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Molecular detection of Anaplasma bovis, Candidatus Anaplasma boleense and Rickettsia spp. in ticks infesting small ruminants

BMC Veterinary Research volume 20, Article number: 408 (2024) Cite this article

Abstract

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.

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Introduction

Ticks are hematophagous ectoparasites, distributed in different ecoregions [1], where they parasitized terrestrial, semi-terrestrial, domestic and wild animals [2,3,4]. They are known to serve as reservoir and vector for wide varieties of pathogens such as viruses, protozoans (Babesia, Hepatozoon, and Theileria), and bacteria (Anaplasma, Borrelia, Coxiella, Ehrlichia, and Rickettsia) infecting different vertebrate hosts [5]. Ticks have great public concern and posing threats to economy [3, 6, 7].

Ticks are capable of causing life-threatening infections in animals and humans [8]. Globalization, geographic expansion, and changes in climate patterns have increase the dispersal of the hosts vectors of these Rickettsiales pathogens [9]. Two genera, Anaplasma and Rickettsia, consist of medically and veterinary important pathogens posing significant public and veterinary health burden [7, 10, 11]). Based on reliable genetic markers, there have been significant advancement in our knowledge about the diversity of novel tick-borne Rickettsial pathogens, while previously certain species that were considered non-pathogenic have been linked now with different animal and human infections [12].

Anaplasma is a diverse genus comprised of species with varied pathogenicity [13], and many hard ticks such as Rhipicephalus, Ixodes, Haemaphysalis, Dermacentor, and Amblyomma play a vector role in its transmission [13,14,15,16]. Previously, molecular based description revealed the presence of Anaplasma capra, Anaplasma marginale, and Anaplasma platys in Pakistan [3, 17,18,19]. Among Anaplasma spp., Anaplasma bovis was first described in 1931 [20] that has been detected in Rhipicephalus haemaphysaloides, Ixodes turdus, Haemaphysalis coccina, Haemaphysalis longicornis, Haemaphysalis punctata, Haemaphysalis danieli, and Hyalomma asiaticum, collected from domestic and wild animals [21,22,23,24]. The zoonotic potential of A. bovis has been confirmed in a patient blood in China [15]. Candidatus Anaplasma boleense was reported in Hy. asiaticum in China [25], and then in Rhipicephalus microplus feeding on cattle [4], buffaloes and deer [6, 25]. Pathogenicity, vector and non-vector transmission of Ca. A. boleense is still unknown [13].

The genus Rickettsia consists of mostly intracellular bacteria associated with arthropods [26, 27]. The geographic spread of known and novel Rickettsia spp. with an increasing rate to new hosts and regions is alarming [28]. Rickettsia species were classified into the spotted fever group (SFG), typhus group (TG), and ancestral group (AG) [29, 30]. Among these, SFG is the most diverse group having both pathogenic and undetermined Rickettsia spp., in which Rickettsia massiliae, Rickettsia roulti, Rickettsia conorii, Rickettsia hoogstraalii, Rickettsia aeschlimannii, and Candidatus Rickettsia shennongii have been reported in Pakistan [2, 18, 31,32,33]. Candidatus R. shennongii was detected in ticks including Rhipicephalus turanicus, Rh. haemaphysaloides, Rh. microplus, Rhipicephalus sanguineus [2, 18], and the pathogenic potential of Ca. R. shennongii in humans is still undetermined [15]. Species diversity in genus Rickettsia expand continuously and recently novel species have been identified based on reliable genetic markers [12, 34].

In Pakistan the presence of suitable habitat and landscape varieties support tick infestations on vertebrate hosts that act as reservoir for numerous tick-borne pathogens [31, 35, 36]. Keeping in view the importance of these pathogens, the present study aimed to molecularly examine ticks infesting cattle, goats, and sheep for the detection of Anaplasma and Rickettsia species in selected districts of Khyber Pakhtunkhwa (KP), Pakistan.

