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Genetic diversity of Bartonella spp. in rodents and fleas from Poland

Abstract

Bartonella spp. are parasites of mammalian erythrocytes and endothelial cells, and are transmitted by blood-feeding arthropod ectoparasites, including fleas. This study aimed to: (i) identify the main flea species responsible for Bartonella transmission and the specific Bartonella species they carry, (ii) evaluate how host-related factors influence the prevalence of Bartonella, (iii) examine the genetic diversity of Bartonella from different flea species and mammalian hosts, including rodents and European moles, and (iv) determine the haplotypes of Bartonella derived from rodents. Blood samples were collected from seven rodent species and two European moles in Poland (n = 994), and fleas were collected from rodents (n = 833). Bartonella spp. were identified and genotyped through rpoB and gltA genes. Phylogenetic analysis revealed two dominant Bartonella spp. in rodents and fleas: B. grahamii and B. taylorii. Moreover, 17 haplotypes of B. taylorii and 9 of B. grahamii were identified. The sequences of Bartonella sp. from T. europaea clustered in a unique separate group, possibly indicating a novel species. The study confirmed fleas as vectors of Bartonella transmission in rodents and highlighted the significant genetic diversity of Bartonella spp. in both fleas and rodents.

Background

Bartonella species are Gram-negative, facultative intracellular bacteria that infect mammalian erythrocytes and endothelial cells1. These infections can lead to the development of bartonellosis, an important vector-borne disease2,3. Bartonella spp. have a wide range of vertebrate hosts and can cause disease in both animals and humans4,5. At least 13 species/subspecies are known to be zoonotic6. In humans, Bartonella infections are responsible for conditions such as cat scratch disease (caused by Bartonella henselae), trench fever (B. quintana), and Carrion’s disease (B. bacilliformis)7,8,9. Clinical symptoms of these infections include swollen lymph nodes (lymphadenopathy) with prolonged fever, intraerythrocytic bacteremia, neuroretinitis (inflammation of the optic nerve and retina), and endocarditis (infection of the heart’s inner lining)6.

Currently there are over 50 defined species of Bartonella, and more than half are known to infect rodents (National Center for Biotechnology Information Data). Small mammals act as reservoirs for Bartonella spp., playing a significant role in the maintenance and distribution of these pathogens10. High prevalences of Bartonella spp. have been detected in rodents, the most speciose group of mammals11. In Central Europe, the dominant small mammal species in woodlands are Clethrionomys glareolus and Apodemus flavicollis, while Microtus spp. dominate fallow lands12. In our previous studies we have shown that the prevalence of Bartonella infections in bank voles from Poland is 38.7%13, while in Microtus spp., it is higher at 66.8%14. Globally, Bartonella spp. infections in rodents range from 26–67%15,16,17,18,19.

Rodents, shrews, hedgehogs, lagomorphs, bats, carnivores, ungulates, and birds are all common hosts for fleas, which are considered the main vectors of Bartonella spp. among small mammals20. Fleas can transmit Bartonella through various routes, including oral transmission, regurgitation of blood meals, contaminated faecal pellets, and, in some cases, by ingestion of infected fleas by hosts21. Fleas can also switch hosts, potentially coming into contact with domestic animals and humans, making it important to understand which flea species are involved in Bartonella transmission. For example, Ctenophthalmus felis is a known vector for the zoonotic B. henselae22. Bartonella DNA has also been detected in other flea species such as C. agyrtes, C. congener, C. sciurorum, C. solutus, Histrichopsylla talpae, and Megabothris turbidus, with an overall prevalence of 22.9%23. The genes coding for the RNA polymerase beta subunit (rpoB) and citrate synthase (gltA) are commonly used to differentiate Bartonella species due to the variability in these genes16,24. This genetic variation can be used as a criterion for identifying new species or subspecies. Bartonella species from rodents in Western Europe are relatively well-studied, with Bartonella grahamii being one of the most widespread, occurring in Apodemus spp. and arvicolid voles (Clethrionomys spp. and Microtus spp.) throughout Eurasia25,26,27. Conversely, Bartonella taylorii is the most diverse yet least understood species in European rodents, composed of several distinct clades and capable of infecting both rodents and insectivores25,26,27,28.

Additionally, there is significant genetic diversity within the Bartonella genus, with the greatest variation found in rodents and bats29. A recent study in India identified 13 haplotypes based on rpoB sequences from unidentified Bartonella spp. in rodents18, while another study in Poland revealed two haplotypes of Bartonella from Lipoptena fortisetosa, also based on the rpoB gene30.

However, the range of Bartonella species transmitted by different flea vectors remains poorly understood. We hypothesize that the genetic diversity of Bartonella spp. is closely linked to their invertebrate host vectors. This study aims to: (I) identify the main flea species involved in Bartonella transmission and the specific Bartonella species they carry, (II) evaluate the influence of host intrinsic and extrinsic factors on Bartonella prevalence in mammalian hosts, (III) characterize the genetic diversity of Bartonella spp. from various flea species and mammalian hosts, including rodents and European moles, and (IV) determine the haplotypes of Bartonella spp. found in rodents.

Methods

Definition of terms

The taxonomic classification of bank voles has undergone several revisions in recent decades. Bank voles were recently classified under the genus Myodes, but it was later determined that the historically recognized name Clethrionomys is more appropriate. Consequently, this study adheres to the nomenclature recommended by the Mammal Diversity Database31, utilizing the designation Clethrionomys glareolus32. Additionally, recent taxonomic revisions have been reported for other vole species33. Within this framework, we refer to Alexandromys oeconomus, formerly known as Microtus oeconomus, in accordance with findings from Lissovsky et al. (2018)34 and Zorenko et al. (2018)35.

