Rickettsioses are predominantly zoonotic infections that circulate among animal populations and can be transmitted to humans, representing emerging vector-borne diseases spread through infected arthropod vectors. Rickettsia asembonensis displays a broad geographic distribution and is predominantly associated with fleas. Recent reports have identified R. asembonensis in febrile human patients in Malaysia, Peru, and Zambia, underscoring its emerging zoonotic potential. In Thailand, however, investigations of Rickettsia spp. have concentrated mainly on ticks and fleas, with comparatively little information regarding infections in pet dogs and cats. The present study examined Rickettsia spp. in 968 pets, 472 client-owned dogs, and 496 client-owned cats from the Bangkok and Nakhon Pathom regions, using the 17-kDa gene as a molecular marker. Genetic analyses of Rickettsia species were performed using maximum-likelihood phylogeny with IQ-TREE, Bayesian inference, pairwise sequence similarity assessment, and nucleotide sequence type network analysis. A total of 114 pets tested positive, corresponding to a prevalence of 11.8%, with 21.6% (102/472) in dogs and 2.4% (12/496) in cats. Of these, 48 samples were sequenced, leading to the identification of R. asembonensis and either uncultured Rickettsia spp. or the Rickettsia endosymbiont of Haemaphysalis bispinosa. Statistical analysis indicated significantly higher infection rates in pet dogs and cats from the Nakhon Pathom region. Phylogenetic reconstruction with maximum-likelihood and Bayesian-inference approaches revealed distinct clustering of R. asembonensis and uncultured Rickettsia spp. Furthermore, three nucleotide sequence types of R. asembonensis identified here were unique and not shared with isolates from other countries or host species, as demonstrated by phylogenetic and Templeton-Crandall-Sing network analyses. This work provides the first documentation of R. asembonensis in client-owned urban dogs and cats in Thailand and highlights the need for increased awareness among veterinarians and pet owners, given its public health relevance.

The proximity of wildlife reserve areas to anthropized regions facilitates the spread and exchange of infectious agents, including pathogens with zoonotic potential, thereby increasing health-related risks for species already threatened with extinction. This study investigated the presence of Rickettsia in wild carnivores of the Brazilian Cerrado. Blood and ectoparasite samples were collected from specimens of the families Canidae (Cerdocyon thous, Chrysocyon brachyurus, Lycalopex vetulus, Speothos venaticus), Mephitidae (Conepatus semistriatus), Procyonidae (Nasua nasua), and Felidae (Leopardus pardalis, Panthera onca, Puma concolor). These samples were screened for Rickettsia using serological analysis (IFA) and molecular techniques (PCR). A total of 68 blood samples were obtained from carnivore specimens, along with 210 ticks collected from 18 individuals. DNA extracted from whole blood samples and from 189 ticks were subjected to PCR targeting the rickettsial genes gltA and ompA. Tick species were identified as Amblyomma tigrinum, Amblyomma sculptum, Amblyomma parvum, Amblyomma ovale, Amblyomma auricularium, and Rhipicephalus linnaei. None of the whole-blood DNA samples amplified the gltA gene, but 31 out of 189 tick DNA samples amplified fragments of the gltA and ompA genes, which were identified by DNA sequencing to correspond to four different rickettsial agents: 'Candidatus Rickettsia andeanae', Rickettsia amblyommatis, and the human pathogens Rickettsia parkeri sensu stricto (s.s.) and Rickettsia parkeri strain Atlantic rainforest. Serological analysis by IFA was performed on 50 serum samples. Among those collected in situ, 69% (20/29) of C. brachyurus and 77% (7/9) of C. thous showed seroreactivity to Rickettsia spp. In addition, a probable antigen involved in a homologous reaction (PAIHR) was identified as R. parkeri in 20% (6/29) of C. brachyurus samples and 44% (4/9) of C. thous samples. This is the first report providing both serological and molecular evidence of Rickettsia infection in different wild carnivore species and their ticks in the Cerrado biome. The detection of R. parkeri in wild canids highlights the importance of monitoring infectious agents to inform conservation strategies and zoonosis prevention efforts.

