INFECTION AND IMMUNITY, Aug. 1982, p. 779-785 Vol. 37, No. 2 0019-9567/82/080779-07$02.00/0 Reactivation of Rickettsia rickettsii in Dermacentor andersoni Ticks: an Ultrastructural Analysis STANLEY F. HAYES* AND WILLY BURGDORFER Department of Health and Human Services, Public Health Service, National Institutes of Health, National Institute of Allergy and Infectious Diseases, Electron Microscopy Section, Operations Branch, and Epidemiology Branch, Rocky Mountain Laboratories, Hamilton, Montana 59840 Received 19 February 1982/Accepted 7 April 1982 Virulent Rickettsia rickettsii in Dermacentor andersoni lose their pathogenicity and virulence for guinea pigs when subjected to physiological stresses, such as starvation (overwintering), of its tick vector. However, incubation of infected ticks at an elevated temperature (37°C) for 24 to 48 h or feeding for a time (usually >10 h) induces R. rickettsii to revert to a virulent state, a phenomenon defined as "reactivation." Electron microscopy reveals that the microcapsular and slime layers of R. rickettsii undergo changes dependent upon the physiological condi- tions within the tick vector. In engorged ticks, the microcapsular layer is readily identified as a discrete layer, approximately 16 nm thick, composed of globular subunits that have a periodicity of approximately 10 nm. The slime layer external to the microcapsular layer forms a discrete electron-lucent zone around the rickettsia. In starved ticks, neither the microcapsular layer nor slime layer remains a discrete entity. Instead, they are shed and form stringy, shredded, and somewhat flocculent strands of low electron density without periodicity. Incuba- tion at 37°C or feeding of starved infected ticks results in the restoration of a discrete microcapsular and slime layer. These reversible structural modifications are linked to physiological changes in the tick host and correlate with reactivation, i.e., restoration of pathogenicity and virulence of R. rickettsii. As early as 1909, Ricketts observed consider- able variation in the time necessary for infected wood ticks, Dermacentor andersoni to induce typical spotted fever symptoms in guinea pigs as a result of feeding (37). Parker and Spencer (33) and later Spencer and Parker (44-46) reported that injection of triturated, starved, infected ticks into guinea pigs did not lead to disease, but caused seroconversion. However, feeding the ticks for a short time or keeping them at an elevated temperature (24 to 48 h at 37°C before trituration and inoculation into nonimmune guin- ea pigs) resulted in a clinically manifest disease. Spencer and Parker postulated that virulence of Rickettsia rickettsii in the tick vector is linked directly to the physiological state of the tick and defined this phenomenon as "reactivation." Bo- varnik and Allen, studying a similar phenome- non of reversible inactivation at low tempera- tures in Rickettsia prowazekii (3-5), determined that modulating factors responsible for this change in virulence were metabolic in nature, involving NAD, coenzyme A, ATP, and gluta- mate. Price (35-36) and Gilford and Price (15) further characterized reactivation in R. rickettsii by in vitro experiments paralleling those of Bovarnik and Allen. They showed that virulent R. rickettsii can be made avirulent by treatment with p-aminobenzoic acid. This process can be reversed by incubation with NAD (coenzyme I) or coenzyme A but not with NADP (coenzyme II). They also observed that loss of virulence could be blocked with p-hydroxybenzoic acid or NAD and p-aminobenzoic acid mixed together. Weiss et al. (52) later confirmed the findings of Gilford and Price and showed that R. rickettsii possessed metabolic requirements similar to R. prowazekii. Although these studies have enhanced our understanding about infectivity and virulence of R. rickettsii, they have not identified specific structural component(s) associated with this phenomenon. Through electron microscopic studies of R. rickettsii, we have repeatedly observed that physiological conditions within the tick host influence the ultrastructural appearance of the microcapsular (MCL) and slime layer (SL) of R. rickettsii. This paper illustrates these changes and demonstrates their correlation to the phe- nomenon of reactivation. MATERIALS AND METHODS To study the fine structural appearance of R. rickett- sii (Wachsmuth 1974 strain), starved, partially or fully engorged D. andersoni from infected tick lines (main- 779 on October 19, 2020 by guest http://iai.asm.org/ Downloaded from
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INFECTION AND IMMUNITY, Aug. 1982, p. 779-785 Vol. 37, No. 20019-9567/82/080779-07$02.00/0
Reactivation of Rickettsia rickettsii in Dermacentor andersoniTicks: an Ultrastructural AnalysisSTANLEY F. HAYES* AND WILLY BURGDORFER
Department of Health and Human Services, Public Health Service, National Institutes of Health, NationalInstitute ofAllergy and Infectious Diseases, Electron Microscopy Section, Operations Branch, and
Virulent Rickettsia rickettsii in Dermacentor andersoni lose their pathogenicityand virulence for guinea pigs when subjected to physiological stresses, such asstarvation (overwintering), of its tick vector. However, incubation of infectedticks at an elevated temperature (37°C) for 24 to 48 h or feeding for a time (usually>10 h) induces R. rickettsii to revert to a virulent state, a phenomenon defined as"reactivation." Electron microscopy reveals that the microcapsular and slimelayers of R. rickettsii undergo changes dependent upon the physiological condi-tions within the tick vector. In engorged ticks, the microcapsular layer is readilyidentified as a discrete layer, approximately 16 nm thick, composed of globularsubunits that have a periodicity of approximately 10 nm. The slime layer externalto the microcapsular layer forms a discrete electron-lucent zone around therickettsia. In starved ticks, neither the microcapsular layer nor slime layerremains a discrete entity. Instead, they are shed and form stringy, shredded, andsomewhat flocculent strands of low electron density without periodicity. Incuba-tion at 37°C or feeding of starved infected ticks results in the restoration of adiscrete microcapsular and slime layer. These reversible structural modificationsare linked to physiological changes in the tick host and correlate with reactivation,i.e., restoration of pathogenicity and virulence of R. rickettsii.
