tick-microbiome-characterization-next-generation-16s-rrna-amplicon

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

San Francisco State University

The disease chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has caused dramatic amphibian population declines and extinctions in Australia, Central and North America, and Europe. Bd is associated with >200 species extinctions of amphibians, but not all species that become infected are susceptible to the disease. Specifically, Bd has rapidly emerged in some areas of the world, such as in Australia, USA, and throughout Central and South America, causing population and species collapse. The mechanism behind the rapid global emergence of the disease is poorly understood, in part due to an incomplete picture of the global distribution of Bd. At present, there is a considerable amount of geographic bias in survey effort for Bd, with Asia being the most neglected continent. To date, Bd surveys have been published for few Asian countries, and infected amphibians have been reported only from Indonesia, South Korea, China and Japan. Thus far, there have been no substantiated reports of enigmatic or suspected disease-caused population declines of the kind that has been attributed to Bd in other areas. In order to gain a more detailed picture of the distribution of Bd in Asia, we undertook a widespread, opportunistic survey of over 3,000 amphibians for Bd throughout Asia and adjoining Papua New Guinea. Survey sites spanned 15 countries, approximately 36° latitude, 111° longitude, and over 2000 m in elevation. Bd prevalence was very low throughout our survey area (2.35% overall) and infected animals were not clumped as would be expected in epizootic events. This suggests that Bd is either newly emerging in Asia, endemic at low prevalence, or that some other ecological factor is preventing Bd from fully invading Asian amphibians. The current observed pattern in Asia differs from that in many other parts of the world.

Is chytridiomycosis an emerging infectious disease in Asia?

Global changes such as deforestation, climate change, and invasive species have the potential to greatly alter zoonotic disease systems through impacts on biodiversity. This study examined the impact of the invasive pathogen that causes sudden oak death (SOD) on the ecology of Lyme disease in California. The Lyme disease bacterium, Borrelia burgdorferi, is maintained in the far western United States by a suite of animal reservoirs including the dusky-footed woodrat (Neotoma fuscipes) and deer mouse (Peromyscus maniculatus), and is transmitted by the western black-legged tick (Ixodes pacificus). Other vertebrates, such as the western fence lizard (Sceloporus occidentalis), are important tick hosts but are not reservoirs of the pathogen. Previous work found that higher levels of SOD are correlated with greater abundance of P. maniculatus and S. occidentalis and lower N. fuscipes abundance. Here we model the contribution of these tick hosts to Lyme disease risk and also evaluate the potential impact of SOD on infection prevalence of the tick vector. By empirically parameterizing a static model with field and laboratory data on tick hosts, we predict that SOD reduces an important index of disease risk, nymphal infection prevalence, leading to a reduction in Lyme disease risk in certain coastal woodlands. Direct observational analysis of the impact of SOD on nymphal infection prevalence supports these model results. This study underscores the important direct and indirect impacts of invasive plant pathogens on biodiversity, the transmission cycles of zoonotic diseases, and ultimately human health.

Impacts of an introduced forest pathogen on the risk of Lyme disease in California.

Viruses in the family Bunyaviridae infect a wide range of plant, insect, and animal hosts. Tick-borne bunyaviruses in the Phlebovirus genus, including Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) in China, Heartland virus (HRTV) in the United States, and Bhanja virus in Eurasia and Africa have been associated with acute febrile illness in humans. Here we sought to characterize the growth characteristics and genome of Lone Star virus (LSV), an unclassified bunyavirus originally isolated from the lone star tick Amblyomma americanum. LSV was able to infect both human (HeLa) and monkey (Vero) cells. Cytopathic effects were seen within 72 h in both cell lines; vacuolization was observed in infected Vero, but not HeLa, cells. Viral culture supernatants were examined by unbiased deep sequencing and analysis using an in-house developed rapid computational pipeline for viral discovery, which definitively identified LSV as a phlebovirus. De novo assembly of the full genome revealed that LSV is highly divergent, sharing <61% overall amino acid identity with any other bunyavirus. Despite this sequence diversity, LSV was found by phylogenetic analysis to be part of a well-supported clade that includes members of the Bhanja group viruses, which are most closely related to SFSTV/HRTV. The genome sequencing of LSV is a critical first step in developing diagnostic tools to determine the risk of arbovirus transmission by A. americanum, a tick of growing importance given its expanding geographic range and competence as a disease vector. This study also underscores the power of deep sequencing analysis in rapidly identifying and sequencing the genomes of viruses of potential clinical and public health significance.

The genome sequence of Lone Star virus, a highly divergent bunyavirus found in the Amblyomma americanum tick.

