K. TOLDERLUND_ fi M. WEIS BENTZON_ z K. BUNCH-CHRISTENSEN_ 3 B

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					  Bull. Org. mond. Sante l
  Bull. Wld filth Org.     1967,36,747-758
                         J




    BCG-induced Allergy and Immunity in Guinea-pigs
         during the First Year after Vaccination
 K.             WEIS       K.              3
                                                 .
       TOLDERLUND,            fi M.            BENTZON, z    BUNCH-CHRISTENSEN,                   B. MACKEPRANG, °
                                                J. GULD 5&H.   WAALER e

                              It has been traditional practice in many countries to lit revaccination with BCG depend
                         on the outcome of periodically repeated tuberculin testing. Acquired resistance to tubercu-
                         losis and tuberculin skin sensitivity are concomitant results of BCG vaccination, and it is
                         apparently inferred that a waning resistance will be faithfully reflected in a waning tuberculin
                         sensitivity. However, it has been demonstrated in recent years, both in man and in animals,
                         that the waning of tuberculin sensitivity may be prevented merely by a repetition of the
                         tuberculin test, while data presented in this and a following report indicate that tuberculin
                         testing has no such influence on the course of BCG-induced resistance to tuberculosis.


                        INTRODUCTION                                Index) and in guinea-pigs (Magnus, 1957; Tolder-
                                                                    lund et al., 1960). The phenomenon has previously
   It is traditional practice in many countries to let              been described as " boosting " but this term is less
 revaccination   with BCG depend on the outcome of                  fortunate because the tuberculin sensitivity is not
periodically    repeated tuberculin testing: in spite               boosted     beyond     the maximum    level that was
of  theoretical uncertainty about the role played by                reached at an early date after vaccination. Only
delayed-type tuberculin hypersensitivity in the                     after waning has taken place-in   the guinea-pig after
mechanism of acquired resistance to tuberculosis,                   6 to 12 months but in man only after several years
waning    of tuberculin skin sensitivity has remained                        the restitution   be demonstrated.   As any
                                                                   -can
the only   measurable factor that might just possibly              tuberculin test of suitable dosage has this restoring
be an index of waning of acquired resistance in the                effect, waning will not occur at all under conditions
individual. However, the findings in recent years                  of periodical retesting, and can be demonstrated
that waning tuberculin sensitivity after BCG-                      only by refraining from tuberculin testing in the
vaccination   may be restored to its former maximum                individual until a sufficient time has elapsed after the
strength   by a single injection of a low dose of
                                                                   vaccination.
tuberculin further obscure the meaning of the                         One might speculate either (1) that the acquired
tuberculin reaction in this context.
                                                                   resistance is restored through a tuberculin injection
   The restitution of waning tuberculin sensitivity                (just as for the tuberculin sensitivity), in which case
by an injection of tuberculin has been demonstrated
                                                                   periodical tuberculin testing in itself would be a
both in man (Magnus & Edwards, 1955; and
                                                                   much more convenient substitute for revaccination;
unpublished data of WHO & Danish Tuberculosis                      or  (2) that resistance is not thus restored, in which
      ' Deceased 13 April 1964; late Chief, BCG Department,
                                                                   case the tuberculin reaction would not at all reflect
 Statens Seruminstitut, Copenhagen, Denmark.                       the course over the years of BCG-induced resistance
      ' Chief, Biostatistical Department, Statens Seruminstitut,   to tuberculosis and may be completely valueless
Copenhagen, Denmark.
                                                                   as a guide to revaccination. Either interpretation, if
     ' BCG Department, Statens Seruminstitut, Copenhagen,
Denmark.                                                           true, would have important, but very different,
     ' Formerly,    Tuberculosis   Department,  Statens Serum-     consequencesfor the practice of revaccination with
institut, Copenhagen, Denmark.                                     BCG.
     ' Division   of Communicable      Diseases, World Health         The results of two guinea-pig studies relevant to
Organization, Geneva, Switzerland.
   ' Formerly, Division of Communicable Diseases,World
                                                                   this question are reported in this and a following
Health Organization, Geneva, Switzerland.                          article (Tolderlund et al., 1967).

