Detection of a Low Level and Heterogeneous B Cell Immune Response in Peripheral Blood of Acute Borreliosis Patients With High Throughput Sequencing.

The molecular diagnosis of acute Borreliosis is complicated and better strategies to improve the diagnostic processes are warranted. High Throughput Sequencing (HTS) of human B cell repertoires after e.g., Dengue virus infection or influenza vaccination revealed antigen-associated "CDR3 signatures" which may have the potential to support diagnosis in infectious diseases. The human B cell immune response to Borrelia burgdorferi sensu lato-the causative agent of Borreliosis-has mainly been studied at the antibody level, while less attention has been given to the cellular part of the humoral immune response. There are indications that Borrelia actively influence the B cell immune response and that it is therefore not directly comparable to responses induced by other infections. The main goal of this study was to identify B cell features that could be used to support diagnosis of Borreliosis. Therefore, we characterized the B cell immune response in these patients by combining multicolor flow cytometry, single Borrelia-reactive B cell receptor (BCR) sequencing, and B cell repertoire deep sequencing. Our phenotyping experiments showed, that there is no significant difference between B cell subpopulations of acute Borreliosis patients and controls. BCR sequences from individual epitope-reactive B cells had little in common between each other. HTS showed, however, a higher complementarity determining region 3 (CDR3) amino acid (aa) sequence overlap between samples from different timepoints in patients as compared to controls. This indicates, that HTS is sensitive enough to detect ongoing B cell immune responses in these patients. Although each individual's repertoire was dominated by rather unique clones, clustering of bulk BCR repertoire sequences revealed a higher overlap of IgG BCR repertoire sequences between acute patients than controls. Even if we have identified a few Borrelia-associated CDR3aa sequences, they seem to be rather unique for each patient and therefore not suitable as biomarkers.

Integration of Ixodes ricinus genome sequencing with transcriptome and proteome annotation of the naïve midgut.

BACKGROUND: In Europe, Ixodes ricinus ticks are the most important vectors of diseases threatening humans, livestock, wildlife and companion animals. Nevertheless, genomic sequence information is missing and functional annotation of transcripts and proteins is limited. This lack of information is restricting studies of the vector and its interactions with pathogens and hosts. Here we present and integrate the first analysis of the I. ricinus genome with the transcriptome and proteome of the unfed I. ricinus midgut. METHODS: Whole genome sequencing was performed on I. ricinus ticks and the sequences were de novo assembled. In parallel, I. ricinus ticks were dissected and the midgut transcriptome sequenced. Both datasets were integrated by transcript discovery analysis to identify putative genes and genome contigs were screened for homology. An alignment-based and a motif-search-based approach were combined for the annotation of the midgut transcriptome. Additionally, midgut proteins were identified and annotated by mass spectrometry with public databases and the in-house built transcriptome database as references and results were cross-validated. RESULTS: The de novo assembly of 1 billion DNA sequences to a reference genome of 393 Mb length provides an unprecedented insight into the I. ricinus genome. A homology search revealed sequences in the assembled genome contigs homologous to 89% of the I. scapularis genome scaffolds indicating coverage of most genome regions. We identified moreover 6,415 putative genes. More than 10,000 transcripts from naïve midgut were annotated with respect of predicted function and/or cellular localization. By combining an alignment-based with a motif-search-based annotation approach, we doubled the number of annotations throughout all functional categories. In addition, 574 gel spots were significantly identified by mass spectrometry (p<0.05) and 285 distinct proteins expressed in the naïve midgut were annotated functionally and/or for cellular localization. Our systems approach reveals a midgut metabolism of the unfed tick that is prepared to sense and process an anticipated blood meal. CONCLUSIONS: This multiple-omics study vastly extends the publicly available DNA and RNA databases for I. ricinus, paving the way for further in-depth analysis of the most important European disease vector and its interactions with pathogens and hosts.

Prevalence of tick-borne pathogens in Ixodes ricinus and Dermacentor reticulatus ticks from different geographical locations in Belarus.

