INFECTION AND IMMUNITY, Nov. 1977, p. 459-466Copyright © 1977 American Society for Microbiology

Vol. 18, No. 2Printed in U.S.A.

Nocardia Infections in Congenitally Athymic (Nude) Mice andin Other Inbred Mouse StrainsP. I. FOLB,1* A. TIMME,' AND A. HOROWITZ'

Departments of Pharmacology' and Pathology,2 University of Cape Town Medical School, Cape Town,South Africa, and Departments ofMedicine and Pathology, Ch. Sheba Medical Center, Tel Hashomer and

Tel Aviv University Medical School, Tel Aviv, Israel"

Received for publication 20 June 1977

The mortality rate and histopathological features of Nocardia asteroides andNocardia brasiliensis infections in congenitally athymic (nude) mice of ICRand C3H/eB origins were quite different from what we found for Swiss whitemice and other inbred mouse strains (namely, C57/BL/6J, New Zealand Black,BALB/c, CBA/LAC, and C3H/eB). The immunocompetent littermates of thecongenitally athymic mice occupied an intermediate position between theirathymic siblings and Swiss white mice in terms of their responses to both theseorganisms. Macrophage ingestion and destruction of N. brasiliensis, as demon-strated by electron microscopy, was found to occur. The T-lymphocyte appearsto be an essential component in normal mouse resistance to infection by both N.asteroides and N. brasiliensis.

Nocardia infections in humans are becomingincreasingly common after transplantation sur-gery (1, 10) and in patients whose normal im-munity has been undermined by immunosup-pressive drug therapy (5, 11). The precise mech-anisms by which nocardiosis develops in thesesituations have not been defined, although thereis some evidence to suggest that disorders ofcell-mediated immunity may be of particularimportance in the pathogenesis of these infec-tions (6, 10, 11).

In an attempt to define more clearly the roleof the T-lymphocyte in normal host immunityto Nocardia infections, we have studied thenatural history, cumulative mortality, histopath-ological features, and electron microscopic (EM)features of experimental Nocardia asteroidesand Nocardia brasiliensis infections in congen-itally athymic (nude) mice. Comparison wasmade with the model we have established inSwiss white mice (6) and with other inbredmouse strains, namely, C57/BL/6J, New Zea-land Black (NZB), BALB/c, CBA/LAC, andC3H/eB.The EM studies were directed towards deter-

mining whether or not macrophage function isnormal in thymus- and T-lymphocyte-depletednude mice infected with Nocardia.Our studies of nocardia infections in Swiss

white mice (6) have shown that N. asteroidesand N. brasiliensis produce distinctive lesions,which are quite different in their morphologicalappearances. This was in accordance with theearlier findings of Uesaka et al. (12), from whom

we took the terms "asteroides lesion" and "bras-iliensis lesion." An acute suppurative abscesscharacterizes the lesions of N. asteroides. The"brasiliensis lesion" is a granuloma, in which astriking feature is the presence of large numbersof foam-laden macrophages. EM studies havedemonstrated that these macrophages containwithin their cytoplasm organisms in varyingstages of degeneration.

MATERIALS AND METHODSNocardia organisms. The cultures of N. aster-

oides and N. brasiliensis were isolated, respectively,from the sputum and from a subcutaneous abscess ofpatients suffering from clinical illnesses. The actino-mycetes were identified and characterized by estab-lished criteria (7, 9). Confirmation of the N. asteroidesisolate was obtained from Ruth Gordon, Rutgers Uni-versity, New Brunswick, N.J. The N. brasiliensis cul-ture was confirmed by L. Kaufman, Center for DiseaseControl, Atlanta, Ga.Experimental mice. Swiss white mice weighing

between 25 and 30 g were used. Congenitally athymic(nude) mice of both ICR and C3H/eB origins, andtheir normal littermates, and ICR, C57/BL/6J, NZB,BALB/c, CBA/LAC, and C3H/eB mice, weighing be-tween 25 and 30 g, were obtained from the VeterinaryDepartment, Weizmann Institute, Rehovot, Israel.

Inoculations. The mice were inoculated intraperi-toneally in the manner we described previously (6).

