Spotted fever group rickettsiae associated with ixodid ticks in wild environment in Southern Italy

Abstract Ixodidae ticks are vectors and reservoirs of several species of rickettsiae, and tick‐borne rickettsioses are reported worldwide. This study was aimed to verify the distribution of spotted fever group rickettsiae associated with ticks in a wild environment, the National Park of Gargano, where there is proximity between wild and domestic animals, and which is within an endemic area for rickettsiosis. Ticks were collected from animals or vegetation, morphologically identified and tested by a PCR targeting the 17kDa gene, and by a loop‐mediated isothermal amplification (LAMP) targeting ompB gene. Out of 34 tested tick pools, 2 from Dermacentor marginatus, 1 from Ixodes ricinus, and 1 from Rhipicephalus turanicus resulted positive. Nucleotide sequences of amplicons showed high similarity with sequences from Rickettsia slovaca, Rickettsia raoultii, Rickettsia helvetica, and Rickettsia felis. The overall calculated infection rate was 26.19 per 1,000, while it rose up to 107.77 when only D. marginatus was considered. The results highlight the association among Ri. slovaca, Ri. raoultii, D. marginatus and wild boars from which infected ticks were collected. Finally, the study shows the low efficacy of the previously described LAMP method for the detection of Rickettsia spp., when compared to PCR, making urgent the development of most effective LAMP protocols.

associated with the distribution of the host arthropods that act as vectors and/or reservoirs (Fournier & Raoult, 2009;Weinert et al., 2009), and which may transmit the infection to humans.
The disease is currently considered to be mainly diffused in Europe, in particular, along the Mediterranean basin, insomuch that it is historically known as Mediterranean spotted fever (MSF). Rickettsia conorii is the agent considered to be mainly associated with MSF. Among Mediterranean countries, Italy has the highest incidence, recording 4,604 cases from 1998 to 2002, with an average of 921 cases per year (1.6 per 100,000 inhabitants, up to 10 per 100,000 inhabitants in endemic regions such as Sardinia) (Ciceroni, Pinto, Ciarrocchi, & Ciervo, 2006;Madeddu et al., 2016).
However, it is reasonable to assume that the overall incidence of TBR is underestimated: considering the high occurrence of mild forms of TBR, and the lack of specificity of most symptoms, it may be easily misdiagnosed or simply not reported (Parola et al., 2005).
In the last decades, the development of molecular techniques has improved the sensitivity of diagnostic techniques by making possible the detection of Rickettsia spp. DNA directly from biological samples. Consequently, rickettsioses recently reported from Europe were found to be caused by many SFG species (Oteo & Portillo, 2012).
However, very few up-to-date data (among them, Masala et al., 2012 andMancini et al., 2015) are available about the diffusion in the wild environment of SFG rickettsiae in Mediterranean Europe, as most of studies were targeted to human infections.
In light of those considerations, this research was aimed to evaluate the circulation of SFG rickettsiae in ticks collected from the environment in the National Park of Gargano (Italy), to gather details about the distribution and the natural hosts of known rickettsia species in the natural environment. Beyond their environmental relevance, data from that park may be of wider interest because of the close contacts between wild fauna and domestic animals, mainly ovine, which range over a part of its area.
The research was performed using a PCR-based amplification technique and a loop-mediated isothermal amplification (LAMP) method in order to verify the most suitable approach for the investigations from field.

| Tick collection and identification
From July to October 2013, a collection campaign was undertaken to collect ticks in the National Park of Gargano, in the Apulia region, Italy.
The location was chosen because it is an environmentally protected area, but whose boundaries are close to agricultural and animal farms, and this granted a good balance between wild natural and anthropic rural environments. The park is also characterized by a high level of biodiversity of vertebrate and invertebrate species living there.
During the campaign, 158 tick specimens were collected. Among those, 110 were manually removed from domestic and wild dead mammals found in field, and sent to the facilities of the Veterinary Entomology section of the Experimental Zooprophylactic Institute of Apulia and Basilicata (Foggia, Italy). Forty-eight ticks were collected by the dragging method (Rulison et al., 2013) in three different sites of the park during September 2013.
All specimens were placed in vials containing 70% ethanol and identified according to the morphological keys of Manilla (1998) and Estrada-Peña, Bouattour, Camicas, and Walker (2014). After identification, the ticks were divided into pools by species and host (Table 1) for the molecular analyses.

