576 . 851 . 5 (L isterel la monocytogenes) : 576 . 851 . 5 (Erysipelothrix rhwiopathicz),

A COMPARATIVE STUDY O F LISTERELLA AND, ER YSIPELOTHRIX.

MARY BARBER. From the Pathology Unit, Royal Free Hospital and the London County Council

Group Laboratory, Archway Hospital, London.

(PLATES 111.-VI.)

THE superficial resemblance between strains of Listerella and those of Erysipelothrix has been commented on by Topley and Wilson (1936). This study has been carried out in order to make a careful comparison of the two.

I have been working with five strains of Listerella rnonocytogenes and six of Erysipelothrix. The Listerella strains are 58. XXIII, 53. XXIII, H. Pirie, Gibson and Schultz. 58. XXIII was isolated from a guinea-pig and 53. XXIII from a rabbit by Murray, Webb and Swann (1926) during an epidemic in the Field Laboratories, Cambridge, in 1924. H. Pirie was obtained from the National Collection of Type Cultures, where it was sent after its isolation from a gerbille in the Tiger River district in 1925 by Harvey Pirie (1927). The Gibson and Schultz strains are both from human cases. The former was isolated by Dr Gibson (1935) from a fatal case of meningitis in a man aged 37 years in Edinburgh, the latter by Professor Schultz (Schultz et al., 1933-34) from a case of prolonged meningitis with recovery in California. These five strains were compared by Webb and Barber (1937), who failed to find any material differences between them. Strains 53.XXIII and H. Pirie were of low virulence but the others were highly virulent.

Strains 32 and 33 were isolated from the spleens of sows dead of swine erysipelas in 1934 and 1937 respectively and were kindlyplacedat my disposalby Mr Watts, of the Institute of Animal Pathology, Cambridge. Strain 6.35 was isolated from the spleen of a pig dead of an acute infection in 1935 and strain 9.35 from the heart blood of a pigeon which had been scarified with an emulsion of fzeces from a sick pig. These were obtained from the Institute of Animal Pathology, Cambridge, by the courtcsy of Dr Bosworth. Strains Kolle and M.S. 3 were obtained from Dr Balfour-Jones from the National Institute for Medical Research. Kolle was isolated by Professor Kolle of Frankfurt but no history is availabIe. M.S. 3 was isolated in 1934 by Dr. Balfour-Jones (1935) from the liver of a mouse in a spontaneous outbreak of disease in a breeding stock of mice a t the Institute. The virulence of these strains is extremely variable. Strains 9.35 and 6.35 failed to kill mice even in enormous doses. Strains 32 and 33 when first received had an M.L.D. for mice of about 500 million organisms, but after passage it was much smaller. Kolle and M.S. 3 killed only in very large doses when first received, but after passage were fairly virulent.

11

The Erysipelothrix strains are 32, 33, 6.35, 9.35, Kolle and M.S. 3.

12 M . BARBER

Morphology.

The morphology was studied in preparations from cultures from single colonies on plain agar slopes cultivated at 37" C. and at room temperature (17-25" C.). The morphology at 30" C. does not differ appreciably from that at 37" C.

Lhterella. Rods about 0.5 p in width, with rounded ends, arranged singly, in packets, in groups or in chains. No capsules and no spores. Smooth form. After 7-12 hours' incubation a t 37" C., rods from 0.5 to 2.4 p pre- dominate with occasional longer forms. At 24 hours the culture consists almost entirely of coccobacillary forms, 0.5-1.0 p (fig. 1). At 48 hours the coccal forms are slightly more predominant. No change occurs after this. In cultures incubated at room temperature the organisms divide into small forms rather more slowly, a 24- or 48-hour culture resembling a 12-hour culture at 37'C., and even older cultures show bacilli rather than coccal forms. Rough form. Early cultures (7-12 hours) a t 37" C. consist almost entirely of long filaments averaging about 60 p. Some of these filaments can be seen to be dividing into chains of bacilli. At 24 hours, long rods from 4.5 to 8.0 p and filaments from 10 to 30 p, predominate ; a few short rods from 1.5 to 3.0 p are also present (fig. 2). At 48 hours, rods from 1.0 to 5.0 p predominate, short filaments from 10 to 12 p are present in moderate numbers and there are a few coccal forms. No further breakdown takes place after 48 hours. Cultures a t room temperature consist of enormous filaments with little or no tendency to split up into bacilli (fig. 3). Intermediate form. 53. XXIII shows a morphology intermediate between these two after 24 hours' incubation but a t 48 hours the short filaments and long rods almost entirely split up into cocco-bacillary forms resembling the smooth type. Fig. 6 shows the filaments dividing into rods.

