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The application of fluorescent antibody test to Balantidium coli
80 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE.
Vol. 59.
No.
1. January,
1965.
THE APPLICATION OF F L U O R E S C E N T ANTIBODY TEST TO BALANTIDIUM. COLI BY
V. ZAM.AN'*
Department of Parasitology, Faculty of Medicine, University of Singapore Serological investigations on Balantidium coli are few, although the members of the genus Balantidium are widely distributed in nature. The ciliate has been isolated from about 50 different species of animals (MouLTON et al., 1961). KRASCHENINNIKOW and JESKA (1961) investigated the antigenic relationship between different species of the genus, using agar gel-diffusion technique, and the writer has recently described an immobilization reaction against the organism (ZAMAN, 1962). It is felt that the immobilization reaction and the fluorescent antibody test, which is being described in this paper, have an advantage over the tests with soluble antigens. This is because neither the immobilization nor the fluorescent antibody test is affected by bacterial and other extraneous antigens, which are always present in the cultures. With the exception of a report by GURSKIet al. (1961), the ciliates have not yet been recorded as available in an axenic state. The fluorescent antibody test has already been used widely in various fields of microbiology and has given interesting information on the serological relationships of some parasitic protozoa (MCENTE~ART, 1962). Material and m e t h o d s
The cultures : The strain of Balantidium was isolated from the caecal contents of a pig slaughtered at the Singapore abattoir. This strain has been maintained for the last 2 years in Balamuth's medium (BALAMUTHand SANDZA, 1944) in which it grows luxuriantly. It is subcultured every 72 hours and kept at 37°C. The strain is growing with the mixed bacterial flora from the host. preparation of antisera : Adult rabbits were used for preparing the antisera. Before the immunization schedule started they were bled once and the serum was used as control. The animals were immunized by subcutaneous injections of a washed suspension of Balantidium. Each animal received approximately 80,000 cells in 8 divided doses, given at intervals of 4 days. The serum was obtained a week after the last injection. Conjugation of the serum : The globulin fraction of the normal serum and the antiserum was conjugated to fluorescein isothiocyanate~ according to the method described by CHADWICKand FOTHERGILL(1962). The unconjugated dye was removed by passage through a column of Sephadex G25~. The serum was then re-dialysed overnight against * I wish to thank the World Health Organization for sanctioning a travel grant to visit Dr. Morris Goldman's laboratory at the National Institutes of Health, Bethesda, USA, after which this work was done. My thanks are also due to Professor R. S. Desowitz for his encouragement and to Mr. N. Visuvalingam and Mr. Cheong Chee Hock for their technical assistance. "~ Sylvana Company, California, USA. Pharmacia, Uppsala, Sweden
FIG. 1. Shows a Balantidium which has been treated by the conjugated antiserum before fixation. There are aggregates of fluorescent material around the cell. The cilia are adherent to each other and the distal ends are brightly fluorescent. There is also a greater accumulation of fluorescent material at the posterior end. x 1,000
FIG. 2. Shows a Balantidium which has been treated by the conjugated antiserum after fixation. The cell is evenly fluorescent and the pellicle is clearly visible. There are no aggregates of fluorescent material around the cell. x 1,000
v. ZAMAN
81
phosphate buffered saline (0.01 M phosphate, p H 7.5). This was necessary because the carbonate-bicarbonate buffer (pH 9), which was added to the serum during conjugation, was found to have a lytic effect on the live Balantidium. The serum was then stored at --20°C. Microscopy : A Zeiss fluorescent apparatus was used. The source of fluorescent light was a HBO 200 lamp (Osram). The filters were Schott U G I I as an excitor and a red (Code No. 53) plus a yellow (Code No. 44) as barriers. The photography was done on a Tri-X film (Kodak). Performance of the test : The ciliates were treated by the following methods : (a) The active unfixed Balantidium from the cultures were suspended in undiluted conjugated sera for one hour, then washed twice by centrifugation at 500 r.p.m, in phosphate buffered saline (PBS), sealed with a cover slip and examined as a wet preparation. (b) The active unfixed Balantidium from the cultures were suspended in undiluted conjugated sera for 1 hour, then washed twice by centrifugation at 500 r.p.m, in PBS. The cells were then fixed in acetone for another hour and washed twice by centrifugation at 500 r.p.m, in PBS and examined as a wet preparation. (c) The Balantidium were first fixed and then treated with undiluted conjugated sera for 1 hour. The fixatives used were acetone, 95~/o ethyl alcohol, 10~o formalin, 20//0 osmium tetroxide. The cells were fixed in these chemicals for 1 hour at room temperature. They were then washed 3 times in PBS by centrifugation. The washed cells were then suspended in undiluted conjugated sera for 1 hour and washed twice in PBS and examined as a wet preparation. (d) The cells were fixed on glass slides by drying at room temperature, treated with undiluted conjugated sera for 1 hour in a moist chamber, then washed in PBS and examined as a dry preparation. Results
