Rickettsiosis and theileriosis can cause mortalities in camel populations. This study was conducted to achieve 2 objectives: (1) to detect the presence of SFG Rickettsia sp. and Theileria sp. in Hyalomma dromedarii Koch, 1844 (Acari: Ixodidae) ticks and (2) to determine their prevalence in the tick population on the sampled camel farms in Al-Ain, United Arab Emirates (UAE). Camel ticks (H. dromedarii) were collected from a total of 625 one-humped camels (Camelus dromedarius) in 22 sampling locations in Al-Ain, UAE. Tick samples were analyzed by Polymerase Chain Reaction (PCR). An SFG Rickettsia sp., which was 99% similar to Candidatus ‘Rickettsia andeanae’ and Rickettsia endosymbionts, was detected only in 2011 and its prevalence in the sampled ticks was 1.12%, while Theileria annulata was detected in both years with a prevalence of 2.3% and 1.60%, respectively. Additionally, T. annulata was present in all of the sampling zones (east, west, north, and south) of the study area, whereas SFG Rickettsia sp. was limited to 2 zones only (east and south). The geographic distributions of SFG Rickettsia sp. and T. annulata showed no overlap throughout the entire study area except in one location in which both of the disease agents were present. This study is the first published record on the presence of SFG Rickettsia sp. and T. annulata in camel ticks in the UAE. In addition, the current study should serve as a foundation for more studies leading to a better understanding of the reservoir potential of camels and the risk posed by these 2 disease agents to camels and other livestock.
Ticks are vectors to many pathogens and parasites that are of significance to human, domestic animal and wildlife health (Al Khalifa et al. 2006; Diab et al. 2006). Hyalomma (Acari: Ixodidae) ticks are widespread in North Africa, Southern Europe, Middle East, Central Asia and China and may serve as vectors of Crimean-Congo hemorrhagic fever (CCHF), rickettsiae and tropical theileriosis (Wernery & Kaaden 2002; Atkas et al. 2004). Few studies document tick-borne diseases in the Middle East region (Wernery & Kaaden 2002). Rickettsia-like microorganisms were first described from the tick Ornithodoros savignyi collected from camels in Egypt (Roshdy 1968). Several rickettsiae including Anaplasma marginale, Coxiella burnetii, Rickettsia aeschlim were reported from Egypt, Somalia and Nigeria from several Hyalomma spp. including H. dromedarii Koch, 1844 (Wernery & Kaaden 2002; Loftis et al. 2006). Seventeen species of the genus Rickettsia are categorized within the Spotted Fever Group (SFG) rickettsiae (Hackstadt 1996) and several have been documented in the Middle East, although some reports are considered questionable (Wernery & Kaaden 2002). SFG rickettsiae are intracellular, gram-negative bacteria that are transmitted by Hyalomma ticks, and generally they are host-specific and have the ability to infect a variety of atypical hosts, including humans. Theileria annulata (an apicomplexan) is a parasite of potentially important domestic animal health concern in the region because it infects cattle and is transmitted by Hyalomma spp. (Hussein et al. 1991; De Kok et al. 1993; Atkas et al. 2004). The parasite causes acute, often fatal, disease (tropical theileriosis) and occurs from North Africa and Southern Europe, through the Middle East and across Southern Asia. Both diseases could cause great losses in livestock production in the region (Wernery & Kaaden 2002).
The importance of this study is that it explores the presence of 2 pathogens that can infect the one-humped camel (C. dromedarius), which is the most important animal in the UAE. Historically and to date, camels in the UAE produce milk and meat plus they have been used in racing, which is a traditional sport, practiced by many Emiratis. Hyalomma ticks are abundant in the UAE, with heavy infestation of primarily H. dromedarii occurring widely in camels (Wernery & Kaaden 2002; Diab et al. 2006). There are other ticks in the Middle East and North African region including H. anatolicum, H. excavatum, Boophilus microplus and O. savignyi reported from camels, cattle and other livestock (Diab et al. 2006). It was reported that the prevalence of H. excavatum infesting cattle to be 92.35% in Iran, where T. annulata is endemic (Ramzi et al. 2003). In Saudi Arabia, H. anatolicum had a prevalence of 77.3%, with considerable geographic variation in the prevalence of different species of ticks (Diab et al. 2006). Thus, multiple species of ticks could be involved in maintaining a number of pathogens and parasites in the Middle East. This study was conducted to achieve 2 objectives: : (1) to detect the presence of SFG Rickettsia sp. and Theileria sp. in Hyalomma dromedarii ticks and (2) to determine their prevalence in the tick population on the sampled camel farms in Al-Ain, UAE.
