Recently evolved Francisella-like endosymbiont outcompetes an ancient and evolutionarily associated Coxiella-like endosymbiont in the lone star tick (Amblyomma americanum) linked to the Alpha-Gal Syndrome

BackgroundTicks are hematophagous arthropods that transmit various bacterial, viral, and protozoan pathogens of public health significance. The lone star tick (Amblyomma americanum) is an aggressive human-biting tick that transmits bacterial and viral pathogens, and its bites are suspected of eliciting the Alpha-Gal Syndrome, a newly emerged delayed hypersensitivity following consumption of red meat in the United States. While ongoing studies have attempted to investigate the contribution of different tick-inherent factors to the induction of Alpha-Gal syndrome, an otherwise understudied aspect is the contribution of the tick microbiome and specifically obligate endosymbionts to the establishment of the Alpha-Gal syndrome in humans.MethodsHere we utilized a high throughput metagenomic sequencing approach to cataloging the entire microbial communities residing within different developmental stages and tissues of unfed and blood-fed ticks from laboratory-maintained ticks and three new geographical locations in the USA. The quantitative insights into microbial ecology (QIIME2) pipeline were used to perform data analysis and taxonomic classification. Moreover, using a SparCC network construction model, we investigated potential interactions between members of the microbial communities from lab-maintained and field-collected ticks. ResultsOverall, Francisellaceae was the most dominant bacteria identified in the microbiome of both lab-raised and field-collected Am. americanum across all tissues and developmental stages. Likewise, microbial diversity was seen to be significantly higher in field-collected ticks compared to lab-maintained ticks as seen with a higher number of both OTUs and measures of species richness. Several potential positive and negative correlations were identified from our network analysis. We observed a strong positive correlation between Francisellaceae, Rickettsiaceae, and Midichloriaceae in both developmental stages and tissues from lab-maintained ticks, while ovarian tissues had a strong positive correlation of bacteria in the family Xanthobacteraceae and Rhizobiaceae. A negative interaction was observed between Coxiellaceae and Francisellaceae in Illinois, and all the bacteria detected from ticks from Delaware were negatively correlated.ConclusionThis study is the first to catalog the microbiome of Am. americanum throughout its developmental stages and different tissue niches and report the potential replacement of Coxiellaceae by Francisellaceae across developmental stages and tissues tested except in ovarian tissues. These unique and significant findings advance our knowledge and open a new avenue of research to further understand the role of tick microbiome in tick-borne diseases and develop a holistic strategy to control Alpha-Gal syndrome.

Presence of Human Pathogens of the Borrelia burgdorferi sensu lato Complex Shifts the Sequence Read Abundances of Tick Microbiomes in Two German Locations

The distribution of human Lyme borreliosis (LB) is assumed random in Germany, indicating that the human pathogenic species of the Borrelia burgdorferi sensu lato complex (Bb) are similarly distributed as part of the tick microbiome. The aim of this study was to differentiate if the presence of Bb occurs with a defined tick microbiome composition. Furthermore, the effect of location on tick microbiome composition was addressed for two German locations. Therefore, nucleic acid extracts from 82 Borrelia-positive and 118 Borrelia-negative Ixodes ricinus ticks sampled from human hosts in both districts were selected. Nucleic acid extracts were used for human pathogenic Bb species diagnostics based on qPCR and multilocus sequence typing (MLST) and bacterial 16S rRNA gene amplicon sequencing followed by network analyses. As a result, the presence of Bb shifted the sequence read abundances of Candidatus Midichloria, Rickettsia, Pseudomonas, Staphylococcus, and Candidatus Neoehrlichia and their topological roles in the tick microbiome. Moreover, the location was less important in the tick microbiome composition but shifted significantly sequence read abundances of Pseudomonas and Wolbachia as well as the topological role of microbial members. Since the presence of human pathogenic Bb species with other tick-associated pathogens varies regionally, we suggest that a bacterial 16S rRNA gene-based microbiome survey should be implemented in the routine diagnostics for both tick and host if human pathogenic species of Bb were detected. This diagnostic extension will help to optimize therapeutic approaches against Bb infection and co-occurring pathogens.