Extremely slender rods from 0.3 to 0.4 p in width, arranged singly or in packets, groups or chains. No capsule and no spores. Smooth form. 12- to 24-hour cultures consist predominantly of minute rods 0.8-2.0 p in length (fig. 5 ) , with occasional filaments. In older cultures there are rather more filaments. Room temperature cultures show little difference. Rough form. 7- to 12-hour cultures show organisms varying from 1.5 to 11 p. At 24 hours, filaments from 10 to 30 p predominate, while rods (0.8-5.0 p) and short filaments (10 p) are present in about equal numbers. Older cultures show little change. Cultures at room temperature show enormous filamonts which fail to split up into rods (fig. 7). Intermediate form. In 7- to 12-hour cultures a t 37" C., organisms vary from 1.5 to 10 p. At 24 hours filaments from 15 to 25 p predominate, with some rods 1-5-6.0 p (fig. 4). At 48 hours organisms are almost entirely filamentous, averaging about 30 p. At room temperature the organisms vary from rods of 2 to 5 p t o short filaments of 10 to 11 p.

All strains of Listerella and Erysipelothrix show similar staining reactions. They are Gram-positive, although fairly easiljr decolourised. Staining is sometimes irregular but no true granules have been seen with metachromatic stains in plain agar or inspissated serum cultures.

Erysipelothrix strains are non-motile, Listerella strains are all motile.

Eryslpefothrlx.

Staining.

Motility.

Comparison.

Both Listerella and Erysipelothrix organisms are Gram-positive rods with no capsules or spores and have a similar arrangement in

FIG. 1.-Smooth Listerella (68. XXIII) ; 24 hours at 37' C. Gram. x915.

FIQ. 2.-Rough Liutrrslla (H. Pirie) ; 24 hours at 37" C. Gram. x 915.

FIG. 3.-Rough Liutere l la (H. Pirie) ; 24 hours at room temperature. Gram. x 915.

F1~.'4.-intermec~iate Ery8ipelotkt'ia (32) ; 24 hours at 37" 0. Gram. x915.

FIQ. 5.-Smooth Et'ysipeZothrk (33) ; 24 hours at 37" C. Gram. ~ 9 1 5 .

FIG. 6.-Intermediate Listerella (53. XXIII) ; 9 hours at room temperature. x 915.

FIU. 'I.-Rough Yrysipelolhriz (9.35) ; 24 hours at room temperature. Gram. X915.

FIG. I.

FIG. 3.

FIG. j. FIG. G .

FIG. 2.

FIG. 4.

FIG. 7.

LISTERELLA A N D ERYSIPELOTHRIX 13

cultures. Both organisms show a striking difference in morphology between smooth and rough forms. Both rough forms in early cultures at 37" C . consist of long filaments which tend to split up after further incubation ; both produce enormous filaments if cultivated at room temperature.

Erysipelothrix is a much more slender organism than Listerella, whether in the form of rod or filament. The smooth strains of Listerella are smaller in 48-hour or older cultures than in young cultures. The smooth strains of Erysipelothrix show somewhat longer rods and more filaments in older cultures than in young. Listerella is motile, Erysipelothrix non-motile.

Cultural characteristics. The medium used was beef agar

prepared with early addition of peptone as advocated by Wright (1933). For purposes of description 48-hour colonies were chosen, as younger colonies are rather small.

48-hour colonies are typically round, convex and amorphous, with smooth, glistening surface and entire edge, about 1 - 1 6 mm. in diameter. They are transparent by transmitted light, milky white by reflected light, butyrous in consistency and easily emulsifiable (fig. 8). Older colonies show umbonate centres and are somewhat sticky but otherwise remain unchanged. Partially rough colonies are sometimes seen in these strains. The typical colony at 48 hours is slightly larger (1.5-2.0 mm.) and more flattened, with a matt surface and umbonate centre. This edge gradually spreads outwards until a colony of about 5 mm. is reached in about five days (figs. 10 and 11). Very young colonies of this type or colonies grown a t room temperature for 48 hours can be seen by a magnification of 110 to be made up of filaments (fig. 12). A few colonies of intermediate type are sometimes seen. Intermediate strain. 48-hour agar plates of 53. XXIII are made up almost entirely of partially rough colonies. These are about 1.5-2.0 mm. in diameter, with raised centre, slightly flattened periphery, matt surface and dentate edge (fig. 13).

