BACKGROUND: Lyme disease, caused by Borrelia burgdorferi sensu lato (s.l.), is the most common vector-borne disease in the Northern Hemisphere, with Ixodes ticks as its primary vectors. However, many patients do not recall tick bites, fueling speculation about alternative transmission routes, particularly via mosquito bites. This belief is reinforced by studies reporting Borrelia presence in mosquitoes. This study evaluates whether three mosquito species can acquire, maintain, and transmit Borrelia spirochetes. METHODS: Mosquitoes (Aedes aegypti, Culex quinquefasciatus, and Culex pipiens biotype molestus) were fed on Borrelia-infected mice to assess pathogen acquisition. Additional experiments involved ex vivo feeding on Borrelia-enriched blood to examine spirochete persistence in the mosquito gut. The potential for mechanical transmission was tested by simulating interrupted feeding between infected and naive hosts. The role of trypsin in Borrelia survival and infectivity was also investigated. RESULTS: Mosquitoes exhibited low efficiency in acquiring Borrelia from infected hosts. Spirochetes artificially introduced through ex vivo blood meals were rapidly eliminated during digestion, primarily due to trypsin activity. Inhibition of trypsin prolonged spirochete persistence and infectivity in the mosquito gut. Mechanical transmission experiments revealed no evidence of Borrelia transmission from infected to naive hosts. CONCLUSIONS: Our findings demonstrate that mosquitoes lack the biological capacity to efficiently acquire and maintain B. burgdorferi s.l. spirochetes and are unable to transmit them through natural or mechanical means. This study provides compelling evidence against mosquito-borne transmission of Lyme disease and reinforces Ixodes ticks as the sole competent vectors, which is crucial for targeted public health interventions and accurate risk communication.

• Keywords
• Borrelia, Borreliosis, Lyme disease, Mosquito, Tick, Transmission,
• MeSH
• Aedes * microbiology   MeSH
• Borrelia burgdorferi * physiology   MeSH
• Culex * microbiology   MeSH
• Ixodes microbiology   MeSH
• Mosquito Vectors * microbiology   MeSH
• Lyme Disease * transmission   microbiology   MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

We report here on the development of tailored plasmonic AgNPs/C:H:N:O plasma polymer nanocomposites for the detection of the pathogenic bacterium Borrelia afzelii , with high selectivity and sensitivity. Silver (Ag) nanoparticles, generated by a gas aggregation source, are incorporated onto a C:H:N:O plasma polymer matrix, which is deposited by magnetron sputtering of a nylon 6.6. These anchored Ag nanoparticles propagate localized surface plasmon resonance (LSPR), optically responding to changes caused by immobilized pathogens near the nanoparticles. The tailored functionalization of AgNPs/C:H:N:O nanocomposite surface allows both high selectivity for the pathogen and high sensitivity with an LSPR red-shift Δλ > (4.20 ± 0.71) nm for 50 Borrelia per area 0.785 cm2. The results confirmed the ability of LSPR modulation for the rapid and early detection of (not only) tested pathogens.

• Keywords
• C:H:N:O thin film, Lyme disease, ag nanoparticles, borrelia, localized surface plasmon resonance, magnetron sputtering, nanocomposite, nylon, plasma polymer, surface functionalization,
• MeSH
• Metal Nanoparticles * chemistry   MeSH
• Limit of Detection * MeSH
• Nanocomposites * chemistry   MeSH
• Surface Plasmon Resonance * methods   MeSH
• Silver * chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Silver * MeSH

Tick saliva is a rich source of antihemostatic, anti-inflammatory, and immunomodulatory molecules that actively help the tick to finish its blood meal. Moreover, these molecules facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-8, a salivary serpin from the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Iripin-8 displayed blood-meal-induced mRNA expression that peaked in nymphs and the salivary glands of adult females. Iripin-8 inhibited multiple proteases involved in blood coagulation and blocked the intrinsic and common pathways of the coagulation cascade in vitro. Moreover, Iripin-8 inhibited erythrocyte lysis by complement, and Iripin-8 knockdown by RNA interference in tick nymphs delayed the feeding time. Finally, we resolved the crystal structure of Iripin-8 at 1.89 Å resolution to reveal an unusually long and rigid reactive center loop that is conserved in several tick species. The P1 Arg residue is held in place distant from the serpin body by a conserved poly-Pro element on the P' side. Several PEG molecules bind to Iripin-8, including one in a deep cavity, perhaps indicating the presence of a small-molecule binding site. This is the first crystal structure of a tick serpin in the native state, and Iripin-8 is a tick serpin with a conserved reactive center loop that possesses antihemostatic activity that may mediate interference with host innate immunity.

