Molecular Detection of Rickettsia hoogstraalii in Hyalomma anatolicum and Haemaphysalis sulcata: Updated Knowledge on the Epidemiology of Tick-Borne Rickettsia hoogstraalii

Simple Summary Ticks are hematophagous ectoparasites that spread diseases to both animals and humans through their bites. They are notorious for carrying various disease-causing agents, such as viruses, protozoa, and bacteria, which present substantial risks to both human and animal well-being. Continuous changes in the climate can impact both tick distribution and abundance. Understanding of the epidemiology of tick-borne Rickettsia hoogstraalii is limited, with gaps in its molecular detection, genetic characterization, and absence of data, especially from Pakistan. This study aimed to use molecular methods to genetically analyze Rickettsia species, particularly R. hoogstraalii, in Pakistan while also contributing new insights into the pathogen′s global epidemiology. For this purpose, ticks were collected from different hosts, including goats, sheep, and cattle, from six districts of Khyber-Pakhtunkhwa, Pakistan. This study is the first to genetically characterize R. hoogstraalii in Hyalomma anatolicum ticks globally and Haemaphysalis sulcata in Pakistan. This species was first described in 2006 in Croatia and has also been detected in different species of ticks in different countries. The pathogenicity of R. hoogstraalii in vertebrate hosts is not well understood. Encouraging additional research is essential to unveil the involvement of ticks in the transmission and persistence of R. hoogstraalii across various host species. Abstract Ticks are hematophagous ectoparasites that transmit pathogens to animals and humans. Updated knowledge regarding the global epidemiology of tick-borne Rickettsia hoogstraalii is dispersed, and its molecular detection and genetic characterization are missing in Pakistan. The current study objectives were to molecularly detect and genetically characterize Rickettsia species, especially R. hoogstraalii, in hard ticks infesting livestock in Pakistan, and to provide updated knowledge regarding their global epidemiology. Ticks were collected from livestock, including goats, sheep, and cattle, in six districts of Khyber Pakhtunkhwa (KP) Pakistan. Overall, 183 hosts were examined, of which 134 (73.2%), including goats (number = 39/54, 72.2%), sheep (23/40, 57.5%), and cattle (71/89, 80%) were infested by 823 ticks. The most prevalent tick species was Rhipicephalus microplus (number = 283, 34.3%), followed by Hyalomma anatolicum (223, 27.0%), Rhipicephalus turanicus (122, 14.8%), Haemaphysalis sulcata (104, 12.6%), Haemaphysalis montgomeryi (66, 8.0%), and Haemaphysalis bispinosa (25, 3.03%). A subset of 210 ticks was selected and screened for Rickettsia spp. using PCR-based amplification and subsequent sequencing of rickettsial gltA and ompB fragments. The overall occurrence rate of R. hoogstraalii was 4.3% (number = 9/210). The DNA of Rickettsia was detected in Hy. anatolicum (3/35, 8.5%) and Ha. sulcata (6/49, 12.2%). However, no rickettsial DNA was detected in Rh. microplus (35), Rh. turanicus (35), Ha. montgomeryi (42), and Ha. bispinosa (14). The gltA and ompB fragments showed 99–100% identity with R. hoogstraalii and clustered phylogenetically with the corresponding species from Pakistan, Italy, Georgia, and China. R. hoogstraalii was genetically characterized for the first time in Pakistan and Hy. anatolicum globally. Further studies should be encouraged to determine the role of ticks in the maintenance and transmission of R. hoogstraalii in different hosts.


