Infection (2020) 48:85–90 https://doi.org/10.1007/s15010-019-01355-w ORIGINAL PAPER Assessment of Coxiella burnetii presence after tick bite in north‑eastern Poland Karol Borawski1 · Justyna Dunaj1 · Piotr Czupryna1 · Sławomir Pancewicz1 · Renata Świerzbińska1 · Agnieszka Żebrowska2 · Anna Moniuszko‑Malinowska1 Received: 12 June 2019 / Accepted: 4 September 2019 / Published online: 14 September 2019 © The Author(s) 2019 Abstract Purpose The aim of the study is to assess anti-Coxiella burnetii antibodies presence in inhabitants of north-eastern Poland, to assess the risk of Q fever after tick bite and to assess the percentage of co-infection with other pathogens. Methods The serological study included 164 foresters and farmers with a history of tick bite. The molecular study included 540 patients, hospitalized because of various symptoms after tick bite. The control group consisted of 20 honorary blood donors. Anti-Coxiella burnetii antibodies titers were determined by Coxiella burnetii (Q fever) Phase 1 IgG ELISA (DRG International Inc. USA). PCR was performed to detect DNA of C. burnetii, Borrelia burgdorferi and Anaplasma phagocytophilum. Results Anti-C. burnetii IgG was detected in six foresters (7.3%). All foresters with the anti-C. burnetii IgG presence were positive toward anti-B. burgdorferi IgG and anti-TBE (tick-borne encephalitis). Anti-C. burnetii IgG was detected in fve farmers (6%). Four farmers with anti-C. burnetii IgG presence were positive toward anti-B. burgdorferi IgG and two with anti-TBE. Among them one was co-infected with B. burgdorferi and TBEV. Correlations between anti-C. burnetii IgG and anti-B. burgdorferi IgG presence and between anti-C. burnetii IgG presence and symptoms of Lyme disease were observed. C. burnetii DNA was not detected in any of the 540 (0%) patients. Conclusions C. burnetii is rarely transmitted by ticks, but we proved that it is present in the environment, so it may be a danger to humans. The most common co-occurrence after tick bite concerns C. burnetii and B. burgdorferi. Keywords Coxiella burnetii · Borrelia burgdorferi · Anaplasma phagocytophilum · Serology · PCR Introduction burnetii), Rickettsiales or Candidatus Neoehrlichia miku- rensis [1]. Ixodes ricinus ticks, which are common in Poland, transmit Gram-negative C. burnetii is responsible for zoonosis several diferent pathogens: Borrelia burgdorferi sensu lato called Q fever, which most often manifests as fu-like illness (Borrelia species), tick-borne encephalitis virus (TBEV), with fever, general malaise, severe headache, muscle pain, Anaplasma phagocytophilum (A. phagocytophilum) and loss of appetite, dry cough and chills. Also other symptoms Babesia species (Babesia spp.). They may also transmit such as: vomiting, diarrhea, nausea, endocarditis and pneu- other less known pathogens, such as Coxiella burnetii (C. monia may occur. The reservoir of bacteria is: cattle, sheep, goats, dogs and other domestic animals. In vertebrates, infection afects reticulo-endothelial, vas- Karol Borawski and Justyna Dunaj contributed equally to the cular endothelial cells or erythrocytes. C. burnetii bacteria manuscript. were found in the gut and hemolymph of ticks, suggesting * Anna Moniuszko-Malinowska that they are a natural vector [2]. Infection occurs most [email protected] often by inhaling aerosols contaminated with particles of feces, urine or milk of animals. The bacterium is also rarely 1 Department of Infectious Diseases and Neuroinfections, transmitted to humans by ticks. C. burnetii was detected in Medical University of Białystok, Żurawia 14, 15-540 Białystok, Poland more than 40 tick species, mainly in those belonging to the genus Ixodes, Rhipicephalus, Amblyomma and Dermacentor. 2 Regional Centre for Transfusion Medicine, Bialystok, Poland Vol.:(0123456789)1 3 86 K. Borawski et al. Moreover, ticks may play a role of a vector of Q fever. C. Materials and methods burnetii bacteria have the ability to penetrate their digestive tract and multiply in epithelial cells of the intestine and in Material and patient group (Fig. 2) the midgut. Ticks can transmit bacteria through saliva and feces, the latter contaminating the skin and fur of the ani- The study was divided into two separate steps: mal. However, tick’s participation in the epidemiological process in many cases is limited to the passive spread of the – Serological, which assessed the prevalence of anti-C. pathogen in the environment and is not a necessary link in burnetii antibodies in people endangered by tick bites, the epidemical chain [3]. but without symptoms; The incubation time of Q fever is 9–40 days. Q fever is – Molecular, which aimed at detecting C. burnetii DNA in considered one of the most contagious diseases in the world, patients with symptoms suggestive of Q fever. because only one bacterium can be sufcient to cause infec- tion in susceptible patients [4]. The serological study included 184 people divided into Q fever has gained renewed attention after the large out- three groups: break in the Netherlands in 