Analysis of Amblyomma americanum microRNAs in response to Ehrlichia chaff eensis infection and their potential role in vectorial capacity.

Background: MicroRNAs (miRNAs) represent a subset of small noncoding RNAs and carry tremendous potential for regulating gene expression at the post-transcriptional level. They play pivotal roles in distinct cellular mechanisms including inhibition of bacterial, parasitic, and viral infections via immune response pathways. Intriguingly, pathogens have developed strategies to manipulate the host's miRNA profile, fostering environments conducive to successful infection. Therefore, changes in an arthropod host's miRNA profile in response to pathogen invasion could be critical in understanding host-pathogen dynamics. Additionally, this area of study could provide insights into discovering new targets for disease control and prevention. The main objective of the present study is to investigate the functional role of differentially expressed miRNAs upon Ehrlichia chaf feensis, a tick-borne pathogen, infection in tick vector, Amblyomma americanum . Methods: Small RNA libraries from uninfected and E. chaffeensis -infected Am. americanum midgut and salivary gland tissues were prepared using the Illumina Truseq kit. Small RNA sequencing data was analyzed using miRDeep2 and sRNAtoolbox to identify novel and known miRNAs. The differentially expressed miRNAs were validated using a quantitative PCR assay. Furthermore, a miRNA inhibitor approach was used to determine the functional role of selected miRNA candidates. Results: The sequencing of small RNA libraries generated >147 million raw reads in all four libraries and identified a total of >250 miRNAs across the four libraries. We identified 23 and 14 differentially expressed miRNAs in salivary glands, and midgut tissues infected with E. chaffeensis , respectively. Three differentially expressed miRNAs (miR-87, miR-750, and miR-275) were further characterized to determine their roles in pathogen infection. Inhibition of target miRNAs significantly decreased the E. chaffeensis load in tick tissues, which warrants more in-depth mechanistic studies. Conclusions: The current study identified known and novel miRNAs and suggests that interfering with these miRNAs may impact the vectorial capacity of ticks to harbor Ehrlichia . This study identified several new miRNAs for future analysis of their functions in tick biology and tick-pathogen interaction studies.

Ehrlichia effector TRP120 manipulates bacteremia to facilitate tick acquisition.

Ehrlichia species are obligatory intracellular bacteria that cause a potentially fatal disease, human ehrlichiosis. The biomolecular mechanisms of tick acquisition of Ehrlichia and transmission between ticks and mammals are poorly understood. Ehrlichia japonica infection of mice recapitulates the full spectrum of human ehrlichiosis. We compared the pathogenicity and host acquisition of wild-type E. japonica with an isogenic transposon mutant of E. japonica that lacks tandem repeat protein 120 (TRP120) (ΔTRP120). Both wild-type and ΔTRP120 E. japonica proliferated similarly in cultures of mammalian and tick cells. Upon inoculation into mice, both wild-type and ΔTRP120 E. japonica multiplied to high levels in various tissues, with similar clinical chemistry and hematologic changes, proinflammatory cytokine induction, and fatal disease. However, the blood levels of ΔTRP120 E. japonica were almost undetectable within 24 h, whereas the levels of the wild type increased exponentially. Greater than 90% of TRP120 was released from infected cells into the culture medium. Mouse blood monocytes exposed to native TRP120 from culture supernatants showed significantly reduced cell surface expression of the transmigration-related markers Ly6C and CD11b. Larval ticks attached to mice infected with either wild-type or ΔTRP120 E. japonica imbibed similar amounts of blood and subsequently molted to nymphs at similar rates. However, unlike wild-type E. japonica, the ΔTRP120 mutant was minimally acquired by larval ticks and subsequent molted nymphs and, thus, failed to transmit to naïve mice. Thus, TRP120 is required for bacteremia but not disease. These findings suggest a novel mechanism whereby an obligatory intracellular bacterium manipulates infected blood monocytes to sustain the tick-mammal transmission cycle. IMPORTANCE: Effective prevention of tick-borne diseases such as human ehrlichiosis requires an understanding of how disease-causing organisms are acquired. Ehrlichia species are intracellular bacteria that require infection of both mammals and ticks, involving cycles of transmission between them. Mouse models of ehrlichiosis and tick-mouse transmission can advance our fundamental understanding of the pathogenesis and prevention of ehrlichiosis. Herein, a mutant of Ehrlichia japonica was used to investigate the role of a single Ehrlichia factor, named tandem repeat protein 120 (TRP120), in infection of mammalian and tick cells in culture, infection and disease progression in mice, and tick acquisition of E. japonica from infected mice. Our results suggest that TRP120 is necessary only for Ehrlichia proliferation in circulating mouse blood and ongoing bacteremia to permit Ehrlichia acquisition by ticks. This study provides new insights into the importance of bacterial factors in regulating bacteremia, which may facilitate tick acquisition of pathogens.

