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M- - -'—__.._.‘_.“ M M: - ”a“: 101'? : 1'! a” 'fl'hfnj‘d‘}. ’1. . {I y IIIII ' II111.:.‘LII/IMIIIIII'I’IrIfIILI;fI/l III/1h JIYIJIIIMIIIILE ”W M W5 \ bmomc av "' HDAG & SONS' BOOK BINDERY LIBRARY amoans mm Ion, mm“. ‘2 A 5" A Study on the Transmission of Bacterium abortus (Bang) thru Milk. THESIS. Submitted to the faculty of the lichigan Agricultural College in partial fulfillment of the requirements for the degree of Lester of Science. I. Forest Huddleson. August, 1916. W 1 2 ,. ~ a ’. / (t - $544. is“, (at 01 (j .1 U A Study on the Transmission of Bactegigg gbgrtus (Bang) thru Milk. Introduction. Review of Literature. method of Investigation. Experimental Work. The Result of Feeding Infected Lilk to Guinea Pigs. The Result of Feeding Infected flilk to Rabbi t8 0 The Result of Feeding Infected kilk to Hen-Born Calves. General Discussion. Summary. Acknowledgement. References Cited. Plates. 1992135 A Study on the Transmission of Bacterium gbortus (Bang) Thru Milk. Introduction. numerous investigations have shown the infec- tiousness for animals of milk containing pathogenic cr- ganisms. The milk may become infected indirectly thru unsanitary conditions and thru human carriers of disease producing microorganisms. The milk may become infected directly thru infections of the udder, tuberculosis and mammitis being two common infections of the udder in which the organisms may be excreted continually or occasionally in the milk thruout the infection. The investigations relative to the transmission of Bactezigg gbogtug thru milk are as yet in an experimenufl. stage. Very little study has been completed, but numerous suggestions have been made as to the possibility of infec- tious abortion being acquired in this manner. The researches regarding the transmission of Bgcterigm gbggtgg,thru milk have been confined largely to animal inoculation, to observa- tions and to a large collection of clinical evidence. The possibility of infectious abortion being acquired by animals thru the ingestion of raw milk was first suggested by Williams (1). His researches are, however, very incomplete.§ Consider- ing the economic importance of the control of infectious abortion, an eXperimental study of the numerous observa— tions and of the clinical evidence now on record covering the infectiousness of raw milk would aid materially in solving one of the modes of transmitting ggpterigm abortus. Historic Review. Researches concerning the presence of pathogenic organisms in milk and the possibility of a disease result- ing therefrom have been extensive. Fisher(2). many years she, reported a paratyphoid epidemic due to infectedmilk, two cows of a herd supplying the milk were suffering from enteritis. Anderson(5) found the tubercle bacilli in 11 percent of the dairies supplying milk to Washington, D. C. Hess(4) likewise isolated the human type of tubercle bacilli from the milk supplying the city of New York. Griffith(5) making a study of the milk of cows and goats, vaccinated subcutaneously and intraven- ously with live tubercle bacilli. showed by means of guinea pig inoculations that the bacilli, bovine or human, not only made its appearance in the milk 24 hours after inocu- lation, but continued to be excreted therein for long periods subsequently. Capp and Davis(6) reported a septic sore throat epidemic due to milk from a herd of 15 cows, 3 of which were affected with mastitis. That Bacterium abortus is present in the milk from apparently normal cows and is excreted therein for long periomg was first demonstrated by Schroeder and Cotton(7) in 1911. After an extensive examination of a large number of samples of milk, the abortion bacterium.was isolated by cultural methods and identified by means of the complement-fixation test. The most noteworthy feature of their work, was the demonstration of lesions in guinea pigs inoculated intra- abdominally with samples of the milk identical to those produced by a culture of Bactegium abortus inoculated in the same manner. Zwick and Krage(8) successfully demonstrated the presence of Bgcterium abortus in the milk of cows fourteen days, six months and thirteen months after abortion. Fabyan (9) likewise demonstrated the presence of Bagtggium abortue in the cream and milk sediment of the milk from a herd of twelve cows, two of which had previously aborted, by means of guinea pig inoculation. The animals inoculated with material from the cows which had records of abortion revealed characteristic lesions, the other guinea pigs showed no histological changes. Larson and Sedgwick(10) examining the blood serum of four hundred and twenty-five children demon- strated the presence of Bgcterigg gbortus antibodies in seventy-three, or seventeen percent of the total number examined. They were not able to demonstrate antibodies in milk, so concluded that probably the antibodies present in the blood serum of the children were due to an active im- munity. Schroeder(ll) feeding raw, pasteurized and heated milk to young guinea pigs, found that there was a greater mortality and tendency to unthriftiness among the animals fed raw milk, than among those fed pasteurized or heated milk. At the end of the experiment an autOpsy was performed on the entire number fed, many animals receiving raw milk showed lesions characteristic of abortion disease. The first investigators to demonstrate the presence of fiagtggigg gbgrtus antibodies in milk. were Reinhard and Gauss(12). They were able to show the relation between the antibody