ev aT Sea aa THESIS FOR DEGREE OF M. 8. STANLEY GEORGE BANDEEN ee ee a, mebert Catters THESIS Dareders ve Mich. LO. The relation of the bacterial flora of the uterus, to that of the meconium of the calf. Thesis. Submitted to the faculty of the wichigan agricultural vollege in partial fulfiliment of the requirements for the Vegree of waster of Science. Stanley George Bandeen @’ qa la CONTEHIITS Introduction, © neview of Literature. ethod of Investization. uwedia used and wethod of Isolating Organisms. Guinea Pig Inoculations. Agglutination Test. Experimental iWork. Case neports. history. work accomplished. results. Tables. Swuuaary. Acknowledgement. xeferences, Index. 93838 TEE ReLsTICw Cs THe BatCTunIaL #LORa OF [His UTLRUS, TC Thal’ O« THE iiCGOGlhlUs. OF THs VALE Introduction. This project was undertaken with the hope of throwing some light on two important phases of the abortion disease, first on the relation of bacterial infection of the uterus to that of the alimentary canal of the retus, and second, on the persis- tance of the abortion bacilli in the uterus subseaouent to abor- tion and parturition. In the course of the work some data were collected with reference to the bacterial flora of cystic ovaries and of the uterus of sterile cows. neview of Literature. as early as 1885 hocard (1) made a scientific investi- gation of inlectious abortion at the reauest of the rrench «win- ister of agriculture. ode concluded anong other things, that the disease was due to bucterial inivection of the fetus and placenta, though he was unable to designate the specific organisn. It was left for 3ang (2), and his assistant, Stribolt, in 1895, to dis- cover the organism and to ceiwonstrate its definite relationship to the disease. «4 detailed account of this discovery and of the organism is given in their original puolication (1897). Nowak (3) made an inportant contribution to the study of infectious abortion by improving sang's ;ethod of isolation and cultivation of 3ang's bacillus under conditions of reduced w& i oxyren + pressures brewsie -sout with the acid of B. subtilis in sealed jars. s,owak's method is described in a bulletin by Good (4) of Xentucky. The more recent WOrk on infectious abortion deals large- ly with the inethod of diagnosis, orevention, pathological chang- es, and the various organisms found in the uterus. done investi- gators claim that there are other organisss besides sang's bacil- lus that cause abortion. woussu (5) is of the opinion that certain strains of colon organisms will cause abortion. «c*adyean and Stockman(6) Claim that uterine infection with tubercule bacilli as well as with streptococci may result in abortion. Good (7) reports that in one instance, he found the foetal tembranes and amniotic fluid of an aborting cow teeming with the Staphylococcus pyogenes aureus in pure culture. The cow becane weaker and weaker until it became evident that she would die. she was then slaughtered and autopsied. the uterus was fil- led with pus, which was found in numerous other internal organs, Staphylococcus pyogenes aureus being the irvader. It is evident that this organisn entered the uterus causing the expulsion of the fetus and afterwards becoming sufficiently generalizei to kill the cow, Williams (8), in his report on "Researches upon abortion of vattle" (1916), states that the bacillus of Bang is frequently found in the stomach and fetal blood or glands, but is never found in the alimentury tract. he clains this indicates that the pri- mary invasion was throuzh the chorion into the amniotic fluid. dilliams (9) reports that the amniotic fluid is being constantly swallowed by the calf. ‘This being true, a bacterio- logical exanination of the meconiwn should give an idea oy the organisms present in the amniotic fluid in case any are present. In 1916, hagan (10) began work on the meconium of new born calves in connection with the general problem of eontagious abortion and allied diseases. The meconia of a total or fifteen new-born calves were examined. s«ourteen of the calves were in two different herds, six being in the first and eight in the se- cond, one was fro.: an unbori: calf obtained at an abattoir. the meconia from she six Calves in the first herd all proved to be sterile. In the second herd, three were sterile, while the other Pive gave growths. whree of the cultures were of colon bacilli, one was a streptococcus, and one was mixed a streptococcus and a staphylococcus, she abattoir specimen cave 3. coli. Ragan gives the following result of the examination of the uterus of sixteen preznant cows. -«zhe utero-chorionic space eave cultures in fourteen instances, or in about 87 percent, the fetal fluids gave cultures in five cases or in about 33 percent. The work oz: Hagan shows that there is a relation of the organisms found in the utero-chorionic space to those in the amniotic fluid and in the meconium. 4“ question in the minds of many investigators involves the length of time the abortion bacillus renains in fhe uterus after abortion. Schroeder and votton (11) injected cultures of the abortion bacillus into non-pregnant uteri of cows. ‘he or- ganisms were found to all disappear in the course of a few days. Kxamination of the uterine discharge of a cow which had aborted, Shows that the abortion bacilius persists for a period of fwm twenty to thirty days, after which time the organism disappears. It is the opinion of Schroeder and votton that the abundance and period of persistence is intimately related to the magnitude of the lesions in the uterus attendent upon an abortion. Schroeder and votton (12) have verified through their tests that aborted fetuses harbor 3ang's bacilli in their stomach, liver, intestines, lymph glands, spleen and blood, and that in all cases in which the fetus ..as infected with abortion bacilli, the organism was found in the utero-chorionic space. Huddleson (13) reports the findine of Bact. abortus in the stomach, liver, spleen, kidney and blood of aborted fetuses. woussu (5) was the first to make the statemont that ther> were other organisms besides 3ang's bacillus which caused abortion. Since this report of «woussu was made, other investigators have re- ported different organisms found in cattle subsequent to abortion. Egglink, reportec by ward (14), records a bacteriological inves- tigation of 20 cases of tndometritis. In the 20 cases tr re oc- cured: Tubercle bacilli-~---------~-------- £ Bacillus pyogenes--------~ a2 =- 14 Streptococcus-------------------- 12 Volon bacillus-------~-----------.- 6 Staphylococcus-------------------- 5 Bacillus proteus------------------ 3 Bacillus subtilis-----------~------ 1 In eleven cases reported by Wall (15) there occured: Streptococcus-~-------------------- "7 Colon bacillus------------~-------- 5 Bacillus pyogenes----------------- 3 Anaerobic bacilius (in one of these bacillus of malignant edema)--2 Necrosis bacillus------------- 1 Froteus bacillus---------~-/--.. 