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Molecular identity of cat fleas (Ctenocephalides felis) from cats in Georgia, USA carrying Bartonella clarridgeiae, Bartonella henselae and Rickettsia sp. RF2125
Molecular identity of cat fleas (Ctenocephalides felis) from cats in Georgia, USA carrying Bartonella clarridgeiae, Bartonella henselae and Rickettsia sp. RF2125 ˇ ep´an Slapeta, ˇ ˇ Stˇ Jan Slapeta PII: DOI: Reference:
S2405-9390(16)30049-1 doi: 10.1016/j.vprsr.2016.06.005 VPRSR 25
To appear in: Received date: Revised date: Accepted date:
7 April 2016 24 June 2016 30 June 2016
ˇ ˇ ep´an, Slapeta, ˇ Please cite this article as: Slapeta, Stˇ Jan, Molecular identity of cat fleas (Ctenocephalides felis) from cats in Georgia, USA carrying Bartonella clarridgeiae, Bartonella henselae and Rickettsia sp. RF2125, (2016), doi: 10.1016/j.vprsr.2016.06.005
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Molecular identity of cat fleas (Ctenocephalides felis) from cats in Georgia, USA carrying
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Bartonella clarridgeiae, Bartonella henselae and Rickettsia sp. RF2125
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Štěpán Šlapeta and Jan Šlapeta*
School of Life and Environmental Sciences, Faculty of Veterinary Science, McMaster Building
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B14, The University of Sydney, New South Wales 2006, Australia
*Corresponding author: Jan Šlapeta, McMaster Building B14, Faculty of Veterinary Science,
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The University of Sydney, New South Wales 2006, Australia; tel: +61 2 9351 2025; fax: +61 2 9351 7348; email: [email protected]
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Abstract
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The cat flea (Ctenocephalides felis) is the most common ectoparasite of dogs and cats. Close
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association of humans with cats and dogs enables flea-borne disease transmission either directly, via flea bites, or indirectly, via pathogens excreted in flea faeces. The aim of this study was to
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evaluate molecular identity of C. felis from cats in Georgia, USA based on a molecular barcode
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marker gene (cox1) and the frequency at which the fleas were carriers of the vector-borne bacteria, Bartonella and Rickettsia. The multiplexed Bartonella and Rickettsia real-time PCR
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assay (targeting ssrA and gltA, respectively) adopted in this study, together with sequencing of the ssrA enabled rapid identification of Bartonella spp. in cat fleas. Eighteen out of 20 fleas
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examined were positive for Bartonella spp. and all fleas were positive for Rickettsia spp. DNA sequencing of the ssrA confirmed presence B. clarridgeiae and B. henselae. Amplification and
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DNA sequencing of gltA and ompA confirmed presence of Rickettsia sp. RF2125 (Rickettsia
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felis-like organism). All fleas from 21 cats in Georgia, USA were morphologically identical with C. felis. Sequencing of the flea cox1 revealed identity with C. felis from Seychelles, Africa recognised as a subspecies C. felis strongylus, the African cat flea. Analysis of the cox1 sequences of fleas improves understanding of global distribution of cat flea cox1 clades (C. felis) when compared with sequences from Ctenocephalides spp. from Asia, Africa, Europe, Asia and Australia. Coupling flea barcoding approach with the multiplexed real-time PCR assay followed by Bartonella sequencing represents a rational approach for screening and elucidation of fleas’ capacity to vector infectious agents.
Keywords: barcoding, Siphonaptera, real-time PCR, phylogeny, cox1, COI
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1. Introduction
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The cat flea (Ctenocephalides felis) is the most common ectoparasite of dogs and cats in tropical, subtropical, as well as temperate climates. The cat flea is a generalist parasite and
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potentially feeds on any mammal, including humans particularly when exposed to highly infested environments (Dryden and Rust, 1994; Rust, 2005, 2016). Most commonly, however,
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cat fleas are found on domestic and feral or community cats and dogs. Close association of
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humans with cats and dogs enables flea borne disease transmission either directly, via flea bites, or indirectly, via pathogens excreted in flea faeces (Boulouis et al., 2005; Chomel et al., 2006).
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Recently, molecular tools were applied to characterise cat fleas and closely related species
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in the genus Ctenocephalides (Vobis et al., 2004; Šlapeta et al., 2011; Marrugal et al., 2013; Lawrence et al., 2014; Hii et al., 2015; Lawrence et al., 2015). A previously assumed subspecies
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C. felis orientis, Oriental cat flea, was confirmed to represent a full species C. orientis which is more closely related to the dog flea, Ctenocephalides canis than to C. felis (Hopkins and Rothschild, 1953; Menier and Beaucournu, 1998; Lawrence et al., 2015). The use of cytochrome c oxidase subunit I (cox1) marker to ‘barcode’ fleas has demonstrated that the cat flea is not as homogeneous as previously thought (Vobis et al., 2004; Marrugal et al., 2013), consisting of a number of defined clades whose global distribution is yet to be fully understood (Lawrence et al., 2014; Hii et al., 2015; Lawrence et al., 2015). The capacity of different Ctenocephalides species, subspecies or even cox1 clades to vector infectious agents has been suggested on a small scale sample and requires further scrutiny (Hii et al., 2015).
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The aim of this study was to evaluate the molecular identity of Ctenocephalides felis from
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cats in Georgia, USA based on a molecular barcoding of cox1 marker gene sequence and the
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frequency at which the fleas were carriers of the vector-borne bacteria, Bartonella and Rickettsia. The multiplexed Bartonella and Rickettsia real-time PCR assay adopted in this study, together
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with sequencing of the ssrA, a gene that codes transfer-mRNA (tmRNA), enables rapid identification of Bartonella spp. in cat fleas. Analysis of the cox1 sequences of fleas improved
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our understanding of global distribution of cat flea clades (C. felis) when compared with
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sequences from Ctenocephalides spp. from Asia, Africa, Europe, Asia and Australia.
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2. Materials and Methods
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2.1. Flea collection, identification and extraction of the total DNA
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Fleas were collected from cats in Georgia, USA (Figure 1) between October and November 2015. Fleas (n=74) were collected opportunistically from 20 cats during visits to a desexing veterinary clinic (neutered cats in a trap-neuter-return program), and one cat directly from a household (for details on locality see Table 1). Fleas were collected using flea comb and stored in 80% ethanol. Fleas were morphologically identified based on published keys (Hopkins and Rothschild, 1953). Total DNA was extracted whilst retaining flea exoskeletons using Isolate II Genomic DNA kit (BioLine, Australia) as previously described (Whiting et al., 2008; Lawrence et al., 2014). DNA was eluted into 100 µL of Tris buffer (pH=8.5) and stored at -20°C. Previously isolated DNA from Ctenocephalides felis strongylus (n=5) collected on dogs from Seychelles was included in this study (Lawrence et al., 2014).
