Molecular Detection and Identification of Spotted Fever Group Rickettsiae in Ticks Collected from the West Bank, Palestinian Territories

Background Tick-borne rickettsioses are caused by obligate intracellular bacteria belonging to the spotted fever group (SFG) rickettsiae. Although Spotted Fever is prevalent in the Middle East, no reports for the presence of tick-borne pathogens are available or any studies on the epidemiology of this disease in the West Bank. We aimed to identify the circulating hard tick vectors and genetically characterize SFG Rickettsia species in ixodid ticks from the West Bank-Palestinian territories. Methodology/Principal Findings A total of 1,123 ixodid ticks belonging to eight species (Haemaphysalis parva, Haemaphysalis adleri, Rhipicephalus turanicus, Rhipicephalus sanguineus, Rhipicephalus bursa, Hyalomma dromedarii, Hyalomma aegyptium and Hyalomma impeltatum) were collected from goats, sheep, camels, dogs, a wolf, a horse and a tortoise in different localities throughout the West Bank during the period of January-April, 2014. A total of 867 ticks were screened for the presence of rickettsiae by PCR targeting a partial sequence of the ompA gene followed by sequence analysis. Two additional genes, 17 kDa and 16SrRNA were also targeted for further characterization of the detected Rickettsia species. Rickettsial DNA was detected in 148 out of the 867 (17%) tested ticks. The infection rates in Rh. turanicus, Rh. sanguineus, H. adleri, H. parva, H. dromedarii, and H. impeltatum ticks were 41.7, 11.6, 16.7, 16.2, 11.8 and 20%, respectively. None of the ticks, belonging to the species Rh. bursa and H. aegyptium, were infected. Four SFG rickettsiae were identified: Rickettsia massiliae, Rickettsia africae, Candidatus Rickettsia barbariae and Candidatus Rickettsia goldwasserii. Significance The results of this study demonstrate the geographic distribution of SFG rickettsiae and clearly indicate the presence of at least four of them in collected ticks. Palestinian clinicians should be aware of emerging tick-borne diseases in the West Bank, particularly infections due to R. massiliae and R. africae.


Introduction
Tick-borne spotted fever group (SFG) rickettsioses are caused by obligate intracellular Gramnegative bacteria belonging to the genus Rickettsia [1]. Feeding ticks can transmit these microorganisms to humans and animals. Various vertebrates are suspected to serve as reservoirs for Rickettsia species; however, some are susceptible to rickettsial infections and may develop rickettsemia following tick bite [2]. The human disease may present as a fever with clinical symptoms including headache, rash, and occasional eschar formation at the site of the tick bites [3].
Mediterranean spotted fever (MSF), caused by Rickettsia conorii, is transmitted by the brown dog tick, Rhipicephalus sanguineus, which is well adapted to urban environments. Previous studies in Israel have documented the presence of two spotted -fever group (SFG) rickettsiae: the tick-borne rickettsia, Rickettsia conorii israelensis and the flea-borne rickettsia, Rickettsia felis [4], [5]. Rickettsia conorii israelensis has been described in Tunisia, Libya, Sardinia-Italy, and Portugal [6]. Furthermore, a number of other SFG pathogenic rickettsiae including Rickettsia africae, Rickettsia massiliae and Rickettsia sibirica mongolitimonae have been detected in ticks from Israel in addition to some rickettsial species such as Candidatus Rickettsia barbariae and Candidatus Rickettsia goldwasserii which were not associated with diseases, to date [7], [8], [9].
The number of newly described SFG rickettsiae has increased in recent decades [4]. Sequence analysis of PCR-amplified fragments targeting genes encoding the citrate synthase (gltA) [10], Rickettsia-specific outer membrane protein (ompA) [11], the 17kDa lipoprotein precursor antigen gene (17 kDa) [12], and the ribosomal 16S rRNA gene [13] has become a reliable method for the identification of Rickettsia species. Molecular typing of infectious agents is important for better understanding of ecological niches and identifying circulating strains and their virulence. Although various Rickettsia species are found in ticks from Israel; to date, no entomological survey has been carried out in the West Bank, and no clinical data or reports for the presence of tick-borne pathogens are available. Thus, this study aimed at identification of the circulating hard tick vectors and Rickettsia species in naturally infected ixodid ticks collected from the West Bank, using PCR and sequence analysis with special focus on their potential threat for humans and animals.