2016
 748                                     K.   TOLDERLUND    AND   OTHERS



               MATERIAL AND METHODS                         for each of the three batches prepared from 12 to
                                                            26 May 1958,30 animals were vaccinated and
   Guinea-pigs were set aside each week during a
                                                             12 kept as controls. Fifteen animals were vaccinated
54-week period, some vaccinated with that week's
freshly prepared batch of BCG vaccine and some              with the batch prepared on 2 June 1958 and 12 kept
(selected randomly) left unvaccinated. Two weeks            as controls. The total number of guinea-pigs was
                                                            847 less 7 dead from intercurrent diseasebefore the
before the date of challenge, some of the vaccinated
                                                            first round of tuberculin injections.
animals, selected at random, were given a tuber-
                                                                Two animals were placed in each cage, any 2
culin injection to restore tuberculin sensitivity that
                                                            animals in a cage being vaccinated with vaccine from
might have waned. Also, some randomly selected
                                                            two consecutive lots so as to make the readings of the
unvaccinated animals were given a tuberculin
injection. Two days before challenge, a final tuber-        local reactions as " blind " as possible. Any 2 animals
culin test was given to a random sample of each             allocated to one cage-werealso allocated to the same
                                                           treatment combination (both were either tuberculin-
group of animals. Challenge with virulent tubercle
bacilli was given to all animals except those vaccin-      tested or not tested, both were challenged on the
ated animals that had been injected with tuberculin        same day, etc.) with the restriction, however, that
both 2 weeks and 2 days before (some animals had           each treatment combination was to be used in a
to be excluded because of lack of space in the             given number of animals.
                                                               The cages were placed in random order in the i
quarters for infected animals, and this particular
group was thought to be the least relevant for the         animal house, and were rearranged in a new random
protection trial). The duration of survival after          order at the time of the tuberculin injection, and
challenge was taken as an index of acquired resist-        again at the final tuberculin test, as well as after
ance.                                                      challenge.
                                                            Tuberculin testing
The vaccines
                                                               The dose of tuberculin injected 2 weeks before
   At the Statens Seruminstitut, Copenhagen, a
                                                           challenge was 10 TU of the Danish PPD RT 19-20-21
batch of liquid BCG vaccine is prepared every week.        in buffer solution. The injection was made intra-
Each weekly batch is tested for potency by intra-          dermally in the right flank. The same dose of the
dermal injection in a number of guinea-pigs (in the
                                                           same tuberculin was used for the test carried out
abdominal region) with 4 doses of vaccine (0.1 ml          2 days before challenge; this time the injection was
of 1/1,1/10,1/100 and 1/1000 standard strength).           made in the left flank. (A 5-TU test also was given,
Observations are made during the next 10 weeks             but the results are not included in the present report).
on the development and duration of the skin lesions            The hair of all animals was clipped at the injection
at the injection sites (Jensen, 1946). The viability of    site whether they were to have an injection or not.
each batch of vaccine is tested by colony counts           The animals were tested in the (random) order in
on Löwenstein-Jensen medium.                               which the cages were placed in the animal house.
   Fifty-four batches of BCG, prepared from 27 May         Twenty-four hours after the tuberculin had been
1957 to 2 June 1958 inclusive, were used in this           injected, the animals were depilated (on the test site)
study. The batch numbers were 1259 to 1312 inclu-          and the reactions were read in the same order as that
sive. The colony counts for each individual batch          in which the tests had been given.
are included in the appendix table; there is no time           The transverse and longitudinal diameters of the
trend in these colony counts and there are no very         induration were measured. The tuberculin tests
extreme values.                                            were given and read by specially trained-nurses from
The animals                                                the Danish Tuberculosis Index.
   Male albino guinea-pigs were used, weighing             Challenge
400 g to 450 g at the time of vaccination. For each           A 12-day-old culture of a virulent bovine strain
of the BCG batches prepared during the period              (E 6884), grown on Dubos fluid medium, was
27 May 1957 to 10 February 1958,10 animals were            injected in the ear vein in a dose of 0.2 ml of a
vaccinated and another 2 animals were kept as              1/10000 dilution, corresponding to 1150 culturable
unvaccinated controls; during the period 17 Feb-           particles.
ruary to 5 May 1958,16 animals were vaccinated                The animals were left for observation of survival,
for each batch and 2 animals left as controls; and         and after spontaneous death each animal was
                            BCG ALLERGY AND IMMUNITY                         IN GUINEA-PIGS           IN YEAR AFTER VACCINATION                         749

    autopsied for verification of macroscopic evidence                                 size 2 days before challenge is shown, in Fig. 1, for
    of tuberculous foci in the organs. The extent of                                   each weekly batch of animals and for each of four
    macroscopic lesions of apparently tuberculous origin                               treatment combinations. It is seen that the pro-
    was recorded in scores IN (and 0 for no lesions)                                   nounced, progressive waning of tuberculin sensitivity
    according to the scale traditionally used in this                                  in vaccinated animals is largely compensated for by
    Institute (Jensen & Bindslev, 1937).                                               the tuberculin injection 2 weeks before. It is also
                                                                                       seen that such an injection of tuberculin has no
                                    RESULTS
                                                                                       comparable antigenic effect in the unvaccinated
    The basic data for individual animals are given                                    animals.
 in the Appendix Table.                                                                Distributions                       to survival   time
                                                                                                           according
 Tuberculin reactions                                                                     After a preliminary analysis of survival times had
   The tuberculin reactions conformed quite closely                                    shown the duration of survival after challenge to be
 to previously reported findings (Tolderlund, Bunch-                                   rather independent of the interval between vaccina-
 Christensen & Waaler, 1960). The average reaction                                     tion and challenge (or of the age of the animal, the

