T U B E R C L E P R I C E 3 s. 6d. 35/" P E R A N N U M

A STAPLES ~ 1 PUBLIGA TION

The Intracutaneous Injection of Vole Tubercle Bacilli in Tuberculous Persons

BY CHARLES C A M E R O N Professor of Tuberculosis, University of Edinburgh.

A N D

ISABELLA A. P U R D I E

Medical Offcer-in-Charge of Department of Health Laboratory, E.M.S. Hospital, Bangour. From East Fortune Sanatorium Unit, Bangour Hospital and Department of Health Laboratory,

Bangour Hospital.

In July, I942, we received from Dr A. Q. Wells a culture of the vole tubercle bacillus originally isolated by him from a vole which died in his laboratory after a month's cap- tivity. This organism, as is now well known, is only slightly pathogenic for guinea-pigs and rabbits, and in 194o Wells and Brooke (194 o) using caseous material from tuberculous lesions in naturally infected voles, injections of living vole tubercle bacilli, and injections of B.C.G., proved that by the first two methods a considerable degree of protection against later infection with virulent mam- malian tubercle bacilli could be produced. The protection was greater when living vole bacilli in a total dose of 2.o mg. were em- ployed, and it exceeded that effected by total immunizing injections of 3 ~ mg. of B.C.G. This work was interrupted by the war and the experiment lasted only eleven weeks.

Immunising Power of Wells' Bacillus

Later in that year Griffith and Dalling (194o) published the results of an investiga- tion into the pathogenicity of the organism in calves and its immunizing properties in calves and guinea-pigs. They found that its pathogenicity for calves was low and that its

immunizing powers exceeded those of B.C.G. Their results in guinea-pig immunization corresponded closely with those of Wells and Brooke. All of the immunized calves became strongly tuberculin positive, and sensitivity deyeloped as early as the thirteenth day. Since then a fairly extensive literature has grown, and more recently Birkhaug (I946) has published an exhaustive investigation into the immunizing effects of the vole tubercle bacillus and B.C.G. on guinea-pigs. He used 48 animals. 24 received immunizing doses of the vole tubercle bacillus, 12 were given B.C.G. and the remaining I2, along with I2 of the vole-immunized group, were used as a control group. All of the immunized animals reacted strongly to tuberculin one month after the last immunizing injection, but sensi- tivity was stronger in the B.C.G. group. That group had received a total of 3 ~ mg. of B.C.G. and the vole bacillus group had re- ceived a total of 2 rag. of living vole bacilli. It had been Wells' intention in his original experiment to carry the work over a period of eighteen months and Birkhaug left the surviving animals for that period. The degree of resistance to infection, two months after the last immunizing dose of vole bacillus or B.C.G., with o.oooooi mg. of virulent

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human tubercle bacilli was found by Birk- haug to be high, but equal, in the two groups.

The immunizing power of the vole tubercle bacillus in experimental animals is thus not in doubt and when Dr Wells gave us his culture it was his hope that we might be able to investigate its effects in human beings.The obvious line of investigation was among Mantoux-negative persons. These, however, are rare in sanatoria and the effect of the bacillus on uninfected humans was then un- known. We were at that time planning the investigation of some work which had been done in Shanghai by the Clinical Research Group of that City, reports of which had been given to us by Dr I. G. Anderson who had been a member of the group. This work con- sisted of the vaccination of patients suffering from lung tuberculosis by scarification of the skin of the thigh and the inunction of a heavy suspension of tubercle bacilli grown from the patient's own sputum.

One of us (C.C.) is at present investigating this work, but when we received the culture of the vole tubercle bacillus we thought it better to defer it and to try instead the effect of injecting measured doses of that bacillus i/lto patients who were already tuberculous, or were at any rate infected with tubercle bacilli. The work, of which the following is a brief record, was carried out during I943 and I944 and was interrupted frequently by heavy pressure of work in other directions. It was planned with the help and guidance of Professor T. J. Mackie to whom we express our acknowledgment.

