ORIGINAL ARTICLES AND REVIEWS
Fabiola ManciniI; Massimo CiccozziI,II; Alessandra Lo PrestiI; Eleonora CellaI; Marta GiovanettiI; Marco Di LucaI; Luciano TomaI; Riccardo BianchiI; Cristina KhouryI; Giovanni RezzaI; Alessandra CiervoI
IDipartimento di Malattie Infettive, Parassitarie ed Immunomediate, Istituto Superiore di Sanità, Rome, Italy
IIUniversità Campus-Biomedico, Rome, Italy
BACKGROUND: Ticks are vectors and important reservoirs for microbial agents that cause disease in humans and animals. Among these pathogens, the members of Rickettsia species play an important role in public health.
AIM AND METHODS: One hundred twenty-nine ticks belonging to four tick species (Ixodes ricinus,Rhipicephalus turanicus,Dermacentor marginatus, and Haemaphysalis punctata) were collected at different sites of the Insugherata Natural Reserve, localized in the urban area of Rome, Italy. Questing ticks were tested by PCR for Rickettsia spp., amplifying partial gene of ompA.
RESULTS: Forty-six ticks were found to be infected with Rickettsia species. Five SFG rickettsiae were identified: three human pathogens Rickettsia conorii, Rickettsia massiliae and Rickettsia aeschlimannii, and two putative new strains Rickettsia sp. strain RM1 and Rickettsia sp. strain RM2. The phylogenetic analysis of partial gene sequences of ompA, gltA, and 17-kd antigen showed that they clustered with several rickettsiae with unidentified pathogenicity. However, Rickettsia sp. strain RM1 and Rickettsia sp. strain RM2 clustered in a statistically supported clade with R. massiliae, and R. monacensis, respectively.
CONCLUSION: Our findings suggest that Rickettsia species other than R. conorii are implicated in human disease in Italy.
Key words: tick, urban park, Rickettsia SFG, molecular characterization
Ticks are known to be vectors and important reservoirs for microbial agents that cause disease in humans and animals. Among these pathogens, the members of Rickettsia species play an important role in public health. The genus Rickettsia is divided into three groups on the basis of phenotypic criteria: the spotted fever group (SFG), the typhus group (TG), and the scrub typhus group (STG) which is absent in Europe .
The most common and well-known tick-borne rickettsiosis in Europe is the Mediterranean Spotted Fever (MSF). The MSF is due to Rickettsia conorii, but in the last decade other rickettsial species, such as R. slovaca,R. sibirica mongolotimonae,R. helvetica,R. monacensis,R. massiliae,Rickettsia aeschlimannii,R. africae or R. akari, were identified in the Mediteranean basin and have been implicated or potentially involved in human diseases . In the past, identification and differentiation of rickettsiae were based exclusively on serological data, ecology, and epidemiology of these microorganisms. The discovery of these new species has been made possible by the use of molecular identification techniques. Molecular methods can offer a real burst in the investigation of novel Rickettsia species and/or their interaction with new vectors. In particular, PCR based method and DNA sequencing are helpful tools for the identification of rickettsial DNA in a variety of human specimens and arthropods, and allows the simultaneous detection of different microorganisms in the same sample. Monitoring vector distribution and the prevalence of tick-transmitted pathogens is therefore essential to describe and understand the risk of tick-borne disease.
In Italy, from 1998 to 2002, 4604 clinical cases of rickettsiosis with 33 deaths were reported . Italian regions with elevated incidences of rickettsial diseases are Sicily, Sardinia, Lazio, and Calabria, while in other regions, rickettsiosis is sporadic .
Currently, in Italy, several studies are focused on molecular identification and characterization of Rickettsia spp. in ticks and human samples to verify the potential presence of species that have been recently discovered in other parts of Europe [3-8].
In order to provide a useful contribution in this field, we analyzed the presence of Rickettsia spp. in questing ticks collected in an urban park of Rome, during an entomological survey conducted in 2011 . The aims of the present study were i) to investigate the prevalence of Rickettsia spp. in ticks collected in the public park of Rome, highly frequented by daily visitors and used for recreational activities; and ii) to characterize rickettsiae in infected ticks using molecular methods, including PCR, sequence, and phylogenetic analyses. Hereby we report the results of this investigation.
