Molecular Evidence of Hemolivia mauritanica, Ehrlichia spp. and the Endosymbiont Candidatus Midichloria Mitochondrii in Hyalomma aegyptium Infesting Testudo graeca Tortoises from Doha, Qatar

Simple Summary Due to the veterinary and medical importance of pathogens transmitted by Hyalomma aegyptium, we tested ticks removed from Testudo graeca tortoises for the presence of Anaplasma, Ehrlichia, Hemolivia, Babesia and Hepatozoon. Forty-three percent of the examined adult ticks were infected with at least one agent. The most prevalent agent identified was Hemolivia mauritanica (28.6%), followed by Candidatus Midichloria mitochondrii (9.5%) and Ehrlichia spp. (4.7%). Our study reported for the first time H. mauritanica, Ehrlichia spp. and Candidatus M. mitochondrii in H. aegyptium ticks collected from pet spur-thighed tortoises, in Qatar, providing data that adds to the geographical extension of these agents. Abstract Tick-borne agents constitute a growing concern for human and animal health worldwide. Hyalomma aegyptium is a hard tick with a three-host life cycle, whose main hosts for adults are Palearctic tortoises of genus Testudo. Nevertheless, immature ticks can feed on a variety of hosts, representing an important eco-epidemiological issue regarding H. aegyptium pathogens circulation. Hyalomma aegyptium ticks are vectors and/or reservoirs of various pathogenic agents, such as Ehrlichia, Anaplasma, Babesia and Hepatozoon/Hemolivia. Ehrlichia and Anaplasma are emergent tick-borne bacteria with a worldwide distribution and zoonotic potential, responsible for diseases that cause clinical manifestations that grade from acute febrile illness to a fulminant disease characterized by multi-organ system failure, depending on the species. Babesia and Hepatozoon/Hemolivia are tick-borne parasites with increasing importance in multiple species. Testudo graeca tortoises acquired in a large animal market in Doha, Qatar, were screened for a panel of tick-borne pathogens by conventional PCR followed by bidirectional sequencing. The most prevalent agent identified in ticks was Hemolivia mauritanica (28.6%), followed by Candidatus Midichloria mitochondrii (9.5%) and Ehrlichia spp. (4.7%). All samples were negative for Babesia spp. and Hepatozoon spp. Overall, 43% of the examined adult ticks were infected with at least one agent. Only 4.7% of the ticks appeared to be simultaneously infected with two agents, i.e., Ehrlichia spp. and H. mauritanica. This is the first detection of H. mauritanica, Ehrlichia spp. and Candidatus M. mitochondrii in H. aegyptium ticks collected from pet spur-thighed tortoises, in Qatar, a fact which adds to the geographical extension of these agents. The international trade of Testudo tortoises carrying ticks infected with pathogens of veterinary and medical importance deserves strict control, in order to reduce potential exotic diseases.

