STUDIES ON VIBRIO FETUS -- I. CONCENTRATIONS OF PENICILLIN, STREPTOMYCIN AND VARIOUS DYES NECESSARY FOR INHIBITION OF VIBRIO mg IN VITRO. II. COMPARISON OF TWO MORPHO- LOGICALLY DISSIMILAR STRAINS OF VIBRIO w BY MEANS OF ELECTRON MICROSCOPY. by JACK J'ENKS STOCKTON A THESIS Submitted to the School of Graduate Studies oijichigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of MASTER OF SCIENCE Department of Bacteriology 1950 TH 5518 CL,/ TABLE OF INTRODUCTION . . . . . . . . HISTORICAL REVIEW . . . . . . MATERIALS . . . . . . . . . . METHODS AND RESULTS . . . . . Part I Antibiotic Studies . . . . Dye Studies . . . . . . . . Part II CONTENTS Page . . . . . . . . . . . . l . . . . . . . . . . . . . 5 . . . . . . . . . . . . . 7 Comparison by Electron.Microscopy of TonMorpho- logically Dissimilar Strains of Vibrio fetus . . . 23 Colonial characteristics Cellular morphology . . . DISCUSSION . . . . . . . . . SUMMARY . . . . . . .,. . . . BIBLIOGRAEHY . . . . . . . . ACKNOWLEDGMENT O O O O O O O O O O O O O 23 O O O O O O O O O O O O O 24 O O O O O O O O O O O 0 O 33 O O O O O O O O O O O O O 34 11 0341 $93.9 ). J ’. 2 '...v , Table l. 2. 3. LIST OF TABLES Result of Screening Tests to Determine the Ap- proximate Concentration of Penicillin and of Streptomycin Necessary to Inhibit the Growth of E, fetus in Vitro . . . . . . . . . . Result of Tests to Determine the Minimum.Concen- tration of Penicillin and of Streptomycin Nec- essary to Inhibit the Growth oflE. fetus in Vitro O O O O O O O O O O O O O O O 0 O 0 Effect of Thionin on the Growth of I. fetus, E. pyggenes var aureus and E. coli in Thiol Ifledilm O O O O O O O O O O O O O O 0 O 0 Effect of Sodium Azide on the Growth of;[. fetus, E, pyogenes var aureus and E. coli in Thiol Medium. . . . . . . . . . . . . . . . . . Effect of Brilliant Green on the Growth of E, fetus,iE,pyogenes var aureus and E, coli in ThiOl Lied-111m. O O O O O O O O 0 O O O O 0 Effect of Gentian Violet on the Growth of E} fetus,IE, pzogenes var aureus and E, coli in ThiOl Lied-111m . C O O C O O O O O O O O 0 Effect of Basic Fuchsin on the Growth of Z. fetus, M. pyogenes var aureus and E, coli in Thiol I'iedim O 0 O 0 O O O O O O O O 0 O O 0 Page 13 14 18 18 19 19 20 Table Page 8. Result of Simultaneous Inoculation of I, fetus and iE, pyogenes var aureus into Various Concen- trations of Basic Fuchsin Thiol Medium. . . . . 21 9. Result of Simultaneous Inoculation of 1. ME and E, gyogenes var aureus into Various Concen- trations of Gentian Violet Thiol Medium . . . . 21 LIST OF FIGURES Figure Page 1. Electron Micrograph of E, £§£E§,Plastridge Strain, Approximately 55,000 X . . . . . . . . 26 2. Electron Micrograph of Eg‘ggggg Strain 490, Approximately 50,000 X . . . . . . . . . . . . 27 iv INTRO DUC TION Isolation of Vibrio 3333s in pure culture from con- taminated specimens is a very difficult task and attempts in a great majority of instances result in failure. Specimens submitted for bacteriological examination to determine the presence or absence of this organism are commonly contami- nated. Materials such as placentas, uterine and vaginal discharges, vaginal swabs, and aborted fetuses, or organs from aborted fetuses, are those most frequently examined. It is not uncommon to observe the organism in smears made directly from the mentioned specimens. When such smears are fixed in heat and stained with crystal violet, the organism, if present, is readily seen on microscopic examination. Ex- amination of '76 films prepared from vaginal Swabs taken from cows in a herd known to be infected with E. _f_‘_e_’§_1_1_§ revealed 19 cows as possible carriers of this organism. Of these 19 only 2 gave 1. am when cultured. Sixteen of the remain- ing 17 yielded gram-positive cocci, in clumps and in chains, and gram-negative bacilli. One specimen was bacteriologi- cally sterile. Unless 1. £93113 is the only organism present in such a Specimen it is very difficult to obtain a pure culture of it since other bacteria literally crowd it out. 1. W has been cited as a cause of retained pla- centas and breeding difficulties in cows, particularly in l herds free from brucellosis. No suggestion has been made as to possible therapeutic measures that might be employed to eliminate this infectious agent from.the genital tract of infected animals. The present study on the inhibitory effect of various dyes was undertaken to learn if such dyes might prove bene- ficial in the isolation of E§.£g§g§ from contaminated speci- mens. Studies with the antibiotics, penicillin and strepto- mycin, were carried out for the same purpose and also to learn whether or not they might offer an approach to the problem of effective treatment in cases of known infection. The electron micrographs included in this study were made to compare, at high magnification, the morphology of two strains of E} Egggg that microscopically were consider- ably dissimilar. The colonial characteristics likewise showed considerable difference. One strain showed distinct comma-shaped organisms typical of members of the genus Vibrio. Organisms in the other strain appeared.more nearly as straight rods, the definite comma shape being absent in a great majority of individual organisms viewed. HISTORICAL REVIEW The earliest recording of the association of a spi- rillum with abortion in animals was made by Mac Fadyean and Stockman (1913) in England. The particular case recorded involved abortion in ewes. This report was not available but is mentioned in most writings dealing with the history of vibrionic infection in anrmals. Smith (1918) drew atten- tion te the fact that, buried in this report, there is a brief statement that vibrio-like forms had been isolated from.aborting cattle in Ireland and wales in 1911. IMac Fadyean and Stockman named the organism Spirillum.