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ABSTRACT Pathogens must adapt to disparate environments in permissive host species, a feat that is especially pronounced for vector-borne microbes, which transition between vertebrate hosts and arthropod vectors to complete their lifecycles. Most knowledge about arthropod-vectored bacterial pathogens centers on their life in the mammalian host, where disease occurs. However, disease outbreaks are driven by the arthropod vectors. Adapting to the arthropod is critical for obligate intracellular rickettsial pathogens, as they depend on eukaryotic cells for survival. To manipulate the intracellular environment, these bacteria use type IV secretion systems (T4SS) to deliver effectors into the host cell. To date, few rickettsial T4SS translocated effectors have been identified and have only been examined in the context of mammalian infection. We identified an effector from the tick-borne rickettsial pathogen Anaplasma phagocytophilum, HGE1_02492, as critical for survival in tick cells and acquisition by ticks in vivo. Conversely, HGE1_02492 was dispensable during mammalian cell culture and murine infection. We show that HGE1_02492 is translocatable in a T4SS-dependent manner to the host cell cytosol. In eukaryotic cells, the HGE1_02492 localized with cortical actin filaments, which is dependent on multiple sub-domains of the protein. HGE1_02492 is the first arthropod-vector specific T4SS translocated effector identified from a rickettsial pathogen. Moreover, the subcellular target of HGE1_02492 suggests that A. phagocytophilum is manipulating actin to enable arthropod colonization. Based on these findings, we propose the name AteA for Anaplasma (phagocytophilum) tick effector A. Altogether, we show that A. phagocytophilum uses distinct strategies to cycle between mammals and arthropods. IMPORTANCE Ticks are the number one vector of pathogens for livestock worldwide and for humans in the United States. The biology of tick transmission is an understudied area. Understanding this critical interaction could provide opportunities to affect the course of disease spread. In this study, we examined the zoonotic tick-borne agent Anaplasma phagocytophilum and identified a secreted protein, AteA, which is expressed in a tick-specific manner. These secreted proteins, termed effectors, are the first proteins to interact with the host environment. AteA is essential for survival in ticks and appears to interact with cortical actin. Most effector proteins are studied in the context of the mammalian host; however, understanding how this unique set of proteins affects tick transmission is critical to developing interventions.

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.

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.

Bovine babesiosis caused by Babesia bigemina and Babesia bovis is an economically important disease that affects cattle worldwide. Both B. bigemina and B. bovis are transovarially transmitted by Rhipicephalus ticks. However, little is known regarding parasite gene expression during infection of the tick vector or mammalian host, which has limited the development of effective control strategies to alleviate the losses to the cattle industry. To understand Babesia gene regulation during tick and mammalian host infection, we performed high throughput RNA-sequencing using samples collected from calves and Rhipicephalus microplus ticks infected with B. bigemina. We evaluated gene expression between B. bigemina blood-stages and kinetes and compared them with previous B. bovis RNA-seq data. The results revealed similar patterns of gene regulation between these two tick-borne transovarially transmitted Babesia parasites. Like B. bovis, the transcription of several B. bigemina genes in kinetes exceeded a 1,000-fold change while a few of these genes had a >20,000-fold increase. To identify genes that may have important roles in B. bigemina and B. bovis transovarial transmission, we searched for genes upregulated in B. bigemina kinetes in the genomic datasets of B. bovis and non-transovarially transmitted parasites, Theileria spp. and Babesia microti. Using this approach, we identify genes that may be potential markers for transovarial transmission by B. bigemina and B. bovis. The findings presented herein demonstrate common Babesia genes linked to infection of the vector or mammalian host and may contribute to elucidating strategies used by the parasite to complete their life cycle.