index of the blood serum and that of the milk serum by goats inoculated subcutaneously and intravenously and testing same by means of the agglutination and complement-fixation test for long per- iods subsequently. Their results showed that there was a direct relation between the antibody indices of the two serums, but antibodies could be demonstrated to be present in the milk long after they had ceased to appear in the blood. The studies of Evans(13) on the bacterial flora of. the udder revealed Easteriflg aboxtug in 50 percent of the milk samples examined from four dairies in numbers varying from.one hundred to fifty thousand per cubic centimeter. In a recent investigation(l4) she examined one hundred and ninetyatwo samples of milk from five dairies. A Bacterigg abortug-like organism was isolated from twenty-three percent of the samples. Three different types of organisms were distinguished, a variety designated as Esoterium abortus var. lipolytious on account of its tendency to decompose butter fat. Two other varieties resembled cultures isolated from pathogenic sourcea The observations and researches of hilliams(15) have develOped a new phase of the subject which is of economic importance from the standpoint of controlling the disease, that is, the possibility of an infection being acquired thru the ingestion of raw milk. He firmly contends from his researches that Bacterium abortug is transmitted to calves fed upon raw milk, also that the matting and staining of the sexual hairs and granular vaginitis are in direct relation to the infection. Cooledge(16) has successfully shown the difference existing between infected milk and non-infected milk by means of the agglutination test. He further showed that by inoculating one quarter of the udder of a cow through the teat orifice with a culture of Bacterium abortus, agglu- tinins appeared in the quarter inoculated and gradually spread to the other quarters of the udder. Method of Investigation. The literature reviewed has not revealed any definite results as to the possibility of transmitting Eggt. abortus to animals thru the ingestion of milk. With this object in view, experiments were outlined in such a manner with susceptible animals so as to procure more complete data and to show the relationship between the feeding of infected milk and non-infected milk. The experiments were outlined in the following manner: (1) The result of feeding infected milk to guinea pigs; (2) the result of feeding infected milk to rabbits; (3) the result of feeding infected milk to calves; (4) the significance of the matting of the sexual hairs around the sheath of the bull and that below the vulva of heifer calves. The term infected milk upon which the study is based implies milk reacting positively to the agglutination and complement fixation tests using Egg}. gbortus as antigen. The milk was fed to the guinea pigs and rabbits daily, and to the calves twice daily thruout the eXperiment. Every precaution was taken to guard against an infection from an outside source. The milking utensils were thoroughly sterilized, and the udder and tests cleansed with a damp cloth before each milking. The effect of the infected milk fed was studied by means of agglutination and complement-fixation tests on the blood serum of the animals, using gggt. abortgs as antigen. The guinea pigs were fed for a period of several weeks and then autopsied. The blood serum was tested and any anatomical changes noted. The rabbits were bled from the marginal vein of the ear or from the heart at intervals of about two seeks and the blood serum tested. They were autOpsied at the conclusion of the eXperiment and any anatomical changes noted. The calves were bled from the Jugular vein at intervals of about one week and the blood serum tested. Observation and microsc0pic examination of the sexual hairs of the calves were made during the course of the feeding. .7.- Technique Employed. The Complement Fixation Test. The application of the complement fixation test to the diagnosis of infectious abortion has been fully dis- cussed by Hadley and Beach(l7L Surface(18) and many others. The technique used thruout this investigation is a slight modification of the previously described methods for the preparation of the different components and the quantities used. The writer has found that by using a 2.. percent solution of sheep erythrocytes, more satisfactory results could be obtained than by using either a 1 percent or a 5 percent solution as is generally used by most workers. A much better differentiation between partial and complete fixation of complement is obtained over that of a 1 percent solution, while using a 6 percent solution of cells would require the use of a larger quantity of the other necessary components. The following components are necessary in making the complement fixation test. (1) Antigen. (2) Serum to be tested. (3) Complement. (4) hemolysin. (5) Erythrocytes. (6) Sterile physiological salt solution (.Qfl). Preparation of Antigen. The antigen was prepared by growing a culture of 2523, abortug for 41hours upon plain agar. The growth was then washed off with a solution containing phenol (.5 percent) and sodium chloride (.9 percent) and filtered thru cotton. The suspension was then standsrdized so that the turbidity corresponded to tube 5 of KcFarland's Nephelometer (19). The Antigen if kept at ice box temperature will retain the same titer for many months. Preparation of Serum to be Tested. The manner in which the serum to be tested was prepared differed