Eryslpelothrlx : smooth strains. The typical colonies after 48 hours are round, convex and amorphous, with smooth glistening surface and entire or slightly undulate edge. The diameter varies from 0.1 to 0-8 mm. They are water-clear (fig. 8) and fairly easy to emulsify. On further incubation there is only very slight increase in size but central patches of brown pigmentation appear. The colonies of different strains vary slightly in size, those of M.S. 3 being the smallest and ranging from 0.1 to 0 . 4 mm., while Kolle and 6.35 have slightly larger colonies. Strain 33 when first received showed almost all smooth colonies but after repeated subculture it shows a mixture of smooth and rough colonies in about equal proportions. Rough strain. The colonies are typically slightly larger (1.0-1.5 mm.) at 48 hours and more flattened with a matt surface and dentate edge (fig. 14). On further incubation they spread out, until after about 5 days they reach about 4 mm. in diameter. Central pigmentation is very noticeable a t this stage (fig. 15). Very young colonies of this type or colonies incubated at room temperature for 48 hours can be seen by a magniflcation of 110 to be made up of filaments (fig. 16). Intermediate strain. 48-hour plates of strain 32 consist almost entirely of partially rough colonies. These have a

Aerobic agar plate cultures at 37" C.

Listerella : smooth strains.

Rough strains.

A fimbriate edge is just visible with a lens (fig. 9).

14 M . BARBER

raised centre, flattened periphery, matt surface and slightly dentate edge and measure about 0.6-1.0 mm. (fig. 17). No change takes place on further incubation.

After 24 hours, thin, smooth, confluent, raised, semi-transparent growth. After 48 hours, growth is fairly profuse. Rough forms show slightly spreading edge, but otherwise are similar.

After 24 hours, extremely poor growth, partly confluent, colourless, transparent, with slightly irregular surface and slightly dentate edge.

With both Listerella and Erysipelothrix growth in broth is similar except in degree. The smooth strains show a uniform turbidity with slight deposit which disintegrates on shaking. The rough strains show little or no turbidity, with thread-liko masses of deposit which are difficult to disintegrate. The amount of growth is much greater with Listerella than with Erysipelothrix.

All Listerella strains showed a simple filiform growth. Four of the Erysipelothrix strains showed a lamp-brush type of growth and the other two a filiform growth.

Aerobic slope cultures at 37" C. Listerella.

Erysipelothrix.

There is only slight improvement in growth on further incubation. Aerobic broth cultures at 37" C.

Gelatin stabs.

No liquefaction occurred. Loe$Zer's serum. MacConkey's agar. No growth. Peptone water. Listerella strains grow fairly readily, but there was no

growth of any of the Erysipelothrix strains unless serum was added. Resistance. Listerella. All strains survived moist heat at 55' C. for

30 minutes but were killed after 60 minutes a t this temperature. Erysipelothrix. Strains 33 and 6.35 were killed after 10 minutes and

strains 32 and 9.35, Kolle and M.S. 3 after 20 minutes at 55" C. (moist heat). Metabolism. Listerella. Aerobic : only extremely slight growth under

anaerobic conditions. Growth favoured by glucose, slightly by blood or serum. Optimum temperature for growth 30-37" C. Growth takes place between 20 and 44" C.

Erysipelothrix. Micro-aerophilic : grows under aerobic and rather less well under anaerobic conditions. Growth favoured by glucose, slightly by blood or serum. Growth takes place from 20 to 42" C.

Growth is similar to that on agar.

Optimum temperature for growth 30-37' C .

Hemolysin production is shown in table I.

TABLE I. Hemolysin production.

Strains.

L. 58. XXIII L. 53. XXIII L. H. Pirie L. Schultz L. Gibson E. 32 E. 33 E. 6.36 E. 9.35 E. Kolle E. M.S. 3

Blood agar plates (48 hours).

+ + + +

Trace

-

Soluble haemolysin.

For soluble hamolysin a qualitative test only was carried out. One C.C. of an 18-hour broth culture of each strain was mixed with 1 C.C. of washed horse red blood cells and the mixtures were incubated at 37' C. for one hour.

JOIJILNA I , OY PATIIOI.OGY--\'oL. XLVtlI.

