Molecular evidence of potential novel spotted fever group rickettsiae, Anaplasma and Ehrlichia species in Amblyomma ticks parasitizing wild snakes

Background Amblyomma ticks parasitize a wide range of animals in tropical regions. This study describes the identification of Amblyomma ticks from wild snakes in Malaysia and the detection of potential human pathogens such as Rickettsia, Anaplasma, Ehrlichia and bartonellae in the ticks. Findings Twenty one adult ticks (twelve A. varanense and nine Amblyomma helvolum ticks) identified from seven Python molurus snakes in Sepang and a pool of six A. helvolum ticks from a Naja sumatrana snake in Johore, Malaysia were investigated in this study. Amplification of the citrate synthase (gltA), 190-kDa surface antigen gene (ompA), 135-kDa surface antigen (ompB) and surface cell antigen (sca4) genes followed by sequence analysis confirmed the presence of two potential novel spotted fever group rickettsiae in the ticks. Candidatus Rickettsia sepangensis from an engorged A. varanense tick demonstrated high sequence similarity to Rickettsia tamurae; while Candidatus Rickettsia johorensis from two samples (individual and pooled) of A. helvolum and two A. varanense ticks were closely related to Rickettsia raoultii. Anaplasma and Ehrlichia DNA were detected from seven and two ticks, respectively. No bartonellae was detected from any of the ticks. Conclusion The finding in this study suggests that Amblyomma ticks parasitizing wild snakes may serve as reservoir hosts and carriers for rickettsioses, anaplasmosis and ehrlichiosis in this region.


Background
Ticks are the vector for numerous emerging zoonotic diseases which can be severe and life-threatening to humans. In nature, ticks and a wide range of animals may act as reservoirs or amplifiers for human pathogens such as spotted fever group rickettsiae, anaplasma, ehrlichiae and bartonellae. Humans can be accidentally infected with these organisms through tick bites. The ticks belonging to the genus Amblyomma have been implicated as a carrier for several pathogenic rickettsiae including Rickettsia rickettsii, R. aeschlimannii, R. raoultii, and R. tamurae [1], Anaplasma phagocytophilum, Ehrlichia chaffeensis and E. ewingii [2,3]. Additionally, Bartonella DNA has also been detected in A. americanum ticks [4].
Amblyomma ticks parasitize a wide range of animals and are often seen on mammalian hosts, reptiles and amphibians [5,6]. However, information is lacking on tick carriage of emerging human pathogens in the tropical region. In this study, we assessed the occurrence of these microorganisms in Amblyomma ticks parasitizing wild snakes in Malaysia by using molecular approach.
Tick DNA was extracted using QIAamp DNA mini kit (Qiagen, Hilden, Germany) in accordance to the manufacturer's instruction. Four rickettsial-specific genes were targeted for amplification from the tick samples, i.e., citrate synthase gene (gltA), 190-kDa outer membrane protein gene (ompA), 135-kDa outer membrane protein gene (ompB) and surface cell antigen (sca4) [8][9][10][11]. Identification of Anaplasma and Ehrlichia DNA in the samples was performed using a PCR assay targeting 16S rRNA gene of the organisms [12] followed by sequence analysis. For further differentiation of Anaplasma spp., amplification of the full length sequences of 16S rDNA and msp4 genes were performed [13]. A PCR assay targeting citrate synthase (gltA) gene was performed for detection of bartonellae DNA [14]. Cloned PCR2.1-TOPO T/A plasmids (Invitrogen, USA) with amplified gltA fragment from R. honei (strain TT118), ompA and ompB fragments from rickettsial endosymbionts (98% similarity to R. heilongjiangensis and R. raoultii, respectively) of tick samples were used as positive controls. BLAST analysis was performed to search for homologous sequences in the GenBank database. To determine the phylogenetic position of the rickettsiae identified in this study, dendrogram was constructed based on concatenated sequences of gltA (1040-1046 nucleotides) and ompA (407-431 nucleotides) genes using neighbour-joining method of MEGA software [15]. Table 1 shows the amplification of rickettsial gltA gene from three A. varanense (S5, S4-2 and S7-2) and two A. helvolum tick samples (S6-1, P1). The gltA and ompA sequences from the S5 tick was almost similar (99.0% and 97.7%, respectively) with R. tamurae strain AT-1 from A. testudinarium tick in Japan [16]. However, the ompB gene of the rickettsia was unable to be amplified and no significant similarity was obtained for the amplified sca4 fragment.