Molecular detection and characterization of vector-borne pathogens in domestic cats (Felis catus) in Türkiye: The first report of Coxiella burnetii from cats in Türkiye.

Arthropod-borne rickettsial pathogens are affecting both human and animal health. Due to the lack of studies on tick-borne rickettsiae in this region, the present study aimed to investigate the molecular identity of Rickettsia spp. in ticks in Northwestern Iran. This cross-sectional study was conducted in Ardabil Province from January to December 2024. A total of 700 ticks were collected from livestock. DNA was extracted from pooled ticks, and PCR assays targeting the gltA and 16S rRNA genes were performed to detect Rickettsia spp.; positive PCR products were sequenced for confirmation (Pishgam, Iran). A total of 700 ticks belonging to nine species were isolated from livestock. Among these, Rhipicephalus bursa was the most common species. PCR detected Rickettsia DNA in 2 of 26 pools (7.7%), according to accession gene code: PV929760 and PV929761. According to our findings, R. aeschlimannii was detected in ticks from northwestern Iran. Livestock-associated ticks, including Rhipicephalus spp., are considered logical candidates for further study. Our study highlighted the need for continued surveillance using a One Health approach.

ABSTRACT A rickettsial pathogen, Ehrlichia chaffeensis, causes an emerging tick-borne disease called human monocytic ehrlichiosis (HME). E. chaffeensis in ticks expresses a porin OMP-1B, the immunodominant major outer membrane protein, which is required for nutrient uptake across the outer membrane of bacteria. We developed an OMP-1B-encoding mRNA-lipid nanoparticle (hereafter OMP-1B mRNA-LNP) vaccine. Immunization of naïve mice with OMP-1B mRNA-LNP showed significant protection against E. chaffeensis infection following a challenge with E. chaffeensis-infected tick cells. Following vaccination, increased OMP-1B-specific serum IgG, IgG1, and IgG2a titres, as well as E. chaffeensis infection-neutralizing antibodies were detected. An ELISpot assay revealed significant increase in OMP-1B-specific IFN-γ-secreting cells in the blood and spleen samples from mice vaccinated with OMP-1B mRNA-LNP. Flow cytometry analysis of spleen samples showed that OMP-1B antigen-specific IFN-γ-producing CD4+ and CD8+ T cells as well as granzyme B-producing cytotoxic CD8+ T cells were significantly increased in the vaccinated mice. Our results demonstrate that an mRNA vaccine targeting OMP-1B conferred protection against E. chaffeensis infection, with significant humoral immune responses including infection-neutralizing antibodies, balanced Th1/Th2 response, and antigen-specific T helper and cytotoxic T cell activation. These data suggest that the mRNA-LNP approach is a viable strategy for developing efficient anti-rickettsial vaccines.

Targeted mutagenesis in Ehrlichia canis deleting the phage head-to-tail connector protein gene and its assessment as a vaccine candidate preventing canine ehrlichiosis.

Obligately intracellular rickettsiae cause a broad spectrum of disease in humans and animals, ranging from mild illness to life-threatening infections. Multiple species co-circulate along the southern United States of America-northern Mexico border, yet their seroprevalence in susceptible hosts remains incompletely understood. Dogs serve as key amplifying hosts for several of these pathogens, including Rickettsia rickettsii and Rickettsia massiliae, and have been shown to be infected by flea-borne Rickettsia typhi and Rickettsia felis. To better characterize exposure and potential co-infection patterns, we conducted a large binational seroepidemiologic study of 779 dogs from urban households and shelter settings across seven subregions along both sides of the border. Using a custom multiplex micro-immunofluorescence assay, we simultaneously screened for antibodies to R. rickettsii, R. massiliae, R. typhi, and R. felis. Overall, 41.2% of dogs were seroreactive to at least one pathogen, with the highest seroprevalence for R. felis (19.3%), followed by R. massiliae (15.7%), R. typhi (14.5%), and R. rickettsii (9.8%). Co-seroreactivity was common, particularly between R. typhi and R. felis, with 34.2% of R. typhi-seroreactive samples also seroreactive to R. felis, and 16.7% exhibiting high titers to both. In contrast, R. rickettsii and R. massiliae showed limited overlap (15.2% co-seroreactive; 6.4% with high titers), suggesting possible cross-protection or competitive exclusion. Spatial analyses revealed distinct geographic patterns: R. massiliae predominated in western Baja California, R. rickettsii was concentrated in Mexicali and the Rio Grande Valley, and R. felis was widely distributed. Seroreactivity patterns were generally consistent across age and sex but varied modestly between shelter and neighborhood dogs, particularly for R. typhi. These findings highlight the complex ecology of rickettsial pathogens in a binational context, underscore the importance of dogs as sentinels for human risk, and provide a foundation for future studies on vector-host-pathogen interactions, cross-protective immunity, and spatial epidemiological risk.

