STUDIES WITH THE VOLE (MEADOW MOUSE) ACID-FAST BACILLUS

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1 STUDIES WITH THE VOLE (MEADOW MOUSE) ACID-FAST BACILLUS VIRULENCE, PATHOGENICITY, AND SPECIFIC ALLERGIC SENSITIZING AND IMMUNIZING PROPERTIES* H. J. CORPER AND MAURICE L. COHN Research Department, National Jewish Hospital at Denver, Colorado In 94, encouraging reports appeared on the use of the vole acid-fast bacillus, isolated from an English jneadow mouse, for specific tuberculosis immunization in the guinea pig infected with virulent human and bovine tubercle bacilli. The vole bacillus was reputed to be superior to BCG (Bacillus Calmette-Guerin: bovine) and therefore merited further study, particularly because of interruption of the work in Britain by the war. The vole aeid-fast bacillus was isolated by Wells and Oxon and, in their preliminary report in 937, they noted the présence of a disease, presumed to be tuberculosis, in wild voles. The disease is widespread in the British Isles, suggesting that it is not a chance infection. The pathological findings were caseous areas throughout the subcutaneous tissues of the body involving the lymph nodes of the neck, axillae, inguinal région, and back, with ulcération of the skin around the right pinna. Both lungs contained caseous areas with sharply defined edges. The mediastinal and mesenteric lymph nodes were much enlarged and caseous, and the spleen was enlarged. The caseous lésions contained numerous acid-fast bacilli. Growth of the bacilli was obtained on Dorset's egg médium after six weeks. General disease was produced in voles and guinea pigs with résultant death. In rabbits, no death occurred but local lésions were produced. Ail the animais reacted to tuberculin. Wells and Brooke 3 stated in 94 that their results were "inconclusive and statistically insignificant" but that they were "sufficiently striking to warrant a preliminary report especially as the war seriously interfered with the continuation of the experiments." They note also that vaccination of guinea pigs with the vole acid-fast bacillus prior to infection with virulent mammalian tubercle bacilli gives a degree of protection which apparently is far greater than has been recorded by other means. They found the vole acid-fast bacillus slightly virulent for guinea pigs: a subcutaneous injection of 5 mg. may produce progressive disease and death; while a smaller inoculation produces a local abscess with caseation and sometimes ulcération, but the lésion is régressive. For vaccinating, a total of mg. of vole bacilli were used in four weekly subcutaneous injections. In comparison, 3 mg. of BCG were used in four weekly injections in guinea pigs. Intercurrent diseases killed a large number of the animais. In one séries (vole), the initial 6 animais were reduced to 9; in a second (vole), 6 to ; and a third (BCG), 3 to 8. In those that survived, the vole appeared to produce the best immunity and BCG did not prove efficient. In 94 Griffith, Dalling, and Pagel 4 reported unexpectedly good results with the vole bacillus in calves and better than those achieved when BCG was used as a vaccine. They suggest further testing and the intravenous injection of one dose, 5 mg., and then testing the calves again after six months. In their guinea pig vaccination experiments, they found ail the vaccinated animais more résistant to tuberculosis than the controls. Ail had living bovine tubercle bacilli in their tissues and "there is little doubt that thèse bacilli would hâve caused progressive fatal disease when the immunizing effect of the vole bacilli had faded." The vole strain is able to excite a tuberculous reaction in a calf, chronic in nature, * This investigation was aided by a gift from Morton May in memory of Florence G. May. 8

2 THE VOLE ACID-FAST BACILLUS 9 which tends to disappear without leavingmany traces. Two control calves became infected while five of nine vaccinated calves were found to hâve trivial tuberculous lésions. They found no advantage in giving more than one dose of vole. Brooke 6 presented a study of the vole bacillus in 94, the object of which was to classify and study the organism. He suggested the name "mycobacterium tuberculosis variety mûris" and stated that it is apparently a new type of mammalian tubercle bacillus. It is an aérobic bacillus growing slowly at from 36 to 38 C. Growth upon ail médiums is slow, colonies becoming visible only in from to days but often not before six weeks. The bacillus grows on various egg média and on per cent glycerol sérum agar. Liquid média are unsatisfactory. The organism is not enhanced in growth by glycerol but is somewhat inhibited by it. Egg yolk normal saline médium was most suitable. The slowness of expérimental disease with vole is notable, and usually the great multiplication of bacilli obscures the small amount of cellular reaction. The amount of subcutaneous necrosis caused by the vole bacillus is impressive, reminding one of leprosy. The guinea pig, rabbit, and calf are very résistant to moderate doses of the vole bacilli, whose effects are generally restricted to the site of subcutaneous inoculation. Sometimes massive doses by this route may cause death. No significant différence could be found between absorption of agglutinins for vole, or human or bovine tubercle bacilli, but differentiation was possible from avian, rat leprosy, M. phlei, and M. smegmatis. Vole tuberculin appeared to show différences from fixed mammalian tubercle bacilli tuberculin in biological skin tests. Vole bacilli sensitized guinea pigs give reactions with O.T., or purified protein derivative, and vole tuberculin. The culture of the vole bacillus used throughout our studies was received May, 94, and was obtained through the courtesy of Dr. A. Q. Wells. He advised us that this culture, "Vole D 5," was isolated on May 7, 938, from the caseous glands in the neck of a vole which had been trapped in Wales. Following the instructions of Dr. Wells, we first grew the organisms on a variety of egg médiums without glycerol, but they grew equally well on our glycerol-containing médiums on which we also perpetuated them. In corroboration of Wells' and his colleagues' previous reports, we found that glycerol broth agar and ail liquid médiums (Sauton's, Long's, glycerol broth, and Wong-Weinzirl's médiums) were unsuited for obtaining growth. In ail cases, even on the best solid médiums, growth was slow and not profuse. Heavy seedings with spreads of transplants also proved unsatisfactory for obtaining maximum growth on solid médiums, but better results were obtained from the planting of fine dilute suspensions in saline. In ail of our animal inoculation studies, a six weeks old culture at 37 C. was used since earlier growths rarely produced enough bacillary culture mass. Réaction of vole bacilli to reagents Since no comparative studies could be found on the effect of the common reagents used for destroying contaminating microorganisms in tubercle bacillus isolations, a vole-cidal time test was performed with the results recorded in table. We 6 ' 7 had reported previous studies of this subject on human, bovine, and avian tubercle bacilli. The method used was the same as that previously recorded in similar experiments 8. It is to be noted from the results recorded in table that the vole bacillus is similar to human and bovine tubercle bacilli in its reaction to acid and alkaline reagents; i.e., thèse reagents are relatively innocuous to the vole bacilli in the time of test, while acetic acid destroys them. Avian bacilli, as previously recorded, as well as some acid-fast saprophytes proved résistant to acetic acid. Tuberculin reaction In order to note the specificity of intracutaneous tuberculin tests made on viable vole allergically sensitized guinea pigs, tuberculins were prepared with human, bovine, and avian tubercle bacilli grown on Wong-Weinzirl's liquid médium 9. After filtration, the

