Bacteria and protozoa with pathogenic potential in Ixodes ricinus ticks in Viennese recreational areas.

Ixodes ricinus is the most relevant vector for tick-borne diseases in Austria and responsible for the transmission of Borrelia burgdorferi sensu lato (s. l.), which causes Lyme borreliosis in humans; however, also other bacteria and protozoa can be found in ticks and have the potential of infecting people and animals. In this study we collected ticks in popular recreational areas in the city of Vienna in the years 2019 and 2020 and analyzed them for the presence of such putative pathogenic microorganisms. By using reverse line blot (RLB) hybridization we detected DNA of B. burgdorferi s. l., Rickettsia spp., Babesia spp., Candidatus Neoehrlichia mikurensis (CNM) and Anaplasma phagocytophilum. Moreover, we also screened them for the relapsing fever spirochete Borrelia miyamotoi employing real-time PCR. The most frequently detected pathogens were B. burgdorferi s. l. in 28.6% of the ticks in 2019 and 21.3% of the ticks in 2020. The genus Rickettsia was detected in 13.8% of the ticks from 2019 and only in 4.6% from 2020. Babesia spp. were detected in 5.7% in 2019 and 4.2% in 2020. Furthermore, we detected CNM in 4.0% (2019) and 5.6% (2020), A. phagocytophilum in 0.5% (2019) and 1.3% (2020) and finally B. miyamotoi in 3.3% (2019) and 1.7% (2020). Collectively, we show that various microorganisms are prevalent in ticks collected in Vienna and identify hotspots for B. miyamotoi, which we have detected for the first time in the city.

Tick feeding modulates the human skin immune landscape to facilitate tick-borne pathogen transmission.

During cutaneous tick attachment, the feeding cavity becomes a site of transmission for tick salivary compounds and tick-borne pathogens. However, the immunological consequences of tick feeding for human skin remain unclear. Here, we assessed human skin and blood samples upon tick bite and developed a human skin explant model mimicking Ixodes ricinus bites and tick-borne pathogen infection. Following tick attachment, we observed rapidly occurring patterns of immunomodulation, including increases in neutrophils and cutaneous B and T cells. T cells upregulated tissue residency markers, while lymphocytic cytokine production was impaired. In early stages of Borrelia burgdorferi model infections, we detected strain-specific immune responses and close spatial relationships between macrophages and spirochetes. Preincubation of spirochetes with tick salivary gland extracts hampered accumulation of immune cells and increased spirochete loads. Collectively, we showed that tick feeding exerts profound changes on the skin immune network that interfere with the primary response against tick-borne pathogens.

SARS-CoV-2-mRNA Booster Vaccination Reverses Non-Responsiveness and Early Antibody Waning in Immunocompromised Patients - A Phase Four Study Comparing Immune Responses in Patients With Solid Cancers, Multiple Myeloma and Inflammatory Bowel Disease.

Background: Individuals with secondary immunodeficiencies belong to the most vulnerable groups to succumb to COVID-19 and thus are prioritized for SARS-CoV-2 vaccination. However, knowledge about the persistence and anamnestic responses following SARS-CoV-2-mRNA vaccinations is limited in these patients. Methods: In a prospective, open-label, phase four trial we analyzed S1-specific IgG, neutralizing antibodies and cytokine responses in previously non-infected patients with cancer or autoimmune disease during primary mRNA vaccination and up to one month after booster. Results: 263 patients with solid tumors (SOT, n=63), multiple myeloma (MM, n=70), inflammatory bowel diseases (IBD, n=130) and 66 controls were analyzed. One month after the two-dose primary vaccination the highest non-responder rate was associated with lower CD19+ B-cell counts and was found in MM patients (17%). S1-specific IgG levels correlated with IL-2 and IFN-γ responses in controls and IBD patients, but not in cancer patients. Six months after the second dose, 18% of patients with MM, 10% with SOT and 4% with IBD became seronegative; no one from the control group became negative. However, in IBD patients treated with TNF-α inhibitors, antibody levels declined more rapidly than in controls. Overall, vaccination with mRNA-1273 led to higher antibody levels than with BNT162b2. Importantly, booster vaccination increased antibody levels >8-fold in seroresponders and induced anamnestic responses even in those with undetectable pre-booster antibody levels. Nevertheless, in IBD patients with TNF-α inhibitors even after booster vaccination, antibody levels were lower than in untreated IBD patients and controls. Conclusion: Immunomonitoring of vaccine-specific antibody and cellular responses seems advisable to identify vaccination failures and consequently establishing personalized vaccination schedules, including shorter booster intervals, and helps to improve vaccine effectiveness in all patients with secondary immunodeficiencies. Trial registration: EudraCT Number: 2021-000291-11.

