2mm mm m was ABORTWN msugg

Transcription

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4 S BACTERUM ABORTUS PRESENT N THE URNE 0F CATTLE AFFECTED WTH BANG'S ABORTON DSEASE?

5 S BACTERUE ABORTUS PRESENT N THE URNE OF CATTLE AFFECTED WTH BANG'S ABORTON DSEASE? THESS. Submitted to the Facuty of the Michigan State Coege in partia fufiment of the requirements for the degree of Master of Science. 7 By L. E. ;gng Juy 1927.

6 THES'E

7 CONTENTS. NTRODUCTON AND HSTORY. REVEW E LTERATURE. HSTORY OF ANMALS USED N TiS EXPERMENT. EXPERMENTAL WORK. METHOD OF STUDY. TABLE 1. DSCUSSON ON TABLE. METHOD OF STUDY. TABLE. DSCUSSON ON TABLE. METHOD OF STUDY. TABLE. DSCUSSON OF TABLE. CENLRAL DSCUSSON. SUMMARY.' ACKNOWLEDGMENT. BBLOGRAPHY. A '3! 1-0FQQ ) /f

8 S BACTEREH ABORTUS PRESENT N THE URNE OE CATTLE AFFECTED WTH BANG'S ABORTON DSEASE? NTRODUCTON AND HSTORY. Since Bacterium abortus is eiminated in the mik of infected, pregnant and non-pregnant, cows, and in the uterine discharges during and shorty after parturition or abortion, the question naturay arises as to whether or not the micro-parasite is present in.the excreted urine. 7 The fact that some animas aborted their young and that others were unabe to reproduce their kind was known in eariest Bibe times (Genesis XXX, verse 38). Reference was again made to these conditions in the writings of.maeoa, in t was not unti 1793, however, that Eberhard and Gunther of Begiumipubished the first veterinary work on this subject and this was foowed by numerous writings in. the eary nineteenth century which show that the infectiousness of abortion disease was a we known fact at that time (Lawrence 1805, Lafoose and Zfinde 1807, Skeet 1808, Jonati 1837, and Barow 1851). The "Compete Farmer" in 1807 said "t is cansidered certainy contagious, and when it happens the abortion shoud be immediatey burned, and the cow kept as widey apart as possibe from the herd, and not receive the bu that goes with them." St. Cyr (1875) reported the infectiousness of the disease but it remained for.frank (1876) and Lehnert and Brafier (1880) to artificiay inocuate heathy pregnant cows. Nocard (1885) was the first to do

9 scientific investigation athough he faied to isoate the causative organism. This investigation.into abortion.wae.toowed by that of Woodhead, Aitken, McFadyean, and Campbe (1889) and.the Roya Agricutura Society of Engand (1894). t was not unti 1897, through the remarkabe work of Bang and Striebot, that the Specific etioogica agent, Bacterimm abortus, was discovered. Bang's statement was not fuy accepted, however, unti 1906, when he reannounced his discovery before the Nationa Veterinary Association at Liverpoo. His work was confined by the investigations of Preiee (1902) and.mcfadyean and Stockman(909). n.america, Chester, Law, andeoore faied to isoate the organism but MacNea, Khrr, Gitner, and Good (1910) independenty confirmed Bang's discovery. Traum (1914) is the first recorded to isoate the causative organism from aborted swine. Moher and Eichhorn in 1925 reported the pathogenicity of Brucea meitensis for sheep, goats, catte and horses. t is remarkabe, however, that with a the research into contagious abortion by the above mentioned and many other investigators, very itte has been written regarding Bacterium abortus in connection with the urinary organs, and practicay nothing has been mentioned concerning the urine. AthOugh there has been some experimenta work done with the urine of man, goats, rabbits, and guinea pigs, Zeer appears to be the ony one to have worked with that of catte and his experiment wi be discussed ater.

10 REVEW OF LTERATURE. Athough there are histo-pathoogica changes present in the materna cotyedons of infected pregnant femaes, and those that have had recent parturition or abortion, other tissues may be invaded by the Bacterium.abortus or Brucea meitensis without these changes of the norma histoogy of'the part. This is evidenced in the udder. Notwithstanding this ack of esions, the causative micro-parasite may be isoated from the tissues, if present, athough one might encounter some difficuties in so doing. Huddeson (1) reported the isoation of Bacterium abortus in pure cutures from.diseased speens, ivers, and kidneys of guinea pigs fed forty-eight hour agar sants of viruent cutures. Keyer, Shaw, and Feischner (2) found the porcine type of Bacterium.abortus in a tissues except the heart bood or musces. They were aso abe to isoate from the kidneys of a number of inocuated guinea pigs both Bacter-.329 abortus and Brucea meitensis. Schroeder (3) observed that Bacterium abortus produced most changes in guinea pige' ivers, speens, kidneys, testices, bones, eto., athough in a ater paper (4) he caimed that no macroscopic esions are produced in the Speen two months after injection even though the organisms canine isoated. T. G. Evans (5) was abe to produce esions in guinea pigs simiar to those of Schroeder and Cotton, by feeding infected mik. Cotton (6) reported joint esions in guinea pigs from both bovine and porcine strains with the atter appearing to be more viruent. Smiie (7) was abe to recover Bacterium abortus from the kidneys,speen,

11 testices, and bones of inocuated guinea pigs regardess of the point of injection or the size of the infecting dose. Schroeder (4) found Bacterium abortus maintaining itsef in bodies of rabbits for ong periods of time without causing macroscopic changes. Zeer (2) injected rabbits and guinea pigs intravenousy, intraperitoneay, and subcutaneousy, with arge doses of Bacterium abortus and Brucea meitensis. This histo-pathoogica changes after varying intervas of time were mosty negative but he was abe, in a few cases, to isoate the organisms from the speen, uterus, testices, and occasionay the kidney. Generaized invasion of the tissues resuted when monkeys were fed viruent cutures of Bacterium abortus, as was found by Feischner, Vecki, Shaw, and Meyer (8). The abortion organism; according to Schroeder and Cotton (5) has been found in many more regions of the bodies of swine than in those of catte, and Cotton (9) found that the iving micro-organisms coud be isoated fran necrotic areas in the kidney fat and udder tissues, uterine exudates,. and portions of the uterine was of pigs that had been.inocup ated. Hays (10) was unabe to isoate Bacterium.abortus from the kidneys, speen, iver, thyroid, and urethra of two positive barrows. Weeter (11) was unabe to obtain the abortion organism from any of the interna organs of swine six months after the initia and subsequent infection, therefore concuding that the organisms were eiminated. The reports of the Mediterranian Commission and ikewise those of Moher and Hart (12) showed that the Brucea meitensis can be isoated from the ymph nodes,

