Molecular Detection and Isolation of Bartonella Species in Bats and Their Ectoparasites Along the China-Myanmar Border

Abstract

Bartonella are parasitic pathogens that infect many mammals, including humans, and cause significant diseases. This study investigates the presence, genetic diversity, and tissue tropism of Bartonella in bats and their ectoparasites along the China-Myanmar border. Bats and ectoparasites were collected from Yingjiang, Ruili, and Gengma Counties. Nested PCR (nPCR) and quantitative real-time PCR (qPCR) were used to detect and quantify Bartonella in bat tissues. Bartonella was isolated using brain-heart infusion broth and tryptone soy agar medium containing 5% sheep blood (TSA containing 5% sheep blood), and DNA sequences were analyzed with Clustal W and MEGA X. In total, 601 bats from 11 species (four families and seven genera) and 32 ectoparasites (two orders, three families, and four genera) were collected. The qPCR results revealed Bartonella detection rates of 22.96% (138/601) in bats and 62.50% (5/8) in ectoparasites. Using nPCR to detect the Bartonella gltA and rpoB genes in bats, ectoparasites, and strains isolated from bat blood samples, yielding 58 and 10 strains, respectively. When comparing bats, ectoparasites, and isolated strains to other Bartonella in GenBank, the gltA gene was 74.21%-100.00% at the nucleotide level of similarity and 75.70%-100.00% at the amino acid level. In comparison, the rpoB gene was 79.58%-100.00% at the nucleotide level of similarity and 89.71%-100.00% at the amino acid level. By phylogenetic analysis except for Bartonella sp. and uncultured Bartonella sp., we found a clade that was less than 96.0% at the nucleotide level of similarity in the gltA gene and less than 95.4% at the nucleotide level of similarity in the rpoB gene. Based on the threshold values for the delineation of new species of Bartonella, we believe that a new species of Bartonella prevalent in bats was discovered in this study, which we named "Candidatus Bartonella dianxisis". Otherwise, the average copy number of Bartonella in bat tissues (blood, spleen, heart, brain, kidney, lung, liver, and rectum) ranged from 1.15 × 10⁴ to 6.87 × 10⁴ copies/μL, with the highest levels observed in blood and spleen. Our findings highlight the genetic diversity of Bartonella in bats and ectoparasites along the China-Myanmar border and underscore potential public health risks associated with these pathogens.

Keywords: Bartonella; Bartonella isolation; Candidatus Bartonella dianxisis; China–Myanmar border; bats; ectoparasites; genetic diversity; tissue tropism.

Figure 1

Map of sampling locations. The right panel shows the geographical map of China and Myanmar, while the left panel highlights the counties and cities situated along the China–Myanmar border region where sampling was conducted.

Figure 2

Prevalence of Bartonella among different bat species. Dots represent the prevalence rates of Bartonella for each bat species, while bar graphs indicate the 95% confidence intervals (95% CIs). Bat species with sample sizes fewer than 10 were excluded from the analysis.

Figure 3

Phylogenetic classification of Bartonella species based on bat gltA gene sequences. The phylogenetic tree was constructed using the maximum likelihood method under the T92 + G model (Tamura 3-parameter + gamma distribution). Bootstrap values were calculated with 1000 iterations, and values greater than 70% are displayed on the tree.

Figure 4

Phylogenetic classification of Bartonella species based on bat ectoparasites rpoB gene sequences. The phylogenetic tree was constructed using the maximum likelihood method under the T92 + G model (Tamura 3-parameter + gamma distribution). Bootstrap values were calculated with 1000 iterations, and values greater than 70% are displayed on the tree.

Figure 5

Tissue tropism of Bartonella in positive samples. The Bartonella load was measured across various tissues, including blood, spleen, heart, brain, kidney, lung, liver, and intestine, from 18 positive samples. Data are presented as mean ± standard error in copes/μL. Significance testing were conducted using one-way ANOVA (⁣^∗p < 0.05, ⁣^∗∗p < 0.01, ⁣^∗∗∗p < 0.001). The red dotted line indicates the detection limits of qPCR established in this study.