1 dall notes th2t of these only streptococcus and B. pyogenes heve been found as pure infection, the others always be- ine associatei with other microorganisms. wall suggested that the streptoccccus infection sees to be the most acute, as in a very short time it can cause a very deep and diffuse necrosis. ‘the pyogen-s iniection proceeds more slowly and less intensely, but is of longer duration. vomplication with other infections with pu- trefaction of the inflammatory exudate considerably increases the Changes. Yifferent means of diagnosing the presence of abortion disease and of isolatin= sang's bacilius have been used. 4auddleson (16) inoculated guinea pigs intraperitoneally. After an incuba- tion period or about twelve weeks, the antibody formation and the lesions of abortion were determined. vooledge (17), «vans (18), Schroeder and Cotton (19) determined the presence of Bact. abortus to be common in milk by inoculation into suinea pigs. iihenever 3ang's b2cillus was present in the ilk there was gs slow develop- ment of lesions which are characteristic for this organism, The method of the above investigators in isolating Bang's bacillus by puines pig inoculations is a very slow process. he latest method is by Snillie (2) and is considered by him to be much better. Lystic ovaries have been studied to some extent in con- nection with abortion and sterility. thiss, reported by Albrechtsen (21) believes that cysts may be the result of the disturbance oi nutrition of the graafian follicle. Loeb (22) has recently srown that cvarian cysts may be produced in guinea pigs by underfeedins. In his experiments tne feed was reduced to such an e:tent that the guinea pigs lost from -0 to 3 percent of their wright in from 7 to 14 days, and withcut exception cysts were found in the ovaries No cysts developed in the control animals, THis, however, was extreme underfeeding. Fitch (23) reports that in all cases where a cystic corpus luteum wes cultured, vigorous growth was obtaine:. One ovary eave a pure culture of a long chained strep- tococcus. 3. coli has been found in several ovaries. .icroco- ccus pyogenes has been found twice. « rod Which seems to be a slight gas producer has been found three times. albrechtsen (24) believes that ovarian cysts may result from direct infection from the uterus through the tubes, but that they are more fre- quently produced reflexly. wethod of Investigation. Immediately after abortion or apparently normal partur- ition, (cases in which the calf lived) the buttocks and tail of the calf were thoroughly washe« with mecurial Soap and water. A sterile rubber glove was then put on the hand and by means of the first finger inserteci into the rectum a small amount of meconiun was obtained for bacteriological examination. If the fetus was dead at the time of abortion, or died in a few minutes afterwards, it was taken to the laboratory where the meconium could be obtain- ec under strictly aseptic conditions. «at the laboratory, the fetus was opened to expose the stomach and intestines, then a red hot spatula was held on an area of the stomach, as weil as on a part of the colon to be opened. «a Sharp scalpel, that had been previously boiled in water, was used to open the stomach. A different scalpel was used for the opening of the colon. Thé material was taken from the stomaeh by a sterile 10 c.c. pipette which was forced through the smali opening msde by the scalpel. The meconium was taken from the colon by a small spatula. iaterial from each wes placed in a sterile uwsmarch dish. material was collected frou the uterus of the cow with- in a few hours after ebortion or apparently normal parturition, al- so, on the following day, and then once each week for several weeks. ‘The buttocks, tail and vulva of the cow were thoroughly washed with mecuri2l so2av and water. Whe vagina wes washed out with plenty of physiological salt solution. ‘vhe uterus was re- tracted with uterine retractors, and if in any case the cervical canizl was closed or too small for the metal catheter, it was first dilated with a pair of uterine dilators. ‘Yhe introduction of a pair of dilators into the cervical canal should be done with great caution, 2s the congested mucosa is easily perforated. Ordinarily fatal conseauences do not result. after dilation of the cervical canal, a i.etal catheter was introduced into the uterine cavity. the catheter cannot be introduced to any con- Siderable distance unless the end is turned to one side or the other, as the body of the uterus is comparatively short. If the catheter is inserted straight in, it wilsz come in contact with the anterior wa.l sepsrating the right and left horn. the catheter was then connected with a force pump by means of a rubber tube, end the sterile physiological salt so- lution pumpe* into the uterus. a return ilow catheter was not usei in this work. It was necessary to compress the rubber tube and hold the solution in the uterine cavity until the uterus was well masseged per rectum. «hen the uterus was well masseged tne rubber tube wes renoved fron the catheter an@ a sinall amount of the solution aliowed to escape. a sterile flask was used to catch the remainder which was taken to the laboratory. In bad cases of pyometra, with abundance of pus, it w2s not necessary to use the physiological salt solution except for flushing the uterus as a therapeutic measure. Just massag- ing the uterus per rectu.r was enouzh to force out through the catheter a larse quantity of pus, which was collected in a flask and used for the examination. wedia used and »ethod of Isolating Urzganisms, the media used for isolatinzs the organisms found in the material collected were: serum agar slants. serum agor plates. Plain agar slants. Flein agar plates. Bouillon. she serum w2s mude by bleeding a cow or horse asepti- cally, allowine the blood to vorm a clot. ‘the serum that rises to the top was drawn off and creserved with chloroform. ‘he serum agar was made a day or two before using by meltin: tubes of plain agar and ellowins them to cool to 40 or 45 degrees OC, perum was added in sufficient amount to eaual one third of the amount ot sgar. If plates were to be used the iixture was pour- ea into a petri dish ani allowed to harden. whe slants were made in the usual way. Urdinsry plain ager which was meade neu- tral or very slightly acid (1.5 percent normal) to phenolphthalein was used. the same process wes used in isolating the organisms from uterine materiel 2s from the meconiuia or cysts. Whe me- Coniun w2s brozxen up and mixed well with a few cubic centimeters of physiological salt solution. A platinum loop was inserted into the material to be examined and then streaked across an 2far plate four or five times. A second and a third plate were streak- ed in the same manner with the original material on the needle. The pletes were incubated for 48 hours at 37 degrees 0. The first plate might have to be discarded, but the second ¢1.4 third plates had many isolated colonies. sach colony was marked with a blue pencil and the plates were put in a Novy jar with plates of 8. subtilis and incubated at 37 degrees vu. for about four