permit Corynebacterium probablylymphadenitis in sheep and ulcerative lymphangitis in horsesis given the name C. ovis. C. bovis is given the credit for causing pyelonephritis in cattle

A STUDY OF THE CORYNEBACTERIA ASSOCIATEDWITH DISEASES OF DOMESTIC ANIMALS'

I. A. MERCHANTDepartment of Veterinary Hygtene, Iowa State College, Ames, Iowa

Received for publication January 21, 1935

Corynebacteria found in human tissue have been rather com-pletely studied and classified. This has been due largely to theimportant relationship many of them have had with Corynebacte-rium diphtheriae. The Corynebacteria of animal origin, however,have not been studied or classified adequately. Diphtheroidbacteria are the causes of acute and chronic diseases of animals,and some species have been described which are of great impor-tance to animal health. Space does not permit a complete reviewof the literature of these organisms, but a few important contribu-tions should be mentioned.2

Corynebacterium pyogenes is probably the most important, sinceit is the organism found in more types of lesions than any of theothers. This organism has been isolated from suppurative pneu-monia and arthritis in swine and chronic suppurative conditionsin cattle and sheep, particularly mastitis. From the time of theoriginal descriptions of the organism by Lucet (1893), Grips(1902), Kunneman (1903), and Glage (1903), the organism hasbeen isolated from a variety of tissues and animals by numerousinvestigators. Most of the work was done in European countries,particularly Germany. Brown and Orcutt (1920) in the UnitedStates studied it in detail. The most recent and complete studyof C. pyogenes was by Rolle (1929). Ray (1928) recorded finding

1 A thesis presented as partial fulfilment for the degree of Doctor of Philosophyat Iowa State College in 1933.

2 The detailed literature concerning the Corynebacteria associated withanimal diseases may be obtained by consulting the original thesis on file at theIowa State College Library, Ames, Iowa.

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a diphtheroid associated with pulmonary edema of swine, which,because of its location and species of animal infected, may havebeen C. pyogenes.

Since the original studies of Nocard (1896) and Preisz (1894),Corynebacterium pseudotuberculosis has been known to causecaseous lymphadenitis in sheep and ulcerative lymphangitis inhorses. N6rgaard and Mohler (1899) described the occurrenceof caseous lymphadenitis in the United States. This organismalso has been found in suppurative conditions in cattle. Dainesand Austin (1931) found it in skin lesions of tuberculin-reactingcattle.

Corynebacterium renalis was described in detail by Ernst (1905,1906) in his study of the cause of pyelonephritis of cattle. Jonesand Little (1925) also found this organism associated with pyelo-nephritis of cattle.Magnusson (1923) was the first to describe Corynebacterium equi

after isolating it from cases of pneumonia of colts. Dimock andEdwards (1931) added to the knowledge of this organism whenthey described cultures which had been isolated from foals.There has been no consistency in the nomenclature and classi-

fication of the Corynebacteria of animal origin by various authors,and some species have been entirely overlooked.Eberson (1918) probably was the first to place C. pseudotuber-

culosis and C. pyogenes under the generic name Corynebac-terium.Buchanan and Murray (1922) considered only one diphtheroid,

C. pseudotuberculosis, of any consequence in animal disease. C.pyogenes was placed in the genus Hemophilus and assigned thename Hemophilus pyogenes.

Minett (1922) studied 53 strains of Corynebacteria from varioussources, and on the basis of morphology, biology and immunity(agglutination) placed them in the following groups: I. Preisz-Nocard group; II. Wound diphtheroids; III. Skin diphtheroids;IV. Conjunctival diphtheroids, (a) equine, (b) bovine, (c) human;V. Nasal diphtheroids (horses); VI. Urine diphtheroids (a) equine,(b) bovine, (c) human; VII. Bacillus pyogenes; VIII. Bacilluslipolyticus; IX. Bacillus enzymicus; X. Throat diphtheroids

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(human). The article did not give any of the characteristics ofthe above groups.Andrewes et al. (1923) included C. pseudotuberculosis-ovis and

C. pseudotuberculosis-murium in a group which they titled "SpecialDiphtheroid Series." They observed that the latter organismwould find a place in their group 7 with a number of human strainswhich they studied. C. pseudotuberculosis-ovis was not placedin any of their groups.Bergey et al. (1934) placed the diphtheroid organisms from

animals in a group composed of short, thick, straight, barred andoccasionally club-shaped rods. There appear to be many discrep-ancies in the descriptions of these organisms. C. pseudotuber-culosis is the name given to a Gram-negative rod found in rodents.One of the cardinal characteristics of the Corynebacteria is theirpositive reaction to the Gram stain. The above organism isusually considered a Pasteurella. Bergey omits C. pyogenes.The well established C. pseudotuberculosis, which causes caseouslymphadenitis in sheep and ulcerative lymphangitis in horses isgiven the name C. ovis. C. bovis is given the credit for causingpyelonephritis in cattle while the organism C. renale, which hasbeen considered to be the etiologic agent, has been relegated to asecondary place.Thomson and Thomson (1929) proposed five groups of Coryne-

bacteria. C. pseudotuberculosis-murium was placed in group Iwhich was composed of non-chromogenic, profuse-growing,aerobic and facultative anaerobic bacteria. C. pseudotuberculosis-botns was allotted to group III which included the non-chromo-genic delicate growers, aerobic or anaerobic. C. equi was placedin group V, the chromogenic, profuse growers, aerobic or faculta-tive. C. pyogenes was not classified. C. renalis was notmentioned.

