Abstract
Coxiella burnetii is an environmentally stable intracellular bacterium responsible for severe global outbreaks of the zoonotic disease, Q fever. Q fever is transmitted by the inhalation of aerosols contaminated with the birth products and excretions of infected animals, mainly of sheep and goats. A 28-year-old cattle raiser presented with headache and fever without a significant medical history, although his cattle herd had previously been diagnosed with brucellosis. He spent time outdoors but reported no tick bites or eschar. Initial evaluation revealed mild thrombocytopenia and elevated aspartate aminotransferase, alanine aminotransferase, and C-reactive protein levels. On day 2 after disease onset, his blood tested positive for the IS1111 gene of C. burnetii by nested PCR. After amplifying C. burnetii in severe combined immunodeficient mice, the pathogen was isolated using a cell culture system. The isolated CH12 strain was confirmed via PCR, nucleotide sequence analysis, and whole-genome sequencing. This study reports a case of acute Q fever in Korea, in which C. burnetii was isolated and characterized using whole-genome sequencing.
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Key words: Coxiella burnetii, Q fever, Korea, isolation, whole genome sequencing
Introduction
Coxiella burnetii is an obligate intracellular, gram-negative bacterium belonging to the family
Coxiellaceae, and it is recognized as the etiological agent of Q fever in humans and coxiellosis in animals [
1]. With a broad host range that encompasses mammals, birds, reptiles, and arthropods,
C. burnetii is globally distributed and notable for its high environmental stability and extremely low infectious dose, which are factors that contribute to its classification as an emerging zoonotic pathogen [
2]. Human infection typically occurs through the inhalation of aerosolized particles contaminated with the birth products or bodily fluids derived from infected animals, which consequently leads to Q fever. This condition is often asymptomatic or presents as a nonspecific influenza-like illness, although severe and occasionally fatal cases have been documented [
2]. In animals,
C. burnetii infection is frequently subclinical, although it can result in reproductive complications, such as abortion, stillbirth, low birth weight, and weakened offspring [
3].
In Korea, the first case of Q fever was reported in 1992 [
4]. Over the past decade (2014–2024), 814 Q fever cases have been reported, with a peak of 163 cases in 2018, followed by a decline to approximately 50–60 cases annually. Among the cases with known occupations, 19.0% comprised individuals in high-risk animal-related professions, such as livestock farmers, slaughterhouse workers, and veterinary quarantine officers [
5]. In 2020, the first isolation of
C. burnetii was reported from nonspecific febrile patients in Korea [
6]. Here, we report the isolation and characterization of
C. burnetii from the organs of severe combined immunodeficient (SCID) mice that died of hepatosplenomegaly after being injected with Q fever–confirmed patient blood.
Case Report
A 28-year-old man who worked as a cattle raiser presented to the emergency department on December 3, 2022, for further evaluation of his elevated liver function test results. Two days prior, he presented to a local clinic with a headache and fever; he had no other relevant medical history. Two years before presentation, his herd of approximately 100 cattle had been diagnosed with brucellosis. He reported spending time outdoors but had no history of tick bites or eschar. At presentation (on day 2), the patient had a platelet count of 116,000/µl (normal range, 150,000–450,000/µl), C-reactive protein (CRP) level of 8.95 mg/dl (normal range, 0–0.3 mg/dl), aspartate aminotransferase (AST) level of 90 U/L (normal range, 5–40 U/L), alanine aminotransferase level of 106 U/L (normal range, 5–40 U/L), and D-dimer concentration of 1,700 ng/ml FEU (normal range, 0–560 ng/ml FEU). The white blood cell count, procalcitonin, creatine phosphokinase, and total bilirubin levels of the patient were normal. Moreover, the patient exhibited a blood pressure of 110/70 mmHg, pulse of 86 beats/min, respiratory rate of 15 breaths/min, and body temperature of 36.3°C. No abnormal findings on thoracic or abdominopelvic computed tomography were observed. The patient was tested for tick-borne infectious diseases, including Q fever, anaplasmosis, and brucellosis, among others, and serological testing performed at the Gwangju Institute of Health and Environment (Gwangju, Korea). The
Brucella microagglutination test showed a titer of <1:20 (negative), and the
C. burnetii indirect immunofluorescence antibody (IFA) assay revealed phases I and II IgG and IgM titers <1:16, which were interpreted as negative. However, nested PCR assays targeting
IS1111 repeat elements in the blood sample tested positive on day 2 (
Table 1). As the patient refused hospitalization, an outpatient follow-up was scheduled, and 7 days of doxycycline prescribed. At the follow-up visit (on day 6), the patient’s headache had improved, although the AST and alanine aminotransferase levels were 86 and 93 U/L, respectively. Additionally, his platelet count was 181,000/µl. Most of the clinical data suggested that the bacterial infection did not progress. Nevertheless, the patient was asked to return for further evaluation, but his condition declined; thus, long-term evaluation for chronic Q fever and the assessment of delayed seroconversion could not be conducted.
