Molecular detection of Bartonella in ixodid ticks and plateau pika (Ochotona curzoniae) in Shiqu County, China

Background: Bartonella bacteria have been associated with an increasingly wide range of human and animal diseases. They were identi�ed as being globally dispersed as emerging pathogens. Ticks and small rodents are known as hosts of human and animal bartonellosis. They play a signi�cant role in the preservation and circulation of bartonellae in nature. This study investigates the occurrence of Bartonella spp. in ticks and plateau pika in Shiqu county which is located on the Eastern Qinghai-Tibetan plateau in China. Shiqu county is spread over approximately 26,000 square kilometers, with an average altitude of above 4,200 meters and vast area of pastureland. Results: A total of 818 ticks (Dermacentor everestianus, 79.0%, 646/818; Haemphysalis qinghaiensis, 21.0%, 172/818) were collected in 4 villages of Shiqu county. Only Bartonella melophagi was detected in tick samples with a total prevalence of 30.1% (246/818). The infection rates of Bartonella spp. in ticks from Arizha, Maga, Derongma, and Changxgma were 4.8%, 76.8%, 12.5%, and 18.0% respectively. The infection rate of Bartonella spp. in Maga was higher (p< 0.01) than in other villages. Regarding plateau pika, the total infection rate of Bartonella spp was 24.1%, with 20.8% (15/72), 30.9% (25/81), 13.8% (9/65), and 29.4% (20/68) in Arizha, Maga, Derongma, and Changxgma respectively. Finally, B. queenslandensis, B. grahamii, and two unvalidated Bartonella species were detected. No signi�cant difference was observed (p> 0.05) in the infection rates between these study sites. Conclusion: To date, only D. everestianus and H. qinghaiensis were found in Shiqu county with high infection of Bartonella spp. in the ticks and plateau pika. The threats of Bartonella species to the public health should be closely monitored.

They play an essential role in the preservation and movement of bartonellae in nature within arthropodmammal systems.Shiqu county spreads over approximately 26,000 square kilometers, with an average altitude of above 4,200 meters and vast area of pastureland on the Eastern Qinghai-Tibetan plateau.Its population was estimated at 97,000, consisting of individuals with low education and poor health.Yak is the largest population of local livestock (about 600,000), where severe tick infestation is often observed.
Apart from yak, plateau pika (Ochotona curzoniae) is the largest population of local small rodents with close interaction with local people and livestock.The signi cance of ticks (Acari: Ixodida) has long been recognized due to their ability to feed on a large range of host species and to transmit Bartonella pathogens that can infect a variety of vertebrate hosts, including humans.However, little information exists on bartonellae and their hosts and vectors in Shiqu county.This study aims to prove the presence of Bartonella spp. in plateau pika and ticks and to provide preliminary results in view of establishing prevention and control measures for this tick-borne disease.
According to criteria (Bartonella spp.species thresholds: gltA ≥ 96.0% and rpoB ≥ 95.4%) proposed by La Scola, et al [13], only B. melophagi was detected for tick samples (Table 2), whereas for plateau pika, as shown in Table 3, B. grahamii was the predominantly identi ed specie in the four villages, with B. queenslandensis detected only in Maga and 2 unvalidated Bartonella species (Bartonella sp.* and Bartonella sp.**) found in Ariza and Changxgma respectively.Furthermore, gltA and rpoB based phylogenetic analysis supported the classi cation of Bartonella spp.detected in the current study (Fig. 2 and Fig. 3).