                                                                                  FIG. 1
                  RESULTS OF TUBERCULIN TEST 2 DAYS BEFORE CHALLENGE, SHOWING MEAN SIZE
       OF TUBERCULIN REACTIONS, ACCORDING TO INTERVAL BETWEEN VACCINATION AND TUBERCULIN TESTING




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        048                               12       16          20            24      28          32        36         40       44        48      52
                      INTERVAL       BETWEEN                  VACCINATION                  AND    TUBERCULIN                  TESTING         (WEEKS)

                           VACCINATED          ANIMALS,       NO       PREVIOUS      TUBERCULIN          INJECTION.

              0-0                                             TUBERCULIN          INJECTION       TWO     WEEKS       PREVIOUSLY.

              A-      -A   NON    VACCINATED        ANIMALS,            NO    PREVIOUS       TUBERCULIN          INJECTION.

              f-"-A                                                     TUBERCULIN         INJECTION       TWO    WEEKS       PREVIOUSLY.
                           -. -     -K-                 -"-        ,
750                                                               K:        TOLDERLUND                                      AND        OTHERS



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                          BCG ALLERGY AND IMMUNITY          IN GUINEA-PIGS      IN YEAR AFTER VACCINATION                        751

  latter two factors being confounded in the design of Results in terms of survival time
  this experiment), the material was divided into three       It appears from the accompanying        table that
  broad classesof similar size according to the interval   the variance, 52, depends on the vaccination time,
  between vaccination and challenge: (1) 11/2-141/2 in so far as the vaccinated animals are concerned,
  weeks, (2) 151/2-341/2   weeks, and (3) 35-541/ weeks. the oldest animals (with the largest vaccination
  Distributions according to survival time are shown interval) showing the largest variance. There is no
  in Fig. 2, separately for these classes and for each similar tendency for the control groups, how-
  combination of treatment.                                ever.
     Nine animals were found to have little or no             The .x values appear to be entirely independent of
  evidence of tuberculosis at autopsy (scores 0-III).      whether tuberculin has been injected either 2 weeks
  They are indicated by an "x" in Fig. 2. They all or 2 days before or not injected at all; this is in
  died less than 40 days after challenge, and they seem striking contrast to the findings for tuberculin
 to be distributed at random over the experimental sensitivity. There is some dependenceof the x values
 groups. It was agreed in advance to exclude these on the vaccination time, the oldest animals showing
 animals from further analysis, on the assumption the shortest survival time; this tendency is observed
 that they had not died from tuberculosis.                in both the vacccinated and the controls, though it is
    Apart from these animals, the earliest deaths statistically significant only for the former. Con-
 among the vaccinated animals occurred about sidered together with the larger variance in the oldest
 75 days after challenge, and the heaviest accumula- vaccinated groups, this might be taken as an indica-
 tion of deaths was around 115 days; among control        tion that the immunity has become reduced in some
 animals    the first deaths were after about 60 days and but not all of these animals.
 the heaviest accumulation was at 85 days. Yet some           Fig. 3 illustrates the median survival time as a
animals     survived for long periods and 4 were still    function     of interval   between vaccination    and
alive at the conclusion of the experiment, 234 days challenge (or of the age of the animal), separately
after    challenge (these animals shown in Fig. 2 by for vaccinated animals injected with tuberculin 2
an    "s "). The distributions of survival times are weeks before challenge, for vaccinated animals not
clearly    skew and further analysis has been based injected with tuberculin at all, and for all unvaccin-
on    a transformation to reciprocal survival times, ated control animals combined. The contrasting
        it had been checked by means of probit dia- patterns for survival time and tuberculin reactions
after
          (not shown) that this transformation yielded are seen by comparing this graph with Fig. 1.
grams
approximately      normal distributions. For each group,
the average     (x) and an estimate of the mean square Correlation between allergy and immunity in
              been calculated on the basis of the reci-      individual animals
(s2) have
procal    survival  times, with x= 10 000/t, where t is      Correlation diagrams of the tuberculin reaction
the survival time in days. Finally, the median survival   and the survival time in individual, similarly vaccin-
time is calculated as t= 10 000/z.                        ated, animals (not shown here) demonstrated no

