The Use of the Antigenically Variable Major Surface Protein 2 in the Establishment of Superinfection during Natural Tick Transmission of Anaplasma marginale in Southern Ghana.

Many vector-borne pathogens, including Anaplasma spp., Borrelia spp., Trypanosoma spp., and Plasmodium spp., establish persistent infection in the mammalian host by using antigenic variation. These pathogens are also able to establish strain superinfection, defined as infection of an infected host with additional strains of the same pathogen despite an adaptive immune response. The ability to establish superinfection results in a population of susceptible hosts even with high pathogen prevalence. It is likely that antigenic variation, responsible for persistent infection, also plays a role in the establishment of superinfection. Anaplasma marginale, an antigenically variable, obligate intracellular, tickborne bacterial pathogen of cattle, is well suited for the study of the role of antigenically variant surface proteins in the establishment of superinfection. Anaplasma marginale establishes persistent infection by variation in major surface protein 2 (msp2), which is encoded by approximately six donor alleles that recombine into a single expression site to produce immune escape variants. Nearly all cattle in regions of high prevalence are superinfected. By tracking the acquisition of strains in calves through time, the complement of donor alleles, and how those donor alleles are expressed, we determined that simple variants derived from a single donor allele, rather than multiple donor alleles, were predominant. Additionally, superinfection is associated with the introduction of new donor alleles, but these new donor alleles are not predominantly used to establish superinfection. These findings highlight the potential for competition among multiple strains of a pathogen for resources within the host and the balance between pathogen fitness and antigenic variation.

Analysis of the Type 4 Effectome across the Genus Rickettsia.

Rickettsia are obligate intracellular bacteria primarily carried by arthropod hosts. The genus Rickettsia contains several vertebrate pathogens vectored by hematophagous arthropods. Despite the potential for disease, our understanding of Rickettsias are limited by the difficulties associated with growing and manipulating obligate intracellular bacteria. To aid with this, our lab conducted an analysis of eight genomes and three plasmids from across the genus Rickettsia. Using OPT4e, a learning algorithm-based program designed to identify effector proteins secreted by the type 4 secretion system, we generated a putative effectome for the genus. We then consolidated effectors into homolog sets to identify effectors unique to Rickettsia with different life strategies or evolutionary histories. We also compared predicted effectors to non-effectors for differences in G+C content and gene splitting. Based on this analysis, we predicted 1571 effectors across the genus, resulting in 604 homolog sets. Each species had unique homolog sets, while 42 were present in all eight species analyzed. Effectors were flagged in association with pathogenic, tick and flea-borne Rickettsia. Predicted effectors also varied in G+C content and frequency of gene splitting as compared to non-effectors. Species effector repertoires show signs of expansion, degradation, and horizontal acquisition associated with lifestyle and lineage.

Iron Reduction in Dermacentor andersoni Tick Cells Inhibits Anaplasma marginale Replication.

Anaplasma spp. are obligate intracellular, tick-borne, bacterial pathogens that cause bovine and human anaplasmosis. We lack tools to prevent these diseases in part due to major knowledge gaps in our fundamental understanding of the tick-pathogen interface, including the requirement for and molecules involved in iron transport during tick colonization. We determine that iron is required for the pathogen Anaplasma marginale, which causes bovine anaplasmosis, to replicate in Dermacentor andersoni tick cells. Using bioinformatics and protein modeling, we identified three orthologs of the Gram-negative siderophore-independent iron uptake system, FbpABC. Am069, the A. marginale ortholog of FbpA, lacks predicted iron-binding residues according to the NCBI conserved domain database. However, according to protein modeling, the best structural orthologs of Am069 are iron transport proteins from Cyanobacteria and Campylobacterjejuni. We then determined that all three A. marginale genes are modestly differentially expressed in response to altered host cell iron levels, despite the lack of a Ferric uptake regulator or operon structure. This work is foundational for building a mechanistic understanding of iron uptake, which could lead to interventions to prevent bovine and human anaplasmosis.

Both Coinfection and Superinfection Drive Complex Anaplasma marginale Strain Structure in a Natural Transmission Setting.