Study area and ticks
To identify the circulating hard ticks in the West Bank and to evaluate the presence of rickettsial infection in these ticks, one to ten hard ticks per animal host, for a total of 1,123, were collected during January to April, 2014 from dogs, camels, sheep, a horse, a wolf, and a tortoise residing in nine districts in the West Bank. The districts are located in three zones in the central, northern and southern regions of the country (Fig 1). All ticks were gently removed from their hosting animals by forceps or hand, and individually placed into small, labeled plastic tubes containing 70% ethanol for morphological identification. The ticks were identified using standard taxonomic keys [14], [15], [16], [17] and stored at −20°C until DNA extraction.

DNA extraction
A maximum of five ticks of different tick species per hosting animal were randomly selected for DNA extraction. Genomic DNA was individually extracted from a total of 867 tick samples. Prior DNA extraction, individual ticks were washed with phosphate-buffered saline (PBS), air dried for 10 min on tissue paper and separately sliced into small pieces by a sterile scalpel blade then manually homogenized with a sterile micro pestle, resuspended in 200 μl of lysis buffer and 20 μl of proteinase K. After overnight incubation at 56°C with a continuous gentle shaking, the DNA was extracted using the QIAamp DNA tissue extraction kit (Qiagen, Hilden, Germany) following the manufacturer's protocol. Purified DNA was stored at 4°C until use. Three μl of template DNA (approximately 100-200 ng per tick) were used for each PCR.

Molecular detection of Rickettsia species
Screening for the presence of rickettsial DNA was carried out by conventional PCR targeting a 250-bp fragment of the ompA gene using 107F and Rm299 primers as described previously [7] with the following modification: final volume of 25μl using PCR-Ready Supreme mix (Syntezza Bioscience, Jerusalem) including primers at 1μM final concentration. Positive samples were further characterized targeting a 426-bp portion of the 16SrRNA and a 265-bp portion of the 17kDa protein gene as previously described [18], [19]. For Rickettsia species identification, strong positive samples were sent for sequencing. DNA extract of the first ompA positive sample, identified as R. massiliae, by direct sequencing, was used as a positive control and ultra pure water were used as a negative control in each amplification reaction.

Phylogenetic analysis
DNA sequences of the positive PCR products were assembled using Bioedit software, used in a BLAST search (ncbi.nlm.nih.gov/blast) and aligned with sequences of other rickettsial species registered in the GenBank. To infer relationships between the obtained amplicons and other reference sequences published in GenBank, a phylogenetic tree was constructed using MEGA6 program.

Statistical tests
Statistical analysis was done using the SPSS program v20. Two -tailed t-test and Pearson's correlation were performed. P-value <0.05 was considered statistically significant.

Ethics statement
The animal population was residing in different farms throughout the West Bank. Prior to ticks sampling, the animal owners were verbally informed about the goals of the project and the sampling protocol. All owners gave their verbal informed consent to collect ticks from their animals. The study was approved by the ethics committee at the Faculty of Medicine in Al-Quds University-Palestine (EC number: ZA/196/013).

Sampling and identification of ticks
A total of 1,123 hard ticks were collected from 320 animals (234 dogs, 68 sheep, 10 camels, 5 goats, one horse, one wolf and one Mediterranean spur-thighed tortoise). From each infested animal one to ten ticks were collected. Of them, 547 were male ticks (48.7%), 511 females (45.5%) and 65 nymphs (5.8%). All tick samples were identified to the species level as follows:  Table 1).

Screening of ticks for rickettsial DNA
A set of 867 ticks comprising eight different species were screened for rickettsial DNA targeting Rickettsia-specific ompA ( Table 2). A sample was considered positive when PCR yielded a fragment with the expected length (250 bp) of the ompA rickettsial gene. The overall prevalence of rickettsiae infection was 17% (148/867) in all ticks ( Table 2). The detection of rickettsial DNA was significantly higher in female ticks (20.4%) than in male (15.7%) and nymph ticks (4.8%, p<0.01). The overall prevalence of rickettsial DNA was markedly higher in ticks collected from Nablus (34.3%; 66/192) compared to other districts (p<0.01) (Fig 1). The infection rates in Rh. turanicus, Rh. sanguineus, Haemaphysalis adleri, H. parva, Hyalomma dromedarii and H. impeltatum ticks were 41.7, 11.6, 16.7, 16.2, 11.8 and 20% respectively. None of the ticks belonging to Rh. bursa (0/13) and H. aegyptium (0/4), taken from sheep and one tortoise, respectively, were infected (Table 2).  Rh. sanguineus ticks, Furthermore, R. africae-DNA was detected in two ticks, H. impeltatum and H. dromedarii obtained from two different camels in Jericho (Fig 2A). There were no cases in which multiple rickettsiae species were detected in the same infected tick.