Vector-borne pathogens are transmitted between vertebrate hosts and arthropod vectors, two immensely different environments for the pathogen. There is further differentiation among vertebrate hosts that often have complex, species-specific immunological responses to the pathogen. All this presents a heterogeneous environmental and immunological landscape with possible consequences on the population genetic structure of the pathogen. We evaluated the differential genetic diversity of the Lyme disease pathogen, Borrelia burgdorferi, in its vector, the western black-legged tick (Ixodes pacificus), and in its mammal host community using the 5S-23S rRNA intergenic spacer region. We found differences in haplotype distribution of B. burgdorferi in tick populations from two counties in California as well as between a sympatric tick and vertebrate host community. In addition, we found that three closely related haplotypes consistently occurred in high frequency in all sample types. Lastly, our study found lower species diversity of the B. burgdorferi species complex, known as B. burgdorferi sensu lato, in small mammal hosts versus the tick populations in a sympatric study area. 

Comparative genetic diversity of Lyme disease bacteria in Northern Californian ticks and their vertebrate hosts.

Lyme disease is a tick-borne illness caused by the bacterium Borrelia burgdorferi, and approximately 10 to 20% of patients report persistent symptoms lasting months to years despite appropriate treatment with antibiotics. To gain insights into the molecular basis of acute Lyme disease and the ensuing development of post-treatment symptoms, we conducted a longitudinal transcriptome study of 29 Lyme disease patients (and 13 matched controls) enrolled at the time of diagnosis and followed for up to 6 months. The differential gene expression signature of Lyme disease following the acute phase of infection persisted for at least 3 weeks and had fewer than 44% differentially expressed genes (DEGs) in common with other infectious or noninfectious syndromes. Early Lyme disease prior to antibiotic therapy was characterized by marked upregulation of Toll-like receptor signaling but lack of activation of the inflammatory T-cell apoptotic and B-cell developmental pathways seen in other acute infectious syndromes. Six months after completion of therapy, Lyme disease patients were found to have 31 to 60% of their pathways in common with three different immune-mediated chronic diseases. No differential gene expression signature was observed between Lyme disease patients with resolved illness to those with persistent symptoms at 6 months post-treatment. The identification of a sustained differential gene expression signature in Lyme disease suggests that a panel of selected human host-based biomarkers may address the need for sensitive clinical diagnostics during the "window period" of infection prior to the appearance of a detectable antibody response and may also inform the development of new therapeutic targets.

Longitudinal Transcriptome Analysis Reveals a Sustained Differential Gene Expression Signature in Patients Treated for Acute Lyme Disease.

Lyme disease, a zoonotic disease, is the most prevalent vector-borne disease in the Northern Hemisphere. Diversity of the vector (tick) microbiome can impact pathogen transmission, yet the biotic and abiotic factors that drive microbiome diversity are largely unresolved, especially under natural, field conditions. We describe the microbiome of Ixodes pacificus ticks, the vector for Lyme disease in the western United States, and show a strong impact of host blood meal identity on tick microbiome species richness and composition. Western fence lizards, a host that is refractory to the Lyme disease pathogen, significantly reduces microbiome diversity in ticks relative to ticks that feed on a mammalian reservoir host. Host blood meal-driven reduction of tick microbiome diversity may have lifelong repercussions on I. pacificus vector competency and ultimately disease dynamics.

Tick microbiome and pathogen acquisition altered by host blood meal.

Tick-borne diseases, particularly Lyme disease, are emerging across the northern hemisphere. In order to manage emerging diseases and predict where emergence will likely occur, it is necessary to understand the factors influencing the distribution, abundance and infection prevalence of vector species. In North America, Lyme disease is the most common vector-borne disease and is transmitted by blacklegged ticks. This study aimed to explore the abiotic and environmental drivers of density and infection prevalence of western blacklegged ticks (Ixodes pacificus) in southern California, an understudied and densely populated region of North America.

Lyme disease risk in southern California: abiotic and environmental drivers of Ixodes pacificus (Acari: Ixodidae) density and infection prevalence with Borrelia burgdorferi.