Study sites

The rodents were live-trapped in five sites, both woodland and fallow lands in Poland. The study sites have been previously comprehensively described13,36,37,38,39. They are located in Mazury in the north-eastern corner of Poland, in the vicinity of Lake Śniardwy and the towns of Mikołajki, Ryn, and Pisz. Site 1 is referred to as Urwitałt (53.79838, 21.64549), site 2 as Tałty (53.89202, 21.53494), site 3 as Pilchy (53.70569, 21.80617), and site 4 as Łuknajno (53.81395, 21.65199). The fifth site was Białobrzegi, in Central Poland (Mazovia), located about 75 km south of Warsaw (51.66445, 20.947) (Fig. 1).

Fig. 1

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Map of the study sites: (site 1 = Urwitałt, site 2 = Tałty, site 3 = Pilchy, Site 4 = Łuknajno, site 5 = Białobrzegi)

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Collection of rodent samples

Blood samples were collected during our long-term studies of rodent populations in the Mazurian Lake District of Northeastern Poland in the years 2010, 2013, 2014, and 201813,36,37,38,40. At the field stations, all animals were identified, culled, weighed (to the nearest 0.1 g), sexed and relevant morphometric data were recorded. The necropsies of rodents were carried out under terminal isoflurane (Merck, Darmstadt, Germany) anesthesia. The rodents were placed in an anesthetic chamber with an isoflurane concentration of (3–5%) and then culled by cervical dislocation. The methodologies employed for rodent trapping, as well as the procedures for sampling and processing of captured specimens, have all been detailed in previous publications36,37,38,39. For the present study, a total of 994 rodents, encompassing eight species, were examined: C. glareolus (n = 740) in 2010, 2014, and 2018 from forest habitats; Microtus arvalis (n = 124), Microtus agrestis (n = 19), Alexandromys oeconomus (n = 88), and Microtus sp. (n = 2) from fallow land; and Apodemus agrarius (n = 11), A. flavicollis (n = 9), and Apodemus sylvaticus (n = 1) from forest regions in 2013, 2014, and 2017. Additionally, we obtained samples from two dog-killed moles, Talpa europaea, collected in Kury, approximately 50 km northeast of Warsaw (Table 1).

Table 1 Prevalence (%) of Bartonella spp. Based on host identity, study site, and years of study.

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Collection and identification of fleas

In total, 914 fleas were collected from rodents. We identified five species of fleas: Ctenophthalmus assimilis (n = 260), C. agyrtes (n = 229), Megabothris walkeri (n = 189), M. turbidus (n = 182), and H. talpae (n = 54). Fleas were collected from voles trapped in fallow lands in 2013, 2014 and from bank voles trapped in forest areas in 2018. Fleas were fixed in 70% ethanol and identified to the species and sex levels morphologically using the key of Skuratowicz41, and molecularly by amplification and sequencing of the 18S rDNA gene fragment (570 bp)14,16,40. Most of the collected fleas (833 of 914) underwent further analysis (Table 2). The 18S rDNA products from all fleas that tested positive for Bartonella DNA (n = 26) were sequenced, identified, and subjected to phylogenetic analysis. For the phylogenetic analysis, we used all our DNA sequences and 10 reference sequences from GenBank.

Table 2 Prevalence (%) of Bartonella spp. Isolated from fleas collected from rodents, based on study sites and year of study.

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Blood collection and DNA extraction

Blood samples (200 µl) were collected from euthanized animals into tubes containing 0.001 M EDTA and subsequently frozen at − 20 °C. In cases where individuals were found deceased in traps, the entire heart was excised and homogenized in 400 µl of 0.001 M EDTA. Genomic DNA was extracted from both whole blood and heart homogenates utilizing the AxyPrep MiniPrep Blood Kit (AxyGen, USA) and the DNeasy Blood and Tissue Kit (Qiagen, Germany), with all samples stored at − 20 °C. The extracted DNA was subjected to specific polymerase chain reaction (PCR) protocols as outlined in previous studies14,16,40. For the detection of Bartonella spp., the rpoB gene was amplified using two fragments of 333 bp and 860 bp, along with the gltA gene (810 bp). Additionally, for the identification of flea species, the 18S rDNA (570 bp) was targeted. The primers utilized in this study are detailed in Table 3. PCR products were analyzed by electrophoresis on a 1.5% agarose gel and visualized using Midori Green stain (Nippon Genetics Europe GmbH, Germany).

Table 3 Gene markers, primers, and PCR conditions.

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Sequencing and molecular typing

PCR products were sequenced by private companies in both directions (Genomed S.A., Poland, and Eurofins Genomics, Germany). Both reads were checked for quality, and then aligned and edited to form a consensus sequence using BioEdit42. All sequences for each marker were checked in BLAST to identify the donor species. For each marker all obtained sequences were aligned with reference sequences from GenBank using the ClustalW in MEGA11: Molecular Evolutionary Genetics Analysis version 1143, and AliView44. DnaSP 6.12.0345 were used to calculate the number of haplotypes in the rpoB gene sequences. Minimum Spanning Network analysis was performed for the rpoB gene of Bartonella spp. DNA sequences using PopART 1. 46, and confined to sequences obtained in this study.

