In this study, two tick species were found: H.qinghaiensis (only in Maga) and D.everestianus(in all of four sites). D.everestianus was only reported in Northwestern China and Nepal [15] with an altitude of 2600-4700m [16]. Larvae and nymphs of this tick species 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,a typical three-host tick, is only recorded in China [17-21], particularly prevalent in the western plateau, including the provinces of Qinghai, Gansu, Sichuan and Tibet [21]. Its natural hosts include sheep, goat, yak, cattle and hare (Lepus oiostolus). It is known that all stages of the tick could develop in sheep, goat, yak and cattle [21-27]. Comparing to ticks at high altitude, the activity of H.qinghaiensis is more frequently at low altitude. In this study, Arizha, Changxgma and Derongma villages belong to sub-frigid zone, with an altitude of 4300-4600m;Maga village is located in the cold temperate zone, with an altitude of 3799m. There was a significant difference in altitude between Maga and the other three villages, which was probably why H.qinghaiensis was only found in Maga village.
All types of ticks were found to contain Bartonella DNA, although in varying percentages and locations. A survey of ticks from 16 states in the U.S 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 derived from Vienna (with a 7.5% infection rate), and that adult ticks had a higher prevalence than other 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 firstly 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 USA [32]. In this study, this is the first report of DNA of B.melophagi detected in D.everestianus and H.qinghaiensis and it was the first molecular evidence of B.melophagi found in Shiqu county. However, currently there is no evidence supporting the ability of these ticks to transmit B. melophagi to livestock or human. To address this issue, experiments need to be performed to assess vector competency of D.everestianus and H.qinghaiensis to transmit B.melophagi in the future.
Bartonella infection has been mostly reported in Rodentia [33-42], with few cases reported in Lagomorpha. Until now, there has only one report of Bartonella infection in plateau pika with a positive rate of 18.99% [43]. Totally, 15 Bartonella strains were obtained and most of them were closely related to B.taylorii and B. grahamii [43]. In our results, B.grahamii, a pathogenic strain in humans, was detected in all of four villages while B.queenslandensis was detected only in Maga. Nevertheless, as 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 : 1) based on gltA gene, they were clustered with B.rochalimae and B. queenslandensis, respectively; 2) based on rpoB, however, they were clustered with B. melophagi. The causes of this conflicting result can be classified as fowllows: 1) potential presence of multiple Bartonella species in the sample although it is not common based on culturing; 2) different primer sets may have amplification bias towards particular species based on the annealing affinity. These complications may cause the observed Bartonella diversity to differ depending on which marker was used for amplification; 3) homologous recombination, a specific form of LGT (lateral gene transfer) among Bartonella spp. This problem has been documented in several studies of Bartonella strains from cats, rodents and bats based on sequencing multiple protein-coding loci [12, 44-48]. However, culturing, sequencing multiple loci (including 16S rRNA, ftsZ, gltA, groEL, ribC and rpoB and ITS), cloning sequences into vectors before sequencing or deep sequencing approaches can be sufficient to describe a potentially novel Bartonella specie or subspecies and may differentiate these possible scenarios.
In Shiqu, plateau pika, the largest population of local small rodents, has close contact with local people and livestock and can be infested with fleas and ticks, implicating them in transmission cycles of Bartonella spp. In China, Bartonella infections among humans have mainly been 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. So, the relationship of plateau pika and the transmission of Bartonella should be studied closer and more thoroughly with controlled experiments to determine the exact routes of transmission between plateau pika, the transmission between plateau pika and their vectors, as well as the transmission between plateau pika to humans and livestock.