LISTERELLA AND ERYSIPELOZ~HRIX

PLATE IV

FIG. 8.--To left, one colony o f smooth Listerella (58. XXIII) and two of smooth to right, one colony of smooth Listwelln and four of smooth ICT'rysipeloth??z (33) ;

E r y s i p e l o t h i ~ (M.S. 3) ; 48 hours at 37" C. x 20.

F I G . 9. F I ~ . 10.

FIG. 11.

FIGS. 9, 10 and 11.-Colonies of rough Listerella (H. Pirie) 44 hours, 63 hours and 5 days at 37" C. respectively. x 20.

LISTERELLA A N D ERYSIPELOTHRIX 15

Comparison.

Listerella and Erysipelothrix show similar differences between the smooth and rough types of colony. For both species growth is improved by addition of glucose and slightly improved by blood. Neither grows on MacConkey's agar. Both have similar temperature requirements.

Listerella grows much more profusely than Erysipelothrix in all media and will grow even in unenriched peptone water. Single colonies of Listerella on agar are larger and less transparent than Erysipelothrix colonies. All strains of Listerella are more resistant to heat than Erysipelothrix strains. Listerella is aerobic, Erysipelo- thrix micro-aerophilic.

Biochemical reactions. The biochemical activities of the strains are set out in table 11.

Horse serum heated to 65" C. for half-an-hour to destroy the amylase (Goldsworthy et al., 1938) was used to improve the growth of the Erysipelothrix strains.

It will be seen that the Listerella strains all show considerably more biochemical activity than the Erysipelothrix strains.

In no case was gas formed in any of the media.

Antigenic relationships. The antigenic relationships have been tested by agglutination

and agglutinin absorption. Antisera were prepared in rabbits by intravenous injection of a formolised

suspension of organisms standardised by opacity to a density of from 1000 to 3000 million organisms per C.C. Injections were given daily for one or two courses of seven days with a week's rest between and the serum harvested ten days after the last injection. The serum was stored at 4" C. , with the addition of 1 : 10,000 merthiolace.

Table I11 shows the readings obtained with the agglutination These were made after four hours in a water-bath at 55" C .

These tests show no antigenic relationship between Listerella

The absence of antigenic relationship was confirmed by absorp-

test. and confirmed after standing at room temperature overnight.

and Erysipelothrix.

tion experiments (table IV). For absorption with L. 58. XXIII a single dose of organisms was used.

A thick emulsion containing about 100,000 million organisms per C.C. was prepared from the washed deposit of broth cultures. Half a C.C. of this was mixed with 0.5 C.C. of antiserum diluted 1 : 10, heated at 55' C. in a water-bath for 4 hours, stored a t room temperature overnight and then centrifuged. The supernatant fluid (representing an antiserum dilution of 1 : 20) was set up against a suspension of the homologous organism. With E.33 a single dose was found to be insufficient to remove all the

TABLE 11. Biochemical characters.

0 1 : 2 5 1 : 1 0 1 :50 1 : 5 0

1 : 5 1 :50 1 : 2 5 1 : 5 0 1 : l O 1 : 1 0 1 : 5 0 1 : 2 0 1 : l O 1 : 2 0

1 : 2500 1 2500 1 : 2500 1 : 2500

1 : 1( 1 : 1( 1 : 5( 1 : 1(

: 1000 : 1000 : 1000 : 1000 : 1000

1 : 500 1 : 500 1 : 500 1 : 500 1 : 500

0 0 0 0 0 0

1 : 2 0 0 0 0 0 0

1 : 2500 1:500 1 : 1000 1 : 500 1:250 1 : 500

1 : 2500 1 : 50 1:500 1 : 2 0 1 : 2500 1 : 20 1 : 2500 1 : 20 1:1000 0 1 : 1000 1 : 10

0 0

1 : l O 0

0 0 0 0

LISTERE L LA. Eh! YSIPELOTHRIX.

3. XXlII 3. XXIII i. Pirie Colle.

lonosaccharides- Pentoses

Arabinose . . Xylose , . Rhamnose . ,

Glucose . . mvulose . . Galactose . .

Hexoses

pactose . Frehalose .

rrisaccharide- Raffinose .

'olysaccharides- Starch. . . Inulin . . . Dextrin . . Glycogen . .

Ucohols- ..~ ~

Trihydric

Hexahydric Glycerol . .

Mannitol . . Duleitol . . Sorbitol

Hucn4de- Salicin . .

Inosito . . . Litmus milk . . Gelatin . . .

tion- Voqes-l'roekauer re-

action Ammonin Hydrogen sulphide : Catalase . . ,

TABLE 111. Byglutination reactions.