according to the different animals tested and the nature of the serum, as blood serum and milk serum both were examined during the eXperiment. Guinea pigs: The blood serum from the guinea pigs was obtained by slightly anesthetising the animal, clipping the hair directly over the sternum. making a diagonal incision about one~half inch long in the skin covering the sternum. severing the sternum with a pair of small dissecting scissors, at the same time extending them forward far enough to sever the chambers of the heart completely. The blood is then collected in a sterile 25 cubic centimeter centrifuge tube, and the serum allowed to separate from the clot. The clot is then centrifuged and the serum pipetted off and inactivated at 56°C. for thirty minutes. This last Operation is very important on account of the complementary preperties of the serum. many workers do -9- not retard inactivation of importance if the serum stands any length of time before testing. A serum will lose a certain amount of its complementary properties on standing for any length of time, but sufficient complement often remains in the serum to produce aberrant results. After inactivation. the serum is preserved by adding .05 of a cubic centimeter of a 20 percent solution of phenol per 5 cubic centimeters of serum. Rabbits: The rabbits were bled from the marginal vein of the ear or from the heart by means of a small hypo- dermic needle. When bleeding from the ear. xylol should be used to dilate the blood vessels. This may be done by saturating a small piece of cotton with xylol and.massaging the ear gently until the vessels present a dilated appearance. The vessels will remain dilated for a considerable length of time. The xylol will not injure the ear in any way if removed with a damp cloth When the operation is finished. Obtaining blood from the heart is not a complicated Operation. The animal should first be firmly fastened on its back to an animal board, the heigwggfipped closely from over the left side of the thorax ribs, the spot disinfected with 5 percent phenol and a small hypodermic needle inserted into the left ventricle. Anesthesia is not necessary as the animal suffers no pain whatever, and only a few seconds are required to obtain sufficient blood. The blood is cellected in a sterile test tube and allowed to clot. The clot is centrifuged and the serum pipetted off, inactivated and preserved in the same manner as described for the guinea pigs serum. -10- Calveet- The blood of the calves to be tested was obtained by bleeding from the jugular vein by'meune of a large hypodermic needle. The bleeding may be facilitated by dilating the vein. The thumb pressed firmly below the point of puncture will cause a dilation of the vein. The blood is collected in e aterile centrifuge tube,allowed to leparate from the clot, centrifuged, pipetted off, inactivated and preserved in the some manner as described for the guinea pigs serum. The milk serum used in the test was prepared by coagulating milk by adding .01 cubic centimeters of rennet per 10 cubic centimeters of whole milk. The milk was then placed in a water bath at a temperature of 40°C. About forty-five minutes is sufficient to completely coagulate the milk and for the separation of the serum. The milk should be about two degrees agig?r:ifgrwise a considerable length of time is required for the separation of the serum. Ac eoon as the serum is sufiiciently separated from the clot, it is filtered thru filter paper to remove any material which would have a tendency to produce a cloudy serum. It is not neces- sary to inactivate the milk serum as Lane-Claypon(20) has shown that there is very little complement in milk. Two cubic centimeters in the smallest quantity in which complement can be demonstrated. If the milk sermn is to be retained for any length of time it is advisable to add a few drops of 20 percent phenol to prevent microbial growth. .11. Preparation of Complement. The guinea pig produces the most potent complement, therefore the blood serum of this animal was used as comple- ment. The animal was bled as in the previously described method for bleeding guinea pigs, or by bleeding from the heart without killing or anesthetising the animal. This operation was performed in the following manner; The animal was firmly fastened on its back to an animal board, a small space over the left thorax clipped free from hair, the space disinfected with 5 percent phenol and a small hypodermic needle inserted between the fourth and fifth rib into the heart. If the needle enters the left side of the heart the blood will flow from the needle very rapidly. If the right side of the heart is entered instead, the blood flow will not be rapid, but by drape. Six cubic centimeters of blood may be taken from a large guinea pig weekly without the slightest discomfort or injury to the animal. In order to obtain sufficient complement from a large number of tests, the writer used the pooled blood of three guinea pigs. The blood was collected in a sterile centrifuge tube and the serum allowed to separate. The writer has obtained a more potent complement by bleeding the animal and allowing the blood to stand in the ice box from twenty to twenty-four hours before the serum is separated from the clot for use. Complement serum should always be kept at a very low tempera- ture. Its strength diminishes very fast when allowed to remain for a short time at room temperature. Preparation of Sheep Erythrocytes. The blood is drawn from the Jugular vein of a sheep by means of a