First Detection of Theileria sinensis-like and Anaplasma capra in Ixodes kashmiricus: With Notes on cox1-Based Phylogenetic Position and New Locality Records

Simple Summary Ixodes species are the main vectors of bacteria and piroplasm for different vertebrate hosts. Research on these unexplored concerns has been neglected in different regions including Pakistan. Recently, we molecularly characterized Ixodes kashmiricus ticks and associated Rickettsia spp.; however, the cox1 sequence and associated Theileria spp. and Anaplasma spp. for this tick are unknown. This study aimed to genetically identify I. kashmiricus based on the cox1 sequence and associated Theileria spp. and Anaplasma spp. A total of 352 ticks including adult females, nymphs and males were collected from small ruminants. The BLAST results and phylogenetic analysis of the cox1 sequence revealed a close resemblance with the Ixodes ricinus complex sequences. The 18S rDNA and 16S rDNA sequences showed maximum identity with Theileria cf. sinensis or Theileria sinensis and Anaplasma capra, respectively, and they phylogenetically clustered with the same species. This is the first report on the cox1 sequence of the I. kashmiricus tick, new locality records, and associated T. sinensis-like and A. capra. In order to determine the epidemiology of Ixodes ticks and their related pathogens, a widespread tick investigation is required. Abstract Ixodes ticks transmit Theileria and Anaplasma species to a wide range of animals. The spreading of ticks and tick-borne pathogens has been attributed to transhumant herds, and research on these uninvestigated issues has been neglected in many countries, including Pakistan. Recently, we used internal transcribed spacer (ITS) and 16S ribosomal DNA partial sequences to genetically characterize Ixodes kashmiricus ticks and their associated Rickettsia spp. However, the data on its cox1 sequence and associated Theileria spp. and Anaplasma spp. are missing. This study aimed to genetically characterize I. kashmiricus based on the cox1 sequence and their associated Theileria spp. and Anaplasma spp. The I. kashmiricus ticks were collected from small ruminants: sheep (Ovis aries) and goats (Capra hircus) of transhumant herds in district Shangla, Dir Upper and Chitral, Khyber Pakhtunkhwa (KP), Pakistan. Out of 129 examined hosts, 94 (72.87%) (56 sheep and 38 goats) were infested by 352 ticks, including adult females (175; 49.7%) followed by nymphs (115; 32.7%) and males (62; 17.6%). For molecular analyses, 121 ticks were subjected to DNA isolation and PCR for the amplification of the cox1 sequence for I. kashmiricus, 18S rDNA for Theileria spp. and 16S rDNA sequences for Anaplasma spp. The obtained cox1 sequence showed 89.29%, 88.78%, and 88.71% identity with Ixodes scapularis, Ixodes gibbosus, and Ixodes apronophorus, respectively. Phylogenetically, the present cox1 sequence clustered with the Ixodes ricinus complex. Additionally, the 18S rDNA sequence showed 98.11% maximum identity with Theileria cf. sinensis and 97.99% identity with Theileria sinensis. Phylogenetically, Theileria spp. clustered with the T. cf. sinensis and T. sinensis. In the case of Anaplasma spp., the 16S rDNA sequence showed 100% identity with Anaplasma capra and phylogenetically clustered with the A. capra. PCR-based DNA detection targeting the amplification of groEL and flaB sequences of Coxiella spp. and Borrelia spp., respectively, was unsuccessful. This is the first phylogenetic report based on cox1 and new locality records of I. kashmiricus, and the associated T. sinensis-like and A. capra. Significant tick surveillance studies are needed in order to determine the epidemiology of Ixodes ticks and their associated pathogens.