Isolation of Francisella tularensis from Skin Ulcer after a Tick Bite, Austria, 2020.

Ulceroglandular tularemia is caused by the transmission of Francisella tularensis by arthropods to a human host. We report a case of tick-borne tularemia in Austria which was followed by an abscess formation in a lymph node, making drainage necessary. F. tularensis subsp. holarctica was identified by PCR and multilocus sequence typing.

Novel Protozoans in Austria Revealed through the Use of Dogs as Sentinels for Ticks and Tick-Borne Pathogens.

We previously isolated and cultivated the novel Rickettsia raoultii strain Jongejan. This prompted us to ask whether this strain is unique or more widely present in Austria. To assess this issue, we retrospectively screened ticks collected from dogs in 2008. Of these collected ticks, we randomly selected 75 (47 females and 28 males) Dermacentor reticulatus, 44 (21 females, 7 males, and 16 nymphs) Haemaphysalis concinna, and 55 (52 females and 3 males) ticks of the Ixodes ricinus complex. Subsequently, these ticks were individually screened for the presence of tick-borne pathogens using the reverse line blot hybridization assay. In our current study, we detected DNA from the following microbes in D. reticulatus: Anaplasma phagocytophilum, Borrelia lusitaniae, Borrelia spielmanii, Borrelia valaisiana, and R. raoultii, all of which were R. raoultii strain Jongejan. In H. concinna, we found DNA of a Babesia sp., Rickettsia helvetica, and an organism closely related to Theileria capreoli. Lastly, I. ricinus was positive for Anaplasma phagocytophilum, Borrelia afzelii, Borrelia burgdorferi sensu stricto, Borrelia garinii/Borrelia bavariensis, B. lusitaniae, B. spielmanii, B. valaisiana, Candidatus Neoehrlichia mikurensis, Rickettsia helvetica, Rickettsia monacensis, and Theileria (Babesia) microti DNA. The detection of DNA of the Babesia sp. and an organism closely related to Theileria capreoli, both found in H. concinna ticks, is novel for Austria.

Identification and Characterization of "Candidatus Rickettsia Thierseensis", a Novel Spotted Fever Group Rickettsia Species Detected in Austria.

Rickettsia spp. are the second most common pathogens detected in Ixodes ricinus ticks in Austria after Borrelia burgdorferi sensu lato. Species belonging to the spotted fever group (SFG) are the causative agents for tick-borne rickettsiosis across the world. So far, only four SFG Rickettsia spp. were detected in Austria, namely R. helvetica, R. raoultii, R. monacensis and R. slovaca. Here, we describe the identification of a new SFG Rickettsia species detected in an I. ricinus tick. Sequencing of various rickettsial genes revealed a nucleotide sequence similarity of 99.6%, 98.5%, 97.3% and 98.5% to the gltA, ompA, ompB, and sca4 genes, respectively, of known and validated species. Additionally, sequencing of the htrA gene and 23S-5S intergenic spacer region also only showed 99.6% and 99.2%, respectively, similarity to known species. Therefore, and in accordance with current criteria for Rickettsia species discrimination, we hereby describe a new species of the SFG with putative pathogenic potential. We propose the name "Candidatus Rickettsia thierseensis" based on the village Thiersee in the Austrian province of Tyrol, where the carrying tick was found.

Human Borrelia miyamotoi Infection, Austria.

We report a human case of Borrelia miyamotoi infection diagnosed in Austria. Spirochetes were detected in Giemsa-stained blood smears. The presence of B. miyamotoi in the patient's blood was confirmed by PCR, and phylogenetic analysis identified an infection with a strain from Europe.

Lyme Borreliosis with Scalp Eschar Mimicking Rickettsial Infection, Austria.

We report on a patient in Austria with scalp eschar and neck lymphadenopathy. Rickettsial etiology was excluded by culture, PCR, and serologic tests. Borrelia afzelii was identified from the eschar swab by PCR. Lyme borreliosis can mimic rickettsiosis; appropriate tests should be included in the diagnostic workup of patients with eschars.

First broad-range molecular screening of tick-borne pathogens in Ixodes (Pholeoixodes) kaiseri, with special emphasis on piroplasms.

