting the WNV Envelope (E) protein and equine G3PDH to determine relative sensitivity for WNV and host depletion, respectively. To determine the effect of these methods on viral variation, deep sequencing of the E protein was performed. Our results demonstrate that additional separation and enrichment methods resulted in loss of virus in the face of host RNA depletion. DNA sequencing showed no significant difference in total sequence variation between the RNA enrichment protocols. For equine brain infected with WNV, direct RNA extraction followed by host RNA depletion was most suitable. This study highlights the importance of evaluating viral enrichment and separation methods according to tissue type before embarking on studies where quantification of virus and viral variants is essential to the outcome of the study.

Quantifying known and emerging uterine pathogens, and evaluating their association with metritis and fever in dairy cows.

Metritis is caused by polymicrobial infection; however, recent metagenomic work challenges the importance of known pathogens such as Escherichia coli and Trueperella pyogenes while identifying potential new pathogens such as Bacteroides pyogenes, Porphyromonas levii and Helcococcus ovis. This study aims to quantify known and emerging uterine pathogens, and to evaluate their association with metritis and fever in dairy cows. Metritis was diagnosed at 6 ±â€¯2 days postpartum, a uterine swab was collected and rectal temperature was measured. 39 cows were classified into three groups: Healthy (n = 14), Metritis without fever (MNoFever; n = 12), and Metritis with fever (MFever; n = 13). Absolute copy number was determined for total bacteria and for 8 potentially pathogenic bacteria using droplet digital PCR. Both MNoFever and MFever cows had higher copy number of total bacteria, Fusobacterium necrophorum, Prevotella melaninogenica, Bacteroides pyogenes, Porphyromonas levii, and Helcococcus ovis than Healthy cows. MNoFever and MFever groups were similar. There was no difference among groups in copy number of Escherichia coli, Trueperella pyogenes, and Bacteroides heparinolyticus, and they all had low copy numbers. Our work confirms the importance of some bacteria identified by culture-based studies in the pathogenesis of metritis such as Fusobacterium necrophorum and Prevotella melaninogenica; however, it challenges the importance of others such as Escherichia coli and Trueperella pyogenes at the time of metritis diagnosis. Additionally, Bacteroides pyogenes, Porphyromonas levii, and Helcococcus ovis were recognized as emerging pathogens involved in the etiology of metritis. Furthermore, fever was not associated with the total bacterial load or specific bacteria.

Transformation of Anaplasma phagocytophilum.

BACKGROUND: Tick-borne pathogens cause emerging zoonoses, and include fastidious organisms such as Anaplasma phagocytophilum. Because of their obligate intracellular nature, methods for mutagenesis and transformation have not been available. RESULTS: To facilitate genetic manipulation, we transformed A. phagocytophilum (Ap) to express a green fluorescent protein (GFP) with the Himar1 transposase system and selection with the clinically irrelevant antibiotic spectinomycin. CONCLUSION: These transformed bacteria (GFP/Ap) grow at normal rates and are brightly fluorescent in human, monkey, and tick cell culture. Molecular characterization of the GFP/Ap genomic DNA confirmed transposition and the flanking genomic insertion locations were sequenced. Three mice inoculated with GFP/Ap by intraperitoneal injection became infected as demonstrated by the appearance of morulae in a peripheral blood neutrophil and re-isolation of the bacteria in culture.

Evaluation of E. ruminantium genes in DBA/2 mice as potential DNA vaccine candidates for control of heartwater.

Heartwater caused by the rickettsia Ehrlichia ruminantium (E. ruminantium) is an acute and fatal tick-borne disease of domestic and some wild ruminants. A user-friendly vaccine does not exist. We selected and tested nine genes of E. ruminantium for protection against challenge in a DBA/2 mouse model, in order to identify candidate genes for incorporation into a recombinant vaccine. Of the nine DNA vaccine constructs tested, four DNA constructs 14HWORF1/VR1012, 14HWORF2/VR1012, 27HWORF1/VR1012, and HSP58/VR1012 were not protective and were excluded from the study. The remaining five DNA constructs-MAP2/ VR1012, 1HWORF3/ VR1012, 4HWORF1/ VR1012, 18HWORF1/ VR1012, and 3GDORF3/ VR1012-offered partial protection against lethal challenge demonstrated by reduced mortalities compared to control groups. Protection was augmented when DNA primed mice were boosted with a respective homologous recombinant protein. Protection in these five groups was associated with the induction of cell-mediated or T helper 1 (Th1) type of immune responses characterized by the production of large amounts of interferon-gamma and interleukin-2 in in vitro proliferation assays using E. ruminantium antigens for stimulation. These responses were enhanced when the DNA-vaccinated DBA/2 mice were boosted with specific homologous recombinant protein vaccination. In a preliminary follow-up study, protection conferred by DNA vaccination with individual gene constructs was not enhanced when the protective constructs were administered in combination (including the map-1 gene of E. ruminantium). Further evaluation of these and other untested DNA constructs is necessary to optimize their expression in vivo in the presence of molecular adjuvants, such as the IFN-gamma gene, GM-CSF gene, IL-12 gene, and CpG motifs to fully evaluate their protective value.