Anaplasma marginale is a tick-borne pathogen that causes bovine anaplasmosis, which affects cattle around the world. Despite its broad prevalence and severe economic impacts, limited treatments exist for this disease. Our lab previously reported that a high proportion of Rickettsia bellii, a tick endosymbiont, in the microbiome of a population of Dermacentor andersoni ticks negatively impacts the ticks’ ability to acquire A. marginale. To better understand this correlation, we used mixed infection of A. marginale and R. bellii in D. andersoni cell culture. We assessed the impacts of different amounts of R. bellii in coinfections, as well as established R. bellii infection, on the ability of A. marginale to establish an infection and grow in D. andersoni cells. From these experiments, we conclude that A. marginale is less able to establish an infection in the presence of R. bellii and that an established R. bellii infection inhibits A. marginale replication. This interaction highlights the importance of the microbiome in preventing tick vector competence and may lead to the development of a biological or mechanistic control for A. marginale transmission by the tick.

New treatments for the diseases caused by apicomplexans are needed. Recently, we determined that tartrolon E (trtE), a secondary metabolite derived from a shipworm symbiotic bacterium, has broad-spectrum anti-apicomplexan parasite activity. TrtE inhibits apicomplexans at nM concentrations in vitro, including Cryptosporidium parvum, Toxoplasma gondii, Sarcocystis neurona, Plasmodium falciparum, Babesia spp. and Theileria equi. To investigate the mechanism of action of trtE against apicomplexan parasites, we examined changes in the transcriptome of trtE-treated T. gondii parasites. RNA-Seq data revealed that the gene, TGGT1_272370, which is broadly conserved in the coccidia, is significantly upregulated within 4 h of treatment. Using bioinformatics and proteome data available on ToxoDB, we determined that the protein product of this tartrolon E responsive gene (trg) has multiple transmembrane domains, a phosphorylation site, and localizes to the plasma membrane. Deletion of trg in a luciferase-expressing T. gondii strain by CRISPR/Cas9 resulted in a 68% increase in parasite resistance to trtE treatment, supporting a role for the trg protein product in the response of T. gondii to trtE treatment. Trg is conserved in the coccidia, but not in more distantly related apicomplexans, indicating that this response to trtE may be unique to the coccidians, and other mechanisms may be operating in other trtE-sensitive apicomplexans. Uncovering the mechanisms by which trtE inhibits apicomplexans may identify shared pathways critical to apicomplexan parasite survival and advance the search for new treatments.

Tick midgut is the primary infection site required by tick-borne pathogens to initiate their development for transmission. Despite the biological significance of this organ, cell cultures derived exclusively from tick midgut tissues are unavailable and protocols for generating primary midgut cell cultures have not been described. To study the mechanism of Anaplasma marginale-tick cell interactions, we successfully developed an in vitro Dermacentor andersoni primary midgut cell culture system. Midgut cells were maintained for up to 120 days. We demonstrated the infection of in vitro midgut cells by using an A. marginale omp10::himar1 mutant with continued replication for up to 10 days post-infection. Anaplasma marginale infection of midgut cells regulated the differential expression of tick α-(1,3)-fucosyltransferases A1 and A2. Silencing of α-(1,3)-fucosyltransferase A2 in uninfected midgut cells reduced the display of fucosylated glycans and significantly lowered the susceptibility of midgut cells to A. marginale infection, suggesting that the pathogen utilized core α-(1,3)-fucose of N-glycans to infect tick midgut cells. This is the first report using in vitro primary D. andersoni midgut cells to study A. marginale-tick cell interactions at the molecular level. The primary midgut cell culture system will further facilitate the investigation of tick-pathogen interactions, leading to the development of novel intervention strategies for tick-borne diseases.

Bovine anaplasmosis, caused by Anaplasma marginale, is one of the most important tick-borne diseases of cattle. Anaplasma marginale is known to be present in the Mnisi community, Mpumalanga Province, with frequent cases of anaplasmosis reported. This study investigated the infection dynamics in calves (n = 10) in two habitats in the study area over 12 months. A duplex real-time PCR assay targeting the msp1β gene of A. marginale and the groEL gene of A. centrale confirmed the presence of A. marginale in five calves in a peri-urban area from the first month, but in only two calves at the wildlife–livestock interface and only after six months. These results were confirmed by 16S rRNA microbiome analysis. Over 50 A. marginale msp1α genotypes were detected in the calves along with five novel Msp1a repeats. Calves in the peri-urban area were more likely to be infected with A. marginale than calves in the wildlife–livestock interface. Cattle management, acaricide treatment, and cattle density could explain differences in infection prevalence in the two areas. Our results revealed that most calves were superinfected by distinct A. marginale strains within the study period, indicating continuous challenge with multiple strains that should lead to robust immunity in the calves and endemic stability in the area.