large hypodermic needle. The blood is collected in a sterile Erlenmeyer flask containing small pieces of glass. The flask is shaken vigorously for several minutes in order to defibrinate the blood. The blood is then strained thru sterile gauze into a large centrifuge tube to remove the glass and fibrin and centrifugalized for several minutes in order to separate the cells from the serum. The supernatant liquid is syphoned off, and physiological salt solution (.9 percent) added and again centrifugslized. This washing is repeated three times in order to completely wash out all remaining serum from the erythrocytes. A 2.5 percent solution of erythrocytes is used in all the titrations. Preparation of Hemolysin. lhe heaolysin was prepared by either the slow or the quick method. The slow method consists in injecting intraperitoneally into a rabbit 8, 12 and 15 cubic centi- meters of concentrated corpusclec reapectively at intervals of five days. Five days after the last injection the blood is tested for its hemolytic property. The quick method is most desirable as it involves less time and a smaller quantity of corpuscles. The quick method consiSts in three intravenous injections into the marginal vein of a rabbit of two cubic centimeters of concentrated corpuscles at intervals of three - 15 - days. Three days after the last injection the rabbit is bled from the marginal vein of the ear, and the blood serum obtained tested for its hemolytic properties according to the method described in Table II. If .001 of a cubic centi- meter will completely hemolyze .5 cubic centimeters of a 2.5 percent suSpension of sheep erythrocytes in 30 minutes, the serum is considered satisfactory for use. The rabbit may now beslightly anesthetised and bled from the heart. The blood is collected in a deep culture dish and the serum allowed to separate from the clot. The serum is collected in small vials, inactivated at 56°C. for 30 minutes, preserved by adding .05 cubic centimeters of phenol per 5 cubic centi- meters of serum and sealed with paraffin. A hemolytic serum prepared in this manner and kept in a dark cool place will retain the some titre for many months. Titration of Components. The serum obtained from the guinea pig by the previously described method is diluted 1:3 with sterile physiological salt solution (.9 percent). The mixture is then titrated to determine the smallest quantity necessary to completely hemolyse .5 cubic centimeters of a 2.5 percent solution of sheep erythrocytes mixed with a constant quantity of hemolysin maintained in a water bath at 37°C. for 30 min- utes. The amount of complement required to cause complete hemolysis is not constant with all guinea pigs, but usually .05 cubic centimeters of the dilute serum is sufficient. In the final test, two times the original titer was used. This - 14 - is necessary because the antigen sbsorbs s small amount d complement. which leads to sbsrrsnt rlsults. The titration of the complement is shown in stls 1. \ Table I. 8hssp Tubs Complement Bomolysin HaCl Erythrocytes Results Ho. Dil. 1:5? 1% .9% 2.5% 1 .1 cc. 5 x :5” 1.5 cc. .5 cc. ,1 Homolysis 2 .06 cc. '5 x t. 1.5 cc. .5 cc. I ' 5 .05 cc. 5 x t. 1.5 cc. .5 cc. f“ ' 4 .04 cc. 3 x t. 1.5 cc. "5 cc. 3 No Hemolysis 5 .1 cc. 1.5 cc. .5 cc. 3 ” ' 6 .5cc. 1.5 cc. .5 cc. 3 ' ' 7 1.5 cc. .5 cc. g “ ' H Is) t. . the titer found on titration. From tho tsble it may be soon that .05 cubic cents» meters is the smallest quantity csusing complete hemolysis. Two times this amount is used in the finsl test. Tube 5 is a control to show that the complement scrum will not causs hemolysis. Tube 6 is s control to show that tho hemolytic serum will not cause hemolysis. Tube 7 is s control to show that the salt solution will not cause homolysis. Thsss con- trols are very essential so as to avoid any possible error. Titration of hemolysin. It is necessary to know the smsllsst quantity of hemolysin that will complstsly homolyso .5 cubic centimeters of a 2.5 percent solution of sheep erythrocytes mixed with a constant quantity of complement. The hemolysin prepared in the previously described . 15 - manner is prepared in a 1 percent physiological salt solutien for titration. The titration of the hemolysin is shown in Table II. Table II. fim Sheep Tube Hemolysin Complement HaCl Sol. Erythro- Results. No. 1% D11. 1:3 .9% cytes l .1 cc. 2 1 t3“) 1.5 cc. .5 cc. € Hemolysis 2 005 cc. 2 I to 105 CC. 05 Cc. "u‘\‘ it 5 .04 cc. 2 x t. 1.5 cc. .5 cc. g‘ ' 4 .03 cc. 2 x t. 1.5 cc. .5 cc. g No Hemolylis 5 .2 cc. 1.5 cc. .5 cc. ; ' ” (a) t. . the titer found on titration. In the above table .04 is the smallest quantity which will give complete hemolysis. Tube 5 is a control to show that a large amount of hemolysin will not itself cause hemolysis. In the final test it was necessary to employ three times the original titer to insure a sufficient quantity for the solution of the corpuscles. Titration of Antigen. 