Molecular Detection of Spotted Fever Group Rickettsia (Rickettsiales: Rickettsiaceae) in Ticks of Iran

Introduction

Ticks (Ixodida order) harbor many symbiotic microorganisms, including some Rickettsia species ( Benson et al., 2004 ; Heise et al., 2010 ; Noh et al., 2017 ). As intracellular bacteria, Rickettsiae are symbionts in the broad sense with an intimate but not necessarily beneficial relationship with their arthropod hosts that may be opportunistic or pathogenic under various conditions, affecting vector competency ( Perlman et al., 2006 , Telford and Parola, 2007 ). According to molecular phylogenetics, the term Rickettsia is currently applied to arthropod-borne bacteria belonging to the genus Rickettsia within the family Rickettsiaceae in the order of the Rickettsiales. This genus is currently made of 24 recognized species and contains several dozens of as-yet uncharacterized strains ( Fournier and Raoult, 2007 ). Rickettsia species are obligate intracellular bacteria that infect various vertebrate hosts, including humans ( Noh et al., 2017 ). They are the most frequent organisms detected within the bacterial community of ticks ( Moreno et al., 2006 ). Most human pathogenic Rickettsiae belong to the spotted fever group (SFG), including Rickettsia rickettsii, Rickettsia akari, Rickettsia conorii, Rickettsia parkeri, Rickettsia sibirica,Rickettsia australis, Rickettsia japonica, Rickettsia slovaca, and Rickettsia africae ( Braig et al., 2009 ). The number of known pathogenic Rickettsia species has been increasing over time ( Owen et al., 2006 ; Li et al., 2016 ). Within 1984-2004, nine novel Rickettsia species or subspecies were identified causing tick-borne rickettsioses, including six initially isolates from ticks ( Raoult and Parola, 2007 ). Because ticks are regarded as the reservoir host of SFG Rickettsia ( Raoult and Roux, 1997 ), they have been frequently examined for the presence of Rickettsia ( Perlman et al., 2006 ). All tick-borne pathogenic Rickettsia species are transmitted by hard ticks. Some individual ticks can be concurrently infected with more than one Rickettsia species ( Braig et al., 2009 ). Rickettsiae can be considered potential pathogens, particularly if they have been detected in the tick’s salivary glands ( Parola et al., 2005 ). When ovaries and oocytes of an adult female tick are infected, Rickettsia might be transovarially transmitted to at least some offspring ( Raoult and Roux, 1997 ). The frequency of Rickettsia transmission through tick bite depends on tick-host specificity, abundance of the tick vector, prevalence of the infection within tick organs, and frequency of tick-human contact ( Parola et al., 2005 ). After the revolution in bacterial taxonomy by the innovation of polymerase chain reaction (PCR), genome sequencing has become a tool in research and clinical applications ( Loeffelholz and Deng, 2006 ; Nolte and Wittwer, 2016 ). The advantages of PCR include simplicity, speed, low cost, and ability to detect microorganisms without cultivation ( Clay and Fuqua, 2010 ; Nolte and Wittwer, 2016 ). In addition to the importance of 23S ribosomal ribonucleic acid (rRNA) and 16S rRNA for the molecular detection of Rickettsia, the faster evolution of citrate synthase (gltA), a citrate synthase-encoding gene, shows that this gene is more sensitive to change than 16S rRNA ( Fournier et al., 1998 ). To date, 50 hard and soft tick species have been recorded for the fauna of Iran ( Kamali et al., 2001 ). In the current study, a molecular survey of Rickettsia agents was carried out for the first time using genus-specific 23S rRNA, 16S rRNA, and gltA genes for the identification of Rickettsia in the ticks collected from Iran.

Material and Methods

Study area, Collection, and Identification of Ticks. Tick specimens were collected from domestic animals in nine provinces of Iran, namely Azarbayjan-‎e Sharqi, Hamedan, Kerman, Kermanshah, Khorasan-e ‎‎Shomali, Khuzestan, Kurdistan, Lorestan, and Semnan. Table 1 tabulates the data related to the collection of the specimens. The ticks were transported to the laboratory in a glass tube and identified at the level of species based on taxonomic keys ( Estrada-Peña et al., 2004 ; Hosseini-Chegeni and Tavakoli, 2013 ; Pomerantzev, ‎1950‎ ) under a light stereomicroscope (SZX12-Olympus, JAPAN). Then, the tick specimens were stored at -20 °C for further examination.