Pathological studies. Intraperitoneal N. aster-oides infections were produced in the following mousestrains: Swiss white, ICR, ICR nude (nu/nu) and theirnormal littermates (nu/+), C3H/eB nude (nu/nu) andtheir normal littermates (nu/+), NZB, BALB/c,C57/BL/6J, and CBA/LAC. In each case 10 mice

459

on March 20, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

460 FOLB, TIMME, AND HOROWITZ

were studied. With the exception of the nude mice,four mice were examined from each group at intervalsof 11 and 21 days, and two mice were examined at 60days after inoculation. The mice were anesthetizedand killed with ethyl ether. In the two groups of nudemice, five were examined from each group at 11 days,and the remainder of those surviving were examinedat 21 days. A full postmortem examination in eachcase, including those mice that died during the course

of the experiment, was performed. Portions of theorgans, including the brain, were taken for histologicalexamination. These experiments were repeated in an

identical manner with inoculations of N. brasiliensis.Routine staining of the histological sections and

postmortem bacteriological examinations were per-

formed as we described previously (6).EM studies. Selected lesions produced by N. bras-

iliensis in ICR nude mice and their normal littermateswere removed for EM study. The EM studies were

confined to lesions at 11 days after inoculation. Themethod used for the fixation, preparation, and cuttingof the specimens was the same as that describedpreviously (6). A Siemens 1A electron microscope was

used.Cumulative mortality studies. In a separate ex-

periment, 10 mice from each of the following groups

were inoculated intraperitoneally with 10 mg (wetweight) of N. asteroides cells: Swiss white, ICR, ICRnu/nu and their normal littermates, C:3H/eB, C3H/eBnu/nu and their normal littermates, NZB, BALB/c,C57/BL/6J, and CBA/LAC. In the case of the Swisswhite and the nude mice and their littermates, thisexperiment was repeated once. A control group of 10mice was injected intraperitoneally with 0.5 ml ofsterile saline. (This dose of inoculum was chosen sincehigher doses of the organism had produced a uniformlyhigh mortality rate.)The experiment was repeated in an identical man-

ner with inoculations of N. brasiliensis.

RESULTSThere were no differences in cumulative mor-

tality rate or in histopathological features pro-duced by N. asteroides and N. brasiliensis, re-

spectively, in Swiss white mice and in the ICR,C3H/eB, NZB, BALB/c, C57/BL/6J, andCBA/LAC mice. (This does not refer to thenude varieties of the ICR and C3H/eB mice;Table 1.) The characteristic asteroides and bras-iliensis lesions, which were demonstrated to oc-

cur consistently in the Swiss white mice (6),were found with the same consistency in eachof these genetic mouse strains.

In the groups of ICR and C3H/eB nude mice,the cumulative mortality produced by both N.asteroides and N. brasiliensis infections differedconsiderably from that which had been notedfor the Swiss white mice and the other inbredstrains referred to above. Within 28 days ofinoculation both N. asteroides and N. brasilien-sis had produced 100% mortality in the nudemice. The cumulative mortality in the nude

mouse groups over the first 28-day period dif-fered from that in all the other mouse groupsat a significance level ofP < 0.001. There was nodifference in cumulative mortality in the normallittermates (nu/+) of the ICR and C3H/eB nudemice after inoculations of N. asteroides and N.brasiliensis or in the Swiss white mice and theother inbred strains (Table 1).

N. asteroides produced a fulminating systemicdisease in the ICR and C3H/eB strains of nudemice. There was no localization of the organismat the original site of inoculation in the perito-neum. A disseminated pyemic process involvingvirtually every organ of the body was well estab-lished by day 11 after inoculation. Invariablythe organs involved with abscess formation in-cluded skin and subcutaneous tissues, lymphnodes, lungs, liver, skeletal muscle, and retroor-bital area. Remarkably, no case of metastaticspread to the brain or meninges was noted. Thebrain appeared to be protected from the dissem-inated effects of the infection. The microscopicfeatures of the microabscesses produced did notdiffer appreciably from those found in the Swisswhite mouse (6).The morbid anatomical features of the infec-

