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
OSCAR P. LARGHI Am EDWIN JIMENEZ CH.2 Pan American Zoonoses Center, Pan American Health Organization, P.O. Box 23, Ramos Mejia, Buenos Aires, Argentina

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

6. Larghi. Immunol. Bagnaroli. R. Marchevsky. Susceptibilidad de ratones lactantes y adultos al virus rabico demostrado por immunofluorescencia. A. Ward III. as well as by the human samples and the staff of the Instituto Antirrnbico. for at room temperature for 10 min).612 LARGHI AND JIMENEZ CH. V.. The specificity of that result was confirmed by inoculating the brain sample into suckling mice. Res. 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. 1958. P. 2. 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." were positive and 140 were negative. M. J. The use of fluorescein-labeled anti-Brucella suis globulin for demonstrating Brucella antigen in animal tissues. during air-drying. A. A suspension of this brain more likely in unfixed fixed smears. 1969. W.formed. B. On one occasion. E. P. 23:592-595. The intensity of the stain and the amount of We also gratefully acknowledge the capable assistance of Juan C. Fluorescent antibody staining of street and fixed rabies virus antigens. Gesamte Virusforsch. and R. because rabies virus is sensitive to organic slide. Purification of rabies antibodies in horse serum and diagnostic importance of the fluorescent antibody technique. M. R. Barnett. 105 brains minishing velocity. Goldwasser. 3. Reyton. 68:388-392. In all tests. 90% and FA tests. Beriial. In their study. Buenos Aires. and the sions. Lower intensity of staining could result in erroneous diagnosis on slides with minimal antigen. results more quickly to the physician considering Effect of modified incubation. and E. Vet. D. Neutralizing and fluorescent antibody response in man after antirabies treatment with suckling rabbit brain vaccine. Mayer and Heidelberger (14) for pneumococcal tion among the results of mouse inoculation tests polysaccharides might occur. R.. Of the 161 rabies treatment for the bitten persons. Sanit. was inoculated into 10 suckling mice. were somewhat lower than with the standard LITERATURE CITED staining method in 40% of the positive impres1. In some cases. Also in one case. 5. Flynt. M. 1970. Res. Proc. sions. Argentina. J. Beutner. known to be more sensitive to rabies virus than adult mice (1. Vet. and G. 7. and J. 1962. 98:219-223. B. 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. 15).. E. with either fixed or unfixed impres. 15:377-386. J. samples used in this part of the present study. more antigen was seen in the unfixed positive impressions than in the fixed ones. Panamer. J. Moody. and B. Etchebarne. and N. The remainFischman and Ward (5) have found infective ing brain sample was negative by adult mouse rabies virus in impressions fixed with acetone. UGI (2 mm) and BG38 (4 mm). H. a positive result was obtained with the unfixed smear whereas the corresponding fixed one was negative. Relationship of characteristics of unabsorbed antihuman IgG conjugates to their specific and non-specific staining properties in an indirect test for antinuclear factors. and F. Although the reason for finding less antigen with the acetone-fixed impressions is not known.. World Health Organ. but we had no problem in differentiating the positive samples from the negative ones. Sepulveda. and 2 of this problem could bethan overcome exposing the these animals subsequently died of rabies. 39:587-606. Mouse inoculation test. was found in 245 of 246 brain samples used remainder "took place with progressively diin this part of the study. Exp. 0. the amount Gamet (12). R. for the animal samples used in this study. as smears to ultraviolet light prior by to staining and demonstrated by FA on their brains. the occurrence of infective virus is unfixed slide was used. 1968. 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. mouse inoculation. Hospital Mufniz. inoculation and by FA test with the acetone-fixed and.. 30:2205-2208. it could be due to damage to the rabies antigen by acetone. Marcus.. Kissling. 1965. antigen detected with the rapid staining method Areitio and Luis Lazaro. APPL. but was positive by the FA test in which the solvents (9). suckling mice were also used. 1960. Complete correla. MICROBIOL. 84:6-10. Biol. Gispen. Bol. Quantitative studies of immunofluorescent staining. Soc. If desired. Mor6n. E. R. G. 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). Ofic. In general. 4. H. Biegeleisen. less antigen and lower intensity . and barrier filter K 430. Arch. Sasthof. Amer. Infectivity of fixed impression smears prepared from rabies virus-infected brain. 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. Z. as described by LUpine and In all but two of the positive cases. R. Amer. Bull.of the reaction was completed in 3 sec. Fischman..

L. Indirect fluorescent antibody tests for parasitic disease. R. and R. R. 20:579-588. Trop. P. J. R. V. Amer. . 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. Med. Serokawa. Sugay. Sulzer. Proc. 2:24-34. 10. La rage. Lennette. and M. Rabies diagnosis.. Biol. E. and E. and D. Rabies diagnosis. U. Antirabies vaccine of tissue culture origin. 1968.. Gamet. In P. M. and S. Miller. Sao Paulo 35:43-47. Immunol. A. 0. Chem. 21. K. Nakamura. 17-25. McQueen. 16. 91:362-368. World Health Organization. K. 17. Comparative study on susceptibility of adult and suckling mice. Health Lab.. (ed.. Atanasiu et al. Heidelberger. E. The diagnosis of rabies by fluorescent antibody method (FRA) employing immune hamster serum. Geneva. C. K. 9. H. 1969. Atanasiu et al. J. Mouse inoculation test. Magoffin. 1966. L. and T. Krawczyfiski. R. and W. 1959. 1960. 18:199-205. 11. E. Geneva. 1965. 1963. M. 143:567-574. R. 1967. World Health Organization. 19:204-216. World Health Organ. Med. In P. and techniques for preparation of animal tissues. Livestock Sanitary Ass. Brzosko. Wilson. R. (ed. A.. Woodie. Microbiol. B. Shipment of specimens.S. Lepine. Meet. 63:356-363. 1942. W. Tierkel. 14. Laboratory techniques in rabies. 18. Nilsson. Reese. 1966. Carski.VOL. J.. Kissling. An evaluation of a thick-smear antigen in the IFA test for malaria antibodies. J.. H. Hall. 1971 ACCELERATED FA TEST FOR RABIES DIAGNOSIS 613 8. J. Hyg. Paris. Special application of fluorescent antibody techniques. 69-80. D. Fluorescent antibody staining of rabies virus antigens in the salivary glands of rabid animals. Kissling. Pasqualin. p. 12..). Annu. 15. Exp. L'Expansion Editeur. Laboratory techniques in rabies. Velocity of combination of antibody with specific polysaccharides of pneumococcus. S. p. 1969. L. Koprowski. Arch. Mayer. 2nd ed. Goldwasser. Biol. 13. and A. E. J. D. 2nd ed.). Bull. Inst. Sci. M.

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