APPLI MICROBIOLOGY, Apr. 1971, p.

611-613 Copyright 1971 American Society for Microbiology

Vol. 21, No. 4 Printed in U.S.A.

Methods for Accelerating the Fluorescent-Antibody Test for Rabies Diagnosis1

Received for publication 4 December 1970

The time required to perform the fluorescent-antibody test for rabies was reduced by eliminating acetone fixation of the brain impressions and by incubating the conjugate-impression reaction at room temperature for only 10 min. Elimination of the preliminary acetone fixation had no effect on the diagnosis of impression smears from 246 mammalian brains by immunofluorescence. Staining at 37 C for 30 min and staining at room temperature for 10 min were found to be equally effective in the examination of impression smears from 161 brain samples. The procedure, as modified, shortens the time required for the diagnosis of rabies by immunofluorescence from about 5.5 hr to approximately 45 min.
from heads sent to this laboratory for the diagnosis of rabies. A total of 246 brain samples obtained from 195 dogs, 41 cats, 4 laboratory mice, 2 human beings, 1 cow, 1 goat, 1 monkey, and 1 rabbit were used to study performance. the effect of alterations of the fixation method on the Although the use of several fixatives, times of rabies FA test. fixation, and times and temperatures of incubaAlso, 161 brain samples were used for the study of tion has been reported (4, 6-8, 11, 17), the the effect of variations in the incubation schedules on standard FA test calls for air-drying of the im- the FA test. These samples were from 128 dogs, 28 pressions for 30 min, 4 hr of fixation in acetone, cats, 2 laboratory mice, 1 cow, 1 goat, and 1 monkey. FA test. The conjugate used was prepared accordand 30 min of incubation for the conjugateimpression reaction (8), a total of approximately ing to the technique of Lennette et al. (11) and showed a titer of 1:80. The standard FA technique of Gold5.5 hr. wasser et al. (8) was used, and the results were comOther antigens do not require fixation for use pared with those obtained by FA tests in which the in the FA test (2, 3, 16), and it has been demon- methods of fixation and incubation were modified. strated that the initial antigen-antibody combinaModified fixation method. Two microscope slides tion occurs within seconds (14). It seemed, there- with two impressions each were prepared from the fore, worthwhile to attempt to apply these Ammon's horn of each brain sample and were airprinciples to the rabies FA test to accelerate the dried. One slide was left unfixed and the other was fixed in acetone at -20 C for 4 hr as recommended diagnostic process. In this study, the results obtained with varia- by Goldwasser et al. (8). Both were then stained, by tions in the fixation and incubation schedules of the standard staining method, at 37 C for 30 min. Modifid incubation method. Two pairs of impresthe rabies FA test were compared with those were obtained from each brain as above and obtained by the standard technique of Gold- sions were kept unfixed. One of the slides was stained with wasser, Kissling, and Carski (8) and by the mouse conjugate by the standard staining method at 37 C for inoculation test (10). 30 min and the other was stained by the rapid staining method at room temperature (22 to 25 C) for 10 min. MATERIALS AND METHODS After staining, the slides were rinsed as usual. The Brain samples. Brain samples were obtained ac- stained slides were coded so that the person observing cording to the technique described by Tierkel (18) them under the microscope would not know their sources or the fixation and staining methods. The in1 A preliminary report on this study was presented at the II tensity of the specific stain and the amount of antigen Jornadas Argentinas de Microbiologia, 22-26 November 1970, present in the positive smears were graded on a 1 to 4 Buenos Aires, Argentina. scale. A monocular Leitz microscope, model SM, was 2 Holder of a PAHO/WHO training feliowship. Present adused together with an HBO 200 lamp, exciting filters dress: Ministerio de Salubridad P,iblica, San Jose, Costa Rica. 611

The fluorescent-antibody (FA) test is sensitive and specific for rabies diagnosis (13). The only inconvenience of the rabies FA test in comparison with the Sellers' stain is the time required for its

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APPL. MICROBIOL.

UGI (2 mm) and BG38 (4 mm), and barrier filter K 430. Mouse inoculation test. Suspensions from each brain used in this study were prepared as described by Koprowski (10) and inoculated intracerebrally into 10 mice (3 to 4 weeks old). On one occasion, suckling mice were also used.

