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Tick-borne bacteria of the genera Rickettsia and Coxiella cause several emerging veterinary and human infectious diseases. Ticks of the genus Hyalomma are medically important vectors due to their potential role in the transmission of pathogens to vertebrate hosts. There is an inadequate knowledge on tick-borne Rickettsia spp. and Coxiella spp. in ticks infesting transhumant camels in Pakistan. In this study, we conducted a molecular survey for screening of Rickettsia spp. and Coxiella spp. in ticks infesting camels. Seven hard tick species including Hyalomma dromedarii, Hyalomma anatolicum, Hyalomma scupense, Hyalomma isaaci, Hyalomma turanicum, Hyalomma asiaticum, and Rhipicephalus sanguineus s.l were confirmed on camels in three distinct physiographic regions of Khyber Pakhtunkhwa, Pakistan. A subset of morphologically identified ticks were subjected to molecular assays for the genetic characterization of ticks and the detection and genetic characterization of Rickettsia and Coxiella species using standard genetic markers. Ticks screened for pathogens resulted in the detection of Rickettsia aeschlimannii and Candidatus Rickettsia shennongii and Coxiella burnetii. The molecular analysis further reveals the presences of an undetermined Rickettsia aeschlimannii-like species, that is making a distinct phylogenetic clade with R. aeschlimannii. The detection of pathogens in camel ticks poses potential health hazards as these ticks frequently bites humans. Molecular screening of Rickettsia spp. and Coxiella spp. associated with camel ticks is a preliminary step toward the surveillance of evaluating their zoonotic threats in the region., (© 2024. The Author(s).)

Anaplasma spp. and Rickettsia spp. are intracellular vector-borne pathogens and harbored by a wide range of ticks and vertebrate hosts. Aim of this study was to molecularly characterize Anaplasma spp. and Rickettsia spp. in different ticks collected from livestock hosts in nine districts of Khyber Pakhtunkhwa (KP), Pakistan. In total, 862 ticks were collected from cattle, goats and sheep. Highest tick's infestation was observed on cattle 56.14% (32/57), followed by goats 45.45% (40/88), and sheep 42.05% (45/107). Rhipicephalus microplus (305/862, 35.38%) was predominant species, followed by Haemaphysalis sulcata (243/862, 28.19%), Hyalomma anatolicum (133/862, 15.42%), Haemaphysalis bispinosa (120/862, 13.92%), and Hyalomma kumari (61/862, 7.07%). A subset of 135 ticks were screened for Anaplasma spp. and Rickettsia spp. based on the amplification of partial 16 S rDNA and outer-membrane protein A (ompA) fragments, respectively. In total, 16 ticks (11.85%) were positive for Anaplasma spp. and Rickettsia spp. Obtained 16 S rDNA sequences for Anaplasma spp. detected in Ha. bispinosa and Ha. sulcata showed 99.98% identity with Anaplasma bovis, while other detected in Rh. microplus showed 99.84% identity with Candidatus Anaplasma boleense. Similarly, detected ompA sequence in Ha. sulcata showed 100% identity with Rickettsia sp. and 97.93% with Rickettsia slovaca, and another sequence detected in Rh. microplus showed 100% identity with Candidatus Rickettsia shennongii. In phylogenetic trees, these sequences clustered with corresponding species from Pakistan, China, Turkey, South Korea, South Africa, and Herzegovina. This is the first study reporting detection of A. bovis in Ha. bispinosa and Ha. sulcata, Ca. A. boleense in Rh. microplus collected from goats, and R. slovaca-like in Ha. sulcata. Our results enforce the need for regular surveillance of Rickettsiales in hard ticks infesting livestock in the region., (© 2024. The Author(s).)