Materials and Methods
This study is a cross-sectional prospective exploring the presence of 2 pathogens in camels' ticks that were collected from Al-Ain farms. The inclusion criterion is the approval of the farm owner to enter the farm and collect the ticks. The number of camels that were sampled from each farm represented a percentage of the total number of camels reared at each farm.
STUDY LOCATION AND TICK COLLECTION
Al-Ain area was theoretically divided into 4 sampling zones (east, west, north, south). Camel farms were chosen from each zone randomly. The selected farms represented all of the major camel breeding locations in the study area. Al-Ain is located about 120 km south-southeast of Abu Dhabi (N 24° 11′ E 55° 45′). The inland location of Al-Ain makes its environment drier and warmer compared to Abu Dhabi or Dubai. In 2010, ticks were collected from individual camels (n = 133) of 14 farms. In 2011, a larger number of camels (n = 492) was sampled for ticks from 20 farms. In both years, collection was done during spring and summer (Mar-Aug). In each farm, 15–20 ticks were removed manually from each one of the randomly selected camels. Ticks were placed in 50 mL blue cap plastic tubes (Sterilin, UK). The tubes containing ticks were put on ice inside a cooler and were taken to the Entomology Laboratory at UAE University where they were frozen at -20 °C until further processing. All ticks were morphologically identified to the species level by using a tick taxonomic key (Walker et al. 2003). The morphological identification was confirmed by molecular methods.
DNA EXTRACTION FROM TICKS
As previously mentioned ticks were collected from 625 camels and were placed in tubes. From each tube, one unsexed engorged adult tick was picked for DNA extraction. Each tick was washed individually in 500 μL 70% ethanol followed by 500 μL sterile double distilled H2O to remove any contamination attached to its body. To extract genomic DNA, each tick separately was manually crushed with a sterile ceramic mortar and was placed in a sterile 1.5 mL Eppendorf tube. DNA was extracted from the tick homogenate using the QIAamp Tissue Kit (Qiagen, Hilden, Germany), following the protocol of the manufacturer. To save time and labor, a second extraction method was applied using a Maxwell 16 automated DNA extractor (Promega, USA) following the manufacturer's protocol. The 625 extracted DNA samples were stored in a -80 °C freezer.
POLYMERASE CHAIN REACTION AMPLIFICATION
The template DNA was amplified in a 25 μL reaction mixture containing about 80 ng of tick DNA, 20 pmol of each primer pair, and 12.5 μL 2X PCR Master Mix (Promega, USA). PCR amplifications were carried out in a Swift MaxPro thermocycler (ESCO, Singapore).
In order to confirm the morphological identification of the ticks the primer pair Fish1F: 5′-TCAACCAACCACAAAGACATTGGCAC-3′ and Fish1R: 5′-TAGACTTCTGGGTGGCCAAAGAATCA-3′ (Ward et al. 2005) was used to amplify the cytochrome oxidase c sub-unit I (COI) of the mitochondrial DNA. Amplification started with an initial denaturation for 2 min at 95 °C followed by 30 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 54 °C, and extension for 90 s at 72 °C. A final extension step was performed for 10 min at 72 °C.
Rickettsia pathogen was detected according to a published method (Blair et al. 2004). Briefly, the detection involved a nested PCR of the outer membrane protein (ompA) gene. In the first PCR, the amplification of a 590 bp fragment was obtained by using the forward primer 5′-ATGGCGAATATTTCTCCAAAA-3′ and the reverse primer 5′-GTTCCGTTAATGGCAGCATCT-3′. In the second PCR, the amplification of a 540 bp fragment was obtained by using the forward primer 5′-AAGCAATACAACAAGGTC -3′ and the reverse primer 5′-TGACAGTTATTATACCTC -3′. Following initial denaturation for 1 min at 94 °C, 35 cycles of denaturation for 30 s at 94 °C, annealing for 1 min at 50 °C, and extension for 4 min at 68 °C were performed. A final extension step was done for 20 min at 72 °C. Every PCR included a negative control (no-template DNA) to make sure there was no contamination. Also, a positive control (Rickettsia endosymbiont of Amblyomma maculatum ompA, Gen-Bank# JX134638) was used to insure that the conditions were optimum and the primers were properly annealing to the target region on the template DNA. Theileira genus-specific primers were used in the PCR to produce a specific band size in the positive samples. A 560 bp fragment of the ssrRNA gene was amplified from DNA by using the forward primer 5′-GTCTTGTAATTGGAATGATGG-3′ and the reverse primer 5′-CCAAAGACTTTGATTTCTCTC-3′. An initial denaturation step at 94 °C for 2 min was followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s and extension at 72 °C for 60 s. A final extension was done at 72 °C for 7 min, followed by a hold step at 4 °C. Every PCR included a negative control (no-template DNA). The PCR products were resolved by electrophoresis in a 1.5% agarose gel, stained with ethidium bromide, and photographed by UVDI gel documentation system (Major Science, Taiwan). By the end of each PCR amplification process the amplicons of the positive samples (i.e. producing the expected band size on electrophoresis gel) were saved for DNA sequencing. The amplicons were purified by a Qiagen PCR purification kit (Qiagen, Hilden, Germany) following the manufacturer's protocol.