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.

Advances in genome sequencing have accelerated the growth of sequenced genomes but at a cost in the quality of genome annotation. At the same time, computational analysis is widely used for protein annotation, but a dearth of experimental verification has contributed to inaccurate annotation as well as to annotation error propagation. Thus, a tool to help life scientists with accurate protein annotation would be useful. In this work we describe a website we have developed, the Protein Annotation Surveillance Site (PASS), which provides such a tool. This website consists of three major components: a database of homologous clusters of more than eight million protein sequences deduced from the representative genomes of bacteria, archaea, eukarya, and viruses, together with sequence information; a machine-learning software tool which periodically queries the UniprotKB database to determine whether protein function has been experimentally verified; and a query-able webpage where the FASTA headers of sequences from the cluster best matching an input sequence are returned. The user can choose from these sequences to create a sequence similarity network to assist in annotation or else use their expert knowledge to choose an annotation from the cluster sequences. Illustrations demonstrating use of this website are presented.

Abstract Background Feline babesiosis, sporadically reported from various countries, is of major clinical significance in South Africa, particularly in certain coastal areas. Babesia felis, B. leo, B. lengau and B. microti have been reported from domestic cats in South Africa. Blood specimens from domestic cats (n = 18) showing clinical signs consistent with feline babesiosis and confirmed to harbour Babesia spp. piroplasms by microscopy of blood smears and/or reverse line blot (RLB) hybridization were further investigated. Twelve of the RLB-positive specimens had reacted with the Babesia genus-specific probe only, which would suggest the presence of a novel or previously undescribed Babesia species. The aim of this study was to characterise these organisms using 18S rRNA gene sequence analysis. Results The parasite 18S rRNA gene was cloned and sequenced from genomic DNA from blood samples. Assembled sequences were used to construct similarity matrices and phylogenetic relationships with known Babesia spp. Fifty-five 18S rRNA gene sequences were obtained. Sequences from 6 cats were most closely related to published B. felis sequences (99–100% sequence identity), while sequences from 5 cats were most closely related to B. leo sequences (99–100% sequence identity). One of these was the first record of B. leo in Mozambique. One sequence had 100% sequence identity with the published B. microti Otsu strain. The most significant finding was that sequences from 7 cats constituted a novel Babesia group with 96% identity to Babesia spp. previously recorded from a maned wolf (Chrysocyon brachyurus), a raccoon (Procyon lotor) from the USA and feral raccoons from Japan, as well as from ticks collected from dogs in Japan. Conclusions Babesia leo was unambiguously linked to babesiosis in cats. Our results indicate the presence of a novel potentially pathogenic Babesia sp. in felids in South Africa, which is not closely related to B. felis, B. lengau and B. leo, the species known to be pathogenic to cats in South Africa. Due to the lack of an appropriate type-specimen, we refrain from describing a new species but refer to the novel organism as Babesia sp. cat Western Cape.

Abstract 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.

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2), which still infects hundreds of thousands of people globally each day despite various countermeasures, has been mutating rapidly. Mutations in the spike (S) protein seem to play a vital role in viral stability, transmission, and adaptability. Therefore, to control the spread of the virus, it is important to gain insight into the evolution and transmission of the S protein. This study deals with the temporal and geographical distribution of mutant S proteins from sequences gathered across the US over a period of 19 months in 2020 and 2021. The S protein sequences are studied using two approaches: (i) multiple sequence alignment is used to identify prominent mutations and highly mutable regions and (ii) sequence similarity networks are subsequently employed to gain further insight and study mutation profiles of concerning variants across the defined time periods and states. Additionally, we tracked the variants using visualizations on geographical maps. The visualizations produced using the Directed Weighted All Nearest Neighbors (DiWANN) networks and maps provided insights into the transmission of the virus that reflect well the statistics reported for the time periods studied. We found that the networks created using DiWANN are superior to commonly used approximate distance networks created using BLAST bitscores. The study offers a richer computational approach to analyze the transmission profile of the prominent S protein mutations in SARS-CoV-2 and can be extended to other proteins and viruses.

