Anaplasma platys and Rickettsia massiliae in Rhipicephalus sanguineus sensu stricto ticks collected on dogs in the Patagonian region of Argentina.

The aim of this study was to examine the presence of tick-borne rickettsial bacteria in Rhipicephalus sanguineus sensu stricto ticks collected from dogs in the Patagonian region of Argentina. Fourteen stray dogs from Valcheta, Río Negro province, Argentina were examined for the presence of R. sanguineus s.s. ticks. Ninety ticks were collected and identified to species level. DNA was extracted and analysed by conventional PCR assays for the presence of tick-borne bacteria belonging to the genera Anaplasma, Ehrlichia and Rickettsia. Thirty-three tick pools were tested by different PCR assays of which 3 were positive for Anaplasmataceae bacteria. From the 3 Anaplasmataceae positive samples, 2 partial 16S rDNA sequences were generated and belonging to Anaplasma platys, the causative agent of canine cyclic thrombocytopenia. Two tick samples were positive in Rickettsia specific PCR assays and were identified by phylogenetic analysis as Rickettsia massiliae, a member of the spotted fever group rickettsiae. The results of this study demonstrate the molecular detection of 2 rickettsial bacteria in R. sanguineus s.s. in a region of Argentina where no data were available so far.

Anaplasma phagocytophilum invasin AipA interacts with CD13 to elicit Src kinase signaling that promotes infection.

Host-microbe interactions that facilitate entry into mammalian cells are essential for obligate intracellular bacterial survival and pathogenesis. Anaplasma phagocytophilum is an obligate intracellular bacterium that invades neutrophils to cause granulocytic anaplasmosis. The invasin-receptor pairs and signaling events that induce Anaplasma uptake are inadequately defined. A. phagocytophilum invasion protein A orchestrates entry via residues 9-21 (AipA9-21) engaging an unknown receptor. Yeast two-hybrid screening suggested that AipA binds within C-terminal amino acids 851-967 of CD13 (aminopeptidase N), a multifunctional protein that, when crosslinked, initiates Src kinase and Syk signaling that culminates in endocytosis. Co-immunoprecipitation validated the interaction and confirmed that it requires the AipA N-terminus. CD13 ectopic expression on non-phagocytic cells increased susceptibility to A. phagocytophilum infection. Antibody blocking and enzymatic inhibition experiments found that the microbe exploits CD13 but not its ectopeptidase activity to infect myeloid cells. A. phagocytophilum induces Src and Syk phosphorylation during invasion. Inhibitor treatment established that Src is key for A. phagocytophilum infection, while Syk is dispensable and oriented the pathogen-invoked signaling pathway by showing that Src is activated before Syk. Disrupting the AipA-CD13 interaction with AipA9-21 or CD13781-967 antibody inhibited Src and Syk phosphorylation and also infection. CD13 crosslinking antibody that induces Src and Syk signaling restored infectivity of anti-AipA9-21-treated A. phagocytophilum. The bacterium poorly infected CD13 knockout mice, providing the first demonstration that CD13 is important for microbial infection in vivo. Overall, A. phagocytophilum AipA9-21 binds CD13 to induce Src signaling that mediates uptake into host cells, and CD13 is critical for infection in vivo. IMPORTANCE: Diverse microbes engage CD13 to infect host cells. Yet invasin-CD13 interactions, the signaling they invoke for pathogen entry, and the relevance of CD13 to infection in vivo are underexplored. Dissecting these concepts would advance fundamental understanding of a convergently evolved infection strategy and could have translational benefits. Anaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis, an emerging disease for which there is no vaccine and few therapeutic options. We found that A. phagocytophilum uses its surface protein and recently identified protective immunogen, AipA, to bind CD13 to elicit Src kinase signaling, which is critical for infection. We elucidated the AipA CD13 binding domain, which CD13 region AipA engages, and established that CD13 is key for A. phagocytophilum infection in vivo. Disrupting the AipA-CD13 interaction could be utilized to prevent granulocytic anaplasmosis and offers a model that could be applied to protect against multiple infectious diseases.

Orientia tsutsugamushi infection reduces host gluconeogenic but not glycolytic substrates.

Orientia tsutsugamushi a causal agent of scrub typhus, is an obligate intracellular bacterium that, akin to other rickettsiae, is dependent on host cell-derived nutrients for survival and thus pathogenesis. Based on limited experimental evidence and genome-based in silico predictions, O. tsutsugamushi is hypothesized to parasitize host central carbon metabolism (CCM). Here, we (re-)evaluated O. tsutsugamushi dependency on host cell CCM as initiated by glucose and glutamine. Orientia infection had no effect on host glucose and glutamine consumption or lactate accumulation, indicating no change in overall flux through CCM. However, host cell mitochondrial activity and ATP levels were reduced during infection and correspond with lower intracellular glutamine and glutamate pools. To further probe the essentiality of host CCM in O. tsutsugamushi proliferation, we developed a minimal medium for host cell cultivation and paired it with chemical inhibitors to restrict the intermediates and processes related to glucose and glutamine metabolism. These conditions failed to negatively impact O. tsutsugamushi intracellular growth, suggesting the bacterium is adept at scavenging from host CCM. Accordingly, untargeted metabolomics was utilized to evaluate minor changes in host CCM metabolic intermediates across O. tsutsugamushi infection and revealed that pathogen proliferation corresponds with reductions in critical CCM building blocks, including amino acids and TCA cycle intermediates, as well as increases in lipid catabolism. This study directly correlates O. tsutsugamushi proliferation to alterations in host CCM and identifies metabolic intermediates that are likely critical for pathogen fitness.IMPORTANCEObligate intracellular bacterial pathogens have evolved strategies to reside and proliferate within the eukaryotic intracellular environment. At the crux of this parasitism is the balance between host and pathogen metabolic requirements. The physiological basis driving O. tsutsugamushi dependency on its mammalian host remains undefined. By evaluating alterations in host metabolism during O. tsutsugamushi proliferation, we discovered that bacterial growth is independent of the host's nutritional environment but appears dependent on host gluconeogenic substrates, including amino acids. Given that O. tsutsugamushi replication is essential for its virulence, this study provides experimental evidence for the first time in the post-genomic era of metabolic intermediates potentially parasitized by a scrub typhus agent.

Addictive manipulation: a perspective on the role of reproductive parasitism in the evolution of bacteria-eukaryote symbioses.

Wolbachia bacteria encompass noteworthy reproductive manipulators of their arthropod hosts. which influence host reproduction to favour their own transmission, also exploiting toxin-antitoxin systems. Recently, multiple other bacterial symbionts of arthropods have been shown to display comparable manipulative capabilities. Here, we wonder whether such phenomena are truly restricted to arthropod hosts. We focused on protists, primary models for evolutionary investigations on eukaryotes due to their diversity and antiquity, but still overall under-investigated. After a thorough re-examination of the literature on bacterial-protist interactions with this question in mind, we conclude that such bacterial 'addictive manipulators' of protists do exist, are probably widespread, and have been overlooked until now as a consequence of the fact that investigations are commonly host-centred, thus ineffective to detect such behaviour. Additionally, we posit that toxin-antitoxin systems are crucial in these phenomena of addictive manipulation of protists, as a result of recurrent evolutionary repurposing. This indicates intriguing functional analogy and molecular homology with plasmid-bacterial interplays. Finally, we remark that multiple addictive manipulators are affiliated with specific bacterial lineages with ancient associations with diverse eukaryotes. This suggests a possible role of addictive manipulation of protists in paving the way to the evolution of bacteria associated with multicellular organisms.

Long-Term Protective Immunity against Ehrlichia chaffeensis Infection Induced by a Genetically Modified Live Vaccine.

