Direct Immunofluorescence Tests with Counterstains for Detection of Chiamydia psittaci in Infected Avian Tissues J. Tessler, S. S. Stone and L. A. Page*
Different methods of preparation and serological evaluation of rabbit globulins for use in fluorescent antibody conjugate and different methods of counterstaining with fluorescent antibody tests were evaluated for detection of Chlamydia psittaci in infected turkey tissues. The agar gel precipitin reaction was that chosen for testing and selecting antiserums to be used for fluorescein isothiocyanate conjugation. The fluorescent antibody staining was most pronounced with conjugate made from globulins precipitated with ammonium sulfate. A direct fluorescent antibody method with Evans blue counterstain correctly identified "coded" specimens of C. psittaci-infected and noninfected turkey air sacs. However, naphthalene black was superior to Evans blue as a counterstain when infected pericardial sacs were tested.
RESUME Cette etude visait 'a deceler Chlamydia psittaci dans les tissus de dindes inoculees avec cet agent, par la voie intra-musculaire. Les auteurs experimenterent a cette fin diverses m'thodes de preparation et d'evaluation de globulines de lapins, destinees 'a la preparation d'un conjugue qui servirait a proceder a l'aide de l'immunofluorescence; ils determinerent aussi l'efficacite de diverses methodes de coloration differentielle. Ils choisirent l'epreuve d'immunodiffusion
sur gelose pour eprouver et selectionner les antiserums qu'ils conjugueraient avec de l'isothiocyanate de fluoresceine. L'immunofluorescence donna de meilleurs resultats lorsqu'ils utilisaient un conjugue prepare avec des globulines precipitees 'a l'aide de sulfate d'ammonium.
L'utilisation conjointe d'une methode d'immunofluorescence directe et d'une coloration differentielle au bleu Evans permit d'identifier correctement des echantillons connus des sacs aeriens de dindes experimentales et temoins. Les lesions du pericarde ressortirent cependant mieux avec la coloration differentielle au noir de naphtal'ene.
INTRODUCTION Although the direct fluorescent antibody technique (FAT) has been described for identifying bacteria (7,9,10) including chlamydia (12) in animal tissues, it has not been suitable, in our experience, when used with turkey tissues infected with Chlamydia psittaci. The major problems have been nonspecific fluorescence and autofluorescence. In the present study, we examined several parameters of the FAT to minimize these problems and to develop a routine FAT for diagnosing chlamydial infections of turkeys.
MATERIALS AND METHODS ANIMALS
*National Animal Disease Center, North Central Region, Agricultural Research, Science and Education Administration, U.S. Department of Agriculture, Ames, Iowa 50010. No endorsements implied herein. Submitted November 20, 1978.
Volume 43 - April, 1979
Broad-breasted White turkeys, eight to 15 weeks old, were obtained as day-old poults from a chlamydiae-free commercial source and raised in isolation quarters
with controlled entry and exit of air and water.
rated against starting 0.0175 M phosphate buffer, pH 6.3 according to Riggs et al (17), and loaded on a DEAE cellulose column that had been equilibrated with INFECTIVE AGENT the same buffer. The "fall through" peak (FP-1) was eluted from the column, and The South Carolina turkey virulent the protein was monitored at 280 nm. This strain of C. psittaci (15) was inoculated fraction (FP-1) was gamma globulin. The intramuscularly; 1 x 105 embryo LD50 (16) second fraction (FP-2) was eluted from per turkey were used. the column with 0.4 M NaHPO4 buffer, pH 5.2, containing 2M NaCl and was predominantly macroglobulin. Both fractions ANTISERUM PRODUCTION were concentrated to the original volume filtration on XM50,3 which retains The Iowa Turkey strain (IT-1) (15) of by molecules in excess of 50,000 daltons. C. psittaci adapted for tissue culture was Each of the globulin precipitates was grown in cultures of the McCoy cell line adjusted to 1% protein spectrophotometric(mouse fibroblasts obtained from Dr. E. R. 14.0 was used as the extinction factor Alexander, University of Washington) ally; 280 nm for 1% protein. treated with cytochalasin B1 (18). After at The globulins were conjugated with incubation for 72 hours in plastic flasks, fluorescein isothiocyanate (FITC) by dialthe cultures were rapidly frozen and ysis (2); 0.1 mg of FITC/ml of 0.5 M thawed three times at -70°C (in ethyl carbonate buffer, pH 9.5 was used. The alcohol and dry ice). Cultures were pooled globulins were dialyzed the FITC and centrifuged for one hour at 34,800 x for 18-20 hours at 40C. against Unreacted FITC G at 40C. The supernatant fraction was was removed with Sephadex G-50 columns, removed and pellets were reconstituted and the conjugated globulins were conwith medium K-36 (20) to make a 1OX centrated to the original serum volumes concentration. The concentrated C. psittaci by filtration through XM50 membranes. was mixed with an equal volume of Freund complete adjuvant.' Adult New Zealand white rabbits were IMMUNOELECTROPHORESIS injected intramuscularly in the leg with 1 ml of inoculum. The animals were hyperGlobulin fractions were assayed by imimmunized by injections of the same ino- munoelectrophoresis (IEP), using a Noble4 culum at monthly intervals for seven agar gel prepared in pH 8.6 borate buffer months. They were narcotized ten days (19). The precipitin bands were developed after the last inoculation and exsanguin- with goat antirabbit serum and photoated. graphed after 18 hours of incubation at room temperature. PREPARATION OF FLUORESCENT ANTIBODY (FA) SEROLOGY Two procedures for isolating the globulins from serums were used. In the first procedure, two ammonium sulfate precipitations were used; first 50% and then 40% ammonium sulfate. The resulting precipitate was dissolved in water and dialyzed against 0.85% saline until the dialysate was free of sulfate ions. The second procedure was conducted as previously described (8) except that globulins were precipitated from serum by 50 % ammonium sulfate precipitation, equiliblAldrich Chemical Co., Milwaukee, Wisconsin. 2Difco Laboratories, Detroit, Michigan.