         STATISTICS       FOR DISTRIBUTIONS    OF SURVIVAL TIME SEPARATELY FOR EACH EXPERIMENTAL TREATMENT

                                                              Vaccinated animals            -              Controls
     Tuberculin      Injections        Interva l
                                                       n         x        s'          t         n      x            s'      t

                                         35-54.5       60       91.9     448       108.8         8    111.6         434   89.6
No tuberculin Injection                15.5-34.5       61       82.7     368       120.9        12    125.9         893   79.4
                                        1.5-14.5       97       85.2     277       117.4        21    111.9         666   89.4

                                         35-54.5       59       88.9     880       112.5         7    116.0         282   86.2
Tuberculin Injection 2weeks            11.5-14.5
before   challenge                                                                 117.8        14    118.8         384   84.2
                                                       70       84.9     204-

                                        35-54.5        42       91.7     405       109.1        11    135.0         574   74.1
Tuberculintest2days                                                                                             1
before   challenge
                                       ' 1.5-14.5      53       88.1     272       113.5        20    120.7         461   82.9

                                        35-54.5                                                  9    123.6         823   80.9
Tuberculin injection 2 weeks                                                                          109.7         637   91.2
                                      15 5-34 5                  No challenge                   111
and 2 days before challenge             .    .
                                       1.5-14.5                                                       114.3         329   87.5
    752                                                                                                    K.            TOLDERLUND                       AND    OTHERS



                                                                                                                                         FIG. 3
                    MEDIAN SURVIVAL TIME, ACCORDING TO INTERVAL BETWEEN VACCINATION                                                                                                                                     AND CHALLENGE


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                                                                                                                                                                                                      38               42         III 46      50
                                                      INTERVAL                              BETWEEN                         VACCINATION                                   AND             CHALLENGE                                 (WEEKS)

                                                 VACCINATED                             ANIMALS,                    NO PREVIOUS                           TUBERCULIN                  INJECTION.
                                                        -"-                                  -.    -         TUBERCULIN    INJECTION                                             TWO              WEEKS             PREVIOUSLY.
                                                                                                           ,
                                                 NON              VACCINATED                           ANIMALS,    ALL  COMBINATIONS                                             OF TUBERCULIN.




    correlation at all between the two. This confirms                                                                                                 nated a year   before are allergic to tuberculin in a very
    previously reported findings of Jespersenet al. (1962).                                                                                           real sense: while they do not react to a single tuber-
                                                                                                                                                      culin test, they do react to the second of two tests
                                                            DISCUSSION
                                                                                                                                                      in a way completely different from that in which they                                            i
                                                                                                                                                      would had they not been vaccinated. The restitution
    The very clear-cut results of this experiment are                                                                                                 of allergy   is known to come about very fast, much
 in close agreement with results previously published                                                                                                 sooner than it would take for either vaccination or
 from the Statens Seruminstitut (Magnusson et al.,                                                                                                    challenge   (with a limited number of bacilli) to induce
  1960), and they immediately bring to mind the old                                                                                                   tuberculin sensitivity in an animal never sensitized
 disagreement about the role played by tuberculin                                                                                                     before. Magnus (1957) found waned sensitivity fully
 hypersensitivity in the mechanism of acquired                                                                                                        restored one week after the first test, and there are
 resistance. It appears from the present data that                                                                                                    no data available to prove that it does not occur even
 acquired resistance is independent of tuberculin                                                                                                     much    faster. One could well imagine that even the
 sensitivity in this particular experimental situation,                                                                                               small dose of virulent bacilli given in this experiment
 and it might be tempting to conclude that immunity                                                                                                   might restore the tuberculin sensitivity within     a day
 can be present without hypersensitivity. However,                                                                                                    or two, that is, before, there has been much multi-
 it should not be overlooked that the animals vacci-                                                                                                  plication of the virulent bacilli.
                     BCG ALLERGY AND IMMUNITY             IN GUINEA-PIGS      IN YEAR AFTER VACCINATION                       753

    A clearer instance of dissociation between resist-               vaccinated, and is not restored (apparently not
  ance to tuberculosis and tuberculin sensitivity is                 influenced at all) by the injection of tuberculin.
  described in the second report (Tolderlund et al.,                    Whatever the more profound connexion between
  1967): several years after vaccination, the tuberculin             acquired resistance to tuberculosis and delayed-type
 sensitivity can still be restored, by the injection of              hypersensitivity, it is at least clear that the reaction
 tuberculin, to the same level as that found in newly                to a tuberculin skin test cannot be trusted as an
 vaccinated animals of the same age. But the resist-                 unequivocal indicator of waning of acquired resist-
 ance to tuberculosis has waned, by this time, to an                 ance, but this is a problem that will be discussed
 intermediate level, inferior to that of the newly                   in greater detail in the following article.