Human monocytic ehrlichiosis, an emerging tick-borne disease, is caused by Ehrlichia chaffeensis. Infections with the pathogen are also common in the canine host. Our previous studies demonstrated that functional disruption within the E. chaffeensis phage head-to-tail connector protein gene results in bacterial attenuation, creating a modified live attenuated vaccine (MLAV). The MLAV confers protective immunity against intravenous and tick transmission challenges one month following vaccination. In this study, we evaluated the duration of MLAV protection. Dogs vaccinated with the MLAV were challenged with wild-type E. chaffeensis via intravenous infection at 4-, 8-, and 12-months post-vaccination. Immunized dogs rapidly cleared the wild-type pathogen infection and tested positive for bacteremia less frequently than unvaccinated controls. While immune responses varied among dogs, vaccinees consistently mounted IgG and CD4+ T-cell responses specific to E. chaffeensis throughout the assessment period. Our findings demonstrate that MLAV-mediated immune protection persists for at least one year against wild-type bacterial infection, marking a major advancement in combating this serious tick-borne disease. The data presented here serve as the foundation for further studies, elucidating the molecular mechanisms underlying virulence and vaccine development and aiding in preventing the diseases caused by E. chaffeensis and other tick-borne rickettsial pathogens.

Genetic diversity and prevalence of emerging Rickettsiales in Yunnan Province: a large-scale study.

BACKGROUND: Rickettsia and related diseases have been identified as significant global public health threats. This study involved comprehensive field and systematic investigations of various rickettsial organisms in Yunnan Province. METHODS: Between May 18, 2011 and November 23, 2020, field investigations were conducted across 42 counties in Yunnan Province, China, encompassing small mammals, livestock, and ticks. Preliminary screenings for Rickettsiales involved amplifying the 16S rRNA genes, along with additional genus- or species-specific genes, which were subsequently confirmed through sequencing results. Sequence comparisons were carried out using the Basic Local Alignment Search Tool (BLAST). Phylogenetic relationships were analyzed using the default parameters in the Molecular Evolutionary Genetics Analysis (MEGA) program. The chi-squared test was used to assess the diversities and component ratios of rickettsial agents across various parameters. RESULTS: A total of 7964 samples were collected from small mammals, livestock, and ticks through Yunnan Province and submitted for screening for rickettsial organisms. Sixteen rickettsial species from the genera Rickettsia, Anaplasma, Ehrlichia, Neoehrlichia, and Wolbachia were detected, with an overall prevalence of 14.72%. Among these, 11 species were identified as pathogens or potential pathogens to humans and livestock. Specifically, 10 rickettsial organisms were widely found in 42.11% (24 out of 57) of small mammal species. High prevalence was observed in Dremomys samples at 5.60%, in samples from regions with latitudes above 4000 m or alpine meadows, and in those obtained from Yuanmou County. Anaplasma phagocytophilum and Candidatus Neoehrlichia mikurensis were broadly infecting multiple genera of animal hosts. In contrast, the small mammal genera Neodon, Dremomys, Ochotona, Anourosorex, and Mus were carrying individually specific rickettsial agents, indicating host tropism. There were 13 rickettsial species detected in 57.14% (8 out of 14) of tick species, with the highest prevalence (37.07%) observed in the genus Rhipicephalus. Eight rickettsial species were identified in 2375 livestock samples. Notably, six new Rickettsiales variants/strains were discovered, and Candidatus Rickettsia longicornii was unambiguously identified. CONCLUSIONS: This large-scale survey provided further insight into the high genetic diversity and overall prevalence of emerging Rickettsiales within endemic hotspots in Yunnan Province. The potential threats posed by these emerging tick-borne Rickettsiales to public health warrant attention, underscoring the need for effective strategies to guide the prevention and control of emerging zoonotic diseases in China.

Nutritional symbiosis in ticks: singularities of the genus Ixodes.

Symbiosis with intracellular bacteria is essential for the nutrition of ticks, particularly through the biosynthesis of B vitamins. Yet, ticks of the genus Ixodes, which include major vectors of human pathogens, lack the nutritional symbionts usually found in other tick genera. This paradox raises questions about the mechanisms that Ixodes ticks use to prevent nutritional deficiencies. Nonetheless, Ixodes ticks commonly harbor other symbionts belonging to the order Rickettsiales. Although these obligate intracellular bacteria are primarily known as human pathogens, Rickettsiales symbionts often dominate the Ixodes microbial community without causing diseases. They also significantly influence Ixodes physiology, synthesize key B vitamins, and are crucial for immatures. These findings underscore unique associations between Rickettsiales and Ixodes ticks distinct from other tick genera.

OrthoPhyl-streamlining large-scale, orthology-based phylogenomic studies of bacteria at broad evolutionary scales.

There are a staggering number of publicly available bacterial genome sequences (at writing, 2.0 million assemblies in NCBI's GenBank alone), and the deposition rate continues to increase. This wealth of data begs for phylogenetic analyses to place these sequences within an evolutionary context. A phylogenetic placement not only aids in taxonomic classification but informs the evolution of novel phenotypes, targets of selection, and horizontal gene transfer. Building trees from multi-gene codon alignments is a laborious task that requires bioinformatic expertise, rigorous curation of orthologs, and heavy computation. Compounding the problem is the lack of tools that can streamline these processes for building trees from large-scale genomic data. Here we present OrthoPhyl, which takes bacterial genome assemblies and reconstructs trees from whole genome codon alignments. The analysis pipeline can analyze an arbitrarily large number of input genomes (>1200 tested here) by identifying a diversity-spanning subset of assemblies and using these genomes to build gene models to infer orthologs in the full dataset. To illustrate the versatility of OrthoPhyl, we show three use cases: E. coli/Shigella, Brucella/Ochrobactrum and the order Rickettsiales. We compare trees generated with OrthoPhyl to trees generated with kSNP3 and GToTree along with published trees using alternative methods. We show that OrthoPhyl trees are consistent with other methods while incorporating more data, allowing for greater numbers of input genomes, and more flexibility of analysis.

Survey of Rickettsia spp. in ticks (Acari: Ixodidae) infesting opossums (Didelphis albiventris) and capybaras (Hydrochoerus hydrochaeris) from an urban park in southeastern Brazil.

Ticks are hematophagous arthropods and, during feeding, may transmit pathogens to vertebrate hosts, including humans. This study aimed to investigate the presence of Rickettsia spp. in ticks collected between 2010 and 2013 from free-ranging capybaras (Hydrochoerus hydrochaeris) and opossums (Didelphis albiventris) that inhabit Sabiá Park in Uberlândia, Brazil. Overall, 1,860 ticks were collected: 1,272 (68.4%) from capybaras (487 of the species Amblyomma sculptum, 475 adults and 12 nymphs; 778 Amblyomma dubitatum, 727 adults and 51 nymphs; and seven larva clusters of the genus Amblyomma); and 588 (31.6%) from opossums (21 A. sculptum, one adult and 20 nymphs; 79 A. dubitatum, all nymphs; 15 Ixodes loricatus, 12 adults and three nymphs; 457 Amblyomma sp. larva clusters; 15 Ixodes sp. larva clusters; and one Argasidae larva cluster). Out of 201 DNA samples tested for the presence of Rickettsia spp. DNA using polymerase chain reaction (PCR) 12 showed amplification of a gtlA gene segment that was specific to Rickettsia bellii, a bacterium non-pathogenic to humans. As there has been a report showing serological evidence of infections caused by Rickettsia species of the spotted fever group (SFG) in capybaras and opossums in the park, including Rickettsia rickettsii, the etiological agent of Brazilian spotted fever, and considering the presence of A. sculptum ticks, which are aggressive to humans, as well as these vertebrate hosts, which are amplifiers of R. rickettsii, it is important to monitor the presence of SFG rickettsiae in the Sabiá Park, which is visited daily by thousands of people.

New insights into the systematics of the afrotropical Amblyomma marmoreum complex (Acari: Ixodidae) and the genome of a novel Rickettsia africae strain using morphological and metagenomic approaches.