Agar gel precipitin (AGP) and complement fixation (CF) tests were used to detect antibodies in the rabbit serums as previously described (14). IMPRESSION SMEARS
Turkeys were killed when they appeared to be clinically infected with C. psittaci (13). Impression smears of noninfected and infected air sacs and pericardial sacs 3Amicon, Lexington, Massachusetts. 4Difco Laboratories, Detroit, Michigan.
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were made on glass slides (13). Impression smears were also made of diseased air sacs and pericardial sacs from turkeys infected with Mycoplasma gallisepticum, Escherichia coli and Pasteurella multocida. Smears were air dried and then fixed with acetone at room temperature for three minutes, dried and stored at 4°C until tested. Sixty impression smears from C. psittaci-infected turkey tissues and 25 smears from noninfected tissues were tested for establishment of a diagnostic staining procedure. Thirty-two turkeys of mixed C. psittaciinfected and noninfected histories were euthanatized and examined for gross lesions (13) to evaluate staining procedures. STAINING PROCEDURES
P. multocida were tested. In addition pericardial sacs infected with E. coli and P. multocida were reacted with conjugate. Normal rabbit conjugate - Serum from nonimmunized rabbits was conjugated as described above. Antibody blocking - The specificity of the conjugate was tested by the initial treatment of known positive material with unconjugated antiserum followed by the conjugated serum after the method of Goldman (6).
RESULTS MINIMAL CRITERIA FOR CHOICE OF ANTISERUM
Antiserums having a CF titer of 1:64 In preliminary experiments, counterstains were not used. Then 0.005% to 2% or higher, or a positive AGP reaction at solutions of Congo red, Evans blue and a dilution of 1:8 once conjugated with naphthalene black were tested to determine FITC had the highest FA activity and the optimal concentrations of each dye for hence were the most suitable as a source depressions of nonspecific fluorescence and of gamma globulin for conjugation. The DEAE fraction (FP-1) in Fig. 1 had an autofluorescence. Staining of air sacs - Acetone-fixed air AGP titer of 1:1 (CF titer of 1:256). The sac impressions were washed in phosphate ammonium sulfate precipitated globulin buffered saline (PBS), pH 7.4, then had an AGP titer of 1:2 (CF 1:192), but washed in distilled water and air dried. the latter produced the most reactive FA The FITC conjugate at 1:10 dilution was conjugate. In contrast, the macroglobulin applied to the slides and they were in- fraction from DEAE chromatography was cubated in a humidified chamber at 370C negative in the AGP test (CF titer 1:24) for 30 minutes. Excess conjugate was de- and produced a 1+ FA reaction in C. psitcanted, and slides were washed with PBS taci-infected air sac and pericardial sac. In Fig. 1, patterns of precipitin reactwo times, air dried and overlaid with 0.005% Evans blue for thirty seconds. The tions of the three globulin preparations are excess dye was decanted and slides washed compared with those of normal rabbit once in PBS and twice in distilled water. + They were air dried and coverslips were NORMAL applied using 50% PBS in 50% glycerin RABBIT as mounting media. The slides were examined with a Leitz5 fluorescence microscope. Transmitted ultraviolet light, primary (NH4)2 SO4 PPT filter BG#12, barrier filter #510 and a dry darkfield condenser were used. Staining of pericardial sacs - The pro- F-(NH4)2S04-FA cedure described above was followed, except F-P-1 that 0.005% naphthalene black was directly mixed with the conjugate. The conjugate and dye were placed on the smears for 20 F-P-2 minutes before the washing procedure. Impressions from turkey air sacs infected with M. gallisepticum, E. coli and GOAT ANTI-RANBIT SERUM
5E. Leitz & Co., Rockleigh, New Jersey.
Fig. 1. Immunoelectrophoretic pattern of rabbit antiChlamydia psittaci globulin fractions before and after conjugation with fluorescein isothiocyanate. Fractions are designated according to the text.
serum. The ammonium sulfate precipitate before and after conjugation contained slowly migrating proteins in addition to the fast cathodic migrating albumin. In contrast, the FP-1 gamma globulin fraction obtained by DEAE chromatography was predominantly IgG, with only a trace of one slowly migrating band visible. OPTIMUM CONCENTRATION OF COUNTERSTAINS
Concentrations of Evans blue higher than 0.005% were not suitable because they masked the specific FA reaction. The best concentration of naphthalene black was 0.005 %; higher percent concentrations of naphthalene black obscured the FA reaction. Congo red hid the fluorescence at all concentrations tested. The nonspecific fluorescence and autofluorescence were removed by the counterstains. Figure 2 shows cytoplasmic fluorescence in the impression smear of an infected air sac; Evans blue was used as a counterstain.