                                                            RESUME

      Il est d'usage, dans de nombreux pays, de subordonner            tuberculinique (toujours a la meme dose). Les bacilles
   la revaccination par le BCG an resultat d'une epreuve               tuberculeux virulents etaient injectes par voie intra-
   tuberculinique periodique: malgre 1'incertitude theorique           veineuse ä raison de 1150 elements cultivables par dose.
   dans laquelle nous sommes quant an role de I'hyper-                La duree de la survie apres cette injection virulente a ete
  sensibilite retardee ä la tuberculine dans le mecanisme             prise comme indicateur de la resistance acquise.
   de la resistance acquise ik la tuberculose, le declin de la            Les reactions tuberculiniques ont ete etroitement
  sensibilite cutanee est reste le seul facteur mesurable             conformes aux observations rapportees par Tolderlund,
  susceptible de refleter 1'affaiblissement de cette resistance.      Bunch-Christensen & Waaler en 1960. La mesure des
  Neanmoins, les observations recentes selon lesquelles               dimensions moyennes de la reaction deux jours avant
  cette sensibilite postvaccinale, apr8s son declin, pent We          l'injection virulente, pour chaque lot hebdomadaire
  retablie ä son maximum anterieur par une seule injection            d'animaux et pour chacun des quatre schemas de traite-
  d'une faible dose de tuberculine complique encore                   ment, montre que le net declin progressif de la sensibilite
  1'interpretation de ]a reaction tuberculinique dans ce              tuberculinique chez les animaux vaccines est largement
 contexte.                                                           compense par l'injection de tuberculine pratiquee deux
     on pent envisager deux eventualites. Si, tout comme ]a          semaines avant 1'injection virulente. On constate egale-
                ä la tuberculine, la resistance acquise est          ment que cette injection n'a pas d'effet antigenique
 sensibilite
 restauree     par une injection de tuberculine, il serait bien      comparable chez les animaux non vaccines.
                    dans ce cas de substituer ii la revaccination         Neuf animaux qui ne presentaient ä l'autopsie que peu
 plus commode
 une    epreuve tuberculinique periodique. Si cette resistance       ou pas de signes de tuberculose sont morts moins de
 n'est    pas ainsi restauree, la reaction tuberculinique ne         40 jours apres l'injection virulente et ont ete exclus de
 refleterait   nullement, an cours des annees, 1'evolution de        l'analyse, en tant que victimes presumees d'une affection
 la resistance induite par le BCG et serait denuee de                autre que la tuberculose. 'Les distributions des durees de
 toute valeur en tant qu'indice de la necessite d'une               survie sont nettement dissymetriques et l'analyse ulte-
 revaccination. Dans l'une et 1'autre de ces eventualites,          rieure a ete faite sur les inverses des temps de survie
 les consequences pour la pratique de la revaccination par           apres avoir verifie que cette transformation donnait des
 le BCG seraient considerables, mais profondement dif-               distributions sensiblement normales.
ferentes. Les resultats d'une de deux etudes ä ce sujet                  La duree moyenne de survie a ete determine en fonc-
faites sur des cobayes sont exposes ci-apres.                       tion soit de l'intervalle de temps entre ]a vaccination et
     Pendant 54 semaines consecutives, on a pris chaque             l'injection virulente, soit de l'äge de l'animal, separe-
semaine un lot de cobayes que Pon a separe aleatoirement            ment pour les animaux vaccines ayant requ de la tuber-
en deux groupes; Ies sujets dc l'un des groupes etaient             culine deux semaines avant l'injection virulente, pour les
vaccipes par application intradermique d'une dose                   animaux n'ayant jamais requ de tuberculine et pour
standard humaine de vaccin BCG frais; ceux de 1'autre               l'ensemble de tons les animaux temoins non vaccines.
groupe n'etaient pas vaccines. Deux semaines avant de               Elle s'est revelee absolument independante de I'injection
pratiquer 1'injection virulente d'epreuve, certains des             de tuberculine: eile etait ]a meme que celle-ci soit faite
animaux vaccines, choisis au hasard, etaient soumis ä               deux semaines ou deux jours avant l'injection virulente
une injection de tuberculine (10 UT de PPD RT 19-20-21)             ou ne le soit pas; le contraste entre ces constatations et les
destinee it restaurer le niveau de tuberculino-sensibilite          observations concernant la sensibilite tuberculinique
eventuellement abaisse. De meme certains animaux non                est frappant.
vaccines, choisis au hasard, recevaient une injection de                 Les diagrammes de comparaison entre la reaction
tuberculine. Deux jours avant 1'injection virulente                 tuberculinique et la duree de survie parmi les animaux
d'epreuve, les animaux d'un echantillon aleatoire de                soumis it un meme schema de vaccination, et pris ]ndi-
chacun des groupes etaient soumis ä une derni8re epreuve            viduellement, ont revele l'absence de toute correlation.
                                                                                                                                          APPENI
                                                                                                                 BASIC DATA FOR INDIVIDU

                                                                                                     Vaccinated animals

                                                                            Further treatment
 Vacci-     Colony
 nation     count                                                                                                            Final
  date      (10°/m9)                 Challenge                             Tuberculin Injection
                                                                              + challenge                               tuberculin test
                                                                                                                          + challenge
                          CCCCCC                                 ACACAC                                     BCBCB

   1958

  3 June      26.8       125   115   185     84   101   109                                                 13 122          7 125          18 11
                          93   125   101    106                                                              4 100          8 126

 27 May       25.4        94   136   138    106   113   143       7   171         7   129       13   144    17    133      25   122        20   10(
                         124   135   131     92   173   105      15   127         9   120        8   122    30    113      14    94        21   110

 20 May       28.0       111   122   113    125    91   130     23    147        23   125       12   102    33    115      32   109        33   13
                         136   131   195    156   115    91     16    130        17    78       16   119    23    132      32   122        31   121

 13 May       29.4       131   128   116    122   126   118     27    135        26   128       28   115    34     91      35   123        32   121
                         101    93   173    102   118   115     28    102        34    91       29   108    29     97      37    89        24   137

  6 May       41.8             112   120    146   107           33    106        28   119                                                       131
                         _b                                     34    169        39             35   186   37     145      32   82         34    31
                                                                                      133

29 April      27.2       92    117                              35    112        30    95
                                     106    123   166                                           31   129   31 115          34 150          34 10:
                                                                37    113        32   102

22 April      25.2       123   130   132                        29    123        33   100
                                             97   162           35                              36   109   23 150          34 -b           35   99
                                                                      115        30    78

 15 April     17.8       104   155                              34    138        25   115
                                     114> 234     130                                           29   154   30      89      32 120          30 110
                                                                32    124        33   111

  8 April     24.8      102    110                              42    146        34   132
                                     155   124    115                                           33   112   24      75      27 162          30 119
                                                                31    121        36   110

  1 April     23.8      107    148                              28    127        31   123
                                     102    77     95                                           33   101   35 102          31   97        35 172
                                                                33    101        35   -c
28 March      28.0      105                                     28    148
                               132                              31               30    98       34   109   31 131          34 139
                                                                      120

25 March     28.8       103    142   106    97                  25 100
                                                  138
                                                                34 116           36 125         28   113   33 104          27 127         26 219

18 March     23.6       156    113   133                        29 124          32 155
                                           128    140
                                                                32 162
                                                                                                28   107   26 -b           28   93        30 109
                                                                                32 105
11 March     29.8       107     76    86   122    145           33 153           21 138         32   100                                  31 110
                                                                30 96            31 111                    20 123          20   95

 4 March     14.0       151     96   124    93    156           35 143          25 114          35   114                                  28 121
                                                                27 123          29 109                     25      94      28   93

25 Feb.                 114                                      75             26 143
             24.6              226   129   139    138
                                                                32 122          23 86
                                                                                                30   159   23 114         31 142          20 121

18 Feb.      21.4        97    171   106   105    202           32 104          29 96           28   110                                  25    79
                                                                31 153          25 110                     34 121         24 118

11 Feb.      16.6       104    150   118                        24 116          13 108          28 100                    31    97
                                                                                                           24 129
 4 Feb.      24.0       113    103   185                        24 139          24 109          28 118                    25 101
                                                                                                           22 173
28 Jan.      27.2       102    124   131                        24 114          27>234          16   91                   20 186
                                                                                                           25 185

21 Jan.      11.3       113    84    129                        26 114          21 183          30 112     16 101         32    27
14 Jan.      36.8       127    92    115                        32    94        28 123          32 109     10 115         25 104
 7 Jan.      18.6       135    112   194                        24 127          26 132          27 126     15     94      15 187



      A: reaction (sum of 2 diameters) to tuberculin Injection 2 weeks before challenge.
      B: reaction (sum of 2 diameters) to tuberculin test 2 days before challenge.
      C: Survival after challenge, in days.
      When 2 or 3 figures on the same line are close together, they refer to the same animal.
 ABLE
;UINEA-PIGS IN THE STUDY a

                             I                                                 Non-vaccinated animals

                                                               Further treatment

 Tuberculin Injection +                                                                                                  Tfnalculin injection
                                                        Tuberculin injection           Final tuberculin test
  final tuberculin test                Challenge                                                                        I final            test
                                                           + challenge                     + challenge                       + challenge
                                                                                                                                       ge
                                                                                                                                     nge
A   B    A     B   A     B        C        CC            A    C      A    C        B   C     B    C       B    C   A      BC          ABC