Vector-borne pathogens commonly establish multistrain infections, also called complex infections. How complex infections are established, either before or after the development of an adaptive immune response, termed coinfection or superinfection, respectively, has broad implications for the maintenance of genetic diversity, pathogen phenotype, epidemiology, and disease control strategies. Anaplasma marginale, a genetically diverse, obligate, intracellular, tick-borne bacterial pathogen of cattle, commonly establishes complex infections, particularly in regions with high transmission rates. Both coinfection and superinfection can be established experimentally; however, it is unknown how complex infections develop in a natural transmission setting. To address this question, we introduced naive animals into a herd in southern Ghana with a high infection prevalence and high transmission pressure and tracked the strain acquisition of A. marginale through time using multilocus sequence typing. As expected, the genetic diversity among strains was high, and 97% of animals in the herd harbored multiple strains. All the introduced naive animals became infected, and three to four strains were typically detected in an individual animal prior to seroconversion, while one to two new strains were detected in an individual animal following seroconversion. On average, the number of strains acquired via superinfection was 16% lower than the number acquired via coinfection. Thus, while complex infections develop via both coinfection and superinfection, coinfection predominates in this setting. These findings have broad implications for the development of control strategies in high-transmission settings.

Segmental Variation in a Duplicated msp2 Pseudogene Generates Anaplasma marginale Antigenic Variants.

Anaplasma marginale is a prototypical highly antigenically variant bacterial pathogen dependent on the sequential generation of major surface protein 2 (Msp2) outer membrane variants to establish persistent infection. Msp2 is encoded by a single expression site, and diversity is achieved by gene conversion of chromosomally encoded msp2 pseudogenes. Analysis of the full complement of msp2 pseudogenes in the St. Maries strain revealed identical sequences in different loci. The Florida strain shared the same locus structure, but in the loci where the St. Maries strain had two identical pseudogenes, the Florida strain had one whose sequence was identical to the St. Maries sequences, while the sequence of the second pseudogene differed. Consequently, we hypothesized that the msp2 pseudogene repertoire arose via gene duplication, allowing structural variation to occur in one copy but the utility of the other to be retained. Using comparative genomics, we first established that duplication of msp2 pseudogenes is common among A. marginale strains: all seven examined strains had at least one duplicate pair in which either the genes in the pair were maintained as identical copies or the genes contained segmental changes. We then demonstrated that a minimal segmental change in a duplicated pseudogene locus is sufficient for immune escape from the broad antibody response generated in a natural host, as is a completely divergent pseudogene sequence in an otherwise conserved locus. The results support a model in which a locus first duplicates, resulting in a second identical copy, and then progressively incorporates changes to generate an msp2 repertoire capable of generating sufficient antigenic variants to escape immunity and establish persistent infection.

The Anaplasma ovis genome reveals a high proportion of pseudogenes.

BACKGROUND: The genus Anaplasma is made up of organisms characterized by small genomes that are undergoing reductive evolution. Anaplasma ovis, one of the seven recognized species in this genus, is an understudied pathogen of sheep and other ruminants. This tick-borne agent is thought to induce only mild clinical disease; however, small deficits may add to larger economic impacts due to the wide geographic distribution of this pathogen. RESULTS: In this report we present the first complete genome sequence for A. ovis and compare the genome features with other closely related species. The 1,214,674 bp A. ovis genome encodes 933 protein coding sequences, the split operon arrangement for ribosomal RNA genes, and more pseudogenes than previously recognized for other Anaplasma species. The metabolic potential is similar to other Anaplasma species. Anaplasma ovis has a small repertoire of surface proteins and transporters. Several novel genes are identified. CONCLUSIONS: Analyses of these important features and significant gene families/genes with potential to be vaccine candidates are presented in a comparative context. The availability of this genome will significantly facilitate research for this pathogen.

A nearest-neighbors network model for sequence data reveals new insight into genotype distribution of a pathogen.

