spotted fever rickettsiae. Mediterranean spotted fever, a tick-transmitted rickettsi- osis endemic in southern Europe, Africa, and Asia (24), has seldom been ...
JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1993, p. 2225-2227
Vol. 31, No. 8
0095-1137/93/082225-03$02.00/0 Copyright © 1993, American Society for Microbiology
Prevalence of Antibodies to Spotted Fever Group Rickettsiae along the Eastern Coast of the Adriatic Sea SUZANA RADULOVIC,l* DAVID H. WALKER,1 KIMBERLY WEISS,' BORIS DZELALIJA,2 AND MIRO
MOROVIC2
Department of Pathology, University of Texas Medical Branch, 11th Street and Te-xas Avenue, G. 129, Galveston, Texas 77555-0609, 1 and Department of Infectious Diseases, Medical Center of Zadar, Zadar, Croatia Received 21 January 1993/Accepted 10 May 1993
A seroepidemiological survey in coastal Croatia detected antibodies reactive with Rickettsia conorii in 4.2% of sera by immunofluorescence assay and in 5.0%o of sera by enzyme immunoassay. Western immunoblotting demonstrated antibodies to the 120-kDa surface protein in all 20 positive serum samples examined and to rickettsial lipopolysaccharide in 3 of these serum samples. Humans in this area are clearly being exposed to spotted fever rickettsiae.
Mediterranean spotted fever, a tick-transmitted rickettsiosis endemic in southern Europe, Africa, and Asia (24), has seldom been reported in the Yugoslavian literature (25). The incidence of Mediterranean spotted fever appears to be increasing in the Mediterranean basin (11), including the Adriatic coast of Croatia (15). A spotted fever group (SFG) rickettsiosis was first described for this area by Tartaglia in 1935 (22). According to serosurveys in the Mediterranean basin, the prevalence of antibodies to SFG rickettsiae is as high as 73.5% by the indirect immunofluorescence assay (IFA) (8). In 1984, antibodies to SFG rickettsiae were demonstrated by complement fixation in the sera of 5% of 730 healthy persons from eight islands in North Dalmatia (15), and in 1990 the IFA showed a substantial seroprevalence among rural and urban populations (5). The area of investigation consists of a typical stony karst region, covered with dense shrubs and bushes of Mediterranean type in places altered by human changes. The vegetation and microclimatic factors provide a splendid habitat for small mammals, reptiles, and a large population of ticks (4, 12, 13). Sera collected from 477 healthy subjects living in the Adriatic coastal region including Istra (n = 42), Zadar (n = 57), Split (n = 103), and Dubrovnik (n = 275) were tested by IFA, enzyme immunoassay (EIA), and Western immunoblot assay (WB) to detect antibodies reactive with Rickettsia conorii antlgens. R. conorii (Moroccan and Malish-7 strains) isolates obtained from the American Type Culture Collection were cultivated in confluent Vero cells, which were applied to microscope slides, air dried, and fixed with absolute acetone (14). Sera were serially diluted in phosphate-buffered saline (PBS) containing 3% nonfat dry milk, incubated on the antigen slides in a moist chamber for 30 min at 37°C, and washed three times with PBS. Fluorescein isothiocyanateconjugated goat anti-human immunoglobulin G (IgG) (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) diluted 1:100 was incubated on the slides for 30 min at room temperature and washed in PBS. For microtiter EIA, wells of polystyrene U-bottom micro* Corresponding author. Electronic mail address: bitnet: dwalker @beach.utmb.edu.
