Variation among strains of Borrelia burgdorferi in host tissue abundance and lifetime transmission determine the population strain structure in nature.

Pathogen life history theory assumes a positive relationship between pathogen load in host tissues and pathogen transmission. Empirical evidence for this relationship is surprisingly rare due to the difficulty of measuring transmission for many pathogens. The comparative method, where a common host is experimentally infected with a set of pathogen strains, is a powerful approach for investigating the relationships between pathogen load and transmission. The validity of such experimental estimates of strain-specific transmission is greatly enhanced if they can predict the pathogen population strain structure in nature. Borrelia burgdorferi is a multi-strain, tick-borne spirochete that causes Lyme disease in North America. This study used 11 field-collected strains of B. burgdorferi, a rodent host (Mus musculus, C3H/HeJ) and its tick vector (Ixodes scapularis) to determine the relationship between pathogen load in host tissues and lifetime host-to-tick transmission (HTT). Mice were experimentally infected via tick bite with 1 of 11 strains. Lifetime HTT was measured by infesting mice with I. scapularis larval ticks on 3 separate occasions. The prevalence and abundance of the strains in the mouse tissues and the ticks were determined by qPCR. We used published databases to obtain estimates of the frequencies of these strains in wild I. scapularis tick populations. Spirochete loads in ticks and lifetime HTT varied significantly among the 11 strains of B. burgdorferi. Strains with higher spirochete loads in the host tissues were more likely to infect feeding larval ticks, which molted into nymphal ticks that had a higher probability of B. burgdorferi infection (i.e., higher HTT). Our laboratory-based estimates of lifetime HTT were predictive of the frequencies of these strains in wild I. scapularis populations. For B. burgdorferi, the strains that establish high abundance in host tissues and that have high lifetime transmission are the strains that are most common in nature.

Selective whole-genome amplification reveals population genetics of Leishmania braziliensis directly from patient skin biopsies.

In Brazil, Leishmania braziliensis is the main causative agent of the neglected tropical disease, cutaneous leishmaniasis (CL). CL presents on a spectrum of disease severity with a high rate of treatment failure. Yet the parasite factors that contribute to disease presentation and treatment outcome are not well understood, in part because successfully isolating and culturing parasites from patient lesions remains a major technical challenge. Here we describe the development of selective whole genome amplification (SWGA) for Leishmania and show that this method enables culture-independent analysis of parasite genomes obtained directly from primary patient skin samples, allowing us to circumvent artifacts associated with adaptation to culture. We show that SWGA can be applied to multiple Leishmania species residing in different host species, suggesting that this method is broadly useful in both experimental infection models and clinical studies. SWGA carried out directly on skin biopsies collected from patients in Corte de Pedra, Bahia, Brazil, showed extensive genomic diversity. Finally, as a proof-of-concept, we demonstrated that SWGA data can be integrated with published whole genome data from cultured parasite isolates to identify variants unique to specific geographic regions in Brazil where treatment failure rates are known to be high. SWGA provides a relatively simple method to generate Leishmania genomes directly from patient samples, unlocking the potential to link parasite genetics with host clinical phenotypes.

Evolution of intermediate latency strategies in seasonal parasites.

Traditional mechanistic trade-offs between transmission and parasite latency period length are foundational for nearly all theories on the evolution of parasite life-history strategies. Prior theoretical studies demonstrate that seasonal host activity can generate a trade-off for obligate-host killer parasites that selects for intermediate latency periods in the absence of a mechanistic trade-off between transmission and latency period lengths. Extensions of these studies predict that host seasonal patterns can lead to evolutionary bistability for obligate-host killer parasites in which two evolutionarily stable strategies, a shorter and longer latency period, are possible. Here we demonstrate that these conclusions from previously published studies hold for non-obligate host killer parasites. That is, seasonal host activity can select for intermediate parasite latency periods for non-obligate killer parasites in the absence of a trade-off between transmission and latency period length and can maintain multiple evolutionarily stable parasite life-history strategies. These results reinforce the hypothesis that host seasonal activity can act as a major selective force on parasite life-history evolution by extending the narrower prior theory to encompass a greater range of disease systems.

