Nanopore Sequencing Using the Full-Length 16S rRNA Gene for Detection of Blood-Borne Bacteria in Dogs Reveals a Novel Species of Hemotropic Mycoplasma

Huggins, Lucas G.; Colella, Vito; Atapattu, Ushani; Koehler, Anson V.; Traub, Rebecca J.

doi:10.1128/spectrum.03088-22

Cited by 16 publications

(30 citation statements)

References 93 publications

(139 reference statements)

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“…The final component of the C. orientis microbiome sequenced here, B. clarridgeiae, has been detected frequently in Ctenocephalides spp. fleas and the blood or other tissues of cats and dogs worldwide [25, 130–132], with a number of reports across South-East Asia [133–136], although to the best of our knowledge no confirmed zoonotic infections from this part of the world have been documented. However, only two other genomes are available (one complete and circularized [14]), which are derived from feline blood isolates from France and the USA.…”

Section: Discussionmentioning

confidence: 99%

Metagenomics of culture isolates and insect tissue illuminate the evolution of Wolbachia, Rickettsia and Bartonella symbionts in Ctenocephalides spp. fleas

et al. 2023

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While fleas are often perceived simply as a biting nuisance and a cause of allergic dermatitis, they represent important disease vectors worldwide, especially for bacterial zoonoses such as plague (transmitted by rodent fleas) and some of the rickettsioses and bartonelloses. The cosmopolitan cat (Ctenocephalides felis) and dog (Ctenocephalides canis) fleas, as well as Ctenocephalides orientis (restricted to tropical and subtropical Asia), breed in human dwellings and are vectors of cat-scratch fever (caused by Bartonella spp.) and Rickettsia spp., including Rickettsia felis (agent of flea-borne spotted fever) and Rickettsia asembonensis , a suspected pathogen. These Rickettsia spp. are members of a phylogenetic clade known as the ‘transitional group’, which includes both human pathogens and arthropod-specific endosymbionts. The relatively depauperate flea microbiome can also contain other endosymbionts, including a diverse range of Wolbachia strains. Here, we present circularized genome assemblies for two C. orientis-derived pathogens ( Bartonella clarridgeiae and R. asembonensis ) from Malaysia, a novel Wolbachia strain (wCori), and the C. orientis mitochondrion; all were obtained by direct metagenomic sequencing of flea tissues. Moreover, we isolated two Wolbachia strains from Malaysian C. felis into tick cell culture and recovered circularized genome assemblies for both, one of which (wCfeF) is newly sequenced. We demonstrate that the three Wolbachia strains are representatives of different major clades (‘supergroups’), two of which appear to be flea-specific. These Wolbachia genomes exhibit unique combinations of features associated with reproductive parasitism or mutualism, including prophage WO, cytoplasmic incompatibility factors and the biotin operon of obligate intracellular microbes. The first circularized assembly for R. asembonensis includes a plasmid with a markedly different structure and gene content compared to the published plasmid; moreover, this novel plasmid was also detected in cat flea metagenomes from the USA. Analysis of loci under positive selection in the transitional group revealed genes involved in host–pathogen interactions that may facilitate host switching. Finally, the first B. clarridgeiae genome from Asia exhibited large-scale genome stability compared to isolates from other continents, except for SNPs in regions predicted to mediate interactions with the vertebrate host. These findings highlight the paucity of data on the genomic diversity of Ctenocephalides-associated bacteria and raise questions regarding how interactions between members of the flea microbiome might influence vector competence.

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Section: Discussionmentioning

confidence: 99%

Metagenomics of culture isolates and insect tissue illuminate the evolution of Wolbachia, Rickettsia and Bartonella symbionts in Ctenocephalides spp. fleas

et al. 2023

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“…The final component of the C. orientis microbiome sequenced here, B. clarridgeiae , has been detected frequently in Ctenocephalides spp. fleas and the blood or other tissues of cats and dogs worldwide [17, 123-125], with a number of reports across South-East Asia [126-129], although to the best of our knowledge no confirmed zoonotic infections from this part of the world have been documented. However, only two other genomes are available (one complete and circularised [130]), which are derived from feline blood isolates from France and the USA.…”

Section: Discussionmentioning

confidence: 99%

Metagenomics of culture isolates and insect tissue illuminate the evolution ofWolbachia,RickettsiaandBartonellasymbionts inCtenocephalidesspp. fleas

Beliavskaia

Tan

Sinha

et al. 2023

Preprint

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While fleas are often perceived simply as a biting nuisance and cause of allergic dermatitis, they represent important disease vectors worldwide, especially for bacterial zoonoses such as plague (transmitted by rodent fleas) and some of the rickettsioses and bartonelloses. The cosmopolitan cat (Ctenocephalidesfelis) and dog (C. canis) fleas, as well asC. orientis(restricted to tropical and subtropical Asia), breed in human dwellings and are vectors of cat-scratch fever (caused byBartonellaspp.) andRickettsiaspp. of the so-called "transitional group". The latter includesR. felis(agent of flea-borne spotted fever) andR. asembonensis, an emerging pathogen. The relatively depauperate flea microbiome can also contain arthropod-specific endosymbionts, including a diverse range ofWolbachiastrains. Here, we present circularized genome assemblies for twoC. orientis-associated pathogens (Bartonella clarridgeiaeandR. asembonensis) from Malaysia, a novelWolbachiastrain (wCori), and theC. orientismitochondrion; all obtained by direct metagenomic sequencing of flea tissues. Moreover, we isolated twoWolbachiastrains from MalaysianC. felisinto tick cell culture and recovered circularized genome assemblies for both, one of which (wCfeF) is newly sequenced. We demonstrate that the threeWolbachiastrains are representatives of different major clades ("supergroups"), two of which appear to be flea-specific. TheseWolbachiagenomes exhibit unique combinations of features associated with reproductive parasitism or mutualism, including prophage WO, cytoplasmic incompatibility factors, and the biotin operon of obligate intracellular microbes. The first circularized assembly forR. asembonensisincludes a plasmid with a markedly different structure and gene content compared to the published plasmid; moreover, this novel plasmid was also detected in cat flea metagenomes from the US. Analysis of loci under positive selection in the transitional group revealed genes involved in host-pathogen interactions that may facilitate host switching. Finally, the firstB. clarridgeiaegenome from Asia exhibited largescale genome stability compared to isolates from other continents, except for SNPs in regions predicted to mediate interactions with the vertebrate host. These findings highlight the paucity of data on the genomic diversity ofCtenocephalides-associated bacteria and raise questions regarding how interactions between members of the flea microbiome might influence vector competence.

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“…Through selection of the highest qPCR C q value obtained for a sample positive to B. vogeli or H. canis that was also positive by nanopore sequencing, the lowest number of 18S rRNA gene copies that was detected for either of these pathogens by the novel method was calculated, as has previously been done by the authors in (Huggins et al, 2022).…”

Section: Comparison Of Molecular Assaysmentioning

confidence: 99%

“…Nevertheless, to date, most studies have utilized short‐read NGS platforms, limiting the size of amplicons that can be sequenced and, therefore, the taxonomic resolution achievable (Ghafar et al, 2021; Huggins, Colella, et al, 2021; Mohamed et al, 2021; Squarre et al, 2020). Long‐read NGS platforms, provided by Oxford Nanopore Technologies (ONT) devices, resolve these challenges as the length of amplicon that can be sequenced is theoretically uncapped (Ciuffreda et al, 2021; Huggins et al, 2022; Leggett & Clark, 2017; Wang et al, 2021). ONT′ flagship device, the MinION™ Mk1B, is a small, portable and cheap sequencer that has already leveraged its long‐read capabilities to detect a diverse spectrum of pathogens from viruses, bacteria and helminths within a wide range of sample types, including blood, skin, urine, faeces, sputum and brain tissue (Huggins et al, 2022; Kai et al, 2019; Lanszki et al, 2022; Moon et al, 2018; Peserico et al, 2020; Rozas et al, 2022; Sears et al, 2022; Yandle et al, 2023; Zhang et al, 2022).…”

Section: Introductionmentioning

confidence: 99%

Metabarcoding using nanopore long‐read sequencing for the unbiased characterization of apicomplexan haemoparasites

Huggins,

Colella,

Young

et al. 2023

Molecular Ecology Resources

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Apicomplexan haemoparasites generate significant morbidity and mortality in humans and other animals, particularly in many low‐to‐middle income countries. Malaria caused by Plasmodium remains responsible for some of the highest numbers of annual deaths of any human pathogen, whilst piroplasmids, such as Babesia and Theileria can have immense negative economic effects through livestock loss. Diagnosing haemoparasites via traditional methods like microscopy is challenging due to low‐level and transient parasitaemia. PCR‐based diagnostics overcome these limitations by being both highly sensitive and specific, but they may be unable to accurately detect coinfections or identify novel species. In contrast, next‐generation sequencing (NGS)‐based methods can characterize all pathogens from a group of interest concurrently, although, the short‐read platforms previously used have been limited in the taxonomic resolution achievable. Here, we used Oxford Nanopore Technologies' (ONT) long‐read MinION™ sequencer to conduct apicomplexan haemoparasite metabarcoding via sequencing the near full‐length 18S ribosomal RNA gene, demonstrating its ability to detect Babesia, Hepatozoon, Neospora, Plasmodium, Theileria and Toxoplasma species. This method was tested on blood‐extracted DNA from 100 dogs and the results benchmarked against qPCR and Illumina‐based metabarcoding. For two common haemoparasites, nanopore sequencing performed as well as qPCR (kappa agreement statistics > 0.98), whilst also detecting one pathogen, Hepatozoon felis, missed by the other techniques. The long‐reads obtained by nanopore sequencing provide an improved species‐level taxonomic resolution whilst the method's broad applicability mean it can be used to explore apicomplexan communities from diverse mammalian hosts, on a portable sequencer that easily permits adaptation to field use.

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Nanopore Sequencing Using the Full-Length 16S rRNA Gene for Detection of Blood-Borne Bacteria in Dogs Reveals a Novel Species of Hemotropic Mycoplasma

Cited by 16 publications

References 93 publications

Metagenomics of culture isolates and insect tissue illuminate the evolution of Wolbachia, Rickettsia and Bartonella symbionts in Ctenocephalides spp. fleas

Metagenomics of culture isolates and insect tissue illuminate the evolution of Wolbachia, Rickettsia and Bartonella symbionts in Ctenocephalides spp. fleas

Metagenomics of culture isolates and insect tissue illuminate the evolution ofWolbachia,RickettsiaandBartonellasymbionts inCtenocephalidesspp. fleas

Metabarcoding using nanopore long‐read sequencing for the unbiased characterization of apicomplexan haemoparasites

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