AUTHOR=Himsworth Chelsea G. , Byers Kaylee A. , Fernando Champika , Speerin Laura , Lee Michael J. , Hill Janet E. 

TITLE=When the Sum of the Parts Tells You More Than the Whole: The Advantage of Using Metagenomics to Characterize Bartonella spp. Infections in Norway Rats (Rattus norvegicus) and Their Fleas

JOURNAL=Frontiers in Veterinary Science

VOLUME=7

YEAR=2020

URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2020.584724

DOI=10.3389/fvets.2020.584724

ISSN=2297-1769

ABSTRACT=<p>Urban Norway rats (<italic>Rattus norvegicus</italic>) are a reservoir for <italic>Bartonella</italic> spp. - a genus of zoonotic bacteria transmitted by hematophagous vectors, particularly fleas. Rats and fleas may be infected with more than one <italic>Bartonella</italic> species; however, mixed infections may be difficult to detect using culture and/or mono-locus PCR. We set out to characterize <italic>Bartonella</italic> spp. using <italic>gltA</italic> PCR and Sanger sequencing on blood (<italic>n</italic> = 480) and <italic>Nosopsyllus fasciatus</italic> flea pools (<italic>n</italic> = 200) obtained from a population of urban Norways rats from Vancouver, Canada. However, when contamination of a subset of flea pools necessitated the use of a second target (<italic>ssrA</italic>) and the results of <italic>gltA</italic> and <italic>ssrA</italic> were discordant, a metagenomic approach was used to better characterize the <italic>Bartonella</italic> spp. present in these samples and our objective transitioned to comparing data obtained via metagenomics to those from PCR/sequencing. Among the <italic>Bartonella</italic> spp.-positive rats (<italic>n</italic> = 95), 52 (55.3%), and 41 (43.6%) had Sanger sequences consistent with <italic>Bartonella tribocorum</italic> and <italic>Bartonella vinsonii</italic>, respectively. One rat had a mixed infection. All sequences from <italic>Bartonella</italic> spp.-positive flea pools (<italic>n</italic> = 85), were consistent with <italic>B. tribocorum</italic>, and re-analysis of 34 bloods of varying <italic>Bartonella</italic> spp. infection status (based <italic>gltA</italic> PCR and sequencing) using <italic>ssrA</italic> PCR showed that the assay was capable of identifying <italic>B. tribocorum</italic> but not <italic>B. vinsonii</italic>. Metagenomics analysis of a subset of PCR-positive blood samples (<italic>n</italic> = 70) and flea pools (<italic>n</italic> = 24) revealed that both <italic>B. tribocorum</italic> and <italic>B. vinsonii</italic> were circulating widely in the study population with 31/70 (44.3%) rats and 5/24 (2.1%) flea pools infected with both species. <italic>B. vinsonii</italic>, however, made up a smaller relative proportion of the reads for samples with mixed infections, which may be why it was generally not detected by genus-specific PCR and Sanger sequencing. Further analysis of 16S−23S ITS sequences amplified from a subset of samples identified the <italic>B. vinsonii</italic> strain as <italic>B. vinsonii</italic> subsp. <italic>berkhoffii</italic> type II. This demonstrates the value of a metagenomic approach for better characterizing the ecology and health risks associated with this bacterium, particularly given that the less dominant species, <italic>B. vinsonii</italic> is associated with greater pathogenicity in people.</p>