| Detection of rickettsia DNA by PCR
Ticks from each pool were crushed by the mean of sterile pestle and mortar, and the homogenates were subjected to total DNA extraction using the DNeasy blood and tissue extraction kit (Qiagen, Milan, Italy) according to the manufacturer's instructions. Five microliter of each DNA solution were used as a template in the amplification reactions. Detection of SFG Rickettsia spp. was performed according to Webb, Mitchell, Malloy, Dasch, and Azad (1990), who set up a PCR protocol targeting the 17 kDa outer membrane protein gene using primers 5′-GCTCTTGCAACTTCTATGTT-3′ and 5′-CATTGTTCGTCAGGTTGGCG-3′. Thermal cycle was as follows: initial denaturation at 95°C for 5 min and 35 cycles of denaturation at 95°C for 30 s, annealing at 57°C for 2 min, elongation at 72°C for 1 min.
Whenever the protocol was not enough to discriminate at species level, the PCR protocol described by Roux, Fournier, and Raoult (1996), targeting the ompA gene (primers 5′-ATGGCGAATATTTCTCCAAAA-3′ and 5′-GTTCCGTTAATGGCAGCATCT-3′), was applied. Thermal cycle consisted of an initial denaturation at 95°C followed by 35 cycles of 95°C for 30 s, 46°C for 45 s, and 72°C for 40 s. In both cases, the REDTaq ReadyMix PCR Reaction Mix (Sigma Aldrich, Milan, Italy) was used.
All the gathered PCR products were purified by the mean of the QIAquick Spin PCR Purification kit (Qiagen) according to the manufacturer's instructions, and the nucleotide sequences of both strands were determined by the BigDye Terminator DNA sequencing kit (Thermo Scientific, Milan, Italy). Sequencing primers were the same used for PCR. The nucleotide sequences of amplicons were submitted in GenBank under the accession numbers KY576905-KY576908 (17 kDa-protein gene) and KY576909-KY576910 (ompA). The nucleotide sequences were compared with those present in GenBank by Nucleotide BLAST (Johnson et al., 2008) to confirm the identification.

| Detection of rickettsia DNA by LAMP
The detection of SFG Rickettsiae by LAMP was carried out according to the protocol of Pan, Zhang, Wang, and Liu (2012), based on the amplification of a portion of the ompB gene. The sequences of oligonucleotides are provided in Table 2. The protocol was slightly modified using the Isothermal Master Mix with carboxyfluorescein (Optigene, Horsham, UK) and ROX as a passive reference dye. The reactions were carried out at 65°C for 40 min in a StepOne Real-Time PCR system (Applied Biosystems, Milan, Italy). For each group of LAMP reactions, a sample with nuclease-free water was added as negative control; and a reaction with DNA of Ri. conorii (kindly provided by the Unité des Rickettsies, Marseille, France) was set up as positive control.

| Analysis of LAMP primers
In order to verify the annealing of the LAMP primers, the sequences of the ompB gene from Ri. helvetica (GenBank accession number KP866151), Ri. felis (CP000053), Ri. raoultii (CP019435), and Ri. slovaca (CP003375) were aligned by the clustalW algorithm implemented in CLC sequence viewer 7.7.1 (Qiagen, Aarhus, Denmark). The match of the six oligonucleotide sequences with their target was manually checked.

| Infection rate calculation
The maximum likelihood estimation (MLE) of the infection rate (IR) and the respective 95% confidence interval were calculated using the PooledInfRate software (Biggerstaff, 2005). Infection rate has been expressed as number of infected ticks per 1,000.

| Detection of rickettsiae in ticks by PCR
Out of the 34 pools of ticks, 4 (11.76%) resulted positive to PCR, returning the expected amplicons. Among positive pools, one consisted of free-living I. ricinus, two of D. marginatus (collected from wild boar), and one of Rh. turanicus (from a sheep) (

| Detection of rickettsiae in ticks by LAMP/ Analysis of LAMP primers
The LAMP approach confirmed positivity for both the D. marginatus felis and Ri. helvetica (Figure 1 and Table 2). For these species, the divergence between primer and target sequences was not negligible, as they often mismatched within the last five nucleotides at the 3′ terminus.
F I G U R E 1 Schematic view of the ompB gene representing the target for the Rickettsia-specific LAMP reaction. Arrow directions reflect the 5′-3′ orientation of the oligonucleotides. Divergent nucleotides are in red T A B L E 2 In silico analysis of the annealing of the LAMP primers to their target region within the ompB gene of the rickettsia chromosome

| Infection rate calculation
The calculated values of the infection rates are listed in Table 1.  On the other hand, infection rate was considerably higher for D.