BACKGROUND: Sequence similarity networks are useful for classifying and characterizing biologically important proteins. Threshold-based approaches to similarity network construction using exact distance measures are prohibitively slow to compute and rely on the difficult task of selecting an appropriate threshold, while similarity networks based on approximate distance calculations compromise useful structural information. RESULTS: We present an alternative network representation for a set of sequence data that overcomes these drawbacks. In our model, called the Directed Weighted All Nearest Neighbors (DiWANN) network, each sequence is represented by a node and is connected via a directed edge to only the closest sequence, or sequences in the case of ties, in the dataset. Our contributions span several aspects. Specifically, we: (i) Apply an all nearest neighbors network model to protein sequence data from three different applications and examine the structural properties of the networks; (ii) Compare the model against threshold-based networks to validate their semantic equivalence, and demonstrate the relative advantages the model offers; (iii) Demonstrate the model's resilience to missing sequences; and (iv) Develop an efficient algorithm for constructing a DiWANN network from a set of sequences. We find that the DiWANN network representation attains similar semantic properties to threshold-based graphs, while avoiding weaknesses of both high and low threshold graphs. Additionally, we find that approximate distance networks, using BLAST bitscores in place of exact edit distances, can cause significant loss of structural information. We show that the proposed DiWANN network construction algorithm provides a fourfold speedup over a standard threshold based approach to network construction. We also identify a relationship between the centrality of a sequence in a similarity network of an Anaplasma marginale short sequence repeat dataset and how broadly that sequence is dispersed geographically. CONCLUSION: We demonstrate that using approximate distance measures to rapidly construct similarity networks may lead to significant deficiencies in the structure of that network in terms centrality and clustering analyses. We present a new network representation that maintains the structural semantics of threshold-based networks while increasing connectedness, and an algorithm for constructing the network using exact distance measures in a fraction of the time it would take to build a threshold-based equivalent.

Evidence confirming the phylogenetic position of Anaplasma centrale (ex Theiler 1911) Ristic and Kreier 1984.

In 1911, Sir Arnold Theiler isolated and described a parasite that was very similar to Anaplasma marginale but which was more centrally located within the erythrocytes of the host cells, and was much less pathogenic than A. marginale. He named the parasite A. marginale variety centrale. The name Anaplasma centrale, referring to the same organism, was published in Validation List No. 15 in 1984, but the publication was based on an erroneous assumption that Theiler had indicated that it was a separate species. Many authors have subsequently accepted this organism as a separate species, but evidence to indicate that it is a distinct species has never been presented. The near full-length 16S rRNA gene sequence, and the deduced amino acid sequences for groEL and msp4 from several isolates of A. marginale and A. centrale from around South Africa were compared with those of the A. marginale type strain, St Maries, and the A. centrale Israel strain and other reference sequences. Phylogenetic analyses of these sequences demonstrated that A. centrale consistently forms a separate clade from A. marginale, supported by high bootstrap values (≥90 %), revealing that there is divergence between these two organisms. In addition, we discuss distinctive characteristics which have been published recently, such as differences in Msp1a/Msp1aS gene structure, as well as genome architecture that provide further evidence to suggest that A. centrale is, in fact, a separate species. Our results, therefore, provide evidence to support the existing nomenclature, and confirm that A. centrale (ex Theiler 1911) Ristic and Kreier 1984 is, indeed, a distinct species.

Comparative genomics reveals multiple pathways to mutualism for tick-borne pathogens.

BACKGROUND: Multiple important human and livestock pathogens employ ticks as their primary host vectors. It is not currently known whether this means of infecting a host arose once or many times during evolution. RESULTS: In order to address this question, we conducted a comparative genomics analysis on a set of bacterial pathogens from seven genera - Borrelia, Rickettsia, Anaplasma, Ehrlichia, Francisella, Coxiella, and Bartonella, including species from three different host vectors - ticks, lice, and fleas. The final set of 102 genomes used in the study encoded a total of 120,046 protein sequences. We found that no genes or metabolic pathways were present in all tick-borne bacteria. However, we found some genes and pathways were present in subsets of tick-transmitted organisms while absent from bacteria transmitted by lice or fleas. CONCLUSION: Our analysis suggests that the ability of pathogens to be transmitted by ticks arose multiple times over the course of evolution. To our knowledge, this is the most comprehensive study of tick transmissibility to date.

RepeatAnalyzer: a tool for analysing and managing short-sequence repeat data.

