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, Borreliaburgdorferi 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 five 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.
Hinweise
Karol Borawski and Justyna Dunaj contributed equally to the manuscript.
Introduction
Ixodes ricinus ticks, which are common in Poland, transmit several different pathogens: Borrelia burgdorferi sensu lato (Borrelia species), tick-borne encephalitis virus (TBEV), Anaplasma phagocytophilum (A. phagocytophilum) and Babesia species (Babesia spp.). They may also transmit other less known pathogens, such as Coxiella burnetii (C. burnetii), Rickettsiales or Candidatus Neoehrlichia mikurensis [1].
Gram-negative C. burnetii is responsible for zoonosis called Q fever, which most often manifests as flu-like illness with fever, general malaise, severe headache, muscle pain, loss of appetite, dry cough and chills. Also other symptoms such as: vomiting, diarrhea, nausea, endocarditis and pneumonia may occur. The reservoir of bacteria is: cattle, sheep, goats, dogs and other domestic animals.
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In vertebrates, infection affects reticulo-endothelial, vascular endothelial cells or erythrocytes. C. burnetii bacteria were found in the gut and hemolymph of ticks, suggesting that they are a natural vector [2]. Infection occurs most often by inhaling aerosols contaminated with particles of feces, urine or milk of animals. The bacterium is also rarely transmitted to humans by ticks. C. burnetii was detected in more than 40 tick species, mainly in those belonging to the genus Ixodes, Rhipicephalus, Amblyomma and Dermacentor. Moreover, ticks may play a role of a vector of Q fever. C. burnetii bacteria have the ability to penetrate their digestive tract and multiply in epithelial cells of the intestine and in the midgut. Ticks can transmit bacteria through saliva and feces, the latter contaminating the skin and fur of the animal. However, tick’s participation in the epidemiological process in many cases is limited to the passive spread of the pathogen in the environment and is not a necessary link in the epidemical chain [3].
The incubation time of Q fever is 9–40 days. Q fever is considered one of the most contagious diseases in the world, because only one bacterium can be sufficient to cause infection in susceptible patients [4].
Q fever has gained renewed attention after the large outbreak in the Netherlands in 2007–2009, indicating its importance as an emerging public health threat [5].
The data concerning C. burnetii epidemiology in Poland is scarce. There are only individual cases of patients with Q fever described in Poland. The last epidemics occurred in 1983 in the Lublin region, then in 2005–2011 25 individuals presented clinical symptoms of acute Q fever and DNA of C. burnetii was found in 8 human blood samples obtained from 3 farm workers and 5 family members [6]. According to the annual report of the National Institute of Public Health–National Institute of Hygiene, only a single case of Q fever has been recognized since 2010.
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The main aim of the study is to assess the prevalence of anti-C. burnetii antibodies in the inhabitants of the Podlaskie Voivodship (Fig. 1), which is considered as endemic area of tick-borne diseases. Another goal is to assess the risk of Q fever development after tick bite. Moreover, we assessed the percentage of co-infection with C. burnetii and other pathogens.
The categories of patients, the tests performed and the obtained results
Serological, which assessed the prevalence of anti-C. burnetii antibodies in people endangered by tick bites, but without symptoms;
Molecular, which aimed at detecting C. burnetii DNA in patients with symptoms suggestive of Q fever.
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The serological study included 184 people divided into three groups:
Group Ia—82 foresters from the Podlaskie Voivodship remaining in cooperation with the Department of Infectious Diseases and Neuroinfections: 4 women and 78 men;
Group IIa—82 patients—farmers living in the Podlaskie Voivodship, hospitalized in the Department of Infectious Diseases and Neuroinfections in 2015–2018 due to various symptoms after tick bite: 36 women and 46 men;
Group III—control group—20 honorary blood donors from the Regional Centre for Transfusion Medicine, Bialystok, Poland, who have never been bitten by ticks.
The molecular study included 560 people:
Group Ib—540 patients, hospitalized in the Department of Infectious Diseases and Neuroinfections in 2015–2018 because of various symptoms after tick bite. For molecular study, the control group was the same as for serological tests (20 blood donors—Group III).
Blood samples for molecular and immunoserological diagnostics were collected from patients. Clinical analysis of all patients was performed based on medical documentation and a personal questionnaire form prepared specifically for this study. We recorded the presence or absence of the presenting symptomatology including fever, headache, dizziness, musculoskeletal pain, back pain, neck pain, malaise, photophobia, nausea/vomit, cranial nerve paresis and duration of fever determined by review of clinical notes made by physicians following diagnosis.
The study was approved by the Bioethical Commission of Medical University of Bialystok (R-I-002/329/2018). All patients signed a written informed consent form for the study.
Methods: serological analyses
Anti-Coxiella burnetii antibodies titers were determined by ELISA: C. burnetii (Q fever) Phase 1 IgG ELISA (DRG International Inc. USA).
TBE was confirmed by detection of specific antibodies with enzyme-linked immunosorbent assay (ELISA) using the kit of Virion/Serion (Wurzburg, Germany) according to the manufacturer’s instructions.
