Identification of tickborne pathogens in cattle and sheep ticks from Kyrgyzstan using next-generation sequencing

Abstract

Background: Various tickborne diseases and pathogens in livestock have been reported in Kyrgyzstan; however, comprehensive molecular analyses from ticks and their tickborne pathogen diversity in the region are lacking. This study aimed to identify tick species and bacterial pathogens infesting cattle and sheep across Kyrgyzstan using amplicon-based next-generation sequencing (NGS).

Methods: In 2022, ticks were collected from livestock across seven provinces and subjected to molecular analyses. Genomic DNA was extracted from ticks for species identification based on cytochrome c oxidase I (COI) gene sequence analyses. Pathogens were screened using amplicon NGS targeting the V3-V4 region of the 16S rRNA gene, followed by confirmation using polymerase chain reaction (PCR) and Sanger sequencing.

Results: A total of 546 ticks belonging to two families, five genera, and 12 species were identified. The dominant species were Dermacentor spp. (30.2%), Hyalomma marginatum (17.2%), Hyalomma scupense (13.4%), and Haemaphysalis punctata (11.7%). Furthermore, 11.7% of ticks tested positive for bacterial pathogens, including spotted fever group Rickettsiae (8.6%), Anaplasma (2.7%), Ehrlichia (0.2%), and Bartonella (0.2%). Coxiella burnetii and Francisella tularensis were not detected.

Conclusions: This is the first nationwide study on bacterial pathogens in ticks in Kyrgyzstan and the first reports of spotted fever group Rickettsiae and Bartonella in the country. These findings improve our understanding of tickborne disease epidemiology and highlight the utility of NGS as an efficient screening method for capturing pathogen diversity in arthropod vectors.

Keywords: Anaplasma; Bartonella; Ehrlichia; Next-generation sequencing; Spotted fever group Rickettsiae; Tick; Tickborne pathogen.

Fig. 1

Map showing the composition of tick species in seven provinces. The size of the pie charts corresponds to the number of ticks collected in each region, while the size of each slice within the pie chart represents the proportions of different tick species. The map has been adapted from [24]

Fig. 2

Phylogenetic analysis for tick molecular identification based on partial nucleotide sequences of the COI gene fragment (597 bp). The sequences identified in this study are indicated in bold (blue color). The phylogenetic tree was constructed using the maximum likelihood method based on the general time reversible model. Rate variation among sites was modeled using a gamma distribution with invariant sites (G + I). The numbers on the branches indicate bootstrap percentages based on 1,000 replications

Fig. 3

Phylogenetic analysis of tickborne bacterial pathogens (Anaplasma spp., Ehrlichia spp., spotted fever group Rickettsiae, and Bartonella spp.). Phylogenetic analysis of a Anaplasma spp., b Ehrlichia spp., and d Bartonella spp. based on 16S rRNA gene fragments (878, 385, and 1,020 bp). Phylogenetic analysis of c spotted fever group Rickettsiae based on the fragments of the 17 kDa antigen coding gene (398 bp). The sequences identified in this study are indicated in bold (blue color). The phylogenetic tree was constructed using the maximum likelihood method based on the Kimura 2-parameter model. Rate variation among sites was modeled using a gamma distribution in (a–c), and a gamma distribution with a proportion of invariant sites (G + I) in (d). The numbers on the branches indicate bootstrap percentages based on 1,000 replications