Active-learning pedagogies have been repeatedly demonstrated to produce superior learning gains with large effect sizes compared with lecture-based pedagogies. Shifting large numbers of college science, technology, engineering, and mathematics (STEM) faculty to include any active learning in their teaching may retain and more effectively educate far more students than having a few faculty completely transform their teaching, but the extent to which STEM faculty are changing their teaching methods is unclear. Here, we describe the development and application of the machine-learning-derived algorithm Decibel Analysis for Research in Teaching (DART), which can analyze thousands of hours of STEM course audio recordings quickly, with minimal costs, and without need for human observers. DART analyzes the volume and variance of classroom recordings to predict the quantity of time spent on single voice (e.g., lecture), multiple voice (e.g., pair discussion), and no voice (e.g., clicker question thinking) activities. Applying DART to 1,486 recordings of class sessions from 67 courses, a total of 1,720 h of audio, revealed varied patterns of lecture (single voice) and nonlecture activity (multiple and no voice) use. We also found that there was significantly more use of multiple and no voice strategies in courses for STEM majors compared with courses for non-STEM majors, indicating that DART can be used to compare teaching strategies in different types of courses. Therefore, DART has the potential to systematically inventory the presence of active learning with ∼90% accuracy across thousands of courses in diverse settings with minimal effort.

Classroom sound can be used to classify teaching practices in college science courses.

Lyme disease is the most common tick-borne disease in temperate regions of North America, Europe and Asia, and the number of reported cases has increased in many regions as landscapes have been altered. Although there has been extensive work on the ecology and epidemiology of this disease in both Europe and North America, substantial uncertainty exists about fundamental aspects that determine spatial and temporal variation in both disease risk and human incidence, which hamper effective and efficient prevention and control. Here we describe areas of consensus that can be built on, identify areas of uncertainty and outline research needed to fill these gaps to facilitate predictive models of disease risk and the development of novel disease control strategies. Key areas of uncertainty include: (i) the precise influence of deer abundance on tick abundance, (ii) how tick populations are regulated, (iii) assembly of host communities and tick-feeding patterns across different habitats, (iv) reservoir competence of host species, and (v) pathogenicity for humans of different genotypes of  Filling these knowledge gaps will improve Lyme disease prevention and control and provide general insights into the drivers and dynamics of this emblematic multi-host-vector-borne zoonotic disease.This article is part of the themed issue 'Conservation, biodiversity and infectious disease: scientific evidence and policy implications'.

Lyme disease ecology in a changing world: consensus, uncertainty and critical gaps for improving control.

An increasing number of emerging tick-borne diseases has been reported in the United States since the 1970s. Using metagenomic next generation sequencing, we detected nucleic acid sequences from 2 novel viruses in the family Bunyaviridae and an emerging human rickettsial pathogen, Rickettsia philipii, in a population of the Pacific Coast tick, Dermacentor occidentalis in Mendocino County sampled annually from 2011 to 2014. A total of 250 adults of this human-biting, generalist tick were collected from contiguous chaparral and grassland habitats, and RNA from each individually extracted tick was deep sequenced to an average depth of 7.3 million reads. We detected a Francisella endosymbiont in 174 ticks (70%), and Rickettsia spp. in 19 ticks (8%); Rickettsia-infected ticks contained R. rhipicephali (16 of 250, 6.4%) or R. philipii (3 of 250,1.2%), the agent of eschar-associated febrile illness in humans. The genomes of 2 novel bunyaviruses (>99% complete) in the genera Nairovirus and Phlebovirus were also identified and found to be present in 20-91% of ticks, depending on the year of collection. The high prevalence of these bunyaviruses in sampled Dermacentor ticks suggests that they may be viral endosymbionts, although further studies are needed to determine whether they are infectious for vertebrate hosts, especially humans, and their potential role in tick ecology.

Metagenomic-based Surveillance of Pacific Coast tick Dermacentor occidentalis Identifies Two Novel Bunyaviruses and an Emerging Human Ricksettsial Pathogen.

The fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis, has been linked to catastrophic amphibian declines throughout the world. Amphibians differ in their vulnerability to chytridiomycosis; some species experience epizootics followed by collapse while others exhibit stable host/pathogen dynamics where most amphibian hosts survive in the presence of Bd (e.g., in the enzootic state). Little is known about the factors that drive the transition between the two disease states within a community, or whether populations of species that survived the initial epizootic are stable, yet this information is essential for conservation and theory. Our study focuses on a diverse Peruvian amphibian community that experienced a Bd-caused collapse. We explore host/Bd dynamics of eight surviving species a decade after the mass extinction by using population level disease metrics and Bd-susceptibility trials. We found that three of the eight species continue to be susceptible to Bd, and that their populations are declining. Only one species is growing in numbers and it was non-susceptible in our trials. Our study suggests that some species remain vulnerable to Bd and exhibit ongoing population declines in enzootic systems where Bd-host dynamics are assumed to be stable.

Epizootic to enzootic transition of a fungal disease in tropical Andean frogs: Are surviving species still susceptible?