£§§g§. Smith (1918) and SmithzumHTaylor (1919) found a spi- rillum, morphologically identical with that described by Mac Fadyean and Stockman, in the stomach contents of aborted bo- vine fetuses. This finding in the State of New Jersey was the first such infection recorded in the United States. They named the organism Vibrio Egfigg. Since these two early recordings the reporting of the infection in cattle has been confined largely to the period since 1940. Reports of infection in cattle in the United States since Smith's recognition have appeared as follows: Schroeder (1920), Traum.(l925) and Barger (1928) in Califor- nia; Gilman (1959) in.New*Ybrk; Plastridge (1941) in Connecti- cut; Rhoades and.Hardenbrook (1947) in Illinois; webster and 3 Thorp (l949) in Michigan and Bell (1950) in Virginia. Plas- tridge and Williams (1948) have drawn attention to the prob- able widespread distribution of the infection in the United States as a result of tests made by them on specimens sent in by veterinarians from herds in Maine, New Hampshire, Mas- sachusetts, Pennsylvania, Virginia, Georgia and New York. Moore (1950) stated that, to his knowledge, the disease has been found in the states of Ohio, Indiana, Wisconsin, Mis- souri, Kansas, Nebraska, and Oklahoma. Vibriosis in sheep has been reported as follows: Carpenter (1920) and Baker and Stone (1959) in New York State; Welsh and Marsh (1924) in Montana; Graham and Thorp (1950) in Illinois; Ryff (1940) in Michigan; Lee and Scriv- ner (1941) in Wyoming, and Ryff and Lee (1945) in Wyoming. Curtis (1915) reported the isolation of a curved mo- tile bacillus from the uterine discharges of two women pa- tients, one whose infedtion followed instrument-induced abortion. The other patient's labor, at full tenn, was com- plicated as a result of infection with this organism. Cur- tis assigned this rod to the genus Vibrio. Ward (1948) re- ported the finding of E. £31533 in a pustule on the cheek of a male laboratory worker who had been engaged in research involving 1. 39.31% for some weeks. Also isolated from this lesion was a large gram-positive, non-sporulating rod and a few short gram-negative rods. The capability of 1. 3.212.151 to maintain itself within the body of human males was cited 4 'with the possibility of the larger gram-positive rod being responsible for the small lesion and the smaller organism being restricted to the role of a secondary invader. The need for some effective means of isolatingj1.,£g- Egg from contaminated specimens has been evident for a num» ber of years. Lee and Scrivner (1941) pointed out that vag- inal discharges and fetal membranes are not satisfactory for the isolation of E} £§£g§_because of the excessive contami- nation invariably present. Ryff and Lee (1945) stated that, aside from Ryff's description of Hammond's use of crystal violet veal infusion agar plates in the isolation of 25 32: Egg, no attempt has been made to develop a selective medium for the isolation of this organism although the material of- fered for examination is frequently such that overgrowth flwith contaminants will result. Hagan (1943) gave a method that has'been used to isolate the organism when mixed with other bacteria, as, for example, when it occurs in a soiled placenta. He stated that after intraperitoneal inoculation of a guinea pig the organism would often survive for 3 or 4 days and might then be re-isolated from the spleen. There are no lesions evident and later the organisms disappear. Chambers (1948) reported his experiences using peni- cillin in the treatment of non-breeding cows. This report gave no account of the possible causative organisms but the author classified his results as good. It has been suspected (Moore, 1950) that E} fetus might be the cause of consider- 5 able breeding difficulties in cattle. This author called attention to the need for some method of treating this con- dition. webster and Thorp (1949) stated that E, Egggg is one of the organisms that must be considered in brucella- free herds plagued with abortions, retained placentas and a low conception rate. At the present time Difco's "Thiol" medium.is the me- dium.of choice for the cultivation of E5 Egggg, This is a semisolid.medium. The organism can be recovered with more regularity by the inoculation of suspected.material into this medium.than by the inoculation of other semisolid, li- quid or solid media. On occasion it has been necessary to incubate the thiol medium at 37 C for as long as 7 days be- fore growth became evident. Bacterial contaminants, fre- quently found in suspected specimens, grow profusely and rapidly in this medium. Overgrowth of the vibrios occur! when such contamination is present. MATERIALS To determine the inhibiting powers of penicillin and streptomycin