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Chandra S, Forsyth M, Lawrence AL, Emery D, Šlapeta J (2017). Cat fleas (Ctenocephalides felis) across the Tasman Sea: presence of Rickettsia felis and Bartonella clarridgeiae from fleas on dogs and cats in New Zealand veterinary clinics. VETERINARY PARASITOLOGY 234, 25–30. [http://dx.doi.org/10.1016/j.vetpar.2016.12.017]
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Cat fleas (Ctenocephalides felis) from cats and dogs in New Zealand: Molecular
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characterisation, presence of Rickettsia felis and Bartonella clarridgeiae and comparison
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with Australia
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Shona Chandra1, Maureen Forsyth2, Andrea L. Lawrence1,3, David Emery1, Jan Šlapeta1*
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School of Life and Environmental Sciences, Faculty of Veterinary Science, University of
Sydney, NSW, Australia 2
Merial New Zealand, Auckland, New Zealand
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Department of Medical Entomology, The University of Sydney and Pathology West,
ICPMR, Westmead Hospital, Westmead, NSW, Australia
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* Corresponding author: McMaster Building B14, Faculty of Veterinary Science, University
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of Sydney, NSW, Australia. Email address:
[email protected] (J. Šlapeta)
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Abstract
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The cat flea (Ctenocephalides felis) is the most common flea species parasitising both
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domestic cats and dogs globally. Fleas are known vectors of zoonotic pathogens such as
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vector borne Rickettsia and Bartonella. This study compared cat fleas from domestic cats and
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dogs in New Zealand’s North and South Islands to Australian cat fleas, using the
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mitochondrial DNA (mtDNA) marker, cytochrome c oxidase subunit I and II (cox1, cox2).
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We assessed the prevalence of Rickettsia and Bartonella using genus specific multiplexed
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real-time PCR assays. Morphological identification confirmed that the cat flea (C. felis) is the
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most common flea in New Zealand. The examined fleas (n = 43) at cox1 locus revealed six
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closely related C. felis haplotypes (inter-haplotype distance 1.1%) across New Zealand. The
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New Zealand C. felis haplotypes were identical or near identical with haplotypes from
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southern Australia demonstrating common dispersal of haplotype lineage across both the
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geographical (Tasman Sea) and climate scale. New Zealand cat fleas carried Rickettsia felis
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(5.3%) and Bartonella clarridgeiae (18.4%). To understand the capability of C. felis to vector
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zoonotic pathogens, we determined flea cox1 and cox2 haplotype diversity with the tandem
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multiplexed real-time PCR and sequencing for Bartonella and Rickettsia. This enabled us to
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demonstrate highly similar cat flea on cat and dog populations across Australia and New
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Zealand.
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Keywords
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Bartonella, cox1, Ctenocephalides felis, genetic diversity, PCR, real-time PCR, Rickettsia
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1. Introduction
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The cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae) (Bouché, 1835) is the most
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common household ectoparasite affecting domestic cats and dogs globally (Linardi and Costa
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Santos, 2012; Pilgrim, 1992; Rust and Dryden, 1997). In New Zealand, C. felis is an
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established cosmopolitan species and has exhibited a high prevalence in the North Island
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(Kelly et al., 2005b; Pilgrim, 1980, 1992). Prevalence studies have previously been limited to
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the North Island and one study in 1984 found that of the 75 cats surveyed, 92.6% carried C.
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felis (Guzman, 1984; Kelly et al., 2005b; Pilgrim, 1980, 1992). Recently, a nationwide survey
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determined the prevalence of C. felis on 76% of cats and 46% on dogs (Woollett et al., 2016).
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Genetic diversity at the molecular DNA level has been established for C. felis in coastal
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Australia and Fiji, however there is no data for genetic diversity recorded in New Zealand
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(Lawrence et al., 2014; Lawrence et al., 2015b; Šlapeta et al., 2011).