days. The cultural methods of iiowak (3) were used. a macroscopic examination of the colonies was made with a good hand lens or the 110i; power objective of the microscope. Transfers were made from all the different kind of colonies to agar Slents. vthe different kind of colonies formed on the serum agar plates were transfered to serum agar slants and plain agar Slants. ‘the small transparent, dew drop colonies that developed on the plates while in the lhovy jar were transfered to bouillon and incubated for two days. then a transfer from this wes made to another bouillon tube which was 2lso incubated for one or two days at 37 degrees U. A transfer wes made from the second tube of bouillon to a plein agar slant. tater in the work, it was found that dextrose agar slants gave better growth for Bact. abortus than the plain agar. +t has been found, by running duplicate tests using plain agar in one case, and agar slants and bouillon, :s described above in the other test, that the latter method gave a much better growth in a much shorter period of time and with less chance of losing tne orgunism, ‘whe serum 2gar was used for isolatins 3B. pyogenes. isolated: the followins medic were used to Plain agar. Vextrose 22ar. serum agar. serum slants. Blood e&gar. «auannit sugar. Litmus lactose agar. Gelatin. Pork celatin afar. Litmus milk. Plain milk. Lactose bouilion. identify the organi wus ) ) Placed in fermentatiion tubes Dextrose bouillon. ! and neutral red added. ) sacchrose pouillon. Bouillon. LSCulin bile. Potato slants. Glycerine afr. Soluble starch agar. Dulcite agar. yvorbin agar. arabinose 29ar. Inulin agar. Inulose agar. The agar for the last six media, was made of agar-arar, water, salt and peptone, with one percent of the desired sugar added. any organisms especially the different tyoes of the colon group will ferment the ordinary plain agar, -~ill- Guinea tig Inoculstions. another method used to determine the presence of Bang's bacillus in the uterus and meconium was the injection of guinea pigs with the riaterial collected. ‘Two cubic centimeters of a so- lution of meconium was injected intraperitoneally into each of two guinea pigs. Yhis was continued for about four washings covering a period ot four weexs. ‘the blood of the guinea pigs was tested for abortion every month after injection, for a period of three months. from twelve to fourteen weeks after the injec- tion, the pigs were killed and examined for abortion lesions, The blood was tested by the complement fixation and agglutination tests. the techniogue ex»ployed in the complement fixation test is that of Hadly and Beach (25) of wisconsin. Table I, Agelutination Test. 1 Vv Ty v ' Tube ' Antigen 'SBerum ' Dilution ' ' 6.C. ‘undiluted ' ‘drops t T | T rol ' 2 ' 2 ' 1-20 q v | T ' 2 ' 2 ' 1 ' 1-40 ' ms "serum dilut | ' ‘ "ed 1-20 ' , ' ’ drop ’ q v OE T ' 3 ' 2 I 8 " 1-100 ’ r 1 ’ 4 "2 ' 4 11-200 ! r 1 ? ~ ' 5! 2 ' 2 ' 1-400 t Tv v qv r 6 ' 2 ' 1 ' 1-800 Shake all tubes well and incubate over nicht at 37 degrees OC, -~12- In abortion work it is not necessary to use the first tube which is a dilution or 1-20. In the above table a dropper system was used. ihe cap- illary pipettes were graduated to deliver twenty drops to the cubic centimeter, ixperimental Work, The experimental work was begun in June 1918. wach an- imal worked with is taken up individually. A history of the an- imal, if any could be obtained, is given. Case 995 was purchased at the age of one year (1913). Blood test august 20, 191é, negative. Bred “ay 11, 1914. November 15, 1913, she received @ 10 c.c. culture of dead Bact. abortus, also, in December 1913, January and ebruary 1914. Complement fixation and agglutination tests made after these injections showed a positive reaction until way 29, 1914. November 20, 1916 and December 10, 1916, she was given subcuta- eously 5 c.c. and 10 c.c. dead culture of cact. abortus. tebru- ary 12, 1917, she was given 20 c.c. of live culture of Bact. abortus subcutaneously. She developed a positive reaction «ebru- ary 12, 1917 and has remained positive until the present time, Valved January 15, 1916. Bred Uctober 17, 1917. Valved June 24, 1918 the 249th day of the period of gestation. ollowing this, a uterine infection developed. The discharge from the uterus which was mixed with mucus, was of a thick yellowish non-purulent nature. «he contents from the first ~13- washing (the day of parturition) were centrifuged and 0.5 cubic centimeters of the sediment injected into each of twe guinea pigs. A microscopic examination of the first washing revealed many short rods, a few long rods, and cocci, also, a few lymphocytes. July 3, 1918, after collecting material for examination, the uterus was treated with a 25 percent Lugol's iodine solution. A microscopic examination of the uterine washing at this time revealed many short rods and cocci, also, some lymphocytes and epithelial cells. July 8, 1918, the discharge was more the nature of mucus and was not as abundant as when previously treated. After collecting material for examination, the uterus was again treated with 25 percent Lugol's iodine solution. Results obtained from the guinea pigs; One pig died of an infection of B. coli communior. ‘the second pig was bled and then autopsied. ‘The pig had a slightly enlarged spleen, but Bang's bacillus could not be isolated. ‘the complement fixation and ag- glutination tests of the guinea pig blood was negative with Bact. abortus antigen. The organisms isolated from uterine washings were: B. subtilis. B. coli communior. Staphylococcus pyogenes aureus. Staphylococcus pyogenes bovis. Streptococcus pyogenes. B, 121.2421032 (see table VI) the cow was bred July 26, 1918. Doctor Hallman examined this cow for pregnancy January él, 1919, xternal os was open to such an extent that the thumb could be introduced as far as the second fold. She was killed for ~ 14- beef January 30, 1919, and the following notes were taken. TFreg- nant in left horn. FVosterior presentation. The head of the calf was bent backward and to the left, with the point of the head ly- ing on the left shoulder. VCervical canal was slightly open at the posterior eni. ‘the canai was quite well filled with mucus of preg- nancy which was protruding back into the vagina. In the body of the uterus and posterior ends of both horns, the chorion was attached to the uterine mucosa by a diffuse development of cotyledonous tissue. This cotyleaonous tissue consisted of tufts varying in size frmm one centimeter to nodules three centimeters in diameter by five centi- meters in length. she area involved was avproxi:.ately fourty centi- meters long. along the dorsal wall of the pregmant horn there was apparently @ diminition in the number o? cotyledons. In other por- tions of the uterine cavity the development of cotyledons was appar- ently normal. ‘the cotyledons varied from three to eight centimeters in length. i1n the utero-chorionic cavity there was a small quan- tity of the so called abortion exudate. w«aterial was taken from the utero-chorionic space, amnionic fluid, and meconium of fetus for bacteriological examination. suo organisms could be grown from any of the above. Case 995 B., the calf of cow 995, born on the 249th day of gestation, was very weak, but lived. .