Borrelia afzelii infection in the rodent host has dramatic effects on the bacterial microbiome of Ixodes ricinus ticks

The microbiome of blood-sucking arthropods can shape their competence to acquire and maintain infections with vector-borne pathogens. We used a controlled study to investigate the interactions between Borrelia afzelii , which causes Lyme disease in Europe, and the bacterial microbiome of Ixodes ricinus , its primary tick vector. We applied a surface sterilization treatment to I. ricinus eggs to produce dysbiosed tick larvae that had a much lower bacterial abundance and a changed bacterial microbiome compared to the control larvae. Dysbiosed and control larvae were fed on B. afzelii -infected mice and uninfected control mice and the engorged larvae were left to molt into nymphs, which were tested for B. afzelii infection and their bacterial microbiome by 16S rRNA amplicon sequencing. Surprisingly, larval dysbiosis had no effect on the vector competence of I. ricinus for B. afzelii , as the nymphal infection prevalence and the nymphal spirochete load were the same between the dysbiosed group and the control group. The strong effect of egg surface sterilization on the tick bacterial microbiome largely disappeared once the larvae molted into nymphs. The most important determinant of the bacterial microbiome of I. ricinus nymphs was the B. afzelii infection status of the mouse on which the nymphs had fed as larvae. Nymphs that had taken their larval blood meal from an infected mouse had a less abundant but more diverse bacterial microbiome compared to control nymphs. Our study demonstrates that vector-borne infections in the vertebrate host shape the microbiome of the arthropod vector. IMPORTANCE Many blood-sucking arthropods transmit pathogens that cause infectious disease. For example, Ixodes ricinus ticks transmit the bacterium Borrelia afzelii , which causes Lyme disease in humans. Ticks also have a microbiome, which can influence their ability to acquire and transmit tick-borne pathogens like B. afzelii . We sterilized I. ricinus eggs with bleach, and the tick larvae that hatched from these eggs had a dramatically reduced and changed bacterial microbiome compared to control larvae. These larvae were fed on B. afzelii -infected mice and the resultant nymphs were tested for B. afzelii and their bacterial microbiome. We found that our manipulation of the bacterial microbiome had no effect on the ability of the tick larvae to acquire and maintain populations of B. afzelii . In contrast, we found that B. afzelii infection had dramatic effects on the bacterial microbiome of I. ricinus nymphs. Our study demonstrates that infections in the vertebrate host can shape the tick microbiome.

Infection with Borrelia afzelii and manipulation of the egg surface microbiota have no effect on the fitness of immature Ixodes ricinus ticks

Arthropod vectors carry vector-borne pathogens that cause infectious disease in vertebrate hosts, and arthropod-associated microbiota, which consists of non-pathogenic microorganisms. Vector-borne pathogens and the microbiota can both influence the fitness of their arthropod vectors, and hence the epidemiology of vector-borne diseases. The bacterium Borrelia afzelii, which causes Lyme borreliosis in Europe, is transmitted among vertebrate reservoir hosts by Ixodes ricinus ticks, which also harbour a diverse microbiota of non-pathogenic bacteria. The purpose of this controlled study was to test whether B. afzelii and the tick-associated microbiota influence the fitness of I. ricinus. Eggs obtained from field-collected adult female ticks were surface sterilized (with bleach and ethanol), which reduced the abundance of the bacterial microbiota in the hatched I. ricinus larvae by 28-fold compared to larvae that hatched from control eggs washed with water. The dysbiosed and control larvae were subsequently fed on B. afzelii-infected or uninfected control mice, and the engorged larvae were left to moult into nymphs under laboratory conditions. I. ricinus larvae that fed on B. afzelii-infected mice had a significantly faster larva-to-nymph moulting time compared to larvae that fed on uninfected control mice, but the effect was small (2.4% reduction) and unlikely to be biologically significant. We found no evidence that B. afzelii infection or reduction of the larval microbiota influenced the four other life history traits of the immature I. ricinus ticks, which included engorged larval weight, unfed nymphal weight, larva-to-nymph moulting success, and immature tick survival. A retrospective power analysis found that our sampling effort had sufficient power (> 80%) to detect small effects (differences of 5% to 10%) of our treatments. Under the environmental conditions of this study, we conclude that B. afzelii and the egg surface microbiota had no meaningful effects on tick fitness and hence on the R0 of Lyme borreliosis.

Competition between strains of Borrelia afzelii in the host tissues and consequences for transmission to ticks

Pathogen species often consist of genetically distinct strains, which can establish mixed infections or coinfections in the host. In coinfections, interactions between pathogen strains can have important consequences for their transmission success. We used the tick-borne bacterium Borrelia afzelii, which is the most common cause of Lyme disease in Europe, as a model multi-strain pathogen to investigate the relationship between coinfection, competition between strains, and strain-specific transmission success. Mus musculus mice were infected with one or two strains of B. afzelii, strain transmission success was measured by feeding ticks on mice, and the distribution of each strain in six different mouse organs and the ticks was measured using qPCR. Coinfection and competition reduced the tissue infection prevalence of both strains and changed their bacterial abundance in some tissues. Coinfection and competition also reduced the transmission success of the B. afzelii strains from the infected hosts to feeding ticks. The ability of the B. afzelii strains to establish infection in the host tissues was strongly correlated with their transmission success to the tick vector. Our study demonstrates that coinfection and competition between pathogen strains inside the host tissues can have major consequences for their transmission success.

Investigating BB0405 as a novel Borrelia afzelii vaccination candidate in Lyme borreliosis

BB0405 is a surface exposed Borrelia burgdorferi protein and its vaccination protected mice against B. burgdorferi infection. As BB0405 is highly conserved across different B. burgdorferi sensu lato species, we investigated whether vaccination with recombinant BB0405 or through intradermal bb0405 DNA tattoo vaccination could provide protection against different Borrelia species, specifically against Borrelia afzelii, the predominant B. burgdorferi sensu lato genospecies causing Lyme borreliosis across Eurasia. We immunized C3H/HeN mice with recombinant BB0405 or with a codon-optimized bb0405 DNA vaccine using the pVAC plasmid and immunized corresponding control groups mice with only adjuvant or empty vectors. We subsequently subjected these immunized mice to a tick challenge with B. afzelii CB43-infected Ixodes ricinus nymphs. Upon vaccination, recombinant BB0405 induced a high total IgG response, but bb0405 DNA vaccination did not elicit antibody responses. Both vaccine formulations did not provide protection against Borrelia afzelii strain CB43 after tick challenge. In an attempt to understand the lack of protection of the recombinant vaccine, we determined expression of BB0405 and showed that B. afzelii CB43 spirochetes significantly and drastically downregulate the expression of BB0405 protein at 37 °C compared to 33 °C, where as in B. burgdorferi B31 spirochetes expression levels remain unaltered. Vaccination with recombinant BB0405 was previously shown to protect against B. burgdorferi sensu stricto. Here we show that vaccination with either recombinant BB0405 (or non-immunogenic bb0405 DNA), despite being highly conserved among B. burgdorferi sl genospecies, does not provide cross-protection against B. afzelii, mostly likely due to downregulation of this protein in B. afzelii in the mammalian host.

Identification of Tick Ixodes ricinus Midgut Genes Differentially Expressed During the Transmission of Borrelia afzelii Spirochetes Using a Transcriptomic Approach