on.E} £333§Din vitro, these antibiotics were tested in a f1uid.medium.and in a solid medium. No fluid medium was known that would support growth of E} Egggg, and it was necessary to develop one that would satisfactorily grow this organism. Stockman (1919) described the growth of I} §9§E§_in a broth.medium, the composition of which is not mentioned. He stated that subculture from broth to broth usually failed, but, with the addition of a small portion of raw potato to the liquid medium, the culture could be kept up in series. Ryff and Lee (1945) mentioned that growth was obtained in nutrient broth, but that better development was obtained by the addition of 1.0% tryptose to Difco veal in- fusion medium, which contains a small percent of agar. Plastridge and Williams (1943) described a medium.containing 0.3% agar in which growth occurred approximately 0.5 to 1.0 mm subsurface. This medium.was considered unsatisfactory for the testing of antibiotic action since growth of the or- ganism occurred in a limited zone only, and hence would be subjected to the inhibiting action of the antibiotics only in a small portion of the total volume. Huddleson (1948) drew attention to the excellent growth obtained by the use of Difco's ”Thiol" mediumt This medium contains 0.1% agar '7 and like the semi-solid medium of Plastridge and Williams yields growth in a limited zone, slightly sub-surface. A.number of f1uid.media were tried to determine if they would support sufficient growth of this organism for the purpose desired. A.medium of the following composition was found to be the most satisfactory; Chicken Infusion Broth . . . . 500 ml Bacto-Peptone . . . . . . . . . 10 gm Distilled water q.s. . . . . . 1000 ml The pH was adjusted to 6.8 and the medium.was autoclaved at 121 C for 20 minutes. When this medium was seeded and incubated aerobically at 37 C a slight amount of growth occurred on the original transfer from.Plastridge's semi-solid medium, Subculture from this f1uid.medium, with incubation under aerobic condi- tions, was unsuccessful. When the original transfer and sub- sequent transfers were incubated at 37 C under 10% 003 all tubes consistently showed growth. Growth was faintly evi- dent at the end of 24 hours incubation, decidedly evident at 48 hours and at 72 hours the broth was quite turbid. At the end of 96 hours a coating of the bottom.of the tubes was ap- parent. Blood agar plates were used as the solid medium. These were made from Difco's Blood Agar Base and 5% bovine blood. Thiol medium supports excellent and abundant growth of E} 22222.8nd is being used almost exclusively in attempt- ing primary isolation of this organism. For these reasons the dye studies were performed employing this medium,‘ By incorporation of various concentrations of a single dye into this medium.the inhibitory action of several dyes for 1, £2: tus,‘Micrococcus pyogenes var aureus, and Escherichia coli ‘was studied. Stock solutions of the dyes were 1% concentra- tions in distilled water. The cultures used in this study and their origin are as follows: Vibrio fetus, Plastridge Strain. Obtained from Dr. W. N. Plastridge, Storr's- Experiment Station, University of Connecticut, Storrs, Connecticut. Vibrio fetus, Strain 490. Isolated from.an aborted bovine fetus from.the Michigan State College Dairy Herd, East Lansing, Michigan. Vibrio Egggg, Strain B-24. Isolated from an aborted bovine fetus submitted for bacteriological exam, ination to the Department of Bacteriology, Michi- gan State College. Micrococcus pyogenes ggg aureus. .A coagulase posi- tive micrococcus isolated from a lesion on the face of a man at the Department of Bacteriology, Michigan State College. 9 Escherg’chia coli. Isolated from a human stool speci- men submitted to the Department of Bacteriology, Michigan State College. The streptomycin and the penicillin used in this study were commercial lots obtained from Merck and Co., Inc., Rahway, New Jersey. The penicillin was crystalline penicillin G sodium and the streptomycin was the calcium chloride complex. Thiol medium and Bacto-Penase were ob- tained from the Difco Laboratories, Detroit, Michigan. 10 METHODS AND RESULTS Antibiotic Studies The fluid medium was tubed in 9.5 m1 amounts. Trial had shown that autoclaving at 121 C for 20 minutes and sub- sequent cooling to room temperature resulted in a loss of approximately 0.25 ml of medium per tube. Solutions of the antibiotics were prepared so that the addition of 0.5 ml to the medium resulted in the unit concentration desired. The test cultures were maintained on Plastridge's semi-solid medium. Growth from 4-day—old cultures was sus- pended in 4 ml of the fluid medium and vigorously shaken for fifteen minutes to assure the breaking up of bacterial clumps. An inoculum of 0.25 ml of this suspension was used to seed each tube, thus bringing the total volume in each tube to 10 ml. Counts by darkfield examination Showed ap- proximately 480,000,000 organisms per 0.25 ml of inoculum. Each concentration of the antibiotics was tested in lots of ten tubes. Tubes were incubated at 37 C and under approximately 10% 002. They were observed daily for evidence of growth and were agitated at each examination. Incubation was con- tinued for a total of 96 hours. If growth was apparent at the end of this