LOUIS LEVY AND LYDIA P. MURRA Y. Leprosy Research Unit, Pub lic Health Service Hospital, San Francisco, California 94118, U.S.A.

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1 Lepr. Rev. (1976) 47,13-23 Studies f the Muse Ft Pad Technique fr Cultivatin f Mycbacterium leprae. 2. The Relatinship Between Incubatin Perid and Generatin Time LOUIS LEVY AND LYDIA P. MURRA Y Leprsy Research Unit, Pub lic Health Service Hspital, San Francisc, Califrnia 94118, U.S.A. The results were reviewed f muse inculatin with My cbacterium leprae recvered frm 417 skin bipsy specimens. The incubatin perid (IP), the number f mnths between inculatin and the first appearance f a significant number f AFB in a mnthly sectin, was fund t be c10sely related t the generatin time CC), the average number f days per dubling. Specimens frm treated patients gave larger values f IP and C, cnsistent with killing f My c. leprae during effective treatment. Frty-fur specimens are described that appeared t prvide incula nly marginally sufficient t infect a muse. The results f this review cnfirm the validity f the use f the IP and C as criteria f infectivity f incula f My c. leprae fr the muse ft pad, and justify the practice f emplying bth measurements in the cnduct f the technique. Intrductin Tw measurements are made in the perfrmance f Shepard's ft pad technique (Shepard, 1960, 1962). One muse is sacrificed each mnth fr measurement f the "incubatin perid" (IP), the number f mnths that have elapsed between inculatin f the mice with a small number «1 04 ) f Mycbacterium leprae and the demnstratin f acid-fast bacilli (AFB) within 30 t 40 cells in histlgical sectins f the inculated ft pad tissues. After evidence f multiplicatin has been nted in a mnthly sectin, a harvest f Myc. leprae is perfrmed, usually frm the pled tissues f 4 ft pads. The "generatin time" (G) is the average number f days per dubling, calculated as if all f the inculated bacilli multiplied at a cnstant rate between incubatin and harvest. Bth measuremen ts are used in determining the rate at which Myc. leprae are killed during treatment f patients with leprmatus leprsy (Cllabrative Effrt, 1975; Levy et ai., 1972; Shepard et ai., 1968, 1972a, 1972b, 1974). * Received fr publicatin 16 Octber, 1975.

2 14 L. LEVY ANO L. P. MURRA Y Usually, bth the /P and G are bserved t increase at abut the same rate during effective chemtherapy. Occasinally, hwever, evidence f multiplicatin is bserved in a mnthly sectin that cannt be cnfirmed by harvest. And cnversely, a harvest sme times yields evidence f multiplicatin despite there having been n such evidence in the mnthly sectins btained during ne year after inculatin f the mice. In rder t understand these apparently anmalus results, we have reviewed the data btained in ur labratry during the past 8 years, and have examined the relatinship between measurements f the /P and G fr 417 skin bipsy specimens frm which My c. leprae were recvered and inculated int mice. Materiais and Methds Since September, 1967, the Myc. leprae recvered frm mre than 500 skin bipsy specimens btained frm leprsy patients have been inculated int mice in this labratry. Of these specimens, 417 were selected fr analysis because the average inculum was at least 10 3 but less than 104 Myc. lepraelft pad, and because data frm bth mnthly sectins and harvests f AFB frm ft pad tissues were available. Recvery f rganisms frm bipsy specimens, inculatin f mice and harvests f AFB frm muse ft pad tissues were accmplished by published methds (Shepard, 1960; Shepard and McRae, 1968). Abut half f the ft pad tissues btained fr histpathlgical examinatin were prepared by decalcificatin f tissue blcks cut thrugh the entire ft (Shepard, 1962). The remaining ft pad tissues were prepared by a technique suggested by S. R. Pattyn, Prince Lepld Institute fr Trpical Medicine, Antwerp, Belgium, in which the sft tissue was dissected frm the metatarsals in a single piece that was fixed and subsequen tly sectined. Results Each f 417 specimens is represented by a pint in Fig. 1, which is a histgram shwing the distributin f values f G fr ali values f the IP ranging frm 4 t > 12 mnths. Because mice were nt always sacrificed earlier than 4 mnths after inculatin, the few specimens shwing histpathlgical evidence f multiplicatin as early as the third mnth have been pled with thse yielding an IP f fur mnths. Mice were nt sacrificed t prvide material fr histpathlgical examinatin later than ne year after inculatin. In the 158 cases in which histpathlgical evidence f multiplicatin was nt encuntered within ne year after inculatin, a harvest was perfrmed frm the inculated ft pad tissues f all remaining mice t a maxirnum f 8 ft pads. In additin t the pints representing each specirnen, the median value f G is shwn fr each value f the IP. As shwn in Fig. 1, there appears t be a reasnably linear relatinship between the median value f G in each distributin and the crrespnding value f the IP. Because the IP is a discrete rather than a cntinuus variable, this relatinship cannt be examined by the linear regressin technique. Hwever, ne may readily cnstruct the 2 x 2 table shwn in Table 1, which demnstrates that the 417 specimens are abut evenly divided between thse with IP > 8 mnths and thse with IP';;;; 8 mnths. Likewise, the specimens are evenly divided between thse

