Molecular Evidence of Bartonella melophagi in Ticks in Border Areas of Xinjiang, China

Bartonella are gram-negative intracellular bacteria; certain species of Bartonella can cause diseases in mammals and humans. Ticks play a major role in the transmission of Bartonella. Xinjiang is the largest province in China according to land area and has one-third of the tick species in China; the infection rate of Bartonella in ticks in the Xinjiang border areas has not been studied in detail. Therefore, this study investigated tick infections by Bartonella in Xinjiang border areas, and the purpose of the study was to fill in gaps in information regarding the genetic diversity of tick infections by Bartonella in Xinjiang. We tested 1,549 tick samples from domestic animals (sheep and cattle) for Bartonella using ribC-PCR. Positive samples from the ribC-PCR assay for Bartonella spp. were further subjected to PCR assays targeting the ITS, rpoB and gltA genes followed by phylogenetic analyses. Bartonella DNA was detected in 2.19% (34/1,549) of tick samples, and the ITS, rpoB and gltA genes of ribC gene-positive samples were amplified to identify nine samples of Bartonella melophagi. In this study, molecular analysis was used to assess the presence and genetic diversity of B. melophagi in ticks collected from sheep and cattle from Xinjiang, China. This study provides new information on the presence and identity of B. melophagi in ticks from sheep and cattle.


INTRODUCTION
Bacteria of the genus Bartonella are obligate gram-negative intracellular bacteria that primarily infect mammalian erythrocytes, macrophages, monocytes, and endothelial and dendritic cells (1,2). B. bacilliformis was the only named species of Bartonella before 1990. At present, more than 36 species have been discovered, 17 of which are related to human and animal diseases (3).
Bartonella uses humans, cats, dogs, mice, horses, cows, rabbits and other wild animals around the world as hosts (3,21,22). It can be transmitted by insect vectors, such as ticks, human body louse, cat fleas, and sand flies (3,22,23). Ticks are a widely distributed vector insect worldwide and can transmit and carry a variety of zoonotic pathogens, including viruses, bacteria, parasites, spirochetes and Rickettsia (24,25).
As the largest province in China, Xinjiang has a vast territory, a diverse ecological environment, and rich species. At present, Xinjiang has one-third of the tick species found in China (26). Ticks in Xinjiang have been shown to transmit multiple pathogenic infections, such as Babesia, Anaplasma, Rickettsia, Theileria and Tularemia (27)(28)(29)(30); however, there are only a few studies on Bartonella infections in ticks in Xinjiang. Previous studies confirmed that sheep keds (M. ovinus) in Xinjiang were infected with B. melophagi (31). Therefore, we aimed to investigate Bartonella infection in ticks collected from the border areas of Xinjiang and its genetic diversity to fill in the existing lacunae.

DNA Extraction and Molecular Analysis
Tick samples were placed in 50 mL sterilized centrifuge tubes and washed individually twice with 75% ethanol followed by rinsing with double-distilled water (ddH 2 O) until the liquid was clear. DNA was extracted from each sample using a QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol, and the extracted DNA was stored at −20 • C. Initial screening of Bartonella DNA was performed using ribC-PCR.
To further characterize the positive samples in this study, positive samples by ribC-PCR assay for Bartonella spp. were further subjected to PCR assays targeting the ITS, rpoB and gltA genes, followed by phylogenetic analyses (33)(34)(35)(36). A negative control was prepared with ddH 2 O; a positive control was prepared in our laboratory using DNA extracted from Hy. asiaticum infected by B. melophagi. The PCR products of the partial gltA, rpoB, ribC and ITS genes were purified using an E.Z.N.A. R gel extraction kit (Omega, USA) and cloned into the pGEM-T Easy vector (Promega, USA).

Sequencing and Phylogenetic Analysis
The nucleotide sequences were confirmed by bidirectional sequencing at TSINGKE Biotech, China. The nucleotide sequences were compared with the reference sequences of GenBank (www.ncbi.nlm.nih.gov/nuccore/) by SeqMan (www.dnastar.com/); the forward and reverse primers of the sequences after bidirectional sequencing were selected using SeqMan; extra sequences at the ends of the forward and reverse primers were removed, and corrected calibration was performed after manual adjustment as needed. The sequences were aligned with sequences downloaded in GenBank using MAFFT (37). ModelFinder (38) was used to select the best model using the BIC criterion. Phylogenetic inference was founded on maximum likelihood (ML) analysis through the IQ-TREE (39) program of PhyloSuite V1.2.2 (40). The trees were edited in Figtree v1.4.3 (https://github.com/rambaut/figtree/releases). The confidence values for each branch of the phylogenetic tree were determined by using 1,000 repeat analyses.

RESULTS
A total of 1,549 tick samples were collected from livestock in various regions of the Xinjiang border ( Table 1) Table 2).
In the phylogenetic inference based on gltA and rpoB genes, the gltA and rpoB sequences obtained in this study were clustered into a branch of Bartonella associated with ruminants and supported by 98 and 100% bootstrap analysis. The gltA and rpoB sequences of B. melophagi in this study were closest to AY724768.1 and EF605288.1, respectively (Figures 2, 3).

