UC SOUTHERN REGIONAL LIBRARY FACILITY u gfj J n nilary 18, 'BUREAU A 001120189 4 32 A. 1). MH1.VIN, CHIEF OF BUREAU. FURTHER EXPERIMENTS CONCERNING THE PRODUCTION OF IMMUNITY FROM HOG CHOLERA. BY M. DORSET. M. D., C. N. MCBRYDE, M. D., AND W. B. NILES, D. V. M., Of the Biochemic Dirision. KM WASHINGTON: GoVI RNMl'.N I' I'RINTINt; ii|-HICE. (90 Issued January 18, 1908. U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY. BULLETIN 102. A. D. MELVIN, CHIEF OF BUREAU. FURTHER EXPERIMENTS CONCERNING THE PRODUCTION OF IMMUNITY FROM HOG CHOLERA. BY M. DORSET, M. D., C. N. MCBRYDE, M. I). AND W. B. NILES, 1). V. M., Of the Biochemic Division. WASHINGTON: GOVERNMENT I'RINTINC, OFFICE. 190*. THE BUREAU OF ANIMAL INDUSTRY. Chief: A. D. MELVIX. Assistant Chief: A. M. FAKRINGTON. Chief Cleric: E. B. JONES. Biochcmic Dirision: M. DORSET, chief; JAMES A. EMERY, assistant chief. Dairy Dirision: ED. II. WEBSTER, chief; C. B. LANE, assistant chief. Inspection Division: RICE P. STEDDOM, chief; MORRIS WOODEN, R. A. RAMSAY, and ALBERT E. BEHNKE, associate chiefs. Pathological Dirision: JOHN R. MOHLER, chief; HENRY J. WASHBURN, assistant chief. Quarantine Division: RICHARD W. HICKMAX, chief. Division of Zoology: B. H. RANSOM, chief. Experiment Station: E. C. SCHROEDER, superintendent; W. E. COTTON, assistant. Animal Husbandman: GEORGE M. ROMMEL. Editor: JAMES M. PICKENS. Librarian: BEATRICE OBERLY ROGERS. Biochemic Division. Chief: M. Dorset. Assistant Chief: James A. Emery. Meat inspection laboratories: Central laboratory, T. M. Price in charge; Philip Cas- tleman, E. H. Ingersoll, F. D. Hardesty, A. V. Fuller, E. J. Ralph, assistants. Chem- ists in branch laboratories: Ralph Hoagland, A. E. Graham, C. H. Swanger, A. H. Roop, W. B. Smith, E. A. Boyer, Clarence T. N. Marsh, W. P. Colvin. W. C. Powick. Hog cholera investigations: In charge of Chief of Division; C. N. McBryde, bacteri- ologist; W. B. Xiles, inspector in charge of field experiments; Frank W. Tilley, assist- ant bacteriologist. Bacteriological investigations of meat food products: G. N. McBryde, bacteriologist, in charge. Investigations of dips and disinfectants: Robert M. Chapin, chemist; C. N. McBryde, bacteriologist; A. M. West, assistant bacteriologist; J. B. Munro. assistant chemist. Preparation of tuberculin andmallein: In charge of Chief of Division; A. M. West and H. J. Shore, assistant bacteriologists; W. S. Stamper, H. S. McAuley, Roy E. Burnett, assistants. 2 LETTER OF TRANSMITTAL. U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY, Washington, D. C., September 25, 1907. SIR: I have the honor to transmit herewith a paper entitled "Fur- ther Experiments Concerning the Production of Immunity from Hog Cholera," by Doctors M. Dorset, C. N. McBryde, and W. B. Niles, of the Biochemic Division of this Bureau, and to recommend its publication as a bulletin in the Bureau series. This paper embodies the records of experiments carried out during the seasons of 1903, 1904, 1905, and 1906, this work being a continua- tion of that reported in Bulletin 72 of this Bureau. The results obtained show quite clearly that a comparatively certain method of protecting hogs from hog cholera has been secured. The experiments, however, have not as yet been extensive enough, nor have they been applied under the varying conditions of practice to a sufficient extent to warrant claims concerning the practical value of the methods described in this paper, though it is strongly believed that they should be of great service in combating hog cholera in this country. Very respectfully, A. D. MELVIN, Chief of Bureau. Hon. JAMES WILSON, Secretary of Agriculture. 3 CONTENTS. Page. Introduction 5 Preliminary experiments 7 Experiments of 1903 and 1904 7 Experiments of 1905 9 Immunes used for production of serum in 1905 10 Vaccination experiments in 1905 12 Experiments of 1906 17 Plan of the experiments 17 General plan for the production of serum in 190(5 18 Description of the Scribner outbreak 20 Scribner immunes 20 Description of the Syphax outbreak 23 Syphax immunes 24 Vaccination experiments in 1906 .' 27 Tests of serum from hogs hyperimmunized with blood from the Scrib- ner outbreak 28 Tests of serum from hogs hyperimmunized with blood from the Syphax outbreak 51 Production of a protectiye serum by using a reduced dose of diseased blood 68 Experiments with serum from nonhyperimmunized immunes 71 Experiments with suckling pigs 74 Curative value of hyperimmune serum 81 Duration of protective power in blood of immunes, and keeping quality <;f hyperimmune serum 86 General summary of .results 89 Transmission of disease by hogs treated by serum-simultaneous method . . 90 Immunity in hogs after serum-simultaneous vaccination 91 Immunity in hogs treated with serum alone 92 Curative value of hyperimmune serum 92 Results with serum from immunes treated by different methods with dif- ferent disease-producing bloods 93 Comparison of the quick and slow methods of producing hyperimmune serum 93 The comparative potency of sera secured by hyperimmunization with the Scribner and Syphax strains of disease 93 Comparative potency of sera from hogs possessing natural immunity and from those possessing acquired immunity 94 Conclusions 95 4 FURTHER EXPERIMENTS CONCERNING THE PRODUC- TION OF IMMUNITY FROM HOG CHOLERA. INTRODUCTION. In a previous publication from this Bureau it has been shown that the disease known as hog cholera, encountered in epizootic form in Iowa and other parts of the United States, is caused by a filterable and probably ultramicroscopic virus which exists in the blood of sick hogs, and that Bacillus cholerse, suis, although present in most cases of hog cholera, in all likelihood plays the part of a secondary invader. This modification of our views regarding the etiology of hog cholera necessarily carried with it changes in plans for combating the disease. Most of the methods for combating the disease which have hereto- fore been proposed were very naturally based upon the general belief that B. cholerse, suis was the cause of hog cholera, and many different methods of preparing vaccines and antitoxic sera through the use of that organism were devised and subjected to extensive laboratory as well as practical tests. Although a certain degree of success was appar- ently attained in some instances, none of these methods have been found to act with sufficient reliability to warrant their use in practice. In the case of vaccines prepared from cultures of B. cholerse suis a certain amount of immunity against that organism may have been at times induced in the treated hogs, and a similar result may have been secured by the administration of antitoxins prepared through the use of the same organism, for in the earlier publication above referred to it has been shown that B. cholerse suis usually invades the body of the hog after injection with the filterable virus, and, as stated there, it is reasonable to believe that the final result of the disease is in many cases materially influenced by the presence of that organism. Any vaccine or serum which protects against B. cholera? suis would therefore be expected to aid the body in its struggle against the twofold invasion of filterable virus and B. choleric suis. We believe that the partial success attained at times by the use of sera and vaccines prepared from B. choleric suis is to be explained upon this ground. In view of the fact, however, that the filterable virus is the primary invader, and that it appears to be quite sufficient in itself to cause Hullctin 72, llurrau of Animal Industry, I'. S. Dcpt. of Agriculture. 5 6 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. the death of most hogs, any substance which protects only from B. cholerse, suis must necessarily be regarded simply as a palliative, which, in the light of past experiences, is without a value that is in any way commensurate with the cost of applying it. It thus seems evident that all efforts looking to the prevention or treatment of hog cholera should be directed primarily toward secur- ing some means of protecting the hog from invasion by the filterable virus, or of overcoming that virus if invasion has already taken place. Since it was first definitely determined that B. cholerse suis is not the primary cause of hog cholera the work of the Bureau of Animal Industry has been along the lines just suggested. Much difficulty has been experienced in carrying out this work, due in great measure to our inability to cultivate the filterable virus artificially and to the fact that of all animals exposed to this virus only hogs have proven susceptible. We have thus been restricted to the use of the blood and tissues of sick hogs in our attempt to secure a satisfactory serum or vaccine. As stated in Circular 43 of this Bureau, issued February 12, 1904, " the basis of the immunity experiments, therefore, has been the use of attenuated and disease-producing liquid or dried blood, or the use of this blood mixed with blood obtained from immune animals, in which animals the immunity has been increased by the injection of large doses of disease-producing blood obtained from hogs known to have the disease; or, in other words, disease-producing blood and anti- toxic blood separate and combined have been successfully used." a The efforts to attenuate the virus in blood from diseased hogs by drying, by heat, and by chemical agents have not led to satisfactory results. This appears to be due in part at least to the unequal resistance of the virus in different lots of diseased blood to these agencies. At times the protection' afforded by diseased blood attenuated artificially has appeared to be complete; at others a vaccine prepared in identically the same manner has either caused death, thus showing an entire lack of attenuation, or else the hogs have shown no ill effects from the vaccination but have succumbed promptly when exposed to hog cholera, showing that the supposed vaccine had no protective power whatever. It may be that later investigations will show that it is possible to obtain a satisfactory vaccine for hog cholera by attenuating the virus, but our own results w r ere so discouraging that efforts in this direction were abandoned and our entire attention turned to the production of a serum in the manner hereinafter described. Subsequent to the publication of Circular 43, Boxmeyer and McClintock (Journal of Infectious Diseases, vol. 2, No. 2, March, 1905), described a few experiments along these lines and seemed to obtain better results by using a combination of disease- producing blood and immune serum than by using an artificially attenuated virus. RESULTS OF EARLY EXPERIMENTS. 7 PRELIMINARY EXPERIMENTS. The earlier experiments, which had for their object the production of a serum which would protect from the filterable virus, were not wholly successful, some of them indeed resulting in almost complete failure; but as these failures are quite instructive on account of the light they throw upon the details of producing the serum, it seems desirable to describe them very briefly before taking up the more recent work. As it was known that immune hogs could withstand perfectly injections of disease-producing blood which were more than suffi- cient to destroy nonimmunes, the plan originally adopted was to inject hogs immune from hog cholera with disease-producing blood in increasing doses, the object of this treatment being, of course, to impart a protective power to the blood of the immune, previous experiments having shown that blood from nonhyperimmunized immunes possessed very little if any such power. The records of two experiments carried out with serum from immune hog 844 treated in this manner are given below. EXPERIMENTS OF 1903 AND 1904. Immune hog 844 was an adult animal weighing approximately 150 pounds, which had recovered from an attack of hog cholera. This immune was injected subcutaneously with increasing doses of disease-producing blood which had been shown to contain the filterable virus. 6 The amount of disease-producing blood injected was increased to 400 c. c. before blood was drawn from the immune. The following statement shows the dates and amounts of disease- producing blood used for injection of immune hog 844, c with records of blood drawings: July 24, 1903, 1 c. e. disease-producing blood injected. August 4, 1903, 4 c. c. disease-producing blood injected. August 19, UK):}, 12 c. c. disease-producing blood injected. September 1, 190.'}, 30 c. c. disease-producing blood injected. September 20, 1903, 50 c. c. disease-producing blood injected. October 14, 1903. 250 c. c. disease-producing blood injected. October 20, 1903, 250 <-. c. disease-producing blood injected. October 29, 1903, 400 c. c. disease-producing blood injected. November 9, 1903, 100 c. c. blood drawn from tail. The term "disease-producing blood" is applied in this paper to blood from hogs affected with hog cholera, which bld was shown to be capable of causing the disea.se when injected into nonimmune hogs. 6 See experiments with blood from Herd I. Bulletin 7'J. Bureau of Animal Industry, pp. 43-65. c We acknowledge our indebtedness to Dr. K. ('. Schrocder. who had these injections, as well as those described under experiments A ami B. made for us at the Experiment Station of this Bureau at Bethesda, Md. PRODUCTION OF IMMUNITY FROM HOG CHOLERA. November 19, 1903, 400 c. c. disease-producing blood injected. December 10, 1903, 480 c. c. disease-producing blood injected. January 4, 1904, 150 c. c. blood drawn from tail. January 5, 1904, 490 c. c. disease-producing blood injected. January 28, 1904, 480 c. c. disease-producing blood injected. February 9, 1904, 185 c. c. blood drawn from tail. It is interesting to note that even the largest doses of disease- producing blood did not seriously disturb the general health of the immune, the only ill effects noted after the injection of the blood being swelling at the point of injection and occasionally a temporary loss of appetite. Two experiments to test the protective value of the blood drawn from hog 844 were carried out. TABLE 1. Experiment A with blood drawn November 9, -1903, from immune hog 844. No. of hog. Material injected. Inoculation. Exposure. Date. Result. Date. Result. 1002.... 1004.... 1003. . . . 20 c. c. defibrinated blood of hog 844, subcutaneously. 20 c. c. defibrinated blood of hog 844+ 1 c.c. disease-producing blood. 1 c. c. disease-producing blood Nov. 11,1903 do Remained well. do Dec. 18,1903 .do.. Died; hog cholera. Remained well. do Died; hog cholera. TABLE 2. Experiment B with blood drawn February 9, 1904, from immune hog 844- No. of hog. Material injected. Inoculation. Exposure. Date. Result. Date. Result. 1086. . . . 1087 20 c.c. blood of hog 844+1 c.c. disease- producing blood. do Feb. 11,1904 do Remained well. do Mar. 11,1904 do . Died; hog cholera. Remained well. S i c k ; r e- covered . Died; hog cholera. 1088. . . . 1089 do do do do do.... do .....do ...do... 1090.... 1091 1 c. c. disease-producing blood alone . . . do do do Died; hog cholera. do.... .:. Experiment A. By referring to Table 1 it will be seen that only three hogs were used in this experiment. Of these, after injection, the one that received only immune blood remained well; the one which received both immune blood and "disease-producing blood also remained well, while the third animal which received only dis- ease-producing blood died of hog cholera. When the two surviving hogs were injected approximately thirty days later with disease- producing blood to test their immunity the one which had been treated previously with immune blood alone died, while the other which had received the mixture of immune blood and disease-pro- ducing blood remained well. Experiment B. This experiment was designed as a control on the results of experiment A, except that it was considered unnecessary to use the immune serum alone, as it appeared not to produce any METHODS OF HYPERIMMUNIZING HOGS. 9 lasting immunity. One point of difference between experiments A and B should be noted : Experiment B was carried out with immune blood drawn February 9, 1904, whereas in experiment A the drawing of November 9, 1903, was used. The two experiments agree in showing that the serum from the hyperimmunized immune was capable of protecting hogs from hog cholera when the infection took place simultaneously with the administration of the immune serum. It is evident, however, that the duration of the immunity secured by these injections was not of sufficient length to render the method, as used at that time, suit- able for practical purposes. Other experiments with blood from immune hog 844 and from other hyperimmunized hogs about the same time did not lead to more promising results, and the experi- ments were therefore laid aside temporarily on account of the pressure of other work. EXPERIMENTS OF 1905. In the spring of 1905 this work was resumed with the intention of determining whether it was possible to secure by means of the above- described method, or some variation of it, an immunity which would be more lasting than that obtained in previous experiments. Four immunes were used to supply serum. These hogs were all adult animals, more than a year old, and had all passed through exposure to hog cholera without exhibiting noticeable symptoms of disease. It is difficult to classify these hogs as possessing either "natural" immunity or "acquired" immunity, as they may have suffered a light attack of disease which passed by unnoticed. It is quite certain, however, that they possessed considerably greater powers of resistance than is ordinarily the rule in hogs. Two methods were resorted to for hyperimmunizing these hogs. For the sake of convenience the first plan has been designated the "slow method" and the second the "quick method," depending upon the manner of injecting the disease-producing blood. The slow method consisted of three successive injections of disease- producing blood, followed by a drawing of blood from the immune, and then more injections of disease-producing blood, followed by more drawings from the immune; in other words, it followed in a general way the plan used in raising the potency of the blood serum of immune cattle for securing a protective serum for "rinderpest." The quick method is along the line of Xicolle's so-called "me'thode brutale" of producing an antirinderpest scrum, and was planned to consist of one very large dose of disease-producing blood administered subcutaneously, and followed after an interval of ten days or more by blood drawings from the immune. It was planned to use a different strain of disease-producing blood for injecting each immune, but this could not be done owing to the impossibility of securing a 10 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. sufficient quantity of blood from different outbreaks at the time the injections were to be made. All of the immunes, therefore, were injected with diseased blood which might be considered a mixture of several different strains, but which was virulent in all cases (as proven by the injection of controls) unless otherwise stated. In view of the virulence of this blood the behavior of the immunes after injection seems truly remarkable, for not one of them showed any ill effects whatever other than a transitory stiffness, due undoubt- edly to the local inflammation caused by the large injections. Immune hog Q No. 2 received 1,440 tunes the usual lethal dose of hog-cholera blood without any effect whatever being noticeable except a little stiffness. As far as the immune is concerned, therefore, there seemed to be no reason why the single large dose should not be used instead of the much slower process of administering the disease- producing blood in increasing doses. IMMUNES USED FOB PRODUCTION OF SEBUM IN 1905. A very brief history of each of the immunes is given below. It should be noted that the virulence of the disease-producing blood injected was tested each time by the injection of one or more non- immune hogs, and in each case it proved to be of high pathogenic power unless otherwise stated. HYPERIMMUNIZED IMMUNE Q No. 1 (QUICK METHOD). This hog passed through an exposure to hog cholera in the summer of 1904 without showing any symptoms of disease, and was not sub- jected to further exposure until the summer of 1905, when it weighed approximately 130 pounds. On September 12, 1905, it was injected subcutaneously with 900 c. c. of blood that was supposed to be viru- lent, but nonimmune hogs that were given small doses of the same blood did not sicken, so the treatment had to be repeated. On October 16, 1905, this hog was given 1,275 c. c. of virulent blood. No reaction worthy of note followed this injection. Blood draw- ings from this immune were made as follows : November 1, 1005, 140 c. c. defibrinated blood obtained. November 9, 1905, 125 c. c. defibrinated blood obtained. November 16, 1905, 150 c. c. defibrinated blood obtained. December 27, 1905, 200 c. c. defibrinated blood obtained. The first drawing of this blood was used in the vaccination of hogs in the 1905 experiment. HYPERIMMUNIZED IMMUNE Q No. 2 (QUICK METHOD). This hog passed through an exposure to hog cholera in 1904 with- out showing any symptoms of disease. On November 1, 1905, when the hog weighed 145 pounds, it was injected subcutaneously with 1,440 c. c. of disease-producing blood. Some soreness and stiffness followed this injection, but otherwise the animal remained well. IMMUNES USED FOB PRODUCTION OF SEBUM. 