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NB (neuroborreliosis) was confirmed by detection of specific anti-B.burgdorferi antibodies in serum and CSF with enzyme-linked immunosorbent assay (ELISA) using the kit of Borrelia IgM, IgG (DRG, Germany) confirmed by Western blot (DRG, Germany) according to the manufacturer’s instructions and with intrathecal synthesis of anti-B. burgdorferi antibodies (EcoLine; Virotech).
Methods: DNA extraction
DNA was extracted with the Qiagen DNAeasy Blood and Tissue Mini kit. Whole blood was gently mixed (200 µl) and skin biopsies were enzymatically digested at 56 °C before extraction. Purified DNA isolates were frozen at − 20 °C.
Methods: molecular techniques (PCR)
Molecular analysis for Coxiella burnetii
The Hum PCR Coxiella burnetii detection kit (Bioingentech Ltd., Chile) for in vitro diagnostics was used for C. burnetii molecular detection.
One-tube type of conventional qualitative PCR was performed. To each PCR tube with 2.7 µl of HumPCR C. burnetii premixture, 6 µl of Free Water and 2 µl of sample DNA, negative or positive control, was added. Internal control samples was prepared with 2.7 µl of Internal Control Mixture, 6 µl 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 tubes on the top of the mixture, 8 µl of Mineral Oil was added.
PCR was performed on the SensoQuest LabCycler (SensoQuest, Germany) in compatibility to Bioingentech’s instruction: initial denaturation at 94 °C for 2 min, amplification for 30 cycles (denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s, extension at 72 °C for 30 s) and final extension at 72 °C for 5 min.
Amplification products were separated on 1.5% agarose gel (Sigma-Aldrich, Germany) containing Midori Green (5 µg/1 ml; Nippon Genetics, Japan) in electrophoresis at 100 V for 45 min. Amplicons were visualized by means of UV illumination in Gel Logic System 100 (Kodak Imaging System, Inc., USA). Positive samples were those with amplification products with the length of 340 bp fragments of C. burnetii gene. Additionally, 140 bp-long fragments of internal standard were detected in all samples.
Molecular analysis for B. burgdorferi and A. phagocytophilum
Molecular detection of Borrelia species was performed by using The Borrelia burgdorferi PCR kit (GeneProof, Czech Republic) which amplifies a specific DNA sequence of a 276 bp fragment of the flagellin encoding gene by a nested one-tube PCR on the SensoQuest LabCycler (SensoQuest, Germany) [7]. To further confirm the results, PCR amplification was performed with a real-time PCR assay targeting the 16S rRNA gene as previously described [8].
For A. phagocytophilum in vitro detection, a gene fragment encoding a part of the small ribosomal 16S rRNA subunit (546 bp) was amplified (Blirt-DNA Gdańsk, Poland). Analyses were conducted by nested PCR on a SensoQuest LabCycler (SensoQuest, Germany) [7].
Methods: statistical analysis
The statistical analysis was performed using the Statistica 10.0 program. Kruskal–Wallis and Spearman rank correlation testes were used. P values < 0.05 were considered statistically significant.
Results
Results of serological tests
Among 184 people included in the study, 82 were foresters: 4 women and 78 men, and 82 patients were farmers hospitalized in the Department of Infectious Diseases and Neuroinfections in 2015–2018 due to various symptoms after tick bite: 36 women and 46 men.
Group Ia
Anti-B. burgdorferi IgG antibodies were identified in 81 (98%) of the 82 foresters included in this study (ELISA confirmed with Western blot). IgM anti-B. burgdorferi-specific antibodies were present in serum of ten (12.2%) patients. In 80 (97%) patients, anti-TBE antibodies were detected after the vaccination (Fig. 2).
Anti-C. burnetii IgG were 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.
Group IIa
Forty-one patients were hospitalized because of TBE, while 38 because of Lyme disease: 10 with EM and 10 with NB and 18 with musculoskeletal symptoms (among them 8 were co-infected with B. burgdorferi and tick-borne encephalitis virus). In three patients, anaplasmosis was diagnosed.
Anti-C. burnetii IgG were detected in five farmers (6%). Four farmers with anti-C. burnetii IgG presence were positive toward anti-B. burgdorferi IgG and two with anti-TBE (Fig. 2). Among them, one was co-infected with B. burgdorferi and tick-borne encephalitis virus. No co-infection with A. phagocytophilum was observed.
Group III
In the control group, no anti-C. burnetii IgG, anti-B. burgdorferi IgG, anti-TBE IgG and DNA of A. phagocytophilum were detected.
No statistical significance was detected between the frequency of anti-C. burnetii IgG between both groups; however, there were differences between group Ia and CG, IIa and CG (p < 0.05).
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Analysis of correlations
The most common symptoms reported by patients from group IIa were headache—57.3% (47/82) and fever (over 38 °C)–51.2% (42/82). Less common manifestations were: nausea—25.6% (21/82) and vertigo—18.2% (21/183). Symptoms which appeared rarely were vomit—12.1% (10/82), facial nerve paresis—7.3% (6/82) and muscle pain—3.6% (3/82). Fever lasted from 1 to 20 days (mean: 4.04 ± 4.03; median 3 days). No correlation between symptoms reported by patients and anti-C. burnetii antibodies was detected.
