Find more content written by:  Richard W. Truman1; James L. Krahenbuhl2
  • Volume 69 , Number 1
  • Page: 1–2
ORIGINAL ARTICLE

Viable M. leprae as a research reagent

Richard W. Truman1; James L. Krahenbuhl2




ABSTRACT

Mycobacterium leprae remain a rare research resource. They cannot be cultivated on artificial media, and the only established means to quantify viability of M. leprae has been by its relative growth in the foot pads of conventional mice (MFP). The MFP method is technically difficult and requires several months to yield results. More effective methods are needed. We examined the association between M. leprae's ability to oxidize 14C-palmitate in axenic culture and the MFP growth results of a large number of suspensions. Oxidative activity was assessed by radiorespirometry (RR) using the Buddemeyer-type biphasic culture vessels containing 7H12 liquid medium and 14C-palmitate, or with com­mercially prepared BACTEC 12B vessels containing the same medium. The RR results were highly correlated (r = 0.71) with the growth level that each M. leprae suspension achieved by the MFP technique. In using this technique to examine the effects that many common laboratory practices have on M. leprae viability, we found that viability varies markedly between bacillary suspensions derived from different hosts and tissues. The highest viabilities were obtained with bacilli from moderately enlarged nude MFP(<1 g). Viability tended to be lower among very large nude MFP or long-duration infections and from armadillo tissues. After their harvest from host tissues, leprosy bacilli lost viability quickly. Suspensions stored in 7HI2 liquid medium retained <1% of their viability within 3 weeks of harvest, and freezing bacillary preparations or incubating them at 37°C resulted in nearly an immediate equivalent loss in metabolic activity and viability. M. leprae viability is maintained best when bacilli are stored for only short periods of time at 4°C-33°C. Palmitate oxidation is a rapid, reliable and objective means by which to estimate the viability of M. leprae and can be used effectively as a surrogate for the conventional MFP technique in many studies.



RÉSUMÉ

Mycobacterium leprae demeurent une ressource rare pour la recherche. Ils ne peuvent cire cultivés sur milieu artificiel, et la seule méthode pour quantilier la viabilité se M. leprae a été de mesurer sa croissance relative dans la plante de patte de souris conventionnelles (PPS). La méthode PPS est techniquement difficile et demande plusieurs mois avant de pouvoir obtenir in résultat. Des méthodes plus efficaces seraient les bienvenues. Nous avons examiné l'association entre la capacité de A7. leprae à oxyder le l4C-palmitate dans des cultures axéniques et les résultats d'un taux de croissance des PPS d'un grand nombre de suspensiones. L'activ­ité oxydante lui évaluée par radio-respirométrie (RR) en utilisant le flaçon de culture biphasique de Buddemeyer contenant le milieu liquide de culture 7H12 et du 14C-palmitate ou des (laçons BACTEC 12B pré­parés commercialement contenant le même milieu de culture. Les résultats de RR furent hautement corrélés (r = 0,71 ) avec les niveaux de croissance que chaque suspension de M. leprae a atteinte par la technique des PPS. Nous avons utilisé cette technique pour examiner l'impact sur la viabilité de M. leprae de quelques pratiques de laboratoire et nous avons constaté que la viabilité varie beaucoup entre des suspensions de bacilles provenant de tissus et d'hôtes différents. Les viabilités les plus élevées furent obtenues de suspensions bacil­laires provenant de PPS de souries nues de taille moyenne (inférieure à 1 gramme). La viabilité tendait à être moindre parmi les PPS de grande à très grande taille ou celles avec une infection de longue durée et parmi les tissus infectés de tatous à neuf bandes. Après leur isolement à partir des tissus lésés de l'hôte, les bacilles lépreux pendent rapidement leur viabilité. Les suspensions stockées 3 semaines dans le milieu de culture liquide 7H12 après collection des lépromes ont fortement perdu en viabilité, ne gardent que moins de 1% de leur viabilité initiale. La congélation ou l'incubation à 37°C de préparations bacillaires a entraîné une parte équivalente d'activité métabolique et de viabilité. Le meilieur moyen de conserver une bonne via­bilité de M. leprae est de les conserver pendent une courte période entre 4 et 33°C. L'oxydation du palmitate est un moyen rapide, objectif et sûr d'estimer la viabilité de M. leprae et peut être utilisé comme méthode complémentaire à la technique PPS conventionnelle pour l'estimation de la viabilité dans de nombreuses études.