12 mammary gands, and speen of goats and aso indicated distinct hyperemia of the kidneys and iver, enarged speen and genera enargement of ymph nodes. Z. Khaed_(15) and Cesari (14) were abe to isoate the Brucea meitensis, in fata goat cases, from the kidneys, enarged mesenteric gands, Speen, iver, saiva, mik, and bood (10 per cent). Bacterium abortus was obtained in pure cutures from the heart bood and a organs of the body of a arge number of aborted ambs (15). Out of 140 cases of pyeitis of the human femae, Kidd of London (16) found one due to paramata fever and Cesari (14) states that the kidneys constitute the avenue of eimination for Brucea meitensis. Schroeder (17) found, as a resut of a arge number of tests, that young catte rarey harbor abortion bacii but they may be present in the gastro-hepatic ymph gands, iver, and stomach fuids of newy born, viabe caves. Hadey (18) asserts that the germs of abortion may exist in arge numbers in the stomachs or intestines of newy born caves, but shorty after birth these bacii disappear as there are no tissues in the caves' bodies suitabe for the existence of the germs. Carpenter (19) found ony one fetus with kidney infection and aso found (20) that the abortion bacii invaded the ymph gands adjacent to the head as we as those aong the intestines, athough they did not persist in these gands upon discontinuing the feeding of infected materia. Heifers rarey carry the abortion bacii permanenty in their bodies uness they have found odgement in the udder and suprammary ymph gands. As a rue this occurs ony

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14 6. after the gands have activey engaged in.mik secretion, athough Schroeder and Cotton (21) have, in isoated cases, been abe to cuture the organism from udders that had never actated. Primariy the favorite habitat of Bacterium abortus is neither in the parent nor the offspring but rather in the.medium through which the two are connected. A attempts to isoate the organism from the kidneys, speen, iver, ungs, serous membranes, synovia fuids, bone marrow, brain, spina cord, musce, uterus, vagina, faopian tubes, ovaries, etc., and ymph gands from a portions of their bodies, have faied as caimed by Schroeder (17). Kavarzik (22) caimed that autopsy may revea punctiform or streaked hemorrhages in the serous membranes of the gastro-intestiona cana, and in the urinary badder, or more or ess pronounced acute sweing of the speen and ymph gands. Schoreder and Cotton (21), after examining hundreds of tests with.mik from numerous cows, are of the opinion that the udder is the ony habitat of Bacterium abortus in the bodies of non-pregnant cows, and that the abortion bacii do not maintain themseves in the bodies of cows esewhere than in their udders and gravid uteruses (21). Hadey (18) is of the opinion that the udder is the ony organ in the body of non-pregnant cows where the bacii can ive, and Cotton (25) decares the bacius is unique in that it requires embryonic tissue for its deveopment and is ony present in animas during this reativey short period of their existanee. Huddeson (24), Moher and Traum, Buck, Creech and Ladson, and Hart (25), and Khaed (15) caim the bacius can be

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16 isoated from the speen, iver, mik, and ymph gands, as we as the cotyedons, uterus, and uterine discharges of pregnant cows. ZMeyer, Shaw and Feischner (2) were abe to isoate Brucea meitensis from the urine of inocuated guinea pigs, and simiar resuts were obtained by Durham, Eyre, and Nicoe and Consei (2). Zeer (2), upon subcutaneous, intraperitonea, and intravenous injections of guinea pigs and rabbits with Bacterium abortus and Brucea meitensig was abe to demonstrate that the organisms were present in the urine. Z. Khaed (15) was unabe, on various occasions, to cuture either Bacterium abortus or Brucea meitensis frmm the urine of inocuated goats athough he did find the organisms in the bood. He was abe, however, to demonstrate that Brucea meitensig was present in the urine of fata cases in goats. The Reports of the Mediterranian.Commission and of Moher and Hart and Cerari (4) indicated the occasiona finding of'brucea meitensis in the urine of goats. Huddeson (26) reported the presence of Bacterium abortus and Brucea meitensis in the urine of infected humans, athough ony occasiony isoated and cutured from Singe sampes. The presence of Brucea meitensis in human urine aso by Cesari (4), Stitt(27), Park and Wiiams (28), and Buchanan (29). As previousy stated very itte work appears to have been done on the urine of infected animas of the bovine species,; and Zeer (50) appears to be the ony one to have worked on that. He inocuated subcutaneousy a cow with ten agar sant cutures in twenty mie of sat soution. Seventeen cutura

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18 .8 examinations, ranging from one to eighty-nine days, were made from sampes of bood, mik, saiva, feces, and urine, with negative resuts. The cow was kied one hundred twenty-ehght days after inocuation and various organs cutured with negative resuts. Cow number two was inocuated simiary with twenty agar sant cutures in thirty mie of sat soution and sixteen sampes of the same materia were examined cuturay from three to sixty-seven days with negative resuts. This cow's organs upon autopsy proved negative cuturay. Cow number three was inocuated with twenty agar sant cutures in thirty mie of sat soution and twenty-one sampes of simiar substances were examined cuturay from one to sixty-five days and proved negative. The post mortmm findings were the same. Cow number four was inocuated with ten agar sant cutures and at various intervas sampes of the same materia was examined previousy, excepting the mik, were cutured with negative resuts. One hundred sixty-one days after inocuation the cow was destroyed and guinea pigs were inocuated with the materias from her speen, uterus, ovaries, udder, and supreemammary ymph gands with negative resuts. t might be we to note that since these catte were inocuated the conditions differed from those existing where animas were infected naturay. According to Zwick and Wedemann (51) Bacterium abortus, when present in urine or dry cow manure,wi die within twenty-four hours. This differs from the resuts obtained in the present investigation since the writer was abe to grow the organism for seventy-two hours, this growth being even more uxuriant than that obtained in twentyfour hours. There was no notation as to the type, viruency, or amount of organisms that Zwick and Wedemann used in their ex-

19 periment. G. Ranchbar (52) was abe to keep Bacterium abortus aive for nine days in the urine of mice. p//' HSTORY OF ANMALS USED N THS EXPERMENT. Cow number 216 was born August 16, 1922, and is a positive reactor to the Bacterium abortus aggutination test, habing had a five pus reaction since Juy On November 25, 1925, she was bred and carried her fetus for two hundred sixty-eight days, with a norma parturition on August 19, She was again bred November 25, 1924, and this time carried her fetus ten days onger, having a norma parturition August 28, On her third pregnancy, fire was bred November 27, 1925, and carried her fetus unti August 10, 1926, two hundred fiftysix days, when she aborted. The caf died at birth and the dam ceaned in eight hours. Bacterium abortus was isoated from the fetus, pacenta, and from a four quarters of the udder. The mik is positive to the aggutination test. Cow number 256 was born.apri 14, 1925, and was a four pus reactor in August 1925, and has since been a partia to a five pus reactor. She was bred Juy 8, 1924, and carried her fetus two hundred sixty-four days to which she gave norma parturition on March 29, The second breeding was on June 27, 1925, and the fetus was carried unti Apri 5, 1926 (two hundred eighty days), to be born norma. This dam.was bred the third time, August 15, 1926, and norma parturition occurred May 12, Bacterium abortus was isoated from.the coostrum on.apri 5, 1926, as was aso streptococci. Ho abortion bacii were found in the pacenta or feta membranes but they were