As a core pastoral region of the Qinghai-Tibet Plateau, Qinghai Province faces substantial threats to livestock production from tick-borne diseases. This study aimed to investigate the prevalence of six bacterial pathogens in dominant tick species from Qinghai Province, to provide baseline epidemiological data for local tick-borne disease surveillance. A total of 1025 questing ticks were collected from key pastoral regions of Qinghai Province during April to May in 2024 and 2025. All ticks were morphologically identified as belonging to 1 family (Ixodidae), 2 genera, and 4 species. Dermacentor nuttalli was the dominant species with a relative dominance of 66.83% (685/1025, 95% CI: 63.92-69.61%), followed by Haemaphysalis qinghaiensis at 30.83% (316/1025, 95% CI: 28.11-33.69%), Dermacentor silvarum at 1.95% (20/1025, 95% CI: 1.27-2.98%), and Dermacentor niveus at 0.39% (4/1025, 95% CI: 0.15-1.01%). PCR detection was performed for six target pathogens, with an overall Brucella spp. DNA detection rate of 0.78% (8/1025, 95% CI: 0.40-1.53%) and an overall Rickettsia spp. detection rate of 16.29% (167/1025, 95% CI: 14.16-18.67%). Statistical analysis showed that the prevalence of Brucella spp. and Rickettsia spp. differed significantly between the two dominant tick species (Fisher's exact test/χ2 test, all p < 0.001). No Brucella or Rickettsia pathogens were detected in D. silvarum and D. niveus. Notably, detection of Brucella spp. DNA does not confirm the presence of viable bacteria or tick vector competence. This study fills the regional data gap of tick-borne pathogens in Qinghai, and provides reference for the prevention and control of local tick-borne zoonotic diseases.

Ticks are blood-feeding arthropods that carry diverse pathogenic and nonpathogenic microorganisms, yet their dynamics within tick tissues remain poorly understood. We compared the microbial communities in the haemolymph, saliva, ovaries, midgut, and salivary glands of individual Amblyomma gemma and Hyalomma rufipes sampled from dromedary camel using V1-V2 16S rRNA gene metabarcoding. The haemolymph exhibited the highest bacterial diversity, followed by the saliva and ovaries, while the midgut and salivary glands had lower diversities. Keystone taxa exhibited varied connectivity across different tissues and played a crucial role in structuring microbial communities in both tick species. We exclusively detected Rickettsia africae in Am. gemma and Rickettsia aeschlimannii in Hy. rufipes. Both Rickettsia species showed negative correlations with dominant endosymbionts and environmental bacteria across multiple tissues. Coxiella endosymbionts were detected solely in Am. gemma and were most abundant in the salivary glands, while Francisella endosymbionts were dominant in Hy. rufipes’ salivary glands. Francisella endosymbionts were less abundant when R. aeschlimannii was high. In Am. gemma’s haemolymph and saliva, Wolbachia endosymbionts were prevalent, where they were inversely associated with R. africae at the individual tick level. In Hy. rufipes, Wolbachia endosymbionts were present in the ovaries, but not in the midgut. Candidatus Midichloria mitochondrii was found in all tissues of Hy. rufipes and predominantly in saliva, but was only detected in the ovaries, midgut, and salivary glands of Am. gemma. Positive interactions were found between Ca. Midichloria mitochondrii and R. aeschlimannii in Hy. rufipes’ saliva and ovaries, and Ca. Midichloria mitochondrii and Francisella endosymbionts in Am. gemma’s ovaries. Rickettsiella spp. were found in the saliva and haemolymph but not the midgut in both tick species. Environmental bacteria, such as Pseudomonas, Acinetobacter, Staphylococcus, and Corynebacterium, whilst abundant in the haemolymph and saliva were scarce in other tissues. Tick tissue significantly influences bacterial composition, with tissue-specific interactions between pathogens and endosymbionts suggesting bacterial functional specialization. The complex interactions observed between key endosymbionts and Rickettsia pathogens across tissues, including negative correlations with Coxiella, Francisella, and Wolbachia endosymbionts, and positive correlations between Ca. Midichloria mitochondrii and R. aeschlimannii, require further investigation. Identified keystone taxa may serve as targets for anti-microbiota vaccines.

