Masters Thesis

Molecular investigations of two spotted fever group rickettsia phylotypes in Ixodes pacificus, Dermacentor occidentalis and domestic dogs

Spotted fever group (SFG) rickettsiae have been shown to cause many human and animal diseases. SRG rickettsial phylotypes G021 and G022 were recently detected in Ixodes pacificus ticks from Napa and California using quantitative real time polymerase chain reaction (qPCR). Prevalence, genotyping, and geographic distribution of SFG Rickettsia species phylotypes in Ixodes pacificus, Dermacentor occidentalis, and mammalian hosts in Califonia have yet to be investigated. A qPCR outer membrane protein A (ompA) gene assay detected and quantified SFG Rickettsia species phylotypes G022 and G021 in I. pacificus (n=265). Of 265 I. pacificus tested, 10 (3.8%) ticks were positive for phylotype G022, while 265 ticks (100%) had phylotype G021. The prevalence of phylotype G022 did not differ between counties (χ2 = 1.42, Pχ2> 0.05) or vegetation habitat types (χ2 = 2.85, Pχ2>0.05). However, the burden of SFG Rickettsia speecies phylotype G021 differed between collection sites (P<0.05) and vegetative type (P<0.05). Habitat vegetation information of the tick collection sites suggested that the density of Rickettsia species phylotype G021 is linked to a predominantly wooded habitat in which its host may reside (P<0.05). Although genomes of I. pacificus and its associated microflora have not been completely assessed, the elevated density and ubiquitous prevalence of SFG Rickettsia species phylotype G021 strongly suggests that it is a bacterial endosymbiont of I. pacificus. The ompA qPCR assay detected Rickettsia species phylotype G021 in coagulated dog blood (n=3/199), I. pacificus (n=18/265) and D. occidentalis (n=3/8) that were collected off shelter dogs. The ompA gene sequencing and alignment between coagulated dog blood, as well as I. pacificus and D. occidentalis ticks collected off dogs revealed 100% nucleotide sequence identity among all samples. OmpA sequencing and alignment between Rickettsia species phylotype G021, all coagulated dog blood, I.pacificus and D.occidentalis samples revealed a 99% sequence identitiy with 100% amino acid sequence identity. Multi-spacer typing (MST) of the intergenic regions revealed four SFG Rickettsia species genotypes (M1, M2, M3, and M4) for mppA-purC locus, two genotypes (R1 and R2) for RpmE-tRNAfMet locus, and two genotypes (D1 and D2) for DksA-XerC locus. MST concatenation with ompA revealed 7 genotypes (T1, T2, T3, T4, T5, T6, and T7), with genotype T7 sharing 100% sequence identity between coagulated dog blood (n=3/199) and D.occidentlis ticks (n=3/8). This suggests that Rickettsia species from D. occidentalis ticks maybe responsible for transmitting a unique Rickettsia species genotype (not found in I.pacificus) to dogs and should be further investigated. MST sequencing of the three intergenic loci concatenated with ompA revealed multiple genotypes, suggesting subspecies diversity amongst Rickettsia species phylotype G021. Future genome sequencing, isolation in cell culture, and mammalian antibody titers are needed to further classify Rickettsia species phylotypes G021 and G022.