Kelser (1933) considered three Corynebacteria of importance inanimal diseases. C. pseudotuberculosis, C. pseudotuberculosis-rodentium and C. pyogenes. He agreed with Bergey in placing aGram-negative organism with the Corynebacteria. He placedC. renalis with C. pyogenes. These two organisms are quitedifferent, as will be shown in this paper. The descriptions of

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C. renalis by Enderlen, Ernst and Jones and Little do not suggestC. pyogenes described by Poels, Brown and Orcutt.Ford (1927) gave C. pyogenes and C. renalis specific recognition

but included C. pseudotuberculosis-ovis in a group of bacteriawhich cause pseudotuberculosis.

Topley and Wilson (1929) included C. renalis with C. pyogenes.They recognized C. pseudotuberculosis-ovis.

Gaiger and Davies (1932) mentioned C. pyogenes and C. bovisas the Corynebacteria which cause animal diseases.

SOURCES OF CULTURES

No attempt was made in this study to isolate all possiblediphtheroid bacteria from animal tissues. It is presumed thatmany exist as saprophytes, as do many which are found in humantissue; consequently it is probable that Corynebacteria existwhich may not show the characteristics of those included in thisstudy. An attempt was made to secure all strains of diphtheroidswhich have any etiologic relationship with animal disease. Repre-sentative types were obtained from other laboratories and somewere isolated by the author. It is believed that the culturesisolated by the author caused the lesions from which they came.The animal diphtheroids which have been studied sufficiently

to have received specific designation are as follows:

Corynebactertum pyogenesCorynebacterium pseudotuberculosisCorynebacterium renalisCorynebacterium equi

Most of the cultures which are described in this study belong tothe above four species. The history of each culture is given intable 1.

STAINING METHODS

Smears for staining were made from cultures twenty-four toforty-eight hours old. Various Gram staining methods were used.The one which gave the most satisfactory results was the methoddescribed by Kolmer and Boerner (1931), in which Weigert'sgentian-violet solution and acetone were used. The Ziehl-

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STUDY OF CORYNEBACTERIA 99

TABLE 1History of cultures

CULTURE OSTin LESION FROM WHOM OBTAINED

Type I. Corynebacterium pyogenes

8664 Bovine Purulent metritis Isolated by author8986 Bovine Suppurative mastitis Isolated by author8987 Bovine Suppurative arthritis Isolated by author4823 Bovine Pneumonia American Type Culture col-

lection9292 Ovine Suppurative mastitis Isolated by author

Type II. Corynebacterium pseudotuberculosis

63 Bovine Purulent cervicitis S. H. McNutt-Iowa163 Bovine Purulent cervicitis S. H. McNutt-Iowa167 Bovine Purulent cervicitis S. H. McNutt-Iowa

4-C-29 Bovine Tuberculosis ? of skin Daines and Austin-Utah12-C-29 Bovine Tuberculosis ? of skin Daines and Austin-Utah15-C-30 Bovine Tuberculosis ? of skin Daines and Austin-Utah18-0-31 Bovine Tuberculosis ? of skin Daines and Austin-Utah27-C-29 Bovine Tuberculosis ? of skin Daines and Austin-Utah8728 Bovine Tuberculosis ? of skin 0. G. Larson-Utah8772 Bovine Tuberculosis ? of skin 0. G. Larson-Utah9126 Bovine Purulent vulvitis Isolated by author809 American Type Culture col-

lection

Type III. Corynebacternum renalis

1l1 Bovine Purulent cervicitis S. H. McNutt-Iowa174 Bovine Pyelonephritis S. H. McNutt-Iowa

1310 Bovine Pyelonephritis Little and Jones-NewJersey

9064 Bovine Pyelonephritis Isolated by author34 Bovine Purulent cervicitis S. H. McNutt-Iowa112 Bovine Purulent cervicitis S. H. McNutt-Iowa149 Bovine Purulent cervicitis S. H. McNutt-Iowa152 Bovine Purulent cervicitis S. H. McNutt-Iowa182 Bovine Purulent cervicitis S. H. McNutt-Iowa173 Bovine Pyelonephritis S. H. McNutt-Iowa169 Bovine Purulent cervicitis S. H. McNutt-Iowa74 Bovine Purulent cervicitis S. H. McNutt-Iowa

8592 Bovine Pyelonephritis Isolated by author8713 Bovine Pyelonephritis Isolated by author8714 Bovine Pyelonephritis Isolated by authorW-2 Bovine Purulent cervicitis S. H. McNutt-Iowa


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TABLE 1-Concluded

CULTURE OST LHSION FROM WHOM OBTAINHD

Type III. Corynebacterium renalis-Concluded

183 Bovine Purulent cervicitis S. H. McNutt-Iowa164 Bovine Purulent cervicitis S. H. McNutt-Iowa165 Bovine Purulent cervicitis S.H. McNutt-Iowa