BACKGROUND: Short-sequence repeats (SSRs) occur in both prokaryotic and eukaryotic DNA, inter- and intragenically, and may be exact or inexact copies. When heterogeneous SSRs are present in a given locus, we can take advantage of the pattern of different repeats to genotype strains based on the SSRs. Cataloguing and tracking these repeats can be difficult as diverse groups of researchers are involved in the identification of the repeats. Additionally, the task is error-prone when done manually. RESULTS: We developed RepeatAnalyzer, a new software tool capable of tracking, managing, analysing and cataloguing SSRs and genotypes using Anaplasma marginale as a model species. RepeatAnalyzer's analysis capability includes novel metrics for measuring regional genetic diversity (corresponding to variety and regularity of SSR occurrence). As a part of its visualization capabilities, RepeatAnalyzer produces high quality maps of the geographic distribution of genotypes or SSRs over a region of interest. RepeatAnalyzer's repeat identification functionality was validated for all SSRs and genotypes reported in 21 publications, using 380 A. marginale isolates gathered from the five publications within that list that provided access to their isolates. The tool produced accurate genotyping results in every case. In addition, it uncovered a number of errors in the published literature: 11 cases where SSRs were misreported, 5 cases where two different SSRs had been given the same name, and 16 cases where two or more names had been given to a single SSR. The analysis and visualization functionalities of the tool are demonstrated using several examples. CONCLUSIONS: RepeatAnalyzer is a robust software tool that can be used for storing, managing, and analysing short-sequence repeats for the purpose of strain identification. The tool can be used for any set of SSRs regardless of species. When applied to A. marginale, our test case, we show that genotype lengths for a given region follow a normal distribution, while SSR frequencies follow a power-law-like distribution. Further, we find that over 90 % of repeats are 28 to 29 amino acids long, which is in agreement with conventional wisdom. Lastly, our analysis reveals that the most common edit distance is five or six, which is counter-intuitive since we expected that result to be closer to one, resulting from the simplest change from one repeat to another.

Characterization of Anaplasma marginale subsp. centrale Strains by Use of msp1aS Genotyping Reveals a Wildlife Reservoir.

Bovine anaplasmosis caused by the intraerythrocytic rickettsial pathogen Anaplasma marginale is endemic in South Africa. Anaplasma marginale subspecies centrale also infects cattle; however, it causes a milder form of anaplasmosis and is used as a live vaccine against A. marginale There has been less interest in the epidemiology of A. marginale subsp. centrale, and, as a result, there are few reports detecting natural infections of this organism. When detected in cattle, it is often assumed that it is due to vaccination, and in most cases, it is reported as coinfection with A. marginale without characterization of the strain. A total of 380 blood samples from wild ruminant species and cattle collected from biobanks, national parks, and other regions of South Africa were used in duplex real-time PCR assays to simultaneously detect A. marginale and A. marginale subsp. centrale. PCR results indicated high occurrence of A. marginale subsp. centrale infections, ranging from 25 to 100% in national parks. Samples positive for A. marginale subsp. centrale were further characterized using the msp1aS gene, a homolog of msp1α of A. marginale, which contains repeats at the 5' ends that are useful for genotyping strains. A total of 47 Msp1aS repeats were identified, which corresponded to 32 A. marginale subsp. centrale genotypes detected in cattle, buffalo, and wildebeest. RepeatAnalyzer was used to examine strain diversity. Our results demonstrate a diversity of A. marginale subsp. centrale strains from cattle and wildlife hosts from South Africa and indicate the utility of msp1aS as a genotypic marker for A. marginale subsp. centrale strain diversity.

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.

Novel Waddlia Intracellular Bacterium in Artibeus intermedius Fruit Bats, Mexico.

An intracellular bacterium was isolated from fruit bats (Artibeus intermedius) in Cocoyoc, Mexico. The bacterium caused severe lesions in the lungs and spleens of bats and intracytoplasmic vacuoles in cell cultures. Sequence analyses showed it is related to Waddlia spp. (order Chlamydiales). We propose to call this bacterium Waddlia cocoyoc.

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.

Antigenic variation and transmission fitness as drivers of bacterial strain structure.

Shifts in microbial strain structure underlie both emergence of new pathogens and shifts in patterns of infection and disease of known agents. Understanding the selective pressures at a population level as well as the mechanisms at the molecular level represent significant gaps in our knowledge regarding microbial epidemiology. Highly antigenically variant pathogens, which are broadly represented among microbial taxa, are most commonly viewed through the mechanistic lens of how they evade immune clearance within the host. However, equally important are mechanisms that allow pathogens to evade immunity at the population level. The selective pressure of immunity at both the level of the individual host and the population is a driver of diversification within a pathogen strain. Using Anaplasma marginale as a model highly antigenically variable bacterial pathogen, we review how immunity selects for genetic diversification in alleles encoding outer membrane proteins both within and among strains. Importantly, genomic comparisons among strains isolated from diverse epidemiological settings elucidates the counterbalancing pressures for diversification and conservation, driven by immune escape and transmission fitness, respectively, and how these shape pathogen strain structure.

Identification of multilocus genetic heterogeneity in Anaplasma marginale subsp. centrale and its restriction following tick-borne transmission.

