Molecular detection of vector-borne bacteria in bat ticks (Acari: Ixodidae, Argasidae) from eight countries of the Old and New Worlds

Despite the increasingly recognized eco-epidemiological significance of bats, data from molecular analyses of vector-borne bacteria in bat ectoparasites are lacking from several regions of the Old and New Worlds. During this study, six species of ticks (630 specimens) were collected from bats in Hungary, Romania, Italy, Kenya, South Africa, China, Vietnam and Mexico. DNA was extracted from these ticks and analyzed for vector-borne bacteria with real-time PCRs (screening), as well as conventional PCRs and sequencing (for pathogen identification), based on the amplification of various genetic markers. In the screening assays, Rickettsia DNA was only detected in bat soft ticks, whereas Anaplasma phagocytophilum and haemoplasma DNA were present exclusively in hard ticks. Bartonella DNA was significantly more frequently amplified from hard ticks than from soft ticks of bats. In addition to Rickettsia helvetica detected by a species-specific PCR, sequencing identified four Rickettsia species in soft ticks, including a Rickettsia africae-like genotype (in association with a bat species, which is not known to migrate to Africa), three haemotropic Mycoplasma genotypes in Ixodes simplex, and Bartonella genotypes in I. ariadnae and I. vespertilionis. Rickettsiae (from both the spotted fever and the R. felis groups) appear to be associated with soft rather than hard ticks of bats, as opposed to bartonellae. Two tick-borne zoonotic pathogens (R. helvetica and A. phagocytophilum) have been detected for the first time in bat ticks. The present findings add Asia (China) to the geographical range of R. lusitaniae, as well as indicate the occurrence of R. hoogstraalii in South Africa. This is also the first molecular evidence for the autochthonous occurrence of a R. africae-like genotype in Europe. Bat haemoplasmas, which are closely related to haemoplasmas previously identified in bats in Spain and to “Candidatus Mycoplasma haemohominis”, are reported here for the first time from Central Europe and from any bat tick.


Background
Bats (order Chiroptera) are the only mammals which actively fly. Among the consequences of this trait, bats show a geographically widespread distribution and may even undergo short to long distance seasonal migration [1]. Additionally, the evolution of flight in bats yielded inadvertent consequences on their immune functioning, and therefore bats are special in their capacity to act as reservoir hosts for intracellular pathogens [2]. Bats frequently reach high population densities in or near urban habitats, and their ticks may blood-feed on humans [3,4], which further increases their veterinary-medical importance.
The presence of DNA from vector-borne bacteria in bat ticks appears to be most extensively studied in Europe. In western Europe, Rickettsia and Ehrlichia species have been molecularly identified in soft ticks (Argas vespertilionis) of bats (in France [5] and the UK [6]). Another study carried out in central Europe (Poland) failed to detect Borrelia burgdorferi (s.l.), rickettsiae and Anaplasma phagocytophilum in the bat-associated hard tick species, Ixodes vespertilionis [7]. Nonetheless, literature data on molecular analyses of vector-borne bacteria in bat ticks are lacking from several regions of the Old and New Worlds. Therefore, during this study, bat ticks collected in countries representing less-studied regions (eastern and southern Europe, central and southeast Asia, eastern Africa, central America) were screened for the presence of DNA from four important genera of vector-borne bacteria, which include zoonotic species.
Bat tick DNA extracts (n = 514) were screened for the presence of Rickettsia helvetica, other Rickettsia spp., A. phagocytophilum, haemotropic Mycoplasma spp. and Bartonella spp. with real-time PCRs (Additional file 1: Table S1). This was followed by conventional PCRs and sequencing of various genetic markers (Additional file 2: Table S2), and phylogenetic analyses (Additional file 3: Text S1) except for R. helvetica and A. phagocytophilum.
Prevalences were compared with Fisherʼs exact test.