incubation period, the contents of the tubes showing growth were examined microscopically. If growth was 11 not evident at the end of the incubation period, the inocu- lated material was transferred.to thiol medium.or Bacto- Penase was added to the original medium, the tubes reincu- bated for an additional 96 hours, at the end of which time they were again examined for growth. Control tubes containing 9.75 ml of fluid medium were inoculated with 0.25 ml of the suspension used to inoculate the tubes containing the antibiotics. They were incubated exactly as were the test cultures. Screening tests to obtain an approximate range of ef- fectiveness of each antibiotic were conducted. Table 1 shows the results of these tests. Penicillin concentrations of 0.5 unit per m1 and above were found to inhibit bacterial growth in all instances whereas in concentrations of 0.0125 unit per m1 and lower, growth occurred in all but 2 tubes. Streptemycin in concentrations of 1.01ug per ml and above inhibited growth in all instances whereas concentrations of 0.1,pg per m1 allowed growth in all tubes. All controls showed growth. Similar procedures were followed to determine the minimum.concentration necessary for inhibition. Table 2 shows the results of these studies. Penicillin in concen- trations of 0.3 unit per m1 and above inhibited growth in all tubes. In a concentration of 0.2 unit per ml, 4/10 tubes of Plastridge's Strain, 2/10 of Strain 490 and 0/10 of Strain B-24 showed growth. B-24 was a recent isolate and 12 TABLE 1 RESULT OF SCREENING TESTS TO DETERMINE THE APPROXIMATE CONCENTRATION OF PENICILLIN AND OF STREPTOI‘IYCIN NECESSARY TO INHIBIT THE GROWTH OF E. FETUS IN VITRO *COncentration of Antibiotic Strain of Test Organism Used Penicillin u/ml if Plastridge 490 B—24 2.5 O/IOa 0/10 0/10 0.5 0/10 0/10‘ 0/10 0.0125 8/10 10/10 10/10 Streptom.cin .ng/ml 5.0 0/10 0/10 0/10 1.0 0/10 0/10 0/10 0.1 10/10 - 10/10 10/10 10 controls of each strain all shewed growth at 96 hours. 8Indicates the number of tubes showing growth over the number of tubes inoculated. bThe potency and dosage of streptomycin were former- ly stated in "S" units, 1,000 of which correspond to 1 mg of streptomycin base. All potencies and dosages are now expressed in terms of weight of base. did not grow as profusely in the fluid medium controls as did the other two strains. Streptomycin showed.complete inhibition in concentra— tions of 0.5)ug per m1. In concentrations of 0.3,ug per ml 10/10 of Plastridge's Strain grew, 9/10 of Strain 490 grew and 6/10 of Strain B-24 grew. In concentrations of 0.4,ug per ml only Plastridge's Strain grew (8/10). 13 TABLE 2 RESULT OF TESTS TO DETERMINE THE MINIMUM CONCENTRATION OF PENICILLIN AND OF STREPTOMYCIN NECESSARY TO INHIBIT THE GROWTH OF E. FETUS IN VITRO Configgggigigg 0f Strain of Test Organism Used Penicillin u/ml Plastridge 490 B-24 0.4 0/10 0/10 0/10 0.3 0/10 0/10 0/10 0.2 4/10 2/10 0/10 0.1 10/10 10/10 10/10 0.05 10/10 10/10 10/10 Streptomycin “'Jus/ml"“"“ 0.9 0/10 0/10 0/10 0.8 0/10 0/10 0/10 0.7 0/10 0/10 0/10 0.6 0/10 0/10 0/10 0.5 0/10 0/10 0/10 0.4 a/10 0/10 0/10 0.5 10/10 9/10 6/10 0.2 10/10 10/10 10/10 10 controls of each strain all Showed growth at 96 hours. To learn if the antibiotics tested were bactericidal or merely bacteriostatic two procedures were employed to in- activate the antibiotics following 72 hours incubation. The dilution of the antibiotic selected for trial was the mini- 14 I. mum.dilution giving complete inhibition in all tubes. To half of the tubes of this dilution was added 0.1 m1 of Bacto- Penase. This amount of Bacto-Penase will inactivate 1,000 units of penicillin. One hundred micrograms of streptomycin are inactivated by this quantity of Bacto-Penase. One- hundredth.m1 was added for the sake of convenience and to insure complete inactivation of the antibiotics. To learn if BactoéPenase might have some inhibitory action on the test organism, 0.2 ml amounts were added to tubes containing approximately 10 m1 of the fluid medium. These tubes were then seeded with the test organism exactly as were the tubes containing the various concentrations of the antibiotics. It was learned that Bacto-Penase is not inhibitory for any strain of 1. mg tested. Inoculations were made from the remaining tubes, con- taining the antibiotics in minimum.inhibiting concentration, into thiol medium. Thiol medium will inactivate small amounts of penicillin and streptomycin and was developed for the cultivation of specimens containing inhibiting coneen- trations of these two antibiotics (Huddleson, 1948). The cultures were incubated for an additional 72 hours. Examination at the end of this period revealed that there had been no growth in any tube of minimum effective concentration of the antibiotics tested. Other concentra- tions, greater than the minimum inhibiting concentration, “were treated in a similar manner. Tubes containing penicil- l5 lin showed growth in three isolated cases, one each in the following concentrations: 2.5 units per ml, 1.0 unit per m1 and 0.5 unit per ml. Tubes containing streptomycin showed no growth in any instance. The minimum concentration of penicillin and of strep- tomycin necessary for inhibition of E. M on blood agar plates was variable. For penicillin some trials showed a minimum