8124 Equine Pus in semen Isolated by author

Type IV. Corynebacterium equi

15655 Equine Pneumonia Dimock and Edwards-Ken-tucky

Type V. Non-chromogenic-viscid

175 Bovine Mastitis-milk S. H. McNutt-Iowa176 Bovine Mastitis-milk S. H. McNutt-Iowa

Type VI. Non-chromogenic-profuse growers-moist

181 Bovine Purulent cervicitis S. H. McNutt-Iowa184 Bovine Purulent cervicitis S. H. McNutt-Iowa

Type VII. Orange colored variants

112a Variant of culture 112 Isolated by author184a Variant of culture 184 Isolated by author

27-C-29a Variant of culture 27-C-29 Isolated by author95 American Type Culture collection

8774 American Type Culture collection

Corynebacterium diphtheriae-Park Strain 8

296 American Type Culture collection

Neelsen acid-fast stain was used. Cultures grown on modifiedPetroff's medium and in milk were stained by the Ziehl-Neelsenmethod on the second, fifth, tenth and thirtieth days. Albert'sstain was used to demonstrate metachromatic granules. Culturesfor this stain were grown on Loeffler's serum because this mediumapparently caused the formation of granules more readily thanany other.

CULTURE MEDIA AND TESTS

Liver-infusion serum agar was used as the stock medium forthis study. It was found that a liver-infusion broth base was


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more suitable than lean meat or heart-infusion broth. Not onlywas growth more abundant on this medium, but chromogenesiswas more marked; in fact, a few cultures produced pigment onlyon this medium. To prepare this medium, two per cent granu-lated agar was added to liver-infusion broth. After sterilizationin the autoclave and cooling to 450C., bovine serum which wassterilized by filtration, was added to the agar making 10 per centof the total volume. The reaction of the medium was pH 6.6.This medium was found to be suitable for the isolation of thecultures obtained by the author and it produced suitable growthof all the cultures which were studied.

Various types of solid media have been described for the isola-tion of the diphtheria bacillus and related microorganisms.Douglas (1922) described a medium composed of nutrient agar,potassium tellurite and sterile trypsiniized serum. He found thatcolonies of Corynebacteria had a distinctive black color on thismedium. In this study all the cultures grew with colonies ofvarious shades of blackness, slate to jet black. The use of potas-sium tellurite in medium as a diagnostic agent for diphtheroidsmay lead to confusion. King and Davis (1914) studied the use ofthis salt as an indicator of microbial life. They described themacroscopical and microscopical appearance of most of thecommon species of pathogenic bacteria. It was found that allof the more common micro6rganism reacted with potassiumtellurite forming characteristic black compounds. The reactionwas probably due to a reduction of the tellurite. They found adilution of 1:50,000 of the salt seemed to be most suitable as ageneral microbic indicator. The author found that many of thecommon bacteria produced black colonies on Douglas medium.Allison and Ayling (1929) described an improved medium forisolating C. diphtheria. They added copper sulphate to trypsin-ized-serum tellurite agar and found this medium inhibited thegrowth of streptococci and staphylococci, which were the organ-isms commonly found in the throat of man. The writer foundthis medium to be unsatisfactory for the cultures he studied. Avery heavy seeding was necessary to get any growth of manycultures and the growth of C. pyogenes was entirely inhibited.

JOURNAL OF BACTERIOLOGY, VOL. 30, No. 1

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An excellent medium for the isolation of C. pyogenes wasLoeffler's medium to which was added 1 cc. of 1 per cent potassiumtellurite to each 100 cc. of medium. This medium was particu-larly suitable for C. pyogenes because the serum was liquefied andthe tiny black colonies contrasted with the white color of themedium could be seen in the liquefied depressions. (See fig. 1.)

Hiss serum water, pH 7.0, was used for the fluid medium. Forthe fermentation reactions 1 per cent of the carbohydrate to betested was added to the medium. Andrade's indicator was usedto detect acid formation. Readings were made after 1, 2, 3, 5,7,10, 15, 20 and 30 days. When acid was formed in any of thetubes, agar slants were inoculated to determine the purity of theculture. Inoculations to determine growth were conducted onthe tenth day on all cultures.Litmus skim-milk was used to demonstrate the action of the

microorganisms on milk.The action of the various cultures on solidified serum was

determined by growth on Loeffler's serum. Cow, instead of horse,serum was used.

Gelatin liquefaction was studied by growing the cultures inserum gelatin. Fifteen per cent granulated gelatin was added toliver-infusion broth to which serum, 10 per cent, was added.The cultures were incubated at 370C. and observed every day forten days. They were placed in a refrigerator for one hour eachday in order to detect liquefaction.

Hemolysis was detected by growing the cultures in blood agar.The medium was prepared by adding sterile defibrinated rabbit'sblood to a 2-per-cent heart-infusion agar, pH 7.2. Tubes wereinoculated when cooled to 450C. and agitated gently to insure aneven distribution of the bacteria in the medium. The mediumwas poured into Petri dishes in a thin layer. The final readingunder 100 magnification was made on the fourth day.Hydrogen sulphide production was ascertained by growing on

lead acetate agar (Difco). Voges-Proskauer and methyl-redreactions were determined by growing the cultures in V.P.-M.R.medium (Difco), nitrite formation by growing the cultures onnitrate agar (Difco) using the test given for this medium and indol

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formation by growing the cultures in tryptophane broth (Difco)and applying the Gor6 test. Sterile cow serum, 10 per cent, wasadded to all the above media to assure uniform growth.The oxygen relationship of the cultures was obtained by grow-

ing them in a semi-solid serum liver-infusion agar. Instead of a2-per-cent agar, as used in the stock medium, a 1-per-cent agarwas used. Tubes were filled one-half full with this medium.Two stab inoculations were made in each tube. They wereincubated at 370C. for 10 days, observations being made each day.