As early as 1909, Ricketts observed consider-able variation in the time necessary for infectedwood ticks, Dermacentor andersoni to inducetypical spotted fever symptoms in guinea pigs asa result of feeding (37). Parker and Spencer (33)and later Spencer and Parker (44-46) reportedthat injection of triturated, starved, infectedticks into guinea pigs did not lead to disease, butcaused seroconversion. However, feeding theticks for a short time or keeping them at anelevated temperature (24 to 48 h at 37°C beforetrituration and inoculation into nonimmune guin-ea pigs) resulted in a clinically manifest disease.Spencer and Parker postulated that virulence ofRickettsia rickettsii in the tick vector is linkeddirectly to the physiological state of the tick anddefined this phenomenon as "reactivation." Bo-varnik and Allen, studying a similar phenome-non of reversible inactivation at low tempera-tures in Rickettsia prowazekii (3-5), determinedthat modulating factors responsible for thischange in virulence were metabolic in nature,involving NAD, coenzyme A, ATP, and gluta-mate. Price (35-36) and Gilford and Price (15)further characterized reactivation in R. rickettsiiby in vitro experiments paralleling those ofBovarnik and Allen. They showed that virulentR. rickettsii can be made avirulent by treatment
with p-aminobenzoic acid. This process can bereversed by incubation with NAD (coenzyme I)or coenzyme A but not with NADP (coenzymeII). They also observed that loss of virulencecould be blocked with p-hydroxybenzoic acid orNAD and p-aminobenzoic acid mixed together.Weiss et al. (52) later confirmed the findings ofGilford and Price and showed that R. rickettsiipossessed metabolic requirements similar to R.prowazekii.
Although these studies have enhanced ourunderstanding about infectivity and virulence ofR. rickettsii, they have not identified specificstructural component(s) associated with thisphenomenon.Through electron microscopic studies of R.
rickettsii, we have repeatedly observed thatphysiological conditions within the tick hostinfluence the ultrastructural appearance of themicrocapsular (MCL) and slime layer (SL) of R.rickettsii. This paper illustrates these changesand demonstrates their correlation to the phe-nomenon of reactivation.
MATERIALS AND METHODSTo study the fine structural appearance of R. rickett-
sii (Wachsmuth 1974 strain), starved, partially or fullyengorged D. andersoni from infected tick lines (main-
tained at Rocky Mountain Laboratories) were dissect-ed in minimal essential media. Their tissues wereprepared for electron microscopy as previously de-scribed (18). Tick feeding was done on capsuled, whiteHartley guinea pigs. Capsules were metal and attachedto the shaved bellies of the guinea pigs by 4-in. (10.2cm) adhesive tape girdling each animal. In someinstances 2 mM ATP, 5 mM pyruvate, and 0.1 mMcoenzyme A were added to the modified Ito fixative(18) by the methods of Weidner and Trager (51) to seewhether this would stabilize sloughing MCL and SLcomponents. Blocks of tick tissues embedded in Spurrresin were sectioned with a DuPont diamond knife.Sections were picked up on flamed, naked, 300-meshgrids, stained with uranyl acetate and lead citrate, andexamined in a Hitachi 11E-U-1 electron microscope at75 kV. Photographs were recorded on Kodak glasselectron image plates (3.25 by 4.0 in.; 8.25 by 10.2 cm).
