Mixed infection of Rickettsiella phytoseiuli and Coxiella burnetii in Dermacentor reticulatus female ticks: Electron microscope study

JOURNAL

OF INVERTEBRATE

PATHOLOGY

%,407-416(1990)

Mixed infection of Rickettsieh phytoseiuli and Coxiella burnetii in Dermacentor reticulatus Female Ticks: Electron Microscope Study J. ~EHACEK

G.&JTAKOVAAND

Institute of Experimental Phytopathology and Entomology, Slovak Academy of Sciences, 900 28 Ivanka pri Dunaji, and Institute of Virology, Slovak Academy of Sciences, 842 46 Bratislava, Czechoslovakia Received October 24, 1988; accepted September 15, 1989 Mixed infection of Rickettsiella phytoseiuli and Coxiella burnetii was investigated in hemolymph and organs of experimentally infected females of Dermacentor reticulatus ticks. Following intracoelomic infection, both agents, with the exception of Gene’s organ, multiplied well in the cells of the tick host’s organs. Two out of six developmental stages of R. phytoseiuli, i.e., crystal-forming and small dark particles, in dual infection with C. burnetii revealed marked morphological alterations. C. burnetii in the presence of R. phytoseiuli penetrated into the cortical layer of the synganglion and into the alveoli of the second and third type of salivary glands, but did not occur in the single infection. 8 1990 Academic PXSS. IN. KEY WORDS: Dermacentor reticulatus; Rickettsiella phytoseiuli; Coxielia burnetii; mixed infection; ultrastructure.

INTRODUCTION

concomitant infection with both agents has a negative effect on their hosts. The electron microscope method was used to study the morphology of the propagation and interaction of R. phytoseiuli and C. burnetii in hemolymph and cells of various organs in dually infected D. reticulatus females.

Rickettsiella phytoseiuli is a putative new species of the genus Rickettsiella (Sufakova, 1977a, b; Weiss and Moulder, 1984). It was successfully isolated and propagated in half-engorged ticks, Dermacentor reticulatus, where it multiplied as in its natural host, mite Phytoseiulus persimilis, comprising six morphologically well-differentiated stages (&tfakova and Rehacek, 1988, 1989). R. phytoseiuli, in contrast to other species of Rickettsiella in arthropods, did not cause any harm to its natural host, i.e., its viability, fertility, etc. were normal as observed in the colonized infected mites at the Institute of Experimental Phytopathology and Entomology in Ivanka pri Dunaji since 1983. Mixed microbial arthropod infections represent an interesting biological phenomenon from the standpoint of mutual interaction of microorganisms in the arthropods. The present investigations were undertaken to determine whether tolerance, interference, or stimulation occurs between R. phytoseiuli and C. burnetii in dually infected ticks D. reticulatus and whether this

MATERIALS

AND METHODS

As inoculum for the infection of ticks, the first passage of the isolate of R. phytoseiuli in D. reticulatus ticks (Sufakova and Behacek, 1988) which had been stored at -70°C for 6 months was used. The inoculum of C. burnetii used was from strain Nine Mile, phase I, third passage in yolk sacs of embryonated hens eggs. The dose of C. burnetii was about lo3 EID,, per tick, the concentration of rickettsiella was estimated to be higher than of C. burnetii based on comparisons of smears on light microscopical slides. The ticks D. reticulatus were collected in a meadow forest near Bratislava. After verification by the hemocyte test that they contained no rickettsiae and rickettsia-like organisms, they were semiengorged on lab407 0022-201

l/90 $1 so

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oratory rabbits and used as recipients for both rickettsiella and coxiella infection by intracoelomal route of inoculation. The ticks were inoculated with either R. phytoseiuli (a) or 6. burnetii (b) (monoinfections). In double infection, experimental ticks were inoculated initially with R. phytoseiuli and 14 days thereafterwith C. burnetii (c) or the reverse(d) or simultaneously with both agents(dual infection) (e). From each of these combinations three females were involved in the electron microscope study. For the study of developmentof the infection, the ticks were used following the 21st day after the first infection. The hemolymph, salivary glands, Malpighian tubules, synganglion,ovaries, Gene’s organ, digestivetract, fat body, and trachealcomplex were usedin this study. The dissected organs and hemolymph were placed into fixative solution (Ito and Kamovsky, 1968) for about 20hr at 4°C. Other proceduresfor the preparationof the material for electron microscopy were introduceda previous paper by Suf&kova(1988).The ultrathin sections were evaluatedin a TESLA BS 500 electron microscope and photographedon ORWO EU 2 glassplates. RESULTS I. Monoinfection