Worldwide, ticks are important vectors of human and animal pathogens. Besides Lyme Borreliosis, a variety of other bacterial and protozoal tick-borne infections are of medical interest in Europe. In this study, 553 questing and feeding Ixodes ricinus (n = 327) and Dermacentor reticulatus ticks (n = 226) were analysed by PCR for Borrelia, Rickettsia, Anaplasma, Coxiella, Francisella and Babesia species. Overall, the pathogen prevalence in ticks was 30.6% for I. ricinus and 45.6% for D. reticulatus. The majority of infections were caused by members of the spotted-fever group rickettsiae (24.4%), 9.4% of ticks were positive for Borrelia burgdorferi sensu lato, with Borrelia afzelii being the most frequently detected species (40.4%). Pathogens with low prevalence rates in ticks were Anaplasma phagocytophilum (2.2%), Coxiella burnetii (0.9%), Francisella tularensis subspecies (0.7%), Bartonella henselae (0.7%), Babesia microti (0.5%) and Babesia venatorum (0.4%). On a regional level, hotspots of pathogens were identified for A. phagocytophilum (12.5-17.2%), F. tularensis ssp. (5.5%) and C. burnetii (9.1%), suggesting established zoonotic cycles of these pathogens at least at these sites. Our survey revealed a high burden of tick-borne pathogens in questing and feeding I. ricinus and D. reticulatus ticks collected in different regions in Belarus, indicating a potential risk for humans and animals. Identified hotspots of infected ticks should be included in future surveillance studies, especially when F. tularensis ssp. and C. burnetii are involved.

Pathogen prevalence in ticks collected from the vegetation and livestock in Nigeria.

Ticks are important disease vectors that can cause considerable economic losses by affecting animal health and productivity, especially in tropical and subtropical regions. In this study, we investigated the prevalence and diversity of bacterial and protozoan tick-borne pathogens in ticks collected from the vegetation and cattle in Nigeria by PCR. The infection rates of questing ticks were 3.1% for Rickettsia species, 0.1% for Coxiella burnetii and 0.4% for Borrelia species. Other pathogens, such as Babesia, Theileria, Anaplasma, and Ehrlichia species, were not detected in ticks from the vegetation. Feeding ticks collected from cattle displayed infection rates of 12.5% for Rickettsia species, 14% for Coxiella burnetii, 5.9% for Anaplasma species, 5.1% for Ehrlichia species, and 2.9% for Theileria mutans. Babesia and Borrelia species were not detected in ticks collected from cattle. Mixed infections were found only in feeding ticks and mainly Rickettsia species and Coxiella burnetii were involved. The diversity of tick-borne pathogens in Nigeria was higher in feeding than in questing ticks, suggesting that cattle serve as reservoirs for at least some of the pathogens studied, in particular C. burnetii. The total estimated herd infection rates of 20.6% for a Rickettsia africae-like species, 27% for Coxiella burnetii, and 8.5% for Anaplasma marginale/centrale suggest that these pathogens may have considerable implications for human and animal health.

Detection of new Francisella-like tick endosymbionts in Hyalomma spp. and Rhipicephalus spp. (Acari: Ixodidae) from Bulgaria.

We report on the identification of two new Francisella-like endosymbionts (FLEs) found in three different tick species from Bulgaria. The FLEs were characterized by 16S rRNA and tul4 gene sequencing and seem to lack the molecular marker RD1. These two new taxa seem to be facultative secondary endosymbionts of ticks.

Detection of Babesia Sp. EU1 and members of spotted fever group rickettsiae in ticks collected from migratory birds at Curonian Spit, North-Western Russia.