AGGLUTINATION TECHNIC

The dry growth of some of the diphtheroids made it ratherdifficult to obtain uniform suspensions. This was especially trueof C. pseudotuberculosis. Various methods to overcome thisdifficulty have been suggested. Bull and Dickinson (1931)described a method whereby they were able to get uniform sus-pensions of living bacteria by the use of bile salts in the dilutingfluid and by shaking and centrifuging. Minett (1922a) was ableto get a uniform suspension by grinding the growth, which hadbeen dried over CaCl2, in a mortar and then slowly adding salinesolution.Uniform suspensions of the cultures used in this study were

made as follows. Young actively growing cultures were trans-ferred to freshly made serum agar slants. After twenty-fourhours' incubation at 370C. the cultures were washed off with 1per cent sodium chloride solution and transferred to thick-walledtest tubes in which were placed glass beads. The suspensionswere shaken for one hour in a mechanical shaker. They werethen filtered through compact cotton pads. The cotton padsused in a milk sediment-tester proved satisfactory. The tubeswere then centrifuged at low speed for fifteen minutes. Thelarger clumps were thrown to the bottom of the tubes. Thesupernatant suspensions were poured into flasks. Saline wasadded to the residue in the tubes which were again shaken andcentrifuged. This procedure was continued until all the bacteriawere in a uniform suspension. The suspensions were of thedensity of tube 2 of McFarland's nephelometer.

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Suspensions made in the above manner were used to inoculaterabbits intravenously. None of them were attenuated exceptC. diphtheriae, which was subjected to 60'C. for one hour. Therabbits were given the following doses three days apart, 0.5 cc.,1 cc., 2 cc., 3 cc., 4 cc., 5 cc., for the production of agglutinatingserum. The cultures inoculated into rabbits were chosen uponthe basis of colony characteristics and fermentation reactions.They were considered to be different species or different strainsof the same species. The health of the rabbits was not visiblyimpaired except in two cases. The rabbits which were given C.pyogenes, 8986, and the variant 112a developed subcutaneousabscesses and arthritis.The agglutination tests were made by the rapid plate method.

It was found that a number of the cultures settled during theforty-eight hours required for the macroscopic tube test. Thesuspensions for the plate test were made by centrifuging the dilutesuspensions at high speed until as many of the cells as possiblewere thrown down. In the dry cultures all were precipitated butin the moist types of growth it was impossible to get all the bacte-ria out of suspension. The precipitated bacteria were thenplaced in suspension in 1 per cent sodium chloride solution. Thesuspensions were standardized to the density of 4 mm. by a Gate'snephelometer and 0.5 per cent phenol was added as a preservative.Equal amounts, 0.04 cc., of suspension and serum were used forpreliminary agglutination reactions conducted with the differentsera and with each culture. Those which showed a positivereaction were then tested using a standard amount of suspension,0.04 cc., and varying amounts of serum; 0.08 cc., 0.04 cc., 0.02 cc.,0.01 cc. and 0.005 cc.The tests were conducted on a glass plate placed on a box in

which there was an electric light. The background was black.When agglutination occurred it was present within one minutein all cases. The reactions were very definite.

RESULTS

MorphologyThe Corynebacteria are noted for their variability in size and

shape. This becomes more apparent when they are grown on the

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various types of media. Some of the classical characteristics ofthese bacteria are their swollen, as well as pointed ends, striations,coccoid shapes, and metachromatic granules. They are non-motile, and do not produce spores nor capsules. In liquid mediathey have a decided tendency to form clumps. A parallel (pali-sade) arrangement of the cells is often observed. This arrange-ment resembles that of streptococci, but the cells are ovoid,arranged side by side. Some of the Corynebacteria in this studycan be recognized by their morphology.

C. pyogenes was the smallest. The morphology of this type isrepresented in figure 2. It is seen that various shapes rangingfrom coccoid to bacillary are present. Club-like ends, as well aspointed ends, are visible. On serum agar the cells were large andthere was more of a tendency toward the coccoid shape, althougha few swollen bacillary forms were observed. The organismgrowing in milk assumed the bacillary shapes characteristic ofthose in the direct smear. In serum bouillon C. pyogenes wasmore ovoid than in milk. The formation of groups and theparallel arrangement of the cells was noted. Growth of strain(8986) on Loeffler's serum did not show metachromatic granuleswhen smears were stained with Albert's stain.

C. pseudotuberculosis was distinctly coccoid when grown uponserum agar. Upon careful observation, however, it was seenthat the cells were more ovoid. Parallel arrangements were alsoseen. Granules were formed when the organism was grown onLoeffler's serum medium (fig. 3). The cells were longer on thismedium than those on serum agar. The bacteria grown in milkand serum bouillon, were ovoid in shape and tended to formclumps.

C. renalis was the largest of the four species of diphtheroidsincluded in this study. This organism had a distinct bacillaryshape in all media. The direct smear (fig. 4) from kidney exudatereveals striated bacilli. When grown on serum agar the cellswere generally quite large and uniform, but segmented shapeswere seen also. Numerous large metachromatic granules wereformed when C. renalis was grown on Loeffler's serum medium.Clumping of this organism in serum bouillon and milk occurred.