'g-VAQ;J$_ Before performing the final test it is necessary to know the smallest quantity of antigen required to fix complement in the presence of a specific antibody. Since the antigen, when used in large quantities has the preperty of absorbing complement, it is necessary to know the smallest Quantity that will absorb complement in the absence of a specific antibody. In titrating the antigen, serum containing specific antibodies must be employed. This Specific serum may be obtained - 15 . by injecting a rabbit intraperitoneally with 5 cubic centi- meters of a 48 hour old bouillon culture of 333;. abortge. Two injections are given at intervals of five days. Five days after the last injection the rabbit is anesthetised and bled from the heart, the blood collected in a deep culture dish and the serum allowed to separate from the clot. The serum is inactivated at 56°C. for 50 minutes, preserved with phenol (.05 cc. of s 20 percent solution per 5 cc. of serum) placed in Small vials and sealed with paraffin. An immune serum prepared in this manner and kept in a dark cool place will retain the same titer for many months. The titration of antigen is shown in Table III. Table III. ‘§hsep Tube Immune Comple- NaCl Hemoly- Erythro- No. Antigen serum ment .9% sin 1% cytes Results ' 107.3 Di 1 .035) 2 . 523 I V i [ l .01 cc. .1 cc. 2 x t.(a) 1.5cc.: a ‘3 3: t5“) .5 cc. ‘5 .IoaHemoljeie 2 .02 cc. .1 cc. 2 x t. 1.5cc. g 5 x t. .5 co. 3 .p” " a .05 cc. .1 cc. 2 x t. 1.5cc. ‘.3 x t. .5 cc. 0 9N0 " 4 .1 cc. .1 cc. 2 x t. 1.5cc. g x t. .5 cc. g Nwl . “ 5 .1 cc. 2 x t. 1.5cc. ”5 x t. .5 cc. Q E>hemolysis 6 .01 cc. 2 x t. l.5cc.- 5 x t. .5 cc. 5 Q " 7 .02 cc. 2 x t. 1.5cc. i5 x t. .5 cc. 3 fl ” 8 .05 cc. 2 x t. 1.5cc. 5 x t. .5 cc. % m ' 9 .1 cc. 2 x t. 1.5cc. 5 x t. .5 ch ? ' 10 .5 cc. 2 x t. 1.5cc. a x t. .5 cc. j iInc.” (a) t. e the titer found on titration. 1n the above table tube 5 .h... the smallest quantity of antigen that will fix complement in the presence of a specific antibody.‘ Tube 541s a controltoshow that the immune serum will not prevent hemolysis in the absence of the antigen. Tubes from 6 to 10 inclusive show the amount of antigen that may be -17. employed without causing absorption of complement. Tube 10 shows the smallest amount of antigen that will absorb comple- ment in the absence of the specific antibody. The writer has obtained satisfactory results in the final test by using one-fourth the quantity of antigen necessary to absorb complement. The Complement Fixation Test. The object of the complement fixation test is to determine the presence of a specific antibody in s given serum and to what concentration it may be present. The outline followed in the complement fixation test is shown in Table IV. Table IV. gheep Tubs Serum Compls- Hemolyw Erythro- No. tested Antigsga msnt ' HsCl sin 1% cytes Results Dil.(l:5) .es » 2.5% 7’ I Z» _ “f A; _ l .1 cc. .1 cc. 2 x is? 1.5cc. ~ 3 x t‘?) .5 co. ° HoHssolysis 2 .04 cc. .1 cc. 2 x t. 1.5cc.. 3 x t. ,5 cc. - ' ' 5 .02 cc. .1 co. 2 x t. 1.5cc. {5 x t. .5 co. . Inc. ' 4 .005oc. .1 cc. 2 x t. 1.5cc. 3 x t. .5 cc. Hemolysis <5. .1 cc. .’ 2 x t. 1.5cc. 5 x t. .5 cc. ' at» .1 cc. 2 x t. 1.5cc. .5 cc. No ' (I) Add l/I complement Absorption Titer. (b) Add sheep cells on first incubation. (c) t. s the titer found on titration. In the above table, tube 2 shows the smallest quantity of serum which will completely fix complement. A number of serums, especially rabbit serum have been found to inhibit hemolysis in the absence of the antigen. - 18 - Tube 5 is a control to show that the serum itself will not inhibit hemolysis. Tube 6 is a control to show that the serum being tested will not cause hemolysis in the absence of the antigen and the hemolysin. The Agglutination Test. In order to determine if there was any possible . relation between the lytic antibodies and the agglutinating antibodies in a sermn, the agglutination test was used in conjunction with the complement fixation test. Preparation of Antigen. 0 The antigen was prepared in the some manner as in the complement fixation test. The suspension was prepared so that the turbidity compared with tube 1.5 of McFarland's Rephelameter. Four cubic centimeters of the bacterial sus- pension is placed in each of the small test tubes used and the following quantities of blood serum or milk added; .1, .05, .025, .01, and .005. From these approximate dilutions of 1:50, 1:100, 1:200, izsou and lleJO were obtained. The turbidity of the milk ddes not interfere with the readings of the final results. EXperimental work. (1) The result of feeding infected milk to guinea pigs. The susceptibility of guinea pigs to infectious abortion has been demonstrated by nmnerous investigators, - 19 - chiefly by the inoculation route. Schroeder(19) states in a discussion of a paper read before the American Association of medical milk Commis- sion that he and Cotton have not only produced the abortion disease in guinea pigs by injecting them with milk, but have successfully caused the disease in guinea pigs by feeding them seemingly normal milk from apparentlyflggns that had become chronic carriers of the abortion bacillus. Table 5 shows the result of feeding infected milk to eighteen normal female guinea pigs, {gfiizhing-six controls, two of which were fed upon non-infected milk, two upon non- infected milk plus five cubic centimeters of a 48 hour bouilla1 culture of Eggt. abortus and two received no milk. A.milk having a high antibody index was fed daily to the eighteen guinea pigs. The milk was tested every fourteen days to determine its antibody content. The lesions which were found present in the liver of the guinea pigs when autOpsied cannot be said to be char- acteristic lesions caused by @333. abortus. Lany of the guinea pigs which were killed for complement sermn during the past year showed the same kind of pathological changes in the liver. The pathological changes in the liver consisted of irregular brownish yellow or as from 1 to 5 mm. in area scattered over the surface. There seemed to be no uniformity in size of the lesions. She size of the liver did not seem to be affected. The histological