Simple Summary: Due to the veterinary and medical importance of pathogens transmitted by Hyalomma aegyptium, we tested ticks removed from Testudo graeca tortoises for the presence of Anaplasma, Ehrlichia, Hemolivia, Babesia and Hepatozoon. Forty-three percent of the examined adult ticks were infected with at least one agent. The most prevalent agent identified was Hemolivia mauritanica (28.6%), followed by Candidatus Midichloria mitochondrii (9.5%) and Ehrlichia spp. (4.7%). Our study reported for the first time H. mauritanica, Ehrlichia spp. and Candidatus M. mitochondrii in H. aegyptium ticks collected from pet spur-thighed tortoises, in Qatar, providing data that adds to the geographical extension of these agents.
Abstract: Tick-borne agents constitute a growing concern for human and animal health worldwide. Hyalomma aegyptium is a hard tick with a three-host life cycle, whose main hosts for adults are Palearctic tortoises of genus Testudo. Nevertheless, immature ticks can feed on a variety of hosts, representing an important eco-epidemiological issue regarding H. aegyptium pathogens circulation. Hyalomma aegyptium ticks are vectors and/or reservoirs of various pathogenic agents, such as Ehrlichia, Anaplasma, Babesia and Hepatozoon/Hemolivia. Ehrlichia and Anaplasma are emergent tick-borne bacteria with a worldwide distribution and zoonotic potential, responsible for diseases that cause clinical manifestations that grade from acute febrile illness to a fulminant disease characterized by multi-organ system failure, depending on the species. Babesia and Hepatozoon/Hemolivia are tick-borne parasites with increasing importance in multiple species. Testudo graeca tortoises acquired in a large animal market in Doha, Qatar, were screened for a panel of tick-borne pathogens by conventional PCR followed by bidirectional sequencing. The most prevalent agent identified in ticks was Hemolivia mauritanica (28.6%), followed by Candidatus Midichloria mitochondrii (9.5%) and Ehrlichia spp. (4.7%). All samples were negative for Babesia spp. and Hepatozoon spp. Overall, 43% of the examined adult ticks were infected with at least one agent. Only 4.7% of the ticks appeared to be simultaneously infected with two agents, i.e., Ehrlichia spp. and H. mauritanica. This is the first detection of H. mauritanica, Ehrlichia spp. and Candidatus M. mitochondrii in H. aegyptium ticks collected from pet spur-thighed tortoises, in Qatar, a fact which adds to the geographical extension of these agents. The international trade of Testudo tortoises carrying ticks infected with pathogens of veterinary and medical importance deserves strict control, in order to reduce potential exotic diseases.

Introduction
Ticks are known as important vectors of many viral, bacterial and protozoan infectious microorganisms capable of producing disease in both humans and animals [1]. As hematophagous arthropods, while taking a blood meal, they can transmit pathogens to susceptible hosts, supporting the enzootic cycles of many infectious agents in various ecosystems and being regarded as major human and veterinary public health problems [2]. Nevertheless, these arthropods also harbor intracellular bacteria that are apparently not detrimental to humans, animals or even to ticks themselves. Symbionts, such as Candidatus Midichloria mitochondrii, are obligately intracellular bacteria, and in some cases are closely associated with the presence of known pathogens, such as Rickettsia parkeri [3]. Symbiotic, commensal and pathogenic microorganisms harbored by ticks can positively influence pathogen transmission or interfere with their maintenance in the tick [4]. For example, Coxiella-like endosymbionts seem to impair the transmission of Ehrlichia chaffeensis by Amblyomma ticks [5], whereas the presence of Francisella sp. endosymbionts increases the colonization success of pathogenic Francisella novicida in Dermacentor andersoni ticks [6].
Hyalomma aegyptium is a three-host life cycle hard tick endemic in North Africa, Balkan countries, the Middle East, Caucasus, Central Asia, Afghanistan and Pakistan, whose adult stage main hosts are Palearctic tortoises of the genus Testudo [7][8][9]. However, adult ticks, together with the less host-specific nymphs and larvae, also feed on various vertebrates, such as domestic animals (dogs, cattle, pigs, horses), wild animals (birds, boar, deer, foxes, jackals, hamsters, hares, hedgehogs, mustelids, squirrels) and humans [10][11][12][13][14][15]. This wide host range yields a variety of pathogen transmission scenarios between the numerous hosts, becoming a concern under an eco-epidemiological point of view.
Due to the veterinary and medical importance of pathogens transmitted by H. aegyptium ticks and their wide host range, ticks from Testudo graeca acquired in an animal market in Doha, Qatar, were screened for several pathogens, namely, Ehrlichia, Anaplasma, Babesia and Hepatozoon/Hemolivia.

Study Area
A country located on the eastern side of the Arabian Peninsula, Qatar has a desert climate with an arid and hot summer characterized by temperatures ranging between 25 • C and 46 • C. Rainfall is scarce (75.6 mm per year), falling with erratic patterns from October to March. Doha is the country's capital and its largest city.