3 H. J. CORPER AND MAURICE L. COHN amount of tuberculoprotein was adjusted to equal concentrations (.4 mg. per ce. in the standard) in the test dilutions (.4 and.4 mg. per ce). Since the vole bacilli would not grow satisfactorily on any liquids we tested, we were unable to include this tuberciilin in our séries. It is noted from the findings recorded in table that the vole injected (subcutaneously or intravenously) guinea pigs reacted well to the intracutaneous injection of both human TABLE The "Cidal" Urne of varions reagents on the vole acid-fast bacillus AMOUNTOFBACILLI IN FINESUSPENSION PERCUBICCENTIMETERUSEDFORTESTING REAGENTANDCONCENTRATIONUSED* lmg.. mg. Immed. hour 6 hours Immed. hour 6 hours Sulphuric acid, 6% Oxalic acid, 5% Acetic acid, 5% Sodium hydroxide, 3% * Ail solutions were neutralized before planting on the médiums. ** The numéral indicates the number of weeks after planting when macroscopic growth first appeared. The exponent after the numéral indicates that only the number of tubes given as exponent out of a total of 3 tubes planted revealed growth during a 3 month observation period. FIG.. THE RESULTS OF INTRAVENOUS INJECTION OF THE VOLE BACILLUS Left : Organs of a guinea pig killed 6 days after the intravenous injection of mg. avirulent human tubercle bacilli. Note absence of lésions. Center: Organs of a guinea pig that died 6 days after the intravenous injection of mg. vole bacilli. Note pulmonary vole lésions and splenomegaly. Right: Organs of white rat killed 3 months after the intravenous injection of mg. vole bacilli. Note multiple pulmonary lésions which contained numerous acid-fast bacilli. or bovine tuberculins but not to avian tuberculin. The hypersensitivity produced by mg. viable vole bacilli was about the same as that produced by an équivalent amount of viable avirulent human or bovine (BCG) tubercle bacilli. Pathogenicity of the vole bacillus In the guinea pig, the avirulent human and bovine tubercle bacilli given intravenously are fairly well tolerated when introduced in amounts of from to 5 mg. in fine suspension. Although they show definite reactions resulting from the présence of thèse bacilli in the

4 THE VOLE ACID-FAST BACILLUS TABLE Spécifie tuberculin allergie skin hypersensitiveness of guinea pigs injected with viable vole bacilli AMOUNT,ROUTEOF INJECTION,ANDINTERVAL BETWEENINJECTIONANDTUBERCULINTEST month aft-er subeutaneous injection of mg. viable vole. 3 months after subeutaneous injection of mg. viable month** after intravenous injection of mg. viable 3 month after intravenous injection of mg. avirulent 3 months after intravenous injection of mg. avirulent 3 TYPEANDCONCENTRATIONOFTUBERCULIN Human Bovine Avian mg.* mg. mg. mg. mg. mg. * The amounts recorded are contained in ce. Only. ce. was used for test. ** Since ail the intravenously injected guinea pigs given vole bacilli died within two months, no tuberculin tests beyond the one month interval could be recorded. *** The tuberculin skin reactions were graded in the customary manner: = a négative response. = a definite and slight reaction; = unquestionable positive; 3 = a positive with pronounced reaction but no necrosis; and 4 = a positive pronounced reaction with necrosis. TABLE 3 Pathogenicity of vole bacillus given intravenously to guinea pigs TIMEOFDEATHFOLLOWJNG INJECTION OFBACILLIANDAMOUNTUSED POSTMORTEM mg.* vole intravenously 4 days mg. vole intravenously 4 days mg. vole intravenously 45 days mg. vole intravenously 47 days mg. vole intravenously 5 days mg. vole intravenously 6 days mg. vole intravenously 6 days... mg. vole intravenously 65 days. mg. vole intravenously. Killed in 3 months Splenomegaly (.5 x 5 cm.), hemoperitoneum Splenomegaly (.5 x 5 cm.) Splenomegaly ( x 4 cm.), small hepatic focal lésions, toxic diffuse dermatitis Splenomegaly (.5 x 5 cm.), diffuse congestion, focal pulmonary lésions Splenomegaly (8x5 cm.), hemorrhagic congestion, serous pleurisy Splenomegaly (4 x 5.5 cm.), congested, focal hepatic and pulmonary lésions Splenomegaly (.5 x 5 cm.), congested, focal pulmonary lésions Splenomegaly (3x6 cm.), congested, focal pulmonary lésions No gross pathologie changes were noted. Sections revealed an interstitial and nodular pulmonary pneumonitis * None of the guinea pigs receiving mg. intravenously survived beyond 65 days. The illustrations were chosen from a large séries of guinea pigs. 3 4