                                  81      138      77                              8   74     9 111       16 77
                                  78       89      87                              6   65     8 79         9 90
 5 20     8   15    7 20         116       64      84    12 66        7 86         8 129      3    69      9 84     9      7    90    8 10- 97
4   21   13   21   19   25                                9   78                                                    3    15    104

13 29    20 27     21 25          91       90            11 67       17 83         6   80     8    84      5 78    11     8    94     7 10 92
14 37    15 21     24 26                                 13 71        9 95         7   84
26 29    29 33     35 30          73       86      88     6 79       11 87         4   75    10    90      6 93    13 10 80          11     6 79
24 33    35 33     29 25                                  8 86                                                     13 4 103

29 33    29 32     35 32                                 10 95                     5   75

28 31    25 37     31 35         168                                                                                9 12        90

28 32    38 29     28 32          80                                                                               10      4 127

31 32    33 32     26 33                                13 107                     9 138

11 29    36 35     31 36          93                                                                               13     5     76

14 40    32 39      7 29                                20 89                      5   67


"9 37    32 39                                          30 119                     9   78


9 34     30   31   28   31        76                                                                               14    10    70


'6 31    26 32     33 36         113                                                                               7      8    79


1 37     32 36     31 37         165                                                                               16     7    95


3 31     28 31     23 33         68                                                                                5      5    70


8 32     31 32     28 31         104                                                                               7 10        12


8 34     32 38     26 28                                 7 106                     7   75

5 30     29 36                   60                                                                                5 12 149
'. 33
 4       27 33                   71                                                                                6      9    76
8 36     26 32                   77                                                                                6      2    83

 9
'. 33    26 32                   62                                                                                11     5    161
5 39     29 34                   192                                                                               10     6    97
1 33     28 32                                           8 30                      7 105



         b Excluded from experiment due to inadequate challenge dose.
         C Died In Interval between tuberculin injection and challenge.
                                                                                                                                       APPEND
                                                                                                             RAI.    DATA    FOR INDIVIDUA
                                                                                                                                             ý
                                                                                                   Vaccinated animals

                                                                            Further treatment
 Vacci-       Colony
 nation        count                                                                                                      Final
  date       (10'/mg)                 Challenge                       Tuberculin Injection                           tuberculin test
                                                                         + challenge                                   + challenge

                           CCCCCC                                ACACAC                                  BCBCBC

   1957

 31 Dec.       12.8       35     85   123                       21 146       30 129          28 105      24 145         26 110
 23 Dec.       18.6      131    115   139                       25 102       23    44        34 123     21 134          22    88
 17 Dec.       27.0       181   123   142                       23 208       23 122           7 128     26 126          26 103
 10 Dec.       24.8       99     96    92                       33 142       22    86        23 106     22      94      27 122
  3 Dec.       38.2      132    154    82                       25 101       15    92         9 106     21 123          14 147
26 Nov.        20.6       84     29                             26 109       13 193          29    92   22      92      15 109
                                      -c
 19 Nov.       21.0      142    147   120                       15 114       11    94        27    87   12 115          32    99
 12 Nov.       27.8      121    104    98                       26 139       21 141          31 145     11      88      28 114
  5 Nov.       26.2      128    102   114                       36 232       31 109          21 150     11 112          21 168
29 Oct.        19.0      152    162   152                       23 106       28 104          32    39   12      95      11 134
22 Oct.        30.8       78    163   115                       19    88      6 108          23 135     27 140          16 126
 15 Oct.       27.2      139    200   126                       26 107                                  10      78      20 117
                                                                             26 104          22 115
 11 Oct.       23.6      130    104   104                                                                                     91
                                                                28 165       24 113          9     97    7 162          18
  8 Oct.       21.6      171     97   123                       25 121       18 181          28    89   13 111          22 127
  1 Oct.       28.4      100    107   155                       21 111        8   143        23 110     16 107          24 103
 24 Sept.      37.6      104                                                                                                           21   99
                                 87   141                       23>234       25 153                     14 102          14    98
 17 Sept.      30.6      153                                                                                                           23 165
                                112   120                       22 136        7 162                      7 131          19 110
 10 Sept.      38.4       89     86                             10 134       33 203                     22 108          20 121
  3 Sept.      28.4       93                                                                                            16    88
                                 98                             18 104       24 228          10 127     24     122
27 Aug.        18.0      169                                                                                                 149
                                141   122                       28    88     10 115          6     58    5      91      27
20 Aug.        21.2      170    126                                                                                     10    95
                                       69                        9 105       13 103          21 101      8     119
13 Aug.        12.0      124                                                                                            11    98
                                110    87                       10    95     12    44        4    186    9     147
 6 Aug.        22.2       95     83                                                                                      9   111
                                      109                        7 104       18    92        29 195      8     142
30 July        24.3       98     91    88                       11 142       14 136          16 114      8     110      12    38