Cells prepared according to method (a) The cells fluoresced brightly when the conjugated antiserum was used, but showed no fluorescence with conjugated normal serum. The staining with the conjugated antiserum was inhibited by pre-treating the preparation with unconjugated specific antiserum, but not by pre-treating with non-immune serum. The reaction of the cells in conjugated antiserum was the same as that of the cells treated with unconjugated antiserum (ZAMAN, 1962). They were still alive, but immobilized. In other words there was no propulsion of the cells, although the ciliary activity continued. Most of the cilia were, however, adherent to each other and covered with aggregates of fluorescent material. The distal ends of the cilia seemed to show a greater accumulation of the fluorescent material than the proximal ends. There was also a greater accumulation of the fluorescent material at the posterior end of the organism.
Cells prepared according to method (b) The appearance was identical with that of the cells prepared according to method (a) except that there was no ciliary activity because of the fixation. This preparation was photographed and a typical cell is shown in Fig. 1. The controls showed no fluorescence.
Cells prepared according to method (c) With all the fixatives used it was found that the controls, consisting of slides treated with normal conjugated serum, did fluoresce. However, the intensity of fluorescence
82
THE APPLICATION OF FLUORESCENT ANTIBODY TEST TO
Balantidium coli
was much greater in the test slides, compared with the controls. Of the fixatives tried, acetone was found to be the best in terms of brightness. Morphologically the cells appeared somewhat different from those prepared according to methods (a) and (b). There were no large aggregates of fluorescent material attached to the organism. The pellicle was evenly fluorescent and the cilia were not adherent to each other. A typical cell from an acetone-fixed specimen is shown in Fig. 2. Cells prepared according to method (d) Although the controls were again fluorescent, the intensity of fluorescence was much greater in the test slides. The main drawback of this method was that the cells were grossly distorted, and in many places the peUicle had ruptured. Discussion
The results obtained are similar to those reported by BEALE and KACSER (1957) in their initial fluorescent-antibody work on Paramecium. It seems that when the living cells are used the reaction is highly specific, but after treatment with fixative and dry fixation there is a certain amount of non-specific staining. However, none of these methods completely destroyed the antigenic specificity. The morphological differences observed between the unfixed and the fixed cells is again in accordance with observations made by BEALEand KACSER (1957). It was suggested by them that the differences were due to the exudation of antigens during the living state, which combined with the antibody to give rise to visible aggregates. This seems to be the most plausible explanation and should apply to Balantidium as well. The reaction is now being used in conjunction with the immobilization test to study the antigenic differences between various species of Balantidium. It is hoped that the results of this study will be published shortly. Summary The fluorescent antibody test on Balantidium coli is described. Experience with unfixed and fixed specimens has shown that there is no non-specific staining with the unfixed specimens, the controls remaining completely negative ; with the fixed specimens, however, the controls show a certain amount of non-specific fluorescence. It is, however, possible to differentiate the test slides from the controls because of the difference in the intensity of the fluorescence. In terms of brightness, acetone gave the best results among the five different fixatives tried. References
W. & SANDZA, J. G. (1944). Proc. Soc. exp. Biol., N.Y., 57, 161. BEALE, G. H. & KACSER,H. (1957). J. gen. Microbiol., 17, 68. CHADWICK, C. S. & FOTHERGILL,J. E. (1962). Fluorescent Protein Tracing (Ed. R. C. Nairn) p. 19, Edinburgh : E. & S. Livingstone. GIdRSKI, D. R., JOHN, J. L. & PIERCE, S. (1961). ft. ProtozooL (Supplement), 8, 11. KRASI-IENINNII
Vol. 59.