RESUMEN

Mycobacterium leprae sigue siendo un raro espécimen de investigation. No se ha podido cultivar en médios de cultivo artificiales y cl único medio establecido para cuantificar la viabilidad de M. leprae es su crecimiento relativo en la almohadilla plantar dei ratón (APR). El método de la APR es técnicamente dilicil y requiere vários meses para obtener resultados. Se necesitan métodos más efectivos. En este trabajo, examinamos la asociación entre la habilidad de M. leprae para oxidar el 14C-palmitato en un cultivo axénico y su capacidad de crecimiento en la APR. La oxidación del palmitato se estableeió por radiorespirometría (RR) usando un sistema bifásico tipo Buddemeyer con medio 7H12 y 14C-palmitato, o con el sistema comercial BACTEC con el mismo medio. Los resultados de la RR mostraron una alta correlación (r = 0.71) con el nivel de crecimiento de M. leprae en la APR. Usando esta técnica para examinar los electos que tienen rnuchas prácticas de laboratorio sobre la vi­abilidad de M. leprae, encontramos que la viabilidad varia ampliamente entre las suspensiones de bacilos derivadas de diferentes huéspedes y tejidos. Las viabilidades más altas se obtuvieron con bacilos de las APR moderadamente engrosadas de ratones desnudos (<1 g). La viabilidad tendió a ser menor entre los bacilos de las APR nuiy engrosadas o en aquellas con mucho tiempo de infección y en las lesiones del tejido de armadillos. Después de su aislamiento de los tejidos infectados, el bacilo de la lepra perdió rápidamente su viabilidad. Las suspensiones mantenidas en el medio líquido 7H12 retuvieron menos del 1% de su viabilidad a las 3 semanas de su aislamiento y el congelamiento de las suspensiones, o su incubación a 37°C, condujeron a la perdida casi inmediata de su actividad metabólica y de su viabilidad. La viabilidad de M. leprae se preserva mejor cuando los bacilos se mantienen entre 4°C y 33°C por períodos coi tos de tiempo. La oxidación dei palmitato es una técnica rápida, corliable y objetiva para determinar Ia viabilidad de M. leprae y puede usarse como una alternativa de Ia técnica de la APR en muchos estúdios.





The viability of bacterial preparations can have a marked influence on experimen­tal results. Replication and growth of bacilli has always been considered the gold stan­dard for bacterial viability. Unfortunately, Mycobacterium leprae replicate only in liv­ing tissues, and the level of growth achieved by organisms inoculated into mouse foot pads (MFP) has been the only means to quantify viability of M. leprae. Such procedures require a high level of technical expertise and can yield results only several months after the bacilli already have been used in experiments. As a conse­quence, very few laboratories quantify M. leprae viability.

In recent times, a number of in vitro bio­chemical assays have been employed as screening aids for anti-M. leprae drug de­velopment. These assays do not measure replication of M. leprae in vitro but index their ATP content or uptake of radiolabeled carbon sources in axenic media. Since maintenance of metabolic activity is a hall­mark of viability, detecting changes in the 'Received for publication on 4 October 2000. Ac­cepted for publication in revised form on 17 January 2001. -R. W. Truman, Ph.D.; J. L. Krahenbuhl. Ph.D., Laboratory Research Branch, Hansen's Disease Pro­grams at LSU-SVM. P.O. Box 25072. Baton Rouge, Louisiana 70894. U.S.A. Reprint requests to Dr. Truman at the above ad­dress or FAX 1-225-578-9856; e-mail: metabolic status of bacteria incubated in the presence of candidate drugs suggests a loss in viability and presumptive efficacy for the drug.