A fast machine-learning-guided primer design pipeline for selective whole genome amplification.

Addressing many of the major outstanding questions in the fields of microbial evolution and pathogenesis will require analyses of populations of microbial genomes. Although population genomic studies provide the analytical resolution to investigate evolutionary and mechanistic processes at fine spatial and temporal scales-precisely the scales at which these processes occur-microbial population genomic research is currently hindered by the practicalities of obtaining sufficient quantities of the relatively pure microbial genomic DNA necessary for next-generation sequencing. Here we present swga2.0, an optimized and parallelized pipeline to design selective whole genome amplification (SWGA) primer sets. Unlike previous methods, swga2.0 incorporates active and machine learning methods to evaluate the amplification efficacy of individual primers and primer sets. Additionally, swga2.0 optimizes primer set search and evaluation strategies, including parallelization at each stage of the pipeline, to dramatically decrease program runtime. Here we describe the swga2.0 pipeline, including the empirical data used to identify primer and primer set characteristics, that improve amplification performance. Additionally, we evaluate the novel swga2.0 pipeline by designing primer sets that successfully amplify Prevotella melaninogenica, an important component of the lung microbiome in cystic fibrosis patients, from samples dominated by human DNA.

Development of an mRNA-lipid nanoparticle vaccine against Lyme disease.

Lyme disease is the most common vector-borne infectious disease in the United States, in part because a vaccine against it is not currently available for humans. We propose utilizing the lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) platform to generate a Lyme disease vaccine like the successful clinical vaccines against SARS-CoV-2. Of the antigens expressed by Borrelia burgdorferi, the causative agent of Lyme disease, outer surface protein A (OspA) is the most promising candidate for vaccine development. We have designed and synthesized an OspA-encoding mRNA-LNP vaccine and compared its immunogenicity and protective efficacy to an alum-adjuvanted OspA protein subunit vaccine. OspA mRNA-LNP induced superior humoral and cell-mediated immune responses in mice after a single immunization. These potent immune responses resulted in protection against bacterial infection. Our study demonstrates that highly efficient mRNA vaccines can be developed against bacterial targets.

Deployment of a Reservoir-Targeted Vaccine Against Borrelia burgdorferi Reduces the Prevalence of Babesia microti Coinfection in Ixodes scapularis Ticks.

In the Northeast and upper Midwest of the United States, Babesia microti and Borrelia burgdorferi use Ixodes scapularis ticks as vector and Peromyscus leucopus mice as major reservoir host. We previously established, in a 5-year field trial, that a reservoir-targeted outer surface protein A vaccine reduces the prevalence of B. burgdorferi-infected ticks. We accessed ticks and mouse blood samples collected during the trial, extracted total DNA, and amplified the B. microti 18S rRNA gene. Vaccine deployment reduced the prevalence of ticks coinfected with B. microti and that of mice infected with B. microti. Breaking the enzootic cycle of B. burgdorferi may reduce the incidence of babesiosis.

Associations of Anaplasma phagocytophilum Bacteria Variants in Ixodes scapularis Ticks and Humans, New York, USA.

Anaplasmosis, caused by the tickborne bacterium Anaplasma phagocytophilum, is an emerging public health threat in the United States. In the northeastern United States, the blacklegged tick (Ixodes scapularis) transmits the human pathogenic genetic variant of A. phagocytophilum (Ap-ha) and a nonpathogenic variant (Ap-V1). New York has recently experienced a rapid and geographically focused increase in cases of anaplasmosis. We analyzed A. phagocytophilum-infected I. scapularis ticks collected across New York during 2008-2020 to differentiate between variants and calculate an entomological risk index (ERI) for each. Ap-ha ERI varied between regions and increased in all regions during the final years of the study. Space-time scan analyses detected expanding clusters of Ap-ha located within documented anaplasmosis hotspots. Ap-ha ERI was more positively correlated with anaplasmosis incidence than non-genotyped A. phagocytophilum ERI. Our findings help elucidate the relationship between the spatial ecology of A. phagocytophilum variants and anaplasmosis.

