Diversity and host specificity of Borrelia burgdorferi's outer surface protein C (ospC) alleles in synanthropic mammals, with a notable ospC allele U absence from mixed infections.

Interactions among pathogen genotypes that vary in host specificity may affect overall transmission dynamics in multi-host systems. Borrelia burgdorferi, a bacterium that causes Lyme disease, is typically transmitted among wildlife by Ixodes ticks. Despite the existence of many alleles of B. burgdorferi's sensu stricto outer surface protein C (ospC) gene, most human infections are caused by a small number of ospC alleles ["human infectious alleles" (HIAs)], suggesting variation in host specificity associated with ospC. To characterize the wildlife host association of B. burgdorferi's ospC alleles, we used metagenomics to sequence ospC alleles from 68 infected individuals belonging to eight mammalian species trapped at three sites in suburban New Brunswick, New Jersey (USA). We found that multiple allele ("mixed") infections were common. HIAs were most common in mice (Peromyscus spp.) and only one HIA was detected at a site where mice were rarely captured. ospC allele U was exclusively found in chipmunks (Tamias striatus), and although a significant number of different alleles were observed in chipmunks, including HIAs, allele U never co-occurred with other alleles in mixed infections. Our results suggest that allele U may be excluding other alleles, thereby reducing the capacity of chipmunks to act as reservoirs for HIAs.

Lipid rafts can form in the inner and outer membranes of Borrelia burgdorferi and have different properties and associated proteins.

Lipid rafts are microdomains present in the membrane of eukaryotic organisms and bacterial pathogens. They are characterized by having tightly packed lipids and a subset of specific proteins. Lipid rafts are associated with a variety of important biological processes including signaling and lateral sorting of proteins. To determine whether lipid rafts exist in the inner membrane of Borrelia burgdorferi, we separated the inner and outer membranes and analyzed the lipid constituents present in each membrane fraction. We found that both the inner and outer membranes have cholesterol and cholesterol glycolipids. Fluorescence anisotropy and FRET showed that lipids from both membranes can form rafts but have different abilities to do so. The analysis of the biochemically defined proteome of lipid rafts from the inner membrane revealed a diverse set of proteins, different from those associated with the outer membrane, with functions in protein trafficking, chemotaxis and signaling.

Hypercholesterolemia and ApoE deficiency result in severe infection with Lyme disease and relapsing-fever Borrelia.

The Lyme disease (Borrelia burgdorferi) and relapsing-fever (Borrelia hispanica) agents have distinct infection courses, but both require cholesterol for growth. They acquire cholesterol from the environment and process it to form cholesterol glycolipids that are incorporated onto their membranes. To determine whether higher levels of serum cholesterol could enhance the organ burdens of B. burgdorferi and the spirochetemia of B. hispanica in laboratory mice, apolipoprotein E (apoE)-deficient and low-density lipoprotein receptor (LDLR)-deficient mice that produce large amounts of serum cholesterol were infected with both spirochetes. Both apoE- and LDLR-deficient mice infected with B. burgdorferi had an increased number of spirochetes in the joints and inflamed ankles compared with the infected wild-type (WT) mice, suggesting that mutations in cholesterol transport that result in high serum cholesterol levels can affect the pathogenicity of B. burgdorferi. In contrast, elevated serum cholesterol did not lead to an increase in the spirochetemia of B. hispanica. In the LDLR-deficient mice, the course of infection was indistinguishable from the WT mice. However, infection of apoE-deficient mice with B. hispanica resulted in a longer spirochetemia and increased mortality. Together, these results argue for the apoE deficiency, and not hypercholesterolemia, as the cause for the increased severity with B. hispanica. Serum hyperlipidemias are common human diseases that could be a risk factor for increased severity in Lyme disease.

Borrelia burgdorferi†HtrA: evidence for twofold proteolysis of outer membrane protein p66.

