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Infections with Bartonella spp. in free-ranging cervids and deer keds (Lipoptena cervi) in Norway
Comparative Immunology, Microbiology and Infectious Diseases 58 (2018) 26–30
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Infections with Bartonella spp. in free-ranging cervids and deer keds (Lipoptena cervi) in Norway
T
Irma Razanskea, Olav Rosefa,b, Jana Radzijevskajaa, Kamile Klepeckienea, Indre Lipatovaa, ⁎ Algimantas Paulauskasa, a b
Vytautas Magnus University, Vileikos str. 8, LT-44404 Kaunas, Lithuania Rosef Field Research Station, Frolandsveien, 2665, 4828 Mjåvatn, Norway
A R T I C LE I N FO
A B S T R A C T
Keywords: Moose Red deer Roe deer Deer keds
Bartonella bacteria are arthropod-borne and can cause long-term bacteremia in humans and animals. The predominant arthropod vectors and the mode of transmission for many novel Bartonella species remain elusive or essentially unstudied. The aim of this study was to investigate the prevalence of Bartonella spp. in Norwegian cervids and deer keds (Lipoptena cervi) and to characterise the bacteria by sequencing of the partial gltA gene and 16 S–23 S rRNA intergenic spacer region (ITS) in order to evaluate a possible transmission route. A total of 260 spleen samples and 118 deer keds were collected from cervids by hunters in the Southern part of Norway. Bartonella DNA was detected in 10.5% of spleen samples of roe deer (n = 67), in 35.1% red deer (n = 37), in 35.9% moose (n = 156), and in 85% pools of adult wingless deer ked (n = 59). Two Bartonella lineages were identified based on phylogenetic analysis of the gltA gene and ITS region sequences.
1. Introduction Bartonella spp. are facultative intracellular, aerobic Gram-negative bacilli belonging to the alpha-2 subgroup of the class Proteobacteria [1,2]. Bartonellae are hemotropic, transmitted from host to host by a diverse range of hematogenous arthropod vectors and considered to be emerging pathogens in humans and animals [3]. In 1992, the Bartonella genus was comprised of a single species Bartonella bacilliformis [4]. Over the past 20 years, there has been a rapid increase in the number of Bartonella species, and since the reclassification of the genus Bartonella in 1993, the number of species has grown from 1 to 45 currently designated members [5]. To date, 35 species and three subspecies in this genus have been officially recognised [6] of which at least 16 Bartonella species have been described as pathogenic for humans [5,7,8]. Various Bartonella spp. were found to be specifically adapted to distinct mammalian hosts where they cause long-term intra-erythrocytic infections. Bartonella spp. infections are often chronic or asymptomatic in their reservoir hosts. Bartonella spp. have been isolated or detected in a wide range of wild and domestic mammals [5,9]. A novel Bartonella sp. was isolated from dromedary camel (Camelus dromedarius) [10]. Domesticated and wild ruminants were also found to harbour several Bartonella spp.: Bartonella bovis, Bartonella schoenbuchensis, Bartonella capreoli, Bartonella chomelii, Bartonella henselae, and Bartonella melophagi [11,12]. Although Bartonella-infected
⁎
domesticated and wild ruminants usually do not demonstrate obvious clinical signs, but endocarditis due to B. bovis infection has been diagnosed in cows (Bos taurus) [13,14]. Bartonella spp. have been detected in a wide range of blood-sucking arthropods, such as lice, mites, fleas, deer keds, various species of biting flies and ticks [15]. Transmission of Bartonella spp. by ticks, including B. henselae as the etiological agent of cat scratch disease in humans, is discussed, but has so far not been fully proven [7,16]. However, the predominant arthropod vectors and the mode of transmission for many novel Bartonella species remain elusive or essentially unstudied. Three Bartonella species are known to infect and replicate in the digestive tract of their respective vectors: B. quintana in body lice (Pediculus humanus corporis) [17], B. henselae in cat fleas (Ctenocephalides felis) [18] and B. schoenbuchensis in the deer keds (Lipoptena cervi) [19]. In the last few years, the vector potential of deer ked (Diptera: Hippoboschidae), a haematophagous ectoparasite of cervids and domesticated animals, has been investigated [20]. A prerequisite for the vector competence of the deer ked is vertical transmission of the pathogen from the mother to its progeny and transstadial transmission from pupa to winged adult. In Norway, the first case of L. cervi was reported in 1983 [21]. During the last few decades, L. cervi has shown a remarkable increase in abundance in the Nordic countries [22]. The predominant hosts of L. cervi are cervids, but the insect may attack a wide range of animals
Corresponding author. E-mail address: [email protected] (A. Paulauskas).