C. equi showed great variability in shape when grown on various

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media. This organism was quite ovoid on serum agar but anoccasional bacillary shape was seen (fig. 5). The same coccoidform was found on Loeffler's serum and no granules were formed.When grown in serum bouillon and milk, enormously large cellscould be found. When smears were first made from these media,contamination was suggested. Careful plating and staining,however, revealed that swollen bacillary shapes in fluid mediawere definitely characteristic of this organism.The morphology of the organisms in Types V and VI did not

differ from that of C. pseudotuberculosis.

StainingAll of the strains of Corynebacteria used in this study were

Gram-positive. However, in the case of C. pyogenes a number ofGram-negative cells were found in cultures over three days old.Metachromatic granules were revealed in the majority of thecultures by Albert's stain after the cultures were grown on Loef-fler's serum. More granules were seen when the cultures werenot over forty-eight hours old. In older cultures the granuleswhich were present were large. Smears from the cultures used inthis study were also stained by the Ziehl-Neelsen acid-fast stainafter they had grown on modified Petroff's medium and in milk.None of the cultures studied was acid fast.

Macroscopic growth characteristicsThe types of diphtheroids described here showed different

growth characteristics. C. pyogenes grew on serum agar in a verysmall dew-drop like colony (fig. 6) very similar to the colonies ofstreptococci. The edge of the colony was entire. Small granuleswere observed in the center and the outer part of the colony wasvery finely granular (fig. 7). As the culture aged, the coloniesincreased in size and became opaque and dry. One strain, 8664,which showed all of the biologic characteristics of C. pyogenes wasisolated from a case of bovine metritis. This culture grewprofusely on all media, was smooth and moist and produced alight yellow-colored pigment.The growth of C. pseudotuberculosis was very dry on all media.

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When the colony was small (fig. 8) it was granular and the edgewas often uneven. Other colonies the same size showed a centralgrowth from which extended secondary colonies. This gave apitted, and, in some cases, a mammillated appearance to thecolonies when viewed under lower magnification. As the cultureaged, it became granular and dry (fig. 9). The entire growthcould easily be lifted from the surface of the medium. The colorrange of the cultures was from a cream to an orange.

C. renalis grew in a moist, cream-colored colony. The edgeswere irregular, but secondary growth was not observed (fig. 10).The culture became dryer as it aged but never was as dry as C.pseudotuberculosis.The growth of C. equi was very distinctive. Small, young

colonies were finely granular with smooth edges (fig. 11). Theorganism grew rapidly, forming a heavy, moist, red-colored streakalong the needle track. The growth was distinctly mottled(fig. 12).

Cultures 175 and 176 from milk, placed in Type V, were charac-terized by adhering so tenaciously to the medium that the growthwas removed with difficulty. Otherwise they resembled C.renalis. The cultures 181 and 184 in Type VI developed rapidlyand were moist and opaque.In serum bouillon, C. pyogenes formed light powdery flakes on

the walls and a finely granular sediment in the bottom of the tube.The liquid remained clear. A thick, granular pellicle was formedby C. pseudotuberculosis in serum bouillon. As this pellicle agedit fell to the bottom of the tube. In serum bouillon, C. renalisproduced a white powdery growth which collected along the wallsand in the bottom of the tube and in some cultures a thin pelliclewas present. The liquid remained clear. Serum bouillon wasuniformly clouded by C. equi. Very little sediment was formedand no pellicle was produced. Type V, milk cultures, grew inserum bouillon, forming a light powdery sediment. The culturesof Type VI, profusely growing, non-chromogenic organisms,produced a dense turbidity in serum bouillon and formed a thinmembranous pellicle.

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Biologic reactionsAll cultures of the Corynebacteria grew under aerobic condi-

tions upon media to which serum was added. C. pyogenes,however, also grew well under anaerobic conditions in a semi-solid serum agar. C. pyogenes was the only species which liquefiedsolidified-bovine serum and gelatin. The reaction of the differenttypes was variable in milk. C. pyogenes caused coagulation ofmilk within forty-eight hours. Digestion of the curd then beganand continued slowly until nothing but a very small mass re-mained in a water-clear whey. During the digestion process themilk had an acid reaction. C. pseudotuberculosis grew readily inmilk but did not produce any change. The action of the strainsof C. renalis was variable. Nine of the twenty strains studiedcaused a digestion of the milk casein with a resulting alkalinereaction but no coagulation. The digestion continued until themedium was of a blue transparent nature. No change occurredin eleven of the cultures. The cultures in Types IV and V didnot alter milk. The two cultures of Type VI coagulated the milkand reduced the litmus. There was no digestion of the curd.

C. pyogenes was the only species studied which hemolyzed redblood cells. Indol was not produced by any cultures. None ofthe types gave a Voges-Proskauer reaction. Only Type VI gave apositive reaction to methyl red. Nitrite was formed by C. equionly. Hydrogen sulphide was not present in any of the cultureson lead acetate medium.