Abstract 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.

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.

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.

With the advent of cheaper, high-throughput sequencing technologies, the ability to survey biodiversity in previously unexplored niches and geographies has expanded massively. Within Anaplasma, a genus containing several intra-hematopoietic pathogens of medical and economic importance, at least 25 new species have been proposed since the last formal taxonomic organization. Given the obligate intracellular nature of these bacteria, none of these proposed species have been able to attain formal standing in the nomenclature per the International Code of Nomenclature of Prokaryotes rules. Many novel species’ proposals use sequence data obtained from targeted or metagenomic PCR studies of only a few genes, most commonly the 16S rRNA gene. We examined the utility of the 16S rRNA gene sequence for discriminating Anaplasma samples to the species level. We find that while the genetic diversity of the genus Anaplasma appears greater than appreciated in the last organization of the genus, caution must be used when attempting to resolve to a species descriptor from the 16S rRNA gene alone. Specifically, genomically distinct species have similar 16S rRNA gene sequences, especially when only partial amplicons of the 16S rRNA are used. Furthermore, we provide key bases that allow classification of the formally named species of Anaplasma.

Abstract Background Only a few studies have examined the presence of Anaplasma marginale and Anaplasma centrale in South Africa, and no studies have comprehensively examined these species across the whole country. To undertake this country-wide study we adapted a duplex quantitative real-time PCR (qPCR) assay for use in South Africa but found that one of the genes on which the assay was based was variable. Therefore, we sequenced a variety of field samples and tested the assay on the variants detected. We used the assay to screen 517 cattle samples sourced from all nine provinces of South Africa, and subsequently examined A. marginale positive samples for msp1α genotype to gauge strain diversity. Results Although the A. marginale msp1β gene is variable, the qPCR functions at an acceptable efficiency. The A. centrale groEL gene was not variable within the qPCR assay region. Of the cattle samples screened using the assay, 57% and 17% were found to be positive for A. marginale and A. centrale, respectively. Approximately 15% of the cattle were co-infected. Msp1α genotyping revealed 36 novel repeat sequences. Together with data from previous studies, we analysed the Msp1a repeats from South Africa where a total of 99 repeats have been described that can be attributed to 190 msp1α genotypes. While 22% of these repeats are also found in other countries, only two South African genotypes are also found in other countries; otherwise, the genotypes are unique to South Africa. Conclusions Anaplasma marginale was prevalent in the Western Cape, KwaZulu-Natal and Mpumalanga and absent in the Northern Cape. Anaplasma centrale was prevalent in the Western Cape and KwaZulu-Natal and absent in the Northern Cape and Eastern Cape. None of the cattle in the study were known to be vaccinated with A. centrale, so finding positive cattle indicates that this organism appears to be naturally circulating in cattle. A diverse population of A. marginale strains are found in South Africa, with some msp1α genotypes widely distributed across the country, and others appearing only once in one province. This diversity should be taken into account in future vaccine development studies.

Abstract Background Nearly a quarter of emerging infectious diseases identified in the last century are arthropod-borne. Although ticks and insects can carry pathogenic microorganisms, non-pathogenic microbes make up the majority of their microbial communities. The majority of tick microbiome research has had a focus on discovery and description; very few studies have analyzed the ecological context and functional responses of the bacterial microbiome of ticks. The goal of this analysis was to characterize the stability of the bacterial microbiome of Dermacentor andersoni ticks between generations and two populations within a species. Methods The bacterial microbiome of D. andersoni midguts and salivary glands was analyzed from populations collected at two different ecologically distinct sites by comparing field (F1) and lab-reared populations (F1-F3) over three generations. The microbiome composition of pooled and individual samples was analyzed by sequencing nearly full-length 16S rRNA gene amplicons using a Pacific Biosciences CCS platform that allows identification of bacteria to the species level. Findings In this study, we found that the D. andersoni microbiome was distinct in different geographic populations and was tissue specific, differing between the midgut and the salivary gland, over multiple generations. Additionally, our study showed that the microbiomes of laboratory-reared populations were not necessarily representative of their respective field populations. Furthermore, we demonstrated that the microbiome of a few individual ticks does not represent the microbiome composition at the population level. Conclusions We demonstrated that the bacterial microbiome of D. andersoni was complex over three generations and specific to tick tissue (midgut vs. salivary glands) as well as geographic location (Burns, Oregon vs. Lake Como, Montana vs. laboratory setting). These results provide evidence that habitat of the tick population is a vital component of the complexity of the bacterial microbiome of ticks, and that the microbiome of lab colonies may not allow for comparative analyses with field populations. A broader understanding of microbiome variation will be required if we are to employ manipulation of the microbiome as a method for interfering with acquisition and transmission of tick-borne pathogens.