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2.2. Amplification of the mitochondrial encoded cytochrome c oxidase subunit I and flea
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phylogeny
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A 5' fragment of the cytochrome c oxidase subunit I (cox1) coding for COX1 protein was PCR amplified using primers: LCO1490, 5’- GGT CAA CAA ATC ATA AAG ATA TTG G-3’
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and Cff-R (S0368) 5’-GAA GGG TCA AAG AAT GAT GT-3’ (Lawrence et al., 2014).
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Reactions of 30 μl contained MyTaq Red Mix (BioLine, Australia) with approximately 10–50 ng of genomic DNA template (2 μl) in a Veriti Thermal Cycler (Life Sciences, Australia). The cycling conditions were as follows: denaturing at 95°C for 1 min followed by 35 cycles of 95°C
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for 15 s, 55°C for 15 s, 72°C for 10 s, and a final elongation for 5 min at 72°C. Sterile PCR-
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grade water was used as negative control, and a positive control consisting of flea DNA known to amplify at these conditions was also run along-side. PCR products that yielded an
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unambiguous single band product (~600bp) were directly and bidirectionally sequenced at Macrogen Inc. (Seoul, Korea). All sequences were assembled, aligned with related sequences and analysed using CLC Main Workbench 6.9.1 (CLC bio, Denmark) and deposited in GenBank (National Center for Biotechnology Information, NCBI) under the Accession Numbers: KX021755-KX021774.
Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013). Multiple sequence alignment was constructed with all available Ctenocephalides sequences belonging to defined clades (Lawrence et al., 2014; Hii et al., 2015; Lawrence et al., 2015). Sequence divergences were calculated using the Kimura 2 parameter (K2P) distance model, and
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phylogenetic tree was inferred using minimum evolution and the bootstrap support inferred from
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2.3. Diagnostic Rickettsia and Bartonella real-time PCR
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500 replicates.
A diagnostic TaqMan probe real-time PCR assay targeting the gltA (citrate synthase) gene
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of Rickettsia spp. was applied for the detection of Rickettsia (Stenos et al., 2005). A diagnostic
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TaqMan probe real-time PCR assay targeting ssrA gene was applied to specifically detect Bartonella (Diaz et al., 2012). The assay was multiplexed using Rickettsia oligonucleotide probe labelled with HEX and Bartonella oligonucleotide probe labelled with FAM. Bartonella assay
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used ssrA-F (S0508), 5’-GCT ATG GTA ATA AAT GGA CAA TGA AAT AA-3’, ssrA-R
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(S0509), 5’-GCT TCT GTT GCC AGG TG-3’ and a probe S0510, 5’-FAM-ACC CCG CTT AAA CCT GCG ACG-3’-BHQ1 (Diaz et al., 2012). CS-F (S0576), 5’-TCG CAA ATG TTC
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ACG GTA CTT T-3’, CS-R (S0577) 5’-TCG TGC ATT TCT TTC CAT TGT G-3’ and a probe CS-P (S0578), 5’-HEX-TGC AAT AGC AAG AAC CGT AGG CTG GAT G-3’-BHQ1 (Stenos et al., 2005). Oligonucleotides and labelled oligonucleotides were synthesised by Macrogen Inc. (Seoul, Korea). The reaction was carried out in 20 µl volumes and included a final concentration of 400 nM and 100 nM of primers and probes, respectively, along with 4 µl of template DNA with SensiFAST Probe No-ROX Kit (BioLine, Australia). Duplicate PCRs were performed using the following conditions: 3 mins at 95°C followed by 40 cycles of 5 s at 95°C and 15 s at 60°C. Each run included a no template negative control (NTC) and positive control plasmid DNA carrying a Bartonella and Rickettsia target DNA insert. Bartonella plasmid control included a region of the ssrA gene of Bartonella henselae with TAA AGC TAC GAC AAC GAG TAC
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TAA coding for SYDNEY flanked by stop codons in a pMA-T backbone (GeneArt, Life
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Technologies, Australia) as previously described (Lawrence et al., 2015). The amplification on
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CFX96 Touch™ Real-Time PCR Detection System was analysed with the corresponding software (BioRad, Australia). The arbitrary real-time PCR threshold was set to a single threshold
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at 100 rfu.
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The Bartonella assay amplifies approximately 300 bp of ssrA gene sufficient to resolve
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Bartonella species (Diaz et al., 2012). All Bartonella real-time PCR reactions returning satisfactory Ct values (Ct <35) were repeated as single Bartonella real-time PCR assay and sequenced using amplification primers at Macrogen Inc. (Seoul, Korea). Sequences were
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assembled, aligned with related sequences and analysed using CLC Main Workbench 6.9.1
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(CLC bio, Denmark) and deposited in GenBank (National Center for Biotechnology Information,
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NCBI) under the Accession Numbers: KX021743- KX021754.
2.4. Diagnostic Rickettsia felis gltA and ompA nested PCR
A diagnostic conventional nested PCR assays targeting the gltA gene of R. felis and rickettsial outer membrane protein B (ompB) gene were applied for the detection of R. felis as previously described (Hii et al., 2015). A fragment (654 bp) of gltA was amplified using primer pair gltA-F1 (S0659), 5’-GCA AGT ATT GGT GAG GAT GTA ATC-3’ / gltA-R1 (S0660), 5’CTG CGG CAC GTG GGT CAT AG-3’ followed by nested primer pair gltA-F2 (S0661), 5’GCG ACA TCG AGG ATA TGA CAT-3’ / gltA-R2 (S0662) 5’-GGA ATA TTC TCA GAA CTA CCG-3’. A fragment (879 bp) of ompA was amplified using primer pair omp-A-F1 (S0663)
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5’-CGA TAG TGT TAC AAG TAC CGG-3’ / omp-A-R1 (S0664) 5’-GCA TCT TCC ATT
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AAC TCA AGC-3’ followed by nested primer pair ompA-F2 (S0665) 5’-CGG TAC AAT CAT
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TGC AAC TGG-3’ / ompA-R2 (S0666) 5’-GCT ATA TCT TCA GCA AAT AAC G-3’. Reactions of 25 μl contained MyTaq Red Mix (BioLine, Australia) and 2 μl of genomic DNA
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template were amplified in a Veriti Thermal Cycler (Life Sciences, Australia). Template for the secondary PCR was 2 µl of the primary PCR reaction. PCR grade water served as negative
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control. PCR cycling conditions comprised an initial activation step at 95°C for 2 min, followed
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by 40 cycles of 95°C for 15 s, 63°C (primary gltA PCR) or 59 °C (primary ompA PCR)for 30 s and 72°C for 30 s with a final extension step of 72°C for 5 min. The secondary PCR amplification for gltA was performed as for the primary PCR except that the annealing
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temperature was 59°C, and for ompA secondary PCR was identical to the primary. PCR
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amplicons were sequenced using amplification primers at Macrogen Inc. (Seoul, Korea). Sequence of region amplified with the above mentioned primers (gltA and ompA) from the
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genome R. felis URRWXCal2 (CP000053) served as reference (Ogata et al., 2005). Sequences were assembled, aligned with related sequences and analysed using CLC Main Workbench 6.9.1 (CLC bio, Denmark) and deposited in GenBank (National Center for Biotechnology Information, NCBI) under the Accession Numbers: KX431974-KX431985.