Introduction
Ticks are obligate blood-sucking ectoparasites distributed all over the world, especially in tropical and subtropical areas [1][2][3].The most important hard ticks that transmit pathogens and affect domestic and wild animals belong to different genera such as Rhipicephalus, Hyalomma, Haemaphysalis, Ambylomma, and Ixodes [4].These hematophagous ectoparasites play a significant role in transmitting pathogens, encompassing bacteria, protozoans, and viruses that lead to zoonotic outcomes threatening human and animal health [5,6] Rickettsia species are obligatory intracellular Gram-negative bacteria that are divided into major groups: the spotted fever group (SFG), the typhus group, the bellii group, and the limioniae group [7,8].Among these, tick-borne SFG Rickettsia spp.include a large number of zoonotic agents and are considered important pathogens causing SFG [5,9,10].Rickettsia spp. of the SFG are mostly transmitted by hard ticks (Ixodidae) to vertebrate hosts [5].In addition, the human pathogenicity of several rickettsial species has been described, and rickettsial species with undetermined pathogenicity have been observed in ticks [5,11,12].
Rickettsia hoogstraalii is a member of the SFG with unknown pathogenicity and is closely related to Rickettsia felis, an emerging pathogen known to be spread through arthropods, especially ticks and fleas [13][14][15][16].Rickettsia hoogstraalii was first reported in 2006 in Ha. sulcata ticks in Croatia [17] and later on was detected in various tick species of the genera Heamaphysalis, Rhipicephalus, Argas, Dermacentor, Carios, Ixodes, and Africaniella in Croatia, Pakistan, Georgia, Spain, Cyprus, India, Ethiopia Turkey, Italy, Greece, Iran, USA, Namibia, Zambia, Romania, China, Africa, and Anatolia .Notably, it has been detected in Ha. montgomery, infesting goat and sheep from Pakistan [15].

Ticks Collection and Identification
Ticks were collected between June 2021 and August 2022 from asymptomatic livestock hosts (cattle, goats, and sheep) at different sites.Ticks were collected from the bodies of the hosts, regardless of their particular location within the planned survey zones or times, whenever they were found in different farms, open fields, or free-roaming animals in pastures.With the use of forceps, 1-10 ticks per animal were collected from each host while examining their entire body.The collected specimens were washed with distilled water followed by 70% ethanol to remove contaminants and stored in Eppendorf tubes with 99.98% ethanol.Morphological identification was performed using a StereoZoom microscope (HT StereoZoom), following taxonomic keys [55,56] and stored in 2 mL microtubes for further molecular analysis.

Ticks Collection and Identification
Ticks were collected between June 2021 and August 2022 from asymptomatic livestock hosts (cattle, goats, and sheep) at different sites.Ticks were collected from the bodies of the hosts, regardless of their particular location within the planned survey zones or times, whenever they were found in different farms, open fields, or free-roaming animals in pastures.With the use of forceps, 1-10 ticks per animal were collected from each host while examining their entire body.The collected specimens were washed with distilled water followed by 70% ethanol to remove contaminants and stored in Eppendorf tubes with 99.98% ethanol.Morphological identification was performed using a StereoZoom microscope (HT StereoZoom), following taxonomic keys [55,56] and stored in 2 mL microtubes for further molecular analysis.

Sequencing and Phylogenetic Analysis
The obtained sequences were trimmed using Seqman 5.0 (DNASTAR, Inc., Madison, WI, USA) to remove poor sequencing reads and primer contaminations.All the obtained sequences were identical; hence, a single consensus sequence was obtained.The consensus sequences were submitted to the Basic Local Alignment Search Tool (BLASTn; National Center for Biotechnology Information [NCBI)]).Higher-identity sequences were aligned using BioEdit alignment editor v. 7.0.5 [61] and were subjected to ClustalW Multiple alignment [62].The individual phylogenetic trees of gltA and ompB were constructed in accordance with the maximum likelihood method in Molecular Evolutionary Genetics Analysis (MEGA-XI) software [63], using the MUSCLE algorithm [64].A similar outcome was observed for all the available methods.However, due to its ability to evaluate different phylogenetic trees and models under a statistical framework, the maximum likelihood method is recommended as the actual method for the best evolutionary analysis [65].Statistical analysis of the nodes was performed using bootstrap resampling analysis, which involved 1000 replicates.This approach provided a rigorous assessment of the reliability of the tree branching patterns and relationships [63].The acquired fragments of gltA and ompB were used to determine their final positions in the dataset.

