SF 796 .ri6 Copy 1 Issued April 7, 1911. U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY.— BULLETIN 136. A. D. iMELVIN, Chief of Bureau. THE DIAGNOSIS OF GLANDERS BY COMPLEMENT FIXATION. BY JOHN R. MOHLER, V. M. D., Chief of the Pathological Division, AND ADOLPH EICHHORN, D. V. S., Bacteriologist, Pathological Division. [Reprint, October, 1911, with slight revision.] WASHINGTON: GOVERNMENT PRINTING OFFICE. 1911. GIass_ S-E44i». Book \^y 7 Issued April 7, 1911. U. S. DEPARTMENT OF AGRICULTURE, , ^ BUREAU OF ANIMAL INDUSTRY.— Bulletin 136. / » ; A. D. MELVIN, Chief of Bureau. THE DIAGNOSIS OF GLANDERS BY COMPLEMENT FIXATION. BY JOHN R. MOHLER, V. M. D., '1 Chief of the Pathological Division, AND ADOLPH EICHHORN, D. V. S., Bacteriologist, Pathological Division. [Reprint, October, 1911, with slight revision.] WASHINGTON: GOVERNMENT PRINTING OFFICE. 1911. M^3 ^ ^^ ^^ ^^ THE BUREAU OF ANIMAL INDUSTRY. Chief: A. D. Melvin. Assistant Chief: A. M. Farrington. Chief Clerk: Charles C Carroll. Animal Hushandry Division: George M. Rommel, chief. Biochemic Division: M. Dorset, cliief. Dairy Division: B. H. Rawl, chief. Inspection Division: Rice P. Steddom, chief; R. A. Ramsay, Morris Wooden, and Albert E. Behnke, associate chiefs. Pathological Division: John R. Mohler, chief. Quarantine Division: Richard W. Hickman, chief. Zoological Division: B. H. Ransom, chief. Experiment Station: E. C. Schroeder, superintendent. Editor: James M. Pickens. 2 n, 0* ffi. LETTER OF TRANSMITTAL. U. S. Department of Agriculture, Bureau of Animal Industry, Washington, D. C, March 20, 1911. Sir: I have the honor to transmit herewith a paper entitled " The Diagnosis of Glanders b}^ Complement Fixation," by Drs. John R. Mohler and Adoph Eichhorn, of the Pathological Division of this bureau. Since the discovery of the glanders bacillus in 1883 many efforts have been made to find a reliable method of making an early diag- nosis of the disease. The mallein test and later the agglutination test have been and are at present in general use, but neither of these is sufficiently reliable to be entirely satisfactory. Schiitz and Schu- bert, German investigators, recently called attention to the value of complement fixation as affording a more reliable method of diag- nosing glanders, and within the past year this method has been carefully studied and tested in the Pathological Division of this bureau. It will be seen from the details given in the accompanying paper that the authors have found the complement fixation test to be highly reliable as a diagnostic agent for glanders, and they present a thor- ough exposition of the method resulting from their searching experi- ments, including practical tests in a recent outbreak of glanders at Washington, D. C. In view of the great economic and scientific importance of the subject, and as no work upon this new method has so far been pub- lished in the United States, I recommend the immediate publication of the paper in the bulletin series of this bureau, in order that the value of the method and the technique necessary for its application may be made more fully available in this country. Respectfully, A. D. Melvin, Chief of Bureau. Hon. James Wilson, Secretary of Agriculture. CONTENTS Page. Introduction 5 Hemohsis 7 Method of obtaining hemolytic amboceptor (rabbit serum ) 9 Titration of hemolytic rabbit serum 12 Method of obtaining complement (guinea-pig serum) 14 Titration of complement 14 Specific complement fixation (deviation) 16 Method of obtaining serum of animals to be tested 18 Inactivating the serum 19 Preparation of the antigen (glanders bacilli extract ) 20 Titration of the extract 21 The complement-fixation test 23 Application of the test 24 Controls _ 25 Interpreting results of tests 27 Controlling glanders in an infected stable 29 Results of practical tests with complement fixation 29 ILLUSTRATIONS. Page. Plate I. Diagrammatic representation of complement fixation 8 II. Titration of hemolytic amboceptor (rabbit serum) 12 III. Titration of complement (guinea-pig serum) 16 IV. Titration of antigen (glanders bacilli extract) 20 V. Final test showing positive reaction to glanders 26 4 THE DIAGNOSIS OF GLANDERS BY COMPLEMENT FIXATION. INTRODUCTION. The early diagnosis of glanders constitutes one of the most im- portant and difficult tasks which confronts the veterinarian engaged in sanitary work. This of course does not apply to the clinical cases of glanders, as in such cases the diagnosis is usually made without much difficulty from the characteristic symptoms and lesions present. In those instances, however, where there are no positive indications of the disease, it is impossible to establish a diagnosis by j^hj'^sical examination, and only through the aid of some special diagnostic method or test can there be any hope of determining the presence or absence of the disease. Horses affected with occult or latent glanders, and in which the disease is not suspected, are undoubtedly great fac- tors in the propagation of the infection. Indeed, there are many glandered horses which do not show positive symptoms until the later stages of the disease. Since the discovery of the glanders bacillus in 1883 by Loeffler and Schiitz the diagnosis of glanders has been the subject of numerous investigations, and as a result great progress has been made in its determination. After the isolation of the infective agent of the disease the diagnosis was confined to the demonstration and culti- vation of the organism, or to the reproduction of the disease by inoc- ulations of exudates or parts of diseased organs from affected horses into susceptible animals. The first important step toward determining obscure and latent cases of glanders was made by the discovery of mallein. With the aid of this biological product