The spotted fever group Rickettsia (SFGR) species are mostly transmitted by hard ticks (Ixodidae), besides hard tick species, these are also spread by soft ticks (Argasidae) and other blood-feeding arthropods^1,5,6. The spotted fever group Rickettsia species have worldwide distributions, and these species may lead to infection in domestic and wild animals and humans^1,6. The spotted fever group Rickettsia species often cause non-specific clinical symptoms in hosts, such as eschar, local lymphadenopathy on the tick-bite site, headache, fatigue, pyrexia, muscle pain (localized or generalized), anorexia, and nausea. The clinical symptoms show up in five to seven days after tick-bite. The course of the disease may change according to the species that caused the infection, the time of diagnosis, and the immune status of the patient^1,2,7-9.

Different identification methods (staining, culture, serological, and molecular) are used for the detection of Rickettsia species in the hosts⁵. Staining (Giemsa, Diff-Quik, and acridine orange stains) and culture (cell, embryonated eggs, and lab animals) can be used for the detection of pathogens, but these methods have low sensitivity and specificity, moreover, culture methods take a long time and are laborious ⁵. Serological methods (Weil-Felix, indirect immunoperoxidase (IIP), complement fixation, immunofluorescent assay (IFA), and enzyme-linked immunosorbent assay (ELISA), etc.) are preferred by researchers for the detection of Rickettsia species in hosts. However, these methods have several disadvantages, such as less sensitivity and specificity, laborious to perform, and cross-reaction with other Rickettsia species^5,10. Compared to other techniques, molecular techniques have many advantages over other identification techniques, like high sensitivity and specificity and identification from different clinical samples¹⁰. Molecular techniques provide contributions to understanding the epidemiology of Rickettsia species, identifying different Rickettsia species in various hosts that never reported them, and reporting novel Rickettsia genotypes/species. Therefore, researchers have recommended the use of these techniques in studies on Rickettsia species^1,5,10.

Türkiye is a suitable country for many vector arthropods thanks to its climate and vegetation diversity. Many vectors and vector-borne pathogens have been detected in the country so far^11-19. Rickettsia species have been also researched in the country and Rickettsia aeschlimannii (Ri. aeschlimannii), Ri. raoultii, Ri. slovaca, Ri. africae, Ri. helvetica, Ri. massiliae, Ri. felis, Ri. conorii subsp. conorii, Ri. sibirica mongolitimonae, Ri. hoogstraalii, Ri. monacensis, Candidatus Rickettsia barbariae Candidatus Rickettsia goldwasserii, and Candidatus Rickettsia vini have been reported in different hosts^20-28. However, literature research showed that no data on Rickettsia species among water buffalo herds in Türkiye. This study aimed to investigate the Rickettsia species in buffaloes in Sivas province using molecular methods and to reveal the phylogenetic characteristics of the identified species by DNA sequence analysis.

Study Area and Materials: Türkiye consists of seven geographical regions: Marmara, Aegean, Mediterranean, Central Anatolia, Black Sea, Eastern Anatolia, and South Eastern Anatolia. Sivas province is located in the Central Anatolia region and the intersection between Central Anatolia, Black Sea, and Eastern Anatolia regions (Figure 1). The city is the second-largest province in the country with a geographical area of approximately 28,400 km2. Sivas is an important region for water buffalo breeding due to its large pasture lands.

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Figure 1: Location of Türkiye in the world maps, and location of sampling site in the Türkiye map.

In this study, DNA belonging to 364 water buffalo blood samples was used. These materials were collected by Sahin et al.¹⁸ for the other study, and stored in appropriate conditions in the research laboratory. Detailed information about the samples can be found in the study by Sahin et al. (18). Briefly, these blood samples were obtained from 119 buffalo herds in seven districts (Sivas city center, Koyulhisar, Zara, Yildizeli, Sarkisla, Susehri, and Ulas) of Sivas province (Table 1).

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Tablo 1: Age, sex and location of water buffalo blood samples

Molecular Research of Rickettsia spp. in Water Buffalo Blood Samples: The gDNAs were screened in terms of Rickettsia species with PCR assay using 190-70 (5’-ATGGCGAATATTTCTCCAAAA-3’) and 190.701 (5’-GTTCCGTTAATGGCAGCATCT-3’) amplified to ompA gene²⁹.

PCR assay was performed to a total volume 25 μL, including 10× PCR buffer (Invitrogen™, Carlsbad, USA), MgCl2 (50 mM) (Invitrogen™, Carlsbad, USA), 200 μM of each dNTP (Cat.No.: R0181, Thermo Scientific™, Lithuania), 1 μL (10 pmol/μL) of each of the primers, 1.25 U of Taq DNA polymerase (5U/μL) (Ref.No.: 100021276, Invitrogen™, Carlsbad, USA), 2.5 μL template DNA, and DNase-RNase-free sterile water.