Phylogenetic analysis

Phylogenetic relationships among the rpoB and gltA gene sequences, representing samples of various Bartonella species, were assessed using Bayesian Inference, implemented in MrBayes v3.2.647. Due to the large size of the dataset, we fitted a GTR + G model with all six rate parameters free and with variation in the rate of evolution among sites in the alignment. Two independent runs of 10 000 000 generations were sampled every 1000 generations and 25% of the initial samples were discarded as a burn-in phase. The convergence of two runs and effective sample sizes for the model’s parameters were checked using Tracer v. 1.748.

The Maximum Likelihood method implemented in MEGA 1149,50 was used to infer phylogenetic relationships among 18S rDNA sequences obtained from fleas positive for Bartonella spp. We assumed a Kimura’s 2-parameter model49,50 and branch support was evaluated by 1000 bootstrap replicates summarized on the best scoring tree.

Statistical analysis

Prevalences were estimated based on PCR screening and microscopic analyses and were analysed by maximum likelihood techniques based on log-linear analysis of contingency tables in the software package SPSS (version 16.0.1, SPSS, Inc., Chicago, IL, USA). We first fitted full factorial models that incorporated flea species (five levels), sex of the flea (two levels, males and females), year of study (four levels), and study sites (five levels) as factors, with infection as a binary factor (presence/absence [P/A] of parasite). Beginning with the most complex model, which involved all possible main effects and interactions, and starting with the highest-level interaction those combinations that did not contribute significantly to explaining variation in the data were eliminated in a stepwise fashion (backward selection procedure in SPSS). Eventually, a minimum sufficient model was obtained, for which the likelihood ratio of χ2 was not significant. This indicated that the model was sufficient in explaining the data and, where relevant, these values are provided in the legends to the figures. The importance of each term (i.e. interactions involving P/A of infection) in the final model was assessed by the probability that its exclusion would significantly alter the model, and both χ2 and P values for these interactions are given in the text.

Ethical statement

All procedures were conducted under a license from the First Warsaw Local Ethics Committee for Animal Experimentation in Poland (Ethical license numbers: 73/2010, 148/2011, 304/2012 and 706/2015). All rodents were culled by Prof. Anna Bajer (authorized to implement experimental procedures and the culling of animals for scientific objectives by the Polish Laboratory Animal Science Association, (License number, 13/2015)

This study was carried out in strict accordance with the recommendations in the Guidelines for the Care and Use of Laboratory Animals of the Polish National Ethics Committee on Animal Experimentation, and according to the Polish national law for field studies involving the trapping and culling of unprotected vertebrates for scientific purposes (Resolution No. 12/2022 of the Polish National Ethics Committee on Animal Experimentation, 11 March 2022). The study was performed according to the ARRIVE guidelines 2.0.

Results

Prevalence of infection and infestation

Based on our screening of blood smears and the subsequent molecular analysis (PCR), the overall prevalence of Bartonella spp. infections in rodents was 42.3% (421/994) (Table 1) while, the overall prevalence of Bartonella spp. in fleas was 11.3% (109/833) (Table 2). Moreover, the total prevalence and mean abundance of fleas on rodents were 60.4% (303/509) and 2.94 fleas per individual, respectively (Table 4). The most common flea species was C. assimilis (n = 260), while the mole flea H. talpae was the rarest (n = 54).

Table 4 Flea infestations (%) on rodents in the three years of study.

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Effect of intrinsic and extrinsic factors on Bartonella prevalence

In our fieldwork in 2013 and 2014 we trapped Microtus spp. (two species) and one Alexandromys sp. from open areas (fallow land), while in 2018 we trapped C. glareolus from forest areas. Hence, we performed statistical analyses separately for these two areas. In the Microtus spp. dataset, we investigated how host species, host sex, flea species, and flea sex (intrinsic factors) and the year of study (an extrinsic factor) impact the prevalence of Bartonella spp. infections. The log-linear analysis revealed a significant interaction between flea species, year of study, and P/A of Bartonella spp. infections (χ24 = 10.1, P = 0.037). In 2013, Bartonella spp. were detected only in two flea species (C. assimilis and C. agyrtes), whereas in 2014, Bartonella was identified in all species of fleas with varying prevalence (Table 2; Fig. 2A).

Fig. 2

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(A) Prevalence of Bartonella spp. (%) in flea species. (B) Prevalnce of Bartonella spp. (%) by flea species and year of study. (C) Prevalence of Bartonella spp. (%), by flea sex and year of study. (D) Prevalence of Bartonella spp. (%), by study sites. (E) Prevalence of Bartonella spp. (%), by age class of host (rodents) and year of study.

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The second significant interaction was between flea sex, year of study and P/A of Bartonella spp. infections (χ21 = 5.6, P = 0.018). In 2013 only, males of C. assimilis and C. agyrtes tested positive for Bartonella spp. whereas in 2014 males of C. agyrtes, M. walkeri and M. turbidus showed higher prevalence of Bartonella spp. than females. However, there was no significant effect of host species on the prevalence of Bartonella spp. (Fig. 2B).

Analysis of Bartonella spp. prevalence in C. glareolus (2018), revealed a significant effect of study site on the prevalence of Bartonella spp. infections (χ22 = 9.7, P = 0.007), the highest prevalence of 24.7% being recorded in Tałty (Fig. 2). Furthermore, prevalence of Bartonella spp. differed significantly between flea species (χ24 = 9.5, P = 0.045); the highest prevalence of Bartonella spp. infections was in M. turbidus (26.9%) while the lowest was in C. agyrtes (9.7%) (Fig. 2D).