Discussion
Two tick species were identi ed in this study: H. qinghaiensis (in Maga only) and D. everestianus (in all four sites).D. everestianus was reported only in Northwestern China and Nepal [15] with an altitude of 2,600 -4,700 m [16].Larvae and nymphs of this tick specie often infest lagomorphs and rodents, while adult ticks usually utilize medium-large sized, modest and wild mammals as hosts, including hares, sheep, yaks, and horses [15,16].However, H. qinghaiensis is only reported in China [17][18][19][20][21], particularly prevalent in the western plateau, including the provinces of Qinghai, Gansu, Sichuan, and Tibet [21].Its natural hosts include sheep, goats, yaks, cattle, and hares (Lepus oiostolus).All life stages of the tick could develop in sheep, goats, yaks, and cattle [21][22][23][24][25][26][27].Contrary to ticks, H. qinghaiensis mostly performs its activity at low altitude.Arizha, Changxgma, and Derongma belong to the sub-frigid zone, whereas Maga village is located in the cold temperate zone.Due to the signi cant difference in altitude between Maga and the other three villages, H. qinghaiensis was only found in Maga.
All types of ticks were found to contain Bartonella DNA, although in varying percentages and locations.A survey regarding ticks from 16 states of the United States revealed that the overall prevalence of B. henselae in Ixodes ticks was 2.5% [28].In Austria, Bartonella spp.(B.henselae, B. doshiae, and B. grahamii) were detected in 2.1% of I. ricinus, with the highest rate in ticks from Vienna (with an infection rate of 7.5%) and higher prevalence in adult ticks than other life stages [29].Furthermore, a recent One Health perspective review on Bartonella indicated that the overall presence of Bartonella in ticks (combining evidence from multiple surveillance studies) was about 15% [30].In our results, a total prevalence of 30.1% in ticks (especially in Maga, 76.8%) indicated the severity in Shiqu county.
B. melophagi, a human bacterial pathogen, was rst isolated from sheep blood in 2007 [31], and the same bacteria were then isolated from the blood of two female patients with pericarditis and skin lesions in the United States of America [32].As a result, the rst report of DNA of B. melophagi detected in D. everestianus and H. qinghaiensis was obtained, which was the rst molecular evidence of B. melophagi in Shiqu county.However, there is no current evidence supporting the ability of these ticks to transmit B. melophagi to livestock or human.To address this issue, experiments should be performed to assess the vector competency of D. everestianus and H. qinghaiensis to transmit B. melophagi in the future.
Bartonella infection has been mostly reported in Rodentia [33][34][35][36][37][38][39][40][41][42], with few cases reported in Lagomorpha.Until now, there has been only one report of Bartonella infection in plateau pika with a positive rate of 18.99% [43].A total of 15 Bartonella strains were obtained, and most of them were closely related to B. taylorii and B. grahamii [43].Based on our research, B. grahamii, a pathogenic strain in humans, was detected in all four villages, while B. queenslandensis was detected only in Maga.Nevertheless, similar to B. coopersplainsensis, the zoonotic potential of B. queenslandensis has not been reported.Additionally, for two unvalidated Bartonella species (Bartonella.sp*and Bartonella.sp**)found in Ariza and Changxgma respectively, sequences analysis showed that: 1) based on gltA gene, they were clustered with B. rochalimae and B. queenslandensis respectively and 2) based on rpoB, however, they were clustered with B. melophagi.There are possible explanations for why this con icting result may occur.For instance, the potential presence of multiple Bartonella species in the sample although it is not commonly based on culturing.Secondly, different primer sets may also have ampli cation bias towards particular species based on the annealing a nity, which may cause the observed Bartonella diversity to differ, depending on the primer sets used for ampli cation.Lastly, homologous recombination, a speci c form of LGT (lateral gene transfer) among Bartonella spp., have been reported in other studies based on sequencing multiple protein-coding loci [12,[44][45][46][47][48].However, culturing, sequencing multiple loci (including 16S rRNA, ftsZ, gltA, groEL, ribC and rpoB, and ITS), cloning sequences into vectors before sequencing, or implementing deep sequencing approaches may discover a potentially novel Bartonella specie or subspecies and may differentiate these possible scenarios.
In Shiqu, plateau pika, the largest population of local small rodents, is in close contact with local people and livestock and can be infested with eas and ticks, implicating them in transmission cycles of Bartonella spp.In China, Bartonella infections among humans have been mainly reported in the central plain area such as Jiangsu, Zhejiang, Anhui, and Hubei province.No cases or suspected cases have been reported in the Qinghai-Tibetan plateau.Therefore, the relationship of plateau pika and the transmission of Bartonella should be further studied.A thorough analysis with controlled experiments should be conducted to determine the exact routes of transmission between plateau pika, the transmission between plateau pika and their vectors, as well as the transmission from plateau pika to humans and livestock.