20 10 found in one quarter of her udder. Cow number 259 was born Juy 51, 1922, and since her abortion has been a positive reactor to the aggutination test. Up to this time she was negative. Shexwas first bred Juy 51, 1924, and did not become pregnant so she was again bred August 22, The fetus was carried two hundred seventy-three days and the dam.had a norma parturition on May 22, Her second pregnancy started August 24, 1925, and the fetus was carried two hundred twenty-nine days to be aborted Apri 10, She was again bred Juy 7, 1926, with a norma partue rition Apri 14, 1927 (two hundred twenty-seven days). There has been a partia aggutination of her mik to the Bacterium abortus. Cow number 147 was born October 18, 1921, and is a positive reactor. There is no record of retained pacenta membranes but she aborted on her first and second pregnancies. This, her fifth, resuted in abortion on December 1, She has been bred four different times since the above date, the ast being Apri 4, Cow number 191 was born.may 7, 1924, and is a positive reactor. On her first pregnancy there was an abortion and on August 7, 1926, she aborted in the pasture. There have been four different breeding dates, the ast June 25, Cow number 195 was born May 18, 1924, and is a positive reactor. There was no previous history of abortion. She aborted a dead caf on August 5, 1926, and the pacenta was retained. Bacterium.abortus was isoated from.the pacenta membranes, and the mik is positive to the aggus tination test.

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22 11 Cow number 95 was born.march 12, 1920, and is a positive reactor. fourth pregnancies. Her history shows abortion on her first and On her fifth pregnancy, the third abortion occurred whie in the pasture, November 17, The pacenta membranes were retained, and the caf was dead at deivery. Bacterium abortus has been isoated from the membranes and mik. Cow number 84 is a positive reactor and a chronic spreader. Her previous pregnancy resuted in an abortion as did the ninth on February 14, 1926, with death of the caf on deivery. The same occurred October 5, 1926, and this time Bacterium abortus was isoated from the feta membranes. The mik was positive to the Bacterium abortus aggutination test. Later this anima became very emaciated andmwas kied May 17, Upon autopsy, it was found that a hay wire had penetrated through the diaphragm at three different points. There were three fistuous tracts with sight peurisy, traumatic pneumonia, and some pericarditis. Cow number 171 was born February 7, 1925, and is a positive reactor. She was first bred on Juy 7, 1924, and carried her fetus two hundred eighty-three days unti Apri 15, 1925, with a norma parturition. She was twice bred again Juy 20, 1925, and August 16, 1926 when She became pregnant. This time the fetus was carried two hundred fortyethree days and aborted Apri 15, The caf was dead on deivery with feta pneumonia. After this abortion, she was bred three times, May 51, 1926, June 24, 1926, and August 15, This time she carried her fetus two hundred sixty-one days to abort a dead caf May 6, Bacterium abortus was isoated from the fetus and:feta1 membranes.

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24 .12 Cow number 1665 is ten years od and is a positive reactor. March 17, 1920, she gave birth to a norma caf and the same occurred Juy 4, On Juy 27, 1921, she was bred and on December 28, 1921, there was an abortion. Again she aborted on November 17, 1922, osing a seven.months od fetus, and retained the pacenta membranes, which required manua remova. Metritis was present in this case. The fetus, pacenta, and mik showed Bacterium abortus. On March 26, 1925, she was again bred but aborted an eight and one-haf months od fetus and retained the pacenta which required manua remova. This occurred December 15, There were symptoms of severe metritis which continued for a ong time. OnMHay 50, 1925, this dam had a norma parturition and ceaned propery. The pacenta showed no indications of Bacterium abortus. Again on August 1, 1926, she had another norma parturition. No Bacterium abortus was present in the pacenta membranes athough she has had the organisms in a four quarters since March 25, Cow number 55 was born February 8, 1925, and is a positive reactor to the aggutination test for Bacterigm abortus infection. She was artificiay fed cutures of the abortion bacii on September 7, 1926, and reacted to the aggutination.test on September 29, This anima was bred November 50, 1926, but aborted an eight months od fetus. The pacenta came through norma invoution. Bacterium abortus was isoated from the feta membranes, ungs, iver, kidneys, and abdomina fuid. Cow number 175 was born June 15, 1925, and is s positive reactor. Her first pregnancy resuted in an abortion

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26 13 which took pace on June 12, 1926, two hundred fifty-three days after being bred. be removed manuay. The pacenta was retained and had to Bacterium abortus was isoated from the pacenta. This anima faied to conceive upon subsequent breedings and diedearch 21, 1927, from traumatic pericarditis. Cow number 99 was born Apri 14, 1922, and is a positive reactor. There is no history of previous retained pacentas athough she aborted on her second pregnancy. On her third pregnancy she aborted, Apri 6, 1926 and.retained her pacenta. Bacterium.abortus was isoated from her pacenta membranes and her mik was positive to the Bacterium.abortus, aggutination test. Norma caving occurred March 17, Cow number 95 was born February 16, 1920, and is a positive reactor. Her first two caves born June 15, 1927, and December 12, 1925, were dead at birth. After this there were eight breeding dates before conception took pace that. resuted in a norma parturition on March 18, Her caf born February 25, 1927, died shorty after birth with nave i. The ast breeding date is May 10, Cow number 1659 is about eeven years od, and is a positive reactor. She was naturay infected in 1922 and Bacterigm abortus has been ocated in her udder ever since. This anima has never aborted nor have the organisms been found in her pacenta membranes. She has had four or five caves since being in the experimenta herd. Cow number 161 was born March 15, 1920, and is a positive reactor. Her first pregnancy, on November 19, 1922, terminated with a favorabe parturition. She was bred May 22,1923

