1. Introduction
The genus Bartonella includes Gram-negative pleomorphic bacteria infecting endothelial cells and erythrocytes of mammalian hosts [1]. More than 35 species of Bartonella have been described [2], and the use of molecular surveys is continuously increasing the list of genetic variants identified within this genus. Some species cause well-documented human diseases, such as B. quintana, the agent of trench fever, and B. henselae, the cause of the commonly diagnosed cat-scratch disease. Other species are considered emerging pathogens associated with a broad range of clinical conditions, including endocarditis [3].
Bartonella transmission to humans is widely believed to be mediated by haematophagous arthropods [4], and an increasing number of surveys indicate rodents as reservoirs of Bartonella species [5]. Yet, rodents are not usually listed as reservoirs of B. henselae [5,6]. Usually, B. henselae is identified in humans and cats and their arthropods; however, two reports of the molecular identification of this species in rodents were previously described. In particular B. henselae was identified in Apodemus sylvaticus in Denmark [7], and in Rattus rattus in New Zealand [8]. Here, a molecular survey of Bartonella spp. DNA in wild rats and mice from Pianosa Island in the Tuscan Archipelago National Park of Central Italy is described. To the authors’ best knowledge, this is the first report of the presence of Bartonella spp. in rodents from Italy, and the third report, worldwide, of the detection of B. henselae in these hosts.
2. Materials and Methods
This study used carcasses of 15 rats (Rattus spp.) and 16 field mice (Apodemus spp.) trapped using T-Rex Snap Rat and Sherman Traps placed all along Pianosa Island, Italy, as part of a rodent control program (project LIFE13 NAT/IT/000471—RESTO CON LIFE “Island conservation in Tuscany, restoring habitat not only for birds”). Traps were emptied within 24 h of setting and the carcasses preserved at −20 °C. The carcasses were thawed overnight at room temperature and the spleens collected by incision of the left abdominal wall changing scalpel and gloves between carcasses. The collected spleens were re-frozen at −20 °C until analyzed.
The spleens were thawed at room temperature and DNA was extracted using kits (GenElute Mammalian Genomic Miniprep Kit, Sigma Aldrich, St. Louis, MO, USA), according to the manufacturer’s instructions. Initial screening for Bartonella spp. was performed by means of a quantitative Real-Time PCR (qPCR) designed to amplify a ~200 bp segment of the Bartonella spp. 16S-23S rRNA intergenic transcribed spacer (ITS), using previously described primers [9]. Amplification was performed using the CFX ConnectTM Real-Time PCR Detection System (BioRad, Hercules, CA, USA), in a PCR mixture consisting of 10 µL of iTaq Universal SYBR® Green Supermix (BioRad, Hercules, CA, USA), 300 nM of each primer, 1 µL of DNA to a final volume of 20 µL with nuclease-free water. The protocol included a 4-min step at 94 °C followed by 45 cycles at 95 °C for 5 s and 60 °C for 20 s. Samples with a quantification cycle (Cq) less than 35 were considered positive, and the amplicons were sent for bidirectional sequencing to a commercial sequencing provider (BMR Genomics, Padova, Italy) using the same primers.
All the samples yielding ITS sequences consistent with Bartonella spp. were further analyzed by conventional PCR (cPCR) at two loci: ~340 bp segment of citrate synthase gene (gltA), and ~600 bp- segment of ITS locus. cPCRs were performed using previously described primers [9,10,11], using 10 µL of a master mix (HotStarTaq; Qiagen, Hilden, Germany), 400 nM of each primer and 2 µL of DNA template. For the 600bp-ITS, cPCR conditions were heating to 95 °C for 15 min, followed by 35 cycles at 95 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, and a final extension at 72 °C for 5 min. The gltA gene fragment was amplified by heating to 95 °C for 15 min, followed by 45 cycles at 95 °C for 5 s and 60 °C for 45 s. PCR products were electrophoresed in 2% agarose gels, stained by GelRed Nucleic Acid Gel Stain (Biotium Inc., Fremont, CA) and visualized under UV. Bartonella sp. FG4-1 DNA was included as positive control and nuclease-free water as negative control in each PCR batch testing. Amplicons showing bands on gels consistent in size with the amplified locus were sequenced as above, and the raw sequences edited using Geneious Prime version 2019.0.4 [12] and compared online to the sequences stored in the National Centre for Biotechnology Information of the United States of America using BLAST (
3. Results
The results of the survey are summarized in Table 1. Spleen DNA from 25/31 rodents (81%) yielded Cq values of < 35 by qPCR (200bp-ITS), and the sequences of all the amplicons aligned with a high degree of similarity to sequences of Bartonella genus (data not shown). A total of 10/25 (40%) yielded Bartonella DNA by means of cPCR. GltA amplicons were obtained by cPCR in 6/25 (24%) qPCR-positive samples, and 600bp-ITS amplicons were obtained in 8/25 samples. Four samples yielded amplicons at both loci.