No.
1. January,
1965.
THE APPLICATION OF F L U O R E S C E N T ANTIBODY TEST TO BALANTIDIUM. COLI BY
V. ZAM.AN'*
Department of Parasitology, Faculty of Medicine, University of Singapore Serological investigations on Balantidium coli are few, although the members of the genus Balantidium are widely distributed in nature. The ciliate has been isolated from about 50 different species of animals (MouLTON et al., 1961). KRASCHENINNIKOW and JESKA (1961) investigated the antigenic relationship between different species of the genus, using agar gel-diffusion technique, and the writer has recently described an immobilization reaction against the organism (ZAMAN, 1962). It is felt that the immobilization reaction and the fluorescent antibody test, which is being described in this paper, have an advantage over the tests with soluble antigens. This is because neither the immobilization nor the fluorescent antibody test is affected by bacterial and other extraneous antigens, which are always present in the cultures. With the exception of a report by GURSKIet al. (1961), the ciliates have not yet been recorded as available in an axenic state. The fluorescent antibody test has already been used widely in various fields of microbiology and has given interesting information on the serological relationships of some parasitic protozoa (MCENTE~ART, 1962). Material and m e t h o d s
The cultures : The strain of Balantidium was isolated from the caecal contents of a pig slaughtered at the Singapore abattoir. This strain has been maintained for the last 2 years in Balamuth's medium (BALAMUTHand SANDZA, 1944) in which it grows luxuriantly. It is subcultured every 72 hours and kept at 37°C. The strain is growing with the mixed bacterial flora from the host. preparation of antisera : Adult rabbits were used for preparing the antisera. Before the immunization schedule started they were bled once and the serum was used as control. The animals were immunized by subcutaneous injections of a washed suspension of Balantidium. Each animal received approximately 80,000 cells in 8 divided doses, given at intervals of 4 days. The serum was obtained a week after the last injection. Conjugation of the serum : The globulin fraction of the normal serum and the antiserum was conjugated to fluorescein isothiocyanate~ according to the method described by CHADWICKand FOTHERGILL(1962). The unconjugated dye was removed by passage through a column of Sephadex G25~. The serum was then re-dialysed overnight against * I wish to thank the World Health Organization for sanctioning a travel grant to visit Dr. Morris Goldman's laboratory at the National Institutes of Health, Bethesda, USA, after which this work was done. My thanks are also due to Professor R. S. Desowitz for his encouragement and to Mr. N. Visuvalingam and Mr. Cheong Chee Hock for their technical assistance. "~ Sylvana Company, California, USA. Pharmacia, Uppsala, Sweden
FIG. 1. Shows a Balantidium which has been treated by the conjugated antiserum before fixation. There are aggregates of fluorescent material around the cell. The cilia are adherent to each other and the distal ends are brightly fluorescent. There is also a greater accumulation of fluorescent material at the posterior end. x 1,000
FIG. 2. Shows a Balantidium which has been treated by the conjugated antiserum after fixation. The cell is evenly fluorescent and the pellicle is clearly visible. There are no aggregates of fluorescent material around the cell. x 1,000
v. ZAMAN
81
phosphate buffered saline (0.01 M phosphate, p H 7.5). This was necessary because the carbonate-bicarbonate buffer (pH 9), which was added to the serum during conjugation, was found to have a lytic effect on the live Balantidium. The serum was then stored at --20°C. Microscopy : A Zeiss fluorescent apparatus was used. The source of fluorescent light was a HBO 200 lamp (Osram). The filters were Schott U G I I as an excitor and a red (Code No. 53) plus a yellow (Code No. 44) as barriers. The photography was done on a Tri-X film (Kodak). Performance of the test : The ciliates were treated by the following methods : (a) The active unfixed Balantidium from the cultures were suspended in undiluted conjugated sera for one hour, then washed twice by centrifugation at 500 r.p.m, in phosphate buffered saline (PBS), sealed with a cover slip and examined as a wet preparation. (b) The active unfixed Balantidium from the cultures were suspended in undiluted conjugated sera for 1 hour, then washed twice by centrifugation at 500 r.p.m, in PBS. The cells were then fixed in acetone for another hour and washed twice by centrifugation at 500 r.p.m, in PBS and examined as a wet preparation. (c) The Balantidium were first fixed and then treated with undiluted conjugated sera for 1 hour. The fixatives used were acetone, 95~/o ethyl alcohol, 10~o formalin, 20//0 osmium tetroxide. The cells were fixed in these chemicals for 1 hour at room temperature. They were then washed 3 times in PBS by centrifugation. The washed cells were then suspended in undiluted conjugated sera for 1 hour and washed twice in PBS and examined as a wet preparation. (d) The cells were fixed on glass slides by drying at room temperature, treated with undiluted conjugated sera for 1 hour in a moist chamber, then washed in PBS and examined as a dry preparation. Results