MATERIALS AND METHODS
Questing ticks were collected in the Insugherata Natural Reserve, localized in the north-western sector of Rome and connected to the green zones outside the urban area. The park is characterized by woods and bush. Moreover, the reserve, with its Mediterranean climate hosts a rich fauna: foxes, weasels, and porcupines are very common, whilebadgers occur only sporadically. Many small mammals (Apodemus sylvaticus, Microtus savii, Suncus etruscus, Erinaceus europaeus, Talpa europaea, and Muscardinus avellanarius) and a great variety of birds, reptiles, and amphibians complete the wild fauna of the reserve. Only in the past few years, wild boar have spread from northern boundaries of the park. Tick collections were conducted in three selected sites within the park twice a month from January to December 2011, along transects of 100 m each for a total of 12 fixed transects covered per visit. Questing ticks were collected in all sites by dragging a 1 m2 woolen blanket through the vegetation .
Ticks were identified according to morphological characters ,and stored at -80 ºC.
Ticks were individually dissected and homogenized under sterile conditions. Genomic DNA was extracted using Dneasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to manufacturing protocol. DNA samples were stored at -20 ºC and later used as templates for the PCR amplification.
Rickettsiae DNA detection
Detection of Rickettsia spp. DNA was done with primers RpCS.877p-RpCS.1258n of the citrate synthase gene (gltA) . Two different sets of primers of the groEL gene , were used for the discrimination between SFG and TG (Table 1). PCR products were resolved by electrophoresis on a 1.5% agarose gel, then stained with ethidium bromide, and visualized under UV light.
The genomic DNA of R. conorii and R. typhi, were used as positive controls in specific PCR analyses.
PCR amplification and sequencing of specific rickettsial gene target fragments
As shown in Table 1, PCRs were performed using oligonucleotide primers: CS409d and Rp1258n which amplify a 750 bp fragment of the gltA : Rr 190.70 and Rr 190.701 for the outer surface protein rOmpA (ompA gene), which amplify a 629-632 bp portion ; Rr17.13 and Rr17.495r of the17-kDa protein (17-kd gene), which amplify a 400 bp fragment .
PCR products were purified by the QIAquick PCR purification kit (Qiagen, Hilden, Germany), and amplicons were sequenced in the forward and reverse directions with the same primer pairs used for the PCR amplifications. Sequencing was performed by Bio-Fab research (Italy; http://www.biofabresearch.it), and DNA sequences were compared with available databases in GenBank using the Basic Local Alignment Search Tool (BLAST) on http://blast.ncbi.nlm.nih.gov.
The rickettsial nucleotide sequences of the partial gltA, ompA and 17-kd genes were submitted to the NCBI GenBank.
Three different datasets were built. The first one included the rickettsial nucleotide sequences of the partial ompA gene isolated from Rome, plus 165 representative rickettsial species sequences downloaded from GenBank (www.ncbi.nlm.nih.gov/). The second dataset included the rickettsial nucleotide sequences of the partial gltA gene, isolated from Rome, plus 122 representative rickettsial species sequences downloaded from GenBank (www.ncbi.nlm.nih.gov/). The third dataset included the rickettsial nucleotide sequences of the partial 17-kd gene collected from Rome, plus 76 representative rickettsial species sequences downloaded from GenBank (www.ncbi.nlm.nih.gov/). The sequences of all datasets were aligned using Clustal X software , and then manually edited using Bioedit software . Version 3.7 of the ModelTest program was used to select the evolutionary model that best fitted the sequence data . Maximum likelihood phylogenetic trees were constructed with the GTR + I + G nucleotide substitution model for the first dataset, with the HKY + I + G nucleotide substitution model for the second dataset, and with the K80 + I + G nucleotide substitution model for the third dataset.
The phylogenetic signal of each sequence dataset was investigated by means of the likelihood mapping analysis of 10 000 random quartets, generated using TreePuzzle .
For a quartet, just three unrooted tree topologies are possible. The likelihood of each topology is estimated with the maximum likelihood method and the three likelihoods are reported as a dot in an equilateral triangle (the likelihood map). Three main areas in the map can be distinguished: the three corners representing fully resolved tree topologies, i.e. the presence of treelike phylogenetic signal in the data; the center, which represents star-like phylogeny, and the three areas on the sides indicating network-like phylogeny, i.e. presence of recombination or conflicting phylogenetic signals. When using this strategy, if more than 30% of the dots fall into the center of the triangle, the data are considered unreliable for the purposes of phylogenetic inference.