Results and discussion
Rickettsia DNA was only detected in bat soft ticks (all three evaluated species), whereas Anaplasma phagocytophilum and three haemotropic Mycoplasma genotypes were present exclusively in the hard tick species I. simplex (Table 1). In addition, Bartonella DNA was Table 1 Prevalence of pathogen DNA in bat ticks according to bat host species and country of origin. The latter are referred to with superscript letters (the cumulative number of bat individuals is equal to or less than the number of positives, because one or more ticks could have been collected from a single bat). After the name of the tick species, the number of analyzed DNA extracts is shown, which corresponds to the number of tick individuals (except for A. vespertilionis, in the case of which pooled samples were also used) significantly more frequently detected in hard than in soft ticks of bats (Fisherʼs exact test: P = 0.01).
In particular, R. helvetica was identified in one soft tick (A. vespertilionis) from China. This finding is consistent with former reports of R. helvetica in bat fleas [10] and bat faeces [11] in Hungary. Taking into account the bat host-specificity of these PCR-positive ectoparasites, it is possible that bats are susceptible to R. helvetica, although based on the very low prevalence this may have low epidemiological significance.
In four samples of A. vespertilionis from Hungary, the same Rickettsia genotype was identified, which was reported from bat soft ticks collected in France (GenBank: JN038177, see Table 2) [12]. More importantly, in one A. vespertilionis from Hungary rickettsial DNA was detected, which in the amplified part of the gltA gene had 99.9-100% sequence identity (depending on the nucleotide at position 679: C or T) to sequences of R. africae from Ethiopia (GenBank: CP001612) and from migratory bird fleas reported in neighboring Slovakia (GenBank: HM538186) [13]. Two other markers were also successfully amplified from this sample: the 17 kDa gene sequence was identical with that of several Rickettsia species, whereas the OmpA sequence showed 2 bp differences from that of R. africae (Table 2).
Interestingly, the OmpA sequence from this A. vespertilionis was identical with that of the Rickettsia strain "Atlantic rainforest" (GenBank: MF536975 [14]) and Rickettsia sp. "Atlantic rainforest Aa46" (GenBank: KY113110 [15]), which represent a genetic variant of the human pathogen R. parkeri [14,15] detected so far only in the New World. Nevertheless, we consider the species detected in A. vespertilionis to belong to R. africae because of the following four reasons: (i) the gltA gene is a reliable genetic marker for species identification and phylogenetic comparison of rickettsiae [13,16]; (ii) R. africae was identified based on this gene in previous studies (e.g. [13]); (iii) the gltA phylogenetic analysis confirmed that the rickettsial genotype from A. vespertilionis collected in Hungary clustered with R. africae, but apart from R. parkeri (Fig. 1); and (iv) the OmpA gene of the type strain of R. parkeri (GenBank: U43802) was only 98.3% (469/477 bp) identical with the OmpA sequence obtained here.
The soft tick containing the R. africae-like DNA was collected from Myotis dasycneme, which occurs north of the Mediterranean Basin and is a facultative, middle distance migrant bat species, not known to move between Europe and Africa [1]. Therefore, this result implies the autochthonous occurrence of a R. africae-like genotype in Europe. In the phylogenetic analysis, this genotype was clearly separated (with moderate, 72% bootstrap support value) from the Rickettsia sp. from A. vespertilionis reported in France (Fig. 1).
In addition, R. hoogstraalii was identified in a soft tick from South Africa (Table 2). This rickettsia has only been reported from Europe and North America [17], therefore its occurrence in Africa is new. Similarly, R. lusitaniae was formerly only reported in Europe (Portugal) [18] and Central America (Mexico) [19], the latter being confirmed in the present study (Table 2). However, a gltA genotype highly similar to R. lusitaniae (1 bp difference from JQ771933, i.e. 99.9% identity) was also shown here, for the first time, to occur in Asia (China) ( Table 2). The level of OmpA sequence divergence of this Chinese isolate (MH383149) was the same (3 bp) from R. lusitaniae in Portugal (JQ771935) and from R. lusitaniae in Mexico (GenBank: KX377432).
In summary, bat soft ticks contained the DNA of three Rickettsia species from the spotted fever group (SFG), and two further ones from the Rickettsia felis group (RFG) (Fig. 1).
Anaplasma phagocytophilum DNA was detected here in the hard tick species, I. simplex, in both Hungary and Romania. Previously, Anaplasma sp. DNA was also shown to be present in bat feces in Hungary (GenBank: KP862895). This low prevalence in bat ticks, suggests that bats may be susceptible to this pathogen, but most likely play a subordinate (if any) role in the epidemiology of granulocytic anaplasmosis in the evaluated region.
In Europe, molecular evidence on the occurrence of bat haemoplasmas has hitherto been reported from western countries, i.e. Spain [23] and the Netherlands [11]. Based on blood and fecal samples, respectively, these studies suggested infections of bats with the relevant agents. Haemoplasmas are regarded as predominantly vector-borne [24]. However, bat-associated haemoplasmas have not hitherto been identified in blood-sucking arthropods. Here, three haemotropic Mycoplasma genotypes have been detected in a tick specimen (I. simplex), collected in Hungary (Table 2). Ixodes simplex is specialized to its host, Miniopterus schreibersii [25], from which bat species haemoplasma genotypes having 99.8-99.9% 16S rRNA gene similarity to those from I. simplex collected in Hungary (Table 2) have been reported in Spain [23]. Importantly, these bat-associated haemoplasmas are phylogenetically Rickettsia helvetica and Anaplasma phagocytophilum were detected by using species-specific primers (Additional file 1: Table S1)  close to "Candidatus Mycoplasma haemohominis", as reported [23] and as also shown here (Fig. 2).

Conclusions
Rickettsiae (from both the spotted fever and the R. felis groups) appear to be associated with soft rather than hard ticks of bats, as opposed to bartonellae. Although with low prevalence, two tick-borne zoonotic pathogens (R. helvetica and A. phagocytophilum) have been detected for the first time in bat ticks. The present findings add Asia (China) to the geographical range of R. lusitaniae, as well as indicate the occurrence of R. hoogstraalii in South Africa. This is also the first molecular evidence of a R. africae-like genotype in Europe, in association with a bat host species that is not known to migrate to Africa. Bat haemoplasmas, which are

Additional files
Additional file 1: