Molecular Survey and Genetic Characterization of Anaplasma marginale in Ticks Collected from Livestock Hosts in Pakistan

Simple Summary Ticks transmit different disease-causing agents to humans and animals. Pakistan is an agricultural country, the rural economy mainly relies on livestock farming, and tick infestation is a severe constraint to its livelihood. The genus Anaplasma comprises obligate Gram-negative intracellular bacteria multiplying within the host cells and can be transmitted to humans and animals through the tick vector. The current study aimed to molecularly characterize the Anaplasma spp. in hard ticks infesting livestock in different districts of Khyber Pakhtunkhwa, Pakistan. The present study reported nine species of hard ticks infesting different hosts. The most prevalent tick life stage was adult females, followed by nymphs and adult males. In the phylogenetic tree, 16S rDNA sequences of Anaplasma spp. clustered with sequences of A. marginale. The hard ticks act as a carrier for the transmission of A. marginale. Further extensive country-wide research is required to explore the diverse tick species and the associated pathogens in Pakistan. Abstract Ticks transmit pathogens to animals and humans more often than any other arthropod vector. The rural economy of Pakistan mainly depends on livestock farming, and tick infestations cause severe problems in this sector. The present study aimed to molecularly characterize the Anaplasma spp. in hard ticks collected from six districts of Khyber Pakhtunkhwa, Pakistan. Ticks were collected from various livestock hosts, including cattle breeds (Holstein-Friesian, Jersey, Sahiwal, and Achai), Asian water buffaloes, sheep, and goats from March 2018 to February 2019. Collected ticks were morphologically identified and subjected to molecular screening of Anaplasma spp. by amplifying 16S rDNA sequences. Six hundred seventy-six ticks were collected from infested hosts (224/350, 64%). Among the nine morphologically identified tick species, the highest occurrence was noted for Rhipicephalus microplus (254, 37.6%), followed by Hyalomma anatolicum (136, 20.1%), Rhipicephalus haemaphysaloides (119, 17.6%), Rhipicephalus turanicus (116, 17.1%), Haemaphysalis montgomeryi (14, 2.1%), Hyalomma dromedarii (11, 1.6%), Haemaphysalis bispinosa (10, 1.5%), Hyalomma scupense (8, 1.2%), and Haemaphysalis kashmirensis (8, 1.2%). The occurrence of tick females was highest (260, 38.5%), followed by nymphs (246, 36.4%) and males (170, 25.1%). Overall, the highest occurrence of ticks was recorded in the Peshawar district (239, 35.3%), followed by Mardan (183, 27.1%), Charsadda (110, 16.3%), Swat (52, 7.7%), Shangla (48, 7.1%), and Chitral (44, 6.5%). Among these ticks, Anaplasma marginale was detected in R. microplus, R. turanicus, and R. haemaphysaloides. The 16S rDNA sequences showed high identity (98–100%) with A. marginale reported from Australia, China, Japan, Pakistan, Thailand, Uganda, and the USA. In phylogenetic analysis, the sequence of A. marginale clustered with the same species reported from Australia, China, Pakistan, Thailand, Uruguay, and the USA. Further molecular work regarding the diversity of tick species and associated pathogens is essential across the country.

In Pakistan, the Khyber Pakhtunkhwa (KP) province is a hotspot for developing and reoccurring TBDs of veterinary and public health relevance [5,6,15,16,18,21]. Due to the extension in dairy cattle industries and the introduction of exotic cattle breeds, the surveillance of ticks and TBPs in indigenous and exotic cattle breeds is essential. There is a scarcity of available information regarding the detection of Anaplasma spp. in ticks [5,19]. The current study aimed to investigate different tick species to detect Anaplasma spp. throughout KP.

Ethical Approval
The research study was approved by the Advanced Studies Research Board (ASRB) members of Abdul Wali Khan University Mardan, Pakistan (Dir/A&R/AWKUM/2018/1410). Verbal/oral permission was taken from the livestock owners and farmers during tick collection.

Study Area
Tick specimens were collected from various herds randomly in six districts, including Peshawar (34. (Figure 1).

Ticks Collection and Preservation
Tick specimens were randomly collected from March 2018 to February 2019 during four seasons (spring, summer, fall, and winter). These specimens were collected from livestock hosts having different ages, genders, and host types reared at houses (herd size at house was 1-4 animals) and animal farms (herd size at farm was 20-80 animals). The collections were randomly done from different cattle breeds (Holstein-Friesian, Jersey, Sahiwal, and Achai), Asian water buffaloes, sheep, and goats at various collection sites in the six districts of KP, Pakistan. Ticks were washed with distilled water followed by 70% ethanol to remove the contaminants and tissues from the tick's body. Finally, the specimens were preserved in 100% ethanol for further experimental work.

DNA Sequencing and Phylogenetic Analysis
Amplified amplicons of 16S rDNA of Anaplasma spp. from different tick species infesting Holstein-Friesian, Jersey, and Asian water buffaloes were sequenced in both directions (Macrogen, Inc., Seoul, South Korea). The obtained sequences were trimmed to remove the contaminated and poor reading regions via SeqMan V. 5 (DNASTAR, Inc., Madison, WI, USA). The obtained sequences were subjected to the Basic Local Alignment Search Tool (BLAST) [30] at National Center for Biotechnology Information (NCBI). The homologous sequences were downloaded in FASTA format from NCBI based on their high percentage identity. The downloaded sequences were aligned with the obtained sequences and an outgroup sequence using ClustalW multiple alignments [31] in BioEdit alignment editor V.7.0.5 (Raleigh, NC, USA) [32]. The phylogenetic tree was constructed through the Maximum-Likelihood statistical method and Tamura-Nei model [33] with a 1000 bootstrapping value in Molecular evolutionary genetics analysis (MEGA-X) [34].