Despite high diversity in the Oriental region, ticks of the genus Haemaphysalis have been neglected regarding their genetic data and vector potential. This study aimed to genetically characterize three species of the genus Haemaphysalis : Haemaphysalis cornupunctata , Haemaphysalis kashmirensis, and Haemaphysalis montgomeryi infesting goats and sheep, and Rickettsia spp. associated with these tick species in the Hindu Kush Himalayan range of Pakistan. Altogether, 834 ticks were collected by examining 120 hosts including goats (64/120, 53.3%) and sheep (56/120, 46.6%), in which 86 (71.6%) hosts were found to be tick-infested. The morphologically identified ticks were subjected to DNA extraction and PCR for the amplification of partial 16S rDNA and cox fragments. Rickettsia spp. associated with the collected ticks were detected through the amplification of gltA , ompA and ompB partial fragments. The 16S rDNA of H. cornupunctata and H. montgomeryi showed a maximum identity of 100% with the sequences of the same species, whereas the 16S rDNA of H. kashmirensis showed the highest identity of 93-95% with Haemaphysalis sulcata . The cox sequence of H. montgomeryi displayed 100% identity with the same species. In comparison, the cox sequences of H. cornupunctata and H. kashmirensis showed maximum identities of 87.65-89.22% with Haemaphysalis punctata and 89.34% with H. sulcata , respectively. The gltA sequence of Rickettsia sp. from H. kashmirensis showed the highest identity of 97.89% with Rickettsia conorii subsp. raoultii , while the ompA and ompB fragments from the same DNA samples revealed 100% and 98.16% identity with Rickettsia sp. and " Candidatus Rickettsia longicornii", respectively. Another gltA sequence amplified from H. montgomeryi ticks showed 100% identity with Rickettsia hoogstraalii , while the attempts to amplify ompA and ompB for R. hoogstraalii were unsuccessful. In the phylogenetic tree, the 16S rDNA of H. cornupunctata clustered with the corresponding species while its cox clustered with H. punctata . Both 16S rDNA and cox sequences of H. kashmirensis clustered with H. sulcata . The gltA sequence of Rickettsia sp. was clustered individually in the spotted fever (SF) group of Rickettsia , while the gltA sequence of R. hoogstraalii was clustered with the same species in the transition group of Rickettsia . In the SF group, the rickettsial ompA and ompB sequence clustered with undetermined Rickettsia sp. and " Candidatus Rickettsia longicornii", respectively. This is the earliest study regarding the genetic characterization of H. kashmirensis . This study indicated that ticks belong to the genus Haemaphysalis have the potential of harboring and/or transmitting Rickettsia spp. in the region.

There is limited information on the occurrence of Hyalomma turanicum and Hyalomma asiaticum ticks, as well as associated Rickettsia, Anaplasma, and Ehrlichia species in Pakistan. Addressing this knowledge gap, the current study aimed at morphomolecular confirmation of these ticks and molecular assessment of associated Rickettsiales bacteria (Rickettsia, Anaplasma , and Ehrlichia spp.) in Balochistan, Pakistan. A total of 314 ticks were collected from 74 of 117 (63.2%) hosts, including 41 of 74 (55.4%) goats and 33 of 74 (44.5%) sheep. Subsequently, a subset of microscopically identified ticks was subjected to DNA extraction and PCR to amplify 16S rDNA and cox1 fragments. Additionally, gltA , ompA, and ompB fragments were targeted for Rickettsia spp. and 16S rDNA fragments for both Anaplasma and Ehrlichia spp. The 16S rDNA and cox1 sequences of Hy. turanicum demonstrated 100% identity with those of the same species previously reported from Pakistan. The 16S rDNA and cox1 sequences of Hy. asiaticum exhibited 99.52 and 100% identities, respectively, with corresponding species reported from China, Kazakhstan, and Turkey. The gltA, ompA, and ompB fragments associated with Hy. turanicum showed 100% identities with Rickettsia aeschlimannii reported from Egypt, Italy, Kazakhstan, Kenya, Pakistan, and Senegal. The 16S rDNA sequences of Anaplasma sp. and Ehrlichia sp. associated with both Hy. asiaticum and Hy. turanicum exhibited 99.67 and 100% identities with unknown Anaplasma sp. and Ehrlichia sp. reported from Morocco and Pakistan, respectively. In the 16S rDNA and cox1 phylogenetic trees of ticks, Hy. turanicum and Hy. asiaticum from the current study clustered with their respective species. Similarly, in gltA, ompA, and ompB phylogenetic trees of Rickettsia , R. aeschlimannii of the present study clustered with the same species, whereas Anaplasma sp. and Ehrlichia sp. of this study clustered with undetermined Anaplasma spp. and Ehrlichia spp. in the 16S rDNA phylogenetic tree of Anaplasmataceae. Among the DNA samples from the screened ticks, a coinfection rate of R. aeschlimannii , Anaplasma sp., and Ehrlichia sp. (2 of 80, 2.5%) was observed in Hy. turanicum , whereas individual infection rates were noted as follows: R. aeschlimannii (8 of 80, 10%), Anaplasma sp. (5 of 80, 6.3%), and Ehrlichia sp. (5 of 80, 6.3%). This study marks the first record of molecular characterization of Hy. turanicum and Hy. asiaticum as well as the detection of associated R. aeschlimannii , Anaplasma sp., and Ehrlichia sp. in Balochistan, Pakistan. [ABSTRACT FROM AUTHOR]