The thermal cycling protocol used for PCR was 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, and a final extension of 5 min at 72°C. The resulting PCR products were loaded onto a 1% agarose gel and then electrophoresed at 90 volts for 60 minutes. After electrophoresis, the agarose gel was stained by ethidium bromide for 20 min and visualized with a UV transilluminator for specific amplicons.

Preparation of the Samples for DNA Sequence Analyses: The positive sample detected in this work was sequenced for species identification and phylogenetic analyses. For these purposes, PCR assays were done to amplify ompA using primers 190-70 and 190-701 and ompB gene with the primers 120-2788 and 120-3599^29,30. In these PCR assays, the EZ-FD PCR High Fidelity DNA polymerase kit (Cat.No.: 9K-005-0019, BioBasic) was used, and the PCR was done with reagents supplied by manufacturers and in a total volume of 25μL, including 10× EZ-FD Reaction buffer with 25mM MgCl2 (Lot.No.: O807R0K, BioBasic), 200 μM of each dNTP (Lot.No.: R22256RoX, BioBasic), 1 μL (10 pmol/μL) of each of the primers, 1.25 U of EZ-FD PCR High Fidelity DNA Taq polymerase (2.5U/μL) (Lot.No.: Q6A00120KF, BioBasic), 2.5 μL template DNA, and DNase-RNase-free sterile water. After PCR assays, PCR products were loaded into the agarose gel and checked in terms of appropriate amplicon sizes, and then, these products were sent to DNA sequence analyses.

DNA sequence analyses were performed in the commercial company (BM Labosis, Ankara). Before DNA sequence analyses, all PCR products were purified with the HighPrepTM PCR Clean-up System (Cat. No.: AC-60005, MagBio) following the manufacturer’s instructions. The sequence data were checked with FinchTV (version 1.4.0) software (Geospiza Inc., Seattle, Washington, USA) for chromatogram qualities, and nucleotides that had poor chromatogram qualities were trimmed. The consensus sequences were determined using MEGA-11 software³¹. The identified consensus sequences were deposited to GenBank, and accession numbers were determined.

The maximum likelihood method was used to construct phylogenetic trees using MEGA-11 software³¹ to reveal the genetic variation between the Rickettsia species identified in this study and the Rickettsia species in GenBank. Before constructing the phylogenetic tree, it was determined that the best algorithm to be used in the phylogenetic tree of related pathogens was the Tamura-3 parameter model³² using the Find Best-Fit Substitution Model in MEGA-11 and this algorithm was used in the phylogenetic tree. Bootstrap analysis (1,000 repetitions) was performed.

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Figure 2: Agarose-gel electrophoresis of water buffalo Rickettsia species. L. Ladder, 1. Ri. aeschlimannii positive sample (ompA, gene), 2. Ri. aeschlimannii positive sample (ompB, gene), 3-4. Negative control, 5. Ri. aeschlimannii positive water buffalo sample (ompA, gene), 6. Ri. aeschlimannii positive water buffalo sample (ompB, gene)

The ompA gene sequence analyses of the positive sample revealed 98.93-100% nucleotide similarities between Ri. aeschlimannii isolate identified in this study and Ri. aeschlimannii isolate present in the GenBank. In addition, our Ri. aeschlimannii isolates had 100% nucleotide identities with Ri. aeschlimannii identified in H. marginatum larvae (MG920564) from Türkiye, in H. lusitanicum (MH532238) from Italy, in H. marginatum (MT793816) from Lithuania, in H. impeltatum (HQ335157) from Egypt, and in goat (OR248871) from Ghana.

The BLASTn analyses of the consensus sequence of the ompB gene showed that there were 98.39-100% nucleotide resemblance between our Ri. aeschlimannii isolate and Ri. aeschlimannii isolate identified in various parts of the world. Moreover, 100% nucleotide identities were seen Ri. aeschlimannii isolate determined in the study and Ri. aeschlimannii isolate detected in H. marginatum (MK215218) from Germany, D. reticulatus (OR000446) from Poland, and H. rufipes (OR734630) from Kenya.

The phylogenetic trees based on ompA (Figure 3A) and ompB (Figure 3B) genes revealed our Ri. aeschlimannii isolate placed with the same clade with Ri. aeschlimannii isolates reported in different parts of the world and positioned in different branch from other Rickettsia species.