of the Bacillus mallei a large propor- tion of latent and occult cases of glanders can be diagnosed, partic- ularly when such tests are made by efficient and experienced veteri- narians. There are, however, a considerable number of glanderous animals in which the mallein fails to give a typical reaction, and, on the contrary, a reaction may follow the injection of mallein in the absence of glanders. Thus mallein is not an entirely reliable diag- nostic agent for determining glanders, nor has it ever been considered 5 D DIAGNOSIS OF GLANDERS. as efficacious in the detection of this disease as tuberculin for the diagnosis of tuberculosis. With the application of the agglutination test for glanders it ap- peared that a more satisfactor}^ method had been found for the diag- nosis of all types of infection with this disease. It was first sug- gested by McFadyean in 1896, after this investigator had observed the value of Widal's typhoid-fever agglutination test, but was not generally adopted until the method was perfected by Schiitz and Meissner, whose interesting results were published in 1905. This test has since been extensively employed in jiractically every country where glanders exists, and therefore ample opportunity has been furnished for drawing conclusions relative to its diagnostic value. While there is no doubt that the agglutination test is of great value in all cases of recent infection, the blood in such cases possessing a very high agglutination power (1 to 1,000 and higher), nevertheless extensive experience has proved that horses affected with chronic glanders give occasionally a very low agglutination value, which in some cases is even lower than that of normal blood serum (1 to 400 or even lower). From this condition it appears evident that in cer- tain cases of chronic glanders the disease can be determined only by repeated tests, and a diagnosis in such cases is only possible from the fluctuation of the agglutination value — either an increase or a de- crease — as it is a well-known fact that this value remains stationary in normal horses. Besides this difficulty, there should also be taken into consideration the fact that the blood of normal horses sometimes shows a high agglutination value (1 to 800 and higher), and that changes in the agglutination power have been observed even in animals free of glanders. Furthermore, repeated agglutination tests require con- siderable time, as at least two weeks should elapse between two tests. Therefore the agglutination test alone does not constitute an entirely satisfactory diagnostic method for glanders. However, as its great value has been proved beyond doubt in the early cases of infection, it may well be utilized as an adjunct to any other test which may be applied in connection with the diagnosis of suspected cases of the disease. Hutyra compared the agglutination test with the mallein test from the tables included in the works of Schiitz and Miessner and of Nevermann, and came to the conclusion that the application of the agglutination test alone has not decreased the number of faulty diagnoses. He believes that the principal difference in the results lies in the fact that a large number of horses which were classified as only suspicious by the mallein test are considered as actually in- fected by the agglutination test. HEMOLYSIS. 7 In further efforts to find a method by which an early diagnosis of glanders could be made, various investigators directed their atten- tion to the precipitation reaction. This is based upon the fact that when blood serum comes in contact with a concentrated extract of glanders bacilli the precipitins or receptors, which are formed in the blood of infected animals from the time the infection first occurs, are bound to the bodies in the bacillarj^ extract, producing a precipita- tion which is manifested by cloudiness at the point of contact of the two fluids. This method of diagnosing glanders has recentl}^ been recommended by Pfeiler ^ in Germany and by Konew ^ in Russia, but it has not been applied extensively in practice. This is probably due to the fact that the reading of the reaction is in some cases diffi- cult, due to the indistinct ring which occasionally is formed at the line of contact between the precipitant and the serum. In 1909 Schiitz and Schubert ^ published the results of their im- portant work on the application of the method of complement fixa- tion for the diagnosis of glanders. And since their experiments were followed b}^ splendid results, exceeding by far the results ob- tained from either the mallein or the agglutination test, they recom- mended that this method of diagnosis in combination with the agglu- tination test be taken as the official test in Germany. This method, overcoming as it does the disadvantages of the mallein and aggluti- nation tests, constitutes without doubt the most reliable method for the diagnosis of glanders which we have at our command at the present time. The complement-fixation test is, in fact, the most definite method known for determining specific infections and is as nearly perfect as a biological test can be. It has only recently been introduced in veterinary science and the publications concerning it are at present limited exclusively to foreign periodicals. The prin- ciple of this test is presented in the phenomenon of hemolysis, which was first discovered and studied by Bordet and Gengou, and extended by Ehrlich, Morgenroth, and Sachs. HEMOLYSIS. It is a well-known fact that if red blood corpuscles of one animal are introduced into another of a different species the blood of the latter acquires the power to dissolve the blood corpuscles of the 1 Pfeiler, Willy. Die Ermittelung der Rotzkrankheit durch die Prazipitationsmethode. Archiv fiir Wissenschaftliche und Praktische Tierheilkunde. Band 35, Heft 4/5, pp. 323-337. June 24, 1909. - Konew, D. Prazipitationsreaktion als diagnostische Methode beim Rotz. Vorliiuflge Mitteilung. Centralblatt fiir Bakteriologie. Abt. 1, Orig., Band 55, Heft 3, pp. 251-253. July 9, 1910. 3 Schiitz and Schubert. Die Ermittelung der Rotzkrankheit mit Hilfe der Komplement- ablenkungsmethode. Archiv fiir Wissenschaftliche und Praktische Tierheilkunde. Band 35, Heft 1/2, pp. 44-83. 1909. 8 DIAGNOSIS OF GLANDERS. foiTner when mixed with them in a reagent ghiss. This reaction is termed hemolysis, which means the dissokition of blood corpuscles, thereby setting the hemoglobin free in the medium in which the corpuscles are suspended. To illustrate this phenomenon, if a rabbit is injected intraperi- toneally, intravenously, or subcutaneously with washed red blood corpuscles of a sheep, the blood of the rabbit will develop antibodies which possess a dissolving action for the sheep blood corpuscles; that is, the rabbit blood will contain specific hemolysins. The acquired hemolytic property of the blood depends on two substances. One of these is present in the blood of every animal, and is known as the complement. It is thermo-labile, which means that it is rendered inactive after the blood or serum has been heated to 5G° C. for half an hour. The other body, which is formed as a result of the injection of blood corpuscles, is thermo-stabile ; that is, it resists heating even higher than 56° C.. and is know^n as immune body, fixative, sensitizer, or hemolytic amboceptor. The name am- boceptor is derived from the fact that it has an affinity on the one hand for the blood corpuscles of the species of animal with which the animal has been injected, and on the other for the complement, this union taking place only after the first-mentioned affinity has been satisfied. These two substances, together with the corpuscles to be dissolved, comprise the hemolytic system, and their combination leads to hemolysis. (See PI. I, A.) This means that an opaque suspension of blood corpuscles is rendered semitransparent or " laked." The hemolysis, strictly speaking, does not represent a complete solution, but only an action of the hemol3^sin on the stroma of the erythrocytes, which permits the escape of the hemoglobin of the red blood corpuscles. The injection of blood corpuscles of one animal into another of a different species gives rise to the development of antibodies which confer upon the blood serum the hemolytic action. This phenomenon is somewhat similar to the production of receptors in the formation of antitoxins which are thrown off, but these receptors alone are not able to dissolve the red blood corpuscles, requiring also the presence of a ferment. This ferment, however, is a. constant constituent of the blood and is known as the complement. That both of these substances are constantly present in the hemo- lytic serum can be demonstrated in the following manner: If the hemolytic serum is heated to 56° C. for half an hour, thereby de- stroying the complement, this serum will no longer possess a hemo- lytic action; that is, it will no longer dissolve red blood corpuscles. This heating of the serum is known as inactivation. On the other hand, if to such inactivated serum there be added fresh ' untreated (Jul. 136, Bureau of Animal Industry, U. S. Dept. of Agriculture. Plate I. A. fc vi:^ + XrS = fleJi/ocdce//. ceatorr/fMit/ (/„i^n .freMocJc^// (aTnl7^"Si Jlem^Uis^,// result. (Sheep) niJ nmiccepfiir (no/Kmo/L,3). B. xfac^rto. rNoloct^Ho/u^i^) S8 Hoije ■serum frymolement 90 /i)!/f, .a/anders l-omplemenr. Jjocte'n'a. (Gc/fin, Positive reaction with glandered horse serum. 1 „ METHOD OF OBTAINING THE HEMOLYTIC AMBOCEPTOR. 9 serum, which in itself has no henK)l3'tic properties, hemolysis will result. Thus by the addition of this ''resh serum a reactivation is accomplished. This is explained by the fact that through the heat- ing of the serum one of the substances necessary for the hemolysis has been destroyed, which is the complement. After the complement has been destroyed by heating it can be replaced by the addition of any normal serum, because it is known that the complement is present in all blood. However, the guinea-pig serum appears to be the most satisfactory in the application of hemolysis, inasmuch as it is very rich in complement and only a very small quantity is required to be added to the inactivated hemol3'tic serum in order to produce hemolysis. Accordingly, the substances necessar}^ for hemolysis are (1) the hemolytic amboceptor, which is the serum of a rabbit that has been injected with washed sheep blood corpuscles, (2) the complement in the form of normal guinea-pig serum, and (3) washed red blood corpuscles of a sheep. In the jireparation of these difl'eren( substances it is necessary to fix standards of practical constancy by proceed- ing along definite lines in the following manner: METHOD OF 0BTAI^■I^■G THE HEMOLYTIC A^IBOCEPTOK (rABBIT SERUm). Strong, vigorous rabbits are selected, and tliey are injected in- traperitoneally with a suspension of washed red blood corpuscles from a sheep. Three injections are made at intervals of seven days with 7 c. c, 10 c. c, and 12 c. c. of these blood corpuscles suspended in like quantities of phj'siological salt solution.