Moreover, there was a significant interaction between the age class of bank voles, year of the study and infection with Bartonella spp. (X24 = 15.4, P = 0.004). The highest prevalence of Bartonella spp. was 43.5% in 2014 (Table 2; Fig. 2E). In years 2014 and 2018 the highest prevalence of Bartonella spp. was recorded in the third age class (the oldest) of bank voles, whereas in 2010 the highest was in the first age class (Table 2; Fig. 2E).

Identification of Bartonella species from rodents, moles and fleas

Following sequencing of rpoB gene PCR products (333–860 bp), we obtained 104 sequences of Bartonella spp. from seven species of rodents, 53 sequences from fleas and 2 sequences from moles (Table 5). Of the 104 sequences from rodents, 67 were identified as B. grahamii, which was present in four species of rodents (A. flavicollis, A. sylvaticus, A. oeconomus, and C. glareolus). Thirty-three sequences were identified as B. taylorii, which was present in five species of rodents (A. flavicollis, A. sylvaticus, M. agrestis, Microtus sp. and C. glareolus; Table 6). Three isolates showed 96–98% homology to a newly described species, Bartonella bilalgolemii51, and clustered in one subgroup containing B. rochalimae from dogs in the USA (DQ676489) and a Bartonella rochalimae-like isolate from M. arvalis from Poland (MG839175), (Fig. 3A; Table 6). Additionally, two Bartonella sequences from moles were identical and could not be identified to species level, because they showed only a low homology of 95% to B. taylorii from A. sylvaticus from the UK (CP083693).

Table 5 Number of sequences of Bartonella spp. Isolated from rodents, moles, and fleas.

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Table 6 Molecular identification of Bartonella spp. In rodents, European moles, and fleas.

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Fig. 3

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(A) The phylogenetic tree of Bartonella spp. inferred from sequence variation of 860-bp rpoB gene fragment. The tree is 50%-majority rule consensus obtained using MrBayes (Bayesian Inference). Numbers along nodes represent a posteriori probability. The clades of B. taylorii and B. grahamii were collapsed into simplified forms for clarity. Custers of sequences obtained from Białobrzegi and Kury are highlighted in green and orange, respectively. (B) The phylogenetic tree of Bartonella spp. inferred from sequence variation of 860-bp rpoB gene fragment. The B. grahamii clade is shown in greater detail. (C) The phylogenetic tree of Bartonella spp. inferred from sequence variation of 860-bp rpoB gene fragment. The B. taylorii clade is shown in greater detail.

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Moreover, we obtained 19 DNA sequences of B. grahamii from four species of fleas: C. assimilis, M. turbidus, M. walkeri and H. talpae. Seventeen sequences of B. taylorii were obtained from all five species of fleas and one unidentified sequence/species of Bartonella isolated from H. talpae (Table 6).

Phylogenetic analysis of the rpoB gene

Phylogenetic analyses were conducted on 135 obtained sequences of Bartonella spp. and 64 reference sequences from GenBank (Fig. 3A–C). Phylogenetic analysis by the MrBayes method, revealed five major clades: Clade I, which included B. grahamii, contained seven diverse branches/groups related to the country of origin, with our sequences clustered on three of these branches (Fig. 3B). One branch/group contained Polish B. grahamii sequences from C. glareolus, A. oeconomus, M. arvalis, A. flavicollis, M. turbidus, and M. walkeri. The second group contained isolates from different hosts and countries and showed more diversity than the first group. The third group contained two sequences from (A) sylvaticus that clustered with one sequence from C. glareolus, all from Poland (Fig. 3B). Interestingly, the B. grahamii clade also clustered with B. elizabethae, a zoonotic pathogen from the UK (Fig. 3A–C).

Sequences of B. taylorii formed clade II and showed higher diversity than B. grahamii. This clade contained 6 branches/groups, one of which contained identical sequences of B. taylorii from Poland and Lithuania, from the flea C. agyrtes, and three species of rodents (A. flavicollis, M. agrestis and M. arvalis), and formed a sister branch with two subgroups of B. taylorii from C. glareolus, Microtus spp., and C. agyrtes (Fig. 3C). The second group of B. taylorii contained two subgroups; one sequence from C. glareolus and the second B. taylorii from Microtus spp. Interestingly, the third group of B. taylorii contained four sequences from fleas C. assimilis and C. agyrtes which grouped with sequences from M. arvalis obtained previously by our team from the same place- Łuknajno lake area14,27 (Fig. 3C). The next subgroup of B. taylorii sequences from C. glareolus was located as a sister branch to the subgroup of B. taylorii sequences from fleas (C. assimilis and C. agyrtes). The last subgroup of B. taylorii sequences from A. flavicollis from Poland and Lithuania clustered with B. taylorii from C. agyrtes fleas collected from C. glareolus from Poland. The two Bartonella sequences from T. europaea and one from A. sylvaticus formed two separate branches close to the B. taylorii group, possibly indicating a novel species of Bartonella (Fig. 3A–C). Three sequences of Bartonella sp. isolated from A. oeconomus and A. agrarius from Białobrzegi and one Bartonella sp. from M. arvalis formed a sister group to B. bilalgolemii recently described in Apodemus uralensis from Turkey51, furthermore these groups closely clustered to B. rochalimae from Canis lupus familiaris from the USA and from Homo sapiens from Peru (Fig. 3A–C). Clades III, IV and V contained reference sequences of Bartonella spp. from different hosts and countries.