Simple Summary: Dermacentor ticks have a wide geographic range with an uneven distribution in the globe. They are not scientifically well known because they survive in hard topographic and harsh climatic regions along with elevated mountains. Many mammals serve as a primary host for Dermacentor ticks, like many other tick species. The present study aimed to provide the first morphological and molecular confirmation of Dermacentor marginatus and its related pathogens like Anaplasma marginale and Rickettsia raoultii in Pakistan. In this study, a total of 26 specimens (19 males and 7 females) were collected from goats and morphologically identified. A subset of 18 specimens were subjected for the molecular characterization of ticks and associated pathogen detection. In the BLAST and phylogenetic analyses, D. marginatus and their associated pathogen sequences showed close resemblance with their corresponding species. In the present study, we reported the first genetic characterization of D. marginatus and associated A. marginale and R. raoultii in Pakistan. Due to the difficult access and harsh climate, it is important to investigate the ticks and related pathogens in the northern parts of Pakistan due to their zoonotic threats. Ticks of the genus Dermacentor Koch, 1844 (Acari: Ixodidae) are poorly known systematically due to their habitation in harsh topographic environments and high mountains. Dermacentor ticks are diversely distributed in the Palearctic, Nearctic, and Oriental regions. There is no available information on the occurrence of Dermacentor marginatus in Pakistan; thus, the current investigation aimed the first morphological and molecular confirmation of this species and associated Anaplasma marginale and Rickettsia raoultii. Ticks were collected from goats (Capra hircus) and morphologically identified. Genomic DNA was extracted from 18/26 (69.23%) tick specimens, including 11 males and 7 females (1 unfed and 6 fed females). Extracted DNA was subjected to PCR for the amplification of genetic markers like 16S rDNA and cox1 for ticks, 16S rDNA for Anaplasma spp., and gltA and ompB for Rickettsia spp. A total of 26 D. marginatus ticks composed of 19 males (73.07%) and 7 females (26.9%) [1 (3.84%) unfed and 6 (23.07%) fed females] were collected from goats. According to amplicons via BLAST analysis, the 16S rDNA sequence showed 97.28–98.85% identity and the cox1 sequence showed 95.82–98.03% identity with D. marginatus. Additionally, the 16S rDNA sequence for Anaplasma sp. was detected in D. marginatus that showed 100% identity with Anaplasma marginale. Rickettsial gltA and ompB sequences for Rickettsia sp. showed 100% identity with Rickettsia raoultii. In phylogenetic analysis, ticks' 16S rDNA and cox1 sequences clustered with the same species. In phylogenetic analysis, A. marginale based on 16 rDNA clustered with A. marginale, while gltA and ompB sequences clustered with R. raoultii. This is the first study on the genetic characterization of D. marginatus and associated A. marginale and R. raoultii in Pakistan. The northern areas of Pakistan, which need to be explored in terms of ticks and associated pathogens due to their zoonotic threats, have been neglected due to the inaccessible climatic conditions. [ABSTRACT FROM AUTHOR]

