Detection and genetic diversity of Bartonella species in small mammals from the central region of the Qinghai-Tibetan Plateau, China

In this study, we aimed to investigate the prevalence and molecular characteristics of Bartonella infections in small mammals from the central region of the Qinghai-Tibetan Plateau. Toward this, small mammals were captured using snap traps in Yushu City and Nangqian County, West China, and the spleen tissue was used for Bartonella culture. The suspected positive colonies were evaluated using polymerase chain reaction (PCR) amplification and by sequencing the citrate synthase (gltA) gene. We discovered that 31 out of the 103 small mammals tested positive for Bartonella, with an infection rate of 30.10%. Sex differences between the mammals did not result in a significant difference in infection rate (χ2 = 0.018, P = 0.892). However, there was a significant difference in infection rates in different small mammals (Fisher’s exact probability method, P = 0.017) and habitats (χ2 = 7.157, P = 0.028). Additionally, 31 Bartonella strains belonging to three species were identified, including B. grahamii (25), B. japonica (4) and B. heixiaziensis (2), among which B. grahamii was the dominant epidemic strain (accounting for 80.65%). Phylogenetic analyses showed that most of the B. grahamii isolates identified in this study may be closely related to the strains isolated from Japan and China. Genetic diversity analyses revealed that B. grahamii strains had high genetic diversity, which showed a certain host and geographical specificity. The results of Tajima’s test suggested that the B. grahamii followed the progressions simulated by a neutral evolutionary model in the process of evolution. Overall, a high prevalence and genetic diversity of Bartonella infection were observed in small mammals in the central region of the Qinghai-Tibetan Plateau. B. grahamii as the dominant epidemic strain may cause diseases in humans, and the corresponding prevention and control measures should be taken into consideration in this area.

www.nature.com/scientificreports/ characteristics of Bartonella in small mammals has important implications for the prevention and control of human bartonellosis. The Qinghai-Tibetan Plateau, referred to as the "Roof of the World", is an inland plateau in Asia; the largest in China and the highest in the world. The Yushu Tibetan Autonomous Prefecture lies in the central region of the Qinghai-Tibetan Plateau and belongs to the Sanjiangyuan Region, the source of the Yangtze, Yellow, and Lantsang rivers (between 31.65° and 36.27° N, 89.40° and 102.38° E), with an average elevation of 4493 m 18 . It has an important ecological status, with the highest concentration of biodiversity area in the world; nearly 30 species of mammals have been reported to inhabit this area. Our team has previously detected Bartonella species infection in small mammals in some areas of the Qinghai-Tibetan Plateau, with infection rates of 18.99% and 38.61% 19,20 . However, investigations of Bartonella species in small mammals in the central region of the Qinghai-Tibetan Plateau have not yet been undertaken. This region's tourism industry was greatly developed following the reconstruction work after the Yushu earthquake. This increased the probability of people being infected with natural infectious diseases. Therefore, in this study, we investigated the prevalence and genetic diversity of Bartonella species in small mammals in the Yushu Tibetan Autonomous Prefecture. Our findings provide insights into the distribution and genetic diversity of Bartonella in small mammals and the scientific basis for the control and prevention of Bartonella infection in humans in this region.

Bartonella infections.
Spleens of the small mammals were collected and used for Bartonella isolation, and the pure colonies obtained were confirmed by polymerase chain reaction (PCR) amplification of the partial citrate synthase (gltA) gene (379 bp). In total, 31 small mammals were positive for Bartonella infection, with an infection rate of 30 In our previous study, phylogenetic analyses of Bartonella species was performed based on the DNA sequences of the gltA, ftsZ, rpoB and ribC revealed the same results 20 . Of these, gltA is the most commonly used in the phylogenetic analyses of Bartonella. Therefore, in this study, we selected gltA to construct a phylogenetic tree using the maximum likelihood (ML) method. All Bartonella strains could be divided into three clusters, i.e., B. grahamii, B. heixiaziensis, and B. japonica; B. grahamii were the dominant Bartonella species in this area (Fig. 2). Bartonella was detected in small mammals from three of the four trapping sites and the distribution of Bartonella species showed slight geographical differences (Fig. 3).
Phylogenetic analyses based on gltA sequences showed that B. grahamii was mainly grouped into four clusters, indicating that B. grahamii might have the different origins. We then obtained the gltA sequences of B. grahamii from GenBank released before July, 2021, and performed the traceability analyses. The majority of B. grahamii strains from A. peninsulae clustered with B. grahamii from A. speciosus in Japan; three strains, i.e., AP1QHYS, MA61QHYS, CR102QHYS, from A. peninsulae, M. arvalis, and Cricetidae clustered with B. grahamii from M. oeconomus in our previous study; three strains, i.e., CR34QHYS, CR36QHYS, CR103QHYS, from Cricetidae and one strain from M. gregalis (MG5QHYS) clustered separately and not with the reference strains (Fig. 4). Subsequently Table 4).