DISCUSSION
Our study reports the prevalence of Bartonella in ticks in the border area of Xinjiang, China. A total of 1,549 tick samples were collected in the current study, and 2.19% (340/1,549) contained Bartonella DNA assayed by ribC-PCR. The gltA, rpoB and ITS genes of the ribC gene-positive samples were selectively amplified, and BLASTn analysis showed that the gltA, rpoB, ITS and ribC genes were all B. melophagi. This may be due to the potential pitfall in this study, because the positive control we selected is Bartonella melophagi. In view of this, more follow-up studies are needed, such as using another Bartonella as a positive control. Previous studies on Bartonella in ticks showed that the positive rates were 2.40% (7/292) in China (41) (17). Previous studies on Bartonella in other ruminant-related blood-eating arthropods showed that the positive rates were 78.79% (26/33) in Hippoboscidae flies from Algeria (9) 4.21% (4/95) and 84.62% (11/13) (19,20), and 84.75% (50/59) and 93.75% (45/48) in L. cervi from Norway and Europe (6,8). There were differences in the positive rates of Bartonella in ticks from different countries and regions, which may be related to the location, number, detection methods and ecological environment of the collected samples. Interestingly, the positive rate of Bartonella is also different in different arthropods. Because  Bartonella species are mainly transmitted through vectors, it is speculated that the prevalence and richness of specific arthropods play a vital role. Xinjiang is the largest province in China and is rich in tick resources. Forty-two species of ticks belonging to nine genera were reported in Xinjiang, accounting for more than 1/3 of the total number of tick species in China (26,46,47); the wide distribution of ticks has a significant impact on the development of animal husbandry and on public health. In recent years, B. melophagi was detected in M. ovinus from northeastern Algeria (20), China (31), central Europe (19), the western United States (48), and northern Oromia, Ethiopia (12). B. melophagi was detected in white-tailed deer (49) and human blood (50) in the United States. Xinjiang is located in the hinterland of Eurasia and has a land border of more than 5,600 km bordering Russia, Kazakhstan, Kyrgyzstan, Tajikistan, Pakistan, Mongolia, India, and Afghanistan. With the proposal of the Belt and Road Initiative of China, import and export trade becomes more frequent, and Bartonella may spread among different countries and regions with the import and export of trade products.
In the phylogenetic inference, the gltA and rpoB sequences obtained in this study were clustered into a branch with Bartonella related to ruminants. In the cluster, the hosts of Bartonella were mainly ruminants, such as cattle, sheep, camel, and deer, and the vectors of Bartonella mainly included deer keds, sheep keds, biting flies, and ticks (Figures 2, 3). However, ticks can be invoked as vectors for several ruminant-related Bartonella. This suggests that certain vectors, especially ticks, may play a key role in the spread of Bartonella. At the same time, ticks have a wide range of hosts, which makes it possible for Bartonella to spread across species through ticks. Ruminant-related Bartonella clustered into one branch, which may be explained by the relationship between Bartonella and the host. A host-specific association between B. washoensis and squirrel was revealed by using multisite sequence analysis. The results showed that B. washoensis may have co-speciated with the host squirrel. The phylogenetic relationships showed that the B. washoensis strain of Spermophilus is mainly related to the host genus rather than to its geographic origin (51). There may be a symbiotic relationship between M. ovinus and B. melophagi (8), and B. melophagi may transfer between M. ovinus and sheep (48). Interestingly, we also detected B. melophagi in ticks collected from cattle ( Table 3). The coparasitism of ticks and M. ovinus in sheep may be considered a route of B. melophagi transmission. At present, there are no reports on associations between B. melophagi and sheep disease. B. melophagi was isolated from two women with a history of animal contact (50), which may suggest that B.
FIGURE 2 | The phylogenetic tree of Bartonella gltA sequences was constructed by using maximum likelihood (ML) and GTR+I+G4+F as evolutionary models. The sequences detected in this study are shown in bold, and the numbers at the nodes correspond to bootstrap values. Sinorhizobium fredii was used as the out group.
Frontiers in Veterinary Science | www.frontiersin.org melophagi is a possible human pathogen. In brief, our study provides evidence for the presence of B. melophagi DNA in ticks collected from cattle and sheep; however, further studies are needed to demonstrate that ticks are the vectors of B. melophagi. Additionally, we need to systematically investigate the prevalence of Bartonella in ticks, domestic mammals and people with a history of animal contact in Xinjiang, China. In general, this study is an important step in the control and public health safety of tick-borne Bartonella disease in mainland China and its neighboring countries.

CONCLUSIONS
In this study, molecular analysis was used to assess the presence and genetic diversity of B. melophagi in ticks collected from sheep and cattle from Xinjiang, China. This study provides new information on the presence and identity of B. melophagi in ticks from sheep and cattle. Ticks are used as the transmission medium of Bartonella, and it is necessary to investigate the prevalence of Bartonella in ticks, animals and people with animal contact.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories.