AGGWTINATIQNLYSIS TEST AND THE AGGLUTENATEQN TEST FGR LEFTGSFIRA FQMONA INFECTION Thesis for Ike Degree of M. S. MICHIGAN STATE UNIVERSITY Mario Barbosa 1958 THESIS EFFECT OF VARIOUS FACTORS UPON THE AGGLUTINATION- LYSIS TEST AND THE AGGLUTINATION TEST FOR mmggg POMONA INFECTION By Mario Barbosa A THESIS Submitted to the College of Veterinary Medicine Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1958 ACKNOWLEDGMENTS The author wishes to express his sincere gratitude to Dr. E. V. Horse, for his patience, guidance and advice during the course of this research and preparation of this manuscript. He also extends his appreciation to Mrs. Athalie Lundberg, for her technical assist- ance. The author is very much indebted to the Rockefeller Foundation and the Escola Superior de Veterinaria da U. R. E. M. 6., Bela-Hori- zonte, Minas Gerais, Brazil, and wishes to convey his sincere appre- ciation to them for making possible his stay in the United.States. TABLE CHAPTER I. INTRODUCTION . . . . . . II. REVIEW OF THE LITERATURE III. MATERIAL AND METHODS . . IV. RESULTS . . . . . . . . Explanation of Symbols Degree of Agglutinati Tables I through VIII V. DISCUSSION AND CONCLUSION VI . SUMY O O O O O 0 O 0 REFERENCES . . . . . . . . . OF CONTENTS Used in Expressing on, Lysis or Both . ‘ O O O O O O O O O 0 PAGE 13 16 18 19 27 31 CHAPTER I INTRODUCTION Human and animal leptospiral infections have, in recent years, received an increasing amount of attention because of their signifi- cance as a world wide veterinary ahd public health problem. This has resulted in an increased interest in the serological diagnosis of leptospirosis. The following methods have been employed for diagnosis: com- plement fixation test,2’ 3: 27 adhesion test,’4 modified macroscopic agglutination tests,6a 25: 32 capillary tube test,39 urine fraction test,31 hemagglutination test,9 hemolytic test,12 and the microsco- pic agglutinationplysis test.36 I However, the microscOpic agglutination-lysis tests are consid- ered to be most accurate and are used extensively by research groups. Macroscopic or plate agglutination tests are favored by diagnostic laboratories in many states. Up to new, no standard procedure has been established to determine a.means of standardization of techniques and interpreta- tions for the various procedures. many laboratories have been using different interpretations of the results of serological methods, and have also employed different techniques in the preparations of anti- gens, dilutions of the serum, and the time and temperature of incu- bation. There is a lack of information on the comparison of controlled conditions as they apply to the microsc0pic agglutination-lysis tests. The purpose of this research was to ascertain the effects of living and formalin killed antigens, various incubation temper- atures and periods of time upon the sensitivity of the microscopic agglutination-lysis tests using Leptospira pomona antisera obtained from man, cattle, sheep, goats, pigs, dogs, guinea pigs and hamsters. CHAPTER II REVIEW OF THE LITERATURE Costa and Troisier10 examined the reactions of the Wassermann test on five sera from patients who had "ictero hemorragic spiro- chetosis." They concluded that the reaction of the test might be positive with “spirochetosis.” Martin and Pettit27 found confirmation in their studies with human sera concerning the fixation of complement in "ictero hemor- ragic" infection. The reaction was positive with the Wassermann antigen and a positive spirochetosis serum. The reciprocal was also true. They used, as antigen guinea pig liver rich in "spirochetes," _, /V{fly,- but positive results were obtained with "spirochetosis" serum as well as with a strongly positive syphilitic serum. Schuffner and Miochtar36 developed the microscopic aggluti- nation-lysis reaction using living leptospirae as an antigen. Incu- bation was at room.temperature and the tests were read employing dark ground illumination. Brown and Davis’4 applied the phenomenon of adhesion onto colloidal particles as means of differentiating serological types of leptospirae. Bessemans and N’elis2 found that fixation of complement was specific. They used thirteen sera from human syphilitics with strong positive'Wassermann reactions. These always gave negative results 'when used against antigens prepared from pure cultures of leptospirae, but poor results when used with alcoholic antigen extracts of guinea pig livers. Gaehtgens17 tested 70 sera of patients showing symptoms sug- gestive of‘Heil's disease using the agglutination and complement fixation tests. Their results showed fairly close agreement between the two methods. However, as a rule, agglutination titers were higher than complement fixation titers. Brown and Camb5 demonstrated the