Polymerase Chain Reaction. Genomic deoxyribonucleic acid (gDNA) was extracted using Phenol-chloroform according to Sambrook and Russell (2001). The fragments of 16S rRNA, 23S rRNA, and gltA were amplified by PCR. In order to specifically and accurately amplify the target agents in ticks’ bodies, six primers were newly designed, including; 16S: Fric16S (5ʹ- CGG AGG AAA GAT TTA TCG CTG ATG -3ʹ), Rric16S (5ʹ- GTT TAC GGC GTG GAC TAC C -3ʹ), 23S: Fric23S (5ʹ- CGT GAG GGA AAG GTG AAA AG -3ʹ), Rric23S (5ʹ- CGC TAC CTT AGG ACC GTC -3ʹ), gltA: FricgltA (5ʹ- GGY TTT ATG TCT MCT GCT TC -3ʹ), and RricgltA (5ʹ- AGC TTC AAG TTC TAT TGC TAT TTG -3ʹ). The PCR reactions for each gene were carried out in a thermocycler Corbett® (Australia). Touchdown temperature profile, including 5 min at 95 °C, 10 cycles (50 sec at 94 °C, 50 sec at 60-50 °C, and 1 min at 72 °C), followed by 20 cycles (50 sec at 94 °C, 50 sec at 50 °C, and 1 min at 72 °C), and final extension step (3 min at 72 °C). Each PCR reaction consisted of 12.5 µl of 2× RedMaster PCR® (Sinaclon®, Iran), 1 µl from each primer (10 pM), 4 µl of gDNA template (50-100 ng/µl), and 6.5 µl of deionized water to the final volume of 25 µl. The positive controls included the positive tick samples with successful DNA extraction, PCR amplification, and sequencing of a Rickettsia species. In addition, distilled water without target DNA was used as negative control.

Electrophoresis, Purification, and Sequencing. The PCR products were visualized by 1% agarose gel electrophoresis, and the selective desired bands of the different gene fragments from different tick species were purified using the GF-1 Gel DNA Recovery Kit® (Vivantis, Malaysia). Then, the purified PCR products were submitted for sequencing to Faza-Biotech® Company (Iran). Subsequently, the sequences were manually edited using FinchTV® software (version 1.4.0). Finally, all the sequences were submitted to GenBank, and accession numbers were assigned.

Phylogenetic analysis. All the sequences were aligned using SeaView software (version 4) ( Gouy et al., 2010 ). Phylogenetic trees were constructed for 16S and gltA sequence data using neighbor-joining as well as 23S sequence data using Bayesian inference methods by MEGA software (version 7) and BEAST software (version 1.8.2) ( Drummond et al., 2012 ). For this purpose, 17, 17, and 18 taxa (including sequences from the present study as well as comparable GenBank data as in- and out-group) were used for the construction of 16S, gltA, and 23S phylogenetic trees, respectively. The constructed clades of 16S, gltA, and 23S phylogenetic trees were reorganized based on 100% bootstrap support values and reasonable genetic distance differences within and between the clade members. The sequences from Orientia tsutsugamushi and Wolbachia (i.e., an endosymbiont species of Drosophila) were included as out-groups in both 16S and 23S phylogenetic trees in addition to Rickettsia australis, R. prowazekii, and R. tarasevichiae in the gltA phylogenetic tree.

Results

Tick species, PCR, and Sequences. Totally, 301 tick specimens were collected from different parts of the study area. A total of 61 individual ticks were analyzed for Rickettsia agents with 36 Rickettsia positive tick samples (Table 1). The ticks, including Argas persicus, Haemaphysalis sulcate, Ha. inermis, and Hyalomma asiaticum, were identified based on taxonomic keys from different geographical regions. Figure 1 illustrates the PCR amplification of a 590-bp fragment of 16S rRNA, 1501-bp fragment of 23S rRNA, and 903-bp fragment of gltA of Rickettsia genus in various tick samples. The 16S rRNA of Rickettsia was detected in A. persicus, Hy. asiaticum, Ha. sulcata, and Ha. inermis collected from Lorestan, Hamedan, Khorasan-e ‎‎Shomali, and Khuzestan provinces, Iran, respectively. Moreover, 23S rRNA was detected in the A. persicus ticks of three different regions in Lorestan province (Aleshtar, Pol-e Dokhtar, Sepiddasht). Furthermore, gltA was also detected in Hy. asiaticum and Ha. sulcata from Hamedan and Khorasan-e ‎‎Shomali provinces, respectively. Twelve sequences, including five 16S, five 23S, and two gltA sequences, were obtained after sequencing. The accession numbers were assigned in GenBank, including KY569634-41 (16S rRNA), KY784689-90 (gltA), and KY857839-40 (23S rRNA).