«econium was obtained for examination. Two c.c. of @ suspension of meconium was injected intraperitoneally tnto each of two guinea pigs. the organisms isolated were: B. coli communior. Staphylococcus pyogenes aureus. On September 26, 1918 the pigs were bled and then autop- Siei. io abortion lesions were found. ‘The complement- - — | -~ 15 - | fixation and agglutination tests of the guinea pigs blood were negative. vase 801 was purchased September 1, 1917, Her blood reaction at that time was negative. She was bred “ovember 3, 1917. «at week intervals, beginning on ay 29, 1918, she received intravenously four injections of 5, 10, 20, and 20 cubic centi- meters respectively of live culture or Bact. abortus. On June 7, 1918, one week after injection of Bact. abortus, she developed a stronz positive reaction to the complement fixation and ag- glutination test rescectively. whe aborted vuly 17, 1918, the 257tn day ot the period or sestation. ‘whe calf was very weak and died in about one hour. ‘she following day the fetal membranes had to be relio Vel« at this time the uterus was treated with Sep tico- solution and bozvic acid. Un vuly <0 and 26 and august lst, the uterus was washed out with physiological salt solution and the Sanples taken to the laboratory for exanination. aA microscopic examination of the various washings revealed many small rods and cocci, also, a few lymphocytes and epithelial cells. In the bacteriological examination, the following organ- isms were isolated: ° 5. coli comunior. Streptococcus pyogenes. Staphylococcus pyogenes bovis. Bact. abortus. Bact. 122.42£2034 (See table VI). Case 8014, the calf of cow 801, was aborted on the co7th day of gestation, and lived only about one hour. some of the meconium and contents of the stomach were obtained for bacteriolocical exanination and for injection into cuinea pies. The bacteriolocical examination of the stomach showed no growth aerobically or anaerobically. ‘he organism isolated from the meconium was Staphylococcus pyogenes bovis. The blood of the guinea pigs was negative to both the complement fixation and agglutination tests for abortion. autops: revealed no lesions of abortion. Vase 802 was purchased September 7, 1917. The blood re- action at the time of purchase was negative. Sred for the first time September 21, 1917 and calved June 27, 1918. %he hes remained negative up to the present time. She developed a uterine infection after parturition, the discharge being of @ bloody nature. ‘the microscopic examination revealed nany cocci and a few snort rods, also, many leucocytes and a few epithelial cells. ‘whe cervix was perforated on July 18, 1918, so no more material was collected. the organisms isolated were: Staphylococcus pyogenes aureus. Staphylococcus pyosenes bovis. Streptococcus pyoreness. the guinea pigs did not show any lesions of abortion. The complement fixation and agglutination tests of the guinea pigs blood were negative. vase 802 is the calf of cow 802, born June 27, 2918. whe calf was strong and healthy. .iieconium was obtained for examination. the organisms isolated were: Staphylococcus pyogenes bovise 3B. coli communior. the guinea pizs which had been injected with meconium were bled and then autopsied. Autopsy revealed no abortion. “are - ~ lesions. ‘he complement fixation and agglutination tests of the blood were negative, Case B. S., a 3rown Swiss cow from a herd near by, aborted July 5, 1918, on the 200th day of gestation. -laterial w2s collected from the uterus only once. +he organisms isolated were: 3. coli communior. B. coli (Sub. vroup I). Bact. 212.2442014 (see table VI) Bact. 111.2421014 (see table VI) 3act. 122.1111032 (see table VI) Bact. 222.2121012 (see table VI) vase 3B, S.a, the fetus of the srown Swiss cow, was aborted on the 200th day of gestation. ileconium and material from the stomach were obtained for examination and injection into guinea pigs. No organisms were found in the stomach. The blood of the guinea pizs was negative and on autopsy no lesiont of abortion could be found. The organisms isolated from the meconium were: Be coli communior. B. coli (Sub Group II) Staphylococcus pyogenes aureus. Bact. 212.2441014 (see table VI). A bacteriological examination of the heart blood of the fetus revealed 8. coli communior. Case 805 was purchased September 7, 1917. Blood re- action at the time of purchase was negative. She was bred November 3, 1917. «at week intervals beginning say 29, 1918, She received subcutaneously 10,20,40, and 40 Gubic centimeters respectively of live culture of Bact. abortus. On June 7, 1918 ~ 18 - she showed a partial reaction to the agglutination test; on June 21, 1918, she showed a marked positive reaction to both the Ccomplemsnt fixation and agslutination tests and has maintained that reaction to the present time. she calved August 7, 1918. the calf was strong and apparentiy healthy. she cleaned in a short time, but developed a bad case of endometritis, with an abundance of dirty brown, thick discharge from the uterus. august 15, 1918, after some muterial was collected for examina- tion, the uterus was treated with 2 oz. of a 25 percent Lugol's iodine solution. Smears were made from the uterine material at the time of the first washins and many small rods and cocci were observed. There ware also some lymphocytes and epithelial cells present. the orgariisms isolated were: Sact. abortus. 3. pyogenes. Streptococcus (rcran negative) Staphylococcus pyogenes albus. Staphylococcus pyogenes bovis. Streptococcus pyogenes. In this case the sact. abortus persisted in.the uterus for 28 days after parturition, -Casge 8054 is the calf of case 806. «he calf was strong and apparently healthy. «meconium was obtained for guinea pig injection and for bacteriological examination. the organisms isolated were: staphylococcus pyogenes aureus. B. coli communior. ~ 19 - The guinea piss were bldd and then autopsied. The autopsy re- vealed no lesions of abortion. The blood was negative by both the complement fixation and agglutination tests. vase 9964 was born uisy £8, 1916. There is no re- cord of her until November 9, 1917, at which time the complement fixation and agglutination tests or her blood were positive. She had a very difficult parturition from an over sized calf warch 31, 1918. « bad case of pyometra developed and B. coli, streptococcus pyogenes, and B. pyogenes were isolated from the uterug by Doctor seaver. she return of the uterus to an ap= parently normal condition within a few weeks w2s probably due to the treatment recommended b: Doctor Hallman, The latter part of July she developed a bad case of endometritis. The discharge was of a dirty brown or reddish brown color, sticky in character, adhesive to the tail and other parts of the body with which it came in contact. ‘The uterus was washed out several times with