To reveal the prevalence of spotted fever group (SFG) rickettsiae and Babesia sp. in Ixodes ricinus (L.) ticks from migratory birds, 236 specimens represented 8 species of Passeriformes and were collected at Curonian Spit in Kaliningrad enclave of North-Western Russia. The ticks (total 126) being detached from four bird species, Turdus philomelos, Fringilla coelebs, Parus major, and Sturnus vulgaris, were investigated by PCR using the primers Rp CS.877p/Rp CS.1258n for the detection of Rickettsia and BJ1/BN2 for Babesia spp. Babesia spp. were detected in 2 of 126 (1.6%) ticks. The partial sequence of 18S rDNA had 100% similarity to human pathogenic Babesia sp. EU1. The SFG rickettsiae were detected in 19 of 126 (15.1%) ticks collected from the above-mentioned bird species. BLAST analysis of SFG rickettsia gltA assigned sequences to human pathogenic Rickettsia helvetica (10.3%), Rickettsia monacensis (3.9%), and Rickettsia japonica (0.8%) with 98%-100% sequence similarity. The SFG rickettsiae and Babesia sp. EU1 in ticks collected from the passerines in Russia were detected for the first time. The survey indicates that migratory birds may become a reservoir for Babesia spp. and SFG rickettsiae. Future investigations need to characterize the role of birds in the epidemiology of these human pathogens in the region.

Prevalence and seasonality of tick-borne pathogens in questing Ixodes ricinus ticks from Luxembourg.

In Europe, ixodid ticks are important arthropod vectors of human and animal pathogens, but comprehensive studies of the prevalence of all relevant pathogens in Central Europe are scarce. As a result of ecological changes, the incidences of tick-borne infections are expected to increase. In this study, 1,394 nymphal and adult Ixodes ricinus ticks sampled monthly during the active season from 33 ecologically distinct collection sites throughout Luxembourg were screened for all human tick-borne pathogens relevant in Central Europe. Species were identified by sequence analysis of detection PCR amplicons. Mean infection rates of ticks were 11.3% for Borrelia burgdorferi sensu lato, 5.1% for Rickettsia sp., 2.7% for Babesia sp., and 1.9% for Anaplasma phagocytophilum. No tick was found to be infected with Coxiella sp., Francisella tularensis subsp., or Tick-borne encephalitis virus (TBEV). A total of 3.2% of ticks were infected with more than one pathogen species, including mixed Borrelia infections (1.5%). Seasonal variations of tick infection rates were observed for Borrelia, Babesia, and Anaplasma, possibly reflecting a behavioral adaptation strategy of questing ticks. A positive correlation between the grade of urbanization and Borrelia infection rate of ticks was observed, suggesting an established urban zoonotic cycle. We also found Hepatozoon canis (0.1%) and Bartonella henselae (0.3%), which so far have not been found in questing Ixodes ricinus ticks in Central Europe.

Sex steroid receptor evolution and signalling in aquatic invertebrates.

In vertebrate reproductive endocrinology sex steroids play a pivotal role via binding to receptors. However, information on the origin and relevance of sex steroids in invertebrates is limited. This review highlights current literature on steroid receptors in aquatic invertebrates and reports on some new findings. It has been shown that invertebrates of the deuterostome clade, such as Acrania and Echinodermata, respond to estrogens and androgens and, at least in Branchiostoma, an estrogen receptor has been cloned. Within the protostomes, most findings are related to aquatic molluscs. Sex steroid receptor-like proteins are abundant in gastropods, bivalves and cephalopods and also sex hormone signalling shows partial similarity to the deuterostomes. In ecdysozoans, however, the impact of sex steroids is still a matter of debate even though there is evidence on the presence of estrogen receptor-like proteins in Crustacea and on physiological effects of estrogens in both Nematoda and Crustacea. Recent findings suggest the presence of an estrogen receptor alpha-like protein of unclear physiological role in Gammarus fossarum (Crustacea). Binding studies revealed the crustacean Hyalella azteca to possess specific binding sites only for androgens but not for estrogens suggesting a possible limitation to functional androgen receptors in this species. Further studies have to be conducted to shed more light into the discussion about the controversy about sex steroid receptors in invertebrates.