Survey of Ticks and Tick-Borne Rickettsial and Protozoan Pathogens in Eswatini

Abstract

Ticks are widespread parasites of vertebrates and major vectors of pathogens to humans, domestic animals, and wildlife. In southern Africa, numerous tick species transmit diseases of economic and health importance. This study aimed to describe the occurrence of ticks and tick-borne pathogens in multiple land-use types and the possible role of ticks in the transmission of pathogen species. Using molecular techniques, we screened 1716 ticks for infection by rickettsial bacteria and protozoans. To characterize pathogen identity, we sequenced multiple loci from positive samples and analyzed sequences within a phylogenetic framework. Across the seven tick species collected as nymphs or adults, we detected Rickettsia, Anaplasma, Ehrlichia, Babesia, Hepatozoon, and Theileira species. We found that some tick species and tick-borne pathogens differed according to land use. For example, we found a higher density of Haemaphysalis elliptica and higher prevalence of Rickettsia in H. elliptica collected from savanna grasses used for livestock grazing near human settlements than savanna grasses in conservation areas. These findings highlight the importance of comprehensive surveillance to achieve a full understanding of the diversity and ecology of the tick-borne pathogens that can infect humans, domestic animals, and wildlife.

Keywords: Anaplasma; Babesia; Ehrlichia; Eswatini; Hepatozoon; Rickettsia; Theileria; land use; ticks.

Figure 1

Phylogenetic analysis of tick sequences using (A) CO1 gene for all ticks in study and (B) concatenated 12S, CO1, and ITS2 gene sequences for Rhipicephalus species represented as maximum likelihood trees. Bootstrap values at the nodes represent the percent agreement among 1000 replicates. The branch length scale represents substitutions per site. The number of individual specimens that were sequenced from each lineage is indicated in parentheses. For (A) the Genbank accession numbers are indicated in brackets and for (B) the Genbank accession numbers for 12S/CO1/ITS2 are: [1] = KY457536/KY457536/KY457500; [2] = KY457538/KY457538/KY457503; [3] = KY457540/KY457540/KY457504; [4] = KY457542/KY457542/KY457508; [5] = KY457544/KY457544/KY457509; [6] = NC_002074/NC_002074/KY945496; [7] = MW080169/MW079312/NA; [8] = NA/KP862674/KP862668; [9] = EU921764/KY678117/KY457506; [10] = KF569940/KY678131/BDU97716. NA = reference sequence not available. * = GenBank sequence unverified.

Figure 2

PCR-RFLP digestion profiles. Undigested ITS2 amplicon and BauI restriction digestion profile for variable sequences of the ITS2 PCR amplicon of R. appendiculatus (B = undigested, C = digested), R. muehlensi (D = undigested, E = digested), R. simus (F = undigested, G = digested), R. maculatus (H = undigested, I = digested), R. microplus (J = undigested, K = digested), and R. decoloratus (L = undigested, M = digested) with 1 kb base pair ladder in A and N.

Figure 3

Phylogenetic analysis of Rickettsia species and genotypes inferred from concatenated ompA, ompB, and gltA gene sequences using the maximum likelihood method with 1000 bootstraps. The tick species from which each Rickettsia isolate was sequenced in this study is represented by the colored pie chart at each tip, followed by the isolate name, the number of samples, and the Genbank accession numbers in square brackets ompA/ompB/gltA). Sequences obtained from GenBank are indicated by their name and accession number. Bootstrap supports, represented as percentages, are indicated on each node. The branch length scale represents substitutions per site.

Figure 4

Phylogenetic analysis of Anaplasmataceae species and genotypes found in ticks based on (A) 16S, (B) rpoB, and (C) groEL genes using the maximum likelihood method with 1000 bootstraps. The tick species from which of each Anaplasma or Ehrlichia isolate was sequenced is represented by the colored pie chart at each tip, followed by the isolate name, the number of samples, and the Genbank accession number in square brackets. Sequences obtained from GenBank are indicated by their name and accession number. Bootstrap supports, represented as percentages, are indicated on each node. The branch length scale represents substitutions per site.

Figure 5

Phylogenetic analysis of (A) Theileria, (B) Babesia, and (C) Hepatozoon species and genotypes found in ticks based on a partial 18S gene using the maximum likelihood method with 1000 bootstraps. The tick species from which each isolate was sequenced is represented by the colored pie chart at each tip, followed by the isolate name, the number of samples, and the Genbank accession number in square brackets. Sequences obtained from Genbank are indicated by their name and followed by their accession number. Bootstrap supports, represented as percentages, are indicated on each node. The branch length scale represents substitutions per site.

Figure 6

Map of study area. (A) Southern Africa showing the location of study area (black box) within Eswatini (red); (B) land-use/land-cover map and sampling locations (black dots) within the study area; and (C) an example of tick sampling by dragging a white cloth across the vegetation.