2007–2009, indicating its impor- tance as an emerging public health threat [5]. • Group Ia—82 foresters from the Podlaskie Voivodship The data concerning C. burnetii epidemiology in Poland remaining in cooperation with the Department of Infec- is scarce. There are only individual cases of patients with Q tious Diseases and Neuroinfections: 4 women and 78 fever described in Poland. The last epidemics occurred in men; 1983 in the Lublin region, then in 2005–2011 25 individuals • Group IIa—82 patients—farmers living in the Podlaskie presented clinical symptoms of acute Q fever and DNA of Voivodship, hospitalized in the Department of Infectious C. burnetii was found in 8 human blood samples obtained Diseases and Neuroinfections in 2015–2018 due to vari- from 3 farm workers and 5 family members [6]. Accord- ous symptoms after tick bite: 36 women and 46 men; ing to the annual report of the National Institute of Public • Group III—control group—20 honorary blood donors Health–National Institute of Hygiene, only a single case of from the Regional Centre for Transfusion Medicine, Bia- Q fever has been recognized since 2010. lystok, Poland, who have never been bitten by ticks. The main aim of the study is to assess the prevalence of anti-C. burnetii antibodies in the inhabitants of the Pod- The molecular study included 560 people: laskie Voivodship (Fig. 1), which is considered as endemic area of tick-borne diseases. Another goal is to assess the • Group Ib—540 patients, hospitalized in the Department risk of Q fever development after tick bite. Moreover, we of Infectious Diseases and Neuroinfections in 2015–2018 assessed the percentage of co-infection with C. burnetii and other pathogens. Fig. 2 Fig. 1 The location of Podlaskie Voivodship The categories of patients, the tests performed and the obtained results 1 3 87 Assessment of Coxiella burnetii presence after tick bite in north-eastern Poland because of various symptoms after tick bite. For molecu- negative or positive control, was added. Internal control sam- lar study, the control group was the same as for serologi- ples was prepared with 2.7 µl of Internal Control Mixture, 6 µl cal tests (20 blood donors—Group III). of Free Water and 2 µl of sample DNA. The total volume of the PCR mixture with template DNA was 10.7 µl. To all PCR Blood samples for molecular and immunoserological tubes on the top of the mixture, 8 µl of Mineral Oil was added. diagnostics were collected from patients. Clinical analysis PCR was performed on the SensoQuest LabCycler (Senso- of all patients was performed based on medical documenta- Quest, Germany) in compatibility to Bioingentech’s instruc- tion and a personal questionnaire form prepared specifcally tion: initial denaturation at 94 °C for 2 min, amplifcation for for this study. We recorded the presence or absence of the 30 cycles (denaturation at 94 °C for 30 s, annealing at 57 °C presenting symptomatology including fever, headache, diz- for 30 s, extension at 72 °C for 30 s) and fnal extension at ziness, musculoskeletal pain, back pain, neck pain, malaise, 72 °C for 5 min. photophobia, nausea/vomit, cranial nerve paresis and dura- Amplifcation products were separated on 1.5% agarose tion of fever determined by review of clinical notes made by gel (Sigma-Aldrich, Germany) containing Midori Green physicians following diagnosis. (5 µg/1 ml; Nippon Genetics, Japan) in electrophoresis The study was approved by the Bioethical Commission at 100 V for 45 min. Amplicons were visualized by means of of Medical University of Bialystok (R-I-002/329/2018). All UV illumination in Gel Logic System 100 (Kodak Imaging patients signed a written informed consent form for the study. System, Inc., USA). Positive samples were those with ampli- fcation products with the length of 340 bp fragments of C. Methods: serological analyses burnetii gene. Additionally, 140 bp-long fragments of internal standard were detected in all samples. Anti-Coxiella burnetii antibodies titers were determined by Molecular analysis for B. burgdorferi and A. ELISA: C. burnetii (Q fever) Phase 1 IgG ELISA (DRG phagocytophilum International Inc. USA). TBE was confrmed by detection of specifc antibodies Borrelia with enzyme-linked immunosorbent assay (ELISA) using Molecular detection of species was performed by The Borrelia burgdorferi PCR kit the kit of Virion/Serion (Wurzburg, Germany) according to using (GeneProof, Czech the manufacturer’s instructions. Republic) which amplifes a specifc DNA sequence of a NB (neuroborreliosis) was confrmed by detection of spe- 276 bp fragment of the fagellin encoding gene by a nested cifc anti-B. burgdorferi antibodies in serum and CSF with one-tube PCR on the SensoQuest LabCycler (SensoQuest, enzyme-linked immunosorbent assay (ELISA) using the kit Germany) [7]. To further confrm the results, PCR amplifca- of Borrelia IgM, IgG (DRG, Germany) confrmed by West- tion was performed with a real-time PCR assay targeting the 16S rRNA ern blot (DRG, Germany) according to the manufacturer’s gene as previously described [8].