A draft of the genome of the Gulf Coast tick, Amblyomma maculatum.

The Gulf Coast tick, Amblyomma maculatum, inhabits the Southeastern states of the USA bordering the Gulf of Mexico, Mexico, and other Central and South American countries. More recently, its U.S. range has extended West to Arizona and Northeast to New York state and Connecticut. It is a vector of Rickettsia parkeri and Hepatozoon americanum. This tick species has become a model to study tick/Rickettsia interactions. To increase our knowledge of the basic biology of A. maculatum we report here a draft genome of this tick and an extensive functional classification of its proteome. The DNA from a single male tick was used as a genomic source, and a 10X genomics protocol determined 28,460 scaffolds having equal or more than 10 Kb, totaling 1.98 Gb. The N50 scaffold size was 19,849 Kb. The BRAKER pipeline was used to find the protein-coding gene boundaries on the assembled A. maculatum genome, discovering 237,921 CDS. After trimming and classifying the transposable elements, bacterial contaminants, and truncated genes, a set of 25,702 were annotated and classified as the core gene products. A BUSCO analysis revealed 83.4% complete BUSCOs. A hyperlinked spreadsheet is provided, allowing browsing of the individual gene products and their matches to several databases.

Hematophagy and tick-borne Rickettsial pathogen shape the microbial community structure and predicted functions within the tick vector, Amblyomma maculatum.

Background: Ticks are the primary vectors of emerging and resurging pathogens of public health significance worldwide. Analyzing tick bacterial composition, diversity, and functionality across developmental stages and tissues is crucial for designing new strategies to control ticks and prevent tick-borne diseases. Materials and methods: Here, we explored the microbial communities across the developmental timeline and in different tissues of the Gulf-Coast ticks (Amblyomma maculatum). Using a high-throughput sequencing approach, the influence of blood meal and Rickettsia parkeri, a spotted fever group rickettsiae infection in driving changes in microbiome composition, diversity, and functionality was determined. Results: This study shows that the core microbiome of Am. maculatum comprises ten core bacterial genera. The genus Rickettsia, Francisella, and Candidatus_Midichloria are the key players, with positive interactions within each developmental stage and adult tick organ tested. Blood meal and Rickettsia parkeri led to an increase in the bacterial abundance in the tissues. According to functional analysis, the increase in bacterial numbers is positively correlated to highly abundant energy metabolism orthologs with blood meal. Correlation analysis identified an increase in OTUs identified as Candidatus Midichloria and a subsequent decrease in Francisella OTUs in Rickettsia parkeri infected tick stages and tissues. Results demonstrate the abundance of Rickettsia and Francisella predominate in the core microbiome of Am. maculatum, whereas Candidatus_Midichloria and Cutibacterium prevalence increase with R. parkeri-infection. Network analysis and functional annotation suggest that R. parkeri interacts positively with Candidatus_Midichloria and negatively with Francisella. Conclusion: We conclude that tick-transmitted pathogens, such as R. parkeri establishes infection by interacting with the core microbiome of the tick vector.

Rickettsia parkeri infection modulates the sialome and ovariome of the Gulf coast tick, Amblyomma maculatum.

The Gulf Coast tick, Amblyomma maculatum, is a vector of several tick-borne pathogens, including Rickettsia parkeri. The ability of R. parkeri to persist within the tick population through transovarial and transstadial transmission, without apparently harming the ticks, contributes to the pathogen's perpetuation in the tick population. Previous studies have shown that the R. parkeri load in A. maculatum is regulated by the tick tissues' oxidant/antioxidant balance and the non-pathogenic tick microbiome. To obtain further insights into the interaction between tick and pathogen, we performed a bulk RNA-Seq for differential transcriptomic analysis of ovaries and salivary glands from R. parkeri-infected and uninfected ticks over the feeding course on a host. The most differentially expressed functional category was of bacterial origin, exhibiting a massive overexpression of bacterial transcripts in response to the R. parkeri infection. Candidatus Midichloria mitochondrii and bacteria from the genus Rickettsia were mainly responsible for the overexpression of bacterial transcripts. Host genes were also modulated in R. parkeri-infected tick organs. A similar number of host transcripts from all analyzed functional categories was negatively and positively modulated, revealing a global alteration of the A. maculatum transcriptome in response to pathogen infection. R. parkeri infection led to an increase in salivary transcripts involved in blood feeding success as well as a decrease in ovarian immune transcripts. We hypothesize that these transcriptional alterations facilitate pathogen persistence and transmission within tick population.