In prokaryotes, members of the High Temperature Requirement A (HtrA) family of serine proteases function in the periplasm to degrade damaged or improperly folded membrane proteins. Borrelia burgdorferi, the agent of Lyme disease, codes for a single HtrA homolog. Two-dimensional electrophoresis analysis of B. burgdorferi B31A3 and a strain that overexpresses HtrA (A3HtrAOE) identified a downregulated protein in A3HtrAOE with a mass, pI and MALDI-TOF spectrum consistent with outer membrane protein p66. P66 and HtrA from cellular lysates partitioned into detergent-resistant membranes, which contain cholesterol-glycolipid-rich membrane regions known as lipid rafts, suggesting that HtrA and p66 may reside together in lipid rafts also. This agrees with previous work from our laboratory, which showed that HtrA and p66 are constituents of B. burgdorferi outer membrane vesicles. HtrA degraded p66 in vitro and A3HtrAOE expressed reduced levels of p66 in vivo. Fluorescence confocal microscopy revealed that HtrA and p66 colocalize in the membrane. The association of HtrA and p66 establishes that they could interact efficiently and their protease/substrate relationship provides functional relevance to this interaction. A3HtrAOE also showed reduced levels of p66 transcript in comparison with wild-type B31A3, indicating that HtrA-mediated regulation of p66 may occur at multiple levels.

Ordered Membrane Domain-Forming Properties of the Lipids of Borrelia burgdorferi.

Co-existing disordered and ordered (raft) membrane domains exist in Borrelia burgdorferi, the causative agent of Lyme disease. However, although B. burgdorferi contains cholesterol lipids, it lacks sphingolipids-a crucial component of rafts in eukaryotes. To define the principles of ordered lipid domain formation in Borrelia, the domain forming properties of vesicles composed of its three major lipids, acylated cholesteryl galactoside (ACGal), monogalactosyl diacyglycerol (MGalD), and phosphatidylcholine (PC) and/or their mixtures were studied. Anisotropy and fluorescence resonance energy transfer measurements were used to assay membrane order and ordered-domain formation. ACGal had the highest potential to form ordered domains. Interestingly, mixtures of ACGal with B. burgdorferi PC formed ordered domains more readily than mixtures of ACGal with MGalD. This appears to reflect the relatively high level of saturation observed for B. burgdorferi PC, as vesicles containing ACGal and PC, but in which the unsaturated lipid dioleoyl PC was substituted for Borrelia PC, failed to form ordered domains. In addition, the properties of ACGal were compared to those of cholesterol. Depending on what other lipids were present, ordered-domain formation in the presence of ACGal was greater than or equal to that in the presence of cholesterol. Giant unilamellar vesicles formed from ACGal-containing mixtures showed rounded domain shapes similar to those in analogous vesicles containing cholesterol, indicative of liquid-ordered state rather than solid-like gel-state domain formation. Over all, principles of ordered-domain formation in B. burgdorferi appear to be very similar to those in eukaryotes, with saturated PC taking the place of sphingolipids, but with ACGal being the main lipid component inducing ordered-domain formation.

The lipid raft proteome of Borrelia burgdorferi.

Eukaryotic lipid rafts are membrane microdomains that have significant amounts of cholesterol and a selective set of proteins that have been associated with multiple biological functions. The Lyme disease agent, Borrelia burgdorferi, is one of an increasing number of bacterial pathogens that incorporates cholesterol onto its membrane, and form cholesterol glycolipid domains that possess all the hallmarks of eukaryotic lipid rafts. In this study, we isolated lipid rafts from cultured B. burgdorferi as a detergent resistant membrane (DRM) fraction on density gradients, and characterized those molecules that partitioned exclusively or are highly enriched in these domains. Cholesterol glycolipids, the previously known raft-associated lipoproteins OspA and OpsB, and cholera toxin partitioned into the lipid rafts fraction indicating compatibility with components of the DRM. The proteome of lipid rafts was analyzed by a combination of LC-MS/MS or MudPIT. Identified proteins were analyzed in silico for parameters that included localization, isoelectric point, molecular mass and biological function. The proteome provided a consistent pattern of lipoproteins, proteases and their substrates, sensing molecules and prokaryotic homologs of eukaryotic lipid rafts. This study provides the first analysis of a prokaryotic lipid raft and has relevance for the biology of Borrelia, other pathogenic bacteria, as well as for the evolution of these structures. All MS data have been deposited in the ProteomeXchange with identifier PXD002365 (http://proteomecentral.proteomexchange.org/dataset/PXD002365).

Lipid exchange between Borrelia burgdorferi and host cells.