Abstract Background Ovine anaplasmosis is a tick-borne disease that is caused by Anaplasma ovis in sheep and goats. The pathogen is widely distributed in tropical and subtropical regions of the world. At present, diagnosis of the disease mainly depends on microscopy or nucleic acid based molecular tests, although a few serological tests have been applied for the detection of A. ovis infection. Results Here we describe the identification of an A. ovis protein that is homologous to the A. marginale appendage-associated protein (AAAP). We expressed a recombinant fragment of this protein for the development of an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of A. ovis. Anaplasma ovis-positive serum showed specific reactivity to recombinantly expressed AAAP (rAAAP), which was further confirmed by the rAAAP ELISA, which also demonstrated no cross-reactivity with sera from animals infected with A. bovis or other related pathogens in sheep and goats. Testing antibody kinetics of five experimentally infected sheep for 1 year demonstrated that the rAAAP ELISA is suitable for the detection of early and persistent infection of A. ovis infections. Investigation of 3138 field-collected serum samples from 54 regions in 23 provinces in China demonstrated that the seroprevalence varied from 9.4% to 65.3%, which is in agreement with previous reports of A. ovis infection. Conclusions An A. ovis derived antigenic protein, AAAP, was identified and the antigenicity of the recombinant AAAP was confirmed. Using rAAAP an indirect ELISA assay was established, and the assay has been proven to be an alternative serological diagnostic tool for investigating the prevalence of ovine anaplasmosis of sheep and goats.

Type IV secretion systems (T4SS) are used by a number of bacterial pathogens to attack the host cell. The complex protein structure of the T4SS is used to directly translocate effector proteins into host cells, often causing fatal diseases in humans and animals. Identification of effector proteins is the first step in understanding how they function to cause virulence and pathogenicity. Accurate prediction of effector proteins via a machine learning approach can assist in the process of their identification. The main goal of this study is to predict a set of candidate effectors for the tick-borne pathogen Anaplasma phagocytophilum, the causative agent of anaplasmosis in humans. To our knowledge, we present the first computational study for effector prediction with a focus on A. phagocytophilum. In a previous study, we systematically selected a set of optimal features from more than 1,000 possible protein characteristics for predicting T4SS effector candidates. This was followed by a study of the features using the proteome of Legionella pneumophila strain Philadelphia deduced from its complete genome. In this manuscript we introduce the OPT4e software package for Optimal-features Predictor for T4SS Effector proteins. An earlier version of OPT4e was verified using cross-validation tests, accuracy tests, and comparison with previous results for L. pneumophila. We use OPT4e to predict candidate effectors from the proteomes of A. phagocytophilum strains HZ and HGE-1 and predict 48 and 46 candidates, respectively, with 16 and 18 deemed most probable as effectors. These latter include the three known validated effectors for A. phagocytophilum.