changes consisted of small focal necrotic areas scattered diffusely thruout the tissue. The areas were filled with polynuclear and endothelial cells. lhere was almost a complete atrophy of the surrounding liver cells. Tabl. 5. Showing the result of feeding infected milk to guinea pigs. H0.Days No. of feed- Reaction of Blood Serum GIG. ing 111- Result of Autopsy lut nation Com . Fix. Pig fected .l .25 .05 .01.005 .1 .04 .02.005 w: A Spleen slightly enlarged. l 58 many lesions in liver. - . - - - - - - - Few scattered 2(e)287 lesions in liver. - - - - - . - - - kany scattered 3(b)287 lesions in liver. - - - - - - - - - 4 38 Died Of pnfiumonia. - O O G D O - c - 5 38 Died of pneumonia. - - - - . - - - - 6 48 Died of pneumonia. - - - - - - - . - 7 80 Ho anatomical changes. + P - - . + + - - 8 124 Re anatomical changes. - - - - - - - - - 9 96 Ho anatomical changes. - - - - - - - - - iany scattered 10(0) 60 lesions in liver. - - - - - - - - - Lany scattered ll 33 lesions in liver. - - - - - - - - - 12 33 Ho anatomical changes. - - - s - - - - - Lany scattered 13 73 lesions in liver. . - - - - - . . - Few scattered 14 73 18810118 in liver. 0 a n n a o o q . hany scattered 15 9b lesions in liver. - — - - - - - - - Few scattered 16 95 lesions in liver. - - ~ - - . - - - many scattered 17 95 lesions in liver. - - - - - - - - - 18 95 Ho anatomical changes. - - - - - - - - - Controls red Ion-Infected Milk. 1 98 Ho anatomical changes. - - - - - - - - 2 98 he anatomical changes. - - - - - . - - - 3 Fedrnuim: No anatomical changes. - - - - - - - - 4 ” ” ” Ho anatomical changes. - - - - - - - - - 5 60 Fed non-infect- ed milk plus cult Beet. abortus. No anatomical changes. + + - - - + + - - Do 60 No anatomical changes .+ + + - - + + + - (a7 Gave birth to one live and two dead fetus on the 109th 63y of feifi (b)Gave birth to three live and one dead fetus on the 266th dEZe Sing (0) Aborted two fetus on 59th day of feeding. - 21 . One abortion and two abnormal births oooured during the time of feeding of infected milk. Guinea pig Ho. 10 aborted two fetus on the 59th day of feeding. Pig ha. 2 gave birth to one live and two still births on the 109th day of feeding. Big 80. 3 gave birth to three live and one still birth on the 266th day of feeding. Two or eleven percent of the guinea pigs viiich were fed upon infected milk develOped Beet. abertus anti- bodies in the blood during the time of feeding The blood serum of guinea pig Ho. 7 gave a positive reaction at the time of eutOpsy. The blood serum of he. 3 gave a positive reaction eight days before autopsy just after parturition. Crtt’ o—f The blood was negative Whggmsutopsgyé" ‘(2) The result of feeding infected milk to rabbits. That rabbits are susceptible to the abortion infec- tion has been demonstrated by Bang(21), Nowak(22) and thlsr and Traum(23). This fact has been demonstrated only by in— oculation eXperimente. No data are available pertaining to infections being acquired orally by rabbits thru the ingestion Merit .§“'§‘;7__;§f abortus itself. Fourteen normal females pregnant and non-pregnant were selected for the CXperiment. The effect of infected milk upon rabbits after continued ingestion was studied by means of agglutination and complement fixation tests on the blood serum. Tne frequency of abortions during pregnancy was also observed. Teble 6 shows the result of feeding infected milk to fourteen normal rabbits ingzidieg four controls, two of which were fed upon non-infected milk, the remaining two . _ . _ _ _ . . . . . . . _ , . . . . . . a . . . . . received no milk for a period of one hundred and twenty-four days. A milk having.a high antibody index was fed daily to the fourteen rabbits. The milk was tested every fourteen days to determine its antibody content. The reactions and partial reactions shown in the table were that to be due to a passive immunity. In order to clear up this point, four of the same rabbits as shown in Table 6 were fed no milk for a period of seventy four days. tests being made on the blood serum every two weeks. From the data, Table 7, one may easily see that there is very little difference in the blood reaction after feeding no milk and that during the feeding of infected milk. Rabbit so. 7 developed a positive reaction after being fed upon infected milk for 124 days. Tie reaction seemed to rennin fairly constant after feeding of milk was discontinued. The milk controls. (rabbits fed upon non-infected milk) and controls that were fed no milk snow the same reac- tions and partial reactions as thos rabbits fed infected milk. The reactions and partial reactions seem to be non- specific. It is a well known fact that old serum or serum containing an excess of hemoglobin often contains antilytic bodies, this could not have been the cause of the non-specific action of the above serums as they were separated from the clot in 8 clear state, inactivated, and titrated twenty-four hours after being drawn. Anticomnlemsntsry bodies cannot be con- sidered, since there was no inhibition of hemolysis in control tubes containing serum and antigen controls. A conclusion may ”either specific antibodies . V be crawn in two different ways, were present, or an absorption of complement occured due to the union of a substance in the serun with the antigen and complement. I to «'5 I In order to determine if the non-specific reaction was due to the presence of Specific antibodies, three normal Tb tonyp'Uuzbt” serums which were titrated and found to fix complement.snd antigen were incubated for two hours. The mixture