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28 14 and as there were no signs of pregnancy, she was again bred June 8, The fetus was carried one hundred eighty-six days to be aborted dead on December 14, Bacterium abortus was isoated at this time from the pacenta membranes. The next pregnancy required two breedings, Apri 11, 1924, and June 22, The fetus was carried unti February 24, 1925 (two hundred seventy-nine days), and terminated with s favorabe parturition. On June 1, 1925, she was bred and this time the fetus was carried unti February 10, 1926 (two hundred sixty-five days), but was dead at deivery. May 15, 1926, she was bred and one hundred eighty-seven days ater (February 18, 1927), norma parturition.took pace. Since the atter date, she has not been in heat and became very poor, waking with a stiff gait. After her death, June 6, 1927, post mortem.reveaed mutipe abscesses of the iver and a chronic diffused nephritis. Cow number 75 was born Apri 28, 1921, and is a positive reactor. Her first conception took pace November 19, 1922, and in one hundred ninety-two days (May 20, 1925), she deivered a dead caf. Her third pregnancy required three servings, Juy 24, 1925, August 14, 1925, and September 25, There was a norma parturition after two hundred seventy-four days (June 25, 1924). Her next pregnancy from August 6, 1924, to May 19, 1925, terminated nromay at the end of two hundred eighty-seven days. Again it required the three services of the sire (August 24, 1925, September 29, 1925, and November 21, 1925) before norma pregnancy occurred. This took pace two hundred sixty-one

29 '15 days (Juy 12, 1926) after the ast breeding. The caf died one month ater. The next pregnancy required three servings, September 28, 1926, January 2, 1927, and February 5, After carrying the fetus one hundred seventy-five days, she aborted (June 21, She died June 22, 1927) and the caf was removed manuay from Bacius coi septicemia. Cow number 252 was born November 11, 1924, and is a positive reactor. There was no history of abortion up to the one that took pace January 27, At this time Bacterium abortus was isoated from the pacenta membranes. Cow number 47 was born October 18, 1921, and is a positive reactor. The history shows there was an abortion on her first and second pregnancies but with no record of retained pacentas. She again aborted upon her fifth pregnancy, December 1, The Bacterium abortus aggutination test was positive for her mik. Seven breeding dates have occurred since the ast abortion, the ast being on May 4, 1926.

30 16 METHOD OF STUDY. Urine of catte infected with Bang's abortion disease was caught in sterie one iter fasks upon the animas' urinating. From these fasks, thirty-five cubic centimeters was centrifugaized at about 2500 revoutions per minute for one hour and the sediment then smeared over the surface of three pates consisting of beef iver infusion agar (26) and a saturated aqueous soution of gentian vioet to make a 1: diution. This inhibited the Gram positive organisms especiay the fast growing ones. After pating, the inocuated pates were incubated at 57 C. under ten per cent 00. conditions for seventy-two hours and then examined for typica1, gcterium abortus coonies. nasmuch as my purpose was to try to isoate Bacterium abortus from the urine of infected catte and since contamination from other organisms woud naturay resut from the urine passing over the foor and inferior commissure of the vuva, no attempts to identify the other organisms found growing on the pates were made. The first eight trips to the barn where these animas were staed were made in the evening, and as resut were negative it was decided to make a future trips in the morning so as to procure the night's urine upon the animas' first morning micturition.

31 Tabe e " Nfiifiaé ai diiiéi-seet"""""""?"""""""""" """"" M" e e Date : Hour ndividua: Series : of 'of tests :of tests: coection : Same ---~-uuuuuuu*uuuuuuwu ~----* : : August 9,1926: 5:00 P.M. 2 : : 3 3 3! n: it! an-.----~ cown- 4 : 2 : August.,926: 5:00 P.1i. 5 : 3!! : H 6 : :! N N :! u : 5 :August 16,1926: 5 00 ram. 8 : : : " ' 9 : 3 f 7' 3 7! t 10 Z 4 EAuguet 19,1925} 5:00 P.1d ! N 12 : H N N 3 N N 15 z!!! : n! 14 5 :August 21,1926; 5:00 P.M. 15 : :!! 16 : : N!! 3 f '7 _ 17 z :!! W 3 N! :---u : _ 18 : 6 :August 25,1926: 5:50 Pam. 19 : : " " : 20 : : " " " : " C 21 E 7 August 25,1926: 4:50 P.M. 22 : : W! : N! 25. : " " : " " :34L 0 : '. '. " z " a 25 E 8 August 50,1925: 4.50 P.M. 26 : : n w w 3 w w 27 : z n w w 3 w w a: : : : 9 : Sept. 1, 1926: 4:00 ANN :! '1! z n! 50 : : " " : " " 51 E 10 Sept. 4, 1926; 4:00 A.M. 52 z : N H N z W W 55 : : " " " : " " 54 : : " " " : " " a. ee ee ee ee ee ee ee e. ee ee ee 0. O. O. O. O. O. O. O... O. O..0 O. O. O. O. Q..0 O. O. O. O. O O..0 O..0 Q. Number: Resut of :of : examianima: nations. nnnnnnn QF : Negative. 171 : : " uuuuu~-*---_u----~ 171 : " 1659 : " 1665 : Question. 84 : Negative : " 1665 : " 84 : ' 171 : " 1659 : : " 84 : Question 171 : Negative 1659 : " 1665 : " 171 : ' 1659 : " 1665 : ' 84 : " 171 : " 1659 : " 1665 : " 84 : 1659 : " 1665 : " 171 x " 1659 : " 1665 : " 84 : " 171 : " 1659 : " 1665 : "

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33 18 Tabe (continued). 2::::::::=::::::::::::=:::=::=::=================:=::=======::=:=:= -NUmber of different: : z, : ; Date - Hour : Number: Resut of ndividua: Series : of : of : of : examitests :of tests: coection : same : anima: nations Sept. 8,1926: 4: 50 A..M Negative. 35 : :. ". 171 : ' 56. : " " " : " " : " 57 : " ". : " " : 1665 : " Sept. 18, 1926: AeMe: t! 39 = : " : " " : w 40 E 15 E Sept.29, 1925: 5:00 A.M. 84 S u 41 : : " " " : " " : 1659 : ' 42 : 7 " " : " " : 1665 : " nn-c-oqc-e--- : -QQOQe-Q- :u-os- Q-Q-o-en-n : -ww-o-u-c-n : nun : 14 : Oct. 9, 1926 : 4:50 ANM.: 171 : " 44 = : " " " : " " : 1659 : n 45 : : f 7! f 3: : f ::::::::::::i::::::::i::::::::::::::i::::::::::i:::::::i::::::::::

34 19 DSCUSSON OF TABLE. Tabe number shows the resuts of forty-five different attempts to isoate the Bacterium abortus from four individua cows positive to the aggutination test, and kept in the same barn during a period of eight weeks. Eight separate urine sampes were coected from cow number 84, ten sampes from cow number 171, and thirteen sampes from cows number 1659 and 1665, respectivey. ndividua urine sampe number six was not cutured since the cotton stopper of the urine fask became contaminated when knocked from the investigator's hands into the gutter. The tabe aso shows a question as to the resuts with urine sampe number fourteen. Ony about fifty cubic centimeters were coected at the time of urination since the first to be passed was missed. Negative resuts were obtained throughout this experiment. p