23 July        24.8       94    148   130                       12 154       25 130          7    131   28      97      17   111

16 July       21.4       107    124   158                       11    95      8 113          7    113   25     103       9   109

 9 July       28.4       100    221   101                        9 180        9    71        12 114     28      75       5   226

 2 July       26.6       118    93    119                        9 145        7    78        6 128       7      79       6    78

25 June       14.6        87    147   81                        26 118        8   112        6     82    9               6   133
                                                                                                               -b
18 June       13.8        95    90    117                       10 113        6 117          7 109       6     101       6    93

12 June       14.2       110    168    11                        6    96     15 135                      4      91       9   189

 4 June       18.6        88    82    142                       13   83       6   103        6    109    6     106      13   103

28 May        20.2        84    89    177                        9   107     12   117        8    118    9     158       5    83



            a A: reaction (sum of 2 diameters) to tuberculin infection 2 weeks before challenge.
              B: reaction (sum of 2 diameters) to tuberculin test 2 days before challenge.
              C: survival after challenge, in days.
              When 2 ou 3 figures on the same line are close together, they refer to the same animal.
ABLE (continued)
UINEA-PIGS IN THE STUDY a

                              I                                                    Non-vaccinated animals

                                                                      Further treatment

    Tuberculin Injection +                                                                                                   Tuberculin Injection
                                         Challenge       Tuberculin Injection                  Final tuberculin test        + final tuberculin test
     final tuberculin test                                  + challenge                            + challenge                   + challenge

AB         ABAB                     CCC                   A       CAC                     B   CBCBC                    A       BCABC



19 35      21 29                                             6 114                    10       78
19 32      22    31                 83                                                                                  7      5    84

15 33      31 38                    74                                                                                  7 15        78
12 29      27 33                                             7     81                 14      90
!1 31      20 21                   77                                                                                  10      5   73

19 36      20 26                                         12 100                           8   74
31 34      22 31                                             6     71                     4 122
!7 32      27 34                   86                                                                                   9      8   79

19 37      11 23                   75                                                                                   6      5 102

6 30       23 33                   77                                                                                   9      7   88

!2 28      25 29                                          9       86                      9>234
7 33           9 22                                      25 110                           9   72
                                                     .
5 30       31 31
2 29       25 31                                     -d                               4       76

8 32       17 37                   73                                                                                   6      5   73

6 29       24 30                                                                     10       90

4 27       14 30                  103                                                                                  7      0    75

4 20       24 31                                                                      6       72

3 22        8    29                                       7      -b                   9       91

1 33       10 32                                          7 101                       6       54

            4 28                   82                                                                                  11 13       84
2 27
3 29       11 29                                         6        80                  9       64

            7    17               101                                                                                  12     4 177
5 29
T 31        8 22                  103                                                                                  7      4    80

3 25       12   28                122                                                                                   8      9   75

1 27       15 33                                         8        74                 14       74

3 22       6    12                                       6        73                  8       91

1 27      15 24                   72                                                                                    8 10       81

!    24    8    30                83                                                                                   14      2   82

i 16      10 17                   15                                                                                    8      8   61

     22   11    30                                   10           87                  6       63

     27   13 15                                          8        95                  7       86
     29    8    28                                       8       104                  4       76


            b Excluded from experiment due to Inadequate challenge dose.
            C Died in interval between tuberculin injection and challenge.
            d Excluded from experiment.
758                                         K.    TOLDERLUND   AND   OTHERS




                                                     REFERENCES

Jensen, K. A. (1946) Acta tuberc. "stand., 20,1                Magnus, K. & Edwards, L. (1955) Lancet, 2,643
Jensen, K. A. & Bindslev, G. (1937) Acta tuberc. scand.,       Magnusson, M., Jespersen, A. & Bentzon, M. NV. (1960)
  11,101                                                         Acta tuberc. stand., 34,34
                                                               Tolderlund, K., Bunch-Christensen, K. & Guld, J. (1967)
Jespersen, A., Bentzon, M. W. & Magnusson, M. (1962)             Bull. Wld Hlth Org., 36,759-769
  Acta path. microbiol. scand., 54,291                         Tolderlund, K., Bunch-Christensen, K. & Waaler,      H.
Magnus, K. (1957) Bull. Wld Hlth Org., 17,249-254                (1960) Bull. Wld Huth Org., 22,177-184

				
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