Efficacy and Immune Correlates of OMP-1B and VirB2-4 Vaccines for Protection of Dogs from Tick Transmission of Ehrlichia chaffeensis.

Ehrlichia chaffeensis, an obligatory intracellular bacterium, causes human monocytic ehrlichiosis, an emerging disease transmitted by the Lone Star tick, Amblyomma americanum. Here, we investigated the vaccine potential of OMP-1B and VirB2-4. Among the highly expressed and immunodominant E. chaffeensis porin P28s/OMP-1s, OMP-1B is predominantly expressed by E. chaffeensis in A. americanum ticks, whereas VirB2-4 is a pilus protein of the type IV secretion system essential for E. chaffeensis infection of host cells. Immunization with recombinant OMP-1B (rOMP-1B) or recombinant VirB2-4 (rVirB2-4) protected mice from E. chaffeensis infection as effectively as Entry-triggering protein of Ehrlichia immunization. Dogs vaccinated with a nanoparticle vaccine composed of rOMP-1B or rVirB2-4 and an immunostimulating complex developed high antibody titers against the respective antigen. Upon challenge with E. chaffeensis-infected A. americanum ticks, E. chaffeensis was undetectable in the blood of rOMP-1B or rVirB2-4 immunized dogs on day 3 or 6 post-tick attachment and for the duration of the experiment, whereas dogs sham-vaccinated with the complex alone were persistently infected for the duration of the experiment. E. chaffeensis exponentially replicates in blood-feeding ticks to facilitate transmission. Previously infected ticks removed from OMP-1B-immunized dogs showed significantly lower bacterial load relative to ticks removed from sham-immunized dogs, suggesting in-tick neutralization. Peripheral blood leukocytes from rVirB2-4-vaccinated dogs secreted significantly elevated amounts of interferon-γ soon after tick attachment by ELISpot assay and reverse transcription-quantitative PCR, suggesting interferon-γ-mediated Ehrlichia inhibition. Thus, Ehrlichia surface-exposed proteins OMP-1B and VirB2-4 represent new potential vaccine candidates for blocking tick-borne ehrlichial transmission. IMPORTANCE Ehrlichia are tick-borne pathogens that cause a potentially fatal illness-ehrlichiosis-in animals and humans worldwide. Currently, no vaccine is available for ehrlichiosis, and treatment options are limited. Ticks are biological vectors of Ehrlichia, i.e., Ehrlichia exponentially replicates in blood-sucking ticks before infecting animals. Ticks also inoculate immunomodulatory substances into animals. Thus, it is important to study effects of candidate vaccines on Ehrlichia infection in both animals and ticks and the immune responses of animals shortly after infected tick challenge. Here, we investigated the efficacy of vaccination with functionality-defined two surface-exposed outer membrane proteins of Ehrlichia chaffeensis, OMP-1B and VirB2-4, in a mouse infection model and then in a dog-tick transmission model. Our results begin to fill gaps in our understanding of Ehrlichia-derived protective antigens against tick-transmission and immune correlates and mechanisms that could help future development of vaccines for immunization of humans and animals to counter tick-transmitted ehrlichiosis.

Real-Time PCR Differential Detection of Neorickettsia findlayensis and N. risticii in Cases of Potomac Horse Fever.

Potomac horse fever (PHF) is an acute and potentially fatal enterotyphlocolitis of horses with clinical signs that include anorexia, fever, diarrhea, and laminitis. Its incidence is increasing despite a commercially available vaccine. PHF is caused by Neorickettsia risticii, and the recently rediscovered and classified N. findlayensis. PHF diagnosis is currently accomplished using serology or nested PCR. However, both methods cannot distinguish the two Neorickettsia species that cause PHF. Further, the current N. risticii real-time PCR test fails to detect N. findlayensis. Thus, in this study, two Neorickettsia species-specific real-time PCR assays based on Neorickettsia ssa2 and a Neorickettsia genus-specific real-time PCR assay based on Neorickettsia 16S rRNA gene were developed. The ssa2 real-time PCR tests differentiated N. findlayensis from N. risticii in the field samples for which infection with either species had been verified using multiple other molecular tests and culture isolation, and the 16S rRNA gene real-time PCR detected both Neorickettsia species in the samples. These tests were applied to new field culture isolates from three Canadian provinces (Alberta, Quebec, Ontario) and Ohio as well as archival DNA samples from suspected PHF cases to estimate the prevalence of N. findlayensis in different geographic regions. The results suggest that N. findlayensis frequently causes PHF in horses in Alberta and Quebec. The development of these tests will allow rapid, sensitive, and specific diagnosis of horses presenting with clinical signs of PHF. These tests will also enable rapid and targeted treatment and help develop broad-spectrum vaccines for PHF.