3 INCUBATlON PERIOD AND GENERATlON TIME IN MYCOBACTERIUM CULTlVATlON (2) (5) (1 5) 90 Tlal n. n N. untrealed I 2 nu "' >-." 60 Q) E -= c: -= c 50 Q) t!) :1' r-;- f-:,- 1 f-'::- J., 1-.*.- :}.:! T... f- r-.:..,. I T <. OI:" f-.- f-.-.- f >12 Incubalin perid (mnlhs) Fig. I. Relatinship f IP and G fr 417 specimens. with G < 40 days and thse with G 40 days. Abut 95% f the specimens with G < 40 days yielded an IP.ç 8 mnths, whereas nly 14% f specimens wíth G 40 days yielded an IP.ç 8 mnths. The prbability that this distributin f 417 results culd have ccurred by chance is vanishingly small; P, as measured by Fisher's exact prbability test (Gldstein, 1964), is abut Thus, bth IP and G appear t measure the same phenmenn. Of the 417 specimens, 155 were btained frm untreated patients. The results f these specimens are nt distributed unifrrnly acrss all f the values fr the IP, but, as might be expected, represent a disprprtinately large share f the specimens yielding smailer values f IP and G. The distributins f the values f IP and G yielded by the specimens btained frm untreated patients are shwn in Table 2. As demnstrated in the upper panel, nly abut 3% f the specimens frm untreated patients but 75% f thse btained during treatment yielded values f the IP > 8 mnths. Similarly, as the distributin in the 10wer panel f Table 2 shws, nly abut 14% f specimens frm untreated patients but 77% f thse frm treated patients yielded values fr G 40 days. The prbability that either f these distributins culd have been encuntered by chance is negligible.

4 16 L. LEVY ANO L. P. MURRA Y TABLE 1 Analysis f relatinship between IP and G fr 417 specimens Number f specimens G (days) <40 ;;;'40 Ttal ';;;8 IP (mnths) >8 Ttal Therefre, the distributins f the values fr IP and G btained frm treated and untreated patients are cnsistent with ur expectatins: the larger values f IP and G are assciated with the specimens btained frm treated patients, cnsistent with killing f My c. leprae during effective chemtherapy. TABLE 2 Distributins f IP and G fr untreated and treated patients Number f specimens Untreated Treated Ttal (a) IP (mnths) ';;; > (b) G (days) Ttal < ;;;' Ttal Of particular interest in this presentatin are fur categries f results summarized in Table 3, ali f which may be explained by incula cntaining numbers f viable rganisms nly marginally adequate t infect mice. Categry A includes the 10 specimens shwn in Fig. 1 with IP <; 12 mnths and G > 60 days but < 1 00 days. AlI f these specimens were btained frm patients under treatment. Cnsistent with the large values f G are the large values f IP and the small numbers f AFB actually cunted in each preparatin and calculated as the mean value per ft pad. Prtins f 6 specimens studied simultaneusly in the labratry f C. C. Shepard, Center fr Disease Cntrl, Atlanta, Gergia, r in the Lenard Wd Memrial Leprsy Research Labratry, Cebu, Philippines,