concentration of 0.25 unit per m1 as inhibitory, while other trials showed good growth at the highest concen- tration tested, namely, 0.4 unit per m1. Tests with strep- tomycin showed a variation of from 0.2,ug per ml to 0.35 mg per ml as minimum.inhibitory concentrations. The highest concentration of streptomycin tested was 0.5}1g per ml. Tests using Micrococcus pyogenes var aureus and tests using Escherichia coli as seed cultures showed growth of both these organisms at all concentrations of the antibiotics tested. It appears that the use of streptomycin or penicil- lin in blood agar plates would not be effective in isolating E} {gigg'from.a specimen contaminated with either the grams positive coccus or the gramrnegative rod mentioned above. E§§_Studies One percent aqueous stock solutions were added to 100 ml amounts of thiol medium in a volume necessary to give the desired final concentration. The medium containing the dye was tubed in approximately 10 ml amounts and autoclaved at 121 C for 20 minutes. Stock cultures of E} Egggg were main- 16 tained in thiol medium and inoculation into the dye contain- ing medium was made by mixing the growth with the upper por- tion of the thiol medium and pipetting 0.1 m1 amounts into the dye containing medium. The tubes were incubated aero- bically at 37 C for 96 hours. Stock cultures of E. pyogenes var aureus and of E. 92;; were maintained in brain heart infusion broth. Amounts of 0.1 ml from a 24 hour broth culture were transferred to the dye containing medium. Results of these studies are recorded in Tables 3 to 7. Three of the compounds tested were found to offer. no promise whatsoever as a means of inhibiting the gram-positive cocci or the gram-negative coliform organisms while allowing the vibrios to grow. Thionin, brilliant green and sodium azide inhibited growthof the three strains of l. £213.12 tested. However, the inhibitory concentration for E. _f_§_t_u_s_ allowed both E. pyogenes var aureus and E. 293.}; to grow abundantly. From the results obtained it is believed that two of the compounds, basic fuchsin and gentian violet, of- fer definite possibilities as regards inhibition of gram- positive cocci that might be present as contaminants. Basic fuchsin allowed growth of the three strains of 1. 39% tested in a majority of instances in concentrations of 1 : 5,000 through 1 : 9,000, while inhibiting IE. pypgenes var aureus in every instance. Gentian violet likewise showed promise since concentrations from 1 : 50,000 to 1'7 — ‘fi TABLE 5 EFFECT OF TRIONIN ON THE GROWTH OF T. FETUS, 131. FYOCFNES VAR AUREUS AND E. COLI IN THIOL MEDIUM I ’: Organism Concentration of Thionin Strain of 3} 32:35 l-5T 1-10T 1-20T 1-3OT l-40T Plastridge 0/2 4/4 4/4 4/4 4/4 490 0/2 4/4 4/4 4/4 4/4 3-24 0/2 4/4 4/4 4/4 4/4 1.5.- pyogenes 2/2 4/4 4/4 4/4 4/4 E, 0011 2/2 4/4 4/4 4/4 4/4 TABLE 4 EFFECT OF SODIUM AZIDE ON THE GROWTH OF 1. 213m, MEDIWM 111, PYOGENES VAR AUREUS AND E. COLI IN 'JHIOL Organism Concentration of Sodium lzide Strain of 1. fetus Plastridge 490 B-24 El! ogenes E0 0011 IF 1-5T l-lOT 1-15T 1-20T 0/2 2/2 2/2 2/2 0/2 2/2 2/2 2/2 0/2 0/2 0/2 2/2 2/2 2/2 2/2 2/3 2/2 2/2 2/2 2/2 18 TABLE 5 EFFECT OF BRILLIANT GREEN ON THE GROWTH OF E. FETUS, M. PYOGENES VAR AUREUS AND E. COLI IN 'I'HIOL MEDIUM 1_'_ Organism 1 Concentration of Brilliant Green Strain of y, fetus l-1OT* 1-20T 1-3OT 1-4OT Plastridge 4/4 4/4 4/4 4/4 490 4/4 4/4 4/4 4/4 3-24 4/4 4/4 4/4 4/4 E. pxogenes 4/4 4/4 4/4 4/4 E. 92}; 4/4 4/4 4/4 4/4 *At concentrations greater than 1-10T the medium was too dark to determine grossly if growth was present. TABLE 6 EFFECT OF GENTIAN VIOLET ON THE CRONTH 0F V. FETUS, E. PYOGENES VAR AUREUS AND E. COLI IN EIIOE MEDIUE -—.__ u t m L— ~__—- .u: “ Organism Concentration of GEEtian Violet Strain of Efetus l-50T 1-55'1‘ 1-60T 1-65T I-VOT 1-100T Plastridge 2/6 2/6 2/6 3/6 3/6 4/4 490 3/6 4/6 5/6 5/6 6/6 4/4 3-24 3/6 4/6 4/6 5/6 6/6 4/4 E- mogenes 0/6 0/6 0/6 0/6 0/6 4/4- .E- coli 4/4 4/4 4/4 4/4 4/4 4/4 19 TABLE 7 EFFECT OF BASIC FUCHSIN ON THE GROWTH OF E} FETUS, ELI. PYOGENES VAR AUREUS AND E. COLI IN THIOL LEDIUM Organism Concentration of Basic Fuchsin Strain of E} fetus }-5T* l-6T l-VT l-8T l-9T l-lOT 1-20T Plastridge 4/4 4/4 4/4 4/4 4/4 4/4 4/4 490 . 4/4 4/4 4/4 3/4 4/4 4/4 4/4 3-24 4/4 5/4 4/4 3/3 4/4 4/4 4/4 M;_pypgenes 0/4 0/4 0/4 0/4 0/4 1/4 4/4 E, coli 4/4 4/4 4/4 4/4 4/4 4/4 4/4 *At concentrations greater than l-5T the medium was too dark to determine grossly if growth was present. 1 : 70,000 inhibited M, pyogenes var aureus uniformly but al- lowed all strains of E} Egggg to grow in a majority of in- stances, with the exception of Plastridge's Strain, which showed poor growth throughout. An attempt was made to learn the outcome of simulta- neous inoculation of _V_. fetus and g. pyogenes var aureus in- to solutions of gentian violet and basic fuchsin which had shown inhibition of the gram-positive coccus but had allowed the vibrio to grow. Five dilutions of basic fuchsin and 5 dilutions of gentian violet were tried. The inoculum.for each was 0.1 ml of a suspension of each organism as previ- ously described. Only one strain of E} ggtgg (B-24) was tried, this being the most recently isolated strain. Re- sults are recorded in tables 8 and 9. 