Carbohydrate fermentationsC. pyogenes was actively saccharolytic. The fermentation

reactions of the strains of this organism studied were quite uni-form. One strain, 8664, fermented xylose, salicin and mannitolin addition to the sugars fermented by all the strains, and culture8986 produced acid in xylose. This reaction was not true of themajority of the strains. C. pseudotuberculosis varied in itsfermentation reactions. The majority of the strains fermentedglucose, levulose, mannose, sucrose and maltose. C. renalis alsowas quite variable in its ability to ferment carbohydrates. Acid

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was formed in glucose by the majority of the cultures; many ofthem fermented levulose and mannose. One strain, 164, wasunique in that it fermented only dextrin. C. equi, 15655, did notferment any of the carbohydrates used in this study. Culture175 Type V caused acid to form in glucose, levulose, galactose andsucrose. The two cultures placed in Type VI were quite activein their attack upon carbohydrates. They produced acid in 11of the 15 sugars.

TABLE 2Carbohydrate fermentation

0

N

CULTURE N ENO0 0 NZNUMBER 44E-

8599 -- ++Type I. C. pyogenes........... 8664 4- ++++++++---

I8986 -+++++++++ -63 --++-+--+ -

Type II. C. pseudotubercu- 163 - - + + - +- + +T0888.................|..4-0-29 -|-|+ ++ + -|+

{18-0-31 --+++++-+ -111 --+.

Type III. C. renalis..........{ 112 - ++173 --++-+164--+-

Type IV.C.eqi15655 ------

Type V. Cultures from milk 175 - - + + + - +Type VI. Non-chromogenic-profuse growers-moist . 181 -- + + + + + + + + + -+ +

Type VII. Orange coloredvariants. . 112a .-

No gas was produced by any of the cultures. The fermentingaction of representative strains is given in table 2.

Agglutination reactionsThe sera used in the agglutination reactions were obtained from

rabbits inoculated with cultures chosen from the different types.In this study the serum is given the number of the culture.

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Serum 34-Corynebacterium renalisSerum 167-Corynebacterium pseudotuberculosisSerum 8664-Corynebacterium pyogenesSerum 8713-Corynebacterium renalisSerum 8728-Corynebacterium pseudotuberculosisSerum 15655-Corynebacterium equiSerum 8772-Corynebacterium pseudotuberculosisSerum 8986-Corynebacterium pyogenesSerum 1310-Corynebacterium renalisSerum 296-Corynebacterium diphtheriaeSerum 112a-Orange variant from 112

The strains of C. pyogenes were uniform in their agglutinationreactions. The five strains studied (table 3) were agglutinatedby sera 8664 and 8986. One strain, 9292, from a ewe, wasagglutinated by two C. renalis sera and one C. pseudotuberculosisserum.

C. pseudotuberculosis and C. renalis showed a close serologicalrelationship (tables 3 and 4). Four C. pseudotuberculosis strainswere agglutinated by C. renalis serum 34, six by C. renalis serum8713, and four by C. renalis serum, 1310. A number of thestrains of C. pseudotuberculosis failed to be agglutinated by C.pseudotuberculosis sera. One serum, 8772, was active in causingagglutination of all of the strains. None of the cultures wasagglutinated by C. pyogenes serum. Two cultures, 8772 and 809,were slightly agglutinated by C. equi serum.The close relationship between C. pseudotuberculosis and C.

renalis is again emphasized by a study of table 4. Two C. renalisstrains were agglutinated by C. pseudotuberculosis serum 167,eight by serum 8728 and fifteen by serum 8772. More of thestrains, however, were agglutinated by sera 34, 8713 and 1310,which were C. renalis. Cultures 183, 164, and 165 were placedwith the C. renalis type because of their fermentation reactions,their morphology, cultural characteristics and because theyoriginated from cases of bovine pyelonephritis. Agglutinationreactions, however, indicated a closer relationship to C. pseudo-tuberculosis. Culture 8124, from the semen of a horse, was placedin this type on the basis of its fermentation and agglutinationreactions.

C. equi was agglutinated only by its own antiserum.

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Since the cultures in Type V could not be made into suitablesuspensions, agglutination reactions were not possible.Type VI cultures were not agglutinated by any of the sera.Agglutination of Type VII, orange-colored variants, is dis-

cussed under heading, "Variation."It is interesting to note that serum from the rabbit inoculated

with culture 296, C. diphtheriae, agglutinated two of the C. renalis

TABLE 3Agglutination reactions

SERUM NUMBER 0

CULTURE

NUMBER C X0 .O

8664 - +---Type I. Corynebacterium pyo- 89876 --+----+

898728 --+-- - - +--- - - - -

genes............. 4823 --+--- -+-9292 + ++ ++ .

63 + - +- +-+ -163 .+.----167 -+-++-+ -

4-C-29. .+.------12-0-29 -----

Type II. Corynebacterium 15-C-30. . + .pseudotuberculosist.rains,.318-0-31an+- - .-----

27-0-29 + : -+ L=-+ ----- - -

8728 -+--+-+---------9126 -+-++-+-8772 :-1-- 4- 4 Ai:+- 4i1-4 - -809 4--- =L-44 +i-4--4 - -

8599, 8600, 8644 and 8744 were lost before agglutination work was started.+ = agglutination in all four dilutions. =agglutination in first two dilu-tions. - = negative.

strains, 34 and 8713, and one strain of C. pseudotuberculosis.Sera produced by the injection of these three cultures agglutinatedC. diphtheriae, 296.

