Seroreactivity to Coxiella burnetii in an Agricultural Population and Prevalence of Coxiella burnetii Infection in Ticks of a Non-Endemic Region for Q Fever in South Korea

Coxiella burnetii infects humans and wild and domesticated animals. Although reported cases on Jeju Island, off the coast of South Korea, are rare, the region is considered to have a high potential for Q fever. We investigated the seroprevalence of antibodies to C. burnetii in 230 farmers living in ten rural areas on Jeju Island between January 2015 and December 2019. Blood samples were collected and examined for C. burnetii Phase I/II IgM and IgG antibodies. Trained researchers collected ticks from rural areas. Clone XCP-1 16S ribosomal RNA gene sequencing was performed to identify Coxiella species from the collected ticks. The overall seroprevalence of antibodies to C. burnetii in farmers was 35.7%. The seroprevalence was significantly higher in fruit farmers. Of the collected ticks, 5.4% (19/351) of the Haemaphysalis longicornis ticks harbored C. burnetti. A high seroprevalence of antibodies to C. burnetii was observed in this region of Jeju Island, confirming that C. burnetti is endemic. Physicians should thus consider Q fever in the differential diagnosis of patients that present with acute fever after participating in outdoor activities.


Introduction
Query fever (Q fever) is a widespread disease caused by Coxiella burnetii [1], an obligate gram-negative intracellular bacterium of the family Coxiellaecae [2]. C. burnetii infects humans and several wild and domesticated animals, and survives in arthropod hosts such as ticks. Domestic ruminants (primarily cattle, sheep, and goats) are the most important reservoir of C. burnetii [3,4]. C. burnetii DNA has been detected in the blood samples of cattle, horses, and goats in South Korea [5]. C. burnetii is transmitted to humans mainly via the inhalation of contaminated aerosols from infected animals. Other routes of transmission to humans include the consumption of contaminated milk and dairy products, skin or mucosal contact, blood transfusion, and sexual transmission [4,6,7]. The vector capacity of ticks to transmit C. burnetii remains unclear [8]. A recent report described a case of severe fever with thrombocytopenia syndrome and Q fever in a girl in South Korea following a tick bite [9], and it has been suggested that C. burnetii infection can be transmitted through tick bites [10]. Seroprevalence surveys in human populations have revealed that the seroprevalence is highest (up to 30%) in African countries with a high density of domestic ruminants [4]. Although Q fever has long been considered a rare and regionally localized disease, it has been reported in almost every region worldwide [4]. C. burnetii was first isolated from hard ticks (Dermacentor andersoni in Montana and Haemaphysalis humerosa in Australia), and has been identified in more than 40 hard tick species, 14 soft tick species, and many other arthropods worldwide [4]. Among the tick species, Haemaphysalis longicornis is the dominant tick species (88.9%) in South Korea [10]. Although the vector capacity of Korean ticks for transmitting C. burnetii is unknown, ticks are known to harbor the organism. In a study of 105 H. longicornis ticks conducted in 2004, two were PCR positive for Coxiella [11], and in a study of 213 ticks collected from horses on Jeju Island, 121 (52.4%) were PCR positive for C. burnetii [12]. In addition, the seroprevalence of C. burnetii infection in cattle on Jeju Island is higher than in mainland South Korea [13].
Q fever has been notifiable in South Korea since 2012, and 162 cases were recorded in the national notifiable diseases surveillance system by the Korea Disease Control and Prevention Agency from 2001 to 2019. Jeju Island, which is located off the coast of South Korea, is an area with dense forest and grassland and is home to several wild animals (deer, roe deer, wild boar, badgers, and field mice) and livestock (horses, beef cattle, dairy cattle, and pigs) [14], which creates a favorable environment for ticks. In the past 20 years, only two cases of Q fever have been reported on Jeju Island [15]. Although reported cases of Q fever are rare in this region [15], is the area is considered to have potential for Q fever to occur [10,12] because of the high prevalence of Coxiella in ticks on horses [12]. Ticks are considered to be a potential vector for C. burnetii, although this has not been confirmed. This study aimed to investigate the seroprevalence of antibodies to C. burnetii in an agricultural population and the prevalence of C. burnetti infection in ticks on Jeju Island.