3. Results
Morphological analysis revealed the presence of only the cat flea (Ctenocephalides felis) among the fleas collected from cats in Georgia, USA (Table 1). To infer their phylogenetic position, DNA from a subset of 20 fleas (C. felis) was extracted and sequenced at the cox1 gene
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(Figure 1). Multiple sequence alignment of existing clades revealed indistinguishable or a highly
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similar cox1 sequences (>99%) of the cat fleas from Georgia, USA to the clade previously
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recognised as the African clade of C. felis strongylus (Clade III, in Lawrence et al. 2014) from Seychelles (Lawrence et al., 2014). Previously, five distinct clades were recognised to represent
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C. felis felis and C. felis strongylus (Lawrence et al., 2014; Lawrence et al., 2015) (Figure 1).
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The multiplexed Rickettsia and Bartonella real-time PCR assay revealed presence of
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Rickettsia DNA in all fleas with an average Ct value of 25.12 (min. 21.19, max. 30.28). Thirteen (65%) out of 20 fleas were positive for Bartonella spp. in duplicate real-time PCRs with an average Ct value of 28.57 (min. 20.80, max. 34.30). In addition five fleas (AL885-1, AL874-1,
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AL873-1, AL870-1, AL877-1) were considered ‘suspect positives’ (late amplifiers) for
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control reactions.
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Bartonella because their Ct values were >35. No amplification was observed in any negative
Direct DNA sequencing of the real-time PCR products of the Bartonella ssrA gene confirmed presence of Bartonella spp. in 12 samples, including 11 real-time PCR positive and 1 real-time PCR suspect (AL874-1) (Table 2). Two sequences (AL969-1, AL879-2) showed overlapping sequence chromatographs. Sequence alignment of ssrA gene sequences revealed that six sequences were 100% identical to B. clarridgeiae (JN982716) and six were 100% identical to B. henselae (JN029785).
Selected Rickettsia-positive fleas (n=6) were further characterised with conventional nested PCR targeting a fragment of gltA (654 bp) and ompA (879 bp) genes. All randomly selected fleas
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(AL866-1, AL868-1, AL874-1, AL877-1, AL880-1, AL885-1) returned positive amplicons of
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expected size. DNA sequencing revealed that all gltA sequences were 100% identical to the
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reference sequence of Rickettsia sp. RF2125 (AF516333) (Parola et al., 2003). The gltA region was 99% identical (7 differences across 612 alignment residues) to the reference sequence of R.
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felis (Ogata et al., 2005). Similarly, ompA sequences were most closely related to ompA sequences from Rickettsia sp. RF2125 (KP687807, KP256359, KP687806, KP256358), there
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was a single mismatch G/T (Hii et al., 2015). The ompA region was 96% identical (34
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differences across 834 alignment residues) to the reference sequence of R. felis (Ogata et al., 2005).
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The discovery that cat fleas collected on cats in Georgia, USA were monophyletic with C.
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felis strongylus from Seychelles prompted us to evaluate the Seychelles fleas for the presence of Rickettsia and Bartonella (Lawrence et al., 2014). None of the five fleas originally collected
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from dogs from Seychelles revealed presence of Bartonella DNA, nevertheless four were positive for Rickettsia DNA in duplicate multiplex real-time PCR (average Ct value of 23.76). In one reaction, a single flea (AL83-1) was a late amplifier (Ct value of 39.54) for Rickettsia DNA. Diluting the DNA 1:10 confirmed Rickettsia DNA in four samples (average Ct value of 26.62), while AL83-1 remained negative for Rickettsia DNA, and no Bartonella DNA was amplified (Supplementary Figure 1).
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4. Discussion
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We have identified only C. felis on cats in Georgia, USA. Molecular barcoding using the cox1 gene sequence revealed that all the cat fleas from several locations in Georgia, USA are
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identical to those found in Africa, and are assumed to represent an African subspecies C. felis strongylus, African cat flea (Lawrence et al., 2014). In general, it is assumed that the subspecies
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C. felis felis is the globally distributed subspecies, dispersed with domesticated cats, dogs, and
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human migration. Previous studies have demonstrated the presence of a cox1 clade that is shared between Europe, Asia and Australia and is considered the ubiquitous on dog and cats in veterinary practices in Australia (Šlapeta et al., 2011; Hii et al., 2015). It was rather surprising
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that despite similar climate between Georgia, USA and Australia, a different cat flea clade is
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sustained. This study is the first to apply cox1 barcoding to cat fleas from USA and compare them to the growing dataset of cat fleas from other countries. Further sampling across different
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states will resolve the extent of the distribution of the Clade III in USA and elsewhere. The role of human migration and the historical African slave trade in the introduction of C. felis strongylus to USA will require further global sampling. The absence of dog fleas (C. canis) in this study is not unexpected since only cats were sampled. The dog flea (C. canis) is known to be present in the USA in lesser frequency then cat flea (C. felis) (Durden et al., 2012).
The cat fleas were collected on a mix of community animals living on a property as well as owned cats. In such situations, contact with human is frequent and increases the chance for fleaborne disease transmission (Boulouis et al., 2005; Durden et al., 2012). Identification of B. clarridgeiae and B. henselae in the subset of fleas confirms previous finding that Bartonella spp.
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are frequently present in cat fleas in USA and humans or dogs may become accidental hosts
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(Breitschwerdt and Kordick, 2000; Chomel et al., 2006; Breitschwerdt, 2008; Breitschwerdt et
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al., 2010; Chomel and Kasten, 2010). In humans, B. henselae is the cause of the ‘cat scratch disease’, however, the evidence that B. clarridgeiae causes disease in human is minimal (Chomel
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et al., 2006). Both B. clarridgeiae and B. henselae spp. are associated with several different systemic illnesses in dogs including chronic endocarditis (MacDonald et al., 2004). A study in
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Florida based on 553 community cats documented 34% prevalence for B. hansellae (Luria et al.,
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2004). Frequent presence of Bartonella spp. in fleas collected on community cats from Georgia is therefore expected. As argued previously, community cats do not represent greater risk to humans or other pets than owned pet cats because their prevalence of infectious agents in
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community cats fall within reported ranges for owned pet cats (Luria et al., 2004).
Cat fleas are commonly identified to carry Rickettsia felis or R. felis-like organisms
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(Angelakis et al., 2016). Identification of R. felis-like organisms (Rickettsia sp. RF2125) in fleas from Georgia, USA indicates that cats and dogs may act as potential reservoir hosts. The zoonotic risk to humans remains unascertained for R. felis-like species (Parola et al., 2003; Hii et al., 2015). The significance of Rickettsia sp. RF2125 and global distribution is incomplete, and our report confirms its presence in USA in what we recognise as C. felis strongylus.