Conclusion
To date, only D. everestianus and H. qinghaiensis were found in Shiqu county with high infection of Bartonella spp. in ticks and plateau pika.Further research should be conducted to determine the risk of Bartonella infections to humans and livestock.

Samples collection
A total of 818 ticks were collected by blanket dragging between June and August 2018, among which, 168, 224,192, and 234 were collected from Arizha, Maga, Derongma, and Changxgma respectively (Fig. 1 C).In the same period, a total of 286 pikas were captured; 72 in Arizha, 81 in Maga, 65 in Derongma, and 68 in Changxgma.Plateau pikas were captured using mouse snap traps.Then, spleens were collected under sterile conditions and stored in liquid nitrogen until use.Each pika's body was deeply buried to avoid being eaten by dogs, cats, and other wild carnivores.
Identi cation of tick species Ticks were carefully removed from blanket and stored in 70% of ethanol at 4°C.The specimens were morphologically identi ed according to the guidelines for tick identi cation [9].Then, molecular identi cation of tick species was performed, targeting the mitochondrial 16S rRNA gene [10].
DNA extraction, PCR, and sequences analysis Ticks were sectioned longitudinally, and one half per each tick was used for DNA extraction.For all spleen samples, an average of 30 mg of tissue was used.The total DNA of all samples were extracted using the TIANamp Genomic DNA Kit (TIANGEN Biotech Co., Ltd, Beijing, China; Cat No: DP304) for tick molecular identi cation and characterization of Bartonella spp.All samples were submitted to previously described PCR assays targeting gltA (379 bp) [11].All gltA-positive samples were further analyzed with PCR targeting rpoB (379 bp) [12].All primers were listed in Table 1.PCR ampli cations were conducted in a 25μl reaction mixture consisting of 1μl of genomic DNA (2 -3 ng), 1μl of each primer (10 μM), 12.5μl of PCR Supermix (Transgen Co., Ltd, Beijing, China; Cat No: AS111-11), and 9.5 μl of nuclease-free water.
Each PCR reaction included a positive control (DNA of B. henselae preserved in laboratory) and a negative control (nuclease-free water).Observed bands were puri ed using the QIAquick Gel Extraction Kit and sent for sequencing (Sangon Biotech Shanghai Co., Ltd).Obtained sequences were analyzed by employing Bioedit v.7.0.2 and were submitted for nucleotide BLAST search through the NCBI database.Sequences with ≥ 95% quality cover and identity were considered as positive for Bartonella spp.and were compared with validated Bartonella species in GenBank/EMBL/DDBJ through the Clustal X program (http://www.clustal.org/clustal2/).Clones that share ≥ 96.0% and ≥ 95.4% similarity in gltA and rpoB sequences with the validated species, respectively, can be considered as the same species [13].

Phylogenetic analysis and statistics
For phylogenetic analysis, Neighbor-Joining phylogenetic trees were constructed based on Bartonella gltA and rpoB sequences, using the Kimura two-parameter model with partial gap deletion and a cutoff of 95% site coverage respectively.The evolutionary distance was calculated, and bootstrap analysis with 1,000 iterations was carried out with the MEGA6 [14].SPSS19.0 (Pearson Chi-square test) was applied to Tables

Figure 1 The
Figure 1

Table 1
Primer sequences used for ticks and Bartonella spp.identification

Table 2
The prevalence of Bartonella spp. in ticks in Shiqu county