The Amblyomma marmoreum complex includes afrotropical species, such as Amblyomma sparsum, a three-host tick that parasitizes reptiles, birds, and mammals, and is a recognized vector of Ehrlichia ruminantium. However, the lack of morphological, genetic and ecological data on A. sparsum has caused considerable confusion in its identification. In this study, we used microscopy and metagenomic approaches to analyze A. sparsum ticks collected from a puff adder snake (Bitis arietans) in southwest Senegal (an endemic rickettsioses area) in order to supplement previous morphological descriptions, provide novel genomic data for the A. marmoreum complex, and describe the genome of a novel spotted fever group Rickettsia strain. Based on stereoscope and scanning electron microscopy (SEM) morphological evaluations, we provide high-quality images and new insights about punctation and enameling in the adult male of A. sparsum to facilitate identification for future studies. The metagenomic approach allowed us assembly the complete mitochondrial genome of A. sparsum, as well as the nearly entire chromosome and complete plasmid sequences of a novel Rickettsia africae strain. Phylogenomic analyses demonstrated a close relationship between A. sparsum and Amblyomma nuttalli for the first time and confirmed the position of A. sparsum within the A. marmoreum complex. Our results provide new insights into the systematics of A. sparsum and A. marmoreum complex, as well as the genetic diversity of R. africae in the Afrotropical region. Future studies should consider the possibility that A. sparsum may be a vector for R. africae.

Microbial diversity of ticks and a novel typhus group Rickettsia species (Rickettsiales bacterium Ac37b) in Inner Mongolia, China.

Ticks can carry multiple pathogens, and Inner Mongolia's animal husbandry provides excellent environmental conditions for ticks. This study characterized the microbiome of ticks from different geographical locations in Inner Mongolia; 905 Dermacentor nuttalli and 36 Ixodes persulcatus were collected from sheep in three main pasture areas and from bushes within the forested area. Mixed DNA samples were prepared from three specimens from each region and tick species. Microbial diversity was analyzed by 16S rRNA sequencing, and α and ß diversity were determined. The predominant bacterial genera were Rickettsia (54.60%), including Rickettsiales bacterium Ac37b (19.33%) and other Rickettsia (35.27%), Arsenophonus (11.21%), Candidatus Lariskella (10.84%), and Acinetobacter (7.17%). Rickettsia bellii was identified in I. persulcatus, while Rickettsiales bacterium Ac37b was found in D. nuttalli from Ordos and Chifeng. Potential Rickettsia and Anaplasma coinfections were observed in the Ordos region. Tick microbial diversity analysis in Inner Mongolia suggests that sheep at the sampling sites were exposed to multiple pathogens.

Title: Diversité microbienne des tiques et nouvelle espèce de Rickettsia du groupe du typhus (bactérie Rickettsiales Ac37b) en Mongolie intérieure, Chine. Abstract: Les tiques peuvent être porteuses de plusieurs agents pathogènes et l'élevage en Mongolie intérieure offre d'excellentes conditions environnementales pour les tiques. Cette étude a caractérisé le microbiome des tiques de différentes zones géographiques de Mongolie intérieure; 905 Dermacentor nuttalli et 36 Ixodes persulcatus ont été collectés sur des moutons dans trois principales zones de pâturage et dans des buissons de la zone forestière. Des échantillons d'ADN mixtes ont été préparés à partir de trois spécimens de chaque région et espèce de tique. La diversité microbienne a été analysée par séquençage de l'ARNr 16S et la diversité α et ß a été déterminée. Les genres bactériens prédominants étaient les Rickettsia (54,60 %), dont la bactérie Rickettsiales Ac37b (19,33 %) et d'autres Rickettsia (35,27 %), Arsenophonus (11,21 %), Candidatus Lariskella (10,84 %) et Acinetobacter (7,17 %). Rickettsia bellii a été identifiée chez I. persulcatus, tandis que la bactérie Rickettsiales Ac37b a été trouvée chez D. nuttalli d'Ordos et Chifeng. Des co-infections potentielles à Rickettsia et Anaplasma ont été observées dans la région d'Ordos. L'analyse de la diversité microbienne des tiques en Mongolie intérieure montre que les moutons présents sur les sites d'échantillonnage sont exposés à plusieurs agents pathogènes.