20 TABLE 8 RESULT OF SIMULTANEOUS INOCULATION OF V. FETUS AND M; PYOGENES VAR AUREUS INTO VARIOUS CONCENTRATIONS OFTASIC FUCHSIN THIOL MEDIUM J“ M Organism Concentration of Basic Fuchsin 1-5‘1‘ l-6T 1-7T l-8T l-9T l-lO‘I' V. f__e_____tus (B-24) b b b and 4/6a 6/68 6/681 6/6 6/6 6/6 L_?I_. pzogenes aldicroscopic examination showed only V. fetus present. icroscopic examination showed V. fefhs In abundance and an occasional clump of gram-positive coch. TABLE 9 RESULT OF SIMULTANEOUS IEOCULATIUN 0F V. FETUS AND M. PyocEEms VAR AUREUS INTO VARIOUS.CONCENTRATIONS o "ENTTIE’VIOLET 'JHIOL MEDIUM i Z" Organism Concentration of Gentian Violet 1-50'I' 1-60T 1-70'1‘ V. f_§___tus (B-24) and 0/6 4/6* 6/6* E. pvogenes *Microscopic examination showed only V. fetus present. It will be seen that a dilution of l : 70,000 of gen- tian violet allowed 1. fetus to grow but inhibited 1g. pyg- genes var aureus in every instance. Basic fuchsin, in con- centrations of 1 : 6,000 to 1 : 10,000, allowed 1. fetus to grow in every instance with inhibition of .111, pyogenes var aureus in concentrations up to l : 8,000. In concentrations 21 of l : 8,000 to l : 10,000 a few clumps of gram-positive cocci were seen on microscopic examination, but the gram- negative vibrios were present in an overwhelming majority. It was noted that, after an additional 48 hours incubation at 37 C, the growth in the tubes containing gentian violet was'much more profuse than that in the tubes containing basic fuchsin. 22 COMPARISON BY ELECTRON MICROSCOPY OF TWO MORPHOLOGICAEUY DISSIMILAR STRAINS OF VIBRIO FETUS Rhoades and Hardenbrook (1947) were the first to pub- lish electron micrographs of V. ggygs. They attempted to show the various forms that have been reported in the lit- erature. The pictures presented here were taken for pur- poses of comparison of two strains of 2} {Eggs designated as Plastridge's Strain and Strain 490. The colonial and cellu- lar morphology of the two strains are distinctly dissimilar. Colonial Characteristics 0bservation.wme made of 5 day old colonies on 5% bo- vine blood agar plates, incubated at 57 C in an atmosphere of approximately 10% 002. Examination‘wa51made using a dis- section.microscope and reflected light. Plastridge Strain: The colonies were raised, moist, glistening, circular, entire and homogenous. They averaged approximately 1 mm in diameter and were nonhesmolytic . Strain ggg: The colonies were raised, moist, glis- tening, circular, entire and slightly granular. They were decidedly smaller than the colonies of the Plastridge Strain, averaging approximately 0.5 mm in diameter. They were nonhemolytic. 23 Cellular Morphology Gram stains were made of 5 day old cultures in thiol medium and were examined using oil immersion. Plastridg§_Strain: The organisms were gram-negative. NUmerous cells showed a densely stained granule located terminally. These granules were slightly larger in diameter than the width of the cell. Individual cells were distinctly comma-shaped and many were joined in pairs forming an S-shaped ar- rangement. Strain £29; The organisms were grampnegative. Gran- ules similar to those described in the Plastridge Strain were seen in Strain 490. Individual cells appeared slightly longer than those of the Plas- tridge Strain. Distinct comma-shaped cells were very few in number. A.majority of the cells were straight or only slightly curved. Merchant (1946) described the granules as appearing in both young and old cultures. Stockman (1919) reported these granules as being filterable but that no growth came from.them. These granules are considerably more in evidence when the organisms are grown in thiol medium.than when grown in.Plastridge's semisolid.medium, The characteristics of both strains of this organism correspond to those of V} Egygg as given in Bergey's Manual 24 of Determinative Bacteriology, 6th Edition, with the excep- tion that the distinct comma-shape is not evident in indi- vidual cells of Strain 490. Serologically both strains ag- glutinate in high titer with antiserum prepared by the in- jection of Strain 490 into rabbits. Specimens for electron microscopy were prepared as follows. Seventyetwo hour cultures of each strain, grown in thiol medium, were suspended in 0.5% neutral formalinized saline and by differential centrifugation the cells were separated from the agar. The final preparation was a thrice 'washed suspension in physiological saline. Specimens were placed on collodion-covered screens and were viewed with an RCA.electron microscope, Model EMU, at the laboratories of the Michigan Department of Health. The original magnifica- tion was 7,000 diameters. 'Magnification of the accompanying figures was arrived at by enlargement of the plates exposed at the original magnification.mentioned. Figure 1 shows the Plastridge Strain at approximately 35,000 magnifications. The individual cells have a distinct comma-shape. One S-shaped form shows a slight constriction at its middle and suggests the occurrence of fission. One individual cell shows a single polar flagellum that appears to curl and go out of View underneath the cell and then emerge on the opposite side. Figure 2 shows a single typical cell of Strain 490 enlarged approximately 50,000 times. The distinct comma- 25 Fig. l.--Electron micrograph of 1. fetus Plastridge. Strain, approximately 55,000 X. 26 Fig. 2.