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In New Zealand and Australia, a high prevalence of C. felis has been documented around
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coastal regions, but the humidity, sandy soils and moderate to high temperatures are more
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favourable for flea survival and proliferation in Australia (Schloderer et al., 2006; Silverman
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et al., 1981). In a study conducted during the 2009 and 2010 flea seasons, 2,500 of the 2,530
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fleas (98.8%) collected were C. felis (Šlapeta et al., 2011). There was very low genetic
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diversity of C. felis in Australia, with only two distinct mitochondrial cytochrome c oxidase
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subunit I (cox1) haplotypes and one cytochrome c oxidase subunit II (cox2) haplotype found
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across the east coast (Lawrence et al., 2014). Despite New Zealand having a characteristically
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variable climate, the North Island is climatically similar to Sydney and surrounding regions
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(Hopkins et al., 2015; Reichgelt et al., 2015). As climate plays such an integral role in the flea
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life cycle, it was important to know if the variable climate between the North and South
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Islands had an effect on flea diversity and distribution. A survey of cox1 and cox2 haplotypes
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of cat fleas from New Zealand would enable understanding their genetic diversity and their
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relatedness across the Tasman Sea to Australian cat fleas.
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New Zealand has the highest cat ownership rate globally, with a total cat population of 1.419
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million owned by 48% of households (Mackay, 2011). Comparatively, there are 700,000 dogs
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owned by 29% of households (Mackay, 2011). The close relationship shared between humans
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and their companion animals increases the transmission opportunities of zoonotic pathogens,
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including Bartonella and Rickettsia (Kelly et al., 2005b). Rickettsia and Bartonella currently
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have a global emerging disease status and are present in Australia and New Zealand (Bitam et
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al., 2010; Pérez-Osorio et al., 2008). In New Zealand, the last known prevalence study
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conducted for Rickettsia and Bartonella species, was limited to the North Island. Of the 114
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cat fleas assayed, 7% were positive for B. clarridgeiae, 11% were positive for B. henselae and
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19% were positive for R. felis (Kelly et al., 2005b).
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The aim of this study was to discover the level of genetic identity of cat fleas (C. felis) across
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the Tasman Sea. To do so, we compared the mtDNA cox1 haplotype diversity of C. felis from
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New Zealand with those from Australia. In this study, we characterised the genetic diversity
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and origin of C. felis and evaluated presence of flea-borne pathogens Rickettsia and
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Bartonella in fleas from veterinary practices across New Zealand’s North and South Islands.
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2. Materials & Methods
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2.1 Flea specimens
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A total of 231 fleas were collected from the North and South Islands of New Zealand for this
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study between January 2014 and March 2016 (Table 1; Supplementary Table 1). All fleas
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were collected opportunistically from owned and stray cats and dogs presented to New
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Zealand veterinary clinics, including SPCA clinics (The Royal New Zealand Society for the
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Prevention of Cruelty to Animals). Fleas from individual animals were collected by
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veterinarians and veterinary nurses and were stored in 70-100% ethanol in the freezer (-20˚C).
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2.2 Morphological identification and DNA extraction of fleas
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Fleas were examined under a stereo microscope (5-20x objectives, BX41, Olympus,
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Australia) and the species, sex and number of flea(s) were recorded and identified
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morphologically with the aid of keys and descriptions (Dunnet and Mardon, 1974; Hopkins
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and Rothschild, 1953).
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Total flea DNA was extracted in accordance with the ISOLATE II Genomic DNA Kit
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(BioLine, Australia) and eluted in 100 µL of elution buffer as previously described (Lawrence
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et al., 2014). Voucher specimens were cleared and mounted in Euparal (Ento Supplies,
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Australia).
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2.3 Amplification of the mitochondrial encoded cytochrome c oxidase subunit I and II
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A 601-nt 5’ fragment of cox1 gene was amplified in a polymerase chain reaction (PCR) using
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a generic invertebrate amplification forward primer, LCO1490 (5’–GGT CAA CAA ATC
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ATA AAG ATA TTG G–3’) and a reverse primer designed in a previous study Cff-R [S0368]
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(5’–GAA GGG TCA AAG AAT GAT GT–3’) (Folmer et al., 1994; Lawrence et al., 2014). A
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727-nt portion of mtDNA including cox2 gene sequence was amplified using F-Leu (5’–TCT
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AAT ATG GCA GAT TAG TGC–‘3) and R-Lys (5’–GAG ACC AGT ACT TGC TTT CAG
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TCA TC–‘3) (Whiting et al., 2008). MyTaqTM Red Mix (BioLine, Australia) was used for
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cox1 amplification as previously described (Lawrence et al., 2014). PCR products were
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bidirectionally sequenced (Macrogen Ltd., Seoul, South Korea). The sequences have been
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deposited to GenBank (cox1: KY048308 - KY048351; cox2: KY048303 - KY048307)
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(National Center for Biotechnology Information, NCBI).
