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. 2022 Aug 17;10(8):1663.
doi: 10.3390/microorganisms10081663.

Spatio-Temporal Patterns of Ticks and Molecular Survey of Anaplasma marginale, with Notes on Their Phylogeny

Shumaila Alam  1 , Mehran Khan  1 , Abdulaziz Alouffi  2 , Mashal M Almutairi  3 , Shafi Ullah  1 , Muhammad Numan  1 , Nabila Islam  4 , Zaibullah Khan  1 , Ome Aiman  1 , Sher Zaman Safi  5 , Tetsuya Tanaka  6 , Abid Ali  1
Affiliations

Spatio-Temporal Patterns of Ticks and Molecular Survey of Anaplasma marginale, with Notes on Their Phylogeny

Shumaila Alam et al. Microorganisms. .

Abstract

Hard ticks (Ixodida: Ixodidae) are medically important ectoparasites that feed on all classes of terrestrial vertebrates. Recently, we molecularly characterized hard ticks and associated Anaplasma spp. in the northern and central regions of Khyber Pakhtunkhwa (KP), Pakistan; however, this knowledge was missing in the southern regions. This study aimed to investigate tick prevalence, host range, genetic diversity, and molecular survey of Anaplasma spp. in a wide range of tick species in two distinct physiographic regions of southern KP. A total of 1873 hard ticks were randomly collected from 443/837 hosts (cattle, Asian water buffaloes, horses, goats, sheep, dogs, and camels) in Lakki Marwat, Bannu, and Orakzai districts of KP. Overall, 12 tick species were morphologically identified, among which Hyalomma dromedarii was the most prevalent species (390/1873, 20.9%), followed by Hy. anatolicum (294, 15.7%), Rhipicephalus microplus (262, 14%), Hy. scupense (207, 11.1%), R. sanguineus (136, 7.3%), R. turanicus (121, 6.5%), Haemaphysalis cornupunctata (107, 5.7%), R. haemaphysaloides (110, 5.9%), Ha. montgomeryi (87, 4.6%), Hy. isaaci (58, 3.1%), Ha. bispinosa (54, 2.9%), and Ha. sulcata (47, 2.5%). The extracted DNA from a subset of each tick species was subjected to PCR to amplify cox1 or 16S rRNA sequences of ticks and 16S rRNA sequences of Anaplasma spp. The tick cox1 sequences showed 99-100% identities with the sequences of the same species, whereas 16S rRNA sequences of R. turanicus, Ha. montgomeryi and Ha. sulcata showed 97-100% identities with the corresponding species. The 16S rRNA sequence of Ha. cornupunctata showed 92% identity with the species from the same subgenus, such as Ha. punctata. The 16S rRNA sequence of Anaplasma spp. showed 100% identity with Anaplasma marginale. Moreover, 54 ticks were found positive for A. marginale with a total infection rate of 17.2%. The highest infection rate was recorded in Hy. dromedarii (31.1%) and the lowest in each R. haemaphysaloides and R. sanguineus (20%). All the cox1 or 16S rRNA sequences in phylogenetic trees clustered with the same species, except Ha. cornupunctata, which clustered with the Ha. (Aboimisalis) punctata. In this study, Ha. cornupunctata was reported for the first time at the molecular level. The genetic characterization of ixodid ticks and molecular detection of associated A. marginale will assist in the epidemiological surveillance of these parasites in the region.

Keywords: Anaplasma marginale; Pakistan; hard ticks; phylogeny; surveillance.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Map showing tick collection sites in southern Khyber Pakhtunkhwa, Pakistan.
Figure 2
Figure 2
Tick infestation on different hosts: Hy. dromedarii from camels (A). Ha. bispinosa from goats (B). R. turanicus from sheep (C). Hy. anatolicum from male Asian water buffaloes (D). R. sanguineus from dogs (E). R. turanicus from goats (F).
Figure 3
Figure 3
Spatial patterns of the collected ixodid ticks in the study regions.
Figure 4
Figure 4
Seasonal abundance of the collected ixodid ticks in the study regions.
Figure 5
Figure 5
Maximum likelihood phylogenetic tree based on cox1 sequences of Hy. dromedarii, Hy. anatolicum, Hy. scupense, Hy. isaaci, Ha. bispinosa, R. microplus, R. sanguineus and R. haemaphysaloides. Haemaphysalis longicornis was used as an outgroup, using supporting values (1000 replicons) at each node. The scale bar indicates the number of substitutions per site. The obtained sequences were represented with black circles.
Figure 6
Figure 6
Maximum likelihood phylogenetic tree based on 16S rRNA sequences of R. turanicus, Ha. cornupunctata, Ha. montgomeryi and Ha. sulcata. Haemaphysalis longicornis was used as an outgroup, using supporting values (1000 replicons) at each node. The scale bar indicates the number of substitutions per site. The obtained sequences were represented with black circles.
Figure 7
Figure 7
Maximum likelihood phylogenetic tree based on the partial 16S rRNA sequence of A. marginale. The Anaplasma boleense was used as an outgroup, using supporting values (1000 replicons) at each node. The scale bar indicates the number of substitutions per site. The obtained sequence was represented with a black circle.
See this image and copyright information in PMC

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