titer plates were coated overnight with 100 ,ul of R. conorii antigens diluted 1:25 in 0.1 M sodium carbonate buffer (pH 9.6). The wells were then washed three times with 0.25 ml of PBS with 0.05% Tween 20 and covered with 100 ,ul of PBS-3% nonfat dry milk for 1 h at 37°C. After the plates were washed, 100 ,ul of test or control serum diluted 1:200 in PBS was added. The plates were reincubated and washed, and 100 pl of conjugate (alkaline phosphatase-labeled goat anti-human IgG) diluted 1:1,000 in PBS was added to each well. After 1 h at 37°C, the wash was repeated, and p-nitrophenyl phosphate substrate (1 mg/ml in 10% diethanolamine buffer, pH 9.8) was added. Following incubation for 45 min at 37°C, the reaction was stopped with 3 M NaOH (50 pl per well), and optical density (OD) was measured with a microplate reader (Bio-Tek Instruments, Winooski, Vt.) at a wavelength of 405 nm. For each specimen, the net OD was calculated. The mean and standard deviation of net ODs from 37 negative controls were 0.06 + 0.036. Any net OD of more than 3 standard deviations above the mean negative value (0.17) was regarded as positive. For the WB assay, purified Malish-7 strain rickettsiae were dissolved in final sample buffer, and the proteins were separated by electrophoresis in a 7% sodium dodecyl sulfate-polyacrylamide gel and transferred to nitrocellulose paper (10). Nonspecific binding sites were blocked with PBS containing 5% nonfat dry milk. Each human serum sample was diluted at 1:200 in PBS-3% milk and incubated with the nitrocellulose strips for 1 h with gentle shaking at room temperature. Alkaline phosphatase-conjugated goat anti-human immunoglobulins (anti-IgG plus anti-IgM; Bio-Rad Laboratories, Richmond, Calif.) diluted 1:100 in PBS-3% milk were incubated with the strips for 1 h. After three 10-min washes in PBS, the bound enzyme was detected with 4 ng of fast red salt per ml-2 ng of naphthol AS-MX phosphate per ml in 50 mM Tris, pH 8, and the strips were washed in distilled water for 10 min to stop the reaction. Of 477 serum samples tested by IFA, 20 (4.2%) contained antibodies to R. conorii at a titer of >64 (Table 1). Two serum samples from Dubrovnik reacted with the Malish-7 strain at the cutoff titer of 64 and did not react with the Moroccan strain. Similarly, one serum sample each from Istra and Zadar reacted at a titer of 64 with only the Moroccan strain. By EIA, 24 (5.0%) were positive for IgG 2225
2226
NOTES
J. CLIN. MICROBIOL.
TABLE 1. Antibodies to different R. cononi strains in sera from inhabitants living along the Adriatic coastal regions of Croatia tested by IFA and EIA No. positive (%) by assay with strain:
Region
No. of serum
Moroccan
samples tested
IFA
Istra Zadar
Split Dubrovnik Total
42 57 103 275 477
1 5 14 0 20
EIA
Malish-7 IFA
EIA
(2.4) 1 (2.4) 1 (2.4) 2 (4.8) (8.8) 6 (10.5) 4 (7.0) 6 (10.5) (13.6) 14 (13.6) 13 (12.6) 14 (13.6) 0 (0) 2 (0.7) 2 (0.7) (0) (4.2) 21 (4.4) 20 (4.2) 24 (5.0)
antibodies. Three serum samples reacted only with the Malish-7 strain. Twenty-one serum samples were positive by both IFA and EIA, whereas one serum sample was positive only by IFA and two serum samples were positive only by EIA. The WB assay demonstrated that all 20 serum samples containing IFA and/or EIA antibodies that were examined reacted with the 120-kDa protein antigen, three serum samples reacted with the rickettsial lipopolysaccharide bands at molecular weights of 20 to 50 kDa, and one serum sample reacted with the 60-kDa antigen. A pool of 37 negative serum samples from Vermont, where there are no SFG rickettsioses, did not react with any rickettsial antigens by WB. A pool of five convalescent serum samples from Spanish Mediterranean spotted fever patients used as the positive WB control reacted with the 120-kDa antigen. In Croatia and many other parts of the world, SFG rickettsiosis is seldom considered or diagnosed, in part because of the difficulty of the clinical diagnosis of SFG rickettsioses and in part because of the frequent lack of availability of specific laboratory diagnostic tests. Thus, recognition of a persistent, endemic SFG rickettsiosis can be delayed for years even when a substantial number of cases