According to the current criteria for speciation of rickettsial species, uncultured rickettsia exhibiting sequence similarity of ≤99.9% for gltA, ≤ 98.8% for ompA, ≤99.2% ompB and ≤99.3% for sca4 genes with a validated Rickettsia species may be given Candidatus status [18]. Hence, the rickettsiae are thus named as Candidatus Rickettsia sepangensis and Candidatus Rickettsia johorensis, respectively, in accordance to the location of their first sample collection. The dendrogram constructed using concatenated sequence of gltA and ompA gene fragments ( Table 2 and Figure 1) confirmed the clustering of Candidatus Rickettsia sepangensis with the type strain of R. tamurae, and Candidatus Rickettsia johorensis with R. raoultii type strains. Several spotted fever group rickettsiae with unknown or potentially pathogenicity for humans have been reported in the Southeast Asia region, mainly in Thailand. R. honei (strain TT-118) and R. thailandii sp. nov. have been identified from Ixodes and Rhipicephalus ticks [19,20]. Closely related species of R. raoultii have also been detected from A. helvolum from a lizard (Varanus salvator) in Thailand [21]. Exposure to infected snake ticks may pose risks to human health as R. tamurae and R. raoultii have been implicated in human infections [22,23]. High antibody prevalence to R. honei (TT118 strain) has been reported in febrile patients in rural areas in Malaysia [24]. However, information on the type of spotted fever group rickettsiae is still lacking.
Anaplasma DNA was amplified from seven ticks ( There is no report on the human infections caused by tickborne pathogens with reptile as a host in Southeast Asia. The presence of SFG rickettsiae (Rickettsia species closely related to R. raoultii, R. tamurae and R. bellii) has been recently shown in A. varanense and A. helvolum in Thailand [25]. Detection of R. honei in a reptilian tick, Bothriocroton hydrosauri (formerly Aponomma hydrosauri) has been reported in Australia [26]. Rickettsia spp. closely related to R. tamurae has also been detected in A. fimbriatum ticks collected from reptiles (yellow-spotted monitor, water python and green-tree snake) in the Northern Territory of Australia [27], and A. exornatum tick from a lizard (Varanus olivaceus) in United States of America [28]. In the South America, Rickettsia sp. strain Colombianensi has been identified from A. dissimile ticks parasitizing iguanas in Colombia [29]. All these findings suggest the existing of a natural cycle of spotted fever group rickettsial infection in ticks and snakes in different geographical regions. A. phagocytophilum has been detected in A. flavomaculatum tick collected from a Varanus exanthematicus lizard imported into Poland [30]. Meanwhile, the detection of Ehrlichia spp. from ticks collected from snakes has not been reported previously and thus, merits further investigation.
A. helvolum ticks have been identified from different snakes including Python sp., Ptyas (Zamensis) korros and Naja naja (Kohls) [7] in Malaysia. A. varanense is also one of the most widespread Amblyomma ticks in large snakes in Southeast Asia [5]. As P. molurus and N. sumatrana snakes are native to Southeast Asia [31,32], ticks parasitizing the snakes could be endemic where the animal hosts are available. Although there is no data about the affinity of the ticks to bite humans yet, the detection of rickettsial agents in the snake ticks poses a risk to both wildlife and human. Further work is required to assess the prevalence of these potential tick-borne pathogens on a larger scale.   Table 2. Bootstraps analysis was performed with 1000 replications. Numbers in brackets are GenBank accession numbers. Scale bar indicates the nucleotide substitutions per sites.