1. Introduction
Ticks are obligate blood-feeding ectoparasites of animals and humans that are distributed globally [
1]. They can affect their hosts either directly by causing tick-associated stress, irritation, allergy, anemia, weight loss and paralysis or indirectly by transmitting numerous pathogenic microorganisms including bacteria, fungi, protozoa, rickettsiae, spirochetes and/or viruses [
1,
2,
3]. In production animals such as cattle, buffaloes, goats and sheep, tick-borne protozoal (babesiosis and theileriosis) and rickettsial (anaplasmosis and cowdriosis) diseases cause major health problems as well as production and economic losses mainly in subtropical and tropical regions [
1].
Pakistan is a subtropical country where the majority of the rural population is dependent upon livestock including small ruminants for their food and livelihood (Pakistani goat and sheep population: 31.2 million
Ovis aries; 78.2 m
Capra hircus), particularly in the Federally Administered Tribal Area (FATA) of the north-western part of the country [
4,
5]. The FATA is located near the Pak-Afghan border and represents one of the least-developed regions in Pakistan due to political unrest and prolonged military crises over the last 50 years. These areas consist of seven tribal agencies (districts) and six frontier regions, which have been recently merged [
6]. Nomadic pastoralism is a common practice in this region and > 70% of the human population derives their livelihood from livestock farming [
7]. Due to the poor infrastructure, limited resources and inadequate access to veterinary services, tick-borne diseases (TBDs) of humans and animals have a major impact in this region [
7].
Although a number of studies have assessed the prevalence of ticks and tick-borne diseases (TTBDs) of small ruminants in different areas of Pakistan [
8,
9,
10,
11,
12,
13,
14,
15,
16,
17,
18,
19], there is a paucity of information from the FATA. Moreover, no study has yet investigated the presence, prevalence and diversity of tick-borne pathogens (TBPs) of small ruminants in this region. Recently, Khan et al. [
20] assessed tick burdens on small ruminants in the FATA using morphological methods and found three main genera of ixodid ticks (
Haemaphysalis,
Hyalomma and
Rhipicephalus) but these ticks and associated TBPs were not further characterized in detail using molecular tools.
Specific identification is pivotal for understanding the epidemiology of, and developing effective control strategies for, TTBDs [
21]. However, morphological methods do not allow the identification of immature, engorged or damaged tick specimens [
22,
23]. By contrast, molecular methods provide an alternative and complementary approach for the identification of ticks [
24], which employ the characterization of genetic markers such as the mitochondrial cytochrome
c oxidase subunit I (
cox1) and 16S ribosomal RNA genes [
25,
26]. As ticks usually harbor and transmit commensals and numerous pathogens, some of which can be of public health significance (e.g.,
Coxiella burnetii and Crimean-Congo hemorrhagic fever virus) [
27,
28,
29], it is important to detect these microorganisms in ticks to ascertain their prevalence. However, conventional diagnostic methods such as microscopic examination of thin and thick smears usually detect few target pathogens or microorganisms and have a lower sensitivity and specificity than molecular approaches [
30]. Therefore, testing ticks as well as their animal hosts using polymerase chain reaction (PCR) based methods for the detection of TBPs and/or commensals provides distinct advantages over conventional detection methods. Recently, a micro-chip-based microfluidic real-time PCR technique was developed to detect and differentiate up to 96 microorganisms per tick in a single PCR procedure [
31]. This method has been proven to be well-suited for rapid and large-scale epidemiological and surveillance studies [
28,
31,
32,
33,
34,
35,
36] and is anticipated to be an invaluable tool for the detection of microorganisms in ticks from regions such as the FATA.
In the present study, we employed both conventional and PCR-based tools to investigate the diversity of tick taxa from small ruminants and their associated TBPs and/or commensals in the FATA of Pakistan.
4. Discussion
This study provides the first insight into the molecular diversity of ticks, TBPs and endosymbionts in ticks from small ruminants in the FATA, Pakistan. The occurrence of
Hyalomma and
Rhipicephalus species characterized in our study is consistent with what has been reported previously from small and large ruminants in selected areas of Pakistan [
8,
15,
50]. However, this study provides the first genetic evidence for
Hs. sulcata,
Hs. punctata,
Rh. haemaphysaloides and
Rh. turanicus from Pakistan.
Within the
Rh. sanguineus group (clades 1–5), the sequences of
Rh. turanicus determined here (clade-3a) clustered with the sequences belonging to the temperate lineage of
Rh. sanguineus (clade-1) (
Figure 2 and
Figure 3) indicating a close similarity between the two species. The NCBI blast results also supported these findings as two of the 16S sequences determined here (GenBank: MT799954 and MT799955) were similar (96–97.2%) to those of
Rh. sanguineus (GenBank: KR870984) and
Rh. turanicus (GenBank: KR870985) from Turkey (data not shown). Likewise, three
cox1 sequences (GenBank: MT800312–MT800314) were similar (90.5–93.9%) to those of
Rh. sanguineus (GenBank: MF426015) and
Rh. turanicus (GenBank: MN853166) from Portugal and Kazakhstan, respectively (data not shown). The taxonomic classification of
Rh. turanicus has been recently studied by Bakkes et al. [
51] who refuted the monophyletic nature of
Rh. turanicus and proposed a new species, i.e.,
Rh. africanus n sp. Moreover, the authors also provided evidence of the existence of two lineages corresponding to southern Europe and the Middle East/Asia with differing climates. We also found a similar pattern here as
Rh. turanicus sequences (from this study and references) clustered into two distinct clades (3a and 3b) (
Figure 2 and
Figure 3). These two clades may represent two separate species (cf. [
51]). For
Rh. haemaphysaloides, the genetic differences (0.2–7.6%) in
cox1 sequences inferred here suggest the existence of two distinct lineages or even distinct species as their genetic similarity is < 95%, the value generally considered to be the threshold of conspecificity for these genes in ticks [
52,
53,
54,
55,
56]. However, due to limited genetic data being available for this tick species, it is challenging to define a threshold for species delineation.