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2.4 Detecting Rickettsia and Bartonella spp. using real-time PCR
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A diagnostic TaqMan® probe real-time PCR targeting the ssrA gene and citrate synthase
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(gltA) gene was used to detect Bartonella and Rickettsia species, respectively (Diaz et al.,
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2012; Stenos et al., 2005). Duplicate multiplex real-time PCR included 4 µL of template DNA
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with SensiFASTTM Probe No-ROX Kit (BioLine, Australia) was run for 40 cycles using
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CFX96 TouchTM Real-Time PCR Detection System (BioRad, Australia) and analysed using
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BioRad CFX Manager (BioRad, Australia) as previously described (Šlapeta and Šlapeta,
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2016). Positive results were determined if both repeats yield satisfactory cycle threshold (Ct)
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values (Ct < 38.00). Suspect results were determined if one or more repeats yield Ct ≥ 38.00
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and negative results were determined if both repeats did not cross the threshold (Ct > 40).
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We regarded ‘suspect’ positives as unreliable as it could have resulted from non-specific
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amplification or potentially environmental contamination. Ideally, DNA should have been
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reisolated, but in our case where the entire flea was used, this approach was not feasible.
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Bartonella positive real-time PCR yields ~300 bp PCR product of ssrA gene that can be
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directly sequenced and gthe obtained sequence allows Bartonella species identification (Diez
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et al. 2012); therefore, positive real-time PCR products were sent to Macrogen for direct
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sequencing (Macrogen Ltd., Seoul, South Korea). Rickettsia positive DNA samples results
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were subject to a further diagnostic conventional nested PCR targeting the outer membrane
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protein A (ompA) gene and Rickettsia felis citrate synthase (gltA) gene for speciation (Stenos
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et al., 2005). MyTaqTM Red Mix (BioLine, Australia) and 2 µL of template DNA was used for
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the 25 µL nested PCR reaction as previously described (Šlapeta and Šlapeta, 2016). PCR
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amplicons were sent to Macrogen (Macrogen Inc., Seoul, South Korea) for bidirectional
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sequencing. The sequences have been deposited in GenBank (ssrA: KY048301 - KY048302;
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gltA: KY048294 - KY048300; ompA: KY048352 - KY048358).
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2.5 Sequence analysis
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Sequences were assembled using CLC Main Workbench 6.8.1 (CLC bio, Qiagen, Denmark).
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Haplotype diversity was calculated and determined within the clades using DNACollapser,
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FaBox (Villesen, 2007). Phylogenetic analysis of C. felis DNA and the composition of the
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nucleotide sequences were determined using MEGA 6.06 and MEGA 7.0 (Kumar et al., 2016;
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Tamura et al., 2013). Phylogenetic comparison was made between New Zealand and
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Australian haplotypes (KF684882-KF684912, KR363640-KR363646, KT376425-
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KT376439; Supplementary Table 2), using MEGA 7.0 to determine likeness between the
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two sample populations (Kumar et al., 2016; Lawrence et al., 2014). Kimura 2 parameter
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(K2P) distance model and inter-population distance was calculated using MEGA 6.06 to
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determine haplotype divergence.
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Speciation of Bartonella and Rickettsia were determined using CLC Main Workbench 6.8.1
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(CLC bio, Qiagen, Denmark). Sequences were assembled and aligned using reference ssrA
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Bartonella sequences and gltA, ompA Rickettsia sequences available from GenBank (National
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Center for Biotechnology Information, NCBI) to including reference gene ssrA for B.
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clarridgeiae (JN982716) and reference genome of R. felis (CP000053).
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3. Results
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3.1 Identical Ctenocephalides felis across the Tasman Sea: the dominant cox1 haplotype is shared between Australia and New Zealand
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Samples were collected from all but three regions from the North and South Islands (Figure
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1; Supplementary Table 1). The fleas collected were predominantly from the North Island,
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with 164 from the North Island and 67 from the South Island. Morphological analysis
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revealed 98.7% (228/231) were cat fleas, C. felis (Figure 1). Three human fleas, Pulex
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irritans (Linnaeus, 1758), were collected in Gore, located in the South Island (Figure 1;
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Supplementary Table 1). Of the 228 C. felis collected from 25 cats and 22 dogs, 177 were
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female and 48 were male (Table 1, sex ratio: 3.653).