Our laboratory already has shown that oxidation of MC-palmitate by M. leprae in axenic culture is useful for drug screening. The technique detects changes in the meta­bolic activity of M. leprae manifest by drugs both in vivo and in vitro, and the as­say has good qualitative association with MFP results (5).

In this study, we examined the utility of 14C-palmitate oxidation for detecting quantita­tive differences in the viability of M. leprae in comparison to conventional mouse foot pad methods. We also used the technique to describe the effect that many common labo­ratory procedures have on M. leprae viabil­ity in order to help highlight appropriate handling practices for viable M. leprae and to increase our understanding of this re­source as a research reagent.

 

MATERIALS AND METHODS

Leprosy bacilli

Nude mouse-derived bacilli. Mycobac­terium leprae (isolate T-53) was maintained in serial passage in the foot pads of athymic BALB/c nu/nu nude mice (Harlan, Indi­anapolis, Indiana, U.S.A.). Mice were inoc­ulated on the plantar surface of both hind feet and harvested when their foot pads were moderately enlarged (0.5 g-1 g), and contained numerous rapidly growing bacilli.

We harvested the bacilli using a procedure described previously ('-4). Generally, the foot pads were cleansed in Betadine® and the skin removed before the highly bacil- liferous tissue was excised and manually homogenized (Wheaton Science Products, Millville, New Jersey, U.S.A.) in 10 ml of 71112 medium. Excess tissue was removed by slow-speed centrifugation, and the bacillary suspension was enumerated by direct count before aliquoting for further study according to a procedure described earlier (l5,19).

Armadillo-derived M. leprae. We made some M. leprae suspensions from the tis­sues of nine-banded armadillos. These ani­mals were experimentally inoculated intra­venously with I x 109 nude mouse-derived leprosy bacilli and had incubated the infec­tion for between 14 to 30 months before be­ing harvested in the very latest stages of fully disseminated leprosy. Bacterial sus­pensions derived from armadillos were pre­pared as fresh tissue homogenates of highly bacilliferous liver, spleen, lymph node or leproma in a manner analogous to the pro­cedure used for nude mouse foot pads de­scribed above. All of the armadillo,tissues contained greater than 1 x 109 M. leprae per gram. The tissues were taken at varying times over a 3-year period and all were free of cultivable contaminants.

Radiorespirometry (RR)

We assessed the ability of each M. leprae suspension to oxidize 14C-palmitate in BACTEC 7H12B medium (Becton Dickin­son Inc., Franklin Lakes, New Jersey, U.S.A.). Suspensions ofM. leprae were incubated in commercially prepared BAC­TEC vials containing 4 ml of the medium, or in closed-cap Buddemeyer-type culture vessels containing 1 ml of the same com­mercially prepared BACTEC medium. Oxi­dation of palmitate by M. leprae resulted in the release of radiolabeled CO 2. The evolved gas was detected either directly, in the case of BACTEC vials, with a BACTEC 460 instrument or indirectly, in the case of Buddemeyer vessels, on a Beck- man LS6000ic (Beckman Counter, Inc., Fullerton, California, U.S.A.) scintillation counter with the aid of absorbent strips sat­urated with sodium hydroxide and scintilla- lion fluid inside each Buddemeyer vessel (4). For BACTEC vials, an average growth index (GI) for each suspension was deter­mined on triplicate vials inoculated with 1 x 107 bacilli after they had incubated for 7 days at 33°C. The mean GI was converted to DPM/106 bacilli using the manufac­turer's formula accompanying each media lot. The activity in Buddemeyer vessels was read directly 7 days after those cultures had incubated at 33°C. For comparative pur­poses, the mean DPM/106 bacilli were de­rived from triplicate sets.

