Antibody-blocking of a tick transporter impairs Anaplasma phagocytophilum colonization in Haemaphysalis longicornis ticks.

The invasive Asian longhorned tick Haemaphysalis longicornis that vectors and transmits several animal pathogens is significantly expanding in the United States. Recent studies report that these ticks also harbor human pathogens including Borrelia burgdorferi sensu lato, Babesia microti, and Anaplasma phagocytophilum. Therefore, studies that address the interactions of these ticks with human pathogens are important. In this study, we report the characterization of H. longicornis organic anion-transporting polypeptides (OATPs) in interactions of these ticks with A. phagocytophilum. Using OATP-signature sequence, we identified six OATPs in the H. longicornis genome. Bioinformatic analysis revealed that H. longicornis OATPs are closer to other tick orthologs rather than to mammalian counterparts. Quantitative real-time PCR analysis revealed that OATPs are highly expressed in immature stages when compared to mature stages of these ticks. In addition, we noted that the presence of A. phagocytophilum upregulates a specific OATP in these ticks. We also noted that exogenous treatment of H. longicornis with xanthurenic acid, a tryptophan metabolite, influenced OATP expression in these ticks. Immunoblotting analysis revealed that antibody generated against Ixodes scapularis OATP cross-reacted with H. longicornis OATP. Furthermore, treatment of H. longicornis with OATP antibody impaired colonization of A. phagocytophilum in these ticks. These results not only provide evidence that the OATP-tryptophan pathway is important for A. phagocytophilum survival in H. longicornis ticks but also indicate OATP as a promising candidate for the development of a universal anti-tick vaccine to target this bacterium and perhaps other rickettsial pathogens of medical importance.

Multiple factors affecting Ixodes ricinus ticks and associated pathogens in European temperate ecosystems (northeastern France).

In Europe, the main vector of tick-borne zoonoses is Ixodes ricinus, which has three life stages. During their development cycle, ticks take three separate blood meals from a wide variety of vertebrate hosts, during which they can acquire and transmit human pathogens such as Borrelia burgdorferi sensu lato, the causative agent of Lyme borreliosis. In this study conducted in Northeastern France, we studied the importance of soil type, land use, forest stand type, and temporal dynamics on the abundance of ticks and their associated pathogens. Negative binomial regression modeling of the results indicated that limestone-based soils were more favorable to ticks than sandstone-based soils. The highest tick abundance was observed in forests, particularly among coniferous and mixed stands. We identified an effect of habitat time dynamics in forests and in wetlands: recent forests and current wetlands supported more ticks than stable forests and former wetlands, respectively. We observed a close association between tick abundance and the abundance of Cervidae, Leporidae, and birds. The tick-borne pathogens responsible for Lyme borreliosis, anaplasmosis, and hard tick relapsing fever showed specific habitat preferences and associations with specific animal families. Machine learning algorithms identified soil related variables as the best predictors of tick and pathogen abundance.

A RANGE-WIDE ECTOPARASITE SURVEY FOR ALLEGHENY WOODRATS (NEOTOMA MAGISTER).

Allegheny woodrats (Neotoma magister) are karst-specializing rodents that are rare or in conservation need in many states within their current range. Parasitism and habitat fragmentation have been suggested as primary reasons for declining populations. The presence, prevalence, and impact of ectoparasites, including fleas, ticks, and bots, is not fully understood rangewide. We collected Allegheny woodrat ectoparasites across 8 states in their range, identifying parasites via morphological and genetic means. Across contributions from 8 states, we discovered 2 woodrat-specific fleas parasitizing Allegheny woodrats: Orchopeas pennsylvanicus (all contributing states, n = 228) and Epitedia cavernicola (Indiana only, n = 9). The former was a new state record in New Jersey and Ohio. Woodrat specialists Ixodes woodi were morphologically identified as the dominant tick species (n = 38), and our contributions to genetic databases may ease confusion in future efforts. Three generalist species of ticks representing 8 individuals were identified as Dermacentor variabilis, Amblyomma americanum, and Ixodes scapularis. Only 2 bot fly species were recognized in Allegheny woodrats: 1 squirrel bot (Cuterebra emasculator) and 10 individuals of Cuterebra sp. not genetically conspecific to any known eastern U.S. rodent bot. The host specificity for fleas is not surprising, given that previous small-scale surveys and ticks primarily appear to be a mix of genus-specific (Ixodes woodi) and generalist species. There remains uncertainty with bots via morphological and genetic analyses. Our survey presents a wide-ranging baseline survey for Allegheny woodrats across their range, emphasizing the diversity (or specificity) of parasite groups for this species. An understanding of Allegheny woodrats and the health impact of ectoparasites is imperative because they face myriad challenges rangewide, especially considering the bot-driven demise of 1 woodrat in our study. Ectoparasites can have a marked impact on already-declining woodrat populations across their range and should not be overlooked in future surveys.