DNA SEQUENCING AND SEQUENCE ANALYSIS
The identification of the SFG Rickettsia (group level) and Theileira (species level) was done based on the similarity between the DNA sequences of the bands produced by the specific primers used in the present study and GenBank sequences. The PCR purified amplicons were sequenced by Source Bioscience (Cambridge, UK) and Macrogen (Seoul, South Korea). Sequences were analyzed by BLAST ( http://blast.ncbi.nlm. nih.gov/Blast.cgi) sequence analysis tool in the GenBank database. Sequences were submitted in the GenBank and received accession numbers. MEGA5 (Tamura et al. 2011) was used to draw a phylogenetic tree for the Theileira species by using the Neighbor-Joining method (Saitou & Nei 1987). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) was shown next to the branches (Felsenstein 1985). The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method (Kimura 1980). A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequence and are in the units of the number of base substitutions per site. The analysis involved 12 nucleotide sequences (the Theileria DNA sequence reported in the present study plus 11 sequences taken from its BLAST search results). All positions containing gaps and missing data were eliminated. There were a total of 510 positions in the final dataset.
The morphological identification of the ticks reported in this study as H. dromedarii was confirmed based on a DNA identification of the sequences of the cytochrome oxidase subunit I (COI). The sequence was submitted in the GenBank and received an accession number (KJ022631).
The 540 bp DNA fragment specific for the SFG Rickettsia sp. (Fig. 1) was not detected in ticks in 2010. However, it was detected in 2011 in 7/492 (1.42%) of the sampled tick population (Table 1). This detection occurred in 3 sampling locations: Al-Arad, Malaket, and Al-Wagan. The sequence of the ompA gene of the SFG Rickettsia sp. reported in this study was submitted in the GenBank and has been assigned an accession number (KF156874). The molecular identification of SFG Rickettsia sp. based on a BLAST analysis (Table 1) showed 99% sequence identity and 100% coverage with Candidatus ‘Rickettsia andeanae’ and several Rickettsia endosymbionts, all of which belong to the spotted fever group.
Positive samples of SFG Rickettsia sp. and T. annulata in H. dromedarii ticks according to farm locations in Al-Ain, UAE.
The molecular identification of Theileria sp. based on a BLAST analysis revealed that it belonged to the species T. annulata. This identification was supported by 99% sequence identity with other T. annulata GenBank sequences and an E-value = 0 (Table 2). Also, the phylogenetic tree showed a high genetic relatedness with 7 GenBank T. annulata DNA sequences and a 95% bootstrap value (Fig. 2). The T. annulata DNA sequence reported in this study was submitted in the GenBank and has been assigned an accession number (KC902832.1). The 560 bp DNA fragment specific for the T. annulata (Fig. 1), was detected in 8/492 (1.62%) of the ticks (Table 1). The T. annulata pathogen was detected in both years in camel ticks (Fig. 1) and its prevalence in the sampled tick population was 2.3% and 1.60% in 2010 and 2011, respectively (Table 1). In 2010, positive samples were discovered in Maragh and Swehan, which were located in the east and west of Al-Ain, respectively. In 2011, the detection occurred in the east (Mezyad and Dwar Al-Shahenat), west (Swehan, Al-Sad and Bede' Fares), north (Bede'Bent Suod), and south (Al-Arad and She'ab Al-Ghaf) of Al-Ain (Table 1). No significant difference was found between the prevalence of SFG Rickettsia sp. and T. annulata in ticks (χ2 = 0.068, df = 1, P = 0.794) at the significance level (P < 0.05).