Orientia tsutsugamushi: comprehensive analysis of the mobilome of a highly fragmented and repetitive genome reveals the capacity for ongoing lateral gene transfer in an obligate intracellular bacterium.

IMPORTANCE: Obligate intracellular bacteria, or those only capable of growth inside other living cells, have limited opportunities for horizontal gene transfer with other microbes due to their isolated replicative niche. The human pathogen Ot, an obligate intracellular bacterium causing scrub typhus, encodes an unusually high copy number of a ~40 gene mobile genetic element that typically facilitates genetic transfer across microbes. This proliferated element is heavily degraded in Ot and previously assumed to be inactive. Here, we conducted a detailed analysis of this element in eight Ot strains and discovered two strains with at least one intact copy. This implies that the element is still capable of moving across Ot populations and suggests that the genome of this bacterium may be even more dynamic than previously appreciated. Our work raises questions about intracellular microbial evolution and sounds an alarm for gene-based efforts focused on diagnosing and combatting scrub typhus.

PREVALENCE OF EHRLICHIA AND ANAPLASMA SPP. IN TICKS COLLECTED FROM CATTLE AND DEER IN ARKANSAS, USA.

Bovine anaplasmosis, caused by Anaplasma marginale, is widespread in cattle in the southeast United States. The pathogen is biologically transmitted by Dermacentor spp. ticks, and mechanically transmitted by biting flies and via fomites. Despite high reported regional seroprevalence, Dermacentor spp. are rare on cattle in the southeast. To identify other putative An. marginale vectors, and to characterize cattle exposure to other tick-borne pathogens, we collected ticks from Arkansas cattle herds in 2020-2022. Recognizing that deer are important hosts for some of the same tick species that parasitize cattle, we also collected ticks from hunter-killed deer in the fall and winter of 2021. Ticks were screened for bacteria in the family Anaplasmataceae using qPCR. Positive samples were further amplified using a PCR assay targeting the groEL gene, and the resulting amplicons were sequenced for identification. A total of 3,794 ticks were collected, the majority of which were Amblyomma americanum. Amblyomma americanum was the most common species on cattle, and Ixodes scapularis was most common on deer. No ticks were positive for An. marginale, though Anaplasma phagocytophilum was detected in deer-collected I. scapularis, as well as in a single engorged Am. americanum from cattle. Amblyomma americanum from cattle were infected with Ehrlichia ewingii, Ehrilichia chaffeensis, and Panola Mountain Ehrlichia. Cattle in Arkansas are exposed to several ehrlichial pathogens and may also be exposed to An. phagocytophilum. The importance of these pathogens, particularly Panola Mountain Ehrlichia, in causing cattle disease in Arkansas deserves further study, as does the importance of mechanical transmission of An. marginale in bovine anaplasmosis epidemiology.

Multiplex PCR for detection of Anaplasma marginale, A. bovis and A. platys in cattle.

Anaplasma genus has been classified under the family Anaplasmataceae of order Rickettsiales, which contains the spp. Anaplasma marginale, Anaplasma bovis, Anaplasma centrale, Anaplasma platys and Anaplasma phagocytophilum is accountable for a broad range of diseases in both man and animal medicine around the globe. A multiplex PCR procedure was designed to identify A. marginale, A. bovis, and A. platys simultaneously by employing species-specific primers targeting the msp4 (Anaplasma marginale), 16S rRNA (Anaplasma bovis), and groEL (Anaplasma platys) genes. The sensitivity of the assay was ascertained by tenfold dilutions of DNA obtained from cattle blood infected with A. marginale, A. bovis, and A. platys was used to evaluate the test sensitivity. A total of 31 Anaplasma genus positive samples were subjected to mPCR by using species specific primers for simultaneous detection of all the three species. All the 31 samples showed amplification of 753 bp fragment specific for A. marginale and 61.29% of the samples showed amplification of 547 bp fragment specific for A. bovis. The 470 bp DNA fragment specific for A. platys was found in 96.7% of samples. The lower limit of detection of the method for A. platys, A. marginale and A. bovis was found to be 4.4, 0.44 and 0.044 ng/µl, respectively.