35 20. METHOD OF STUDY. V/ Since the resuts, as indicated in Tabe, were negative certain questions arose in the investigator's mind: (1) Were the abortion bacii in the urine too few to isoate? (2) Woud they grow sufficienty to isoate if aowed to stand for varying periods of ties at 23 C.? (3) f urine were inocuated with abortion bacii woud they grow? Aong with the individua inocuated urine sampes pated out as expained for the previous tabe, sampes of urine from the same one iter fask were inocuated and incubated as foows: after the thirty-five cubic centimeters of urine were removed from the one iter fask for centrifugaization, ten cubic centimeters were pipetted into each of eight different sterie test tubes and inocuated with one cubic centimeter of a suspension of Bacterium abortus. This bacteria suspension was prepared by washing off the surface growth from a seventy-two hour beef iver infusion agar sant with ten cubic centimeters of physioogica saine soution. The eight tubes were divided into five series of two tubes each and incubated at for varying periods of time: the first set for tweve hours, second set for twenty-four hours, third set for forty-eight hours, and the fifth set for seventy-two hours. Upon the expiration of the first period of incubation one tube of the first set (tweve hours) was centrifugaized at about 2500 revoutions per minute for thirty'minutes before pating the sediment on three beef iver infusion gentian vioet pates. The other tube of the set containing the in-

36

37 21 ocuated whoe urine was pated out on three separate pates, simiary. The contents, sediment and whoe urine, of set two (twenty-four hours) was pated as above upon the competion of twenty-four hours incubation, set three upon forty-eight hours incubation, and set four upon seventy-two hours incubation. After each set was pated out, it was incubated far seventytwo hours under ten per cent 00. conditions. Beef iver infusion agar sants were inocuated upon the appearance of typica transparent dew drop coonies on any of the pates. Those sants that showed typica Bacterium abortus subcutures were washed off with a soution containing nine-tenths per cent sodium choride and five-tenths per cent pheno. This suspension was standardized with the Gate's nepheometer (33) to a density of seven centimeters, after which the ph was adjusted to 6.8. This suspension then comprised the antigen for the aggutination test to be run against a four pus bood serum. nto each of five precipitation tubes were pipetted two cubic centimeters of the standardized antigen and eight-hundredths, four-hundredths, two-hundredths, onehundredths, and five-thousandths cubic centimeters of four pus bood serum, respectivey, into each tube. diutions of one to twenty-five, one to fifty, This made one to one hundred, one to two hundred, and one to five hundred. / The cuture, number 44, of Bacterium abortus V used to inocuate the eight different sets of urine was isoated about two years ago.

38

39 :Number: Date of coection : 12 hour : 24 hour ndividua: Series : of : and ; tests. :of testszanima: hour of same. : CU.: UU.: CU.: UU :00t.16,1926o4:00AeMe3 23 :BESE) 5 ' o Ecot.so,1926.4;15A.M. Question 10 4 ' N N N N H f " : i]. H --;-:---- "" """"""""""""""""""""""""""""6ut "" -""'""""""""""' Number Of different: 3 : B -g :Uninocup Tabe hour : 72 hour :ated : urinary CU.: UU.:sediment. O... CU.: UU. O. 0 :-ece-nzcuenenue: u-s--pcnnoeuue C-H ---C'CD:----fb: -N-CC-:GG--DDD...-H-H-C-.-i-.-.. Negative O. C. O..0 f f 0. O. O. O. d. NL *..VVR. " " " ' ' ee 90 ee 00 so ee.0 ee 00 so ee.0 ea 0. so so 00 ee. ~t.. ' " 60 as as a: *>LJ 1~\k O..0 v0 00 so ee 00 so e. e. O. Oct.25,1926.4;001.m.' H * k O. O. O..0 O «nun-nu. -napqu-qp-e-e 2,4 { s 1' Negative ' 0' 0 00 C O " co co co so no so ee 00 ee so so so so so so co co 1 i? O. O. C. O. O. O. O. O. O. O. O. 0.. O. O. O. O. O. O. fi.$ k *~Pf~\L kkfii. " ".. 9 '0 '0 " 0 " 9' so so so es en es co ee 00 so so so ee.9 so so ee?- " H N N n ~*.rf~:fnt\g n \k O0 90 so so 00 ee so ee es fee.0 es so ee 00.0 ee.0 so ea ee so 00 Nov.13,1926.4:30A6M. n H eo d ' so so as so ee 0. so so so so so so ee () qu23(),, =:()()j& mhio 5 9 v " " " ' ' 00 ee 09 9e ooroo so so so ee 00 es 0 0. " 0' 0' so so 00 co so so so ee 00 ee so 00 ee 00 co co ee \k\l nn--u-qpcncubn. Question Negative? % ec.,1926.4:30a.m. 6?.0 O. O. cc ee " ". " " so so so ee so so ee 00 ee ee so so so ee ee 00 ee EDec.18,1926.4:004.M C.. O. O. 00". 0. 0O.0 O. C. O. O. O. C. O. O.. C..9 O. O. O. O. O. O. 0. O.. O. O. O. 00 LJ O. O.. O. O. C. O. O. O. O. O.. O..0 O. O. O. O.. kf. 22

40 ] n

41 Tabe. (continued)..-.-' cdc-cdcc..."c.. nun-up.-and-nn 'd-dd-nc-u-.-undue-unnuccnqununc..-(dd--dddnc..-nc-dc-ddc-ddoggcnc-q-c-euni-(ube-dc-iu-ecdc-cd-c-cdcd-c-qd- """"""""""""""' """""""""""""""Gutfife"fowtfi" """" """"' Number of different: : : :Uninocu- ated urinary *sedimeht 12 hour : 24 hour :48 hour : 72 hour : CU.: UU.: CU.: UU.: CU.: UU.: CU.: UU :Number: Date of coection ndividua: Series : of : and tests :of tests:anima: hour of same C. CDC-D-D:(Dun-H-:-H-u:C--:-C.- :--- --DCM,H...-CD.-CCC-D qua---nunununc-n unue-ncnue-c - :Negative 1 xé f 84 :Jan. 8,1927,4:00A.M. 33! :2 \L o. e..0 es 0. e. 00 ee O...0 O..0 O. O : ow ee.0 so so so so co co so so ee ee.0 00 e. 9. e to so so ee ee.0 9. so so so e. {he??? Jan.15,1927.4:001 h. '.' n 9 ' : ee so so so ee so 00 ee 0' 8! so ee so co co co ee.0 es.0 oe.- so so e \L GU--C:-H---.Dp-..C.C.C..D-Cece-. 10 EJan.22,1927.4:001;s. :4 LD r- 51 k 00 co ee 00.0 e. on so so ee.0 ee 96 ee 0! H \L t 00 so co co ee co ee.0 es co so so ee \. \ st ee so e. e so so 0'! u H H u H H H M 828 so en es.0 as so ee 00 ee ee es 00 ee 00 e. ee 00 so so.{ ee e. co 0' Key CU. oenfifirfugaized inocuated urine. UU. inocuated whoe urine.? / see discussion of chart. coonies appeared on pates. - no Bacterium abortus coonies on pates. 25