physiological salt solution and the material was used for bacteriological examination, The organisms isolated were: streptococcus pyogenes. B. coli communior. Bact. 222.1131032 (see table VI). ysmears of the material from the uterus revealed many rods and a few chains of streptococci, also, a few epithelial cells and lymphocytes. she was killed for beef Yctober 7, 1918. hotes on the macroscopic examination of the uterus: Between the body of the uterus and the rectum there was an abcess containing about three ounces of thick creamy pus. - 20 -_ The body of the uterus and vagina were firmly adherent to the rectun, The left oviduct was apparently normal. The right oviduct was normal except for the presence of a small cyst about 2£m.m. in diameter situated about midway between the ovarian and uterine end of the oviduct. The left ovary was covered by peritoneum and showed a few immature cystic follicles on the surface. the right ovary was completely ensheathed in fibrous tissue tnat was a continu- ation of the abscess cansule. she cervical canal Was six centi- eters lom-. Vhe body of uterus was two centimeters long, uterine cavity contained apparently no exudate; the :nucosa was moist and glistenine. ‘he color was not entirely uniform, but oarticularly around some of the cotyledons, including the coty- ledons of the left horn, the mucosa was more vascular. ihe cotyleijions varied in size from one quarter to three quarters of @ centimeter in diameter. ‘The apex of the first cotyledon in the horn which was about one and one half centimeters in dia- meter, was distinctly vascular or like a recent hemorrhage (bright red). The gland mucosa otherwise was of a grayish chocolate color. On the tissues of the broad ligament there were several small abeesses varying in size up to the size of a hazelnut. These contained a thick crewmy pus. Unfortunately, it was not possible to make a bacterio- logical examination of the pus as the case was available at the time the use of the laboratory and assistants (sir. Welsh, mr. Kidmans and myself) ware given over to the bacteriological work in connection with the influenza epidemic in the -~. a, 7. C, - £1 - Case 808 was purchased september 7, 1917. Blood re- action at the time of purchase wes negative. september 23, 1917, she was injected subcutaneously with 25 cubic centimeters of live culture of Bact. abortus. October 5, 1917, she ieveloped a marked positive reaction th the complement fixation and agglutinstion tests. This positive re- action was maintained until she was killed. she was bred sovember 1, 1917 for the first time and on several occasions afterwards, but failed to conceive. she was @ nymphomaniac and was, therefore, killea august lz, 1918. The reproductive organs were re.ioved and taken to the laboratory for a bacteriological as well as for a macroscopic examination. Swabs were taken from the uterus; a bacteriological examination of these failed to show any organisms. an examination of a cystic ovary revealed two organ- isms. B. coli communior. B, 121.4442012 (see table VI). Lotes on the macroscopic examination; The vagina con- tained a considersble quentity of clear mucus. The cervix was contracted. here was a slight amount of adhesive mucus in the cervical canal. whe body of the uterus which was four centimeters long was very much thickened and avparently fibrous. The mucosa of the horns was yellowish gray and moist. ‘he cotyledons of the horns were one quarter to one half certimeter long and one quarter centiueter wide. Uverian tubes were ap arently normal. Right ovary which was three centimeters long and oval, - £2 ~ contained a cyst about the size of a hazelnut. lo corpus luteum was present. Left ovary was Slizhtly smaller; it presented a cic- atrix extending around its dorsal border. 40 corpus luteum was present. Vase 13, was born august 30, 1915. Calved in 1917. Valved July 10, 1918. There was no history of abortion, retained placenta, or difficulty in getting with calf. She has shown no signs of estrum since calving. This cow was killed for beef August 9, 1918, and the reproductive organs saved for bacteriological and iacroscopic examination. otes taken on the macroscopic exanination are as foliows: Vagina contained considerable mucus. kxtermal os clos- ed. Cervical canal about nine centimeters long. Body of uterus five centimeters long. Uterine mucosa a light chocolate color. Cotyledons one quarter to one half centimeter long. volor was more yellowish gray than the gland mucosa, ~ucosa of the uterine horns about thé same color as that of the body. votyledons average a little larger. Oviducts apparently normal. night ovary about three.centineters in diameter. ‘here was a2 cystic corpus luteum present; also about 20 small cysts. Left ovary three centimeters long and one and one half centimeter in diameter. showed no regressed corpus luteun, Bacteriological exauination: lo growth from uterus. vystic ovary contained 38. coli communior. ~ 23 - vase 14., was a cow from a beef nerd that reacted to the tuberculin test without showiny Lesions post uortun. the reproductive orctans were re..oved and taxen to the leborstory. ‘the bacteriological exanination of tne uterus gave nezative results. lotes on the macroscopic exanination are as follows: The vagina was apparently normal. usxternal os, which was Closed, was about three centimeters in diameter. vervix about fourteen centi:.eters long. --ucosa was light chocolate color. voty- ledons were one to one half centimeter long and showed a depressed center. --ucosa of the horns was light chocolate color but not uniform. some areas showed a slight congestion. votyledons a- bout one half to one and one nalf centimeters. Oviducts were ap- parently zormal. Risht ovary was about five centineters long. It showed @ protruding corpus luteu: and either a cyst or a graafian fol- licle. Left ovary was about five centimeters long. vase L, il. was @ cow from a neighboring dairy nerd. Her first calf was aborted, after which she had three normal calves. warech 12, 1917 was tne last time she calved. she was bred four tines since tha last calving but failed to conceive. She went fron way 29, 1917 to December 20, 1917 without occur- rence of estrum. ‘the last estrual period was July 8, 1918. An exanuination was made by Yoctor Hallman for ster- ility July 24, 1918. he vagina showed a small anount of almost Clear imcus. he os uteri was contracted and apparently normal. lhe uterus was not enlarged and was apparently normal. The ~ 24 right ovary was norma] but the left ovary showed a protruding corpus luteum. ‘The uterus was douched with physiological salt solution. ‘The vagina and cervix were douched with salt-soda solution. Bacteriological examination of the uterus was negative. The vaginal secretion was very slightly alkaline. A few rods and cocci were present, also, a few epithelial cells but no lencocytes. Cervix was slightly alkaline. smears from the cervix showed considerable mucus, but only a few cocci and small rods were present. There were a few epithelial cells and