11 Drawings of blood were made from hog Q No. 2 as follows: November 24, 1905, 225 c. c. of defibrinated blood obtained. December 1, 1905, 230 c. c. of defibrinated blood obtained. December 8, 1905, 200 c. c. of defibrinated blood obtained. December 27, 1905, 350 c. c. of defibrinated blood obtained. The first drawing of blood from this hog was used in the vaccination of hogs in the 1905 experiments, and the second drawing was used in the 1906 experiment to test the keeping quality of immune serum. HYPERIMMUNIZED IMMUNE 1234 (SLOW METHOD). This hog was first exposed to hog cholera on November 9, 1904, when it was placed in a pen with sick hogs; no illness resulted. On September 29, 1905, at which time it weighed 168 pounds, hog 1234 was injected subcutaneously with 175 c. c. of disease-producing blood. This was followed on October 21 by 420 c. c. of disease-pro- ducing blood, and the third dose, 840 c. c., was given November 5, 1905. About two months later, or, to be exact, on January 13, 1906, the hog received the last dose of disease-producing blood. The virulence of the blood used for each of these injections was proven by the production of disease in nonimmune hogs. Drawings of blood were made from the tail of this hog as follows : November 14, 1905, 100 c. c. defibrinated blood obtained. November 21, 1905, 130 c. c. defibrinated blood obtained. November 28, 1905, 125 c. c. defibrinated blood obtained. January 24, 190G, 450 c. c. defibrinated blood obtained. February 2, 1905, 525 c. c. defibrinated blood obtained. August 31, 1906, 60 c. c. defibrinated blood obtained. The first drawing of serum from hog 1234 was used in the 1905 experiments, while the second drawing and last drawing were used in 1906 Experiments XXVIII and XXIX, hereinafter described. HYPERIMMUNIZED IMMUNE 1212 (SLOW METHOD^. Hog 1212 was injected with blood from a sick hog on August 27, 1904, and was later placed in the exposure pen, but in neither instance did the hog become sick. On September 29, 1905, this immune weighed approximately 160 pounds and was injected with 175 c. c. of disease-producing blood. The injection of disease-producing blood was repeated as follows: October 21, 1905, 420 c. c. November 5, 1905, 820 r. c. January 13. 1906, 840 <. c. Practically no symptoms followed these injections, although non- immunes that received only 5 c. c. of the same blood contracted hog cholera and in most instances died. 12 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. Drawings of blood were made from immune hog 1212 as follows: November 14, 1905, 100 c. c. defibrinated blood obtained. November 21, 1905, 140 c. c. defibrinated blood obtained. November 28, 1905, 10 c. c. defibrinated blood obtained. January 24, 1906, 75 c. c. defibrinated blood obtained. The first drawing of this serum was the only one used for experi- mental purposes, and this was used in the 1905 experiments only. VACCINATION EXPERIMENTS IN 1905. Only the first drawings of hyperimmune blood were used in the experiments described below. The blood was defibrinated, separated from the clot, and mixed with sufficient 5 per cent carbolic acid solu- tion to give a 0.5 per cent solution in the defibrinated blood. In so far as the method of vaccination is concerned, the 1905 exper- iments practically repeat those of 1903 and 1904, though of course the mode of hyperimmunizing the immunes was modified as ex- plained above. It was also hoped to determine whether different immunes varied in their power to yield a potent serum, whether the 11 quick" or the "slow" method of hyperimmunization was prefer- able, and whether by increasing the amount of disease-producing blood given to the immunes the duration of the immunity in treated hogs could be prolonged. The amount of disease-producing blood administered with the immune blood for the purposes of vaccination was also varied for similar reasons. In all treated hogs the diseased blood and the hyperimmune serum were injected simultaneously on different sides of the body beneath the skin. Control animals of the same size and age, and of the same litters also, if available, were in- jected with the same dose of the same disease-producing blood alone in order to demonstrate the protective value of the immune blood. Several weeks after vaccination the hogs which withstood the treat- ment were placed in an "exposure pen." This pen contained hogs sick of hog cholera, and as the pen was comparatively small the ex- posure to which the treated hogs were subjected was quite as severe as any that could be encountered under natural conditions by asso- ciation. The severity of this exposure is made plain by the death of the control animals. It seems unnecessary to give detailed records of the hogs used in this and succeeding experiments. It may be stated, however, that in cases where hogs became sick the clinical records and the autopsy findings in all cases were such as are usually met with in hog cholera, unless otherwise stated. The salient features are set down in Table 3. The term "exposure pen " as used in this paper refers to a pen where hogs sick of hog cholera were kept and hogs placed therein were subjected at all times to close associa- tion with the sick animals. The exposure was entirely by association, as the hogs were not allowed to feed upon the bodies of those that died. RESULTS OF VACCINATIONS IN 1905 EXPERIMENTS. 13 ? i. A r N Q ft. ', hog vaccinations Inoculation. Result. Sick; recovered... . _do__ 1 1 a" 5 1 8 a 12 G ^ S G G -i -3 _c _c _c _c Results of 1 G G o~ ; c-f G : G 1 G 1 c 8 C o" Q ci C C ec H - a HI ^3 o**c o""c y*s c 1 H H H 1 "5 0"5 O c c x f* 6C^ 6 >3 73 X I s s >o g>? g 3 .C 73 .C O - C - O C C C ?g sc's & T3 J=T3 * c - e c no c 3- O O < 1 i i sg> a -" - 6C CtO O bC C bC C ~ ' ~ '^ C '^ 60 60 C '3 -. '3 - 1 II 1 c 5 -SP - 1 11 c 85 S bO S .2 S S G w-a c ^^-3 rt ci t*^^ T3 'C ^ 2 g 5 JJ S S Ptt> ooez; til Q 01 C S : S ' : r- , oo , M o o o : : : : : $ : S : Inoculation. Result. i Remained well .do.. 5 Sick Dec. 7; died Dec. 12, 1905. Sick Dfin. 7: killed DBC. IS. f* ^ i i 1 i '- c ?.^ ^ a - ^ggii i i 1 1 i i I - & j a [5555 ::::::: "3 Q 35 to" ^ C c 9 a Q 8 c .c ft c - e - 8 :::::::::: Material injected. .0 c. c. first drawing of serum from hof 1212; 2 c. c. disease-producing blood o hog 1406. JO c. c. first drawing of serum from hoj 1212; 2 c. c. disease-producing blood o hog 1406. I c. c. disease-producing blood of hog 139 ...do. . . c c : M :::::::::: o O '..:'.': : -a :::::::;:: o o %': I !g i i i ! i i CJ . . . p | ^ . . g :....: :g : : :.5 :::::: ft -o o o o c^ o o o o o o o" ." .^ g''^'^'^' .'^ a bo 1 8 1 S S 8*3*88888*99 || 9 3 & 1 As will be seen from Table 3, 16 hogs were vaccinated by the use of a combination of hyper- immune blood and dis- ease-producing blood, blood from each immune serving for the treatment of 4 hogs in doses of 10 c. c. and 20 c. c. Half of the treated hogs re- ceived 2 c. c. of disease- producing blood and the remainder 1 c. c., simul- taneously with the im- mune blood. For the purpose of control, 8 hogs were injected with disease-producing blood alone, half of these re- ceiving 2 c. c. and the remainder 1 c. c., all injections being made subcutaneously. Of the 16 hogs which were vaccinated, 2 died, 2 became sick but re- covered, and 12 showed no symptoms of illness whatever as a result of the treatment. The ani- mals which became sick were all treated with se- rum from immune hog Q No. 1, the serum from this immune appearing to be barely sufficient to protect from 1 c. c. of disease-producing blood, but not potent enough to protect from 2 c. c. Serum from the other 3 immunes gave perfect protection from 2 c. c. as well as from 1 c. c. of DEGEEE OF IMMUNITY FROM VACCINATION. 15 disease-producing blood when injected simultaneously with it, and the protection afforded by 10 c. c. of this serum seemed to be quite as satisfactory as that secured by the use of twice that amount. The protective power of the serum from hyperimimmized hogs Q No. 2, 1234, and 1212 is made strikingly apparent by the behavior of the control animals, which were injected with disease-producing blood alone. Of the 8 hogs thus injected, 6 died of hog cholera, 1 was killed when in a moribund condition, and the eighth hog also contracted the disease but recovered. From these facts there seems to be no reason for doubting the protective power of the serum from these 3 immunes when injected simultaneously with disease-produc- ing blood. In order to determine whether or not this immunity was of more than fleeting duration the survivors from the vaccinating dose were placed in the exposure pen together with 7 untreated healthy hogs, as shown in Table 3. This exposure to disease by association took place twenty-four days after vaccination in the case of 6 of the treated hogs and twenty-five days after the vaccination of the remaining 8 hogs. The interval between vaccination and exposure was not as long as was desired, but owing to the lateness of the season it was considered best to expose at that time rather than wait until the following summer. It will be seen from the table that not one of the vaccinated hogs contracted disease as a result of this exposure. Of the 7 control hogs which were placed in the exposure pen at the same time as the vacci- nated hogs to prove the virulence of the exposure, 5 contracted hog cholera, while 2 remained well. Of the 5 which contracted the dis- ease, 3 died and 2 were killed when desperately sick to secure blood for other experiments. From the results given above there seems to be no reason to doubt the power of serum from hyperimmunized immunes to protect non- immunes from a fatal dose of disease-producing blood administered simultaneously with the hyperimmune serum. It appears also that in the case of serum from 3 of the immunes, at least, a dose of 10 c. c. was quite as effective as 20 c. c. The serum from both immunes which were hyperimmunized by the slow method proved potent, whereas serum from immune hog Q No. 1 treated by the quick method failed to give satisfactory results. The low protective power of the serum from this immune would appear to be due to some peculiarity on the part of the immune itself, inasmuch as blood from immune hog Q No. 2 treated by the same method proved quite as effective as blood from either of the "slow" immunes. Without more extended trials, therefore, with other immunes, preference could not be given to either one of the methods of hyperimmunization over the other. These experiments indicate also that large doses of disease-producing blood 16 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. may be expected to confer upon the blood of the immune the power to protect nonimmunes for a greater length of time than blood from immunes which receive considerably smaller doses, for in the experi- ments carried out up to this time the immunity exhibited by the vaccinated hogs after treatment may have been due (in some of the treated hogs at least) to the action of the immune blood alone and not necessarily to the combined action of immune and disease-pro- ducing blood. It will be remembered that in the experiments of 1903 and 1904 the immune blood protected the treated animals from the disease- producing blood which was administered simultaneously with it, but some of the vaccinated animals succumbed when exposed to hog cholera about thirty days later. The most rational explanation of this occurrence appeared to lie in the supposition that too much immune blood was administered with the diseased blood. The animals would thus be completely protected from the simultaneous injection of the virus, but owing to this complete protection from the virus the animal body would have.no stimulus to bring about a reac- tion and a consequent production of an active immunity which was aimed at by the simultaneous method of vaccination. Under such circumstances it seemed reasonable to suppose that the vaccinated hogs possessed only a passive and transitory immunity, such as might be expected from the injection of an antitoxin. In the experiments of 1905 we were unable to clear up this point, owing to difficulties experienced in securing suitable material for the work. The experiments were greatly delayed and it was only possible to carry out those which have just been described. Inas- much as no hogs were injected with the immune serum alone in these experiments, the question as to the nature of the immunity afforded by our injections could not be definitely determined. There is reason to believe, however, that this may have been due to the serum alone, at least in the case of those which received 20 c. c. of immune serum, while there can be no doubt that the 2 hogs 1397 and 1398, which became sick but recovered, secured an active immunity. It is entirely possible that the other hogs which received 10 c. c. of immune blood with the disease-producing blood suffered a very mild attack of disease and thus secured an active, permanent immunity. It would be natural to suppose, however, that if 10 c. c. of immune serum gave sufficient protection from the disease-producing blood to prevent any outward manifestation of disease the protection afforded by 20 c. c. would be complete, and that subsequent immunity in these hogs would presumably be due to the immune blood alone. DESCRIPTION OF THE EXPERIMENTS OF 1906. 17 EXPERIMENTS OF 1906. The experiments thus far described show quite clearly that blood from certain hyperimmunized immunes possesses the power to pro- tect nonimmune hogs from a fatal dose of disease-producing blood administered simultaneously with the serum. It has also been shown that hogs which survive a simultaneous injection of serum and disease-producing blood are rendered immune for at least twenty-five days thereafter, even though they show no reaction after the injection. Notwithstanding the success which attended most of these experi- ments, many questions affecting the practicability of this method for combating hog cholera yet remained undetermined. In conducting the experiments during the year 1906, therefore, we have endeavored to decide, in so far as time and material would permit, these other questions in connection with experiments designed to substantiate the results already obtained. PLAN OF THE EXPERIMENTS. The experiments which are to be described hereafter will perhaps be best understood by indicating the general plan which was followed. The questions to be decided concerned first, the immunes, and second, the hogs that were to be vaccinated. Questions concerning the immunes. Without attempting to go into the plans in detail, the 1906 experiments may be said to have had the following objects in view: To determine (1) whether natural immunes or those which have acquired immunity are best suited for serum pro- duction; (2) what percentage of either natural or acquired immunes may be expected to yield a potent serum; (3) whether the "quick method" or the "slow method" of hyperimmunization yields the best results; (4) whether disease-producing blood from different outbreaks of hog cholera is equally well suited for the purposes of hyperimmuni- zation; (5) whether the amounts of disease-producing blood used for hyperimmunization might be reduced without affecting the potency of the serum, and (6) how long after the last injection with disease- producing blood the immunes retain th'e power to furnish a potent serum. Questions concerning vaccination .--Many questions arise in con- nection with the practical application of the serum, but it has not thus far been possible to attempt to answer all of them. The prin- cipal points we had in mind during the 1900 experiments were as fol- lows: To determine (1) the dose of serum required to protect a small hog (25 to 50 pounds) from a simultaneous injection of a fatal dose of disease-producing blood; (2) the duration of the immunity which follows this simultaneous injection; (3) whether it is necessary to 12840 No. 10208 2 18 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. produce a reaction (i. e., visible illness or fever, or both) in order to secure a lasting immunity; (4) whether the administration of suffi- cient serum to prevent a reaction will tend to shorten the duration of the immunity in vaccinated hogs; (5) whether hogs vaccinated by a simultaneous injection of serum and disease-producing blood are likely to be injured or stunted in growth; (6) whether hogs vaccinated by this " serum-simultaneous " method may communicate hog cholera to unvaccinated hogs by association with them; (7) whether a lasting immunity may be produced by the injection of the serum alone; (8) whether the serum alone can be used successfully as a cure; (9) how long the serum will retain its potency, and (10) whether satisfactory results may be obtained by vaccinating suckling pigs. GENERAL PLAN FOB THE PRODUCTION OF SERUM IN 1906. The immunes used. Eight immune hogs were to be hyperimmu- nized. If possible, 4 of these were to be hogs that had acquired im- munity, while the remainder were to be natural immunes. Four of these 8 hogs (2 natural immunes and 2 that possessed acquired im- munity) were to be hyperimmunized by the use of one strain of disease, while the remaining 4 were to be given disease-producing blood from an entirely different source. Method of hyperimmunization. There were certain general features in the process of immunization which applied alike to all immunes treated by the same method. First of all, in order to make sure of a firm immunity and to avoid losses later, a preliminary injection of 20 c. c. of disease-producing blood was given each immune. After this the regular process of hyperimmunization was begun. Four of the immunes were hyperimmunized by the quick method and 4 by the slow method described under the 1905 "experiments. The immunes treated by the quick method were injected with one dose of disease-producing blood, the amount administered being equal to 1,000 c. c. of disease-producing blood to each 100 pounds of body weight. Exactly three weeks after this injection blood was drawn from the tails of these immunes and preserved for experi- mental use. This blood drawing from the immunes was repeated at intervals of seven or eight days until three drawings had been made, and in most cases, as shown by the detailed records which follow, a fourth drawing was made approximately a month after the third drawing. The immunes treated by the slow method were injected three times. The first dose was in the proportion of 100 c. c. of disease- producing blood to 100 pounds of body weight; the second dose, which followed the first after from ten to fourteen days, was in the proportion of 250 c. c. per 100 pounds body weight, and the third and last dose, which was given approximately twelve days after the GENERAL PLAN FOB PRODUCTION OF SERUM. 19 second, was in the proportion of 500 c. c. per 100 pounds of body weight. Blood drawings from the immunes hyperimmunized by the slow method were begun nine or ten days after the last injection of disease-producing blood and repeated at intervals of seven days. The object of these repeated blood drawings from the immunes was of course to secure blood for the purpose of ascertaining how long after the last injection of disease-producing blood the immune retained the power to furnish potent serum. These different draw- ings of immune blood that is, some of them were tested on non- immune hogs, as shown in the experiments hereinafter described. Method of collecting and preserving the immune blood. The immune blood was drawn in all cases from the tails of the immunes and was collected in a wide, sterilized dish. It was allowed to coagulate, and the clot which formed was then subjected to pressure in order that all of the serum might be separated from it. Instead of a clear serum, therefore, we secured always a serum containing large num- bers of red blood corpuscles and which might be more properly called defibrinated blood. The time required for the red cells to settle and the very considerable loss of serum which would have resulted if we had attempted to use clear serum only, owing to lack of facilities for using a large centrifuge, made it necessary to employ the serum containing red cells. In applying a similar method to cattle the use of a serum containing red cells would no doubt be con- sidered objectionable, owing to the danger of transmitting some intercurrent disease, such as Texas fever, through such injections. Such contingencies were not likely to occur in the case of hogs, how- ever, and, furthermore, any possible danger of this kind was no doubt avoided through the addition of an antiseptic to the serum immediately after it was drawn. After the serum had been com- pletely separated from the clot by pressure and by straining through a sterilized cloth it was mixed with a 5 per cent aqueous solution of carbolic acid in the proportion of 9 parts of serum to 1 of carbolic- acid solution, thus giving a 0.5 per cent solution of carbolic acid in the serum. This served to keep the serum perfectly, provided it was placed at once in sterilized bottles until used. It should be remem- bered that all of the hy peri mm une serum used in the experiments described hereafter consisted, in reality, of 1 part of carbolic-acid solution and 9 parts of serum and red cells. Sources of disease-producing blood trrth details of the Tiyperimmuniza- tion. As already stated, disease-producing blood from two entirely distinct sources was used for the treatment of the immunes. One of these strains of disease was obtained near Scribner, Nebr., and is referred to as the "Scribner disease." The other strain of disease was secured near Alexandria, Va., and has been designated the "Syphax disease." Blood from both of these sources proved quite virulent 20 PRODUCTION^ OF IMMUNITY FROM HOG CHOLERA. when administered to nonimmune hogs; the blood from the Scribner outbreak, however, appeared to produce a somewhat more acute type of disease than the blood from the Syphax outbreak. The immunes hyperimmunized with blood from the Scribner outbreak are designated "Scribner immunes," and, depending upon the method used for hyperimmunization, the individuals have been called " Scrib- ner quick immunes " or " Scribner slow immunes," as the case might be. The same plan is followed in speaking of the " Syphax immunes." DESCRIPTION OF THE SCRIBNER OUTBREAK. This outbreak of hog cholera occurred near Scribner, Nebr., in the fall of 1905. When this locality was first visited by one of us it was found that hog cholera of a virulent type existed on some half dozen farms. Several of these were visited and the sick animals were found to exhibit the usual symptoms of hog cholera, and at autopsy the usual lesions, such as intestinal ulcers and ecchymoses in the various organs, were noted. From one of these infected herds, in which the final loss was about 74 per cent, blood was secured for experimental purposes. This blood was collected in a sterile vessel, defibrinated, and kept on ice until it was used at the Bureau experi- ment farm near Ames, Iowa, for the injection of hogs 1395, 1393, and 1394, which received, respectively, 1 c. c., 2.5 c. c., and 5 c. c., subcu- taneously on November 18, 1905. These hogs became sick six days after injection, and hogs 1393 and 1395 died on December 3, 1905. The other hog, 1394, was killed on December 1 to secure blood for other experiments. Autopsies which were held on 2 of these hogs revealed the usual lesions of hog cholera. The blood of hog 1394, after dilution, was filtered through a Cham- berland bougie, and the filtered blood was proven to possess the same degree of virulence as the unfiltered blood. A portion of the unfiltered blood of hog 1394 was placed in sealed glass bulbs and preserved on ice until May, 1906, when, upon injection, it proved to be quite virulent, and hogs inoculated with this disease or exposed to it furnished all of the blood used in hyperimmunizing the so-called " Scribner immunes." SCRIBNER IMMUNES. HYPERIMMUNIZED IMMUNE 1286 (QUICK METHOD). This hog was exposed to hog cholera on August 17, 1905, together with another of about the same weight (50 pounds). As a result of this exposure hog 1286 exhibited only slight indications of sickness and soon recovered, although the other hog exposed at the same time died, showing at autopsy the usual lesions of hog cholera. In order to test further the immunity of hog 1286 it was placed in the station THE SCRIBNER OUTBREAK. 