Titrc of antiserum prepared against I Erysipelothr is. I Normal Organism.

Listerella 58. XXIII 53. XXIII H. Pirie . Schultz . Gibson .

Erysipelo - thrlx

32 33 6.35 9.35 Kolle . N . S . 3 .

serum.

6.35. ~ Kolle. I Gibson. 32. 33 53. XXIII. S X I

58' I -1-1-

1 : 250( 1 : 2501 1 : 1001 1 : 250( 1 : 2501

1 : 10 0

1 : 50 1 : 25 1 : 25 1 : 25

1 : 20 1 : 25

0 1 : 25 1 : 25

1 : 250( 1 : loo( 1 : loo( 1 : loo( 1 : 500 1 : loo(

1 : 10 0 0 0 0

1 : loo( 1 : 500( 1 : loo( 1 : loo( 1 : 1001 1 : loo(

1 : 25001 1 : 1(

1 : 2 5 I 0 1 :25 , 0

LISTERELLA A N D ERYSIPELOTHRIX 17

homologous antibodies. A preliminary mixture prcpared as for L. 58. XXIII was set up, incubated a t 37" C. for four hours and centrifuged. This process was repeated twice, using packed organisms to give a similar final con- centration of serum.

TABLE IV.

Results of agglutinin absorption tests.

L. 5S.XXIII serum (titre 1 : 1000) Absorbed with I,. 58. XXIII Absorbed with E. 33 .

.

E. 32 serum (titre 1 : 2500-1 : 5000) Absorbed with L. 58. XXIII Absorbed with E. 33 .

E. 33 serum (titre 1 : 5000)

'Absorbed with E. 33 .

.

Absorbed with L. 58. X X I I I .

Titre after absorption.

0 1 : 1000

1 : 5000 0

1 : 5000 0

Production of a circulating monocytosis in rabbits. Emulsions in saline were prepared from the washed deposit of broth

cultures and doses which were close to the minimum lethal dose were injected intravenously. As will be seen in table V this dose varied considerably with the different strains. The white cells were counted on three separate days before injection and daily after injection until the maximum mono- cytosis had been passed. Differential counts were made from Leishman- stained films.

As the production of a circulating monocytosis by Listerella is an established fact and has been previously demonstrated for the strains used in this paper, 58. XXIII was the only Listerella strain used as a control. All the Erysipelothrix strains were injected. Strains 6-35 and 9-35 have been under laboratory condi- tions for several years and are now avirulent. These two had no effect. The results obtained with the others are shown in table V. It will be seen that the four strains of Erysipelothrix as well as the one strain of Listerella produce a significant rise in rnonocytes within 5 or 6 days after injection. Rabbits 38/159, 381169 and 38/93 showed an increase in the total white cell count 7-12 days after injection. Rabbit 38/169 (strain E. 32) developed paralysis after 13 days and was killed.

Pathogenicity. The virulence of the different strains of both Listerella and

Erysipelothrix is extremely variable. Certain strains failed to kill laboratory animals even in enormous doses. These are L. H. Pirie and E. 9.35 (the rough strains of each group), L. 53. XXIII (partially rough) and E. 6.35 (smooth). The most virulent strains are L. 58. XXIII (smooth) and E. 32 (partially rough). The

JOUBN. OF PATH.-VOL. XLVIII. B

18

4,620 I 56 ~ 6,160

1,900 34 1,700 5,014 24 2,616 6,020 21 , 2,730

11,313 36 7,542 5,220 33 2,945

11.1. BARBER

3 1 330 28 I 1,400 30 3,270 25 3,250 10 2,095 5 435

TABLE V

11,000 5,000

10,900 13,000 20,950 8,700

Leucocyte reactions.

42 38 46 54 54 60

Pra-injection count . . 16,400 After 110 hours . . 8,400 ,, 134 ,, . 21,200 ,, 158 ,, . 24,000

,, 13 days . . 23,750 ,, 184 ,, . 20,000

30 4,920 39 3,276 37 7,904 54 12,960 55 11,000 44 10,450

Pre-injection count . . After 84 hours . .

,, 108 ,, . ,, 132 ,, . ,, 9 days . . ,, 48 ,. .

10,200 16,500 10,900 13,900 29,000 11,500

Pre-injection count . . After 84 hours . . ,, 132 .. . ,, 156 ,. .

I , 35 > ) . ,) 22 da.ys . .