https://doi.org/10.1016/j.cimid.2018.06.003 Received 14 October 2017; Received in revised form 20 February 2018; Accepted 10 June 2018 0147-9571/ © 2018 Elsevier Ltd. All rights reserved.
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the MUSCLE [33]. The phylogenetic trees were constructed using Neighbor-Joining [34] method in MEGA 6 [35] software.
[23]. Investigations from Finland and Southeastern Norway showed occurrence of Bartonella spp. in deer keds and moose blood (Alces alces) [20,24]. In Norway, Duodu et al. [20] tested 41 moose samples for the presence of Bartonella spp. and found a higher prevalence of Bartonella DNA in moose within the deer ked zone (70%) than in animals outside the deer ked zone (37%). Currently, L. cervi is expanding its range in Norway, but the possible role as reservoir for Bartonella spp. remains unknown. In the present study, we aimed to investigate the prevalence of Bartonella spp. in three species of Norwegian free-ranging cervids: moose, red deer and roe deer, and deer keds and to characterise the bacteria by sequencing of the partial gltA gene and 16 S–23 S rRNA intergenic spacer region (ITS) in order to evaluate a possible transmission route.
3. Results Deer keds collected from free-ranging cervids in Norway, based on morphological characteristics and cox-1 sequence identity were identified as L. cervi. The sequences (accession no. MG918092-MG918097) of the cox-1 gene in six samples from different species of animals showed 100-99% identity with the cox-1 gene of L.cervi (accession no. MF496025) deposited in GenBank. Forty-nine pools (83%) and 50 pools (85%) out of 59 of adult wingless deer keds pools using Bartonella gltA gene and 16 S–23 S rRNA ITS specific primers were positive respectively. Seven spleen samples (10.5%) out of 67 of roe deer, 13 samples (35.1.%) out of 37 of red deer and 56 (35.9%) out of 156 moose samples were positive (Table 2). Sequences of the amplified products of gltA gene ranged from 280 to 300 bp and products of 16 S–23 S rRNA intergenic region from 620 to 700 bp in lenght. Sequence analysis confirmed the presence of Bartonella DNA in screened samples of all studied cervid species and deer keds. The phylogeny demonstrated two distinct sequence groups. According to the gltA gene samples of moose were similar to B. bovis (showed a 97% sequence similarity) and these sequences differed from each other at three nucleotide positions (3/283), while Bartonella isolates from red deer and deer keds collected from red deer were clustered with B. shoenbuchensis, B.chomelii, B.melophagii and B.capreoli (showed a 99-97% sequence similarity) (Fig. 1) and sequences differed from each other at one nucleotide position (1/283). Partial sequences of gltA gene of both lineages differed at fifteen nucleotide positions (15/283). Two Bartonella lineages in Norwegian cervids and deer keds were detected based on 16 S–23 S rRNA ITS region sequence analysis (Fig. 2). The lineage of Bartonella sp. closely related to B. bovis (showed a 9894% sequence similarity) were detected in all tested cervid species, but not in deer keds. Sequences of this lineage differed from each other at twenty one nucleotide positions (21/671). In the meantime, another lineage of Bartonella sp. closely related to B. schoenbuchensis, B. chomelii and B. capreoli was detected in red deer and deer keds collected from red deer and moose (showed a 99-96% sequence similarity). Sequences differed from each other at two nucleotide positions (2/671). Partial sequences of 16 S–23 S rRNA ITS region of both lineages differed at seventy three nucleotide positions (73/671). Analysis of concatenated fragments of gltA gene and 16 S–23 S rRNA ITS confirmed that isolates of free-ranging cervids and deer keds separate into two lineages. One lineage is closely related to Bartonella shoenbuchensis and another is related to Bartonella bovis (Fig. 3).
