Nataliia Rudenko

Differential Expression of Ixodes ricinus Tick Genes Induced by Blood Feeding or Borrelia burgdorferi Infection

Abstract Ixodes ricinus L. is the principal European vector of Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis. Subtractive hybridization was used to isolate tick genes that were induced in whole ticks after blood meals on uninfected and B. burgdorferi-infected guinea pigs. Novel cDNA clones with similarity to cytochrome c oxidase, salivary secreted protein, actin, and a cysteine protease propeptide were induced after a blood meal. Novel cDNA clones with similarity to thioredoxin peroxidases, dolichyl-phosphate β-glucosyltransferase, glutathione S-transferase, defensin, ML domain-containing protein, and von Willebrand factor were induced after B. burgdorferi infection. Virtual Northern analysis was used to verify that these genes were differentially expressed in ticks after a pathogen-infected blood meal and to detect their tissues of expression. The characterization of genes that are induced after an infected blood meal is essential for gaining an understanding of the molecular mechanisms that underlie vector-pathogen interactions.

Borrelia carolinensis sp. nov., a New (14th) Member of the Borrelia burgdorferi Sensu Lato Complex from the Southeastern Region of the United States

ABSTRACT Approximately 118 Borrelia isolates were cultured from a variety of rodents, birds, and ticks collected in the southern United States. In addition to a highly diverse group of Borrelia bissettii strains and a homogenous group of Borrelia burgdorferi sensu stricto strains, a group of 16 isolates with unusual characteristics was found. The isolates were cultured from ear biopsy samples of the rodents Peromyscus gossypinus and Neotoma floridana trapped at five localities in South Carolina. A multilocus sequence analysis of the rrf-rrl intergenic spacer, 16S rRNA, fla, ospA, and p66 genes were used to clarify the taxonomic status of the new group of B. burgdorferi sensu lato isolates. Thirteen species of the B. burgdorferi sensu lato complex were used as controls. Unique restriction fragment length polymorphism patterns of the rrf-rrl intergenic spacer region and fla gene were recognized. Unique signature nucleotides were also found in the 16S rRNA gene. A phylogenetic analysis shows that the 16 new isolates cluster together but separately from the other species in the B. burgdorferi sensu lato complex. Our data strongly support the recognition of the 16 isolates as a new B. burgdorferi sensu lato species. We propose to name this genospecies “Borrelia carolinensis” with respect to the place of its currently known geographic location.

Multilocus sequence analysis of Borrelia bissettii strains from North America reveals a new Borrelia species, Borrelia kurtenbachii.

Using multilocus sequence analyses (MLSA), we investigated the phylogenetic relationship of spirochaete strains from North America previously assigned to the genospecies Borrelia bissettii. We amplified internal fragments of 8 housekeeping genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA) located on the main linear chromosome by polymerase chain reaction. Phylogenetic analysis of concatenated sequences of the 8 loci showed that the B. bissettii clade consisted of 4 closely related clusters which included strains from California (including the type strain DN127-Cl9-2/p7) and Colorado that were isolated from Ixodes pacificus, I. spinipalpis, or infected reservoir hosts. Several strains isolated from I. scapularis clustered distantly from B. bissettii. Genetic distance analyses confirmed that these strains are more distant to B. bissettii than they are to B. carolinensis, a recently described Borrelia species, which suggests that they constitute a new Borrelia genospecies. We propose that it be named Borrelia kurtenbachii sp. nov. in honour of the late Klaus Kurtenbach. The data suggest that ecological differences between B. bissettii and the new Borrelia genospecies reflect different transmission cycles. In view of these findings, the distinct vertebrate host-tick vector associations and the distributions of B. bissettii and B. kurtenbachii require further investigation.

Delineation of a New Species of the Borrelia burgdorferi Sensu Lato Complex, Borrelia americana sp. nov.