Study Setting and Participant Selection and Recruitment
This study was conducted from January 2015 to December 2019 in an agricultural population living in the rural areas of Jeju Island. Blood samples were collected from the participants at the Center for Farmers' Safety and Health at a teaching hospital on Jeju Island with the support of the Safety from Agricultural Injuries in Farmers (SAIF) organization. The island has a humid, subtropical climate (mean summer temperature: 24.7 • C; and mean winter temperature: 7.1 • C) and had a population of 696,000 in 2019, of whom approximately 12% (83,133) were farmers [16].
The study participants were farmers residing on Jeju Island. First, ten rural villages (-myeon and -eup according to the South Korean administrative division system) were selected based on the type of agriculture practiced and the distance from urban areas, and a sample of 500 farmers (approximately 50 farmers per village) was selected. Second, researchers contacted these farmers by telephone, and 423 consented to participate. Third, the researchers visited each village and conducted face-to-face interviews with the participants. Of the 423 farmers who consented to be interviewed, 352 agreed to provide a blood sample and were enrolled in the study. A 10-mL blood sample was collected from each participant in three serum-separating tubes and one ethylenediaminetetraacetic acid tube. Of the samples collected, 230 had an adequate volume for multiple tests. The remaining 122 samples were excluded because of an insufficient serum volume. All participants provided written informed consent.
Data regarding participants' sociodemographic status, medical history, Charlson comorbidity index (CCI) score, occupation, type of farming, history of tick bites, history of Q fever, and other relevant information were collected by interviewers using a standardized questionnaire. The CCI score was based on participants' previous disease diagnoses. The collected blood samples were immediately centrifuged at the interview site and the serum was frozen on the same day and stored at −70 • C until further analysis.

Antibody Detection in Sera
The serum samples were sent to Eon Laboratories (Incheon, Korea) to be tested for antibodies to C. burnetii, according to the manufacturer's instructions [17,18]. Each serum sample was tested twice for anti-C. burnetii Phase I/II IgM (1:16 dilution) and IgG (1:16 and 1:256 dilutions) using a commercial indirect immunofluorescence antibody (IFA) assay (Focus Diagnostics, Cypress, CA, USA). The relative sensitivity and specificity of this assay have been reported to be 100% [17,18].

Coxiella burnetii Identification in Ticks
Well-equipped trained researchers collected ticks from the natural environment of Jeju Island between June 2016 and January 2019. The tick sampling sites were located in five rural areas: Aewol-eup (AW), Seon Hul-ri (SH), Jeo Ji-ri (JJ), Ha Do-ri (HD), and Bo Mok-ri (BM) (Figure 1). Ticks were manually collected from each site twice a month during the first and third weeks of each month (January to December) by dragging a white cloth through woodlands in each area for 2 h. Tick species and their developmental stages were identified morphologically using an Olympus SD-ILK-200-2 stereomicroscope (Olympus Corporation, Tokyo, Japan, https://www.olympus-lifescience.com, accessed on 10 May 2016) [19]. Total H. longicornis nucleic acid was extracted using a QIAamp RNA Mini kit (QIAGEN Inc., Hilden, Germany, https://www.qiagen.com, accessed on 10 May 2016) according to the manufacturer's instructions. Clone XCP-1 16S ribosomal RNA gene sequencing was used to identify Coxiella species in the ticks.
Pathogens 2021, 10, x FOR PEER REVIEW 3 of 11 each participant in three serum-separating tubes and one ethylenediaminetetraacetic acid tube. Of the samples collected, 230 had an adequate volume for multiple tests. The remaining 122 samples were excluded because of an insufficient serum volume. All participants provided written informed consent. Data regarding participants' sociodemographic status, medical history, Charlson comorbidity index (CCI) score, occupation, type of farming, history of tick bites, history of Q fever, and other relevant information were collected by interviewers using a standardized questionnaire. The CCI score was based on participants' previous disease diagnoses. The collected blood samples were immediately centrifuged at the interview site and the serum was frozen on the same day and stored at −70 °C until further analysis.

Antibody Detection in Sera
The serum samples were sent to Eon Laboratories (Incheon, Korea) to be tested for antibodies to C. burnetii, according to the manufacturer's instructions [17,18]. Each serum sample was tested twice for anti-C. burnetii Phase I/II IgM (1:16 dilution) and IgG (1:16 and 1:256 dilutions) using a commercial indirect immunofluorescence antibody (IFA) assay (Focus Diagnostics, Cypress, CA, USA). The relative sensitivity and specificity of this assay have been reported to be 100% [17,18].

Coxiella burnetii Identification in Ticks
Well-equipped trained researchers collected ticks from the natural environment of Jeju Island between June 2016 and January 2019. The tick sampling sites were located in five rural areas: Aewol-eup (AW), Seon Hul-ri (SH), Jeo Ji-ri (JJ), Ha Do-ri (HD), and Bo Mok-ri (BM) ( Figure 1). Ticks were manually collected from each site twice a month during the first and third weeks of each month (January to December) by dragging a white cloth through woodlands in each area for 2 h. Tick species and their developmental stages were identified morphologically using an Olympus SD-ILK-200-2 stereomicroscope (Olympus Corporation, Tokyo, Japan, https://www.olympus-lifescience.com, accessed on 10 May 2016) [19]. Total H. longicornis nucleic acid was extracted using a QIAamp RNA Mini kit (QIAGEN Inc., Hilden, Germany, https://www.qiagen.com accessed on 10 May 2016) according to the manufacturer's instructions. Clone XCP-1 16S ribosomal RNA gene sequencing was used to identify Coxiella species in the ticks.

Definitions
A positive Phase I/II IgG or IgM antibody reaction at a dilution of ≥1:16 was considered to be seroreactive [4,20]. The C. burnetii-seropositive group (CPG) included participants with positive C. burnetii Phase I/II IgM or IgG results, whereas the C. burnetiiseronegative group (CNG) included participants with negative results.

Statistical Analysis
Statistical analyses were performed using SPSS 20.0 (IBM Corp., Armonk, NY, USA). The results of categorical variables were summarized as frequencies and proportions. The results of continuous variables were summarized as means with standard deviations or as medians with interquartile ranges. Categorical variables were compared using chi-square tests, and continuous variables were compared using the Mann-Whitney U test. p-values of <0.05 were considered to be statistically significant.

Comparison of the Clinical Characteristics of the Coxiella burnetii-Positive and -Negative Groups
Of the 230 participants, 82 were included in the CPG and 148 were included in the CNG ( Table 1). The mean participant age was 59.5 ± 14.7 years and 55.3 ± 19.8 years in the CPG and CNG, respectively, and 56.5% and 50.0% of the CPG and CNG, respectively, were male. The seroprevalence of antibodies to C. burnetii was higher in older participants than in younger participants, but the difference was not statistically significant (p = 0.07). There Pathogens 2021, 10, 1337 6 of 11 was no significant association between anti-C. burnetii antibody status and comorbidities or history of tick bites. Fruit-farming was significantly more common in the CPG than in the CNG (81.7% vs. 67.6%, p = 0.02). The total farming period, daily farming period, and possession of livestock did not differ significantly between the two groups. No participants in either group reported consuming raw milk. In addition, the types of personal protective equipment used did not differ significantly between the two groups.