Borrelia burgdorferi, the agent of Lyme disease, has cholesterol and cholesterol-glycolipids that are essential for bacterial fitness, are antigenic, and could be important in mediating interactions with cells of the eukaryotic host. We show that the spirochetes can acquire cholesterol from plasma membranes of epithelial cells. In addition, through fluorescent and confocal microscopy combined with biochemical approaches, we demonstrated that B. burgdorferi labeled with the fluorescent cholesterol analog BODIPY-cholesterol or (3)H-labeled cholesterol transfer both cholesterol and cholesterol-glycolipids to HeLa cells. The transfer occurs through two different mechanisms, by direct contact between the bacteria and eukaryotic cell and/or through release of outer membrane vesicles. Thus, two-way lipid exchange between spirochetes and host cells can occur. This lipid exchange could be an important process that contributes to the pathogenesis of Lyme disease.

Proving lipid rafts exist: membrane domains in the prokaryote Borrelia burgdorferi have the same properties as eukaryotic lipid rafts.

Lipid rafts in eukaryotic cells are sphingolipid and cholesterol-rich, ordered membrane regions that have been postulated to play roles in many membrane functions, including infection. We previously demonstrated the existence of cholesterol-lipid-rich domains in membranes of the prokaryote, B. burgdorferi, the causative agent of Lyme disease [LaRocca et al. (2010) Cell Host & Microbe 8, 331-342]. Here, we show that these prokaryote membrane domains have the hallmarks of eukaryotic lipid rafts, despite lacking sphingolipids. Substitution experiments replacing cholesterol lipids with a set of sterols, ranging from strongly raft-promoting to raft-inhibiting when mixed with eukaryotic sphingolipids, showed that sterols that can support ordered domain formation are both necessary and sufficient for formation of B. burgdorferi membrane domains that can be detected by transmission electron microscopy or in living organisms by Förster resonance energy transfer (FRET). Raft-supporting sterols were also necessary and sufficient for formation of high amounts of detergent resistant membranes from B. burgdorferi. Furthermore, having saturated acyl chains was required for a biotinylated lipid to associate with the cholesterol-lipid-rich domains in B. burgdorferi, another characteristic identical to that of eukaryotic lipid rafts. Sterols supporting ordered domain formation were also necessary and sufficient to maintain B. burgdorferi membrane integrity, and thus critical to the life of the organism. These findings provide compelling evidence for the existence of lipid rafts and show that the same principles of lipid raft formation apply to prokaryotes and eukaryotes despite marked differences in their lipid compositions.

Raft-like membrane domains in pathogenic microorganisms.

The lipid bilayer of the plasma membrane is thought to be compartmentalized by the presence of lipid-protein microdomains. In eukaryotic cells, microdomains composed of sterols and sphingolipids, commonly known as lipid rafts, are believed to exist, and reports on the presence of sterol- or protein-mediated microdomains in bacterial cell membranes are also appearing. Despite increasing attention, little is known about microdomains in the plasma membrane of pathogenic microorganisms. This review attempts to provide an overview of the current state of knowledge of lipid rafts in pathogenic fungi and bacteria. The current literature on characterization of microdomains in pathogens is reviewed, and their potential role in growth, pathogenesis, and drug resistance is discussed. Better insight into the structure and function of membrane microdomains in pathogenic microorganisms might lead to a better understanding of their pathogenesis and development of raft-mediated approaches for therapy.

The HtrA protease of Borrelia burgdorferi degrades outer membrane protein BmpD and chemotaxis phosphatase CheX.

Borrelia burgdorferi, the spirochaetal agent of Lyme disease, codes for a single HtrA protein, HtrABb (BB0104) that is homologous to DegP of Escherichia coli (41% amino acid identity). HtrABb shows physical and biochemical similarities to DegP in that it has the trimer as its fundamental unit and can degrade casein via its catalytic serine. Recombinant HtrABb exhibits proteolytic activity in vitro, while a mutant (HtrABbS198A) does not. However, HtrABb and DegP have some important differences as well. Native HtrABb occurs in both membrane-bound and soluble forms. Despite its homology to DegP, HtrABb could not complement an E. coli DegP deletion mutant. Late stage Lyme disease patients, as well as infected mice and rabbits developed a robust antibody response to HtrABb, indicating that it is a B-cell antigen. In co-immunoprecipitation studies, a number of potential binding partners for HtrABb were identified, as well as two specific proteolytic substrates, basic membrane protein D (BmpD/BB0385) and chemotaxis signal transduction phosphatase CheX (BB0671). HtrABb may function in regulating outer membrane lipoproteins and in modulating the chemotactic response of B. burgdorferi.

