Rickettsiae in the common pipistrelle Pipistrellus pipistrellus (Chiroptera: Vespertilionidae) and the bat soft tick Argas vespertilionis (Ixodida: Argasidae)

Background Increasing molecular evidence supports that bats and/or their ectoparasites may harbor vector-borne bacteria, such as bartonellae and borreliae. However, the simultaneous occurrence of rickettsiae in bats and bat ticks has been poorly studied. Methods In this study, 54 bat carcasses and their infesting soft ticks (n = 67) were collected in Shihezi City, northwestern China. The heart, liver, spleen, lung, kidney, small intestine and large intestine of bats were dissected, followed by DNA extraction. Soft ticks were identified both morphologically and molecularly. All samples were examined for the presence of rickettsiae by amplifying four genetic markers (17-kDa, gltA, ompA and ompB). Results All bats were identified as Pipistrellus pipistrellus, and their ticks as Argas vespertilionis. Molecular analyses showed that DNA of Rickettsia parkeri, R. lusitaniae, R. slovaca and R. raoultii was present in bat organs/tissues. In addition, nine of the 67 bat soft ticks (13.43%) were positive for R. raoultii (n = 5) and R. rickettsii (n = 4). In the phylogenetic analysis, these bat-associated rickettsiae clustered together with conspecific sequences reported from other host and tick species, confirming the above results. Conclusions To the best of our knowledge, DNA of R. parkeri, R. slovaca and R. raoultii was detected for the first time in bat organs/tissues. This is also the first molecular evidence for the presence of R. raoultii and R. rickettsii in bat ticks. To our knowledge, R. parkeri was not known to occur in Asia. Our results highlight the need to assess rickettsial agents in a broader range of bat species and associated tick species.


Background
Bats (order Chiroptera), including at least 1400 species [1], are the only mammals which actively fly [2]. Among the consequences of this trait, bats show a geographically widespread distribution and may even undergo shortto long-distance seasonal migration [2]. Bats are special in their capacity to act as reservoir hosts for intracellular pathogens [3]. Several species of bat-associated soft (Acari: Argasidae) and hard (Acari: Ixodidae) ticks, i.e. Carios kelleyi, Argas vespertilionis, A. transgariepinus, Ornithodoros sp., Ixodes vespertilionis, I. ariadnae and I. simplex, were shown to carry DNA of vector-borne bacteria and protozoans [4,5]. Among these tick species, A. vespertilionis (which has a wide distribution in Europe, Africa and Asia) was reported to bite humans [6]. In addition, increasing molecular evidence supports that bats and/or their ticks may harbor vector-borne zoonotic bacteria, such as bartonellae and borreliae [7,8]. However, the occurrence of rickettsiae in bats and bat ticks appears to be poorly studied, particularly in Eurasia.
Rickettsiae are Gram-negative, obligatory intracellular bacteria, which may cause disease in animals and humans, and are associated with arthropod vectors (such as ticks, lice, fleas or mites), both as transmitters and reservoirs [9]. Concerning rare reports on rickettsiae from bats and their ticks, previously Rickettsia africae was detected in the blood of bats in the Caribbean region [10] and several rickettsiae were identified in bat ticks from Central America, Europe, Africa and Asia [11]. However, to the best of our knowledge, a simultaneous analysis of bat tissues and bat ticks for the presence of rickettsiae in central Asia has not been carried out. Our hypothesis is that bats might be susceptible to infection with some rickettsial species. In our study, we aimed at evaluating the susceptibility of bats to rickettsial infection and the involvement of these flying mammals and their ticks in Rickettsia transmission cycles in central Asia.

Sample collection and identification
Fifty-four bat carcasses were collected from an idle classroom in Shihezi University, Xinjiang Uygur Autonomous Region (XUAR) in northeastern China during 2015-2019. The heart, liver, spleen, lung, kidney, small intestine and large intestine of bat carcasses were removed, similarly to what has been reported in studies of bat haemoparasites [12]. Genomic DNA was extracted from these organs, as well as from ticks collected from the bats using the 96 Flux Automatic Nucleic Acid Extraction Instrument (Bio Teke, Beijing, People's Republic of China) with a matching commercial kit (Cell & Tissue Kit, Bio Teke) according to the manufacturer's instructions. To confirm the morphological identification of bats, the cytochrome b (cytb) gene was analyzed [13] and a representative cytb sequence was deposited in the Gen-Bank database under the accession number MF106222.
Simultaneously, a total of 67 tick larvae were collected from bat bodies. The ticks were morphologically identified according to the standard taxonomic keys as previously described [14]. From five ticks, the 16S rDNA gene was amplified following a previously reported protocol [15]. The corresponding 16S rDNA sequence was deposited in the GenBank database under the accession number MF106219.

Detection of rickettsiae, sequencing and phylogenetic analyses
Four genetic markers, including 17 kDa antigen (17-kDa), citrate synthase (gltA), and outer membrane proteins A and B (ompA and ompB) genes were assessed within each sample to investigate the presence of rickettsiae [15]. The primers and PCR cycling conditions in this study are shown in Additional file 1: Text S1 and Table S1. Each PCR assay included a negative control (distilled water instead of tick DNA template) and a positive control (containing sequence-verified DNA from R. raoultii obtained from the tick Dermacentor nuttalli collected in XUAR) [15]. Purification and sequencing of the PCR products were performed as described above [16,17].
Sequences were manually edited, aligned and compared to reference GenBank sequences by nucleotide BLASTn program (https ://blast .ncbi.nlm.nih.gov). A phylogenetic tree was constructed using the maximumlikelihood and neighbor-joining algorithms implemented in MEGA 6.0 software [18].