Prevalence of tick-borne pathogens in ticks collected from the wild mountain ungulates mouflon and chamois in 4 regions of France.

Ticks are major vectors of various pathogens of health importance, such as bacteria, viruses and parasites. The problems associated with ticks and vector-borne pathogens are increasing in mountain areas, particularly in connection with global climate change. We collected ticks (n = 2,081) from chamois and mouflon in 4 mountainous areas of France. We identified 6 tick species: Ixodes ricinus, Rhipicephalus bursa, Rh. sanguineus s.l., Haemaphysalis sulcata, H. punctata and Dermacentor marginatus. We observed a strong variation in tick species composition among the study sites, linked in particular to the climate of the sites. We then analysed 791 ticks for DNA of vector-borne pathogens: Babesia/Theileria spp., Borrelia burgdorferi s.l., Anaplasma phagocytophilum, A. marginale, A. ovis, and Rickettsia of the spotted fever group (SFG). Theileria ovis was detected only in Corsica in Rh. bursa. Babesia venatorum (2 sites), Borrelia burgdorferi s.l. (B. afzelii and B. garinii; 2 sites) and Anaplasma phagocytophilum (3 sites) were detected in I. ricinus. Anaplasma ovis was detected at one site in I. ricinus and Rh. sanguineus s.l. SFG Rickettsia were detected at all the study sites: R. monacensis and R. helvetica in I. ricinus at the 3 sites where this tick is present; R. massiliae in Rh. sanguineus s.l. (1 site); and R. hoogstraalii and Candidatus R. barbariae in Rh. bursa in Corsica. These results show that there is a risk of tick-borne diseases for humans and domestic and wild animals frequenting these mountain areas.

Title: Prévalence d'agents pathogènes vectorisés chez des tiques collectées chez des ongulés sauvages (mouflons, chamois) dans 4 zones montagneuses en France. Abstract: Les tiques sont des vecteurs majeurs de différents agents pathogènes d'importance sanitaire, tels que des bactéries, des virus et des parasites. Les problématiques liées aux tiques et aux pathogènes vectorisés augmentent en zones de montagne, en lien notamment avec le réchauffement climatique. Nous avons collecté des tiques (n = 2 081) sur des chamois et des mouflons dans 4 zones montagneuses en France. Six espèces ont été identifiées : Ixodes ricinus, Rhipicephalus bursa, Rh. sanguineus s.l., Haemaphysalis sulcata, H. punctata et Dermacentor marginatus. Nous avons observé une forte variation de la composition en espèces de tiques entre les sites d'étude, en lien notamment avec le climat des sites. Nous avons ensuite recherché les ADN d'agents pathogènes vectorisés sur 791 tiques : Babesia/Theileria spp, Borrelia burgdorferi s.l., Anaplasma phagocytophilum, A. marginale, A. ovis, et de Rickettsia du groupe des fièvres boutonneuses (SFG). Theileria ovis a été détecté uniquement en Corse chez Rh. bursa. Babesia venatorum (2 sites), Borrelia burgdorferi s.l. (B. afzelii and B. garinii; 2 sites) et Anaplasma phagocytophilum (3 sites) ont été détectés chez I. ricinus. Anaplasma ovis a été détecté dans un site chez I. ricinus et Rh. sanguineus s.l.. Les Rickettsia SFG ont été détectées dans tous les sites d'étude : Rickettsia monacensis et R. helvetica chez I. ricinus dans les 3 sites où cette tique est présente; R. massiliae chez Rh. sanguineus s.l. (1 site); et R. hoogstraalii et Candidatus R. barbariae chez Rh. bursa en Corse. Ces résultats montrent un risque de transmission de maladies par les tiques pour les personnes et les animaux domestiques et sauvages fréquentant ces zones de montagne.

Genetic structure and Rickettsia infection rates in Ixodes ovatus and Haemaphysalis flava ticks across different altitudes.

Ixodid ticks, such as Ixodes ovatus and Haemaphysalis flava, are important vectors of tick-borne diseases in Japan, such as Japanese spotted fever caused by Rickettsia japonica. This study describes the Rickettsia infection rates influenced by the population genetic structure of I.ovatus and H. flava along an altitudinal gradient. A total of 346 adult I. ovatus and 243 H. flava were analyzed for the presence of Rickettsia by nested PCR targeting the 17kDA, gltA, rOmpA, and rOmpB genes. The population genetic structure was analyzed utilizing the mitochondrial cytochrome oxidase 1 (cox1) marker. The Rickettsia infection rates were 13.26% in I. ovatus and 6.17% in H. flava. For I. ovatus, the global FST value revealed significant genetic differentiation among the different populations, whereas H. flava showed non-significant genetic differentiation. The cox1 I. ovatus cluster dendrogram showed two cluster groups, while the haplotype network and phylogenetic tree showed three genetic groups. A significant difference was observed in Rickettsia infection rates and mean altitude per group between the two cluster groups and the three genetic groups identified within I. ovatus. No significant differences were found in the mean altitude or Rickettsia infection rates of H. flava. Our results suggest a potential correlation between the low gene flow in I. ovatus populations and the spatially heterogeneous Rickettsia infection rates observed along the altitudinal gradient. This information can be used in understanding the relationship between the tick vector, its pathogen, and environmental factors, such as altitude, and for the control of tick-borne diseases in Japan.

Prevalence of Powassan Virus Seropositivity Among People with History of Lyme Disease and Non-Lyme Community Controls in the Northeastern United States.

Introduction: Lyme disease (LD) affects ∼476,000 people each year in the United States. Symptoms are variable and include rash and flu-like symptoms. Reasons for the wide variation in disease outcomes are unknown. Powassan virus (POWV) is a tick-borne flavivirus that causes disease ranging from asymptomatic infection to encephalitis, neurologic damage, and death. POWV and LD geographic case distributions overlap, with Ixodes species ticks as the common vectors. Clinical ramifications of coinfection or sequential infection are unknown. Objectives: This study's primary objective was to determine the prevalence of POWV-reactive antibodies in sera samples collected from previously studied cohorts of individuals with self-reported LD history residing in the Northeastern United States. As a secondary objective, we studied clinical differences between people with self-reported LD history and low versus high POWV antibody levels. Methods: We used an enzyme-linked immunosorbent assay (ELISA) to quantify IgG directed at the POWV envelope (E) protein domain III in 538 samples from individuals with self-reported LD history and 16 community controls. The samples were also tested with an ELISA assay to quantify IgG directed at the POWV NS1 protein. Results: The percentage of individuals with LD history and possible evidence of POWV exposure varied depending on the assay utilized. We found no significant difference in clinical symptoms between those with low or high POWV IgG levels in the in-house assay. Congruence of the EDIII and NS1 assays was low with only 12% of those positive in the in-house EDIII ELISA testing positive in the POWV NS1 ELISA. Conclusions: The results highlight the difficulty in flavivirus diagnostic testing, particularly in the retrospective detection of flavivirus exposure. The findings suggest that a prospective study with symptomatic patients using approved clinical testing is necessary to address the incidence and clinical implications of LD and POWV co-infection or sequential infection.

Effects of rodent abundance on ticks and Borrelia: results from an experimental and observational study in an island system.