21 exposure pen where a number of sick hogs were kept. This hog failed to show symptoms of disease and was not exposed further until the summer of 1906, when it weighed 125 pounds. June 13, 1906, hog 1286 received subcutaneously 20 c. c. of Scrib- ner disease-producing blood, and at the same time a control hog was injected with 10 c. c of the same blood. The control died, while hog 1286 remained well. June 25, 1906, hog 1286 was injected subcutaneously with 1,250 c. c. of Scribner disease-producing blood secured from several hogs. At the same time 5 nonimmune hogs were injected to serve as checks on the virulence and also to furnish blood for treating other immunes. All of these checks became very sick and were destroyed, while hog 1286 remained well except for slight soreness following the injection. Blood was drawn from the tail of hog 1286 as follows: July 16, 1906, 356 c. c. defibrinated blood obtained. July 23, 1906, 300 c. c. defibrinated blood obtained. July 30, 1906, 250 c. c-. defibrinated blood obtained. This blood was used in Experiments I and II described in another part of this paper. HYPERIMMUMZED IMMUNE 1313 (QUICK METHOD). This hog was injected subcutaneously with 25 c. c. of diluted dis- eased blood on September 30, 1905. Hog 1313 did not become noticeably sick as a result of the injection, although the blood was of a fair degree of virulence. A later exposure to disease in the exposure pen was likewise without effect upon this hog. No further exposure to hog cholera was made until the summer of 1906, as described below. June 13, 1906, hog 1313 received subcutaneously 20 c. c. of Scrib- ner disease-producing blood ; a nonimmune hog received at the same time 10 c. c. of the same blood. The check died of hog cholera, but hog 1313 remained well. July 5, 1906, hog 1313, which weighed 130 pounds, was injected subcutaneously with 1,300 c. c. of Scribner disease-producing blood. Two nonimmune hogs injected at the same time with 2 c. c. each of the same disease-producing blood became very sick and were killed to furnish blood for the other experiments. Hog 1313 was a little sore on the day following the injection; this soon passed away, but on July 8 and for several days thereafter the hog was quite sluggish and disinclined to eat. By July 13, however, recovery was com- plete. Blood was drawn from the tail of hog 1313 as follows: July 2(i, 1906, 400 c. c. defibrinated blcxxl obtained. August 2, 1906, 465 c. r. defibrinated blood obtained. August 9, 1906, 450 c. c. defibrinated bl(x>d obtained. September 6, 1906, 400 c. c. defibrinated blood obtained. 22 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. The blood from this immune was used in Experiments III and IV, which are described in the following pages : HYPERIMMUNIZED IMMUNE 1383 (SLOW METHOD). This hog was injected subcutaneously on November 17, 1905, with 5 c. c. of blood from a hog sick of hog cholera. This blood as a result of a number of inoculations was found to cause the death of somewhat more than 50 per cent of the inoculated hogs. Hog 1383 showed slight sickness after injection, but soon recovered and also remained well when placed in the exposure pen about three weeks later. In June, 1906, this hog was used for the production of serum, as described below. June 13, 1906, hog 1383 was injected subcutaneously with 20 c. c. of Scribner disease-producing blood. A nonimmune hog was in- jected at the same time with 10 c. c. of the same blood. The non- immune died, but hog 1383 remained well. June 25, 1906, hog 1383 weighed 105 pounds and was injected sub- cutaneously with 105 c. c. of Scribner disease-producing blood. Five nonimmune hogs injected at the same time with 10 c. c. each of the same blood became very sick and were killed to furnish blood for other experiments; hog 1383 remained well. The blood used for this injection was the same as that used for the hyperimmuni- zation of hog 1286, and the same checks served for both immunes. July 5, 1906, hog 1383 received a second injection consisting of 265 c. c. of Scribner disease-producing blood. The blood used was the same as that used for the injection of immune 1313 on the same date, and, as stated previously, produced hog cholera in nonimmune animals in doses of 2 c. c. Hog 1383 was made somewhat sore by the injection, but was well again after two or three days. July 14, 1906, hog 1383 was given a third injection of Scribner dis- ease-producing blood, 525 c. c. being administered subcutaneously. Two nonimmunes injected with 2 c. c. each of the same blood as con- trols on the virulence of the blood died of hog cholera. Hog 1383 remained well. This injection completed the hyperimmunization of hog 1383, and blood drawings were made from this animal as follows : July 24, 1906, 392 c. c. defibrinated blood obtained. July 31, 1906, 360 c. c. defibrinated blood obtained. August 7, 1906, 400 c. c. defibrinated blood obtained. August 14, 1906, 360 c. c. defibrinated blood obtained. September 4, 1906, 82 c. c. defibrinated blood obtained. The blood from this immune proved to be exceptionally potent, and it was therefore used in a number of different experiments. HOGS USED TO OBTAIN HYPERIMMUNE SERUM. 23 HYPERIMMUNIZED IMMUNE 1403 (SLOW METHOD). By referring to Table 3 it will be seen that this hog was one of those vaccinated by the serum-simultaneous method in the 1905 experi- ments. The hog showed no symptoms of disease, either as a result of vaccination or of the subsequent exposure to hog cholera in the expo- sure pen on December 27, 1905. No further exposure to disease was made until June, 1906, when hyperimmunization was begun. The weight of the hog at this time was 90 pounds. June 13, 1906, hog 1403 was injected subcutaneously with 20 c. c. of Scribner disease-producing blood. A control injected at the same time with 10 c. c. of the same blood died, whereas hog 1403 remained well. June 25, 1906, hog 1403 was injected subcutaneously with 90 c. c. of the same disease-producing blood as that used for injecting immunes 1286 and 1383 on the same date. Hog 1403 was not visibly affected by the injection. July 5, 1906, hog 1403 was injected with 225 c. c..of Scribner disease-producing blood. The same blood was used at the same time for injecting immunes 1313 and 1383 and four control hogs as previ- ously stated. Immune 1403 was sluggish and disinclined to eat for several days following the injection, but by July 9 had apparently recovered. July 14, 1906, hog 1403 was injected for the last time with 450 c. c. of Scribner disease-producing blood, subcutaneously, blood from the same source being used at the same time for the injection of immune 1383 and two controls, as stated above. Hog 1403 showed about the same disturbance as was noted after the second injection, but was well again in four or five days. Blood drawings were made from immune 1403 as follows: July 24, 190G, 395 c. c. defibrinated blood obtained. July 31, 1906, 390 c. c. defibrinated blood obtained. August 7, 190(5, 400 c. c. defibrinated blood obtained. August 14, 1906, 360 c. c. defibrinated blood obtained. September 14, 190(5, 360 c. o. defibrinated bld obtained. DESCRIPTION OF THE SYPHAX OUTBREAK. This outbreak of hog cholera occurred near Alexandria, Va. At the time the first symptoms of illness were observed, on September 6, 1905, there were 37 hogs in the herd. The disease did not progress very rapidly, but nevertheless all of the hogs were finally attacked by the disease. At the time the herd was first visited by representa- tives of this Bureau only 9 hogs remained alive, and some of these were killed, while others died, the final result being that 3 hogs out of the 37 that were attacked recovered. The lesions found at autopsy 24 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. were such as are usually met with in hog cholera, except that the pul- monary lesions in some of the animals were rather more prominent than we have ordinarily found to be the case. Blood from one of the sick animals in this herd served as the starting point for the strain of disease from which blood was secured to hyperimmunize the "Syphax immunes." Filtered blood from this Syphax herd produced hog cholera when injected into nonimmune hogs, even though it was found to be free from all of the ordinary bacteria. SYPHAX IMMUNE S. HYPERIMMUNIZED IMMUNE 1392 (QUICK METHOD). This hog, with 5 others, was injected subcutaneously on November 17, 1905, with 5 c. c. of hog-cholera blood. All became sick as a result of this inoculation and 4 died, hog 1392 and one other finally recovering. Hog 1392 was placed in the exposure pen in December, 1905, and remained well. This hog exhibited quite typical symptoms of hog cholera after the first blood injection, and the immunity sub- sequently exhibited must be regarded as having been acquired through this attack of disease. June 13, 1906, hog 1392 received 20 c. c. of virulent Scribner dis- ease-producing blood in common with all of the immunes which were later hyperimmunized with blood from either the Scribner or Syphax outbreaks. Hog 1392 remained well. July 13, 1906, hog 1392, which at that time weighed 130 pounds, received subcutaneously 1,300 c. c. of Syphax disease-producing blood. Five control hogs which were injected at the same time with 5 c. c. of the same blood became very sick and, were killed to furnish blood for other experiments. Hog 1392 was somewhat stiff and sore on the two days following the injection, but otherwise remained well. Drawings of blood from immune 1392 were made as follows: August 3, 1906, 375 c. c. defibrinated blood obtained. August 10, 1906, 435 c. c. defibrinated blood obtained. August 17, 1906, 500 c. c. defibrinated blood obtained. September 17. 1906, 100 c. c. defibrinated blood obtained. HYPERIMMUNIZED IMMUNE 1274 (QUICK METHOD). This hog was first exposed to hog cmSlera on July 22, 1905, by the subcutaneous injection of 0.25 c. c. of hog-cholera blood. Another hog injected at the same time died, but hog 1274 did not become sick, nor did this hog show symptoms of disease when placed in the expo- sure pen some weeks later. The records indicate that this hog pos- sessed quite a high degree of natural immunity. June 13, 1906, hog 1274 was injected subcutaneously with 20 c. c. of Scribner disease-producing blood along with the other immunes. No ill effects were noticed as a result of this inoculation. THE SYPHAX OUTBREAK. 25 July 26, 1906, this hog, which weighed at that time 125 pounds, was injected subcutaneously with 1,250 c. c. of Syphax disease- producing blood, and 4 control hogs were injected at the same time with from 2 c. c. to 5 c. c. each of the same blood. All of the controls became very sick and were killed to furnish blood for other experi- ments. Hog 1274 showed no marked effects from the injection. Drawings of blood from this hog were made as follows. August 16, 1906, 450 c. c. defibrinated blood obtained. August 23, 1906, 450 c. c. defibrinated blood obtained. September 1, 1906, 378 c. c. defibrinated blood obtained. October 1, 1906, 700 c. c. defibrinated blood obtained. HYPERIMMUNIZED IMMUNE 1310 (SLOW METHOD). This hog, with one other, was injected with hog-cholera blood on September 29, 1905; the mate to hog 1310 showed slight indisposition after the injection, but hog 1310 remained well. Both hogs were later placed in the exposure pen. Hog 1310 did not thrive there, but never became plainly sick. After removal from the exposure pen this hog regained its vigor, and on July 13, 1906, at which time the hyperimmunization was begun, its weight was 180 pounds. June 13, 1906, the preliminary injection of 20 c. c. of virulent Scribner disease-producing blood was made. No visible reaction followed this injection. July 13, 1906, hog 1310 was injected subcutaneously with 180 c. c. of Syphax disease-producing blood from the same source as that used for the injection of immune hog 1392 on the same date. Hog 1310 remained well, although the control hogs injected at the same time contracted hog cholera. July 26, 1906, hog 1310 was injected subcutaneously with 400 c. c. of Syphax disease-producing blood from the same source, as that used for the injection of immune hog 1274 on the same date. Hog 1310 showed practically no disturbance of health after this injection, although the control animals injected with a much smaller amount of the disease-producing blood contracted hog cholera. August 7, 1906, hog 1310 was injected subcutaneously with 900 c. c. of Syphax disease-producing blood. Two nonimmune hogs injected subcutaneously with 2 c. c. and 5 c. c. of the same blood contracted hog cholera and died. With the exception of a little soreness, hog 1310 remained well. Blood was drawn from this immune as follows: August 16, 1906, 450 c. <. defibrinatod blood obtained. August 23, 1906, 450 c. <. defibrinated blcxxl obtained. September 1, 1906. 540 r. r. defibrinated blood obtained. October 1, 1906, 450 c. c. defibrinated blood obtained. 26 PRODUCTION OP IMMUNITY FROM HOG CHOLERA. HYPERIMMCNIZED IMMUNE 1297 (SLOW METHOD). This hog weighed approximately 55 pounds on September 13, 1905, at which time it was injected subcutaneously with hog-cholera blood. No sickness followed this injection and a later exposure in the ex- posure pen was likewise without result as far as could be observed, though a mild attack at this time may have passed unobserved. June 13, 1906, this hog received, with the other immunes, a prelim- inary injection of 20 c. c. of Scribner disease-producing blood without any symptoms of disease being caused. June 29, 1906, hog 1297 was injected subcutaneously with 175 c. c. of Syphax disease-producing blood. Two nonimmune hogs in- jected at the same time with 2 c. c. each of the same blood died of hog cholera. Hog 1297 remained well. July 13, 1906, hog 1297 was injected subcutaneously with 440 c. c. of Syphax disease-producing blood from the same source as that used for the injection of immunes 1310 and 1392 on the same date. All of the checks injected with the same lot of blood became sick and either died or were killed to furnish blood for other experiments. Hog 1297 remained well except for slight soreness following the injection. July 26, 1906, hog 1297 was again injected with Syphax disease- producing blood, the dose being 850 c. c. of blood from the same source as that used for the injection of immunes 1274 and 1310 on the same date. As previously stated, controls injected at the same time became sick and were killed to furnish blood for other experi- ments. Blood was drawn from immune 1294 as follows: August 4, 1906, 390 c. c. defibrinated blood obtained. August 11, 1906, 300 c. c. defibrinated blood obtained. August 18, 1906, 500 c. c. defibrinated blood obtained. September 17, 1906, 360 c. c. defibrinated blood obtained. It has been previously stated that it was intended to use, for the production of hyperimmune serum, 4 naturally immune hogs and 4 which had acquired immunity through an attack of hog cholera. When it came to the actual working out of this plan, however, as is shown in the preceding pages, we were only able to secure 1 hog (1392) that had recovered from an undoubted attack of hog cholera; 2 others (1286 and 1383) had shown slight though not characteristic symptoms of that disease, while the fourth (1403), among those that were considered as having acquired immunity, had been vaccinated in 1905, but had not shown symptoms of illness at any time after being vaccinated. In regard to this last-mentioned hog, it should be noted that it has been found to be frequently the case that sus- ceptible hogs may show no symptoms following vaccination and yet may secure through this treatment a firm and lasting immunity, and we believe this to have been the case with hog 1403. The remaining 4 immunes, 1274, 1297, 1310, and 1313, were not vaccinated, bu-t VACCINATION EXPERIMENTS. 27 nevertheless they showed no symptoms of disease when exposed to hog cholera, and we may therefore with a reasonable degree of assur- ance look upon these hogs as being naturally immune. From what has just been said it becomes evident that the plan to test the rela- tive capability of ''natural" and "acquired" immunes to produce a potent protective serum could not be carried out on as extensive a scale as was desired, but nevertheless it was hoped that considerable information might be acquired from a comparison of serum from hog 1392 with serum from the hogs that appeared to possess a natural immunity. VACCINATION EXPERIMENTS IN 1906. The experiments as originally planned were necessarily modified more or less owing to the existence of conditions which could not be foreseen, but notwithstanding the fact that the original plan was not carried out in all instances it may perhaps lead to a better under- standing of the* experiments which are now to be described if this plan is briefly outlined. The experiments with serum from each of the hyperimmunized immunes were to consist of two sections, (1) preliminary' experiments and (2) secondary experiments. Preliminary experiments. These experiments were designed in the main to determine the potency of the immune serum. A number of nonimmune hogs were to be injected with 2 c. c. each of disease- producing blood. A few of these were to be left untreated as con- trols, while the others were to receive simultaneously with the disease- producing blood varying doses of the immune serum. Uninoculated hogs were to be placed in the pens with the treated as well as the untreated hogs to determine the likelihood of disease being trans- mitted by the treated hogs. Any survivors from the preliminary experiments were to be reserved for exposure to hog cholera at a later date. Secondary experiments. These were intended to determine many questions of practical interest, the preliminary experiments having already established the degree of potency of the different sera when injected simultaneously with diseased blood. In these secondary experiments four lots of hogs were to be treated. Lot 1. These hogs were to receive disease-producing blood with the minimum dose of serum required to protect. Lot 2. These were to receive disease-producing blood with double the minimum amount of serum required to protect. I.*>t 3. These were to receive the minimum amount of serum alone. Lot 4. This lot was to receive the maximum amount of serum alone. Controls were of course to be injected with disease-producing blood alone, and, as in the preliminary experiments, hogs were to be placed 28 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. in the pens with the treated as well as the untreated ones to deter- mine the likelihood of disease being conveyed to others by contact. The survivors from these experiments were to be divided into two lots, one to be exposed within thirty days after vaccination, the other to be placed in a quarantine pasture and not subjected to exposure until the expiration of three or more months. As will be explained later, the exposure to hog cholera could not be carried out in the manner planned, owing to an outbreak of disease among the hogs in the quarantine pasture. In addition to carrying out these preliminary and secondary experi- ments, serum from certain of the immunes was to be used in special experiments with suckling pigs, and also for the purpose of testing the curative value and the keeping quality of the serum, and other questions of more or less practical importance. Except in the case of these special experiments the tests with the serum from the Syphax and Scribner immunes are described separately. TESTS OF SERUM FROM HOGS HYPERIMMUNIZED WITH BLOOD FROM THE SCRIBNER OUTBREAK. The description of the outbreak from which this strain of disease was derived has already been given on page 20, and the history of the immunes with the details of hyperimmunization and time of the various blood drawings has also been given. SERUM FROM IMMUNE 1286 (QUICK METHOD). EXPERIMENT I. Preliminary experiment with first drawing of serum. This experiment was carried out in accordance with the plan origi- nally decided upon for all preliminary experiments. Eight hogs were injected; 2 of these received disease-producing blood simultaneously with 5 c. c. of serum, 2 received disease-producing blood with 10 c. c. of serum, 2 disease-producing blood with 15 c. c. of serum, and 2 disease-producing blood alone. These four lots of hogs were placed in separate pens with an uninoculated check in each pen. The hogs which received only 5 c. c. and 10 c. c. of immune serum with the diseased blood became distinctly sick, but recovered; the uninocu- lated checks in the pens with these did not contract disease, however, in either case; the 2 hogs which received 15 c. c. of immune serum with the disease-producing blood did not become sick, and their pen check also remained well. The 2 hogs which received disease- producing blood alone sickened and died promptly after injection, and at autopsy exhibited the usual lesions of hog cholera; the unin- oculated pen check with these last 2 hogs contracted disease from them and showed lesions of hog cholera when destroyed on August 4, 1906. EXPERIMENTS WITH SERUM FROM SCRIBNER IMMUNES. 29 1 "*. rft i 1 . c 'E | |M > D i 8 a 1 < 1 1 i C C c C f i 1^ 71 E e "3 10 .. c c * e C lesions ^. ^ PC ft I 1 e c 1 a i t> +. a . 