Effect of short photoperiod on the development of Haemaphysalis longicornis (Ixodida: Ixodidae).

The invasive tick, Haemaphysalis longicornis Neumann, is now present across most of the mid-Atlantic States in the eastern United States. This tick ends its seasonal activity in late October to early November, with larvae being the last life-stage observed questing. Previous research has revealed that the activity of H. longicornis is influenced by photoperiod: short daylight lengths trigger diapause in nymphs, marking it as the primary overwintering stage. However, whether engorged larvae can enter diapause or are affected by short daylight is unclear. In this study, we tested in the laboratory whether the photoperiod Affects the development of H. longicornis engorged larvae and engorged nymphs under constant temperature and humidity. The results showed that engorged larvae molted significantly faster (3 days faster) when the photoperiod was 9 h of light as opposed to 14 h. In contrast, changes in the photoperiod did not affect the molting of engorged nymphs. Our results demonstrate that engorged larvae respond to short daylight length, by molting faster. These results suggest that engorged larvae are unlikely to overwinter under field conditions and support the expectation that nymphs are the primary overwintering stage for H. longicornis in the United States.

Seasonal Dynamics of Tick Species in the Ecotone of Parks and Recreational Areas in Middlesex County (New Jersey, USA).

People often use parks and other forested areas for outdoor activities such as hiking and walking their dogs. Areas of primary use are paths or grassy meadows on the edges of the forests that constitute transitional areas between different plant communities (aka ecotones). In this study, we monitored the seasonal dynamics of questing ticks in forest/meadow and forest/path ecotones in five areas in Middlesex County, New Jersey (NJ). We found anthropophilic species such as Ixodes scapularis, Amblyomma americanum, and Dermacentor variabilis coexisting with Haemaphysalis longicornis, an invasive tick species first detected in NJ in 2017. Surveillance was conducted weekly from March to November 2020, and collected ticks were identified. The most abundant tick species was H. longicornis (83%), followed by A. americanum (9%), I. scapularis (7%), and D. variabilis (<1%). The seasonal dynamics of A. americanum and I. scapularis in the ecotone were similar to previous surveys in forest habitats. The presence of anthropophilic ticks, particularly I. scapularis, suggests the need for specific control approaches to target these habitats. In addition, the extraordinarily high numbers of H. longicornis collected in ecotones (1.70 ticks/m2) and frequent reports of this species on dogs highlight the importance of monitoring its expansion due to its potential as a vector of animal and human diseases.

Baseline susceptibility of Haemaphysalis longicornis to organophosphate, carbamate, and pyrethroid acaricides.

BACKGROUND: The Asian longhorned tick, Haemaphysalis longicornis, continues to expand its range in North America, and synthetic acaricides are likely to play an increasing role in managing this species. Acaricide resistance is common in some tick species that infest livestock. However, baseline acaricide susceptibility has not previously been examined in this invasive tick. RESULTS: We used a standard larval packet test to evaluate the susceptibility of the Asian longhorned tick to acaricides currently or formerly used in tick control: propoxur, carbaryl, bifenthrin, permethrin, and coumaphos. Discriminating concentrations were estimated at 6.5, 27.9, 988, 2242, and 808 ppm, respectively. The half-maximal lethal concentration (LC50 ) values for propoxur, carbaryl, permethrin and coumaphos were compared with data available for other tick species and showed that H. longicornis was more susceptible to propoxur, carbaryl and coumaphos, and had a similar susceptibility to permethrin. CONCLUSIONS: The results indicate that resistance to these acaricides is not currently a concern for H. longicornis in the United States. However, responsible integrated management and early detection of resistance can help ensure the long-term efficacy of products used for controlling this tick species. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Rickettsia and relapsing fever Borrelia in Alectorobius kelleyi (Ixodida: Argasidae) from peri domestic bats in the northeastern United States.

