879

SPECIAL ARTICLES

A SURVEY OF THE

IMMUNISATION OF CATTLE AGAINST

TUBERCULOSIS.

(C) Living Bovine Vaccines.*

The think group comprises vaccines of living bacilli I

of bovine strain!;.The bulk of this work has been done by Calmette

and his co-workers with the well-known strain ofattenuated bovine bacillus-B C G. Chaussinand 1

has recently brought together most of the work andthe present writer is indebted to his book for muchof the summary which follows.

In 1906 Calmette and Guerin stated that if cattlewere given one dose of virulent tubercle bacilli bythe mouth the resulting tuberculosis would some-times heal. The vaccination produced in this wayconferred on the animals a definite degree of resist-ance to fresh infection at a later date. The immunitywas not complete, however, and if the doses of

infecting bacilli were repeated tuberculosis resulted.After a series of tests with a tubercle strain of equineorigin the writers isolated a bovine strain which theycultivated on a medium of potato heated with pureox bile with 5 per cent. glycerin, the cultures beingkept at a temperature of 30" C. in an excess of bile. 1By repeated subcultures this strain lost its virulencefor the ox, the ape, and for the ordinary rodentsused in laboratory work.2 The strain was originallylethal to young cattle when injected intravenouslyin 3 mg. doses, but after 30 subcultures its virulence ihad been reduced so that a smaller dose could be 1tolerated. At this stage Calmette injected intra- Ivenously eight heifers with doses of 1 mg. and 5 mg.of B C C at an interval of one month. Thirty daysafter the second injection the animals were given,intravenously, 3 mg. of virulent tubercle bacilli, aninth animal of the same age and herd being giventhe same dose at the same time as a control. Thecontrol animal showed the usual clinical picture ; atthe end of the sixteenth day there was sudden hyper-pyrexia which persisted up to the time of death. INone of the vaccinated heifers showed any hyper-pyrexia, and all remained perfectly well. Seven ofthe animals were killed one, two, three, four,eight, 12, and 18 months respectively after the

i

beginning of the experiment, and, at the autopsies,no trace of tuberculosis was discovered. The bronchiallymphatics of each animal, however, when ground upand injected into guinea-pigs produced tuberculosis.During the years 1912 to 1915 Calmette repeated

the experiment on ten heifers. Six of them were

given, intravenously, on one occasion 20 mg. ofattenuated bacilli of bovine origin and four were keptas controls. Calmette stalled them with infected Icows in a stable which was arranged so as to be z,specially suitable for natural infection to take place.Three of the vaccinated heifers were revaccinated

intravenously with a dose of 20 mg. one year later,two of these receiving a further injection of 20 mg.after another 12 months. Thirty-two months afterthree of the controls were proved, at autopsy, to havepulmonary and lymphatic tuberculous lesions, whilstthe fourth control remained free. Of the three heiferswhich were not revaceinated two showed at autopsy

* Sections (A) Dead Vaccines, and (B) Living Non-BovineVaccines appeared last week.

small tubercles in the lungs and the bronchial, media-stinal, and mesenteric glands, and the third showed nolesions as proved by inoculation into a guinea-pig.The heifer vaccinated twice and the two vaccinatedthree times showed no lesions at autopsy, and guinea-pig tests were negative.

Calmette next vaccinated 12 heifers, aged from7 to 8 months, subcutaneously. In this case hedecided to use stronger doses. Six heifers were given100 mg. of vaccine and the other six 50 mg. Eightheifers of the same age were used as controls. Intoall these animals he injected a resistance test dose of5 mg. of virulent bovine bacilli. The results were asfollows : six of the controls died, between the thirty-second and the fifty-eighth days, of massive or acutemiliary tuberculosis ; the other two were killed after60 days and showed miliary tuberculosis of the lungs.On the other hand, 11 of the 12 vaccinated animalsshowed no naked-eye tuberculous lesions at autopsymade from two to twelve months after inoculation,although the glands of seven of them proved infectiveto guinea-pigs. In the twelfth heifer, vaccinatedwith only 50 mg., sclerotic lesions without visibletuberculosis resulted.From these experiments Calmette deduced that

the duration of immunity bore a definite relationshipto the size of the dose of B C G vaccine.From 1921 to 1926 Guerin, Richart, and Boissiere

performed some experiments in Gurville (Seine-Inferieure) where tuberculosis infection persistedin spite of all hygienic measures employed. Thecalves were vaccinated at birth with a dose of 1 to2 mg., intravenously, up to 1923, and after that timesubcutaneously with a dose of 50 mg. All the animalswere revaccinated each year. In 1927 the resultswere as follows: In the cattle-sheds at Gurvillethere were 58 cows and heifers, vaccinated andrevaccinated, who were quite well. Twenty-fiveoxen and five cows, born and reared in that infectedenvironment, were slaughtered and showed no lesions.Of these 30 cattle 16 were born of tuberculous mothersand 13 of suspected mothers, and all were fed for thefirst month on raw milk from the mother. Duringthe experiments all isolation and disinfection measureswere suspended.