numer- ous lymphocytes. September 10, 1918, she was bred and conceived. Case D, B. was examined by Doctor Hallman for sterility, July 16, 1918. The date of the last calving was January 15, 1917. She had been served st irregular intervals for more than a year Since last calving. Her general condition was good and state of lactation was dry at the time of examination. Os uteri was com- paratively dry, cicatrix on lower left side. Uterus was not en- larged and tone was firm. Right ovary was about two centimeters long and left ovary was the same size with small cysts about the size of a pea. dSecretions from the vagina and cervix were acid. snears from the vagina revealed quite a few epithelial cells and an occasional lymphocyte. «A few rods usually in clumps and a few cocci were present. some of the epithelial cebls show- ed a few cocci in the process of phagocytosis. Smears fron the cervix showed numerous epithelial cells a few lymphocytes and a few cocci. The cervix was flushed with salt-soda solution. The uterus was flushed with sterile physiolozical salt solution. Some of this salt solution was collectec in a sterile flask for ~ 25 - an examination. Then the uterus was flushed with a salt-soda solution. One organism was isolated from the uterus; Bact. 211.1421012 (see table VI). August 8, 1918, the vagina and cervix were flushed with salt-soda solution and then the cow was bred and conceived. Case ».. C. never had a normal calf. She had two dis- tinct abortions one of which was her last calving on June 16, 1917. clear fluid, also, several small cysts spherical in shave, eft ovary three and ore half cen- tineters lorg, slightly flettened; cyst one ce:tineter in dismeter. (ne orgai.isz, 35. coli cOmcunior was isolated from tne cyst, vase 9973 was born «ebruary 4, 19156. -+he cow was nega- tive to the complexent fixation and agglutination tests. January 1s, 1917, she received £5 c.c. of a live culture or Bact. abortus. October 12, 1918 and Uctober 15, 1918, sre received 5 c.c. of a live culture of Bact abortus on feed. she calved «ovexmber 15,1918 and has remained negative to avortion up to tne present time. anniotic fluid was collected at tre time oz varturition. .saShires frou the uterus were collected three tijzes. -zollowing this, the os was closed so no more waski:.-s were obtained. the amniotic fluid was sterile, All the uterine washi:es were sterile. ‘he blood of the guinea pigs was negative to abortion. vase 997B1, @ strons realthy calf from cow 9975, was born Lovenber 15, 1918. the meconium was examined and 2 c.c. injected into each of two guinea pigs. ‘the blood of the guinea pigs was negative to abortion. The meconium was sterile. vase 999 was purchased at the aga of one year. 3lood re- action at the time of purchase was negative to abortion. Uctober 10, 1913 and “ovember 8, 1913, she receiveéc 10 and 10 c.c. of a live culture of Bact. abortus subcutaneously. She developed a reaction October 29, 1913 and remained positive until ~ay 29, 1914. She calved #ebruary 3, 1916. 4 52 December 13, 1916 she received 10 c.c. of a live culture of tact. abortus intravenously. Calved vDecenuber 51, 1916; premature. January 5, 1917 she developed a positive reaction to abortion. valved sovenber 5, 1917. Bred webruary 15, 1918: calved sovember 26, lyld. “his cow cleaned in a snort time, ‘Shree uterine wash- ings were exaisined and then the cow was sold. Staphylococcus pyogenes aureus was found. she blood of the guinea pigs was neg- ative to the complement fixation and agglutination tests for a- bortion. vase 999 D., a strong calf from cow 999 was born soven- ber 16, 1918, ‘he meconium was examined and 2 c.c. was injected into each of two guinea vnigs. The blood of the guinea pigs re- mained negative and no lesions o. abortion were found, Staphylococcus pyogenes aureus was found in the meconium. Lase 806 was purchased September 7, 1917. ‘The reaction at the time of purchase was negative to abortion. On September 25, 1917, she received intravenously 10 c.c. of a killed culture of Bact. abortus. OUctober 5, 1917, she received intravenously 30 c.c. of a live culture of Bact. abortus. October 5, 1917, she developed a full positive reaction to abortion. This cow had not been in heat up to January 5, 1918. Observations had shown that after an electric dissociation treat- ment wows would come in heat. On January 5, 1918, she was given an electric dissociaticn treatment for sterility by voctor rallman. Seginning -ebruary 22, 1918, she was given live culture of Jact. abortus or feed every day for one week, ~- $3 =~ She was bred february 27, 1918 and calved November 25, 1918. On the date of parturition a washing was taken from the uterus. swovember 27, 1918, there was a discharge of pus from the uterus. It diminishea until by the fourth week it was entirely absent. The organisms isolated were: B, coli comrunior. Streptococcus pyogenes. The guinea pigs that were injected with uterine washings were killed but no lesions of abortion were present. The blood was negative to woth complement fixation and agglutination tests for abortion, Case 806 4., the calf of cow 806 was born isovember 25, 1918. lieconium was obtained for a bacteriological examination and for injection into guinea pigs. B. coli communior was isolated from the meconiun. The guinea pigs were bled and then autopsied after the in- cubation period, The blood of the pigs was negative to abortion tests. All organs were apparently normal. Case 11A14 was purchased January 1917 at the age of two years. Keaction at the time of purchase was positive. She abort- ed her first calf January 1917. January 19, 1918, she gave birth to an apparently normal calf. She was bred April 7, 1918 and calved January 13, 1919. She cleaned in a few hours after calving. washings were taken from the uterus for examination and for injecting into guinea pigs. ~ 54 - There was no discharge from the uterus for two weeks, When there appeared a dirty yellowish discharge which contained epithelial cells, cocci and a few leucocytes. There were no organisms grown from the contents of the first three washings. A streptococcus was isolated fram the next three washines. The results of the guinea pig inoculation were negative Vase 114142, the calf from 11414 was born January 13, 1919. The meconium was collected and 2 c.c. of a suspension of it was injected into each of two guinea pigs. warch 24, 1919 the pigs were killed. All the organs were apparently normal. ‘The blood was negative to abortion tests The meconium was sterile. vase 50 was a clinic case of Doctor Haliman. The cow calved the last week in wovember 1818. lio attention was given her until December 24, 1918. On this date the diagnosis’ of pyometra was made, The uterus was irrigated with physiological Salt solution and then 2 oz. of 50 percent Lugol's solution was introduced into the uterus. irequent treatments were given. January 31, 1919, a 5 percent formalin solution was introduced into the uterus. ‘The same treatment was given on *ebruary 7, 1919. Un cebruary 10, 1919, Doctor haliman