The soft ticks (Argasidae) are known vectors of human and animal pathogens around the globe and are relatively understudied. Our aim was to assess the presence of Rickettsia and Borrelia bacteria in Alectorobius kelleyi (Argasidae) parasitizing synanthropic bats in the highly urbanized northeastern United States. By collaborating with parasitologists, bat scientists and wildlife rehabilitators we were successful in obtaining A. kelleyi from five states. Since Argasid larvae will attach to their hosts for many days, most A. kelleyi examined (92%) were larvae collected from sick or injured big brown bats, Eptesicus fuscus, undergoing care at rehabilitation centers. In addition, we obtained adult A. kelleyi captured in residential living areas and trapped in attics. An in-depth analysis of a A. kelleyi found to be infected with a spotted fever group Rickettsia (SFGR) revealed a dual infection with a R. belli-like taxon (ancestral group) as well as an SFGR closely related to R. peacockii, likely the same previously found in A. kelleyi from Iowa and Kansas. We found that 36% of the A. kelleyi tested carried the SFGR. Furthermore, we detected a relapsing fever spirochete, likely Candidatus Borrelia johnsonii, in 25% of the A. kelleyi from Pennsylvania. While it is unclear if these bacteria constitute a health risk to either bats or humans, our study indicates that human exposure to ectoparasites infesting peridomestic wildlife should be considered in the epidemiology of tick-borne diseases.

Molecular method for the characterization of Coxiella burnetii from clinical and environmental samples: variability of genotypes in Spain.

BACKGROUND: Coxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes), rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes. RESULTS: To assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets) PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct characterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples. CONCLUSIONS: This newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied, while sheep, goats, wild boar, rats and ticks share genotypes with the human population.

Field applications of granular and liquid pyrethroids, carbaryl, and IGRs to control the asian longhorned tick (Haemaphysalis longicornis) and impacts on nontarget invertebrates.

Few documented control strategies exist for the invasive tick, Haemaphysalis longicornis, despite its potential to reach extremely high numbers and vector human and animal pathogens. In 2020, we evaluated the effects of single applications of five granular and liquid acaricides on H. longicornis in a public park in northern New Jersey. Acaricides tested included pyrethroids (lambda-cyhalothrin, bifenthrin), a carbamate (carbaryl), and the insect growth regulators (IGRs) pyriproxyfen and novaluron. We also monitored the impact of each treatment on non-target soil and above-ground invertebrate species using pitfall and sticky traps, respectively. We recorded over 70,000 H. longicornis ticks in the study area from July to October 2020. An average of 99% control was achieved with lambda-cyhalothrin spray and 95% with granular bifenthrin. In contrast, granular carbaryl did not significantly reduce any life stages of H. longicornis. The IGR (pyriproxyfen/novaluron) resulted in a significant 45% reduction of the larval stage following treatments in July. No other stages were significantly impacted by pyriproxyfen alone or in combination with novaluron. Analysis of non-target species revealed that the community composition of soil-dwelling arthropods was strongly impacted by pyrethroid treatments and, to a lesser extent, by the carbamate treatment. The granular pyrethroid bifenthrin had more pronounced effects and impacted a broader range of non-target groups in the pitfall traps than the liquid pyrethroid lambda-cyhalothrin. Arthropod groups that were negatively impacted included Isopoda, Formicidae, Coleoptera, Araneae, Acari, and Grylloidea. Collembola numbers, however, were elevated in both pyrethroid treatments. The community composition of arthropods collected on the above-ground sticky traps was strongly impacted only in the liquid lambda-cyhalothrin treatment. The primary groups impacted in the sticky trap analysis were Collembola and Hemiptera. Community composition in traps remained distinct in the pyrethroid treatments through the entire survey period up to 62 days post-treatment. The results of this study indicate that pyrethroid acaricides were highly effective at controlling H. longicornis, while other compounds, including carbaryl and IGRs, did not achieve consistent levels of control. Further research is needed to find effective and environmentally sustainable alternatives. Integrated management programs can include the judicious use of pyrethroids to control H. longicornis.

Repellency of novel catnip (Nepeta cataria) cultivar extracts against Ixodes scapularis and Haemaphysalis longicornis (Acari: Ixodida: Ixodidae).

Tick bites are a major public health concern due to the vector role that many tick species have in transmitting human pathogens. Synthetic repellents such as N­diethyl-meta-toluamide (DEET) remain the standard for repellency. Still, there is a need for natural commercial alternatives with similar or better properties than DEET. We evaluated the repellency of two extracts, CR3 and CR9, derived for newly developed catnip cultivars on two tick species, Ixodes scapularis and Haemaphysalis longicornis. Dose-response in vitro assays showed that CR3 and CR9 extracts have similar repellency properties to DEET. At a 20% concentration, both CR3 and CR9 extracts exhibited a repellency of 100%. Catnip extracts maintained their repellency properties for at least 8 h. In a two-choice assay, I. scapularis, but not H. longicornis, was more sensitive to CR3 than DEET. In addition, CR3 reduces the life span of I. scapularis, suggesting that it has an acaricidal effect on ticks. In summary, CR3 and CR9 catnip extracts are promising tick repellents that should be further developed, alone or in combination with other tick repellents, and tested for their use as tick repellents for humans.