VARIATION

A possibility that variation may occur in diphtheroids wasmentioned by Daines and Austin (1931). They wondered


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112 I. A. MERCHANT

whether the organisms in their group 2 belonged to the samespecies as group 3. Group 2 was composed of typical Preisz-Nocard bacilli and in group 3 were placed the acid-fast strainswhich produced deep orange-colored, moist colonies. They ask,"Could these groups be smooth and rough variants of the same


Type III. Corynebacterium renalis

CULTURENUMBER

111174

13109064

3411214915218217316974

859287138714W-2183164165

8124

SERUM NUMBER

34 1 167 1 8664 1 8713 8728 1565 8772

++

+

+

+

+4

+

+

+

+

+

+4

+4

+

+++

+ = agglutination in all four dilutions.tions. - = negative.

8986 1310 296 112a

+

+

+

+

-

+

+++

+

NOR-MEAL

SBRUM

SALTSOLU-TION

4 = agglutination in first two dilu-

species? Or is it possible that there is a symbiosis between thesetwo groups and that they are different species?"During the course of this study an interesting type of variation

was observed. When growth tests were being made of thecarbohydrate fermentations an orange-colored colony appeared,with the typical growth of cultures 112, 184 and 27-C-29 fromlactose. The carbohydrate had been incubated for 10 days; the


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growth tests on Petri dishes had been incubated 4 days and hadstood at room temperature 10 days. Variants were also obtainedfrom old milk cultures. These variants were designated 112a,184a and 27-C-29a. They presented the same morphologicappearance as their parent cultures. The colony characteristics,however, were different. The growth of 112 was semi-dry, roughand cream-colored while 112a was moist, smooth and orange-pigmented. The young colonies under high magnification(X 220) did not exhibit a great deal of difference; both were finely


SERUM NUMBER 0CULTURE__PNUMBER C ||s|

Type IV. Corynebacterium equi.. 15655 --------+. - -

The growth of these cultures was ofType V. Cultures from milk.. 175 such a tenacious nature that suit-

176 able suspensions could not bemade for agglutination.

Type VI. Non-chromogenic- 181 _ - - - - - - - - - -

profuse growers-moist. 1 184 _ _ _ - -

( 112a .. + - -

Type VII. Orange-colored 184a - - - - - - -+ _ -

95 - - - - -

8774 ----|------- -|Corynebacterium diphtheriae-Park Strain 8.296+.....+..+..+2

+ = positive in all four dilutions. -= negative.

granular. Under low magnification (X 10) pit-like depressionscould be seen in the growth of 112. The growth appeareduneven. The pit-like depressions were absent in culture 112aand the colonies appeared to coalesce. Strain 184 was a moist-growing culture and was white. Variant 184a was moist but hadan orange color. Strain 27-C-29 was a typical rough, dry cultureof C. pseudotuberculosis while 27-C-29a was smooth, moist andorange in color. The three cultures, 112a, 184a, and 27-C-29a,were devoid of power to ferment the carbohydrates used in this


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I. A. MERCHANT

study. Two strains, 112a and 184a, were agglutinated by serum112a. It was noted that this serum caused the agglutination of5 C. renalis strains (table 5) and a slight agglutination of 2 C.pseudotuberculosis strains (table 5).Two other cultures, 95 and 8774, were placed in this group of

variants. Culture 95 was obtained from the American Typeculture collection and was called C. ovis (C. pseudotuberculosis).Culture 8774, appeared to be similar in all respects to culture 95.Both had a morphology similar to C. pseudotuberculosis, but theygrew profusely, were moist and smooth and had a deep orange-colored pigment.No acid-fast staining characteristics were noted in smears

made from any of the cultures of Type VII grown on modifiedPetroff's medium and in milk.

CONCLUSIONS

1. Four distinct species of Corynebacteria are associated withdiseases of domestic animals. They are, C. pyogenes (Glage)Eberson, C. pseudotuberculosis (Preisz) Eberson, C. renalis (Ender-len) Ernst and C. equi Magnusson.

2. The most distinctive characteristics of C. pyogenes are:morphology; aerobic as well as anaerobic growth; reproductiononly in media containing blood, blood serum or milk; hemolysinproduction; coagulation of milk with subsequent digestion of thecurd; liquefaction of gelatin and coagulated blood serum; produc-tion of acid in, glucose, levulose, galactose, mannose, sucrose,lactose, maltose and dextrin and by some strains, in xylose.

3. C. pseudotuberculosis grows in dry granular colonies; doesnot alter milk, gelatin nor coagulated blood serum; produces acidin glucose, levulose, mannose, sucrose and maltose.

4. C. renalis grows in moist isolated colonies; digests milkcasein producing an alkaline reaction but has no action on gelatinnor solidified blood serum. It ferments glucose and some strainsalso ferment levulose and mannose.

5. C. equi is characterized by its ability to produce a red-colored pigment, its profuse viscid growth, reduction of nitratesand inability to ferment carbohydrates.

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6. C. pseudotuberculosis and C. renalis are the only two of thefour to show a marked serological relationship.

7. Orange-colored variants were isolated from three cultures.These variants resembled their parent cultures in morphologybut were smooth and pigmented and failed to ferment carbo-hydrates whereas the parent cultures were usually rough, cream-colored and demonstrated some fermenting ability.