BACKGROUND: Lyme borreliosis is the most common tick-borne disease in Europe and is often caused by Borrelia afzelii, which is transmitted by Ixodes ricinus ticks. The prevalence and abundance of infected ticks fluctuate in time and space, influencing human infection risk. Rodents are reservoir hosts for B. afzelii and important feeding hosts for larval ticks. In the study reported here, we examined how variation in rodent abundance is associated with B. afzelii infection prevalence in ticks, the density of nymphs (DON) and the density of infected nymphs (DIN) in the following year. We further analysed the relationships between the abundance of infected rodents and nymphal infection prevalence (NIP) and DIN. METHODS: We conducted a study that combined experimental and observational approaches on 15 islands (10 small islands and 5 large islands) in Finland. On all of the islands, ticks and rodents were monitored and sampled during the summer of 2019, with the monitoring of tick abundance and sampling continuing into the spring of 2020. On five of the 10 small islands, captured rodents were removed from the island ("removal" islands), and on the other five small islands, captured rodents were released back to the trapping site after marking and sampling ("control" islands). On the five large islands, captured rodents were released back to the trapping site after marking and sampling. The presence of B. afzelii from nymph and rodent samples was examined. RESULTS: The results of the experimental study showed that neither treatment (removal), rodent abundance index nor abundance index of infected rodents in 2019 was associated with DON, NIP or DIN in 2020. Based on data from the observational study, the NIP in 2020 decreased with increasing rodent abundance index and abundance index of infected rodents in 2019. However, the DIN in 2020 was not associated with the rodent abundance index or the abundance index of infected rodents in 2019. In addition, in the observational study, DON in 2020 increased with increasing rodent abundance index. CONCLUSIONS: Our results suggest that low rodent abundance during the tick activity period is not sufficient for reducing the disease hazard and, hence, rodent removal may not be a feasible control measure in natural ecosystems.

The Prevalence of Tick-Borne Encephalitis Virus in Wild Rodents Captured in Tick-Borne Encephalitis Foci in Highly Endemic Lithuania.

Wild rodents are considered to be one of the most important TBEV-amplifying reservoir hosts; therefore, they may be suitable for foci detection studies. To investigate the effectiveness of viral RNA detection in wild rodents for suspected TBEV foci confirmation, we trapped small rodents (n = 139) in various locations in Lithuania where TBEV was previously detected in questing ticks. Murine neuroblastoma Neuro-2a cells were inoculated with each rodent sample to maximize the chances of detecting viral RNA in rodent samples. TBEV RNA was detected in 74.8% (CI 95% 66.7-81.1) of the brain and/or internal organ mix suspensions, and the prevalence rate increased significantly following sample cultivation in Neuro-2a cells. Moreover, a strong correlation (r = 0.88; p < 0.05) was found between the average monthly air temperature of rodent trapping and the TBEV RNA prevalence rate in cell culture isolates of rodent suspensions, which were PCR-negative before cultivation in cell culture. This study shows that wild rodents are suitable sentinel animals to confirm TBEV foci. In addition, the study results demonstrate that sample cultivation in cell culture is a highly efficient method for increasing TBEV viral load to detectable quantities.

An optimized artificial blood feeding assay to study tick cuticle biology.

Ticks, ectoparasitic arachnids, are prominent disease vectors impacting both humans and animals. Their unique blood-feeding phase involves significant abdominal cuticle expansion, sharing certain similarities with insects. However, vital aspects, including the mechanisms of cuticle expansion, changes in cuticular protein composition, chitin synthesis, and cuticle function, remain poorly understood. Given that the cuticle expansion is crucial for complete engorgement of the ticks, addressing these knowledge gaps is essential. Traditional tick research involving live animal hosts has inherent limitations, such as ethical concerns and host response variability. Artificial membrane feeding systems provide an alternative approach, offering controlled experimental conditions and reduced ethical dilemmas. These systems enable precise monitoring of tick attachment, feeding parameters, and pathogen acquisition. Despite the existence of various methodologies for artificial tick-feeding systems, there is a pressing need to enhance their reproducibility and effectiveness. In this context, we introduce an improved tick-feeding system that incorporates adjustments related to factors like humidity, temperature, and blood-feeding duration. These refinements markedly boost tick engorgement rates, presenting a valuable tool for in-depth investigations into tick cuticle biology and facilitating studies on molting. This refined system allows for collecting feeding ticks at specific stages, supporting research on tick cuticle biology, and evaluating chemical agents' efficacy in the engorgement process.

Differential susceptibility of geographically distinct Ixodes ricinus populations to tick-borne encephalitis virus and louping ill virus.