Combination of existing sensitive TaqMan molecular assays for Rickettsia and Bartonella enabled us to test for both pathogens in a single tube. Both assays are highly sensitive and specific (Stenos et al., 2005; Diaz et al., 2012). The Rickettsia assay detects spotted fever and typhus group rickettsiae, such as Rickettsia akari, R. australis, R. conorii, R. honei, R. rickettsii,
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R. typhi, or R. felis, as well as, potentially symbiotic/non-pathogenic rickettsiae (Stenos et al.,
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2005; Hii et al., 2015). The Bartonella assay targeting ssrA gene was designed for species
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identification, because the real-time PCR product is of sufficient length (~300bp) to allow sequencing and species identification (Diaz et al., 2012). The combination of both assays offer
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rapid screening of vectors, such as fleas, as demonstrated in this study. Bartonella spp. are recognised and differentiated from each other using comparison and application of multilocus
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genotyping, nevertheless for vector screening purposes, the ssrA gene was sufficient to identify
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the species of Bartonella. The limitation of this approach is that if two Bartonella spp. are present both, ssrA will be amplified, rendering sequencing results unambiguous as demonstrated for the two samples with the overlayed ssrA sequence chromatograph. In such cases cloning of
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the real-time PCR product followed by sequencing of multiple copies is an option to overcome
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this limitation. In conclusion, we believe that coupling the flea cox1 approach with the multiplexed real-time PCR assay followed by Bartonella sequencing represents a rational
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approach for screening and elucidation of fleas’ capacity to vector infectious agents.
Acknowledgements
We thank Janet Martin, Kelly Bettinger and Striepen’s family (especially Spike) for technical and logistic assistance with collecting flea specimens, Victoria Morin-Adeline and Andrea Lawrence for technical assistance with real-time PCR of Seychelles fleas and mounting fleas, respectively. We would like to thank Boris Striepen, Center for Tropical and Emerging Global Disease, University of Georgia, Athens during JŠ sabbatical.
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Figure Legend
Figure 1. Phylogenetic analysis of the genus Ctenocephalides. The evolutionary history of the
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cox1 was inferred using the Minimum Evolution (ME) method. The percentage of replicate trees
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in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches (>50%). The tree is drawn to scale, with branch lengths in the same units as
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those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method and are in the units of the
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number of base substitutions per site. The analysis involved 41 nucleotide sequences of cox1 mtDNA. All fleas (C. felis, n=20) from cats in Georgia, USA (inset) has identical cox1 and they
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are represented by a single sequence. There were a total of 601 nucleotide positions in the final
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alignment. Evolutionary analyses were conducted in MEGA6. Bradiopsylla echidnae served as an outgroup. Clade I-IV according to Lawrence et al. (2014) and Clade I* was identified in Lawrence et al. (2015).
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Table 1. Summary of cat fleas (Ctenocephalides felis) collected on cats from Georgia, USA
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and Bartonella species identification
Bartonella (real-time PCR) ssrA assay ǁ
Number Identifier*
Cat gender of fleas
Ct value
Result «
Bartonella spp.
20.81
Positive
B. clarridgeiae
20.80
Positive
B. clarridgeiae
4
31.18
Positive
B. henselae
5
31.43
Positive
n/a
4
38.72
Suspect
-
Atlanta, GA
Female
5
AL867-1
Atlanta, GA
Female
5
AL868-1
Atlanta, GA
Female
AL869-1
Atlanta, GA
Male
AL870-1
Atlanta, GA
Female
AL871-1
Atlanta, GA
Female
6
33.77
Positive
B. henselae
AL872-1
Atlanta, GA
Female
3
20.85
Positive
B. clarridgeiae
AL873-1
Atlanta, GA
Female
1
39.03
Suspect
-
2 Females
7
35.39
Suspect
B. clarridgeiae
#
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#
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AL866-1
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#
Location
Elberton, GA
AL875-1
Athens/Watkinsville, GA
Male
2
29.04
Positive
B. clarridgeiae
AL876-1
Athens/Watkinsville, GA
Male
3
29.62
Positive
B. henselae
Athens/Watkinsville, GA
Male
1
39.42
Suspect
-
Athens/Watkinsville, GA
Male
8
28.24
Positive
B. henselae
Athens/Watkinsville, GA
Male
2
34.10
Positive
n/a
Athens/Watkinsville, GA
Male
4
23.94
Positive
B. clarridgeiae
AL881-1
Athens/Watkinsville, GA
Male
3
AL882-1
Athens/Watkinsville, GA
Male
3
AL883-1
Athens/Watkinsville, GA
Male
1
AL884-1
Athens/Watkinsville, GA
Male
7
34.30
Positive
B. henselae
Athens, GA
Male
1
39.46
Suspect
-
AL878-1 AL879-2 #
AL880-1
#
AL885-1
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#
AL877-1
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AL874-1
Negative 33.32
Positive
B. henselae
Negative
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Notes:
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November 18, 2015, AL885 were collected on November 24, 2015.
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*AL866-AL874 were collected on October 12, 2015, AL875-AL884 were collected on
ǁ multiplex real-time PCR assay (Rickettsia and Bartonella), all samples positive for Rickettsia
« positive if Ct value <35, suspect of Ct value >35
confirmed presence of R. felis-like organisms (Rickettsia sp. RF2125) using altA and ompA
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#
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(see Supplementary Table 1)
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amplification and sequencing
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AL866-1, AL868-1, AL874-1, AL877-1, AL880-1, AL885-1
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phylogenetic diversity of the cat flea (Ctenocephalides felis) at two mitochondrial DNA markers.
Lawrence, A.L., Hii, S.F., Jirsová, D., Panáková, L., Ionică, A.M., Gilchrist, K., Modrý, D., Mihalca,
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identification of cat fleas (Ctenocephalides felis) and dog fleas (Ctenocephalides canis) vectoring Rickettsia felis in central Europe. Vet Parasitol 210, 215-223.
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Raoult, D., 2005. The genome sequence of Rickettsia felis identifies the first putative conjugative
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Telford, S.R., 3rd, Wongsrichanalai, C., Raoult, D., 2003. Identification of Rickettsia spp. and Bartonella spp. in from the Thai-Myanmar border. Ann N Y Acad Sci 990, 173-181.
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Rust, M.K., 2005. Advances in the control of Ctenocephalides felis (cat flea) on cats and dogs. Trends Parasitol 21, 232-236.
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Rust, M.K., 2016. Insecticide resistance in fleas. Insects 7.
Šlapeta, J., King, J., McDonell, D., Malik, R., Homer, D., Hannan, P., Emery, D., 2011. The cat flea
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Stenos, J., Graves, S.R., Unsworth, N.B., 2005. A highly sensitive and specific real-time PCR assay for the detection of spotted fever and typhus group Rickettsiae. Am J Trop Med Hyg 73, 1083-1085.