Phylogenetic analysis
Phylogenetic analysis based on ompA sequences revealed three main clusters: R. massiliae, C. R. barbariae and C. R. goldwasserii. Cluster I, representing the R. massiliae group (n = 28). In this cluster, the nucleotide ompA sequences of R. massiliae identified in this study were identical to each other and to the respective R. massiliae reference sequence (accession no. KJ663746.1) deposited in the NCBI GenBank (Fig 2A). The same samples also showed one cluster and had 100% similarity to the R. massiliae reference strains (NR074486.1 and JN871727.1) based on the 16sRNA and 17kDa analysis, respectively (Fig 2B and 2C). The DNA sequence of Rickettsia canadensis (CP003304.1) that do not belong to the SFG [20], was used as an out group in the analysis of ompA gene while the sequences of Anaplasma phagocytophilum (EU 436157.1) and Rickettsiaceae bacterium (CP009217.2) were used as out groups in the analyses of the 16SrRNA and 17kDa genes, respectively. On the basis of the ompA sequences, DNA sequences of four amplicons (1.4G, 1.4H, 14.8 A, 14.8 B) had several nucleotide differences and showed 96-98% sequence identity to the ompA sequences of R. massiliae identified in this  Tick-Borne Rickettsioses in the Palestinian Territories study and to the reference strain of R. massiliae (KJ663746.1). These samples showed 92% and 89% sequence identity to the ompA sequences of the reference strain sequences of R. aeschlimannii (KF791253.1) and R. raoultii (KR608786.1), respectively. The four samples were further characterized by 17kDa and 16S rRNA genes, the partial 17kDa gene sequence of these samples revealed 94% and 93% similarity to the reference strain sequences of R. raoultii (KT261760.1) and R. conorii (JN182793.1), respectively. Two of them (14.84A and 14.8B) formed a separate branch with in this group complicating the further confirmation of this Rickettsia spp. (Fig 2C). However, none of these samples (n = 4) were successfully sequenced based on 16SrRNA gene. Cluster II represents the C. R. goldwasserii group. The ompA sequences had 100% similarity to the C. R. goldwasserii reference sequence (HM136928.1) whilst the 17kDa sequences of the same samples had 100% similarity to the corresponding sequence of an incompletely described Rickettsia sp. Belarus (JQ711214.1) (Fig 2C). The 16SrRNA sequences showed 99% similarity to that of R. rickettsii (NR103923). Cluster III represents the C. R. barbariae group; the ompA and 16SrRNA sequences had 100% similarity to the Candidatus Rickettsia barbariae reference sequences (JF700253.1 and EU272189.1), respectively (Fig 2A and 2B). When 17kDa sequences were obtained from the same samples, they showed 99% sequence similarity to the homologous fragments of R. raoultii (KT261760.1) and to the other unidentified Rickettsia sp. (KM386654.1) as revealed by BLAST analysis.
A sub-cluster of two ompA sequences was identified as R. africae and showed 100% sequence similarity with a homologous fragment of R. africae (JF700254) detected in Hyalomma detritum from the Golan Heights [9]. The 17kDa sequences of these two samples had also 100% nucleotide identity to that of R. africae (KF646137.1) but they were not detected by the 16S rRNA PCR.