1 . e 8 = C r = - o " 5 ~ c. ll rt G fl * ^ c i C w a s r- r 2 s i - X O ^ ? : =3 J - i C (= (X c tf 1 .-- aS ~ c c e e c G > " c T 5 X ~ _ , c i 1 r 5 1 3 8 s 1 X PS C T e a E '5 < c c C r j * M =3 g >5 t C z ~ ^ 55 e 1 K 7 y OB i 3 - 1 _c c c c e c c G T e T c 1 a e 4 t c . b 7 = g * i . I_ ~s T L ; . 3 ll I ~ c O JC o N ll -_ | 1 i" 3 JS at f g C > ' 1 g j c c 1 :: -: i Ji *5 ! 1 - 5- Ji - Si || T c ^ 1 3 1 | < i f-^ g aZ a a: - -/. OS a 1-1 o 00 I * " c c c C r* G t ^ t c II ~= ] 4 4 i : 1 i * \ serum and2c. c. sease - producing ood of hog 1505. do. . . e- C a E E 1 c sease - producing ood of hog 1505. do. . . P - E E 1 c sease - producing ood of hog 1505. do ... f \ ~ B : : -- ; -: 1 6 i .- . i a 3 i 8 o^ 3 : S3 (, * S3 i L - "1 " - E .S CQ t/2 o> ... 1 ft c +J "C ft 9 CO :f >i i ! ! H 5 ^ 3 ^ (N o c 60> > M~ 3 ; 3 3 ; ^ ^ 1 1 Pasture exposure. Result. ^ * 1 T3 11 Remained well . do Sick and died N 1906. Siok find dipd Nr 0) 1 M Unthrifty; died 14, 1906. 1 T T < 1 a i : a Remaine do . "~ 9 . Q 1 ft us rt c . -c 1 if i i ii 1 | '- ' :3 ^* o u? ; rt c 5 5 : o g : o + :o o : :o O O Inoculation. Result. 8 o S i> C. o ? ^ 35 b : A- JD Sickened Aug. 9: died killed Sept. 3, 19 . Remained well... c t| : : * - "Sb " .X, +J QJ O -Xj ir "H o ^> 8 g si'S o' o' o 3 S -O-O-C i 5 j 3 5 o M d) 11 5 |1 n exposure. Result. Remained well. . . ...do... c - e c : ~ c c : ; i ^ : -o i 1 1 0) * : *> g O Q) : S5 73 S : a ; M * . ^ I * : fr. i : | 5 OS P a = c SR c - c c C c "t : : > G ': ': * '. i '. o ,ure exposure. Result. Remained welL . . ...do... c - 1 e T e c Died Nov. 7, 1906. Sickened, but rec ered. Remained well. . . Not exposed | fit S oS Q 10" rt c c e c c c e e c o o o O -O 73 O i i i I : : : = 10 "* 3 > ,*8 OT! .. noculation. Result. Remained well. ...do.. c - c c e c c ; oc i i i 1 : ; : 1 d o d J O T3 T3 : : : "2 : : : a Oct. 13, 1906. . Sick Oct. 8 to N 1906; recovere . Sickened Oct. 18 Nov. 1, 1906. hH i o > . d Q S c 4-> "C . - c c c e T : : ? +. o : T3 1 : : : j , , ' 1 ' " " t j j i i 5 a 9 5c.c. serum and 2c.c. d i se a se-producing blood of hog 1664. . ..do. . . c. c e* - 9 = S i C & C d i se ase-producing blood of hog 1664. ...do. .. e e r. = E i c '- d i s e a se-producing blood of hog 1664. ...do. . . 1 | 1 1 is^^Sdf ^5^g>Sl ^a-g-o^-o gililil s^^S^Sc. C^; C|^ C^ . PH AH PH c^ I ; | 60 1 * ^! ** ; 1 ' t'g Id ^ MH3 ^g .S | p , ] A I S "2 <5 5 5 p- S S i J? *c 6 ^ 1 i i r t - !t^ ':' T* ? i 110 00 >-< c*5 c<5 -^r u" S3 S? EXPERIMENTS WITH SERUM FROM SCRIBNER IMMUNES. 37 ' 4 *> r < Ulcer in eecum. Hemorrhages and ) 1 a c c 7 j a ^ 0! E C 2 llemorrhagic lesions. a 2 c t 2 .c - e e | l| I 7 oi~ i ^ 3 * . > S fcoEfco = s issfa g 1 IB B 3 no autopsy. Hemorrhagic lesions. No rnnrk-oH Winns Hemorrhages and ulcers. Hemorrhagic lesions. Do. exposure. Result. Remained well . . . ...do... : 3 _ ~ Remained well . . . .do... g h C.C c o 2'3 1 ' *1 O 0) Z CH | 1 Nov. 16, 190(). Sickened and died Nov. 14, 1906. 1 S PH 1 1 II II 1 II i Illl 1 i I > a | I ?< I ^ o "C o C C . a3 ?3e$ S Co o oo o II : B *? c *? a ": * osi ^x * A . H,ON,O >-, . ^ c ^ ; : c : jz 1* "S i, j-v 'asture exposure. i , 1906 Remained but unthri , 1906 Remained w , 1906 Remained w , 1906 Became untt r ' ^ 1 ) 4 c 1 3 o v. : s ' c S VT3 o :o : . . . . c . . . . > . . . . c 1 1 'S 73 73 -C -s?s [noculation 1 "3 C B x 3 K r o n -5 j: C IX Nov. 1< . Remaine .do. . . . . c C CT3T3-O 3 |'|| 0000 00 C 73 O-O-3T3 73 TJ -5 C 9 ~ ic cc ;/; J i> ! I o o : i e o c 9 -9 C o O O O C C -3-S-CTT o o o O O O O O O 1 O -O-S-C-C -3 o o 1 i J i ' . f ; -o|S ; t e_j .... c. B .... SJ i g - : i | X 1 IK! | 8 6C ' ~ i gb "x **'' s : "" : : r ~ J i *"? oC : : g : c ; : -*5? *9 i~ ; * 3 H ^ I i i S^Zo ^.OMTS * t ''"" : o c 9 ~S o ; t *Z '' f 2 .S 73 ^ ' . 5;_c _c 5^^ < S*^ t "^ ~! "" o ** "^ W= ^" ll : S 8t5 ^ . c = c, s. ~~ ; 1 1 c 4 1 3 SS ^ 'F^yft'- 5pr*5 r* 1 I t S S S ^ .= 1 l : f ~ s F r t t' t*' ? F'r ? 1*^^^ 8? , 3 g -T 5? $ 1 38 PRODUCTION OP IMMUNITY FROM HOG CHOLERA. and 6 in the Scribner exposure pen fifteen clays after vaccination. None of these hogs became distinctly sick, though hogs 1780 and 1788, which were exposed in the pasture, became unthrifty. Three of the hogs that survived the exposure in the pasture were placed in the Scribner exposure pen two months and twelve days after vac- cination in order to further test their immunity. Of these 3 hogs 1, No. 1780, finally died more than three months after vaccination. The only lesion of hog cholera found at the autopsy of this hog was a single button ulcer in the cecum. In view of the lack of acute lesions and the unthriftiness which preceded the last exposure, it seems probable that this hog had contracted hog cholera before being placed in the Scribner exposure pen, and indeed the unthrifti- ness of this hog in the pasture may have been caused by a light attack of hog cholera following vaccination, the one ulcer found at autopsy being the only trace left of that attack. Four of the hogs that were treated with serum alone were placed in the Scribner exposure pen nineteen days after vaccination, while the remaining 4 were first exposed three months and twenty-one days after vaccination. Of the first lot, 2 remained well, while 2 died of hog cholera; 2 checks exposed at the same time died of hog cholera. The amount of serum injected did not seem to affect the result of this exposure, as one of the survivors had received 10 c. c. and the other 20 c. c. of serum. Of the 4 that were first exposed three months and twenty-one days after injection of the serum, all died, and the pen check exposed at the same time also died of hog cholera. Summary of experiments with serum from immune 1313. In all, 18 hogs were treated by the serum-simultaneous method, the serum being used in doses of 5 c. c., 10 c. c., 15 c. c. and 20 c. c. Of these, 2 became sick and died, though it was impossible to make a positive diagnosis of hog cholera in the case of one of them. In the case of one other hog (1780) there is also some doubt whether death resulted from vaccination or from subsequent exposure to disease, but for convenience in summarizing, and because no symp- toms of illness were observed after vaccination, the death of the hog is counted as due to exposure. To determine the virulence of the disease-pro'ducing blood used in the serum-simultaneous vaccinations, 4 hogs were injected with dis- ease-producing blood alone. All of these became sick and 3 died. Seven uninoculated hogs were placed in pens with the vaccinated hogs, and all remained well. Two similar unvaccinated hogs which were placed in pens with those injected with disease-producing blood alone died of hog cholera. None of the hogs injected with serum alone were made sick by the injection. In testing the immunity of the treated hogs, 16 that were vacci- nated by the serum-simultaneous method were exposed to hog cholera EXPERIMENTS WITH SERUM FROM SCRIBNER IMMUNES. 39 from fifteen to nineteen days after vaccination, and all remained well except 1780, which became unthrifty. Three of these hogs which had been exposed originally in the pasture were again exposed by being placed in the Scribner exposure pen seventy-three days after vaccination. One of these (1780) finally died as described above ; the other 2 remained well. Of 8 hogs that were treated with serum alone, none became sick from treatment, but 6 died when exposed to infection from nineteen days to three months and twenty-one days after vaccination. It is worthy of note that the 2 survivors were, with 2 others that suc- cumbed, exposed only nineteen clays after vaccination. Of 7 unvac- cinated check hogs exposed along with the treated hogs, 6 became sick and 5 died. The following conclusions may be drawn from the experiments with first drawing of serum from immune 1313: (1) Serum from immune 1313 was sufficiently potent to protect the great majority of the injected hogs from a fatal dose of disease- producing blood adminsitered simultaneously with the serum. (2) Hogs treated by the serum-simultaneous method, even though they became slightly sick, did not convey the disease to nonimmunes by association. (3) Hogs vaccinated by a simultaneous injection of serum and disease-producing blood remained immune for at least fifteen days after vaccination. 01 (4) Immunity subsequent to vaccination in hogs which received the maximum dose of serum with disease-producing blood was quite as firm as in those that received the minimum dose in the same manner. (5) The immunity conferred by the serum of hog 1313 when administered alone in doses of 10 c. c. and 20 c. c. did not persist in full force for as long as nineteen days, and at the end of three months and twenty-one days it seemed to have completely disappeared. SERUM FROM IMMUNE 1383 (Si.ow METHOD). EXPERIMENT V. Preliminary experiment with Jirst drawing of serum. This first experiment with the first drawing of blood from immune 1.383 was carried out in exactly the same manner as the preliminary experiments with the serum-simultaneous method, the serum being given in doses of 5 c. c., 10 c. c., and 15 c. c. Two hogs were injected with disease-producing blood alone, and 1 uninoculated hog was placed in each of the four pens used in this experiment. Table 8 shows that none of the vaccinated hogs became sick after treatment, nor did any of the check hogs in the same pens contract disease from a Other experiments havcnhown that immunity following serum-simultaneous injec- tions lasta much lunger than this. 40 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. i j s u '5 i I i i j p . | 1 J 1 h i H C *n *-" B i i i ? O c ~ c 5 5 ! i 3 '^Q S ~ c a ^ < i u A s 09 X _^ s j 3 1 'f ^ s d O o> 0) 3 ft q c B 00 O Q. 1 * C ~ o '3 '^ tf tf 1 1 cT i a *"^ C c e * ' Cs p > * .^ g o ^ ^ ** i 3 T3 q _ i 3 -3 c o 3 1 1 Ip -- "I! 1 C 3 i 1 i 3"f r e r- s" Q rH e ^\ ~' -; a X o '3 c 6 * e "= c c C O o, ^t? o- "o^ |! > c o E 0) pi c o a >-^ ^ 5 ft 3 1 1 0> tl 8 c c c ; o o c c P c M ~ s ~ c 3 -C t 5 5 _< .i gi 5 e < T3 _ i . i'-. ^^ B * 6 j 5 ^ !_ (D 3 f ^_; ^ g 4 ^" "* ^" - i aj O PS . < ^ - 'S ^ c c c C c ; ' 60 f ^ 5 I = -z c -z O- O -C - J if ^ 8 a> < , .S ** L 8 p! a a fl 1 | I a! 'S S c C o C 5 5 c P x " - c c - a 1-5 H i o6C c U c U) b I Q S bo | > i i- 8 ? N oS 3 rt c-o t oj Ci -z e Ss lo ^ - 'Is C a - i - ^ 1 - 2 j! . e ^ 4 $ i | si s 5 c. c. serum disease-pi blood oft An 1 e i c disease-pi blood of h : 1 c c fr -| |2j ~~ < e '"' T? "^ 4 A | 1 1545 and 1 J 30 |g ? s > if > e 3 ' s O * "C C* 1 i i ? S g s s s jj U) t a C o JH >' Q u5 u ' : J : 1 ! ta >.- 9 > 1C S 5 i 5 > " i i i > ? 5 3 tf r ^ 5 EXPERIMENTS WITH SERUM FROM SCRIBNER IMMUNES. 41 them. Both of the hogs which were injected with the same dose of the same disease-producing blood as that used on the vaccinated hogs but without the serum, contracted hog cholera and died, and the pen check with these 2 hogs contracted hog cholera from them, but recovered. The 6 vaccinated hogs with their 3 pen checks were placed in the quarantine pasture and were there accidentally exposed to disease about two and one-half months after vaccination; as a result of this exposure all 3 of the checks died of hog cholera, while the vaccinated hogs remained well. Five of the vaccinated hogs had their immunity further tested by being placed in the Scribner exposure pen a little more than three and one-half months after vaccination. They again remained well, although the disease to which they were exposed was quite virulent. The protection afforded against a fatal dose of disease-producing blood by even 5 c. c. of serum from immune 1383 was apparently complete. Instead of proceeding with a secondary experiment in the usual manner it was decided to determine the minimum amount of this serum required to protect against a fatal dose of diseased blood. EXPERIMENT VI. Second experiment with first drawing of serum. No hogs were injected with serum alone in this experiment, all vaccinations being made by the serum-simultaneous method. The doses of serum used were reduced to 2i c. c., 5 c. c., and 7i c. c., and only 1 c. c. of disease-producing blood was used. This dose of blood was only half of that heretofore employed, but notwithstand- ing this it proved to be quite sufficient to infect unprotected hogs, for all of the hogs which received 1 c. c. of the disease-producing blood alone died of hog cholera, and the pen check exposed to them likewise contracted the disease and died. Of the 6 vaccinated hogs, Nos. 1600 and 1601, which received only 2\ c. c. of serum with the disease-producing blood, became slightly sick after vaccination; the remaining vaccinated hogs which received larger doses of serum showed no symptoms of dis- ease, and none of the pen checks exposed to the vaccinated hogs became sick. All of the vaccinated hogs with the pen checks wore moved to the quarantine pasture and were there exposed to hog cholera approximately two months after vaccination. Two of the 3 pen checks died as a result of this exposure and at autopsy typical lesions of hog cholera were found. The third check evidently pos- sessed a high degree of natural resistance to the disease, for it did not become distinctly sick in the pasture nor in the exposure pen, where it was placed later. 42 1 a o 1 03 .H 3 p 3 Sfi a "3 g Intestin; 6 1 M o fc fc 2 53 H o cv- fc < G I||SC 5' "w 5 6 6 ij 1 i 1 | S 9 tf 1 3 i a 1 1 ^ S QB | 1 1 i g PH 1 1 1 1 1 00 i a 2 2 g ' tc" 8 c G X c c c S1< i I I c ' 7T - _ ; 1 a s^^ : -c . c _. T g i iiKi s * o; o,^ '= ~ 5 == r 11 1 a p - - 3 I ! - >, c I i ( I } D i 1 i a ? 3) I | 3 J : j | < 1 4 4 , ~ - * < i c c > 00 i *" j - 3 EC a ' O : 8 P 1 f 1 t a : : : : .' "5 is : : : : : B II I ' ' S | d d o'o'd o a S tf 1 1 i : It : c, d J . . 050 . N 00 rtM ft T)13 t**^'''' 1 : : lit : o ;: : : o . . . c . . . 00 C ft S : Q) . ... K o ' ',',', 1 g :: I : : oj a-c 0.' ""I i: I ' 03 rt oo o : : fi o-o -s ; : : : c j e e j - . e ' "5 o~ ; rt c i 1 1-1 c c o o c a 73 o Inoculation. I 1 1 1 ' : b . 6 to 18, 190(i; recov- ered. Remained well ...do.. O 0. - o 00 ?. ? ~ o o O T) do Sickened Aug Aug. 16, 190( Sickened A UP y. =i ?!T ti, ? ^ ^ < i 5 w 2 oS O c i c - C c o c o -c : o ,' s oo" c ~ : i - D 3 S 3 lOc. c. serum and 2 c. c. disease - producing blood of hog 1531. .do... c c, ec ci 10 j^ *o . c c c c 1 !? 1 *a ss 3 1 u- c- Q " C-l CM S 8 S CO |f i~ '~ i' f. 3 d t UiiUi EXPERIMENTS WITH SERUM FROM SCRIBNER IMMUNES. 49 EXPERIMENT X. Second experiment with first drawing of serum from immune 1403. In Experiment X 12 hogs were treated by the serum-simultaneous method, half of these being given 10 c. c. and the other half 20 c. c. of serum. Eight hogs were given serum alone in doses of 10 c. c. and 20 c. c., and 4 hogs were given disease-producing blood alone in the same dose as was used for the serum-simultaneous vaccinations. Checks were placed in the pens with both of the serum-simultaneous lots of hogs and also with those injected with disease-producing blood alone. Table 13 shows that none of the vaccinated hogs became sick, nor did they communicate disease to the exposed pen checks. All of the hogs which received disease-producing blood alone died, and they commu- nicated hog cholera to the check which was placed in the pen with them. For the purpose of testing the subsequent immunity of the vacci- nated hogs, these were divided into two lots. One lot was to be ex- posed to hog cholera within a few weeks after vaccination, while the other was not to be exposed for several months. The first lot was exposed in the Scribner exposure pen, 4 of the hogs treated by serum alone being placed there thirteen days after vaccination. These were followed eleven days later by 6 of those treated by the serum- simultaneous method (see Table 13) and 1 of the pen checks. This pen check died of hog cholera; 1 of the hogs vaccinated with serum alone became very slightly sick, but recovered. The other vaccinated hogs remained well, but in order to secure further data concerning the behavior of hogs immune from one strain of disease when ex- posed to another all of these vaccinated hogs were later placed in the Syphax exposure pen, where they all remained well. The other lot of vaccinated hogs in this experiment, with 2 checks, was placed in the quarantine pasture and there exposed to hog cholera seven weeks after vaccination. Both checks contracted hog cholera; one died, and the other was killed when in a moribund con- tion, and at autopsy showed typical lesions of that disease. One of the hogs (1638) treated with serum alone also became sick following this exposure and died of hog cholera, as did hog 1628, which was treated with 20 c. c. of serum plus 2 c. c. of disease-producing blood. None of the other vaccinated hogs became sick, and nil of them, except hog 1620, which was unthrifty, were placed in the Scribner exposure pen about four months after vaccination. None of them contracted hog cholera from this exposure. Summary of c rperimfnls with scrum from immune Only the first drawing of serum was used. In all 18 hogs were treated by the serum-simultaneous method, the doses of serum given in conjunction with 2 c. c. of disease-producing blood being 10 c. c., 12840 No. 10208 - 1 50 PEODUCTION OF IMMUNITY FROM HOG CHOLERA. TABLE 13. Secondary experiment with first drawing of scrum from hog 1403 ("slow" immune Scribner). Autopsy. L n Hemorrhagic le- sions. Do. "3 "2 i "2 i "2 3 ""* S " S - ^ -^ H . >- /'."-. i- /' 00*" OC O^OCO J<5 R H R W W bner and Syphax strains of disease. Pen exposure. Result. Remained well . . . .. do . X . j j j| : : 0) ' c - 1 1 : : Sg-d! : i || 73 1 1 ! 73 QJ Q) ffl o o : 'S o'S d c d o 7373 Remained 730 *: 11? |o CO : : /. \ n of c +i T: c sf as" c . -*j 7^ is i ii is II 1 1 d ^ ; ^ ^ o ^ c "O . . j_j T) . 'C ' O O p^ ' O II "ft 9 CO 00 ft CO Pasture exposure. Result. QJ ' ' ' ea qj : | ! j )6 Remained well . . . . Became unthrift> . . Remained well . . is : : |j| j] 'o ^- : "- ; g73S ;73" : 73_- : : ; o i : ' QJ O 2 QJ ' QJ a s ' ' -7. * rum alone. :go a) 1 - 1 .ft o ST: 2 c. c. disease-produ ing blood of hog 15 ....do ...do... Pen check with ho] 1640 to 1643. 8 ITS " : i ; 8 : ooo .Sooooo o,- coo.;oc O-O-COj^C 73 737373 73~ 737373^7371 t Weight (pounds.) = ^^99889 SS3S 98 ^^^ sss 3S us a s CJ M t 3 8 !$ a -c iig cc 6 CO CC CDCOCCCCCOCOCCtO TESTS OF SERUM FROM SYPHAX IMMUNES. 51 15 c. c., and 20 c. c. Of these 18 hogs 1 became slightly sick as a result of vaccination but soon recovered, while the others remained well. As controls on the virulence of the disease-producing blood, as well as on the protective power of the serum, 7 hogs were injected with disease-producing blood alone in the same dose as that given the vaccinated hogs. All of these controls died of hog cholera. To test the danger of vaccinated hogs conveying the disease to nonimmunes, 6 checks were exposed in the pens with those treated by the serum- simultaneous method. None of these checks became sick, although the 2 checks exposed to hogs injected with disease-producing blood alone contracted hog cholera, as was to be expected. Of the 18 hogs treated by the serum-simultaneous method which were exposed to infection at various times after vaccination, 1 con- tracted hog cholera and died; none of the others became sick. Of 8 hogs treated with the serum alone, 1 contracted hog cholera when exposed seven weeks after vaccination; the others remained well. Of 6 unvaccinated hogs exposed along with the treated animals, all died of hog cholera. These experiments warrant the following conclusions: (1) Serum from immune 1403 when given in sufficient doses affords perfect protection from an otherwise fatal dose of disease-producing blood administered simultaneously. (2) Hogs vaccinated by the serum-simultaneous method, with rare exceptions, remain immune for at least seven weeks after vaccination. (3) Hogs treated by the serum-simultaneous method, if not made ill by the treatment, do not convey hog cholera to others by association. (4) For seven weeks after vaccination immunity in hogs treated with serum alone is as firm as in those treated by the serum-simul- taneous method. (5) Hogs immunized from one strain of hog cholera are also im- mune from other strains of the same disease. TESTS OF SERUM FROM HOGS II YPERIMMUNIZED WITH BLOOD FROM THE SYPHAX OUTBREAK. SKKI M FKOM IMMCNK 1WJ (QiicK MKTIIOD). KxPKKIMENT XI. Firnl (iniwintj of SITU in . The results obtained in Kxpcriment XI did not differ materially from those of similar experiments carried out with serum from im- munes treated with the Scribncr disease-producing blood. The smallest dose of serum used was 10 c. c.. and although the 2 hogs given this dose did not become visibly sick from the scrum-simul- taneous vaccination, the fact that 1 of those given 1"> c. c. as well as 1 52 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. 7 o> f I Autopsy. " =3* ? ^ o) c E ai c fi 1 S 1 |a 1 | Ic'd -SB ttd l| SS|illl 99 a S fl 0) 9 S B a 3 en Pen exposure. Result. Remained well ...do... do c s "S Q c oT -. c > r d O o Pasture exposure. Result. o c o3 Remained well . . c - o 'C C a T i - s 4 c a ^ ^ e t Became unthrifty died Dec. 28, 190( S j as ft US' *" c c 4J o e C C -C c - oT +* m e T c - Inoculation. Result. Remained well dn S c a t * | -Z 1906; recovered. Remained well s s Aug. 29, 1906. Sickened Aue. 27: died cnT3 * eg _ "1 1 M.. 1 3o C < 'S *- E 1 S ^ c s c Sickened Aug. 21 ; d Aug. 31, 1906. Sickened Aiie. 21:d S ^ OS ** C 6b <= 3 < c t o -) o c o T; 1 V ^ c tf r Material injected. 10 c. c. serum and 2 c. c. d i s e a s e-producing hlood of hog 1560. ...do. . . 15 c. c. serum and 2 c. c. d i s e a s e-producing blood of hog 1560. do disease-producing blood of hog 1560. ...do... Pen check with hogs 1588 and 1589. Pen check with hogs 1591 and 1592. Pen check with hogs 1594 and 1595. 2 c. c. disease-produc- ing hlood of hog 1560. ...do... Pen check with hogs 1597 and 1598. +j i * B ft, S 8 3 S ? S -$ Z A oc C5 -* cs ^r c Op 00 O5 7- r Oa C5 u5 u} o 10 Hi s d i j ft s; a >. 3 S. 62 - ; ft M ii o be i 'S i P a. ** d 1* 1 TESTS OF SERUM FROM SYPHAX IMMUNES. 53 i 1 * ! j i 18 I . . : =q =3 =5 =3*2 : =3 ri'S 1 O *> aj 0) Q .3. . * i * is : i i* a ~ S'*'!! ' a si" !! "^ 'jco co "300 ' g^'5 c <- ; '3 '3 B -* : S S" 53 'gSE.^so ; 6 ^-^i^ - 1 pj - /. : -/ 7 Sf : Remained well . . ** " o oo ** o o o * o : ^ ^ . 0-0 . 1 ; i Us t so ft : 05 : * 0*0 c : : : : :^ : : : : : : :> : : * ; o O : j ft 8 ; i i i ;5 a : 1 M M K s i i 1 ::: : :| ^ : : : ::3 ri s : : =3 3 : : : : r. -o : : 3 ...... ... . .