2. Materials and methods 2.1. Sample collection A total of 67 roe deer (Capreolus capreolus), 37 red deer (Cervus elaphus) and 156 moose (Alces alces) spleens were collected by hunters during culling seasons 2014 to 2016 in an area where deer keds are present, and in an area where deer keds are known to be absent [22,25,26]. All animals belonged to the free-living population in south of Norway. Wingless deer ked imagines (L. cervi) (n = 118) were collected from the skin of the carcasses in the deer ked zone. 2.2. DNA extraction and PCR detection of Bartonella spp. Deer ked individuals were pooled (in groups of 2 by one host species) and then homogenised in liquid nitrogen. Total DNA was extracted from spleen and deer keds using Genomic DNA Purification Kit (Thermo Fisher Scientific, Lithuania). Identification of deer ked species and sex were carried out using morphological identification keys [27] and molecular methods (cytochrome oxidase subunit I (cox-1) gene) [28] to confirm species. For detection of Bartonella spp., conventional and nested PCR amplification of partial gltA gene [29] and 16 S–23 S rRNA ITS region [30,31] were used (Table 1). PCR products were analysed by 1.5% agarose gel electrophoresis under the UV light 2.3. Sequence analysis A part of PCR products of gltA gene and 16 S–23 S rRNA ITS region were sequenced. The sequences were subjected to BLASTn identity searches in the GenBank database. Multiple sequence alignments were performed using Clustal W [32] to highlight the differences between the gltA gene fragment and 16 S–23 S rRNA ITS region sequences and to perform the comparisons with related sequences found in the NCBI database. Fragments of gltA gene and 16 S–23 S rRNA ITS region were joined to form a concatenated sequence. Sequence data were aligned by
4. Discussion Blood feeding arthropods, such as sandflies, human lice, cat fleas, some rodent fleas, human fleas, deer keds or various species of biting flies have been confirmed to be competent vectors for transmission of Bartonella species [15]. In the present study, we detected Bartonella DNA both in the free-ranging cervids and in deer ked. The highest prevalence of Bartonella spp. was found in deer keds feeding on red deer (92.5%; 49/53) and the lowest prevalence in roe deer (10.5%; 7/67). Our study demonstrated the presence of two lineages of Bartonella spp. in the cervids and one lineage of Bartonella sp. in deer keds. Bartonella spp. are closely related with various ruminant hosts, for example B. schoenbuchensis, B. capreoli, B. bovis, have been isolated from wild roe deer, and B. schoenbuchensis and B. bovis were found in red deer, while B. bovis and B. chomelii were recovered from domestic cattle [36–38]. In the present study, Bartonella strains detected in moose from both zones (outside and inside deer ked zone) were closely related to B. bovis. However, Bartonella strain detected in one of the 3 tested pools of deer
Table 1 PCR targets and primers used in this study for detection of Bartonella spp. Gene
Forward and reverse sequences
Amplicon size (bp)
References
gltAa
5'- GGGGACCAGCTCATGGTGG-3' 5'- AATGCAAAAAGAACAGTAAACA-3 5'-ACCTCCTTTCTAAGGATGAT-3' 5'-AAAGACCAGCTTCTCGAGAT-3' 5'-CTCTTTCTTCAGATGATGATCC-3' 5'- GCGGTTAAGCTTCCAATCATA-3'
∼380
[29]
∼900 1600
[30,31]
16S-23S rRNA ITS regionb a b
Conventional PCR. Nested PCR. 27
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Table 2 Bartonella spp. from moose, roe deer, red deer and deer keds.
