High Diversity and Low Coinfections of Pathogens in Ticks from Ruminants in Pakistan

Abstract

Emerging tick-borne infections pose growing public health threats, causing global disease burdens and economic losses. In this study, tick-borne pathogens were detected in ticks collected from ruminants in 19 sites of Khyber Pakhtunkhwa Province, Pakistan, between 2023 and 2024. A total of 989 ticks, belonging to five species, i.e., Hyalomma marginatum, Rhipicephalus microplus, Rhipicephalus sanguineus, Rhipicephalus haemaphysaloides, and Haemaphysalis bispinosa, were tested by specific PCR followed by Sanger sequencing. In total, fourteen pathogens including two Anaplasma species, three Ehrlichia species, three Rickettsia species, one Babesia species, and five Theileria species were identified, with an overall infection rate of 20.2% (95% CI: 17.7-22.7%). Phylogenetic analyses revealed two undefined Ehrlichia species: Candidatus Ehrlichia hyalommae was exclusively detected in Hy. marginatum ticks, while Candidatus Ehrlichia rhipicephalis was only found in R. microplus. Additionally, an undefined Rickettsia, provisionally named Candidatus Rickettsia pakistanensis, was identified, which is phylogenetically close to R. sibirica in North Asia and R. africae in Africa, suggesting its potential pathogenicity to humans. Although coinfections of two pathogens were observed, the coinfection rates were quite low. The findings revealed a significant diversity of tick-borne pathogens in Pakistani ticks, which may pose risks to livestock and humans.

Keywords: Anaplasma; Babesia; Ehrlichia; Pakistan; Rickettsia; Theileria; high diversity; phylogenetic analysis.

Figure 2

Phylogenetic trees of Anaplasma were constructed using the maximum likelihood (ML) method with 1000 bootstrap replicates based on (A) 16S rRNA, (B) gltA, and (C) groEL genes. The evolutionary positions of samples that tested positive for at least two of the three gene fragments are highlighted in red text.

Figure 3

Phylogenetic trees of Ehrlichia were constructed using the maximum likelihood (ML) method with 1000 bootstrap replicates. Separate trees were generated for (A) 16S rRNA and (B) groEL genes. (C) A concatenated tree was built based on 16S rRNA and groEL nucleotide sequences. The evolutionary positions of samples that tested positive for all two gene fragments are highlighted in red text.

Figure 4

Phylogenetic trees of Rickettsia were constructed using the maximum likelihood (ML) method with 1000 bootstrap replicates. Separate trees were generated for (A) OmpA, (B) 17 kDa, and (C) gltA genes. (D) A concatenated tree was built based on OmpA, 17 kDa, and gltA nucleotide sequences. The evolutionary positions of samples that tested positive for at least two of the three gene fragments are highlighted in red text.

Figure 5

Phylogenetic trees of Theileria and Babesia strains based on their 18S rRNA gene sequences. Sequences obtained in this study are highlighted in red text.

Figure 1

Distribution of tick samples in Khyber Pakhtunkhwa Province, Pakistan. The red stars indicated in the diagram designate 19 specific sampling sites located across three districts within the Khyber Pakhtunkhwa Province of Pakistan.