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061 (B)

Identify, Authenticate and Characterize Rickettsia prowazekii


D.R. Merrill1, J. Bannon2, B. Benton1, J. Benson1, K. Langenbach1, C.D. Buck3, R. O. Baker1
1BEI

Resources/ATCC, Manassas VA, 2FBI, Quantico, VA, 3MRI, Frederick, MD.

Abstract
Background: The objective was to rapidly and accurately detect Rickettsia by PCR and further authenticate and characterize Rickettsia prowazekii within BEIR and ATCC. Detection of emerging or re-emerging infectious diseases is critical for the reduction of suffering, death and financial burden resulting from widespread Rickettsia infections. For public health reasons and due to its inclusion on the NIAID Priority Pathogen List under Category B, this research focuses on the human pathogen, Rickettsia prowazekii. Methods: Three pan-Rickettsial PCR primers and three R. prowazekii specific primers were developed by ATCC. The first goal was to design primers that would detect all Rickettsia species (pan-Rickettsia primers) and then use another combination that detects only R. prowazekii (specific primers) with no cross-reactivity among other Rickettsia species or bacteria. Results: Initial testing of pan-Rickettsia primers successfully identified members of the Anaplasma, Bartonella, Chlamydia, Coxiella, Ehrlichia, and Wolbachia genera only. Next, using pan-Rickettsial primers and sequence analysis, the identification focused on members of the Family Rickettsiaceae only. Finally, using R. prowazekii specific primers, we confirmed the species of 5 different R. prowazekii isolates with another 50 being tested. We are currently adapting these assays to a real-time PCR format for quantification and to possibly identify samples containing mixed species. Conclusions: Using the pan-Rickettsial PCR primers, we were able to evaluate and verify the Rickettsia samples in our collection, as well as further characterize R. prowazekii using the R. prowazekii specific PCR primers. Having access to the collections at BEIR and ATCC results in development of high quality, specific and sensitive reagents, such as those described above. These characterized and highly qualified reagents will be made available to the scientific community for research purposes through the BEI program.

Results
Figure 1: Confirmation of primer specificity with known Rickettsia samples
Type 1 Viruses Type 4 Viruses

Figure 4a: pan-Rickettsial primers with unknown samples

Figure 4b: R. prowazekii specific primers with unknown samples

3B. 3A. Negative Control Vero Cells NR-80

Known Rickettsia samples 1) NR-57 2) NR-58 Rickettsia prowazekii Rickettsia prowazekii Rickettsia prowazekii Rickettsia prowazekii Rickettsia typhi Rickettsia rickettsii 7) VR-738 8) VR-891 9) VR-1403 10) VR-1405 11) VR-1524 Rickettsia typhi Rickettsia rickettsii Rickettsia prowazekii Rickettsia prowazekii Rickettsia heilongjiangii Figure 5: Representative tracing of a R. prowazekii specific qPCR assay under development
2B. NR-84

Introduction
The genus Rickettsia is infamous for its pathogenicity in humans and animals, and is responsible for various forms of typhus and spotted fevers. Rickettsia prowazekii and Rickettsia typhi, the causative agents of Rocky Mountain Spotted Fever and epidemic typhus, respectively, are gram negative, obligate intracellular bacteria that are transmitted to humans as a result of being bit by an infected human body louse.1 The Rickettsia are also infectious by inhalation or contact with the mucous membranes of the mouth and eyes. Epidemic typhus, the prototypical infection of the typhus group of diseases can be caused by Rickettsia prowazekii. In the US approximately 15 documented sporadic cases of active infection with Rickettsia prowazekii have been reported, however, internationally, epidemic typhus occurs more frequently in Central and South America, Africa, northern China, and certain regions of the Himalayas. Epidemic typhus has the most severe clinical presentation of the typhus group of Rickettsial infections. In severe cases, gangrene may occur and lead to loss of digits, limbs, or other appendages. The vasculitic process of this disease may also lead to CNS dysfunction, ranging from dullness of mental activity to coma, multi-organ system failure, and death. The mortality rate in untreated persons ranges from 20% in healthy individuals to as high as 60% in elderly or debilitated persons. Since the advent of widely available treatment, mortality rates have fallen to approximately 3-4%.2 R. prowazekii sometimes remains latent and recurs years later; this form is called BrillZinsser disease.3 This form of typhus typically has milder symptoms with lower mortality rates. Typhus has been called camp fever, jail fever, and war fever, names that suggest overcrowding, under washing, and lowered standards of living are primary factors in disease spread. In most parts of the world, humans are the only reservoir host for R. prowazekii. IFA and EIA tests can be used to confirm a diagnosis of typhus, but they do not identify the various Rickettsial species. The complement fixation (CF) test is a serological test that can be used to demonstrate which specific Rickettsial organism is causing disease by detection of specific antibodies but is labor intensive. In an effort to assist in rapidly confirming whether Rickettsia is present we developed various assays which can be used to identify all Rickettial species and can further confirm the presence of R. prowazekii.

3) NR-59 4) NR-60 5) VR-144 6) VR-149

Standard curve R. prowazekii Standard curve Other Rickettsia R. prowazekii Other Rickettsia

Figure 2: Immunofluorescence of Rickettsia prowazekii (BEI NR-57) on Vero (ATCC CCL-81) in 15 days at 37C using a R. prowazekii specific monoclonal antibody

Materials and Methods


Immunofluorescence Assays For IFA, 20-30l of each sample was placed on an IFA slide and allowed to incubate at room temperature in a biological safety cabinet for 30 minutes. The slide was fixed in acetone. After rinsing in PBS, R. prowazekii specific antibody (PanBio, Inc. Baltimore, MD) was added to the wells at 1:400 dilution and allowed to bind for 30 minutes. The slide was rinsed with PBS and anti-Human IgG (Sigma, St. Louis, MO) was added to each well and incubated for 30 minutes and rinsed again with PBS and a 1:50 solution of Evans Blue counterstain (Sigma, St. Louis, MO) was allowed to bind for 30 minutes. The slide was rinsed again with PBS, overlayed with Fluorescent Mounting Medium (DAKO Cytomation, Carpinteria, CA) and viewed using an epi-fluorescent microscope with a FITC filter. Nucleic acid extraction and PCR Assays All genomic material was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Inc., Valencia, CA) according to the manufacturers instructions. Extracted nucleic acid was stored at -80C until use. Primers were developed by BEI/ATCC using multiple sequence alignments of published sequences for several different Rickettsia. Using pan-Rickettsial primers and R. prowazekii specific primers, DNA was amplified using the Clontech Advantage 2 PCR Kit (Clontech Labs, Inc., Mountain View, CA) and Clontech Titanium Taq DNA Polymerase. The reaction was setup according to manufacturers instruction with a final reaction volume of 50l. The reaction was as follows: 5l of SA buffer, 1.0l dNTPs, 1.0l of Titanium Taq, 1.0l of each 10M primer set, 36.0l of dH20, and 5l of template. The assays were run on the BioRad iCycler thermal cycler using the following parameters: initial denaturation at 94C for 30 seconds followed by 35 cycles of denaturation at 94C for 30 seconds, 57C for 30 seconds, 70C for 30 seconds with a final elongation step of 70C for 1 minute. Sequencing 2l of genomic DNA was submitted internally for PCR amplification and full 16S rDNA genes were sequenced using published primers and protocols then sequenced using the ABI3000 Sequencer (Applied Biosystems, Foster City, CA) and those sequences were subsequently blasted against sequences in the NCBI database. The phylogenetic tree was constructed using the Mavid computer program (http://baboon.math.berkeley.edu/mavid). q-Polymerase Chain Reaction We developed two qPCR assays for the detection of R. prowazekii. The specificity of the assays allowed us to authenticate and characterize samples that were deposited into the BEI and ATCC collections. Primer Express 2.0 software, (Applied Biosystems, Foster City, CA) was used to identify the most the suitable primer and probe combinations within our gene of interest. The gene was chosen due to a suitable degree of variability among Rickettsial species. Primers were purchased from Integrated DNA Technologies (IDT Coralville, IA) and the probes were purchased from Applied Biosystems. The master mix was prepared using BioRads iTaq kit (Palo Alto, CA) which consisted of 50mM MgCl2, 10X iTaq buffer, and Taq polymerase (catalog # 170-8870) and 10mM dNTPs (catalog # 170-8874). The reaction was setup according to manufacturers instruction with a final reaction volume of 50l. The reaction was as follows: 5l of buffer, 1.5l of MgCl2, 1.0l dNTPs, 0.25l of iTaq, 1.0l of each 10M primer, and 1.0l of 5M probe, 34.5l of dH20, and 5l of template. The assays were run on the BioRad IQ5 thermal cycler. Thermal cycling parameters consisted of an initial denaturation at 95C for 3 minutes followed by 45 cycles of denaturation at 95C for 15s and annealing/elongation at 63C for 15s.

NC

PC

Conclusions
The pan-Rickettsia assay was able to successfully detect all 11 members of our Rickettsia organism control panel. This panel consisted of several Rickettsia species whose identity was independently verified by IFA and full gene 16S rDNA sequencing. Similarly the R. prowazekii specific assay successfully identified all 6 of the R. prowazekii samples in our control panel. Using the pan-Rickettsial PCR primers, we were able to evaluate and verify a subset of the Rickettsia and related organisms found in the BEI and ATCC collections and were equipped to further characterize our suspected R. prowazekii holdings using the specific PCR primers. We have found that the pan-Rickettsial and R. prowazekii PCR assays provide a relatively rapid and accurate way of typing Rickettsia with an emphasis on discriminating select agents. In an effort to move towards more quantitative molecular assays for detection and differentiation of pathogenic material, we have developed a R. prowazekii specific assay that selectively detects all the R. prowazekii samples in our control panel. We are currently developing our plasmid based quantitation standard for use in this assay. Taken together these data demonstrate novel molecular assays that allow for the rapid detection of all Rickettsia species including the select agent members and advance R. prowazekii identification and characterization. These characterized and highly qualified reagents and assays will be made available to the scientific community for research purposes through the BEI program.

Figure 3: Phylogenetic tree of Rickettsia species prepared utilizing 16S sequences generated in-house. All inhouse 16S data were verified by comparison against those sequences found in the NCBI database.
100 99

R. rickettsii R. prowazekii R. typhi R. australis R. felis R. conorii R. sharonii R. monogolotimonea R. heilongjiangii R. hulinii

References
1) Okulicz, J.F.,M.S.Rasnake, A.A. Hansen, B.A.Cunha: Typhus, eMedicine, May 12, 2006. 2) The Center for Food Security & Public Health, Iowa State University: Typhus Fever- Rickettsia prowazekii. Jan. 2004. http://www.cfsph.iastate.edu/Factsheets/pdfs/typhus_fever.pdf>. 3) Fix, D.F., MJCR/MDMD 403: Medical Microbiology Southern Illinois University Carbondale, Rickettsia. April 1996. http://www.cehs.siu.edu/fix/medmicro/ricke.htm>. 4) Todar, K., Todars Online Textbook of Bacteriology: The Rickettsiae. University of Wisconsin-Madison, Department of Bacteriology. 2005. http://textbookofbacteriology.net/Rickettsia.html>.

Acknowledgements
Special thanks to Catherine Martini, Kristine Peterman, Ted Mullins and Jason Alexander without whose efforts I would not have been able to gather the samples that were tested. I would also like to thank Sydney Lee for her sequence work for generating the Phylogenetic Tree. This project has been funded in whole with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. N01-AI-30067.

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