Recently, the occurrence of Ixodes (Pholeoixodes) kaiseri has been reported for the first time in several European countries, but data on the molecular analysis of this hard tick species are still lacking. Therefore, in this study DNA extracts of 28 I. kaiseri (collected from dogs and red foxes in Germany, Hungary and Romania) were screened with reverse line blot hybridisation (RLB), PCR and sequencing for the presence of 43 tick-borne pathogens or other members of their families from the categories of Anaplasmataceae, piroplasms, rickettsiae and borreliae. Rickettsia helvetica DNA was detected in one I. kaiseri female (from a red fox, Romania), for the first time in this tick species. Six ticks (from red foxes, Romania) contained the DNA of Babesia vulpes, also for the first time in the case of I. kaiseri. Molecular evidence of R. helvetica and B. vulpes in engorged I. kaiseri does not prove that this tick species is a vector of the above two pathogens, because they might have been taken up by the ticks from the blood of foxes. In addition, one I. kaiseri female (from a dog, Hungary) harboured Babesia sp. badger type-B, identified for the first time in Hungary and Central Europe (i.e. it has been reported previously from Western Europe and China). The latter finding can be explained by either the susceptibility of dogs to Babesia sp. badger type-B, or by transstadial survival of this piroplasm in I. kaiseri.

Chlamydia trachomatis serovars in urogenital and ocular samples collected 2014-2017 from Austrian patients.

Infection of humans with Chlamydia trachomatis, a bacterial pathogen with a unique intracellular replication cycle, may cause a variety of clinical manifestations. These are linked to various serovars of the pathogen; trachoma to serovars A-C, oculogenital infections to serovars D-K, and lymphogranuloma venereum to serovars L1-L3. Nineteen serovars are known as human pathogens. The aim of the study was to determine the serovars of 401 C. trachomatis DNA positive extracts from original clinical specimens of patients in Austria including cervical and urethral swabs, urine, genital secretions and conjunctival swabs - collected from 2014 to 2017. Sequence analysis of the omp1 gene, encoding major outer-membrane protein was performed on each sample. In 50.1% of samples serovar E was identified and serovars F, D/Da and G/Ga were found in 16.2%, 9.7% and 9.0%, respectively. Remaining serovars were J (6.0%), K (4.7%), H (2.7%), B/Ba (1.0%), and I/Ia (0.5%). In 19 patients follow up samples could be tested. The majority of C. trachomatis serovars were associated with urogenital tract infections (D-K), however, one of them - serovar B/Ba - is linked to both, ocular and genital tract infection.

Severe Myasthenic Manifestation of Leptospirosis Associated with New Sequence Type of Leptospira interrogans.

We report the rapid development of a myasthenic crisis as the first-time manifestation of myasthenia gravis. The symptoms developed in the course of acute leptospirosis associated with a new sequence type of Leptospira interrogans. Antibiotic treatment led to rapid amelioration of myasthenia.

CXCL13 concentrations in cerebrospinal fluid of patients with Lyme neuroborreliosis and other neurological disorders determined by Luminex and ELISA.

The aims of the study were to determine and compare the concentration of CXCL13 in cerebrospinal fluid (CSF) of patients with Lyme neuroborreliosis (LNB) and various other neurological disorders applying a Luminex based assay and ELISA, and to find factors associated with CXCL13 concentration. CSF samples obtained from four clinically well-defined groups of patients (proven LNB, suspected LNB, tick-borne encephalitis (TBE), and aseptic meningitis/meningoencephalitis other than TBE) - 25 samples per group - were analyzed. The performance of the Luminex recomBead CXCL13 assay (Microgen, Neuried, Germany) and ELISA (Euroimmun, Lübeck, Germany) was assessed by receiver operating characteristics. CXCL13 cut-off values were presented as functions of CSF lymphocyte/monocyte counts. Demographic variables, CSF findings, and history of erythema migrans were assessed as possible predictors for CXCL13 CSF concentrations by a general linear model. The calculated cut-off values determined by the maximum of the Youden index were >131 pg/mL for recomBead and >259 pg/mL for the ELISA. RecomBead showed a sensitivity of 88% (68.8-97.5%) and a specificity of 94% (83.5-98.7%). For the ELISA the corresponding values were 84% (63.9-95.5%) and 98% (89.4-99.9%). The CXCL13 concentration positively correlated with CSF lymphocyte/monocyte count and Borrelia-specific intrathecal antibody index (p < 0.05). High CXCL13 concentrations were only found in the group with proven LNB. CXCL13 levels above cut-off value were established in some patients with viral meningitis/meningoencephalitis but were not detected in patients with suspected LNB without pleocytosis. Applying a linearized cut-off of the CXCL13 concentration in the CSF which is dependent on the CSF cell count is a novel approach in the laboratory diagnosis of LNB.

