Identification of Borrelia burgdorferi ospC genotypes in canine tissue following tick infestation: implications for Lyme disease vaccine and diagnostic assay design

Abstract

In endemic regions, Lyme disease is a potential health threat to dogs. Canine Lyme disease manifests with arthritis-induced lameness, anorexia, fever, lethargy, lymphadenopathy and, in some cases, fatal glomerulonephritis. A recent study revealed that the regional mean for the percentage of seropositive dogs in the north-east of the USA is 11.6%. The outer surface protein C (OspC) of Lyme disease spirochetes is an important virulence factor required for the establishment of infection in mammals. It is a leading candidate in human and canine Lyme disease vaccine development efforts. Over 30 distinct ospC phyletic types have been defined. It has been hypothesized that ospC genotype may influence mammalian host range. In this study, Ixodes scapularis ticks collected from the field in Rhode Island were assessed for infection with B. burgdorferi. Ticks were fed on purpose bred beagles to repletion and infection of the dogs was assessed through serology and PCR. Tissue biopsies (n=2) were collected from each dog 49 days post-tick infestation (dpi) and the ospC genotype of the infecting strains determined by direct PCR of DNA extracted from tissue or by PCR after cultivation of spirochetes from biopsy samples. The dominant ospC types associated with B. burgdorferi canine infections differed from those associated with human infection, indicating a relationship between ospC sequence and preferred host range. Knowledge of the most common ospC genotypes associated specifically with infection of dogs will facilitate the rational design of OspC-based canine Lyme disease vaccines and diagnostic assays.

Keywords: Borrelia burgdorferi; Canine; Lyme disease; OspC; Ticks; Vaccine.

Fig 1

Phylogenetic analysis of OspC sequences amplified directly from skin biopsy samples collected from dogs after infestation with ticks. ospC was PCR amplified directly from skin biopsies and the amplicons were cloned into E. coli. Recombinant plasmids were isolated from several independent colonies for each cloning reaction and the sequence of the inserts was determined. The nomenclature used for the determined sequences is as described in the text. Note that individual sequences cloned from a single PCR reaction are differentiated by a hyphenated number. The translated sequences were aligned and a dendrogram constructed. ospC type identity was determined by comparative analysis with previously determined ospC sequences of known phyletic type. ospC type identity is indicated to the right by bracketing. The scale bar indicates substitutions per sequence position.

Fig 2

Phylogenetic analysis of OspC sequences derived from clonal populations of Borrelia burgdorferi cultivated from biopsies from dogs. Biopsy samples were placed in media and spirochetes cultivated. The cultures were sub-surface plated, individual colonies were picked, ospC was amplified and the ospC sequences determined. The scale bar indicates substitutions per sequence position.