Potomac horse fever in Ontario: Clinical, geographic, and diagnostic aspects.

Clinical findings, geographic locations, laboratory diagnoses, and culture isolation of Neorickettsia spp. in Potomac horse fever (PHF) cases diagnosed in Ontario between 2015 and 2019 are described. Forty-six confirmed PHF cases occurred from late June to early September. Of 41 horses admitted to the Ontario Veterinary College, 28 (68%) survived and 13 (32%) were euthanized due to poor prognosis or financial constraints. Most cases were in southern Ontario along the Canada-USA border. Blood and fecal samples from 43 suspect PHF cases were submitted to 2 laboratories for polymerase chain reaction (PCR) testing for Neorickettsia risticii. Agreement between both laboratories for detection of N. risticii DNA was excellent for feces [κ = 0.932, 95% confidence interval (CI): 0.80 to 1], and fair for blood samples (κ = 0.494, 95% CI: 0.13 to 0.85). Neorickettia spp. were isolated from 16 of 41 (39%) blood samples. DNA analysis confirmed 14 isolates were N. risticii and 2 were N. findlayensis, a novel species of Neorickettsia recently demonstrated to cause PHF.

La fièvre équine du Potomac en Ontario : aspects cliniques, géographiques et diagnostiques. Les résultats cliniques, emplacements géographiques, diagnostics de laboratoire et isolement par culture de Neorickettsia spp. dans les cas de fièvre équine du Potomac (PHF) diagnostiqués en Ontario entre 2015 et 2019 sont décrits. Quarante-six cas confirmés de PHF sont survenus de la fin juin au début septembre. Sur 41 chevaux admis au Ontario Veterinary College, 28 (68%) ont survécu et 13 (32%) ont été euthanasiés en raison d'un mauvais pronostic ou de contraintes financières. La plupart des cas se trouvaient dans le sud de l'Ontario, le long de la frontière canado-américaine. Des échantillons de sang et de matières fécales provenant de 43 cas suspects de PHF ont été soumis à deux laboratoires pour des tests de réaction d'amplification en chaîne par la polymérase (PCR) pour Neorickettsia risticii. La concordance entre les deux laboratoires pour la détection de l'ADN de N. risticii était excellente pour les selles [κ = 0,932, intervalle de confiance (IC) à 95% : 0,80 à 1] et passable pour les échantillons sanguins (κ = 0,494, IC à 95% : 0,13 à 0,85). Neorickettia spp. ont été isolés à partir de 16 des 41 échantillons de sang (39%). L'analyse de l'ADN a confirmé que 14 isolats étaient N. risticii et deux étaient N. findlayensis, une nouvelle espèce de Neorickettsia récemment démontrée comme causant le PHF.(Traduit par Dr Serge Messier).

Iron robbery by intracellular pathogen via bacterial effector-induced ferritinophagy.

Iron is essential for survival and proliferation of Ehrlichia chaffeensis, an obligatory intracellular bacterium that causes an emerging zoonosis, human monocytic ehrlichiosis. However, how Ehrlichia acquires iron in the host cells is poorly understood. Here, we found that native and recombinant (cloned into the Ehrlichia genome) Ehrlichia translocated factor-3 (Etf-3), a previously predicted effector of the Ehrlichia type IV secretion system (T4SS), is secreted into the host cell cytoplasm. Secreted Etf-3 directly bound ferritin light chain with high affinity and induced ferritinophagy by recruiting NCOA4, a cargo receptor that mediates ferritinophagy, a selective form of autophagy, and LC3, an autophagosome biogenesis protein. Etf-3-induced ferritinophagy caused ferritin degradation and significantly increased the labile cellular iron pool, which feeds Ehrlichia Indeed, an increase in cellular ferritin by ferric ammonium citrate or overexpression of Etf-3 or NCOA4 enhanced Ehrlichia proliferation, whereas knockdown of Etf-3 in Ehrlichia via transfection with a plasmid encoding an Etf-3 antisense peptide nucleic acid inhibited Ehrlichia proliferation. Excessive ferritinophagy induces the generation of toxic reactive oxygen species (ROS), which could presumably kill both Ehrlichia and host cells. However, during Ehrlichia proliferation, we observed concomitant up-regulation of Ehrlichia Fe-superoxide dismutase, which is an integral component of Ehrlichia T4SS operon, and increased mitochondrial Mn-superoxide dismutase by cosecreted T4SS effector Etf-1. Consequently, despite enhanced ferritinophagy, cellular ROS levels were reduced in Ehrlichia-infected cells compared with uninfected cells. Thus, Ehrlichia safely robs host cell iron sequestered in ferritin. Etf-3 is a unique example of a bacterial protein that induces ferritinophagy to facilitate pathogen iron capture.