of Rickettsiae

in Ticks

a. Monoinfection of R. phytoseiuli in D. reticulatus females. The results obtained completely confirmedprevious data on cultivation of R. phytoseiuli in D. reticulatus (Sufakova and RehaCek, 1989).With the exception of Gene’s organ which was not infected, hemolymph and all remaining organs showed all six known developmental forms or stages of R. phytoseiuli, i.e., dense (elementary), intermediate, bacterial, giant, and crystal-forming in which small dark particles (initially particles) had developed (Figs. I and 2). We consider thesesmall dark particles as the first developmental phase. When the small dark particles are releasedfrom the crystal-forming

REHACEK

rickettsiae,they apparentlygrow, gaintheir membranecomplex, andchangein this way to the densetype of rickettsiae. They seem to be transformedto intermediateand later to bacterial types of rickettsiae. The bacterial types of rickettsiae can multiply by binary fission. Some bacterial rickettsiae increasegreatly to form the giant type of rickettsiae. Theseform a crystal inside and so they are converted into crystal-forming types of rickettsiae in which then small dark particles develop (Sufakova, 1988). b. Monoinfection ofC. bumetii in D. reticulatus females. C. burnetii infection was less extensive than the R. phytoseiuli monoinfection in D. reticulatus females. Gene’s organ and synganglionwere uninfected. In salivary glandsonly alveoli of the first type contained this microorganism. The morphology and developmental cycle of C. burnetii is describedby McCaul and Williams (1981). 2. Dual Infection of R. phytoseiuli and C. bumetii in D. reticulatus Females

c. R. phytoseiuli inoculated first, 14 days prior to C. bumetii infection. Both R. phytoseiuli and C. burnetii were found in hemolymph and all organs, where the first rickettsiae were seen in monoinfection; both rickettsiae were not found in Gene’s organ. Everywhere were both rickettsiae were seenthe more intensive infection was that of R. phytoseiufi. Both rickettsiaewere seenin the host cells, often close together, and, with sporadic exceptions, they were located in small and large vacuoles. It should be emphasizedthat it is difftcult to differentiate the occurrence of dense and bacterialforms of R. phytoseiuli from small cell variants (SCVs) and large cell variants (LCVs) of C. burnetii in one associatecell (McCaul and Williams, 1981). The most comprehensiveinfection of both rickettsiae was in the fat body, in the cortical layer of synganglion (Fig. 3), and in the tracheal complex (Fig. 4). Less extensive infection occurredin salivary glands,where all three acini types were infected. A similar feature

MIXED

INFECTION

OF R. phytoseidi AND C. burnetii

FIGS. 1 AND 2. Monoinfection of Rickettsiella phytoseiuli in the tick Dermacentor reticulatus. Dense (DR) and intermediate (IR) rickettsiae (Fig. l), bacterial (BR), giant (GR), crystal-forming (CR) rickettsiae with small dark particles (SP) inside them (Fig. 2). Bar = 500 nm.

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SU-~AKOVAANDRJ~-ACEK

..’ .:

FIGS.3 AND 4. Dual infection (cl. Rickettsiella phytoseidi (Rp) and Coxiella burnetii cortical layer of synganglion (Fig. 3) and tracheal complex (Fig. 4). Bar = 2 grn.

