Tubercle, Lond., (1964), 45, 235

S T A B I L I T Y O F T H E V I R U L E N C E O F I N D I A N T U B E R C L E B A C I L L I

By BALBIR SINGH*

fi'om the Department of Microbiology, Maulana Azad Medical College, New Dehli, India

SUMMARY

Nine freshly isolated strains of Indian tubercle bacilli were passaged on successive occa- sions through guinea-pigs, after maintaining them in the laboratory for various intervals.

Little or no change in virulence for the guinea-pig of these strains was observed.

The heterogenous character of strains of. tubercle bacilli obtained from Indian patients with regard to their virulence for guinea-pigs was reported originally by Dhurandhar (1941) and Dhaya- gude & Shah (1948). The majority of the strains were moderately virulent, some were highly virulent and others showed low virulence. These observations were confirmed by Frimodt-M611er (1955; 1956), and by Mitchison and others (1960) and Singh (1964), who compared the virulence of the standard strain H37Rv and strains of American and European origin. The present report is concer- ned with the stability of the virulence of Indian strains, following passage through the guinea-pig.

StrahTs Methods

Nine strains of Mycobacterium tuberculosis, sensitive to PAS, streptomycin and isoniazid, were freshly isolated from the sputum of the same number of patients, seven by guinea-pig inoculation and the remaining two by culture. For isolation by guinea-pig inoculation, the concentrate obtained from 2.5 ml. of decontaminated, smear-positive (1-5 acid-fast bacilli per oil immersion field) sputum was injected into a guinea-pig of about 300 g. weight and an autopsy was done at 70 days, except in the event of an earlier death. Cultures on L6wenstein-Jensen medium were made from suspected lesions.

Virulence Tests The methods of studying guinea-pig virulence have been described previously (Singh, 1964).

Briefly, cultures were transferred to Dubos & Davis Tween-albumin medium (Medical Research Council, 1948). A 10-day old growth was then injected into 6 guinea-pigs, divided into three sets of two animals, each of which received approximately l, 0.I or 0.01 mg. (moist weight) of organisms, respectively. One guinea-pig from each set was autopsied after 4 weeks and the other after 10 weeks, except in the case of an earlier death. The extent of disease in these animals and in the guinea-pig used for isolation of the strain was assessed in terms of the Feldman indices for macroscopic lesions and for histological appearances (Feldman, 1943). Strains yielding indkces for macroscopic lesions of 10-30, 40-70 or 80-100 were arbitrarily classified as of low, medium or high virulence, respectively.

Subsequent Passage ~ Strains recovered from the guinea-pigs were maintained on L~Swenstein-Jensen slopes, stored at

4 ~ C. and subcultured every 6 weeks. At intervals the cultures were transferred to Tween-albumin

* Present address: Willingdon Hospital, New Dehli.

236 TUBERCLE

liquid medium for further virulence tests. The purity of these strains was investigated on Dubos & Davis oleic acid-albumin agar plates, which were also used for obtaining a viable count" none of the cultures contained less than 106 viable units per ml.

Results

The results of the serial virulence tests carried out on the strains from the 9 patients are set out in Table 1. The period of passage is the interval from the date of autopsy of the guinea-pig from which the strain was recovered to the date of its injection into the next group of guinea-pigs; it includes the periods of culture on L6wenstein-Jensen medium, of storage at 4 ~ C. and of subculture in Tween- albumin medium. When the strain was obtained by direct sputum culture it is the period from the date of culture of the sputum to the injection into guinea-pigs.

The guinea-pig inoculated with the sputum of patient 1 yielded Feldman indices for gross patho- logy and for histopathology of l0 and 20, respectively, and this strain was therefore of low virulence. After 5 months, the group of 6 guinea-pigs all yielded indices of gross pathology of 20 or less. After another 10 months, similar results were obtained. A final virulence test after a further 7 months also yielded indices of gross pathology of 20 or 10. No alteration in virulence thus occurred during serial passage of the strain.

TABLE [.--FELDMAN INDICES OBSERVED ON REPEATED PASSAGE IN GUINEA-PIG OF 9 STRAINS OF TUBERCLE BACILLI

Patient No.

Passage Interval 10 weeks (months) after

inoculation o f sputum

.

10 (20)~;

45 (50)

10 (20)

Feldman index*--macroscopic lesions . . . . .

4 weeks after inoculation o f 10 weeks after hroculation of

1 rag. 0"I rag. 0"01 rag. 1 1 rag. 0"1 mg. 0"01 rag.

20 20 20 (20) (40) (30) 10 20 10

(20) (30) (20) 20

(30)

30 30 10 (30) (30) (20) 30 10 10

(60) (20) (10)

20 20 10 (50) (20) �9 (io)

lo (~o)

I0 10 10 (10) (30) (10) 10 10 10

(30) (10) (10) 10

(20)

30 30 10 (30) (10) (10) 10 10 10

(10) (20) (10)

30 30 30 (50) (45) (45)

30 (60)

(Continued on next page

INDIAN TUBERCLE BACILLI 237

TABLE l .--( Continued)

Patient No.

10 weeks after

inoculation of sputum

80

l

Passage hlterval (months)

Feldman iltdex*mmacroscopic lesions _

4 weeks after inoculation o f - 10 weeks after inoculation o f . . . .

rag. 0"1 rag. 0.01 rag. 1 rag. 0"1 rag. 0.01 rag.

40 io 1o (30) (30) 30

(3O)

30 30 lO (55) (55) (30)

60

65 30 40 (75) (35) (40) 80 20 20

(75) (45) (40)

90 60 20 (60) (60) (30)

(60)

30 30 10 (40) (20) (lO)

~o (20)

20 I0 10 (55) (55) (10)

3O (40)

70 30 20 (90) (50) 80 35 20

(100) (45) (40)

90 (80)

100 (ioo)

70 10 (70) (10)

100 0o0) lOO

(too)

100 90 70 (100) (80) (60) 100

(100)

90 (')0)

90 70 50 (80) (70) (40)

* Feldman Index--Maximal value for extensive involvement of site of inoculation and regional glands, spleen, liver and lung respectively was 10, 35,15 and 30, the total being 100.

I Animal killed accidentally 20 days after inoculation.

:1: Figures in parenti'~esis are the Feldman Index for histological appearances.

w Culture isolated directly from spleen.

238 TUBERCLE

Strains 2, 3, 4 and 5 were of low or medium virulence. No change in virulence was observed dur- ing passage of these strains.

Strain 6 gave an index for gross pathology of 30 in the guinea-pig inoculated with sputum. How- ever, the strain recovered from this animal gave indices ranging from 20 to 70 in the next virulence test, done 2 months later, and the range was from 20 to 80 in a final virulence test done after a further 10 months. There is some possibility that this strain may have increased in virulence during passage.

Strains 7, 8 and 9 were of fairly high virulence when first tested. Their virulence remained similar in subsequent tests.

Discussion

In this investigation the 9 strains showed a wide range of virulence, 6 being of low or moderate virulence in their first guinea-pig passage and 3 being of fairly high virulence. In only one of the 6 strains of low or moderate virulence was there any evidence of an increase in virulence during the succeeding passages in guinea-pigs. Thus it seems that the virulence of Indian tubercle bacilli is usually a stable character.

In assessing the virulence of the strains, appreciable variation was found between guinea-pigs. An occasional animal yielded an inconsistent result, as described by Bjerkedal (1960). Only a sub- stantial increase in vir~:!ence could therefore be detected by the methods used.

The writer is grate~.~1 to ~l:,e Indian Council for Medical Research who financed this enquiry, to Dr. D. N. Gupta, Professor :'~: Patholog.~' i%~aulana Azad Medical College, New Delhi, who investigated the histopathology of the tissues ~r~d to Colonel B. I,. Tancja, Principal, Maulana Azad Medical College, and Medical Superintendent, Irwin Hospital, for providing lt~, ~eccssary facilities to carry on this work.

REFERENCES ]~JERKEDAL, Z. (1960) Amer..I. Hyg., 72, 130. DHAYAGUDE, R. G. • SHAH, B. R. (1948). Indian J. reed. Res., 36, 79. DHURANDHAR, C. I . (1941). Indian J. reed. Res., 29, 531. FELDMAN, W. H. (1943). Amer. Rev. Tuberc., 48, 242. FRIMODT-M/SLLER, J. 0955). Technical Report of the Scientific Advisory Board, p. 271, Indian Council for Medical

Research, New Delhi. FRIMODT-M(SLLER, J. (1956). Technical Report of the Scientific Advisory Board, !9. 135. Indian Council for Medical

Research, New Delhi. MEDICAL RESEARCH COUNCIL (1948). Lancet, ii, 862. MITCHISON, D. A., WALLACE, J. G., BHATIA, A. L., SELKON, J. B., SUBBAIAH, Z. V., • LANCASTER, M. C. (1960).

Tubercle, Lond., 41, 1. SINGH, B. (1964). Amer. Rev. resp. Dis., 89, 1.