5 H. J. COHPER AND MAURICE L. COHN blood, their présence in the body does not shorten the normal life span appreciably; and the conspicuous pathological finding is that of a variable splenomegaly, which gradually retrogresses with time. There is no évidence of multiplication of thèse bacilli in the guinea pig since they may even disappear completely from the internai organs after a year to a year and a half. With thèse findings for comparison, it was possible to study the pathogenicity of the vole bacillus for guinea pigs, with the results presented in table 3. In contrast to the avirulent human and bovine tubercle bacilli, the vole acid-fast bacilli proved pathogenic in mg. amounts given intravenously in that they produced death of the guinea pigs within 65 days in ail cases. Distinctive lésions are found in the internai organs, the lungs and liver, of the animais and also a splenomegaly. Splenomegaly can resuit likewise from intravenously injected bacilli which are avirulent. With the amount of mg. of bacilli only a slight splenomegaly results with avirulent human and bovine tubercle bacilli, while it may be qui te pronounced with the vole bacilli. At the time of death of the guinea pigs injected intravenously with mg. of vole bacilli, microscopic examination of histologie sections stained with hematoxylin and eosin showed a definite interstitial pneumonitis in the lung with progressive nodular lésions composed of young A B C D FIG.. DEVELOPMENT OF VOLE BACILLUS SKIN LÉSIONS IN GUINEA PIGS a) 3 days after the intracutaneous injection in duplicate (top to bottom) of mg.,. mg., and. mg. respectively. b) The same animal after days. ; c) After 5 days. d) After 35 days. Note progressive nature of the lésions. monocytic cells predominantly. Sections of the spleen also showed numerous monocytic nodules in the pulp and bordering the malpighian corpuscles, while the liver revealed multiple small discrète tubercles throughout. The kidneys disclosed no definite tuberculosis. The guinea pigs given. mg. vole intravenously and killed at from three to four months, revealed mainly interstitial pneumonitis and monocytic nodules in the lungs. When mg. vole was given subcutaneously, and the guinea pigs killed in from three to four months, the internai organs revealed no appréciable tuberculous changes microscopically. When mg. of viable vole bacilli was injected subcutaneously into guinea pigs, the results were variable in that most of the animais showed no changes within from two to three months aside from the lésion at'the local site and the first tributary glands. However, when the culture was first received, a number of guinea pigs were inoculated and permitted to live over days. One of thèse, killed at days because of his poor gênerai condition, revealed numerous lymphoid glandular-like lésions along the posterior abdomen, around the spleen and the kidneys on both sides, and continued with the glands tributary to the site of injection. Some of thèse lésions were also found in the mesentery. On smear, the pus from thèse contained numerous acid-fast bacilli. Another similar guinea pig examined on the 6th day revealed similar lésions (positive for acid-fast bacilli) predominantly in the mesentery without other internai organ involvement, apparently following lymphatic channels. However, thèse findings were not consistent in ail the guinea pigs examined after one

6 THE VOLE ACID-FAST BACILLUS 3 hundred days. After six months' artificial cultivation, the strain appeared partially to lose this tendency to form lymphoid lésions in the guinea pig following subcutaneous inoculation. In the rabbit, only the mg. intravenous injections proved pathogenic in that the animais usually died within two to four weeks following injection ; and the prédominant lésions occurred as small pin-point focallesions in the lungs. Microscopic examination of stained sections bore out the gross findings in that the lungs showed predominantly a nodular and diffuse monocytic pneumonitis. The other organs, liver, spleen, and kidney, showed no significant tuberculous changes. mg. intravenously in rabbits was without appréciable effect, producing no macroscopic lésions and no fatal issues. In the white rat, mg. of vole intravenously produced a slight splenomegaly and punctate pulmonary lésions in which FlG. 3. A COMPAKISON OF THE SKIN LESIONS PRODUCED BY AVIRULENT HuMAN TuBEBCLE BACILLI AND VOLE BACILLI IN THE SAME