Materials and methods

Study area

This study was conducted in nine districts of KP province, Pakistan: Mardan (34.194697° N, 72.050557° E), Peshawar (34.039825° N, 71.566832° E), Nowshera (34.0105° N, 71.9876° E), Abbottabad (34.1688° N, 73.2215° E), Mansehra (34.3313° N, 73.1980° E), Haripur (33.9946° N, 72.9106° E), Dir Lower (34.9161° N, 92 71.8097° E), Dir Upper (35°12′30.06 ° N, 71°5231.22° E), and Swat (34.8065° N, 27.3548° E), from May 2022 to June 2023. This province situated in the northwest of Pakistan has climatic conditions suitable for different ticks and tick-borne pathogens, having arid plains, humid hilly areas with varied altitude and seasons. The geographic coordinates of each collection site in study area were collected from Google Maps using Global Positioning System to design the distribution map through ArcGIS 10.3.1 (Fig. 1).

Fig. 1
figure 1

Map showing tick’s collection sites in selected district of Khyber Pakhtunkhwa, Pakistan

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Ticks collections, preservation and identification

Ticks were collected during May 2022 to June 2023 from cattle, goats, and sheep in 9 districts (Fig. 1). In the survey region, ticks were collected from each infested host once, in pastures, farms, open field, and free roaming animals. To carry further molecular work, after washing with distilled water, ticks were stored in 1.5 ml Eppendorf tubes filled with 100% ethanol. Ticks were morphologically identified, using a stereo zoom microscope (SZ61, Olympus, Tokyo, Japan) with standard taxonomic keys [37,38,39,40].

DNA extraction and polymerase chain reaction

Total 135 ticks (one male, one female, and one nymph per species from each district) were selected randomly and subjected individually for DNA extraction, using phenol-chloroform method [41]. Extracted genomic DNA was quantified using NanoDrop (NanoQ, Optizen, Daejeon, Republic of Korea). To amplify the 16 S rDNA fragment of Anaplasma spp. and outer membrane protein A (ompA) of Rickettsia spp., genomic DNA was subjected to conventional PCR (GE-96G, BIOER, Hangzhou, China). The PCR was carried out in Thermal Cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA), containing a reaction mixture of 25 µL comprised of 13 µL Dream Taq PCR MasterMix (2×) (Thermo Fisher Scientific, Inc., Waltham, MA, USA); 2 µL of primers (1 µL each forward and reverse) (Table 1), 2 µL (50 ng/µL) genomic DNA, and 8 µL nuclease-free water. The thermo-cycling conditions for 16 S rRNA and ompA genes were used as described previously [6, 34, 42, 43]. Nuclease free water was taken as a negative control, while DNA of A. marginale and R. massiliae were used as a positive control for Anaplasma spp. and Rickettsia spp., respectively. The amplified PCR products were run on agarose gel (1.5%) and observed through gel documentation (BioDoc-It™ Imaging Systems, UVP, LLC, Upland, CA, USA).

Table 1 Primers used for the detection of Anaplasma and Rickettsia DNA in ticks
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DNA sequencing and phylogenetic analysis

The amplified PCR products of 16 S rDNA and ompA fragments were sent for bidirectional sequencing (Macrogen, Inc., Seoul, Republic of Korea) using Sanger sequencing method. The obtained sequences were trimmed using SeqMan v. 5 (DNASTAR, lnc., Madison/WI, US) to remove poor reading and primer region sequences and subjected to Basic Local Alignment Search Tool (BLAST) [45], at the National Center for Biotechnology Information (NCBI). For subsequent phylogenetic trees construction, homologous sequences and an appropriate outgroup were downloaded from GenBank and aligned with obtained sequences using BioEdit alignment editor v 7.0.5 using ClustalW Multiple alignment [46]. Phylogenetic trees were constructed in Molecular Evolutionary Genetics Analysis (MEGA-X), using Maximum Likelihood method with Tamura-Nei model [47], and 1,000 bootstrap replicates.