Figure 1. 1% agarose gel electrophoresis stained with Cyber Safe® showing different gene fragments of Rickettsia amplified in this study, including citrate synthase (903 bp), 16S ribosomal ribonucleic acid (rRNA) (590 bp), and 23S rRNA (1501 bp), detected from different tick species; gene samples from left to right, including negative and positive control, and then various Rickettsia samples isolating from ticks

16S, 23S, and gltA Phylogenetic Trees. Phylogenetic trees were constructed using MEGA software (version 7) and BEAST software (version 1.8.2), including in-group and out-group Rickettsia taxa (Figure 2). The constructed phylogeny indicated that the Rickettsia spp. of Iranian ticks are clustered with the GenBank data of 16S rRNA Rickettsia sequences (i.e., R. aeschlimannii, R. africae, R. conorii, R. japonica, R. kotlanii, R. massiliae, R. peacockii, R. prowazekii, R. raoultii, and R. slovaca), 23S rRNA (i.e., R. africae, R. akari, R. australis, R. conorii, R. felis, R. heilongjiangensis, R. japonica, R. massiliae, R. parkeri, R. philipii, R. prowazekii, R. rickettsia, and R. slovaca), and gltA (i.e., Candidatus R. antechini, C. R. uilenbergi, R. aeschlimannii, R. amblyommii, R. massiliae, R. mongolotimonae, R. montanensis, R. parkeri, R. raoultii, R. rhipicephali, and R. sibirica). No intraspecies variation in terms of genetic distance was observed among 16S, 23S, and gltA sequences of Rickettsia clade. As much as 14% and 9% genetic distance was noticed between Rickettsia 16S sequences with Wolbachia and Orientia tsutsugamushi, respectively. Moreover, the genetic distance rates of 13% and 21% were observed between Rickettsia 23S sequences with Orientia tsutsugamushi and Wolbachia, respectively. The RickettsiagltA sequence data showed 7%, 7%, and 11% of genetic distance difference with R. australis, R. prowazekii, and R. tarasevichiae (as out-groups), respectively. All different sequences of the Rickettsia clade should be considered single species according to each 16S, 23S, and gltA gene fragments.

Figure 2. Phylogenetic tree of Rickettsia spp. inferred from 16S ribosomal ribonucleic acid (rRNA) (A), 23S rRNA (B), and citrate synthase (gltA) (C) sequence data constructed using neighbor-joining (A and C) as well as Bayesian inference (B) methods with 1,000 replicates; main clade in the right side of tree separated with colored rectangular shapes; taxa arranged as species name following GenBank accession number; taxon of the present study indicated as bold by genus name; nodes indicated with bootstrap (A and C) and posterior probability (B) values; Orientia tsutsugamushi, Wolbachia (i.e., an endosymbiont species of Drosophila) (A and B), Rickettsia australis, Rickettsia prowazekii, and Rickettsia tarasevichiae (C) examined as out-groups