sensitivity of the adhesion test and compared it with the agglutination test. They found that adhesion disappears at about the same dilution as the end point of agglutination and hence concluded that adhesion test could be specifi- cally used for leptospirosis diagnosis. Erber16 found centrifugation of older antigen cultures removed debris and facilitated the reading of agglutination lysis tests. The culture and serum were mixed, saline added and incubated at 37 C for one hour. Pot32 employed a macroscopic agglutination test in cases of 'weil's disease using killed antigen. The antigen was treated with phenol which disrupted the leptospirae. He centrifuged and resus— pended the sediment in a fluid free of serum protein. This solution was brought to required density by addition of saline and 0.2 per cent formalin. He tested 26 human sera which were positive for Schuffner's agglutination-lysis test. Positive results were obtained in 25 cases. I Using 100 human sera, Pot andIDornickx33 made a comparison between the complement fixation test and agglutination-lysis test results. They concluded that low titers, approximately 1:100, with the agglutination-lysis test were without diagnostic value and might lead to incorrect conclusions. 37 Smith andeulloch studied the macroscopic agglutination test. They used h - 10 day old leptospirae cultures'as antigen, to which were added various quantities of formalin. They also observed the in- fluence of heat on the effect of agglutination. They stated that optimal results required incubation at a temperature between 30 and 37 C. Human sera, urine, as well as guinea pig sera were employed. Brown6 employed a rapid presumptive serological test for the diagnosis of Weil's disease. A dense saline suspension of Leptospira icterohaemorrhagiae was placed on a slide. This suspension was for- mplized to a concentration of 0.2 per cent. Serum dilutions were then / ‘ added. .After 10 minutes at room temperature readings were made. Brown's technique for antigen preparation was modified by Lederle Laboratories1 in l9hl and was adapted for the plate agglutination test. This test is not used at the present time. 3 Boerner and Luckens have reported a method of preparing anti- gen fOr the complement fixation test using broth cultures of Lept - spira icterohaemorrhagiae and Leptogpira canicola. These preparations showed no anticomplementary effects. A comparison.was made between the complement fixation and agglutination-lysis tests. The complement fixation test demonstrated leptospiral antibodies when the agglutina- tion-lysis test results were as low as 1:300. Starbuck and'W'ard38 made a comparative study of the macroscopic agglutination test with the standard.microscopic test. Using positive human and rabbit sera against Leptospira icterohaemorrhagiae, it was found that the titers obtained with the macroscopic test were always much lower than those obtained with the microscopic test. Gardner and Wylelg examined 1120 human sera utilizing the macroscopic tube agglutination method. The antigen used was a sus- pension of formalinised (0.25%) killed organisms. Positive sera gave visible results after standing overnight at room temperature. Gardner20 described a simple rapid microscopic technique for leptospiral agglutination. He compared the macroscopic agglutination test with the rapid microscopic technique using killed cultures as antigen. The ratio of positive to negative results was very similar for both tests. A similar method was employed by Kruger.26 Randall 22 31.,35 employed sonic vibrated leptospirae as anti- gen in the complement fixation reaction and.mede a comparison with the microscopic agglutination test on hwman sera, using living anti- gen. Their results indicate that there is a parallelism between the amount of the complement fixing antibody and agglutinins; however, they noted that it was necessary to multiply the indicated dilution of the sera in the complement fixation test by six for obtaining a comparison with the dilution in the agglutination test. Newman?o 'used killed antigen (formalin 0.15%) and incubated at 37 C for 3 hours. In this study the use of the agglutination test for laboratory diagnosis of leptospirosis in dogs was evaluated. During the second week of infection the test was only 50 per cent accurate; however, after the third to fourth week the test was 100 per cent accurate. This indicated that the accuracy of the aggluti- nation test increases with chronicity of infection. Even though the test is rapid and reasonably accurate its usefulness is limited. Yorkhl described the complement fixation test for bovine leptospirosis using antigen prepared from embryonated eggs. He also made a comparison between the complement fixation test and the agglu- tination-lysis test with 55 bovine sera. He obtained a close corre- lation between dilution and points for the two tests. However, he mentioned several advantages that the complement fixation test has over the agglutination-test such as: a more objective test, distinct difference between positive and negative sera, and a more useful method when a laboratory has equipment for routine complement fixation tests. Hoaggtwgluzh described a method for the preparation of macroscopic leptospiral agglutinating antigens, prepared in a manner that they became more sensitive, specific and stable under longer periods of storage. These workers made a comparison with microscopic agglutination-lysis tests using sera from cattle, pigs and dogs. It was found that the degree of correlation between these methods, at significant antibody levels, was approximately 70 per cent. York and Johnsonhh compared three basic tests: agglutination- lysis, plate agglutination and complement fixation. They used sera from cattle infected with Leptospiranpgmgga and dogs infected with, Leptospira canicola. For the agglutination-lysis test, the mixed antigen and serum were incubated at 37 C for one hour. In the comp plement fixation test the antigen was mixed with the serum and incu- bated at 37 C for one hour. The hemolytic system was then added. The authors found that the agglutination-lysis and complement fixa- tion tests were equally reliable and sensitive for diagnostic work. However, they also found that the plate agglutination test is less sensitive, but with commercially prepared antigen false positive reactions were absent. Stoenner39 evaluated the technique of the capillary tube test employing sera from human, cattle and dog. The antigen used.was a suspension of formalin killed leptospirae in hypertonic buffered sodium chloride solution. Serum dilutions and antigen were mixed in capillary tubes. This method was compared with the standard micro- scopic agglutination-lysis and was found to be favorable in sensiti- vity and specificity. Gochenour 32 21.,21 studied 29 human cases of acute lepto- spirosis and they found that serological examination by the complement fixation test using sonic vibrated antigen gave good results. Stoenner,"0 evaluated the plate test and capillary tube tech- nique on 7066 bovine sera and found both compare favorably with the agglutination-lysis test in specificity and sensitivity. The hemagglutination procedure developed by Chang and MbComb9 produced lower titers than those obtained with the agglutination lysis test. hl Stoenner stated that incubation of serum.and antigen.varies from laboratory to laboratory from one hour at 37 C to overnight at room temperature. Usually sera are diluted in tenfold increments, but in some laboratories the sera are tested by either 2, h, or 5 fold dilutions. In most instances, cultures with four to five days growth are used as antigen, but in some cases cultures grown for 30 days have been used and produced favorable results. The use of various media influence the number of leptospirae which can be culti- vated; therefore, when using undiluted cultures the numbers of organ- ism used as sources of antigen.may vary due to the dark field method of evaluating cell content. In a series of studies comparing the effect of some variables of the agglutination lysis test on titers of sera he reported that of all the variables and modifications, the most important factors having effect on the titers, were density of antigen and method of preparing serial dilutions. 0f lesser impor— tance, but not insignificant, was the variation due to strain of Leptospira, time of serum-antigen incubation or the age of culture. He stated, "In tests in which antigen of low cell content and ten- fold serial dilutions of serum were used, geometric mean titers were thirtybeight fold greater than titers obtained in tests in which dense antigen and twofold serial dilutions were employed." The author states that the technique of agglutination-lysis test should be standardized so that results from various laboratories will be comparable. N'owicki31 demonstrated that the urine of humans or animals infected with leptospirae contains a specific lytic agent. ‘When the urine was concentrated ten times, the lytic agent was recovered very easily and was used for in;zit£g tests. Positive results were ob- tained in 20-30 hours after infection. Cultures of living lepto- spirae or those preserved with Chinosol were used as antigen. The advantages of this test are: ease of technique, early diagnosis and differentiation between active infection and recovered cases. 10 Following the work of Chang and McComb9, Cox11 found that leptospirae extracted with ethanol could sensitize sheep erythrocytes to agglutination with homologous leptospirae antisera from rabbits. It was also noted that sensitized sheep erythrocytes were susceptible to lysis in high dilutions of homologous antisera and complement. ' Howarth25 also described a macroscopic tube agglutination test. He tested sera of different animals and human using killed formplized antigen. The serum.antigen dilution was kept for 12 hours at 37.5 C then for 6 hours at room temperature. After this time readings were made. A positive test was indicated by the appearance of grossly visible fluffy clumps. This technique was compared with the micro- scOpic agglutination-lysis, using sera with high or low end point dilutions, and showed a close correlation. Fifteen per cent of the sera tested by the macroscopic agglutination method had end point dilution values which were one dilution lower than with the aggluti- nation-lysis test. StoennerL2 employed these following tests: complement fixation, capillary tube and agglutination-lysis, for testing sera from herds of cattle recently infected by Leptospira pomona and also having a history of serological evidence of past infection. He found that the titers were highest with the agglutination-lysis test. He also showed that both the agglutination-lysis and capillary tube tests were comp parable for detecting residual antibodies from herds with previous infectiong'whereas, the complement fixation test was limited in this respect. I I‘\ ($3: 11 12 Cox described the preparation of his hemolytic antigen from nonpathogenic Leptospira biflexa and described the use of the hemo- 1ytic reaction in testing leptospirosis in.human sera. Cox gt 31.,13 compared the hemolytic test with the microscOpic agglutination-lysis test using hSS human sera which had been stored at-IK)C for several years. The results obtained from their studies indicated that the microscopic agglutination test might be replaced by the hemolytic test when diagnosing human leptospirosis. Newborne29 conducted experiments to compare the rapid plate test and capillary tube test of Stoenner to the agglutination-lysis test using dog and pig sera. From the results of this study the rapid plate method seemed to be the better method for screening sera. The advantages which were gained from this were twofold: (a) improved correlation of serological surveys due to standardization of the anti- gen; (b) enabled the practitioner to perform rapid plate screening tests in the field. Bryan7 tested 15,092 serum.specimens from different herds for Leptospira 222223 antibodies. He develOped a rapid plate test using antigen prepared with Giemsa's stain. This test gave positive results with sera that showed titers of 1:100 or higher by the agglutination- 1ysis test. Hirschberg23 modified a plastic depression tray for use in dark field microscopic agglutination-lysis test. A comparison of the results by this method corresponded favorably with the ordinary test tube method incubated 2 hours at 50 C or refrigerator overnight. 12 Salton gt 21.,18 recently developed a macroscopic slide test for leptospirosis diagnosis. They found this technique to compare favorably with microscopic agglutination test with regards to its sensitivity. Because of the simplicity of the test, the authors suggest that this method is well adapted for small laboratories. One of the advantages of this test is that several antigens may be come bined into pools containing as many as four antigens per pool, which make possible the examination of large numbers of sera. CHAPTER III MATERIAL AND METHODS The following sources of sera were used throughout this study: ten samples of human sera which were positive for Leptospira 222293 were obtained from Col. M. B. Starnes, V.C., Walter Reed.Army Insti- tute of Research, Washington, D.C. Representative samples of sera, positive for Leptospira pomona were obtained from the following animal species: cattle, sheep, goats, dogs, guinea pigs and hamsters. These sera had been stored at -20 C. The serum donors were animals which had been utilized by various workers in the Department of Microbiology and Public Health in their research. Turbid sera were centrifuged at 5000 rpm for 15 minutes in the Servall angle centrifuge, Model A. At least 5 negative serum.samples from the same animal species under examination were included as controls for the serological tests. Master dilutions for each sample were made by adding 0.05 ml serum to 0.5 ml of sterile "Chang's buffer," which is prepared by dissolving h.0 g NaZHPOh. 