Approaches for Reverse Line Blot-Based Detection of Microbial Pathogens in Ixodes ricinus Ticks Collected in Austria and Impact of the Chosen Method.

Ticks transmit a large number of pathogens capable of causing human disease. In this study, the PCR-reverse line blot (RLB) method was used to screen for pathogens in a total of 554 Ixodes ricinus ticks collected from all provinces of Austria. These pathogens belong to the genera Borrelia, Rickettsiae, Anaplasma/Ehrlichia (including "Candidatus Neoehrlichia"), Babesia, and Coxiella The pathogens with the highest detected prevalence were spirochetes of the Borrelia burgdorferisensu lato complex, in 142 ticks (25.6%). Borrelia afzelii (80/142) was the most frequently detected species, followed by Borrelia burgdorferisensu stricto (38/142) and Borrelia valaisiana (36/142). Borrelia garinii/Borrelia bavariensis, Borrelia lusitaniae, and Borrelia spielmanii were found in 28 ticks, 5 ticks, and 1 tick, respectively. Rickettsia spp. were detected in 93 ticks (16.8%): R. helvetica (39/93), R. raoultii (38/93), R. monacensis (2/93), and R. slovaca (1/93). Thirteen Rickettsia samples remain uncharacterized. "Candidatus Neoehrlichia mikurensis," Babesia spp. (B. venatorum, B. divergens, B. microti), and Anaplasma phagocytophilum were found in 4.5%, 2.7%, and 0.7%, respectively. Coxiella burnetii was not detected. Multiple microorganisms were detected in 40 ticks (7.2%), and the cooccurrence of Babesia spp. and "Candidatus Neoehrlichia mikurensis" showed a significant positive correlation. We also compared different PCR-RLBs for detection of Borrelia burgdorferisensu lato and Rickettsia spp. and showed that different detection approaches provide highly diverse results, indicating that analysis of environmental samples remains challenging.IMPORTANCE This study determined the wide spectrum of tick-borne bacterial and protozoal pathogens that can be encountered in Austria. Surveillance of (putative) pathogenic microorganisms occurring in the environment is of medical importance, especially when those agents can be transmitted by ticks and cause disease. The observation of significant coinfections of certain microorganisms in field-collected ticks is an initial step to an improved understanding of microbial interactions in ticks. In addition, we show that variations in molecular detection methods, such as in primer pairs and target genes, can considerably influence the final results. For instance, detection of certain genospecies of borreliae may be better or worse by one method or the other, a fact of great importance for future screening studies.

The domestic pig as a potential model for Borrelia skin infection.

The skin lesion erythema migrans is a characteristic early manifestation of Lyme borreliosis in humans. However, the pathomechanisms leading to development of this erythema are not fully understood. Models that mimic human skin would enhance research in this field. Human and porcine skin structures strongly resemble each other. Therefore, we attempted to induce erythema migrans lesions in experimental Borrelia burgdorferi sensu lato infection in the skin of domestic pigs. The formation of erythema migrans-like lesions was observed after intradermal injection of these spirochetes, with the lesions forming very clearly in 2/6 animals when a strain of B. garinii was used. However, no molecular or clinical proof of systemic infection of the pigs with B. afzelii, B. burgdorferi sensu stricto, or B. garinii could be achieved.

Novel Rickettsia raoultii strain isolated and propagated from Austrian Dermacentor reticulatus ticks.

BACKGROUND: Continuous culture of tick cell lines has proven a valuable asset in isolating and propagating several different vector-borne pathogens, making it possible to study these microorganisms under laboratory conditions and develop serological tests to benefit public health. We describe a method for effective, cost- and labor-efficient isolation and propagation of Rickettsia raoultii using generally available laboratory equipment and Rhipicephalus microplus cells, further demonstrating the usefulness of continuous tick cell lines. R. raoultii is one of the causative agents of tick-borne lymphadenopathy (TIBOLA) and is, together with its vector Dermacentor reticulatus, emerging in novel regions of Europe, giving rise to an increased threat to general public health. METHODS: Dermacentor reticulatus ticks were collected in the Donau-Auen (Lobau) national park in Vienna, Austria. The hemolymph of ten collected ticks was screened by PCR-reverse line blot for the presence of rickettsial DNA. A single tick tested positive for R. raoultii DNA and was used to infect Rhipicephalus microplus BME/CTVM2 cells. RESULTS: Sixty-five days after infection of the tick-cell line with an extract from a R. raoultii-infected tick, we observed intracellular bacteria in the cultured cells. On the basis of microscopy we suspected that the intracellular bacteria were a species of Rickettsia; this was confirmed by several PCRs targeting different genes. Subsequent sequencing showed 99-100 % identity with R. raoultii. Cryopreservation and resuscitation of R. raoultii was successful. After 28 days identical intracellular bacteria were microscopically observed. CONCLUSIONS: R. raoultii was successfully isolated and propagated from D. reticulatus ticks using R. microplus BME/CTVM2 cells. The isolated strain shows significant molecular variation compared to currently known sequences. Furthermore we show for the first time the successful cryopreservation and resuscitation of R. raoultii.