Mouse foot pad studies

The level of growth that each M. leprae suspension achieved in conventional mouse foot pads was considered the standard for viability. The MFP technique was per­formed according to the method described by Shepard with minor modifications (l5-l9). In brief, 30 µl aliquots of bacillary suspen­sions containing 1 x 104M. leprae diluted in Hanks balanced salt solution (HBSS; Gibco, Grand Island, New York, U.S.A.) were inoculated into both hind foot pads of conventional BALB/c mice (Harlan). The foot pads were harvested at 90, 120, or 150 days post-inoculation in order to assess the level of growth achieved by the bacillary suspensions. In experiments where mice were harvested at multiple time intervals, control samples of freshly harvested M. leprae diluted to concentrations of 1 x 104, 1 x 103 and 1 x 104 bacilli in 30 µl HBSS also were inoculated into mouse foot pads. These diluted control samples were used to simulate the growth that might be expected after a -90% and -99% effective loss of vi­ability in those individual preparations (9). For enumeration of MFP growth, four mice were harvested in each group with counts made on paired samples and the observa­tions averaged.

Storage and handling conditions

We compared the ability of several M. leprae suspensions to maintain viability during storage. After harvesting M. leprae from nude mouse foot pads, bacilli were aliquoted to 20 ml closed-cap serum vials (Wheaton) containing 4 ml of Middlebrook 7HI2 at a concentration of 1 x 108 M. leprae/ml. Immediately after dispensing the bacilli to storage vials, aliquots of the sam­ple were removed to determine its original baseline activity using RR and MFR After­ward, the vials were held at -8°C, 4°C, 25°C, 33°C, and 37°C for up to 3 weeks. Additional aliquots of identical amounts of bacilli were removed again at 7- and 21-day intervals to retest the samples for changes in viability during storage. The RR and MFP results derived from individual stored samples were compared to the baseline re­sults originally obtained with each fresh suspension and expressed as a percent of the original activity. Some RR and MFP re­sults also were correlated directly in a linear regression model to discern the association of palmitate oxidation with MFP growth. All statistical comparisons were made on a personal computer using the SAS program package (Statistical Analysis Systems, Cary, North Carolina, U.S.A.).

 

RESULTS

Maintenance of metabolic activity with storage

Suspensions of M. leprae prepared from different nude mouse foot pads are not uniform in their ability to oxidize 14C-palmitate, but exhibit a broad range of detectable metabolic activities. The original activity tends to wane over time. We used 10 different suspensions to examine the ability of Ai. leprae to maintain their metabolic activity when stored for periods of 1 to 3 weeks in 7H12 liquid medium at temperatures rang­ing from -80°C to 37°C. As shown in Figure 1, the oxidative metabolism of palmitate declined markedly in each sample over the 3 weeks of storage as compared to the activity originally detected in the fresh suspension. Regardless of the storage conditions used, most samples retained <10% of their original activity within 3 weeks.

 

Fig. 1. Scattergrams depicting the radiorespirometric activity of different M. leprae suspensions (N = 10) upon initial harvest from nude mouse foot pads (fresh) and after storage in 71112 liquid medium at temperatures rang­ing from 0°C to 37°C. Each harvest is represented by its own symbol. Paired scatters at each temperature demon­strate the activity of that preparation with storage for 7 days and 21 days, respectively. Figure shows that M. lep­rae vary in activity with each harvest and viability wanes with storage.

 

The oxidative activity of M. leprae was preserved best by suspensions held at 4°C, 25°C and 33°C. On average, suspensions held at 4°C retained about 60% of their oxidative activity after I week. That activity waned markedly during additional time in storage. Suspensions held at 25°C and 33°C also tended to maintain higher metabolic activities than suspensions held at other temperatures, but those samples also showed a marked decline in oxidative activ­ity during the 3 weeks in storage. Suspensions held at 33°C lost an average of 20% of their metabolic activity within 1 week, and 60% of their overall activity in 3 weeks. Suspensions held at 25°C showed a similar pattern of decline. The percent of loss tended to be greatest among suspensions whose fresh samples had the highest original baseline activities.