Discussion
In this study, the seroprevalence of antibodies to C. burnetii was 37.5% in farmers. The seroprevalence of antibodies to C. burnetii was highest among fruit farmers living in the southwestern part of Jeju Island, and was higher in older participants than in younger participants.
Only two cases of Q fever on Jeju Island were reported in the Korean national notifiable infectious diseases system during the period 2001-2020 [15]; however, based on the high seroprevalence of antibodies to C. burnetii found in this study, C. burnetii infection is endemic in the region. A diagnosis of C. burnetii can only be confirmed by performing an IFA test (Phase I and Phase II) in the Department of Laboratory at the Korea Disease Control and Prevention Agency (KDCPA). Physicians send a sample to the KDCPA with a patient declaration form, and they receive the result a few weeks later. Physicians may find it difficult to diagnose Q fever in patients with acute febrile illnesses because there are no readily available commercial diagnostic tests for C. burnetii infection, and there is a lack of seroepidemiological data in South Korea, so the level of awareness among physicians is low.
The seroprevalence in these farmers was considerably higher than that reported in previous seroepidemiological surveys in another asymptomatic rural population (1.5%) and in cattle slaughterhouse workers (9.1%) in South Korea [21,22]. The high seroprevalence was unexpected because the agricultural workers have less opportunity to being exposed to C. burnetii-containing aerosols compared with slaughterhouse workers who may be exposed to feces, urine, milk, and vaginal discharge of animals infected with C. burnetii during the slaughtering process [6]. C. burnetii-infected ticks may remain infected for an extended time period-between 200 and 1000 days and more than 10 years in several cases-and the excretion of C. burnetii in tick feces occurs on the skin of the animal host while feeding. C. burnetii may persist for prolonged periods in the soil, and bacterial aerosols can be delivered for at least 30 km through air [4]. For the above reasons, most routes of human infection occur after the inhalation of aerosols containing C. burnetii from infected animals and their products. Individuals with Q fever can develop the disease without animal contact because transmission to humans is influenced by geographical factors such as the subtropical climate, the specific natural environment, and the heavy wind on Jeju Island [23].
Physicians may not consider Q fever in the differential diagnosis in patients with acute febrile illness because the symptoms and signs of Q fever are nonspecific; acute fever is common due to severe fever with thrombocytopenia syndrome (in the spring and the summer) and scrub typhus (in the autumn) in individuals who participate in outdoor activity [23][24][25][26], and most infected individuals have no history of animal contact or occupational exposure [27].
In recent published reports, of the individuals diagnosed with Q fever, 2.0% (48/2434) were diagnosed with acute Q fever endocarditis, and C. burnetii-associated interstitial lung disease was rare [28,29]. In addition, C. burnetii infection may be associated with lymphadenitis and cholecystitis [30]. On Jeju Island, transthoracic echocardiography should be performed in patients with confirmed C. burnetii infection and in patients with unexplained acute fever [31,32], because Q fever can lead to severe complications. Fruit farmers and inhabitants of Jocheon-eup and Jungmun-dong were at particularly high risk of infection in this study. Patients with acute febrile illness who reside in these areas should have a diagnostic assessment for Q fever.
The composition of the agricultural population of Jeju Island differs from that on the mainland of South Korea. Among farmers of Jeju Island, the proportion of fruit farmers is relatively high and that of livestock farmers is relatively low (62.5% and 2.2%, respectively) compared with the mainland farming population (16.9% and 5.3%, respectively) [16].
Another recent study conducted on Jeju Island found a high seroprevalence (52.4%) of antibodies to Coxiella in horses [12]. Jeju Island is known for its high number of horses. Its geographical location and climate make it ideal for horse breeding, and 56.0% of the horses bred nationally in South Korea are bred on Jeju Island [14]. There are approximately 390,000 horseback riders living on Jeju Island. Horseback riders may have a high risk of C. burnetii infection from contact with infected horses or aerosols of horse feces.