Ethical considerations

The permission to execute, an ethical approval for the study was given by the Advance Studies Research Board (ASRB: Dir/A&R/AWKUM/2022/9396), Abdul Wali Khan University, Mardan KP, Pakistan. After explaining the importance of the study verbal consent was obtained from each of the cattle owners.

Statistical analysis

The recorded data such as the number of animals hosts, collected ticks, their different life stages in nine districts, and associated pathogens was organized in spread sheet and analyzed for descriptive statistics (Microsoft Excel v. 2016, Microsoft 365®). Host’s infestation was calculated by dividing infested hosts by total examined hosts. The differences were considered significant at p-value < 0.05 with 95% confidence interval (CI) by applying chi-square test among variable using the GraphPad Prism v. 8 (Inc., San Diego, CA, USA).

Results

Tick and hosts data

Overall, 862 ticks were collected from 117 (46.42%) out of 252 examined hosts. The infested hosts were 32 cattle, 40 goats, and 45 sheep. Highest infestation was observed in district Peshawar (21/32, 65.62%), followed by Mardan (17/30, 56.66%), Nowshera (12/24, 50%), Haripur (14/31, 45.16%), Abbottabad (13/29, 44.82%), Dir Lower (14/34, 41.17%), Dir Upper (9/23, 39.13%), Mansehra (8/22, 36.36%), and Swat (9/27, 33.33%). Based on morphology, ticks belonging to three genera (Haemaphysalis, Rhipicephalus, Hyalomma) were identified in which 235 (27.27%) were nymphs, 348 (40.37%) were females and 279 (32.36%) were males. Among identified ticks, highest infestation was observed for Rh. microplus (305/862, 35.38%), followed by Ha. sulcata (243/862, 28.19%), Hyalomma anatolicum (133/862, 15.42%), Haemaphysalis bispinosa (120/862, 13.92%), and Hyalomma kumari (61/862, 7.07%). Rh. microplus, Ha. sulcata and Hy. anatolicum were found on goats, sheep, and cattle, while Ha. bispinosa and Hy. kumari were found only on goats and sheep (Table 2).

Table 2 Distribution, prevalence and detection rate of Anaplasma spp. and Rickettsia spp. in different ticks collected from different hosts
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Detection of Anaplasma spp. and Rickettsia spp

Out of 862 collected ticks (male, female, nymph), 135 were analyzed for the molecular detection of Anaplasma spp. and Rickettsia spp. Overall detection rate for both Anaplasma spp. and Rickettsia spp. was 11.85% (16/135). Among the five screened tick species, highest infection rate was noted for Rh. microplus (12/135, 8.88%) followed by Ha. sulcata (3/135, 2.22%), and Ha. bispinosa (1/135, 0.74%), while no positive case was found in Hy. anatolicum and Hy. kumari. The highest detection rate was found in district Haripur (4/135, 2.96%) followed by Mardan (3/135, 2.22%), Peshawar (2/200, 1.48%), Mansehra (2/200, 1.48%), Dir Upper (2/200, 1.48%), Dir lower (2/200, 1.48%), and Abbottabad (1/135, 0.74%) (Table 2).

Sequencing and phylogenetic analysis

Total 16 DNA sequences (12 for ompA and 4 for 16 S rDNA) were obtained from different ticks. A. bovis was detected in Ha. bispinosa and Ha. sulcata collected from goats, and Ca. A. boleense in Rh. microplus collected from goats and sheep. Based on 16 S rDNA, two fragments were amplified using two set of primers, EHR16SD-EHR16SR and Eh-Out1 forward -Eh-Out2 reverse. In the case of EHR16SF, EHR16SR, a final 280 bp contig was obtained for A. bovis which was unsuccessfully amplified for Ca. A. boleense. In case of Eh-Out1-Eh-Out2, a final 622 bp contig was obtained for Ca. A. boleense which was unsuccessfully amplified for A. bovis. Therefore, two separate phylogenic trees were constructed for Anaplasma spp.