ABSTRACT Analysis of borrelia isolates collected from ticks, birds, and rodents from the southeastern United States revealed the presence of well-established populations of Borrelia burgdorferi sensu stricto, Borrelia bissettii, Borrelia carolinensis, and Borrelia sp. nov. Multilocus sequence analysis of five genomic loci from seven samples representing Borrelia sp. nov. isolated from nymphal Ixodes minor collected in South Carolina showed their close relatedness to California strains known as genomospecies 1 and separation from any other known species of the B. burgdorferi sensu lato complex. One nucleotide difference in the size of the 5S-23S intergenic spacer region, one substitution in 16S rRNA gene signature nucleotides, and silent nucleotide substitutions in sequences of the gene encoding flagellin and the gene p66 clearly separate Borrelia sp. nov. isolates from South Carolina into two subgroups. The sequences of isolates of each subgroup share the same restriction fragment length polymorphism patterns of the 5S-23S intergenic spacer region and contain unique signature nucleotides in the 16S rRNA gene. We propose that seven Borrelia sp. nov. isolates from South Carolina and two California isolates designated as genomospecies 1 comprise a single species, which we name Borrelia americana sp. nov. The currently recognized geographic distribution of B. americana is South Carolina and California. All strains are associated with Ixodes pacificus or Ixodes minor and their rodent and bird hosts.

Molecular detection of Borrelia bissettii DNA in serum samples from patients in the Czech Republic with suspected borreliosis

Until recently, three spirochete genospecies were considered to be the causative agents of Lyme borreliosis (LB) in Europe: Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii. However, the DNA of Borrelia valaisiana, Borrelia lusitaniae, Borrelia spielmanii and Borrelia bissettii has already been detected in samples of human origin, or the spirochetes were isolated from the patients with symptoms of LB. Molecular analysis of 12 selected serum samples collected in the regional hospital confirmed the presence of B. bissettii DNA in cases of single and multiple infection in patients with symptomatic borreliosis or chronic borrelial infection. The presence of B. bissettii as a single strain in patients provides strong support of the fact that B. bissettii might be a causative agent of the disease. After the first isolation of B. bissettii from the samples of human origin in Slovenia, following the detection of this species in cardiac valve tissue of the patient with endocarditis and aortic valve stenosis in the Czech Republic, here we present additional molecular data supporting the involvement of B. bissettii in LB in Europe.

Mutations in the NS2B and NS3 genes affect mouse neuroinvasiveness of a Western European field strain of tick-borne encephalitis virus

An attenuated strain (263) of the tick-borne encephalitis virus, isolated from field ticks, was either serially subcultured, 5 times in mice, or at 40 degrees C in PS cells, producing 2 independent strains, 263-m5 and 263-TR with identical genomes; both strains exhibited increased plaque size, neuroinvasiveness and temperature-resistance. Sequencing revealed two unique amino acid substitutions, one mapping close to the catalytic site of the viral protease. These observations imply that virus adaptation from ticks to mammals occurs by selection of pre-existing virulent variants from the quasispecies population rather than by the emergence of new random mutations. The significance of these observations is discussed.

Tick-Pathogen Interactions and Vector Competence: Identification of Molecular Drivers for Tick-Borne Diseases

Ticks and the pathogens they transmit constitute a growing burden for human and animal health worldwide. Vector competence is a component of vectorial capacity and depends on genetic determinants affecting the ability of a vector to transmit a pathogen. These determinants affect traits such as tick-host-pathogen and susceptibility to pathogen infection. Therefore, the elucidation of the mechanisms involved in tick-pathogen interactions that affect vector competence is essential for the identification of molecular drivers for tick-borne diseases. In this review, we provide a comprehensive overview of tick-pathogen molecular interactions for bacteria, viruses, and protozoa affecting human and animal health. Additionally, the impact of tick microbiome on these interactions was considered. Results show that different pathogens evolved similar strategies such as manipulation of the immune response to infect vectors and facilitate multiplication and transmission. Furthermore, some of these strategies may be used by pathogens to infect both tick and mammalian hosts. Identification of interactions that promote tick survival, spread, and pathogen transmission provides the opportunity to disrupt these interactions and lead to a reduction in tick burden and the prevalence of tick-borne diseases. Targeting some of the similar mechanisms used by the pathogens for infection and transmission by ticks may assist in development of preventative strategies against multiple tick-borne diseases.