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Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30, 2725-2729. Vobis, M., D'Haese, J., Mehlhorn, H., Mencke, N., Blagburn, B.L., Bond, R., Denholm, I., Dryden, M.W., Payne, P., Rust, M.K., Schroeder, I., Vaughn, M.B., Bledsoe, D., 2004. Molecular phylogeny of isolates of Ctenocephalides felis and related species based on analysis of ITS1, ITS2 and mitochondrial 16S rDNA sequences and random binding primers. Parasitol Res 94, 219-226. Whiting, M.F., Whiting, A.S., Hastriter, M.W., Dittmar, K., 2008. A molecular phylogeny of fleas (Insecta: Siphonaptera): origins and host associations. Cladistics 24, 677-707.
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Figure 1
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Molecular barcoding places the cat fleas from Georgia, US in a clade with African cat fleas Fleas are confirmed to carry Rickettsia and Bartonella, specifically B. clarridgeiae, B. henselae and Rickettsia sp. RF2125 (Rickettsia felis-like organism) Individual, published and highly sensitive TaqMan real-time PCR for Rickettsia and Bartonella are combined in a multiplex real-time PCR assay
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Highlights
S2405-9390(16)30049-1 doi: 10.1016/j.vprsr.2016.06.005 VPRSR 25
To appear in: Received date: Revised date: Accepted date:
7 April 2016 24 June 2016 30 June 2016
ˇ ˇ ep´an, Slapeta, ˇ Please cite this article as: Slapeta, Stˇ Jan, Molecular identity of cat fleas (Ctenocephalides felis) from cats in Georgia, USA carrying Bartonella clarridgeiae, Bartonella henselae and Rickettsia sp. RF2125, (2016), doi: 10.1016/j.vprsr.2016.06.005
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Molecular identity of cat fleas (Ctenocephalides felis) from cats in Georgia, USA carrying
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Bartonella clarridgeiae, Bartonella henselae and Rickettsia sp. RF2125
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Štěpán Šlapeta and Jan Šlapeta*
School of Life and Environmental Sciences, Faculty of Veterinary Science, McMaster Building
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B14, The University of Sydney, New South Wales 2006, Australia
*Corresponding author: Jan Šlapeta, McMaster Building B14, Faculty of Veterinary Science,
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The University of Sydney, New South Wales 2006, Australia; tel: +61 2 9351 2025; fax: +61 2 9351 7348; email: [email protected]
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Abstract
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The cat flea (Ctenocephalides felis) is the most common ectoparasite of dogs and cats. Close
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association of humans with cats and dogs enables flea-borne disease transmission either directly, via flea bites, or indirectly, via pathogens excreted in flea faeces. The aim of this study was to
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evaluate molecular identity of C. felis from cats in Georgia, USA based on a molecular barcode
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marker gene (cox1) and the frequency at which the fleas were carriers of the vector-borne bacteria, Bartonella and Rickettsia. The multiplexed Bartonella and Rickettsia real-time PCR
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assay (targeting ssrA and gltA, respectively) adopted in this study, together with sequencing of the ssrA enabled rapid identification of Bartonella spp. in cat fleas. Eighteen out of 20 fleas
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examined were positive for Bartonella spp. and all fleas were positive for Rickettsia spp. DNA sequencing of the ssrA confirmed presence B. clarridgeiae and B. henselae. Amplification and
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DNA sequencing of gltA and ompA confirmed presence of Rickettsia sp. RF2125 (Rickettsia
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felis-like organism). All fleas from 21 cats in Georgia, USA were morphologically identical with C. felis. Sequencing of the flea cox1 revealed identity with C. felis from Seychelles, Africa recognised as a subspecies C. felis strongylus, the African cat flea. Analysis of the cox1 sequences of fleas improves understanding of global distribution of cat flea cox1 clades (C. felis) when compared with sequences from Ctenocephalides spp. from Asia, Africa, Europe, Asia and Australia. Coupling flea barcoding approach with the multiplexed real-time PCR assay followed by Bartonella sequencing represents a rational approach for screening and elucidation of fleas’ capacity to vector infectious agents.
Keywords: barcoding, Siphonaptera, real-time PCR, phylogeny, cox1, COI
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1. Introduction
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The cat flea (Ctenocephalides felis) is the most common ectoparasite of dogs and cats in tropical, subtropical, as well as temperate climates. The cat flea is a generalist parasite and
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potentially feeds on any mammal, including humans particularly when exposed to highly infested environments (Dryden and Rust, 1994; Rust, 2005, 2016). Most commonly, however,
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cat fleas are found on domestic and feral or community cats and dogs. Close association of
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humans with cats and dogs enables flea borne disease transmission either directly, via flea bites, or indirectly, via pathogens excreted in flea faeces (Boulouis et al., 2005; Chomel et al., 2006).
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Recently, molecular tools were applied to characterise cat fleas and closely related species
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in the genus Ctenocephalides (Vobis et al., 2004; Šlapeta et al., 2011; Marrugal et al., 2013; Lawrence et al., 2014; Hii et al., 2015; Lawrence et al., 2015). A previously assumed subspecies
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C. felis orientis, Oriental cat flea, was confirmed to represent a full species C. orientis which is more closely related to the dog flea, Ctenocephalides canis than to C. felis (Hopkins and Rothschild, 1953; Menier and Beaucournu, 1998; Lawrence et al., 2015). The use of cytochrome c oxidase subunit I (cox1) marker to ‘barcode’ fleas has demonstrated that the cat flea is not as homogeneous as previously thought (Vobis et al., 2004; Marrugal et al., 2013), consisting of a number of defined clades whose global distribution is yet to be fully understood (Lawrence et al., 2014; Hii et al., 2015; Lawrence et al., 2015). The capacity of different Ctenocephalides species, subspecies or even cox1 clades to vector infectious agents has been suggested on a small scale sample and requires further scrutiny (Hii et al., 2015).
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The aim of this study was to evaluate the molecular identity of Ctenocephalides felis from
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cats in Georgia, USA based on a molecular barcoding of cox1 marker gene sequence and the
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frequency at which the fleas were carriers of the vector-borne bacteria, Bartonella and Rickettsia. The multiplexed Bartonella and Rickettsia real-time PCR assay adopted in this study, together
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with sequencing of the ssrA, a gene that codes transfer-mRNA (tmRNA), enables rapid identification of Bartonella spp. in cat fleas. Analysis of the cox1 sequences of fleas improved
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our understanding of global distribution of cat flea clades (C. felis) when compared with
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sequences from Ctenocephalides spp. from Asia, Africa, Europe, Asia and Australia.