Based on ompA, two Rickettsia spp. were detected, R. slovaca-like and Ca. R. shennongii in Ha. sulcata and Rh. microplus, respectively, collected from sheep (Table 2). Identical sequences for 16 S rDNA and ompA were considered as a single consensus sequence. The BLAST analysis of 16 S rDNA sequences obtained from Ha. bispinosa and Ha. sulcata showed maximum identity 99.98% with A. bovis reported in South Korea (MK028574) and China (MF289479). Another Anaplasma sequence obtained from Rh. microplus showed maximum 99.84% identity with the Ca. A. boleense reported from South Africa (MK814450) and China (KU586169). Similarly, the BLAST analysis of Rickettsia sp. detected in Ha. sulcata showed 100% identity with Rickettsia sp. reported from Pakistan (MN548866) and 97.93% with R. slovaca reported from Turkey (MK726320), China (KX506733), Herzegovina (KT805229), and Iran (MW779484). Another Rickettsia species detected in Rh. microplus showed maximum 100% identity with Ca. R. shennongii reported from Pakistan (OP820485 and OQ632789) and 99.07% from China (OL856104).

In phylogenetic tree, obtained A. bovis sequence clustered with same species reported from South Korea and China (Fig. 2), while that of Ca. A. boleense clustered with same species reported from China and South Africa (Fig. 3). Similarly, the obtained sequences for R. slovaca-like grouped in a sister clade with Rickettsia sp. reported from Pakistan and R. slovaca reported from Turkey, and Herzegovina, while that of Ca. R. shennongii grouped with same species reported from China and Pakistan (Fig. 4). The obtained sequences were deposited to GenBank under the accession number PP177470, PP177468, PP194444, PP194443.

Fig. 2
figure 2

Phylogenetic tree based on 16 S rDNA sequence for Anaplasma bovis using Maximum likelihood method (Tamura–Nei model). Anaplasma camelii was taken as the outgroup, using supporting values (1000 replicons) at each node. The sequence (PP177470) obtained in the present study are shown by underline fonts

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Fig. 3
figure 3

Phylogenetic tree for Anaplasma spp. based on 16 S rDNA sequences, using Maximum likelihood method (Tamura–Nei model). The Anaplasma camelii, Anaplasma platys and Anaplasma odocoilei was taken as the outgroup. The 1000 bootstraping values were followed. The sequence (PP177468) obtained in the present study are shown by underline fonts

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Fig. 4
figure 4

Phylogenetic tree based on ompA sequences of Rickettsia spp. using Maximum likelihood method (Tamura–Nei model). The Rickettsia akari and Rickettsia tamurae was taken as the outgroup, using supporting values (1000 replicons) at each node. The sequences (PP194443, PP194444) obtained in the present study are shown by underline fonts

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Discussion

Ticks are potential vectors of important pathogens, posing threats to public and veterinary health. The geo-climatic conditions of Pakistan are suitable for tick’s survival and propagation of associated pathogens [18, 48, 49]. In Pakistan, studies have been conducted regarding ticks and tick-borne pathogens however, unidentified diversity of Anaplasma spp. and Rickettsia spp. have been often neglected. Therefore, using suitable genetic markers, insights into this knowledge gap is needed to report unidentified tick-borne pathogens. The present study reported the detection of two Anaplasma spp. (A. bovis and Ca. A. boleense) and two SFG Rickettsia spp. (R. slovaca-like and Ca. R. shennongii) in various ticks from different regions.

Cattle were found infested by Rh. microplus, Ha. sulcata, and Hy. anatolicum. In previous studies in the region, Rh. microplus, Hy. anatolicum, Hyalomma dromedarii, Hyalomma scupense, Hyalomma isaaci, Rh. haemaphysaloides, and Haemaphysalis montgomeryi infestation have been reported on cattle [18, 33, 35, 36, 50. The infestation of cattle, goats, and sheep with Rh. microplus may be due to habitat sharing and their abundance in the region, while broad infestation ranges of Ha. sulcata (cattle, goats and sheep) and Hy. kumari (goats and sheep), may be linked to the completion of their lifecycle on three hosts. Goats and sheep have been reported infested by ticks including Rh. microplus, Rh. turanicus, Rh. sanguineus, Rh. haemaphysaloides, Ha. sulcata, Ha. bispinosa, Ha. cornupunctata, Haemaphysalis kashmirensis, Ha. danieli, Ha. montgomeryi, Hy. kumari, Hy. anatolicum, and Hy. dromedarii in different region of Pakistan [32, 35, 36, 49,50,51,52]. Hyalomma kumari has been found to infest nearly all domestic animals in the study areas where rainfall is high [32].