of the stain were observed in the positive smears stained at room temperature for 10 min than in those stained at 37 C for 30 min. Lower intensity of staining could result in erroneous diagnosis on slides with minimal antigen, but we had no problem in differentiating the positive samples from the negative ones. A kinetic study of the rabies antigen-antibody reaction has not been perRESULTS but a situation similar to that found by Effect of modified fixation. Complete correla- formed, Mayer and Heidelberger (14) for pneumococcal tion among the results of mouse inoculation tests polysaccharides might occur. In their study, 90% and FA tests, with either fixed or unfixed impres- of the reaction was completed in 3 sec, and the sions, was found in 245 of 246 brain samples used remainder "took place with progressively diin this part of the study. In all tests, 105 brains minishing velocity." were positive and 140 were negative. The remainFischman and Ward (5) have found infective ing brain sample was negative by adult mouse rabies virus in impressions fixed with acetone, inoculation and by FA test with the acetone-fixed and, because rabies virus is sensitive to organic slide, but was positive by the FA test in which the solvents (9), the occurrence of infective virus is unfixed slide was used. A suspension of this brain more likely in unfixed fixed smears. If desired, was inoculated into 10 suckling mice, and 2 of this problem could bethan overcome exposing the these animals subsequently died of rabies, as smears to ultraviolet light prior by to staining and demonstrated by FA on their brains. during air-drying, as described by LUpine and In all but two of the positive cases, the amount Gamet (12). of rabies antigen demonstrable in the unfixed The use of the described FA technique would slides was equal to or greater than the amount enable the diagnostic laboratory to report the in the acetone-fixed slides. results more quickly to the physician considering Effect of modified incubation. Of the 161 rabies treatment for the bitten persons. samples used in this part of the present study, 73 were positive and 88 were negative by both FA ACKNOWLEDGMENTS staining methods (staining at 37 C for 30 min or We thank Laura Astarloa, Hospital Mufniz, Buenos Aires, for at room temperature for 10 min), as well as by the human samples and the staff of the Instituto Antirrnbico, mouse inoculation. Mor6n, Argentina, for the animal samples used in this study. The intensity of the stain and the amount of We also gratefully acknowledge the capable assistance of Juan C. antigen detected with the rapid staining method Areitio and Luis Lazaro. were somewhat lower than with the standard LITERATURE CITED staining method in 40% of the positive impres1. Bagnaroli, R. A., 0. P. Larghi, and N. Marchevsky. 1970. sions. Susceptibilidad de ratones lactantes y adultos al virus
rabico demostrado por immunofluorescencia. Bol. Ofic. Sanit. Panamer. 68:388-392. 2. Beutner, E. H., M. R. Sepulveda, and E. V. Barnett. 1968. Quantitative studies of immunofluorescent staining. Relationship of characteristics of unabsorbed antihuman IgG conjugates to their specific and non-specific staining properties in an indirect test for antinuclear factors. Bull. World Health Organ. 39:587-606. 3. Biegeleisen, J. Z., M. D. Moody, B. B. Marcus, and J. W. Flynt. 1962. The use of fluorescein-labeled anti-Brucella suis globulin for demonstrating Brucella antigen in animal tissues. Amer. J. Vet. Res. 23:592-595. 4. Etchebarne, M., P. G. Beriial, and G. R. Reyton. 1960. Purification of rabies antibodies in horse serum and diagnostic importance of the fluorescent antibody technique. J. Immunol. 84:6-10. 5. Fischman, H. R., and F. E. Ward III. 1969. Infectivity of fixed impression smears prepared from rabies virus-infected brain. Amer. J. Vet. Res. 30:2205-2208. 6. Gispen, R., and B. Sasthof. 1965. Neutralizing and fluorescent antibody response in man after antirabies treatment with suckling rabbit brain vaccine. Arch. Gesamte Virusforsch. 15:377-386. 7. Goldwasser, R. A., and R. E. Kissling. 1958. Fluorescent antibody staining of street and fixed rabies virus antigens. Proc. Soc. Exp. Biol. 98:219-223.

DISCUSSION The method described for reducing the performance time of the FA test for rabies by using unfixed brain impressions stained with rabies conjugate at room temperature for 10 min showed the same sensitivity and specificity as the standard FA and mouse inoculation tests. In general, more antigen was seen in the unfixed positive impressions than in the fixed ones. Also in one case, a positive result was obtained with the unfixed smear whereas the corresponding fixed one was negative. The specificity of that result was confirmed by inoculating the brain sample into suckling mice, known to be more sensitive to rabies virus than adult mice (1, 15). Although the reason for finding less antigen with the acetone-fixed impressions is not known, it could be due to damage to the rabies antigen by acetone. In some cases, less antigen and lower intensity

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8. Goldwasser, R. A., R. E. Kissling, and T. R. Carski. 1959. Fluorescent antibody staining of rabies virus antigens in the salivary glands of rabid animals. Bull. World Health Organ. 20:579-588. 9. Kissling, R. E., and D. R. Reese. 1963. Antirabies vaccine of tissue culture origin. J. Immunol. 91:362-368. 10. Koprowski, H. 1966. Mouse inoculation test, p. 69-80. In P. Atanasiu et al. (ed.), Laboratory techniques in rabies, 2nd ed. World Health Organization, Geneva. 11. Lennette, E. H., J. D. Woodie, K. Nakamura, and R. L. Magoffin. 1965. The diagnosis of rabies by fluorescent antibody method (FRA) employing immune hamster serum. Health Lab. Sci. 2:24-34. 12. Lepine, P., and A. Gamet. 1969. La rage. L'Expansion Editeur, Paris. 13. McQueen, J. L. 1960. Rabies diagnosis. Special application of fluorescent antibody techniques. Proc. Annu. Meet. U.S. Livestock Sanitary Ass. 63:356-363.

14. Mayer, M., and M. Heidelberger. 1942. Velocity of combination of antibody with specific polysaccharides of pneumococcus. J. Biol. Chem. 143:567-574. 15. Nilsson, M. R., W. Sugay, 0. L. Pasqualin, and S. B. K. Miller. 1968. Rabies diagnosis. Comparative study on susceptibility of adult and suckling mice. Arch. Inst. Biol. Sao Paulo 35:43-47. 16. Sulzer, A. J., M. Wilson, and E. C. Hall. 1969. Indirect fluorescent antibody tests for parasitic disease. V. An evaluation of a thick-smear antigen in the IFA test for malaria antibodies. Amer. J. Trop. Med. Hyg. 18:199-205. 17. Serokawa, D., K. Krawczyfiski, and W. Brzosko. 1967. The use of immunofluorescence for the detection of street rabies virus in the central nervous system of mice in the incubation period of the disease. Exp. Med. Microbiol. 19:204-216. 18. Tierkel, E. S. 1966. Shipment of specimens, and techniques for preparation of animal tissues, p. 17-25. In P. Atanasiu et al. (ed.), Laboratory techniques in rabies, 2nd ed. World Health Organization, Geneva.