Cells prepared according to method (a) The cells fluoresced brightly when the conjugated antiserum was used, but showed no fluorescence with conjugated normal serum. The staining with the conjugated antiserum was inhibited by pre-treating the preparation with unconjugated specific antiserum, but not by pre-treating with non-immune serum. The reaction of the cells in conjugated antiserum was the same as that of the cells treated with unconjugated antiserum (ZAMAN, 1962). They were still alive, but immobilized. In other words there was no propulsion of the cells, although the ciliary activity continued. Most of the cilia were, however, adherent to each other and covered with aggregates of fluorescent material. The distal ends of the cilia seemed to show a greater accumulation of the fluorescent material than the proximal ends. There was also a greater accumulation of the fluorescent material at the posterior end of the organism.
Cells prepared according to method (b) The appearance was identical with that of the cells prepared according to method (a) except that there was no ciliary activity because of the fixation. This preparation was photographed and a typical cell is shown in Fig. 1. The controls showed no fluorescence.
Cells prepared according to method (c) With all the fixatives used it was found that the controls, consisting of slides treated with normal conjugated serum, did fluoresce. However, the intensity of fluorescence
82
THE APPLICATION OF FLUORESCENT ANTIBODY TEST TO
Balantidium coli
was much greater in the test slides, compared with the controls. Of the fixatives tried, acetone was found to be the best in terms of brightness. Morphologically the cells appeared somewhat different from those prepared according to methods (a) and (b). There were no large aggregates of fluorescent material attached to the organism. The pellicle was evenly fluorescent and the cilia were not adherent to each other. A typical cell from an acetone-fixed specimen is shown in Fig. 2. Cells prepared according to method (d) Although the controls were again fluorescent, the intensity of fluorescence was much greater in the test slides. The main drawback of this method was that the cells were grossly distorted, and in many places the peUicle had ruptured. Discussion
The results obtained are similar to those reported by BEALE and KACSER (1957) in their initial fluorescent-antibody work on Paramecium. It seems that when the living cells are used the reaction is highly specific, but after treatment with fixative and dry fixation there is a certain amount of non-specific staining. However, none of these methods completely destroyed the antigenic specificity. The morphological differences observed between the unfixed and the fixed cells is again in accordance with observations made by BEALEand KACSER (1957). It was suggested by them that the differences were due to the exudation of antigens during the living state, which combined with the antibody to give rise to visible aggregates. This seems to be the most plausible explanation and should apply to Balantidium as well. The reaction is now being used in conjunction with the immobilization test to study the antigenic differences between various species of Balantidium. It is hoped that the results of this study will be published shortly. Summary The fluorescent antibody test on Balantidium coli is described. Experience with unfixed and fixed specimens has shown that there is no non-specific staining with the unfixed specimens, the controls remaining completely negative ; with the fixed specimens, however, the controls show a certain amount of non-specific fluorescence. It is, however, possible to differentiate the test slides from the controls because of the difference in the intensity of the fluorescence. In terms of brightness, acetone gave the best results among the five different fixatives tried. References
W. & SANDZA, J. G. (1944). Proc. Soc. exp. Biol., N.Y., 57, 161. BEALE, G. H. & KACSER,H. (1957). J. gen. Microbiol., 17, 68. CHADWICK, C. S. & FOTHERGILL,J. E. (1962). Fluorescent Protein Tracing (Ed. R. C. Nairn) p. 19, Edinburgh : E. & S. Livingstone. GIdRSKI, D. R., JOHN, J. L. & PIERCE, S. (1961). ft. ProtozooL (Supplement), 8, 11. KRASI-IENINNII