Molecular diagnosis of African tick bite fever using eschar swabs in a traveller returning from Tanzania.

African tick bite fever is an emerging infectious disease among travellers caused by the pathogen Rickettsia africae. Most travel-associated cases have been reported from countries in southern Africa. So far it has rarely been reported among travellers to eastern Africa and our patient is one of the first described cases imported from Tanzania. A woman presented with fever, chills, headache, myalgia and a rickettsial eschar on her ankle after returning from Tanzania. The diagnosis of African tick bite fever is often based on clinical grounds due to a lack of reliable diagnostic tests at commencement of symptoms. In this patient direct molecular detection of R. africae was performed by PCR from a sample obtained non-invasively with a swab from the rickettsial eschar. A positive PCR result was achieved although the patient had already started antibiotic treatment with doxycycline. In conclusion, this non-invasive method enables early diagnosis of African tick bite fever by direct molecular detection of R. africae and might improve the management of undifferentiated fever in travellers from Africa.

Detection of Bartonella spp. in Ixodes ricinus ticks and Bartonella seroprevalence in human populations.

Ticks are vectors for many bacterial, protozoan and viral pathogens and are potential vectors for Bartonella species. Hunters and foresters, therefore, may be regarded as high-risk groups for Bartonella infections. The aims of this study were (i) to identify Bartonella species in questing Ixodes ricinus ticks collected in all provinces of Austria, and (ii) to determine the prevalence of antibodies to Bartonella species in hunters and blood donors in eastern Austria. A total of 515 larval, nymphal and adult I. ricinus, collected throughout Austria in 2005, were selected from the tick library at the Institute for Hygiene and Applied Immunology of the Medical University of Vienna and screened in a specific real-time PCR that targeted a region of the ssrA gene of Bartonella species. The overall Bartonella infection rate was 2.1% (11/515) and the highest rate, 7.5% (4/53), was found in ticks from Vienna. This finding was confirmed by screening a further 60 I. ricinus collected from Vienna in 2013: of these, 6.7% (4/60) were positive for Bartonella spp. The rate of infection was always higher in adult ticks. Sequence analysis in the Bartonella-positive ticks identified several species, including B. henselae, B. doshiae and B. grahamii. To our knowledge this is the first time that these species have been identified in I. ricinus in Austria. Prevalence of IgG antibodies against B. henselae and B. quintana was determined in serum samples from hunters (100) and blood donors (100): in hunters 23% were positive for B. quintana and in 2 samples (2%), antibodies to both B. quintana and B. henselae were detected; in blood donors 22% were positive for B. quintana, 1% for B. henselae and 5% for both. These results indicate that exposure to ticks does not constitutes a relevant risk for Bartonella infection.

Development of a serum-free liquid medium for Bartonella species.

The genus Bartonella comprises numerous species with at least 13 species pathogenic for humans. They are fastidious, aerobic, Gram negative, and facultative intracellular bacteria which cause a variety of human and non-human diseases. This study focused on the development of a serum-free liquid medium for culture of Bartonella species. Some liquid media are available commercially but all of them use undefined supplements such as fetal calf serum or defibrinated sheep blood. Our intention was to create a reproducible liquid medium for Bartonella species that can simply be prepared. We tested several supplements that could potentially support the growth of Bartonella species. Slight growth improvement was achieved with glucose and sucrose. However, hemin in particular improved the growth rate. At a temperature of 37 °C, a CO2 concentration of 5 %, a humidified atmosphere, and the use of the supplements glucose, sucrose, and hemin, we developed a medium that does not need serum as an undefined supplement any more. In conclusion, the newly developed medium supports growth of Bartonella species equal to the commercially available media but with the advantage that it has a serum-free formulation. It can be prepared fast and easy and is a useful tool in studying these bacteria.