tion resulting from inoculation of N. brasiliensisin the nude mice were identical to those pro-duced by N. asteroides, described above. Thecharacteristic brasiliensis lesion that was notedon histological examination in Swiss white mice(6) did not develop in nude mice. There wereoccasional exceptions to this general statement,when a typical brasiliensis lesion or an identifi-able forme fruste was noted in the nude mice.Generally, multiple pyemic lesions characterizedby necrosis and an acute purulent reaction werefound. Hyaloid bodies and the marked lympho-cytic reaction, which were characteristic of the"brasiliensis lesion," were not apparent. Numer-ous organisms were identified in the center ofthe necrotic area. There was early capsule for-mation at the periphery of the lesions by 11days, and this was further developed by 21 days.In common with the brasiliensis lesion in theSwiss white mouse, numerous large macro-phages were noted at the periphery of the lesions(6).The electron microscopic studies confirmed

that the N. brasiliensis organisms were bothfree-lying and ingested by the macrophages. Theultrastructural features of the former were sim-ilar to those described for N. asteroides, exceptthat the cell wall was not as thick as has beenillustrated for N. asteroides (2) (Fig. 1). Oncethe organisms had been phagocytosed they be-came surrounded by the single membrane of thephagosome (Fig. 2). Dense granular or fibrillarmaterial partly filled the residual space of the

INFECT. IMMtTN.

on March 20, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

NOCARDIA INFECTIONS IN NUDE MICE

TABLE 1. Cumulative mortality studies

ICR-nu/+ (littermates)

No. of mice inoc-ulated

202010

1010

202010

202010

Cumulative mortalitv' at:

11 davs 21 davs 28 davs

0 0 02 2 20 0 0

0 0 00 0 0

4 11 208 13 200 0 0

4 6 61 2 20 0 0

C:,H/eB N. asteroidesN. brasiliensis

CGH/eB-nu/nu

C3H/eB-nu/+ (litter-mates)

NZB

BALB/c

N. asteroidesN. brasiliensisSaline

N. asteroidesN. brasiliensisSaline

N. asteroidesN. brasiliensis

N. asteroidesN. brasiliensis

N. asteroidesN. brasiliensis

N. asteroidesN. brasiliensis

C57/BL/6J

CBA/LAC

1010

202010

202010

1010

1010

1010

1010

0 0 10 0 0

9 18 2011 16 200 0 2

0 2 30 4 40 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

2 2 20 0 0

aCumulative mortality of various mouse strains after intraperitoneal inoculation of 10 mg (wet weight) ofN. asteroides or N. brasiliensis suspended in 0.5 ml of normal saline.

phagosome and adhered to the wall of the or-

ganism. Dense round or oval bodies, which were

presumed to be lysosomes, were commonly pres-ent in the vicinity of the organisms, but fusionof these structures with the phagosomes wasnot observed (Fig. 2 and 3). The earliest degen-erative changes noted in the organisms werevariable reduction in the number of lipid vacu-oles and polyphoshate granules (Fig. 2). Thesechanges became more pronounced, so that lipidvacuoles, dense granules, and ribosomes couldno longer be identified, and the cells containedonly thin strands of nuclear material and meso-somes (Fig. 3). The cell wall generally becameappreciably thickened. In the more advancedstages of disintegration, only osmophilic debrisor membranous fragments were found in theinterior of the cells, but the cell membrane re-

mained surprisingly intact in some cases (Fig.4). Ultimately, only dense cell walls could beidentified (Fig. 5). In some macrophages severalorganisms separated by septa were found.

In the littermates of the congenitally athymicmice, a picture intermediate between the ICRand C3H/3B mice and their nude counterpartswas found with respect to cumulative mortality,dissemination of the primary infection, and his-topathological changes. In the case of N. brasi-liensis, the typical brasiliensis lesion was foundin only 2 of 10 of both the ICR and C3H/3Blittermates. Dissemination of infection from theprimary peritoneal source of the infection wasfound in 5 of 10 of the ICR littermates and in 6of 10 of the C3H/eB littermates. However, foam-laden macrophages, which are a feature of thebrasiliensis lesion in Swiss white mice (6), were

Mouse strain Inoculation

Swiss white

ICR

ICR-nu/nu

N. asteroidesN. brasiliensisSaline

N. asteroidesN. brasiliensis

N. asteroidesN. brasiliensisSaline

N. asteroidesN. brasiliensisSaline

461VOL. 18, 1977

on March 20, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

462 FOLB, TIMME, AND HOROWITZ

FIG. 1. Electron micrograph of a free-lying N. brasiliensis, 11 days after inoculation. The cell wall (arrow)stains more densely than the trilaminar cell membrane immediately internal to it. x150,000. Nude (nu/nu)mouse.