Cloning and expression of the gene encoding the major surface protein 5 (MSP5) of Anaplasma phagocytophilum and potential application for serodiagnosis.

BACKGROUND: Anaplasma phagocytophilum (formerly known as the human granulocytic ehrlichia, Ehrlichia equi and Ehrlichia phagocytophila) is an obligate intracellular organism causing clinical disease in humans and various species of domestic animals. OBJECTIVES: The objectives of this investigation were to sequence and clone the major surface protein 5 (MSP5) of A phagocytophilum and to evaluate the suitability of this antigen in the serologic diagnosis of anaplasmosis in humans and dogs. METHODS: The msp5 gene of A phagocytophilum was sequenced, cloned, and expressed in Escherichia coli. The predicted amino acid sequence homology of the various MSP5/major antigenic protein 2 orthologs was compared among various Anaplasma and Ehrlichia species. Recombinant MSP5 of A phagocytophilum was used in an ELISA to detect antibodies in serum samples from humans and dogs infected with the organism. RESULTS: Serum samples from 104 individuals previously diagnosed with A phagocytophilum infection, as well as samples from clinically healthy humans, were tested. In addition, multiple samples from 4 dogs experimentally infected with 2 different geographic isolates of A phagocytophilum and 5 dogs naturally infected with a Swiss isolate were tested using ELISA. Using this group of immunofluorescent antibody test-positive and immunofluorescent antibody test-negative samples, we found the overall agreement between assays to be >90%. CONCLUSIONS: These results indicate that recombinant MSP5 has potential for use as a diagnostic test antigen to detect infection with A phagocytophilum in both dogs and humans. However, sequence similarities among orthologs of MSP5 in related species of anaplasma and ehrlichia suggest that cross-reactivity among these pathogens is likely if the entire peptide is used as a test antigen.

Persistent Infections and Immunity in Ruminants to Arthropod-Borne Bacteria in the Family Anaplasmataceae.

Tick-transmitted gram-negative bacteria in the family Anaplasmataceae in the order Rickettsiales cause persistent infection and morbidity and mortality in ruminants. Whereas Anaplasma marginale infection is restricted to ruminants, Anaplasma phagocytophilum is promiscuous and, in addition to causing disease in sheep and cattle, notably causes disease in humans, horses, and dogs. Although the two pathogens invade and replicate in distinct blood cells (erythrocytes and neutrophils, respectively), they have evolved similar mechanisms of antigenic variation in immunodominant major surface protein 2 (MSP2) and MSP2(P44) that result in immune evasion and persistent infection. Furthermore, these bacteria have evolved distinct strategies to cause immune dysfunction, characterized as an antigen-specific CD4 T-cell exhaustion for A. marginale and a generalized immune suppression for A. phagocytophilum, that also facilitate persistence. This indicates highly adapted strategies of Anaplasma spp. to both suppress protective immune responses and evade those that do develop. However, conserved subdominant antigens are potential targets for immunization.

Prevalence of Tick-Borne Pathogens in Host-Seeking Amblyomma americanum (Acari: Ixodidae) and Odocoileus virginianus (Artiodactyla: Cervidae) in Florida.

Amblyomma americanum (L.), the lone star tick, is an aggressive tick that is expanding its geographic range within the United States. This tick is the vector for the human and veterinary pathogens Ehrlichia chaffeensis and Ehrlichia ewingii and is associated with other microbes of unspecified pathogenicity including Rickettsia amblyommii, Panola Mountain Ehrlichia, and Borrelia lonestari In Florida, there has been sparse contemporary data on the prevalence of these organisms in host-seeking lone star ticks. To determine the prevalence of this tick and associated microbes in North Central Florida state parks, ∼1,500 lone star tick specimens were collected between 2010 and 2012 analyzed by polymerase chain reaction (PCR) sequencing. Additionally, 393 white-tailed deer, Odocoileus virginianus (Zimmerman), samples were analyzed for pathogen prevalence using molecular methods and serology. In lone star ticks, 14.6, 15.6, and 57.1% were positive for E. chaffeensis, E. ewingii, and Rickettsia spp. DNA, respectively. Panola Mountain Ehrlichia or B. lonestari DNA were each detected in nearly 2% of tick specimens. In white-tailed deer, 7.3% were PCR positive for E. chaffeensis, 6.0% for E. ewingii, and 3.2% for rickettsial species. Approximately 45% of white-tailed deer specimens had antibodies to Ehrlichia spp., and <1% had antibodies to Borrelia burgdorferi In summary, E. chaffeensis, E. ewingii, and spotted fever group rickettsia are highly prevalent in host-seeking lone star ticks and in white-tailed deer in Florida. The molecular and serological evidence of these microbes underscore their zoonotic potential in this region.