Ticks
Ticks included in this study were previously collected and screened for the presence of Rickettsia spp. in 2019 [16]. Briefly, a total of 21 ticks were removed from two pet tortoises (T. graeca), which had been acquired from one of Qatar's largest animal markets just before presentation at Parkview Pet Center Veterinary Clinic for a health check and ectoparasitic control in May 2018, Doha. The animal market had a total of 20 animal stores, four of which sold tortoises (averaging 10-15 tortoises per store). The removed ticks were previously identified to the species level as Hyalomma aegyptium [16] using the morphological criteria already described and further confirmed by PCR using mitochondrial genes (12S and 16S rDNA) as molecular targets [23,24].

Detection of Ehrlichia/Anaplasma, Babesia and Hepatozoon/Hemolivia DNA in Ticks
Tick extracted DNA by the alkaline hydrolysis [25] was tested for the presence of Ehrlichia, Anaplasma, Babesia and Hepatozoon/Hemolivia by conventional PCR in the Pathology and Immunology Department of the Institute of Biomedical Sciences Abel Salazar, Porto University, according to previously described protocols (Table 1). For PCR, the KAPA HiFi HotStart ReadyMix, KAPA Biosystems (Woburn, MA, USA) was used according to the manufacturer's instructions. The amplification was performed in Bio-Rad T100 TM Thermal Cycler. Aliquots of each PCR product were electrophoresed on 1.5% agarose gel stained with Xpert Green Safe DNA gel stain (Grisp, Porto, Portugal) and examined for the presence of the specific fragment under UV light. DNA fragment size was compared with a standard molecular weight, 100 bp DNA ladder (Grisp, Porto, Portugal). Distilled water was used as negative control.

Sequencing and Phylogenetic Analysis
All Ehrlichia-positive and Hemolivia-positive amplicons obtained were sequenced for genetic characterization. Amplicons were purified with Exo/SAP Go (Grisp, Porto, Portugal), and bidirectional sequencing was performed with the Sanger method at the genomics core facility of the Institute of Molecular Pathology and Immunology of the University of Porto. Sequence editing and multiple alignments were performed with the BioEdit Sequence Alignment Editor v7.1.9 software package, version 2.1 (Ibis Biosciences). The sequences obtained were subjected to the basic local alignment search tool (BLAST) [29][30][31] using the non-redundant nucleotide database (http://blast.ncbi.nlm.nih.gov/Blast.cgi).     When screening the 21 ticks for the 18S rRNA gene, 5 were found positive for H. mauritanica. Further characterization of the 18S rRNA sequences showed a nucleotide identity between 99.70% and 99.84% with H. mauritanica sequences from the blood of Tes-tudo graeca from Syria (GenBank accession no. KF992707) and Greece (GenBank accession no. KF992710). Phylogenetic analysis was performed for 18S rRNA sequences and confirmed clustering with H. mauritanica reference strains (Figure 3). identity between 99.70% and 99.84% with H. mauritanica sequences from the blood of Testudo graeca from Syria (GenBank accession no. KF992707) and Greece (GenBank accession no. KF992710). Phylogenetic analysis was performed for 18S rRNA sequences and confirmed clustering with H. mauritanica reference strains (Figure 3). No amplification was obtained for Babesia spp. nor Hepatozoon spp. One (4.8%) of the ticks was co-infected with Ehrlichia spp. and H. mauritanica.

Results
The following accession numbers were assigned to the sequences obtained in this work: MW092747 and MW092748 (16S rRNA gene fragment of Candidatus M. mitochondrii), MW092750 (16S rRNA gene fragment of Ehrlichia spp.) and MW092776 to MW092781 (18S rRNA gene fragment of H. mauritanica).

