Bartonella infections are rare in blood-fed Ixodes scapularis and Ixodes pacificus ticks collected from rodents in the United States

Abstract

Background

Ixodes scapularis and Ixodes pacificus are important vectors of multiple pathogens in the United States. However, their role in transmission of Bartonella spp., which are commonly reported in rodents and fleas, has been debated. Our previous investigation on Bartonella spp. in host-seeking I. scapularis and I. pacificus showed Bartonella spp. were absent in the ticks, suggesting the two species are unlikely to contribute to Bartonella transmission. It is unclear whether the absence of Bartonella spp. in the host-seeking ticks was attributable to ticks not being exposed to Bartonella in nature or being exposed but unable to acquire or transstadially transmit the bacterium. To assess the likelihood of exposure and acquisition, we tested Ixodes spp. ticks collected from rodents for Bartonella infections.

Methods

Blood-fed I. scapularis ticks (n = 792; consisting of 645 larvae and 147 nymphs), I. pacificus ticks (n = 45, all larvae), and Ixodes angustus ticks (n = 16, consisting of 11 larvae and 5 nymphs) collected from rodents from Minnesota and Washington were tested for Bartonella spp. using a quadruplex polymerase chain reaction (PCR) amplicon next-generation sequencing approach that targets Bartonella-specific fragments on gltA, ssrA, rpoB, and groEL. In parallel, rodents and fleas collected from the same field studies were investigated to compare the differences of Bartonella distribution among the ticks, fleas, and rodents.

Results

Bartonella spp. were commonly detected in rodents and fleas, with prevalence of 25.6% in rodents and 36.8% in fleas from Minnesota; 27.9% in rodents and 45.2% in fleas from Washington. Of all tested ticks, Bartonella DNA was detected by gltA in only one larval I. scapularis tick from Minnesota.

Conclusions

The high prevalence of Bartonella spp. in rodents and fleas coupled with extremely low prevalence of Bartonella spp. in blood-fed ticks suggests that although Ixodes ticks commonly encounter Bartonella in rodents, they rarely acquire the infection through blood feeding. Notably, ticks were at various stages of feeding on rodents when they were collected. Laboratory transmission studies are needed to assess acquisition rates in fully blood-fed ticks and to assess transstadial transmission efficiency if ticks acquire Bartonella infections from feeding to repletion.

Graphical Abstract

Background

The Gram-negative Bartonella bacteria are widely distributed in a variety of mammalian hosts. Particularly, rodents of many species have been reported to be reservoirs for more than 30 Bartonella species [1–6]. Some Bartonella spp., such as Bartonella elizabethae, B. vinsonii subsp. arupensis, and B. grahamii have been associated with human illnesses and considered zoonotic pathogens [7–11]. Host specificity has been observed for many Bartonella spp. For example, B. elizabethae is Rattus rat-associated [6], while B. vinsonii subsp. arupensis is specific to Peromyscus sp. mice [4, 12].

Bartonella spp. are mainly transmitted by hematophagous arthropods, including sandflies (Lutzomyia verrucarum), human body lice (Pediculus humanus corporis), cat fleas (Ctenocephalides felis), and some rodent fleas [13–17]. The role of other arthropods, such as ticks, in the natural cycle of Bartonella spp. and the transmission of these bacteria has been debated. Molecular surveys from different regions of the world on Bartonella infections in host-seeking ticks, mainly Ixodes ticks, have reported conflicting results, with detection of Bartonella DNA in high or low proportions or none of the tested ticks [18–30].

The blacklegged tick (Ixodes scapularis) and the western blacklegged tick (Ixodes pacificus) are widely distributed in the eastern and far western US, respectively. These ticks frequently bite people [31] and serve as vectors of the Lyme disease spirochete and several other human pathogens [32–34]. We recently tested ~ 2600 host-seeking I. scapularis and I. pacificus nymphs and adults collected across a broad geographic region in the US for Bartonella spp. [35]. Consistent with some previous reports [22, 24, 29], we did not detect any Bartonella DNA in the ticks. Our findings suggested that I. scapularis and I. pacificus ticks are unlikely to contribute to transmission of Bartonella spp. These findings raised several questions. Specifically, which Bartonella species do Ixodes ticks encounter in nature? When feeding on an infected host, how efficiently do they acquire Bartonella infection and how efficiently are bacteria transmitted transstadially?