7H20, 0.8 g KHZPQh and 8.0 g HaCl in dis- tilled water to make two liters. The buffer had.a final pH of 7.0. The buffer consisted of the ingredients of Chang's medium8 less the liver extract, tryptose and rabbit serum. Subsequent tenfold dilu- tions up to 10-.8 were made from this original dilution. The antigen used.was the "Johnson" strain of Leptospira Ramona prepared from h-7 day old cultures which had been grown at 30 C in "Antigen vials" (500 x 1000 mm) containing approximately 50 ml of Chang's fluid mediumg enriched with 0.01 per cent hemoglobin (Difco) and sterile rabbit serum to give a final concentration of 10 per cent. Antigen density was estimated with a dark field microscope and adjusted to approximately 250 cells per field at 590 x magnification or approximately 108 leptospirae per ml. ApprOpriate dilutions were made with sterile Chang's medium and then centrifuged in an.Inter- national Clinical Centrifuge Model C.L. for 15 minutes at 1500 rpm. The formalinized antigen was prepared by the addition of a 0.1 per cent formaldehyde solution to the diluted culture. This culture was centrifuged in the same manner as was done with the live antigen. The formalinized antigen was used within one hour after preparation. A modified darkfield type illumination was used by inserting a star diaphragm into the Abbe condenser and employing 100 x optical magnification.28 The agglutinationslysis tests, using living antigens, were incubated in a thermostatically controlled.water bath at 37 C for 2 hours. A similar test was also conducted in parallel but was incu- bated at room temperature, approximately 22 C for 16-18 hours. The formalinized antigen test was also incubated at room temperature for 16-18 hours. In the comparison of these three methods of preparing the re- agents for agglutination lysis, the modified microscopic agglutina- tion-lysis tube test was used.28 The highest dilution which gave any evidence of agglutination or lysis or both was noted as end point for the DGSDBQ 15 In all of the tests employed in this work the amount of anti- gen added to the dilution of serum was 0.1 ml. The dilution factor created by adding the antigen was not included in the serum dilutions as expressed in this thesis. . CHAPTER IV RESULTS The data shown in Tables I through VIII indicated that regard- less of incubation time or temperature,.formalinization of leptospirae had a deleterious effect on agglutination with leptospiral antiserum. When living antigen was used, however, the agglutination or lysis reaction occurred with higher dilutions of serum.than when for- malinised antigen was used. It can be noted from.the tables that time and temperature of incubation were important factors in deter- mining the end point titers of the sera.‘ The salient feature of these results was that agglutination or lysis and points were always higher when living antigen was incubated with the sera at room temperature for 16-18 hours than when allowed to react at 37 C for 2 hours. In some cases agglutination was not seen in the lower dilution (usually 1:10) of antiserum. However, the remaining dilutions lysed the leptospirae to varying degrees to the end point titer. In general, when formalinized antigen was used, lysis never occurred. In the lower dilutions of antiserum.the cells were adherent in the form of clumps consisting of a loose network. This agglutina- tion resembled the "H" type seen in §almonella. When living antigen was used with lower dilutions of the anti- serum, the phenomenon of agglutination always took place and preceded the formation of "lysis balls" or "degenerative granules” although the 17 number of leptospirae*was greatly diminished in comparison with the control. Loose floccular clumps, as seen, with formalinized antigen, were never found because the leptospirae had become lysed. 18 EXPLANATION OF SYMBOLS USED IN EXPRESSING DEGREE OF AGGLUTINATION, LXSIS OR BOTH IN TABLES I THROUGH VIII (+) Indicates 100 per cent agglutination, lysis or both. (+P) Indicates 75 Per cent agglutination, lysis or both. 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A AI I AI I A AI I A OH I H A I A I u A A I I I u A I AI A I I. A I u I u A AI I A I A AI I n m I A I H I A I I A I A I AI I A I n AI I A I H A AI I AI I A AI AI M o I A I A I I A AI I I I w AI I H m I A I .I. I A AI I I A I A AI I A a H A I I I H A A I I I A AI I AI m I H A I AI I H A AI I AI I A AI I A N I u A AI I u A I AI I A I I a m A m m a m N H b LF 0 m I; h N H P F b Lm b m N H 553 ** H z: m .mfil Illafifi wosmfisfifi i 83E @950 I on: dummaa 560 case *4mmm mmsmz A0 megammm HHH> Handy CHAPTER V DISCUSSION AND CONCLUSION The results of this work provide further evidence to current thinking that there is a great need for standardization of the micro- scopic agglutination-lysis test in the diagnosis of leptospirosis. The test is widely used in various laboratories. Many variations in the time and temperature of incubation, sources of antigen and methods of antigen preparation are described by various workers. Meet inves- tigators agree that the agglutination-lysis test represents