invariably identified, even when other featuresof the typical granuloma were absent.The progressive degenerative changes in N.

brasiliensis organisms, which were demon-strated by EM to take place in the macrophagesof the littermates, proved to be similar to thosethat were noted in the nude mice. However,certain organisms showed a different change intheir structure, and these findings were confinedto the littermates only. These organisms weremore slender and lance shaped, and the cellcontent was more uniformly dense staining,without the normal lipid vacuoles and densegranules being obvious. The wall of the organismthickened (Fig. 6). The ultimate fate of theseorganisms could not be determined, but it ap-peared to be the same as that already shown inN. brasiliensis infection in Swiss white mice (6).

DISCUSSIONThese findings demonstrate that there is an

altered natural history and mortality produced

by both N. asteroides and N. brasiliensis incongenitally athymic (nude) mice, c:mparedwith Swiss white mice and the other inbredstrains examined. Both organisms invariablyproduced disseminated infection and a high mor-tality in nude mice. The loss of the ability ofnude mice to produce a granuloma in responseto N. brasiliensis suggests that the T-lympho-cyte has a central role in the development ofthe granuloma. The results obtained in theC57/BL/6J, NZB, ICR, BALB/c, CBA/LAC,and C3H/eB mice indicate that other geneticfactors do not influence the pathological picturethat we have described previously for Swisswhite mice (6).The littermates (nu/+) occupied an interme-

diate position between the nude (nu/nu) andother mice studied, in terms of mortality andhistological response to inocula of both N. aster-oides and N. brasiliensis. It is possible that thereduction in the number of 0-bearing lympho-cytes in littermates of nude mice (8) might ac-

FIG. 2. N. brasiliensis, 11 days after inoculation. Intact organisms are seen within phagosomes. There isgranular or fibrillar material adherent to the cell wall (arrow). x45,000. Nude (nu/nu) mouse.

FIG. 3. N. brasiliensis, 11 days after inoculation. Thin strands of nuclear material and a mesosome (m)are seen in this organism, but ribosomes and dense granules are absent. Lysosomal dense bodies are notedin the proximity. x45,000. Nude (nu/nu) mouse.

INFECT. IMMUN.

on March 20, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

FIG. 2.

FIG. 3.463

on March 20, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

FIG. 4.

FIG. 5.464

IltsA".

t,7

on March 20, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

NOCARDIA INFECTIONS IN NUDE MICE

WvWa,

FIG. 6. N. brasiliensis infection in the littermate (nu/+) of a nude mouse, 11 days after inoculation. Theorganism has assumed a lanceolate appearance, and no normal internal structure can be noted. The arrowindicates a thickened cell wall. x40,000.

count for this rather unexpected finding in whatnormally are regarded as negative controls fortheir abnormal siblings. It would appear thatthe nu/+ littermate has limited value as a con-trol in the study of experimental nocardiosis.

In the case of N. brasiliensis infection, theEM studies showed that the capacity of themacrophages of nude mice and their littermatesboth to phagocytose the organism and subse-quently to produce its degradation was compa-rable to that demonstrated in Swiss white mice.This finding is in contradistinction to what hasbeen described in experimental Listeria mono-cytogenes infection, where the presence of sen-sitized T-lymphocytes has been shown to di-rectly influence the macrophages in their capac-ity to ingest and kill the organism (4), althoughmacrophages of nude mice are capable of beingactivated during the course of L. monocytogenes

infection (3). Our findings, as do those of Cheersand Waller (3), have interesting implications interms of their demonstration of possible auton-omy of the macrophage from the T-lymphocytesystem in nude mice, and they further highlightthe caution required in interpreting the antibac-terial immune status of nude mice as hosts.The thickening that we have demonstrated

by EM to occur in the cell wall of N. brasiliensisafter phagocytosis by macrophages has beendescribed by Beaman (2). We were surprised tofind that the trilaminar pattern of the cell mem-brane remained clearly discernible in portionseven when the organisms had reached an ad-vanced stage of disintegration. This suggeststhat the cell wall is remarkably resistant tolysosomal attack. The nature of the condensedmaterial surrounding the organisms in the pha-gosomes is not clear. The ultrastructural findings

FIG. 4. N. brasiliensis in an advanced stage of degeneration, 11 days after inoculation. The trilaminarimage of the cell membrane is indicated by the small arrow, and fibrillar material adherent to the thickenedcell wall is indicated by the open arrows. X150,000. Nude (nu/nu) mouse.