Host Phenology Can Select for Multiple Stable Parasite Virulence Strategies in Obligate Killer Parasites.

AbstractThe timing of seasonal host activity, or host phenology, is an important driver of parasite transmission dynamics and evolution. Despite the vast diversity of parasites in seasonal environments, the impact of phenology on parasite diversity remains relatively understudied. For example, little is known about the selective pressures and environmental conditions that favor a monocyclic strategy (complete a single cycle of infection per season) or a polycyclic strategy (complete multiple cycles). Here, we present a mathematical model that demonstrates that seasonal host activity patterns can generate evolutionary bistability in which two evolutionarily stable strategies (ESSs) are possible. The ESS that a particular system reaches is a function of the virulence strategy initially introduced into the system. The results demonstrate that host phenology can, in theory, maintain diverse parasite strategies among isolated geographic locations.

Borrelia burgdorferi strain and host sex influence pathogen prevalence and abundance in the tissues of a laboratory rodent host.

Experimental infections with different pathogen strains give insight into pathogen life history traits. The purpose of the present study was to compare variation in tissue infection prevalence and spirochete abundance among strains of Borrelia burgdorferi in a rodent host (Mus musculus, C3H/HeJ). Male and female mice were experimentally infected via tick bite with one of 12 strains. Ear tissue biopsies were taken at days 29, 59 and 89 postinfection, and seven tissues were collected at necropsy. The presence and abundance of spirochetes in the mouse tissues were measured by quantitative polymerase chain reaction. To determine the frequencies of our strains in nature, their multilocus sequence types were matched to published data sets. For the infected mice, 56.6% of the tissues were infected with B. burgdorferi. The mean spirochete load in the mouse necropsy tissues varied 4.8-fold between the strains. The mean spirochete load in the ear tissue biopsies decreased rapidly over time for some strains. The percentage of infected tissues in male mice (65.4%) was significantly higher compared to female mice (50.5%). The mean spirochete load in the seven tissues was 1.5× higher in male mice compared to female mice; this male bias was 15.3× higher in the ventral skin. Across the 11 strains, the mean spirochete loads in the infected mouse tissues were positively correlated with the strain-specific frequencies in their tick vector populations. The study suggests that laboratory-based estimates of pathogen abundance in host tissues can predict the strain composition of this important tick-borne pathogen in nature.

Epidemiology and Spatial Emergence of Anaplasmosis, New York, USA, 2010‒2018.

Human granulocytic anaplasmosis, a tickborne disease caused by the bacterium Anaplasma phagocytophilum, was first identified during 1994 and is now an emerging public health threat in the United States. New York state (NYS) has experienced a recent increase in the incidence of anaplasmosis. We analyzed human case surveillance and tick surveillance data collected by the NYS Department of Health for spatiotemporal patterns of disease emergence. We describe the epidemiology and growing incidence of anaplasmosis cases reported during 2010-2018. Spatial analysis showed an expanding hot spot of anaplasmosis in the Capital Region, where incidence increased >8-fold. The prevalence of A. phagocytophilum increased greatly within tick populations in the Capital Region over the same period, and entomologic risk factors were correlated with disease incidence at a local level. These results indicate that anaplasmosis is rapidly emerging in a geographically focused area of NYS, likely driven by localized changes in exposure risk.

Evolutionary Genetics of Borrelia.

The genus Borrelia consists of evolutionarily and genetically diverse bacterial species that cause a variety of diseases in humans and domestic animals. These vector-borne spirochetes can be classified into two major evolutionary groups, the Lyme borreliosis clade and the relapsing fever clade, both of which have complex transmission cycles during which they interact with multiple host species and arthropod vectors. Molecular, ecological, and evolutionary studies have each provided significant contributions towards our understanding of the natural history, biology and evolutionary genetics of Borrelia species; however, integration of these studies is required to identify the evolutionary causes and consequences of the genetic variation within and among Borrelia species. For example, molecular and genetic studies have identified the adaptations that maximize fitness components throughout the Borrelia lifecycle and enhance transmission efficacy but provide limited insights into the evolutionary pressures that have produced them. Ecological studies can identify interactions between Borrelia species and the vertebrate hosts and arthropod vectors they encounter and the resulting impact on the geographic distribution and abundance of spirochetes but not the genetic or molecular basis underlying these interactions. In this review we discuss recent findings on the evolutionary genetics from both of the evolutionarily distinct clades of Borrelia species. We focus on connecting molecular interactions to the ecological processes that have driven the evolution and diversification of Borrelia species in order to understand the current distribution of genetic and molecular variation within and between Borrelia species.