An intracellular nanobody targeting T4SS effector inhibits Ehrlichia infection.

Infection with obligatory intracellular bacteria is difficult to treat, as intracellular targets and delivery methods of therapeutics are not well known. Ehrlichia translocated factor-1 (Etf-1), a type IV secretion system (T4SS) effector, is a primary virulence factor for an obligatory intracellular bacterium, Ehrlichia chaffeensis In this study, we developed Etf-1-specific nanobodies (Nbs) by immunizing a llama to determine if intracellular Nbs block Etf-1 functions and Ehrlichia infection. Of 24 distinct anti-Etf-1 Nbs, NbD7 blocked mitochondrial localization of Etf-1-GFP in cotransfected cells. NbD7 and control Nb (NbD3) bound to different regions of Etf-1. Size-exclusion chromatography showed that the NbD7 and Etf-1 complex was more stable than the NbD3 and Etf-1 complex. Intracellular expression of NbD7 inhibited three activities of Etf-1 and E. chaffeensis: up-regulation of mitochondrial manganese superoxide dismutase, reduction of intracellular reactive oxygen species, and inhibition of cellular apoptosis. Consequently, intracellular NbD7 inhibited Ehrlichia infection, whereas NbD3 did not. To safely and effectively deliver Nbs into the host cell cytoplasm, NbD7 was conjugated to cyclized cell-permeable peptide 12 (CPP12-NbD7). CPP12-NbD7 effectively entered mammalian cells and abrogated the blockade of cellular apoptosis caused by E. chaffeensis and inhibited infection by E. chaffeensis in cell culture and in a severe combined-immunodeficiency mouse model. Our results demonstrate the development of an Nb that interferes with T4SS effector functions and intracellular pathogen infection, along with an intracellular delivery method for this Nb. This strategy should overcome current barriers to advance mechanistic research and develop therapies complementary or alternative to the current broad-spectrum antibiotic.

Recent advances in understanding tick and rickettsiae interactions.

Ticks are haematophagous arthropods with unique molecular mechanisms for digesting host blood meal while acting as vectors for various pathogens of public health significance. The tick's pharmacologically active saliva plays a fundamental role in modulating the host's immune system for several days to weeks, depending on the tick species. The vector tick has also developed sophisticated molecular mechanisms to serve as a competent vector for pathogens, including the spotted fever group (SFG) rickettsiae. Evidence is still inadequate concerning tick-rickettsiae-host interactions and saliva-assisted transmission of the pathogen to the mammalian host. Rickettsia parkeri, of the SFG rickettsia, can cause a milder version of Rocky Mountain spotted fever known as American Boutonneuse fever. The Gulf Coast tick (Amblyomma maculatum) often transmits this pathogenic rickettsia in the USA. This review discusses the knowledge gap concerning tick-rickettsiae-host interactions by highlighting the SFG rickettsia and the Am maculatum model system. Filling this knowledge gap will provide a better understanding of the tick-rickettsiae-host interactions in disease causation, which will be crucial for developing effective methods for preventing tick-borne diseases.

An Entry-Triggering Protein of Ehrlichia Is a New Vaccine Candidate against Tick-Borne Human Monocytic Ehrlichiosis.

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes human monocytic ehrlichiosis, an emerging disease transmitted by the Lone Star tick, Amblyomma americanum. E. chaffeensis outer membrane protein entry triggering protein of Ehrlichia (EtpE) is necessary for bacterial entry into human cells. We investigated the role of EtpE in transmission of the bacteria from tick to human cells and whether or not vaccination with EtpE can prevent transmission of ehrlichiae from ticks to mammals. An antiserum against the recombinant C terminus of EtpE (rEtpE-C), which binds a mammalian cell-surface receptor and triggers bacterial entry, significantly inhibited E. chaffeensis transmission from infected tick cells to human monocytes in culture. Each of five specific-pathogen-free dogs were vaccinated with rEtpE-C along with an immunostimulating complex or were sham vaccinated with the complex alone. Dogs vaccinated with rEtpE-C developed high antibody titers against rEtpE-C and produced interferon-γ-secreting cells, as assessed with the ELISpot assay. All 10 dogs were challenged with A. americanum adult ticks infected as nymphs by syringe inoculation with E. chaffeensis Upon challenge, both the vaccinated and control dogs became infected by day 1 post-tick attachment, but the majority of rEtpE-C-vaccinated dogs rapidly cleared the infection from the bloodstream as soon as day 7, whereas most of sham-vaccinated dogs remained infected at day 35. Peripheral blood leukocytes from vaccinated dogs had significantly elevated interferon-γ mRNA levels and secreted significantly elevated interferon-γ soon after tick attachment. Thus, the EtpE-C vaccine represents the first ehrlichial protein vaccine demonstrated to reduce bacterial infection in mammals upon challenge with infected ticks.IMPORTANCE The incidence of tick-borne diseases has risen dramatically in the past two decades and continues to rise. Discovered in 1986 and designated a nationally notifiable disease in 1998 by the Centers for Disease Control and Prevention, human monocytic ehrlichiosis, which is caused by the bacterium Ehrlichia chaffeensis, is one of the most prevalent, life-threatening, emerging tick-borne zoonoses in the United States. We investigated the role of the E. chaffeensis protein EtpE in transmission of the bacterium from tick to human cells and in vaccinated dogs with EtpE to assess the efficacy of vaccination against E. chaffeensis-infected tick challenge. Our results help fill gaps in our understanding of E. chaffeensis-derived protective antigens that could be used in a candidate vaccine for immunization of humans to counter tick-transmitted ehrlichiosis.