Preparation of the Organism The original culture was sub-cultured at monthly intervals on Dorset's egg medium without glycerine, and a two months' old sub-culture was used for the preparation of all of the series of injections. In the prepara- tion of these the culture growth was removed and desiccated, and the dried organism was suspended in sterile double-distilled water without preservative. All suspensions were prepared on the evening before injection and cultures were made for contaminant organ- isms. The suspension in bulk was kept over-

T U B E R C L E December 1946

night in the refrigerator. These bulk suspen- sions were put up in rubber-capped bottles and o. 5 c.c. of the suspension used in Group I contained o.oi rag. by dry weight of vole tubercle bacilli. In later series the injected dose was contained in o.2 c.c. of the suspen- sion. Five groups of patients were used.

Material (I) The first group consisted of i6 patients

of ages varying from I6 to 55, the majority being between 2o and 3 o . All had advanced tuberculosis of the lungs and all had tubercle bacilli in sputum. A preliminary Mantoux test with I.IOOO Old Tuberculin was made and a few days later o.oi mg. by dry weight of living vole tubercle bacilli suspended in o. 5 c.c. of sterile double-distilled water was in- jected intradermally into the anterior aspect of the right thigh. The comparatively large volume (o. 5 c.c.) of the injecting dose was chosen to increase accuracy of dosage. In later series we used a volume of o.2 c.c. to eliminate the element of t rauma introduced by intradermal injection of a comparatively large volume of fluid. That element actually did not exist as the injections in subsequent series behaved in a way precisely similar to the first series of injections.

The intensity of the Mantoux reactions was graded roughly as + , + + , + + + based on diameter reactions ranging from 4 mm. to above 4 ~ ram., the first 4-io, the second I o-2o, and the third 2o and above. All reactions were read on the second day and if necessary at the end of seventy two hours, and tlae grading was not done on the purely arbitrary basis of diameter measurement; e.g. a re- action with dense pallid central oedema and commencing vesiculation would be classed as + + + even if its total diameter was below 2o mm. Diameter variations depend partly on the notorious difficulty of getting exactly the same quantity of tuberculin into the skin of each patient, but generally there was no difficulty in grading the reactions accurately.

Reactions The reaction to the injections of vole

tubercle bacilli was almost uniform. In 13 of the 16 patients there appeared at the site of

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injection on the second day a tiny painless red papule surrounded by a small area of erythema. The papules increased slowly in size during the following seven to ten days, but did not exceed in diameter io-I2 mm. In 5 patients the papules, after reaching a maximum diameter in ten days, receded, and within six weeks disappeared leaving small, slightly livid, crusted areas of induration. All of these papules had shown very small central areas of necrosis, but none advanced to pustule formation. In I I patients the papule formed a pustule. 8 of these were tiny and all formed between the seventh and tenth days. The pustules discharged and left small in- dolent ulcers all of which healed within six weeks, leaving varying but small areas of depressed scar formation. 3 patients devel- oped bigger pustules which appeared at about the same time as the others (seven to ten days). 2 of these (Nos. 15 and 16) were actually indolent abscesses lying in areas of red induration measuring 3 ~ and 6o mm. re- spectively. Both discharged and left small ulcers which clinically looked like tubercu- lous ulcers, and both healed within twelve weeks. None of the ulcers or papules were excised for examination.

There was no associated glandular reaction and there was no constitutional disturbance in any patient. The intensity of the reaction followed the Mantoux reaction closely and graphs plotted on the seventh and fourteenth days showed almost coincident curves. The grading of these reactions was again arbi- trary, but it was made without any reference to the Mantoux reaction. It was found that the average papule was below io mm. in diameter, while bigger reactions between IO and 2o mm. were found in some, and above ~o mm. in a few, patients. These were classed respectively as + , + + , and § + + . They were rough clinical dispositions but their later conformation with the Mantoux reactions was interesting. For example the two patients who developed small abscesses had + + + Mantoux reactions.

Heat-Killed Bacilli This close association between the two

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reactions was so miking that on Professor Mackie's suggestion we resolved to try the effect of the injection of bacilli killed by heat.