Results
All bats were identified as Pipistrellus pipistrellus, and their ticks as A. vespertilionis. Out of 378 bat organs/tissues and 67 bat ticks, 6 bats and 9 ticks were positive for the four Rickettsia genetic markers (17-kDa, gltA, ompA and ompB). Sequencing identified R. parkeri in the heart, liver and kidney of a bat, R. lusitaniae in the heart, liver and small intestine of a bat, R. slovaca in the lung and kidney of two bats and R. raoultii was only found in the liver of two bats (Fig. 1, Table 1). Concerning bat ticks, R. raoultii was detected in five and R. rickettsii in four specimens. Interestingly, four ticks positive to R. raoultii were removed from a R. raoultii-positive bat.
Regarding sequence comparisons based on the four genetic markers, R. parkeri in this study had sequence identities within the range of 99.7-100% when compared with the sequence of R. parkeri from the tick Amblyomma ovale in Colombia (GenBank: CP040325); Rickettsia lusitaniae showed 98.6-100% identity compared with the sequence of R. lusitaniae from A. vespertilionis infesting P. pipistrellus in China [11]; Rickettsia slovaca had 99.8% sequence identity with a conspecific bacteria from Dermacentor marginatus in Turkey; Rickettsia raoultii showed 99.1-100% identity with R. raoultii from D. nuttalli infesting Spermophilus undulatus in northwestern China [13]; and Rickettsia rickettsii was 99.7-99.9% identical to R. rickettsii from D. variabilis in the USA [19]. The detailed similarities and divergences of the sequences in this study are shown in Table 2 and Additional file 2: Table S2.

Discussion
Based on a recent review, R. lusitaniae, R. slovaca, R. raoultii and R. rickettsii have already been found to occur in Asia [20]. To the best of our knowledge, R. parkeri was detected for the first time on the Eurasian continent in the present study. Previously, R. conorii and R. orientalis have been identified in human urine and urine of albino Swiss mice [21,22] and R. helvetica has been detected in  bat feces [23]. Here, the kidneys from common pipistrelle bats were positive for R. parkeri and R. slovaca, while the small intestine was positive for R. lusitaniae, and the lung was positive for R. slovaca. These findings suggest that P. pipistrellus might become infected with R. parkeri, R. lusitaniae and R. slovaca. Importantly, PCR-positivity of the kidneys and the small intestine warrant further studies to investigate, if rickettsiae also pass with the faeces and urine of bats, because some of the rickettsiae (including R. rickettsii detected here in bat ticks) are known to cause infection via aerosol transmission [24]. In our previous studies we provided molecular evidence for the presence of R. raoultii in road-killed marbled polecat (Vormela peregusna) and its infesting tick Haemaphysalis erinacei [18]. Here, we detected R. raoultii in two bat livers and bat ticks for the first time. Interestingly, the gltA, ompA and ompB sequences from bat tissues were 100% identical with those from the PCR-positive bat ticks. Argas vespertilionis has a broad geographical distribution in the Old World, parasitizing several bat species, such as Eptesicus serotinus and P. pipistrellus [25,26]. Thus, the present findings suggest that, in relevant regions of Eurasia, R. raoultii may co-circulate between the bat P. pipistrellus and the bat tick A. vespertilionis.
In 1994, Jaenson et al. [27] reported that two persons living near Stockholm were bitten by the bat tick A. vespertilionis in their bedroom, and consequently clinical signs (fever, ulceration, erythema and edema) developed.
It has also been reported that certain soft tick species can be infected and are capable of transmitting human pathogenic rickettsiae, as exemplified by R. slovaca and R. rickettsii in Argas persicus and Ornithodoros spp., respectively [28,29]. These literature data underline the significance of the present findings and justify the need to evaluate further the actual epidemiological risks associated with the presence of R. raoultii, R. slovaca, R. parkeri and R. rickettsii in bats and their ticks.
Bats are susceptible to several vector-borne disease agents, including Trypanosoma cruzi, Babesia vesperuginis and Polychromophilus murinus [30]. Furthermore, some of these microorganisms may cause pathological changes in bats, as exemplified by B. vesperuginis, inducing anemia, splenomegaly, hemoglobinuria, and elevated reticulocyte and leukocyte counts [31]. In addition, the pathogenic role of rickettsiae is documented in several mammalian species (e.g. in the pine vole, Microtus pinetorum, these bacteria elicit tremor, fur ruffling and heavy breathing [32]). However, the clinico-pathological role of rickettsial infection in bats remains to be elucidated.
Here, 11.11% of bats (6/54) were infected with R. parkeri, R. lusitaniae, R. raoultii or R. slovaca. In addition, bat soft ticks contained the DNA of R. raoultii and R. rickettsii. These results warrant future studies, investigating (i) the routes of infection for bats (i.e. whether bat soft ticks are competent vectors or not; and if so, whether rickettsiae are transmitted by them transstadially and/

Conclusions
To our knowledge, this study provides the first report of R. parkeri, R. slovaca and R. raoultii in bats. Rickettsia raoultii and R. rickettsii were detected for the first time in bat soft ticks. Our findings contribute to the knowledge on the geographical distribution, and tick and vertebrate hosts for rickettsiae.