Considering all the identified ticks and
marginatus (107 per 1,000). Studies from Germany and Slovakia reported a prevalence of Ri. slovaca in D. marginatus and D. reticulatus up to 5%, and a prevalence up to 40.7% in Poland, and higher rates were recorded when Ri. raoultii is also considered (Karbowiak et al., 2016).
In a previous investigation, 58.3% of tested D. marginatus specimens collected from humans were found positive in Southern Italy (Otranto et al., 2014), a rate consistent with data by Selmi et al. (2009), who reported high infection rate for this tick species.
Rickettsia slovaca and Ri. raoultii are causative agents of a syndrome known as DEBONEL/TIBOLA (Dermacentor-borne necrosis erythema and lymphadenopathy/Tick-borne lymphadenopathy) (Raoult et al., 2002). It is a newly recognized emerging disease, as its incidence has been increasing in Europe during the last decade (Portillo et al., 2015).
Such trend is also confirmed in Italy, where the increasing detection of Ri. slovaca and Ri. raoultii counterweights the general decrease in traditionally MSF-associated rickettsiae in both humans and ticks (Parola et al., 2009;Selmi, Bertolotti, Tomassone, & Mannelli, 2008 (Brown & Macaluso, 2016) but, more recently, the pathogen was also detected in several arthropods, including soft and hard ticks (Angelakis et al., 2016), the latter included in Rhipicephalinae subfamily (Abarca, López, Acosta-Jamett, & Martínez-Valdebenito, 2013). To the best of our knowledge, Ri. felis was never detected from Rh. turanicus, although it has been constantly recovered from fleas (Capelli et al., 2009;Persichetti et al., 2016). Therefore, although limited to only one positive pool, the finding of Ri. felis may be relevant in terms of public health. The infected tick was collected from a sheep, but, since no other Ri. felis was detected in this study, it is not possible to suggest a possible route for the transmission of such microorganism, from wild to domestic animals or vice versa.
However, this finding may represent another proof of the increasing diffusion of Ri. felis in a very heterogeneous group of potential vectors.
In conclusion, considering the wide diffusion of rickettsiae and their arthropod hosts, there is a non-negligible risk for people who live and work in proximity of infected or infested animals. Therefore, laboratory detection of SFG rickettsiae may be essential to recognize the pathogens in animals or vectors in the wild environment as well as in the clinical practice, in order to promptly diagnose, or even prevent, the infections in humans.
In this perspective, the LAMP approach for the detection of tick-associated rickettsiae could be very useful, as it has the great advantage to be rapid and specific. LAMP is a promising technique, developed by Notomi et al. (2000) and briefly described in Raele, Pugliese, Galante, Latorre, and Cafiero (2016). In fact, it may return results in very short time, if compared with serological or PCR-based techniques; the LAMP is also affordable for most of diagnostic laboratories (Raele et al., 2016), and it has already successfully been used for ticks endosymbionts (Raele, Galante, Pugliese, De Simone, & Cafiero, 2015).
However, the LAMP protocol used in this study failed to detect Ri. helvetica and Ri. felis in tick pools, while the PCR targeting the 17 kDa-protein gene was more effective. This is probably linked to the variability in the ompB gene, target of this LAMP protocol. In fact, the designed oligonucleotides were found not to correctly match with the targets in Ri. felis and Ri. helvetica, presenting mismatches in the 3′ terminus. This leads to an unstable annealing of oligonucleotides that impedes the molecule to prime the polymerase activity.
Therefore, it should be strongly advisable to set up a new LAMP protocol based on more conserved region among Rickettsia genus, in order to gain a useful tool, which can greatly help the detection or diagnosis of those often neglected rickettsioses.