DIAGNOSTIC RELIABILITY OF THE FA TECHNIQUE
Four out of six "coded" air sacs infected with C. psittaci were detected by the FA reaction (Table I). In addition, the counterstain Evans blue at 0.005% helped define the FA by providing a dark contrasting background. One out of 14 "coded" C. psittaci-positive samples were not detected by the FA test (Table II). One out of 13 "coded" C. psittaci-negative samples was judged positive by FA (Table II). The naphthalene black made a good contrasting background. Conjugates with counterstains were used on smears of M. gallisepticum, E. coli and P. multocida, and all were negative. In addition, normal rabbit globulin conjugate did not stain the smears of infected C. psittaci. Blocking antibody tests confirmed the specificity of the FA reaction.
DISCUSSION IMPORTANCE OF IgG PRECIPITINS TO FINAL FA REACTIVITY
The data in our study agrees with Nairn (11) that measurement of precipitating IgG antibodies is the best method for determining the suitability of an antiserum for conjugation with FITC. The trace of albumin seen in the globulin precipitated by ammonium sulfate did not alter the efficiency of conjugation with FITC. Moreover, the AGP titer of the globulin fractionated by the (NH4) 2S04 was higher than the titer of globulin prepared by the Levy and Sober (8) method. Immunoglobulin other than IgG and the macroglobulin are included in the fraction precipitated by ammonium sulfate (1). Such a mixture of globulins may increase the avidity of the immunological reaction. IMPORTANCE OF OPTIMAL COUNTERSTAIN CONDITIONS _-
Fig. 2. Impression smear of air sacs infected with Chlamydia psittaci. Stained with globulin conjugated with fluorescein isothiocyanate and counterstained with Evans blue.
In our experiments, the use of Evans blue as a counterstain in concentration greater than 0.005%, as recommended by Nichols and McComb (12), obscured the fluorescence. Moreover, we applied Evans
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TABLE I. Comparison Between Fluorescent Antibody (FA) Test of Air Sac Tissue and Gross Pathological Observations to Detect Chiamydia psittkci in Turkeys
Gross Pathological Infectious State of Turkey FA Tests Observations" Known C. psitlaci Negative ......................................... 1/1 1/1 Known C. psittaci Positive .......................................... 1/1 1/1 "Coded" C. psittaci Positive .......................................... 4/6 6/6 = = -Numerator number of tissues diagnosed correctly. Denominator number of tissues tested TABLE II. Comparison Between Fluorescent Antibody (FA) Test of Pericardial Sac Tissue and Gross Pathological Observations to Detect Chlamydid psittaci in Turkeys
Infectious State of Turkey
Known C. psittaci Negative ......................................... Known C. psittaci Positive ........................................... "Coded" C. psittaci Positive ........................................... "Coded" C. psiltaci
Gross Pathological Observationsa
5/5 5/5 13/14 12/13
5/5 5/5 14/14 13/13
number of tissues diagnosed correctly. Denominator = number of tissues tested
blue to tissue smears for only 30 seconds whereas they (12) applied this counterstain for 20 minutes. Therefore, for diagnosis the timing and concentration of counterstains may have to be adjusted according to the infective agent as well as the specific animal tissue under test. Furthermore, because of chemical impurities, different lots of dye may require concentration adjustments to obtain optimal working solutions. The dye concentration was critical also for naphthalene black. We found that increasing the concentration of naphthalene black above 0.005% obscured the FA reaction. Gardner and McQuillin (5) reported that this particular counterstain reduced the level of autofluorescence and levels of nonspecific fluorescence. Finally, contrary to the work of Nichols and McComb (12), who used 0.5% Congo red, our tests with Congo red showed that even smaller amounts of the dye obscured FA.
later found that Emmons and Riggs (4) compensated for the sampling error by processing four samples from the same bird in FA diagnosis. Apparently, the infected pericardial sac was more suitable than the air sac for FA diagnosis. Donaldson et al (3) showed earlier that infected' turkey trachea also was a poor tissue to be used for diagnosis of C. psittaci by FA. Unfortunately, nonspecific fluorescence and autofluorescence precluded the use of our FA conjugates without counterstains. If higher titered specific conjugates were available and were used in greater dilution, nonspecific fluorescence in clinical material could possibly be reduced or eliminated.
ACKNOWLEDGMENTS We thank Mr. J. A. Wells, Mr. M. D. McFarland and Ms. J. K. Eichman for their technical assistance.
RELIABILITY OF THE FA TECHNIQUE REFERENCES
The false-negative reaction found in the infected air sacs and pericardial sac might be due to a sampling error in that we used only two samples from each turkey. We
Volume 43- April, 1979
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