Anti-Microbiota Vaccines Modulate the Tick Microbiome in a Taxon-Specific Manner

The lack of tools for the precise manipulation of the tick microbiome is currently a major limitation to achieve mechanistic insights into the tick microbiome. Anti-tick microbiota vaccines targeting keystone bacteria of the tick microbiota alter tick feeding, but their impact on the taxonomic and functional profiles of the tick microbiome has not been tested. In this study, we immunized a vertebrate host model (Mus musculus) with live bacteria vaccines targeting keystone (i.e., Escherichia-Shigella) or non-keystone (i.e., Leuconostoc) taxa of tick microbiota and tested the impact of bacterial-specific antibodies (Abs) on the structure and function of tick microbiota. We also investigated the effect of these anti-microbiota vaccines on mice gut microbiota composition. Our results showed that the tick microbiota of ticks fed on Escherichia coli-immunized mice had reduced Escherichia-Shigella abundance and lower species diversity compared to ticks fed on control mice immunized with a mock vaccine. Immunization against keystone bacteria restructured the hierarchy of nodes in co-occurrence networks and reduced the resistance of the bacterial network to taxa removal. High levels of E. coli-specific IgM and IgG were negatively correlated with the abundance of Escherichia-Shigella in tick microbiota. These effects were not observed when Leuconostoc was targeted with vaccination against Leuconostoc mesenteroides. Prediction of functional pathways in the tick microbiome using PICRUSt2 revealed that E. coli vaccination reduced the abundance of lysine degradation pathway in tick microbiome, a result validated by qPCR. In contrast, the gut microbiome of immunized mice showed no significant alterations in the diversity, composition and abundance of bacterial taxa. Our results demonstrated that anti-tick microbiota vaccines are a safe, specific and an easy-to-use tool for manipulation of vector microbiome. These results guide interventions for the control of tick infestations and pathogen infection/transmission.

Anti-microbiota vaccines modulate the tick microbiome in a taxon-specific manner

Anti-tick microbiota vaccines have been shown to impact tick feeding but its specificity has not been demonstrated. In this study we aimed to investigate the impact of immune targeting of keystone microbiota bacteria on tick performance, and tick microbiota structure and function. Vaccination against Escherichia coli, the selected keystone taxon, increased tick engorgement weight and reduced bacterial diversity in Ixodes ricinus ticks compared to those that fed on mice immunized against Leuconostoc mesenteroides, a non-keystone taxon or mock-immunized group. The abundance of Escherichia-Shigella, but not Leuconostoc was significantly reduced in ticks fed on E. coli-immunized mice and this reduction was correlated with a significant increase in host antibodies (Abs) of the isotype IgM and IgG specific to E. coli proteins. This negative correlation was not observed between the abundance of Leuconostoc in ticks and anti-L. mesenteroides Abs in mice. We also demonstrated by co-occurrence network analysis, that immunization against the keystone bacterium restructure the hierarchy of the microbial community in ticks and that anti-tick microbiota vaccines reduced the resistance of networks to directed removal of taxa. Functional pathways analysis showed that immunization with a live bacterial vaccine can also induce taxon-specific changes in the abundance of pathways. Our results demonstrated that anti-tick microbiota vaccines can modulate the tick microbiome and that the modification is specific to the taxon chosen for host immunization. These results guide interventions for the control of tick infestations and pathogen infection/transmission.

Applications of Blocker Nucleic Acids and Non-Metazoan PCR Improves the Discovery of the Eukaryotic Microbiome in Ticks

