Development and Validation of an Improved PCR Method Using the 23S-5S Intergenic Spacer for Detection of Rickettsiae in Dermacentor variabilis Ticks and Tissue Samples from Humans and Laboratory Animals.

Madhavi L Kakumanu,William L Nicholson,Charles S Apperson,Steven R Meshnick,Loganathan Ponnusamy,Haley T Sutton,B W Fenwick

doi:10.1128/jcm.02605-15

Abstract

A novel nested PCR assay was developed to detect Rickettsia spp. in ticks and tissue samples from humans and laboratory animals. Primers were designed for the nested run to amplify a variable region of the 23S-5S intergenic spacer (IGS) of Rickettsia spp. The newly designed primers were evaluated using genomic DNA from 11 Rickettsia species belonging to the spotted fever, typhus, and ancestral groups and, in parallel, compared to other Rickettsia-specific PCR targets (ompA, gltA, and the 17-kDa protein gene). The new 23S-5S IGS nested PCR assay amplified all 11 Rickettsia spp., but the assays employing other PCR targets did not. The novel nested assay was sensitive enough to detect one copy of a cloned 23S-5S IGS fragment from “Candidatus Rickettsia amblyommii.” Subsequently, the detection efficiency of the 23S-5S IGS nested assay was compared to those of the other three assays using genomic DNA extracted from 40 adult Dermacentor variabilis ticks. The nested 23S-5S IGS assay detected Rickettsia DNA in 45% of the ticks, while the amplification rates of the other three assays ranged between 5 and 20%. The novel PCR assay was validated using clinical samples from humans and laboratory animals that were known to be infected with pathogenic species of Rickettsia. The nested 23S-5S IGS PCR assay was coupled with reverse line blot hybridization with species-specific probes for high-throughput detection and simultaneous identification of the species of Rickettsia in the ticks. “Candidatus Rickettsia amblyommii,” R. montanensis, R. felis, and R. bellii were frequently identified species, along with some potentially novel Rickettsia strains that were closely related to R. bellii and R. conorii.

Highlights

Rickettsia species are Gram-negative, obligate intracellular bacteria vectored by a diverse array of arthropods [1]
The 23S-5S intergenic spacer (IGS) is a noncoding region of DNA that is present in Rickettsia species [25, 26]
We coupled the 23S-5S IGS PCR assay with reverse line blot (RLB) hybridization for high-throughput screening and simultaneous identification of Rickettsia in ticks

Summary

Introduction

Rickettsia species are Gram-negative, obligate intracellular bacteria vectored by a diverse array of arthropods [1]. To understand the role of novel pathogens in driving the recent increase in SFG rickettsioses, investigations of the diversity of Rickettsia species in D. variabilis ticks should be carried out [17]. While PCR assays targeting different regions of the Rickettsia genome have been used successfully to detect rickettsiae in environmental samples, inconsistencies in the amplification rates of different gene targets in the same samples is not uncommon [8, 23]. This problem could potentially result from a lack of specificity of the primers for potentially novel species or to low copy numbers of the target genes. We coupled the 23S-5S IGS PCR assay with reverse line blot (RLB) hybridization for high-throughput screening and simultaneous identification of Rickettsia in ticks

Objectives

Methods

Results

Conclusion