Outside deer ked zone Inside deer ked zone
Sample type
No of samples/pools tested
No of positive samples/pools (%) gltA
No of positive samples/pools (%) 16S-23S rRNA ITS
Moose spleen Red deer spleen Moose spleen Red deer spleen Roe deer spleen Wingless fed deer ked imagines from moose Wingless fed deer ked imagines from red deer Wingless fed deer ked imagines from roe deer
29 17 127 20 67 6 (3 pools) 106 (53 pools) 6 (3 pools)
5(17.2 %) 1(5.9 %) 51(40.2 %) 6(30 %) 5(7.5 %) 0 49 pools (92.5 %) 0
5(17.2 %) 3(17.6 %) 51(40.2 %) 10(50 %) 7(10.4 %) 1 pool (16.7 %) 49 pools (92.5 %) 0
were found in moose. Moose, hunted exclusively within the deer ked range, and deer keds were infected with a Bartonella lineage closely related to B. chomelii, B. schoenbuchensis, and B. capreoli, whereas moose outside the deer ked distribution range were infected with Bartonella lineage showing similarity to B. bovis. The highest prevalence of Bartonella DNA in red deer (50%) and moose (40.2%) was detected inside the deer ked zone. The prevalence rates of Bartonella spp. in moose and red deer hunted outside the deer ked zone were 17.2% and 17.6%, respectively (Table 2). Different
keds from moose belonged to another Bartonella clade associated with B. chomelii and B. schoenbuchensis. Bartonella sp. similar to B. bovis were also identified in roe deer and red deer (from outside deer ked zone) (Figs. 1,2). Both red deer and wingless deer keds adults collected from red deer in the present study were infected with Bartonella species closely related to B. schoenbuchensis, B. capreoli and B. chomelii. Such association suggests that deer keds may be potential vectors for the transmission of this Bartonella species to red deer. In previous studies conducted in Norway [20] and Finland [24,39], two Bartonella lineages
Fig. 1. Phylogenetic tree of Bartonella isolates (●) based on fragments of the gltA gene and generated using the Neighbor-Joining clustering method in MEGA 6 software (1,000 replicates; bootstrap values indicated at the nodes). Bartonella bacilliformis sequence was used as an outgroup. 28
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Fig. 2. Phylogenetic tree of Bartonella isolates (●) based on fragments of 16 S–23 S rRNA intergenic spacer region, generated using the Neighbor-Joining clustering method in MEGA 6 software (1,000 replicates; bootstrap values indicated at the nodes). Bartonella bacilliformis sequences were used as an outgroup.
found in 13.4% of the tested roe deer. Isolates were identical or showed a high similarity to B. shoenbuchensis, while one isolate was closely related to B. capreoli [44]. Our results showed that Bartonella from roe deer were closely related to B. bovis. The results of previous studies done by Duodu et al. [20] with moose and deer keds in Southeastern Norway as well as of the present study with moose, roe deer, red deer and deer keds from the south of Norway indicate that in the Norwegian cervid populations, Bartonella strains from two different lineages circulated, from which one, closely related to B. chomelii, B. schoenbuchensis, and B. capreoli is transmitted by L. cervi, while for other lineage the possible vectors still remain unknown. Further research is needed to determine the vectors of
positives rates by conventional PCR of gltA gene and nested PCR of 16 S–23 S rRNA ITS region in different animals can be explained by the higher sensitivity of the nested PCR and simultaneously the level of bacteraemia. Animal infection with the same Bartonella sp. lineage both outside and inside the deer ked zone can be explained by seasonal migration of cervids [40–43]. Northern deer populations are typically partially migratory; however, the relationship between migratory movements and parasites has received little attention. Migration often involves movement from a low-elevation winter range towards a summer range at a higher elevation [43]. A study in Poland showed that DNA from genus Bartonella was
Fig. 3. Phylogenetic relationships based on the concatenated sequences of 16 S–23 S rRNA intergenic spacer region and gltA gene fragment, generated using the Neighbor-Joining clustering method in MEGA 6 software (1,000 replicates; bootstrap values indicated at the nodes). GenBank accession numbers are indicated in parentheses as (16 S–23 S rRNA and gltA). Bartonella spp. sequences obtained in this study are marked (●).
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transmission of other Bartonella species to free-ranging cervids. Conflicts of interest
[21] [22]
The authors declare that they have no conflicts of interests. Acknowledgement
[23]
We thank the hunters for providing samples and Aust-Agder Fylkeskommune for financial support.