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For genotyping using cox1, we randomly selected 43 C. felis; 24 fleas were from the North
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Island and 19 fleas were from the South Island. There were six cox1 haplotypes (NZ1-6;
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overall mean inter-haplotype distance, 1.1%; number of polymorphic sites, 26). The most
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commonly seen haplotype (NZ1, 41.9%, 18/43) had a distinct C/T polymorphism at residue
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443 (across the 601-nt sequence) from the reference C. felis cox1 sequence (KF684884). The
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haplotype NZ2 (32.6%, 14/43) was identical to the reference cox1 sequence (KF684884) –
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haplotype h1 in Lawrence et al. (2014) from Australia. Haplotypes NZ1 and NZ2 were 99.8%
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(600/601) identical across the 601 nucleotides of the cox1 gene. In addition, a single P.
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irritans was genotyped at cox1.
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All New Zealand cox1 haplotypes of C. felis were closely related to each other, forming
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monophyletic Clade 1 (Figure 2). To compare the cox1 haplotype diversity with C. felis from
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Australia, we retrieved all (n = 53) C. felis cox1 sequences from GenBank (National Center
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for Biotechnology Information, NCBI). Multiple sequence alignment and phylogenetic
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analysis revealed that all New Zealand C. felis cox1 haplotypes and the majority of Australian
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cox1 haplotypes fall within the major Clade 1 (Figure 2). Clade 2, represented by fleas from
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Cairns region in Queensland, Australia was not detected in New Zealand (Figure 2).
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For genotyping using cox2, we selected five C. felis from the North Island, representing three
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distinct cox1 haplotypes (AL244-1, AL245-1, AL246-1, AL249-1, AL250-1). There was only
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one cox2 haplotype, which was identical to the h1 cox2 haplotype (HQ696926) from
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Australia (Šlapeta et al. 2011).
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3.2 Ctenocephalides felis from New Zealand possess DNA of Bartonella clarridgeiae and Rickettsia felis
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The multiplex Bartonella and Rickettsia real-time PCR of 38 C. felis found Bartonella and
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Rickettsia DNA in two (5.3%, 2/38; average Ct-value 32.95, range 28.86 – 37.11) and seven
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(18.4%, 7/38; average Ct-value 27.43, range 22.39 – 36.27) C. felis, respectively
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(Supplementary Table 3). There were three (7.9%, 3/38) samples and five (13.1%, 5/38)
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samples which were late amplifiers (Ct > 38.00), and were regarded as ‘suspect’ positive for
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Bartonella and Rickettsia, respectively. Negative control reactions, using PCR-grade water,
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revealed no observable amplifications.
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The two positive Bartonella samples came from one owned dog and one owned cat. Of the
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seven positive Rickettsia samples, three samples were from dogs (2 owned, 1 stray) and four
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were from cats (2 owned, 2 stray). All ‘suspect’ positive Bartonella and Rickettsia fleas were
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from cats (Supplementary Table 3).
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Bartonella ssrA gene DNA sequencing of the real-time PCR products (n = 2) revealed 100%
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identity with Bartonella clarridgeiae (JN982716). Rickettsia real-time PCR positive DNA
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samples (n = 7) were successfully characterised using conventional PCR for Rickettsia spp.
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(gltA, ompA) and the obtained new DNA sequences were 100% identical with gltA and ompA
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genes of Rickettsia felis (CP000053) (Supplementary Table 3).
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4. Discussion
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The genetic diversity between the cat flea in Australia and New Zealand are very similar at
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the mitochondrial loci. The New Zealand cat fleas belong to the Clade 1 that is the
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predominant clade for cat fleas in Australia (Lawrence et al., 2014; Lawrence et al., 2015a).
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The cat fleas in Clade 1 are also commonly found in Europe (Lawrence et al., 2015b).
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An initial population bottleneck restricting gene flow could have been the cause of the low
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genetic diversity of New Zealand cat fleas and Australia. The cox1 gene was selected for this
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study as it is applied widely across insects for molecular barcoding studies (Hebert et al.,
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2003). For fleas, it was determined that both cox1 and cox2 could be used to speciate fleas of
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the genus Ctenocephalides, however the cox1 marker demonstrated a higher level of
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nucleotide diversity (Lawrence et al., 2014).
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Cat fleas likely migrated to both Australia and New Zealand with domestic dogs (Canis lupus
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familiaris) or cats (Felis catus) (Hopkins and Rothschild, 1953; Russell et al., 2013).