^ The sheep blood is obtained by bleeding a vigorous sheep from the jugular. The side of the neck is clipped and shaved, and the part over the jugular disinfected with 75 per cent alcohol. Then a sterilized small-calibered trocar is inserted into the jugular, and the blood is collected in a sterile bottle containing a few glass beads. After the desired ((uantity of blood is obtained i( is shaken for 10 minutes in order to defibrinate it. After defibrination, it is filtered through a double layer of sterile gauze into the glass tube in which the washing is to take place. The glass tube containing the blood is then filled with salt solution and placed in a centrifuge wdiich has a speed of 2,500 to 3,000 revolutions per minute. After the red blood corpuscles are thrown down the clear fluid above the corpuscles is pipetted off. Then the blood corpuscles are again thoroughly mixed ,t with salt solution in the proportion of 1 to 9, and the centrifugaliza- tion is repeated. This washing should be can-ied out three or even four times in order to eliminate all the serum adhering to the red 1 T'nlpss otherwise stated, the term " salt solution " In this bulletin refers to an 0.85 por I cent solution of sodium chlorid. 10040°— Bull. 136—11 2 10 DIAGNOSIS OF GLANDERS. blood corpuscles. Such washed blood corpuscles can then be used for the injection of rabbits in the preparation of hemolytic amboceptors, as well as for the test pro^Dcr. The washing of the sheep blood corpuscles must be thoroughly carried out, inasmuch as the presence of even traces of serum adher- ing to the corpuscles may cause difficulty in obtaining satisfactory results. If rabbits were injected with red blood corpuscles contain- ing a small quantity of serum, the rabbits would develop, not only antibodies, or immune bodies, but also coagulins and anticomple- ments, and the presence of these substances would give rise to diffi- culties in demonstrating the presence or absence of a complete hemol- ysis. Furthermore, if blood corpuscles containing even traces of serum were used in the tests it might produce a fixation of the com- plement and thereby give rise to errors. Such errors would occur particularly if the hemolytic action of the rabbit serum was not very high. The washed blood corpuscles should be used for the injection of the rabbit on the day the blood is drawn. For testing purposes, however, it will keep for two or three days in the ice chest. The rabbits to be injected are shaved on the posterior part of the abdomen, and the skin is disinfected with 75 per cent alcohol. They are then held with the head down by an assistant in order to prevent the puncturing of the intestines. The blood corpuscles to be in- jected are mixed with an equal quantity of salt solution and heated in a water bath to body temperature. Intravenous injections have been recommended by some investigators, but it was found in our work that intraperitoneal injections gave very satisfactory results, and furthermore, there is very little danger of losing rabbits from various complications by this method. After three injections with the quantities and at the intervals stated above, a small amount of blood is taken from the rabbit on the fifth or sixth day after the last injection. This blood is then titrated in order to determine whether its hemolytic action is of sufficient strength for future work. If the blood serum is found to be of a sufficiently high titre, the rabbit may be either bled to death, or, what is far more satisfactory, about 15 c. c. of blood may be drawn from the veins of the ear. By the latter method the rabbit may be used continuousl}' for the production of hemolj^tic serum. If it is desired to obtain the blood by bleeding the rabbit to death, the animal is anesthetized by a mixture of chloroform and ether, the hair on the neck is shaved, the skin is disinfected, and an incision made on one side of the neck in order to sever both the jugular vein and the carotid artery. Should the flow of blood cease on this side, the other side may also be cut. The blood is collected in centrifuge METHOD OF OBTAINING THE HEMOLYTIC AMBOCEPTOR. 11 tubes, and after the bleeding has been completed centrifugalization of the blood is accomplished and the supernatant serum drawn off with a pipette. Bleeding of the rabbit through the veins of the ear is best accom- plished in the following way: After washing the ear and closely clipping the hair over the veins on the outside of the ear, the skin is disinfected with 75 per cent alcohol. Then a pledget of cotton is soaked in hot water (about 45° or 50° C.) and wound around the base of the ear in order to produce a hyperemia of the blood vessels. When a sufficient dilation of the vessels is observed, the middle and posterior auricular veins are severed, and the blood is then collected in centrifuge tubes. If the blood ceases to flow, the cotton should be removed from the base of the ear, and after placing it in hot water it is again applied to the ear and the hy2)eremia is thereby reestab- lished. In case the coagulation of the blood has prevented its flow at the place where the veins were severed, the wound may be scraped with a knife, and usually the blood will commence to flow freely again. The collected blood is then treated in the same manner as described above — that is, centrifuged — and the supernatant serum pipetted off. Should this hemolytic rabbit serum be used before it is 3 days old, it must be inactivated by heating to 56° C. for one-half hour. After this time the hemolytic rabbit serum is preserved with one-half of 1 per cent of carbolic acid — that is, to each 9 c. c. of serum 1 c. c. of a 5 per cent carbolic-acid solution is added. Serum preserved in this way ma}^ be kept for two or three months. However, it should be retitrated every two or three weeks, as occasionally the titre of the serum drops. Such carbolized hemolytic serum does not require inactivation. "\"\1ien preparing hemolytic serum it is best to start with several rabbits, as occasionally one may die as a result of anaphylaxis, and, again, some rabbits are not adapted for the production of hemolysins. The titre of the hemolytic serum from a rabbit does not remain stationary, but for two or three weeks after the last injection it gradually