Genomic Characterisation of Novel Veterinary Pathogens: Anaplasma & Bartonella species

Brierley, Sean

Abstract

Background: Bartonella sp. and Anaplasma phagocytophilum (Ap) are vector-borne
bacterial pathogens with significant veterinary and public health implications. While
Bartonella species persist in the bloodstream of various mammals causing long term
bacteraemia, Ap is an intracellular pathogen causing granulocytic anaplasmosis.
Despite their importance, genomic data on novel Bartonella species and UK Ap
strains remains limited. Additionally, the low abundance and intracellular nature of
Ap complicate direct sequencing from host tissues. Expanding genomic resources
and refining enrichment methods are essential for improving pathogen
characterisation and understanding host-pathogen interactions.
Objectives: Characterise a novel species of Bartonella, generate the first complete
genome representations of Anaplasma phagocytophilum (Ap) isolated in the UK and
develop optimised enrichment methodologies for high-resolution sequencing of Ap
directly from infected host tissue.
Methods: Three Bartonella strains isolated from field voles (Microtus agrestis) and
seven Ap strains isolated from domestic ruminants were sequenced using Illumina
short-read and Oxford Nanopore long-read systems. Genomic analyses included
phylogenetic reconstruction based on concatenated core gene alignments,
pangenomic profiling, and average nucleotide identity calculations. Enrichment
strategies encompassing differential lysis (Molzym), CpG methylation depletion
(NEB), biotinylated RNA bait capture (Agilent SureSelect), and adaptive sampling
(ONT) were systematically evaluated on roe deer spleen samples infected with Ap.
Alignment files were investigated to assess genome coverage and identify capture
biases. An optimised approach was applied to the spleen of an Ap-infected common
shrew (Sorex araneus) with the aim of characterising the currently uncultured,
genetically divergent small mammal-associated (ecotype III) strain of the species.
Results: Whole genome analyses identified the three Bartonella strains as a novel
lineage 3 species, proposed as Bartonella bennettii most notably containing a
chromosomally integrated vbh/TraG type IV secretion system of plasmid origin.
Phylogenetic analysis of UK Ap isolates placed them within the European ecotype I
cluster, while revealing potential subdivisions. The pangenome identified core and
accessory genes, with ANI values suggesting species boundaries within Ap.
Enrichment protocols combining Monarch HMW DNA extraction and NEB
microbiome depletion yielded optimal pathogen representation. Gap analysis
highlighted capture biases and the potential of the technology to capture complete
Ap genomes, especially in the context of long read systems. The small mammal-
associated ecotype III strain was partially captured with non-specific ecotype I baits
identifying the limits of the capture technology. Linkage analysis of groEL genes
supported existing ecotype classifications, whereas whole genome phylogenetics
indicated potential reclassification into four epidemiologically separated species in a
global context.
Conclusions: B. bennettii was characterised through genomic analyses, providing
insights into the diversity and evolution of virulence factors in the Bartonella genus.
Additionally, the first complete Ap genomes from the UK were generated, providing
insights into genomic diversity and phylogenetic relationships. Optimised enrichment
strategies were developed for high-resolution metagenomic sequencing, overcoming
challenges posed by low bacterial loads and complex metagenomic samples. Whole
genome analysis suggests the European ecotypes are representative of global Ap
diversity, with ANI supporting the existence of four epidemiologically separate
species within Ap. Continued genomic characterisation is crucial for understanding
the drivers of host specificity, zoonotic potential, and epidemiological dynamics
within these diverse blood-borne parasites.

• Thesis Type: Thesis
• Online Publication Date: Mar 27, 2025
• Deposit Date: Mar 10, 2025
• Publicly Available Date: Apr 28, 2025
• Award Date: Mar 27, 2025