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2. Materials and Methods
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2.1. Flea collection, identification and extraction of the total DNA
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Fleas were collected from cats in Georgia, USA (Figure 1) between October and November 2015. Fleas (n=74) were collected opportunistically from 20 cats during visits to a desexing veterinary clinic (neutered cats in a trap-neuter-return program), and one cat directly from a household (for details on locality see Table 1). Fleas were collected using flea comb and stored in 80% ethanol. Fleas were morphologically identified based on published keys (Hopkins and Rothschild, 1953). Total DNA was extracted whilst retaining flea exoskeletons using Isolate II Genomic DNA kit (BioLine, Australia) as previously described (Whiting et al., 2008; Lawrence et al., 2014). DNA was eluted into 100 µL of Tris buffer (pH=8.5) and stored at -20°C. Previously isolated DNA from Ctenocephalides felis strongylus (n=5) collected on dogs from Seychelles was included in this study (Lawrence et al., 2014).
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2.2. Amplification of the mitochondrial encoded cytochrome c oxidase subunit I and flea
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phylogeny
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A 5' fragment of the cytochrome c oxidase subunit I (cox1) coding for COX1 protein was PCR amplified using primers: LCO1490, 5’- GGT CAA CAA ATC ATA AAG ATA TTG G-3’
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and Cff-R (S0368) 5’-GAA GGG TCA AAG AAT GAT GT-3’ (Lawrence et al., 2014).
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Reactions of 30 μl contained MyTaq Red Mix (BioLine, Australia) with approximately 10–50 ng of genomic DNA template (2 μl) in a Veriti Thermal Cycler (Life Sciences, Australia). The cycling conditions were as follows: denaturing at 95°C for 1 min followed by 35 cycles of 95°C
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for 15 s, 55°C for 15 s, 72°C for 10 s, and a final elongation for 5 min at 72°C. Sterile PCR-
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grade water was used as negative control, and a positive control consisting of flea DNA known to amplify at these conditions was also run along-side. PCR products that yielded an
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unambiguous single band product (~600bp) were directly and bidirectionally sequenced at Macrogen Inc. (Seoul, Korea). All sequences were assembled, aligned with related sequences and analysed using CLC Main Workbench 6.9.1 (CLC bio, Denmark) and deposited in GenBank (National Center for Biotechnology Information, NCBI) under the Accession Numbers: KX021755-KX021774.
Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013). Multiple sequence alignment was constructed with all available Ctenocephalides sequences belonging to defined clades (Lawrence et al., 2014; Hii et al., 2015; Lawrence et al., 2015). Sequence divergences were calculated using the Kimura 2 parameter (K2P) distance model, and
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phylogenetic tree was inferred using minimum evolution and the bootstrap support inferred from
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2.3. Diagnostic Rickettsia and Bartonella real-time PCR
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500 replicates.
A diagnostic TaqMan probe real-time PCR assay targeting the gltA (citrate synthase) gene
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of Rickettsia spp. was applied for the detection of Rickettsia (Stenos et al., 2005). A diagnostic
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TaqMan probe real-time PCR assay targeting ssrA gene was applied to specifically detect Bartonella (Diaz et al., 2012). The assay was multiplexed using Rickettsia oligonucleotide probe labelled with HEX and Bartonella oligonucleotide probe labelled with FAM. Bartonella assay
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used ssrA-F (S0508), 5’-GCT ATG GTA ATA AAT GGA CAA TGA AAT AA-3’, ssrA-R
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(S0509), 5’-GCT TCT GTT GCC AGG TG-3’ and a probe S0510, 5’-FAM-ACC CCG CTT AAA CCT GCG ACG-3’-BHQ1 (Diaz et al., 2012). CS-F (S0576), 5’-TCG CAA ATG TTC
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ACG GTA CTT T-3’, CS-R (S0577) 5’-TCG TGC ATT TCT TTC CAT TGT G-3’ and a probe CS-P (S0578), 5’-HEX-TGC AAT AGC AAG AAC CGT AGG CTG GAT G-3’-BHQ1 (Stenos et al., 2005). Oligonucleotides and labelled oligonucleotides were synthesised by Macrogen Inc. (Seoul, Korea). The reaction was carried out in 20 µl volumes and included a final concentration of 400 nM and 100 nM of primers and probes, respectively, along with 4 µl of template DNA with SensiFAST Probe No-ROX Kit (BioLine, Australia). Duplicate PCRs were performed using the following conditions: 3 mins at 95°C followed by 40 cycles of 5 s at 95°C and 15 s at 60°C. Each run included a no template negative control (NTC) and positive control plasmid DNA carrying a Bartonella and Rickettsia target DNA insert. Bartonella plasmid control included a region of the ssrA gene of Bartonella henselae with TAA AGC TAC GAC AAC GAG TAC
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TAA coding for SYDNEY flanked by stop codons in a pMA-T backbone (GeneArt, Life
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Technologies, Australia) as previously described (Lawrence et al., 2015). The amplification on
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CFX96 Touch™ Real-Time PCR Detection System was analysed with the corresponding software (BioRad, Australia). The arbitrary real-time PCR threshold was set to a single threshold
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at 100 rfu.
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The Bartonella assay amplifies approximately 300 bp of ssrA gene sufficient to resolve
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Bartonella species (Diaz et al., 2012). All Bartonella real-time PCR reactions returning satisfactory Ct values (Ct <35) were repeated as single Bartonella real-time PCR assay and sequenced using amplification primers at Macrogen Inc. (Seoul, Korea). Sequences were
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assembled, aligned with related sequences and analysed using CLC Main Workbench 6.9.1
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(CLC bio, Denmark) and deposited in GenBank (National Center for Biotechnology Information,
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NCBI) under the Accession Numbers: KX021743- KX021754.
2.4. Diagnostic Rickettsia felis gltA and ompA nested PCR
A diagnostic conventional nested PCR assays targeting the gltA gene of R. felis and rickettsial outer membrane protein B (ompB) gene were applied for the detection of R. felis as previously described (Hii et al., 2015). A fragment (654 bp) of gltA was amplified using primer pair gltA-F1 (S0659), 5’-GCA AGT ATT GGT GAG GAT GTA ATC-3’ / gltA-R1 (S0660), 5’CTG CGG CAC GTG GGT CAT AG-3’ followed by nested primer pair gltA-F2 (S0661), 5’GCG ACA TCG AGG ATA TGA CAT-3’ / gltA-R2 (S0662) 5’-GGA ATA TTC TCA GAA CTA CCG-3’. A fragment (879 bp) of ompA was amplified using primer pair omp-A-F1 (S0663)
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5’-CGA TAG TGT TAC AAG TAC CGG-3’ / omp-A-R1 (S0664) 5’-GCA TCT TCC ATT
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AAC TCA AGC-3’ followed by nested primer pair ompA-F2 (S0665) 5’-CGG TAC AAT CAT
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TGC AAC TGG-3’ / ompA-R2 (S0666) 5’-GCT ATA TCT TCA GCA AAT AAC G-3’. Reactions of 25 μl contained MyTaq Red Mix (BioLine, Australia) and 2 μl of genomic DNA
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template were amplified in a Veriti Thermal Cycler (Life Sciences, Australia). Template for the secondary PCR was 2 µl of the primary PCR reaction. PCR grade water served as negative
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control. PCR cycling conditions comprised an initial activation step at 95°C for 2 min, followed
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by 40 cycles of 95°C for 15 s, 63°C (primary gltA PCR) or 59 °C (primary ompA PCR)for 30 s and 72°C for 30 s with a final extension step of 72°C for 5 min. The secondary PCR amplification for gltA was performed as for the primary PCR except that the annealing
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temperature was 59°C, and for ompA secondary PCR was identical to the primary. PCR
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amplicons were sequenced using amplification primers at Macrogen Inc. (Seoul, Korea). Sequence of region amplified with the above mentioned primers (gltA and ompA) from the
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genome R. felis URRWXCal2 (CP000053) served as reference (Ogata et al., 2005). Sequences were assembled, aligned with related sequences and analysed using CLC Main Workbench 6.9.1 (CLC bio, Denmark) and deposited in GenBank (National Center for Biotechnology Information, NCBI) under the Accession Numbers: KX431974-KX431985.