Previous studies on Bartonella spp. in ticks have mostly focused on host-seeking ticks with few studies focused on blood-fed ticks derived from animal hosts [36–43]. Most of the studies on blood-fed ticks showed low to moderate Bartonella prevalence in the ticks, especially Ixodes ticks. For example, in Poland, Bartonella spp. were detected in 5.2% of Ixodes ricinus ticks collected from deer [36]; in the UK, 1.3% of I. ricinus ticks collected from cats showed Bartonella infection [39]; a study from the US reported 13.3% of I. scapularis ticks being infected with Bartonella species based on testing of 15 ticks collected from white-footed mice (Peromyscus leucopus) in southern Indiana using 454 pyrosequencing technology [43].

Peromyscus mice, including P. leucopus and P. maniculatus are common sources of blood meals for I. scapularis and I. pacificus ticks in the US [44]. These mice are known to be infected frequently with Bartonella spp., specifically, B. vinsonii subsp. arupensis [12], a species that has been associated with human bartonellosis [8, 10, 11]. In the present study, we analyzed a large number of blood-fed larvae and nymphs of I. scapularis, I. pacificus, and Ixodes angustus collected from P. leucopus, P. maniculatus, and rodents of other species trapped in Minnesota and Washington for the presence of Bartonella DNA to study the dynamics of Bartonella infection in the ticks and as a follow-up of our previous study [35]. Meanwhile, we tested rodent blood and fleas collected from rodents to learn the status of Bartonella distribution and to identify which Bartonella spp. were circulating in our study sites.

Methods

Study sites and sample collections

The ticks, rodents, and fleas included in the present investigation were residuals of samples from two previous field studies conducted by CDC and other investigators at Camp Ripley (CARI), Chippewa National Forest (CHIP), Itasca State Park (ITAS), Saint Croix State Park (SCSP), and William O’Brien State Park (WIOB) in Minnesota in June 2016 and in San Juan County, Washington in April 2019 (Fig. 1). The study sites are of forested recreational settings in Minnesota and forested rural residential setting in Washington. In both studies, rodents were live-trapped using Sherman traps (H. B. Sherman Traps, Inc., Tallahassee, FL) and Tomahawk traps (Tomahawk Live Trap, Hazelhurst, WI) as previously described [45]. Rodents were identified to species morphologically using Peterson Field Guide [46] or genetically by ctyB using primers Pero_F and Pero_R [45], and blood was collected through submandibular venipuncture under isoflurane anesthesia, or post-mortem by cardiocentesis. Ticks and fleas were collected from the animal hosts and morphologically identified to species, sex, and life stage using taxonomic keys [47–50]. Notably, ticks were at various stages of engorgement when they were collected. All animal procedures were approved by the CDC Division of Vector-Borne Diseases (DVBD) Institutional Animal Care and Use Committee. All samples were shipped to CDC-DVBD in Fort Collins, Colorado.

Fig. 1.

Study sites. Rodents were trapped in Camp Ripley (CARI), Chippewa National Forest (CHIP), Itasca State Park (ITAS), Saint Croix State Park (SCSP), and William O’Brien State Park (WIOB) in Minnesota, June 2016, and San Juan County, Washington, April 2019

DNA of rodent blood was extracted previously using the cador Pathogen 96 QIAcube HT Kit on the QIAcube HT (Qiagen, Chatsworth, CA) and stored at -80 ℃. Residual DNA was used for the present study; DNA of Washington ticks had been extracted previously using the MagMAX™ Pathogen Kit on the KingFisher DNA extraction system (Thermo Fisher Scientific, Waltham, MA, USA). Residual DNA was used for the present study; DNA of Minnesota ticks and all fleas from both states were freshly extracted for use in the present study using the MagMAX™ CORE Kit on the KingFisher DNA extraction system (Thermo Fisher Scientific, Waltham, MA, USA). Negative extraction controls were included to detect contaminations.

PCR amplification, library preparation, next-generation sequencing, and bioinformatics analysis

All DNA samples, including tick DNA, flea DNA, and blood DNA, were tested for the presence of Bartonella spp. using a quadruplex PCR amplicon next-generation sequencing assay that targets gltA, ssrA, rpoB, and groEL using Bartonella-specific primers [51]. Presumptive presence of Bartonella DNA is defined when one target sequence has been obtained, and Bartonella species is confirmed when sequences of at least two targets have been obtained [51].