Oxygen deprivation influences the survival of Listeria monocytogenes in gerbils.

Listeria monocytogenes is a facultative anaerobic foodborne pathogen capable of surviving harsh environments. Recent work has indicated that anaerobic conditions increase the resistance capability of certain strains to environmental stressors. The goal of the study was to conduct a preliminary study to determine whether exposure to anaerobic conditions prior to infection increases the ability to survive in vivo. Gerbils were inoculated with one of five doses of the L. monocytogenes strain F2365 by oral gavage: phosphate-buffered saline (control), 5 × 106 colony forming units aerobic culture (low aerobic), 5 × 108 aerobic culture (high aerobic), 5 × 106 anaerobic culture (low anaerobic), or 5 × 108 anaerobic culture (high anaerobic) dose of F2365. Gerbils inoculated with a high aerobic or anaerobic dose exhibited significant weight loss. Gerbils administered either the low or high anaerobic dose had at least 3 log10 of L. monocytogenes present in fecal samples, which contrasted with gerbils that received the low aerobic dose. Animals that received the high anaerobic dose had a significant increase in bacterial loads within the liver. Histologic examination of the L. monocytogenes positive livers exhibited locally extensive areas of hepatocellular necrosis, though the extent of this damage differed between treatment groups. Microbial community analysis of the cecum from gerbils infected with L. monocytogenes indicated that the abundance of Bacteroidales and Clostridiales increased and there was a decrease in the abundance of Spirochaetales. This study suggests that anaerobic conditions alter the localization pattern of L. monocytogenes within the gastrointestinal tract. These findings could relate to how different populations are more susceptible to listeriosis, as oxygen availability may differ within the gastrointestinal tract.

The tick endosymbiont Candidatus Midichloria mitochondrii and selenoproteins are essential for the growth of Rickettsia parkeri in the Gulf Coast tick vector.

BACKGROUND: Pathogen colonization inside tick tissues is a significant aspect of the overall competence of a vector. Amblyomma maculatum is a competent vector of the spotted fever group rickettsiae, Rickettsia parkeri. When R. parkeri colonizes its tick host, it has the opportunity to dynamically interact with not just its host but with the endosymbionts living within it, and this enables it to modulate the tick's defenses by regulating tick gene expression. The microbiome in A. maculatum is dominated by two endosymbiont microbes: a Francisella-like endosymbiont (FLE) and Candidatus Midichloria mitochondrii (CMM). A range of selenium-containing proteins (selenoproteins) in A. maculatum ticks protects them from oxidative stress during blood feeding and pathogen infections. Here, we investigated rickettsial multiplication in the presence of tick endosymbionts and characterized the functional significance of selenoproteins during R. parkeri replication in the tick. RESULTS: FLE and CMM were quantified throughout the tick life stages by quantitative PCR in R. parkeri-infected and uninfected ticks. R. parkeri infection was found to decrease the FLE numbers but CMM thrived across the tick life cycle. Our qRT-PCR analysis indicated that the transcripts of genes with functions related to redox (selenogenes) were upregulated in ticks infected with R. parkeri. Three differentially expressed proteins, selenoprotein M, selenoprotein O, and selenoprotein S were silenced to examine their functional significance during rickettsial replication within the tick tissues. Gene silencing of the target genes was found to impair R. parkeri colonization in the tick vector. Knockdown of the selenogenes triggered a compensatory response from other selenogenes, as observed by changes in gene expression, but oxidative stress levels and endoplasmic reticulum stress inside the ticks were also found to have heightened. CONCLUSIONS: This study illustrates the potential of this new research model for augmenting our understanding of the pathogen interactions occurring within tick hosts and the important roles that symbionts and various tick factors play in regulating pathogen growth.