Meanwhile Guerin investigated the date of the

appearance of immunity after vaccination withB C G in cattle. He showed that immunity was stillincomplete on the tenth day but became completeabout the twentieth day. In France 49 veterinarysurgeons who had vaccinated 2315 cattle replied toa questionaire sent out by Guerin. From these

replies Guérin found that 103 animals had died fromcauses other than tuberculosis and 121 had been sentto slaughter. At autopsy true tuberculous lesionswere shown in eight cases, whilst 113 showed no lesions.All these animals had been born in infected sheds

and had remained there without any of the pre-cautions having been observed.

Ascoli and his collaborators vaccinated, up to 1927,2414 young cattle subcutaneously. None of thevaccinated animals which had been killed or haddied showed tuberculous lesions. De Assis and

Dupont immunised, between 1926 and 1928, 64 cattlewhich had not been isolated after vaccination andwhich had not been fed on sterilised milk. Theseauthors record three autopsies-two on vaccinatedanimals and one on a control. The two vaccinatedanimals killed at the age of 20 months showed notuberculous lesions. On the other hand, the control,

880 IMMUNISATION OF CATTLE AGAINST TUBERCULOSIS.

having lived under the same conditions as the othertwo and being killed at the age of 16 months, showedcaseous tuberculosis of the left parietal pleura, as

well as a number of tuberculous nodules on thevisceral pleura, and glandular lesions. The authorsemphasised the need for isolation during the monthfollowing the vaccination, and for feeding, duringthis time, with milk free from virulent bacilli. Gerlachvaccinated and revaccinated, for three years, 373calves living in an infected environment and failedto discover any trace of tuberculosis. Lignieresobtained a very marked resistance against naturalinfection in cattle vaccinated with 50 mg. B C Gsubcutaneously compared with non-vaccinated con-trols, but found that this resistance often decreasedvery rapidly. Maya vaccinated 874 calves fromJuly, 1927, to December, 1928, and reported satis-factory results (details not given by Chaussinand).Mello, after various experiments on 22 calves, con-cluded that 50 mg. B C G, given immediately afterbirth, produced in the animals absolute resistance,one month afterwards, to a resistance test of smalldoses of virulent bacilli. On the other hand, largedoses of virulent bacilli always gave rise to seriouslesions even among the vaccinated. In this case,however, the lesions were less widespread and thesurvival longer than in non-vaccinated animals.He also noted that the immunity appeared from20 to 25 days after vaccination.Obukhovski obtained the following results. Six

cows vaccinated subcutaneously (100 mg.) remainedin good health in spite of an intravenous resistancetest dose of known virulence. Four to seven and ahalf months later one animal showed a tuberculousinduration of the right lung, four others an engorge-ment of the mediastinal glands, and the last no signof tuberculosis. On the other hand, the seven controlanimals, infected in the same way, were found clini-cally to be tuberculous, and at autopsy, two to sevenmonths after infection, showed progressive tuber-culosis of the lungs and the bronchial glands. Fourcattle, vaccinated in the same way as the above,received 16 to 18 months later an intravenous resist-ance test dose of 10 mg. of virulent bacilli. A control ’,,and one of the vaccinated animals died, after 25 and27 days respectively, of miliary tuberculosis. Thethree other vaccinated animals were still in goodhealth four months after the virulent inoculation.

Rankin treated 25 calves with a subcutaneousinjection of 60 mg. B C G. One year later theseanimals were given an intravenous resistance test

injection of 5 mg. of virulent bacilli. Of the controls95 per cent. presented gross tuberculous lesions ; ofthe vaccinated animals only one died of generalisedtuberculosis, the remaining 24 showing healed lesionsor none. In the course of another experiment Rankinvaccinated 25 cattle with 50 mg. B C G. Theseanimals were placed in contact with tuberculousanimals for eight months, during which time theywere fed on milk from tuberculous animals. At

autopsy it was found that 32 per cent. of the vacci-nated animals showed slight non-progressive tuber-culous lesions, and the remainder no lesions. Of thecontrols 72 per cent. showed tuberculous lesions,mainly caseous and progressive. Tzekhnovitzervaccinated subcutaneously 13 calves with a dose of86 to 300 mg. B C G, followed seven to eight monthslater by a resistance test dose of 5 mg. of bovinebacilli. Four control heifers died of miliary tuber-culosis, and all the vaccinated animals, with oneexception, lived, but no subsequent autopsy recordsare quoted. Chaussinand quotes Forsner, Jundelland Magnusson as having obtained less conclusive

results, but he considers that their experiments wereperformed under unsatisfactory conditions. Someof the vaccinated animals, for example, had beenin contact with tuberculous animals before the,

experiment began and had not given a tuberculintest before vaccination.