This study report first time detection of A. bovis in Ha. sulcata and Ha. bispinosa infesting goats. Previously, A. bovis has also been detected in Rh. sanguineus, Ha. longicornis, Rh. haemaphysaloides, Rh. turanicus, Ha. qinghaiensis, Dermacentor abaensis, and Ha. punctata in China, Israel, Taiwan, Korea, and Spain [21,22,23, 53, 54], while in cattle blood in Pakistan [55]. Previously, A. bovis has been reported in tick belonging to the genus Haemaphysalis from Asia and America [56]. Herein, the association of A. bovis with two new Haemaphysalis tick species infesting goats, suggest zoonotic threats to livestock holders. The detection of A. bovis in ticks infesting different host emphasized further insight to screen other tick species for this pathogen in the region.

Candidatus Anaplasma boleense was detected for the first time in Rh. microplus infesting goats and sheep in Pakistan. Previously, the detection of this Anaplasma sp. in tick genra such as Haemaphysalis, Rhipicephalus, and Hyalomma, and blood samples from domestic animals [13, 15, 57], indicate the role of these ticks as possible vectors, showing the broad hosts range and geographical distribution of Ca. A. boleense. In the survey region, the detection of new Anaplasma spp. predict their broad diversity than expected and highlight the importance of one-health approach to establish effective surveillance programs particularly about vector, hosts, pathogenicity, and geographical distribution. Ticks infected with Anaplasma spp. may be of significant health concern; for instance, the zoonotic potential of A. phagocytophilium, A. bovis, A. ovis, A. platys, and A. capra has broad medical relevance [24, 58]. Genetic characterization of Anaplasma spp. have been effectively achieved through mitochondrial 16 S rDNA sequences [59]. The obtained sequences of A. bovis and Ca. A. boleense showed maximum identity and in phylogenetic trees clustered with corresponding species. In previous studies Ca. A. boleense was not fully characterized and has been referred as A. phagocytophilium-like 2 and A. phagocytophilium-like clade B in China [60].

Candidatus Rickettsia shennongii was detected in Rh. microplus. This Rickettsia sp. has been reported in Rh. haemaphysaloides in China [15], and recently, in Pakistan in Rh. turanicus, Rh. haemaphysaloides, Rh. sanguineus, and Rh. microplus [2, 18]. The detection of Ca. R. shennongii in expanded geographic range and different tick species infesting livestock provide evidences that the reported ticks have possible role in its spreading thus, further insights are needed to assess its possible zoonotic role. The member of SFG Rickettsia spp., R. slovaca-like was detected for the first time in Pakistan in Ha. sulcata infesting sheep. Further surveillance studies are needed to explore their host and geographic range, and its possible zoonotic outcomes. Phylogenetic analysis of Rickettsia spp. relies on ompA gene that has higher degree of intraspecific variations [61]. Based on ompA gene, to be considered as a valid Rickettsia sp., the obtained sequence similarity should not be less than 98.8% with the most homologous validated species [62]. The obtained ompA sequence of R. slovaca-like showed maximum identity of 97.93% with R. slovaca reported from different countries. The detection of two geno-variants candidate species (Ca. A. boleense and Ca. R. Shennogii) and R. slovaca-like point out the diversity of undescribed Rickettsiales in Pakistan. Thus, regular surveillance is needed to explore emerging rickettsial pathogens, transmission, zoonotic potential, and impacts on hosts.