3. Results
Morphological analysis revealed the presence of only the cat flea (Ctenocephalides felis) among the fleas collected from cats in Georgia, USA (Table 1). To infer their phylogenetic position, DNA from a subset of 20 fleas (C. felis) was extracted and sequenced at the cox1 gene
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(Figure 1). Multiple sequence alignment of existing clades revealed indistinguishable or a highly
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similar cox1 sequences (>99%) of the cat fleas from Georgia, USA to the clade previously
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recognised as the African clade of C. felis strongylus (Clade III, in Lawrence et al. 2014) from Seychelles (Lawrence et al., 2014). Previously, five distinct clades were recognised to represent
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C. felis felis and C. felis strongylus (Lawrence et al., 2014; Lawrence et al., 2015) (Figure 1).
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The multiplexed Rickettsia and Bartonella real-time PCR assay revealed presence of
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Rickettsia DNA in all fleas with an average Ct value of 25.12 (min. 21.19, max. 30.28). Thirteen (65%) out of 20 fleas were positive for Bartonella spp. in duplicate real-time PCRs with an average Ct value of 28.57 (min. 20.80, max. 34.30). In addition five fleas (AL885-1, AL874-1,
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AL873-1, AL870-1, AL877-1) were considered ‘suspect positives’ (late amplifiers) for
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control reactions.
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Bartonella because their Ct values were >35. No amplification was observed in any negative
Direct DNA sequencing of the real-time PCR products of the Bartonella ssrA gene confirmed presence of Bartonella spp. in 12 samples, including 11 real-time PCR positive and 1 real-time PCR suspect (AL874-1) (Table 2). Two sequences (AL969-1, AL879-2) showed overlapping sequence chromatographs. Sequence alignment of ssrA gene sequences revealed that six sequences were 100% identical to B. clarridgeiae (JN982716) and six were 100% identical to B. henselae (JN029785).
Selected Rickettsia-positive fleas (n=6) were further characterised with conventional nested PCR targeting a fragment of gltA (654 bp) and ompA (879 bp) genes. All randomly selected fleas
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(AL866-1, AL868-1, AL874-1, AL877-1, AL880-1, AL885-1) returned positive amplicons of
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expected size. DNA sequencing revealed that all gltA sequences were 100% identical to the
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reference sequence of Rickettsia sp. RF2125 (AF516333) (Parola et al., 2003). The gltA region was 99% identical (7 differences across 612 alignment residues) to the reference sequence of R.
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felis (Ogata et al., 2005). Similarly, ompA sequences were most closely related to ompA sequences from Rickettsia sp. RF2125 (KP687807, KP256359, KP687806, KP256358), there
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was a single mismatch G/T (Hii et al., 2015). The ompA region was 96% identical (34
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differences across 834 alignment residues) to the reference sequence of R. felis (Ogata et al., 2005).
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The discovery that cat fleas collected on cats in Georgia, USA were monophyletic with C.
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felis strongylus from Seychelles prompted us to evaluate the Seychelles fleas for the presence of Rickettsia and Bartonella (Lawrence et al., 2014). None of the five fleas originally collected
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from dogs from Seychelles revealed presence of Bartonella DNA, nevertheless four were positive for Rickettsia DNA in duplicate multiplex real-time PCR (average Ct value of 23.76). In one reaction, a single flea (AL83-1) was a late amplifier (Ct value of 39.54) for Rickettsia DNA. Diluting the DNA 1:10 confirmed Rickettsia DNA in four samples (average Ct value of 26.62), while AL83-1 remained negative for Rickettsia DNA, and no Bartonella DNA was amplified (Supplementary Figure 1).
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4. Discussion
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We have identified only C. felis on cats in Georgia, USA. Molecular barcoding using the cox1 gene sequence revealed that all the cat fleas from several locations in Georgia, USA are
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identical to those found in Africa, and are assumed to represent an African subspecies C. felis strongylus, African cat flea (Lawrence et al., 2014). In general, it is assumed that the subspecies
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C. felis felis is the globally distributed subspecies, dispersed with domesticated cats, dogs, and
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human migration. Previous studies have demonstrated the presence of a cox1 clade that is shared between Europe, Asia and Australia and is considered the ubiquitous on dog and cats in veterinary practices in Australia (Šlapeta et al., 2011; Hii et al., 2015). It was rather surprising
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that despite similar climate between Georgia, USA and Australia, a different cat flea clade is
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sustained. This study is the first to apply cox1 barcoding to cat fleas from USA and compare them to the growing dataset of cat fleas from other countries. Further sampling across different
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states will resolve the extent of the distribution of the Clade III in USA and elsewhere. The role of human migration and the historical African slave trade in the introduction of C. felis strongylus to USA will require further global sampling. The absence of dog fleas (C. canis) in this study is not unexpected since only cats were sampled. The dog flea (C. canis) is known to be present in the USA in lesser frequency then cat flea (C. felis) (Durden et al., 2012).
The cat fleas were collected on a mix of community animals living on a property as well as owned cats. In such situations, contact with human is frequent and increases the chance for fleaborne disease transmission (Boulouis et al., 2005; Durden et al., 2012). Identification of B. clarridgeiae and B. henselae in the subset of fleas confirms previous finding that Bartonella spp.
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are frequently present in cat fleas in USA and humans or dogs may become accidental hosts
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(Breitschwerdt and Kordick, 2000; Chomel et al., 2006; Breitschwerdt, 2008; Breitschwerdt et
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al., 2010; Chomel and Kasten, 2010). In humans, B. henselae is the cause of the ‘cat scratch disease’, however, the evidence that B. clarridgeiae causes disease in human is minimal (Chomel
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et al., 2006). Both B. clarridgeiae and B. henselae spp. are associated with several different systemic illnesses in dogs including chronic endocarditis (MacDonald et al., 2004). A study in
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Florida based on 553 community cats documented 34% prevalence for B. hansellae (Luria et al.,
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2004). Frequent presence of Bartonella spp. in fleas collected on community cats from Georgia is therefore expected. As argued previously, community cats do not represent greater risk to humans or other pets than owned pet cats because their prevalence of infectious agents in
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community cats fall within reported ranges for owned pet cats (Luria et al., 2004).