Tick-borne encephalitis virus (TBEV) is an emerging pathogen in the Netherlands. Multiple divergent viral strains are circulating and the focal distribution of TBEV remains poorly understood. This may, however, be explained by differences in the susceptibility of tick populations for specific viruses and viral strains, and by viral strains having higher infection success in their local tick population. We investigated this hypothesis by exposing Dutch Ixodes ricinus ticks to two different TBEV strains: TBEV-NL from the Netherlands and TBEV-Neudoerfl from Austria. In addition, we exposed ticks to louping Ill virus (LIV), which is endemic to large parts of the United Kingdom and Ireland, but has not been reported in the Netherlands. Ticks were collected from two locations in the Netherlands: one location without evidence of TBEV circulation and one location endemic for the TBEV-NL strain. Ticks were infected in a biosafety level 3 laboratory using an artificial membrane feeding system. Ticks collected from the region without evidence of TBEV circulation had lower infection rates for TBEV-NL as compared to TBEV-Neudoerfl. Vice versa, ticks collected from the TBEV-NL endemic region had higher infection rates for TBEV-NL compared to TBEV-Neudoerfl. In addition, LIV infection rates were much lower in Dutch ticks compared to TBEV, which may explain why LIV is not present in the Netherlands. Our findings show that ticks from two distinct geographical populations differ in their susceptibility to TBEV strains, which could be the result of differences in the genetic background of the tick populations.

SCGB1D2 inhibits growth of Borrelia burgdorferi and affects susceptibility to Lyme disease.

Lyme disease is a tick-borne disease caused by bacteria of the genus Borrelia. The host factors that modulate susceptibility for Lyme disease have remained mostly unknown. Using epidemiological and genetic data from FinnGen and Estonian Biobank, we identify two previously known variants and an unknown common missense variant at the gene encoding for Secretoglobin family 1D member 2 (SCGB1D2) protein that increases the susceptibility for Lyme disease. Using live Borrelia burgdorferi (Bb) we find that recombinant reference SCGB1D2 protein inhibits the growth of Bb in vitro more efficiently than the recombinant protein with SCGB1D2 P53L deleterious missense variant. Finally, using an in vivo murine infection model we show that recombinant SCGB1D2 prevents infection by Borrelia in vivo. Together, these data suggest that SCGB1D2 is a host defense factor present in the skin, sweat, and other secretions which protects against Bb infection and opens an exciting therapeutic avenue for Lyme disease.

Surveillance of tick-borne bacteria infection in ticks and forestry populations in Inner Mongolia, China.

Ticks are one of the most important vectors that can transmit pathogens to animals and human beings. This study investigated the dominant tick-borne bacteria carried by ticks and tick-borne infections in forestry populations in Arxan, Inner Mongolia, China. Ticks were collected by flagging from May 2020 to May 2021, and blood samples were collected from individuals at high risk of acquiring tick-borne diseases from March 2022 to August 2023. The pooled DNA samples of ticks were analyzed to reveal the presence of tick-borne bacteria using high-throughput sequencing of the 16S rDNA V3-V4 region, and species-specific polymerase chain reaction (PCR) related to sequencing was performed to confirm the presence of pathogenic bacteria in individual ticks and human blood samples. All sera samples were examined for anti-SFGR using ELISA and anti-B. burgdorferi using IFA and WB. A total of 295 ticks (282 Ixodes persulcatus and 13 Dermacentor silvarum) and 245 human blood samples were collected. Rickettsia, Anaplasma, Borrelia miyamotoi, and Coxiella endosymbiont were identified in I. persulcatus by high-throughput sequencing, while Candidatus R. tarasevichiae (89.00%, 89/100), B. garinii (17.00%, 17/100), B. afzelii (7.00%, 7/100), and B. miyamotoi (7.00%, 7/100) were detected in I. persulcatus, as well the dual co-infection with Candidatus R. tarasevichiae and B. garinii were detected in 13.00% (13/100) of I. persulcatus. Of the 245 individuals, B. garinii (4.90%, 12/245), R. slovaca (0.82%, 2/245), and C. burnetii (0.41%, 1/245) were detected by PCR, and the sequences of the target genes of B. garinii detected in humans were identical to those detected in I. persulcatus. The seroprevalence of anti-SFGR and anti-B. burgdorferi was 5.71% and 13.47%, respectively. This study demonstrated that Candidatus R. tarasevichiae and B. garinii were the dominant tick-borne bacteria in I. persulcatus from Arxan, and that dual co-infection with Candidatus R. tarasevichiae and B. garinii was frequent. This is the first time that B. miyamotoi has been identified in ticks from Arxan and R. solvaca has been detected in humans from Inner Mongolia. More importantly, this study demonstrated the transmission of B. garinii from ticks to humans in Arxan, suggesting that long-term monitoring of tick-borne pathogens in ticks and humans is important for the prevention and control of tick-borne diseases.