GUINEA PIGS Left: weeks after the intracutaneous injection (from top to bottom) of mg.,. mg., and. mg. The lésions on the left were produced by the avirulent human bacilli and on the right by the vole bacilli. Note that the vole lésions are slightly larger. Right: month after the intracutaneous injection (from top to bottom) of. mg.,. mg., and. mg. The lésions on the left were produced by avirulent human tubercle bacilli and on the right by the vole bacilli. Note larger vole lésions. numerous bacilli could be demonstrated ; but the vole bacilli did not kill the rats in this amount within ninety days.. mg. intravenously proved innocuous to white rats. In the American wild field mouse, mg. given intravenously or subcutaneously proved highly toxic, killing the animais within from 4 to 48 hour's with no visible lésions, however.. mg. intravenously or subcutaneously proved innocuous to thèse wild mice. Skin lésions produced by the vole bacillus In order to note the local effect of vole bacilli in graded amounts, a séries of guinea pigs was given an intracutaneous injection of.,., and. mg. of vole bacilli in. ce. on one side of abdomen and similar amounts of avirulent human tubercle bacilli on the other for comparison. In another séries, the amounts given were.,., and. mg. In earlier studies, similar comparisons were made between avirulent human and bovine (BCG) tubercle bacilli in man and animais '. The reactions to the intracutaneous injection of avirulent human tubercle bacilli were similar to those occurring to avirulent bovine tubercle bacilli (BCG) in normal guinea pigs. Readings and photographie records Qf the séries

7 4 H. J. CORPER AND MAURICE L. COHN reported hère, comparing the vole bacilli and the avirulent human tubercle bacilli, were made at regular intervais from several days to one week. Thèse comparisons showed a slightly larger reaction and resulting nodules to the vole bacilli in normal guinea pigs. Such différences were especially noticeable two weeks after the intracutaneous injections and persisted for at least six weeks. From thèse and foregoing experiments on subcutaneous injections, we feel that when the vole acid-fast bacilli, strain D 5, was received, it still poss'essed a low grade of virulence with slow progression following subcutaneous or intracutaneous injection of relatively large amounts (about mg.) and a decided toxicity when administered intravenously to guinea pigs in relatively large amounts (about mg.). FlG. 4. TUBEBCULO-IMMUNITY PBODUCED BY THE VOLE BACILLUS Left: Infection control killed months after the subcutaneous injection of., mg. of virulent human tubercle bacilli. Note organic tubercles in spleen and lungs. Right: Vole immunized animal which died months after the same injection of virulent human tubercle bacilli as above control but having received mg. vole bacilli subcutaneously two months prior. Note absence of organic tuberculosis but présence of multiple lymphatic vole lésions along the posterior abdomen extending to and in front of the kidneys. Spécifie immunizing value of vole bacillus against infection with human tubercle bacilli Since ail previous comparative studies on the évaluation of immunity of the vole bacillus were performed in a way that allowed no definite déductions, it was considered advisable to carry on such comparisons with avirulent human and bovine (BCG) tubercle bacilli with which we hâve had extensive expérience. In comparing viable avirulent human (Corper) with avirulent bovine (BCG) tubercle bacilli in amounts which in themselves were not harmful, we were unable to prove either superior to the other thus far. Our only reason for preferring the human bacilli is the fact that their specificity for immunizing against human infections would appear to be préférable on an a priori reasoning. The experiments thus far recorded with the vole bacillus hâve compared mg. of vole with 3 mg. of BCG. There is no évidence that such unlike comparisons are proper, and the large amount of BCG thus used approaches closely the threshold of primary toxicity of thèse bacilli to the guinea pigs thus inoculated and are capable of interferring with the development of normal immunity. It is for this reason that practically ail our spécifie immunizing studies wi^h viable avirulent tubercle bacilli hâve limited themselves to amounts not