FIG. 5. N. brasiliensis in the final stages of degradation, 11 days after inoculation. Only thickened cellwall can be identified. A septate appearance is noted. x45,000. Nude (nu/nu) mouse.

VOL. 18, 1977 465

on March 20, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

466 FOLB, TIMME, AND HOROWITZ

suggest that microfilaments may contribute tothe formation of this material. The significanceof the lanceolate transformation of some orga-nisms in the littermates is obscure.The characteristics of the end stage of the

organism of N. brasiliensis depicted in Fig. 6(littermate) differ from those of the nude mouseand are similar to those we have observed forSwiss white mice (6). In Swiss white mice andtheir littermates, we have noted two forms ofdegeneration of nocardia organisms. In nudemice the degenerating organisms appear to takeone form only. The significance of this observa-tion with regard to the pathogenesis or immunebasis of N. brasiliensis infection is not clear.

It is clear that much work remains to be donebefore we can understand basic mechanisms ofnormal host defense against Nocardia orga-nisms.

ACKNOWLEDGMENTThis work was supported by a grant awarded to P.I.F. by

the Chief Scientist, Ministry of Health, Israel.

LITERATURE CITED1. Bach, M. C., A. Sahyoun, J. L. Adler, R. M. Schles-

inger, J. Breman, P. Madras, F.-K. Pleng, and A.P. Monaco. 1973. Influence of rejection therapy onfungal and nocardial infections in renal-transplant re-cipients. Lancet i: 180-184.

2. Beaman, B. L. 1973. An ultrastructural analysis of nocar-

dia during experimental infections in mice. Infect. Im-mun. 8:828-840.

3. Cheers, C., and R. Waller. 1975. Activated macrophagesin congenitally athymic "nude" mice and in lethallyirradiated mice. J. Immunol. 115:844-847.

4. Emmerling, P., H. Finger, and J. Bockemuhl. 1975.Listeria monocytogenes infection in nude mice. Infect.Immun. 12:437-439.

5. Folb, P. I., G. Altmann, D. Merzbach, and E. Ipp.1976. Nocardiosis in Israel. Isr. J. Med. Sci. 12:150-153.

6. Folb, P. I., R. Jaffe, and G. Altmann. 1976. Nocardiaasteroides and Nocardia brasiliensis infections in mice.Infect. Immun. 13:1490-1496.

7. Georg, L. K., L. Ajello, C. McDurmont, and T. S.Hosty. 1961. The identification of Nocardia asteroidesand Nocardia brasiliensis. Am. Rev. Respir. Dis.84:337-347.

8. Gershwin, M. E., B. Merchant, M. C. Gelfand, J.Vickers, A. D. Steinberg, and C. T. Hansen. 1975.The natural history and immunopathology of outbredathymic (nude) mice. Clin. Immunol. Immunopathol.4:324-340.

9. Gordon, M. A. 1974. Aerobic pathogenic Actinomyceta-ceae, p. 175-188. In E. H. Lennette, E. H. Spaulding,and J. P. Truant (ed.), Manual of clinical microbiology,2nd ed. American Society for Microbiology, Washing-ton, D.C.

10. Krick, J. A., E. B. Stinson, and J. S. Remington.1975. Nocardia infection in heart transplant patients.Ann. Intern. Med. 82:18-26.

11. Palmer, D. L., R. L. Harvey, and J. K. Wheeler. 1974.Diagnostic and therapeutic considerations in Nocardiaasteroides infection. Medicine (Baltimore) 53:391-401.

12. Uesaka, I., K. Oiwa, K. Yasuhira, Y. Kobara, and N.M. McClung. 1971. Studies on the pathogenicity ofNocardia isolates for mice. Jpn. J. Exp. Med. 41:443-457.

INFECT. IMMUN.

on March 20, 2020 by guest

http://iai.asm.org/

Dow

nloaded from