10,700 13,600 12,700 13,000 12,200 12,400

5 11 16 11 9

535 1,496 2,032 1,430 1,098

Polymorphs. Lymphocytes. Monocytes.

Strain L. 58 XXIII : 1000 million organisms per kg. intravenously. Rabbit 38/159.

Pre-injection count * . After 84 hours .

,, 108 ,. ,, 132 ,, ), l2days . ,, 19 9 ,

Strain E. 32 : 300 million organisms per kg. intravenously. Rabbit 381189. I 5 6

13 22 15 10

65 1 10,680 55 1 4,620 50 10,600 24 1 5,760 30 6,000 46 1 10,925

820 504

2,756 5,280 3,000 2,375

Strain E. 33 : I500 million organisms per kg. intravenously. Rabbit 38/93.; I 63 43 38 50 52 45

408 1,155 2,616 1,529 4,060

345

6,426 1 33 7,095 i 50 4,146 38 6,950 1 39 15,080 34 5,175 1 51

I

3,366 1 4 8,260 1 7

5,865

Strain E. Koile : 0.2 mg. per kg. intravenously. Rabbit 37/31. I

Pre-injection count . . After 45 hours . ,, 65 ,, 112 ,> ,, 204 ,, ,, 16days .

11,200 11,050 19,500 13,200 14,600 15,000

40 29 46 34 39 54

4,480 3,200 8,970 4,488 5,694 8,100

I 55 6,166 60 1 6,630 42 8,190 55 1 7,260 44 I 6,424 43 1 6,450

5 11 12 11 17 3

560 1,220 2,340 1,452 2,482

450

1 Strain E. M.S. 3 : 8,000 million organisms per kg. intravenously. Rabbit 38/94.

33 37 39 37 46 25

3,521 5,032 4,953 4,810 5,612 3,100

62 I 6,634 52 7,072 45 5,715

6,760 5,490

70 8,680 I _ _ _ I I 1 I I I I

* In all cases the pre-injection count recorded is the one showing the highest monocqte count.

FIG. 12.-Colony of rough FIG. 13.-Colony of in- Listerella (H. Pirie) ; 48 termediate Listerella hours at room tempera- ( 5 3 . XXIII ) ; 44 hours tmre. x 110. a t 37" C. x20.

FIG. 13.

Frcs. 14 and 13.-Colonies of rough Erysipe- lothrix (9.35) ; 48 hours and 6 days a t 37" C. respectively. x 20.

PLATE V

FIG. 14.

Fro. 16.-Colonies of rough Ery.sipelottivix (9 .35) ; 48 hours at room tem- perature. x 110.

.mumkr, OF PATHOLOGY-T~OL XLVIII.

LISTERELLA AND ERPSIPBLOTHRIX

FIG. 17.-Colony of intermediate B ~ J - .qipelothrix ( 3 1 ) ; 48 hoiirs at, 37" C. x 20.

LISTERELLA AND ERYSIPELOTHRIX 19

lesions produced by these two virulent strains and the remaining moderately virulent strains did not vary appreciably.

As mice are readily killed by Listerella and Erysipelothrix the lesions produced have been studied largely in these animals. The following account refers to 68 mice used in this investigation and 60 used in a previous study (Webb and Barber).

The organisms were injected by the intraperitoneal and subcutaneous routes. Doses were prepared by emulsifying in 0.85 per cent. saline the washed deposit of 18- to 24-hour broth cultures. They were standardised by bacterial counts. Some h k t e r e l h emulsions were prepared by spading off and weighing moist 18- to 24-hour agar plate cultures and suspending in 0.85 per cent. saline, but the growth of Erysipelothrix is too poor for this method. No attempt was made to work out an accurate M.L.D. for the different strains. L. 58. XXIII and E. 32 after one or two passages readily killed in doses of 100 million organisms. The other strains killed in doses varying from 500 million to 30,000 million organisms.