lowers until it reaches that of a normal animal. If, how- ever, such a rabbit is reinjected with washed corpuscles after several weeks, the hemolysins will again appear after a short time. Hemo- lysins are kept more or less in reserve in the cells of such an animal. The renewed injection acts as a stimulant, and these bodies are quickly thrown off into the blood, while in a normal animal they form slowly, first being formed in the cells. Thus, the advisability of keeping rabbits which have been bled from the veins of the ear for further production of hemolytic amboceptors can readily be seen. 12 DIAGNOSIS OF GLANDERS. TITRATION OF HEMOLYTIC RABBIT SERUM. The hemolytic rabbit serum is titrated in order to establish the smallest quantity of serum that will produce hemolysis in the pres- ence of a certain quantity of complement and the suspension of washed blood corpuscles of a sheep. The amount of hemolytic serum that will produce complete hemolysis in tAvo hours at 37° C. is an amboceptor unit. For the preliminary work of titration 10 test tubes are taken, and dilutions of the hemolytic serum are made with salt solution in proportions of 1 to 100, 200, 100, 500, etc., up to 4,000. These dilu- tions are made from basic dilutions of 1 to 100 and 1 to 1,000 as may be seen from Table 1. Table 1. — Titration of rabbit scrum {Jiemolytic amboceptor). Tul>e. NaCl solution.! Amboceptor. Comple- ment. 2 Blood cor- puscles. 3 Remarks. 1 2 3 4 5 fi 7 8 9 10 c. c. 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 1 0. c. of — 1: 100 1: 200 1: 400 1: 500 1: 000 1: 800 1:1,000 1:1, .500 1:2,000 1:4,000 c. c. 0.5 .5 .5 .5 .5 .5 .5 .5 .5 .5 c. c. 11 3.5 3.0 4.0 .5 Complement control (no hemolysisshould occur). 12 1: 100 Amboceptor control (no hemolysisshould occur). 13 Salt-solution control (no hemolysisshould occur). 1 0.85 per cent NaCl .solution. 2 0.5 c. c. of a 10 per cent solution of the complement. 35 per cent suspension of sheep-blood corpuscles washed in salt solution. From each batch of serum taken from the rabbit a basic dilution is made in the proportion of 1 to 100, 'JOO, -100, etc., up to 4,000. From these basic dilutions the third column in Table 1 is made, as indicated below : A dilution of — 200 is m:ide by adding 1 c. c. of 1 to 400 is luado by adding 1 c. c. of 1 to 500 is made by adding 1 c. c. of 1 to 600 is made by adding 1 c. c. of 1 to 800 is made by adding 1 c. c. of 1 to 1,000 is made by adding 1 c. c. of 1 to 1,500 is made by adding 2 c. c. of 1 to 1,000 dilution to 1 c. c. NaCl solution. 2,000 is made by adding 1 c. c. of 1 to 1,000 dilution to 1 c. c. NaCl solution. 4.000 is made by adding 1 c. c. of 1 to 1,000 dilution to 3 c. c. NaCl solution. It will be observed that the last three are made from the 1 to 1,000 dilution. 100 dilution to 1 c. c. NaCl solution. 100 dilution to 3 c. c. NaCl solution. 100 dilution to 4 c. c. NaCl solution. 100 dilution to 5 c. c. NaCl solution. 100 dilution to 7 c. c. NaCl solution. 100 dilution to 9 c. c. NaCl solution. BuL. 136, Bureau of An;mal Industry, U.S. Dept. of Agriculture Plate TITRATION OF HEMOLYTIC RABBIT SERUM. 13 The titration proper is then made in the following manner: Ten additional test tubes are each filled with 2.5 c. c. of salt solution, to which is then added the hemolytic serum (amboceptor) in quantities of 1 c. c. of the different dilutions to each tube. Thus, in the first tube we add 1 c. c. of the dilution of 1 to 100 ; in the next, 1 c. c. of the dilution of 1 to 200; and in the third, 1 c. c. of the dilution of 1 to 400, etc. Afterwards the complement of the guinea-pig serum is added in quantities of 0.5 c. c. of a 10 per cent solution to each tube, and finally 1 c. c. of a 5 per cent suspension of sheep corpuscles in salt solution is placed in each tube. Besides these 10 tubes, there are also three control tubes, one to show that the complement alone (without the amboceptor) will not l^roduce hemolysis, the second that the amboceptor alone without the complement will not produce hemolysis, and the third that the salt solution alone will not produce hemolysis. Thus, in the first control tube we add 3.5 c. c. of salt solution, 0.5 c. c. complement, and 1 c. c. suspension of sheep corpuscles. In the second control tube we add 3 c. c. salt solution, 1 c. c. amboceptor of the 1 to 100 dilution, and 1 c. c. suspension of sheep corpuscles. In the third control tube we add 4 c. c. salt solution and 1 c. c. sheep corpuscles. It is advisable to place the tubes for the titration test in the lower row and the control tubes in the upper row of the test-tube rack. As can be seen from the various quantities added to the tubes, the final volume is alwaj's uniformly 5 c. c. Thus, the different amounts of the blood derivatives used are always made up to 5 c. c. by the addition of salt solution. After adding the substances in the test tubes in the order given, the tubes are well shaken, placed in a rack, and the rack put in an incu- bator at 37° C. for two hours. Then it is removed from the incubator and the results are read. (See PI. II.) The highest dilution in which complete hemolysis has taken place represents the titre of the hemolytic amboceptor. Thus, if complete hemolysis has taken place in the tubes where the dilution used in the rabbit serum Avas 1 to 2,000, the hemolytic amboceptor of that serum is represented by 2,000. This titre, however, is not used in the glanders test, but rather its double strength, which would be 1 to 1,000. The titre of the hemolj^tic amboceptor for use in glanders diagnosis should not be less than 1 to 1,000, and therefore if the rabbit serum should jDrove of a low^er titre it should not be used for this test. A low titre of the hemolytic amboceptor would disturb the results of the test, inasmuch as in such low