8 THE VOLE ACID-FAST BACILLUS 5 FlG. 5. TuBERCITLO-IMMUNITY PRODUCED BY THE VOLE BACILLI Left: Infection control guinea pig killed months after the subcutaneous injection of., mg. virulent human tubercle bacilli. Note organic tubercles in spleen and lungs. Center: Spécifie tuberculo-immunity produced 6 weeks prior to the same virulent human tubercle bacillus infection as the above animal by the subcutaneous injection of., mg. of avirulent human tubercle bacilli. Note absence of organic tuberculosis. Right : Organs of guinea piggivenasubcutaneousinjectionof., mg. of vole bacilli 6 weeks prior to the same virulent infection as the infection control above. Note absence of organic tuberculosis but présence of large subcutaneous abscess mass at site of vole bacillus injection. TABLE 4 Spécifie immunizing value of vole acid-fast bacilli as compared wilh avirulent human and bovine (BCO) tubercle bacilli in guinea pigs IMMUNIZING INJECTIONSIXBCUTANEOITSLY MONTHSPRIORTOVIRULENTINFECTION Infection control* mg. vole. mg. vole., mg. vole mg. avirulent human tubercle bacilli. mg. avirulent human tubercle bacilli., mg. avirulent human tubercle bacilli mg. avirulent bovine (BCG) tubercle bacilli. mg. avirulent bovine (BCG) tubercle bacilli...^., mg. avirulent bovine (BCG) tubercle bacilli TUBERCULIN REACTION 3 DAYSPRIORTO VIRULENT INFECTION ORGANICINVOLVEMENTFOUND 6DAYSAFTER VIRULENTINFECTIONSUBCUTANEOUSLYWITH.,MG.VIRULENTHUMANTUBERCLEBACILLI 3 revealed definite vole involvement and showed splenic tubercles with definite vole involvement and 3 showed splenic tubercles with definite vole abscesses at site of inoculation guinea pig revealed a small splenic tubercle * Each recorded finding présents the average results of 5 guinea pigs used in each set of the experiment. ** The involvement is graded from to 4 indicating the amount of organic tuberculosis found when ail the animais were killed two months after virulent infection. = no visible tuberculosis, and 4 = a massive generalized disease.

9 6 H. J. CORPER AND MAURICE L. COHN exceeding greatly mg. the amount where primary toxicity was not manifest in any way upon the guinea pigs. In table 4 the résulta are shown of immunizing with vole bacilli, avirulent human, and avirulent bovine (BCG) tubercle bacilli. In the tabulation of the results of spécifie immunization (table 4), there were certain features which could not be detailed in a table but which are important in the évaluation of the vole immunization as compared with avirulent human or bovine tubercle bacilli. Thèse were disclosed more definitely in a duplicate preliminary experiment which preceded that presented in table 4. In this experiment, the pathogenicity of the vole bacillus was far more pronounced. With the mg. and. mg. immunizing doses, a much more pronounced involvement occurred which displayed itself in two ways: more extensive vole involvement during the four months following vole immunizing injection; and then the effect of such involvement upon the virulent tuberculosis infection, resulting in splenic tuberculosis. Another feature of the pathogenic tendency of the vole bacillus for the guinea pig was the pronounced local abscesses even when so small a dose as., mg. was injected subcutaneously an évidence of multiplication of the vole bacilli since., mg. in itself is well below the visible lésion amount. Excluding thèse other pathogenic tendencies of the vole bacilli, it appeared to produce as much immunity as equal original amounts of the avirulent human or bovine tubercle bacilli. But since the factor of multiplication of the vole bacilli must be considered and since they proved no better in immunizing value, it does not seem justified to consider them more efficient in any way than the already available viable avirulent human or bovine (BCG) tubercle bacilli. Another reason for our study of the vole bacillus was the possibility that it might contain a more efficient immunizing antigen more adapted to isolation than the available avirulent human or bovine tubercle bacilli. Since