Differentiation of Rickettsia felis and Rickettsia felis-Like Organisms via Restriction Fragment Length Polymorphism Analysis

Lucas Blanton, Bethany R. Quade, Donald Bouyer

Research output: Contribution to journalArticle

Abstract

Rickettsia typhi and Rickettsia felis are flea-borne pathogens, which cause murine typhus and flea-borne spotted fever, respectively. Recently, two other flea-borne rickettsiae (phylogenetically similar to R. felis) have been discovered - Rickettsia asembonensis and Candidatus Rickettsia senegalensis. Currently, species-specific identification of detected organisms requires sequencing- or probe-based PCR assays. Our aim was to develop an efficient and inexpensive method to differentiate R. felis and R. felis-like organisms through restriction fragment length polymorphism (RFLP) analysis. Outer membrane protein B sequences of the aforementioned flea-borne rickettsiae were analyzed using DNASTAR Lasergene Core software to focus on the region amplified by the primers 120.2788 and 120.3599. Restriction enzyme digestion sites were identified, and in silico digestions of each species were compared through simulated agarose gels. The enzyme NlaIV was determined to be the most effective at creating a unique banding pattern within the area of interest. To confirm the predicted performance of NlaIV digestion, we tested the DNA of known PCR positive Ctenocephalides felis fleas collected from cats and opossums within Galveston, Texas. DNA from these fleas was amplified using the sca5 primer set 120.2788 and 120.3599. The PCR products were then digested with NlaIV, subjected to polyacrylamide gel electrophoresis, and visualized through ethidium bromide staining. The banding patterns were then compared with the computer-generated digestion patterns. All samples demonstrated a banding pattern consistent with the predicted pattern for the known species, as confirmed by previous sequencing. This RFLP assay was developed to be an efficient and cost-effective method to screen samples for R. felis, R. asembonensis, and Candidatus R. senegalensis. We believe this assay can aid in the epidemiological and ecological studies of flea-borne rickettsiae.

Original languageEnglish (US)
Pages (from-to)637-639
Number of pages3
JournalVector-Borne and Zoonotic Diseases
Volume19
Issue number8
DOIs
StatePublished - Aug 1 2019

Fingerprint

Rickettsia felis
Siphonaptera
Rickettsia
Restriction Fragment Length Polymorphisms
Digestion
Ctenocephalides
Polymerase Chain Reaction
Rickettsia typhi
Endemic Flea-Borne Typhus
Opossums
Ethidium
DNA
Enzymes
Computer Simulation
Sepharose
Epidemiologic Studies
Polyacrylamide Gel Electrophoresis
Membrane Proteins
Fever
Software

Keywords

  • Candidatus Rickettsia senegalensis
  • flea-borne rickettsioses
  • murine typhus
  • Rickettsia asembonensis
  • Rickettsia felis

ASJC Scopus subject areas

  • Microbiology
  • Infectious Diseases
  • Virology

Cite this

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title = "Differentiation of Rickettsia felis and Rickettsia felis-Like Organisms via Restriction Fragment Length Polymorphism Analysis",
abstract = "Rickettsia typhi and Rickettsia felis are flea-borne pathogens, which cause murine typhus and flea-borne spotted fever, respectively. Recently, two other flea-borne rickettsiae (phylogenetically similar to R. felis) have been discovered - Rickettsia asembonensis and Candidatus Rickettsia senegalensis. Currently, species-specific identification of detected organisms requires sequencing- or probe-based PCR assays. Our aim was to develop an efficient and inexpensive method to differentiate R. felis and R. felis-like organisms through restriction fragment length polymorphism (RFLP) analysis. Outer membrane protein B sequences of the aforementioned flea-borne rickettsiae were analyzed using DNASTAR Lasergene Core software to focus on the region amplified by the primers 120.2788 and 120.3599. Restriction enzyme digestion sites were identified, and in silico digestions of each species were compared through simulated agarose gels. The enzyme NlaIV was determined to be the most effective at creating a unique banding pattern within the area of interest. To confirm the predicted performance of NlaIV digestion, we tested the DNA of known PCR positive Ctenocephalides felis fleas collected from cats and opossums within Galveston, Texas. DNA from these fleas was amplified using the sca5 primer set 120.2788 and 120.3599. The PCR products were then digested with NlaIV, subjected to polyacrylamide gel electrophoresis, and visualized through ethidium bromide staining. The banding patterns were then compared with the computer-generated digestion patterns. All samples demonstrated a banding pattern consistent with the predicted pattern for the known species, as confirmed by previous sequencing. This RFLP assay was developed to be an efficient and cost-effective method to screen samples for R. felis, R. asembonensis, and Candidatus R. senegalensis. We believe this assay can aid in the epidemiological and ecological studies of flea-borne rickettsiae.",
keywords = "Candidatus Rickettsia senegalensis, flea-borne rickettsioses, murine typhus, Rickettsia asembonensis, Rickettsia felis",
author = "Lucas Blanton and Quade, {Bethany R.} and Donald Bouyer",
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T1 - Differentiation of Rickettsia felis and Rickettsia felis-Like Organisms via Restriction Fragment Length Polymorphism Analysis