Statistical Analyses
The descriptive statistical analyses were performed in Microsoft Excel V. 2016 (Microsoft 365 ® ). Chi-square tests were performed in the GraphPad Prism V. 5 (GraphPad Software, Inc., San Diego, CA, USA), and the analysis was considered significant at a p-value < 0.05.

Variables Associated with a Tick Infestation
A total of 350 hosts of different ages, genders, and host types were observed for the presence of ticks in different seasons. Based on the hosts' gender, the highest tick occurrence was noted in female hosts compared to male hosts. Based on age, hosts were stratified into three age groups; the highest tick occurrence was recorded in the <3 years age group, followed by the 1-3 years age group and >1-year age group. Among the observed animals, the highest occurrence was recorded in Holstein-Friesian, followed by Jersey, Sahiwal, Asian water buffaloes, goats, sheep, and Achai. The occurrence of ticks was highest in summer, followed by spring, fall, and winter seasons. District-wise, the occurrence of ticks was highest in the Peshawar district and lowest in Chitral. All variables, including genders, ages, hosts, seasons, and areas associated with tick occurrence, were highly significant ( Table 2).

Phylogenetic Analysis of Anaplasma marginale
The multiple obtained identical sequences were considered as a consensus sequence. The BLAST results of the obtained 16S rDNA (1109 bp) sequences showed 98-100% identity with the A. marginale reported from Australia (CP006847 and AF414874), China (KX987330, DQ341369, FJ389579, and HM439433), Japan (FJ226454), Pakistan (MK680804, MK680806, and MK680807), Thailand (KT264188), Uganda (KU686785, KU686774, and KU686775), and the USA (AF311303, CP000030, and AF309866). Thirty-two sequences were downloaded for 16S rDNA of A. marginale from NCBI. The phylogenetic tree obtained a 16S rDNA sequence of A. marginale clustered with the identical species sequences reported from Australia, China, Thailand, Pakistan, Uruguay, and the USA (Figure 2). The 16S rDNA sequence of A. marginale was uploaded to NCBI under the accession number ON306400.

Discussion
Pakistan's hot and humid environment is ideal for the growth of ticks and their associated pathogens [5,21]. Previous studies have reported numerous tick species infesting diverse hosts in different areas of the country [5,15,18,20,35]. More than twenty different hard tick species are biological vectors of A. marginale, causing bovine anaplasmosis [36]. In Pakistan, there is a scarcity of molecular approaches to detecting A. marginale in ticks, primarily identified through microscopy of blood smear analysis [37,38]. During this study, A. marginale was genetically characterized in collected ticks from diverse livestock hosts in Pakistan. The current study identified hard ticks comprised of nine medically essential tick species infesting cattle breeds (Holstein-Friesian, Jersey, Sahiwal, and Achai), Asian water buffaloes, sheep, and goats. Among these nine tick species, three species in the genus Rhipicephalus were found positive for A. marginale, with A. marginale being dominant in R. microplus. This tick has been identified as a significant vector of A. marginale in several tropical and subtropical countries [39].
A low tick occurrence was recorded in local cattle breeds (Achai), which may be due to the natural resistance of Achai towards the tick infestation. The female hosts were found to have a significantly higher occurrence of ticks than male hosts, which is consistent with the previous findings [20]. Female hosts may have a high occurrence due to hormonal changes, because the high level of prolactin and progesterone hormone in female hosts make them more vulnerable to tick infestation [40]. A high tick occurrence has been recorded in adult hosts compared to the younger ones, which is in accordance with the findings of previous reports [17,35]. Free grazing practices and large surfaces of the adult hosts make them more susceptible to tick attachment, in contrast to the younger ones that get less of a tick burden due to less grazing practices, the low surface area of their bodies, and their strong immune system [41]. Annual patterns of tick activity are influenced by seasonal temperature variations, affecting the dynamics of ticks and TBDs. Fluctuation in different seasons may result in varying tick occurrence in the same region [42]. The summer season provides the best conditions for developing and expanding ticks. The winter season hampers the infestation of ticks because all stages of ticks hibernate in cold climatic conditions, and these findings support the previous reports in the region [15,35]. The highest tick occurrence was recorded in the Peshawar district, followed by Mardan, and the least was recorded from the Chitral district. These results may correspond to favorable environmental conditions associated with ticks rearing [18,35,43].
For identifying Anaplasma spp., molecular techniques such as PCR have significant advantages over traditional serological and blood smear testing because PCR is the most sensitive and reliable diagnostic method [44]. The molecular phylogeny of A. marginale from the study area was developed by amplifying 16S rDNA sequences, as this marker is of prime importance in characterizing Anaplasma spp. [40]. The sequence obtained in this study shared a 98-100% identity with available sequences in GenBank. Phylogenetic analysis revealed that A. marginale from the northern regions of Pakistan clustered with related isolates reported from Australia, China, Thailand, Pakistan, Uruguay, and the USA (Figure 2). Previous research on the molecular phylogeny of A. marginale based on 16S rDNA sequences from Pakistan validates our findings [20,45].
Ticks infected with A. marginale may be of significant concern to both animals and humans due to the increased risk of infection, complicating clinical care. These findings highlight the need for a larger-scale tick surveillance program in understanding various TBDs, and their zoonotic and pathogenic potential.

Conclusions
The present study provides information regarding the occurrence of hard ticks as carriers for A. marginale in Pakistan. Anaplasma marginale was detected in three tick species: R. microplus, R. turanicus, and R. haemaphysaloides. These findings will inform the veterinary and livestock community regarding the diversity of tick species and associated A. marginale.
Further research is needed to explore the variety of ticks and tick-associated pathogens in Pakistan.