5 INCUBATION PERIOD AND GENERATION TIME IN MYCOBACTERIUM CULTIVATION 17 were fund t prduce multiplicatin f Myc. leprae in mice. The specimen yielding the median G f 68.0 days gave an IP f 10 mnths. A harvest f Myc. leprae frm the pled tissues f 4 ft pads perfrmed 355 days after inculatin prduced an average f 1.86 x los AFB/ft pad. Assuming a lag phase f 30 days' duratin, the actual dubling time during 10garithmic multiplicatin was 62.3 days if all 5000 f the inculated Myc. leprae multiplied, and 18.6 days if nly ne infective rganism per ft pad was prvided by the inculum. The smaller value is much elser t published estimates f the dubling time f 12 t 13 days (Levy, 1970; Shepard and McRae, 1965), suggesting that specimens yielding large values f G are thse prviding prprtins f Myc. leprae infective fr mice n greater than 5: 5000, because the minimal infective dse is abut 5 infective rganisms (Levy et ai., 1974; Shepard and McRae, 1965). Categry B cntains the 13 specimens shwn in the right-hand bar f Fig. 1 t yie1d IP > 12 mnths but G < 100 days. Ten specimens were btained frm patients during treatment and 2 frm untreated patients; the treatment status f the remaining patient is unknwn. Except fr the IP > 12 mnths, the results f study f these specimens are much like thse f Categry A. Prtins f 7 specimens were als studied in the Cebu labratry ; 5 yielded evidence f multiplicatin. The specimen yielding the median value f G in this categry gave, f curse, IP > 12 mnths. That is, 9 f the 20 mice inculated with 4.87 x 10 3 rganisms/ft pad recvered frm this specimen had been sacrificed during the year fllwing inculatin; nt ne demnstrated evidence f multiplicatin f Myc. leprae n histpathlgic examinatin f the inculated ft pad tissue. Because muitiplicatin shuld have been apparent within ne year, ne r mre f the 9 mice had nt received the minimal infective dse. A harvest f Myc. leprae perfrmed frm a pl f 8 inculated ft pads 364 days after inculatin yielded a mean f 2.20 x 105 AFB per ft pad. Again assuming a 30-day lag phase and that ali f the multiplicatin in each ft pad prceeded frm a single infective rganism, the actual dubling time during 10garithmic mu ltiplicatin may be ca1culated t have been 18.8 days. But it is likely that nt ali f the inculated ft pads received a minimal infective dse. If nly ne f the 8 ft pads had actually been infected, a single infective rganism dubling at a rate f nce every 16 days thrugh 21 dublings culd have prduced ali f the rganisms recvered at harvest. The single infected ft pad wuld have been diluted by the 7 uninfected ft pads t yield the mean number f AFBjft pad fund. The eleven specimens cmprising Categry C were selected frm the grup f 145 specimens with IP > 12 and G > 100 (see Fig. 1) because the number f My c. leprae per ft pad ca1culated frm the resuit f a harvest was greater than 104. AlI f these specimens had been btained frm patients during treatment. Perhaps mre infrmative than the average number f AFB/ft pad is the actual number f AFB cunted (the furth clumn in Table 3); this number was 14 fr 2 specimens, 12 and 10 each fr 1 specimen, 7 fr 2 specimens, and 6, 4, 3, 2 and 1 each fr 1 specimen. The bservatin f 1 t 3 rganisms des nt suggest that the Myc. leprae have multiplied, whereas the bservatin f 10 t 14 rganisms almst certainly indicates that multiplicatin has ccurred. Prtins f 7 f these 11 specimens were studied in Cebu; evidence f multiplicatin was fund fr 5 f the specimens-thse fr which 14, 12, 10 and 4 AFB were actually cunted in San Francisc. One f the 2 specimens yielding 7 AFB and that yielding 3

6 00 TABLE 3 Specimens with large values af G Categry N. f Results f study in San Francisc Results f study elsewhere specimens IP N. AFB/ N. AFB/ G N studied N. shwing 60 fields ft pad multiplicatin median median median median range range range range :> (mnths) (x 10 3 ) (days) Z r A =" :s: c:: B 13 > ;<l ;<l :> -< C 11 > r r m < -< D O < <