Detailed procedures follow those published elsewhere [35, 51]. Briefly, a primary PCR reaction containing 12.5 μl TEMPase 2 × master mix (AMPLICON, Denmark), the four pairs of Bartonella-specific primers (final concentration of 300 nM each), and 5 μl tick DNA was first amplified. Positive and negative controls were included in each PCR run to evaluate performance and detect contamination. Upon completion of amplification, the PCR products were purified with AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA) followed by index PCR using dual unique barcode indices (Nextera XT Index Kit V2, Illumina, San Diego, CA, USA) and then purified with MagSi-DNA allround magnetic beads (BOCA Scientific, Westwood, MA, USA). The purified products were then pooled, quantified, normalized, and denatured to generate the final library to be loaded into a MiSeq Nano v2 (500 cycles) reagent cassette (Illumina, San Diego, CA, USA) to start sequencing on an Illumina MiSeq instrument (Illumina, San Diego, CA, USA).

After sequencing was completed, raw sequences were analyzed with a custom Nextflow bioinformatics pipeline described by Osikowicz et al. [52]. Briefly, quality control analysis and primer trimming were first performed followed by error correction, paired read merging, and amplicon sequence variant (ASV) grouping. The observed ASVs were then aligned to reference sequences with the nucleotide Basic Local Alignment Search Tool (BLASTn) [53, 54]. The minimum read cut-off for a sample to be considered positive was set to 50 reads. A 95% sequence similarity and 90% minimum sequence alignment length were used to align the observed ASVs to the reference sequences. Sequences that represent different Bartonella species for each target were obtained from GenBank and used as reference sequences.

Statistical analysis

Chi-square analyses were performed using the Chi Square Test Excel Function to determine whether Bartonella prevalences differed among the study sites in Minnesota and to compare Bartonella prevalence between tick-infested and tick-free rodents from both Minnesota and Washington.

Results

Summary of rodent and ectoparasite samples

In Minnesota, blood was obtained from 215 individual rodents, with 41, 57, 25, 61, and 31 from site CARI, CHIP, ITAS, SCSP, and WIOB, respectively (Table 1). The rodents belonged to nine species with P. leucuous as the most prevalent species (58.1%; 125/215). Other common species included Clethrionomys gapperi (17.2%; 37/215) and P. maniculatus (9.8%; 21/215) (Table 2). Of all rodents, 159 (74%) were infested with ticks. A total of 792 ticks consisting of 645 larvae and 147 nymphs were tested in the present study (Table 3). All ticks collected in Minnesota were identified as I. scapularis. Among the ticks, 140 were collected from 37 Bartonella-infected rodents (see the following section “Bartonella spp. in rodents, fleas, and ticks from Minnesota” for data on Bartonella-infected rodents). Fleas collected from rodents from site SCSP were included in this study, with a total of 68 fleas of six species collected from 30 rodents (Table 4).

Table 1.

Bartonella distribution in rodents at different site in Minnesota (June 2016)

Site	No. rodents tested	No. Bartonella-positive rodents	Bartonella prevalence (%)
Camp Ripley (CARI)	41	5	12.2
Chippewa National Forest (CHIP)	57	19	33.3
Itasca State Park (ITAS)	25	8	32
Saint Croix State Park (SCSP)	61	18	29.5
William O’Brien State Park (WIOB)	31	5	16.1
Total	215	55	25.6

Table 2.

Bartonella distribution in rodents of different species captured in Minnesota (June 2016) and Washington (April 2019)

Rodent species	Collection state	No. tested	Proportion (%)	Bartonella testing		Bartonella species
				No. positive	Prevalence (%)	B. vinsonii subsp. vinsonii	B. vinsonii subsp. arupensis	B. grahamii
Clethrionomys gapperi	Minnesota	37	17.2	12	32.4	3		9
Glaucomys volans	Minnesota	3	1.4	0	0
Microtus pennsylvanicus	Minnesota	1	0.5	0	0
Peromyscus leucopusa	Minnesota	125	58.1	35	28		35
Peromyscus maniculatusa	Minnesota	21	9.8	8	38.1		8
Sciurus carolinensis	Minnesota	1	0.5	0	0
Tamias striatus	Minnesota	19	8.8	0	0
Tamiasciurus hudsonicus	Minnesota	4	1.9	0	0
Zapus hudsonicus	Minnesota	4	1.9	0	0
Subtotal	Minnesota	215	100	55	25.6
Peromyscus maniculatus	Washington	43	100	12	27.9		12

^aPeromyscus leucopus and Peromyscus maniculatus collected in Minnesota were genetically differentiated by ctyB using primers Pero_F and Pero_R [45]

Table 3.