Listereffa. Death generally occurred in 12-90 hours. The organism was readily recovered from spleen and heart blood. No local lesion occurred apart from very slight local cedema after subcutaneous injection. Con- junctivitis occurred sometimes. The most constant post-mortem finding, provided the animal survived for more than 12 hours, was focal necrosis of the liver. The foci were multiple, pin-point, greyish yellow and scattered throughout the organ. Histologically these foci are of two types, one consisting of a collection of cells, mainly mononuclear, in a coarse reticulum (fig. 18), the other of a necrotic area with comparatively few cells (fig. 19). Gram’s stain generally showed the presence of organisms in these foci, even when the rest of the liver was free. Clumps of organisms phagocytosed by Kupffer cells were often seen. The spleen varied in size but always contained large numbers of organisms. There were generally large areas of cellular reaction, made up mostly of mononuclear cells. Foci of necrosis similar to those occurring in the liver were occasionally seen in the kidneys and suprarenals and these organs frequently contained large numbers of organisms. On one or two occasions similar lesions were seen in the heart. In the lungs no lesions were ever seen apart from a generalised congestion and cedema, and no organisms were demonstrated.

Erysipelothrix. Death occurred in from 12 hours to 7 days after injection. The organisms were readily recovered from the spleen and heart blood. Conjunctivitis, from the discharge of which the organisms could be isolated, almost always occurred. No local lesion beyond a very slight local cedema after subcutaneous injection was seen. Areas of focal necrosis in the liver closely resembling those produced by Listerella were sometimes seen (figs. 20 and 21). These were not a constant feature and, when present, were less numerous than in Listerella infection. The spleen always contained organisms, and there was usually a generalised leucocytic infiltration, but no areas of cellular reaction resembling those occurring with Listerella were found. Organisms were frequently present in the kidneys and adrenals but no lesions were seen in these organs. The lungs showed widespread congestion and often contained large numbers of organisms, many of them in clumps and apparently phagocytosed.

Rabbits. Listerella infection of rabbits has been described at length by Murray, Webb and Swann. In the present experiments 6 rabbits were given the two most virulent strains of Erysipelothrix

Mice.

20 M . BARBER

(E. 32 and 33). The doses were prepared as for mice and given intravenously. Two rabbits were given E. 32 in doses of 1700 million and 500 million organisms per kg. body weight respectively. Two were given E. 33 in doses of 4000 million and 1800 million organisms per kg. All four died. Two rabbits were given smaller doses and survived. The disease did not differ appreciably from that occurring in mice. The organism was readily recovered from spleen and heart blood. No local lesion was produced. Conjunctivitis occurred in every fatal case. One or two focal lesions in the liver similar to those described in mice were seen. In two cases areas of mononuclear cell reaction occurred in the spleen resembling those seen in Listerella infection. Organisms were present in the liver, spleen and kidneys.

Guinea-pigs. Doses were prepared as for mice and injected intraperitoneally .

Listerella. Two guinea-pigs were injected with L. Gibson in doses of 50 and 25 mg. and 8 with L. 58. XXIII in doses ranging from 40 to 2.5 mg. : all died. The organisms were recovered from the spleen in every case and from the heart blood in some. Post rnortem the changes resembled those seen in mice.

Six guinea-pigs were injected with the most virulent strains of Erysipelothrix (E. 32 and 33). All survived and showed no symptoms of disease, although large doses ranging from 30,000 million to 4000 million organisms were used.

Doses were prepared as for mice and injected sub- cutaneously.

Eight pigeons were injected with L. 58. XXIII. Doses ranging from 100 to 5.4 mg. were prepared from agar plates and from 150,000 million to 6000 million organisnis (50-2 mg.) from broth cultures. Three pigeons were given L. Gibson in doses of 100 mg., 50 mg. and 6000 million organisms ( 2 mg.) respectively. All 11 pigeons survived and no symptoms of disease developed even after the enormous dose of 100 mg. One pigeon was injected with 200 mg. of L. 53. XXIII and died after 21 hours. Three pigeons were then injected with L. 53. XXIII in doses of 200, 100 and 40 mg. respectively. All three survived and showed no symptoms of disease. The one isolated death after only 21 hours was therefore assumed to be toxic and not due to Listerella infection.

Erysipelothrix. As the virulence of Erysipelothrix for pigeons is well known, I have used only E. 32 and the mouse strain E. M.S. 3. E. 32 after one or two passages readily killed pigeons in doses of 100 million organisms. Four pigeons were injected with E. M.S. 3 in doses of from 40,000 million to 9000 .million organisms. All survived and showed no symptoms of disease. This strain was isolated in 1934 and is now relatively avirulent, the M.L.D. for mice being about 1000 million organisms.

Erysipelothrix.

Pigeons.

Listerella.

PLATE VL

FIG. Iti.-Typicnl focus of cellular reaction in liver of Inowe infected with Listerella

FIG. 19.-More necrotic foci in liver of rnoiisc H. and E. infccted with Listerella (Gibson).