(2) This series consisted of 32 patients of ages ranging from 14 to 42.6 were below the age of 2o and, of these, 2 were aged 14. The majority were between 25 and 35. I2 were drawn from the first series and 2o were fresh patients. 25 suffered fromAuberculosis of the lungs and among these were the 12 patients from the first series. 5 had disseminated tuberculous lesions (bones, joints, kidneys, etc.), i had isolated spinal caries, and I had diffuse lymphatic tuberculosis. No patient in these last three groups was gravely ill. All were tested in the same way with i. i ooo Old Tuberculin. All received intradermally on the anterior aspect of the right thigh a dosd of a susper~sion representing o.o~ mg. of living vole tubercle bacilli and on the same site on the left thigh a similar dose of the same culture killed by heat. The whole series reacted in a uniform manner, and the I2 patients drawn from the first series were in no way different from the 2o new patients. Again the reactions followed the Mantoux reactions closely and there was little difference between the living and the dead vaccines. There was one slight difference between the first and this second series in the conformity of the Mantoux and vole tubercle bacillus reactions.

�9 In the second series while the immediate reaction followed the Mantoux reaction closely the latter was often no guide to the size of the final papule and ulceration. We have no explanation to offer. The fact of greatest interest is that there was very little difference in the reactions of the living and dead injections. In 3 patients the dead in- jection actually gave a bigger reaction. In 6 the reactions were the same. In the re- mainder, that of the living injection was slightly greater. The same applied to the degree of softening. In 2o patients both papules softened, and in ~ the dead injection produced the bigger pustule. In 5 the right leg papule alone softened and in 7 neither softened. In all the softening was of the

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pustule type and the healing of the resulting small ulcers followed the course of the first series. Among those which softened the date varied from the fourth day after injection to the nineteenth, the majority being between the seventh and twelfth days. In 7 the right leg papule (living injection) and in 2 the left softened first, and I I softened coincidentally. In summation, there was a tendency to a slightly greater reaction to the living than to the dead organism, but the character o[" the reaction was the same. In no patient was there any glandular enlargement or systemic disturbance. The i2 patients in Series 2 who had had a previous injection received their Series 2 injections fifty-two days after the first series injection. At that time 4 of the i2 had small residual healing ulcers from the first injection. These all healed during the course of evolution of the second injection.

Varying Dosage It was evident from these two series that

tubercle bacillus sensitivity was playing a large part in the reactions, and a third series of injections was planned to grade the re- actions to varying doses of living bacilli.

(3) This series included 9 patients of ages ranging from 19 to 4 I. None had had previous injections. All had lung tuberculosis of the chronic ulcerative type, and all received intradermally on the anterior aspect of the right thigh three injections in vertical series of o.ooooI, o.oooi and o.ooi mg. by dry weight of the livfng organism in sus- pension. All had previous Mantoux tests performed with i .iooo Old Tuberculin.

The reactions from the smaller doses in this series were generally less than in the previous two series, and with the exception of I patient who gave the greatest react ionma small one-- to the intermediate dose, the reactions increased with the dose. In 2 patients the tiny papules disappeared at the end of three weeks. In each of these No. 3 papule (o-ooi mg.) had been the biggest. The reactions of the remaining 7 patients were read finally at the end of four weeks. All of the papules were then fading. In I, three papules remained measuring 3, 5 and IO mm.

T U B E R C L E D e c e m b e r i946 in order of dosage. In i other there were three very tiny papules, the largest corres- ponding to the o.oooI mg. dose and measur- ing 7 ram. I patient had two. papules measuring 5 and 7 ram. corresponding to the o.oooI and o.ooi rag. doses, and in 4 patients small papules from the o.ooI mg. dose only persisted. Thus with the exception of one patient who, with very slight reactions, still gave the biggest to the intermediate dose, the reactions followed the dosage scale accurately.

(4) A fourth series of 6 patients, none of whom had been previously used and all of whose Mantoux reactions had been pre- viously tested, were each given in the same manner as the preceding series three intra- dermal injections ofo.ooI , o.oI and o.I rag. of the living organism. The ages of these patients ranged from 14 to 35.2 had chronic lung tuberculosis, 2 had abdominal tuber- culosis, I had hip-joint tuberculosis, and I had a psoas abscess of undetermined origin. This last patient was Mantoux-negative and we shall discuss her condition and reaction in detail later. The reactions of this series were generally the same as, but greater than, those in Series 3. All of the patients developed three papules and all of the papules incregtsed in proportion to the size of the dose of in- jection. Three of the reactions to the o.i mg. dose were prompt and extensive (local). All of the papules and ulcers healed, and none of the patients had any glandular enlargement or more than transient constitutional dis- turbance.

(5) A fifth series consisted of 6 patients of ages ranging from I6 to 27. 5 were young girls aged I6, I8, I9, I9, and 2I. All suffered from tuberculosis of the lungs which in 2 was of a sub-acute type. The sixth patient had extensive tarsal tuberculosis. These patients were each given three intradermal injections of o-i mg. Two of the doses were prepared from suspensions heated to I OO ~ C. and 65 ~ C. for one hour. The third was from an un- heated fresh suspension. The Mantoux re- action to i . iooo Old Tuberculin was de- termined as usual. The 5 young girls with

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lung tuberculosis reacted to all three injections with papules which became pustules within a week. All had + + Mantoux reactions and the papulo-pustules varied between 7 and 37 mm. in diameter, the variation in each case being within a few millimetres, e.g. from 3 ~ to 37, 3 ~ to 35, 25 to 3 ~ (2 cases) and in one from 7 to 12. In most the largest reaction was from the living organism. There was no difference between the heated injections. The sixth patient (with tarsal disease) had given a very violent Mantoux reaction (6o mm.) which underwent extensive vesiculation and looked like a first degree burn. In her case all of the areas of injection reacted with similar, but pustular, vesiculation surrounded by wide zones of angry haemorrhagic ery- thema. These subsided quickly and all healed leaving areas of livid staining. During the reaction she had some malaise which passed off in two days. There was no glandular en- largement.

It was our intention to give a series of sub- cutaneous injections, but pressure of other work interrupted this and that was not done. The object of this communication is merely to record the results of what was done.

None of the patients was affected adversely by the injections which appeared to have no effect, good or bad, on the course of their disease. In the majority the papules, or ulcers if pustule formation had taken place, healed within six weeks. All healed within twelve weeks.

Discussion This investigation was obviously of limited scope and proved generally one thing only, that injection of vole tubercle bacilli, living or dead, into tuberculin-positive human beings causes a modified Koch's phenomenon. To that extent it confirms what is already known-- that antigenically the vole organism is similar to the human and bovine tubercle bacillus. The close correspondence of the degree of reaction to its injections with the grade of Mantoux reaction, and the com- paratively small difference between the effects of the living and dead organism, prove that the reactions are largely allergic.

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The organism does however live for periods in the lesions. In Case 7 of Series i, smears from a very small papulo-pustule made on the seventeenth day after injection showed the presence of vole tubercle bacilli in clumps, and a culture on Dorset medium yielded a growth of the organism. It was impossible to say if any proliferation 0 f organisms had taken place in the tissues. The bacillus was found in the direct smears in the form of numerous clumps which were prob- ably due to imperfect trituration after desic- cation.

Case 49 in Series 4 developed, seven days after the injections, a pustular bleb 15 ram. in diameter from the o.i mg. injection. Smears from this bleb showed a few acid-fast bacilli lying in the midst ofa predominatingly polymorphonuclear cellular exudate and the organism was recovered by culture.

Smears from a pustule in Case 37 in Series 3 which formed fifteen days after the o.ooi mg. dose also showed the organism.

The rapid regression and healing of all lesions, and the absence of glandular involve- ment, show that in tuberculin-positive hu- mans the organism in the doses given is localized and rapidly eliminated either by necrosis and ejection or by the immunity response of the tissue cells and fluids.

Mantoux-Negative Case Interesting speculations are invited by

Case 46 in Series 4. She was a girl of I4 and was admitted with a sinus discharging pus above the middle third of the right inguinal ligament, and an ill-deft.ned swelling of the right half of her abdomen. She failed to react to two series of Mantoux tests carried to the I " I O dilution of Old Tuberculin and she was included in the series as a tuberculin- negative individual. She received the three injections given to this series and she gave an immediate-type reaction with early papule formation on the second day. All of the re- actions were, however, very mild and all disappeared in four weeks. Twenty-four days after the injections she was again tested with Old Tuberculin and gave a + + reaction to the I:IOOO dilution. By this time the sinus

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had practically healed, A few weeks later she became fevered and ill and a typical right psoas abscess formed. There was no detectable bone focus for the abscess which was ob- viously infected, and it was exposed by reflection of the peritoneum and drained. B. coli were cultured from the pus, but cultures on L.J. medium yielded a eugonic growth of tubercle bacilli. The organism was not the vole bacillus and was presumably a tubercle bacillus of human type. The further clinical course is interesting but is not apposite to the subject matter of this communication, the immediate interest lying in the failure of the patient to react to tuberculin in the presence of sensitivity, although mild, to the vole tubercle bacillus, and to the rapid conversion of the Mantoux reaction after the vole tubercle bacillus injections.

Clinical experience has convinced many that a negative tuberculin reaction does not absolutely exclude tuberculosis. We ate not thinking here of the dying or of those whose primary infection has long since healed, but of patients who are believed on clinical grounds to have tuberculous disease but who fail to show any response to intradermal injections of tuberculin. In all tuberculin testing there is the tacit assumption that all tissues of the infected body are allergic in equal degree. On this assumption the skin "s the obvious indicator and is used accordingly. That assumption is valid in the great majority of infected patients, but net in all.

Andenaes' Work Andenaes (1945) in recent work on tubercle bacillus allergy in tuberculin anergetics has published the results of a series of 148 cu- taneous inoculations of B.C.G. by Birkhaug's superficial puncture method in 37 tuberculin- positive and in i i i tuberculin-negative persons. The results in the tuberculin-positives are very similar to the results in the 57 patients in our series. All of his 37 patients reacted within twenty-four to forty-eight hours with papule formation in the punctures. 28 of the 37 were observed for three to four weeks. The papules reached their maximum

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size in the second week with an average diameter of 6" 9 mm. In all, within a week, the papules showed a central apple-jelly- spotted area of necrosis and in two-thirds of the number (18) pustule formation occurred. This process reached its maximum at the end of the second week. An ulcer then formed-- average size 2.6 mm.- -and healed in two weeks with the formation of a bluish-red scar. In ten of the 28 the papules underwent absorption without suppuration leaving very small areas of scar formation.

There were two intermediate groups. One group of 17 people who reacted doubtfully to the Pirquet test but positively to I mg. of Old Tuberculin by the Mantoux method was also injected with B.C.G. in the same way as the preceding group. They all gave an immediate reaction by papule formation, but the reactions were milder than those in the group of definite tuberculin reactors. 14 were observed for three to four weeks. In all central apple-jelly spots appeared but only 5 of the number suppurated, and all of the pustules were small.

A second group of IO people who reacted only to io mg. of Old Tuberculin by the Mantoux method was inoculated with B.C.G. by the same method. The inoculation papules appeared at once, but apple-jelly spots ap- peared in one only and pustule formation did not take place in any.

Of the 111 tuberculin-negative persons who were similarly inoculated with B.C.G., 8 had early papule formation with diameters of 4-6 ram. which persisted for two to three weeks and gradually disappeared without pustule formation. In II others a rapid but less typical formation took place. The pa- pules did not exceed 3-5 mm. Involution began after a few days and within a fort- night the papules had disappeared.

49 gave the typical normergic reaction. Initial swelling and redness in the puncture sites subsided after three to five days, but during the second week intensely red papules appeared. The papules became elevated and indurated during the third week measuring from 2-4 mm. in diameter and I-2 mm. in

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height. In the course of four to seven weeks they began to desquamate and gradually disappeared without showing any ,tendency to ulceration. In 43 cases no reaction was observed, partly because the observation time was too short to allow evolution of the normergic reaction and partly because the inoculation apparently failed.