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Infections with Bartonella spp. in free-ranging cervids and deer keds (Lipoptena cervi) in Norway
T
Irma Razanskea, Olav Rosefa,b, Jana Radzijevskajaa, Kamile Klepeckienea, Indre Lipatovaa, ⁎ Algimantas Paulauskasa, a b
Vytautas Magnus University, Vileikos str. 8, LT-44404 Kaunas, Lithuania Rosef Field Research Station, Frolandsveien, 2665, 4828 Mjåvatn, Norway
A R T I C LE I N FO
A B S T R A C T
Keywords: Moose Red deer Roe deer Deer keds
Bartonella bacteria are arthropod-borne and can cause long-term bacteremia in humans and animals. The predominant arthropod vectors and the mode of transmission for many novel Bartonella species remain elusive or essentially unstudied. The aim of this study was to investigate the prevalence of Bartonella spp. in Norwegian cervids and deer keds (Lipoptena cervi) and to characterise the bacteria by sequencing of the partial gltA gene and 16 S–23 S rRNA intergenic spacer region (ITS) in order to evaluate a possible transmission route. A total of 260 spleen samples and 118 deer keds were collected from cervids by hunters in the Southern part of Norway. Bartonella DNA was detected in 10.5% of spleen samples of roe deer (n = 67), in 35.1% red deer (n = 37), in 35.9% moose (n = 156), and in 85% pools of adult wingless deer ked (n = 59). Two Bartonella lineages were identified based on phylogenetic analysis of the gltA gene and ITS region sequences.
1. Introduction Bartonella spp. are facultative intracellular, aerobic Gram-negative bacilli belonging to the alpha-2 subgroup of the class Proteobacteria [1,2]. Bartonellae are hemotropic, transmitted from host to host by a diverse range of hematogenous arthropod vectors and considered to be emerging pathogens in humans and animals [3]. In 1992, the Bartonella genus was comprised of a single species Bartonella bacilliformis [4]. Over the past 20 years, there has been a rapid increase in the number of Bartonella species, and since the reclassification of the genus Bartonella in 1993, the number of species has grown from 1 to 45 currently designated members [5]. To date, 35 species and three subspecies in this genus have been officially recognised [6] of which at least 16 Bartonella species have been described as pathogenic for humans [5,7,8]. Various Bartonella spp. were found to be specifically adapted to distinct mammalian hosts where they cause long-term intra-erythrocytic infections. Bartonella spp. infections are often chronic or asymptomatic in their reservoir hosts. Bartonella spp. have been isolated or detected in a wide range of wild and domestic mammals [5,9]. A novel Bartonella sp. was isolated from dromedary camel (Camelus dromedarius) [10]. Domesticated and wild ruminants were also found to harbour several Bartonella spp.: Bartonella bovis, Bartonella schoenbuchensis, Bartonella capreoli, Bartonella chomelii, Bartonella henselae, and Bartonella melophagi [11,12]. Although Bartonella-infected
⁎
domesticated and wild ruminants usually do not demonstrate obvious clinical signs, but endocarditis due to B. bovis infection has been diagnosed in cows (Bos taurus) [13,14]. Bartonella spp. have been detected in a wide range of blood-sucking arthropods, such as lice, mites, fleas, deer keds, various species of biting flies and ticks [15]. Transmission of Bartonella spp. by ticks, including B. henselae as the etiological agent of cat scratch disease in humans, is discussed, but has so far not been fully proven [7,16]. However, the predominant arthropod vectors and the mode of transmission for many novel Bartonella species remain elusive or essentially unstudied. Three Bartonella species are known to infect and replicate in the digestive tract of their respective vectors: B. quintana in body lice (Pediculus humanus corporis) [17], B. henselae in cat fleas (Ctenocephalides felis) [18] and B. schoenbuchensis in the deer keds (Lipoptena cervi) [19]. In the last few years, the vector potential of deer ked (Diptera: Hippoboschidae), a haematophagous ectoparasite of cervids and domesticated animals, has been investigated [20]. A prerequisite for the vector competence of the deer ked is vertical transmission of the pathogen from the mother to its progeny and transstadial transmission from pupa to winged adult. In Norway, the first case of L. cervi was reported in 1983 [21]. During the last few decades, L. cervi has shown a remarkable increase in abundance in the Nordic countries [22]. The predominant hosts of L. cervi are cervids, but the insect may attack a wide range of animals
Corresponding author. E-mail address: [email protected] (A. Paulauskas).