Genetics of Borrelia burgdorferi.

The spirochetes in the Borrelia burgdorferi sensu lato genospecies group cycle in nature between tick vectors and vertebrate hosts. The current assemblage of B. burgdorferi sensu lato, of which three species cause Lyme disease in humans, originated from a rapid species radiation that occurred near the origin of the clade. All of these species share a unique genome structure that is highly segmented and predominantly composed of linear replicons. One of the circular plasmids is a prophage that exists as several isoforms in each cell and can be transduced to other cells, likely contributing to an otherwise relatively anemic level of horizontal gene transfer, which nevertheless appears to be adequate to permit strong natural selection and adaptation in populations of B. burgdorferi. Although the molecular genetic toolbox is meager, several antibiotic-resistant mutants have been isolated, and the resistance alleles, as well as some exogenous genes, have been fashioned into markers to dissect gene function. Genetic studies have probed the role of the outer membrane lipoprotein OspC, which is maintained in nature by multiple niche polymorphisms and negative frequency-dependent selection. One of the most intriguing genetic systems in B. burgdorferi is vls recombination, which generates antigenic variation during infection of mammalian hosts.

Evolution and population genomics of the Lyme borreliosis pathogen, Borrelia burgdorferi.

Population genomic studies have the potential to address many unresolved questions about microbial pathogens by facilitating the identification of genes underlying ecologically important traits, such as novel virulence factors and adaptations to humans or other host species. Additionally, this framework improves estimations of population demography and evolutionary history to accurately reconstruct recent epidemics and identify the molecular and environmental factors that resulted in the outbreak. The Lyme disease bacterium, Borrelia burgdorferi, exemplifies the power and promise of the application of population genomics to microbial pathogens. We discuss here the future of evolutionary studies in B. burgdorferi, focusing on the primary evolutionary forces of horizontal gene transfer, natural selection, and migration, as investigations transition from analyses of single genes to genomes.

swga: a primer design toolkit for selective whole genome amplification.

MOTIVATION: Population genomic analyses are often hindered by difficulties in obtaining sufficient numbers of genomes for analysis by DNA sequencing. Selective whole-genome amplification (SWGA) provides an efficient approach to amplify microbial genomes from complex backgrounds for sequence acquisition. However, the process of designing sets of primers for this method has many degrees of freedom and would benefit from an automated process to evaluate the vast number of potential primer sets. RESULTS: Here, we present swga , a program that identifies primer sets for SWGA and evaluates them for efficiency and selectivity. We used swga to design and test primer sets for the selective amplification of Wolbachia pipientis genomic DNA from infected Drosophila melanogaster and Mycobacterium tuberculosis from human blood. We identify primer sets that successfully amplify each against their backgrounds and describe a general method for using swga for arbitrary targets. In addition, we describe characteristics of primer sets that correlate with successful amplification, and present guidelines for implementation of SWGA to detect new targets. AVAILABILITY AND IMPLEMENTATION: Source code and documentation are freely available on https://www.github.com/eclarke/swga . The program is implemented in Python and C and licensed under the GNU Public License. CONTACT: ecl@mail.med.upenn.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Interactions between host immune response and antigenic variation that control Borrelia burgdorferi population dynamics.