occurring (21). Serologic surveys have been employed to estimate the importance of various infectious diseases. This approach offers the possibility of greater sensitivity and specificity than does reviewing medical records or questioning persons regarding a past history of SFG rickettsiosis. Serologic surveys in the area considered to be endemic for boutonneuse fever have included Portugal (1), Spain (6, 8, 20), France including Corsica (16, 17), Italy including Sicily (23), Israel (19), Egypt (3), the Central African Republic (7), Ivory Coast (18), and Zimbabwe (9). Problems with serosurveys include the selection of an assay that will detect persistent antibodies and selection of a cutoff value that provides useful sensitivity and specificity. The use of the three most specific techniques available (IFA, EIA, and WB) yielded a high degree of agreement. It appears that SFG rickettsiae are endemic in this area. Inhabitants and visitors, either military personnel or tourists, are at risk of infection. Although this investigation has documented the presence of antibodies to SFG rickettsiae in a healthy population more convincingly than previous reports, the question of what SFG rickettsial species stimulated the antibodies remains unanswered. The possibilities include the diverse strains of R. conorii (24), the Israeli SFG rickettsia, R. akani, R. sibirica, R. slovaca, R. helvetica, and the two recently identified novel SFG rickettsial serotypes from Rhipicephalus turanicus ticks (2). Isolation and identification of rickettsiae from patients will provide definitive etiologic data, and recovery and identification of rickettsiae are
from ticks and mites will suggest the possibilities for the other rickettsial species that might be involved. Ultimately, serologic specificity will be achieved only when an assay of antibodies to species-specific rickettsial epitopes becomes available. This research project was supported in part by a grant from the National Institute of Allergy and Infectious Diseases (Al 21242) and by a grant from the Research Parliament of Slovenia. We thank Margie Benitez and Carol Lehr for secretarial assistance in the preparation of the manuscript. REFERENCES 1. Bacellar, F., M. S. Nuncio, J. Rehacek, and A. R. Filipe. 1991. Rickettsiae and rickettsioses in Portugal. Eur. J. Epidemiol. 7:291-293. 2. Beati, L., J. P. Finidori, B. Gilot, and D. Raoult. 1992. Comparison of serologic typing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein analysis, and genetic restriction fragment length polymorphism analysis for identification of rickettsiae: characterization of two new rickettsial strains. J. Clin. Microbiol. 30:1922-1930. 3. Botros, B. A. M., A. K. Soliman, M. Darwish, S. El Said, J. C. Morrill, and T. G. Ksiazek. 1989. Seroprevalence of murine typhus and fievre boutonneuse in certain human populations in Egypt. J. Trop. Med. Hyg. 92:373-378. 4. Cvjetanovic, B. 1956. 0 vrstama, razprostranjenosti i sezonskom javljanju krpelja u Dalmaciji s osvrtom na javljanje tickparalize. Veterinaria (Sarajevo) 5:589-596. 5. Dzelalija, B., M. Morovic, and S. Novakovic. 1991. Rickettsial antibody in North Dalmatia: antibodies to Rickettsia cononi, Rickettsia akari, Rickettsia typhi, Rickettsia slovaca and Coxiella bumetii among urban and rural population, p. 457-464. In J. Kazar and D. Raoult (ed.), Rickettsiae and rickettsial diseases. Publishing House of the Slovak Academy of Sciences, Bratislava, Czechoslovakia. 6. Garcia, G. A., and E. Najera-Morrando. 1984. E studio epidemiologico de las rickettsiosis en la provincia de Sevilla, basado en las reacciones serologicas de immunofluorescencia indirecta. Rev. Sanid. Hig. Publica (Madrid) 58:83-98. 7. Gonzales, J. P., P. Fiset, A. J. Georges, J. R. Saluzzo, and C. L. Wisseman, Jr. 1985. Approche serologique sur l'incidence des rickettsioses en Republique Centrafricaine. Bull. Soc. Pathol. Exot. 78:153-156. 8. Herrero-Herrero, J. I., R. Ruiz-Beltran, A. M. Martin-Sanchez, and E. J. Garcia. 1989. Mediterranean spotted fever in Salamanca, Spain. Epidemiological study in patients and serosurvey in animals and healthy human population. Acta Trop. 46:335350. 9. Kelly, P. J., P. R. Mason, L. A. Matthewman, and D. Raoult. 1991. Seroepidemiology of spotted fever group rickettsial infections in humans in Zimbabwe. J. Trop. Med. Hyg. 94:304-309. 10. Li, H., and D. H. Walker. 1990. Biological characterization of major polypeptides on surface of spotted fever group rickettsiae. Ann. N.Y. Acad. Sci. 590:389-394. 11. Mansueto, S., G. Tringali, and D. H. Walker. 1986. Widespread, simultaneous increase in the incidence of spotted fever group rickettsioses. J. Infect. Dis. 154:539-540. 12. Mikacic, D. 1965. Krpelji primorskog pojasa Jugoslavije. Razprostranjenost i dinamika pojedinih vrsta u toku godine. Vet. Arh. 35:155-170. 13. Oswald, B. 1940. 0 prepoznavanju krpelja (Lxodidae) u Jugoslaviji. Vet. Arh. 10:297-300. 14. Philip, R. N., E. A. Casper, R. A. Ormsbee, M. G. Peacock, and
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