Simple Summary: Ticks are chelicerate arthropods that feed on blood and infest all vertebrates except fish and transmit different disease-causing agents including Rickettsia spp. to domestic and wild animals as well as humans. In the present study, we aimed to molecularly screen and genetically characterize Rickettsia spp. in various tick species infesting camels, sheep, and goats from five districts (Kohat, Dera Ismail Khan, Lower Dir, Bajaur, and Mansehra) of Khyber Pakhtunkhwa province, Pakistan. A total of 8/148 (5.4%) ticks, including four Hyalomma turanicum, two Haemaphysalis cornupunctata, one Haemaphysalis montgomeryi, and one Haemaphysalis bispinosa, were found positive for Rickettsia sp. The phylogenetic analysis of detected Rickettsia sp. based on three genetic markers (gltA, ompA, and ompB) revealed 100% identity with Rickettsia raoultii, clustered with its corresponding species reported in China, Russia, USA, Turkey, Denmark, Austria, Italy, and France. Further comprehensive studies on molecular and serosurveillance of various Rickettsia spp. in different ticks should be conducted in the region to understand the zoonotic threats due to these pathogens. Ticks are hematophagous ectoparasites that transmit different pathogens such as Rickettsia spp. to domestic and wild animals as well as humans. Genetic characterizations of Rickettsia spp. from different regions of Pakistan are mostly based on one or two genetic markers and are confined to small sampling areas and limited host ranges. Therefore, this study aimed to molecularly screen and genetically characterize Rickettsia spp. in various tick species infesting camels, sheep, and goats. All the collected tick specimens were morphologically identified, and randomly selected tick species (148) were screened molecularly for the detection of Rickettsia spp. by amplifying three rickettsial DNA fragments, namely, the citrate-synthase gene (gltA), outer-membrane protein A (ompA), and outer-membrane protein B (ompB). After examining 261 hosts, 161 (61.7%) hosts were found infested by 564 ticks, including 287 (50.9%) nymphs, 171 (30.3%) females, and 106 (18.8%) males in five districts (Kohat, Dera Ismail Khan, Lower Dir, Bajaur, and Mansehra). The highest occurrence was noted for Hyalomma dromedarii (number = 72, 12.8%), followed by Haemaphysalis sulcata (n = 70, 12.4%), Rhipicephalus turanicus (n = 64, 11.3%), Rhipicephalus microplus (n = 55, 9.7%), Haemaphysalis cornupunctata (n = 49, 8.7%), Hyalomma turanicum (n = 48, 8.5%), Hyalomma isaaci (n = 45, 8.0%), Haemaphysalis montgomeryi (n = 44, 7.8%), Hyalomma anatolicum (n = 42, 7.5%), Haemaphysalis bispinosa (n = 38, 6.7%), and Rhipicephalus haemaphysaloides (n = 37, 6.6%). A subset of 148 ticks were tested, in which eight (5.4%) ticks, including four Hy. turanicum, two Ha. cornupunctata, one Ha. montgomeryi, and one Ha. bispinosa, were found positive for Rickettsia sp. The gltA, ompA, and ompB sequences revealed 100% identity and were phylogenetically clustered with Rickettsia raoultii reported in China, Russia, USA, Turkey, Denmark, Austria, Italy, and France. Additionally, various reports on R. raoultii from Palearctic and Oriental regions were summarized in this study. To the best of our knowledge, this is the first report regarding genetic characterization and phylogenetic analysis of R. raoultii from Pakistan. Further studies to investigate the association between Rickettsia spp. and ticks should be encouraged to apprise effective management of zoonotic consequences. [ABSTRACT FROM AUTHOR]

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. 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. [ABSTRACT FROM AUTHOR]

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. 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. [ABSTRACT FROM AUTHOR]

Ixodid ticks are responsible for the transmission of various intracellular bacteria, such as the Rickettsia species. Little Information is available about the genetic characterization and epidemiology of Rickettsia spp. The current study was designed to assess the tick species infesting various livestock hosts and the associated Rickettsia spp. in Pakistan. Ticks were collected from different livestock hosts (equids, cattle, buffaloes, sheep, goats, and camels); morphologically identified; and screened for the genetic characterization of Rickettsia spp. by the amplification of partial fragments of the gltA, ompA and ompB genes. Altogether, 707 ticks were collected from 373 infested hosts out of 575 observed hosts. The infested hosts comprised 105 cattle, 71 buffaloes, 70 sheep, 60 goats, 34 camels, and 33 equids. The overall occurrence of Rickettsia spp. was 7.6% (25/330) in the tested ticks. Rickettsia DNA was detected in Rhipicephalus haemaphysaloides (9/50, 18.0%), followed by Rhipicephalus turanicus (13/99, 13.1%), Haemaphysalis cornupunctata (1/18, 5.5%), and Rhipicephalus microplus (2/49, 4.1%); however, no rickettsial DNA was detected in Hyalomma anatolicum (71), Hyalomma dromedarii (35), and Haemaphysalis sulcata (8). Two Rickettsia agents were identified based on partial gltA, ompA, and ompB DNA sequences. The Rickettsia species detected in Rh. haemaphysaloides, Rh. turanicus, and Rh. microplus showed 99–100% identity with Rickettsia sp. and Candidatus Rickettsia shennongii, and in the phylogenetic trees clustered with the corresponding Rickettsia spp. The Rickettsia species detected in Rh. haemaphysaloides, Rh. turanicus, Rh. microplus, and Ha. cornupunctata showed 100% identity with R. massiliae, and in the phylogenetic trees it was clustered with the same species. Candidatus R. shennongii was characterized for the first time in Rh. haemaphysaloides, Rh. turanicus, and Rh. microplus. The presence of SFG Rickettsia spp., including the human pathogen R. massiliae, indicates a zoonotic risk in the study region, thus stressing the need for regular surveillance. [ABSTRACT FROM AUTHOR]

Simple Summary: Ticks are blood-feeding ectoparasites that transmit life-threatening pathogens to humans and animals. Only 10% of all identified tick species have been screened for different tick-borne pathogens. Hyalomma ticks are associated with a wide range of pathogens including Theileria species, Babesia species, Anaplasma species, Ehrlichia species, and Rickettsia species. Moreover, ticks of genus Hyalomma are vectors for the Crimean-Congo hemorrhagic fever (CCHF), a serious threat endemic in Pakistan. In Pakistan, different tick species have been found positive for rickettsial agents; however, Hyalomma kumari ticks have never been investigated for any potential pathogens. In this work, H. kumari ticks were collected from goats and sheep, and morphologically and molecularly identified using different genetic markers. The identified ticks were screened for rickettsial agents using genetic markers that resulted in the detection of Rickettsia conorii for the first time in this tick. A proper surveillance program should be designed to effectively avoid any zoonotic consequences associated with these rickettsial pathogens. As a vector of wide range of pathogenic agents, ticks pose health threats to wild and domestic animals, and humans. Information is unavailable about the prevalence and spatial survey of Hyalomma kumari ticks and associated Rickettsia spp. in Pakistan. Concerning this knowledge gap, the present study aimed to molecularly detect Rickettsia species associated with H. kumari infesting small ruminants in Khyber Pakhtunkhwa (KP), Pakistan. A total of 409 H. kumari ticks were collected from 163/295 infested hosts with an infestation rate of 55.25%. A total of 204 females, 158 males, and 47 nymphs were collected. Goats were heavily infested by 224 ticks having an infestation rate of 58.33% (98/168), whereas sheep were infested by 185 ticks having a lesser infestation rate of 51.18% (65/127). Genomic DNA extracted from ticks was used for the amplification of tick (cox I, 16S rRNA, ITS-2) species and Rickettsia (gltA, ompA, and ompB) partial genes. Eighty-three ticks were subjected to PCR, and 8/83 (9.6%) were found positive for rickettsial agents. The cox I and 16S rRNA sequences of H. kumari showed 98.90–99.74% identity with H. kumari sequences reported from Pakistan, and phylogenetically clustered to the corresponding species reported from Pakistan and India. The obtained rickettsial gltA, ompA, and ompB sequences showed 100% identity with Rickettsia sp. of the Rickettsia conorii reported from Pakistan. In the phylogenetic trees, rickettsial sequences clustered with uncharacterized Rickettsia sp. from Pakistan and R. conorii from Israel, Russia, South Africa, and India. The present molecular based detection of H. kumari-associated R. conorii will facilitate effective surveillance in the region. [ABSTRACT FROM AUTHOR]