Phylogenetic analysis of the gltA gene

Phylogenetic analysis of the gltA gene, by the MrBayes method was conducted on 11 DNA sequences of Bartonella spp. obtained from fleas (collected in 2018) and 21 reference sequences from GenBank and revealed three clades/ groups (Fig. 4).

Fig. 4

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The phylogenetic tree of Bartonella spp. inferred from sequence variation of 810-bp gltA gene fragment. The tree is 50%-majority rule consensus obtained using MrBayes (Bayesian Inference). Numbers along the nodes represent a posteriori probability. Clusters of sequences obtained from T. europaea from Kury are highlighted in red rectangular on the first clade.

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Clade I encompassed B. taylorii sequences and consisted of three subgroups. In the first subgroup four sequences of B. taylorii obtained from C. agyrtes clustered with B. taylorii sequences from Sweden and Poland. In the second subgroup, one B. taylorii sequence from H. talpae clustered with B. taylorii from Russia, China, and Japan. Interestingly, our Bartonella sequences obtained from T. europaea formed the third subgroup with Bartonella sp. from T. europaea from the Netherlands (Fig. 4).

Clade II is divided into two subgroups; one contained B. rattaustraliani, B. coopersplainsensis and B. henselae sequences from Australia and the second subgroup contained six unidentified Bartonella spp., which are related to unidentified species of Bartonella from different countries.

Clade III encompasses B. grahamii sequences, including three sequences of B. grahamii obtained from M. turbidus fleas, which formed a separate sister subgroup to B. grahamii from A. agrarius from Russia (Fig. 4). Moreover, one Bartonella sequence from H. talpae formed a separate branch within the B. grahamii clade (Fig. 4).

Association between flea and rodent infections with Bartonella

We compared the genetic identity of Bartonella detected in fleas, and infection status and species of the rodent hosts from which the fleas were collected. In seven out of 22 Bartonella -positive fleas the same species of Bartonella was identified in both fleas and rodent hosts (Table 7). The DNA of B. grahamii and B. taylorii was detected in C. assimilis, M. turbidus, M. walkerii and H. talpae fleas, while only B. taylorii DNA was found in C. agyrtes (Table 7).

Table 7 Comparison of Bartonella spp. Isolated from the collected fleas and their hosts.

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Flea identification by 18S rDNA sequencing

To confirm morphological identification of flea species, we used a fragment of the 18S rDNA gene (570 bp) for the molecular and phylogenetic analyses. All Bartonella -positive fleas used for Bartonella genotyping (n = 26) were sequenced and subjected to phylogenetic analysis (Fig. 6). For the phylogenetic analysis, all the DNA sequences from this study and 10 reference sequences from GenBank were used. The phylogenetic analysis based on the 18S rDNA gene enabled recognition of three clades of fleas representing three genera: clade I encompassed sequences of Ctenophthalmus spp., clade II sequences of Hystrichopsylla spp. and clade III - Megabothris spp. No further resolution of flea species was possible, so based on morphological identification, and because of the lack of reference sequences for fleas, all the sequences obtained from fleas were deposited in the GenBank database (Fig. 5).

Fig. 5

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The phylogenetic tree of the 18 S rDNA of fleas was inferred by using the Maximum Likelihood method and Kimura 2-parameter model by MEGA11.

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Haplotypes of Bartonella grahamii and Bartonella taylorii

Minimum Spanning Network analysis of the rpoB sequences of B. grahamii (n = 81) obtained in this study revealed nine haplotypes (Gr1-Gr9), differing by 1–4 SNPs (Additional file 1). Haplotype Gr1 was the main haplotype encompassing 70 out of 81 sequences (87.5%), identified in four species of rodents (C. glareolus, A. sylvaticus, A. flavicollis, and A. oeconomus) and in two species of fleas (M. turbidus and M. walkeri) (Fig. 6A). Moreover, haplotype Gr1 involved sequences from all study sites (Fig. 6B). Haplotype Gr2 encompassed two sequences (2.5%) from C. glareolus from Urwitałt and Tałty and differed by one SNP from the dominant haplotype Gr1. Interestingly, haplotype Gr3 encompassed two sequences (2.5%) from A. sylvaticus from Białobrzegi and differed from the dominant haplotype Gr1 by four SNPs. All other haplotypes grouped around haplotype Gr1 and were separated by 1–4 SNPs (Fig. 6A,B).

Despite this generally low genetic diversity, there was some host, vector and site-specific segregation of less common haplotypes, for example haplotypes Gr6, Gr7 and Gr8 were identified in C. glareolus from Urwitałt, whereas haplotypes Gr4 and Gr5 were present only in M. turbidus fleas from the Łuknajno site (Fig. 6A,B).

Fig. 6

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The Minimum Spanning Network of the rpoB gene showing the relationship between the haplotypes. (A) Bartonella grahamii haplotypes based on the host and vector species; (B) Bartonella grahamii haplotypes based on the study sites; (C) Bartonella taylorii haplotypes based on the host and vector species; (D) Bartonella taylorii haplotypes based on the study sites.