Pathogenic Spotted Fever Group (SFG) Rickettsia species, including Rickettsia parkeri, replicate in endothelial cells and macrophages in vitro and during infections in murine models of disease. We demonstrated that infection of human macrophage-like cells with a related SFG Rickettsia, R. conorii, resulted in a significant increase in mitochondria-associated proteins. However, the role of mitochondrial functions in Rickettsia pathogenesis is unknown. Here, we found that R. parkeri exploits mitochondrial dynamics to promote intracellular replication in mouse and human macrophages by activating the mitochondrial fission regulator, the dynamin-related protein 1 (DRP1). R. parkeri proliferated in macrophages, which coincided with a significant increase in mitochondria fission, mitochondria content, and host cell ATP production, primarily due to mitochondrial respiration compared to uninfected cells. In addition, R. parkeri infection also led to a temporal increase in DRP1 serine phosphorylation that was dependent on rickettsial de novo protein synthesis. Importantly, R. parkeri growth was significantly impacted in DRP1-deficient macrophages. These results suggest that the modulation of mitochondrial fission, content, and function is important for replication and survival of pathogenic SFG Rickettsia species in macrophages. Our data highlight that hijacking mitochondrial dynamics and function is essential for intracellular replication of Rickettsia species and may be a shared mechanism utilized by related obligate intracellular pathogens for growth.

Heat shock proteins (HSPs) are highly conserved molecular chaperones that play a crucial role in maintaining cellular protein homeostasis during stress. In bovine hemoparasitic infections caused by Theileria, Babesia, Anaplasma, Trypanosoma, and Ehrlichia, HSPs are not only essential for parasite survival but also actively influence host immune responses. This review summarizes current molecular insights into the expression and functions of major HSP families (HSP27, HSP60, HSP70, and HSP90). These proteins are deeply involved in parasite virulence, intracellular persistence, antigenic variation, and modulation of host signaling pathways. Comparative observations indicate a conserved reliance on the HSP70-HSP90 axis in protozoan parasites, whereas HSP60 plays a dominant immunogenic role in rickettsial pathogens. We also explored the translational potential of HSPs as diagnostic biomarkers, therapeutic targets, and vaccine candidates. Finally, key knowledge gaps are highlighted, emphasizing the importance of integrative multi-omics approaches to better understand host-parasite chaperone interactions.