Discussion
The overall prevalence of SFG rickettsiae detected in ixodid ticks in nine Palestinian districts was 17%. Ticks detected in this study belonged to three genera (Rhipicephalus, Haemaphysalis and Hyalomma) and collected from different host animals. The findings of this study highlight the importance of hard ticks for human health in the West Bank. Rhipicephalus sanguineus was the most prevalent tick species found in this study. It parasitized a wide range of mammals including dogs, sheep, goats and horses; however the main vector for SFG rickettsiae detected in this study was Rh. turanicus. Our results document the detection of two important human pathogens in ticks from the West Bank, R. massiliae and R. africae. R. massiliae was the most prevalent rickettsial species detected in this study. This pathogen was first isolated from Rh. sanguineus in Marseille in 1992 [21], and since then it has been detected in ticks within the genus Rhipicephalus in Greece, Spain, Portugal, Switzerland, Sardinia (Italy), Morocco and Israel [22], [23], [4], [24], [25], [7]. The first isolation of R. massiliae was reported from a human patient in Sicily in 1985 and identified in 2005 [26]. The clinical presentation of R. massiliae infection has been previously described [23], [27], [28]. Common clinical signs include fever, night sweats, headache, maculopapular rash and necrotic eschar at the tick bite site. In agreement with other studies, all ticks infected with this pathogen in this study belonged to the genus Rhipicephalus except for two which belonged to the genus Haemaphysalis obtained from the same dog in Jenin district. However, detection of R. massiliae in Haemaphysalis punctata ticks was previously reported in southeast England [29].
In this study, most of R. massiliaeinfected ticks were removed from dogs (77%) and to a lesser extent from sheep (23%), increasing the risk of transmitting rickettsial infections to the animal owners, i. e. dog owners, shepherds and farmers. The phylogenetic tree based on partial DNA sequences of ompA, 16SrRNA and 17 kDa showed higher genetic variability among the R. massiliae strains using ompA gene than 16SrRNA and 17 kDa loci. The second SFG pathogenic Rickettsia found in this study was R. africae, the agent of African tick bite fever. The detection of R. africae, in Hyalomma spp. collected from camels in the West Bank confirms the results of a previous report associating R. africae with Hyalomma ticks in Egypt and Israel [30], [8], [9]. Livestock movements and migratory birds may play a role in the geographic spread of R. africae [31]. We observed a strong geographic correlation between the overall prevalence of rickettsial DNA in ticks collected from Nablus (Northern district) compared to the prevalence of infected ticks collected from other districts in the north, Ramallah (centre) and Hebron (south). Future studies with high representative number of ticks are required to address the comparative importance of geographic distribution on the infection rate of these ticks in the West Bank.
Candidatus. R. goldwasserii was also detected in Rhipicephalus ticks collected from dogs in the northern region of the West Bank. This Rickettsia was first detected in two Haemaphysalis ticks (H. adleri and H. parva) from golden jackals in Israel. Phylogenetic analysis based on concatenated four gene fragments (gltA-ompA-sca4-ompB) indicated that the nucleotide sequences of these SFG rickettsiae belonged to a novel phylogenetic lineage related to C. R. siciliensis detected in Rh. turanicus ticks. [32]. In the present study, the identification of C. R. goldwasserii in Rh. sanguineus and Rh. turanicus, in addition to the already known Haemaphysalis spp, expands the current knowledge concerning tick species that host C. R. goldwasserii in our region. Based on ompA phylogeny, high genetic homology was observed among the C. R. goldwasserii group identified in this study. However, these samples were found to be 100% similar to the corresponding sequence of a not well characterized Rickettsia sp. Belarus and 99% similar to that of R. rickettsii based on 17kDa and 16srRNA respectively. Thus, for a more accurate classification of this uncultivated SFG Rickettsia, further testing and phylogenetic analysis with additional genes is needed since no sequences of these two latter genes of C. R. goldwasserii were available in the GenBank. This is the first study to report the presence of C. R. barbariae in 9.6% of Hyalomma ticks in the West Bank. The presence of C. R. barbariae has been previously reported in several Rhipicephalus spp. in Portugal, Italy, France, Cyprus and later in Rhipicephalus ticks flagged from the vegetation in Israel [24], [33], [2], [9]. However, no ompA sequence differences were observed in C. R. barbariae DNA detected in Hyalomma or Rhipicephalus ticks collected from camels, dogs and sheep residing in different localities throughout the West Bank. In conclusion, the findings presented in this study provide evidence for the presence of R. massiliae and R. africae in different ixodid ticks collected from various regions in the West Bank. In addition to Rhipicephalus species, members of the genus Hyalomma and Haemaphysalis may also play an important role in the epidemiology of SFG Rickettsia spp. Clinicians in the West Bank and neighboring countries should consider a range of SFG diseases in the differential diagnoses of patients present with fever of unknown origin and clinical signs compatible with rickettsioses.