collected some of the discharge from the uterus and sent it to the laboratory for examination. streptococcus pyogenes was isolated. It will be noted that the streptococcus was isolated after the drastic treat- ment which had been given every tew days.since December £4, 1918, and especially after the formalin treatment, ~ 35 - vase 999 a was born “ebruary 3, 1916, She gave a negative reaction to abortion tests. She calved January 23, 1918. Wags bred warch 16, 1918, and calved again December 24 1918. She cleaned in about two hours but developed a metritis. Uterine washings were taken for examination. Two guinea pigs were injected with < c.c. each of the various washings. The cow remained negative to the abortion tests before and after parturition. The organisms isolated were: B. coli communior. Streptococcus, the streptococcus failed to crow on the media during the first two days of incubation at 37 degrees C., but appeared as tiny pin point blue colonies at the end of the third day of incubation in the w»ovy jar. 4 stain made from the colonies taken from the sovy jar showed the orgn:nisms to be short chain, gran negative strepto- cocci. this streptococcus was isolated twice out of the five washings. the guinea cigs injected with the washings of 999 A were 211 negative to avortion tests. shere were no macroscopic lesions of abortion. Case 999 B., the calf of 999 A, was born December 24, 1918, iieconium was obtained for examination. Guinea pigs were injected. The blood of the guinea pigs was negative to the -~ 26 - &bortion tests. No macroscopic lesions of abortion were present. B. coli communior was isolated from the meconiun. Case 997 was purchased at the age of one year. On October 10, 1913 and on November 8, 1913, she received 10 c.c. of a live culture of Bect. abortus. She developed a positive reaction to the abortion tests and maintained it until way 29, 1914. She calved i#ebruary 4, 1916. On December 18, 1916, she received 10, c.c. of a live culture of Bact. abortus. She developed @ positive readtion January 5, 1917, and maintained it. She calved December 21, 1916. was bred April 8, 1918, and calved again January 18, 1919. Uterine washings were examined and 2 c.c. were inject- ed into guinea pigs. There were no organisms isolated from the uterine washings. The blood of the guines igs was negative to fhe a- bortion tests. There were no macroscopic lesions. vase 997 C., the calf of 997 was born Yanuary 18, 1919. The calf was strong and apparently in a healthy condition. -.econium was plated out but gave negative results. The guinea pigs that were injected gave negative results to the a- bortion test. vase 807 was purchased september 7, 1917. ‘The blood reaction at the time of purchase was negative to the abortion tests. September 13, 1917, she was given 10 c.c. of a culture of killed Bact. abortus subcutaneously. she developed a positive reaction september 20, 1918, which lasted until sovember 16, 1917. Since tnis time she has inaintained a negative reaction to the a- bortion tests. September 23, 1917, she was given 25 O00. of a culture of live 3act. abortus subcutaneously. Observations have shown that an animal will come in heat shortly after an electric dissociation treatment. Since tis cow would not come in heat, she was given an electric treatment for sterility by Doctor “allman. (January 2, 1918). On April 2, 1918, she received 5 c.c. culture of live Bact. abortus intravaginally and was immediately bred but failed to conceive. She was given the same treatment april 22, 1918. way 31, 1918, and on June 9, 1918, On June 10, 1918, she conceived. The writer noticed that she was discharging shreds of tissue and a dirty yellowish putrid pus about tiovember 20, 1918. This continued until December 27, 1918, when she aborted, The Placenta came away in small pieces ..ixe. with an abundance of dirty yellowish putrid pus. ‘Yhe discharge contained numerous small rods, cocci, epithelial cells and leusocytes. #ashings were taken from the uterus evzry week until sebruary 5, 1919. The organisms isolsted were: Be. coli communior. streptococcus. The guinea pigs injected with 2 c.c. of the various washings oi the uterus failed to show any lesions of aborti-n. the blood of the guinea pigs was negative to the abortion tests, ~ 38 - The streptococcus failed to grow the first two days of “incubation of the plates at 37 degrees & On the third day of incubation in the ovy jar, the colonies appeared as very small, pin point, bluish colonies. shis orgarism was isolated six times in this case. April 3, 1919, a 24 hour growth of this organism on veal agar slant was washed off with physiological salt solution and 1 c.c. of the suspension injected subcutaneously into a preg- nant guinea pig. April 6, 1919, the pig aborted. On April 7, 1919 the pig was autopsied. The spleen was once and a half its normal size. ‘the streptococcus was isolated from the splea@, blood, and uterus of the guinea pig. Vase 807 A, the fetus of cow 807, was aborted on the cOOth day of gestation. The fetus was taken to the laboratory for examination. aA streptococcus which grew the same as the one isolated from the uterug of case 807 was isolated from the blood, liver, spleen, stomach and meconium. a gram stain of the blood, liver, and spleen showed short chains of & cocci which were gram negative. The liver and spleen were very friable. The male guinea pigs injected with 2 c.c. of a suspension of meconium did not Show any lesions of abortion and the blood was negative to the abortion tests. ‘the pigs used for the injection of the stomach contents were apparently normal. The organism caused abortion in @ pregnant guinea pig in about three days. Case 20, a grade shorthorn, was about six years old when her lest calf was born in January 1917. The cow was bred in late spring or early summer and was ~ 39 . thought to pe with calf during the summer. There was no evidence of abortion but the animal bezan to show signs of estrum, at first irregularly but durins tne last summer and fall quite regu- larly. she was bred persistently for some time with failure to conceive. Low was Killed for beef webruary 11, 1919. The reproductive organs were sant to the laboratory where a bacteriological and a macroscopic examination was made. Results of the bacteriological examination were: The uterus was sterile. ‘The cystic ovary contained B. coli communior. liotes on the macroscopic examination: kxternal cervix was four centimeters in diameter. ix- ternal os was closed. ‘here was quite a quantity of clear only Slightly adhesive mucus in the external os and covering the pos- terior end of the cervix. vervical canal was seven centimeters long. sransverse folds were aprarently normal but there was an appreciable quantity of rather adhesive exudate present. The body of the uterus was five centimeters long. .ucous membranw was of &@ light gray uniform color except two small areas averaging about ‘two centimeters by one half centimeter near internal os. These were apparently more vascular than that surrounding the gland