Occasionally, SCID mice, which lack functional B cells, T cells, and adaptive immunity, have been used to isolate and study bacterial pathogens, including
C. burnetii [
7,
8]. Therefore, pathogen isolation was performed using SCID mice. To amplify the bacteria, patient-derived blood was inoculated into SCID mice via the intraperitoneal route [
9]. On day 90 post-infection, the infected mice died and developed severe splenomegaly and hepatomegaly. After autopsy, mouse spleens were collected and homogenized for cell culture and DNA extraction. Considering the slow replication kinetics of
C. burnetii and its ability to establish persistent infections, particularly in immunocompromised hosts, extended infection periods are often necessary. In SCID mice, the lack of adaptive immunity permits long-term bacterial maintenance; thus, a long duration was selected to allow for adequate bacterial expansion and infection.
Cultures of the Vero E6 cell line (ATCC CRL-1586), which was maintained in RPMI-1640 medium (Welgene) supplemented with 2% fetal bovine serum (Welgene) and 1% amphotericin B (Welgene), were infected using the homogenized spleen material. The cultures were maintained in an incubator set at 34°C with 5% CO
2. The positive isolation cultures were maintained in the original cell culture dishes, with the medium refreshed weekly, and PCR testing conducted on the supernatant. The detection of
C. burnetii in cell culture dishes was performed using
IS1111-targeting quantitative PCR [
10]. Nested PCR was also used to screen
C. burnetii targeting the
omp1 gene: for the primary PCR, the outer primer set, WJ147 (forward, 5′-AGTAGAAGCATCCCAAGCATTG-3′) and WJ148 (reverse, 5′-TGCCTGCTAGCTGTAACGATTG-3′), was used, producing a 501-bp fragment; for the secondary PCR, the inner primer set, WJ149 (forward, 5′-GAAGCGCAACAAGAAGAACAC-3′) and WJ150 (reverse, 5′-TTGGAAGTTATCACGCAGTTG-3′), was used, generating a 334-bp amplicon [
11]. To confirm the identity of
C. burnetii, Sanger sequencing was performed for both the 16S rRNA and
com1 genes [
12,
13]. The 16S rRNA gene was amplified using primers WJ124 (forward, 5′-AGGAGGTGATCCANCCRCA-3′) and WJ195 (reverse, 5′-AGAGTTTGATCCTGGCTCAG-3′), and the
com1 gene amplified with primers WJ958 (forward, 5′-ACAAACGTTACAACCCAGCCCTG-3′) and WJ959 (reverse, 5′-GCGGAAGAGGTCGTATCAGTGAG-3′). The obtained sequences were identical to that of the
C. burnetii MSU Goat Q177 strain. For whole-genome sequencing (WGS), genomic DNA was extracted from the homogenized spleens of inoculated mice using the FavorPrep Blood/Cultured Cell Genomic DNA Extraction Mini Kit (Favorgen Biotech). Subsequently, DNA libraries were prepared using a TruSeq Nano DNA kit (Illumina), and a quality check then conducted using FastQC version 0.11.5 (Babraham Bioinformatics). Using Trimmomatic version 0.36 (Usadellab), the raw data were trimmed to eliminate low-quality reads and then indexed. Information that can be used in many DNA sequence analyses, such as genome size prediction, genome coverage confirmation, and repeat sequence ratio calculations, was assembled using Jellyfish version 2.2.10. For annotation of the
C. burnetii CH12 genome, the WG annotation pipeline (Macrogen) was used with strain CH12 as a reference (accession No. JBQPHR000000000).
