System of 52 Strainsof Spotted Fever Group Rickettsiae from ...

JOURNAL OF CLINICAL MICROBIOLOGY,

JUlY 1990, P. 1597-1599

Vol. 28, No. 7

0095-1137/90/071597-03$02.00/0 Copyright © 1990, American Society for Microbiology

Isolation by a Sensitive Centrifugation Cell Culture System of 52 Strains of Spotted Fever Group Rickettsiae from Ticks Collected in France O.

PÉTER,t D. RAOULT,*

AND

B. GILOT

Centre National de Référence des Rickettsioses, Centre Hospitalier Universitaire La Timone, Marseille, France Received 9 November 1989/Accepted 23 January 1990

Boutonneuse fever caused by Rickettsia conorii is transmitted mainly by the brown dog tick, Rhipicephalus sanguineus. We collected 540 ticks in Marseille, France, and tried to isolate as many strains of rickettsia as possible. Ticks were evaluated for the presence of rickettsia by the hemolymph test and by a new culture system, the centrifugation-shell vial technique. We avoided contamination in the culture system. Prior to ticks being submitted to the hemolymph test, they were disinfected. Only 5.6% (27 of 478) of the cultures were contaminated. A drop of hemolymph from each of 478 R. sanguineus ticks was cultured in two shell vials, and another drop was stained by the Gimenez method or indirect immunofluorescence. Since Gimenez staining in our hands was not satisfactory, comparison of the hemolymph test and culture is based on the results of indirect immunofluorescence. Thus, 50 of 369 (13.5%) examined ticks were hemolymph test positive, and 44 (11.9%) cultures were positive. After disinfection, another pool of 62 ticks were examined by the hemolymph test. The ticks were kept individually in a sterile environment. A few days later, the hemolymph of these ticks was collected again and cultured. The contamination rate was not significantly higher (6.4%) than in the above-described conditions. It allowed us to isolate eight more strains. Thus, we recommend screening ticks with the hemolymph test and culturing only the hemolymph test-positive ticks.

Mediterranean spotted fever is caused by Rickettsia conorii. It is endemic in Mediterranean countries where Rhipicephalus sanguineus, the brown dog tick, is the main vector. In Marseille, France, within the urban area, the disease is endemic (14) and the seroprevalence approaches 80% in dogs (13) and 20% in humans (12). In order to determine the prevalence of ticks infected with spotted fever group (SFG) rickettsiae and eventually to enable identification of nonpathogenic strains, it would be useful to isolate a number of rickettsiae from ticks. The isolation of SFG rickettsiae has previously been accomplished by the inoculation of infected tick tissues in embryonated hen eggs, laboratory animals (e.g., guinea pigs and meadow voles) (3), or Vero cells (1, 8, 10, 15). Wike and Burgdorfer (16) reported direct isolation of rickettsiae from ticks. The centrifugation-shell vial technique was first used for the rapid detection of cytomegalovirus (7) and herpes simplex virus (11) in clinical specimens. Recently, it was adapted to the timely diagnosis of Mediterranean spotted fever by the rapid cultivation of rickettsiae from the blood of patients (5, 9). With this technique, rickettsial growth is detected within 2 days, thus dramatically accelerating the establishment of a definitive diagnosis. Experience has shown (unpublished data) that 48 h is sufficient to detect infection in this system without a loss of sensitivity, compared with the 72 h as published previously (5, 9). We applied this technique to the detection and establishment in culture of SFG rickettsiae from the hemolymph of ticks. This study was divided into two parts. Part 1 was the direct detection of rickettsiae in the hemolymph (2) compared with the detection in the culture system. In part 2, we performed the hemolymph test (HT), which was followed a few days later by the culture of a * Corresponding author. t Present address: Clinical Microbiology, Institut Hôpitaux Valaisans, CH-1951 Sion, Switzerland.