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Companion cats and dogs have similar historical introduction in these countries and were
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introduced with European settlement (Brockie, 2007; Keane, 2008; King, 1985; Swarbick,
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2008; Van Dyck and Strahan, 2008). In Australia, cats were introduced between the 1600s
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and 1804, whereas companion dogs were likely introduced in 1788 (Van Dyck and Strahan,
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2008). Similarly, in New Zealand, cats were initially introduced in 1773 with European
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exploration, as they were used to control rats on ships and companion cats had become well
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established from 1820 (Brockie, 2007; King, 1985). Companion dogs were likely introduced
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around 1820, which coincided with the population decline of the Polynesian dog, Kurī (Canis
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lupus familiaris), which became extinct by the 1870s (Keane, 2008; Swarbick, 2008). Both
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the historical introduction of host species and haplotype diversity is similar between
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Australian and New Zealand cat fleas. Because of this, we can assume that cat fleas were
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most likely been introduced with their host species (domestic cats and dogs) during European
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settlement, which could explain the genetic similarities despite being separated by the Tasman
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Sea.
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The closely related species, dog fleas (Ctenocephalides canis) was not identified in the
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current survey. Historically New Zealand had a high prevalence (74.5%, n = 327) of C. canis
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on dogs (Guzman, 1984; Pilgrim, 1980). Recent studies, however, documented general
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absence of C. canis in the contemporary settings in Australia and New Zealand (Kelly et al.,
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2005b; Lawrence et al., 2014; Šlapeta et al., 2011).
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Our study supports the possibility for dogs and cats as the reservoir hosts for B. clarridgeiae
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and R. felis, because their DNA was found in fleas collected on both dogs and cats. Cat fleas
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(C. felis) are known vectors for B. henselae, B. clarridgeiae and B. koehlerae (Barrs et al.,
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2010; Breitschwerdt, 2008; Hii et al., 2011; Koehler et al., 1994; Regnery et al., 1992).
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Moreover, C. felis is the dominant vector for R. felis (Angelakis et al., 2016; Bitam et al.,
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2010; Reif and Macaluso, 2009). Presence of R. felis in fleas from the owned dogs and one
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stray dog is in agreement with the proposed role of dog as the reservoir hosts for R. felis (Hii
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et al., 2011).
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In humans, B. henselae is commonly associated with cat scratch disease, while the role of B.
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clarridgeiae in human disease is yet to be fully established (Chomel et al., 2006; Kordick et
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al., 1997). In fleas sampled from the New Zealand’s North Island, B. clarridgeiae and B.
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henselae were detected in 7% and 11% of cat fleas (n = 114), respectively (Kelly et al.,
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2005b). Our findings for B. clarridgeiae confirmed previous reports, but the lack of B.
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henselae was surprising given it had been previously detected in the North Island (Kelly et al.,
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2005b). The lack of B. henselae detected could have been due to the low sample size of this
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study. Our study confirmed the prevalence of R. felis in New Zealand cat fleas, previously
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established to be found in 19% of cat fleas collected about a decade ago (Kelly et al., 2005b).
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New Zealand is yet to diagnose a human case of cat-flea typhus (caused by R. felis). Due to
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the non-specific febrile symptoms, it is not uncommon for cat-flea typhus to be misdiagnosed
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as murine typhus, caused by Rickettsia typhi (Kelly et al., 2004; Kelly et al., 2005a; Lim et al.,
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2012). In 2012, there were four reported cases of murine typhus, which may have in fact been
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misdiagnosed cat-flea typhus (Lim et al., 2012).
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In conclusion, we have determined genetic diversity of a population of C. felis in New
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Zealand across both the North and South Islands. The combination of establishing mtDNA
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cox1 haplotype diversity with the multiplexed real-time PCR and sequencing for Bartonella
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and Rickettsia is suitable to understand the capability of C. felis to vector zoonotic pathogens.
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We demonstrated the close phylogenetic identity of C. felis in Australia and New Zealand,
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suggesting shared introduction of the C. felis cox1 clade.
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5. Conflicts of interest
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Maureen Forsyth is an employee of Merial New Zealand. Merial New Zealand donated most
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of the samples from North Island, New Zealand. Merial New Zealand had no role in the
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interpretation and decision to publish. The authors declare no further competing interests.