-a 4> >-s -3 -n-C : : : a o . . . g-o g og O.. ..C^MOCO .. CO C^^.. G~ ^ii' ' ' ^ ^v ^o ... I loo K P3 oo . . cc Sickened; 3, 1906. | o;: :; rt ooco ;; rt ;:" B . . ... -B-O-O-B ... * *j o o : : : : :o : : : : : : o o : : o o O HI Hi jm N MUM c I 5 1' I : : : : :^ 1 : : : : : : : : : : : : -c auf? co_o oct^.'Sooo' oo o' co c'o o c a S ' : :M 4 : : : : : : : : : : : C T3 4. U ~. ' V. &ti c^ ^ j, O t, _t II 3,3.3, ceo c_c co co o cc o; ; : 1 o : i^ 60 : : : : : : : : : : : : a : g ~'_i''' ..* .- qj .. 2 S : "B!;2 ::::: : : : - : - : :S'56c : : : : :* ' : ~2 : S . : s s : g I 1 : igll ::::: i | *g : * S y i* '- 3 a*. g ? j- j< ::Pi=::: ::g :: :: '* : z ^ ; 1 i i t cc-gccoo oc^c cc ,3 oo c't.-c t -3 -T ^ X C = -5 -O -5-S ."3 -0-B ~ T3 -B T aj! "= PS O q;T) s. x ~ ; :a"" J ; ;l i a i i ;|- ; 1 "" ! 1-1 g = 54 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. given 20 c. c. of serum did become sick makes it quite probable that less than 10 c. c. of this serum could not be used with 2 c. c. of virulent blood with safety. (See Table 14.) None of the hogs treated by the serum-simultaneous method died, although 2 became sick, as already stated; notwithstanding the sick- ness of the 2 vaccinated hogs, the untreated pen checks exposed to them did not become sick. Both of the hogs injected with disease- producing blood alone became sick and died of hog cholera, and they also communicated disease to their pen check. Following out the usual plan, the surviving hogs from this experi- ment were placed in the quarantine pasture. These hogs had been vaccinated two months when the accidental outbreak of disease occurred in the pasture, but none of them became sick from the exposure. Two of the 3 checks which were placed in the pasture with the treated animals died of hog cholera ; the third check became unthrifty and died, but apparently did not suffer from hog cholera. Four of the vaccinated hogs were later transferred to the Scribner exposure pen and again remained well. EXPERIMENT XII. Second experiment with first drawing of serum from immune 1392. In Experiment XII 12 hogs were treated by the serum-simultane- ous method, 8 were treated with serum alone, and 2 were injected with disease-producing blood alone. Four uninoculated hogs were placed in the pens with the serum-simultaneous hogs, and 1 with the 2 that received disease-producing blood only. As a result of the treatment, as far as could be observed none of the vaccinated hogs showed symptoms of illness, and the checks in the pens with them also remained well. With the hogs that received disease-producing blood alone the result was quite different, for both contracted hog cholera and one died; the pen check that was exposed to these hogs also became sick and died of hog cholera. (See Table 1 5.) For the purpose of testing the immunity possessed by the vacci- nated hogs at different lengths of time after vaccination, these hogs were divided into two lots, 6 of the serum-simultaneous vaccinated hogs being placed in the Syphax exposure pen seventeen days after vaccination and 4 of the hogs treated with serum alone in the same pen eleven days after vaccination. Table 15 shows the results of these exposures. Of the 6 hogs treated by the serum-simultaneous method and exposed seventeen days after vaccination, all remained well; of the 4 hogs vaccinated with serum alone and exposed in the Syphax exposure pen eleven days after vaccination, 1 (1704), after being sick a long time, died about two months later, probably of hog cholera, although the lesions found at autopsy were slight and not such as would be expected from a chronic case of hog cholera. The other 3 hogs (1703, 1706, and 1708), vaccinated with serum alone, TESTS OF SEBUM FROM SYPHAX IMMUNES. - 55 remained well and were later exposed to the Scribner disease without being made sick. Two of the uninoculated check hogs were placed in the exposure pen with the serum-simultaneous hogs, and both died of hog cholera. The exposure of the second lot of hogs in this experiment took place in the quarantine pasture thirty days after vaccination. Of the 6 serum-simultaneous hogs that were exposed there, 1 (1696) did not thrive, and finally died of hog cholera seven weeks after vac- cination, while the others remained well. The death of hog 1696 was probably due to a mild form of disease caused by the original serum-simultaneous vaccination, as the hog at autopsy showed the lesions of the chronic type of hog cholera, though it had been exposed to disease in the pasture only three weeks at the time of its death. Of the 4 hogs treated with serum alone, which were accidentally exposed in the quarantine pasture thirty days after vaccination, 1 (1707) died of hog cholera. The others did not contract the disease. Both of the pen checks that were placed in the pasture with the vaccinated hogs died of hog cholera. Summary of experiments with serum from immune 1392. A summary of the results obtained through the use of the first drawing of serum from immune 1392 shows that of 18 hogs treated by the serum-simultaneous method, the serum being used in doses of 10 c. c., 15 c. c., and 20 c. c., 1 became sick and 1 (1696; see above) died from the treatment. Of 4 hogs injected with the same amount of the same disease-producing blood as that used for the serum- simultaneous treatment, all became sick and 3 died. All of the surviving treated hogs with the surviving checks were exposed to infection after vaccination. All 17 of the serum-simulta- neous hogs, exposed seventeen to thirty days after vaccination, re- mained well; 2 of the 8 hogs treated with serum alone died of hog cholera when exposed eleven to thirty days after treatment, while of 4 checks exposed in the same manner as the treated hogs, all died of hog cholera. As a result of these experiments it will be soon that (1) The serum of hog 1392, in doses of 10 c. c. or more, was sufficient to protect hogs from a fatal dose of disease-producing blood admin- istered simultaneously with the serum. (2) Hogs treated by simultaneous injections of serum from hog 1392, together with disease-producing blood, remained immune for at least thirty days after vaccination. (3) Hogs treated by the serum-simultaneous method, even when they became distinctly sick, did not convey disease to unprotected animals which associated with them. 56 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. T pa EH / 1 I ia. c g-o S2 c c '3 6 6 M '3 cj as o-o c^S O-o o +j g g l?l '" o.b ill 6 ,"S 7X ^ ^.i C Oo J W CTJ i M) O 19 c p^ 33 8 TESTS OF SEBUM FROM SYPHAX IMMUNES. 57 (4) The immunity produced by injections of serum alone from hog 1392 did not seem to be as complete as that produced by a simulta- neous injection of serum and disease-producing blood. (5) Hogs immunized with serum prepared through the use of one strain of disease were immune when exposed to another strain of disease. SERUM FROM IMMUNE 1274 (QUICK METHOD). EXPERIMENT XIII. Preliminary experiment with first drawing of serum. The preliminary experiment with the first drawing of serum from immune 1274 consisted of serum-simultaneous vaccinations only, a certain number of controls being, of course, used. The results of this experiment were not entirely successful, as may be seen from Table 16. First of all, we find that serum from hog 1274 probably has distinct protective power when injected simultaneously with a fatal dose of disease-producing blood. This is indicated by the death of the 2 hogs that received disease-producing blood alone, and the survival of all those treated with serum. At the same time it is surprising that the 2 hogs that received 10 c. c. of serum with the disease-producing blood should have become distinctly sick, whereas the 2 that received only 5 c. c. of serum remained well. This result could only be explained on the ground of unusual susceptibility on the part of hogs 1718 and 1719, or because hogs 1715 and 1716, which received only 5 c. c. of serum, were exceptionally resistant to the disease. In the light of this experiment only, the latter supposition seems to be the most reason- able explanation of the conflicting results, for when the vaccinated hogs with their pen checks were exposed to hog cholera in the quaran- tine pasture three weeks after vaccination only one of the three pen checks (1723) died of hog cholera. It is true that one other sickened, but it recovered, and the third check never became distinctly sick in the quarantine pasture. The subsequent death of this check (1717) in the exposure pen can not be attributed to hog cholera. It appears, therefore, that the lot of hogs used in this experiment taken as a whole were probably more than normally resistant to hog cholera, and for this reason too great stress should not be laid upon the results. At the same time the serum must have given the vaccinated hogs a cer- tain degree of protection, for both of the controls injected with dis- ease-producing blood alone became very sick, one of them died, ami the other was killed to secure blood for other experiments, and at autopsy both showed characteristic lesions of hog cholera. In con- trast to this is the behavior of the hogs which received the same dis- ease-producing blood in combination with the serum, not one of these having died from vaccination. When this lot of hogs was ex- posed to disease in the quarantine pasture one of the pen checks died of hog cholera, one became sick but recovered, while the other never showed marked symptoms of illness. With regard to the treated hogs in the quarantine pasture, we find that one of those which had 58 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. received but 5 c. c. of serum with the disease-producing blood died, but a positive diagnosis of hog cholera could not be made from the autopsy findings. The 2 hogs that were made sick by vaccination remained more or less unthrifty, but neither contracted hog cholera; the other 3 vaccinated hogs remained well in the quarantine pasture as well as in the Syphax exposure pen where they were placed after removal from the pasture. EXPERIMENT XIV. Second experiment with first drawing of serum from immune 1874- The second experiment with serum from hog 1274 is incomplete, as the vaccinated hogs were not exposed to natural infection after vaccination. The results of the vaccination are of interest, how- ever, for comparison with those of the preceding experiment in which the 2 hogs treated by the serum-simultaneous method with 10 c. c. of serum became distinctly sick. In Experiment XIV none of the 12 hogs which were given serum with disease producing blood became sick, although both of the hogs treated with the. blood alone died of hog cholera. Owing to the lateness of the season and to the fact that our plans to hold vaccinated hogs for several months before exposing them had been interfered with considerably by the accidental out- break of hog cholera in our quarantine pasture, it was decided to reserve all of the vaccinated hogs in Experiment XIV, together with their pen checks, for exposure during the summer of 1907. TABLE 17. Secondary experiment with first drawing of serum from hog 1274 ("quick" immune. Syphax) . No. of hog. Weight. Material injected. Inoculation. Remarks. Date. Result. 1938.... 1939.. 1940.. 1941.. 1942.. 1943.. 1946.. 1947.. 1948.. 1949.. 1950.. 1951.. 1954. . 1955. . 1956.. 1957. . 1958.. 1959. . I960.. 1961.. 1944. . 1945.. 1952. . 1953.. 1962.. 1963.... 1964. . . . Pounds. 55 50 70 45 40 40 40 40 50 40 40 75 75 40 50 60 50 80 75 40 40 40 40 40 30 70 40 10 c. c. serum and 2 c. c. dis- ease-producing blood of hog 1925. ....do... Dec. 20,1906 do Remained well . Not exposed. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Do. Hemorrhages. Do. Hemorrhages and ulcers. ...do... do.... do do .do. . do .do . do do .. ..do do do do 20 c. c. serum and 2 c. c. dis- ease-producing blood of hog 1925. do do. do ...do... ...do... do do do ..do.. . ...do. do do do . .do do do .. .do 10 c. c. serum alone do . do ..do... 1. do do . do do .do do ...do do 20 c. c. serum alone .do ... do .. ..do do .do do ...do... do do . .do. do Pen check with hogs 1938 to 1943. do .do do Pen check with hogs 1946 to 1951. do .do ...do... 2 c. c. disease-producing Dec. 20, 1906 blood of hog 1925. do do Sick Dec. 27, 1906; died Jan. 3, 1907. Sick Dec. 27, 1906; died Jan. 7, 1907. Sick Jan. 8, 1907; died Jan. 12, 1907. Pen check with hogs 1962 and 1963. TESTS OF SERUM FROM SYPHAX IMMUNES. 59 Summary of results obtained with first drawing of serum from immune 1274. Eighteen hogs were vaccinated by the serum-simultaneous method, serum in doses of 5 c. c., 10 c. c., 15 c. c., and 20 c. c. being used in con- junction with 2 c. c. of disease-producing blood. Of these 18 hogs 2 became sick; none died. Four hogs were injected with disease-producing blood alone; all became sick; 3 died, and 1 was killed when in a moribund condition. Eight hogs were injected with serum alone and all remained well. Only 6 of the vaccinated hogs were later exposed to infection; 1 of these died, but without exhibiting either before death or at autopsy positive signs of hog cholera. Of 3 pen checks which were exposed at the same time as the 6 vaccinated hogs, 1 died of hog cholera, the other became sick but recovered, while the third check did not become plainly ill. As a result of the experiments with the first drawing of serum from hog 1274, we may conclude that (1) The serum when given in sufficient dose will protect non- immunes from a simultaneous injection of a fatal dose of disease- producing blood. (2) The serum-simultaneous injections in which the largest dose of hyperimmune serum was used seemed to afford quite as complete protection from hog cholera when exposure took place three weeks after vaccination as was afforded by the injection of disease-producing blood with a smaller dose of serum. (3) An insufficient dose of serum with disease-producing blood ap- peared to result in more or less permanent injury to the hogs treated in that manner. SERUM FROM IMMUNE 1297 (Si,o\v METHOD). EXPERIMENT XV. First drairinij of serum. The preliminary experiment with the serum from immune 1207 shows that 5 c. c. of that serum was not sufficient to insure protection from a fatal dose of disease-producing blood administered with the serum, for, as may be seen from Table 18, hog 1742 died of hog cholera after vaccination. The other hog injected in the same manner as hog 1742, however, remained well, as did the pen check that associated with these two hogs. None of the hogs treated with doses of 10 c. c. and 15 c. c. of serum plus disease-producing blood showed any symp- toms of illness, but both of the hogs that received disease-producing blood alone became sick, and one of them died. The pen check that was exposed to the hogs that were injected with disease-producing blood alone contracted hog cholera from them and died. With the exception of hogs 1743 and 1744, all of the surviving vaccinated hogs with their pen checks were placed in the pasture on October 17 to 60 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. H > L 3 < HemorrhaKic le- 1 I 1 . | co C O 2 lesions. Ulcers. Hemorrhaeic le- ) : 00 i 1 c = 5t ulcers. =3 4-> 1 tt 5 - B tf C c c c E X a B $ u PH _c H-> 'a E O a - e t: e c c c C ~ o o c 3-35 . n f > K .1 . ._ S^ w c 1 . "^ ~d fc.s |t ".i o 55 S a V. a 5 * 'S Q 1 4i 5 V. g i c e c < ~ c c : ^ : I e X ' ' g g J H | a 4 c c IN c E at E E C c disease - producing blood of hog 1064. Ho es C C | i c c e c. c. disease-produc- ing blood of hog 1664. Ho p T P 0( | T I c c X c. c. disease-produc- ing blood of hog 1004. Ho OE 1 i X ^ ^ B a 1742 and 1743. Pen check with hogs 1745 and 1746. Pen check with hogs 1748 and 1749. 2 c. c. Hispji se-nroHur- ing blood of hog 1604. ep | C , ^ f2 1751 and 1752. 3 i 9 t Pounds. .V} 5 5 i? S 5 % 5 5 i S I O 55 I- t^ 4 t - i r- -r -T r - r S *? t t>- t g f. 3 i^ TESTS OF SERUM FROM SYPHAX IMMUNES. 61 await future exposure. The accidental outbreak of hog cholera in this pasture made its appearance about the time that the hogs were placed there, and they may therefore be regarded as having been exposed to disease on October 17, or twenty-two days after vacci- nation. Both pen checks that were exposed at the same time with the vaccinated hogs became sick; one died, but the other recovered. None of the 4 treated pigs became sick in the pasture, and during a subsequent exposure in the Scribner exposure pen they likewise remained well. Hogs 1743 and 1744, one a vaccinated hog and the other a pen check, were placed on October 29, 1906, in a separate pasture that was used for hogs that had been made sick by vaccin- ation or that had been removed from the exposure pen. Athough there was undoubtedly ample opportunity for these hogs to contract hog cholera in this pasture, neither of them did so, but hog 1743 was killed by other hogs. The pen check 1744 was placed in the Scribner exposure pen on January 23, 1907, to further test its immunity, with the result that it survived without showing disease. EXPERIMENT XVI. Second experiment ivith first drawing of serum. In the second experiment with serum from immune 1 297 the usual plan was followed. Twelve hogs were treated by the serum-simulta- neous method, 6 of them being given 10 c. c. of serum, and 6, 20 c. c. of serum with 2 c. c. of disease-producing blood in each case; 4 hogs were injected with 10 c. c. of serum alone and 4 with 20 c. c. of serum alone; 2 hogs were injected with 2 c. c. of the disease-producing blood alone. Two uninoculated checks were placed in the pens with each of the lots treated by the serum-simultaneous method, and 1 was placed in the pen with the hogs that were injected with disease-pro- ducing blood alone. As may be seen from Table 19, all of the animals that were injected with serum alone or with serum and disease-pro- ducing blood combined, togetHer with their pen checks, remained well after treatment. Both of the hogs that were injected with disease- producing blood alone contracted hog cholera, and their pen check contracted the disease from them and died of hog cholera. In order to test further the immunity of the vaccinated hogs, half of those vaccinated by the serum-simultaneous method, together with half of those treated with serum alone, were placed in the Scribner exposure pen three weeks after vaccination. All 4 of the surviving pen checks were exposed at practically the same time. The scrum- simultaneous vaccinated hogs all remained well as a result of this exposure, but 3 of the 4 hogs treated with scrum alone died of hog cholera, and 3 of the 4 untreated check hogs also died. The other treated hogs that were not exposed with the lot just described were exposed as follows: Five hogs treated by the senim- simultaneous method were exposed in the Scribner exposure pen fifty-one days after vaccination. Four hogs that were treated with 62 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. ft, f O> .S SP ll ill w a oco oooo o o o ooooooo QQQ QQQQ O Q Q OQQQQQO -o-o-o-o-o^ ' fn QS(5( ^ 'S IQ a 03-0 II : :' ! !^l O -O -O -O -O -C T3 C C C C C C C ^ 2 SSSSS3 K TESTS OF SERUM FROM SYPHAX TMMUNES. 63 serum alone were exposed in the same pen twelve weeks after vacci- nation. The virulence of the disease to which the serum-simultane- ous treated hogs were subjected is shown by the fact that 7 check hogs (1992-1998, Table 19) placed in the exposure pen five days later all died of hog cholera, but notwithstanding this very severe test, all of the serum-simultaneous vaccinated hogs remained well. Of the 4 hogs vaccinated with serum alone and exposed twelve weeks after treatment 3 died, as was to be expected, since the hogs treated simi- larly and exposed only three weeks after treatment did not show any appreciable degree of resisting power. Summary of results obtained with serum from immune 1297. Eighteen hogs were vaccinated by the serum-simultaneous method, the serum being used in doses of from 5 c. c. to 20 c. c. Only 1 of these hogs died from the treatment, and this hog (1742) received but 5 c. c. of the serum. Four hogs were injected with the same dose of the same disease-producing blood as was given the serum-simultane- ous treated hogs, and as a result all became sick and 3 died. Of 6 pen checks exposed to the serum-simultaneous treated hogs after vac- cination all remained well, although both of the checks exposed to hogs treated with diseased blood alone contracted hog cholera and died. In regard to immunity in the treated hogs subsequent to vaccina- tion it is seen that of the 16 serum simultaneous treated hogs all remained well when exposed from three to seven weeks after vaccina- tion. Of 8 hogs treated with serum alone and exposed to hog cholera from three to twelve weeks after vaccination we find that 6 died of hog cholera, while of 4 checks not inoculated and exposed to the same infection 3 died. With regard to the experiments with the first drawing of serum from immune 1297, the following conclusions may be drawn: (1) When administered in sufficient dose this serum will com- pletely protect hogs from an otherwise fatal dose of disease-producing blood given simultaneously with the serum. (2) The immunity produced by a serum-simultaneous vaccination remained quite firm for at least seven weeks. (3) This immunity was quite as firm in the hogs that received 20 c. c. of serum with disease-producing blood as in those that received only 10 c. c. (4) It is not necessary for hogs to show visible symptoms of illness after vaccination in order to secure immunity that will last at least seven weeks. (5) The immunity in serum-simultaneous vaccinated hogs was not due to serum alone, as shown by the death of animals injected with serum alone when exposed to hog cholera. 64 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. J "3 > 1 g B O H a 00 o ft as '3 c a ~ o a) o tf I 1 1 * S & C 8 o * c . "" s ^ s- o N o o " C f 1 o 0) '3 ^ S ^ 9 c c I 1 5 k a 1 1 I i ts ira rt c . -c c c - d s o P : 1 ? ~ O 'C T "> \ ~^ 10 c 4 c - c o ' g "Ba =3 '- g o 3 s| s ~ S d .9 S ~ 3 A oco c. ;. 'S uS? C v "SSg ~ ai g * ft ft Sg ^) ffli! 4< -na) oOQ o W c. ft CO 8 CQ ac r7. IB PH K cc SH j W V. 06 i : e nfl C C o c q ^ -c G T O "C Pi 02 Material injected. 