Human parvovirus B19 (B19V) infection during pregnancy can lead to a range of adverse outcomes such as miscarriage, premature delivery, fetal hydrops, severe anemia, myocarditis, heart failure, and even fetal demise, posing significant risks to maternal and fetal health. The aim of this study was to establish a more efficient method for detecting B19V in amniotic fluid and to explore and optimize early diagnosis and treatment strategies for fetal B19V infection. Intrauterine transfusion (IUT) was performed due to the occurrence of severe fetal anemia and hydrops. Amniotic fluid was obtained for genetic detection. Metagenomic next-generation sequencing (mNGS) and bioinformatic analysis were performed on the amniotic cells to identify the viral genome. In this study, the B19V genome was identified in the amniotic cells of the suspected case, with three viral coding sequences mapped. The coverage density reached 99.9% of the viral sequences. No other pathogen sequences, including bacteria, fungi, parasites, chlamydia, mycoplasma, rickettsia and other viruses, were identified. Our study confirmed the diagnosis of fetal B19V infection in a suspected case via amniotic fluid virus genome detection. It is the first time to exhibit the clinical application of mNGS to systematically detect the B19V genome in amniotic fluid in prenatal practice, and to achieve good results in combination with clinical management. The study highlighted the importance of comprehensive management of B19V fetal infection and demonstrated the advantages and wide application prospects of mNGS in intrauterine infection diagnosis.

Dogs accessing natural areas in Brazil are exposed to tick species typically associated with wild carnivores, some of which are vectors of pathogenic Rickettsia. Infested dogs may transport ticks into households and act as amplifying hosts for these agents. Iguaçu National Park (INP), a major inland Atlantic Forest reserve in Brazil, harbors at least nine tick species and five Rickettsia species. This study evaluated tick infestation in 116 rural dogs from properties along most of the 420 km perimeter of INP and 22 urban dogs from forest-adjacent areas in Foz do Iguaçu. Sera from 39 dogs were tested against five Rickettsia antigens using an indirect immunofluorescence assay. A total of 169 ticks were collected, including Rhipicephalus linnaei (n=87), Amblyomma ovale (n=62), Amblyomma coelebs (n=5), and Rhipicephalus microplus (n=3), with an overall infestation prevalence of 28.3%. Seroprevalence against Rickettsia spp. was 42.9% in rural dogs and 18.1% in urban dogs. The highest titers were 1:512 in rural dogs (against R. parkeri, R. amblyommatis, and R. rhipicephali) and 1:256 in urban dogs (against R. rickettsii), while median titers were zero. Both tick infestation and Rickettsia exposure were lower than those reported in dogs from other Atlantic Forest regions. These findings suggest an overall limited exposure of free-ranging dogs to ticks and Rickettsia spp. Further investigation is needed to understand the causes of this low exposure, including the potential influence of the jaguar population and other wild carnivores in INP, which may restrict dog access to forested areas.

Ehrlichia canis is primarily a Rhipicephalus sanguineus tick-borne rickettsial pathogen initially identified as causing canine monocytic ehrlichiosis, and infections in people have also been reported in Venezuela, Mexico, and parts of Europe. It is of high importance to have a vaccine suitable in protecting the canine host, which will aid in lessening E. canis infections also in people. Gene inactivation mutations in the phage head-to-tail connector protein genes ( phtcp ) from E. chaffeensis and A. marginale caused attenuated growth, and prior infection with the mutated bacteria induced protective immunity against wild-type bacterial infections in natural hosts, independent of blood-borne infection or tick-transmission infection. In the current study, we describe the development of targeted mutagenesis for the first time in E. canis genome and with a novel modification to avoid introducing antibiotic resistance cassettes to delete the phtcp ortholog from E. canis . The mutated E. canis was then assessed for its in vivo growth and the induction of host immunity exerted following the mutant infection aiding to protect against wild-type infection challenge in the canine host. We assessed systemic pathogen loads, hematological parameters, IgG immune responses, and plasma cytokines following the mutant infection relative to uninfected dogs. Similarly, the assessments were carried out following wild-type pathogen infections in dogs with or without prior mutant infection challenges. The study demonstrates that prior infection of dogs with the mutant induces immunity to prevent infection establishment by wild-type E. canis. Similarly, the mutant infection resulted in clear biological differences compared to the wild-type infection. This study establishes that the molecular genetic methods are broadly applicable to pathogens belonging to the family Anaplasmataceae and that the modified live vaccines with phtcp gene orthologs are valuable in reducing the diseases caused by the tick-borne rickettsial pathogens belong to Anaplasmataceae, including E. canis .

