Coxiella burnetii is widespread in ticks (Ixodidae) in the Xinjiang areas of China

Background The gram-negative Coxiella burnetii bacterium is the pathogen that causes Q fever. The bacterium is transmitted to animals via ticks, and manure, air, dead infected animals, etc. and can cause infection in domestic animals, wild animals, and humans. Xinjiang, the provincial-level administrative region with the largest land area in China, has many endemic tick species. The infection rate of C. burnetii in ticks in Xinjiang border areas has not been studied in detail. Results For the current study, 1507 ticks were collected from livestock at 22 sampling sites in ten border regions of the Xinjiang Uygur Autonomous region from 2018 to 2019. C. burnetii was detected in 205/348 (58.91%) Dermacentor nuttalli; in 110/146 (75.34%) D. pavlovskyi; in 66/80 (82.50%) D. silvarum; in 15/32 (46.90%) D. niveus; in 28/132 (21.21%) Hyalomma rufipes; in 24/25 (96.00%) H. anatolicum; in 219/312 (70.19%) H. asiaticum; in 252/338 (74.56%) Rhipicephalus sanguineus; and in 54/92 (58.70%) Haemaphysalis punctata. Among these samples, C. burnetii was detected in D. pavlovskyi for the first time. The infection rate of Rhipicephalus was 74.56% (252/338), which was the highest among the four tick genera sampled, whereas the infection rate of H. anatolicum was 96% (24/25), which was the highest among the nine tick species sampled. A sequence analysis indicated that 63 16S rRNA sequences could be found in four newly established genotypes: MT498683.1 (n = 18), MT498684.1 (n = 33), MT498685.1 (n = 6), and MT498686.1 (n = 6). Conclusions This study indicates that MT498684.1 might represent the main C. burnetii genotype in the ticks in Xinjiang because it was detected in eight of the tick species studied. The high infection rate of C. burnetii detected in the ticks found in domestic animals may indicate a high likelihood of Q fever infection in both domestic animals and humans.


Background
Coxiella burnetii, an obligate gram-negative intracellular bacterium, can cause Q fever disease in humans, survive in the environment for long periods of time, and is often found in the phagolysosome of infected mammalian cells [1,2]. Given its impact on global public health, it has attracted significant attention for research purposes [3]. In humans, infection with C. burnetii causes acute symptoms that include vomiting, headache, pneumonia, fever, and hepatitis, as well as chronic symptoms related to hepatitis, osteomyelitis, endocarditis, and intravascular infection [4,5]. In animals, infection with C. burnetii causes various reproductive problems, including delivery of weak offspring, infertility, postpartum metritis, stillbirth, and abortion [6].
Q fever was first detected in workers at a slaughterhouse in Brisbane, Australia, in 1935 by E.H. Derrick, who named the illness "question fever" [7]. It has also been reported in humans in other countries, including Great Britain, the Netherlands, Spain, Germany, and Switzerland [8][9][10][11][12]. It was first reported to be in China during the 1950s, with C. burnetii antibodies being reported in humans from 32 prefectures in 15 provinces of China [13]. Slaughterhouse workers, veterinarians, and farmers are currently at high risk of contracting this relatively rare zoonotic disease [14].
Ticks are widely distributed around the world and are among the most important vectors of human disease, second only to mosquitoes; they are also the main carrier of pathogens in wild animals and livestock [15]. Coxiella maintains a symbiotic relationship with ticks and can infect ticks at all tick life stages [16,17]. It has been isolated from more than 40 species of hard ticks and at least 14 species of soft ticks, indicating the importance of ticks in its transmission [18]. Animals become infected with C. burnetii via tick bites, whereas humans become infected mainly via contact with tick excreta, manure, direct contact with birth products, and air [1,19]. Although the direct transmission of C. burnetii to humans through tick bites has not been reported in detail [20], C. burnetii has been reported in the milk, birth products, faeces, and urine of the infected animals, to which humans can be exposed and thus become infected with C. burnetii by airborne transmission [1]. Xinjiang is the provincial-level administrative region with the largest land area in China. Its boundary is connected with many countries and there are endemic tick species. In the current study, molecular biological methods were used to detect Q fever in tick species collected from the border area of Xinjiang, China, to reveal the species and pathogen-carrying status of the ticks in this region, to analyse the cross-border spread of Q fever. Through this molecular epidemiological survey, the risk of cross-border transmission of tick-borne Q fever and its spread to mainland China was assessed, to provide basic information on the development of effective prevention and control measures for this important tick-borne disease.

Results
In total, 1507 tick samples were collected from livestock in different regions of the Xinjiang border (Table 1) and the C. burnetii in sheep from Swedish (Y11500.1), respectively. As shown by the phylogenetic analysis, the MT498684.1 genotypes belonged to group A, whereas the MT498686.1 and MT498685.1 genotypes belonged to group B, MT498683.1 genotypes belonged to group C (Fig. 1).