It is especially noteworthy that in each of the replicate experiments, M. leprae suspensions held at 37°C, or subjected to freeze-thaw cycles, showed very rapid loss of metabolic activity. Virtually all of the detectable activity toward palmitate was lost within only a few days when M. leprae cultures were incubated at 37°C. Bacillary suspensions held at -80°C and thawed once also registered only l%-5% of the metabolic activity detected in their fresh suspensions. Repeated freeze-thaw cycles eliminated virtually all of their detectable oxidative activity. In addition, retaining the bacilli in whole tissues during freezing did little to preserve their metabolic activity. Bacillary suspensions prepared from four different 0.5-g nude mouse tissue samples frozen to -80°C for 24 hr retained an aver­age of only 7% ± 3.6% of the oxidative activity exhibited by bacillary suspensions prepared from portions of the same tissues before freezing.

Loss of infectivity with time and temperature

We also examined the influence of storage on M. leprae viability as determined with the conventional MFP technique. Bacillary suspensions derived from three different nude mice were held from 0 to 3 weeks at -80°C, 4°C and 33°C before ali-quots were removed and tested for viability. As shown in Figure 2, stored suspensions lost viability quickly and failed to grow in the mouse foot pads to the same levels as the full-strength, fresh control samples.

 

Fig. 2. Average percent of growth in conventional mouse foot pads at various time intervals achieved by M. leprae suspensions (N = 3) stored in 7H12 liquid medium at -80°C, 4°C and 33°C for 7, 14 and 21 days as compared to the baseline growth achieved by the same fresh bacillary suspensions prior to storage. Fresh control samples were inoculated at full strength (104foot pad) and at concentrations simulating a -90% and -99% effective loss in viability (lO3foot pad and lO2foot pad, respectively). Stored samples failed to grow to the same levels as full strength fresh samples.

 

In comparing the level of growth achieved by stored suspensions to control samples at various time points, we can estimate the relative loss in viability incurred during storage. Bacillary suspensions held in storage at either 4°C or 33°C for up to 2 weeks grew in MFP to levels generally comparable to fresh suspensions which contained only 10% of the number of viable bacilli. Samples which had been frozen to -80°C and thawed even once or repeated times showed either no detectable growth in MFP at all or grew to levels comparable to fresh bacillary suspension controls which contained only <1% of the viable bacilli as the full-strength fresh sample. Regardless of the storage temperature used, suspensions held for 3 weeks grew poorly in MFP and failed to achieve growth levels compa­rable to control inocula, which contained <1% of the viable bacilli as the original full-strength fresh samples.

Correlation of MFP and RR

To discern the association between oxidative metabolism of 14C-palmitate and the actual viability of M. leprae suspensions, we compared RR data with the 120-day MFP result in a linear regression model. We used the values obtained from suspensions stored for up to 2 weeks in the previous storage experiment, along with data from 10 additional M. leprae harvests which we prepared from other nude mice in our laboratory during the same time period (total N = 47). As shown in Figure 3, values for the oxidative metabolism of 14C-palmitate among the different M. leprae suspensions were highly correlated with the level of growth they achieved in the MFP (r = 0.71). Although the RR and MFP results were de­rived independently over a period of several months, the majority of the observations fell well within the 95% confidence interval inscribed around the linear regression line. The relative level of palmitate oxidation by M. leprae suspensions appears to be highly proportional to the growth they can achieve in the MFP at the 120-day interval.

 

Fig. 3. Linear regression model comparing radiorespirometric activity of M. leproe suspensions (N = 47)with conventional MFP growth resultis obtained for each suspension at 120 days postinfection in the mouse (r =0.71). The data in each axis are log transformed.

 

Viability of M. leprae obtained from different tissues

To better understand parameters for in vivo propagation likely associated with harvesting highly viable M. leprae, we examined the oxidative activity of bacilli derived from a number of different nude mouse foot pads and armadillo tissues. As shown in Figure 4, the average metabolic activity for M. leprae from the different armadillo tis­sues tended to be lower than the activity of bacilli harvested from nude mice. The activity was lowest among bacilli derived from armadillo liver, and was only moderately higher for bacilli obtained from armadillo spleen and lymph node tissues. The average activity for bacilli from armadillo leproma was not significantly different from the other armadillo tissues. However, leproma-derived bacilli also showed the greatest variability in oxidative activity of all the types of tissues tested.