dilutions too much serum would have to be used, and as the serum contains other substances in addi- tion to amboceptors it Avould have an influencing effect on the hemol3'sis. 14 DIAGNOSIS OF GLANDERS. It is advisable to preserve the carbolized hemolytic amboceptor in small vials containing 1 to 2 c. c. of the rabbit serum, sealing them with paraffin. By this procedure the contents of a vial are used up more quickly and without frequent exposure. The titration test of the hemolytic amboceptor is given in consecu- tive order in Table 1, and the method of carrying out the tests relative to the addition of the different substances is likewise given in con- secutive order in all the tables. Thus the number of test tubes used is indicated, as well as the various substances and the quantities to be added, as they follow each other. It is our opinion that by this method the description of the tests can be followed without much difficulty, and by keeping these tables in the foreground the tests themselves may be applied readily, even by those who have had but little experience in this work. Of course accuracy in technique is the most important factor in the success of this line of serum diagnosis, and too much emphasis can not be laid upon this fact. METHOD OF OBTAINING COMPLEMENT ( GUINEA-PIG SERUM ). The complement represents the blood serum of a healthy guinea pig, and is obtained by bleeding the animal by severing the carotid and the jugular. The guinea pig is anesthetized with a mixture of chloroform and ether, and the neck is shaved and then disinfected with a 75 per cent solution of alcohol. The animal is held by an assistant Avith its head down, while the operator uses his left hand to pull taut the skin over the region of the throat and his right hand to make an incision on one side of the neck by which the carotid and the jugular are sev- ered. The centrifuge tube is immediately held under the opening, in order to collect all the blood. After the flow ceases, the same tech- nique is practiced on the other side of the neck. Care should be taken to avoid the cutting of the trachea. The blood thus collected, which usually amounts to 10 or 12 c. c, is placed in the ice chest, and after one or two hours the serum sepa- rates from the clot. The serum is then drawn off, and the rest is placed in a centrifugal machine, in order to obtain all the serum present in the clot. The guinea-pig serum should always be used fresh, and it is never advisable to use it after the second day, as the complement becomes considerably^ reduced upon standing. TITRATION OF COMPLEMKNT. It is desirable to titrate the complement of every guinea pig, as such practice will insure more accurate work and better results in the glanders test. By titration of the complement we aim to estab- METHOD OP OBTAINING COMPLEMENT. 15 lish the complement unit which is the smallest quantity of comple- ment necessary to produce complete hemolysis in the presence of one amboceptor unit and a suspension of blood corpuscles of sheep. The smallest quantity is then taken as its test value. For the titration of the complement six test tubes are used, in addition to the three for controls. A 10 per cent basic dilution is made of the complement in the first test tul^e; that is, to 2.7 c. c. of salt solution 0.3 c. c. of complement is added. From this basic dilution the other dilutions are made by consecutively reducing the quantity of complement in the different test tubes. Thus, in the second test tube 0.5 c. c. of the 10 per cent basic dilution is added, in the third 0.4 c. c, in the fourth 0.3, and so on. Of course we add to each of these test tubes sufficient quantities of salt solution to make the required 3 c. c. Thus, into the second test tube we add 2.5 c. c, in the third 2.6 c. c, etc., of salt solution. After the complement has been added in quantities stated above, it is followed by the amboceptor. One cubic centimeter of the hemolytic amboceptor of which the titre has previously been de- termined is added, and finall}'^ 1 c. c. of a 5 per cent emulsion of blood corpuscles. The purpose of the controls is to establish in the first that the complement alone without the amboceptor does not produce hemolysis; in the second, that the amboceptor alone without the complement produces no hemolysis; and in the third, that the salt solution alone does not produce hemolysis. The first control tube contains 3.5 c. c. salt solution, 0.5 c. c. of the 10 per cent basic dilu- tion, and 1 c. c. of suspension of corpuscles; in the second control 3 c. c. of salt solution, 1 c. c. of the amboceptor dilution, and 1 c. c. of suspension of blood corpuscles; in the third control, -1 c. c. of salt solution and 1 c. c. of suspension of blood corpuscles are used. After shaking all the tubes, the rack is placed in the incubator for two hours, and removed in order to read the results. (See PI. III.) The highest dilution of complement in the tube in which the hemoly- sis is complete indicates the titre of the complement. For instance, if hemolysis is complete in the tube where 0.3 c. c. of the 10 per cent basic dilution of the complement has been used, and hemolysis is in- complete in the tube in which 0.2 c. c. of a 10 per cent basic dilution has been used, then the titre of the complement is 0.03 c. c, inasmuch as we have started with a 10 per cent basic dilution. Thus, in the tests in this instance it would be necessary to use a 3 per cent com- plement solution. In Table 2, the titration of the complement is given as in Table 1 for the hemolytic amboceptor, and this can be followed without much difficultv. Particular care, however, should be taken to use exact quantities as designated in the table. 16 DIAGNOSIS OF GLANDERS. Table 2. — Titration of complement. Tube. NaCl so- lution.' Comple- meiit.2 Ambo- ceptor.' Blood cor- puscles.