Discussion
As the second largest group of vectors in the world, ticks are hosts to pathogens of a variety of important zoonoses [21][22][23], such as Forest encephalitis, Q fever, Lyme disease, Spot fever, tularemia, and babesiosis [24,25]. In recent years, new tick-borne diseases, such as human granulocytic anaplasmosis (HGA), severe fever with thrombocytopenia syndrome (SFTS), and Guertu virus (GTV), have been reported [26,27]. Ticks transmit pathogens primarily by biting the host [28,29], but also by aerosol transmission (C. burnetii) [5].  [40]; the H. anatolicum (13.33%, 2/15) in Cyprus [34]. The differences in infection rate between this study and others may be related to the number of samples, collection location, detection method, and ecological environment. The temperate continental climate provides a good habitat for a wide variety of ticks in Xinjiang. Xinjiang has a great variety of mammalian and avian species, which could serve as hosts of diverse tick species. Therefore, we speculate that climate and invertebrate vector abundance factors may be related to infection.
We did not detect C. burnetii in H. scupense (0/2; 0.00%), an outcome that may be related to the small sample size (2 samples of H. scupense). In addition, the presence of C. burnetii in D. pavlovskyi was first reported here. The high infection rate of C. burnetii in D. silvarum, H. asiaticum, and R. sanguineus seems to be related to the symbiosis and vertical transmission between them [41,42]. Although there is no related article reporting the existence of symbiotic and vertical propagation of C. burnetii in D. nuttalli, D. pavlovskyi, D. niveus, H. rufipes, H. anatolicum, or H. punctata, the findings lead us to suspect that C. burnetii has these relationships to these ticks, particularly because some reports have shown that C. burnetii has a high infection rate and vertical transmission relationship among some ticks [43][44][45][46]. Confirmation of our hypothesis requires more literature support and research to prove; here we are merely stating the supposition. Overall, our results indicate that C. burnetii is widespread in the border areas of Xinjiang.
The presence of C. burnetii worldwide, including in the environment and in both vertebrate and invertebrate hosts. Phylogenetic analyses indicated that MT498684.1 (from D. nuttalli, D. pavlovskyi, D. silvarum, D. niveus, H. rufipes, H. anatolicum, H. asiaticum and R. sanguineus) belonged to group A, MT498686.1 (from D. nuttalli) and MT498685.1 (from H. punctata) belonged to group B, MT498683.1 (from R. sanguineus and H. asiaticum) belonged to group C (Fig. 1). MT498683.1, MT498686.1, and MT498685.1 genotypes and Coxiella sp. from ticks was clustered into a branch (A and B), whereas MT498684.1 genotypes forms a branch with C. burnetii from different sources (cows, human, sheep, and ticks) (Fig. 1) [20]. However, C. burnetii has been reported in the milk, birth products, faeces, and urine of infected animals, to which humans can be exposed and thus be infected [1]. At particularly high risk are veterinary personnel, stockyard workers, farmers, hide tannery workers and others who work closely with animals. Sporadic cases of C. burnetii in humans are reported each year, although occasionally there are large outbreaks in humans [16,17]. For example, between 2007 and 2011, a Q fever epidemic occurred in the Netherlands, affecting 4107 people and causing the death of > 50,000 dairy goats. It was thought that most of these infected people developed Q fever by inhaling air in which C. burnetii had been released during the birthing season of both goats and sheep (February-May) (http://www.rivm.nl/Onderwerpen/Q/Q_koorts) [9,47].
In the 1950s, Q fever was first reported in China [48,49]. The first Chinese strain of C. burnetii (Qi Yi) was isolated from a confirmed Q fever patient in 1962 [50]. In the past few decades, C. burnetii DNA has been detected in blood samples of human ( [39,55,56].
In summary, this study reports, for the first time, the Q fever infection in ticks in the border areas of Xinjiang, China, indicated that the abundant tick species and high infection rates of C. burnetii in the border areas of Xinjiang pose potential threats to domestic animals and humans. Xinjiang, located in northwest China, is bordered by Pakistan, Tajikistan, Mongolia, Kyrgyzstan, India, Afghanistan, Russia and Kazakhstan. Ticks are widely distributed in wild animals and domestic animals across the region, providing increased opportunities for cross-border transmission of C. burnetii as global trade intensifies. Thus, there is a need for farmers to adhere to livestock testing and to implement tick control strategies. It is of great significance for public health and safety to reduce the risk of cross-border transmission of the pathogen and its spread to the mainland of China.

Conclusions
This study confirmed, for the first time, that C. burnetii is widely distributed in ticks in Xinjiang, China, indicating that domestic animals and humans in this region may be at risk of being infected with C. burnetii. Therefore, there is a need for further research on C. burnetii cross-border transmission via ticks. More importantly, there is a need to monitor domestic animals and humans in the region and in tick control in the region to the greatest extent possible to ensure animal and public health safety.

Sample collection and morphological identification of the ticks
A total of 1507 ticks were collected from cattle and sheep at 22 sampling sites in ten border regions in the spring of Xinjiang, China from 2018 to 2019 (Fig. 2).
The collected samples were stored in 50 mL centrifuge tubes and delivered to the laboratory. The ticks were identified based on morphological criteria following the descriptions provided by Deng GF (1991) [57]. Tick samples were collected with permission from the farmer.

DNA extraction from tick samples
Tick samples were placed in 50 mL sterile centrifuge tubes and washed individually twice with 75% ethanol, followed by ddH 2 O rinsing until the liquid was clear. For each sample, DNA was extracted using a QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol, and the extracted DNA was stored at − 20°C.

PCR amplification and sequencing
As described in previous studies [58][59][60][61], primers were designed with conserved regions of the C. burnetii IS1111 (This is a multi-copy gene encoding a transposase) and 16S rRNA gene sequences (Table 3); the expected product from the C. burnetii primers used for IS1111 amplification was 517 bp, and for the 16S rRNA

Statistical analysis
In this study, Analyses were performed using SPSS Statistics IBM 17 (© IBM Corporation, Somers, New York, USA). Chi-square (χ 2) test was used to conduct statistical analysis on different Genus and species with C. burnetii infection.