Sequences and Phylogenetic Analysis
After a BLAST search of the NCBI database, the gltA sequence revealed 100% identity and 100% query identity with R. hoogstraalii reported in Italy and Pakistan.On the other hand, the ompB (773 bp) sequence of R. hoogstraalii revealed 99.2-99.7%high identity and 100% query to the reported sequences from China and the USA.In the phylogenetic tree of gltA, the obtained sequences clustered with those of R. hoogstraalii from Italy (KY418024 and KY418025) and Pakistan (OQ160792) (Figure 2).In the phylogenetic tree of ompB, the obtained sequence clustered with R. hoogstraalii from Georgia (EF629536 and MH717095) and China (MZ367030) (Figure 3).

Discussion
Ticks cause economic losses to the livestock industry and transmit various pathogens, including SFG Rickettsia spp., to humans and wild and domestic animals.There is a huge variety of Rickettsia spp., of which few have been proven to be zoonotic [5].Rickettsia

Discussion
Ticks cause economic losses to the livestock industry and transmit various pathogens, including SFG Rickettsia spp., to humans and wild and domestic animals.There is a huge variety of Rickettsia spp., of which few have been proven to be zoonotic [5].Rickettsia hoogstraalii is a member of the SFG Rickettsia, but there are no available reports on its pathogenicity in vertebrates [33].The diagnosis of Rickettsia in ticks, not only for the identification of infected ticks but also for the assessment of exposure risk to humans, is important [66,67].Previously, various studies have documented the occurrence of diverse Rickettsia spp. in various ticks infesting different hosts in Pakistan, but there is a lack of information regarding the occurrence and genetic characterization of R. hoogstraalii.To fill this gap, we detected and genetically characterized R. hoogstraalii in hard ticks infesting livestock.The collected ticks were taxonomically identified as Rh.microplus, Rh. turanicus, Hy. anatolicum, Ha. bispinosa, and Ha.sulcata and screened for the detection of rickettsial DNA.Among these, R. hoogstraalii was identified in Hy. anatolicum and Ha.sulcata based on gltA and ompB sequences for the first time in Pakistan.
Tick species such as Rh.microplus, Rh. turanicus, Rh. sanguienus, Rh. haemaphysaloides, Hy. anatolicum, Hy. dromedarii, Ha. sulcata, Ha. bispinosa, Ha. kashmirensis, Ha. cornupunctata, and Ha.montgomeryi have been found to infect different livestock hosts (especially cattle, goats, and sheep) in different regions of Pakistan [2,8,15,44,[48][49][50]. Rhipicephalus microplus and Hy.anatolicum, which are the most prevalent in the area, were found most frequently [2,8].The environmental conditions in the different survey districts varied from one another.The annual mean temperature of study areas such as Lakki Marwat, Bannu, Bajaur, Upper-Dir, Karak, and Buner were 30-42 • C and 4-17 • C, recorded in the summer and winter, respectively, (worldweatheronline.com: accessed on 1 March 2023).High summer temperatures in the target area were correlated with increased tick infestation compared to winter; therefore, high tick infestation was noted during summer.Moreover, several tick species have been found to exhibit low incidences of infestation as a result of lower temperatures in certain districts.These results are consistent with previous regional reports [2,68,69].
Hyalomma anatolicum and Ha.sulcata infesting cattle, goats, and sheep were found positive for R. hoogstraalii.Previously, R. hoogstraalii has been detected in various tick genera, including Haemaphysalis, Rhipicephalus, Argas, Dermacentor, Carios, Ixodes, and Africaniella, infesting goats, sheep, cattle, cows, mouflons, lizards, bat, dog, and birds in different countries .Recently, R. hoogstraalii was reported to contain a short fragment of gltA in Ha. montgomeryi infesting goats and sheep in Pakistan [15].Rickettsia hoogstraalii has been detected in all life stages of different ticks, such as adult females, males, larvae, and nymphs [15,28,33,34].So far, information about the detection of R. hoogstraalii in Hy. anatolicum and Ha.sulcata ticks infesting livestock hosts such as cattle, goats, and sheep were unavailable.Herein, R. hoogstraalii was detected for the first time in Hy. anatolicum globally and in Ha. sulcata in Pakistan.This study presents the first molecular evidence of R. hoogstraalii in nymphs and adult female ticks of Hy. anatolicum and Ha.sulcata.It also suggests that these ticks may play a possible role in the spread of R. hoogstraalii.R. hoogstraalii DNA was detected both in nymph and adult female ticks.Consequently, there are chances that this Rickettsia was ingested from the blood of the infected hosts.Rickettsia spp.-infected ticks may pose unknown health risks to livestock owners, indicating that other tick species in the area might be potential vectors of these infectious agents [5].
Genetic markers such as gltA, ompA, and ompB have been used to distinguish several Rickettsia spp. at the species level [58,60,70].Thus, the characterization of R. hoogstraalii has been validated through the use of these standard markers [15,16,20].We molecularly detected R. hoogstraalii based on the gltA and ompB sequences.Using these genetic markers, the obtained sequences were closely related to R. hoogstraalii in the Palearctic and Neotropic regions.Additionally, sequence analysis of gltA and ompB showed that R. hoogstraalii is closely related to R. felis, making it a distinct species in the spotted fever group [48].PCRbased detection of this species was also attempted based on the ompA fragment; however, the amplification was unsuccessful, as reported previously [28].Amplification failure is common for ompA, which may be the absence of targeted genes, as demonstrated by the rickettsial transition group, or due to primer mismatch [35,71].There is no available information regarding the pathogenicity of R. hoogstraalii in vertebrate hosts including humans [33], and its zoonotic outcomes are yet to be determined in Pakistan.Further studies are required to elucidate the pathogenicity of R. hoogstraalii in mammals.