WGS was performed to determine the genomic characteristics and phylogenetic position of the isolate. In the WGS analysis, 1,986,754 reads were obtained (299,383,989 bp in total), and the N50 was 55,499. Results showed that the CH12 genome (accession No. SAMN50566325) was 2,040,512 bp long, with 2,009 coding sequences, 56 contigs, 3 rRNA genes, 43 tRNA genes, and a GC ratio of 42.35%. The whole-genome sequences, including 24 reference strains, were aligned using BioEdit version 7.7.1, and a phylogenetic tree constructed using the maximum-likelihood method. Bootstrap analysis was performed with 1,000 pseudo-replicates. Results of the phylogenetic analysis showed that strain CH12 was closely related to the MSUGoatQ177 and CbuKQ154 strains (
Fig. 1).
The study protocol was approved by the Institutional Review Board of the Jeonbuk National University Hospital (approval No. 2022-10-012-004). All protocols involving the use of live animals were reviewed and approved by the Institutional Animal Care and Use Committee (approval No. NON2022-042) and the Institutional Biosafety Committee (approval No. JBNU 2022-09-001). Experiments involving the C. burnetii CH12 strain, patient blood, and SCID mice were performed in biosafety level 3 or animal biosafety level 3 facilities at the Korea Zoonosis Research Institute, Jeonbuk National University (Iksan, Korea).
Discussion
Diagnosing Q fever is generally challenging because the disease often exhibits a self-limiting proliferative phase that spontaneously resolves within approximately 2 weeks. In the first 2 cases of Q fever reported in Korea, both IFA and PCR were performed during the initial diagnostic phase; however, the results were negative [
6]. Moreover, the laboratory analyses showed elevated alkaline phosphatase, AST, and CRP levels, and 1 of the 2 patients had mild thrombocytopenia. As detectable antibody levels typically develop only several weeks after infection, diagnosis is often retrospectively confirmed through the serological testing of convalescent-phase samples, which poses a significant diagnostic challenge for Q fever [
6,
14]. Cases of Q fever have also been reported in China and Vietnam [
14,
15], where hepatomegaly and elevated alkaline phosphatase, AST, CRP, and procalcitonin levels have been observed. Moreover, in the case specifically reported in Vietnam, leukocytopenia and thrombocytopenia were observed [
15].
In the present case,
C. burnetii DNA and specific antibodies were simultaneously detected at an early stage of infection (
Table 1). This finding markedly differs from those of previously reported Korean cases, in which PCR failed to confirm infection, and a definitive diagnosis was achieved only 9–14 weeks later via IFA positivity in convalescent-phase serum. In contrast, in the current case, both the pathogen and antibodies were identified only 2 days after the onset of fever, a distinct and noteworthy feature. Given that
C. burnetii typically replicates slowly and elicits a delayed antibody response, this clinical case was unusual. Several hypotheses could explain this phenomenon. First, given the patient’s occupational exposure—raising livestock and having daily contact with animals—continuous or repeated exposure to the pathogen might have triggered the unusually rapid onset of acute Q fever. Although speculative, exposure to highly infectious materials, such as placentas or birth products containing high concentrations of
C. burnetii, might have resulted in infection despite the presence of pre-existing antibodies. Second, the patient might have already developed Q fever antibodies before the febrile episode. Although the exact time at which the patient began livestock farming could not be determined due to loss to follow-up, medical records indicated a previous diagnosis of brucellosis 2 years earlier, suggesting that the patient had likely been engaged in livestock work for several years. Although the present report presents a diagnosis of acute Q fever, intermittent prior exposure to
C. burnetii cannot be ruled out. Furthermore, a limitation of this study is that no follow-up was conducted, which prevented the assessment of seroconversion or further analyses, such as those performed in previous studies, consequently leading to inherently limited interpretations. However, the early detection of
C. burnetii DNA and IFA may provide a basis for the diagnosis of acute Q fever. Compared to previously reported Q fever cases, the present patient similarly showed elevated liver enzyme, CRP, and procalcitonin levels. Thrombocytopenia was also observed in the present study and in a Chinese case; however, the occurrence of thrombocytopenia in bacterial infections rather than in viral infections is uncommon. Although the limited number of clinical cases makes the establishment of a definitive link between Q fever and thrombocytopenia difficult, this case could serve as a reference that indicates a potential connection. Therefore, additional serological and molecular testing would be helpful for a more thorough assessment and precise differential diagnosis.