Discussion

The present preliminary study was designed for the determination of genus Rickettsia among tick species distributed in Iran for the first time. Four tick species in two families and three genera were identified which were infected with Rickettsia according to 16S rRNA, 23S rRNA, and gltA molecular evidence. To date, three Argas, nine Hyalomma, and six Haemaphysalis species were reported from Iran ( Hosseini-Chegeni et al., 2013 ; Hosseini-Chegeni and Tavakoli, 2013 ; Hosseini-Chegeni et al., 2014 ; Hosseini-Chegeni et al., 2015 ). In the present study, rickettsial sequences were identified in A. persicus, Ha. sulcata, Ha. inermis, and Hy. asiaticum in different geographical regions of Iran. Sumrandee et al. (2016) reported a 13% prevalence of Rickettsia within Amblyomma, Dermacentor, Haemaphysalis, and Rhipicephalus (Boophilus). The bacterial diversity associated with Dermacentor niveus Neumann ticks in the natural environment was investigated by 16S rRNA, gltA, and other genes in China ( Zhuang et al., 2014 ). The authors detected Proteobacteria (including Rickettsia ) as a dominant microflora in the ticks collected from the field. Up to 21% infection rate of SFG Rickettsia in Amblyomma ticks was reported from Thailand ( Sumrandee et al., 2014 ). The prevalence of Rickettsia infection based on the detection of the gltA gene varied in different species of ticks, ranging from 6-40% ( Sumrandee et al., 2016 ). Two tick species were examined for the identification of Rickettsia species in a national park in Poland. The results indicated the pathogen prevalence rates of 27.5% and 42.8% for Ixodes ricinus (L.) and Dermacentor reticulatus (Fabricius), respectively. In the present study, 23S rRNA, 16S rRNA, and gltA genes were used to differentiate Rickettsia from genera Orientia tsutsugamushi and Wolbachia (i.e., an endosymbiont species of Drosophila) (Fam. Rickettsiaceae). According to Roux et al. (1997), the phylogeny inferred from the gltA gene is more reliable than 16S rRNA. They could discriminate two subgroups in the SFG Rickettsia based on the gltA phylogenetic tree. The 16S rRNA sequences were not useful for the taxonomy of Rickettsia because greater than 97% similarity exists between any two taxa ( Fournier et al., 1998 ; Lee et al., 2003 ; Parola et al., 2005 ). Therefore, to precisely detect rickettsiae at the species level, the investigation of expression of other genes, including gltA, outer membrane protein A, outer membrane protein B, 120-kDa cell surface antigen 4, and 60-kDa heat shock protein, is recommended. Argas persicus is the main ectoparasite of poultry in Iran and is adapted for living inside the nest/shelter of its host ( Telmadarraiy et al., 2004 , Hosseini-Chegeni and Tavakoli, 2013 ). This tick species was positive for Rickettsia spp. in the current study. According to Pader et al. (2012), 57% of A. persicus tick pools were positive for Rickettsia in Ethiopia. Argas persicus has been reported to occasionally parasitize humans. Although Rickettsia pathogens have been detected in A. persicus, it does not mean that this species is an important vector for human rickettsial diseases ( Estrada-Peña and Jongejan, 1999 ). Argas persicus was also reported as the vector of Rickettsia-like symbiotes ( Suitor Jr and Weiss, 1961 ), R. slovaca from Armenia ( Rehacek et al., 1977 ), and R. hoogstraalii from Ethiopia ( Pader et al., 2012 ). Haemaphysalis sulcata is widely distributed in Iran and is mostly detected in livestock, including goats and sheep, unlike Ha. inermis with more restricted distribution ( Hosseini-Chegeni et al., 2014 ). In the present study, Ha. sulcata and Ha. inermis were positive for Rickettsia . Up to 26% prevalence of Rickettsia felis-like bacteria was reported from Ha. sulcata in Southern Croatia ( Duh et al., 2006 ). Moreover, Sarih et al. (2008) detected a 77% infection prevalence of Rickettsia in Ha. sulcata in Morocco. Generally, Haemaphysalis ticks rarely bite humans and consequently are of little significance in the epidemiology of human pathogens ( Duh et al., 2006 ). Haemaphysalis inermis was newly identified as the most important potential vector for Rickettsia helvetica in Hungary ( Hornok et al., 2010 ). In other parts of the world, rickettsial agents were reported from Haemaphysalis ticks, namely R. aeschlimannii from Ha. inermis and Rickettsia endosymbiont of Ha. sulcata from Spain ( Portillo et al., 2008 ) and R. hoogstraalii from Ha. sulcata ( Duh et al., 2010 ; Tomassone et al., 2017 ). A limited number of studies have been carried out on the detection of Rickettsia in Hyalomma ticks. In the current study, Rickettsia was detected in Hy. asiaticum, a widely distributed tick species in Iran ( Hosseini-Chegeni et al., 2013 ; Telmadarraiy et al., 2015 ). An antigenically and genotypically unique SFG Rickettsia was isolated from Hy. asiaticum in China ( Yu et al., 1993 ). Kordová and Rehacek (1964) reported the multiplication of Rickettsiaprowazekii in Hy. asiaticum and considered this species as a potential vector. According to the obtained results of the present study, SFG Rickettsia is the main group of Rickettsia that can be detected from the three genera of ticks in Iran.

The SFG Rickettsia is the main group of Rickettsia that can be detected from the three genera of ticks in Iran. It is suggested to carry out further genetic studies on other genes to determine the accurate status of Rickettsia species transmitted through vector ticks in Iran.

Ethics

We hereby declare all ethical standards have been respected in preparation of the submitted article.

Authors’ Contribution

Study concept and design: Hosseini-Chegeni, A. Telmadarraiy, Z.

Acquisition of data: Hosseini-Chegeni, A., Tavakoli, M.

Analysis and interpretation of data: Hosseini-Chegeni, A.

Drafting of the manuscript: Hosseini-Chegeni, A., Faghihi, F.

Critical revision of the manuscript for important intellectual content: Faghihi, F.

Statistical analysis: Hosseini-Chegeni, A.

Administrative, technical, and material support: Tavakoli, M., Tavakoli, M., Telmadarraiy, Z.

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