.,

(Cb) in the

MIXED

INFECTION

OF R. phytoseiuli

was demonstrated in hemolymph. The distal parts of the Malpighian tubules were infested only sporadically; proximal and central areas were never totally infected. The same data were obtained in ovaries, where small vacuoles with R. phytoseiuli and C. burnetii occurred only in the lateral and central oviducts. In dual infection, the single presence of R. phytoseiuli or C. burnetii in vacuoles can be differentiated morphologically according to the morphological types of cells: C. burnetii has only two types, i.e., .SCVs and LCVs, and R. phytoseiuli has six types, from which, in our study, dense, bacterial, and crystal-forming cells with small dark particles were prevalent. Most of crystalforming rickettsiae displayed marked morphological changes-crystals were disintegrated into smaller parts and most of small dark particles within crystal-forming cells were broader and longer. However, no lysis of crystals was seen (Fig. 3). d. C. burnetii inoculated first, 14 days prior to R. phytoseiuli inoculation. Comparing this combination with the treatment c, we found that the intensity of the infection of both pathogens was the same and both pathogens failed to attack the Gene’s organ. In contrast to its monoinfection when it did not infect the brain and alveoli of the second and third types of salivary gland, C. burnetii, in dual infection with R. phytoseiuli, also attacked the alveoli of the second and third type in the case when rickettsiella did not occur in its close proximity (Fig. 5). The same situation also occurred in synganglion, where C. burnetii penetrated into the cortical layer only in the case when the tick was also inoculated with R. phytoseiuli. C. burnetii appeared dominant in hemolymph (Fig. 6) and in fat body (Fig. 7); nevertheless, the infestation of R. phytoseiuli in both of these tissues was also high. The presence of C. burnetii and R. phytoseiuli in Malpighian tubules, ovaries, and digestive tract was the same as in the reverse combination of the application of pathogens into D. reticulatus tick where

AND

C. burnetii

411

their occurrence was lower compared to that in hemolymph and fat body. A similar feature of dual infection was seen in the tracheal complex, where both pathogens were more frequent. We were able to detect rickettsiae in the lumen of salivary glands (Fig. 8), but it was impossible to determine whether they were dense forms of R. phytoseiuli or SCVs of C. burnetii. Also, in this combination of the inoculation of C. burnetii and R. phytoseiuli into D. reticulatus, morphological alterations of crystalforming rickettsiae and small dark particles of R. phytoseiuli were observed. Compared with inoculation procedure c, in addition to the disintegration of crystals in crystalforming rickettsiae, the lysis of crystals was also observed. e. Simultaneous application of R. phytoseiuli and C. burnetii in D. reticulatus ticks. As in both previous combinations of R. phytoseiuli and C. burnetii infection in D. reticulatus ticks, only Gene’s organ remained uninfected. With simultaneous inoculation with both rickettsiae, the fat body, synganglion, and tracheal complex were the most infected organs. In the fat body it was almost impossible to see pathogen-free cells (Fig. 9). Similar features were seen in the cortical layer of synganglion and in the tracheal complex (Fig. 10). In contrast to dual infection (d), salivary glands were the maximally infected organs (Figs. 12, 13). The infection in hemolymph was very extensive, both rickettsiae occurred either free in the hemolymph or infected hemocytes (Fig. 11). Less frequent infection was similar to that of the previous inoculation (6) as in Malpighian tubules, ovaries, and digestive tract. R. phytoseiuli and C. burnetii occurred prevalently in host cells in vacuoles of small and large dimensions and only in some cells of the tracheal complex the occurrence of both pathogens was detected outside of vacuoles (Fig. 10). As in previous cases, the occurrence of both pathogens is possible to differentiate only by their developmental stages. Also, in this

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AND REHACEK

FIGS. 5-8. Dual infection (4. Coxiella burnetii in the alveoli of the second type of salivary gland (Fig. 5), hemolymph (Fig. 6), and fat body (Fig. 7) of tick. Dense rickettsiae Rickettsiella phytoseiuli or small cell variant C. burnetii in central salivary duct of tick (Fig. 8). Cu, cuticle; R, rickettsiae; LCV, large cell variant; SW, small cell variant. Bar = 2 bm.

MIXED

INFECTION

OF

R. phyfoseiuli AND

C.

burnetii

FIGS. 9-l 1. Simultaneous dual infection (e). Rickedella phytoseiuli (Rp) and Coxiella burnetii (Cb) in the fat body (Fig. 9), tracheal complex (Fig. lo), and hemocyte (Fig. 11) of tick. Disintegrating crystal-forming cells R. phytoseiuli (CR), giant cells R. phytoseiuli (GR), nucleus (N). Bar = 2 pm.

SU+AKOVAAND

REHACEK

(Rp) and CoxieNa burnetii FIGS. 12 AND 13. Simultaneous dual infection(e). RickettsielZaphytoseiuli (Cb) in the salivary gland of tick (Fig. 12) and destruction and lysis of the crystal-forming cells of R. phyroseiuli (CR) (Fig. 13). Bar = 2 Wm.