Maximum likelihood phylogenetic trees were constructed with Phyml . Statistical robustness and reliability of the branching order within the phylogenetic trees were confirmed by bootstrap analysis.
A total of 325 questing ticks were collected, and a representative sample, randomly selected, of 129 ticks was processed for Rickettsia spp. analyses. Rhipicephalus turanicus was the most abundant species (66%) with 29 males and 56 females, followed by Ixodes ricinus (26%) with 11 males and 22 females, Dermacentor marginatus (5%) with 1 males and 6 females, and Haemaphysalis punctata (3%) with 1 males and 3 females. R. turanicus showed a seasonal pattern from spring to early summer, while I. ricinus and D. marginatus resulted active from October to May and from October to April, respectively. H. punctata was rare, with a seasonal activity in autumn-winter .
Rickettsia spp. detection
Out of the 129 ticks screened through the gltA gene target , 46 (36%) samples were positive for Rickettsia spp. In particular, rickettsial DNA was found in R. turanicus (22/85; 26%), I. ricinus (23/33; 70%), and D. marginatus (1/7; 14%), while any H. punctata tick was positive for the presence of the pathogen (Table 2). I.ricinus was about 2.5 times more likely to be infected by Rickettsia spp. than R. turanicus. Specific groEL PCR reactions for TG and SFG discrimination , determined that all rickettsiae belonged to SFG.
Rickettsia spp. identification
To determine the diversity of SFG rickettsiae, DNA from the 46 positive individual ticks was subjected to partial amplification and sequencing of gene encoding, ompA as previously reported . The sequences obtained were compared to other bacterial sequences present in the GenBank database. A total of five different Rickettsia SFG species were identified.
As shown in Table 2, a 100% identity to the ompA fragment sequences was obtained for: R. monacensis (R. monacensis isolate 3IRF MA, GenBank accession number KF258154), from 13 I. ricinus,7 R. turanicus, and 1 D. marginatus;R. massiliae (R. massiliae MTU5, GenBank accession number CP000683), from 6 R. turanicus, and 4 I. ricinus; R. conorii (R. conorii strain Malish 7, GenBank accession number AE006914), from 2 I. ricinus, and 1 R. turanicus; R. aeschlimannii (R. aeschlimannii EL-Arish-18, GenBank accession number HQ335158), from 3 I. ricinus, and 1 R. turanicus. In contrast, a 99% identity to the partial ompA sequence of Rickettsia sp. strain TwKm01 (GenBank accession number EF219467) was found from 7 R. turanicus, and 1 I. ricinus. This percentage of identity is due to an insertion (216 nucleotide position) of three consecutive nucleotides (GAT), with an introduction of a putative predicted aspartic aminoacid in the sequence of the rOmpA protein.
Sequences from these eight ticks were identical to one another and, in support of a better characterization, the gtlA and the 17-kDa antigen partial gene sequences were also performed and compared in the GenBank [13, 15].
For seven samples, sequence analysis of gtlA showed 99% identity to the partial gtlA sequence of Rickettsia sp. strain TwKm01 (GenBank accession number EF219463), and were named Rickettsia sp. strain RM1. All sequences displayed a nucleotide exchange (C→T) at 845 nucleotide position with an aminoacid replacement (His→Tyr) in the gtlA protein sequence. Otherwise, one sample presented a 100% identity to the partial gtlA sequence of Rickettsia sp. strain IRS4 (Gen-Bank accession number AF141906) and were named Rickettsia sp. strain RM2.
Partial sequence analysis to the 17-kDa antigen of the Rickettsia sp. strain RM1 showed a 99% identity with sequences of the Rickettsia sp. TwKM01 from Taiwan (GenBank accession number AY445821) and R. rhipicephali (GenBank accession number U11020), with a nucleotide exchange (G→A) or (T→C) at position 217 or 140 in the nucleotide sequences, respectively. In contrast, the Rickettsia sp. strain RM2 displayed a 99% identity with the 17-kDa sequence of the Rickettsia sp. 777c (GenBank accession number EU283838) due to a several nucleotide switches (G→A at position 96, A→G at position 157, T→C at position 168, A→G at position 385) in the nucleotide sequence with 2 aminoacids replacement (Met→Ile at position 16, and Ser→Gly at position 37).
The phylogenetic noise of each data set was investigated by means of likelihood mapping. The percentage of dots falling in the central area of the triangles was 28% for the first dataset, 11.2% for the second dataset, and 15.6% for the third dataset: as none of the datasets showed more than 30% of noise, all of them contained a sufficient phylogenetic signal (Figure 1 a, b, c).
Maximum likelihood phylogenetic tree of the first dataset (partial ompA gene) was shown in Figure 2. All rickettsia isolates clustered together in a statistically supported cluster, which included the following reference sequences: a sequence Rickettsia sp. TwKM01 from Taiwan (GenBank accession number EF219467), two rickettsia sequences from China (Rickettsia sp. ZJW4-3/2007, FJ176299; Rickettsia sp. ZJ43/2007, EU258735), and three rickettsia sequences from Cyprus (CyRtu43H, JF803899; CyRtu 43D, EU448158; CyRtu 43S, EU448159).
The maximum likelihood phylogenetic tree of the second dataset (partial gltA gene) was shown in Figure 3. The maximum likelihood analysis identified seven rickettsia isolates (sequence labelled as RM1) in a statistically supported cluster with a strain from Taiwan, Rickettsia sp. strain TwKm01 (GenBank accession number EF219463). Moreover, this cluster was included in a statistically supported clade with representative Rickettsia sequences such as strains of R. massiliae (GenBank accession number: KF826286, HM050293, JN043507, U59719), Rickettsia sp. Bar 29 (GenBank accession number U59720), Rickettsia sp. PoTiR600 from Portugal (GenBank accession number HM149282), a candidatus Rickettsia kulagini strain Kertch (GenBank accession number DQ365806), two strains of R. rhipicephali (GenBank accession number: U59721, DQ865206) and Rickettsia sp. R300 from Brazil (GenBank accession number AY472038).
One rickettsia isolate (sequence labelled as RM2) was found in a statistically supported clade including reference sequences such as Rickettsia sp. IRS4 collected in Slovakia (GenBank accession number AF141906), Rickettsia sp. PoTiR6dt (GenBank accession number EF501756) collected in Portugal, R. monacensis strain IR/Munich (GenBank accession number DQ100163), Rickettsia sp. CH -73-2 from Switzerland (GenBank accession number EU359298), R. monacensis from China (GenBank accession number: EU665236, EU665235), R. monacensis strain CN45kr from South Korea, Rickettsia sp. PoTiR5td from Portugal (GenBank accession number EF501755) and Rickettsia sp. IRS3 from Slovakia (GenBank accession number AF140706). Outside of this clade there was a rickettsia reference sequence labelled Rickettsia sp. 12G1 (GenBank accession number KF831358) isolated from Ecuador.
Maximum likelihood phylogenetic tree of the third dataset (17-kDa antigen partial gene sequences) was shown in Figure 4. Rickettsia sp. strain RM1 isolates were found in a statistically supported cluster, which included Rickettsia sp. TwKM01 from Taiwan (GenBank accession number AY445821), R. massiliae MTU5 (GenBank accession number CP000683), R. rhipicephali (GenBank accession number U11020), R. rhipicephali (GenBank accession number DQ865207) and Rickettsia sp. R300 (GenBank accession number AY472039), both isolated in Brazil isolated, and.
Maximum likelihood analysis identified the Rickettsia sp. strain RM2 isolate in a statistically supported cluster with a strain of Rickettsia sp. InR/D372 from Korea (GenBank accession number KC888948), two strains from Italy Rickettsia sp. IrITA2 and IrITA3 (GenBank accession number AJ427882 and AJ427883), a strain R. monacensis IrR/Munich (GenBank accession number EF380355), one strain Rickettsia sp. clone Pampulha from Brazil (GenBank accession number JN190456), a Rickettsia sp.TR-39 from USA (GenBank accession number DQ480762), two strains of rickettsia endosymbiont of I. scapularis from USA (GenBank accession number KC003472 and KC003474), one strain of Rickettsia tamurae collected from Japan (GenBank accession number AB114825), a strain Rickettsia sp. Ae-8 (GenBank accession number DQ365986), and a strain Rickettsia sp. 777c from Australia (GenBank accession number EU283838).
Worldwide, ticks are important vectors of human and animal pathogens and a variety of tick-borne infections are considered of medical interest. Several European studies conducted in ticks revealed that the prevalence of Rickettsia SFG ranges from about 3% to 15% [21, 22].