Bartonella infections in blood-fed Ixodes ticks collected from rodents captured in Minnesota (June 2016) and Washington (April 2019)

Tick species	Collection state/site	From all rodents						From Bartonella-infected rodents
		Life stage		Bartonella testing		Prevalence (%)	Life stage			Bartonella testing		Prevalence (%)
		Larva	Nymph	No. tested	No. positive		Larva		Nymph	No. tested	No. positive
Ixodes scapulars	Minnesota/CARI	224	107	331	1a	0.3	24		2	26	1a	3.8
Ixodes scapulars	Minnesota/CHIP	75	6	81	0	0	21		0	21	0	0
Ixodes scapulars	Minnesota/ITAS	116	6	122	0	0	57		3	60	0	0
Ixodes scapulars	Minnesota/SCSP	146	18	164	0	0	29		0	29	0	0
Ixodes scapulars	Minnesota/WIOB	84	10	94	0	0	4		0	4	0	0
Subtotal	Minnesota	645	147	792	1a	0.1	135		5	140	1a	0.7
Ixodes pacificus	Washington/San Juan	45	0	45	0	0	10		1	11	0	0
Ixodes angustus	Washington/San Juan	11	5	16	0	0	5		2	7	0	0
Subtotal	Washington	56	5	61	0	0	15		3	18	0	0

^aPresumptive presence of Bartonella species but species identification cannot be confirmed as Bartonella sequence was obtained only by gltA

Table 4.

Bartonella prevalence and Bartonella species in fleas collected from rodents captured in Minnesota (June 2016) and Washington (April 2019)

Flea taxonomy	Collection state	Bartonella testing			Bartonella species
		No. tested	No. positive	Prevalence (%)	B. vinsonii subsp. arupensis	B. grahamii	B. volans
Ctenophthalmus pseudagrytes	Minnesota	1	0	0
Megabothris spp.	Minnesota	9	3	33.3		3
Opisodasys pseudarctomys	Minnesota	1	1	100			1
Orchopeas howardi	Minnesota	10	1	10			1
Orchopeas leucopus	Minnesota	23	8	34.8	8
Orchopeas spp.	Minnesota	24	12	50			12
Subtotal		68	25	36.8	8	3	14
Catallagia spp.	Washington	5	2	40	2
Ceratophyllidae (Family)	Washington	1	1	100	1
Hystrichopsylla schefferi	Washington	2	0	0
Monopsyllus wagneri	Washington	2	1	50	1
Opisodasys keeni	Washington	14	6	42.9	6
Opisodasys spp.	Washington	15	9	60	9
Othersa	Washington	3	0	0
Subtotal		42	19	45.2	19

^aFlea species was not identified

In Washington, blood was collected from 43 individual rodents. All rodents were P. maniculatus (Table 2). Of the mice, 19 were infested with ticks, and 20 were infested with fleas. A total of 56 ticks including 45 I. pacificus larvae and 16 I. angustus (11 larvae and 5 nymphs) were collected and tested (Table 3). Among the ticks, 17 (10 I. pacificus larvae, 5 I. angustus larvae, and 2 I. angustus nymphs) were collected from 7 Bartonella-infected rodents (see the following section “Bartonella spp. in rodents, fleas, and ticks from Washington” for data on Bartonella-infected rodents). A total of 42 fleas of seven species were collected and tested (Table 4).