Pathogenic Rickettsia species are obligate intracellular bacteria that must reside in a mammalian host or arthropod vector cell to survive. Although these bacteria transition between different intracellular environments during infection, they encode few putative transcription factors, and their gene regulatory networks are largely unknown. Because of their inextricable relationship with eukaryotic cells, transcriptional profiling of the pathogen is complicated by the abundance of contaminating host RNA, especially in infection conditions or stages where the bacterial burden is inherently low. Here, we employ a hybrid capture technique (PatH-Cap) to improve library preparation by enriching bacterial transcripts while depleting host and rRNA molecules. Using PatH-Cap, we explored transcriptional changes throughout the first 24 hours of infection, including infection initiation – an infection stage which has been difficult to profile with standard library preparation methods. We then clustered genes based on their temporal trends, revealing cohorts of genes whose expression is up- or downregulated at different stages of infection. We also highlighted the diverse temporal expression trends of genes with known roles in growth and pathogenesis, including translation and cell division genes, secreted effectors, and secretion system components. Lastly, we identified 310 antisense RNA molecules, many of which also showed strong temporal trends. This work demonstrates that sensitive transcriptional profiling approaches like PatH-Cap hold great promise for dissecting gene expression networks driving infection in intracellular pathogens that have historically posed significant technical challenges.

Ticks are important vectors of rickettsial pathogens, particularly in regions where livestock, wildlife, and humans overlap. Despite increasing reports of rickettsioses in India, molecular evidence of Rickettsia species in questing ticks from the Western Ghats of Kerala remains scarce. Questing ticks were collected from forest-edge and fringe habitats in Wayanad, Idukki, and Thrissur districts using standardized dragging. Species were identified morphologically, pooled, and screened for Rickettsia DNA by PCR targeting gltA and ompA genes. Amplicons were sequenced, and phylogenetic analysis was performed using reference sequences. Among 237 ticks belonging to six species, 24 % (12/50 pools) were PCR-positive for Rickettsia. The highest prevalence was observed in Haemaphysalis spinigera (50 %), followed by Haemaphysalis bispinosa (30 %) and Haemaphysalis turturis (9.5 %). Sequence analysis revealed the presence of Rickettsia massiliae and Rickettsia vini like species. Importantly, sequences closely related to "Candidatus Rickettsia jingxinensis" were detected for the first time in India, in H. turturis. Several isolates showed genetic divergence from known strains, suggesting the circulation of uncharacterized Rickettsia species or strains in tick population. This study provides the first molecular evidence of "Ca. R. jingxinensis" in H. turturis from India and demonstrates a high prevalence of Rickettsia spp. in questing ticks of Kerala. Given the pathogenic potential of some of these species, enhanced surveillance and incorporation of rickettsioses into differential diagnoses of febrile illnesses are needed in South India.

Tick-associated microorganisms (Borrelia, Rickettsia, Hepatozoon, and Hemolivia) in Amblyomma varanense ticks parasitizing Asian water monitors (Varanus salvator): Insights from a hotspot in Ratchaburi Province, Western Thailand

Q fever, caused by Coxiella burnetii, is an underdiagnosed zoonotic disease with significant public health implications. Serological tests remain the diagnostic cornerstone but often fail in early infection. This study investigates the utility of polymerase chain reaction (PCR) targeting the IS1111 gene for molecular detection of C. burnetii in human clinical samples from a resource-limited setting. A retrospective PCR-based study was conducted on 243 archived clinical specimens collected from febrile patients across 11 districts of North India. DNA was extracted and subjected to conventional PCR targeting the IS1111 insertion sequence. Samples positive for C. burnetii were further evaluated for coinfection with rickettsial pathogens using real-time and nested PCRs. Sequencing and phylogenetic analysis were performed on positive samples to determine genetic relationships. C. burnetii DNA was detected in 4 of 243 samples (1.64%). Half the positive cases belonged to the patients in 1-15 year age group, and two had coinfections with Orientia tsutsugamushi. One case showed coinfection with spotted fever group rickettsia. Clinical features included fever (100%), myalgia (75%), and rash (25%). Phylogenetic analysis revealed that the isolates clustered with the Z3055 reference strain, indicating a close genetic relationship to known zoonotic strains. PCR targeting IS1111 is a valuable diagnostic tool for early detection of C. burnetii, particularly in settings where serological testing is delayed or unavailable. Detection in pediatric patients and coinfection with other rickettsiae underscore the need for broader diagnostic consideration of Q fever in endemic febrile illnesses.