the best single serological diagnostic tool we have today. Slight variations in end point titers do occur for the same serum.when tested using the 16-18 hour period at room temperature method as compared with the 2 hour period at 37 C. These studies indicate that the use of a longer period of in- cubation, 16-18 hours at'roomItemperature, more nearly approaches the true end point titer of the serum. These findings are in accord with the underlying principle that the combination of antibody with respec- tive antigen requires adequate time to react completely even under optimal conditions. The establishment of equilibrium for combination of antigen with antibody requires at least 5 minutes at 0 C. Accord- ing to Dreyer and.Douglas,lh equilibrium.is not attained.at room temperature even within h hours. The combination of the ever dimin- ishing free residues may take several hours to reach final equilibrium. Taking this principle into consideration one might account for the 28 low end point titers reached when using 37 C for 2 hours. Hartwigk and Stoebbe,22 recently stated that the most desirable time for reading the agglutination-lysis test is after 2h hours at room temperature. During the course of this thesis study, it was ob- served that after 18 hours of incubation the reagents frequently became contamined with.microorganisms from the environment, princi— pally in the test in which living antigen was used. Preliminary tests, undertaken during the initial phases of this investigation, showed that l6-18 hours at roam temperature gave the same end point titers as obtained with 2h hours incubation. Hence the former incu- bation time was used throughout the investigation. According to Eaglels the rate of the agglutination reaction increases rapidly between 0-30 C. Above 30 C the increase in reaction time is much slower and may even become insignificant. This pheno- menon may account for the fact that slightly higher titers were ob- served in the tests incubated 16-18 hours at room temperature as compared to those for 2 hours at 37 C. The results of this study show that the living leptospiral antigen was a better indicator than the formalinized organisms for obtaining high end point titers under the conditions of the two tests. This has been the experience of many other investigators; however, Hartwigk and Stoebbe,22 stated that there is a close correlation (78.h%) of end point titers between living and formalinized (0.5%) antigen at room temperature for 2h hours. Many laboratories utilize formalin treated antigen without concern for sensitivity. Other laboratories have found that 29 preparation of reliable formalinized leptospirae suspensions is a difficult problem to solve because these antigens deteriorate in sensitivity with storage. A salient feature of this study was that the density of anti- gen used was kept constant throughout all the agglutination-lysis tests. Furthermore, the leptospirae were kept at a minimum number commensurate with highest titers. This is in accord with the results of Stoenner,h1 who found that tests with antigen of low cell count gave higher titers than a similar antigen with high cell count. The prozone phenomenon did not affect the determinations of this test conducted with live antigen incubated at room temperature 3 for l6-18 hours. However, prozones of 1:101to 1:10 were observed in the tests with living antigen incubated for the shorter period. In view of the many different opinions as to the most suitable method for leptospirosis diagnosis, a questionnaire was prepared by 3b the American.Association of Veterinary Bacteriologists. Copies of this questionnaire are being sent to bacteriologists responsible for actual performance of leptospirosis diagnosis in the various states. The questions asked were relative to the type of tests used, the interpretations and significance of the results obtained. It is hoped that in the future, a better understanding of the problem of standardization and methods for the diagnosis of lepto- spirosis will be forthcoming. CHAPTER VI SUMMARY A study was made of the effect of various factors upon the agglutination-lysis test and the agglutination test for Leptospira £32223 infection. It was found that incubation at room temperature for 16-18 hours yielded higher end point titers than incubation at 37 C for 2 hours, when live antigen was used. A comparison between formalinized antigen and live antigen using the 16-18 hour*method showed that higher end point titers could be obtained with live antigen. The kinetics of antigen-antibody combination at various periods of time and temperature were discussed. 31 REFERENCES l. Anon: Leptospirosis and its Diagnosis with the New Lederle Antigen. Vet. Bull. 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