This study also provides the first molecular evidence for
Hs. sulcata and
Hs. punctata in Pakistan. The molecular-phylogenetic analyses of
Haemaphysalis sequences confirmed and supported morphological characterization. However, the NCBI blast results demonstrated considerably large nucleotide differences from previously reported sequences of the same species (
Hs. sulcata: 16S; 4.7–5.5%,
cox1; 10.4–10.7%,
Hs. punctata: 16S; 6.8%,
cox1; 10.7%) from Turkey and Iran (
Hs. sulcata) and China and Romania (
Hs. punctata) (data not shown). These large differences could be partly due to the limited availability of gene sequences of these ticks. Future studies should focus on the morphological and genetic characterization of
Haemaphysalis ticks collected from different climatic zones of Pakistan. Furthermore, additional genetic markers such as the elucidation of complete mitochondrial genomes [
56,
57] would allow the phylogeny of
Haemaphysalis and other ticks to be resolved.
In this study, microfluidic real-time PCR-based screening of ticks demonstrated a higher prevalence of microorganisms (72.2%) as well as a higher percentage of ticks (71.8%) testing positive for multiple microorganisms (
Table 3 and
Table 4). The results were validated by conventional PCR for microorganisms whose specific identification was not achieved. However, genetic characterization was not successful for some microorganisms (such as the
Ehrlichia species) due to low cycle threshold (Ct) values. Moreover, this study does not establish the mammalian- or tick-origin of detected microorganisms since ticks were collected while feeding on their hosts (i.e., goats and sheep). In addition, the detection of DNA of multiple microorganisms in several ticks does not imply the co-transmission to their hosts. Furthermore, this study does not provide estimates for the prevalence and distribution of different tick species in small ruminants from the FATA as ticks were collected from a smaller population. However, the prevalence of microorganisms was significantly higher in ticks collected from goats (83.9%) compared with those from sheep (56.5%) as shown in
Table 3. Although there have been reports of higher prevalences of ticks [
15] and internal parasites [
58] in goats compared with sheep, most reports on the prevalence of haemoparasites in small ruminants from Pakistan and elsewhere are contrary to this finding [
11,
59,
60,
61,
62]. For example, Iqbal et al. [
11] reported 32 and a 5% prevalence of piroplasms in sheep and goats from the Punjab and KPK provinces of Pakistan, respectively. Likewise, Azmi et al. [
59] and Rjeibi et al. [
61] found higher prevalences of
Theileria and piroplasms in sheep compared with goats from Palestine and Tunisia, respectively. Further molecular testing would be required to establish such differences, if any, as a smaller number of ticks were tested in the present study.
Francisella-like and
Coxiella-like endosymbionts detected in this study are non-pathogenic mutualistic and/or commensal microbes, which play a key role in the tick’s developmental process and pathogen transmission [
63,
64]. We detected a higher prevalence of DNA of
R. massiliae and
R. slovaca in ticks in all five districts with a low prevalence of
R. aeschlimannii (Bajaur and Mohmand) and
R. conorii (Khyber and Mohmand) (see
Table 3). All of these rickettsiae belong to the spotted fever group (SFG) and, thus, have zoonotic potential. The significance and risk of infections of SFG rickettsiae are higher in Asian countries where surveillance and diagnostic facilities are limited and new cases of rickettsial infections are increasing [
65]. Due to a large influx of livestock and immigrants during the Afghan War, it is possible that new potential pathogens might have been imported into this region [
7]. Moreover, most of the families in the FATA are living with their animals and there is a general lack of awareness about TBDs of veterinary and public health importance [
7]. Our findings and previous reports of rickettsial species from ticks in Pakistan [
28] highlight the need to establish a diagnostic surveillance system for zoonotic rickettsial pathogens in this country.
A higher prevalence of
Theileria spp. (33.3%) was also found in ticks from all five districts and the molecular characterization of piroplasms revealed that they belonged to
Th. ovis. However, it is not possible to exclude the possibility of another caprine/ovine
Theileria species as
Th. lestoquardi has been reported from small ruminants in Pakistan such as by Riaz et al. [
16].
Anaplasma ovis was detected in 25.9% of all tick species identified herein and it is most frequently associated with anaplasmosis in small ruminants worldwide [
64]. However, most of these cases are subclinical infections with a low-grade fever [
66]. More recently, a variant of
A. ovis has also been associated with human infection in Cyprus [
67]. However, due to the lack of a proper diagnostic and surveillance system in Pakistan it is difficult to ascertain the economic losses and zoonotic threat due to this and other TBPs detected in this study.
Finding ticks that carry DNA of up to seven microorganisms of veterinary and medical significance indicates the level of risk associated with tick infestation to animals as well as humans. Previously, a similar level of co-occurrence of endosymbionts (i.e.,
Francisella-like and
Coxiella-like) and pathogens (belonging to
Anaplasma,
Babesia,
Bartonella,
Borrelia,
Ehrlichia,
Hepatozoon,
Rickettsia and
Theileria genera) in bovine ticks was reported from the Punjab and Sindh provinces of Pakistan [
28]. Similarly, microbiome analyses of
Dermacentor silvarum and
Ixodes persulcatus ticks from China revealed the presence of up to 29 and 373 bacterial genera, respectively, belonging to endosymbionts and pathogens [
68,
69]. These high levels of co-occurrence of microorganisms in ticks encourage the large-scale implementation of such high-throughput tools in resource-scarce settings where routine surveillance facilities are not accessible.