 

Fig. 4. Histogram depicting mean and standard deviation DPM/106 bacilli in palmitate oxidation assay of M. leprae derived from fresh samples of armadillo liver (N = 17), spleen (N = 16), lymph node (N = 11), leproma nodule (N = 5) and nude mouse foot pads (N = 63). The relative activity and viability of M. leprae vary widely between different hosts and tissues.

 

The oxidative activity observed among nude mouse-derived bacilli was significantly higher than that from armadillos. However, it too showed considerable variability. The nude mouse foot pads we harvested were mostly of medium size, averaging 0.82 g, but data was available on foot pads ranging in size from 4.1 g to 0.1 g. We compared the RR results from these different harvests with the duration of each infec­tion in the mouse and its bacillary yield. As shown in Figure 5, the highest metabolic activities were associated with shorter intervals of infection that yielded low-to-moderate numbers of M. leprae. Bacilli from very large, especially ulcerated foot pads, showed the lowest overall activity. The three-dimensional figure depicts the association of increasing time and bacillary number with declining radiorespirometric activity. No data were available for infections of less than 140 days' duration, and the relationship of metabolic activity or viability with very short-term infections in nude mice is not described. However, with in­creasing duration of infection or bacterial number the viability of M. leprae obtained from nude mouse foot pads declines.

 

Fig. 5. The three-dimensional relationship between the bacterial number (AFB/gram) and duration of infec­tion (days postinfection) with the relative viability of M. leprae harvested from nude mouse foot pads (N = 63) as expressed by their metabolic activity in oxidation of 14C-palmitate in axenic culture. The declination in metabolic activity describes decreasing M. leprae viability in large nude mouse foot pads in late-stage infections.

 

DISCUSSION

These data indicate that the oxidation of 14C-palmitate by M. leprae in axenic culture can be used effectively as a rapid index of viability. The assay detects very subtle differences in the metabolic activity of M. leprae, which we have shown to be highly correlated with the growth that these bacilli achieve in conventional mouse foot pad (MFP) studies. Our findings also show that many of the storage conditions or incubation temperatures commonly used in the laboratory can rapidly, and markedly, affect the viability and metabolic capacity of freshly harvested M. leprae and can impact their potential use as a leprosy research resource. Most notably, a single freeze-thaw cycle or incubation at 37°C for just a few days has extremely deleterious effects. Modern laboratory studies addressing the molecular regulation of M. leprae, its secreted antigens or the differential host response to living and dead leprosy bacilli require the use of highly viable M. leprae. Palmitate oxidation assays can provide a convenient means to assess viability of M. leprae and can be used effectively to aid in the design of laboratory protocols which benefit survival and maintenance of meta­bolic activity by the organisms under study.

M. leprae does not multiply in axenic culture and propagation of bacilli in the MFP has been the only established means to evaluate viability. The procedure is timeconsuming and difficult. The time to first appearance of bacterial growth and its eventual plateau in the MFP is known to be a function of the proportion of viable organisms in the inoculum (9). The differential growth results seen here in the MFP for the various inocula tested are caused by differ­ences in the proportions of viable organ­isms in those samples. The relative number of viable M. leprae contained in different bacillary preparations vary markedly between donor hosts, the type of tissue, and characteristics of the individual infection, as well as the age of the suspension and conditions under which the bacilli are stored and used.

The high statistical correlation seen here between the RR values and MFP growth results of bacillary suspensions confirm the quantitative nature of palmitate oxidation for indexing M. leprae viability. These observations were made on a large number of samples processed over many different days. The degree of correlation expressed between MFP and RR probably was influenced by inter-run variations and other errors in the reproducibility of both assays, especially the MFP procedure. Palmitate oxidation actually may reflect more subtle differences in M. leprae viability than can be detected in the conventional MFP technique. Since the assay describes the relative viability of a suspension at a specific point in time, tests run at multiple time points would likely be useful for describing trends in viability and could likely increase sensitivity of the technique overall.