42

43 24 DSCUSSON OF TABLE. Thirty-four different urine sampes were cutured and studied, from October 16, 1926, unti January 22, 1927, using sixteen different cows. The urine of cows number 75, 161, 236, and 1665 was examined cuturay three times; that of cows number 33, 84, 147, 171, 191, 216, 232, and 239 was cutured twice; and that of cows number 95, 99, 175, and 193 was examined ony once. Ony forty cubic centimeters of urine were coected on sampe number twenty-eight, so it was centrifugaized and the sediment examined. There was a question concerning the resuts of urine sampes number nine and eighteen (cows 239 and 193 respectivey) as they were both ost; the former fask was tipped over and the atter broken. Resuts showed that Bacterium abortus existed and appeared to mutipy in inocuated urine, both centrifugaized and Whoe. There seemed to be more coonies, as a genera thing, in the twenty-four and forty-eight hour growths than in the tweve hour growth, whie the coonies seemed to become fewer during the seventy-second hour. At no time, though, were the codnies profuse on any of the pates. Bacterium abortus seemed more ikey to be found and the coonies greater in number in the urine sampes that had been centrifugaized. A thirty-four sampes of the uninocuated urinary sediment faied to show coonies.

44 '... Q J V "' e.. - V Q Q ',. ' r _ e,. v n O. ' 9 -.

45 25 METHOD OF STUDY. To insure the finding of the microorganism,_ acterium abortus, in non-inocuated cows' urine, having found that the pathogen woud grow and to some extent mutipy in inocuated urine, sampes of urine were mixed with sterie whipping cream and hed for twenty-four, forty-eight, and seventy-two hours at The purpose of this was that the rising fat gobues and body ces woud carry to the surface the abortion bacii if present (26). Fron each one thousand cubic centimeters of cows' urine sufficient amount was paced in a five hundred cubic centimeter Forence fask as to fi it to the neck and to thisvvas added ten cubic centimeters of sterie whipping cream. This mixture was thoroughy shaken and then aowed to stand at 23'0. for seventy-two hours. n twenty-four hours three cubic centimeters of the cream in the neck of the fask were pated, respectivey, on.each of three beef iver ins fusion gentian agar pates. These three pates were then incubated at 37 C. for seventy-two hours under ten per cent 00; conditions.. This procedure was repeated in forty-eight and seventy-two hours. n conjunction with this "urine-cream" method, noninocuated sampes of urine aone were hed for the same periods of time, centrifugaized, and simiary pated. As there were no typica coonies on any of the.pates in either of these two tests, no agar sants and aggutination tests were made. /1

46

47 Feb. 26, :OOA.M. Mar. 12,1927.4:00A.M Tabe e number of differentzumber of: : ream 0:: Centr fused Urine : anima Date Of 001ection:99 b gi9g -nnccn-9 nun-eeeee-gfiéggégggu "f!""-'.'-""u'd D""""'."-".-.'-'."'.'.'.."'-..'.."."'-'-'-'-'-'-'-'-"'."P."""-.""""'.-'-'-"'."'!-"'-" -'.""'..-"'-'-"""."-""-'-'."-'-D'-C.' 7"""""""""""""" "" """"""""" '""'f'uffi""""""""""'"""" ndividua: Series : urine and : 72 tests :of tests:examined hour of same. hour hour hour hour hour : hour 1 47 CDC-QD C CD-'- -.'-- b. 5,1927.4;oon.m.. CDC-.-DCC.--D CDC-.UC-. D CD-:-M-C--DC 93 99E; " Os z»: O. O. O. O. O. O. O. O O. O. O. O. O. O. O. O. O. O. O. 0. O. O. O. C. O '0 * so 00 so so we so ee so so ee ee 00 co so 90 so so so so so ee 0 ee so ee.0 00 ee.0...e ee 00 so ee.0 so.0 no es.0 00 e e. 00 go so so. '.. ' ee 00 ee.0 so 00 ee 00 so so so so we O. O. O. O. O o. Q....Q g... C. O..0 O. O. O. O :n w -1-p :Eeb.19,1927.4:00A.M. EE so so so so so C. O. O. O. O. O. O. O. O..9 O. O. C. O. O. O. O. O. O. O. O. O. O. O. C. O. O.. O. O. O. O. O. O..0 C. O.. O. O. O qu--ddc--pnc-nq--qugq nc-q---b u e. o. o. 00 g. M re 19,1927 e4:ooaeme a n n u n n n n :7 n n n o. o. o so.0 O ee 00 so as e. 00 co so we 00 e. so so ee 00 ee.0 00 ee 5 00 e. e. on 00.e so so so so we so ee.0 0. so e. 0 e. e. e O.

48 'd n......

49 Tabe. OContinued). --c--uuuu<p«nu-u.catnip-ncnununq-u.-nuqugpuncnu-u u...nun-qucnnpunc-coup-qucnucud13-:gnu-c.con-unqnundunncu-c.tutu-dc. CD---D--uh _-Q u g c. nn-s O-DQG-GOO Number of different: Number 01: Cream Urine.. Centrifuge urine : anima 3 Date of coection on stand.. after standing f---q ndividua: Series : urine and 24 z 48 : 72 ; 24 : tests :of tests: examined. hour of same. hour hour : hour : hour : hour hour.0 O. Q.. O. O. O O. O. O. O.. O. O. O. O. O. O. O. O. : 99 Apr. 9,1927.4:00A.M. 147 'to O. O. O..0 no O.. O. O.. 0 O. O.. C. O. O. O. O 'U- -CC. D O CQ- f' C--Dfb..D CCCHD UU.-CCCC::DCDD. O " 95 :Apr.23,1927.4:00A M. O O o. co co no 00.0 o..0 o. no so to o O O O O,0 O. O. O. C. C. O. O.. O. O O O CDC-nqqu-Chip-uute-Cu-n My 14, :00AOMo F on O. O O. O. O. O. O. O. O. O. O. O. O. O. O. O. 0. C O O. O. Q. C. O. O. o. O O O O O O O O O O O O 3 3 nun-unu-c-uounud-c-nu hn-n-ncnqncnunu : --D-QCDCD :-ucp--uqnu-unupun O CDDCCDC CD-D-.- O

50 ; n..... p i.... t

51 28 DSCUSSON OF TABLE. Eight different series of urine cutures were made from February 5, 1927 unti May 14, 1927, covering urine sampes from sixteen different cows. These eight series consisted of thirty different individua urine cutures; five from cow 99; three from.cows 95, 147, and 193; two from cows 47, 93, 176, and 232; and one from cows 33, 75, 84, 161, 191, 236, and The resuts obtained in both the uninocuated "cream-urine" mixture and the sediment of uninocuated urine were negative; ndividua urine sampe number thirteen'was not sufficient to run both tests so the "creampurine" method was used aone. Twice in the "cream-urine" mixture of cow number 33, once in twenty-four hours standing and again in forty-eight hours, coonies were present that had the appearance of Bacterium abortus but on transpanting to beef iver infusion agar sants the characteristic appearance disappeared.