Amblyomma maculatum SECIS binding protein 2 and putative selenoprotein P are indispensable for pathogen replication and tick fecundity.

Selenium, a vital trace element, is incorporated into selenoproteins to produce selenocysteine. Our previous studies have revealed an adaptive co-evolutionary process that has enabled the spotted fever-causing tick-borne pathogen Rickettsia parkeri to survive by manipulating an antioxidant defense system associated with selenium, which includes a full set of selenoproteins and other antioxidants in ticks. Here, we conducted a systemic investigation of SECIS binding protein 2 (SBP2) and putative selenoprotein P (SELENOP) by transcript silencing in adult female Gulf-coast ticks (Amblyomma maculatum). Knockdown of the SBP2 and SELENOP genes depleted the respective transcript levels of these tick selenogenes, and caused differential regulation of other antioxidants. Importantly, the selenium level in the immature and mature tick stages increased significantly after a blood meal, but the selenium level decreased in ticks after the SBP2 and SELENOP knockdowns. Moreover, the SBP2 knockdown significantly impaired both transovarial transmission of R. parkeri to tick eggs and egg hatching. Overall, our data offer new insight into the relationship between the SBP2 selenoprotein synthesis gene and the putative tick SELENOP gene. It also augments our understanding of selenoprotein synthesis, selenium maintenance and utilization, and bacterial colonization of a tick vector.

A study of ticks and tick-borne livestock pathogens in Pakistan.

BACKGROUND: As obligate blood-feeding arthropods, ticks transmit pathogens to humans and domestic animals more often than other arthropod vectors. Livestock farming plays a vital role in the rural economy of Pakistan, and tick infestation causes serious problems with it. However, research on tick species diversity and tick-borne pathogens has rarely been conducted in Pakistan. In this study, a systematic investigation of the tick species infesting livestock in different ecological regions of Pakistan was conducted to determine the microbiome and pathobiome diversity in the indigenous ticks. METHODOLOGY/PRINCIPAL FINDINGS: A total of 3,866 tick specimens were morphologically identified as 19 different tick species representing three important hard ticks, Rhipicephalus, Haemaphysalis and Hyalomma, and two soft ticks, Ornithodorus and Argas. The bacterial diversity across these tick species was assessed by bacterial 16S rRNA gene sequencing using a 454-sequencing platform on 10 of the different tick species infesting livestock. The notable genera detected include Ralstonia, Clostridium, Staphylococcus, Rickettsia, Lactococcus, Lactobacillus, Corynebacterium, Enterobacter, and Enterococcus. A survey of Spotted fever group rickettsia from 514 samples from the 13 different tick species generated rickettsial-specific amplicons in 10% (54) of total ticks tested. Only three tick species Rhipicephalus microplus, Hyalomma anatolicum, and H. dromedarii had evidence of infection with "Candidatus Rickettsia amblyommii" a result further verified using a rompB gene-specific quantitative PCR (qPCR) assay. The Hyalomma ticks also tested positive for the piroplasm, Theileria annulata, using a qPCR assay. CONCLUSIONS/SIGNIFICANCE: This study provides information about tick diversity in Pakistan, and pathogenic bacteria in different tick species. Our results showed evidence for Candidatus R. amblyommii infection in Rhipicephalus microplus, H. anatolicum, and H. dromedarii ticks, which also carried T. annulata.

The microbiome of neotropical ticks parasitizing on passerine migratory birds.

Seasonal migration of passerine birds between temperate North America and tropical Central and South America is an ecological phenomenon. Migration of birds has been associated with the introduction of ectoparasites like ticks or tick-borne pathogens across the avian migration routes. In this study, the microbial diversity was determined in the ticks and bird DNA samples using 454 pyrosequencing of bacterial 16S rRNA gene. Tick DNA samples showed the dominance of genera Lactococcus, Francisella, Raoultella, Wolbachia and Rickettsia across all the ticks, but birds DNA did not share common microbial diversity with ticks. Furthermore, "Candidatus Rickettsia amblyommii" infection in the 91 ticks collected off the songbirds was also quantified by qPCR assay. Interestingly, "Candidatus R. amblyommii" was tested positive in 24 ticks (26% infection), and infection varied from as low as three copies to thousands of copies, but bird blood samples showed no amplification. Our results provide evidence that songbirds serve as transport carrier for immature ticks, and less likely to be a reservoir for "Candidatus R. amblyommii".

A snapshot of the microbiome of Amblyomma tuberculatum ticks infesting the gopher tortoise, an endangered species.