RESULTS(i) Appearance of R. rickettsii within partially
or fully engorged ticks. Figures 1 and 2 show R.rickettsii during a time of optimal physiologicalstability wherein ticks are actively feeding pro-viding essential nutrients, metabolites and tem-
FIG. 2. R. rickettsii; cross-section profile showingthe location of the MCL (arrows) and its approximate-ly 10-nm subunit periodicity. Bar = 0.1 p.m.
perature required for rickettsial growth. The R.rickettsii isolate appears slightly lanceolate orrodlike, and is bounded by a prominent halo(SL) immediately adjacent to a rigid, trilaminarcell wall (TCW) (Fig. 1). Between the SL and theTCW is a slightly electron-dense MCL, approxi-mately 16 nm thick composed of beadlike sub-units (Fig. 1 and 2) with a periodicity of approxi-mately 10 nm. Internal to the TCW is a narrowperiplasmic space which contains some materialof low electron density. This space is borderedon its internal aspect by a plasma membrane,which is not always discerned because it occa-sionally appears to be an integral part of the cellwall. The plasma membrane bounds a moderate-ly electron-dense mottled cytoplasm that con-tains randomly distributed ribosomes and finefibrils presumed to be DNA.
Within the cytoplasm of host cells, a sharpdemarcation exists between the electron lucentSL of R. rickettsii and the electron dense hostcell cytoplasm. In partially or fully engorgedticks, infected host cells are quite normal show-ing healthy ribosomes, vesicles, mitochondriaand membranes.
observed in cells of starved uninfected ticks,4o^ _ (Fig. 6 and 7) resulting in an electron-lucent*- -iM-center of the nucleus. There is also an apprecia-
ble loss of cytosol, ribosomes, mitochondria,and endoplasmic reticulum. Changes also occur
w;t|in the integrity of both nuclear and cellularmembranes. Under these conditions, R. rickett-
//3lt sii is often seen perinuclearly oriented (Fig. 7)L'EH*itg.a i '}t and adjacent to remaining membranes or mito-
7I_."'chondria, or both. These pathological changesare in direct contrast to conditions withinhealthy cells. The inclusion of ATP, coenzymeI, and pyruvate to fixatives did not improve thestabilization of the rickettsial surface compo-nents.
I 5sbthe o -,,;ADISCUSSIONThe pathogenicity of certain spotted fever
group rickettsiae and their variability in viru-lence continue to draw attention to rickettsialstructure(s) that may be responsible for initiatingdisease in animals and humans. Structural sur-face components in the form of polysaccharides,
3
FIG. 3. R. rickettsii in salivary gland tissue of astarved D. andersoni. Note the electron-lucent ap-pearance of both rickettsiae and host cell cytoplasm.Arrow points to volutin-like granule present in a cross-section profile of a rickettsia. The TCW of all rickettsi-ae is very sinuous. Bar = 0.2 p.m.
(ii) Appearance of R. rickettsii in starved ticks.In starved ticks, R. rickettsii exhibit the follow-ing signs of degeneration (Fig. 3 to 5): plasmoly-sis, blebbing of the plasma membrane, a sinuouscell wall with occasional breaks, an increase involume and concurrent decrease in density ofthe periplasmic space, an increase in electrondensity of the cytoplasm (loss of mottled appear-ance) and the formation of polarly located, volu-tin-like, electron-dense inclusions within the cy-toplasm (Fig. 4 and 5). In addition, the discretebeadlike MCL structures (present under physio-logically optimal conditions) dramaticallychange (Fig. 5 to 7) and resemble stringy strandswhose proximal portion appears to fuse with theelectron-dense outer leaflet of the TCW. Thus,the individual organisms appear fuzzy and rag-ged.
Within infected cells of starved ticks the sharp HG. 4. An electron-dense rickettsia in ovariai tis-demarcation between the rickettsial SL and the sue of a starved tick. Note the beginning of MCLhost cell cytoplasm disappears. Infected cells breakdown (black arrows) and the formation of polarlyexhibit an aggregation of the heterochromatin at located volutin-like granules (white arrows). Bar = 0.1the nuclear envelope, a phenomenon not usually ,um.