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Minimum Spanning Network analysis of the rpoB sequences of B. taylorii (n = 45) successfully delineated 17 haplotypes (T1-T17), differing by 1–8 SNPs (Additional file 1). Haplotype T1 was the dominant haplotype encompassing 15 out of 45 sequences (33.3%) and was identified in C. glareolus and C. agyrtes from three sites, Urwitałt, Tałty and Pilchy (Fig. 6C and D). Haplotype T2 encompassed 8 out of 45 sequences (17.7%) and was also found in C. glareolus from three sites. Haplotype T2 differed from T1 by 5 SNPs. Interestingly, haplotype T3 was present only in the fleas C. assimilis and C. agyrtes from site 4 (Łuknajno) and was represented by 4 sequences out of 45 (8.8%) (Fig. 6C and D). The differences between T1, T2 and T3 were 3–5 SNPs, respectively. Moreover, haplotype T4 was isolated from the same species of fleas as haplotype T3 but was present in Pilchy and Łuknajno and differed from haplotype T2 by one single SNP. All other haplotypes of B. taylorii grouped around haplotypes T1 and T2 and were separated by 1–8 SNPs.

Discussion

This study examined the prevalence, diversity, and phylogenetic relationships of Bartonella spp. in rodent and flea communities in Northeastern and Central Poland. We identified Bartonella spp. in eight rodent species and five flea species. The overall prevalence of Bartonella spp. infection in the rodent community was found to be 42.3%, although this figure varied in comparison to previous studies and was influenced by rodent species and environmental factors (study site). In our previous investigation of haemoparasites in bank voles from NE Poland, we reported a prevalence of Bartonella spp. infections of 30.6% in a rodent community comprising A. flavicollis, C. glareolus, M. arvalis, and A. oeconomus52. Subsequent studies indicated a prevalence of 38.7% in C. glareolus13 and 66.8% in the Microtus spp. community (comprising M. arvalis, M. agrestis, and A. oeconomus)14. In contrast, prevalence rates in rodents from other countries have been reported at 54.8% in Lithuania, 26% in the Netherlands, and 43% in Germany17,19,53,54. The notably high prevalence rates of Bartonella in rodents are significant, as these synanthropic host species are frequently found in habitats where they may come into close contact with humans, potentially posing a risk of exposure.

In our study, five flea species were collected from rodents, four of which (C. agyrtes, C. assimilis, M. turbidus, and H. talpae) are also common on rodents in Slovakia23. The overall prevalence of Bartonella spp. in flea populations associated with rodents was 11.3%, which is lower than previously reported rates of 59.4% in Germany, 34% in Slovakia, and 28% in Chile23,54,55. However, the intensity of flea infestation on rodents in our study was comparable to the 2.44 fleas per individual reported in Slovakia23. In 2013, we detected Bartonella spp. DNA in only two flea species, C. assimilis and C. agyrtes, paralleling findings from a previous study on flea communities in dogs in Israel, where Bartonella spp. DNA was identified in two out of four flea species (Ctenocephalides canis and Ctenocephalides felis)56.

Krasnov et al. (2006)57 have suggested that the composition of flea species on host organisms is influenced by both host-flea and host-habitat interactions. Our analysis indicates that the year of study also may have impacted the Bartonella-flea-host interaction, because we detected Bartonella spp. in only two flea species in 2013, while in 2014 and 2018, Bartonella spp. DNA was identified in all five flea species collected from various hosts. This finding is consistent with previous studies conducted in Chile and Tanzania, where Bartonella spp. DNA was found in all tested flea species55,58. Interestingly, Gutiérrez et al. (2018)20 reported only one flea species, Synosternus cleopatrae, with confirmed Bartonella spp. DNA on Gerbillus andersoni and Gerbillus pyramidum in Israel, suggesting a greater specificity of flea-host relationships compared to our findings.

In 2013, we observed a higher prevalence of Bartonella spp. in male fleas compared to females, potentially linked to the higher male-to-female ratio among the sampled fleas (55% vs. 45%, respectively). In contrast, no significant sex-related differences in Bartonella spp. prevalence were noted in 2014 and 2018 across all five flea species tested, aligning with findings from a previous study on fleas from rodents in Slovakia23, where no significant differences in prevalence were detected between sexes among eight flea species.

Our analysis of Bartonella spp. prevalence in C. glareolus in 2018 revealed significant site-specific effects, with the highest prevalence (24.7%) recorded at site 2 (Tałty). In a previous long-term study (1999–2010) of C. glareolus, we reported a higher overall prevalence (45.4%) of Bartonella spp. at this same site, with peak levels observed in 2006. The lower prevalence of Bartonella spp. observed in the current study suggests changes in conditions affecting parasite survival over time, likely influenced by fluctuations in flea populations, although other unidentified factors cannot be ruled out at this stage. We note a trend of decreasing prevalence of Bartonella spp. infection in C. glareolus through the years of our study (i.e. from 2010, 2014 until 2018) and these results are consistent with our previous long-term studies on the haemoparasites from C. glareolus from NE of Poland, and A. dimidiatus from Egipt13,59. We hypothesised that extrinsic factors would have the major influence on haemoparasite communities, notably through the largely unpredictable long-term temporal effect, resulting in distinct between-year dynamics. Populations of rodent host species often fluctuate significantly over time, sometimes in multi-year cycles or unpredictably between years60. These changes impact the populations of their ectoparasites (vectors) and, consequently, the prevalence of vector-borne pathogens. Studies have found a high prevalence of Bartonella spp. in young voles across various species13,61,62,63. This may be due to high exposure to infections, likely through close contact with flea vectors or possible vertical transmission from infected mothers to offspring. In 2014 and 2018 the oldest bank voles showed the highest prevalence of infection (age class 2 and/or 3). The observed pattern of increased infection likelihood with increasing host age supports the idea that, as a rodent lives longer their chances of encountering an infected vector and thus contracting the infection also increase.

