Nocardia caviae and Nocardia asteroides: Comparative bacteriological and mouse pathogenicity studies

J. COMP.

PATH.

1971.

VOL.

h*OCARDIA COMPARATIVE

79

81.

CAVIAE

and

NOCARDIA

ASTEROIDES:

BACTERIOLOGICAL PATHOGENICITY STUDIES

AND

MOUSE

BY

I. M. SMITH Department

and A. H. S. HAYWARD

of Pathology,

The Royal Veterinaly

College, London

INTRODUCTION

Recent

advances

in classification (Gordon and Mihm, 1962a, b) in the genus 1957) permit differentiation between N. asteroida and N. caviae, a species recently recorded in a dog in the U.K. (Kinch, 1968). The experimental pathogenicity of N. asteroides for laboratory rodents has been extensively studied (Cuttino and McCabe, 1949), but no comparative investigation appears to have been recorded of the relative pathogenicity for mice of known strains of the two organisms.

Nocardia (Waksman,

MATERIALS

AND

METHODS

Organisms. All cultures were grown aerobically at 37OC. Strains, from the sources shown in Table 1, were maintained on nutrient agar. Cultures were stored at about +-4OC. and each strain was passaged every 2 to 3 months. Biochemistry, Inoculated slopes were sealed to decrease dehydration. Reactions were checked at intervals for 21 days. Substrates were incorporated at 1 per cent. concentration in ammonium salt base agar (Smith, Gordon and Clark, 1952) to determine acid production. The utilisation of some organic acids as carbon sources was examined by the method of Gordon and Mihm (1957). Nitrate reduction was tested by the method given by Wilson and Miles (1964). Ability to split gelatin was detected by chilling for 2 hours at 4OC., nutrient gelatin cultures incubated at 37OC. Xanthine and tyrosine agars were prepared as described by Gordon and Mihm (1962b) and casein and starch agars as described by Cowan and Steel (1965); hydrolysis of casein was found by inspection and of starch, by iodine solution. Lecithovitellin agar was made by the addition of 20 per cent. sterile commercial egg yolk suspension* and 5 per cent. Fildes extract+ in nutrient broth to agar. In the oxidation-fermentation test (Hugh and Leifson, 1953) a seal of sterile petroleum jelly was substituted for soft paraffin. Oxidase production by growth from nutrient agar slopes incubated for 2 to 4 days was tested using a 1 per cent. solution of tetramethyl-p-phenylenediamine (Kovacs, 1956). Elaboration of catalase was determined in the same culture by the method given by Wilson and Miles (1964) and urease production in Christensen’s medium. Mice. ‘Specific-pathogen-free’ animals of the TO strain kept in isolation were fed with sterilised diet. In each experiment young adults weighing about 25 g. and of both sexes were randomised before inoculation. Samples of mice that died, or were killed with chloroform vapour, were opened with aseptic precautions and cultures from tissues were made with a straight wire inserted a fixed distance into an organ. When a whole organ was cultured it was removed aseptically and disintegrated with ballotini * Oxoid P

Ltd.

N. caviae

80

AND

N. asteroides

IN MOUSE

in 1 ml. of ice-cold phospate buffer (pH 7.2) with a Mickle tissue disintegrator. Samples of the homogenate, or dilutions in broth thereof, were sown on 5 per cent. ox-blood agar. Agitated broth cultures, viable counts and inocula. Cultures were obtained by continuous oscillation of inoculated broths, a procedure also followed by Gorrill and Heptinstall (1954) with N. sebivorans. These growths were wholly turbid without pellicle formation and formed little deposit on standing. Their viable content was estimated from the number of colony-forming units (CFU) that grew on 5 per cent. ox-blood agar in 72 hr. when sown with appropriate dilutions of culture in ice-cold 2 per cent. ox-serum broth. Mice were inoculated with 2 per cent. ox-serum broth cultures agitated for 3 days and any dilutions required were also made in ice-cold broth because 0*15M NaCl, and to a less extent phosphate buffer (pH 7*2), reduced the viability of the strains by up to 20 fold in about 1 hr. Heat-killed organisms were prepared by steaming agitated broth cultures for 1 hr. at 1OOOC. Filtrates were obtained from agitated cultures by filtration through 450 rnp A.P.D. membranes. The sterility of heat-killed cultures and filtrates was checked retrospectively by sowing a liberal sample on 5 per cent. ox-blood agar. Haemolysin test. Thrice-washed rabbit or ox erythrocytes were resuspended in normal saline in concentrations of 4, 2 and 1 per cent. Washed cells were mixed in equal quantities with supernates or filtrates collected from stationary Nocardia cultures, These mixtures were held in a 37OC. water bath for 2 hr. and then at 4OC. overnight. Supernates from centrifuged extracts of thick, wet pellicle growths of N. caviae strains with normal saline containing 200 i.u. of penicillin per ml. were also employed in some tests. A diffusible haemolysin from Escherichia coli (Smith, 1963) and a negative control were always included in the test. Histological examinations. Tissues were fixed in neutral 10 per cent. formol saline and embeded in paraffin wax. Sections (6~) were stained by haematoxylin and eosin and by Gram’s method. RESULTS

Bacteriological Findings Biochemical reactions. Each strain was tested on several occasions with similar

results (Table 2), and the findings agree closely with those of Gordon and Mihm (1962a, b). Of those used only 3 reactions (Table 2) were helpful in distinguishing between all strains of the 2 species. In 4 tests (haemolysis, glucose, nitrate reduction, TABLE SOURCES

OF

N. asteroides Source

1

Nocardia

STRAINS

N. caviae NCTC*

NCTC

8595

6761

>,

>9

Source reference R.V.C. reference

Vet. W+&ge

NCTC RVC + This organism, ( 1957), an example

+ab.

originally named of Jv. astcroides.

$9

RVC

NCTC

5

1934

>a

Dr.

166

12399999 157

= National Collection Type = Royal Veterinary College 3v. sebivorans (Erikson,

Ruth

1954)

Gordon,

Rutgers

University

1237

416

1252

1315

547

168

169

170

171

172

Cultures Collection is, according

to Gordon

and Xlihm

I.

M.

SMITH

AND

A. TABLE

H.

2

BI~~WEM~C.~L REA~TI~NS~ OFSTR.~INS OFJv.astmdes Species N.ascrrteroides

.N. caviae

No.of strains 4

7

G

ii”

Al

I

X

x

x

x

a

81

S. HAYWARD

a

AND

B

.N. caviae

GL

.N

s

1 Other reactions are listed in the text. * Strain 8595 was erratic t Except strains 157 & 168. $ Except strain 157. 8 Except strain 157, 170. G M I X B GL K

= = = = = = =

glucose mannitol inositol xantbine agar 5% ox-blood agar gelatin liquefaction nitrate reduction

= acid = split L = B haemolysis nh = non-haemolytic x = growth only + = positive - = negative a

gelatin liquefication) not all strains of each speciesbehaved identically (Table 2). The behaviour of all the strains in 18 other tests was the same. Only growth occurred with maltose arabinose, dulcitol and sorbitol as substrate and on Loeffler’s serum. No strain split the substrate in sodium malonate, tyrosine, starch, casein or egg yolk agar. All the strains rendered litmus milk and sodium acetate agar alkaline, attacked glucose by oxidation, produced acid in glycerol agar, split urea and were oxidase and catalase producers. No strain grew on sodium benzoate agar. Growth on solid media. The N. asteroides strains produced white aerial hyphae irrespective of the nature of the solid medium on which they were grown and thus differed from N. cauiae. Four of the 6 strains of N. caviae produced distinct zones of complete lysis on 5 to 10 per cent. ox or rabbit-blood agar, or with the same concentrations in agar of washed erythrocytes of these species. Usually these zones were just visible after 24 hours of incubation, although lysis was more obvious around areas of denser growth; after 72 hours clear zones with sharp margins extended about 2 mm. beyond the edge of individual colonies. N. asteroides strains grown in parallel under the same conditions were always nonhaemolytic. Strains of other species including N. blackwellii, N. madurae, N. bras&en&s and N. cuniculi were similarly always non-haemolytic. The haemolytic strains of N. cauiae did not produce haemolysis on horse-blood agar (7.5 per cent.) although dark green to black zones of altered red cells appeared around colonies in the same time period as that required for complete haemolysis on ox- or rabbitblood agar. Of the two non-haemolytic strains of N. cauia, 157 and 170, the former has been described (R. E. Gordon, personal communication) as an asteroider : caviae intermediate; certainly it was not typical of N. caviae in several respects (Table 2). The other, 170, began to grow erratically soon after it was obtained and the results observed may not be typical of the strain. It was, however, definitely non-haemolytic on first receipt.

82

$. caviae

AND

&“. asteroides

IN MOUSE

Because of the unusual morphology of Nocardia the possibility of contamination of the haemolytic N. cauiue strains with some haemolysing organism cannot be discounted. However, when stationary broth cultures, each inoculated with a haemolytic N. caviae and a N. asteroides strain, were sown on ox-blood agar after 1, 2 and 4 days incubation, no haemolysis was detected on the part of any N. asteroides colonies, although frank haemolysis always appeared in association with N. caviae colonies; a number of single, typical colonies of each species from these cultures inoculated onto ox-blood agar behaved in the same way. Haemolysin production. Haemolysis by some actinomycetes was recorded by Waksman (1918), but no more recent reference to the phenomenon among Nocardia has been noted, although it is known among Dermatophilus spp. (Roberts, 1965). The lytic factor did not appear to be a lecithinase or lipase, because growth on lecithovitellin agar was not surrounded by opacity. Staphylococcus aureus and Ctostridium welchii strains were active on each batch of medium used. Attempts to isolate the factor were unsuccessful. Briefly, the haemolytic strains and a non-haemolytic strain of N. caviae (170; Table 2’ were grown as stationary cultures in Todd-Hewitt broth, with and without glycerol (3 per cent.) and/or iron ion, in nutrient broth, 2 per cent. ox-serum broth, peptone water, a minimum growth medium and alkaline meat broth (Smith, 1963‘, for periods up to 14 days. Samples from these cultures beginning a few hours after incubation were tested. Only the E. coli control showed haemolytic activity. TABLE RESPONSE

Species

.V. cauiae * (5 strains)

OF MICE

T O INTRAVENOUS

Mean No. CFlJ/ml.

Inoculum

304.0

was 0.25 ml. of J-day

Nocardia

CULTURES

Dilution

injected:

Undiluted

10-z

Q 9 days

87/90t

31180

5180

0 75

illness:

3/3

31/49

15175

2 75

Death

< 9 days

32138

2128

O/28

0 18

Chronic

illnessf

53’6

12126

l/28

0 18

Death Chronic

,\t asteroides (4 strains)

OF

Respome

of culture 96-O

3

INOCULATION

agitated

* = Excluding strains No. 157, 170. t = No. affected/No. injected. $ = Including “spinning disease”.

2 per cent. ox-serum CFU

broth

= Colony-forming

10-1

1 o-3

cultures. units.

Pathological Findings Virulence for mice. Except for strains 157 and 170, each strain of each species possessed reproducible and approximately similar virulence by intravenous inoculation and findings for each experiment are, therefore, pooled in Table 3. The N. caviae strains were more pathogenic for mice than N. asteroides strains. This conclusion, however, must be qualified since 2 strains of N. caviae are excluded

I. M.

SMITH

AND

A. H. S. HAYWARD

83

from the data in Table 3. Strain 157, which produced CFU counts of
./% caviae

84

.N. asteroides

AND

IN

MOUSE

TABLE 4 DISTRIBUTIOX

Ab. cultured

Inoculum

Living cultures of N. caviae* and .N. asternides

OF Nocardia

IN MICE

No. with post-mortem macroscopic abnormality

Clinical condition

AFTER

INTRAVENOUS

Day death occurred after inoculation

Mean

INOCULATION

No. (approx.)

CFU

recorreredt

from

Heart blood

Lung

Liver

Spleen

I;idn fg

Brain

18

Acute disease

18

1

11

25

135

115

80

10

21

Acute disease

21

2-4

16

4

129

96

127

33

11

Chronic disease and “Spinning disease”

3

6-9

0

0

6

15

31

15

“Spinning disease”

1

21

0

0

1

1

25

0

Normal

0

),

0

0

0

0

0

0

6 10

Killed ,)

* Excluding strains 157, 170. t By straight wire culture. The inoculum was 0.25 ml. of 72 hr. agitated preparations.

ox-serum

broth

cultures

or 0.25

ml. of the control

examination of 18 brains for unilateral lesions was made; only 3 showed foci of inflammation. Similar lesions occurred in mice without nervous signs. Inflammatory lesions were found, however, in the semicircular canals of 10 out of 14 affected mice examined. These lesions varied from accumulations of necrotic material and inflammatory cells (Fig. 4) to the development of definite granulomas containing the bacteria centrally. Distribution of organisms in infected mice. Some mice from each experiment were cultured and the number of CFU of Nocardia present in various tissueswas estimated. The pattern for both species was basically similar and the data are pooled in Table 4. Whereas in mice which died of the more acute disease the organisms tended to be widely dispersed, the tendency for the organisms to persist in the kidney of the more chronically affected mouse is clear. In clinically abnormal mice which did not die, the organism was not recoverable from the brain by direct culture. If, however, whole brain was disintegrated then organisms were recoverable, sometimes in very large numbers, from about a third only of those examined (Table 5). TABLE RECO\-ERY

OF

Nocardia

FROM

MOUSE

BRAINS

5 AFTER

INTRAVENOUS

INOCULATION

.I% of affected mice examined

Day died after inoculation

DC positirle HC positive

DC negatiae HC positive

1:

Killed7-9 2 1

i

41

-___

DC negatiae HC negative 2

Inoculum was 0.25 ml. of 72 hr. agitated DC = direct culture; HC = homogenate

ox-serum culture.

broth

cultures.

I. M. SMITH

AND

85

A. H. S. HAYWARD

o N.caviae NO.169 l ‘N.asteroides NO.5

I

2

1

Days after Fig.

1.

4

8

inoculation

Mean number of colony-forming units (CFU) of JV. caviar 169 and .N. askroidts 5 recorded per gramme from organs of pairs of mice killed after intravenous inoculation with 1.8 x 106 and 6.3 x 106 CFU of the two strains respectively.

The tendency for both species to persist in the kidney but disappear from the brain was investigated in more detail with mice injected intravenously with about 1.8 and 6.3 million CFU of N. cuviue 169 and N. asteroides 5 respectively. As shown in Fig. 1, increases in CFU numbers in the brain were more pronounced and more prolonged after inoculation of strain 169 than with strain 5. This behaviour may be a factor in the apparently greater pathogenicity of the virulent N. caviue strains. However, by the 8th day after inoculation the CFU counts of each species were higher in the kidney than in the brain and did not appear to be decreasing. The biochemical reactions of strains recovered from infected mice were unchanged when compared directly with their parent strain. However, colonial morphology sometimes differed in being more waxy and more regular in outline, but the organisms from such colonies exhibited a typical morphology. DISCUSSION

N. caviue, originally described as the cause of a disease of Sumatran guinea pigs (Snijders, 1924), may only be a rare variant of N. asteroides (R. E. Gordon, personal communication). Hence, haemolysis by some N. caviue strains was of interest. The present sample of strains was too small for any definite conclusion, but if it is confirmed that haemolysis is a feature of most N. cuviae strains, then this character might be useful in distinguishing between N. caviue and N. asteroides, since the number of differential characters available is small (Gordon and Mihm, 1962b; Tsukamura, 1969).

86

jv. caviae

AND

.N. asteroides

IN

MOUSE

The experimental disease produced by some N. caviae strains in mice was essentially the same as that caused by N. asteroides, although N. caviae was perhaps more virulent. This indication requires quantitative confirmation with a larger number of strains of both species, especially as all the present strains have been maintained by artificial culture for periods of greatly varying length. Relative virulence for laboratory animals, however, is rarely sufficient grounds for distinguishing bacterial speciesand, in fact, the diseaseproduced by the pathogenic N. caviae and N. asteroides strains did not differ from that established by intravenous infection with some other Nocardia spp. (unpublished observation). N. asteroides and N. caviae multiplied in the brain after intravenous inoculation. This invasion also occurs after intraperitoneal inoculation of both species,but to a less extent (unpublished data). Although viable N. caviae and N. asteroides organisms were sometimes detectable in the brain their presence was probably not responsible for persistent “spinning disease”. This condition was associated with histologically detectable lesions in the inner ear. As already mentioned, other workers have observed the same condition in mice inoculated intravenously with N. sebivorans (seeTable 1) and Pseudomonas pyocyanea and it also occurred after inoculation of mice with some Mycobacterium spp. (Gorrill, 1956). The results on virulence are similar to data from Gorrill (1956), but they are at variance with a number of previous reports. Thus Mohapatra and Pine (1963) confirmed earlier accounts (Strauss and Kligman, 1951; Strauss, Kligman and Pillsbury, 195 1; Georg, Ajello, McDurmont and Hosty, 196 1) of apparently irregular pathogenicity and lack of invasiveness of N. asteroides. Observations on the use of gastric mucin (Strauss and Kligman, 195 1) were confirmed by Mohapatra and Pine (1963), who showed that intraveous N. asteroides infection in mice could be enhanced by intraperitoneal inoculation of this substance. However, the use of saline as a suspending liquid by these workers may have affected viability of the organisms, and consequently the results. It is of particular interest that “spinning disease” was not recorded, although the strains of N. asteroides examined by Mohapatra and Pine (1963) were non-lethal for mice. SUMMARY

Among other biochemical properties, 5 of 7 N. caviae strains, but none of 4 N. asteroides strains, produced frank haemolysis on agar containing ox or rabbit blood, but not horse blood, although horse red cells were altered. The factor which produced haemolysis was not isolated. Large dosesof both Nocardia specieswere pathogenic by the intravenous route for mice. N. caviae was probably more pathogenic than N. asteroides. Lesions were the result of vascular blockage followed by a neutrophil reaction and the formation either of abscesses or granulomata in several major organs. Clinically, 2 forms of diseaseoccurred. In the first, acute illness was followed by death and organisms were readily recovered in pure culture from several major organs including the brain. In the second, sick mice which survived for more than about 4 days developed “spinning disease”. A number of mice that showed no other illness after inoculation also developed this condition, which was associated with histologically detectable, predominantly unilateral lesionsof the inner ear.

I.

M.

abscesses x 30.

SMITH

AND

in cerebrum,

A.

H.

4 days

S. HAYWARD

Fig.

2.

Circumscribed 166. H. & E.

after

intravenous

Fig.

3.

Granuloma in lung with a central and a wide border of macrophages, H.&E. x 120.

Fig.

4.

Granuloma of inner ear, 14 days after intravenous inoculation the semicircular canal there is a central area of necrotised macrophages. H. & E. x 120.

inoculation

with

JV. c&ae

colony of .N~cardia, surrounded by zone of neutrophils 21 days after intravenous inoculation with N. c&z 172. of N. asteroides 6761. Within neutrophils with a border of

I.

M.

SMITH

AND

A.

H.

S. HAY\\‘ARD

87

ACKNOWLEDGMENTS

Dr. R. E. Gordon, Rutgers University, Brunswick, N. J., and Mr. P. K. C. Austwick, Veterinary Laboratory, Weybridge, Surrey, generously supplied some of the strains used. Mr. H. H. Skinner, Research Institute, Pirbright, Surrey, kindly examined some mouse brains for the presence of choriolymphocytic meningitis virus. Mr. R. Simpkins and Mr. R. Amoah gave technical assistance. REFERENCES

of Medical S. T., and Steel, K. J. (1965). M anual for the Identification Cambridge University Press. Cuttino, J. T., and McCabe, A. M. (1949). Amer. J. Path., 25, 1. Erikson, D. (1954). Add endurn to Gorrill and Heptinstall (1954). GeorE.sL.zj$$ello, L., McDurmont, C., and Hosty, T. S. (1961). Amer. Rev. rest. Cowan,

Bacteria.

Gordol;k. E., and Mihm J M (1957). J. Bact 73, 15; (1962a). J. gen. Microbial., 27, 1; (1962b). A nn. h;.Y. ALad. Sci., 98, 628: Gorrill, R. H., and Heptinstall, R. H. (1954). 1. Path. Butt., 68, 387. Gorrill, R. H. (1956). Ibid., 71, 353. Hugh, R., and Leifson, E. (1953). J. Bact., 66, 24. Kinch, D. A. (1968). J. Path. Bact., 95,540. Kovacs, N. (1956). Nature Lond., 178, 703. Mohapatra, L. N., and Pine, L. (1963). Sabouraudia, 2, 176. Roberts, D. S. (1965). Nature Lond., 206, 1068. Smith, H. W. (1963). J. Path. Bact., 85, 197. Smith, N. R., Gordon, R. E., and Clark, F. E. (1952). U.S. Dept. Agric., Monograph No. 16.

Snijders, E. P. (1924). Geneesk. Tijdschr. Ned. Znd., 64, 47, 75 (cited by Waksman, 1957). Strauss, R. E., and Kligman, A. M. (1951). 1. inf. Dis., 88, 151. Straus2lR. E., Kligman, A. M., and Pillsbury, D. M. (1951). Amer. Rev. Tuberc., 63, Tsukamura, M. (1969). 1. gen. Microbial., 56, 265. Waksman, S. A. (1918). J. inf. Dis., 23,547; (1957). In Bergey’s Manual of Determinative Bacteriology, 7th Ed., Williams and Wilkins, Co. Ltd.,, Baltimore. Wilson, G. S., and Miles, A. A. (1964). Topley and Wilson’s PrtncipZes of Bacteriology and Immunity. 5th Ed., Edward Arnold (Publishers)Ltd.; London. [Received

for publication,

April

17th, 19701