^ Remarks. 1 c. c. 2.7 2.5 2.6 2.7 2.8 2.9 c. c. 0.3 6.5 .4 .3 9 •1 c. c. c. c. Basic dilution of complement. Tubes 2 to (i are for establishing the smallest quan- tity of complement which produces complete hemolysis. This smallest quantity is then taken as its test value. For instance, if smallest quan- tity is 0.03, then to 97 c. c. NaCl solution 3 c. c. complement is added; if it is 0.02, only 2 c. c. Is added to 98 e. c. NaCl solution. 2 3 4 5 6 1 i 1 1 1 7 3.5 3.0 4.0 .5 1 1 1 Complement control (no hemolysis should occur). Amboceptor control (no hemolysis should occur). Salt-solution control (no hemolysis should occur). 8 i 9 ^ 0.85 per cent NaCl solution. -Guinea pig serum in diminishing quantities. "Of previously titrated hemolytic serum, double dissolving quantity. ^ 5 per cent suspension of sheep blood corpuscles washed in salt solution. 6 This amount is taken from the basic dilution in tube 1. The amboceptor should be inactivated when used fresh; that is, before it is three daj^s old. Otherwise it is carbolized with 10 per cent of 5 per cent carbolic-acid solution, and kept in ice box — in this case it is used without inactivation. Place tube rack in incubator for 2 hours and read results. This test should be made as a preliminary to every suspected glanders serum test, as it is always necessary to determine the smallest quantity of complement to be used for the final test. Of course, within 24 hours any number of tests can be made with the same com- plement dilutions. SPECIFIC COMPLEMENT FIXATION (DEVIATION). ComjDlement fixation is a biological reaction in which the phenome- non of hemolj'sis is emjjloyed as the fundamental princijDle. It is so called on account of the fact that the complement has been fixed by the combination of antigen with antibody and thus prevented from participating in the hemolytic process in which it is essential in order to have hemolysis. By this method even small quantities of amboceptors (antibodies) can be demonstrated in a serum. The presence of an infectious principle in the organism of an animal or a man has a stimulating effect on the production of anti- bodies (immune bodies). If a serum containing such immune bodies is inactivated and brought into contact with the antigen in the presence of complement, the complement Avill become firmly fixed by the combined immune body and antigen. (See PI. I, B.) Thus, anchoring takes place between the antigen and the antibody in which the complement becomes fixed. This anchoring is thoroughly estab- lished when the mixture is placed in an incubator for one hour. The addition of the hemolytic amboceptor and blood corpuscles to KL Industry. U. S. Dept. of AoRiculturs Plate III. SPECIFIC COMPLEMENT FIXATION. 17 such an anchored antigen and innnune body will have no effect. (See PI I, D.) Thus, no hemolysis will take place, inasmuch as the com- plement has been fixed by the immune body and the antigen, thereby leaving the hemolytic system incomplete. On the other hand, if the inactivated serum contains no immune bodies, there would be no substance in the serum to anchor the antigen. As a result, therefore, no fixation of complement will occur, this being left free, and on addition of hemolytic amboceptor and blood corpuscles, hemolysis will now take place. (See PI. I, C.) Neither the antigen nor the antibody alone can fix the complement and thereb}^ influence hemo- lysis when the hemoh^tic amboceptor and blood corpuscles are added. HoAvever, in combination the fixation will invariably take place, and on the addition of the hemolytic amboceptor and blood corpuscles hemolysis will not be produced. Since the discover^' of this phenomenon it has been utilized exten- siveh'^ in serum diagnosis, but probably its greatest value has been obtained from the Wassermann reaction for the diagnosis of syphilis. It has also been employed in other diseases with more or less satis- faction, and its great field in bacteriological investigations has not yet been exhausted for the practical diagnosis and determination of immune bodies in serum. In veterinary practice complement fixa- tion is now gradually becoming used for the diagnosis of glanders. This method of diagnosing glanders has given the most favorable results in Germany, and constitutes at the present time the official test for Prussia and other parts of Germany. It has also been used in the diagnosis of other diseases of animals, but not with such suc- cess as in glanders. Particularly in tuberculosis the results were not uniform and otherwise not very promising. The presence of the specific immune bodies (bacteriolytic ambocep- tors) in the serum of glandered horses brings about the fixation of the complement wdien the antigen in the form of glanders bacilli extract is added to the hemolytic system. The serum of glandered horses, therefore, contains antibodies (immune bodies) against glan- ders bacilli, Avhich are specific only for the glanders bacilli and for no other infection. The complement fixation accordingly represents a specific test, as only in the presence of the glanderous immune bodies and glanderous antigen will the reaction take place. If, in- stead of the glanderous immune bodies, other antibodies of another infectious disease be present in the blood serum, they will exert no effect whatsoever on the glanderous antigen ; and, on the other hand, if serum containing glanderous immune bodies is brought in contact with an antigen of another infectious disease, it will also have no effect on the reaction. By this fixation of the complement the hemo- lytic system is left incomplete, and as a result no hemolysis will take place. This fixation of the complement by the antigen and immune 18 DIAGNOSIS OF GLANDERS. bodies of glanders