Direct Evidence of Powassan Virus Vertical Transmission in Ixodes scapularis in Nature.

Powassan virus (POWV) is a tick-borne flavivirus endemic in North America and Russia. Experimental infections with POWV have confirmed horizontal, transstadial, vertical, and cofeeding transmission routes for potential virus maintenance. In the field, vertical transmission has never been observed. During New York State tick-borne pathogen surveillance, POWV RNA and/or infectious POWV was detected in five pools of questing Ixodes scapularis larvae. Additionally, engorged female I. scapularis adults were collected from hunter-harvested white-tailed deer (Odocoileus virginianus) in a region with relatively high tick infection rates of POWV and allowed to oviposit under laboratory conditions. POWV RNA was detected in three female adult husks and one pool of larvae from a positive female. Infectious virus was isolated from all three RNA-positive females and the single positive larval pool. The detection of RNA and infectious virus in unfed questing larvae from the field and larvae from replete females collected from the primary tick host implicates vertical transmission as a potential mechanism for the maintenance of POWV in I. scapularis in nature, and elucidates the potential epidemiological significance of larval ticks in the transmission of POWV to humans.

Tick hemocytes have a pleiotropic role in microbial infection and arthropod fitness.

Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here we use and develop advanced techniques to describe immune cells (hemocytes) from the clinically relevant tick Ixodes scapularis at a single-cell resolution. We observe molecular alterations in hemocytes upon feeding and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We reveal hemocyte clusters exhibiting defined signatures related to immunity, metabolism, and proliferation. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, two I. scapularis hemocyte markers, impacting blood-feeding, molting behavior, and bacterial acquisition. Mechanistically, astakine alters hemocyte proliferation, whereas hemocytin affects the c-Jun N-terminal kinase (JNK) signaling pathway in I. scapularis. Altogether, we discover a role for tick hemocytes in immunophysiology and provide a valuable resource for comparative biology in arthropods.

Age- and Sex-Associated Pathogenesis of Cell Culture-Passaged Kemerovo Virus in IFNAR(-/-) Mice.

Kemerovo virus (KEMV) is a tick-borne orbivirus transmitted by ticks of the genus Ixodes. Previous animal experimentation studies with orbiviruses, in particular the interferon receptor double knock-out (IFNAR(-/-)) mouse model, did not indicate bias that is related to age or sex. We endeavoured to assess the effect of serial and alternated passages of KEMV in mammalian or Ixodes cells on virus replication and potential virulence in male or female IFNAR(-/-) mice, with important age differences: younger males (4-5 months old), older males (14-15 months old), and old females (14-15 months old). After 30 serial passages in mammalian or tick cells, or alternated passages in the two cell types, older female mice which were inoculated with the resulting virus strains were the first to show clinical signs and die. Younger males behaved differently from older males whether they were inoculated with the parental strain of KEMV or with any of the cell culture-passaged strains. The groups of male and female mice inoculated with the mammalian cell culture-adapted KEMV showed the lowest viraemia. While older female and younger male mice died by day 6 post-inoculation, surprisingly, the older males survived until the end of the experiment, which lasted 10 days. RNA extracted from blood and organs of the various mice was tested by probe-based KEMV real-time RT-PCR. Ct values of the RNA extracts were comparable between older females and younger males, while the values for older males were >5 Ct units higher for the various organs, indicating lower levels of replication. It is noteworthy that the hearts of the old males were the only organs that were negative for KEMV RNA. These results suggest, for the first time, an intriguing age- and sex-related bias for an orbivirus in this animal model. Changes in the amino acid sequence of the RNA-dependent RNA polymerase of Kemerovo virus, derived from the first serial passage in Ixodes cells (KEMV Ps.IRE1), were identified in the vicinity of the active polymerase site. This finding suggests that selection of a subpopulation of KEMV with better replication fitness in tick cells occurred.

Investigations of the tick burden on passeriform, water-associated and predatory birds reveal new tick-host associations and habitat-related factors of tick infestation.