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6. Acknowledgements
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This study was supported by Catherine Heather Paton Bequest (The University of Sydney) as
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well as in part by the School of Life and Environmental Sciences, Faculty of Veterinary
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Science, University of Sydney through the internal support for the Honours project of the first
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author. We thank Merial New Zealand, Stephanie Williams (VetSouth Gore), Alistair
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Newbould (Humanimals, Dunedin), Mark Wiseman (The Vet Centre Marlborough,
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Blenheim), Andrea Wilson (Raumati Veterinary Centre, Paraparaumu), and SPCA New
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Zealand clinics as well all veterinarians and veterinary nurses for their help and technical
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assistance with the donation, collection and shipment of the flea specimens. We thank
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Graeme Brown for his insight.
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Legends to Figures
Figure 1. Flea collection points from the North & South Islands of New Zealand. All but three regions were surveyed. Of the 231 fleas collected, 228 were Ctenocephalides felis, while three Pulex irritans were also found. Location of Rickettsia felis and Bartonella clarridgeiae positive samples were mostly located on the North Island and corresponding ID names are displayed.
Figure 2. Map of Australia and New Zealand depicting geographical distribution of C. felis haplotype clades. Evolutionary relationships of 96 cox1 haplotypes of Ctenocephalides felis, comparing Australian and New Zealand haplotypes. The evolutionary history was inferred using the Minimum Evolution method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter. There was a total of 601 nucleotide positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar et al., 2016).
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Table 1. Summary of cat fleas, Ctenocephalides felis, collected from New Zealand between January 2014 and March 2016 characterised morphologically.
Island
North Island
South Island
Location
Host(s)
No. Specimens (♀/♂)
ID Name
Whangarei
2 cats, 2 dogs
30 (20♀/10♂)
AL837, AL839, AL842, AL858
Helensville
1 cat, 1 dog
12 (9♀/3♂)
AL849, AL853
Mount Wellington
1 cat
3 (3♀/0♂)
AL848
Papakura
2 dogs
8 (3♀/5♂)
AL843, AL845
Hamilton
2 cats
22 (20♀/2♂)
AL836, AL851
Ngongotaha
1 cat
7 (7♀/0♂)
AL846
Gisborne
1 cat, 1 dog
7 (6♀/1♂)
AL838, AL854
Hawera
1 cat, 1 dog
13 (10♀/3♂)
AL840, AL852
Hastings
1 cat
8 (8♀/0♂)
AL847
Paraparaumu
4 cats, 6 dogs
50 (42♀/7♂)
AL244, AL245, AL246, AL247, AL248, AL249, AL250, AL251, AL252, AL253
Porirua
1 dog
4 (3♀/1♂)
AL844
Nelson
1 dog
24 (16♀/8♂)
AL850
Blenheim
4 cats, 1 dog
26 (20♀/6♂)
AL1045, AL1046, AL1048, AL1052, AL1053
Oxford
1 cat
1 (0♀/1♂)
AL841
Dunedin
3 cats, 1 dog
10 (9♀/1♂)
AL1042, AL1047, AL1050, AL1051
Gore
3 cats, 2 dog
4 (3♀/1♂)
AL1041, AL1049, AL1054
Note: Of the 228 C. felis collected, 177 were female and 48 were male (sex ratio 3.653).
Northland
New Zealand
Whangarei
AL839-1 AL842-1
200 km
Helensville
AL848-1
Auckland
AL851-1
Mt Wellington Papakura Waikato
Hamilton Ngongotaha Bay of Plenty
North Island
Gisborne
AL854-1 AL838-1
Gisborne Hawke's Bay Taranaki
AL852-1
Hawera
ManawatuWanganui
Hastings
Raumati Beach
Nelson
Wellington
Nelson
Porirua
Tasman
Blenheim
AL844-1
Marlborough
South Island
AL1053-1 West Coast
Oxford Canterbury
Otago Southland
Gore
Dunedin
fleas on dogs and cats in veterinary clinics Ctenocephalides felis Pulex irritans
real time PCR (gltA) and nested PCR (ompA, gltA) Rickettsia felis real time PCR (ssrA) Bartonella clarridgeiae
Ctenocephalides felis number of fleas (cox1)
n=20
Australia
New Zealand
Australia
h5
0
1
h6
0
1
h4
0
1
h7
0
1
h8
0
1
NZ1
18
0
NZ5
2
0
NZ6
1
0
h1/NZ2
14
28
NZ4
1
0
7
0
0
19
0
1
n=1
n=1 n=1
a
n=29
an
Se
n=1
Ta
sm
n=24
n=19
66
New Zealand 63
cox1 haplotypes (601 nt)
0.002
Clade 1
99
NZ3
Clade 2
h2 h3