FIG. 5. R. rickettsii within a salivary gland acinus cell of a starved tick. The MCL and SL appear strand-likeand fuzzy while being shed from the surface of both electron-lucent and electron-dense organisms. Bar = 0.1 p.m.
lipoproteins, glycoproteins, lipopolysaccha-rides, or mucopolysaccharides of other gram-negative microorganisms have been incriminat-ed as structures capable of facilitating thedisease processes (6, 12, 17, 21, 24-26, 30-32,38, 42, 43, 47-50). Similar structures are associ-ated with R. rickettsii and other rickettsial spe-cies. Data suggests that the degree of pathoge-nicity and virulence of rickettsial species arerelated to these same types of structural compo-nents. Anacker et al. (1) described in R. prowa-zekii the existence of a capsule-like layer locatedexternally and immediately adjacent to the elec-tron-dense outer leaflet of the cell wall. Brintonand Burgdorfer (7), in studies of the typhusgroup rickettsia, R. canada, also referred tosuch a layer as a velvety coating approximately7 nm thick attached or adsorbed to the TCW,whereas Ito (22) in studies on polysaccharidicmaterial associated with procaryote and eucary-ote cell surfaces described it as a glycocalyx.Much, however, that is now known about thenature and possible functions of surface-associ-ated structures of rickettsiae is based on thework of Balayeva and Gulevskaya with R.prowazekii (2). These investigators used ether
extraction and electron microscopy to show thewall structure and general morphology of thisagent. They called the extractable layer a micro-capsular layer because it was reminiscent of astructure described by Tomcsik (48) and Katz etal. (25) for bacteria. A similar layer has beenreported by others for Coxiella burnetii, R.rickettsii, R. rhipicephali, R. sibirica, R. typhi,and R. tsutsugamushi, and WB-8-2, an as yetunnamed serotype of the spotted fever groupassociated with the Lone Star tick, Amblyommaamericanum (2, 7-11, 13, 14, 16, 18-20, 22, 23,34, 39-41).The application of electron microscopy and
ruthenium red staining techniques (27-29) havevisually and biochemically established the acidprotein and polysaccharidic nature of the MCLand of the SL. All pathogenic rickettsiae studiedso far by us appear to possess a MCL and SL.On the other hand, the unclassified, nonpatho-genic 369-C rickettsia, isolated originally fromD. variabilis Say by E. J. Bell, does not possessa MCL and exhibits only a thin SL. This rickett-sia is nonpathogenic for humans, guinea pigs,and all rodent models tested so far. It multipliespoorly in embryonated eggs but is readily main-
modification of virulence and pathogenicity inother bacteria such as Aeromonas salmonicada,the fish pathogen responsible for furunculosis infish (26, 49, 50), for Yersinia pestis, the plagueagent (12, 17) and with Campylobacter fetus,associated with abortion in humans and animals(30, 31). A parallel may be drawn to the roughand smooth designated microbes, whose surfacecharacteristics relate directly to their pathoge-nicity and virulence. The ability of R. rickettsiito reestablish its surface structures (MCL andSL) under optimal physiological conditions afterhaving lost them during the periods of stress andstarvation of its arthropod host may indeedprovide the basis for reactivation and restorationof pathogenicity and virulence. Whether all oronly certain parts of the MCL and SL arenecessary for reactivation remains to be investi-gated. It is apparent, however, that these struc-tures are important in pathogenesis and specifi-cally in the interactions between R. rickettsii andthe cells of its arthropod and vertebrate hosts.We feel that the micrographic evidence present-ed here visually reflects structural changes in-volved in reactivation, i.e., changes in thepathogenic and virulent nature of R. rickettsii.
FIG. 6. Same general area as Fig. 5, illustrating theaggregation of nuclear heterochromatin at the nuclearmembrane (arrows) and the loss of cytoplasmic organ-elles and cytosol of the host cell. Bar = 0.2 ,um.
tained in Vero cell tissue cultures (R. N. Philipet al., manuscript in preparation). The absenceof the MCL, alteration of the acid protein, oralteration of the polysaccharidic surface struc-ture, or a combination of these factors, may beresponsible for its lack of pathogenicity andvirulence and may also account for less virulentspotted fever group rickettsiae such as R. mon-tana and R. rhipicephali. Recently Dasch (13)isolated the MCL of R. prowazekii and R. typhiand identified it as species-specific protein anti-gen which is useful in the immunodiagnosis andimmunoprophylaxis of typhus infections. Thedirect relationship of species-specific proteinantigen to pathogenicity and virulence is as yetundetermined. Similarly, the effects of growthlimitations, i.e., starvation-induced restrictionson metabolites and precursors of the MCL andSL of R. prowazekii have not as yet been tested.However, the literature abounds with data thatset precedence for growth-limiting modificationsof surface components and the effect of theseupon pathogenicity and virulence (6, 26, 40, 41,45). A loss or modification of surface compo-nents has been shown responsible for loss or
FIG. 7. Heterochromatin aggregation (arrows) andperinuclear orientation of rickettsiae. The nuclearmembrane is no longer prominent. Bar = 0.2 p.m.
Research is in progress to define the specificcomponent(s) responsible for pathogenicity andvirulence and to determine the factors responsi-ble for their modification or loss.
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