BACKGROUND: Previous studies on the tick infestation of birds in the Carpathian Basin focused on songbirds (Passeriformes). Thus, the primary aim of the present work was to extend the scope of previous studies, i.e. to include aquatic (water-associated) bird species in a similar context, especially considering that these birds are usually long-distance migrants. METHODS: Between March 2021 and August 2023, 11,919 birds representing 126 species were checked for the presence of ticks. From 352 birds belonging to 40 species, 905 ixodid ticks were collected. Tick species were identified morphologically and/or molecularly. RESULTS: Ticks from avian hosts belonged to seven species: Ixodes ricinus (n = 448), I. frontalis (n = 31), I. festai (n = 2), I. arboricola (n = 36), I. lividus (n = 4), Haemaphysalis concinna (n = 382) and Dermacentor reticulatus (n = 2). Nymphs of I. ricinus occurred with a single activity peak around March-May, whereas its larvae typically infested birds in May, June or July. By contrast, H. concinna usually had its activity maximum during the summer (nymphs in June-July, larvae later in July-August). Interestingly, two ornithophilic species, I. frontalis and I. arboricola, were most active around winter months (between October and April). A significantly lower ratio of aquatic birds was found tick-infested than songbirds. Several new tick-host associations were revealed, including I. ricinus from Greylag Goose (Anser anser) and D. reticulatus from Great Egret (Ardea alba) and Sedge Warbler (Acrocephalus schoenobaenus). Ticks were collected for the first time in Europe from two species of predatory birds as well as from Little Bittern (Ixobrychus minutus). Bird species typically inhabiting reedbeds were most frequently infested with H. concinna, and most ticks localized at their throat, as opposed to forest-dwelling avian hosts, on which I. ricinus predominated and ticks were more evenly distributed. CONCLUSIONS: In the evaluated region, aquatic birds appear to be less important in tick dispersal than songbirds. However, newly revealed tick-host associations in this category attest to their hitherto neglected contribution. The results suggest that the habitat type will have significant impact not only on the species composition but also on the feeding location of ticks on birds.

Toxoplasma gondii infection in ticks infesting migratory birds: the blackbird (Turdus merula) and the song thrush (Turdus philomelos).

Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is the zoonosis widespread all over the world. Birds constitute an important group of T. gondii intermediate hosts often attacked by definitive hosts, e.g. domestic cats. Due to confirmation of an additional way of T. gondii transmission via tick bite, the aim of our study was to state and evaluate the infection prevalence of ticks feeding on blackbirds (Turdus merula) and song thrushes (Turdus philomelos). The real-time PCR amplification of the B1 gene fragment was used for detection of T. gondii infection in 157 Ixodes ricinus ticks removed from captured birds. The results showed the thrushes as hosts intensively attacked by ticks (prevalence 88.5% and 70% for blackbirds and song thrushes, respectively), and T. gondii infected individuals were detected. Among all ticks infected, 7 (5.8%; n = 120) were collected from blackbirds, and 2 (5.4%; n = 37) from song thrushes. The thrushes small body sizes and their tendency to urban ecosystems colonization, suggest that they relatively often become a pray of domestic cats, and combined with our findings, are potentially involved in maintenance the T. gondii population, especially in anthropogenic habitats, where the presence of toxoplasmosis is likely to constitute a serious danger to public health.

A tick saliva serpin, IxsS17 inhibits host innate immune system proteases and enhances host colonization by Lyme disease agent.

Lyme disease (LD) caused by Borrelia burgdorferi is among the most important human vector borne diseases for which there is no effective prevention method. Identification of tick saliva transmission factors of the LD agent is needed before the highly advocated tick antigen-based vaccine could be developed. We previously reported the highly conserved Ixodes scapularis (Ixs) tick saliva serpin (S) 17 (IxsS17) was highly secreted by B. burgdorferi infected nymphs. Here, we show that IxsS17 promote tick feeding and enhances B. burgdorferi colonization of the host. We show that IxsS17 is not part of a redundant system, and its functional domain reactive center loop (RCL) is 100% conserved in all tick species. Yeast expressed recombinant (r) IxsS17 inhibits effector proteases of inflammation, blood clotting, and complement innate immune systems. Interestingly, differential precipitation analysis revealed novel functional insights that IxsS17 interacts with both effector proteases and regulatory protease inhibitors. For instance, rIxsS17 interacted with blood clotting proteases, fXII, fX, fXII, plasmin, and plasma kallikrein alongside blood clotting regulatory serpins (antithrombin III and heparin cofactor II). Similarly, rIxsS17 interacted with both complement system serine proteases, C1s, C2, and factor I and the regulatory serpin, plasma protease C1 inhibitor. Consistently, we validated that rIxsS17 dose dependently blocked deposition of the complement membrane attack complex via the lectin complement pathway and protected complement sensitive B. burgdorferi from complement-mediated killing. Likewise, co-inoculating C3H/HeN mice with rIxsS17 and B. burgdorferi significantly enhanced colonization of mouse heart and skin organs in a reverse dose dependent manner. Taken together, our data suggests an important role for IxsS17 in tick feeding and B. burgdorferi colonization of the host.