We clustered 8.76 M protein sequences deduced from 2,307 completely sequenced Proteobacterial genomes resulting in 707,311 clusters of one or more sequences of which 224,442 ranged in size from 2 to 2,894 sequences. To our knowledge this is the first study of this scale. We were surprised to find that no single cluster contained a representative sequence from all the organisms in the study. Given the minimal genome concept, we expected to find a shared set of proteins. To determine why the clusters did not have universal representation we chose four essential proteins, the chaperonin GroEL, DNA dependent RNA polymerase subunits beta and beta′ (RpoB/RpoB′), and DNA polymerase I (PolA), representing fundamental cellular functions, and examined their cluster distribution. We found these proteins to be remarkably conserved with certain caveats. Although the groEL gene was universally conserved in all the organisms in the study, the protein was not represented in all the deduced proteomes. The genes for RpoB and RpoB′ were missing from two genomes and merged in 88, and the sequences were sufficiently divergent that they formed separate clusters for 18 RpoB proteins (seven clusters) and 14 RpoB′ proteins (three clusters). For PolA, 52 organisms lacked an identifiable sequence, and seven sequences were sufficiently divergent that they formed five separate clusters. Interestingly, organisms lacking an identifiable PolA and those with divergent RpoB/RpoB′ were predominantly endosymbionts. Furthermore, we present a range of examples of annotation issues that caused the deduced proteins to be incorrectly represented in the proteome. These annotation issues made our task of determining protein conservation more difficult than expected and also represent a significant obstacle for high-throughput analyses.

Advances in genome sequencing technology and computing power have brought about the explosive growth of sequenced genomes in public repositories with a concomitant increase in annotation errors. Many protein sequences are annotated using computational analysis rather than experimental verification, leading to inaccuracies in annotation. Confirmation of existing protein annotations is urgently needed before misannotation becomes even more prevalent due to error propagation. In this work we present a novel approach for automatically confirming the existence of manually curated information with experimental evidence of protein annotation. Our ensemble learning method uses a combination of recurrent convolutional neural network, logistic regression, and support vector machine models. Natural language processing in the form of word embeddings is used with journal publication titles retrieved from the UniProtKB database. Importantly, we use recall as our most significant metric to ensure the maximum number of verifications possible; results are reported to a human curator for confirmation. Our ensemble model achieves 91.25% recall, 71.26% accuracy, 65.19% precision, and an F1 score of 76.05% and outperforms the Bidirectional Encoder Representations from Transformers for Biomedical Text Mining (BioBERT) model with fine-tuning using the same data.

Abstract Background In North America, ticks are the most economically impactful vectors of human and animal pathogens. The Rocky Mountain wood tick, Dermacentor andersoni (Acari: Ixodidae), transmits Rickettsia rickettsii and Anaplasma marginale to humans and cattle, respectively. In recent years, studies have shown that symbiotic organisms are involved in a number of biochemical and physiological functions. Characterizing the bacterial microbiome of D. andersoni is a pivotal step towards understanding symbiont-host interactions. Findings In this study, we have shown by high-throughput sequence analysis that the composition of endosymbionts in the midgut and salivary glands in adult ticks is dynamic over three generations. Four Proteobacteria genera, Rickettsia, Francisella, Arsenophonus, and Acinetobacter, were identified as predominant symbionts in these two tissues. Exposure to therapeutic doses of the broad-spectrum antibiotic, oxytetracycline, affected both proportions of predominant genera and significantly reduced reproductive fitness. Additionally, Acinetobacter, a free-living ubiquitous microbe, invaded the bacterial microbiome at different proportions based on antibiotic treatment status suggesting that microbiome composition may have a role in susceptibility to environmental contaminants. Conclusions This study characterized the bacterial microbiome in D. andersoni and determined the generational variability within this tick. Furthermore, this study confirmed that microbiome manipulation is associated with tick fitness and may be a potential method for biocontrol.

DNA samples from 74 patients with non-malarial acute febrile illness (AFI), 282 rodents, 100 cattle, 56 dogs and 160 Rhipicephalus sanguineus ticks were screened for the presence of Anaplasma phagocytophilum DNA using a quantitative PCR (qPCR) assay targeting the msp2 gene. The test detected both A. phagocytophilum and Anaplasma sp. SA/ZAM dog DNA. Microbiome sequencing confirmed the presence of low levels of A. phagocytophilum DNA in the blood of rodents, dogs and cattle, while high levels of A. platys and Anaplasma sp. SA/ZAM dog were detected in dogs. Directed sequencing of the 16S rRNA and gltA genes in selected samples revealed the presence of A. phagocytophilum DNA in humans, dogs and rodents and highlighted its importance as a possible contributing cause of AFI in South Africa. A number of recently described Anaplasma species and A. platys were also detected in the study. Phylogenetic analyses grouped Anaplasma sp. SA/ZAM dog into a distinct clade, with sufficient divergence from other Anaplasma species to warrant classification as a separate species. Until appropriate type-material can be deposited and the species is formally described, we will refer to this novel organism as Anaplasma sp. SA dog.

Reconstructing and visualizing phylogenetic relationships among living organisms is a fundamental challenge because not all organisms share the same genes. As a result, the first phylogenetic visualizations employed a single gene, e.g., rRNA genes, sufficiently conserved to be present in all organisms but divergent enough to provide discrimination between groups. As more genome data became available, researchers began concatenating different combinations of genes or proteins to construct phylogenetic trees believed to be more robust because they incorporated more information. However, the genes or proteins chosen were based on ad hoc approaches. The large number of complete genome sequences available today allows the use of whole genomes to analyze relationships among organisms rather than using an ad hoc set of genes. We present a systematic approach for constructing a phylogenetic tree based on simultaneously clustering the complete proteomes of 360 bacterial species. From the homologous clusters, we identify 49 protein sequences shared by 99% of the organisms to build a tree. Of the 49 sequences, 47 have homologous sequences in both archaea and eukarya. The clusters are also used to create a network from which bacterial species with horizontally-transferred genes from other phyla are identified.

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.

Several nucleic acid-based assays have been developed for detecting Anaplasma marginale and Anaplasma centrale in vectors and hosts, making the choice of method to use in endemic areas difficult. We evaluated the ability of the reverse line blot (RLB) hybridisation assay, two nested polymerase chain reaction (nPCR) assays and a duplex real-time quantitative polymerase chain reaction (qPCR) assay to detect A. marginale and A. centrale infections in cattle (n = 66) in South Africa. The lowest detection limits for A. marginale plasmid DNA were 2500 copies by the RLB assay, 250 copies by the nPCR and qPCR assays and 2500, 250 and 25 copies of A. centrale plasmid DNA by the RLB, nPCR and qPCR assays respectively. The qPCR assay detected more A. marginale- and A. centrale-positive samples than the other assays, either as single or mixed infections. Although the results of the qPCR and nPCR tests were in agreement for the majority (38) of A. marginale-positive samples, 13 samples tested negative for A. marginale using nPCR but positive using qPCR. To explain this discrepancy, the target sequence region of the nPCR assay was evaluated by cloning and sequencing the msp1β gene from selected field samples. The results indicated sequence variation in the internal forward primer (AM100) area amongst the South African A. marginale msp1β sequences, resulting in false negatives. We propose the use of the duplex qPCR assay in future studies as it is more sensitive and offers the benefits of quantification and multiplex detection of both Anaplasma spp.

Vaccination with Salmonella enterica serovar Typhimurium lacking DNA adenine methyltransferase confers cross-protective immunity against multiple Salmonella serotypes. The mechanistic basis is thought to be associated with the de-repression of genes that are tightly regulated when transiting from one microenvironment to another. This de-repression provides a potential means for the production of a more highly expressed and stable antigenic repertoire capable of inducing cross-protective immune responses. To identify genes encoding proteins that may contribute to cross-protective immunity, we used a Salmonella Typhimurium DNA adenine methyltransferase mutant strain (UK-1 dam mutant) derived from the parental UK-1 strain, and assessed the transcriptional profile of the UK-1 dam mutant and UK-1 strain grown under conditions that simulate the intestinal or endosomal microenvironments encountered during the infective process. As expected, the transcriptional profile of the UK-1 dam mutant identified a set of genes more transcriptionally active when compared directly to UK-1, and stably transcribed in biologically relevant culture conditions. Further, 22% of these genes were more highly transcribed in comparison to two other clinically-relevant Salmonella serovars. The strategy employed here helps to identify potentially conserved proteins produced by the UK-1 dam mutant that stimulate and/or modulate the development of cross-protective immune responses toward multiple Salmonella serotypes.

The ability to capture genetic variation with unprecedented resolution improves our understanding of bacterial populations and their ability to cause disease. The goal of the pathogenomics era is to define genetic diversity that results in disease. Despite the economic losses caused by vector-borne bacteria in the Order Rickettsiales, little is known about the genetic variants responsible for observed phenotypes. The tick-transmitted rickettsial pathogen Anaplasma marginale infects cattle in tropical and subtropical regions worldwide, including Australia. Genomic analysis of North American A. marginale strains reveals a closed core genome defined by high levels of Single Nucleotide Polymorphisms (SNPs). Here we report the first genome sequences and comparative analysis for Australian strains that differ in virulence and transmissibility. A list of genetic differences that segregate with phenotype was evaluated for the ability to distinguish the attenuated strain from virulent field strains. Phylogenetic analyses of the Australian strains revealed a marked evolutionary distance from all previously sequenced strains. SNP analysis showed a strikingly reduced genetic diversity between these strains, with the smallest number of SNPs detected between any two A. marginale strains. The low diversity between these phenotypically distinct bacteria presents a unique opportunity to identify the genetic determinants of virulence and transmission.

Anaplasma marginale, patógeno de distribución mundial, es transmitido por garrapatas Ixódidas. Comprender su complejo desarrollo dentro de la garrapata vector, permitirá la predicción de brotes y ofrecerá oportunidades para controlar su transmisión. En este trabajo se revisa su ciclo básico de desarrollo junto con los estudios recientes acerca de las diferencias de transmisión entre cepas, que delinean aspectos de la interacción patógeno - vector. Bacterias, virus o protozoarios transmitidos por artrópodos causan enfermedades severas, tanto en humanos como en animales. Las enfermedades infecciosas transmitidas por garrapatas, entre las que incluimos a la Anaplasmosis (A. marginale), babesiosis (Babesia bigemina, B. bovis, B. divergens) y Theileriosis (Theileria annulata, T. parva), se encuentran entre las más importantes en el ámbito mundial, con pérdidas cercanas a los siete mil millones de dólares anualmente; y, a pesar de su impacto, permanecen escasamente bajo control, basado primordialmente en la aplicación de acaricidas, para interrumpir su transmisión. La aparición de garrapatas resistentes a múltiples sustancias acaricidas, representa una amenaza en este tipo de control y, como resultado, hay un resurgimiento de la investigación para el desarrollo de nuevas estrategias para su control. Nuevas opciones para prevenir la transmisión de patógenos de animales por garrapatas, será el resultado de entender las interacciones garrapata patógeno; proceso que culmina con el desarrollo de la infección y transmisión exitosa. En todos los casos de patógenos transmitidos por garrapatas, el desarrollo de la infección se realiza coordinamente a los momentos de adhesión y alimentación del vector sobre el animal. Esto sucede por la interdependencia en la señalización entre el patógeno y el vector al alimentarse y, por ello, será susceptible de intervención.

Bovine anaplasmosis is endemic in South Africa and it has a negative economic impact on cattle farming. An improved understanding of Anaplasma marginale and Anaplasma marginale variety centrale (A. centrale) transmission, together with improved tools for pathogen detection and characterisation, are required to inform best management practices. Direct detection methods currently in use for A. marginale and A. centrale in South Africa are light microscopic examination of tissue and organ smears, conventional, nested, and quantitative real-time polymerase chain reaction (qPCR) assays, and a reverse line blot hybridisation assay. Of these, qPCR is the most sensitive for detection of A. marginale and A. centrale in South Africa. Serological assays also feature in routine diagnostics, but cross-reactions prevent accurate species identification. Recently, genetic characterisation has confirmed that A. marginale and A. centrale are separate species. Diversity studies targeting Msp1a repeats for A. marginale and Msp1aS repeats for A. centrale have revealed high genetic variation and point to correspondingly high levels of variation in A. marginale outer membrane proteins (OMPs), which have been shown to be potential vaccine candidates in North American studies. Information on these OMPs is lacking for South African A. marginale strains and should be considered in future recombinant vaccine development studies, ultimately informing the development of regional or global vaccines.