Central des

second sample of hemolymph in the shell vial. We evaluated the culture contamination rate between immediate and delayed hemolymph cultures (part 1 versus part 2). MATERIALS AND METHODS

Ticks. A total of 503 adult R. sanguineus ticks were collected from houses and dogs in Marseille. Nine Riphicephalus turanicus and 28 Dermacentor marginatus ticks were collected in southeastern France. Prior to performance of the HT, pools of 10 to 20 ticks were disinfected by immersion for 10 min in iodated alcohol (12 ml of tincture of iodine and 6 ml of ethylene glycol per liter of 70% ethanol). The ticks were then rinsed twice for 5 min in distilled water and allowed to dry on sterile filter paper in petri dishes. Cells. Human embryonic lung fibroblasts were grown in minimal essential medium with 10% fetal calf serum. Shell vials (3.7 ml; Sterilin, Feltham, England) with 12-mm round cover slips were seeded with 1 ml of medium containing 50,000 cells and incubated in a 5% C02 incubator for 3 days to obtain a confluent monolayer. HT and inoculation. The first group of 478 R. sanguineus ticks were disinfected and subjected individually to an HT by cutting of a leg under a laminar flow hood. The hemolymph drop was deposited in 300 ptI of brain heart infusion broth, and this mixture was placed into two shell vials. The shell vials were centrifuged at 700 x g for 1 h at 37°C. The inoculum was discarded, and fresh medium (minimal essential medium supplemented with 5% fetal calf serum) was added. The vials were placed in a 5% C02 incubator at 37°C. A second HT was performed with another leg of each tick. The droplet was placed on a microscope slide and allowed to dry until it was stained. A second group of 62 ticks (25 R. sanguineus, 9 R. turanicus, and 28 D. marginatus) were subjected to HT. Then each tick was maintained individually in a sterile test tube with a cotton 1597

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TABLE 1. Comparison between HT by immunofluorescence and the shell vial culture of 369 ticks Result with: No. of ticks

40 8 3 1

2 315

HT + + -

+

IFA on dissected tick tissues

Shell vial culture

+ +

+ _c + +

ND

-

-

a +, Positive; -, negative; ND, not done. b Confirmed by dissection in cases of discrepant results. c Two were contaminated.

stopper. One or two days later, another drop of hemolymph was cultivated in the shell vials, as described above. Staining methods. HT specimens were stained by the Gimenez method (6) or reacted with a human convalescentphase serum to Rickettsia conorii diluted 1/200 in an indirect fluorescent-antibody test (IFA). We used fluorescein isothiocyanate-conjugated goat polyclonal anti-human immunoglobulin serum (Immunotech, Marseille, France). After 7 days of culture, the cover slip from one of the two inoculated vials was stained with an IFA inside the shell vial, as described previously (9), with hyperimmune rabbit serum to Rickettsia conorii. Prior to fixing, a drop of culture medium was inoculated to a Columbia medium plate and incubated at 37°C with 5% CO2 to test for sterility. The HT and cultures were performed independently by two of us, without knowing the results obtained by the other. A result that was immunofluorescent for the shell vial was considered positive when three coccobacillary organisms were observed. Final establishment of the strains was made through serial subcultures in human embryonic lung fibroblasts.

Confirmation of discrepant results. We considered a tick to be positive or negative when the HT and culture results were in accordance. When discrepant results were observed between the HT and culture system, living ticks were dissected for preparation of multiple tissue smears with the hypodermis, salivary glands, and Malpighian tubules. The smears were stained by an IFA as described above. RESULTS The initial use of Gimenez staining proved unsatisfactory; thus, the remaining 369 tick hemolymph samples were examined by an IFA. Inoculation of 478 tick hemolymph samples into shell vials yielded only 27 contaminated cell cultures (5.6%). The comparison between the HT and culture system is based on the results from only the 369 ticks whose hemolymph was stained by IFA. A total of 50 ticks were found infected with rickettsiae by the HT; 44 cultures were positive (Table 1). We observed that cultures inoculated with material from unfed female ticks frequently showed few scattered cells infected with rickettsiae (Fig. la). In contrast, many foci of rickettsia-infected cells (Fig. lb) or cell cultures packed with rickettsiae (Fig. lc) were regularly observed with the hemolymph of engorged females. Cultures varied from being strongly positive to containing fewer than 10 organisms. The 14 discrepant results were as follows: 10 positive HT yielded negative cultures for SFG rickettsia, and four positive cultures were observed from HT-negative ticks. The tissue smears from 11 of these ticks confirmed the results of eight positive and three negative HT. Thus, six cultures gave false-negative results, and two were contaminated (they were weakly positive cultures). Three cultures gave false-positive results. Furthermore, one false-negative HT was found as tissue smears of one tick revealed a typical rickettsial infection. Two ticks were dead and could not be dissected (Table 1). In part 2 of this study, only 4 of the 62 cultures (6.4%)