Bartonella spp. in rodents, fleas, and ticks from Minnesota

Rodents: Bartonella DNA was detected in 55 (25.6%) of the 215 tested rodent blood samples by at least two of the four targets (gltA, ssrA, rpoB, and groEL) used in the quadruplex PCR amplicon next-generation sequencing assay [51]. The Bartonella-infected rodents were distributed at all sites with prevalence ranging from 12.2% to 33.3% among sites (Table 1). Bartonella prevalence in rodents was similar among the five sites (χ² = 8.1, df = 4, p = 0.09). Among rodent species, Bartonella prevalence was the highest in P. maniculatus (38.1%; 8/21) followed by C. gapperi (32.4%; 12/37) and P. leucopus (28%; 35/125). No Bartonella DNA was detected in other rodent species (Table 2). Sequence analysis showed the Bartonella detected in the P. maniculatus (n = 8) and the P. leucopus (n = 35) belonged to B. vinsonii subsp. arupensis, a species specific to Peromyscus mice [4, 12]. The Bartonella detected in the C. gapperi were identified as B. grahamii (9/12) and B. vinsonii subsp. vinsonii (3/12) (Table 2).

From the 159 tick-infested rodents, Bartonella DNA was detected in 37 (23.3%) of them (Table 5). The Bartonella prevalence in tick-infested rodents was not different compared with the prevalence in tick-free rodents (32.1%; 18/56) (χ² = 1.71, p = 0.19), indicating ticks picked hosts randomly to feed and presence of ticks on rodents did not influence the infection status of the rodent.

Table 5.

Comparison of Bartonella prevalence in tick-infested rodents and tick-free rodents

Collection state	Tick-infested rodents			Tick-free rodents
	No. rodents	No. Bartonella-positive rodents	Bartonella prevalence (%)	No. rodents	No. Bartonella-positive rodents	Bartonella prevalence (%)
Minnesota	159	37	23.3	56	18	32.1
Washington	19	7	36.8	24	5	20.8

Fleas: Bartonella DNA was detected in 25 (36.8%) of the 68 tested fleas by at least two targets as stated above with variable prevalence among the flea species (0–100%) (Table 4). Sequence analysis showed that Bartonella detected in the fleas belonged to B. vinsonii subsp. arupensis, B. grahamii, and Bartonella volans (Table 4).

Ticks: Of all 792 I. scapularis ticks, including the 140 collected from Bartonella-positive rodents, Bartonella DNA was detected in only one larva by gltA, suggesting the presumptive presence [51] of Bartonella species in the tick. This larval tick was collected from a P. leucopus mouse from site CARI (Table 3). The gltA sequence showed the Bartonella was B. vinsonii subsp. arupensis; however, the species was not confirmed as all of the other three targets were negative.

Bartonella spp. in rodents, fleas, and ticks from Washington

Rodents Bartonella DNA was detected in 12 (27.9%) of the 43 tested rodent blood samples by at least two targets. Sequence analysis identified the Bartonella as B. vinsonii subsp. arupensis, showing strong host specificity (Table 2).

From the 19 tick-infested rodents, Bartonella DNA was detected in 7 (36.8%) of them (Table 5). Similar to results from Minnesota, the Bartonella prevalence in tick-infested rodents was similar to that in tick-free rodents (20.8%; 5/24) (χ² = 1.35, p = 0.25).

Fleas Bartonella DNA was detected in 19 (45.2%) of the 42 tested fleas by at least two targets with variable prevalence among the flea species (0–100%) (Table 4). Sequence analysis showed that all 19 Bartonella DNA detected in the fleas was B. vinsonii subsp. arupensis (Table 4).

Ticks no Bartonella DNA was detected in any of the 45 I. pacificus ticks (including the 10 collected from Bartonella-infected rodents) or the 16 I. angustus ticks (including the 7 collected from Bartonella-infected rodents) by any target.

Discussion

We investigated Bartonella spp. in Peromyscus mice and rodents of other species captured in Minnesota and Washington and their ectoparasites, fleas and Ixodes ticks. Similar to other reports [1–6, 55, 56], Bartonella spp. were frequently detected in the rodents and their fleas, with 25.6% (Minnesota) and 27.9% (Washington) of rodents infected and 36.8% (Minnesota) and 45.2% (Washington) of fleas infected. Several Bartonella spp., including B. vinsonii subsp. arupensis, B. vinsonii subsp. vinsonii, B. grahamii, and B. volans, were identified in the rodents and/or their fleas. A strong host specificity was observed between Peromyscus mice and B. vinsonii subsp. arupensis as previously reported [4, 10], while fleas of different species may share the same Bartonella species. These results indicate Bartonella spp. are prevalent in the communities we sampled. Although vector competence of fleas has not been evaluated for these Bartonella species, given the high prevalence of Bartonella infection in the fleas, they are suspected of playing a role in the enzootic transmission of the Bartonella species.