Introduction
Genus Ixodes (Acari: Ixodidae: Prostriata) developed during the Mesozoic era's cretaceous period (65-95 million years ago) [1,2].The Ixodes genus comprises more than 265 species, which are divided based on morphology into 18 subgenera [3].Among them, the largest subgenus Ixodes comprises 18 species and includes the most studied ticks [4].Ixodes ticks are known to adopt in particular environmental conditions for survival and development, and these are considered to limit their dispersal [3,5].Climatic conditions and the availability of a suitable host are the two most important factors determining the distribution and abundance of Ixodes ticks.Ixodes ticks have been commonly found in woodland or mixed forest and grassland, which provide moist vegetation and approximately 80% humidity-a critical threshold for the survival and development of these ticks [2,5].
The identification of ticks, particularly those belonging to the genus Ixodes, has been traditionally based on morphological features, such as the shape of the spiracular plates, grooves of the scutum and punctations [2,4,21].However, these methods are often insufficient for accurate identification and differentiation, particularly for Ixodes and other closely related ticks [22,23].Molecular techniques have been alternatively used for the accurate identification and differentiation of different tick species [22,[24][25][26][27][28].Some genetic markers, such as cox1, 16S ribosomal DNA (rDNA) and internal transcribed spacer (ITS), have been shown suitable for the accurate delineation of ticks [21,[29][30][31][32]. Ixodes kashmiricus tick has been reported based on ITS and 16S rDNA sequences, and their associated Rickettsia spp.has been reported based on gltA and ompA sequences [21].However, genetic data based on cox1 sequence for I. kashmiricus and associated Theileria spp.and Anaplasma spp.are missing.Herein, I. kashmiricus ticks were for the first time genetically characterized based on a mitochondrial cox1 sequence and screened for associated Theileria spp.and Anaplasma spp. in Khyber Pakhtunkhwa (KP), Pakistan.

Ethical Approval
This study was approved by the Advance Studies Research Board (ASRB: Dir/A&R/ AWKUM/2022/9396) committee members of Abdul Wali Khan University, Mardan KP, Pakistan.The oral permission was obtained from the owners of the transhumant herds during the host's observation and tick collection.

Study Area and Tick Collection
This study was conducted in district Shangla (34 •  These districts are highly mountainous, with an elevation approximately 3000-3500 m (m), and situated in the north or northwest of KP.The elevation study map was designed in ArcGIS 10.3.1, using the "Global Positioning System" to determine the locations of the collection sites (Figure 1).Tick specimens were collected from small ruminants in transhumant herds during May-July 2022 in district Shangla, Dir Upper and Chitral.The ticks were isolated from the host body carefully via tweezers to avoid any external damage to the specimens.The tick specimens were washed in distilled water followed by 70% ethanol and preserved in 100% ethanol in 1.5 mL Eppendorf tubes for further experiments.

DNA Isolation and PCR
Individually, 121 ticks including 20 males, 44 adult females and 27 nymphs from

DNA Isolation and PCR
Individually, 121 ticks including 20 males, 44 adult females and 27 nymphs from sheep, as well as 16 females and 14 nymphs from goats, were selected and subjected to molecular analyses.Before the DNA isolation, tick specimens were washed with distilled water followed by 70% ethanol and kept in an incubator (30-45 min) until dried.The specimens were cut with sterile scissors and homogenized in 200-300 µL phosphate-buffered saline (pH 7.4, 137 mM NaCl, 2.7 mM KCl, 8 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 ) using a micro-pestle.The genomic DNA was extracted using a phenol-chloroform protocol [34], and the isolated DNA pellet was diluted by the addition of 20-30 µL of "nuclease-free" PCR water.The isolated genomic DNA was quantified via NanoDrop (Nano-Q, Optizen, Daejeon, Republic of Korea) and stored at −20 °C.
All extracted genomic DNA was used for the screening of associated pathogens based on genetic markers such as 18S rRNA for Theileria spp., 16S rRNA for Anaplasma spp., groEL for Coxiella spp.and flaB for Borrelia spp.Each PCR contained a positive control (DNA of Anaplasma marginale, Theileria annulata, Coxiella burnetii and Borrelia anserina for pathogens and genomic DNA of Hy. anatolicum for ticks) and a negative control ("nuclease-free" PCR water instead of DNA).The primers used in this study and their thermocycler conditions are given in Table 1.The PCR-amplified products were electrophoresed on a 1.5% agarose gel and visualized under ultraviolet light in the Gel Documentation System (BioDoc-It™ Imaging Systems UVP, LLC, Upland, CA, USA).PCR-positive samples were purified by using a DNA Clean & Concentrator Kit (Zymo Research, Irvine, CA, USA) by following the manufacturer's instructions.