in the horse serum constitutes the diagnostic test for this disease. In the application of the test it is necessarj^ to have substances con- stituting the hemolytic system, which are the washed blood corpuscles of a sheep, the hemolytic amboceptor (rabbit serum), and complement (normal guinea pig serum). The quantity of the hemolytic ambo- ceptor to be used has been established by titration and described in a preceding part of this publication. In the test for glanders the double strength of the determined titre of the hemolytic amboceptor is used. A slight excess in the quantity of the amboceptor does not alter the outcome of the tests. On the other hand, the establishment of the smallest amount of complement which will i)roduce hemolysis in the presence of the hemolytic amboceptor and blood corpuscles is very essential, and accordingly this should be established by the previously described preliminary test, before tests for glanders are undertaken. By omit- ting this it is possible that an excessive amount of complement would be used which would in some cases affect the final results of the test. An oversupply of complement in the test may not only prove sufficient to be fixed by the immune bodies (antibodies) and antigen, but there might be also enough complement left to produce an incomplete or an almost complete hemolysis. Thus it is evident that it is of great importance to establish by such a preliminarv^ test (see Table 2) the exact quantity of complement to be used. Schiitz and Schubert found that the success of this method of diagnosing glanders depended greatly upon the proper quantity of complement used, and therefore the establisliment of this quantity should not be omitted. The red blood corpuscles of the hemolytic system always constitute a uniform quantity — that is, a 5 per cent suspension of the washed corpuscles in salt solution. As has been stated, this may be kept for testing purposes for two or even three days in the ice chest. The method of obtaining the corpuscles has been described in the early part of this work. In the complement-fixation test, there are also used, besides the hemolytic system, the serum of the horse to be examined and antigen. METHOD OF OBTAINING SERUM OF ANIMALS TO BE TESTED. The blood is drawn from the jugular vein of the suspected horse after a small area over the jugular has been clipped and disinfected with alcohol. The vein is dilated by pressure on the lower part of the neck, and the blood is drawn from the animal by the insertion of a trocar and cannula. It is recommended that the blood should be collected in uniform-sized tubes or bottles, which should be sterilized before using. A sufficient quantity of blood for testing purposes OBTAINING SERUM OP SUSPECTED HORSE. 19 would be 50 to 100 c. c, and after allowing the blood serum in the tube to separate from the clot in a cool, dark place it is ready to be used for the test. If it is desired to forward the blood to a laboratory the tubes may be packed into separate containers or collectively in a box. Every tube should be labeled, and the number of the horse corresponding with the record number should be designated on the label. It is not absolutely essential to have clear serum, as in repeated tests carried out in this laboratory it was found that blood forwarded from Michigan to "Washington gave satisfactory reactions, although the serum was badly discolored as a result of disintegration of the blood corpuscles. If it is desired to preserve the serum, or if from some cause a test can not be applied during the first few days after the blood has been drawn, it should be preserved with a 0.5 per cent solution of carbolic acid. This percentage is best obtained by adding 1 part of a 5 per cent carbolic-acid solution to 9 parts of the serum to be preserved. Such carbolized serum Avill respond to this test after several months. In cases where the mallein test has been used the blood of sus- pected horses to be examined for glanders by complement fixation should not be taken until from 7 to 10 days have elapsed after the last mallein test. This is necessary because of the possibility that the injected mallein may have exerted a stimulating effect on the cells with the production of immune bodies, and if serum is then taken for the test the results ma)^ lead to a faulty diagnosis. However, in suspected cases of glanders the blood of the animals may be drawn and forwarded for examination to a laboratory where the serum diagnosis of glanders is practiced, without the necessity of the appli- cation of the mallein test. INACTIVATING THE SERUM. Of the horse serum to be examined about 2 c. c. is drawn off and placed in a suitable tube or bottle in order to subject it to a tempera- ture of 58° C. in a water bath for one-half hour. This constitutes the inactivation of the serum ; that is, the complement which is present in the serum is destroyed by this heating. Such inactivated serum is ready for use in the testing, but should be used only on the da}^ of its inactivation. In case it becomes necessary to repeat the test, another 2 c. c. of the sample should be inactivated. The method of inactivation referred to above applies only to horses. Miessner and Trapp found that serum of mules inactivated at 58° C. and 59° C. does not in all cases give satisfactor}^ results, as in many instances even the normal serum of these animals checks hemolysis. The numerous tests which were carried out in this labor- atory with serum of horses and mules proved that while the inactiva- tion of the horse serum at 58° C. and 59° C. always gave satisfactory results, the tests made with mule serum under the same conditions were far from uniform. 20 DIAGNOSIS OF GLANDERS. Accordingly, it was deemed advisable to inactivate sera from mules at a hi