ng~~~~~~~~~~~~~~~~~~

FIG. 1. (a) Rare infected human embryonic lung fibroblasts with a few SFG rickettsiae 7 days after inoculation of hemolymph from unfed female R. sanguineus (indirect immunofluorescent staining; magnification, x ,000); (b) Focus of infected human embryonic lung fibroblasts with SFG rickettsiae 7 days after inoculation of hemolymph from partially fed or fully engorged female R. sanguineus (indirect immunofluorescent staining; magnification, x400); (c) as in panel b. with an abundant rickettsial growth invading all of the cell culture (magnification, x 100).

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were contaminated when the ticks were first examined by the HT and subsequently cultured. Of these cultures, we isolated eight strains of SFG rickettsiae from the three tick species (R. sanguineus, R. turanicus, and D. marginatus).

ACKNOWLEDGMENTS We gratefully thank G. Vestris, V. Galicher, M. Enea, and M. C. Lafforge for excellent technical assistance. This work was supported by a grant from the Centre National de la Recherche Scientifique (convention 86208).

DISCUSSION

LITERATURE CITED 1. Anderson, J. F., L. A. Magnarelli, R. N. Philip, and W. Burgdorfer. 1986. Rickettsia rickettsii and Rickettsia montana from ixodid ticks in Connecticut. Am. J. Trop. Med. Hyg. 35:187-191. 2. Burgdorfer, W. 1970. Hemolymph test. A technique for detection of rickettsiae in ticks. Am. J. Trop. Med. Hyg. 19: 1010-1014. 3. Burgdorfer, W., A. Aeschlimann, O. Péter, S. F. Hayes, and R. N. Philip. 1979. Ixodes ricinus vector of a hitherto undescribed spotted fever group agent in Switzerland. Acta Trop. 36:357-367. 4. Burgdorfer, W., D. J. Sexton, R. K. Gerloff, R. L. Anacker, R. N. Philip, and L. A. Thomas. 1975. Rhipicephalus sanguineus: vector of a new spotted fever group rickettsia in the United States. Infect. Immun. 12:205-210. 5. Espejo-Arenas, E., and D. Raoult. 1989. First isolates of Rickettsia conorii in Spain using a centrifugation-shell vial assay. J. Infect. Dis. 159:1158-1159. 6. Giménez, D. F. 1964. Staining rickettsiae in yolk-sac cultures. Stain Technol. 39:135-140. 7. Gleaves, C. A., T. F. Smith, E. A. Shuster, and G. R. Pearson. 1985. Comparison of standard tube and shell vial cell culture techniques for the detection of cytomegalovirus in clinical specimens. J. Clin. Microbiol. 21:217-221. 8. Magnarelli, L. A., J. F. Anderson, R. N. Philip, W. Burgdorfer, and E. A. Casper. 1981. Endemicity of spotted fever group rickettsiae in Connecticut. Am. J. Trop. Med. Hyg. 30:715-721. 9. Marrero, M., and D. Raoult. 1989. Centrifugation-shell vial technique for rapid detection of Mediterranean spotted fever rickettsia in blood culture. Am. J. Trop. Med. Hyg. 40:197-199. 10. Philip, R. N., and E. A. Casper. 1981. Serotypes of spotted fever group rickettsiae isolated from Dermacentor andersoni (Stiles) ticks in western Montana. Am. J. Trop. Med. Hyg. 30:230-238. 11. Pruneda, R. C., and I. Almanza. 1987. Centrifugation-shell vial technique for rapid detection of herpes simplex virus cytopathic effect in Vero cells. J. Clin. Microbiol. 25:423-424. 12. Raoult, D., B. Toga, H. Chaudet, and C. Chiche-Portiche. 1987. Rickettsial antibody in southern France: antibodies to Rickettsia conorii and Coxiella burnetii among urban, suburban and semirural blood donors. Trans. R. Soc. Trop. Med. Hyg. 81: 80-81. 13. Raoult, D., B. Toga, S. Dunan, B. Davoust, and M. Quilici. 1985. Mediterranean spotted fever in the south of France: serosurvey of dogs. Trop. Geogr. Med. 37:258-260. 14. Raoult, D., P. J. Weiller, A. Chagnon, H. Chaudet, H. Gallais, and P. Casanova. 1986. Mediterranean spotted fever: clinical, laboratory and epidemiological features of 199 cases. Am. J. Trop. Med. Hyg. 35:845-850. 15. Weiss, E., and H. R. Dresser. 1960. Centrifugation of rickettsiae and viruses onto cells and its effects on infection. Proc. Soc. Exp. Biol. Med. 103:691-695. 16. Wike, D. A., and W. Burgdorfer. 1972. Plaque formation in tissue cultures by Rickettsia rickettsii isolated directly from whole blood and tick hemolymph. Infect. Immun. 6:736-738. 17. Wike, D. A., G. Tallent, M. G. Peacock, and R. A. Ormsbee. 1972. Studies of rickettsial plaque assay technique. Infect. Immun. 5:715-722.