In 1928 the Health Committee of the League ofNations authorised the President to convene a.

meeting of a few experts to examine the results ofvaccination with B C G against tuberculosis.3 Threecommissions were formed-bacteriological, clinical,and veterinary. The Veterinary Commission reportedas follows : (1) The experimental facts publishedand the unanimous opinion of practitioners who haveused B C G in cattle justify the conclusions thatvaccination carried out according to the techniqueof Calmette and Guerin, among bovines, is a per-fectly harmless procedure ; (2) similar experimentaldata and published observations with regard to theuse of B C G among bovines demonstrate that this.strain of bacteria confers definite immunity againstboth experimental and natural tuberculosis infection.These recognised " pre-immunising " (premunisantes)qualities justify and encourage the extension of theexperimental use of B C G in the prevention ofbovine tuberculosis. There were no British parti-cipants in this Paris conference.At the Eleventh International Veterinary Congress.

held in London in 1930, Watson gave, up to date,the results of experiments which he and his co-workershad been making for the past five years under theCanadian Government. 4 One set of calves used wasfrom a tuberculous herd and the other from anaccredited tuberculosis-free herd. The usual pre-cautions were taken as to segregation, feeding,tuberculin testing, &c. Two vaccines were used-viz., B C G and one made from a moderately viru-lent strain of tubercle bacillus, with an unvaccinatedgroup as a control, making three groups in all. Aftervaccination of the first two groups, all three groupswere put to proof by one or other of the followingmethods : (1) resistance test dose of virulent tuberclebacilli by inoculation ; (2) resistance test dose byingestion ; (3) natural - exposure to tuberculosisunder ordinary farm conditions. The tests extendedin all up to four and a half years. Autopsies on72 cattle showed tubercle in 78 per cent. of cattlevaccinated with B C G, in 90 per cent. of cattlevaccinated with virulent bovine tubercle bacilli, andin 75 per cent. of unvaccinated controls.

In trials of short duration B C G appeared possiblyto retard the development of infection, but thisseemed to be more or less transient, and in spite ofrevaccinations it diminished with time and as thecattle continued under exposure to natural infection.It was finally considered that B C G was contra-indicated as a means of controlling bovine tuber-culosis in Canada where drastic measures ofdestruction of the sources of infection were possible.Buxton and Griffith,5 in a preliminary report

in these columns a few weeks ago, recorded the resultsof tests on 51 calves vaccinated with B C G byvarious methods-viz., 10 by feeding, 4 intratrache-ally, 3 peritoneally, and 34 intravenously. Thesecalves were subsequently tested as to their resistanceto tuberculosis together with 14 untreated calvesby the intravenous inoculation of very virulent bovinetubercle bacilli in doses from 1 to 0-25 mg. of culture.They found that the strain of B C G with whichthey were working was avirulent for the normalcalf and incapable of giving rise to progressive-tuberculosis even when inoculated intravenously inlarge doses. The strain was capable of raising ther

881MEDICINE AND THE LAW.

resistance of a calf to a subsequent virulent experi-mental resistance test infection. This increase ofresistance was less certain after feeding with B C Gthan after intravenous inoculation, and in the authors’experience intravenous inoculation was the most

satisfactory method of giving the vaccine. The degreeof increased resistance conferred upon calves by thislast method varied within wide limits and appearedin general to be directly proportionate to the doseof B C G vaccine. These experiments are beingcontinued. One of the calves tested by these authorsis of particular interest-viz., calf 3. In this calf a

high degree of resistance had been induced by feedingit with B C G. The calf grew into a fine heifer, andtwo years after the test gave birth to a heifer calfwhich is alive and does not react to tuberculin,although the calf was reared entirely on the milk ofits mother.

(D) Vaccines of Uncertain Nature.