Discussion
This report presents the molecular findings for a panel of tick-borne pathogens from a total of 21 H. aegyptium ticks previously removed from two Testudo graeca tortoises acquired in a large animal market in Doha, Qatar.
In 38% of the 21 H. aegyptium collected from T. graeca tortoises, tested for Ehrlichia/Anaplasma, Hemolivia/Hepatozoon and Babesia spp., at least one agent was detected. The most commonly detected agent was H. mauritanica, with 28.6% of the H. aegyptum ticks being positive for it, followed by the endosymbiont Candidatus M. mitochondrii, 9.5%, and bacterium Ehrlichia spp., 4.8%. Hemolivia mauritanica and Ehrlichia spp. co-infection was detected in one H. aegyptium.
The molecular analysis of a 345 bp stretch of the 16S rRNA gene showed that a sequence found in a tick presented the highest identity with Ehrlichia spp. No amplification was obtained for Babesia spp. nor Hepatozoon spp. One (4.8%) of the ticks was co-infected with Ehrlichia spp. and H. mauritanica.
The following accession numbers were assigned to the sequences obtained in this work: MW092747 and MW092748 (16S rRNA gene fragment of Candidatus M. mitochondrii), MW092750 (16S rRNA gene fragment of Ehrlichia spp.) and MW092776 to MW092781 (18S rRNA gene fragment of H. mauritanica).

Discussion
This report presents the molecular findings for a panel of tick-borne pathogens from a total of 21 H. aegyptium ticks previously removed from two Testudo graeca tortoises acquired in a large animal market in Doha, Qatar.
In 38% of the 21 H. aegyptium collected from T. graeca tortoises, tested for Ehrlichia/ Anaplasma, Hemolivia/Hepatozoon and Babesia spp., at least one agent was detected. The most commonly detected agent was H. mauritanica, with 28.6% of the H. aegyptum ticks being positive for it, followed by the endosymbiont Candidatus M. mitochondrii, 9.5%, and bacterium Ehrlichia spp., 4.8%. Hemolivia mauritanica and Ehrlichia spp. co-infection was detected in one H. aegyptium.
The molecular analysis of a 345 bp stretch of the 16S rRNA gene showed that a sequence found in a tick presented the highest identity with Ehrlichia spp.
Ehrlichia spp. are maintained in complex zoonotic systems involving vector ticks and reservoir hosts. These agents affect both humans [33] and animals such as dogs, ruminants [34,35] and even deer [36]. Infected humans [33] and dogs [37] may manifest fever, malaise, leucopenia, thrombocytopenia and abnormal liver function. Tick species that are vectors of these pathogens, such as Amblyomma, Dermacentor, Rhipicephalus, Ixodes, Haemaphysalis and Hyalomma, also parasitize humans, thus posing a considerable risk [38]. Our results demonstrate a lower occurrence of H. aegyptium infected with Ehrlichia spp. (4.7%) when compared with recent work, which has shown an occurrence of 30.2% [38].
Candidatus M. mitochondrii, an α-proteobacterial symbiont first detected in Ixodes ricinus, has a unique intramitochondrial lifestyle [39]. It was the first bacterium shown to reside within the mitochondria and the possible role in ticks is yet to be determined [40]. In the present study, Candidatus M. mitochondrii was detected in H. aegyptium ticks collected on T. graeca from Qatar. As far as we know, this is the first report of the detection of this symbiont in H. aegyptium ticks.
Our study reports for the first-time detection of H. mauritanica, Ehrlichia spp. and Candidatus M. mitochondrii in H. aegytium ticks collected from pet spur-thighed tortoises, in Qatar, a circumstance which contributes to characterizing the geographical distribution of these agents. The current dimension and growth of international wildlife trade is known not only to act as an avenue for the spread of disease [41] but also poses an important risk to global biodiversity, as well as having an impact on social and economic development [42]. Importation of tick-infested tortoise, later found to be carrying zoonotic pathogens, have been reported in the past [16,43].

Conclusions
Our study reports for the first-time the detection of H. mauritanica, Ehrlichia spp. and Candidatus M. mitochondrii in H. aegytium ticks collected from pet spur-thighed tortoises, in Qatar, a circumstance which contributes to characterizing the geographical distribution of these agents and shows the need of strict surveillance and control to reduce potential non-native diseases while assisting animal conservation.