Constant surveillance is imperative to maintain good camel health status and for the early detection of disease emergencies. In this study, a DNA-based detection technique was used to document the presence of SFG Rickettsia sp. and T. annulata in camel ticks. The presence of the pathogen's DNA in a tick sample was considered as an evidence of the existence of the pathogen itself in the sample. The accuracy of the detection process was confirmed when SFG Rickettsia-specific primers (Blair et al. 2004) produced the exact expected band size in the nested PCR and by the BLAST results of the DNA sequence of the amplicon. Because the SFG Rickettsia sp. reported in the current study had 99% sequence identity and 100% coverage with 2 organisms in the GenBank namely Candidatus ‘Rickettsia andeanae’ and several Rickettsia endosymbionts a species name was not assigned to it. More primers need to be used with the ompA primers in future studies to provide an accurate identification at the species level, which was outside the scope of the current study. According to the detection process, the SFG Rickettsia ompA gene occurred in 3 locations: Al-Arad, Malaket, and Al-Wagan. Malaket is located in the eastern part of Al-Ain while Al-Arad and Al-Wagan are in the south. This showed that the pathogen was not found in 2 of the sampling zones (west and north). However, this situation may change in the future if infested camels or camel ticks reach these zones possibly through sales or exchange of camels. Wernery & Kaaden (2002) mentioned that disease has not been reported in old world camels as a result of Rickettsiae and they are not believed to be an important part in the cycle of Rickettsiae. However, the detection of SFG Rickettsia sp. in the present study implies that there is a possibility for this pathogen to be present in domestic camels because it was detected in ticks feeding on their blood. Because SFG Rickettsia is considered a threat to human as well as cattle health, reporting its presence associated with camel ticks has a great significance to the health authorities in UAE and the neighboring region. Furthermore, this may indicate that the rural population of Al-Ain, UAE, can be at risk for tick-borne rickettsioses if pathogenic species were to be present. Certainly, further studies are needed for better understanding of its epidemiological characteristics. T. annulata was found in camel ticks in all of the sampling zones whereas SFG Rickettsia sp. was limited to 2 zones only. The geographic distributions of SFG Rickettsia sp. and T. annulata did not overlap in the entire study area except in one location (Al-Arad) in which both of the pathogens were present. Although Theileria spp. are regarded as common in livestock (e.g. T. camelensis and T. dromedarii in camels, (Wernery & Kaaden 2002)), T. annulata is regarded as a parasite of cattle (Beck et al. 2009; Wernery & Kaaden 2002; Atkas et al. 2004). The prevalence of T. annulata from cattle in Hofuf, Saudi Arabia was 20.9% (Hussein et al. 1991). In an experiment 5 healthy camels were injected with T. annulata but the pathogen could not be detected during the course of the experiment (one month) (Wernery & Kaaden 2002). Thus it was concluded that camels did not play a role in the epidemiology of T. annulata. On the contrary, the present study found T. annulata in ticks feeding on camels' blood, suggesting that the parasite circulates between camels and ticks albeit in low prevalence in Al-Ain region, UAE. This aspect needs to be examined in future studies. Furthermore, in this study some of the camels' farms had sheep and goats enclosed in separate areas, so it is possible that ticks were serving as mechanical or biological vector between camels, sheep and goats. T. annulata poses a greater risk to cattle; hence, a comprehensive study is warranted to explore the possible role of camels' ticks as a vector of T. annulata to cattle when the 2 animal species are reared on the same farm. For both of the SFG Rickettsia sp. and T. annulata, it needs to be mentioned that we were not able to affirm or negate whether the sampled camels showed signs of illness at the time of sampling because ticks were not collected by veterinarians. Still, we can confirm that at the sampling time none of the camels included in this study exhibited major signs of illness warranting isolation from the rest of the camels on the farm.
Molecular identification of SFG Rickettsia sp. and T. annulata based on DNA similarity according to BLAST.
The selection of farms was not based upon a systematic random sampling, which was due mainly to logistic and financial limitations. Nevertheless, the results of this study remain valid because increasing the number of samples will increase the power of detection. Consequently, more accurate estimations of the prevalence will be made. Systematic random design is likely to result in having greater chance to detect the pathogens in all sampling locations. On the other hand, the above limitations have no effect on the molecular reliability of the identification of the pathogens nor on the identification of the collected ticks.
In conclusion, the current study reported the presence of SFG Rickettsia sp. and T. annulata in camel ticks in the UAE. The prevalence of the 2 organisms in the ticks was < 2%, which is important for the public and animal health authorities in the UAE. However, a real risk of tickborne rickettsioses in Al-Ain rural population becomes conceivable if pathogenic species were to be present. Therefore, a study covering the entire UAE camel breeding range with a complete systematic sampling is warranted. Furthermore, we suggest that SFG Rickettsia sp. and T. annulata detection in future studies should be coupled with examination of camels for signs of illness at the time of tick collection. To our knowledge, this is the first published record on the presence of SFG Rickettsia sp. and T. annulata in H. dromedarii ticks in the UAE.
We thank Mariam Abu Shatarah and Fatima Barakat for assistance in DNA extraction and PCR work. This study was supported by a grant from the Emirates Foundation ( www.emiratesfoundation.ae).