Vector-borne pathogens are increasingly found to interact with the vector's microbiome, influencing disease transmission dynamics. However, the processes that regulate the formation and development of the microbiome are largely unexplored for most tick species, an emerging group of disease vectors. It is not known how much of the tick microbiome is acquired through vertical transmission vs. horizontally from the environment or interactions with bloodmeal sources. Using 16S rRNA sequencing, we examined the microbiome of Ixodes pacificus, the vector of Lyme disease in the western USA, across life stages and infection status. We also characterized microbiome diversity in field and laboratory-collected nymphal ticks to determine how the surrounding environment affects microbiome diversity. We found a decrease in both species richness and evenness as the tick matures from larva to adult. When the dominant Rickettsial endosymbiont was computationally removed from the tick microbial community, we found that infected nymphs had lower species evenness than uninfected ticks, suggesting that lower microbiome diversity is associated with pathogen transmission in wild-type ticks. Furthermore, laboratory-reared nymph microbiome diversity was found to be compositionally distinct and significantly depauperate relative to field-collected nymphs. These results highlight unique patterns in the microbial community of I. pacificus that is distinct from other tick species. We provide strong evidence that ticks acquire a significant portion of their microbiome through exposure to their environment despite a loss of overall diversity through life stages. We provide evidence that loss of microbial diversity is at least in part due to elimination of microbial diversity with bloodmeal feeding but other factors may also play a role.

Vertical vs. horizontal transmission of the microbiome in a key disease vector, Ixodes pacificus.

Many efforts to improve science teaching in higher education focus on a few faculty members at an institution at a time, with limited published evidence on attempts to engage faculty across entire departments. We created a long-term, department-wide collaborative professional development program, Biology Faculty Explorations in Scientific Teaching (Biology FEST). Across 3 years of Biology FEST, 89% of the department's faculty completed a weeklong scientific teaching institute, and 83% of eligible instructors participated in additional semester-long follow-up programs. A semester after institute completion, the majority of Biology FEST alumni reported adding active learning to their courses. These instructor self-reports were corroborated by audio analysis of classroom noise and surveys of students in biology courses on the frequency of active-learning techniques used in classes taught by Biology FEST alumni and nonalumni. Three years after Biology FEST launched, faculty participants overwhelmingly reported that their teaching was positively affected. Unexpectedly, most respondents also believed that they had improved relationships with departmental colleagues and felt a greater sense of belonging to the department. Overall, our results indicate that biology department-wide collaborative efforts to develop scientific teaching skills can indeed attract large numbers of faculty, spark widespread change in teaching practices, and improve departmental relations.

Article	Year
Is chytridiomycosis an emerging infectious disease in Asia? PloS one\| PubMed ID: 21887238	2011
Impacts of an introduced forest pathogen on the risk of Lyme disease in California. Vector borne and zoonotic diseases (Larchmont, N.Y.)\| PubMed ID: 22607076	2012
The genome sequence of Lone Star virus, a highly divergent bunyavirus found in the Amblyomma americanum tick. PloS one\| PubMed ID: 23637969	2013
Comparative genetic diversity of Lyme disease bacteria in Northern Californian ticks and their vertebrate hosts. Ticks and tick-borne diseases\| PubMed ID: 25843810	2015
Longitudinal Transcriptome Analysis Reveals a Sustained Differential Gene Expression Signature in Patients Treated for Acute Lyme Disease. mBio\| PubMed ID: 26873097	2016
Tick microbiome and pathogen acquisition altered by host blood meal. The ISME journal\| PubMed ID: 27858931	2017
Lyme disease risk in southern California: abiotic and environmental drivers of Ixodes pacificus (Acari: Ixodidae) density and infection prevalence with Borrelia burgdorferi. Parasites & vectors\| PubMed ID: 28057067	2017
Classroom sound can be used to classify teaching practices in college science courses. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 28265087	2017
Lyme disease ecology in a changing world: consensus, uncertainty and critical gaps for improving control. Philosophical transactions of the Royal Society of London. Series B, Biological sciences\| PubMed ID: 28438910	2017
Metagenomic-based Surveillance of Pacific Coast tick Dermacentor occidentalis Identifies Two Novel Bunyaviruses and an Emerging Human Ricksettsial Pathogen. Scientific reports\| PubMed ID: 28947798	2017
Epizootic to enzootic transition of a fungal disease in tropical Andean frogs: Are surviving species still susceptible? PloS one\| PubMed ID: 29040327	2017
Vertical vs. horizontal transmission of the microbiome in a key disease vector, Ixodes pacificus. Molecular ecology\| PubMed ID: 29178531	2017
Collectively Improving Our Teaching: Attempting Biology Department-wide Professional Development in Scientific Teaching. CBE life sciences education\| PubMed ID: 29326102	2018

Andrea Swei

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