DNA Sequencing and Phylogenetic Analysis
All amplified amplicons of cox1 (5: 1 male, 2 adult females, and 2 nymphs) for ticks, 18S rDNA (2: 1 adult female and 1 nymph) for Theileria spp.and 16S rDNA (4: 2 adult females and 2 nymphs) for Anaplasma spp.were sequenced (Macrogen Inc., Seoul, Republic of Korea) by Sanger sequencing.The obtained sequences were trimmed/edited via SeqMan v. 5 (DNASTAR, Inc., Madison/WI, USA) for the removal of poor reading sequences and subjected to Basic Local Alignment Search Tool (BLAST, https://blast.ncbi.nlm.nih.gov/Blast.cgi,accessed on: 10 July 2022) at the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/,accessed on: 10 July 2022).After BLAST, maximum identity sequences of the most similar/subgenus species were downloaded in FASTA format from the NCBI.Obtained sequences were aligned with the downloaded sequences using ClustalW multiple alignments in BioEdit Sequence Alignment Editor v. 7.0.5 [39].The phylogenies were constructed individually for each DNA sequence of tick and associated pathogens through the Maximum Likelihood statistical method and Kimura 2-parameter model in Molecular Evolutionary Genetics Analysis (MEGA-X) with a bootstrapping value of 1000 [40].The coding sequences like cox1 sequences were aligned by using MUSCLE algorithms [41].

Statistical Analyses
All recorded data such as the numbers of collected ticks and their life stages in the three districts, as well as associated pathogens like Theileria spp.and Anaplasma spp., were arranged in the spreadsheet (Microsoft Excel v. 2016, Microsoft 365 ® ) for descriptive statistical analyses.The differences were considered significant at a p-value less than 0.05 under chi-square tests using the GraphPad Prism v. 8 (Inc., San Diego, CA, USA).
Furthermore, other tick species were not found co-infesting sheep and goats afflicted by I. kashmiricus ticks.During collection from district Chitral, only an adult female of I. kashmiricus was found on sheep.Details of host records, prevalence of ticks, and detection of Theileria and Anaplasma species in the selected districts are summarized in Table 2.

Sequences and Phylogenetic Relationship of Ticks
A sum of five ticks' (one male, two adult females and two nymphs) genomic DNA was amplified via PCR targeting the cox1 sequence.The BLAST analysis of the cox1 sequence of I. kashmiricus showed 89.29% maximum identity with I. scapularis followed by 88.78% with Ixodes gibbosus and 88.71% with Ixodes apronophorus from Canada, Turkey and Russia, respectively.The obtained 16S rDNA sequence for I. kashmiricus was identical to the sequences of the same species from Pakistan (MW578839).Therefore, the 16S rDNA sequence was not included in further analysis.The obtained cox1 sequence of I. kashmiricus was submitted to GenBank under the accession number OR244356.
Phylogenetically, the cox1 sequence was clustered to the species of the subgenus Ixodes ricinus complex such as I. apronophorus (MH784873) reported from Russia.Furthermore, the cox1 sequence formed sister clades with I. ricinus complex such as I. scapularis, I. gibbosus, Ixodes acuminatus, Ixodes redikorzevi, Ixodes laguri, Ixodes inopinatus, Ixodes ricinus, and Ixodes affinis reported from Canada, Turkey, Malta, Romania, Serbia, Tunisia, Italy and the United States (Figure 2).Among all molecularly analyzed ticks, Theileria spp.and Anaplasma spp.DNA were detected in two (1.65%: one adult female and one nymph) and four (3.3%: two adult females and two nymphs) I. kashmiricus ticks, respectively (Table 2).Moreover, other pathogens such as Coxiella spp.and Borrelia spp.based on groEL and flaB markers, respectively, were not amplified by PCR.
The 18S rDNA sequence of Theileria spp.showed 98.11% maximum identity with Theileria cf.sinensis reported from South Africa, which was followed by 97.99-97.87%identity with T. sinensis reported from Malaysia and China.Similarly, the 16S rDNA sequence of Anaplasma spp.showed 100% identity with A. capra reported from the Republic of Korea, China, and Iraq.The obtained 18S rDNA sequence of T. sinensis-like and 16S rDNA sequence of A. capra were submitted to GenBank (OR244360: T. sinensis-like and OR244358: A. capra).The details regarding the detection rate of T. sinensis-like and Anaplasma capra are shown in Table 2.The phylogenetic tree of the 18S rDNA sequence for T. sinensis-like clustered with T. sinensis (JQ037786-JQ037787) reported from South Africa and T. cf.sinensis reported from Malaysia (MT271902 and MT271911) and China (KX115427 and KF559355).It formed a sister clade with the sequences of Theileria sergenti, Theileria buffeli and Theileria orientalis (Figure 3).In the case of 16S rDNA, A. capra clustered to the corresponding species reported from South Korea (LC432114), China (MG869594), and Iraq (ON872236) (Figure 4).98.11% maximum identity.Furthermore, the obtained 18S rDNA sequence of T. sinensis-like showed a minimum nucleotide difference of 16 bp with the sequences of T. sinensis, which showed 1.89% (16/844 bp) genetic difference.Due to the high genetic differences, this species was considered as T. sinensis-like or related to T. sinensis.Similarly, the phylogeny of the obtained 18S rDNA sequence indicated a similar relationship or related to the T. sinensis reported from the Old World.The constructed phylogeny work has a comparable topology to those demonstrated by Loh et al. [18], whereas Theileria spp. is derived from Ixodes spp.
Ixodes ticks collected from cattle and sheep have been reported as a vector of A. phagocytophilum and A. capra [11][12][13][14][15].This study presents the first evidence for A. capra in I. kashmiricus.For the molecular identification of Anaplasma spp., a highly conserved 16S rDNA sequence has been used historically [27,57].Likewise, the 16S rDNA sequence of A. capra was detected in I. kashmiricus, which was reported for the first time.The present study reported T. sinensis-like and A. capra in I. kashmiricus ticks infesting small ruminants that closely related to the corresponding species.The zoonotic pathogenicity of the T. sinensis-like and A. capra was detected in this survey remains to be examined considering the significance of piroplasm and bacterial species as an agent of novel emerging infectious agents carried by I. kashmiricus ticks.