In particular, in one animal, a 346 bp gltA sequence was identical (100%) to the sequence of Bartonella henselae strain Houston-I (GenBank accession number CP020742.1). This gltA sequence was deposited in the GenBank as accession number MK562498.
The sequences of the other five gltA amplicons were identical with each other and shared 96–97% sequence similarity with the gltA gene sequence of B. grahamii (accession number CP001562.1), and other Bartonella spp. (Figure 1). These sequences were deposited in the GenBank under accession numbers MK562495, MK562496, MK562499, MK562500, and MK562501. The ~600 bp- ITS sequences of four samples were 100% similar to the sequence of B. coopersplainsensis (accession number EU111770.1) and were deposited in the GenBank as accession numbers MK562489, MK562490, MK562492 and MK562494. The other four ITS sequences were unique and shared 96-97% similarity with the Uncultured Bartonella sp. Clone MS_Seville-07021427 (GenBank accession number EU218552.1). These four sequences were deposited under accession numbers MK562487, MK562488, MK562491, MK562493, as Uncultured Bartonella sp. 16S-23S ribosomal RNA intergenic spacer, partial sequence.
gltA and ITS sequences of Bartonella identified in the present study were compared to related gltA and ITS Bartonella sequences from GenBank, showing that both gltA and ITS novel sequences were closely related to B. grahamii, B. elizabethae, B. tribocorum, and B. queenslandensis (Figure 1).
4. Discussion
We performed a molecular survey of the occurrence of Bartonella DNA in carcasses of wild rodents collected by means of traps in the isle of Pianosa, Italy. Initial screening by qPCR suggested the presence of Bartonella DNA in ~80% of the samples, and subsequent cPCR identified Bartonella DNA in 40% of the qPCR positive samples. Only four samples were PCR-positive at both loci. These results suggest a greater sensitivity of the qPCR used for screening, as compared with the cPCRs. Many samples were positive at one locus only, highlighting the utility of multiple-locus PCR as a means to increase overall testing sensitivity.
We identified ITS sequences of B. coopersplainsensis in four rats. B. coopersplainsensis was first identified in blood of Rattus leucopus in Australia [13], and later in different rodents in Thailand, China, Japan, Taiwan, and New Zealand [8,14,15,16,17]. The zoonotic potential of B. coopersplainsensis has yet to be established.
We found novel gltA and ITS sequences in five mice (Table 1). Interestingly, the five positive mice yielded identical gltA sequences, excluding the possibility of PCR artifacts. This sequence had not been reported previously in the GenBank and had high degree of identity to B. grahamii gltA locus. This Bartonella species can cause zoonotic infection, supporting the possible role of wild rodent as reservoir of human infections [3]. Exposure to rats and mice has been associated with rodent-specific Bartonella species infections, and infections with B. elizabethae, B. grahamii, and B. vinsonii have been observed [18].
We also identified four novel 600 bp-ITS sequences that showed a high degree of similarity to an Uncultered Bartonella sp. [19] which is closely related to B. elizabethae and was identified in rodents from Spain. The phylogenetic tree built with gltA and ITS cPCR products and compared to other Bartonella sequences deposited in GenBank allowed to evidence that the five Bartonella spp. identified in Apodemus might belong to a novel species closely related to B. grahamii, B. elizabethae, B. queenslandensis, and B. tribocorum, usually identified in rats and mice, and confirming these mammalian as reservoirs of zoonotic Bartonella spp. [18]. Moreover, the phylogenetic analysis confirms that the five sequences of Bartonella identified in rats are very close to B. henselae and B. coopersplainsens species.