52

53 '29 GENERAL DSCUSSON. n order that Bacterium abortus be present in the urine, it woud have to gain admission by one or more of three routes, namey, the urethra, bood stream, or ymph channes. To summarize from the writings of various investigators, Bacterium abortus is found in the urine of guinea pigs and humans with histopathoogica esions apparent ony in the kidney of the guinea pig. Athough these esions are aso present in the kidneys of rabbits and monkeys, no mention, apparenty, has been made of isoating the bacius from.the urine. Neither esions nor bacii are found in the kidneys or urine of goats. n regard to Brucea meitensis, the organism is found in the urine of guinea pigs, goats, and humans, and accompanied by esions of the kidneys. Rabbits and ambs, athough they show esions in the kidneys, appear not to have the Mata fever bacius in their urine. With these comparisons, excepting Bacterium abortus in the human, every case of urine contamination appears to be associated with pathoogica conditions of the kidney. These resuts bring up twoquestions: (1) s Brucea meitensis more viruent than Bacterium abortus? (2) Are the kidneys of sma animas more susceptabe to pathogens than those of arge animas? Khaed (13) states that from his experiments with Bacterium abortus and Brucea meitensis he found the atter is six times more viruent for guinea pigs. Pfenninger (34) caims that "certain experimenta infections of sma animas have a tendency to ocaize in the kidneys" e

54

55 n order that Bacterium abortus be present in the kidney, there has to be either a haematogenous or an ascending type of infection. Athough Bacterium abortus when introduced into the circuatory system of catte wi ocaize in the udder that has functioned or in the pregnant uterus, postmortem esions show that they do not ocaize in the kidneys. They appear to have deveoped an "organ viruence". Since the typhoid bacii pass through the human kidney into the urine, and Bacterium abortus does the same thing in certain animas, possiby through a esion of some sort, one woud be ed to surmise that it woud be possibe for the same to take pace if Bacterium abortus were present in the kidneys of cows. Hemhoz and Miikin (35), on introducing into the circuation aviruent and viruent staphyococcus, aviruent and viruent streptococcus hemoyticus, and aviruent and viruent coon bacii, found that there was no secretion of these organisms in the urine excepting where there was damage to the rena structures. They aso beieve that the endotheia ces of the arge capiary net work aid the kidneys in being activey phagocytic so that they are abe to dispose of arge numbers of bacteria. t woud be possibe for the ascending type of infection to take pace during a urinary stasis, but during norma canditions, with the continuous fushing of the ureters with urine it woud be impossibe for a non-motie organism such as Bacterium abortus, to ascend the umen of the ureters. The amount of urine excreted daiy by the cow is consideraby more than that of any of the animas, incuding man, that has been so far discussed in connection

56

57 31 with Bacterium abortus and Brucea meitensis. Sampson (16) demonstrated that it was not possibe to force fuid into the ureters from the badder. He removed a badder, cut off both ureters, and then fied the badder with a fuid, but was unabe to force any of the fuid through the severed ureters. The badder woud have to receive Bacterium abortus from the kidneys, bood, or the urethra. t is far more probabe, in view of the foregoing discussion, that the infection shoud arise from the atter than the two former. One naturay woud be ed to suppose that in the non-pregnant cow Bacterium abortus either succumbs to some unknown factor upon eaving the udder in the ymph system, since there are a ack of esions in the ymph gands excepting in the supramammary one, or that they do not eave the udder at a since there are no embryonic tissues in the non-pregnant cow. Wiiams (36) beieves that Bacterium abortus pays a negigibe part as the cause of cystitis. ' One woud suspect that Bacterium abortus, if present in the urethra, woud reach the umen of that vesse by way of the meatus urinarus, athough it coud as readiy do so by means of the bood and ymph systems. Sanderson and Rettger (37) were abe to produce infection by inocuating the urethra of guinea pigs, and mice. Trawm (25) out of eight heifers, produced abortion in two and permanent reaction in three by superficia intra-urethra injection of cutures. According to Embeton and Thie (16), bacteria, other than Bacterium abortus and Brucea meitensis, when paced on the urethra mucosa passed into the ymphatics of the wa of the badder and urethra and coected beneath the capsue of the kidney.

58 4,,

59 32 They aso stated that pathogens did not pass into the kidney or enter the urine but passed rapidy into the umbar gands and thoracic duct and from there into the bood stream. nasmuch as this was merey experimenta work Lubberkusen and Fitch (38) added, from their experimenta evidence, that heifers are not readiy infected with Bacterium abortus by way of the urethra. t is the common opinion of many investigators that the bu acts in the minor roe in regard to impanting abortion bacii in the vagina upon coition. Granting though that the mae acts mechanicay in pacing the pathogens in that organ their habitation there woud be very short. Carpenter (39) quotes Danzer and Berthod as finding that the mucous secretion of the vagina has a marked bacteria action and is a barrier against invading bacteria. t is true that infected feta membranes and discharges from the uterus of abortive catte do pass over the foor of the vagina and vuva, but the meatus urinarus is protected by being more or ess covered by the anterior wa of the suburethra diverticuum. k/m n these series of tests the writer examined cuturay the urine of nineteen different catte affected with Bang's abortion disease, over a period of ten months, considering of thirty-two different series of examinations of one hundred nine individua urine sampes. The urine of five cows, numbers 47, 93, 216, 236, and 239, was cutured two different times; three cows, 33, 175, and 191, three different times; four cows, 75, 95, 161, and 193, four times; cow 147, five times; cows 99 and 232, six different times; cow 84, eeven times; cow 171, tweve times; cow 1659, fifteen times; and cow 1665, six teen times without finding a singe Bacterium abortus coony.

60

61 33 SUMMARY. Bacterium abortus can ive and propagate in cows' urine for seventy-two hours. Bacterium abortus can be isoated from cows' urine that has been inocuated and incubated at 37 C. for seventytwo hours under ten per cent 002. Bacterium abortus can be isoated more easiy from cows' urinary sediment than from whoe urine, after the urine has been inocuated and incubated. Bacterium abortus at no time produced profuse coonies on any of the beef iver infusion gentian-vioet agar pates. Bacterium abortus was not found in uninocuated urine of catte infected with Bang's abortion disease.