Rickettsial infections are a leading cause of febrile illness in Southeast Asia and Malaysia, although they are often underreported. Rickettsial pathogens largely fall within the genera Rickettsia and Orientia and classified within the Rickettsiaceae family. In Malaysia, scrub typhus, caused by Orientia tsutsugamushi, is the most frequently reported rickettsial infection. Traditionally, rickettsial organisms have been isolated and cultured from embryonated eggs. However, this method is labor and skill intensive, has limited scalability, and requires specialized equipment, making it less accessible to many laboratories. An alternative approach is to use mammalian/amphibian/invertebrate cell culture as a host for rickettsial propagation. In this study, we evaluated the potential for culturing rickettsial pathogens previously adapted and maintained in embryonated eggs in mammalian cell lines. Two mammalian cell lines (Vero E6 and L929) were inoculated with rickettsial strains previously identified as O. tsutsugamushi (strains Karp [n=8], Kato [n=4], and Gilliam [n=2]). The presence of O. tsutsugamushi was assessed by quantitative qPCR at 14-day intervals. After 90 days of culture, only one of the fifteen isolates (GL94) showed evidence of propagation in L929 cells, whereas O. tsutsugamushi DNA remained below the qPCR detection limit in Vero E6 cells for all isolates tested. Most of the other isolates showed little to no growth, with some exhibiting the presence of other bacteria. The identification and morphology of GL94 were confirmed via transmission electron microscopy (TEM), followed by full-length 16S sequencing. This study highlights the challenges of transitioning rickettsial culture from embryonated eggs to mammalian cell cultures.

The black-legged tick, Ixodes scapularis, transmits several medically important pathogens to humans including Anaplasma phagocytophilum. Our previous studies provided evidence that this bacterium modulates arthropod organic anion transporting polypeptide (IsOATP4056) and tryptophan pathways for its survival and transmission from ticks. In this study, we identified that IsOATP4056 interacts with I. scapularis hypothetical protein (IsHP) to suppress arthropod innate immunity thereby facilitating A. phagocytophilum survival in ticks and tick cells. Co-precipitation with recombinant IsHP and tick protein lysates followed by immunoblotting analysis with anti- IsOATP4056 antibody was performed to reveal whether these two proteins directly interact. RNAi-mediated silencing experiments were performed to understand the roles of IsHP, IsOATP4056 and Aryl hydrocarbon Receptor (AhR) in tick-A. phagocytophilum interactions. Immunoprecipitation and immunoblotting analysis revealed interaction of IsHP with IsOATP4056. RNAi-mediated silencing of ishp expression affected arthropod innate immune response that resulted in significantly increased bacterial burden in tick cells. In contrast, RNAi-mediated silencing of isoatp4056 expression or antibody-mediated blocking of IsOATP4056 upregulated ishp and innate immune response that controlled bacterial burden in ticks and tick cells. Furthermore, we noted that A. phagocytophilum infection or treatment with tryptophan metabolite xanthurenic acid (XA) significantly upregulates Aryl hydrocarbon Receptor (AhR) expression in ticks and tick cells. RNAi-mediated silencing of ahr expression decreased isoatp4056 transcripts and bacterial burden but increased the ishp expression. EMSA results further support that AhR and XA positively regulate isoatp4056 promoter. These results elucidate that A. phagocytophilum infection triggers AhR-mediated regulation of isoatp4056 expression to inactivate IsHP-associated pelle expression for its survival in ticks and tick cells.