Ticks serve as important vectors of a variety of pathogens. Recently, the viral and prokaryotic microbiomes in ticks have been explored using next-generation sequencing to understand the physiology of ticks and their interactions with pathogens. However, analyses of eukaryotic communities in ticks are limited, owing to the lack of suitable methods. In this study, we developed new methods to selectively amplify microeukaryote genes in tick-derived DNA by blocking the amplification of the 18S rRNA gene of ticks using artificial nucleic acids: peptide nucleic acids (PNAs) and locked nucleic acids (LNAs). In addition, another PCR using non-metazoan primers, referred to as UNonMet-PCR, was performed for comparison. We performed each PCR using tick-derived DNA and sequenced the amplicons using the Illumina MiSeq platform. Almost all sequences obtained by conventional PCR were derived from ticks, whereas the proportion of microeukaryotic reads and alpha diversity increased upon using the newly developed method. Additionally, the PNA- or LNA-based methods were suitable for paneukaryotic analyses, whereas the UNonMet-PCR method was particularly sensitive to fungi. The newly described methods enable analyses of the eukaryotic microbiome in ticks. We expect the application of these methods to improve our understanding of the tick microbiome.

Borrelia infection in rodent host has dramatic effects on the microbiome of ticks

BackgroundVector-borne diseases remain major causes of human morbidity and mortality. It is increasingly recognized that the community of microbes inhabiting arthropods can strongly affect their vector competence, but the role of the tick microbiome in Borrelia transmission – the cause of Lyme disease – remains unclear.ResultsHere, we use a large-scale experiment to clarify the reciprocal interactions between Borrelia afzelii and the microbiome of Ixodes ricinus, its primary vector. In contrast to other reports, we find that depletion of the bacterial microbiome in larval ticks has no effect on their subsequent acquisition of B. afzelii during blood feeding on infected mice. Rather, exposure to B. afzelii-infected hosts drives pervasive changes to the tick microbiome, decreasing overall bacterial abundance, shifting bacterial community composition, and increasing bacterial diversity. These effects appear to be independent of the acquisition of B. afzelii by ticks, suggesting they are mediated by physiological or immunological aspects of B. afzelii infection in the rodent host.ConclusionsManipulation of the microbiome of I. ricinus larvae had no effect on their ability to acquire B. afzelii. In contrast, B. afzelii infection in the mouse had dramatic effects on the composition of the gut microbiome in I. ricinus nymphs. Our study demonstrates that vector-borne infections in the vertebrate host shape the microbiome of the arthropod vector.

Can the impact of climate change on the tick microbiome bring a new epidemiological landscape to tick-borne diseases?

This expert opinion focuses on the possible impact of climate change on the tick microbiome, with potential consequences for disease ecology. Within the text, 'microbiome' refers to the microorganisms and their genes, whereas 'microbiota' only refers to the microbes themselves. 'Holobiont' refers to the close association between host and microbes, that together form a discrete ecological unit.

Ixodes scapularis microbiome correlates with life stage, not the presence of human pathogens, in ticks submitted for diagnostic testing

Ticks are globally distributed arthropods and a public health concern due to the many human pathogens they carry and transmit, including the causative agent of Lyme disease, Borrelia burgdorferi. As tick species’ ranges increase, so do the number of reported tick related illnesses. The microbiome is a critical part of understanding arthropod biology, and the microbiome of pathogen vectors may provide critical insight into disease transmission and management. Yet we lack a comprehensive understanding of the microbiome of wild ticks, including what effect the presence of multiple tick-borne pathogens (TBPs) has on the microbiome. In this study we chose samples based on life stage (adult or nymph) and which TBPs were present. We used DNA from previously extracted Ixodes scapularis ticks that tested positive for zero, one, two or three common TBPs (B. burgdorferi, B. miyamotoi, Anaplasma phagocytophilum, Babesia microti). We produced 16S rRNA amplicon data for the whole tick microbiome and compared samples across TBPs status, single vs multiple coinfections, and life stages. Focusing on samples with a single TBP, we found no significant differences in microbiome diversity in ticks that were infected with B. burgdorferi and ticks with no TBPs. When comparing multiple TBPs, we found no significant difference in both alpha and beta diversity between ticks with a single TBP and ticks with multiple TBPs. Removal of TBPs from the microbiome did not alter alpha or beta diversity results. Life stage significantly correlated to variation in beta diversity and nymphs had higher alpha diversity than adult ticks. Rickettsia, a common tick endosymbiont, was the most abundant genus. This study confirms that the wild tick microbiome is highly influenced by life stage and much less by the presence of human pathogenic bacteria.