In Europe, B. grahamii and B. taylorii have been detected in various rodent species, including A. flavicollis, A. agrarius, A. sylvaticus, Apodemus uralensis, Apodemus witherbyi, Micromys minutus, C. glareolus, M. arvalis, M. agrestis, and M. musculus. B. rochalimae has been identified in A. flavicollis, C. glareolus, and M. arvalis (reviewed by Špitalská et al., 201766), while B. doshiae has been detected in A. flavicollis, A. agrarius, M. agrestis, and C. glareolus (reviewed by Buffet et al., 201367). In our current study, 58% of sequences derived from small rodents and fleas were classified as B. grahamii, 32% as B. taylorii, 1.9% as Bartonella spp., and 7.8% as an unidentified Bartonella species. While these findings differ somewhat from a study in Lithuania, which reported B. grahamii in 26% of hosts, B. taylorii in 42%, and lower occurrences of B. rochalimae and other species17, B. grahamii and B. taylorii were clearly the dominant species in both studies. Generally, B. taylorii exhibited greater diversity, occurring in five rodent and five flea species, while B. grahamii demonstrated lower diversity, identified in four rodent and four flea species.

Phylogenetic and Minimum Spanning Network analyses based on the rpoB sequence of B. taylorii revealed 17 haplotypes associated with five rodent species and three flea species. Previous studies have reported high genetic diversity of B. taylorii strains in rodents from Poland, Turkey, and the Netherlands14,27,66,67,68, potentially resulting from frequent recombination events, horizontal gene acquisition, and accumulation of mutations (reviewed by Gutiérrez et al.71). In our study, B. grahamii strains exhibited lower sequence diversity, with only nine haplotypes identified, fewer than the 30 haplotypes reported from Sweden69. Notably, five haplotypes were restricted to C. glareolus, and two haplotypes were confined to M. turbidus. The two Bartonella isolates from European moles (T. europaea) were identical and formed separate branches on phylogenetic trees for the rpoB and gltA genes. These sequences closely resembled an uncultured Bartonella sp. obtained from T. europaea in the Netherlands66. Currently, Bartonella talpae is the only species associated with this host70, but no type strain or molecular data exist for Bartonella spp. from moles. Our DNA sequences cannot yet be assigned to this species until a type strain is isolated and characterized. The Bartonella from our study showed host specificity in moles, which aligns with reports of host specificity in certain rodent and shrew species from China71 and rodents from Egypt16. In contrast, B. grahamii and B. taylorii demonstrated poor host specificity, as they were detected across all studied rodent hosts, consistent with findings from our previous work14,27,52. Furthermore, B. grahamii has been reported in 53 small mammal species to-date, while B. taylorii has been identified in 27 small mammal species (reviewed by Krügel et al., 2022).

Our Bartonella rochalimae-like sequence has previously been reported also from M. arvalis from the Mazurian Lake District14. Interestingly, our Bartonella sp. isolate from A. oeconomus and A. agrarius in Białobrzegi (approximately 340 km from the Mazurian Lake District) clustered together with the Bartonella rochalimae-like sequence from M. arvalis with minimal diversity. While B. rochalimae is generally thought to infect rodents29,47, our phylogenetic analysis showed that sequences from a common vole (M. arvalis) from Poland14, which closely resembled our sequence (99.3–99.8%), formed a sister branch to B. rochalimae isolates from dogs and humans in Peru and the USA, but were closer to B. bilalgolemii described recently from A. uralensis from Turkey. Thus, their species identity needs further investigation and currently this Bartonella variant from rodents can be referred to only as Bartonella sp.

Phylogenetic analysis of gltA sequences from Bartonella spp. obtained from fleas during the 2018 fieldwork largely confirmed the results obtained from analysis of the rpoB gene. The B. taylorii clade exhibited greater diversity than the B. grahamii clade. However, the phylogenetic placement of the Bartonella sp. from T. europaea differed between the rpoB and gltA phylogenetic trees. The two Bartonella sp. rpoB sequences from moles clustered on separate branches distinct from other Bartonella species, while they clustered within the B. taylorii clade on the gltA tree. As previously noted16,27, diversity in the gltA gene correlates with host specificity, suggesting that further research is needed to formally describe the isolate from European moles as a novel Bartonella species. One of the gltA sequences from the Bartonella sp. isolated from H. talpae (site 1) formed a distinct branch, exhibiting low homology (87%) to B. taylorii. Unfortunately, the quality of the rpoB sequence for this sample was inadequate for conclusive species identification. However, H. talpae is known to infest various hosts, including dogs73, cats73, dormice (Glis)74 and bank voles75,76, suggesting it may harbour a different or novel species/strain of Bartonella.

Remarkably, Bartonella spp. DNA was identified in 19 fleas collected from Bartonella -negative hosts, indicating a potential vector role of fleas for Bartonella spp. We identified C. assimilis, M. turbidus, M. walkeri, and H. talpae as vectors for B. grahamii and B. taylorii, while C. agyrtes was specifically associated with B. taylorii. These findings confirm the role of fleas as vectors for Bartonella spp. Although Špitalská et al. (2022) suggested that fleas act as vectors for rodent-infective Bartonella spp., precise information regarding the specific flea species involved and the corresponding Bartonella species/strains has been lacking.