--Electron micrograph of 1. fetus Strain 490, ap- proximately 50,000 X. 2'7 shape, evident in Plastridge Strain, is absent in this in- stance and a nearly straight rod is presented. .A study of this figure with a magnifying lens shows a constriction at the middle of the cell in the grayish halo that surrounds the denser inner cell structure. A very distinct polar fla- gellum is seen and near the point of attachment to the body of the cell the distinct outline becomes less evident and there is a gradual blending into the material seen as a grayish halo surrounding the denser inner cell mass. It ap- pears that the flagellum is a continuation of the outer con- fines of the bacterial cell. Pijper (1949), studying the flagella of Spirillum volutans by means of sunlight dark- ground microscopy, observed that the flagellum seemed to be attached to the cell wall of the organism, but that it did not pierce it. His conclusions, after study of a report by Miss van Iterson (1947) in which electron.micrographs of Vibrio metschnikovii showing flagellation were present, was that in vibrios the supposed flagellum is a continuation of the cell wall, and not, as most motile bacteria, mucous twirls derived from the capsule. Common to the organisms in both Figures 1 and 2 is the grayish halo immediately surrounding the dense inner mass of the organism. This is described by most workers as g shrinking of the cytoplasm away from.the outer confines of the bacterial cell. Absent from.all pictures of these two strains were the granules so noticeable on ordinary micro- 28 scopic examination. The small, smoothly outlined circular areas, seen in the photographs, are holes in the collodion membrane that was used as a retainer for the bacterial spec- imen. 29 DISCUSS ION The use of streptomycin or penicillin as aids in the recovery of _V_. fetus from a contaminated specimen does not appear to be of any practical value. The results obtained with these antibiotics incorporated into blood plates were so variable that no definite concentration could be given as the minimum inhibiting concentration for 1. 3316113. Repre- sentatives of the gram-positive cocci and the gram-negative bacilli, which so commonly contaminate specimens suspected of harboring _V_. £91323: showed growth at the greatest concen- tration of the antibiotics tried, whereas 1. 332?. was in- hibited by this concentration in a majority of instances. It is felt that the fluid medium, in which the minimum in- hibiting concentration of each antibiotic was determined, is not a satisfactory medium for the primary isolation of 1. M. Thiol medium inactivates penicillin and streptomycin and was originally developed as a medium into which speci- mens containing inhibiting concentrations of these antibiot- ics could be inoculated. For these reasons it is believed that the use of penicillin and streptomycin, in attempting to recover _V_. 33.1% in pure culture from a contaminated specimen, would not be helpful. The concentration of streptomycin necessary to inhib- it growth of _V_. £213.22 in the fluid medium was approximately 30 0.5,ng per ml. The concentration of penicillin necessary to inhibit the growth of 2} fetus in the f1uid.medium was ap- proximately 0.5 unit per ml. The use of penicillin solu- tions for vaginal douche in non-breeding cows has been ad- vocated by Chambers (1948). It is believed that the use of either penicillin or streptomycin in solution, given as a utero-vaginal douche, might be indicated as a therapeutic measure in cows known to be infected with E} ggtgg. It seems logical to suggest the use of antibiotics, known to be bactericidal in high dilution_for:V. fetus in vitro, when this organism is shown by examination to be an inhabitant of the bovine female genital tract. Gentian violet in thiol medium, in a concentration of l : 70,000 was shown to inhibit the growth of E. pyogenes 27.21; aureus, While allowing _V_. M to grow. Basic fuchsin in thiol medium, in concentrations ranging from 1 : 5,000 to l : 9,000 likewise allowed 1. £3313 to grow but inhibited 114.. pyogenes var aureus. The use of either of these dye-contain- ing media seams indicated for primary culture where the specimen is contaminated with gramppositive cocci. Presence or absence of such contamination can readily be determined by a gram-stain of the material prior to culturing. Gram- positive cocci are the most frequently found contaminants when working with vaginal swabs that have been taken in an aseptic manner. 0f the dye compounds studied none was found that 31 would inhibit the growth of :E_. 991; in thiol medium while allowing 1. _i_‘_e_t_1_1_§_ to grow. Coliforms are the most frequent- ly found contaminants in such specimens as vaginal dis- charges, placentas and aborted fetuses. The finding of some agent that will hold these rapidly multiplying bacteria in check, while allowing 1. £3133 to grow, would make it pos- sible to culture _V_. 