5 c. c. serum and 2 c. c. disease-produc- ing blood of hog 1641. do 10 c. c. serum and 2 c. c. disease-produc- ing blood of hog 1641 . do 15 c. c. serum and 2 c. c. disease-produc- ing blood of hog 1641. ...do... Pen check with hogs 1671 and 1672. Pen check with hogs 1674 and 1675. Pen check with hogs 1677 and 1678. 2 c. c. disease-produc- ing blood of hog 1641. do 1 i I s iO O ^ IS SS SS?g:S?SS a> ^ ^ ft, If i e g ^y d u U J TESTS OF SERUM FEOM SYPHAX IMMUNES. 65 (6) Hogs immunized with serum prepared from one strain of dis- ease (Syphax) may be expected to resist infection of the same nature from an entirely different source (Scribner). (7) Hogs vaccinated by the serum-simultaneous method, if not made sick by the treatment, will not communicate disease to other hogs. SERUM FROM IMMUNE 1310 (SLOW METHOD). EXPERIMENT XVII. Preliminary experiment with first draving of serum. In this experiment only the serum-simultaneous method of vacci- nation was used, and Table 20 shows the results. Six hogs were vac- cinated by the serum-simultaneous method, doses of 5 c. c., 10 c. c., and 15 c. c. of serum being used. Of the 4 hogs injected with less than 15 c. c. of serum all became sick and 2 died, and the 2 pen checks contracted hog cholera from them. The 2 hogs that were injected with 15 c. c. of serum in combination with the disease-producing blood remained well, while the 2 injected with disease-producing blood alone died and communicated disease to their pen check. Three of the surviving vaccinated hogs in this experiment were exposed to hog cholera in the quarantine pasture and 1 in the Syphax exposure pen. It was to be expected that the hogs that had been sick would be subsequently immune, and this was found to be the case. The chief interest in the exposure of these hogs lay in the behavior of the hogs that received 15 c. c. of serum with disease-producing blood and which did not become ill as a result of vaccination. These 2 hogs (1677 and 1678) were exposed to hog cholera in the pasture thirty- seven days after vaccination. They did not become sick as a result of this exposure, although their pen check which was exposed with them contracted hog cholera and died. A later exposure of these 2 hogs in the Scribner exposure pen three and one-half months after vaccination proved them to be still immune. EXPERIMENT XVIII. Second c.rperiment with first drau'ing of scrum. In this experiment, through an error, doses of 10 c. c. and 20 c. c. of serum were used with disease-producing blood, although the prelim- inary experiment had shown that 10 c. c. of this serum was not sufficient to protect' hogs from a fatal dose of disease-producing blood. It appears, however, that even the dose of 10 c. c. of serum may have increased the resisting power of the vaccinated hogs somewhat. Six hogs were injected with 10 c. c. of serum plus disease-producing blood, (> with 20 c. c. of scrum plus disease-producing blood, I with 10 c. c. of scrum alone, 1 with 20 c. c. of scrum alone, and 2 with 2 c. c. of disease-producing blood alone. Two pen checks were placed with each lot of serum-simultaneous treated hogs and one with the hogs injected with disease-producing blood alone. Table 21 shows that I of the r> hogs treated with 10 c. c. of serum plus disease-producing 128-40 No. 101' OS f> 66 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. >, / 9 ^ a < a C c '5 c "E a | : a S lesions not marked. Do. Hemorrhages and ulcers. .8 co *j5 .C-3 .0 (B C X ^ * ^ O O "Sb tJJD > 'S; II II OO/-S/-N 3 C/~\/- sg CC s gGC O GJ W iz; W 1 Hemorrhages and ulcers. Do. Hemorrhagic lesions. Hn a t~ : a i t \ ft > 666 PQP u Q JO c *-> ~ t~ 2 2 S r- S Q G 5; -H..-x^a).. Jij. 588 t'xposuro Sc f a : S i g : o : ^ 5o OS i-l -H "3 Oj'ffi --- C-ST3 .2.2 CGG ? S : i : S 1 :88 :| : :| :l :8 : 8 : ' 03 n . -ft : N o' 0^6 r - 1 o' cr5 o'' H c cv: 'd !N c 1-1 C c o 1 i ' 3? i * $ ; s i$ ; S ! 1 ' 9 ' '."''. '.^ '. Pounds. 55 5jS SS? S8 SS858SSSRS8SSSi S^^ 9 8 ^9 1 xx x 26 ' 2! >c ^ r^ x 3; 71 c^> "^ " t- ac o c^ co iii i 1 HI TESTS OF SERUM FROM SYPHAX IMMUNES. 67 blood died from the treatment, but, strange to say, the checks in the same pen did not contract the disease from them; the hogs that received 20 c. c. of serum with disease-producing blood all remained well, as did those injected with serum alone. The 2 hogs injected with disease-producing blood alone died of hog cholera, as did the check exposed in the pen with them. The surviving hogs from the protective inoculations, including 4 pen checks (1902, 1903, 1910, and 1911) which had not been sick, were exposed to hog cholera at different lengths of time after vaccina- tion. Of the hogs treated with serum alone, 2 that received 10 c. c. and 2 that received 20 c. c. of serum were placed in the Scribner exposure pen eighteen days after vaccination with 2 of the pen checks; both of the checks and 3 of the 4 serum-treated pigs died of hog cholera. The surviving treated hog was one that had received 20 c. c. of serum. The 4 remaining hogs treated with serum alone were exposed first sixty-eight days after vaccination, and 3 out of 4 died of hog cholera, the surviving hog being again one of those that received 20 c. c. of serum, though it is very doubtful whether the resistance of this hog can be properly attributed to the previous serum injection. The behavior of the serum-simultaneous hogs after exposure to hog chol- era was quite different from that of the hogs treated with serum alone. It will be remembered that 12 hogs were vaccinated by the serum- simultaneous method, and of these 4 died as a result of vaccination, these having received only 10 c. c. of serum with the disease-producing blood. The 2 surviving hogs that received 10 c. c. of serum plus disease-producing blood were placed in the Scribner exposure pe:i with 3 of those treated with 20 c. c. of serum plus disease-producing blood thirty-four days after vaccination; all of these hogs remained well, although the 2 checks (1902 and 1903) exposed at tlu> same time died of hog cholera. The 3 remaining hogs treated with 20 c. c. of serum plus disease-producing blood were first exposed to hog cholera by being placed in the Scribner exposure pen seventy-two days after vaccination. All of them remained well, although imvac- cinated check hogs 2003, 2004, and 200"). subjected to the same exposure died of hog cholera. Summary of experiment* irith first drawing oj mrum jrmn niu>i< l.iin. Results of vaccination. Eighteen hogs were vaccinated by the serum-simultaneous method: of these 10 received 10 c. c. or less of serum, while the remaining s received \~> c. c. or more of serum. Eight out of tho first 10 became sick, and C> died, us a result of vacci- nation, while none of the S hogs that received larger doses of serum became sick. Four hogs that were injected with disease-producing blood alone died of hog cholera as u result of the injection. The S hogs that were injected with serum alone as usual remained well. 68 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. All pen checks with vaccinated hogs remained well, notwithstanding the fact that some were exposed to hogs that died as a result of vaccination. Results of exposure to hog cholera. Twelve hogs treated by the serum-simultaneous method were exposed to hog cholera after vaccination, some being exposed after an interval of twenty-four days while others were not exposed until approximately two and one-half months after vaccination. Of these hogs only 2 had shown symptoms of sickness after vaccination. None of the 12 became sick as a result of exposure. Eight hogs that had been treated with serum alone were exposed to hog cholera, 4 being exposed eighteen days after vaccination, and 4 two months and eighteen days after vaccination; 6 of these died of hog cholera. Five unvaccinated hogs were exposed with the treated hogs as checks, and all of these died of hog cholera. From these experiments we may conclude that (1) The serum from hog 1310, first drawing, will, when given in sufficient dose, protect hogs from a fatal dose of disease-producing blood administered simultaneously with the serum. (2) This simultaneous vaccination confers on the treated hogs an immunity which lasts for at least two and one-half months. (3) The immunity in hogs which showed no symptoms after vac- cination seems to be quite as firm as in those that were made visibly ill. (4) Serum alone did not confer a satisfactory immunity for even three weeks in the dose used. (5) The immunity in serum-simultaneous hogs was not due to the action of the serum alone. (6) Check hogs exposed to hogs which became sick as a result of vaccination contracted disease from them in some cases. PRODUCTION OF A PROTECTIVE SERUM BY USING A REDUCED DOSE OF DISEASED BLOOD. The hogs in the preceding experiments that were hyperimmunized by the " quick method" all received a dose of disease-producing blood in the proportion of 1,000 c. c. of blood to 100 pounds of body. We have carried out only one experiment with serum from an immune hyperimmunized with a smaller dose of disease-producing blood. This hog (1401) received only 500 c. c. of disease-producing blood for hyperimmunization. The history of hog 1401, as well as the experiments carried out with serum from that hog, are described below. EXPERIMENT XIX. Serum from immune 1401. Hog 1401 is one of those that was vaccinated by the serum-simul- taneous method in the experiments of 1905, and its detailed history is as follows: Hog 1401 weighed 60 pounds December 2, 1905, and on this date, together with other hogs, was injected simultaneously with disease- EXPERIMENTS WITH A REDUCED DOSE OF DISEASED BLOOD. 69 producing blood and 10 c. c. of serum from immune 1234 (see p. 11). No symptoms of sickness followed this injection, and the hog was exposed to disease on December 27, 1905. Hog 1401 again remained well and was not further exposed until September 3, 1906, when it was injected subcutaneously with 500 c. c. of Scribner disease-pro- ducing blood. The weight of the hog at this time was approximately 100 pounds. No visible disturbance of health followed this injection. On September 24, 1906, just three weeks after the injection of dis- ease-producing blood, hog 1401 was bled in order to secure serum for experimental purposes. This hog may be classed with the Scrib- ner quick immunes, though the dose of serum used in proportion to the body weight of the immune is only half of that used on the other Scribner quick immunes. Unfortunately, we were not able to carry out as extensive experi- ments with this serum as was desirable, but the number of animals available for experimental purposes and the requirements of other experiments in the way of pens and hogs made it impossible to do more than make a preliminary test of the potency of the serum from immune 1401. The tests of this serum were made in the usuai manner, the serum being used in doses of 5c.c., 10 c. c., and 15 c. c., given simultaneously with a fatal dose of disease-producing blood. Table 22 shows the results of these injections. Both of the hogs injected with only 5 c. c. of serum became sick and 1 died, and they also communicated disease to their pen check, hog 1814. One of the 2 hogs that were injected with 10 c.c. of serum plus disease-producing blood became slightly sick but recovered, while the other remained well; the pen check in this pen contracted hog cholera. Neither of the hogs that received 15 c.c. of serum with the disease-producing blood showed any symp- toms of illness following vaccination, and their pen check likewise remained well. Both of the hogs that were injected with disease- producing blood alone died of hog cholera and communicated the disease to their pen check, hog 1X23. It will thus be seen that of hogs inoculated simultaneously with serum and diseased blood, 3 became sick but only 1 died, and this one received the smallest dose (5 c. c.) of serum. Four of the 5 surviving vaccinated hogs were later exposed to hog cholera to test the duration of the immunity conferred by vaccination. Two of these hogs were placed in the Scribner exposure pen on November 17, 190(5, thirty days after vaccination; the surviving pen check, hog 1X20, was exposed at the same time. The check died, but both of the treated hogs remained well. The 2 other vaccinated hogs were first exposed to hog cholera three and one-half months after vaccination. They both remained well, alt hough unprotected hogs exposed at the same time died of hog cholera. Although we have hyperimmuni/.ed only 1 hog by the quick method with a dose of disease-producing blood equal to 500 c. c. per 100 pounds of body weight, the results obtained with this serum are 70 PRODUCTION OF IMMUNITY FROM HOG CHOLEBA. such that there is good reason to believe that this reduced dose might be sufficient to produce a satisfactory serum. Owing to reasons al- ready stated, we have not as yet been able to decide this point, but from what has been done we may conclude that (1) the serum of hog 1401 is as potent as that of some other immunes injected with larger doses of disease- producing blood, but that it is not equal in potency to that from the majority of immunes which received twice as much disease-producing blood; (2) when given in sufficient dose this serum will protect per- fectly from a fatal dose of disease-producing blood administered simultane- ously with the serum; (3) the immunity produced by the serum-simultane- ous injections lasted at least three and one-half months, even though the treated hogs showed no signs of reaction af- ter injection; (4) it ap- pears possible that the injection of serum did not lower the infectious- ness of the disease to as great an extent as other sera from immunes that were given larger doses of diseased blood, O i tic 5b CD ( a .C.C 11 SJ 1 s p c a. c c C Pi * " KK 1 o J S j | re Sen 5 I c 73 a '3 C - rt o C C ' 6 ~ T TS 73 P. X 'A o tf 5 r PL, 1 5 1 -S t^: ~ i-: - 1 c C Q z > l _ S : 8 ^1 ^ . F-* )>. ? a O9 t , C S; ^ CO I C * :o c ;K * ! [2 -^ - 73 *" S i O -r I .73 "C .S .5 1 " S c 73 T: Ir ilation. c5 c S-d* >ll X' > S -M O'oj C c 1 s ^^ IJ5 S T3 Q5 +> *- ^^^ C' 1 ( 8 e co c? 6 C C =r e ,s CO PS ^ C - a s s^s^ 1-1 S oi c. J*; -* Pace cr S^< i r-l i !g OC 00 c C - e - 1 c Q "3 T: . 7Z o O i " c 1 - * E j p< a c i OC 00 OC *-< OC $ i ^-t t * * ' O *H 03 i 73 T3 7373 c c co 73 _: S^' wv oj 03 o3 O 33 S 73 1^ 73 f; [^ m oo^: rj o o'^ c 00 00 OC ClC i-H l-l i-( f< OC = N* e. e- c - | | H U O 3 73 O 'K - 7 c A A -e'g ti "X- 0373 O rC ^ 3 33 S II |j u- 2 | I 2 P 5 "5| J jf rf.lM o I C '- be 4 i 80 o & o c 2 S -fi -?-73- "B T > C ? c a C ^ : c I c . Sj i | i \ fi | |o a - Q- i M O T3 .2 -1 g SI ^ /- | s IT."* ^ .8 s "S 1 -i _> gllloJ SS ' o z; T 2^cjo"o oS"i rtoS 4 a 5 / CO CO COCO V. CO s a I 1 a 9 "* 1 i H q a 4 H " * " 1 H i $ 03 ft I 5 i i i 1 c C a a 1 c c (>) C I I c c, c producing blood of hog 1711. .do... do < j ? c & CT z 05 = C ; c 5. s producing blood of hog 1711. ...do... ...do.. Pen check with hogs 1801 and 1802. Pen check with hogs 1804 and 1805. 2 c. c. disease-producing blood of i Oct. 10 hog 1711. ...do .do Pen check with hogs 1807 and 1808. t j ' r a 1 > Pounds. 40 ^ 3 S c?S * 1 o a i 1 % 1 1 1 | SEBUM FROM NONHYPERIMMUNIZED IMMUNES. 73 distinctly though not desperately sick, showing the usual symptoms of a rather mild attack of hog cholera. After recovery from this illness hog 1295 was placed in the exposure pen, but did not become sick. This hog was finally removed from the exposure pen, but was not again exposed to hog cholera. Blood was drawn from hog 1295 on Novemberv22, 1906, and after being prepared in the usual manner was used in the experiment next described. EXPERIMENT XXI. Serum from nonhyperimmnnized immune, 1295. This experiment was modeled directly after the preliminary experi- ments with the hyperimmune serum, the serum-simultaneous method only being used, as previous experiments had shown that all of the hyperimmunized hogs furnished serum that would protect, when given in sufficient dose, from a simultaneous injection of disease- producing -blood, and it was evident that a failure to protect from a simultaneous injection of such blood would certainly render unneces- sary an experiment to determine the duration of immunity conferred by serum alone. TABLE 24. Experiment with sertimfrnm hog 12<)~> (nonhyperimmiini:ffl immune}. No. of hog. Weight. Material injected. Inoculation. Autopsy. Date. Result. 1920.... 1921.... 1922 Pounds. .5 c. c. serum and 2 c.c. dis- ease-producing blood (,f hog 1891. . ..do Nov. 23, 1906 do Sick Nov. 29; Dec. 1,1906. Sick Nov. 29; Dec. 2, 1906. Sick Dec. 8; Deo. 16, 1900. Sick Nov. 29; Dec. 2, 1906. Sick Nov. 29. 190t died died died died ;died illed died died died died died died Homorrhagic lesions. Do. Hemorrhu gos and ulcers. Hemorrhagic lesions. Do. No marked le- sions. Hemorrhagic lesions. Do. Hemorrhages and ulcers. Hemorrhagic lesions. Hemorrhages and ulcers. Do. Pen check with hogs 1920 1923.... 1924... 1925 45 45 35 and 1921. 10 c.c. serum and 2 c.c. dis- ease-producing blood of hog 1891. do Nov. _:{, 1906 do Pen check with hogs 1923 Sick Deo. 13; I Doe. 20. 1906. Sick Nov. 29; Dec. 1, 1906. Sick Nov. 29; Dec. 3. 1906. Sick I)oo. .1: Deo. 9. 1906. Sick Nov. 29; Doc. 2. 1906. Sick Nov. 29: Dec. 3. 1906. Sick Dee. S; Deo. 19. 1906. 1926. . . . 1927.... 1928.... 1929.... 1930. . . . 1931 40 40 40 .10 40 35 and 1924. 1.1 c. e. serum and 2 c. c. dis- eased blood of hog iw.ll. ...do Nov. 2:t, 1906 .do . Pen chock with hogs 1926 and 1927. 2 c. c. disease-producing blood of hog 1891. .do Nov. 23, 1906 ...do Pen chock with hogs 1929 and 1930. Table 24 shows without question the lack of potency of the serum from hog 1295. Doses of 15 c. c. of serum did not prove to be any more potent than doses of 5 e. c.. and every hog in this experiment died of hog cholera, the disease being communicated to the pen 74 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. checks in all cases. It does not appear that the serum, even in the largest doses, exercised any retarding influence upon the incubation period or upon the course of the disease once it had appeared. Summary of results obtained with serum from nonhyperimmunized immunes. Serum from 2 immunes was used in doses of from 5 c. c. to 20 c. c., and this serum was injected into nonimmunes simultaneously with a fatal dose of disease-producing blood. Ten were treated by this process; all contracted hog cholera, and 8 died of that disease. Of 4 hogs injected with disease-producing blood alone, all died of hog cholera. Five checks were placed in the pens with the vaccinated hogs, and all contracted disease from them and died. The 2 pen checks exposed in the pen with the hogs that received disease- producing blood only also died of hog cholera. From the results stated above it will be seen that (1) the protec- tion afforded to nonimmune hogs by the injection of serum from nonhyperimmunized immunes is very slight as compared with that afforded by hyperimmune serum; (2) even in doses of 20 c. c. the protection afforded was not sufficient to prevent the death of 1 of the 2 animals that received that dose; (3) the contagiousness of the disease is not noticeably lessened by the injection of serum from a nonhyperimmunized immune; and (4) the serum from non- hyperimmunized immunes is not potent enough to render its use practicable in combating hog cholera. EXPERIMENTS WITH SUCKLING PIGS. The following experiments with young pigs were carried out for the purpose of determining whether very young pigs would react to vaccination with immune serum in the same manner as the larger shoats. It \vas desirable to determine also whether very much smaller doses of serum might not be used on the small pigs, thus effecting a considerable saving in the cost of vaccination. The plan adopted was to vaccinate part of a litter of pigs and leave part unvaccinated, so that when the time came for testing the immunity of the vaccinated pigs we \vould have a sufficient number of checks for exposure along with the vaccinated pigs, and additional infor- mation regarding the danger of hog cholera being transmitted through the agency of vaccinated hogs would also be secured. EXPERIMENTS WITH SUCKLING PIGS. 75 d 2 Q) D O C. 3 3 ' S -o -3 0) 1 9 .s s s | < ~ ~ 2S 'S O O O Q O E SE a 6 a oi - C x e- ^2 - ^ 8 11 t * "si i *. e E^ 1 a X -V Q H I | 1 s s ~ o fo ^ 1 g , 1 ^ tj Cf P = o _ is "-1 C . o E c _o S 3 CD O> 1 5 c c c ^ -a c 5 s S'gr 5 ~ '.\^L ~ r 0. | o .3 s*j ^e o g 1 sc - ^ - - ^^ Ci-* c p2 c. 1. - - a O i ?l O W CO X 03 !/ CO a ? i ? c : 1 C (5 2 D, * i i c S 1- d i u, 5 s ^ c fs V < V c,, i- 3 K = : c c r 1 j | 8. a .c - -5 *^ z SI C --_ c. c. seruii producing ..do. .. | c|c 1 c V e U- ri i 1 - ---"- 5 ? 5 5 ^ ? S, ^.L-./ifiS - ZJ l-l-l. 1- i- i EXPERIMENT XXII. Serum-simul taneous vaccination of suckling pigs, using serum from immune 1383. The 5 suckling pigs used in this ex- periment were 10 days old at the time of vaccination and all were vigorous and healthy. Two older pigs (1730 and 1731) were injected at the same time as the suckling pigs with disease- producing blood alone, in order to test the virulence of the blood. By re- ferring to Table 25 the method of car- rying out the experiment may be best understood. Three of the suckling pigs were given 2 c. c. each of serum with 0.25 c. c. of disease-producing blood sub- cutaneously; the remaining 2 were not, treated, but the entire litter was left together, the sow being given 40 c. c. of serum as a protection against possible infection from the vaccinated pigs. No pigs were injected with 0.25 c. c. of disease-producing blood alone, but the death of pig 1726 and the sick- ness of 1725, in spite of the injection of the serum, indicate that 0.25 c. c. of the disease-producing blood used in this experiment would probably have been sullicient to destroy all of the injected pigs if the serum hud not been used. The virulence of the disease-producing blood in the usual dose, 2 c. c., is shown by the records of hogs 1730 and 1731, and the con- tagiousness of the disease is proven by the sickness and death of uninoc- ulated hog 1732, exposed in the pen with hogs 1730 and 17:51. These ."