Neutrophil Extracellular Traps Entrap and Kill Borrelia burgdorferi Sensu Stricto Spirochetes and Are Not Affected by Ixodes ricinus Tick Saliva

Lyme disease is caused by spirochetes of the Borrelia burgdorferi sensu lato complex. They are transmitted mainly by Ixodes ricinus ticks. After a few hours of infestation, neutrophils massively infiltrate the bite site. They can kill Borrelia via phagocytosis, oxidative burst, and hydrolytic enzymes. However, factors in tick saliva promote propagation of the bacteria in the host even in the presence of a large number of neutrophils. The neutrophil extracellular trap (NET) consists in the extrusion of the neutrophil’s own DNA, forming traps that can retain and kill bacteria. The production of reactive oxygen species is apparently associated with the onset of NETs (NETosis). In this article, we describe NET formation at the tick bite site in vivo in mice. We show that Borrelia burgdorferi sensu stricto spirochetes become trapped and killed by NETs in humans and that the bacteria do not seem to release significant nucleases to evade this process. Saliva from I. ricinus did not affect NET formation by human neutrophils or its stability. However, it greatly decreased neutrophil reactive oxygen species production, suggesting that a strong decrease of hydrogen peroxide does not affect NET formation. Finally, round bodies trapped in NETs were observed, some of them staining as live bacteria. This observation could help contribute to a better understanding of the early steps of Borrelia invasion and erythema migrans formation after tick bite.

Borrelia carolinensis sp. nov., a novel species of the Borrelia burgdorferi sensu lato complex isolated from rodents and a tick from the south-eastern USA

A group of 16 isolates with genotypic characteristics different from those of known species of the Borrelia burgdorferi sensu lato complex were cultured from ear biopsies of the rodents Peromyscus gossypinus and Neotoma floridana trapped at five localities in South Carolina, USA, and from the tick Ixodes minor feeding on N. floridana. Multilocus sequence analysis of members of the novel species, involving the 16S rRNA gene, the 5S–23S (rrf–rrl) intergenic spacer region and the flagellin, ospA and p66 genes, was conducted and published previously and was used to clarify the taxonomic status of the novel group of B. burgdorferi sensu lato isolates. Phylogenetic analysis based on concatenated sequences of the five analysed genomic loci showed that the 16 isolates clustered together but separately from other species in the B. burgdorferi sensu lato complex. The analysed group therefore represents a novel species, formally described here as Borrelia carolinensis sp. nov., with the type strain SCW-22T (=ATCC BAA-1773T =DSM 22119T).

New defensins from hard and soft ticks: similarities, differences, and phylogenetic analyses.

Despite the importance of ticks as vectors of disease very little is known about their immune system. Antimicrobial peptides, including defensins (phylogenetically ancient antibacterial peptides) are major components of innate immunity in ticks that have been shown to provide protection against gram-negative and gram-positive bacteria, fungi, viruses and protozoan parasites. With the aim of studying the evolution of the genes involved in tick defense, we identified the preprodefensin genes from four Ornithodoros tick species (O. papillipes: isoforms A, B, and D; O. tartakovskyi and O. puertoricensis: isoforms A and B; O. rostratus: isoform A) and from two Dermacentor tick species (D. reticulatus and D. marginatus: one isoform) not previously described. Phylogenetic analyses revealed that Ornithodoros defensin isoforms (A, B, C, and D) form 4 separate clades, while hard tick defensins are divided into several branches based on particular tick species.