Slaughterhouse workers and veterinarians are generally at high risk of C. burnetii infection, and most individuals diagnosed with Q fever live in rural areas [10,22,33,34]. Q fever is most commonly diagnosed in the summer and the early autumn [27]. In this study, participants in the CPG were may have been infected by a tick bite or by droplet transmission from an infected animal because the agricultural population did not wear N95 masks or other personal protective equipment during the pre-coronavirus disease pandemic era, and infected domestic animals shed a large amount of C. burnetii. In addition, farmers generally do not wear face masks after their work is finished, and they may have become infected via droplet infection in their natural environment. Protective masks (N95 or higher type) can provide better protection against C. burnetii infection compared to standard surgical masks and personal protective equipment [35].
In the CPG, 17% of the participants were positive for Phase II IgM antibodies, so they may have had an acute C. burnetii infection at the time of the survey. Participants did not have a fever within the two weeks before sample collection, but C. burnetii infection in humans is usually asymptomatic or presents as a mild disease with spontaneous recovery. However, we were unable to diagnose acute C. burnetii infection in study participants because we did not collect serial samples and could not compare the IgM titer between Phases I and II. The median time from symptom onset to serological diagnosis is 21 days, and 60% of cases in South Korea are diagnosed between June and September [27].
Studies conducted in other countries have shown a seroprevalence of 15-30% in African countries [34][35][36][37][38][39][40]; approximately 30.0% in Iran [41]; 5.3% in the rural population of Australia [42]; 2.4% in the Netherlands [43]; 24% among patients with acute febrile illness in France [4], 1.3% in Kazakhstan [44], and 5.8% in Asia [45]. In Japan, the incidence rate of Q fever increased significantly during the period 2000-2004, with a seroprevalence of antibodies to C. burnetii of 22-36% in veterinarians [46]. The study found that Q fever had been underdiagnosed for many years in Japan. In previous seroprevalence surveys conducted in South Korea, the seroprevalence of antibodies to C. burnetii was 9.1% in cattle slaughterhouse workers and 11.0% in dairy cattle farmers [22,47], and the seroprevalence of Phase II IgM and IgG antibody titers to C. burnetii infection of ≥1:16 was 9.1% and 0.2%, respectively. Although there have not been any recent reports of cases of Q fever on Jeju Island, this study provides novel evidence that the seroprevalence of antibodies to C. burnetii is relatively high in the agricultural population of Jeju Island, and there is thus a potential for Q fever.
This study has some limitations. First, we did not perform polymerase chain reaction analysis to determine whether participants in the CPG had a C. burnetii infection, as the participants did not have symptoms of Q fever and the volume of sample collected was insufficient for performing further tests. Second, we did not evaluate the seroprevalence of antibodies to C. burnetii in inhabitants of urban areas of Jeju Island. Third, we did not test the samples for cross-reactivity with other infectious pathogens. However, according to the manufacturer's instructions, neither Phase I nor Phase II C. burnetii antigen cross-reacts with other Rickettsia or bacteria, so false-positive reactions are unlikely [17,18]. Fourth, we did not investigate participants' history of contact with various animals or the type of compost and manure to which they were exposed. Finally, this was not a diagnostic study of primary C. burnetii infection and persistent C. burnetii infection. Because Phase II antibodies are detectable within 7-15 days after the onset of clinical symptoms and then decrease within three to six6 months, antibodies are detectable by the third week after infection in 90% of infected individuals [35,48]. The prevalence of anti-C. burnetii antibody titers of Phase I/II IgM ≥ 1:16, Phase I/II IgG ≥ 1:16, and Phase I/II IgG ≥ 1:256 were analyzed in this cross-sectional study, but we did not perform serial testing to assess changes in the antibody titers.
In conclusion, Jeju Island has been considered to be a non-endemic area for Q fever, and little is known about the incidence of C. burnetii infection in South Korea or of the viability of ticks as possible vectors. Previous studies have not focused on the agricultural population of South Korea, who have many opportunities for tick bite exposure. The study revealed that the agricultural population of Jeju Island has a higher seroprevalence of antibodies to C. burnetii than in regions where Q fever is considered to be endemic, and that C. burnetii is prevalent in ticks on Jeju Island. Therefore, physicians should consider Q fever in the differential diagnosis of patients with acute febrile illnesses and a history of outdoor activity. Further studies of C. burnetii seroprevalence are necessary in South Korea, and active surveillance and monitoring are needed on Jeju Island.