Type 1 secretion system and effectors in Rickettsiales.

Obligate intracellular bacteria in the order Rickettsiales are transmitted by arthropod vectors and cause life-threatening infections in humans and animals. While both type 1 and type 4 secretion systems (T1SS and T4SS) have been identified in this group, the most extensive studies of Rickettsiales T1SS and associated effectors have been performed in Ehrlichia. These studies have uncovered important roles for the T1SS effectors in pathobiology and immunity. To evade innate immune responses and promote intracellular survival, Ehrlichia and other related obligate pathogens secrete multiple T1SS effectors which interact with a diverse network of host targets associated with essential cellular processes. T1SS effectors have multiple functional activities during infection including acting as nucleomodulins and ligand mimetics that activate evolutionarily conserved cellular signaling pathways. In Ehrlichia, an array of newly defined major immunoreactive proteins have been identified that are predicted as T1SS substrates and have conformation-dependent antibody epitopes. These findings highlight the underappreciated and largely uncharacterized roles of T1SS effector proteins in pathobiology and immunity. This review summarizes current knowledge regarding roles of T1SS effectors in Rickettsiales members during infection and explores newly identified immunoreactive proteins as potential T1SS substrates and targets of a protective host immune response.

Orientia tsutsugamushi: analysis of the mobilome of a highly fragmented and repetitive genome reveals ongoing lateral gene transfer in an obligate intracellular bacterium.

The rickettsial human pathogen Orientia tsutsugamushi (Ot) is an obligate intracellular Gram-negative bacterium with one of the most highly fragmented and repetitive genomes of any organism. Around 50% of its ~2.3 Mb genome is comprised of repetitive DNA that is derived from the highly proliferated Rickettsiales amplified genetic element (RAGE). RAGE is an integrative and conjugative element (ICE) that is present in a single Ot genome in up to 92 copies, most of which are partially or heavily degraded. In this report, we analysed RAGEs in eight fully sequenced Ot genomes and manually curated and reannotated all RAGE-associated genes, including those encoding DNA mobilisation proteins, P-type (vir) and F-type (tra) type IV secretion system (T4SS) components, Ankyrin repeat- and tetratricopeptide repeat-containing effectors, and other piggybacking cargo. Originally, the heavily degraded Ot RAGEs led to speculation that they are remnants of historical ICEs that are no longer active. Our analysis, however, identified two Ot genomes harbouring one or more intact RAGEs with complete F-T4SS genes essential for mediating ICE DNA transfer. As similar ICEs have been identified in unrelated rickettsial species, we assert that RAGEs play an ongoing role in lateral gene transfer within the Rickettsiales. Remarkably, we also identified in several Ot genomes remnants of prophages with no similarity to other rickettsial prophages. Together these findings indicate that, despite their obligate intracellular lifestyle and host range restricted to mites, rodents and humans, Ot genomes are highly dynamic and shaped through ongoing invasions by mobile genetic elements and viruses.

Diversity of Rickettsiales bacteria in five species of ticks collected from Jinzhai County, Anhui Province, China in 2021-2022.