62

63 54 ACKNO ELED Qa ENT. n.conousion indebtedness to Doctors. wish to acknowedge my Forest Huddeson and Ward Gitner for assistance and suggestions received during this investigation.

64 35 BBLOGRAPHY. (1) (2) (5) Huddeson, F. The Vaccina mmunization of Guinea-pigs Against Bacterium abortus (Bang) nfection. Mich. Sta.Co., Quart.Bfi.,Vo.7,p.63. Meyer, K.F.Shaw,E.B.Feischner,E.C The Pathogenicity of Brucea meitensis and Bacterium abortus for Guinea-pigs. Jour.nf.Ds.,Vo.31,p.159. Schroeder, E.C. The Cause and Occurrence of Contagious Abortion in Catte. Jour.Amer.Vet.Med.ASSO.,VOeXOV, Pe504e (4) (5) Schroeder, E.C. Recent B.A..EXperiment Station Bovine nfectious Abortion Studies. Jour.Amer.Vet.Med.Asso.,Vo.LX,p.557., Report of the Director Genera for the Canadian Veterinary Service. No. 5 and 6. (67 (9) (e) Smiie, W.E. Discussion on C M.Carpenter's "Resuts of njecting Pregnant Heifers with Brucea abortus soated froijan". Jour.Amer.Vet.Med.Asso.,Vo.LXX,p.459. E.W. An mprovement in the Method of soating and Recovering the Bacius of Catte Abortion through Guinea-pigs. Jour.Exp.Med.,Vo.28,p.585. Gitner,W. Huddeson,.F. and Tweed,R.L The Roe of the Udder and ts Secretion in Bacterium of nfectious Abortion, Jour.Amer.Vet.M6d.A880..VOeL-X, Do 1720 (9) Cotton, W.E. The Character and Possibe Significance of the Bang's Abortion Bacius that Attacks SW1n e Jour.Amer.Vet.Med.Asso.,Vo.LX,p.79.

65

66 (10) Hadey,F. B. and Beach, B. A Experimenta Study of nfectious Abortion in SWinee Wisc.Agr.Exp.Sta., Bu.55. Wester, HeHe nfectious Abortion in Domesticated Animas., nfection of Swine and Rabbits. Jour.nf. Dis., Vo. XXX,p.401. (12) (13) (i4) (15) (16) ( M" ) (18) (19) (20) ( 21f) Moher, JeRe Twenty-Fifth Annua Report of The Bureau of Anima ndustry.,p.279. Khaed, Z A Comparative Study of Bovine nfectious Abortion and Unduent Fever from the Bacterioogica Point of View. Jour. Hygiene, Vo.XX. p.319. Cesari, C Veterinary Prephyaxis of Mediterranean Fever. Jour.Amer.Vet.Med.Asso., Vo. LXV,p.637. Bosworth,T.J. and Gover,R. E Contagious Abortion in Ewes. Vet.Jour., Vo.81,p.39. Hundey, J.M., Jr Pyeitis in the Femae. Jour.Amer Med. Asso., Vo.86, p.86. Schroeder, E.C. ( V \ Bureau of Anima ndustry nvestigations on nfectious Abortion. Jour. Amer.Vet.Med.Asso., Vo.LX,p.542. Hadey, F Contagious Abortion in Catte. WSC.Agr.EXP. Sta-e. E Carpenter, C.M Resut of njecting Pregnant Heifers with Birch, R.R Brucea abortus soated from Man. Jour.Amer.Vet;Med.Asso., Vo.LXX,p.459. and Giman, H.L. The Channe of nvasion of Bacterium abortus with Specia Reference to ngestion. The Cor.Vet., Vo.XV,p.92. Schroeder, E. C. and Cotton, W. E Some Facts About Abortion Disease. Jour.Amer.Vet. Med.Asso., Vo. L, p.321.

67

68 37 Kavarzik Reference. See Hutyra and.marek, V01., Do 7860 (25) (24) (25) (26) (27) (28) (29) m ) (31) (32) (53) (34) Cotton, W.E Abortion Disease in Catte. Jour.Amer.Vet.Med.Asso., Vo.LV.p.504. Huddeson,.F Studies on a Non-viruent Cuture of Bacterium abortus Towards Protective Vac- EhatiOn of Catte Against nfectious Abortion (Bang's Abortion Disease). Mich.Sta.Co.,Tech.Bu. 65. Hart, G.H., Traum,J. and Hays, F.M Bovine nfectious Abortion. Caif.Agri.Exp.Sta., Bu Huddeson,.F., Hasey, D.E. and Torrey, J.P Further Studies on the soation and Cutivation of Bacterium abortus (Bang). Reprinted from the Jour.nf.Dis., Vo.40. Stitt, E.R Practica Bacterioogy, Bood Work and Anima Parasitoogy, pp.104 and 569. Park, W.H. and Wiiams, Anna W See Pathogenic Microorganisms, p.290. Buchanan, R.E See Veterinary Bacterioogy, p Z91er, Ho Waiters Untersuohungen aber das senchenhafte Verwerfen des Rindes. Archiv.wissenschaft praktische.,49,heft 1-5: Zwick and Wedemann See Hutyra antharek, Vo.,p.781. Rauchbaar, G Uber das Verhaton Keimfreier Abortuskuturftrate in Vitro und in Vivo.naug.Diss. Vet.nst., Univ.Leipzig. See Ezp.Sta.Rec., 1924, Vo.50. Gates, FOL A Method of Standardizing Bacteria Suspensions. Jour.Exp;Med., Vo.51, p Pfenninger,W Our Present Knowedge Regarding White Secure and Simiar Diseases in Caves. Jour.Amer.Vet.Med.Asso., Vo.LxV,p.6a.

69 58 (55) Hemhoz, H.F. and Miikin,F Reprint from Amer.Jour.Dis.Chid., 1925, Vo.29. Jour.Amer4Med.Asso., Vo.84. (36) Wiiams, W.L. The Diseases of the Genita Organs of Domesticated Animas,p Sanderson, E.S. and Rettger,L.F The Paths of nfection by the Bacterium abortus. Jour.nf.Dis., Vo.32,p (38) (39) Lubbehusen, Carpenter, R.E. and. FitCh, 00?. A Report on EXperimenta Work on the Bu as a Factor in the Spread of nfectious Abortion. Jour.Amer.Vet.Med.Asso., Vo.LXV,p C.M. The Bacteria Fora of Genita Tract of Catte and ts Reation to Caf nfection. Jour.Amer.Vet.Med.Asso., Vo.LV,p.676.

70 fm1 UM CM.) RUQH 13K;, AM. v-u - i.

71