there was no évidence to verify the foregoing and since growth proved far less satisfactory, it appears that the vole bacillus is not as suitable for this purpose. SUMMARY AND CONCLUSIONS. The vole acid-fast bacillus, strain "D 5," isolated in 938, is similar to human and bovine tubercle bacilli in its reaction to acids and alkalies. However, they grow less profusely on solid médiums and practically not at ail on the usual liquid culture médiums or non-protein synthetic liquid médiums. Their sensitization allergically to tuberculin skin reactions is similar to that of human or bovine tubercle bacilli.. The vole acid-fast bacillus, strain "D 5," 938, was pathogenic (lethal) in relatively large amounts (about mg.) when injected intravenously into guinea pigs, white rats, and mice. In rabbits, mg. intravenously was pathogenic. In contrast, the universally avirulent human or bovine (BCG) tubercle bacilli are non-pathogenic (non-lethal) when given intravenously to thèse animais in relatively large amounts ( to mg. or more). In the guinea pig, progressive lésions were produced by the vole bacillus whether given intravenously or subcutaneously in relatively large amounts. The lésions produced by the vole bacillus are progressive, while those produced by avirulent human or bovine (BCG) tubercle bacilli even in large amounts are retrogressive and resemble those produced by dead bacilli. 3. The vole acid-fast bacillus injected in reasonable amounts produced a protection against infection with virulent human tubercle bacilli about equal, but not superior, to that produced by like amounts of avirulent human or bovine (BCG) tubercle bacilli. While thèse amounts protect against the virulent infec-

10 THE VOLE AGID-FAST BACILLUS 7 tion, the vole infection, especially with the larger amounts used for protection, tends in many instances to show progression in the guinea pig. Therefore, no advantage was noted in the use of viable vole acid-fast bacilli over avirulent human or bovine (BCG) tubercle bacilli for immunizing. The retarded pathogenicity of the vole acid-fast bacillus as indicated by its slow restricted growth and disease production would indicate careful prolonged control observations before attempting to use it for immunizing man to replace the universally avirulent human or bovine tubercle bacilli. REFERENCES () Editorial. The Oxford Tuberculosis Vaccine. J. A. M. A., 6, 59, 94. () WELLS, A. Q., AND OXON, D. M.: Tuberculosis in wild voles. Lancet,,, 937. (3) WELLS, A. Q., AND BEOOKE, W. S.: The effect of vaccination of guinea pigs with the vole acid-fast bacillus on subséquent tuberculous infection. B. J. Exper. Path.,, 4, 94. (4) GRIFFITH, A. S., DALLING, T., AND PAGEL, W.: Inoculation and immunity experiments on calves with the vole strain of acid-fast bacillus. J. Hyg., 4, 673, 94. (5) BKOOKE, WALLACE S.: The vole acidfast bacillus.. Expérimental studies on a new type of Mycobacterium tuberculosis. Âm. Rev. Tuberc, 43, 86, 94. (6) CORPER, H. J., AND UYEI, NAO: Oxalic acid as a reagent for isolating tubercle bacilli and a study of the growth of acid-fast non-pathogens on différent médiums with their reaction to chemical reagents. J. Lab. and Clin. Med., 5, 348, 93. (7) CORPER, H. J., AND UYEI, NAO: Additional observations on isolating tubercle bacilli; the oxalic acid reagent for primary culture. Am. J. Clin. Path.,, 35, 93. (8) COHN, M. L.: The antiseptic effect upon tubercle bacilli of certain recently advocated compounds. J. Bact., 7, 57, 934. (9) CORPER, H. J., COHN, M. L., AND BOWER, C. : A study of the growth of human tubercle bacilli on a nonprotein synthetic médium. J. Lab. and Clin. Med., 5, 98, 94. () CORPER, H. J.: Course of expérimental tuberculous infection in guinea pigs. Pathologie changes produced by massive intravenous injections of virulent and avirulent tubercle bacilli. Arch. Path., 6, 9, 938. () CORPER, H. J., COHN, M. L., AND DAMEROW, A. P.: Studies on the behavior of tubercle bacilli within the body. IV. The cutaneous reaction to avirulent tubercle bacilli and immunity. Am. Rev. Tuberc, 33, 79, 936. () CORPER, H. J., AND CLARK, C: The cutaneous reaction to avirulent tubercle bacilli. Am. J. Clin. Path., 6, 3, 936.