The order Rickettsiales in the class Alphaproteobacteria comprises vector-borne pathogens of both medical and veterinary importance. Ticks, as a group, are second only to mosquitoes as vectors of pathogens to humans, playing a critical role in the transmission of rickettsiosis. In the present study, 880 ticks collected from Jinzhai County, Lu'an City, Anhui Province, China in 2021-2022 were identified as belonging to five species from three genera. DNA extracted from individual ticks was examined using nested polymerase chain reaction targeting the 16S rRNA gene (rrs), and the gene fragments amplified were sequenced to detect and identify Rickettsiales bacteria in the ticks. For further identification, the rrs-positive tick samples were further amplified by PCR targeting the gltA and groEL gene and sequenced. As a result, 13 Rickettsiales species belonging to the genera Rickettsia, Anaplasma, and Ehrlichia were detected, including three tentative species of Ehrlichia. Our results reveal the extensive diversity of Rickettsiales bacteria in ticks from Jinzhai County, Anhui Province. There, emerging rickettsial species may be pathogenic and cause under-recognized diseases. Detection of several pathogens in ticks that are closely related to human diseases may indicate a potential risk of infection in humans. Therefore, additional studies to assess the potential public health risks of the Rickettsiales pathogens identified in the present study are warranted.

A single cryptomonad cell harbors a complex community of organelles, bacteria, a phage, and selfish elements.

Symbiosis between prokaryotes and microbial eukaryotes (protists) has broadly impacted both evolution and ecology. Endosymbiosis led to mitochondria and plastids, the latter spreading across the tree of eukaryotes by subsequent rounds of endosymbiosis. Present-day endosymbionts in protists remain both common and diverse, although what function they serve is often unknown. Here, we describe a highly complex community of endosymbionts and a bacteriophage (phage) within a single cryptomonad cell. Cryptomonads are a model for organelle evolution because their secondary plastid retains a relict endosymbiont nucleus, but only one previously unidentified Cryptomonas strain (SAG 25.80) is known to harbor bacterial endosymbionts. We carried out electron microscopy and FISH imaging as well as genomic sequencing on Cryptomonas SAG 25.80, which revealed a stable, complex community even after over 50 years in continuous cultivation. We identified the host strain as Cryptomonas gyropyrenoidosa, and sequenced genomes from its mitochondria, plastid, and nucleomorph (and partially its nucleus), as well as two symbionts, Megaira polyxenophila and Grellia numerosa, and one phage (MAnkyphage) infecting M. polyxenophila. Comparing closely related endosymbionts from other hosts revealed similar metabolic and genomic features, with the exception of abundant transposons and genome plasticity in M. polyxenophila from Cryptomonas. We found an abundance of eukaryote-interacting genes as well as many toxin-antitoxin systems, including in the MAnkyphage genome that also encodes several eukaryotic-like proteins. Overall, the Cryptomonas cell is an endosymbiotic conglomeration with seven distinct evolving genomes that all show evidence of inter-lineage conflict but nevertheless remain stable, even after more than 4,000 generations in culture.

Co-Infection of Potential Tick-Borne Pathogens of the Order Rickettsiales and Borrelia burgdorferi s. l. and Their Link to Season and Area in Germany.

The prevalence of potential human pathogenic members of the order Rickettsiales differs between Borrelia burgdorferi sensu lato-positive and -negative tick microbiomes. Here, co-infection of members of the order Rickettsiales, such as Rickettsia spp., Anaplasma phagocytophilum, Wolbachia pipientis, and Neoehrlichia mikurensis as well as B. burgdorferi s.l. in the tick microbiome was addressed. This study used conventional PCRs to investigate the diversity and prevalence of the before-mentioned bacteria in 760 nucleic acid extracts of I. ricinus ticks detached from humans, which were previously tested for B. burgdorferi s.l.. A gltA gene-based amplicon sequencing approach was performed to identify Rickettsia species. The prevalence of Rickettsia spp. (16.7%, n = 127) and W. pipientis (15.9%, n = 121) were similar, while A. phagocytophilum was found in 2.8% (n = 21) and N. mikurensis in 0.1% (n = 1) of all ticks. Co-infection of B. burgdorferi s. l. with Rickettsia spp. was most frequent. The gltA gene sequencing indicated that Rickettsia helvetica was the dominant Rickettsia species in tick microbiomes. Moreover, R, monacensis and R. raoultii were correlated with autumn and area south, respectively, and a negative B. burgdorferi s. l. finding. Almost every fifth tick carried DNA of at least two of the human pathogenic bacteria studied here.