A number of methods have been employed as surrogates to MFP testing. M. leprae in skin biopsies become granular af­ter patients initiate drug therapy, and such morphologic changes have been used to estimate qualitative differences in the viability of M. leprae preparations based on their morphologic index (MI = percent of solidstaining normal organisms in a microscopic field) (11,20). Unfortunately, the MI is not re­producible between laboratories and tends to vary between individual runs, technicians and the staining reagents used. So called viable stains, such as FDA-EB, appear to have similar problems in reproducibility between laboratories and in association with MFP results (8,13,18). In addition, FDA-EB staining has not proven to be effective for in vitro drug susceptibility testing, and M. leprae may require in vivo incubation before viability differences can be noted with this procedure.

Other biochemical assays also may be useful for indexing M. leprae viability. Some studies have suggested that the relative concentrations of ATP among M. leprae is broadly associable with the likelihood that they may eventually manifest detectable growth in the MFP. However, the specific cellular concentration of ATP required has not been standardized, and the assay is not known to discern subtle differences in viability (3,6). Similar to palmitate, the uptake of hypoxanthine by M. leprae in axenic culture is reported to be useful for in vitro drug screening. However, its use as a quantitative index of viability was dis­counted early on because hypoxanthine in­corporation appeared to increase continu­ously even among relatively old M. leprae suspensions with declining viability (21). Palmitate oxidation assays show a broader range of activity than hypoxanthine and require considerably fewer bacilli (105-6 compared to 108). However, some culture sys­tems also can erroneously suggest that palmitate is metabolized continuously by aging M. leprae suspensions.

Oxidation of 14C-palmitate in axenic culture results in the evolution of radiolabeled CO 2 which can be detected in a number of different ways. When using the BACTEC 460 instrument, the respiration index for M. leprae in culture rises rapidly during the first 7 days to a plateau which is sustained for another 2 weeks. However, the actual viability of M. leprae in these aging cultures continues to decline. Therefore, the third week GI of BACTEC cultures correlates very poorly with the MFP results of M. leprae suspensions of this age. Interestingly though, when those stored M. leprae suspensions are subcultured they show a similar activity pattern in BACTEC; rising rapidly for 7 days to a plateau that is sustained for another 2 weeks. The peak GI achieved by those aged subcultures, how­ever, is markedly lower than the peak achieved by their fresh counterparts. The lower peak of the stored cultures is consistent with the lower viability of the aged organisms. With BACTEC, the peak GI is highly correlated with the suspension's MFP growth results. The second and third week plateau GIs appear to be just an artifact of the BACTEC 460 culture system. Buddemeyer-type (biphasic) culture sys­tems also show a 7-day peak in oxidative metabolism of 14C-palmitate by M. leprae. BACTEC and Buddemeyer RR results are highly correlated, both individually and with MFP. Therefore, the rate that a substrate is metabolized by M. leprae in axenic culture may be far more important in terms of viability than its apparent duration or total uptake.

Supplies of M. leprae used in experimental studies are usually propagated in either nude mice or armadillos. These hosts serve different roles in propagation and the leprosy bacilli become manifest in their tissues by different routes. Nude mouse-derived M. leprae tend to show higher RR activity than M. leprae obtained from armadillo tissues. Armadillos yield hundreds of times more M. leprae than can be derived from a nude mouse, and they are harvested only after a long incubation period in the latest stages of experimental leprosy in order to obtain those maximum bacillary yields. M. leprae infections of nude mice are more easily scheduled and controlled, and nude mice can be a good source for small quantities of highly viable M. leprae. However, the via­bility of nude mouse-derived M. leprae also varies markedly. M. leprae taken from nude mice in very late stages of experimental infection or derived from very large or ulcerated foot pads also show low viability. Therefore, the intended use of the bacilli really dictates what propagation method is most appropriate.