mu- cosa, The cotyledons were about one half centimeter in diameter; and were depressed in center. Left horn was similiar to the body, fairly uniform with few wascular areas. volor of the horns was Similiar to that of a heifer ::sore than that of a cow that had calved. Left oviduct was normal. teft ovary was four centi:eters in diameter, spherical in shape and contained a protruding corpus luteum two centimeters in diameter, The right ovary was four -~ 40 - centimeters long; contained one cyst-like structure two centimetes. -in diameter and several suall ones. vase 9, was born “ay 16, 1911. The animal was bred on the following dates: reb. 29-13. way 30-13. Dec. 20-13. Jan. 12-14. Feb. 3-14. larch 10-14. April 10-14. april 27-14. way On-14. June 30-14. Aug. 9-14. valved --ay 15-15. there was no record of abortion at any time nor did the records indicate the appearance of estrum between way 30, 1913 and December 20, 1913, artificial inpregnation was atte.pted on the following dates: Dec, 20-13. June 12-14. Heb. 2-14, wlarch 10-14. luarch 30-14. april 10-14. April 27-14. way 514, The yeast treatment was given June 30, 1914 and August 9, 1914, - 4] - This anizal was bred again on the following dates: July 20-15. Sept. 9-15. sept. 18-15. Oct. 4-15. ~ Uct. 21-15. Vec. 21-15. vec, 31-15. Jan. 20-16. Keb, 24-16. ~arch 31-16 Yeast treatment was given. uay 19-16 " " " " June 77-16. " " " " June 23-16. " " " " July 11-16. sept. 9-16. Uct. 12-16. Love 8-16. after washing out vagina with salt solution Jan. 20-17. Dept. 22-17. After using soda solution. Dec. 6-17. march 25-18. Sometime during the spring of 1918, (date not recorded) Doctor Hallman made an examination and pronounced the animal pregnant, apparently about seventy or eighty days advanced. A few weeks later, another examination was made and it was found that the ani..al wzs not pregnant. apparently she had aborted but had been unnoticed b:; the herdsman. +o record was made of the clinical condition at this ti:..e. In a short time the animal was ~-~ 42 « turned out to pasture without further service by bull and re- mained in the pasture urtil October 13, 1918. On this date the following notes were made by voctor halinan: "There is a small quantity of clear mucus in vagina in which are seen a few flakes of pus. Un manipulation of cervix, a tablespoonful or wore of an adhesive light mud colored mucus is forced out of cervical canal. ‘she external os and posterior eni of cervix is open (can insert three finsers), but internal os is closed," "On rectal examination, it is found that the uterus is distended. ‘the walls are very tense simulating pregnancy of the fourth to fifth month, though a fetus cannot be felt. A diagnosis of uterine dropsy wasmade and we proceeded to draw off the fluid. Fourteen to sixteen quarts of a straw colored fluid, in which there is considerable coagulated blood, is drawn from the uterus." "After removal of this fluid, a foetus about twenty fivd or thirty centimeters long can be palpated per rectuin. There is no record of breeding this animal after arch 25. I1t is positive- ly known that she was not bred to the herd bull before turning to pasture. The only explanation of pregnancy is that a bull of some neighbors must have broxen into the pasture during the summer and served the cow. ‘his assumption is verified by the fact that on this same date a heifer, which had not been bred to the herd bull and which was put on pasture at the same time as the aniiial under consideration, was examined and found to be pregnant? "ho effort was made to remove the fetus at this tine, believing that it would be delivered in a few days". On November 8th this animal wag again examined and the following notes were made: "Phere is an adhesive muco-purulent discharge in the anterior part of vegins ond cervical canal. The uterus is very little if any enlarged. Tone is fair. There is 2 small cyst in right ovary. The cervix was cleansed and one ounce of 50 percent Lugol's solution was injected into the uterus.” "February 1, 1919 considerable muco-purulent discharge in the vegina. vervical canal is open. sxternal cervix is con- gested. i0 marked enlargement of the uterus. Tone is fair." Jith the record of this animal, an unfavorable progno- Sis was made and it was decided to sleughter her for beef. She was killed february 11, 1919. The reproductive organs were sent to the laboratory where 8 bacteriological and a macroscopic examination was made. Kesults of the bacteriological examination were; B. coli communior was found in the uterus. The cystic ovary was sterile. Notes on the macroscopic examination were: Lxternal os was dilated; there was a slight hypertrophy of the external fold at the lower and right lateral border. There was also a small quantity of a clear slightly adhesive secretion of the external os. The cervical canal was nine centimeters long and contained a small quantity of slightly adhesive exudate. «ucow: membrane was light drab color. ‘the body of the uterus was two centimeters long and of slightly mottled pinkish color. In the mucous membrane small areas of a lighter grayish color were ob- served. The mucous membrane of the posterior end of the left horn was similiar in appearance to thet of the bogy of the uterus. The cotyledons were three milimeters in diameter and were of a “a - 44 - grayish white color, contrasting strikinely with gland mucosa. In proxinity to one cotyledon near the posterior end of the horn was &@ small nodule one centimeter in diameter, and one centimeter high, which from superficial exacination appeares to be a sinali tumor. she mucous wie:branes of the mid€dle and anterior portious were not uniform in color; some portions apreeared more vascular, other portions were light drab in color. ‘shere was a snall quantity of turbid creas-like exudate. The mucous inmemprane of the right norn was a little nore uniform in color and a little lighter drab than the left horn. sane character of exudate was present. In the anterior ena, @ small piece of a flat bone five centimetcrs long and about one centiieter wide was founa. Looked like it might be a tlat piece of a shaft or long bone. 3. Coli communior and an abundance of leucocytes were in the exudate which surrounded this piece of bone. fhe right oviduct was apparently normal except for the presence of a cyst about one and one half centimeters in diameter at the fenestral end. the right ovary was five centimeters by four centi- meters and contained a cyst-like structure two centimeters in diameter. she left ovary was two and one half by four centi- meters and contained a large cystic (?) corpus luteum four centimeters in diameter. Left oviduct was apparently normal. soeue Snd00004de146, STAOG SouodOAG “qde4s, BT-9S~4 1 ZOTUNMUIOD TTOO °d, 1 Syaod Bouesokd yaege, 1 Souesoha snooo000qder4¢, 1 koe tats aie er Sep ee ee eee ee eee ee oe le Snoatoge “200g, aT=07—), 1 YqMois ON, 1 6T-8T"T . 0466 “4 iL ‘ L A UQMols ON, 6T-8g=T , A 1 tL. i ; ‘ UgMoLe O17, 6T-Te-T : L ‘ = I 2 1 ; : UyMots O17, 6T-8T-T 1 L66 1 i I 1 ' UQMOLsS ON, » 6T~ST