Evidence that two ATP-dependent (Lon) proteases in Borrelia burgdorferi serve different functions.

The canonical ATP-dependent protease Lon participates in an assortment of biological processes in bacteria, including the catalysis of damaged or senescent proteins and short-lived regulatory proteins. Borrelia spirochetes are unusual in that they code for two putative ATP-dependent Lon homologs, Lon-1 and Lon-2. Borrelia burgdorferi, the etiologic agent of Lyme disease, is transmitted through the blood feeding of Ixodes ticks. Previous work in our laboratory reported that B. burgdorferi lon-1 is upregulated transcriptionally by exposure to blood in vitro, while lon-2 is not. Because blood induction of Lon-1 may be of importance in the regulation of virulence factors critical for spirochete transmission, the clarification of functional roles for these two proteases in B. burgdorferi was the object of this study. On the chromosome, lon-2 is immediately downstream of ATP-dependent proteases clpP and clpX, an arrangement identical to that of lon of Escherichia coli. Phylogenetic analysis revealed that Lon-1 and Lon-2 cluster separately due to differences in the NH(2)-terminal substrate binding domains that may reflect differences in substrate specificity. Recombinant Lon-1 manifested properties of an ATP-dependent chaperone-protease in vitro but did not complement an E. coli Lon mutant, while Lon-2 corrected two characteristic Lon-mutant phenotypes. We conclude that B. burgdorferi Lons -1 and -2 have distinct functional roles. Lon-2 functions in a manner consistent with canonical Lon, engaged in cellular homeostasis. Lon-1, by virtue of its blood induction, and as a unique feature of the Borreliae, may be important in host adaptation from the arthropod to a warm-blooded host.

Borrelia burgdorferi Surface Exposed GroEL Is a Multifunctional Protein.

The spirochete, Borrelia burgdorferi, has a large number of membrane proteins involved in a complex life cycle, that includes a tick vector and a vertebrate host. Some of these proteins also serve different roles in infection and dissemination of the spirochete in the mammalian host. In this spirochete, a number of proteins have been associated with binding to plasminogen or components of the extracellular matrix, which is important for tissue colonization and dissemination. GroEL is a cytoplasmic chaperone protein that has previously been associated with the outer membrane of Borrelia. A His-tag purified B. burgdorferi GroEL was used to generate a polyclonal rabbit antibody showing that GroEL also localizes in the outer membrane and is surface exposed. GroEL binds plasminogen in a lysine dependent manner. GroEL may be part of the protein repertoire that Borrelia successfully uses to establish infection and disseminate in the host. Importantly, this chaperone is readily recognized by sera from experimentally infected mice and rabbits. In summary, GroEL is an immunogenic protein that in addition to its chaperon role it may contribute to pathogenesis of the spirochete by binding to plasminogen and components of the extra cellular matrix.

Applications of artificial membrane feeding for ixodid ticks.

Ticks are obligatory hematophagous ectoparasites that feed on a large variety of vertebrates. In the laboratory, animals (mainly mice and rabbits) are used to maintain tick colonies. However, the use of animals to rear ticks can be expensive and requires dedicated animal facilities. In addition, research institutions are committed to the principle of 3Rs (Replacement, Reduction and Refinement), which encourages the use of alternatives to animals when possible. The development of artificial membrane systems has provided an alternative to animals, at least for some tick species. Over the years, different modifications in artificial feeding systems have led to new applications, including acaricide testing, tick-pathogen interaction, and novel approaches to study tick physiology. Although artificial membrane feeding still has some limitations, the method can provide numerous advantages, including the standardization of acaricide treatments under controlled conditions, an alternative to animals for tick rearing, and reduction of cost associated with animals and animal housing facilities. In this review, we summarized the evolution of tick feeding membranes and their applications over time, explaining the modifications incorporated to study tick physiology, tick-pathogen interactions, and acaricide testing.