Loss of Tuberculin Sensitivity The results in the tuberculin-positive group

are very similar to the results of vole tubercle bacillus injection in our series of patients with tuberculous disease. Those in patients who reacted only to very large doses of tuberculin, and in some who were tuberculin-negative, are almost identical with the results in our Case 4 6 which we have discussed, and Andenaes' remarks that his investigation has demonstrated in man the occurrence of tubercle bacillus allergy without hypersensi- tivity to tuberculin. The experiment indi- cates ' that negative reactors to tuberculin constitute no homogeneous group of non-in- fected but a group composed of non-infected as well as infected who have lost their

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tuberculin hypersensitivity.' Why our patient, who had extensive tuberculosis without skin tuberculin sensitivity, developed that sensi- tivity after the vole tubercle bacillus injection is a matter for speculation. Before we ob- tained the positive culture result from the psoas pus we assumed that the injections had been responsible for the conversion.

One cannot argue from infected to non- infected people, but the similarity of the results of injection of the former group with the vole tubercle bacillus and with B.C.G. is apparent. I f future work should show that the vole bacillus has the same low pathogenicity for man as it has for the rabbit, the calf, and the guinea-pig, its fixity of type gives it obvious advantages over B.C.G. for human immunizing purposes.

References Andenaes, O. H. Act. Tub. Scandin., i945, xrx,

2 I I .

Birkhaug, K. Am. Rev. Tub., I946, Lm, 411. Griffith, A. S., and Dalling T. J. Hyg. Camb.,

I94o, XL, 673. Weils, A. Q., and Brooke, W. S. B. Jour. Exper.

Path., 194o , xxI, io 4.

Review ACTA. MED. SCAND. SUPPLEMENT i7i.

ON SPECIFIC TREATMENT OF THROMBOSIS & PULMONARY EM- BOLISM WITH ANTICOAGULANTS. By H. Zilliacus. Results of Five Years' Treatment in the Swedish Hospitals (i94o- I945).

The experiences in Sweden with Heparin and Dicoumarol are decidedly encouraging and should certainly stimulate the greater use of these substances in this country. The results are of equal interest to physician, surgeon and gynaecologist. Briefly, this paper, like that published by Gunnar Bauer in a recent number of the Lancet, emphasizes several salient facts:---

(i) The commencement of most thrombosis in the deep veins of the calf of the leg and the tautness that this condition produces in the calf when the foot is dorsiflexed.

(2) The amenability of this condition to prompt and adequate intravenous Heparin therapy, combined with general active move- ments at an early stage of treatment.

(3) The evil results in the shape of leg ulcers~

induration of the skin and oedema occurring in nine tenths of the patients in whom thrombosis had spread to the femoral vein.

(4) The fact that thrombosis of the femoral vein is liable to occur in nearly 9 ~ per cent of conservatively treated cases of deep thrombosis of the lower limb.

(5) The value of Heparin in preventing pulmonary embolism and in limiting the spread of pulmonary embolism once it has occurred.

Heparin is still, unfortunately, expensive but this extensive series leaves little doubt that it is the most valuable form of therapy in the treat- ment of venous thrombosis, and that untreated venous thrombosis is a time-consuming and very expensive form of ill health.

Dicoumarol, a cheaper preparation, which can be given by mouth, acts on the liver and retards prothrombin formation. Its effect is such that it can be used only in a hospital where daily pro- thrombin levels in the blood can be estimated. Its action is too slow to prevent the spread of an established thrombus or the production of pulmonary emboli, bui its effect is fairly pro- longed. Its liability to cause haemorrhage does not make it suitable for post-operative use,

These Illustrations are the work of Dr. R. Cra~zston Low.

F/r;. I.

FIG. 2. Fxo. 3.

Fro. I (top left).--Series 5, Case No. 57, M.M. , 36 years. Extensive tarsal tuberculosis showirfg the Mantoux reaction (I/XOOO) on second day. Fie. 2 (bottom lef t ) . - -Same case showing vole bacillus injections on 5th day (o. i mg. of dried organism in series heated to Ioo ~ C. to 65 ~ C. and unheated) . FIG. 3 (r ight) . --Series 5, Case No. 53, A.M. , 17 years. Man toux reaction (I / iooo) 4- 4-. Series of vole bacillus injections on 5th day (o. i mg. of dried organism in series heated to too ~ C. to 65 ~ C. and unheated) .