> hogs 1730, 1731, and 1732 were of c5 course placed in a pen to themselves and did not at any time associate with the other hogs in (his experi- ment. The low degree of contagious- ness of the disease following vaccina- 76 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. tion, which has been remarked upon in connection with other experi- ments already described, is again well shown in this experiment, where the uninoculated suckling pigs remained well even though they associated with pig 1726, which died from vaccination. The 4 surviving pigs 1725, 1727, 1728, and 1729 were with the mother transferred to a pasture that was free from hog cholera. All remained well, and approximately four months after vaccination they were weaned. Shortly after this, pigs 1725 and 1728 became sick and died. Neither showed lesions of hog cholera, and as there is nothing to show that disease had ever been introduced into this pasture, we must consider the death of these pigs to have been due to some other cause, the exact nature of which we were not able to determine. For this reason Table 25 does not show the history of pigs 1725 and 1728 subsequent to vaccination. The remaining pigs of this litter, 1727 and 1729, one a vaccinated pig and the other a check, were placed in the Scribner exposure pen on February 20, 1907, just five months after pig 1727 had been vaccinated. As a result of this exposure the check (1729) sickened and died on March 10, 1907, while the treated pig (1727) remained well. The results of this exposure furnish additional proof that the death of pigs 1725 and 1728 in the pasture before exposure was not due to hog cholera, for, upon undoubted exposure to that disease, vaccinated pig 1727, treated in the same manner as pig 1725, remained well, while the check (1729) died, thus indicating that had the disease in the pasture been hog 'cholera the vaccinated pig (1725) would not have suc- cumbed, while pig 1729, which was later found to be susceptible, would have contracted disease at that time. Owing to the loss of 1 of the pigs of this litter from vaccination and of 2 others through an intercurrent disease, as just explained, this experiment does not throw very much light upon the feasibility of vaccinating suckling pigs. It does indicate, however, that the serum of hog 1383, in doses of 2 c. c., is sufficient in most cases to protect pigs weighing 5 pounds from a fatal dose of disease-producing blood administered simultaneously with the serum. It appears also that the serum exerted decided influence in lessening the contagiousness of the disease. The weight that should be attached to the death of check pig 1729 and the survival of vaccinated pig 1727 when exposed together to hog cholera can only be properly considered in connection with the behavior of other pigs under similar conditions and treated in a similar manner. EXPERIMENTS WITH SUCKLING PIGS. 77 Autopsy. . Hemorrhages. i / )| c I. t. -. ~ ) c ^ S> 1 % J |i '- ~ o c s 5 ,- = t^. , S & ' i 1 "= . 5 -^ ~ ~ - - 00 o ft - -- - c > ? 1 '1 ? ! -i 5 "3 --; : 1 S 1 :- r. gf I 'S :- C 1 S 4 z _j 3 ~ e 1 O-d 1 c n *ts X "O *^ . ^ * i 3 ' - = C C -: . c X . - 2^ g | !E~ ~^ " L 2-^1 * 1-1 S _ M 0" X I 1 H* 1 ". -5 3 ^ e S j ^ 7 1'ounds. Sov J c. 5> " . - i ? * ~v x- 5 - 5 *. 1 $5 5 5 I rC V EXPERIMENT XXIII. Serum-simul- taneous vaccination of suckling pigs, using serum from immune 1313. Seven suckling pigs, all of the same litter and about 2 weeks old, were used in this experiment. The weight of these pigs was approximately 9 pounds each, with the ex- ception of 2 of those reserved as checks, which were a very little lighterin weight. Of the vaccinated pigs, 2 were given 3 c. c. of serum from hog 1313 and 2 were given 4 c. c. of the same serum, each receiv- ing at the same time 0.25 c. c. of Scribner disease-producing blood. The remaining 3 pigs were left untreated to serve as controls. Unfortunately, as in Experiment XXII, we did not have a sufficient num- ber of pigs to permit the in- jection of some with 0.25 c. c. of disease - producing blood alone and thus determine the protection afforded by the se- rum at the time of vaccina- tion. For tliis reason we were forced to be content with the injection of larger pigs with a larger dose, 2 c. c., of disease- producing blood. The vacci- nated and the unvaccinated pigs of this litter were left to- gether with the sow after the ~ vaccination: the larger pigs, 1X21 and 1X22, that were in- jected with disease-producing blood only, and their pen check, 1X23, were placed in a pen to themselves. For histor of this immune j>. L'l. 78 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. The death of the 2 hogs (1821 and 1822) from the diseased blood injection and the sickness and death of hog 1823 from association with these two show the virulence and the contagiousness of the dis- ease produced by the blood used in the vaccination of the suckling pigs. All of the vaccinated suckling pigs became slightly sick about twelve days after vaccination. The symptoms exhibited were droopiness and conjunctivitis, and slight diarrhea in some cases; this had all passed away after four or five days. None of the untreated pigs became sick. After the recovery of the vaccinated pigs the entire litter with the mother was placed in a pasture free from disease. They all thrived, and after being weaned were placed in the Scribner exposure pen on February 20, 1907, approximately four months after vaccina- tion. As a result of this exposure all of the checks died and showed at autopsy the typical lesions of hog cholera. In marked contrast is the behavior of the vaccinated pigs, none of which became sick in the exposure pen. The results of this experiment indicate (1) that suckling pigs may be vaccinated by using smaller doses of serum and disease-producing blood than is feasible in the case of larger hogs; (2) that the immunity conferred by the injection of these small doses of serum and disease- producing blood is quite durable, and (3) that the vaccinated pigs are not likely to transmit hog cholera to unprotected pigs that associate with them. EXPERIMENT XXIV. Serum-simultaneous vaccination of suckling pigs, using serum from immune 1274- The pigs used in this experiment were 10 days old and weighed about 6 pounds each. On November 1, 1906, 3 of them were injected simultaneously with 3 c. c. of serum and 0.25 c. c. of disease-producing blood and 3 were left untreated. Two larger hogs (1867 and 1868) were given 2 c. c. of diseased blood alone, and an untreated hog was placed in the pen as a control on the contagiousness of the disease. Table 27 shows very clearly that this experiment was not a success, for 2 of the 3 vaccinated pigs died as a result of the treatment they received and 1 of the checks contracted disease from these and died, thus leaving only 1 treated pig with 2 checks for subsequent exposure. This exposure, which took place three months and twenty days after vaccination, caused the death of 1 of the checks, while the 2 other pigs remained well. Incidentally these results show that 0.25 c. c. of the disease-producing blood used in this experiment was sufficient to cause the death of suckling pigs. EXPERIMENTS WITH SUCKLING PIGS. 79 EXPERIMENT XXV. Immunization of suckling pigs with serum alone from immune 1274- This experiment was designed to determine the protective power of the serum when used alone. A litter of 9 healthy pigs, 1 week old and weighing about 6 pounds each, was used. Five of these pigs were given 5 c. c. each of serum from hog 1274 subcutaneouslyon Novem- ber 14, 1906, and the remaining 4 pigs were left untreated. As was to be expected, no sickness followed the injection of the serum, and the mother, with the entire litter of pigs, was placed in a lot supposed to be free from disease. They all remained well until some time during the month of January, when the checks were found to be sick. Pig 1988 was the first of these to die, but the other 3 checks did not live much longer. The vaccinated pigs all remained well. From the lesions found at the autopsy on the checks there seems to be no reason to doubt that these pigs died of hog cholera. We are unable to determine just when they were first exposed to infection, but it seems probable that this exposure took place about ten or twelve days prior to the death of the first pigs. This would place the date of exposure about January 10, approximately two Hionths after the vaccination. It Is fair to assume, therefore, that the serum alone conferred upon the treated pigs immunity which lasted for two months at least. > 1 i on ; i is 6 rt V NH . Ulcers. c c . Hemorrhages and ulcers. . Hemorrhages. o O j i 1 a "5-1 1 1 X w - Remained w Remained w Died Mar. 1 1 1 3) Q S S o .* * * Inoculation. C y, y 1 1 O "S K C j. 35 (7 I'.KXi. .. Sick, hut recovered Sink D.f 1 rlicri l)f 11 ISKXi. . Remained well ...do... ? - ? ~t i^. ^ S i IT " 5 ~N i a a a) O o e o X | z a 3! i 1 ; ' ; i i ' ' - Cf 3 c. c. jM-ruin ami 0.25 c. c producing Mood of hog :> ! j r ,$ t 5 > 2 c. c. disease-producing hog H31. ; | . t 1 ? i i : i 'a I'ound*. c t. J -, 3 C -~ > 77, 35 3! 7 777 80 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. The five treated pigs were given another exposure by being placed in the Scrib- ner exposure pen on February 20, 1907. They did not thrive in this pen, but none of them contracted hog cholera. Summary of results with suckling pigs. In looking over the four experiments which have just been described and which w r ere carried out with very young pigs it will be seen that serum from three dif- ferent immunes was used, and this was given in varying doses. For this reason it is not possible by merely averaging the losses among vaccinated and un- vaccinated pigs to determine the value of the serum for practical use, for it is probable that if we had used doses of 5 c. c. in all cases there would have been no deaths from vaccination, and in the light of the experiments with older pigs it also seems probable that this increased dose of serum would not have lessened the duration of immunity in the vacci- nated pigs. In spite of the facts just stated, it is at least interesting to consider all together the results of the experiments with suck- ling pigs. With 3 of the litters the serum- simultaneous method was used, while serum alone was used for vaccinating the fourth litter. In all, 10 suckling pigs were treated by the serum-simultaneous method, and all showed more or less marked symptoms of illness after vac- cination, 3 of them finally dying of hog cholera. The remaining 7 recovered, and 6 of these were exposed to hog cholera in an infected pen from three and one- half to five months after vaccination; all remained well. In the same litters with these 10 vaccinated pigs there were 8 untreated pigs reserved as checks. Only 1 of these checks, all of which asso- ciated with the vaccinated pigs, con- j i 4 > !i E 3 < Ilemorrhagic lesions. Hemorrhages and ulcers. No autopsy. Ulcers. 4- H 1 a 8 3 a> P5 ""O ; c-i * c oi 0_ M 0,*, ? 3 O a ii "3 O O O O'-sT-s'-i 3 J W o c; .a.^.^.a fctf CGQQ o a :g :::::::: 1 _x o a 2 5 o P i^ccoooooc . . -a -a -o -o -c -a -o -a IB:::::::: I-S 9 3 a 2> ft a i o noculation. Result. o 'O '.'.'.'.'.'.'.'.'. |i::i::::: gooooocooo g-o-o-o-a-c-o-o-ot) o i A A igititjtti a 2 5 to a3 .' rt o o o o : : : ; o 3 q ^ Q T i 1 'i i i c :;:;;:; c ;.!:;;:: H40 No. 101' OH > 82 PEODUCTION OF IMMUNITY FEOM HOG CHOLERA. It will be understood, however, that in testing the curative power of a serum it is very desirable to carry out the experiments with ani- mals under natural conditions as nearly as possible. For this reason, and also in order that we might compare results, one of the experi- ments to test the curative value of hyperimmune serum was carried out with hogs that had been exposed to infection by association sim- ply, while in the other experiments hogs that had been infected through disease-producing blood injections were used. In this connection it may be of interest, before considering the experiments designed to test the curative value of the serum, to note the results of a single experiment wliich was designed to test the effect of immediate exposure to hog cholera by association with sick animals upon hogs that had been injected with varying sized doses of immune serum. EXPERIMENT XXVI. Immediate exposure of serum-treated hogs to disease by association, Six hogs were injected subcutaneously with serum from immune 1383; two received 5 c. c., two 10 c. c., and t\vo 15 c. c. of the serum. Immediately after injection of the serum all were placed in the Scribner exposure pen. Two checks, not treated, were placed in the same pen several days later, as shown in Table 29. TABLE 29. Experiment to test protective, value of 1383 serum where the treated animals were given immediate exposure by association. N hog? f g". Exposure. Autopsy. Date. Result. Pounds. 1707.... 50 1768 50 1769... 40 1770.... 40 1771.... 45 1772.... 50 1773.... 50 1774.... 50 5 c. c. serum. . . 1 Oct. 3, 1900 Remained well Hemorrhage, s and ulcers. Hemorrha g ic lesions. Do. do . . .do Sickened Oct. 10; died Oct. 15, 1900. Remained well 10 c. c. scrum. do do .do ... do 15 c. c. serum. . . ...do... . .do .do... do do Unvaccinated check . Oct. 8, 1900 Sickened a n d died Nov. 4, 19W>. Sickened and w a s killed Oct. 18, 1900. do . do With one exception (hog 1768, injected with 5 c. c. of serum) the hogs that received serum remained well, while both of the checks contracted hog cholera, one of them being killed to secure blood for other experiments. This experiment should be considered in con- nection with Experiment XXVIII, in which the same serum, instead of being injected simultaneously with the exposure to disease, was injected at varying intervals of time after exposure had taken place. CURATIVE VALUE OF HYPERIMMUNE SERUM. 83 I S i 5 , 2R^ ^ 7 L m I ~3 C S 1 222 3 ^ \ s oco ~ O _ : a ": l~ [ *J .s .s .s -- S si - i o r V - - ~ i "7 ^ - ; - ! - _i4 t^ ^ ^ ~~ : - - _ A CO o _ e : x ?"r ? o - |' S =-== JS ji Si ~ - - - y o o _ - - ^ - i : U . X aacow 09 / i i g> := i^ >.444 |S | i T -^ ! ! X ** x r_ si.'i r - 1 JL -i-; v^ v i C C :S -SS s ' 7 '7 K JK * W { - " ' ._ . . y ^ '" , i ' ' ' Si S S ; S Si ?i Si Si Sj S S? ITs It is also interest- ing to compare these results with those of experiments in which the same serum was used in approximately the same doses, but in which the simultane- ous exposure to dis- ease was made by injecting disease- producing blood. (See Experiments V, VI, VII, and VIII.) EXPERIMENT XXVII. Curative value of serum from hoy 1383; exposure through blood injec- tions. Twelve hogs weighing 50 pounds each were injected subcutaneously with 2 c. c. of blood from a sick hog on Sep- tember 29, 1006. Of these 12 hogs, 2 were not given any serum, but were held as controls on the viru- lence of the diseased blood. One hog (17(i()) was not in- jected, but was placed in the pen with the others to test the contagious- ness of the disease. Two days after infect i o n w i t h d iscase- producing blood 2 of the infected hogs were 84 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. injected with 15 c. c. of serum each; four days after infection 2 more hogs were given a similar dose of serum; six days after in- fection 4 more of the infected hogs were given serum, two being given 30 c. c. each, one 35 c. c., and one 50 c. c.; eight days after the disease-producing blood was administered the 2 remaining hogs of the 10 reserved for treatment were given 50 c. c. each of serum. The accompanying table (Table 30) illustrates very well the method of carrying out the experiment and the results obtained. The 2 hogs that were given the serum two days after injection remained per- fectly well; those that were first given serum four days after infection became slightly sick, but recovered. The hogs that were given serum on the sixth and eighth days after infection were all sick when treated, but the course of the disease was apparently not affected by the treat- ment, although comparatively large doses of serum were used. The same serum, however, when administered simultaneously with disease- producing blood proved effective in quite small doses (5 c. c. See Experiments V and VI). The 2 hogs (1762 and 1765) that were not treated with serum died promptly, as was to be expected, and the pen check also contracted disease and died. The 4 surviving hogs in this experiment were placed in the Syphax exposure pen a few days less than one month after the serum injection; all survived this exposure. If conclusions may be drawn from this one experiment, we would infer that even very large doses of serum will not save hogs when it is injected more than four days after infection, but that prior to that time much smaller doses will suffice. EXPERIMENT XXVIII. Curative valueof serum from hog 1403; exposure by association. Twelve healthy hogs, weighing approximately 60 pounds each, were placed in the Scribner exposure pen on November 5, 1906. One day later 3 of these were removed from the exposure pen and 2 of the 3 were given 20 c. c. each of serum, the third hog being left un- treated as a control. This procedure was repeated three times, 3 hogs being removed from the exposure pen on the second, the fourth, and the sixth days after exposure. In each instance 2 of the 3 hogs were injected subcutaneously with 20 c. c. of serum from hog 1403. Table 31 shows that none of the hogs that were removed from the exposure pen within forty-eight hours after being placed there showed any signs of illness as a result of that exposure; even the controls which received no serum remained well. Of the 3 hogs that were taken out of the exposure pen four days after exposure all became sick, but the 2 that received serum recovered, while the control died. The results were almost precisely similar in the case of the 3 hogs that were removed from the exposure pen six days after having been placed CURATIVE VALUE OP HYPERIMMUNE SERUM. 85 r c ffi c C : < ! _;; S 5 5 i 5 - D 1 ' 1 > ~ _i ' ?' s g | 4 - E 'a i- s w T r - gt, s - a - - " 1 i s = 5 I OS C : c 2 : / j > ^ .! T - i - ? ^ O - s ^ "3 S . Xl 3 i c , c r ^ C S5 - c - - -- - H C 5 . s f ^i xl f S K -/ / EC -/ 1 -'_ - - E 5 ; i i C - - c c - ^ C c e E QQ J* ' " - S s ~* - - 8 x to * s X s X 5 i s ? i -i t. a s - f c e 1 c _: 1 s S ~ * "c p - C : 4 : IF sc i S S I ^ - , '" c c X. o X. ; X. w = 5 ss - S SS R-?. 1 < r. r. there, for in this case also the 2 serum-treated hogs recovered after having been somewhat sick, while the untreated control died of hog cholera. In summing up the re- sults of this experiment, the behavior of hogs 1870 to 1875, inclusive, when later exposed to infection, must be taken into consid- eration, for the deaths of control hogs 1872 and 1875 as a result of this second exposure shows that they were all along susceptible to the disease. This points strongly to the likelihood that none of the first 6 animals in this experiment became infected from the first exposure of one and two days, respectively, in the exposure pen, and the immune serum can not, therefore, be considered as having exerted any cur- ative influence in these cases, although its immu- nizing action is well shown by the exposure of Decem- ber 4, 1906, just referred to. The hogs that were treated with serum four days and six days, respec- tively, after being placed in the exposure pen can not be regarded as hav- ing become infected at once after being placed in the exposure pen, for, as shown by the behavior of the controls in the lots that were removed on the 86 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. first and second days, infection probably did not take place in any of the hogs during the first forty-eight hours in the exposure pen. On the other hand, it is quite evident from the behavior of hogs 1876, 1877, and 1878 that infection did take place some time prior to the end of the fourth day in the exposure pen. While it is impos- sible, as already stated, to fix the time of infection with exactness, we know that it took place on the third or fourth day. It is plain, therefore, that the serum in doses of 20 c. c. saved the life of hogs that had been infected from two to four days previously. DISCUSSION OF CURATIVE ACTION OF IMMUNE SERUM. Earlier experiments have shown that the serum from hyperim- munized immunes, when given in sufficient dose, may be expected to protect nonimmune hogs from a simultaneous infection with hog cholera, and the two experiments just described show that this serum may also be expected to prevent the death of hogs when it is admin- istered in moderate doses within four days after infection actually takes place. Later than this it is probable that the serum in virulent attacks of hog cholera will do little good, even though comparatively large doses are given. For this reason it seems possible that serum from immunes prepared in the manner described in this paper may not prove suitable for general use as a curative agent for hog cholera, although in cases where the disease is recognized soon after its appear- ance in a herd the serum would probably save many hogs that had been exposed only a few days. In stating that the serum should be administered within four days after infection to insure success, we wish to make it plain that we refer to the date of actual infection and not to the time of first exposure, for, as is shown by Experiment XXVIII, several days may elapse before infection takes place after exposure to disease by association. (See records of hogs 1872 and 1875, Experiment XXVIII.) DURATION OF PROTECTIVE POWER IN BLOOD OF IMMUNES, AND KEEPING QUALITY OF HYPERIMMUNE SERUM. The original plan of our experiments included the testing of serum drawn from the immunes at different periods of time after the last injection of disease-producing blood, and we were able in some instances to test the second and third drawings of immune serum, but as these later drawings of serum were apparently quite as potent as the first drawing, the question of the duration of protective power in the blood of the immune had not been decided by the experiments already described. For the purpose of testing this matter still further a special experiment was carried out with a drawing of serum from immune 1234. This hog furnished part of the serum used in the 1305 experiments. (See p. 11.) DURATION OP PROTECTIVE POWER OF SERUM. 