(58. XXIII). H. and E. x 300. x 80.

FIG. 2O.-Vacus of cellular reaction in liver of mouse infected with ErysipelothTiz (M.S. 3. ) . H. and E. x 300. a n d E . ~ 8 0 .

FIG. 21 .-Mare necrotic focus in liver of mouse H. infected with E'rysipelothriz (M.S. 3.) .

LISTERELLA AND ERYSIPELOTHRIX 21

Comparison. Listerella and Erysipelothrix are both pathogenic to rabbits

and mice. Both produce a circulating monocytosis in rabbits and similar areas of focal necrosis in the liver in mice.

The diseases produced by Listerella and Erysipelothrix in mice show many differences. Conjunctivitis is a much more frequent feature of Erysipelothrix than of Listerella infection. After injection with Erysipelothrix there are often no lesions in the liver and when present they are always few in number, whereas after injection with Listerella, multiple pin-point foci throughout the liver are an almost constant feature. Listerella readily kills guinea-pigs, Erysipelothrix does not. With regard to pigeons the position is somewhat equivocal. Swine strains of Erysipelothrix are known to be virulent for pigeons, but the only mouse strain in my possession has failed to kill pigeons. It should perhaps be borne in mind that this strain was isolated in 1934, and now shows a low degree of virulence. A human, a rabbit and a guinea-pig strain of Listerella all appear to be non-pathogenic to pigeons but a fowl strain has not been tried.

Natural distribution. The natural distribution of Listerella infection among animals

appears to be widespread. The organism has been isolated from naturally occurring diseases of rabbits, guinea-pigs, gerbilles, sheep, cattle and fowls. The reported human cases are at present few in number. They are all cases of meningitis or septiczmia, with the possible exception of some cases of infectious mononucleosis reported by Nyfeldt (1929).

Erysipelothrix infection appears to be less widespread. It has been reported as occurring in natural diseases in pigs, mice and sheep. Brunner (1938) has recently reported its isolation from fish but states that the fish only harbour the organisms and show no signs of disease. The recorded cases of Erysipelothrix infection in man are all erysipeloid lesions.

DISCUSSION. The similarities between Listerella and Erysipelothrix are sufficient

to raise the question as to whether they should be classified in the same bacterial genus. The morphology of the two, although not identical, is very closely similar and the correlation in each case between morphology and colony form is very striking. The cultural and metabolic characters show many points of similarity. Listerella is very much more active than Erysipelothrix in biochemical activities but neither produces gas in any of the carbohydrate media. In rabbits and mice it has been shown that Erysipelothrix will

JOURN. OF PATH.-VOL. XLVIIL B 2

22 M. BARBER

produce the two features which have aroused the most attention in Listerella infection in these animals, namely a circulating mono- cytosis in rabbits and areas of focal necrosis of the liver in rabbits and mice. In this connection it is of interest that Egeh~ j (1937) has recorded a circulating monocytosis in swine suffering from swine erysipelas.

Against these similarities must be set a fairly heavy list of differences. Within the limits of this study there appears to be no antigenic relationship between the two organisms. There are many significant differences as well as similarities in cultural characteristics and in metabolism. With regard to pathogenicity, Listerella readily kills guinea-pigs but apparently not pigeons, while Erysipelothrix readily kills pigeons but not guinea-pigs. The production of a circulating monocytosis in rabbits by both organisms is striking but it would be of interest to know if any other organisms have the same effect. Although focal necroses of the liver in rabbits and mice occur with Erysipelothrix they are an inconstant feature and, when present, are few in number, so that really the picture does not closely resemble that of Listerella infection. Finally the distribution and the naturally occurring diseases differ widely.

Conclusion.

Five strains of Listerella and six of Erysipelothrix have been compared. In the present state of our knowledge it is impossible to decide finally upon their nomenclature and classification, but until further work has been done it seems wiser to retain the two generic names Listerella and Erysipelothrix.

This study was carried out at the suggestion of Professor R. A. Webb and under his direction. The work was finished at the Group Laboratory, Archway Hospital, and I am indebted to Dr J. M. Alston for affording me every facility. My thanks are also due to Professor G. S. Wilson for valuable criticism and suggestions.

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J. L., AND DUMARESQ, J. A. MURRAY, E. G. D., WEBB,

R. A., AND SWANN, M. B. R. NYFELDT, A. . . . . . PIRIE, J. H. H. . . . . .

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LISTERELLA AND ERYSIPELOTHRIX 23

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