By contrast, of over 850 blood-fed immature I. scapularis, I. pacificus, and I. angustus ticks collected from Peromyscus mice and other rodents, which contained 157 ticks collected from Bartonella-infected rodents, only one I. scapularis larva was presumptively positive for Bartonella by one target, gltA. When comparing tick-infested rodents and tick-free rodents, Bartonella prevalence was similar, suggesting that ticks picked hosts randomly to feed and tick infestation status did not influence infection status of the sampled rodents.

These findings demonstrate an extremely low acquisition rate of Bartonella spp. in blood-feeding Ixodes ticks. Our results were discordant with those reported by Rynkiewicz and others who detected Bartonella DNA in 13.3% of I. scapularis collected from P. leucopus mice in southern Indiana [43]. Notably, their detections were based on 16S rRNA results. 16S rRNA is a highly conserved target that shares homology with a wide range of soil bacteria. Studies utilizing non-specific targets could potentially yield falsely high prevalence of Bartonella infections in ticks [57, 58]. Furthermore, the presence of microbial DNA within a tick does not conclusively establish the viability of the microbe or the competency of the tick species as a vector [57]. Given the lack of Bartonella DNA in blood-feeding Ixodes spp. larvae and nymphs, we found no evidence to suggest transovarial or transstadial transmission of Bartonella by Ixodes ticks. Indeed, our results suggest that Ixodes ticks rarely acquire Bartonella spp. by blood feeding on bacteremic hosts. However, ticks examined in this study were collected at varying stages of engorgement, and bacteremia likely differed among their rodent hosts. Using these samples, we cannot determine whether acquisition rates would have been higher if ticks had been allowed to feed to repletion on highly bacteremic hosts.

Previous laboratory experiments showed that Ixodes ricinus can acquire Bartonella infection when continuously fed on blood with very high bacteremia (10⁸ – 10⁹ CFU) [59, 60]. However, such high bacterial concentrations are rarely seen in natural infections [57; 61] and may not be relevant under natural conditions. Vector competence has not been demonstrated for I. scapularis or I. pacificus and any Bartonella species that naturally occur in the US. Laboratory transmission studies on I. scapularis and I. pacificus are warranted to elucidate acquisition, survival, and transstadial transmission rates. Our previous study conducted over a much broader geographic range found no Bartonella DNA in host-seeking Ixodes spp. ticks [35]. Such a finding could arise from either a lack of exposure to Bartonella-infected hosts, failure to acquire infection through feeding on infected hosts, high mortality in Bartonella-infected ticks, or inefficient transstadial transmission. Here, we showed that tick-infested rodents were commonly infected with Bartonella sp. (most commonly B. vinsonii subsp. arupensis), but ticks feeding on infected hosts were seldom, if ever, infected with Bartonella. Laboratory transmission studies are needed to assess acquisition rates in Ixodes spp. ticks fed to repletion, and, if ticks are proven to acquire infection, assess survival rates of infected ticks and transstadial transmission efficiency from larva to nymph and nymph to adult.

Conclusions

The high prevalence of Bartonella spp. in rodents and rodent-associated fleas coupled with extremely low prevalence of Bartonella spp. in blood-fed ticks suggests that although Ixodes spp. ticks commonly encounter Bartonella spp. in rodents, they rarely acquire infection through blood feeding. Laboratory transmission studies are needed to assess acquisition rates in fully engorged ticks and to assess transstadial transmission efficiency if ticks acquire Bartonella infections from feeding to repletion. Bartonella vinsonii subsp. arupensis would be a good candidate for transmission study based on the common association between the Bartonella species and Peromyscus sp. mice that Ixodes scapularis and I. pacificus ticks commonly feed on.

Author contributions

YB and RJE conceived and designed the study. YB performed the lab testing. LMO and YB conducted data analysis. JC, EF, CP, LMO, and SM prepared and organized samples. YB prepared the manuscript draft. All authors reviewed and edited the manuscript.

Funding

This research was supported by intramural funding within the Centers of Disease Control and Prevention.

Availability of data and materials

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.