Cat fleas are commonly identified to carry Rickettsia felis or R. felis-like organisms
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(Angelakis et al., 2016). Identification of R. felis-like organisms (Rickettsia sp. RF2125) in fleas from Georgia, USA indicates that cats and dogs may act as potential reservoir hosts. The zoonotic risk to humans remains unascertained for R. felis-like species (Parola et al., 2003; Hii et al., 2015). The significance of Rickettsia sp. RF2125 and global distribution is incomplete, and our report confirms its presence in USA in what we recognise as C. felis strongylus.
Combination of existing sensitive TaqMan molecular assays for Rickettsia and Bartonella enabled us to test for both pathogens in a single tube. Both assays are highly sensitive and specific (Stenos et al., 2005; Diaz et al., 2012). The Rickettsia assay detects spotted fever and typhus group rickettsiae, such as Rickettsia akari, R. australis, R. conorii, R. honei, R. rickettsii,
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R. typhi, or R. felis, as well as, potentially symbiotic/non-pathogenic rickettsiae (Stenos et al.,
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2005; Hii et al., 2015). The Bartonella assay targeting ssrA gene was designed for species
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identification, because the real-time PCR product is of sufficient length (~300bp) to allow sequencing and species identification (Diaz et al., 2012). The combination of both assays offer
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rapid screening of vectors, such as fleas, as demonstrated in this study. Bartonella spp. are recognised and differentiated from each other using comparison and application of multilocus
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genotyping, nevertheless for vector screening purposes, the ssrA gene was sufficient to identify
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the species of Bartonella. The limitation of this approach is that if two Bartonella spp. are present both, ssrA will be amplified, rendering sequencing results unambiguous as demonstrated for the two samples with the overlayed ssrA sequence chromatograph. In such cases cloning of
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the real-time PCR product followed by sequencing of multiple copies is an option to overcome
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this limitation. In conclusion, we believe that coupling the flea cox1 approach with the multiplexed real-time PCR assay followed by Bartonella sequencing represents a rational
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approach for screening and elucidation of fleas’ capacity to vector infectious agents.
Acknowledgements
We thank Janet Martin, Kelly Bettinger and Striepen’s family (especially Spike) for technical and logistic assistance with collecting flea specimens, Victoria Morin-Adeline and Andrea Lawrence for technical assistance with real-time PCR of Seychelles fleas and mounting fleas, respectively. We would like to thank Boris Striepen, Center for Tropical and Emerging Global Disease, University of Georgia, Athens during JŠ sabbatical.
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Figure Legend
Figure 1. Phylogenetic analysis of the genus Ctenocephalides. The evolutionary history of the
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cox1 was inferred using the Minimum Evolution (ME) method. The percentage of replicate trees
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in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches (>50%). The tree is drawn to scale, with branch lengths in the same units as
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those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method and are in the units of the
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number of base substitutions per site. The analysis involved 41 nucleotide sequences of cox1 mtDNA. All fleas (C. felis, n=20) from cats in Georgia, USA (inset) has identical cox1 and they
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are represented by a single sequence. There were a total of 601 nucleotide positions in the final
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alignment. Evolutionary analyses were conducted in MEGA6. Bradiopsylla echidnae served as an outgroup. Clade I-IV according to Lawrence et al. (2014) and Clade I* was identified in Lawrence et al. (2015).
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Table 1. Summary of cat fleas (Ctenocephalides felis) collected on cats from Georgia, USA
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and Bartonella species identification
Bartonella (real-time PCR) ssrA assay ǁ
Number Identifier*
Cat gender of fleas
Ct value
Result «
Bartonella spp.
20.81
Positive
B. clarridgeiae
20.80
Positive
B. clarridgeiae
4
31.18
Positive
B. henselae
5
31.43
Positive
n/a
4
38.72
Suspect
-
Atlanta, GA
Female
5
AL867-1
Atlanta, GA
Female
5
AL868-1
Atlanta, GA
Female
AL869-1
Atlanta, GA
Male
AL870-1
Atlanta, GA
Female
AL871-1
Atlanta, GA
Female
6
33.77
Positive
B. henselae
AL872-1
Atlanta, GA
Female
3
20.85
Positive
B. clarridgeiae
AL873-1
Atlanta, GA
Female
1
39.03
Suspect
-
2 Females
7
35.39
Suspect
B. clarridgeiae
#
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#
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AL866-1
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#
Location
Elberton, GA
AL875-1
Athens/Watkinsville, GA
Male
2
29.04
Positive
B. clarridgeiae
AL876-1
Athens/Watkinsville, GA
Male
3
29.62
Positive
B. henselae
Athens/Watkinsville, GA
Male
1
39.42
Suspect
-
Athens/Watkinsville, GA
Male
8
28.24
Positive
B. henselae
Athens/Watkinsville, GA
Male
2
34.10
Positive
n/a
Athens/Watkinsville, GA
Male
4
23.94
Positive
B. clarridgeiae
AL881-1
Athens/Watkinsville, GA
Male
3
AL882-1
Athens/Watkinsville, GA
Male
3
AL883-1
Athens/Watkinsville, GA
Male
1
AL884-1
Athens/Watkinsville, GA
Male
7
34.30
Positive
B. henselae
Athens, GA
Male
1
39.46
Suspect
-
AL878-1 AL879-2 #
AL880-1
#
AL885-1
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#
AL877-1
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AL874-1
Negative 33.32
Positive
B. henselae
Negative
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Notes:
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November 18, 2015, AL885 were collected on November 24, 2015.
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*AL866-AL874 were collected on October 12, 2015, AL875-AL884 were collected on
ǁ multiplex real-time PCR assay (Rickettsia and Bartonella), all samples positive for Rickettsia
« positive if Ct value <35, suspect of Ct value >35
confirmed presence of R. felis-like organisms (Rickettsia sp. RF2125) using altA and ompA
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#
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(see Supplementary Table 1)
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amplification and sequencing
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AL866-1, AL868-1, AL874-1, AL877-1, AL880-1, AL885-1
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Figure 1
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Molecular barcoding places the cat fleas from Georgia, US in a clade with African cat fleas Fleas are confirmed to carry Rickettsia and Bartonella, specifically B. clarridgeiae, B. henselae and Rickettsia sp. RF2125 (Rickettsia felis-like organism) Individual, published and highly sensitive TaqMan real-time PCR for Rickettsia and Bartonella are combined in a multiplex real-time PCR assay
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Highlights