IgM anti-B. burgdorferi-specific antibodies were present in the serum of 25 (30%) patients and IgG antibodies–in 34 (41%) patients. Seventeen (21%) patients were positive in both classes.
There was correlation between anti-C. burnetii IgG presence and anti-B. burgdorferi IgG presence (r = 0.974; p < 0.05) and between anti-C. burnetii IgG presence and symptoms of Lyme disease (r = 0.231; p < 0.05).
No correlation between anti-C. burnetii IgG presence and anti-TBE antibodies presence was seen.
Results of molecular analyses
In none of the 540 (0%) patients, the DNA of C. burnetii was detected using conventional PCR. The results of molecular tests for B. burgdorferi and A. phagocytophilum infection are presented in Fig. 2.
Discussion
In our study, we concentrated on tick bite as a risk factor of C. burnetii infection. This is not the main route of Q fever spread and therefore our results should be interpreted with care.
The results of our study indicate the possibility of C. burnetii infection after tick bite in Poland. Although we have not detected C. burnetii DNA in the samples, the presence of antibodies against this pathogen in 11 patients confirms the circulation of C. burnetii in the environment. The risk of symptomatic infection seems to be minimal; however, it has to be taken into consideration in the differential diagnosis of fever after tick bite, as the symptoms of Q fever are nonspecific. It is worth underlining that the study was performed in an area where annual incidence of tick-borne diseases such as TBE or LB is 16–28 times higher than in the whole country throughout the years. Moreover, the population included in the study comprised foresters and farmers, so people were occupationally exposed to frequent tick bites. So even in the endemic area for tick-borne diseases and in the group of patients with frequent tick bites, the risk of Q fever is low. It reflects well the data from the National Institute of Health, where registered incidence on Q fever is low—one case in 2014 and five cases in 2009 [9].
Studies performed in specific regions of Poland indicate that C. burnetii prevalence varies in different studies and throughout the country.
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Szymańska-Czerwińska et al., tested 2082 serum samples taken from 936 goats, 933 cattle, 89 sheep and 124 horses, including various horse breeds, and revealed that Polish horses were seronegative, while in the populations of cattle and small ruminants, seropositive animals were present. The percentage of seropositive cattle, goats and sheep was 4.18%, 6.3%, and 13.48%, respectively [10].
The prevalence of C. burnetii in ticks ranged from 0.45 to 3.45% in north-western Poland [11] to 15.9% in south-eastern Poland [12], although the samples, isolated from ticks collected from places where local outbreaks occurred, accounted for 33.3%, which may suggest that ticks can be an important vector for C.burnetii [13].
The results of studies performed on humans were equivocal. Wójcik-Fatla et al. detected the presence of Coxiella burnetii antibodies in 16 out of 373 (4.3%) veterinarians [14]. The study included people from 12 districts of Poland. On the other hand, Szymańska-Czerwińska et al. examined 151 farmers from six regions of Poland and acquired significantly different results. The samples tested with indirect fluorescent antibody (IFA) were positive in 31.12%, with ELISA 39.07% and with complement fixation test (CFT) 15.23%. Of the three test types, IFA results were considered the most sensitive. Real-time PCR confirmed the presence of DNA specific for C. burnetii in ten patients [15]. Moreover, the authors analyzed patients with direct contact with animals and our study concentrated on patients after a tick bite.
In central Poland C. burnetii IgG antibodies have been found in sera of 4.4% of the farmers and in 12% of waste collectors [16].
In south-eastern Poland (Lublin area), the prevalence of anti-C. burnetii antibodies among hunters was higher—16.2% of 104 sera [17]. Another study conducted on a group of farmers inhabiting this region yielded similar results (17.8% of sera positive) [18].
Our study showed the correlation between anti-C. burnetii IgG presence and anti-B. burgdorferi IgG presence, which might suggest that these pathogens are transmitted by the same tick and lead to co-infection.
In persons with occupational risk of tick bites—forestry workers and farmers—anti-B.burgdorferi antibodies are more frequently detected and they are more often co-infected with various tick-borne pathogens than persons from the control group, as shown in previous studies [19]. Clinical analysis of symptomatic cases showed no infection caused by C. burnetii among 540 analyzed patients. It seems that although north-eastern Poland is an endemic region for tick-borne diseases, the prevalence of C. burnetii in humans is rather low and the possibility of Q fever development after tick bite is even lower.
Conclusions
1.
Seroprevalence of C. burnetii antibodies in north–eastern Poland is low, but this bacterium is present in the environment and may cause disease in humans.
2.
The most common co-occurrence after a tick bite concerns C. burnetii and B. burgdorferi.
3.
Q fever development after a tick bite in north-eastern Poland is very unlikely.
Compliance with ethical standards
Conflict of interests
None.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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