• Keywords
• Ixodes ricinus, blood coagulation, crystal structure, parasite, saliva, serpin, tick,
• MeSH
• Complement Activation drug effects   immunology   physiology   MeSH
• Erythrocytes metabolism   MeSH
• Gene Expression genetics   MeSH
• Blood Coagulation drug effects   physiology   MeSH
• Ixodes enzymology   genetics   metabolism   MeSH
• Complement System Proteins metabolism   MeSH
• Lyme Disease MeSH
• Nymph MeSH
• Arthropod Proteins metabolism   MeSH
• Gene Expression Regulation genetics   MeSH
• Serpins metabolism   ultrastructure   MeSH
• Salivary Glands metabolism   MeSH
• Saliva chemistry   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Complement System Proteins MeSH
• Arthropod Proteins MeSH
• Serpins MeSH

A growing global health concern, Lyme disease has become the most common tick-borne disease in the United States and Europe. Caused by the bacterial spirochete Borrelia burgdorferi sensu lato (sl), this disease can be debilitating if not treated promptly. Because diagnosis is challenging, prevention remains a priority; however, a previously licensed vaccine is no longer available to the public. Here, we designed a six component vaccine that elicits antibody (Ab) responses against all Borrelia strains that commonly cause Lyme disease in humans. The outer surface protein A (OspA) of Borrelia was fused to a bacterial ferritin to generate self-assembling nanoparticles. OspA-ferritin nanoparticles elicited durable high titer Ab responses to the seven major serotypes in mice and non-human primates at titers higher than a previously licensed vaccine. This response was durable in rhesus macaques for more than 6 months. Vaccination with adjuvanted OspA-ferritin nanoparticles stimulated protective immunity from both B. burgdorferi and B. afzelii infection in a tick-fed murine challenge model. This multivalent Lyme vaccine offers the potential to limit the spread of Lyme disease.

• Keywords
• Immunology, Infectious diseases, Vaccines,
• Publication type
• Journal Article MeSH

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.

• Keywords
• Borrelia afzelii, Ixodes ricinus, RNAi, massive analysis of cDNA ends (MACE), midgut, tick, transcriptome,
• MeSH
• Borrelia burgdorferi Group genetics   MeSH
• Ticks genetics   microbiology   MeSH
• Ixodes genetics   MeSH
• Lyme Disease microbiology   transmission   MeSH
• Mice, Inbred C3H MeSH
• Mice MeSH
• Nymph microbiology   MeSH
• Transcriptome genetics   MeSH
• Digestive System microbiology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Quantitative and microscopic tracking of Borrelia afzelii transmission from infected Ixodes ricinus nymphs has shown a transmission cycle different from that of Borrelia burgdorferi and Ixodes scapularisBorrelia afzelii organisms are abundant in the guts of unfed I. ricinus nymphs, and their numbers continuously decrease during feeding. Borrelia afzelii spirochetes are present in murine skin within 1 day of tick attachment. In contrast, spirochetes were not detectable in salivary glands at any stage of tick feeding. Further experiments demonstrated that tick saliva is not essential for B. afzelii infectivity, the most important requirement for successful host colonization being a change in expression of outer surface proteins that occurs in the tick gut during feeding. Spirochetes in vertebrate mode are then able to survive within the host even in the absence of tick saliva. Taken together, our data suggest that the tick gut is the decisive organ that determines the competence of I. ricinus to vector B. afzelii We discuss possible transmission mechanisms of B. afzelii spirochetes that should be further tested in order to design effective preventive and therapeutic strategies against Lyme disease.

• Keywords
• Borrelia, Borrelia afzelii, Ixodes ricinus, Lyme disease, tick-borne pathogens, transmission,
• MeSH
• Arachnid Vectors microbiology   physiology   MeSH
• Borrelia burgdorferi Group physiology   MeSH
• Ixodes microbiology   physiology   MeSH
• Humans MeSH
• Lyme Disease microbiology   transmission   MeSH
• Mice, Inbred C3H MeSH
• Mice MeSH
• Nymph microbiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Ticks are important vectors of serious human and animal disease-causing organisms, but their innate immunity can fight invading pathogens and may have the ability to reduce or block transmission to mammalian hosts. Lectins, sugar-binding proteins, can distinguish between self and non-self-oligosaccharide motifs on pathogen surfaces. Although tick hemolymph possesses strong lectin activity, and several lectins have already been isolated and characterized, little is known about the implementation of these molecules in tick immunity. Here, we have described and functionally characterized fibrinogen-related protein (FReP) lectins in Ixodes ticks. We have shown that the FReP family contains at least 27 genes (ixoderins, ixo) that could, based on phylogenetic and expression analyses, be divided into three groups with differing degrees of expansion. By using RNA interference-mediated gene silencing (RNAi) we demonstrated that IXO-A was the main lectin in tick hemolymph. Further, we found that ixoderins were important for phagocytosis of Gram-negative bacteria and yeasts by tick hemocytes and that their expression was upregulated upon injection of microbes, wounding, or after blood feeding. However, although the tick hemocytes could swiftly phagocytose Borrelia afzelii spirochetes, their transmission and burst of infection in mice was not altered. Our results demonstrate that tick ixoderins are crucial immune proteins that work as opsonins in the tick hemolymph, targeting microbes for phagocytosis or lysis.