https://doi.org/10.1016/j.cimid.2018.06.003 Received 14 October 2017; Received in revised form 20 February 2018; Accepted 10 June 2018 0147-9571/ © 2018 Elsevier Ltd. All rights reserved.
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the MUSCLE [33]. The phylogenetic trees were constructed using Neighbor-Joining [34] method in MEGA 6 [35] software.
[23]. Investigations from Finland and Southeastern Norway showed occurrence of Bartonella spp. in deer keds and moose blood (Alces alces) [20,24]. In Norway, Duodu et al. [20] tested 41 moose samples for the presence of Bartonella spp. and found a higher prevalence of Bartonella DNA in moose within the deer ked zone (70%) than in animals outside the deer ked zone (37%). Currently, L. cervi is expanding its range in Norway, but the possible role as reservoir for Bartonella spp. remains unknown. In the present study, we aimed to investigate the prevalence of Bartonella spp. in three species of Norwegian free-ranging cervids: moose, red deer and roe deer, and deer keds and to characterise the bacteria by sequencing of the partial gltA gene and 16 S–23 S rRNA intergenic spacer region (ITS) in order to evaluate a possible transmission route.
3. Results Deer keds collected from free-ranging cervids in Norway, based on morphological characteristics and cox-1 sequence identity were identified as L. cervi. The sequences (accession no. MG918092-MG918097) of the cox-1 gene in six samples from different species of animals showed 100-99% identity with the cox-1 gene of L.cervi (accession no. MF496025) deposited in GenBank. Forty-nine pools (83%) and 50 pools (85%) out of 59 of adult wingless deer keds pools using Bartonella gltA gene and 16 S–23 S rRNA ITS specific primers were positive respectively. Seven spleen samples (10.5%) out of 67 of roe deer, 13 samples (35.1.%) out of 37 of red deer and 56 (35.9%) out of 156 moose samples were positive (Table 2). Sequences of the amplified products of gltA gene ranged from 280 to 300 bp and products of 16 S–23 S rRNA intergenic region from 620 to 700 bp in lenght. Sequence analysis confirmed the presence of Bartonella DNA in screened samples of all studied cervid species and deer keds. The phylogeny demonstrated two distinct sequence groups. According to the gltA gene samples of moose were similar to B. bovis (showed a 97% sequence similarity) and these sequences differed from each other at three nucleotide positions (3/283), while Bartonella isolates from red deer and deer keds collected from red deer were clustered with B. shoenbuchensis, B.chomelii, B.melophagii and B.capreoli (showed a 99-97% sequence similarity) (Fig. 1) and sequences differed from each other at one nucleotide position (1/283). Partial sequences of gltA gene of both lineages differed at fifteen nucleotide positions (15/283). Two Bartonella lineages in Norwegian cervids and deer keds were detected based on 16 S–23 S rRNA ITS region sequence analysis (Fig. 2). The lineage of Bartonella sp. closely related to B. bovis (showed a 9894% sequence similarity) were detected in all tested cervid species, but not in deer keds. Sequences of this lineage differed from each other at twenty one nucleotide positions (21/671). In the meantime, another lineage of Bartonella sp. closely related to B. schoenbuchensis, B. chomelii and B. capreoli was detected in red deer and deer keds collected from red deer and moose (showed a 99-96% sequence similarity). Sequences differed from each other at two nucleotide positions (2/671). Partial sequences of 16 S–23 S rRNA ITS region of both lineages differed at seventy three nucleotide positions (73/671). Analysis of concatenated fragments of gltA gene and 16 S–23 S rRNA ITS confirmed that isolates of free-ranging cervids and deer keds separate into two lineages. One lineage is closely related to Bartonella shoenbuchensis and another is related to Bartonella bovis (Fig. 3).