The population dynamics of pathogens within hosts result from interactions between host immune responses and mechanisms of the pathogen to evade or resist immune responses. Vertebrate hosts have evolved adaptive immune responses to eliminate the infection, while many pathogens evade immune clearance through altering surface antigens. Such interactions can result in a characteristic pattern of pathogen population dynamics within hosts consisting of population growth after infection, rapid population decline following specific immune responses, followed by persistence at low densities during a chronic infection stage. Despite the medical importance of chronic infections, little is known about the conditions of the interactions between variable antigens and the adaptive immune system that cause the characteristic pathogen population dynamics. Using the vls antigenic variation system of the Lyme disease pathogen, Borrelia burgdorferi, as a model system, we investigated conditions of the interaction between the antigenic variation system and the adaptive immune response that can explain the within-host population dynamics of B. burgdorferi using mathematical modelling. This characteristic population dynamic pattern can be explained by models that assume a variable immune removal rate of antibody-bound B. burgdorferi. However, models with a constant immune removal rate could reproduce the rapid population decline of B. burgdorferi populations but not their long-term persistence within hosts using parameter values determined by fitting empirical data. The model predictions, along with the assumptions about the interactions between B. burgdorferi and the immune response, can be tested experimentally to estimate the likelihood that each mechanism affects B. burgdorferi population dynamics in real infections.

Differentiation of reinfection from relapse in recurrent Lyme disease.

BACKGROUND: Erythema migrans is the most common manifestation of Lyme disease. Recurrences are not uncommon, and although they are usually attributed to reinfection rather than relapse of the original infection, this remains somewhat controversial. We used molecular typing of Borrelia burgdorferi isolates obtained from patients with culture-confirmed episodes of erythema migrans to distinguish between relapse and reinfection. METHODS: We determined the genotype of the gene encoding outer-surface protein C (ospC) of B. burgdorferi strains detected in cultures of skin or blood specimens obtained from patients with consecutive episodes of erythema migrans. After polymerase-chain-reaction amplification, ospC genotyping was performed by means of reverse line-blot analysis or DNA sequencing of the nearly full-length gene. Most strains were further analyzed by determining the genotype according to the 16S-23S ribosomal RNA intergenic spacer type, multilocus sequence typing, or both. Patients received standard courses of antibiotics for erythema migrans. RESULTS: B. burgdorferi isolates obtained from 17 patients who received a diagnosis of erythema migrans between 1991 and 2011 and who had 22 paired episodes of this lesion (initial and second episodes) were available for testing. The ospC genotype was found to be different at each initial and second episode. Apparently identical genotypes were identified on more than one occasion in only one patient, at the first and third episodes, 5 years apart, but different genotypes were identified at the second and fourth episodes. CONCLUSIONS: None of the 22 paired consecutive episodes of erythema migrans were associated with the same strain of B. burgdorferi on culture. Our data show that repeat episodes of erythema migrans in appropriately treated patients were due to reinfection and not relapse. (Funded by the National Institutes of Health and the William and Sylvia Silberstein Foundation.).

Natural selection promotes antigenic evolvability.

The hypothesis that evolvability - the capacity to evolve by natural selection - is itself the object of natural selection is highly intriguing but remains controversial due in large part to a paucity of direct experimental evidence. The antigenic variation mechanisms of microbial pathogens provide an experimentally tractable system to test whether natural selection has favored mechanisms that increase evolvability. Many antigenic variation systems consist of paralogous unexpressed 'cassettes' that recombine into an expression site to rapidly alter the expressed protein. Importantly, the magnitude of antigenic change is a function of the genetic diversity among the unexpressed cassettes. Thus, evidence that selection favors among-cassette diversity is direct evidence that natural selection promotes antigenic evolvability. We used the Lyme disease bacterium, Borrelia burgdorferi, as a model to test the prediction that natural selection favors amino acid diversity among unexpressed vls cassettes and thereby promotes evolvability in a primary surface antigen, VlsE. The hypothesis that diversity among vls cassettes is favored by natural selection was supported in each B. burgdorferi strain analyzed using both classical (dN/dS ratios) and Bayesian population genetic analyses of genetic sequence data. This hypothesis was also supported by the conservation of highly mutable tandem-repeat structures across B. burgdorferi strains despite a near complete absence of sequence conservation. Diversification among vls cassettes due to natural selection and mutable repeat structures promotes long-term antigenic evolvability of VlsE. These findings provide a direct demonstration that molecular mechanisms that enhance evolvability of surface antigens are an evolutionary adaptation. The molecular evolutionary processes identified here can serve as a model for the evolution of antigenic evolvability in many pathogens which utilize similar strategies to establish chronic infections.