MIXED

INFECTION

OF R. phytoseiuli

case of simultaneous infection of R. phytoseiuli and C. burn&ii in D. reticulutus ticks, the damage of crystal-forming rickettsiae was noticed. This is the same situation as when C. burnetii in dual infection with R. phytoseiuli was inoculated into ticks first. Crystal-forming rickettsiae showed crumbling of crystals as well as lysis.

AND

C. burnetii

415

ing rickettsiae showed a tendency to divide by binary fission or exhibit an irregular shape in single extensive infection of R. phytoseiuli in experimental host D. reticulatus. In the developmental cycle of R. phytoseiuli in primary host P. persimilis, binary fission occurred only in the bacterial form of the rickettsiae (Sufakova and Rehaeek, 1989). The abnormalities noted above in the DISCUSSION crystal-forming form in single infection of As reported in monoinfection of R. phy- R. phytoseiuli could result from a disturtoseiuli (Sufakova and RehaEek, 1988, bance in the relationship between a host 1989) as well as in C. burnetii (RehaEek and cell and the rickettsiae (Popov, 1985). It apSufakova, 1989) both pathogens also prop- pears that the abnormalities in the crystalagate in dual infections. However, R. phy- forming rickettsiae of R. phytoseiuli and abtoseiuli quantitatively prevailed. In all normal small dark particles resulted from treatments (u-e) the organs attacked most the presence of C. burnetii since they were were fat body, cortical layer of synganfound only in the infected tissues taken glion, and tracheal complex. In treatment e from the dual-infection experiments. the salivary glands also were most infected. These quantitative changes of crystalIn qualitative evaluation of the material, the forming rickettsiae and small dark particles dominant position probably belongs to C. of R. phytoseiuli during their mixed infection with C. burnetii are comparable with burn&ii since some of the crystal-forming rickettsiae with small dark particles were those observed in R. phytoseiuli by facultative mixed infection with virus-like partimorphologically altered in the dual infection experiments (c-e). There appeared to cles (VLPs) in P. persimilis mites. In this case in the presence of VLPs, the more exbe a crumbling of some crystals in different pressed changes in developmental stages of stages of lysis. Some small dark particles were wider and two to three times longer R. phytoseiuli were observed when besides than those in treatment a. In natural infecthose changes in crystal-forming rickettsiae tions of R. phytoseiuli in P. persimilis as and small dark particles an irregular diviwell as in experimental infections in D. re- sion of bacterial rickettsiae, the division of dense rickettsiae, and other minor changes ticulatus, small dark particles were visible were also registered (Sutakova and Ruttin crystal-forming rickettsiae only outside of the crystals but in mixed infections with gen, 1978). C. burnetii, they were present inside as well We are aware that with the use of ultraoutside of the crystals. Other stages of R. structural studies only it is difficult to evaluate the mutual influence of pathogens inphytoseiuli such as dense, intermediate, bacterial, and giant cells showed no dam- vestigated and that for this process it will age. No altered morphology was seen in be necessary to find a new biochemical method. SCVs or LCVs of C. burnetii. It can be hypothesized that in the case of The above-mentioned abnormal crystalforming rickettsiae were not seen in treat- treatment d, the infection of C. burnetii in D. reticulatus was more extensive due to ment a or in the study reported by Sufakova the presence of R. phytoseiuli acting as a and RehaEek (1988, 1989). The changes helper pathogen. noted in double-infection experiments (c-e) On the basis of our results from the were observed in areas where extensive inmixed infection of R. phytoseiuli and C. fections occurred. However, crystal-form-

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in D. reticulatus tricks we can, even if indirectly, confirm the conclusions stated by Avakyan et al. (1983) and Avakyan and Popov (1984),who proposed to classify Coxiella andRickettsiella genera into a secondtransitional groupof microorganisms between Rickettsiaceae and Chlamydiaceae. On the basisof our results with R. phytoseiuli and C. burnetii in addition to resultsknown for Chlamydia (Popov et al., 1978;Avakyan et al., 1983;Avakyan and Popov, 1984;Chi et al., 1987)there arises the question of the possibility of a complex of the genera Chlamydia, Coxiells, and Rickettsiella. burnetii

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