7 INCUBATION PERIOO ANO GENERATION TIME IN MYCOBA CTERIUM CULTIVATION 19 rganisms did nt shw multip1icatin in Cebu. Illustrative f the specimens in this categry is the specimen fr which 10 AFB were cunted in 60 il-immersin fie1ds. N rganisms were encuntered in mnth1y sectins during the year fllwing incu1atin f mice with 5 x 10 3 Myc. leprae/ft pad. A harvest perfrmed frm the p1ed tissues f 8 ft pads 378 days after incu1atin yie1ded 4. O x 104 AFB/ft pad with G = 126 days. Assuming that multip1icatin had ccurred in all f the ft pads, the dub1ing time during 10garithmic mu 1tiplicatin may be shwn t be 23 days, whereas the dubling time becmes 19 days if all f the enumerated rganisms had been cntributed by n1y ne f the 8 ft pads. This categry appears t represent an extensin f Categry B. The furth categry, Categry D, cntains the 10 specimens with IP ';;; 12 mnths but G> 100 days shwn at the tp f Fig. 1. All but ne f these specimens were btained fram patients under treatment. Seven AFB were cunted in the study f the specimen with IP = 9 mnths. A harvest fram 8 mice perfrmed 327 days after incu1atin f 5 x 10 3 AFB/ft pad yie1ded a mean f 3. 1 x 104 rganisms/ft pad with G = 124 days. If all f these rganisms had been cntributed by ne ft pad that had been infected with a single infective rganism, the dubling time during 10garithmic multiplicatin wu1d be 17 days. l11us, Myc. leprae a1mst certain1y multiplied in the mice incu1ated with rganisms fram this specimen in additin t the muse sacrificed fr histpath10gica1 study 9 mnths after incu1atin. N rganisms r n1y ne were fund in harvests f 2 t 12 mice inculated with rganisms recvered frm the remaining 9 specimens. Prtins f 7 f these specimens were studied simultaneus1y in anther 1abratry ; evidence f multiplicatin was fund in 6 f the 7 cases. Seven f these 44 specimens were studied with mre than ne harvest. The results f the harvests, shwn in detai1 in Table 4, suggest that inculatin f mice with rganisms recvered frm each specimen prduced irregular infectin. Cnsidering the first specimen, fr examp1e, the muse sacrificed after 8 mnths t prvide material fr histpathlgica1 examinatin shwed multip1icatin f My c. leprae as did ne r mre f the 4 mice frm which a harvest was made 301 days after inculatin. Hwever, nne f the 9 mice harvested 1ater had been infected (that is, multiplicatin f My c. leprae had nt ccurred). Similarly, ne may cnclude that at 1east 2 mice incu1ated with AFB frm the secnd specimen received the minimal infective dse-the muse yielding the IP f 1 1 mnths and at least ne f the mice used fr harvest 379 days after inculatin, whereas nne f the fur mice fram which a harvest was made n day 343 appears t have been infected. The rganisms recvered frm specimen n. 3 infected the muse sacrificed fr sectins at 1 1 mnths and at least ne f the 6 sacrificed fr harvest at day 42 1, but nne f the 8 mice used fr harvest n day 378. The AFB frm specimen ns 4, 5 and 6 appear t have infected n1y ne muse in each case. The results f study f these 44 specimens have demnstrated that: (1) a harvest may yie1d evidence f mu1tiplicatin f Myc. leprae when mnth1y sectins have nt; (2) a sectin may shw evidence f mu1tiplicatin, whereas harvests d nt; (3) ne harvest may shw evidence f mu1tiplicatin, whereas ne r mre additinal harvests frm the same grup f mice may nt; and (4) evidence f multiplicatin may be fund in ne 1abratry but nt in a secnd when prtins f the same specimens are studied simultaneus1y by methds shwn t be entire1y cmparable (Cllabrative Effrt, unpublished data; Levy et ai., 1970). Thus, a specimen yie1ding G = 60 days appears 1ike1y t be ne

8 N TABLE 4 Specimens with large values f G harvested mre than nce Inculum Harvest N. AFB/ IP N. N. N. AFB/ NO. f Categry ft pad (mnths) days mice 60 fields specimen (xl0 3 ) A O 2 A A B 5.0 > O c 5.0 > D O N. AFB/ ft pad (x ) < < <8.0 G (days) r r t'l <: -< ;J> z t:i r :- ;;;:: c::: ;.l ;.l ;J> -<