Anaplasma marginale subsp. centrale was the first vaccine used to protect against a rickettsial disease and is still in widespread use a century later. As its use preceded development of either cryopreservation or cell culture, the vaccine strain was maintained for decades by sequential passage among donor animals, excluding the natural tick-borne transmission cycle that provides a selective pressure or population "bottleneck." We demonstrated that the vaccine strain is genetically heterogeneous at 46 chromosomal loci and that heterogeneity was maintained upon inoculation into recipient animals. The number of variants per site ranged from 2 to 11 with a mean of 2.8/locus and a mode and median of 2/locus; variants included single-nucleotide polymorphisms, insertions/deletions, polynucleotide tracts, and different numbers of perfect repeats. The genetic heterogeneity is highly unlikely to be a result of strain contamination based on analysis using a panel of eight gene markers with a high power for strain discrimination. In contrast, heterogeneity appears to be a result of genetic drift in the absence of the restriction of tick passage. Heterogeneity could be reduced following tick passage, and the reduced heterogeneity could be maintained in sequential intravenous and tick-borne passages. The reduction in vaccine strain heterogeneity following tick passage did not confer an enhanced transmission phenotype, indicating that a stochastically determined population bottleneck was likely responsible as opposed to a positive selective pressure. These findings demonstrate the plasticity of an otherwise highly constrained genome and highlight the role of natural transmission cycles in shaping and maintaining the bacterial genome.

Transcriptional pathways associated with the slow growth phenotype of transformed Anaplasma marginale.

BACKGROUND: The ability to genetically manipulate bacteria has been fundamentally important for both basic biological discovery and translational research to develop new vaccines and antibiotics. Experimental alteration of the genetic content of prokaryotic pathogens has revealed both expected functional relationships and unexpected phenotypic consequences. Slow growth phenotypes have been reported for multiple transformed bacterial species, including extracellular and intracellular pathogens. Understanding the genes and pathways responsible for the slow growth phenotype provides the opportunity to develop attenuated vaccines as well as bacteriostatic antibiotics. Transformed Anaplasma marginale, a rickettsial pathogen, exhibits slow growth in vitro and in vivo as compared to the parent wild type strain, providing the opportunity to identify the underlying genes and pathways associated with this phenotype. RESULTS: Whole genome transcriptional profiling allowed for identification of specific genes and pathways altered in transformed A. marginale. Genes found immediately upstream and downstream of the insertion site, including a four gene operon encoding key outer membrane proteins, were not differentially transcribed between wild type and transformed A. marginale. This lack of significant difference in transcription of flanking genes and the large size of the insert relative to the genome were consistent with a trans rather than a cis effect. Transcriptional profiling across the complete genome identified the most differentially transcribed genes, including an iron transporter, an RNA cleaving enzyme and several genes involved in translation. In order to confirm the trend seen in translation-related genes, K-means clustering and Gene Set Enrichment Analysis (GSEA) were applied. These algorithms allowed evaluation of the behavior of genes as groups that share transcriptional status or biological function. Clustering and GSEA confirmed the initial observations and found additional pathways altered in transformed A. marginale. Three pathways were significantly altered as compared to the wild type: translation, translation elongation, and purine biosynthesis. CONCLUSIONS: Identification of perturbed genes and networks through genome wide transcriptional profiling highlights the relevance of pathways such as nucleotide biosynthesis, translation, and translation elongation in the growth phenotype of obligate intracellular bacteria. These genes and pathways provide specific targets for development of slow growing attenuated vaccines and for bacteriostatic antibiotics.

Gut transcriptome of replete adult female cattle ticks, Rhipicephalus (Boophilus) microplus, feeding upon a Babesia bovis-infected bovine host.

As it feeds upon cattle, Rhipicephalus (Boophilus) microplus is capable of transmitting a number of pathogenic organisms, including the apicomplexan hemoparasite Babesia bovis, a causative agent of bovine babesiosis. The R. microplus female gut transcriptome was studied for two cohorts: adult females feeding on a bovine host infected with B. bovis and adult females feeding on an uninfected bovine. RNA was purified and used to generate a subtracted cDNA library from B. bovis-infected female gut, and 4,077 expressed sequence tags (ESTs) were sequenced. Gene expression was also measured by a microarray designed from the publicly available R. microplus gene index: BmiGI Version 2. We compared gene expression in the tick gut from females feeding upon an uninfected bovine to gene expression in tick gut from females feeding upon a splenectomized bovine infected with B. bovis. Thirty-three ESTs represented on the microarray were expressed at a higher level in female gut samples from the ticks feeding upon a B. bovis-infected calf compared to expression levels in female gut samples from ticks feeding on an uninfected calf. Forty-three transcripts were expressed at a lower level in the ticks feeding upon B. bovis-infected female guts compared with expression in female gut samples from ticks feeding on the uninfected calf. These array data were used as initial characterization of gene expression associated with the infection of R. microplus by B. bovis.