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Figure 3: Phylogenetic tree based on the ompA (A) and ompB (B) genes sequences of Rickettsia species using the maximum likelihood method. Rickettsia species identified in this study were marked with underlined. Numbers at the nodes represent the bootstrap values with 1,000 replicates. The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-3 parameter model32. Pastorella multocida and Chlamydophila pneumoniae were used as an outgroup in the phylogenetic trees

In the last two decades, many Rickettsia species, some of them novel species, have been identified among various hosts and their vectors in the different parts of the world using molecular-based techniques^1,4. These studies also showed that Rickettsia species have been circulated in domestic ruminants, and DNA of these pathogens was detected in blood samples of cattle, sheep, and goats^37-39. Serological studies were performed in water buffalo, and antibodies against Rickettsia species were detected in these animals⁴⁰. In this study, 364 water buffalo blood samples were researched and one sample was found to be infected with Rickettsia species. This is the first molecular detection of Rickettsia DNA in a water buffalo blood sample. Our work and study performed by Pangjai et al.⁴⁰ demonstrated that water buffaloes may be exposed to Rickettsia species. The prevalence and distribution of vector-borne pathogens such as Rickettsia may vary according to the climatic characteristics of the study regions, the common vector species in the sampling regions, the specificity and sensitivity of the diagnostic methods used in the study, and the number of animals included in the study and their ages. In addition, since new Rickettsia species and strains have been detected in different hosts in recent years^1,4, it is thought that studies should be carried out to determine the epidemiological risk factors of these pathogens.

DNA sequence analyses have been done for different purposes such as the correction of PCR results, revealing phylogenetic analyses and genetic diversity of species, identification of novel species or genotypes of pathogens, and discovery of new host or epidemiological areas of pathogens in the studies^1,14,18. The DNA sequence of one Rickettsia species identified in the work was done by targeting ompA and ompB genes. These sequence results revealed that Ri. aeschlimannii was circulated in a water buffalo herd in Sivas province. The ompA (98.93-100%) and ompB (98.39-100%) gene sequences showed that our Ri. aeschlimannii isolate had high nucleotide similarities with Ri. aeschlimannii isolates identified in different hosts from various parts of the world. With this study Ri. aeschlimannii was detected for the first time in water buffalo using PCR and DNA sequence analyses. Although Ri. aeschlimannii was detected in a water buffalo in the work, large-scale molecular studies involving buffaloes and their ectoparasites in different countries are needed to understand the contribution of these animals to the epidemiology of Ri. aeschlimannii.

Rickettsia aeschlimannii was identified firstly in ticks (H. marginatum) from Morocco⁴¹. Five years later, this pathogen was reported in humans returning to France from Morocco using serological and molecular techniques⁴². In the same and following years, Ri. aeschlimannii was detected in humans from South Africa^43, and Greece⁸. In these studies, clinical manifestations such as necrotic vesicular symptoms on the ankle, high fever, and maculopapular skin rash around the tick-bite sites were reported in patients, and these patients were treated with doxycycline for one week^8,42,43. To date, several studies have been performed, and Ri. aeschlimannii was reported in different hosts (Meriones shawi, camel, human, Hyalomma marginatum, Rhipicephalus sanguineus etc.) from various parts of the world⁴. In this study, the DNA of Ri. aeschlimannii was detected for the first time in a water buffalo blood sample both in Türkiye and world. In Türkiye, Ri. aeschlimannii was identified in different cities from multiple hosts like Hyalomma spp. collected from cattle^27, hares^26, and humans^23,25, H. marginatum from wild boars^26, cattle^23, goats^28, sheep^28, and humans^20,22,23,25, H. aegyptium from human ^20,23,25, and hedgehogs^24, H. excavatum from humans^25, Rhipicephalus spp. from goats^27, Rhipicephalus bursa from humans^20,25, R. turanicus from humans^25, cattle^28, and sheep^23, Haemaphysalis parva from human^25, Hae. punctata from human^25, Hae. sulcata from humans^25, Dermacentor marginatus from humans^25, and Ixodes ricinus from humans²⁵. Above-mentioned studies revealed Ri. aeschlimannii is carried by multiple tick species in Türkiye. Sivas is the second biggest city in Türkiye, and different tick species, some of them vectors of Ri. aeschlimannii, circulate among domestic and wild animals in the city^44-46. In studies conducted by Altay et al.^45,46 several tick-borne pathogens were reported in the ticks collected from cattle and sheep. Considering that Ri. aeschlimannii is a zoonotic pathogen transmitted by ticks, it is thought that buffalo breeders and people living in the region should take the necessary precautions against the pathogen.

In conclusion, Ri. aeschlimannii was reported for the first time with this study in a water buffalo. This molecular report showed that buffaloes, like other domestic ruminants, are hosts/reservoirs for vector-borne zoonotic pathogens. Vector-borne pathogens have a wide range of pathological effects on human and animal health. In addition, the economic loss due to these pathogens can reach billions of dollars, and studies showing the current prevalence and distribution of vector-borne pathogens among hosts are needed to reduce this economic loss.

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