9 INCUBATION PERIOO ANO GENERATION TIME IN MYCOBA CTERlUM CULTIVATION 21 cntaining a prprtin f My c. leprae infective fr mice n 1arger than 5 : Inculatin f mice with suspensins cntaining smalier prprtins f infective rganisms appears t infect the mice irregu1arly. Discussin The purpse f this study was t examine the re1atinship between the 2 measurements f bacteria1 multip1icatin emp10yed in the perfrmance f Shepard's muse f t pad technique fr cu1tivatin f Myc. leprae. Review f the resu1ts f the study f 417 specimens revea1s a dse re1atinship between the 2 measures in the case f 373 specimens (a1mst 90%). Tw hundred thirty-nine specimens yie1ded IP 12 mnths and G < 60 days, and 134 specimens yie1ded IP > 12 mnths and G 100 days, suggesting that the 2 measurements dea1 with the same phenmenn. Mrever, the fact that specimens frm untreated patients accunt fr 152 f the 239 specimens (64%) with IP 12 mnths and G < 60 days and fr n1y 1 f the 134 specimens with IP > 12 mn ths and G 100 days appears t cnfirm the validity f these measurements as criteria f the respnse f patients with 1eprmatus 1eprsy t effective antimicrbia1 treatment. The remaining 44 specimens were arbitrarily divided in t fur categries- 1 O with IP 12 mnths and G> 60 days but < 100 days; 13 with IP > 12 mnths and G < 100 days; 11 with IP > 12 mn ths and G > 100 days but with harvests yie1ding > 1 04 AFB/ft pad; and 10 with IP 12 mnths and G > 100 days. Of the 44 specimens, n1y 3 were btained frm previus1y untreated patients. The 41 specimens btained frm patients during treatment represent 14% f the ttal number f specimens btained during treatment. Nine treatment regimens are represented; these 41 specimens accunted fr 6 t 21 % f the specimens in each regimen, but were nt significantly segregated in any ne regimen. There was perhaps a re1atinship between regimen and the duratin f treatment at the time these specimens were btained. These specimens appear t represent a grup intermediate between thse yie1ding unequivca1 evidence f multip1icatin and thse yie1ding n such evidence, prviding incula cntaining n1y smali prprtins f Myc. leprae infective fr mice. When the prprtin is 1arge enugh (perhaps sme r ali f the specimens in Categry A), ali f the mice receive a minima1 infective dse ; mu1tiplicatin is "slw" -i.e., G is large-because mre dub1ings are required fr multiplicatin t reach detectab1e leveis than when a larger prprtin f the inculum cnsists f infective rganisms. An incu1um cntaining n1y a marginaliy adequate prprtin f infective rganisms may infect mice irregu1ar1y. In fact, this has been demnstrated by Hilsn in his technique f "prprtinal bactericide" (H1mes and Hilsn, 1974), in which the assumptin is implicit that ne can a1ways detect multiplicatin if it has ccurred. The same assumptin underlies the decisin t discntinue examinatin f mnthly sectins after a year has passed; ne year shu1d be 10ng enugh fr multiplicatin t have becme apparent. The mst prblematic f the 4 categries is Categry D, which cnsists f the specimens with IP 12 mnths and G> 100 days. D these results indicate multiplicatin, r may they represent persistence f the inculum which has been distributed in s frtuitus a manner as t be encuntered in a sectin cut at randm thrugh the incu1ated tissue? If the 1atter exp1anatin were true, ne wu1d expect t find such specimens early as weli as late after incu1atin, and

10 22 L. LEVY AND L. P. MURRA Y unsupprted by evidence f multiplicatin in a secnd labratry. Hwever, the IP was 9 mnths r lnge r fr 9 f the la specimens in this categry ; a secnd mnthly sectin was fund t shw numbers f AFB in ne case; evidence f multiplicatin was fund in a secnd labratry in 6 f 7 cases; and a harvest shwed evidence f multiplicatin althugh G was greater than 100 days in anther case. Thus, the demnstratin f numbers f AFB in nly a single mnthly sectin may be taken as evidence f multiplicatin despite G> 100 days. This review has prvided an pprtunity t assess the validity f the cri teria applied t the results f muse ft pad inculatin. D an IP > 12 mnths and G ;:;;' 100 days separate thse specimens cntaining Myc. leprae infective fr the muse frm thse that d nt? If n harvests had been perfrmed after mnthly sectins had failed t reveal multiplicatin f the rganisms fr 12 mnths, 13 f the 272 (5%) specimens demnstrating multiplicatin wuld nt have been s recgnized. The results f a clinicai drug trial wuld nt be changed by cnsidering these 5% f specimens t have indicated n multiplicatin rather than irregular multiplicatin frm a marginally adequate inculum. If the single infected muse had nt frtuitusly been sacrificed fr histpathlgical study in the case f the la specimens (4%) yielding an IP <, 12 mnths with G ;:;;' 100 days, these specimens might have been cnsidered nt t cntain infective Myc. leprae; this wuld nt have changed the results f a drug tria!. One may reasnably inquire why, if the tw measures f multiplicatin f Myc. leprae yield essentially identical results, bth shuld be perfrmed. The sectins are far mre ecnmical f mice and f effrt, but prvide nly a qualitative answer. The harvest f Myc. leprae frm a pl f ft pad tissues prvides a mre quantitative estimate f multiplicatin; a small value f G indicates multiplicatin f Myc. leprae in all f the tissues in the pl frm an inculum cntaining greater than a minimal prprtin f infective rganisms. But harvests are mre time-cnsuming and use mre mice, s that ne wuld like t knw in advance f the harvest that multiplicatin had ccurred. Therefre, the cmbined use f bth measures appears ptimal, using the results f the mnthly sectins t schedule the harvest. The rutine develped by Shepard appears mst ecnmical-namely, t sacrifice a muse mnthly fr histpathlgical examinatin, scheduling a harvest when a sectin shws multiplicatin, and cnsidering the mice uninfected withut further study if sectins shw n multiplicatin fr 12 mnths. Acknwledgements This research was suppited in part by the U.S. Leprsy Pane! f the U.S.-Japan Cperative Medicai Science Prgram administered by the Gegraphic Medicine Branch, Natinal Institute f Al!ergy and Infectius Diseases, Natinal Institutes f Health, Bethesda, Maryland 20014, O.S.A. (Grant R22 AI and Interagency Agreement YO l-ai 10004). References Cllabrative Effrt f the U.S. Leprsy Panel (U.S.-Japan Cperative Medicai Science Prgram) and the. Lenard Wd Memrial. (1975). Rifampin therapy f leprmatus leprsy. Am. J. trp. Med. Hyg. 24, 475. Gldstein, A. (1964). Bistatistics. New Yrk: MacMillan.

11 INCUBATION PERIOD AND GENERATION TIME IN MYCOBA CTERlUM CULTIVATION 23 Hlmes, I.B. and Hilsn, G. R. F. (1974). The rate f bactericida! actin f rifampin n My cbacterium leprae in the muse ftpad. Prc. Sc exp. Bil. Med. 145, Levy, L. (1970). Death f My cbacterium leprae in mice, and the additinal effect f dapsne administratin. Prc. Sc exp. Bil. Med. 135, 745. Levy, L., Mn, N., Murray, L. P., O'Neill, S. M., Gustafsn, L. E. and Evans, M. J. (1974). Studies f the muse ft pad technic fr cultivatin f My cbacterium leprae. I. Fate f inculated rganisms. Int. J. Lepr. 42, 165. Levy, L., Murray, L. P. and Shepard, C. C. (1970). A cmparative study f muse ft pad inculatin f skin bipsy specimens frm patients with leprmatus leprsy in San Francisc and Atlanta. Int. J. Lepr. 38, 54. Levy, L., Shepard, C. C. and Fasal, P. (1972). Clfazimine therapy f leprmatus leprsy caused by dapsne-resistant Mycbacterium leprae. Am. J. trp. Med. Hyg. 21, 315. Shepard, C. C. (1960). The experimental disease that fllws the injectin f human leprsy bacilli in t ft-pads f mice. J. exp. Med. 112, 445. Shepard, C. C. (1962). Multiplicatin f My cbacterium leprae in the ft-pad f the muse. Int. J. Lepr. 30, 291. Shepard, C. C., Levy, L. and Fasal, P. (1968). The death f My cbacterium leprae during treatment with 4,4 -diamindiphenylsulfne (DDS). Am. J. trp. Med. Hyg. 17, 769. Shepard, C. C., Levy, L. and Fasal, P. (J 972a). The death rate f My cbacterium leprae during treatment f leprmatus leprsy with acedapsne (DADDS). Am. J. trp. Med. Hyg. 21, 440. Shepard, C. C., Levy, L. and Fasal, P. (I 972b). Rapid bactericidal effect f rifampin n My cbacterium leprae. Am. J. trp. Med. Hyg. 21,446. Shepard, C. c., Levy, L. and Fasal, P. (J 974). Further experience with the rapid bactericidal effect f rifampin n M. leprae. Am. J. trp. Med. Hyg. 23, Shepard, C. C. and McRae, D. H. (1965). My cbacterium leprae in mice: minimal infectius dse, relatinship between staining quality and infectivity, and effect f crtisne. J. Bact. 89, 365. Shepard, C. C. and McRae, D. H. (1968). A methd fr cunting acid-fast bacteria. Int. J. Lepr. 36, 78.