Tick-borne pathogens can occur not only in natural woodlands, but also in recreational urban areas [23-26]. However, to the best of our knowledge, only one investigation was conducted in public parks in Italy, showing the presence of Bartonella spp., B. burgdorferi s.l., and Rickettsia spp. .
In view of this fact, we planned a one-year survey to investigate Rickettsia spp. Pathogens in ticks collected in the Insugherata Natural Reserve of Rome located in the north-western outskirts of the city. The main tick species found were R. turanicus and I. ricinus , which are well-known vectors of several animal and human pathogens recognized in Italy [28-31].
The results of our study in questing ticks demonstrated an expected occurrence of Rickettsia SFG, as previously described .
Although the presence of rickettsia in ticks is expected, our results document for the first time the detection of these agents, in an urban park of Italy. In particular, three human pathogens (R. conorii, R. massiliae and R. aeschlimannii), and two putative new strains with unknown pathogenicity Rickettsia sp. strain RM1 obtained from seven individual R. turanicus, and Rickettsia sp. strain RM2 found in one I. ricinus tick, were detected.
The sequence analyses of partial gene sequences of ompA, gltA and 17-kd antigen of Rickettsia sp. strain RM1 showed a high identity with Rickettsia sp. strain TwKM01 from Taiwan (99% identity with all partial genes sequenced), while Rickettsia sp. strain RM2 exhibited identity with three different strains, Rickettsia sp. strain TwKM01 from Taiwan (99% identity with ompA partial gene), Rickettsia sp. IRS4 collected in Slovakia (100% identity with gtlA partial gene) and Rickettsia sp. 777c from Australia (99% identity with 17-kd partial gene).
Although the ompA phylogenetic analyses showed that, the Rickettsia sp. strain RM1 and strain RM2 were most closely related to several rickettsiae with unidentified pathogenicity, the clusters obtained with the gtlA and 17kd sequence analyses were included in statistically supported clades with representative other rickettsiae can cause human diseases. In particular, Rickettsia sp. strain RM1 was included in a clade with R. massiliae, while Rickettsia sp. strain RM2 clustered with R. monacensis.
Even if not all rickettsiae detected in this study may be considered human pathogens, their infectivity and potential pathogenicity remains to be further examined. Actually, several Rickettsia spp., originally detected in ticks and characterized as unknown pathogenicity, were subsequently demonstrated to be human pathogens, as reported for R. massiliae and R. monacensis [4, 8].
Moreover, the nonspecific feeding habits of these ticks with the involvement of a wide variety of vertebrates, which are potential reservoirs for several tick-borne pathogens, highlight the potential risk of transmission of multiple infections. In the context of public health, the clinical implications of tick-borne polymicrobial infections may be crucial for planning prophylactic measures and for limiting the probability of misdiagnosis.
Our data suggest that atypical cases of rickettsiosis due to agents other than R. conorii might occur. Microbiologists and clinicians should be alerted about the presence of new species of rickettsiae in our Country, especially in public parks located in an urban area. For that reason, continuing entomological surveys supported by clinical investigations and identification of rickettsiae in patients, through blood specimens and swabbing eschars analyses, could be an essential aspect to characterize distinct tick-borne rickettsioses occurring in Italy.
However, the epidemiological significance of these results must be taken with prudence, because the presence of a pathogen in ticks does not necessarily mean certain transmission to susceptible hosts. In spite of this, our investigation may be important and helpful for further epidemiological studies of tick-borne pathogens in urban areas in Italy and for the risk prevention associated with tick-borne pathogens transmission to humans and animals.
We would like to thank Luca Marini and the local authority, Roma-Natura, for providing us with the opportunity to carry out this study in the Insugherata Natural Reserve.
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Address for correspondence:
Dipartimento di Malattie Infettive, Parassitarie ed Immunomediate, Istituto Superiore di Sanità
Viale Regina Elena 299
00161 Rome, Italy
Received on 11 February 2015. Acceptedon 11 May 2015.
Financial support: This study was partially supported by a research grant from the Italian Ministry of Health (CCM 2013-2014: "Sorveglianza di laboratorio di infezioni batteriche sottoposte a sorveglianza europea e da agenti di bioterrorismo").
Conflict of interest statement: No competing financial interests exist.
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