Conclusions
This study provides preliminary information regarding the occurrence of R. hoogstraalii in Hyalomma and Haemaphysalis ticks including Hy. anatolicum and Ha.sulcata.To our knowledge, tick-borne R. hoogstraalii was detected and genetically characterized for the first time in globally in Hy. anatolicum and for the first time in Pakistan in Ha. sulcata.These findings indicate that ticks that infest goats, sheep, and cattle ultimately pose unknown health risks to livestock holders who mostly share living habitats.These findings enhance our understanding of the occurrence of R. hoogstraalii in ticks parasitizing livestock in Pakistan.To prevent zoonotic outcomes, it is important to examine the vector potential of different ticks for other rickettsial pathogens.

Figure 1 .
Figure 1.Map showing the locations (black triangles) of tick collection in specific districts of Khyber Pakhtunkhwa (KP), Pakistan.

Figure 1 .
Figure 1.Map showing the locations (black triangles) of tick collection in specific districts of Khyber Pakhtunkhwa (KP), Pakistan.

Figure 2 .
Figure 2. A maximum likelihood phylogenetic tree of R. hoogstraalii was constructed based on the gltA fragment.R. canadensis was used as an outgroup.The bootstrap values (1000-replication) are shown at each node.The obtained sequence (OR392758) of the present study is marked in bold and underlined font.

Figure 2 .
Figure 2. A maximum likelihood phylogenetic tree of R. hoogstraalii was constructed based on the gltA fragment.R. canadensis was used as an outgroup.The bootstrap values (1000-replication) are shown at each node.The obtained sequence (OR392758) of the present study is marked in bold and underlined font.

Figure 3 .
Figure 3.A maximum likelihood phylogenetic tree of R. hoogstraalii was constructed based on the ompB partial fragment.R. prowazekii was used as the outgroup.Bootstrap values (1000-replication) are shown at each node.The obtained sequence (OR392759) of the present study is marked in bold and underlined font.

Figure 3 .
Figure 3.A maximum likelihood phylogenetic tree of R. hoogstraalii was constructed based on the ompB partial fragment.R. prowazekii was used as the outgroup.Bootstrap values (1000-replication) are shown at each node.The obtained sequence (OR392759) of the present study is marked in bold and underlined font.

Table 1 .
List of primers used in the present study for the amplification of Rickettsia spp.

Table 3 .
Information regarding ticks, hosts, locality, and the molecular detection of R. hoogstraalii in this study.