Recent epidemiological studies suggest that human Q fever transmitted by cattle is relatively uncommon compared with that transmitted by sheep or goats [
16]. For example, a systematic review analyzing 81 unique human Q fever outbreaks worldwide found that exposure to ruminants and their products was the leading risk factor. Sheep were implicated in approximately 34.6% of outbreaks (28/81), goats in 14.8% (12/81), and cattle in 8.6% (7/81), indicating that human Q fever is far less frequently associated with cattle than with sheep or goats [
17]. In this context, the present case offers an opportunity to investigate the epidemiological role of livestock in Q fever transmission.
The morphology of the isolated
C. burnetii in Vero cells was similar to that of previously reported Korean isolates [
6]. Based on the WGS results, the CH12 isolate showed the highest sequence homology with the Korean isolates, KZQ2 and KZQ3, exhibiting 99.94% nucleotide identity. Phylogenetic analysis revealed that the most closely related strains were MSUGoatQ177 and CbuKQ154, with 99.93% sequence similarity (
Fig. 1). The Schperling, CH12, KZQ2, KZQ3, MSUGoatQ177, and CbuKQ154 isolates formed a distinct, small clade that was separated from the other
C. burnetii isolates in the phylogenetic tree. As only a limited number of
C. burnetii isolates have been obtained and characterized in Korea and other Asian regions, a comprehensive analysis of the genetic diversity and regional distribution patterns remains challenging. Therefore, continuous efforts are necessary to isolate and characterize the genome of
C. burnetii to better understand its molecular epidemiology and strain-specific features. Moreover, further studies are required to better understand the pathogenicity and transmission mechanisms of
C. burnetii.
Notes
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Author contributions
Conceptualization: Choi Y, Kim D, Choi YJ, Kim DM, Jang WJ, Kang JG. Data curation: Choi Y, Choi SW, Jeong DE. Formal analysis: Choi Y, Kim D, Choi YJ. Funding acquisition: Kang JG. Investigation: Choi Y, Kim D, Choi YJ, Choi SW, Jeong DE, Cho NH. Methodology: Choi Y, Kim D, Choi YJ. Project administration: Kim DM, Jang WJ, Kang JG. Resources: Lee KJ, Kim DM, Jang WJ, Kang JG. Software: Lee JY, Choi SW, Jeong DE. Supervision: Lee KJ, Kim DM, Jang WJ, Kang JG. Validation: Cho NH, Lee JY, Choi Y, Kim D, Choi YJ. Visualization: Choi Y, Kim D, Choi YJ, Lee JY. Writing – original draft: Choi Y, Kim D, Choi YJ. Writing – review & editing: Kim DM, Jang WJ, Kang JG.
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Conflict of interest
The authors have no conflicts of interest to declare.
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Funding
This research was supported by the National Institute of Health research project (project No. 2022ER210801).
Fig. 1.Phylogenetic tree of Coxiella burnetii based on complete genome sequences. Bold letters indicate the C. burnetii sequences obtained from a patient in this study.
Table 1.Laboratory findings and antibody titers
Table 1.
|
Symptom onset date |
Period between symptom onset and sampling |
Antibiotic administration day from symptom onset |
Laboratory finding |
Nested PCR |
IFA |
|
WBC (103/µl) (neutrophil/lymphocyte, %) |
Platelet (103/µl) |
AST (U/L) |
ALT (U/L) |
CRP (mg/dl) |
Procalcitonin (ng/ml) |
Coxiella IS1111 |
Phase 1 IgG |
Phase 1 IgM |
Phase 2 IgG |
Phase 2 IgM |
|
1 |
1 |
0 |
4.39 (NA) |
130 |
78 |
81 |
8.1 |
NA |
NA |
NA |
NA |
NA |
NA |
|
2 |
2 |
1 |
4.24 (65.8/25.0) |
116 |
90 |
106 |
8.95 |
0.35 |
+ |
<1:16 |
<1:16 |
<1:16 |
<1:16 |
|
6 |
6 |
5 |
6.47 (40.8/40.2) |
181 |
86 |
93 |
NA |
NA |
NA |
NA |
NA |
NA |
NA |
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, Dayoung Kim3,†