Genetic manipulation of an Ixodes scapularis cell line.

Although genetic manipulation is one of the hallmarks of model organisms, its applicability to non-model species has remained difficult due to our limited understanding of their fundamental biology. For instance, manipulation of a cell line originated from the black-legged tick Ixodes scapularis, an arthropod that serves as a vector for several human pathogens, has yet to be established. Here, we demonstrate the successful genetic modification of the commonly used tick ISE6 line through ectopic expression and clustered regularly interspaced palindromic repeats [(CRISPR)/CRISPR-associated protein 9 (Cas9)] genome editing. We performed ectopic expression using nucleofection and attained CRISPR-Cas9 editing via homology-dependent recombination. Targeting the E3 ubiquitin ligase x-linked inhibitor of apoptosis (xiap) and its substrate p47 led to an alteration in molecular signaling within the immune deficiency network and increased infection of the rickettsial agent Anaplasma phagocytophilum in I. scapularis ISE6 cells. Collectively, our findings complement techniques for the genetic engineering of I. scapularis ticks, which currently limit efficient and scalable molecular genetic screens in vivo.IMPORTANCEGenetic engineering in arachnids has lagged compared to insects, largely because of substantial differences in their biology. This study unveils the implementation of ectopic expression and CRISPR-Cas9 gene editing in a tick cell line. We introduced fluorescently tagged proteins in ISE6 cells and edited its genome via homology-dependent recombination. We ablated the expression of xiap and p47, two signaling molecules present in the immune deficiency (IMD) pathway of Ixodes scapularis. Impairment of the tick IMD pathway, an analogous network of the tumor necrosis factor receptor in mammals, led to enhanced infection of the rickettsial agent Anaplasma phagocytophilum. Altogether, our findings provide a critical technical resource to the scientific community to enable a deeper understanding of biological circuits in the black-legged tick I. scapularis.

Seasonal pattern of questing ticks and prevalence of pathogenic Rickettsia and Anaplasmataceae in Khao Yai national park, Thailand.

BACKGROUND: Tick-borne diseases (TBD) are considered neglected diseases in Thailand with disease burden likely underestimated. To assess risk for emerging TBD in Thailand, the seasonality of questing tick and pathogen prevalence were studied in Khao Yai National Park, a top tourist destination. METHODS: During 2019, questing ticks around tourist attractions were systematically collected bimonthly and analyzed for Rickettsia and Anaplasmataceae bacterial species by polymerase chain reaction and DNA sequencing. RESULTS: Larvae and nymphs of questing ticks peaked in Khao Yai National Park during the late rainy-winter season, though no specific trends were observed in adult ticks. Winter (November to February) was the highest risk for human tick-bites due to higher numbers of both ticks and visitors. Of the total 5916 ticks analyzed (651 pools), Anaplasma phagocytophilum, Neoehrlichia mikurensis, Ehrlichia ewingii, and Ehrlichia chaffeensis were detected at low rates (≤0.05%). There was a higher prevalence of human rickettsioses (0.2-7%) in ticks surveyed with Rickettsia tamurae, Rickettsia raoultii, and Rickettsia montana the major species. Amblyomma ticks had the highest prevalence of Rickettsia (85%, 35/44 Amblyomma adults), in which only R. tamurae and R. raoultii were found in Amblyomma with mixed species infections common. We report the first detection of R. africae-like and N. mikurensis in Ixodes granulatus adults in Thailand, suggesting I. granulatus as a potential vector for these pathogens. CONCLUSION: This study demonstrated the risk of emerging TBD in Thailand and underscores the need for tick-bite prevention among tourists in Thailand.