The quality of viable M. leprae as a re­search reagent is markedly influenced by storage conditions and incubation temperatures. We and a number of other investigators have observed that without regard to the storage conditions used, M. leprae suspensions lose nearly all of their detectable metabolic activity and a significant proportion of their total viability within 3 weeks of harvest from host tissues. A similar degree of loss is incurred within just hours if the bacilli are incubated at 37°C or frozen even a single time (2-10-12-16-17). These findings can bring into question the conclusions drawn in many scientific studies which used so-called "viable" M. leprae derived from frozen stocks, or incubated the bacilli in experiments at temperatures clearly detrimental to maintenance of M. leprae viability. The optimal temperature lor in vivo growth of M. leprae has been shown to be 33°C (14). Viable M. leprae must be used soon after their harvest from host tissue and are best when stored for only short periods of time at 4°C.

We currently share viable M. leprae with other laboratories by shipping freshly harvested bacillary suspensions on wet ice via express courier. The delivery range is limited and this is a cumbersome method of supply. The leprosy bacilli remain usable for only for a short period of time, and better preservation and handling methods are needed. Freezing M. leprae in whole tissues appears to have little benefit for the maintenance of their viability. Preliminary results in our laboratory suggest that program freezing bacilli in whole organs or in dimethyl sulfoxide (DMSO) is somewhat superior to snap freezing bacillary sus­pensions without protectants, but only mar­ginally so (unpublished observations). The potential utility for modern cryoprotectants or more innovative program freezing protocols that might improve maintenance of M. leprae viability during storage or shipment still merits additional investigation.

M. leprae have been isolated from dust, droplets, moist soil and dried sputum (7). Observations on the transient viability of M. leprae in the laboratory always seem to contrast with reports of recovering M. leprae from harsh conditions in the natural environment. However, survival of bacilli should not be confused with maintenance of high viability. Reports on recovery of lep­rosy-like bacilli from the environment are based largely on detecting small quantities of acid-fast organisms in the MFP—either on initial isolation or in successive blind passage. Relatively few M. leprae are needed to infect the MFP (11), and the number of viable organisms that might be required for growth to become detectable or to eventually reach plateau levels in the MFP can vary by tens of thousands (9). Since lepromatous hosts are known to shed large numbers of leprosy bacilli (7), survival and occasional recovery of fractional numbers of organisms from the natural environment is not really inconsistent with the highly transient viability of M. leprae described here.

Palmitate oxidation is a convenient, easy to perform assay. The substrate is used rapidly and reliable activity is detectable with as few as 10s 11M. leprae. The reagents are generally available commercially, but assay results will vary with different media components and culture conditions. The relative amount of 14CO2 derived from palmitate in axenic culture is influenced by the saturation of the label, competing carbon sources, temperature, the pH of the medium and the atmosphere of the culture chamber. Use of palmitate oxidation to index M. leprae viability in other laboratories will require standardization of reagents or revalidation of these test results. Nonetheless, oxidation of 14C-palmitate is an objective reliable means to rapidly assess the viability of M. leprae, and the technique likely will benefit a number of studies in the future.

Acknowledgment. We appreciate the technical assistance given by Roena Domingue, J. P. Pasqua. Balgil Randawa and Rita Sanchez during the period of this project. This work was supporled in part by funds from the National Institutes of Allergy and Infectious Disease, contract number Y02-AI-5015-01.

 

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1. Ph.D., Laboratory Research Branch, Hansen's Disease Programis at LSU-SVM. P.O. Box 25072. Baton Rouge, Louisiana 70894. U.S.A.
2. Ph.D., Laboratory Research Branch, H:ansen's Disease Programis at LSU-SVM. P.O. Box 25072. Baton Rouge, Louisiana 70894. U.S.A.

Reprint requests to Dr. Trumam at the above address or FAX 1-225-578-9856; e-mail: rtrumal@Isu.edu

Received for publication on 4 October 2000.
Accepted for publication in revise form on 17 January 2001.

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