REFERENCESALLISON, V. D., AND AmING, T. H. 1929 Jour. Path. and Bacteriol., 32, 299.ANDRzwEs, F. W. ET AL. 1923 Diphtheria, Its Bacteriology, Pathology and

Immunology. His Majesty's Stationery Office, London.BERGEY, DAVID H. ET AL. 1934 Bergey's Manual of Determinative Bacteri-

ology. Williams & Wilkins Co., Baltimore, Fourth Edition.BROWN, J. HOWARD, AND ORCUTT, MARION L. 1920 Jour. Exp. Med., 32, 219-

248.BUCHANAN, R. E., Arm MuRRAY, CHAS. 1922 Veterinary Bacteriology, W. B.

Saunders Co., Philadelphia, Third Edition.BULL, L. B., AND DICKINSON, C. G. 1931 Australian Jour. Exp. Biol. and Med.

SC., 8, 45-52.DAINMS, L. L., AND AUSTIN, HAROLD 1931 Proc. Soc. Exper. Biol. and Med.,

29, 3-5.DIMoCK, W. W., AND EDWARDS, P. R. 1931 Jour. Amer. Vet. Med. Assoc., 79

(N.S. 32), 809-812.DOUGLAS, S. R. 1922 Brit. Jour. Exper. Path., 3, 263.EBERSON, F. 1918 Jour. Inf. Dis., 23, 1-42.ERNST, W. 1905 and 1906 Centralbl. F. Bakteriol., I. Abt., Orig., 39, 549; 40, 79.FORD, W. H. 1927 Text-book of Bacteriology. W. B. Saunders Co., Phila-

delphia.GAIGER, S. H., AND DAVIES, G. 0. 1932 Veterinary Bacteriology and Path-

ology. Alex Eger, Chicago.GLAGE, F. 1903 Ztschr. f. Fleisch. u. Milchhyg., 13, 166-175.GRIPS, W. 1902 Deutsch. tierarzt, Wchnschr., 10, 213-216.HALL, I. C., A.ND STONE, R. V. 1916 Jour. Inf. Dis., 18, 195-208.JoNES, F. S., AND LITTLE, R. B. 1925 Jour. Exp. Med., 42, 593-607.KELSER, R. A. 1933 Manual of Veterinary Bacteriology, Williams & Wilkins

Co., Baltimore, Second Edition.KING, W. E., Aim DAVIS, LEWIS. 1914 Amer. Jour. Pub. Health, 4, 917-932.KOLMER, J. A., AND BOERNER, FRED 1931 Approved Laboratory Technic.

D. Appleton and Co., New York.KUNNEMAN, D. 1903 Arch. f. Wisschft. u. Prak. Tierhlknde, 29, 128-157.LUCET, A. 1893 Ann. de l'Inst. Pasteur, 7, 325-330.MAGNUSSON, H. 1923 Arch. f. Wiss. Prakt. Tier., 50, 22-38.MINETT, F. C. 1922a Jour. Comp. Path. and Therap., 35, 71-107.MiErTT, F. C. 1922b Jour. Comp. Path. and Therap., 35, 291-302.NOCARD, ED. 1896 Ann. de l'Inst. Pasteur, 10, 609-629.


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116 I. A. MERCHANT

N6RGAARD, V. A., AND MOHLER, JOHN R. 1899 16th Ann. Rep., Bur. An. Ind.,U. S. Dept. Agr.

PE1EISZ, HUGo 1894 Ann. de l'Inst. Pasteur, 8, 231-255.RAY, J. D. 1928 Vet. Med., 23, 490.ROLLE, M. 1929 Biology der Bacterium pyogenes. Inaug. Diss., Hannover.

M. and W. Shafer, Hannover.THOMSON, DAVID, AND THOMSON, ROBERT 1929 Annals, Pickett-Thomson

Research Laboratory, Bailliere, Tindall and Cox, London, Vol. 2.TOPLEY, W. W. C., AND WILSON, G. S. 1929 The Principles of Bacteriology and

Immunity. Edw. Arnold & Co., London.

PLATE 1

FIG. 1. C. pyogenes-Loeffler's serum with potassium tellurite, 48 hours.Natural size.

FIG. 2. C. pyogenes-direct smear, lung of calf. Gram stain. X 1400.FIG. 3. C. pseudotuberculosis-Loeffler's serum. Albert's stain. X 1400.FIG. 4. C. renalis-direct smear, kidney exudate. Gram stain. X 1400.FIG. 5. C. equi-serum agar. Gram stain. X 1400.FIG. 6. C. pyogenes, 48 hours, serum agar. X 10.FIG. 7. C. pyogenes, 48 hours, serum agar. X 220.FIG. 8. C. pseudotuberculosi8, 48 hours, serum agar. X 220.FIG. 9. C. pseudotuberculostis, 4 days, serum agar. X 2j.FIG. 10. C. renali8, 48 hours, serum agar. X 220.FIG. 11. C. equi, 48 hours, serum agar. X 220.FIG. 12. C. equi, 48 hours, serum agar. X 10.


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JOURNAL OF BACTERIOLOGY, VOL. XXX

,.", ', ,v;

2 3

6

i

.Xi

9 10 11 12

(I. A. Merchant: Study of Corynebacteria)

J.

'

4I

.4^

5.1"w :

7 8

PLATE 1


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Documents

permit Corynebacterium probablylymphadenitis in sheep and ulcerative lymphangitis in horsesis given the name C. ovis. C. bovis is given the credit for causing pyelonephritis in cattle