was than ccntrifugelized and the clear serum drawn off and tested. The serums possessed the complement fixing preperties in the same dilutions as before. sonospecific reactions of serum have been studied with particular care by hoguchi(24) and to them he has given the name of proteotrOpic or false complement fixing bodies. holmer and Irist(25) found non-specific substances present in the blood serum of rabbits and dogs against bac- terial antigens such as staphldcocci, colon and typfioidbmdufi Table 8 shows the result of feeding non-infected milk plus a. culture of 31.33;. goggw to six normal rabbits. The rabbits developed antibodies is a result of feeding the mixture. The antibodies are not persistent. They gradually deveIOp to a maximum, remain for a short time and gradually disappear while the mixture is continually being fed. ho anatomical changes occured in any of the rabbits Cl'Ft‘G-r’ eutOpsied during, or si_thewtiee the feeding of infected milk or non-infected milk plus s culture of gagt. sbortus.was discontinued. Lech of the rabbits used were bred one or more times during the feeding of infected milk or non-infected milk plus a culture of gggt. gbgrtus. Ho abortions occured in any of m {3) The result of feeding infected milk to new- born calves. That calves may acquire an infection thru the ingatlm) of milk has neither been definitely proven to occur, nor has it bee proven not to occur. The possibility of a calf acquiring an infection in utere has been given very little consideration in previous researches upon the subject. The literature reveals no im- portent esperimental data concerning infection in utero. In order to obtein unquestionable experimental data, calves (excepting those fed non-infected milk) were separated from their reopective dang shortly after bir h in order to prevent a possible infection fr0m infectious material which might be deposited on the surface of the udder or test. The infectious materiel being ingested at the time of sucking. The calves were bled from the jugular vein and the serum test ‘ . .WGS mode tba. for the presence of fleet. ggprtus antibodies before rEceivflni any milk whatever. Six calves were fed upon naturally infected milk and six calves were fed upon non-infected hilk. The calves fed upon non-infected milk were not separated from their respective da.s after birth due to unavoidable circumstances. They were allowed to retain and suck their dame for three days and then separated. a control was fed pasteurized natarslly infected milk in order to compare the results-with those fed infected milk. A scound CanLPJI was fed nos-infected milk plus 5 cubic céntimeters of a 48 hour old bouillon culture of gggt..§gggtgg with each fecdinb in order to cougars the effect of a naturally infectcd milk witn that of an artificially infected milk. One gallon of the milk was fed twice duly thruout the exPeriment. As accurately es'possible, the history of each dam was obtained, 32’;or.'in._3t the nu-rber of hertz-l pcrturitions, the number of abortions aid the reaction of the blood serum to the a 5glutinatio.1 and complehent fixation tests at the time of the past parturition or abortion. Table 9 shows the history of the dams of the calves used in the experiment. The blood of coa 995 was negative up to the time of calving. Two days after calving, the blood and milk both became positive and have remained positive up to the time of this M? ting. Guinea gigs inoc ulatcd intra-abdominally with 5 cubic centimeters of the milk frow tech quarter showed characteristic Lect. at:1tus lesi us when eutoysied twelve ween later. The blood serim of the guinea piie gave a positive reaction to the agglutination ami complement fixa- tion tests. ioct. abogtgg was isolated frco the diseased spleens of the éuinee pigs. This cow hes he.d one ce.lf e.nd no ebo: tio:.e. l}ie blood one milk of cows 997,.998, 999, 1000, 160 0‘0 , 1305 and 1296 more negative before and after the last parturition. The blood and ail of cow 1&0? were not tested meediatoly after parturition 5-4 before the last pertzr itioo. both the milk and bleed gave a negative reaction. This cow has had six ashes. The calf of the second pro-.nancy was aborted. No data was obtained on the blojd reaction of cow 1301 before the last pa m1rition but 11m: ii: tely after the (2) blood gave a partial reaction and th milk gave a positive reaction. The Jill Cintizued to react for four weeks at which tine she died from a.n inflzazotion of the udder. This cow ce>u+_1=F1eo mozu1sv Obapamom obauamom ode; puma 0% «Hdwccflu One N ww okfipumom -9$aa«mom ode: puma on one“ eta m hm ohapamom obupfimom chauumom ass osoz m ww obfioumom ohauamom obavwmom 0:02 039 m oooa chapfimom obuuamom ocea away 02 neon conga m weed opgpemoz opasueoe acme pass 02 name new «an o poo“ 013" 03102 ob." newoz coma amen. oz 0mm“ Boom h vooa chapemmz abfiuemez ocea game oz 0:02 ago N need obuudmom obapewoz oven umoa ca name can Mum w toad any Obaadmez hddwnwnu aka : MOOH m. opaseeoe opfiuweoz meme puma oz omwm oases m moon 1% opspfimom A.amswv Hmfipuam oven one“ 02 once open p aooa chapemez chapemez obfiaemoz econ 0:0 N OOOH obuuewoz o>fipemoz chauewoz oeoz one N mow obfiaemom abauemoz obavewez 0:02 one u poo ohfiuamom obapumom chapemoz omen one N woo .obwvamom obnoauom obfivomoz anon one N mom coaauon< doapuon< ho soupauzauom Swank msusooem awake ho Gadvauzvuem amend Mada no coauoeom aspen voodm no coaooeom anon enemas. Esuom uaofiouon< anvufim Hesuoa .02 cOOHm no scavoeom ho nonEsz no hopssz om< sen oops08auooxm meacooh nu coup uobdoo