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

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.

Small Regulatory RNAs of Rickettsia conorii

“…Although Rhipicephalus sanguineus is the natural vector of R. conorii, A. americanum ticks can also acquire and transmit R. conorii 16. All four novel Rc _sRs, identified by RNA-Seq and confirmed by Northern blots above, were expressed during the infection of both HMECs and AAE2 cells, indicating their presence and biogenesis in R. conorii .…”

Tick Ecdysteroid Hormone, Global Microbiota/Rickettsia Signaling in the Ovary versus Carcass during Vitellogenesis in Part-Fed (Virgin) American Dog Ticks, Dermacentor variabilis

“…To measure relative abundance of Rickettsia spp., qPCR was performed using RCK/23-5N1F and RCK/23-5N1R primers for the 23S-5S ITS gene [40] and the GAPDH reference gene [39]. A total of 2 µL of the isolated DNA (100 ng) from each tick sample was added to the qPCR mix as described, followed by thermal cycling and detection.…”

Active surveillance of pathogens from ticks collected in New York State suburban parks and schoolyards

“…assay were selected for amplification and sequencing of the 23S-5S intergenic spacer region. Nested PCR was performed as described by Kakumanu et al (2016).…”