87 K ' - EXPERIMENT XXIX. Potency of scrum drawn seven and one- half months after last treatment of immune. This experiment practi- cally repeats the test of se- rum from immune 1234 that was carried out in 1905. Two hogs were in- jected with 10 c. c. of se- rum and 2 c. c. of disease- producing blood each, and 2 with 20 c. c. of serum plus the usual dose of disease- producing blood; at the same time 3 hogs were in- jected with 2 c. c. each of disease - producing blood alone, and 1 healthy hog was placed in the pen with these in order to make sure of the infectiousness of the disease. Table 32 shows the man- ner of carrying out this experiment as well as the results obtained. The sig- nificance of the experiment may perhaps be best appre- ciated by a comparison of the action of serum from the same hog drawn nearly a year before, shortly after an injection of disease-pro- ducing blood. The action of blood from nonhyper- immuni/ed i in mimes, as shown in Experiments XX and XXI, should also be kept in mind. Table 32 shows (hat nil of the vaccinated hogs be- came sick as a result of the treatment, but they all re- covered. The 3 hogs (Hi))!, 88 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. 1662, and 1663) that received the same dose of the same disease-pro- ducing blood alone likewise became sick and all died, and they com- municated the disease to their pen check. The vaccinated hogs were exposed to hog cholera during the pas- ture outbreak about six weeks after vaccination. They all remained well and when placed in the Scribner exposure pen three months and nineteen days after vaccination were still completely immune to the disease. Inasmuch as 10 c. c. and 20 c. c. of serum drawn from immune 1234 in 1905 gave complete protection from a fatal dose of disease-pro- ducing blood, and as the last drawing, made seven and one-half months after the last treatment with disease-producing blood, gave only partial protection (the serum in this case prevented death, but did not prevent illness), we may conclude that the protective power of the blood of immune 1234 had diminished somewhat seven and one- half months after the last disease-producing blood injection. But although the potency was diminished, it was still present in a marked degree, and although, owing to the limited number of tests that have been made, we can not be absolutely certain, it is at least reasonable to believe that this power was due to the previous injections of disease- producing blood and not to a natural protective power existing nor- mally in the blood of the immune. (See experiments with blood from nonhyperimmunized immunes, p. 71.) This question is one of prac- tical importance, and further experiments along this line will be car- ried out. EXPERIMENT XXX. The keeping quality of immune serum. A quantity of hyperimmune serum prepared in 1905 remained Unused in the experiments of that year, and as it was very desirable to know how long the serum, prepared with carbolic acid, would retain its potency, we have utilized some of this 1905 -serum in experiments designed to test this point. The serum used was from 1905 immunes Q No. 2 and 1234, and was drawn from these hogs on December 1, 1905, and November 21, 1905, respectively. Immediately after being drawn these sera were mixed with a 5 per cent solution of carbolic acid in such proportion that the serum contained 0.5 per cent carbolic acid. After the addition of this preservative the serum was placed in sterilized bottles and kept in a cool place until June 6, 1906, when it was used in the following experiment. Two hogs, W 1925 and W 1926, were injected subcutaneously with 5 c. c. of Scribner disease-producing blood, and at the same time hog W 1925 was given 20 c. c. and hog W 1926 was given 17 c. c. of hyperimmune serum from hog 1234. Hogs W 1928 and W 1929 were given 5 c. c. each of the same disease-producing blood, but KEEPING QUALITY OP SERUM. 89 TABLE 33. Experiment to test keeping quality of immune sera. Autopsy. Hemorrhagic lesions. Do. ] lemorrhages and ulcers. Ulceration of cecum. NOTI- This ..p.Titn..nt was carrie, hog "1234 by th.' slow method and hog Q No. 2 by the quick method. The sera used in the above experiment were prepared from second drawings of blood. Ten exposure Scribner. . 1 f i M 00 o ~ o 3 Result li Slightly sick Aug recovered. . Remained well. .. Sickened Aug. ti; 12, 190(i. . . Remained well. ...do... c -- 53 8 : c ^ a x ~ -~ -r o _o_c Inoculation. Vn -,f Result. >; died July . i: died .lulv . 2 =: do Sickened .Iune2t 9. 19011. Sifk..n..(l .lune >( Remained \s ...do... T c c -5 1 i i c e "O ~~ ~ c : 5 y i . s s 5? js ... 20 e.c. serum of 1234 and 5 c.c. disease- producing blood of hog 1910. 17 e. c. serum of 1234 and 5c. c. disease- producing blood of hog litlO. . . . Pen check with hogs 1925 and 192ti 20 e. e. serum of Q No. 2 and 5 c. c. disease-producing blood of hog 1910. ** ~ I- 3 ^- c, is 5 4- ^ f ". - O i, 3 I l -j zj i?ja = p ; : : ' f- Zf- Z ' received 20 c. c. each of hyper- immune serum from immune Q No. 2. An untreated hog was placed in each of the two pens with the vaccinated hogs. As controls, 2 hogs (W 1931 and W 1932) were each given 5 c.c. of the same disease -producing blood as was used for injecting the 4 serum-treated hogs. These 2 hogs were placed in a sepa- rate pen. As may be seen from Table 33, none of the 4 hogs that re- ceived the simultaneous injec- tions of serum and disease-pro- ducing blood showed any symp- toms of illness after injection, while both of the hogs that were given the disease - producing blood alone sickened and died of hog cholera. The 4 vaccinated hogs, with their 2 pen checks, were exposed to hog cholera about seven weeks after vacci- nation. As a result of this ex- posure, both check animals died of hog cholera, while 1 of the o vaccinated hogs became slightly sick, but soon recovered. The 3 other vaccinated hogs did not show any signs of illness. This experiment shows that hyperimimine serum preserved with carbolic acid may retain its potency for at least six months. GENERAL SUMMARY OF RE- SULTS. In the experiments of 1905 and 19011, 13 immune hogs (in- cluding hog 1 101) were used for the production of serum, and all of these, with one exception 90 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. (immune Q No. 1, 1905), after hyperimmunization have furnished serum that in doses of 15 c. c. or more gave protection from a simultaneous injection of disease-producing blood. When smaller doses of serum were given, differences in the potency of the various sera could be observed. The doses of immune serum given with disease-producing blood varied from 1\ to 20 c. c., and, as was to be expected, some of the hogs that received the smaller doses were not sufficiently protected. In summarizing the results of these experiments, therefore, the percentage of loss which resulted from vaccination is not to be regarded as representing what would occur in the practical application of the method, for, as already stated, when a sufficient dose of serum is used no deaths are caused by the immunizing treatment. Even though, for the reasons just stated, the average results of our experiments are not to be considered as representing what should be obtained in practice, a summary of these results is not without interest as showing in a general way the beha- vior of serum-treated hogs as compared with unprotected hogs when both are exposed to disease through injections of disease-producing blood. In all, 168 hogs were given a simultaneous injection of serum and disease-producing blood, the dose of serum varying from 1\ c. c. to 20 c. c. Of these, 35, or 21 per cent, showed visible symptoms of illness after injection, and 15, or approximately 9 per cent of the total number, died. As a contrast to this we find that 54 hogs, were injected with the same dose of the same disease-producing blood alone. Of these, 54, or 100 per cent, became sick, and 50, or 92.5 per cent, died of hog cholera. These figures, we believe, show beyond doubt that by hyperimmunizing immunes in the manner described in this paper a highly potent serum may be secured. We are con- fident also, as a result of the experiments we have described, that had we used the larger doses of serum in all cases or else reduced the dose of disease-producing blood the percentage of deaths follow- ing vaccination would have been so small as to be without practical significance. TRANSMISSION OF DISEASE BY HOGS TREATED BY SERTJM- SIMTJLTANEOUS METHOD. To test the possibility of disease being conveyed to normal hogs by hogs that had been vaccinated, 58 shoats were placed in the pens with the vaccinated hogs. Of these pen checks none became sick unless some of the vaccinated hogs with which they associated became severely ill; indeed in a number of instances the pen checks remained well, even though they associated with vaccinated hogs exhibiting undoubted symptoms of hog cholera. From this it appears that the immune serum not only possesses the power to protect hogs DISCUSSION OF RESULTS OF EXPERIMENTS. 91 from a simultaneous injection of disease-producing blood, but that even in cases where too little serum is given it yet has a marked ten- dency to lessen the infectiousness of the disease. Of the 58 pen checks exposed to vaccinated hogs, 6 contracted hog cholera and 5 died. As a control experiment, 19 shoats were placed in the pens with the hogs that were injected with disease-producing blood alone. Of these, all became sick, 17 died, 1 was killed to secure blood for other experiments, and 1 recovered. In other words, of the pen checks exposed to hogs injected with disease-producing blood alone, 94 per cent died, whereas only 8 per cent of the checks exposed to vacci- nated hogs succumbed. IMMUNITY IN HOGS AFTER SERTJM-SIMTJXTANEOTJS VACCINATION. In describing the individual experiments attention has been called to the accidental exposure to hog cholera of those vaccinated hogs that were being reserved for a late exposure in order to test the dura- tion of the immunity conferred by the serum-simultaneous and the serum-alone methods. The pasture in w^hich this accidental outbreak of disease occurred contained a large number of hogs which had been treated at different times, and as a consequence this exposure took place only a few weeks after vaccination in the case of some of the hogs, while in the case of others the interval between vaccination and expo- sure was as great as three months. Aside from the hogs that were acci- dentally exposed in this way, a considerable number were placed in the exposure pen within three weeks after vaccination, the object being to compare the immunity at this time with that exhibited by others treated in the same way but exposed very much later. For the reasons given, this comparison could not be made in all cases, and all results of exposure to hog cholera are therefore considered together in this summary, it being understood that exposure took place from three weeks to three months after vaccination. Of the hogs treated by the serum-simultaneous method, I.'W wore exposed to hog cholera, and 4, or nearly 3 per cent, diod as a result, while of 68 unvaccinated hogs exposed under the same conditions, 5(>, or approximately 82 per cent, died. In regard to the 4 vaccinated hogs that are stated to have died from exposure after vaccination, it should bo noted that there is room for considerable doubt as to the cause of death. In only one of those hogs (1780) were typical lesions of hog cholera, found at autopsy: and as this hog remained unthrifty after vaccination, and as its mato (17M) treated in the same manner died from vaccination, it is not unlikely that the intestinal lesions found at tho autopsy of hog 17X0 resulted from vaccination and not from exposure. Neither of the other three hogs exhibited losions of hog cholera at autopsy, and two of thorn (1552 and 1550) passed through exposure in tho pasture and only sue- 92 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. cumbed when placed in the exposure pen. The conditions in this ex- posure pen were not conducive to thriftiness, for no attempt was made to disinfect or clean the pen, as this might have defeated its object, which was to furnish a certain and severe exposure to hog cholera. Before leaving this subject, we wish to state our conviction that as small as was the loss among vaccinated hogs exposed to hog cholera, this would have been even less had the exposure taken place under the conditions usually met with in practice. IMMUNITY IN HOGS TREATED WITH SERUM ALONE. In all, 58 hogs were treated with serum alone in the experiments of 1906. None of these showed any ill effects from the treatment, and 49 were subsequently exposed to hog cholera. Of these, 23, or 47 per cent, survived exposure, whereas of 68 untreated hogs which served as controls only 18 per cent survived. It is thus evident that the protection afforded by the hyperimmune serum when given alone, while it did not equal that afforded by the serum-simultaneous method, was nevertheless sufficient to protect nearly 50 per cent of the treated animals against subsequent exposure. As regards the duration of the protection afforded by the serum when given alone, we find that of 49 animals treated with serum alone, 26 were exposed within three weeks after receiving the serum, and 23 were exposed after one to three and one-half months. Of the first lot, exposed within three weeks, 9, or 34.6 per cent, died as a result of the exposure, while of the 23 exposed after one to three and one-half months, 17, or 73 per cent, succumbed. From these results it would seem that the protection afforded by the serum when administered alone is not as lasting as when administered in combi- nation with diseased blood. CURATIVE VALUE OF HYPERIMMUNE SERUM. The exact methods of carrying out the experiments has been de- scribed elsewhere. Twelve hogs that were certainly infected with hog cholera were treated with serum after the elapse of different lengths of time, and the results showed that doses of 15 or 20 c. c. were sufficient to save the life of the treated hogs, provided the serum was given within four days after the date of infection. If given later than this, even much larger doses (35 to 50 c. c.) were not suffi- cient to save the infected hogs from the severe exposure to which they were subjected. DISCUSSION OF RESULTS OF EXPERIMENTS. 93 RESULTS WITH SEBUM FROM IMMUNES TREATED BY DIFFERENT METHODS WITH DIFFERENT DISEASE-PRODUCING BLOODS. In the previous summaries simply the general results have been considered, without reference to any differences that may have been found in the action of serum prepared by the different methods or by the use of different strains of disease-producing blood, and also with- out regard to differences in the immunes themselves. In discussing these features we wish to call attention again to the fact that the figures given are not to be taken as representing what may be ex- pected in actual practice, for it is probable that all of the serum would have given better results if the doses had been somewhat larger in certain cases. COMPARISON OF THE -QUICK AND SLOW METHODS OF PRODUCING HYPERIMMUNE SERUM. The comparative potency of the sera prepared by the quick and slow methods may no doubt be best shown by comparing the results obtained by injecting nonimmune hogs simultaneously with serum and a fatal dose of disease-producing blood. In the experiments of 1905 and 1906, 86 hogs were treated in this way with serum pre- pared by the quick method. Of these 86 hogs, 7, or approximately 8 per cent, died from the injection. There were treated with serum prepared by the slow method 82 hogs, all of which were injected at the same time with disease-producing blood. Of these, nearly 10 per cent died as a result of the injection. The slight difference in favor of the quick method we are inclined to attribute to other causes than to the method used for producing the serum, and it seems probable that one method will be found as suitable as the other for producing a potent serum, though of course in either case the potency might be heightened by increasing the dose of disease-producing blood given the immune. The potency of the scrum secured being equal, the quick method would probably be found more desirable for prac- tical use. THE COMPARATIVE POTENCY OF SEKA SECURED HY II Y PERIMMUNIZA- TIOX WITH THE SCRIBNEU AND SYPIIAX STRAINS OF DISEASE. Serum from 8 hogs was used, 4 having been hyperi minimized with Scribner disease-producing blood, and 1 with Svplmx Mood. In all, 74 hogs were treated with scrum from the Scribner immunes at the same time that they were injected with disease-producing blood: as a result, 4, or approximately . r >l per cent, died. In the case of the serum from the Syphax immunes, 7'J hogs were given disease- producing blood simultaneously with serum, and, as a result, 8, or II Ui include." the hop." treated with scrum from immune 1-101. 94 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. per cent, died. The apparent lower potency of the Syphax hyper- immune serum is caused by the distinctly weaker power of the serum from 1 hog (1310), a dose of 10 c. c. of this serum being too small to protect nonimmunes, although larger doses were entirely satis- factory. Inasmuch as the average potency of the serum from the other 3 immunes hyperimmunized with the Syphax blood was quite as great as that of those treated with the Scribner blood, we are inclined to believe that the lower potency of serum from hog 1310 was due to some peculiarity of this hog and not to a lower hyperimmunizing power of the Syphax blood. COMPARATIVE POTENCY OF SERA FROM HOGS POSSESSING NATURAL IMMUNITY AND FROM THOSE POSSESSING ACQUIRED IMMUNITY. Attention has already been directed to the difficulty experienced in deciding whether a particular animal should be classed as one possess- ing acquired immunity or natural immunity. In fact the whole ques- tion seems to be simply one of degree of resisting power on the part of the hog. In any outbreak of hog cholera it is probable that at least a few hogs will survive. Some of these may have been markedly sick and have recovered only after a long siege of the disease, while others may have been only slightly indisposed, and still others may not have exhibited visible symptoms of disease. It seems hardly cor- rect to speak thereafter of one of these hogs as a natural immune, and to regard another as having acquired immunity through the attack of disease. We are rather inclined, in the light of our present knowledge of -the disease, to regard all hogs as being naturally susceptible but varying greatly in the degree of resisting power which they possess. If this assumption be correct, it would be expected that a protective serum might be obtained by the hyperimmunization of any immune hog, the differences in the serum from different hogs being simply in degree of potency and depending probably upon the intensity of the reaction produced by the injection of diseased blood. In actual practice we have been able to secure but one immune that had actually been sick of hog cholera that was vigorous and suitable for hyperimmunization. The serum from this immune (1392), as the records show, is not more potent than that obtained by similar meth- ods of hyperimmunization from other hogs that had never shown visible symptoms of illness when exposed to hog cholera. In fact the sera from the different immunes, with the exception of hogsQ No. 2 and No. 1310, were quite uniform in potency. The lesser potency of the sera from the two hogs just mentioned can not be explained upon the ground of natural immunity, but probably was caused by some individual peculiarity which we do not understand. CONCLUSIONS. 95 The remaining experiments, not mentioned in this general summary, have already been discussed at some length and it seems unnecessary to refer to them further at this time. Before closing this discussion, however, we wish to suggest to anyone who may have occasion to repeat these experiments that it is essential to make sure that the outbreak from which blood is secured for hyperimmunization exhib- its all of the characteristics of hog cholera" and likewise that the blood used in conjunction with the serum for serum-simultaneous vaccination is taken from a hog suffering from undoubted hog cholera. In addition, more than one hog should be hyperimmunized in order that individual peculiarity on the part of one hog may not lead to erroneous conclusions. As the methods herein described involve the use of a disease-producing virus we believe that only those trained in bacteriological methods should attempt to use them, and in all cases before the serum is employed in a practical way the amount required to give protection from a fatal dose of disease-producing blood should be determined experimentally. CONCLUSIONS. (1) When hogs immune from hog cholera are injected with suitable amounts of virulent blood taken from hogs sick of hog cholera the blood serum of the immunes acquires the power to protect nonim- mune hogs from an otherwise fatal dose of disease-producing blood administered simultaneously with the serum. (2) Immunes which have never exhibited symptoms of disease after exposure to hog cholera (natural immunity) may furnish equally as potent serum as those which have recovered from an at- tack of the disease (acquired immunity). (3) Hyperimmunization may be accomplished equally as well by the administration of one large dose of disease-producing blood us by repeated injections of smaller doses. (4) Hyperimmunization may be accomplished with blood from any virulent strain of hog cholera. (5) Hyperimmunized hogs probably retain for several months the power to furnish a potent serum. ((>) Serum from properly hyperiminunized hogs in doses of iM) c. <-. should protect noniinniunes weighing from "J") to .">() pounds from a fatal dose of disease-producing blood administered simultaneously. (7) Immunity in hogs treated by the serum-simultaneous method lasts at least three and one-half months and probably longer. (X) In serum-simultaneous vaccination it is not necessary to pro- duce illness in the vaccinated hog in order to secure an immunity last ing for at least three months. oSc'f Bulletin 7'J, Bumiu nf Animal Industry, p. 1-. 96 PRODUCTION OF IMMUNITY FROM HOG CHOLERA. (9) If a sufficient dose of serum is given, hogs are not injured by the serum-simultaneous vaccination. (10) Hogs vaccinated by the serum-simultaneous method do not communicate disease to others unless they themselves become sick. (11) Serum administered alone may not furnish complete protec- tion for a longer period than three weeks. (12) The serum probably can be used successfully as a curative agent if administered in the early stages of the disease. O