313113 from many specimens that are now utterly useless. Electron micrographs of two morphologically dissimilar strains of E. £63113 revealed considerable difference between these two strains at a magnification of approximately 35,000 diameters. Both strains were isolated from aborted fetuses and their characteristics correspond to those of _V_. £33393 given in Bergey's Manual of Determinative Bacteriology, 6th Edition. The comma-shape so descriptive of organisms in the genus Vibrio is regularly present in the Plastridge Strain but is mostly absent in Strain 490. The flagellum of Strain 490 appears to be a continuation of the cell wall and thus is the same as that of another member of the genus Vibrio, namely 1. metschnikovii (Pijper, 1949) . 32 SULE‘LARY The minimum concentration of streptomycin and of pen- icillin necessary for inhibition of 1. _fstss in vitro, using a fluid medium, was found to be 0.5pg per ml and 0.3 unit per ml respectively. Gentian violet in thiol medium in a concentration of l : 70,000 allowed the growth of three strains of 1. _fsjsss but inhibited the growth of s; pyogenes var aureus. Basic fuchsin in concentrations of 1 : 5,000 to 1 : 7,000 was found to give similar results. The amount of growth evident by gross observation was decidedly greater It the end of 6 days in the thiol tubes containing gentian violet than in those containing basic fuchsin. No dye studied inhibited _E_:_. 921; while allowing 1. ms to grow. Electron micrographs of two morphologically dissimi- lar strains of E. fstss showed a very marked difference in individual cellular morphology at magnifications of approxi- mately 35,000 diameters. 313 REFERENCES Baker, D. W., and Stone, W. S. 1959 A study of Spirillum ovis in a group of ewes maintained under observation for Ewe successive lambing seasons. Cornell Vet., gs, 52-54. Barger, E. H. 1927 Report of a case of abortion induced by Vibrio fetus. J. A. v. M. Ag, 17-?- (11.8. 25) , 4:68-740 Bell, W. B. 1950 Vibrio fetus infection in Virginia. J. A. V} M. A., 116, 56-57. Breed, R. S.,'Murray, E. G. D., and Hitchens, A, P. 1948 Bergey's Manual of Determinative Bacteriology. 6th ed. The Williams and Wilkins Company, Baltimore. Carpenter, C. M; 1920 Researches upon a Spirillum associ- ated with abortion in ewes. Rpt. N. Y. State Vet. Coll., 1918-19, 129. _ Chambers, E. E. 1948 Penicillin in the treatment of non- breeding cows. N. Am. Vet., g2, 640-41. Curtis, A. H. 1915 A motile curved anaerobic bacillus in uterine discharges. J. Infectious Diseases, lg, 165-69. Gilman, H. L. 1959 Some causes of abortion in cattle free from Bang's disease. Cornell Vet., gs, 155-64. Graham, R., and Thorp, F. Jr. 1950 Vibrionic abortion in sheep. J. A. V} M. A., Z§.(n.s. 29), 568-75. Hagan, W. A. 1945 The infectious diseases of domestic ani- mals. Comstock Publishing 00., Inc., Ithaca, New York. Refer to p. 154. Huddleson, I. F. 1948 A satisfactory medium for the isola- tion, cultivation and maintenance of viability of Vibrio fetus (bovine). J. Bact., s9, 508. Lee, A» M5, and Scrivner, L. H. 1941 Experimental work on recent outbreaks of abortion in ewes. Ami J. Vet. Res., a, 50-540 Mac Fadyean and Stockman 1915 Report of the departmental committee appointed by the Board of Agriculture and Fisheries to inquire into epizootic abortion. Part III, Abortion in sheep. Cd. 7156. Appendix to Part III, Cd. 34 7157. wyman and Sons, London. Cited in: Smith, T. 1918. Merchant, I. A. 1948 Veterinary Bacteriology, 5rd ed. The Iowa State College Press, Ames. Refer to p. 501. Moore, G. R.- 1950 Clinical aspects of Vibrio fetus infec- tion in cattle. J. A. V. M. A., 116, 190-92. Pijper, A. 1949 The flagella of Spirillum volutans. J. Bact., §Z, 111-17. Plastridge, W. N., and Williams, L. H. 1948 Vibrionic abortion in cattle: Second report. Cornell Vet., 58, 165-800 ' Rhoades, H. E., and Hardenbrook, H. Jr. 1947 The occur- rence of vibrionic abortion in an Illinois dairy herd. Cornell Vet., 51, 8-15. Ryff, J. F. 1940 Vibrionic abortion in Michigan Sheep. J. A. V. M. A., 2.2-, 452-55. Ryff, J. F., and Lee, A. M. 1945 Vibrio fetus in sheep. m. J. Vet. Res., 9, l49"580 Schroeder, E. C. 1920 A review of several publications on infectious abortion disease. J. A. V. M. A., £1 (n.s. 10), 270-81. ‘ Smith, T. 1918 Spirilla associated with disease of the fetal membranes in cattle (infectious abortion). J. Exp. Med., gs, 701-20. Smith, T., and Taylor, M. S. 1919 Some morphological and biological characters of the spirilla (Vibrio fetus N. Sp.) associated with disease of the fetal membranes in cattle. J. EXp. Med., sg, 229-512. Stockman, S. 1919 Vibrionic abortion. J. A. V. M. A., §§ (nos. 8) ’ 4:99-504. Traum, J. 1925 The importance of Bacterium abortum of Bang and other microorganisms in boVTneIinfectious aBortion. Calif. Agr. Exp. Sta. Bull. 555. van Iterson, W. 1947 Some electron-microscopical observa- tions on bacterial cytology. Biochem. Biophys. Acta, 1, 527-48. Cited in:: Pijper, A. 1949. 35 Ward, B. Q. 1948 The apparent involvement of Vibrio fetus in an infection in man. J. Bact., §_5_, 115- . Webster, H. D., and Thorp, F. Jr. 1949 Vibrionic abortion of cattle in Michigan. M. S. 0. Vet., _9_, 1949. Welch, H., and Marsh, H. 1924 Vibrionic abortion in sheep. J. A. V. L1. Ag, @- (HoSo 18), 203-100 36 I wish to express my sincere gratitude to Dr. H. J. Stafseth for his counsel and assistance in the preparation of this manuscript. For the original electron micrographs, enlargements of which are presented here, I am indebted to Mr. Morris Rhian and to the Bureau of Laboratories of the Michigan Department of Health, Lansing, Michigan. I "l Inc-II I. e d