Lyme borreliosis is an emerging tick-borne disease caused by spirochetes Borrelia burgdorferi sensu lato. In Europe, Lyme borreliosis is predominantly caused by Borrelia afzelii and transmitted by Ixodes ricinus. Although Borrelia behavior throughout tick development is quite well documented, specific molecular interactions between Borrelia and the tick have not been satisfactorily examined. Here, we present the first transcriptomic study focused on the expression of tick midgut genes regulated by Borrelia. By using massive analysis of cDNA ends (MACE), we searched for tick transcripts expressed differentially in the midgut of unfed, 24h-fed, and fully fed I. ricinus nymphs infected with B. afzelii. In total, we identified 553 upregulated and 530 downregulated tick genes and demonstrated that B. afzelii interacts intensively with the tick. Technical and biological validations confirmed the accuracy of the transcriptome. The expression of five validated tick genes was silenced by RNA interference. Silencing of the uncharacterized protein (GXP_Contig_30818) delayed the infection progress and decreased infection prevalence in the target mice tissues. Silencing of other genes did not significantly affect tick feeding nor the transmission of B. afzelii, suggesting a possible role of these genes rather in Borrelia acquisition or persistence in ticks. Identification of genes and proteins exploited by Borrelia during transmission and establishment in a tick could help the development of novel preventive strategies for Lyme borreliosis.

Infection With Borrelia Afzelii Reduces Moulting Time of Lxodes Ricinus Ticks

Arthropod vectors carry vector-borne pathogens that cause infectious disease in vertebrate hosts, and arthropod-associated non-pathogenic microorganisms. Both types of micro-organisms can influence the fitness of their arthropod vectors, and hence the epidemiology of vector-borne diseases. The bacterium Borrelia afzelii, which causes Lyme disease in Europe, is transmitted among vertebrate reservoir hosts by Ixodes ricinus ticks, which also harbour a large diversity of non-pathogenic bacteria. The purpose of this study was to test experimentally whether B. afzelii and tick-associated non-pathogenic bacteria influence the fitness of I. ricinus. Eggs obtained from field-collected adult female ticks were washed with bleach, which reduced the abundance of non-pathogenic bacteria in the hatched I. ricinus larvae by 28-fold compared to larvae that hatched from eggs washed with water. The dysbiosed and control larvae were subsequently fed on B. afzelii-infected or uninfected control mice, and the engorged larvae were allowed to moult into nymphs under laboratory conditions. I. ricinus larvae that fed on B. afzelii-infected mice had a significantly faster larva-to-nymph moulting time compared to larvae that fed on uninfected control mice. In contrast, we found no evidence that B. afzelii infection or larval dysbiosis influenced the four other life history traits of the immature I. ricinus ticks, which included engorged larval weight, unfed nymphal weight, larva-to-nymph moulting success, and immature tick survival. A power analysis found that our sampling effort had sufficient power (>80%) to detect small effects (difference of 5% – 10%) of either B. afzelii infection or larval dysbiosis. Under the laboratory conditions of our study, B. afzelii appears to be a mostly passive passenger in I. Ricinus ticks.

Direct immunofluorescence as a low-budget method for brain tissue inspection in standardization of neuroborreliosis animal model in NMRI mice

Objective. To test if the direct immunofluorescence can be used for the detection of Borrelia afzelii in brain tissue during the standardization of the animal model of neuroborreliosis in NMRI mice. Methods. The study was performed on 15 mice of NMRI strain. All mice were subcutaneously inoculated with 100 ml of BSK-H medium containing the local isolate of Borrelia afzelii. Animals were sacrificed after inoculation at III (n = 4), IV (n = 6) and V (n = 5) weeks, by cervical dislocation. In the sampled brains of mice, the presence of Borrelia was detected by direct immunofluorescence (DIF) and chain polymerization reaction (PCR). Results. The first brain tested positive for Borrelia three weeks after the inoculation. The bacteria were detected in 1 out of 4 brains (25%). After that, there was a growth in the percentage of positive results. The data showed that 3 out of 6 brains (50%) were found positive on Borrelia presence by the end of the fourth week. Whereas, in 3 out of 5 brains (60%) Borrelia was detected five weeks following the inoculation. Conclusion. According to the preliminary results, direct immunofluorescence appeared to be a practical, low budget method for following the kinetics of neuro-infection. NMRI mice could be considered as an adequate animal model for neuroborreliosis. Thus, more research is needed on the topics of infection kinetics for the period after fifth week post inoculation, as well as sensitivity and specificity of direct immunofluorescence.