• Keywords
• Borrelia, Ixodes, RNAi, complement, fibrinogen-related protein, ixoderin, lectin, tick,
• MeSH
• Phagocytosis MeSH
• Hemocytes immunology   MeSH
• Hemolymph immunology   MeSH
• Ixodes genetics   immunology   MeSH
• Lectins genetics   metabolism   MeSH
• Immunity, Innate * MeSH
• Arthropod Proteins genetics   metabolism   MeSH
• RNA Interference MeSH
• Gene Silencing MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Lectins MeSH
• Arthropod Proteins MeSH

The impact of Ixodes ricinus salivary gland extract (SGE) on inflammatory changes in the skin and draining lymph nodes of mice, elicited by the infection with the important human pathogen, B. afzelii, was determined using flow cytometry. SGE injected together with spirochetes reduced the numbers of leukocytes and gammadelta-T lymphocytes in infected epidermis at early time-points post infection. In draining lymph nodes, the anti-inflammatory effect of SGE was manifested by the decrease of total cell count compared with that in mice treated with inactivated SGE. Changes in subpopulations of immunocompetent cells apparently reflected the effect of SGE on the proliferation of spirochetes in the host. The significance of tick saliva anti-inflammatory effect for saliva activated transmission of B. afzelii is shown.

• MeSH
• Borrelia burgdorferi Group growth & development   MeSH
• Ticks immunology   MeSH
• Skin immunology   pathology   MeSH
• Lyme Disease immunology   pathology   transmission   MeSH
• Lymph Nodes immunology   pathology   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Lymphocyte Count MeSH
• Lymphocyte Subsets MeSH
• Salivary Glands immunology   MeSH
• Tissue Extracts immunology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Tissue Extracts MeSH

In Europe the Borrelia burgdorferi sensu lato complex is represented by five distinct genospecies: Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, and Borrelia lusitaniae. These taxonomic entities are known to differ in their specific associations with vertebrate hosts and to provoke distinct clinical manifestations in human patients. However, exceptions to these rules have often been observed, indicating that strains belonging to a single genospecies may be more heterogeneous than expected. It is, therefore, important to develop alternative identification tools which are able to distinguish Borrelia strains not only at the specific level but also at the intraspecific level. DNA from a sample of 370 Ixodes ricinus ticks collected in the Czech Republic was analyzed by PCR for the presence of a approximately 230-bp fragment of the rrfA-rrlB intergenic spacer of Borrelia spp. A total of 20.5% of the ticks were found to be positive. The infecting genospecies were identified by analyzing the amplified products by the restriction fragment length polymorphism (RFLP) method with restriction enzyme MseI and by single-strand conformation polymorphism (SSCP) analysis. The two methods were compared, and PCR-SSCP analysis appeared to be a valuable tool for rapid identification of spirochetes at the intraspecific level, particularly when large samples are examined. Furthermore, by using PCR-SSCP analysis we identified a previously unknown Borrelia genotype, genotype I-77, which would have gone unnoticed if RFLP analysis alone had been used.

• MeSH
• Borrelia burgdorferi Group classification   genetics   MeSH
• DNA, Bacterial genetics   isolation & purification   MeSH
• Phylogeny MeSH
• Genetic Variation * MeSH
• Genotype MeSH
• Ixodes microbiology   MeSH
• Humans MeSH
• DNA, Ribosomal Spacer genetics   MeSH
• Molecular Sequence Data MeSH
• Polymerase Chain Reaction methods   MeSH
• Polymorphism, Restriction Fragment Length MeSH
• Polymorphism, Single-Stranded Conformational * MeSH
• Sequence Analysis, DNA MeSH
• Bacterial Typing Techniques MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic MeSH
• Names of Substances
• DNA, Bacterial MeSH
• DNA, Ribosomal Spacer MeSH

Salivary gland extract (SGE) from Ixodes ricinus ticks inhibited the killing of Borrelia afzelii spirochetes by murine macrophages. SGE also reduced the production of two major defense molecules of phagocytes, superoxide and nitric oxide. It is likely that the suppression of macrophage microbicidal mechanisms contributes to the inhibitory effect of tick saliva on the killing of B. afzelii spirochetes, thus facilitating the transmission of this important pathogen.

• MeSH
• Borrelia immunology   MeSH
• Ixodes physiology   MeSH
• Macrophages immunology   MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Saliva physiology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH