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Urban Norway rats ( Rattus norvegicus ) are a reservoir for  Bartonella  spp. - a genus of zoonotic bacteria transmitted by hematophagous vectors, particularly fleas. Rats and fleas may be infected with more than one  Bartonella  species; however, mixed infections may be difficult to detect using culture and/or mono-locus PCR. We set out to characterize  Bartonella  spp. using  gltA  PCR and Sanger sequencing on blood ( n  = 480) and  Nosopsyllus fasciatus  flea pools ( n  = 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 ( ssrA ) and the results of  gltA  and  ssrA  were discordant, a metagenomic approach was used to better characterize the  Bartonella  spp. present in these samples and our objective transitioned to comparing data obtained via metagenomics to those from PCR/sequencing. Among the  Bartonella  spp.-positive rats ( n  = 95), 52 (55.3%), and 41 (43.6%) had Sanger sequences consistent with  Bartonella tribocorum  and  Bartonella vinsonii , respectively. One rat had a mixed infection. All sequences from  Bartonella  spp.-positive flea pools ( n  = 85), were consistent with  B. tribocorum , and re-analysis of 34 bloods of varying  Bartonella  spp. infection status (based  gltA  PCR and sequencing) using  ssrA  PCR showed that the assay was capable of identifying  B. tribocorum  but not  B. vinsonii . Metagenomics analysis of a subset of PCR-positive blood samples ( n  = 70) and flea pools ( n  = 24) revealed that both  B. tribocorum  and  B. vinsonii  were circulating widely in the study population with 31/70 (44.3%) rats and 5/24 (2.1%) flea pools infected with both species.  B. vinsonii , 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  B. vinsonii  strain as  B. vinsonii  subsp.  berkhoffii  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,  B. vinsonii  is associated with greater pathogenicity in people.

frontiers in veterinary science

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