The gopher tortoise tick, Amblyomma tuberculatum, has a unique relationship with the gopher tortoise, Gopherus polyphemus, found in sandy habitats across the southeastern United States. We aimed to understand the overall bacterial community associated with A. tuberculatum while also focusing on spotted fever group Rickettsia. These tortoises in the Southern Mississippi region are a federally threatened species; therefore, we have carefully trapped the tortoises and removed the species-specific ticks attached to them. Genomic DNA was extracted from individual ticks and used to explore overall bacterial load using pyrosequencing of bacterial 16S rRNA on 454-sequencing platform. The spotted fever group of Rickettsia was explored by amplifying rickettsial outer membrane protein A (rompA) gene by nested PCR. Sequencing results revealed 330 bacterial operational taxonomic units (OTUs) after all the necessary curation of sequences. Four whole A. tuberculatum ticks showed Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes as the most dominant phyla with a total of 74 different bacterial genera detected. Together Rickettsiae and Francisella showed >85% abundance, thus dominating the bacterial community structure. Partial sequences obtained from ompA amplicons revealed the presence of an uncharacterized Rickettsia similar to the Rickettsial endosymbiont of A. tuberculatum. This is the first preliminary profile of a complete bacterial community from gopher tortoise ticks and warrants further investigation regarding the functional role of Rickettsial and Francisella-like endosymbionts in tick physiology.

Rickettsia parkeri colonization in Amblyomma maculatum: the role of superoxide dismutases.

BACKGROUND: The Gulf Coast tick (Amblyomma maculatum) is an arthropod vector of Rickettsia parkeri, the causative agent of American boutonneuse fever and an infectious agent of public health significance. In this study, we evaluated the biological significance of the superoxide dismutases (SODs) of A. maculatum in hematophagy and R. parkeri colonization within the tick host. METHODS: An RNA interference approach was used to measure the functional roles of tick SODs (Cu/Zn-SOD and Mn-SOD) in R. parkeri colonization of the tick vector. Total microbial load, R. parkeri infection rate, and compensatory mechanisms by tick genes were examined using quantitative polymerase chain reaction (PCR) and quantitative reverse-transcriptase PCR assays. SOD enzymatic activity assays and malondialdehyde (MDA) lipid peroxidation were employed to determine the redox states in the tick tissues. RESULTS: Knockdown of the Cu/Zn-SOD gene caused the upregulation of Mn-SOD in transcript levels. Single and dual knockdowns of the SOD genes caused an increase in MDA lipid peroxidation while SOD enzymatic activities did not show a significant change. Mn-SOD knockdown resulted in a substantial increase in the microbial load; however, Cu/Zn-SOD transcript depletion prompted an upsurge in the midgut bacterial load, and significantly decreased the bacterial load in salivary gland tissues. Additionally, Cu/Zn-SOD transcript silencing led to significantly fewer R. parkeri DNA copy numbers in both tick tissues (midguts and salivary glands). CONCLUSIONS: SOD enzymes play an important function in the regulation of bacterial communities associated with tick vectors and also in the defense mechanisms against the damage caused by reactive oxygen species within the tick. Knockdown experiments increased the levels of total oxidative stress in ticks, revealing the interplay between SOD isozymes that results in the transcriptional regulation of tick antioxidants. Moreover, the tick's Cu/Zn-SOD aids in the colonization of R. parkeri in tick tissues providing evidence of A. maculatum's vectorial success for a spotted fever group rickettsial pathogen.

An insight into the microbiome of the Amblyomma maculatum (Acari: Ixodidae).

The aim of this study was to survey the bacterial diversity of Amblyomma maculatum Koch, 1844, and characterize its infection with Rickettsia parkeri. Pyrosequencing of the bacterial 16S rRNA was used to determine the total bacterial population in A. maculatum. Pyrosequencing analysis identified Rickettsia in A. maculatum midguts, salivary glands, and saliva, which indicates successful trafficking in the arthropod vector. The identity of Rickettsia spp. was determined based on sequencing the rickettsial outer membrane protein A (rompA) gene. The sequence homology search revealed the presence of R. parkeri, Rickettsia amblyommii, and Rickettsia endosymbiont ofA. maculatum in midgut tissues, whereas the only rickettsia detected in salivary glands was R. parkeri, suggesting it is unique in its ability to migrate from midgut to salivary glands, and colonize this tissue before dissemination to the host. Owing to its importance as an emerging infectious disease, the R. parkeri pathogen burden was quantified by a rompB-based quantitative polymerase chain reaction (qPCR) assay and the diagnostic effectiveness of using R. parkeri polyclonal antibodies in tick tissues was tested. Together, these data indicate that field-collected A. maculatum had a R. parkeri infection rate of 12-32%. This study provides an insight into the A. maculatum microbiome and confirms the presence of R. parkeri, which will serve as the basis for future tick and microbiome interaction studies.