mo ween no hnouuam mcfihonm canon -28- has had five normal calves. Kc abortions have occurred. The blood and milk of cow 1003 was negative before parturition. Two days after parturition she died of milk fever. This cow has had two normal calves. The calf of the second pregnancy was aborted. No data was obtained on the blood and milk reaction of cow 1004 before parturition. After parturition the blood was negative, but the milk was positive for one week. This cow has had six normal calves. The calf of the second pregnancy was aborted. The milk of cow 1008 has given a positive reaction for more than two years. The blood was not tested before the last parturition, but immediately afterward gave a posi~ tive reaction. This cow has had three normal calves. Ho abortions have occurred. The milk and blood of cow 1009 was positive before the last parturition. Both milk and blood were positive when tested after the last parturition. This cow has had two normal calves. No abortions have occurred. The milk of cows 86, 87 and 88 has shown a high antibody index for more than a year and a half, and was sus- pected of containing Bast. AbortugJ which was later demon- strated to be true. The milk of these cows, including cow 995, was the ”infected milk” which was fed to the guinea pigs, rabbits and calves. Repeated trials to isolate figgt, Abortus directly from the milk fed to the animals failed, but by inoculating guinea pigs with the milk and allowing twelve weeks for in- cubation; lesions were produced in the spleen of guinea pigs -29- from which E ct. nbortus Wis isolated. Bull calf 995 A was fed upon naturally infected milk for a period of thirteen.weeks. The blood of the calf was tested before feeding, and was found to be negative. A slight reaction deveIOped the first neck after feeding. A high antibody index deve10ped at the end of the second week, and remained constant until the end of the third week. then tested at the end of the fourth week, the blood was negative. The blood remained negfltive during the remainder of the time feeding was continued. At no time during the feeding did the sexual h irs around the sheath show nny tendency to become matted. Approximately three weeks after feeding of milk was dis- continued, the senual heirs showed a brownish red staining and matting. A small portion of the matted hairs were clipped off and washed with saline solution. The mixture was then centrifugslized and the sediment exmnined micro- scepically. The microscOpicsl findings consisted of’a fen epithelial cells, uric acid crystals and many bacteria. he pus cells were found in the sediment. . , heifer calf 996d n~s fed upon nnturally infecte‘ @ilh for a period of ten weeks. The blood of the calf was tests before feeding, and was found to be negative. The blood retained negative throughout the ti:e of feeding. At no tine during the feeding did the sexual hfiirs below the vulva show a tendency to become netted, nor was there thy dischsrge of muse-pus from the vulvs. .eoznfipzoomau mee.MHaE no deem oped mane no ADV .eHIdea chem Add I I I I I I I I I oHIoHIp . . . name I I I I I I I I I QHImHIQ a a u flENN I I n W I I I I I WHIM I0 3 a - SPON I I I I I I I I I QHIHHIm a a a fivhfl I R 4 4 I Q 4 4 4 I I I I I I I I I wHINNIé a a any: fivdd I I I I I I I I I wfiImdlfi - a - Sand I a 4 4 I n + e 4 I I I I I I I I I wHIw I: a a . spud I I I I I I I I I cHIH I: a a . head I I 4 4 I a 4 4 + I I I I I I I I I oaImuIm . . . need I I I I I I I I I wfilwdlm a a a fivo I I I 4 I Q 4 fl 4 I I I I I I I I I QHIHHIM a n a flvw I I I I I I I I I 0HI: Im a a u fivh mIII+III++IIIIIIIIISISIN .. ....§ 3 I I I I I I I I I oHIeaIm . . . sum . I I + + I I + + + I I I I I I I I I WHINHIN a g a #0: I m 4 4 I I I I Q OHIm IN a a - GHM I m 4 4 I a + + + I n 4 .H I I I I a WdIomIH . . . one I n 4 I I I I a HINNIH.mnHeooH beams xooB.H 4 4 + 4 e a 4 + e I I I I I I I I I oaImHIa essence ogoeom moo.~o.:oodo noooao.mo.mm.d. moo.mo.:ood.moo.do.mo.mN.H. Open .u«m.msoo nadaecavaamm< .Kam.msoc :ofipenfiusnem< .cOR madam MHHE ho nonpoeem Efihom coedm Mo noqudom Adv < mwc «Ado Hdfim .uamo chem sex e 0» Mafia empoomsn sHHeusvez wnaeooh no adzmom on» mauso3m .ou odpma DUC‘ -51- Bull calf 997d nee fed upon.nsturelly infected milk for a period of two weeks. The calf died of acute intoxica- tion during the third week of feeding. lhe blood reaction was negative before feeding milk. A slight reoction devel— Oped at the end of the first week. At the end.of the seconl week the blood was again negative. She sexual heirs around the sheath showed no signs of matting. Heifer calf lOOOd was fed upon n turslly infected milk for a period of fourteen weeks. The blood was negative before the feeding of milk, and centinued to rem in negative throughout the period of feeding. At no time during the feeding did the sexual heirs below the vulva show a tendency to becoze matted, nor was tiers any dis- charge oflnuco-pus from the vulva. Bull colf 1001A Wis fed upon naturally infected milk for a period of five nooks. The self was slaughtered for veal at th, find.of the fifth week. The blood of the chlf gave e positive reaction before feeding milk and con~ tinned to give 3 positive reaction up to the end of the third week. The blood gave n negative reaction on the fourth week and continued to remain negative. The Sexufil heirs around the sheath becane slight- ly m tted and stained black the second week of feeding. A micrOSCOpic examination‘wes m‘de of the sediment wsshed from the heirs. 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