2. Materials and methods 2.1. Sample collection A total of 67 roe deer (Capreolus capreolus), 37 red deer (Cervus elaphus) and 156 moose (Alces alces) spleens were collected by hunters during culling seasons 2014 to 2016 in an area where deer keds are present, and in an area where deer keds are known to be absent [22,25,26]. All animals belonged to the free-living population in south of Norway. Wingless deer ked imagines (L. cervi) (n = 118) were collected from the skin of the carcasses in the deer ked zone. 2.2. DNA extraction and PCR detection of Bartonella spp. Deer ked individuals were pooled (in groups of 2 by one host species) and then homogenised in liquid nitrogen. Total DNA was extracted from spleen and deer keds using Genomic DNA Purification Kit (Thermo Fisher Scientific, Lithuania). Identification of deer ked species and sex were carried out using morphological identification keys [27] and molecular methods (cytochrome oxidase subunit I (cox-1) gene) [28] to confirm species. For detection of Bartonella spp., conventional and nested PCR amplification of partial gltA gene [29] and 16 S–23 S rRNA ITS region [30,31] were used (Table 1). PCR products were analysed by 1.5% agarose gel electrophoresis under the UV light 2.3. Sequence analysis A part of PCR products of gltA gene and 16 S–23 S rRNA ITS region were sequenced. The sequences were subjected to BLASTn identity searches in the GenBank database. Multiple sequence alignments were performed using Clustal W [32] to highlight the differences between the gltA gene fragment and 16 S–23 S rRNA ITS region sequences and to perform the comparisons with related sequences found in the NCBI database. Fragments of gltA gene and 16 S–23 S rRNA ITS region were joined to form a concatenated sequence. Sequence data were aligned by
4. Discussion Blood feeding arthropods, such as sandflies, human lice, cat fleas, some rodent fleas, human fleas, deer keds or various species of biting flies have been confirmed to be competent vectors for transmission of Bartonella species [15]. In the present study, we detected Bartonella DNA both in the free-ranging cervids and in deer ked. The highest prevalence of Bartonella spp. was found in deer keds feeding on red deer (92.5%; 49/53) and the lowest prevalence in roe deer (10.5%; 7/67). Our study demonstrated the presence of two lineages of Bartonella spp. in the cervids and one lineage of Bartonella sp. in deer keds. Bartonella spp. are closely related with various ruminant hosts, for example B. schoenbuchensis, B. capreoli, B. bovis, have been isolated from wild roe deer, and B. schoenbuchensis and B. bovis were found in red deer, while B. bovis and B. chomelii were recovered from domestic cattle [36–38]. In the present study, Bartonella strains detected in moose from both zones (outside and inside deer ked zone) were closely related to B. bovis. However, Bartonella strain detected in one of the 3 tested pools of deer
Table 1 PCR targets and primers used in this study for detection of Bartonella spp. Gene
Forward and reverse sequences
Amplicon size (bp)
References
gltAa
5'- GGGGACCAGCTCATGGTGG-3' 5'- AATGCAAAAAGAACAGTAAACA-3 5'-ACCTCCTTTCTAAGGATGAT-3' 5'-AAAGACCAGCTTCTCGAGAT-3' 5'-CTCTTTCTTCAGATGATGATCC-3' 5'- GCGGTTAAGCTTCCAATCATA-3'
∼380
[29]
∼900 1600
[30,31]
16S-23S rRNA ITS regionb a b
Conventional PCR. Nested PCR. 27
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Table 2 Bartonella spp. from moose, roe deer, red deer and deer keds.