Interestingly, DNA of B. henselae was identified in one rat, corroborating previous observations in New Zealand [8] and Denmark [7]. This was obtained by amplifying a fragment of the gltA locus, which has the largest number of sequences in the GenBank. The cPCR of the 600bp-ITS of this sample did not produce an amplicon, perhaps due to DNA degradation. This is the third report of the presence of B. henselae DNA in rodents, worldwide. Hence, rodents might have a greater role in the eco-epidemiology of cat scratch disease than previously thought. The circulation of B. henselae in rodents would need to be corroborated by culture [20]. It should be underlined that Bartonella culture from wild rodent carcasses is often hindered by the decomposition of the carcasses and the in vitro overgrowth of fast growing post-mortem contaminants.
5. Conclusions
We identified DNA of B. henselae, B. coopersplainsensis, and of a potential novel Bartonella species in spleen of rodents in the isle of Pianosa, Italy. Rodents may play a previously underestimated reservoir role in the eco-epidemiology of cat scratch disease.
Author Contributions
Conceptualization, E.B. and P.P.; methodology, P.P. and S.D.; validation, F.E.S.; investigation, S.Z. and S.D.; resources, F.G.; data curation, S.D.; writing—original draft preparation, S.D.; writing—review and editing, A.G.; supervision, E.F.; project administration, E.B.; funding acquisition, B.B. All authors have read and agreed to the published version of the manuscript.
Funding
This study was financially supported by Fondazione CRT, project “Isolamento, tipizzazione e diversità genetica di Bartonella spp. nei roditori urbani e della fauna selvatica in Regione Piemonte” (RF: 2017.2022).
Acknowledgments
We are grateful to Domenico Palmerini and Alessandra Sereno for their technical support. Animals were trapped within the project RESTO CON LIFE—“Island conservation in Tuscany, restoring habitat not only for birds” (LIFE13 NAT/IT/000471). We are grateful to Prof. Shimon Harrus, Hebrew University of Jerusalem, Koret School of Veterinary Medicine, for providing positive control DNA.
Conflicts of Interest
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Figure and Table
Enlarge this image.Figure 1. Phylogenetic trees based on the gltA (340 bp) (A) and ITS (512 bp) (B) partial sequences of Bartonella identified in the present study (GenBank accession number in bold) and from GenBank (description and GenBank accession number in bracket). The phylogenetic tree was constructed selecting Tamura-Nei as genetic distance and Neighbor-Joining as tree building method. Bootstrap calculation was carried out with 1000 resamplings with support threshold ≥ 70%. Phylogenetic trees were built using Geneious Prime software version 2019.0.4.
Detailed results of the Bartonella spp. positive samples found in this study. GenBank accession numbers are reported. For each sample, the closest sequence (description, accession number, query cover %, e-value and identity %) are reported in brackets.
| Host Genus | qPCR-Positive Samples (n) | DNA Sequences | |
|---|---|---|---|
| GltA | ITS | ||
| Rattus | 1 | MK562498 |
NA |
| Rattus | 4 | NA | MK562489; MK562490; MK562492; MK562494 |
| Apodemus | 4 | MK562495 |
MK562487 |
| MK562496 |
MK562488 |
||
| MK562499 |
MK562491 |
||
| MK562501 |
MK562493 |
||
| Apodemus | 1 | MK562500 |
NA |
| Rattus | 9 | NA | NA |
| Apodemus | 6 | NA | NA |
gltA, citrate synthase gene; ITS: 16S-23S rRNA gene intergenic transcribed spacer; NA: not amplified.
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.
Details
; Zanet, Stefania 1 ; Ferroglio, Ezio 1 ; Giannini, Francesca 2 ; Frine, Eleonora Scaglione 1 ; Grinberg, Alex 3 ; Biolatti, Bartolomeo 1 ; Bollo, Enrico 1
1 Dipartimento di Scienze Veterinarie, Università degli Studi di Torino, 10095 Grugliasco (TO), Italy;
2 Parco Nazionale Arcipelago Toscano, 57037 Portoferraio (LI), Italy;
3 School of Veterinary Science, Massey University, 4442 Palmerston North, New Zealand;