Due to the absence of reference sequences for fleas in GenBank, it was challenging to confirm flea identification based on 18S rDNA. Nevertheless, we believe our morphological identifications are accurate, and thus, all obtained DNA sequences of the fleas have been deposited in the GenBank database. There is a dearth of phylogenetic studies on fleas, particularly regarding species infesting rodents; however, some research has focused on cat fleas using the Internal Transcribed Spacer 1 (ITS1) and Internal Transcribed Spacer 2 (ITS2)77, as well as mitochondrial genes such as cytochrome c oxidase subunit I (cox1) and II (cox2)78.

In this study, the haplotypes of the rpoB gene from B. grahamii and B. taylorii were analyzed, enabling a comparison of haplotype diversity, structure, and distribution between both Bartonella species across different sites, hosts, and vectors. Minimum Spanning Network analysis for B. grahamii revealed nine haplotypes, with the dominant haplotype (Gr1, 87.5%) present in four rodent species and two flea species, with sequences originating from all study sites. This result suggests that the Gr1 haplotype is the predominant haplotype found in NW Poland; however, additional B. grahamii sequences from other locations in Poland are necessary to corroborate this conclusion. Interestingly, among the five haplotypes of B. grahamii from C. glareolus (Gr2, Gr6, Gr7, Gr8, and Gr9), three (Gr6, Gr7, and Gr8) originating from Urwitałt, one haplotype was shared between Urwitałt and Tałty, and the last haplotype was from Pilchy. The unique haplotype (Gr3) was exclusively identified in A. sylvaticus from Białobrzegi. These findings support our hypothesis that host identity and study site significantly influence the genetic diversity of Bartonella. Among fleas, two unique haplotypes of B. grahamii were identified in M. turbidus (Gr4 and Gr5), both from Łuknajno, echoing findings from a study on Lipoptena fortisetosa (Diptera, Hippoboscida) in SE Poland, where two unique haplotypes of Bartonella sp. were detected from the same vector and site30.

Minimum Spanning Network analysis of B. taylorii revealed 17 haplotypes, with two dominant haplotypes (T1 = 33.3% and T2 = 17.7%) encompassing sequences from C. glareolus and C. agyrtes from three sites: Urwitałt, Tałty, and Pilchy. The detection of haplotype T1 in both C. glareolus and C. agyrtes suggests a correlation between host, bacterium, and vector, indicating that host identity and vector specificity may influence the genetic diversity of B. taylorii. The remaining haplotypes (T7, T8, T9, T10, T11, T12, T13, T14, T15, T16, and T17) are unique and related to their respective hosts or vectors, with six haplotypes identified from Tałty, three from Białobrzegi, and one from Łuknajno (Additional file 1).

A previous investigation of Bartonella haplotypes in rodents from Poland27 provided strong evidence for recombination within the rpoB gene among all B. grahamii haplotypes and all B. taylorii haplotypes, isolated from four rodent species (A. flavicollis, M. arvalis, A. oeconomus, and C. glareolus). The analysis of the gltA gene revealed six haplotypes of B. grahamii and 24 haplotypes of B. taylorii.

Conclusions

We identified nine haplotypes of B. grahamii and 17 haplotypes of B. taylorii from rodents and fleas at five study sites in Poland. Two Bartonella species, the first isolated from T. europaea and the other from A. sylvaticus, and the second from A. agrarius and A, oeconomus are proposed as novel species (Fig. 3). Furthermore, we conclude that the fleas C. agyrtes and H. talpae are vectors for B. taylorii, while C. assimilis and M. walkeri serve as vectors for B. grahamii, and M. turbidus is a vector for both B. grahamii and B. taylorii.

Data availability

Data availabilityAll relevant data are included in the article. All DNA sequences of Bartonella spp. and fleas have been deposited in GenBank (https://www.ncbi.nlm.nih.gov/nucleotide/) under accession numbers provided in main text and Additional file 1.

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Acknowledgements

We thank Mgr Grzegorz Górecki and mgr Anna Zaborowska for their help and hospitality at The Masurian Center For Biodiversity And Nature Education in Urwitałt.

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Authors and Affiliations

Department of Eco-Epidemiology of Parasitic Diseases, Institute of Developmental Biology and Biomedical Sciences, Faculty of Biology, University of Warsaw, Miecznikowa 1, Warsaw, 02-096, Poland

Mohammed Alsarraf, Katarzyna Tołkacz, Mustafa Alsarraf, Dorota Dwużnik-Szarek, Monika Juśko, Dina Al-qazaz, Paulina Drabik & Anna Bajer

Institute of Evolutionary Biology, Faculty of Biology, Biological and Chemical Research Centre, University of Warsaw, Żwirki i Wigury 101, Warsaw, 02-089, Poland

Łukasz Banasiak

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5A, Warsaw, 02-106, Poland

Katarzyna Tołkacz

Department of Tropical Parasitology, Medical University of Gdańsk, Powstania Styczniowego 9B, Gdynia, 81-519, Poland

Maciej Grzybek

Department of Forest Entomology and Pathology, Faculty of Forestry and Wood Technology, Poznań University of Life Sciences, Poznań, Poland

Jolanta Behnke-Borowczyk

School of Life Sciences, University of Nottingham, University Park, Nottingham, NG7 2RD, UK

Jerzy M. Behnke

Department of Microbiology, Molecular Genetics and Genomics, Centre for Advanced Materials and Technologies, CEZAMAT, 19 Poleczki St, Warsaw, 02-822, Poland

Mustafa Alsarraf

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Mohammed Alsarraf

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