Conclusion

The effective control management of tick-borne pathogens solely based on understanding their epidemiological surveillance and public awareness. This study reported A. bovis, Ca. A. boleense, and R. slovaca-like for the first time in hard ticks infesting cattle, goat and sheep in Pakistan, posing threats to animal’s and animal-keepers health. The detected pathogens indicate the broad diversity and zoonotic threats associated to tick-borne pathogens in the region. Further attentions should be focused to improve public awareness and assist the policy makers in designing effective control programs to avoid any zoonotic consequences associated with these pathogens.

Data availability

All relevant data is within the manuscript. In this study the obtained sequences have been submitted to GenBank under accession numbers PP177468 for Ca. A. boleense, PP177470 for Anaplasma bovis, PP194443 for Ca. R. shennongii, and PP194444 for R. slovaca-like.

Abbreviations

KP:

Khyber Pakhtunkhwa

ompA :

Outer membrane protein A

BLAST:

Basic Local Alignment Search Tool

SFGR:

Spotted Fever Group Rickettsia

MEGA:

Molecular Evolutionary Genetics Analysis

NCBI:

National Center for Biotechnology Information

PCR:

Polymerase Chain Reaction

rDNA:

Ribosomal DNA

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Acknowledgements

The authors are grateful of Higher Education Commission (HEC) and the Pakistan Science Foundation (PSF), and appreciate their financial support by providing the necessary funding for this research. The researchers supporting project number (RSP2024R494), King Saud University, Riyadh, Saudi Arabia.

Funding

Higher Education Commission (HEC) and the Pakistan Science Foundation (PSF) contributed for financial support by providing the necessary funding for this research. The researchers supporting project number (RSP2024R494), King Saud University, Riyadh, Saudi Arabia.

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Authors and Affiliations

  1. Department of Zoology, Abdul Wali Khan University Mardan, Mardan, 23180, Pakistan

    Zaibullah Khan, Farman Ullah, Shafi Ullah, Gauhar Rehman & Abid Ali

  2. Department of Chemistry, Abdul Wali Khan University Mardan, Mardan, 23180, Pakistan

    Mohammed Ibrahim

  3. Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, 890-0065, Japan

    Momin Khan & Tetsuya Tanaka

  4. Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh, 11451, Saudi Arabia

    Mashal M. Almutairi

  5. King Abdulaziz City for Science and Technology, Riyadh, 12354, Saudi Arabia

    Abdulaziz Alouffi

  6. Laboratory of Animal Microbiology, Graduate School of Agricultural Science/Faculty of Agriculture, Tohoku University, Sendai , 980-8572, Japan

    Tetsuya Tanaka

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  1. Zaibullah Khan
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Contributions

The experimental idea was designed by Abid Ali. Zaibullah Khan, Shafi Ullah, and Farman Ullah collected the tick samples and performed the experiments. Abid Ali, Shafi Ullah, Zaibullah Khan, Mashal M. Almutairi, Abdulaziz Alouffi, Mohammed Ibrahim, Momin Khan, Gauhar Rehman, Tetsuya Tanaka, and Farman Ullah performed different analysis. All authors performed interpretation and refinement. All authors have read and agreed to the published version of the manuscript.

Corresponding author

Correspondence to Abid Ali.

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Ethical approval

The owners of the form were informed before animals handling for tick’s collection. Animals were handled with care to minimize discomfort. All the employed procedure in this research adhere to the ethical guidelines implemented by the Advance Studies Research Board (ASRB: Dir/A&R/AWKUM/2022/9396), Abdul Wali Khan University, Mardan KP, Pakistan.

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Not applicable.

Consent to participate

Before tick’s collection informed consent was obtained from all cattle owners.

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The authors declare no competing interests.

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Khan, Z., Ullah, F., Ullah, S. et al. Molecular detection of Anaplasma bovis, Candidatus Anaplasma boleense and Rickettsia spp. in ticks infesting small ruminants. BMC Vet Res 20, 408 (2024). https://doi.org/10.1186/s12917-024-04259-6

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BMC Veterinary Research

ISSN: 1746-6148

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