First arrived takes all: inhibitory priority effects dominate competition between co-infecting Borrelia burgdorferi strains.

BACKGROUND: Within-host microbial communities and interactions among microbes are increasingly recognized as important factors influencing host health and pathogen transmission. The microbial community associated with a host is indeed influenced by a complex network of direct and indirect interactions between the host and the lineages of microbes it harbors, but the mechanisms are rarely established. We investigated the within-host interactions among strains of Borrelia burgdorferi, the causative agent of Lyme disease, using experimental infections in mice. We used a fully crossed-design with three distinct strains, each group of hosts receiving two sequential inoculations. We used data from these experimental infections to assess the effect of coinfection on bacterial dissemination and fitness (by measuring the transmission of bacteria to xenodiagnostic ticks) as well as the effect of coinfection on host immune response compared to single infection. RESULTS: The infection and transmission data strongly indicate a competitive interaction among B. burgdorferi strains within a host in which the order of appearance of the strain is the main determinant of the competitive outcome. This pattern is well described by the classic priority effect in the ecological literature. In all cases, the primary strain a mouse was infected with had an absolute fitness advantage primarily since it was transmitted an order of magnitude more than the secondary strain. The mechanism of exclusion of the secondary strain is an inhibition of the colonization of mouse tissues, even though 29% of mice showed some evidence of infection by secondary strain. Contrary to expectation, the strong and specific adaptive immune response evoked against the primary strain was not followed by production of immunoglobulins after the inoculation of the secondary strain, neither against strain-specific antigen nor against antigens common to all strains. Hence, the data do not support a major role of the immune response in the observed priority effect. CONCLUSION: The strong inhibitory priority effect is a dominant mechanism underlying competition for transmission between coinfecting B. burgdorferi strains, most likely through resource exploitation. The observed priority effect could shape bacterial diversity in nature, with consequences in epidemiology and evolution of the disease.

Public health impact of strain specific immunity to Borrelia burgdorferi.

BACKGROUND: Lyme disease, caused by Borrelia burgdorferi, is the most common tick-borne infection in the United States. Although humans can be infected by at least 16 different strains of B. burgdorferi, the overwhelming majority of infections are due to only four strains. It was recently demonstrated that patients who are treated for early Lyme disease develop immunity to the specific strain of B. burgdorferi that caused their infection. The aim of this study is to estimate the reduction in cases of Lyme disease in the United States that may occur as a result of type specific immunity. METHODS: The analysis was performed based on three analytical models that assessed the effects of type specific immunity. Observational data on the frequency with which different B. burgdorferi strains cause human infection in culture-confirmed patients with an initial episode of erythema migrans diagnosed between 1991 and 2005 in the Northeastern United States were used in the analyses. RESULTS: Assuming a reinfection rate of 3 % and a total incidence of Lyme disease per year of 300,000, the estimated number of averted cases of Lyme disease per year ranges from 319 to 2378 depending on the duration of type specific immunity and the model used. CONCLUSION: Given the assumptions of the analyses, this analysis suggests that type specific immunity is likely to have public health significance in the United States.

Reservoir targeted vaccine against Borrelia burgdorferi: a new strategy to prevent Lyme disease transmission.

A high prevalence of infection with Borrelia burgdorferi in ixodid ticks is correlated with a high incidence of Lyme disease. The transmission of B. burgdorferi to humans can be disrupted by targeting 2 key elements in its enzootic cycle: the reservoir host and the tick vector. In a prospective 5-year field trial, we show that oral vaccination of wild white-footed mice resulted in outer surface protein A-specific seropositivity that led to reductions of 23% and 76% in the nymphal infection prevalence in a cumulative, time-dependent manner (2 and 5 years, respectively), whereas the proportion of infected ticks recovered from control plots varied randomly over time. Significant decreases in tick infection prevalence were observed within 3 years of vaccine deployment. Implementation of such a long-term public health measure could substantially reduce the risk of human exposure to Lyme disease.