Immunization with Anaplasma centrale Msp2 HVRs Is Less Effective than the Live A. centrale Vaccine against Anaplasmosis.

Bovine anaplasmosis, caused by Anaplasma marginale, is the most prevalent tick-transmitted pathogen of livestock globally. In many parts of the world, Anaplasma centrale, a related organism, is used as a live blood-borne vaccine as it causes either no or only a mild clinical disease. Anaplasma centrale does not prevent infection with A. marginale but does prevent acute disease. Anaplasma centrale is prohibited from being used in the U.S. due to the risk of transmitting emerging pathogens. Both of these organisms encode proteins known as major surface protein 2 (Msp2), which is the most immunodominant protein for the organism. Both organisms persist in their host by evading clearance, i.e., the adaptive immune response, by recombining the hypervariable region (HVR) of msp2 with pseudogene alleles. The study goal was to test whether the Msp2 HVRs encoded by A. centrale are a sufficient source of immune stimulation to provide the clinical protection exhibited by the blood-borne vaccine. Calves were inoculated with recombinantly expressed A. centrale HVRs. Control groups were inoculated with saponin or infected with the A. centrale live vaccine and compared with the test group. A Western blot analysis demonstrated that the HVR immunizations and A. centrale live vaccine stimulated an immune response. All animals in the study became infected upon challenge with A. marginale-infected ticks. The saponin-immunized control group had a high PPE (5.4%) and larger drops in PCVs (14.6%). As expected, the A. centrale-immunized animals were protected from acute disease with lower (0.6%) parasitemia and lower drops in PCV (8.6%). The HVR-immunized group had intermediate results that were not statistically significantly different from either the negative or positive controls. This suggests that the HVR immunogen does not fully recapitulate the protective capacity of the live vaccine.

Unravelling the diversity of Anaplasma species circulating in selected African wildlife hosts by targeted 16S microbiome analysis.

Organisms in the genus Anaplasma are obligate intracellular alphaproteobacteria. Bovine anaplasmosis, predominantly caused by Anaplasma marginale, is the most prevalent tick-borne disease (TBD) of cattle worldwide. Other Anaplasma species are known to cause disease; these include A. ovis, A. platys in dogs, A. capra in goats and humans, and A. phagocytophilum in humans. The rapid advancement of next-generation sequencing technologies has led to the discovery of many novel sequences ascribed to the genus Anaplasma, with over 20 putative new species being proposed since the last formal organization of the genus. Most 16S rRNA gene surveys for Anaplasma were conducted on cattle and to a lesser extent on rodents, dogs, and ticks. Little is known about the occurrence, diversity, or impact of Anaplasma species circulating in wildlife species. Therefore, we conducted a 16S rRNA gene survey with the goal of identifying Anaplasma species in a variety of wildlife species in the Kruger National Park and neighbouring game reserves, using an unbiased 16S rRNA gene microbiome approach. An Anaplasma/Ehrlichia-group specific quantitative real-time PCR (qPCR) assay revealed the presence of Anaplasma and/or Ehrlichia species in 70.0% (21/30) of African buffalo, 86.7% (26/30) of impala, 36.7% (11/30) of greater kudu, 3.2% (1/31) of African wild dog, 40.6% (13/32) of Burchell's zebra, 43.3% (13/30) of warthog, 22.6% (7/31) of spotted hyena, 40.0% (12/30) of leopard, 17.6% (6/34) of lion, 16.7% (5/30) of African elephant and 8.6% (3/35) of white rhinoceros samples. Microbiome sequencing data from the qPCR positive samples revealed four 16S rRNA sequences identical to previously published Anaplasma sequences, as well as nine novel Anaplasma 16S genotypes. Our results reveal a greater diversity of putative Anaplasma species circulating in wildlife than currently classified within the genus. Our findings highlight a potential expansion of the Anaplasma host range and the need for more genetic information from other important genes or genome sequencing of putative novel species for correct classification and further assessment of their occurrence in wildlife, livestock and companion animals.