Discussion
Bartonella species are distributed throughout the world. They are highly prevalent in small mammals and are generally transmitted by bloodsucking arthropod vectors 21 . Previous studies have revealed that Bartonella infection varies in different regions and animals 16,17   In this study, we observed the prevalence and molecular characteristics of Bartonella species in small mammals from the central region of the Qinghai-Tibetan Plateau. The infection rate of Bartonella species in small mammals was 30.10%, which was similar to that of 38.61% in Qaidam Basin as determined in our previous study 20 , and higher than that in most areas of China 40 . Bartonella species were detected in five out of ten species of small mammals, including A. peninsulae, M. arvalis, Cricetidae, M. gregalis, and M. oeconomus, and we found differing infection rates among them. Additionally, the infection rate varied significantly by habitats, but not by sex, which is concurrent with the results of a previous study 20 .
Bartonella species are fastidious, slow-growing, facultative intracellular bacteria that are difficult and time consuming to culture. In our previous study, we used spleen, liver, and brain tissue for Bartonella culture and found that the positive rates in different tissues of small mammals did not differ significantly 19,20 . Here, the spleen tissue of small mammals was used for Bartonella culture, and 31 Bartonella strains were obtained. BLAST and phylogenetic analyses showed that 31 Bartonella strains corresponded to three species of Bartonella-(B. grahamii, B. japonica, and B. heixiaziensis). Importantly, 80.65% isolates (25/31) were B. grahamii and detected in all five species of the small mammals studied, suggesting that it was the dominant Bartonella strain. B. grahamii is associated with neuroretinitis and cat scratch disease (CSD) in immunocompromised individuals 10,11 , suggesting that Bartonella species may have the ability to cause human disease in this area. In addition, four isolates

Phylogenetic analyses.
The sequences generated in this study have been submitted to GenBank (accession numbers MZ126613-MZ126643). The nucleotide sequences of the isolated sequences were compared against the Bartonella species sequences hosted on GenBank using BLAST at the National Center for Biotechnology Information Website (http:// blast. ncbi. nlm. nih. gov/ Blast. cgi). The gltA sequences of B. grahamii hosted on GenBank released before July, 2021 were collected for traceability analyses. Furthermore, one strain isolated from the same host in the same laboratory at the same time was selected randomly as the reference strain. A phylogenetic tree was created using the maximum-likelihood method with the Kimura 2-parameter model in MEGA version 7.0, and bootstrap values were calculated with 1000 replicates 42,43 . Brucella abortus was used as the outgroup.
Genetic diversity analyses. The polymorphism of nucleotide sequences, including the number of polymorphic sites (S), the number of haplotypes (H), the nucleotide diversity (π), the mean number of nucleotide differences (κ), and the haplotype diversity (Hd), was analyzed using DNASP 6.12.03. A sliding window interval of 25 bp was used to determine which segment of the target gene sequence had the highest nucleotide diversity (π) by analyzing 100 bp each time. Tajima's test was performed to determine whether the target gene sequence followed the progressions simulated by a neutral evolutionary model in the process of evolution. Then, the sequences were analyzed based on a median-joining network using the Population Analysis with Reticulate Trees (PopART) software version 1.7 with the default setting (epsilon = 0).