The shell vial culture has been successfully used for diagnosis of viral diseases (7, 11). Recently, Marrero and Raoult (9) and Espejo-Arenas and Raoult (5) demonstrated in a small series of cases that it could be easily adapted to the diagnosis of boutonneuse fever. Indeed, blood from febrile patients in whom rickettsiosis is suspected may be inoculated into this cell culture system. Results were available within 72 h, and strains were isolated and established in the laboratory for future investigation. In the overall study of SFG rickettsiae in Marseille, we tried to isolate strains of SFG rickettsia from R. sanguineus. So far, the isolation process required inoculation of infected ticks into embryonated hen eggs, sensitive animals (guinea pigs or meadow voles), or Vero cells. Shell vial culture was evaluated as a new isolation procedure which had never been applied to rickettsial isolation from ticks. The time for recovery of rickettsiae from ticks in the shell vial assay is longer than from human or guinea pig blood (7 days from hemolymph and only 2 days from blood). The comparison of the HT and shell vial culture was based on results obtained with IFA staining. Indeed, in our hands, the Gimenez method gave poorly stained rickettsiae which did not allow a correct evaluation of both systems. The shell vial culture gave excellent results in detecting 80% of the positive ticks. When compared with Vero cell culture as reported in the literature, this system is much more efficient: Anderson et al. (1) isolated five SFG rickettsiae from 25 IFA HT-positive ticks, Philip and Casper (10) isolated 120 rickettsiae from 177 such ticks, and Magnarelli et al. (8) isolated 4 rickettsiae from 14 such ticks. The explanation may be the increased infectivity of centrifuged samples (15, 17). In one instance, we isolated rickettsiae from an HT-negative tick. We were unable to compare the shell vial assay to animal inoculation because inoculation of 540 animals was cost prohibitive. Moreover, some of the SFG rickettsiae are not easily isolated in animals (3). The HT is more sensitive and less time-consuming than the shell vial culture as a screening test. This was the reason that in part 2 of this study, ticks were screened by a stained HT with IFA, followed 1 or 2 days later by the culture of a second drop of hemolymph. Since the contamination rate remained low, we recommend this procedure and that only the hemolymph from rickettsiainfected ticks be cultured in the shell vials. Shell vial culture is definitely very efficient in the isolation of rickettsiae from ticks and is without any doubt a good alternative to the animal inoculations. Its sensitivity seems to be better than that of the usual Vero cell culture, and it does not require specific equipment. It is specially adapted to large isolation campaigns in tick population and makes it possible to isolate both pathogenic and nonpathogenic rickettsiae.