The fonrth group comprises vaccines the precisenature of ivhich has not been divulged.From time to time methods of immunisation

against tuberculosis are introduced in which themethods of preparation of the vaccine used are notdisclosed. Probably the best known of these is thevaccine introduced by Mr. H. Spahlinger in 1913.In 1926 Lovell 6 quoted a preliminary report on thevaccination of cattle against tuberculosis compiledfrom notes by Wansbrough Jones and Watts. In1916 some 20 cows were chosen and immunised bythe vaccine prepared by Spahlinger. The vaccineconsists of " potent and virulent strains of tuberclebacilli," and was administered for six months in

gradually increasing doses, after which the cows

were rested for six months. At the end of this periodthey were given a resistance test dose of living tuberclebacilli in varying quantities and of different degreesof virulence, but all of these strains were known tobe virulent to animals. No ill-effects were noted.In 1918 some of these cows (numbers not stated)were killed and post-mortem examination madewithout tubercle being discovered. At the time of the

report the remaining cows appeared to be quite well.At yearly intervals one cow was being slaughteredand examined, and no tuberculous lesion of any kindhad yet been found. No further details were given.At the end of last year a report was issued on

a further experiment by Mr. Spahlinger on thevaccination of cattle. The vaccine employed on

this occasion is of different manufacture fromthat previously used and is said to be preparedfrom dead germs, but no further details are given.The injections varied in quantity from " a meretrace to large doses." Six months after vaccinationall the calves were given resistance tests of massivedoses of living bovine tubercle bacilli, and all exceptone of the calves was reported to be alive and ingood health 19 weeks later. Four control calves,given the same resistance test dose, had died withineight weeks of tuberculosis.

Conclusions.

Reading the records now it is obvious that manyof the early experiments on the immunisation ofcattle were carried out under unsatisfactory experi-mental conditions, and the criteria adopted were sodifferent that a comparison of the results is oftenquite impossible. It seems clear that immunisationby means of tuberculin, bacillary products, or deadbacilli can only be maintained for a short time

(probably only a few months), and even then theresults are uncertain. Similarly vaccination with

strains of tubercle bacilli from cold-blooded animalshave not shown results sufficiently good to encouragefurther tests. The vaccination tests made early thiscentury with living tubercle bacilli of human strainsat first seemed hopeful, but the recovery of thebacilli in the milk at a subsequent date when theheifers calved destroyed the hope of successfulvaccination by this means. As pointed out byMcFadyean at the time, this objection would pro-bably not apply to the same degree with vaccinesof living avian bacilli, but the few results recordedhave not been followed up. As shown in the pre-liminary note of Buxton and Griffith recently pub-lished in our columns, and in most of the evidencequoted above, the new method of Calmette of usinga vaccine of a modified living bovine strain (B C G)looks at the moment more hopeful, though here againthe extensive tests of Watson in Canada have beendecidedly unfavourable.Whether the results are going to be vitiated, as

in the earlier immunisation work, by the subsequentappearance of living bacilli in the milk of the vac-cinated animals it is too early to say. Meanwhile afresh factor has arisen in the liability of B C G torevert to a virulent state when grown on mediaother than the original glycerinated ox-bile-potato.These untoward results have been obtained on smalllaboratory animals and, as far as we know, havenot yet been encountered in cattle. Here again itis too early to come to any definite conclusion.

In conclusion it may be useful to summariseCalmette and Guerin’s own recommendations for thevaccination of calves. They appear to prefer thesubcutaneous route, vaccinating during the first15 days and revaccinating each year, care beingtaken that the animals are excluded from infectionfor one month after vaccination. By careful attentionto these details the authors believe that it is possibleto eradicate tuberculosis from an infected herd in aperiod of five years.

REFERENCES.

1. Chaussinand, R. : La vaccination contre la tuberculose parle B C G, Paris, 1931.

2. Calmette, A., and Guérin C.: C. R. Acad. des Sc., 150,Nov. 2nd, 1909; and 151, July 4th, 1910 ; and Ann.Past. Instut., Feb. 27th, 1913, and April 28th, 1914.

3. League of Nations : Health Committee, Report to Council,1928, iii., 16, 28 ; and Minutes of Thirteenth Session,1929, iii., i., 36.

4. Watson, E. A.: Comparative Study of Vaccination withLiving Tubercle Bacilli and with Special Reference toB C G. Report of the Eleventh International VeterinaryCongress, London, 1930.

5. Buxton, J. B., and Griffith, A. S. : THE LANCET, Feb. 21st,1931, p. 393.

6. Lovell, R. Goulburn : Why Tuberculosis Exists : How itcan be and has been Cured and Prevented. London. 1926.

7. THE LANCET, 1930, ii., 1354.

MEDICINE AND THE LAW.

Coroner and Post-mortem Examination.

A MEDICAL practitioner directed by a coroner toundertake a post-mortem examination should becareful to understand and comply with his instructions.At Eltham last month a young sapper of the RoyalEngineers, stationed at Woolwich, was run over bya motor-car and killed. A house physician, who wasdirected to conduct the post-mortem examination,stated at the inquest that he found extensive headinjuries and a fractured right thigh. The coroner forSouth-East London reprehended the witness for nothaving made the full examination of lungs, heart, andother organs, which he had been subpoenaed to under-take. Asked by the coroner why he had failed to makethe examination as ordered, the house physician said