Spotted Fever GroupRickettsiaInfection and Transmission Dynamics inAmblyomma maculatum

ABSTRACTTick vectors are capable of transmitting several rickettsial species to vertebrate hosts, resulting in various levels of disease. Studies have demonstrated the transmissibility of both rickettsial pathogens and novelRickettsiaspecies or strains with unknown pathogenicity to vertebrate hosts during tick blood meal acquisition; however, the quantitative nature of transmission remains unknown. We tested the hypothesis that if infection severity is a function of the rickettsial load delivered during tick transmission, then a more virulent spotted fever group (SFG)Rickettsiaspecies is transmitted at higher levels during tick feeding. UsingAmblyomma maculatumcohorts infected withRickettsia parkerior “CandidatusRickettsia andeanae,” a quantitative PCR (qPCR) assay was employed to quantify rickettsiae in tick salivary glands and saliva, as well as in the vertebrate hosts at the tick attachment site over the duration of tick feeding. Significantly greater numbers ofR. parkerithan of “Ca. Rickettsia andeanae” rickettsiae were present in tick saliva and salivary glands and in the vertebrate hosts at the feeding site during tick feeding. Microscopy demonstrated the presence of both rickettsial species in tick salivary glands, and immunohistochemical analysis of the attachment site identified localizedR. parkeri, but not “Ca. Rickettsia andeanae,” in the vertebrate host. Lesions were also distinct and more severe in vertebrate hosts exposed toR. parkerithan in those exposed to “Ca. Rickettsia andeanae.” The specific factors that contribute to the generation of a sustained rickettsial infection and subsequent disease have yet to be elucidated, but the results of this study suggest that the rickettsial load in ticks and during transmission may be an important element.

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Minimal Duration of Tick Attachment Sufficient for Transmission of Infectious Rickettsia rickettsii (Rickettsiales: Rickettsiaceae) by Its Primary Vector Dermacentor variabilis (Acari: Ixodidae): Duration of Rickettsial Reactivation in the Vector Revisited

Journal of Medical Entomology ◽

10.1093/jme/tjz191 ◽

2019 ◽

Author(s):

Michael L Levin ◽

Shelby L Ford ◽

Kris Hartzer ◽

Lnna Krapiunaya ◽

Hannah Stanley ◽

...

Keyword(s):

Salivary Glands ◽

Guinea Pigs ◽

Pathogenic Bacteria ◽

Spotted Fever ◽

Dermacentor Variabilis ◽

Control Group ◽

Spotted Fever Group ◽

Susceptible Host ◽

Rickettsia Rickettsii ◽

Tick Attachment

Abstract It has been reported that starving ticks do not transmit spotted fever group Rickettsia immediately upon attachment because pathogenic bacteria exist in a dormant, uninfectious state and require time for ‘reactivation’ before transmission to a susceptible host. To clarify the length of reactivation period, we exposed guinea pigs to bites of Rickettsia rickettsii-infected Dermacentor variabilis (Say) and allowed ticks to remain attached for predetermined time periods from 0 to 48 h. Following removal of attached ticks, salivary glands were immediately tested by PCR, while guinea pigs were observed for 10–12 d post-exposure. Guinea pigs in a control group were subcutaneously inoculated with salivary glands from unfed D. variabilis from the same cohort. In a parallel experiment, skin at the location of tick bite was also excised at the time of tick removal to ascertain dissemination of pathogen from the inoculation site. Animals in every exposure group developed clinical and pathological signs of infection. The severity of rickettsial infection in animals increased with the length of tick attachment, but even attachments for less than 8 h resulted in clinically identifiable infection in some guinea pigs. Guinea pigs inoculated with salivary glands from unfed ticks also became severely ill. Results of our study indicate that R. rickettsii residing in salivary glands of unfed questing ticks does not necessarily require a period of reactivation to precede the salivary transmission and ticks can transmit infectious Rickettsia virtually as soon as they attach to the host.

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Optimization and Evaluation of a Multiplex Quantitative PCR Assay for Detection of Nucleic Acids in Human Blood Samples from Patients with Spotted Fever Rickettsiosis, Typhus Rickettsiosis, Scrub Typhus, Monocytic Ehrlichiosis, and Granulocytic Anaplasmosis

Journal of Clinical Microbiology ◽

10.1128/jcm.01802-19 ◽

2020 ◽

Vol 58 (9) ◽

Author(s):

Megan E. Reller ◽

J. Stephen Dumler

Keyword(s):

Quantitative Pcr ◽

Scrub Typhus ◽

Spotted Fever ◽

Febrile Illness ◽

Epidemiologic Studies ◽

Qpcr Assay ◽

Spotted Fever Group ◽

Analytical Sensitivity ◽

Content Type ◽

Real Time Quantitative Pcr

ABSTRACT Spotted fever group rickettsioses (SFGR), typhus group rickettsioses (TGR), scrub typhus (caused by Orientia tsutsugamushi), ehrlichiosis, and anaplasmosis often present as undifferentiated fever but are not treated by agents (penicillins and cephalosporins) typically used for acute febrile illness. Inability to diagnose these infections when the patient is acutely ill leads to excess morbidity and mortality. Failure to confirm these infections retrospectively if a convalescent blood sample is not obtained also impairs epidemiologic and clinical research. We designed a multiplex real-time quantitative PCR (qPCR) assay to detect SFGR, TGR, O. tsutsugamushi, and infections caused by Anaplasma phagocytophilum and Ehrlichia chaffeensis with the ompA, 17-kDa surface antigen gene, tsa56, msp2 (p44), and vlpt gene targets, respectively. Analytical sensitivity was ≥2 copies/μl (linear range, 2 to 2 × 105) and specificity was 100%. Clinical sensitivities for SFGR, TGR, and O. tsutsugamushi were 25%, 20%, and 27%, respectively, and specificities were 98%, 99%, and 100%, respectively. Clinical sensitivities for A. phagocytophilum and E. chaffeensis were 93% and 84%, respectively, and specificities were 99% and 98%, respectively. This multiplex qPCR assay could support early clinical diagnosis and treatment, confirm acute infections in the absence of a convalescent-phase serum sample, and provide the high-throughput testing required to support large clinical and epidemiologic studies. Because replication of SFGR and TGR in endothelial cells results in very low bacteremia, optimal sensitivity of qPCR for these rickettsioses will require use of larger volumes of input DNA, which could be achieved by improved extraction of DNA from blood and/or extraction of DNA from a larger initial volume of blood.

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Tick-borne rickettsial diseases

10.1093/med/9780198570028.003.0010 ◽

2011 ◽

Cited By ~ 1

Author(s):

Emmanouil Angelakis ◽

Didier Raoult

Keyword(s):

Spotted Fever ◽

Reservoir Host ◽

Spotted Fever Group ◽

Rickettsia Species ◽

The Family ◽

Risk Areas ◽

Vertebrate Hosts ◽

Group 2 ◽

Rickettsial Diseases ◽

Rickettsia Bellii

Bacteria of the genus Rickettsia belong to the family Rickettsiaceae in the order Rickettsiales and have for long been described simply as short, Gram-negative, strict intracellular rods that retain basic fuchsin when stained by the method of Gimenez (Raoult and Roux 1997). These bacteria are associated with ticks, lice, fleas or mites. To date the Rickettsia genus contains 24 recognized species classified into three groups based on their antigenic, morphological, and ecologic patterns: 1) the typhus group, 2) the spotted fever group and 3) Rickettsia bellii (Fournier and Raoult 2007). Most spotted fever group (SFG) rickettsiae are closely associated with ticks belonging to the family Ixodidae (also called “hard” ticks) (Parola et al. 2005). Ticks can act as vectors, reservoirs, and/or amplifiers of SFG rickettsiae and require optimal environmental conditions which determine the geographic distribution of the vectors and consequently the risk areas for rickettsioses. Many Rickettsia species are strictly associated with one genus of ticks and the transmittion to people is made through the tick bite, which generally implies that the Rickettsia can localize to their salivary glands. Therefore, since larvae, nymphs, and adults may all be infective for susceptible vertebrate hosts, the ticks must be regarded as the main reservoir host of rickettsiae. Humans are not considered as good reservoirs for Rickettsiae, as they are seldom infested with ticks for long periods and rickettsiaemia has normally short duration, especially with antibiotic intervention.

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Susceptibility of Inbred Mice to Rickettsia parkeri

Infection and Immunity ◽

10.1128/iai.00109-12 ◽

2012 ◽

Vol 80 (5) ◽

pp. 1846-1852 ◽

Cited By ~ 22

Author(s):

Britton J. Grasperge ◽

Kathryn E. Reif ◽

Timothy D. Morgan ◽

Piyanate Sunyakumthorn ◽

Joseph Bynog ◽

...

Keyword(s):

Inbred Mice ◽

Spotted Fever ◽

Inbred Mouse ◽

Transmission Model ◽

Mouse Strains ◽

Spotted Fever Group ◽

Limited Information ◽

Rickettsia Parkeri ◽

Content Type ◽

Boutonneuse Fever

ABSTRACTRickettsia parkeri, a member of the spotted fever groupRickettsia, is the causative agent of American boutonneuse fever in humans. Despite the increased recognition of human cases, limited information is available regarding the infection of invertebrate and vertebrate hosts for this emerging tick-borne disease. Toward the development of a viable transmission model and to further characterize the pathology associated withR. parkeriinfection, inbred mouse strains (A/J, BALB/c, C3H/HeJ, and C3H/HeN) were intravenously and intradermally inoculated with 105low-passage-numberR. parkeri(Portsmouth strain), and infection, gross pathology, and histopathology were scored. Additionally, a quantitative real-time PCR (qPCR) was performed to estimate rickettsial load in heart, lung, spleen, and liver tissues of infected mice at 19 days postinoculation. Of the A/J, BALB/c, and C3H/HeN mice, none displayed universal pathology consistent with sustained infection. Compared to age-matched control mice, the intravenously inoculated C3H/HeJ mice exhibited marked facial edema and marked splenomegaly upon gross examination, while the intradermally inoculated mice developed characteristic eschar-like lesions. The C3H/HeJ mice also exhibited the greatest concentrations of rickettsial DNA from heart, lung, liver, and spleen samples when examined by qPCR. The similarity of the pathology of human disease and sustained infection suggests that the C3H/HeJ strain of mice is a promising candidate for subsequent experiments to examine the tick transmission, dissemination, and pathology ofR. parkeririckettsiosis.

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Tick salivary glands: function, physiology and future

Parasitology ◽

10.1017/s0031182004006468 ◽

2004 ◽

Vol 129 (S1) ◽

pp. S67-S81 ◽

Cited By ~ 100

Author(s):

A. S. BOWMAN ◽

J. R. SAUER

Keyword(s):

Salivary Glands ◽

Blood Meal ◽

Cell Biology ◽

Expressed Sequence Tag ◽

Extended Period ◽

Feeding Site ◽

Route Of Transmission ◽

The Many ◽

Vector Competency ◽

Tick Salivary Glands

The salivary glands are the organs of osmoregulation in ticks and, as such, are critical to the biological success of ticks both during the extended period off the host and also during the feeding period on the host. Absorption of water vapour from unsaturated air into hygroscopic fluid produced by the salivary glands permit the tick to remain hydrated and viable during the many months between blood-meals. When feeding, the tick is able to return about 70% of the fluid and ion content of the blood-meal into the host by salivation into the feeding site. This saliva also contains many bioactive protein and lipid components that aid acquisition of the blood-meal. The salivary glands are the site of pathogen development and the saliva the route of transmission. The importance of the multifunctional salivary glands to tick survival and vector competency makes the glands a potential target for intervention. Here we review the cell biology of tick salivary glands and discuss the application of new approaches such as expressed sequence tag projects and RNA interference to this important area in the field of tick and tick-borne pathogen research.

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Vector Competence of Geographical Populations ofOrnithodoros turicatafor the Tick-Borne Relapsing Fever SpirocheteBorrelia turicatae

Applied and Environmental Microbiology ◽

10.1128/aem.01505-18 ◽

2018 ◽

Vol 84 (21) ◽

Cited By ~ 5

Author(s):

Aparna Krishnavajhala ◽

Brittany A. Armstrong ◽

Job E. Lopez

Keyword(s):

Salivary Glands ◽

Vector Competence ◽

Relapsing Fever ◽

Tick Saliva ◽

Eastern Population ◽

Content Type ◽

Western Population ◽

Geographical Populations ◽

Globally Distributed ◽

Two Populations

ABSTRACTVector competence refers to the ability of an arthropod to acquire, maintain, and successfully transmit a microbial pathogen. Tick-borne relapsing fever (TBRF) spirochetes are globally distributed pathogens, and most species are transmitted by argasid ticks of the genusOrnithodoros.A defining characteristic in vector competence is an apparent specificity of a species of TBRF spirochete to a given tick species. In arid regions of the southern United States,Borrelia turicataeis the primary cause of TBRF. Interestingly, there are two populations of the tick vector distributed throughout this region.Ornithodoros turicatais a western population that ranges from California to Texas. There is a gap through Louisiana, Mississippi, and Alabama where the tick has not been identified. An isolated eastern population exists in Florida and was designated a subspecies,O. turicata americanus.A knowledge gap that exists is the poor understanding of vector competence between western and eastern populations of ticks forB. turicatae.In this study, we generated uninfected colonies ofO. turicatathat originated in Texas and Kansas and ofO. turicataamericanus. B. turicataeacquisition, maintenance through the molt, and subsequent transmission were evaluated. Our findings revealed significant differences in murine infection after feeding infectedO. turicataandO. turicataamericanusticks on the animals. Interestingly, the salivary glands of both tick populations were colonized withB. turicataeto similar densities. Our results suggest that the salivary glands of the tick colonies assessed in this study impact vector competence of the evaluatedB. turicataeisolates.IMPORTANCESeveral knowledge gaps exist in the vector competence of various geographical populations ofO. turicatathat transmitB. turicatae. A western population of this tick is distributed from California to Texas, and an eastern population exists in Florida. Utilizing western and eastern populations of the vector, we studied acquisition and transmission of twoB. turicataeisolates. Regardless of the isolate used, infection frequencies were poor in mice after the eastern population feeding on them. Since salivary gland colonization is essential forB. turicataetransmission, these tissues were further evaluated. Interestingly, the salivary glands from the two populations were similarly colonized withB. turicatae.These findings suggest the role of tick saliva in the establishment of infection and that the salivary glands may be a bottleneck for successful transmission.

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Comparison of the proteins in salivary glands, saliva and haemolymph of Rhipicephalus appendiculatus female ticks during feeding

Parasitology ◽

10.1017/s003118200008077x ◽

1994 ◽

Vol 109 (4) ◽

pp. 517-523 ◽

Cited By ~ 29

Author(s):

H. Wang ◽

P. A. Nuttall

Keyword(s):

Salivary Glands ◽

Gel Electrophoresis ◽

Guinea Pigs ◽

Polyacrylamide Gel Electrophoresis ◽

Rhipicephalus Appendiculatus ◽

Host Immunity ◽

Tick Feeding ◽

Tick Saliva ◽

Small Proteins ◽

Common Antigens

SUMMARYTo compare the proteins in salivary glands, saliva and haemolymph of Rhipicephalus appendiculatus female ticks, antisera were prepared from guinea-pigs immunized with soluble denatured salivary gland extracts (SGE). The extracts were derived from R. appendiculatus female ticks that were either unfed (day 0) or partly fed (day 6). The sera were used in immunoblotting, following SDS–polyacrylamide gel electrophoresis, to examine the antigen profiles during the course of tick feeding on guinea-pigs. Day 0 and day 6 SGE antisera appeared to detect common proteins in the different tick samples. For example, haemolymph apparently shared some of the small protein bands (31·5–34 kDa) detected in SGEs. These small proteins appeared in both samples at the same stage of feeding, suggesting that haemolymph and salivary glands not only have common antigens but may also share some functions. Furthermore, a number of protein bands were detected in haemolymph before they were apparent in the salivary glands or saliva. Thus some antigens detected in the salivary glands and saliva may be derived from the haemolymph. The results indicate that the host may be exposed to tick saliva antigens that are also present in the haemolymph. We discuss the significance of these observations with regard to the induction of host immunity to ticks and the development of tick vaccines.

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A Novel TaqMan Assay for Detection of Rickettsia 364D, the Etiologic Agent of Pacific Coast Tick Fever

Journal of Clinical Microbiology ◽

10.1128/jcm.01106-19 ◽

2019 ◽

Vol 58 (1) ◽

Author(s):

Sandor E. Karpathy ◽

Alex Espinosa ◽

Melissa H. Yoshimizu ◽

Jill K. Hacker ◽

Kerry A. Padgett ◽

...

Keyword(s):

Pacific Coast ◽

Spotted Fever ◽

Febrile Illness ◽

Etiologic Agent ◽

Taqman Assay ◽

Spotted Fever Group ◽

Content Type ◽

Clinical Sensitivity ◽

Detection And Identification ◽

Diagnostic Applications

ABSTRACT Pacific Coast tick fever is a febrile illness associated with the bite of Dermacentor occidentalis and results from an infection due to the intracellular pathogen Rickettsia 364D (also known by the proposed name “Rickettsia philipii”). Current molecular methods for the detection of this pathogen rely on the amplification of a conserved spotted fever group rickettsial gene (ompA) followed by DNA sequencing of the amplicon to identify the species. This work describes the development of a Rickettsia 364D-specific TaqMan assay to simplify and accelerate the detection and identification processes. The assay demonstrated a sensitivity of 1 genomic copy per 4-μl sample and is highly specific for Rickettsia 364D. The utility of this assay for ecological and diagnostic samples was evaluated using banked specimens collected in a single-blind manner and yielded a clinical sensitivity and specificity of 100%. In conclusion, we describe the development and evaluation of a novel TaqMan real-time PCR assay for the detection and identification of Rickettsia 364D suitable for ecological and diagnostic applications.

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Proteolytic Cleavage of the Immunodominant Outer Membrane Protein rOmpA in Rickettsia rickettsii

Journal of Bacteriology ◽

10.1128/jb.00826-16 ◽

2016 ◽

Vol 199 (6) ◽

Cited By ~ 7

Author(s):

Nicholas F. Noriea ◽

Tina R. Clark ◽

David Mead ◽

Ted Hackstadt

Keyword(s):

Outer Membrane ◽

Cleavage Site ◽

Spotted Fever ◽

Protein A ◽

Rocky Mountain ◽

Spotted Fever Group ◽

Rocky Mountain Spotted Fever ◽

Content Type ◽

Rickettsia Rickettsii ◽

Carboxy Terminal

ABSTRACT Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, contains two immunodominant proteins, rOmpA and rOmpB, in the outer membrane. Both rOmpA and rOmpB are conserved throughout spotted fever group rickettsiae as members of a family of autotransporter proteins. Previously, it was demonstrated that rOmpB is proteolytically processed, with the cleavage site residing near the autotransporter domain at the carboxy-terminal end of the protein, cleaving the 168-kDa precursor into apparent 120-kDa and 32-kDa fragments. The 120- and 32-kDa fragments remain noncovalently associated on the surface of the bacterium, with implications that the 32-kDa fragment functions as the membrane anchor domain. Here we present evidence for a similar posttranslational processing of rOmpA. rOmpA is expressed as a predicted 224-kDa precursor yet is observed on SDS-PAGE as a 190-kDa protein. A small rOmpA fragment of ∼32 kDa was discovered during surface proteome analysis and identified as the carboxy-terminal end of the protein. A rabbit polyclonal antibody was generated to the autotransporter region of rOmpA and confirmed a 32-kDa fragment corresponding to the calculated mass of a proteolytically cleaved rOmpA autotransporter region. N-terminal amino acid sequencing revealed a cleavage site on the carboxy-terminal side of Ser-1958 in rOmpA. An avirulent strain of R. rickettsii Iowa deficient in rOmpB processing was also defective in the processing of rOmpA. The similarities of the cleavage sites and the failure of R. rickettsii Iowa to process either rOmpA or rOmpB suggest that a single enzyme may be responsible for both processing events. IMPORTANCE Members of the spotted fever group of rickettsiae, including R. rickettsii, the etiologic agent of Rocky Mountain spotted fever, express at least four autotransporter proteins that are protective antigens or putative virulence determinants. One member of this class of proteins, rOmpB, is proteolytically processed to a passenger domain and an autotransporter domain that remain associated on the rickettsial outer membrane. The protease responsible for this posttranslation processing remains unknown. Here we show that another autotransporter, rOmpA, is similarly processed by R. rickettsii. Similarities in sequence at the cleavage site and predicted secondary protein structure suggest that all four R. rickettsii autotransporters may be processed by the same outer membrane protease.

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Identification of Host Proteins Involved in Rickettsial Invasion of Tick Cells

Infection and Immunity ◽

10.1128/iai.02888-14 ◽

2014 ◽

Vol 83 (3) ◽

pp. 1048-1055 ◽

Cited By ~ 3

Author(s):

Natthida Petchampai ◽

Piyanate Sunyakumthorn ◽

Kaikhushroo H. Banajee ◽

Victoria I. Verhoeve ◽

Michael T. Kearney ◽

...

Keyword(s):

Host Cell ◽

Tyrosine Kinases ◽

Molecular Mechanisms ◽

Rho Gtpase ◽

Spotted Fever ◽

Dermacentor Variabilis ◽

Host Cells ◽

Spotted Fever Group ◽

Bacterial Survival ◽

Content Type

Tick-borne spotted fever group (SFG)Rickettsiaspecies are obligate intracellular bacteria capable of infecting both vertebrate and invertebrate host cells, an essential process for subsequent bacterial survival in distinct hosts. The host cell signaling molecules involved in the uptake ofRickettsiainto mammalian andDrosophilacells have been identified; however, invasion into tick cells is understudied. Considering the movement of SFGRickettsiabetween vertebrate and invertebrate hosts, the hypothesis is that conserved mechanisms are utilized for host cell invasion. The current study employed biochemical inhibition assays to determine the tick proteins involved inRickettsia montanensisinfection of tick-derived cells from a natural host,Dermacentor variabilis. The results revealed several tick proteins important for rickettsial invasion, including actin filaments, actin-related protein 2/3 complex, phosphatidylinositol-3′-kinase, protein tyrosine kinases (PTKs), Src family PTK, focal adhesion kinase, Rho GTPase Rac1, and neural Wiskott-Aldrich syndrome protein. Delineating the molecular mechanisms of rickettsial infection is critical to a thorough understanding of rickettsial transmission in tick populations and the ecology of tick-borne rickettsial diseases.

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