Conclusions
A new locality for I. kashmiricus was recorded, and its phylogenetic position based on the cox1 sequence was delineated for the first time.Based on a phylogenetic analysis, the I. kashmiricus tick is closely related and clustered with the species of same subgenus-the I. ricinus complex.Theileria sinensis-like and A. capra were detected in I. kashmiricus for the first time.These findings may help to further understand the epidemiology of the I. kashmiricus tick and its associated Theileria and Anaplasma species, and they may strengthen the need for tick and tick-borne pathogen surveillance programs.

Animals 2023, 13 , x 4 of 15 Figure 1 .
Figure 1.Map showing the locations (black stars) where Ixodes ticks were collected during this study.

Figure 1 .
Figure 1.Map showing the locations (black stars) where Ixodes ticks were collected during this study.

Figure 2 .
Figure 2. Phylogenetic tree of Ixodes species based on the cox1 sequences.The cox1 sequence of Ixodes simplex belonging to the subgenus Eschatocephalus was taken as an outgroup.The obtained cox1 sequence was highlighted with bold and underlined fonts (OR244356).

Figure 2 .
Figure 2. Phylogenetic tree of Ixodes species based on the cox1 sequences.The cox1 sequence of Ixodes simplex belonging to the subgenus Eschatocephalus was taken as an outgroup.The obtained cox1 sequence was highlighted with bold and underlined fonts (OR244356).

Animals 2023, 13 , x 9 of 15 Figure 3 .
Figure 3. Phylogenetic tree of Theileria species based on the 18S rDNA sequences.The 18S rDNA sequence of Theileria annae was taken as an outgroup.The obtained 18S rDNA sequence was highlighted with bold and underlined fonts (OR244360).

Figure 3 .
Figure 3. Phylogenetic tree of Theileria species based on the 18S rDNA sequences.The 18S rDNA sequence of Theileria annae was taken as an outgroup.The obtained 18S rDNA sequence was highlighted with bold and underlined fonts (OR244360).

Animals 2023, 13 , x 10 of 15 Figure 4 .
Figure 4. Phylogenetic tree of Anaplasma species based on the 16S rDNA sequences.The 16S rDNA sequence of "Candidatus Anaplasma sphenisci" was taken as an outgroup.The obtained 16S rDNA sequence was highlighted with bold and underlined fonts (OR244358).

Figure 4 .
Figure 4. Phylogenetic tree of Anaplasma species based on the 16S rDNA sequences.The 16S rDNA sequence of "Candidatus Anaplasma sphenisci" was taken as an outgroup.The obtained 16S rDNA sequence was highlighted with bold and underlined fonts (OR244358).

Table 1 .
List of the primers used to amplify target DNA of the Ixodes kashmiricus and associated Theileria and Anaplasma species.

Table 2 .
Prevalence of identified Ixodes kashmiricus ticks and their life stages and molecular detection of associated Theileria spp.and Anaplasma spp.