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AU - Bouyer, Donald

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N2 - Rickettsia typhi and Rickettsia felis are flea-borne pathogens, which cause murine typhus and flea-borne spotted fever, respectively. Recently, two other flea-borne rickettsiae (phylogenetically similar to R. felis) have been discovered - Rickettsia asembonensis and Candidatus Rickettsia senegalensis. Currently, species-specific identification of detected organisms requires sequencing- or probe-based PCR assays. Our aim was to develop an efficient and inexpensive method to differentiate R. felis and R. felis-like organisms through restriction fragment length polymorphism (RFLP) analysis. Outer membrane protein B sequences of the aforementioned flea-borne rickettsiae were analyzed using DNASTAR Lasergene Core software to focus on the region amplified by the primers 120.2788 and 120.3599. Restriction enzyme digestion sites were identified, and in silico digestions of each species were compared through simulated agarose gels. The enzyme NlaIV was determined to be the most effective at creating a unique banding pattern within the area of interest. To confirm the predicted performance of NlaIV digestion, we tested the DNA of known PCR positive Ctenocephalides felis fleas collected from cats and opossums within Galveston, Texas. DNA from these fleas was amplified using the sca5 primer set 120.2788 and 120.3599. The PCR products were then digested with NlaIV, subjected to polyacrylamide gel electrophoresis, and visualized through ethidium bromide staining. The banding patterns were then compared with the computer-generated digestion patterns. All samples demonstrated a banding pattern consistent with the predicted pattern for the known species, as confirmed by previous sequencing. This RFLP assay was developed to be an efficient and cost-effective method to screen samples for R. felis, R. asembonensis, and Candidatus R. senegalensis. We believe this assay can aid in the epidemiological and ecological studies of flea-borne rickettsiae.

AB - Rickettsia typhi and Rickettsia felis are flea-borne pathogens, which cause murine typhus and flea-borne spotted fever, respectively. Recently, two other flea-borne rickettsiae (phylogenetically similar to R. felis) have been discovered - Rickettsia asembonensis and Candidatus Rickettsia senegalensis. Currently, species-specific identification of detected organisms requires sequencing- or probe-based PCR assays. Our aim was to develop an efficient and inexpensive method to differentiate R. felis and R. felis-like organisms through restriction fragment length polymorphism (RFLP) analysis. Outer membrane protein B sequences of the aforementioned flea-borne rickettsiae were analyzed using DNASTAR Lasergene Core software to focus on the region amplified by the primers 120.2788 and 120.3599. Restriction enzyme digestion sites were identified, and in silico digestions of each species were compared through simulated agarose gels. The enzyme NlaIV was determined to be the most effective at creating a unique banding pattern within the area of interest. To confirm the predicted performance of NlaIV digestion, we tested the DNA of known PCR positive Ctenocephalides felis fleas collected from cats and opossums within Galveston, Texas. DNA from these fleas was amplified using the sca5 primer set 120.2788 and 120.3599. The PCR products were then digested with NlaIV, subjected to polyacrylamide gel electrophoresis, and visualized through ethidium bromide staining. The banding patterns were then compared with the computer-generated digestion patterns. All samples demonstrated a banding pattern consistent with the predicted pattern for the known species, as confirmed by previous sequencing. This RFLP assay was developed to be an efficient and cost-effective method to screen samples for R. felis, R. asembonensis, and Candidatus R. senegalensis. We believe this assay can aid in the epidemiological and ecological studies of flea-borne rickettsiae.

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