Outside deer ked zone Inside deer ked zone
Sample type
No of samples/pools tested
No of positive samples/pools (%) gltA
No of positive samples/pools (%) 16S-23S rRNA ITS
Moose spleen Red deer spleen Moose spleen Red deer spleen Roe deer spleen Wingless fed deer ked imagines from moose Wingless fed deer ked imagines from red deer Wingless fed deer ked imagines from roe deer
29 17 127 20 67 6 (3 pools) 106 (53 pools) 6 (3 pools)
5(17.2 %) 1(5.9 %) 51(40.2 %) 6(30 %) 5(7.5 %) 0 49 pools (92.5 %) 0
5(17.2 %) 3(17.6 %) 51(40.2 %) 10(50 %) 7(10.4 %) 1 pool (16.7 %) 49 pools (92.5 %) 0
were found in moose. Moose, hunted exclusively within the deer ked range, and deer keds were infected with a Bartonella lineage closely related to B. chomelii, B. schoenbuchensis, and B. capreoli, whereas moose outside the deer ked distribution range were infected with Bartonella lineage showing similarity to B. bovis. The highest prevalence of Bartonella DNA in red deer (50%) and moose (40.2%) was detected inside the deer ked zone. The prevalence rates of Bartonella spp. in moose and red deer hunted outside the deer ked zone were 17.2% and 17.6%, respectively (Table 2). Different
keds from moose belonged to another Bartonella clade associated with B. chomelii and B. schoenbuchensis. Bartonella sp. similar to B. bovis were also identified in roe deer and red deer (from outside deer ked zone) (Figs. 1,2). Both red deer and wingless deer keds adults collected from red deer in the present study were infected with Bartonella species closely related to B. schoenbuchensis, B. capreoli and B. chomelii. Such association suggests that deer keds may be potential vectors for the transmission of this Bartonella species to red deer. In previous studies conducted in Norway [20] and Finland [24,39], two Bartonella lineages
Fig. 1. Phylogenetic tree of Bartonella isolates (●) based on fragments of the gltA gene and generated using the Neighbor-Joining clustering method in MEGA 6 software (1,000 replicates; bootstrap values indicated at the nodes). Bartonella bacilliformis sequence was used as an outgroup. 28
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Fig. 2. Phylogenetic tree of Bartonella isolates (●) based on fragments of 16 S–23 S rRNA intergenic spacer region, generated using the Neighbor-Joining clustering method in MEGA 6 software (1,000 replicates; bootstrap values indicated at the nodes). Bartonella bacilliformis sequences were used as an outgroup.
found in 13.4% of the tested roe deer. Isolates were identical or showed a high similarity to B. shoenbuchensis, while one isolate was closely related to B. capreoli [44]. Our results showed that Bartonella from roe deer were closely related to B. bovis. The results of previous studies done by Duodu et al. [20] with moose and deer keds in Southeastern Norway as well as of the present study with moose, roe deer, red deer and deer keds from the south of Norway indicate that in the Norwegian cervid populations, Bartonella strains from two different lineages circulated, from which one, closely related to B. chomelii, B. schoenbuchensis, and B. capreoli is transmitted by L. cervi, while for other lineage the possible vectors still remain unknown. Further research is needed to determine the vectors of
positives rates by conventional PCR of gltA gene and nested PCR of 16 S–23 S rRNA ITS region in different animals can be explained by the higher sensitivity of the nested PCR and simultaneously the level of bacteraemia. Animal infection with the same Bartonella sp. lineage both outside and inside the deer ked zone can be explained by seasonal migration of cervids [40–43]. Northern deer populations are typically partially migratory; however, the relationship between migratory movements and parasites has received little attention. Migration often involves movement from a low-elevation winter range towards a summer range at a higher elevation [43]. A study in Poland showed that DNA from genus Bartonella was
Fig. 3. Phylogenetic relationships based on the concatenated sequences of 16 S–23 S rRNA intergenic spacer region and gltA gene fragment, generated using the Neighbor-Joining clustering method in MEGA 6 software (1,000 replicates; bootstrap values indicated at the nodes). GenBank accession numbers are indicated in parentheses as (16 S–23 S rRNA and gltA). Bartonella spp. sequences obtained in this study are marked (●).
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transmission of other Bartonella species to free-ranging cervids. Conflicts of interest
[21] [22]
The authors declare that they have no conflicts of interests. Acknowledgement
[23]
We thank the hunters for providing samples and Aust-Agder Fylkeskommune for financial support.
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