APPLIED

AND ENVIRONMENTAL MICROBIOLOGY, July 1978, p. 144-159 0099-2240/78/0036-0144$02.00/0 Copyright © 1978 American Society for Microbiology

Vol. 36, No. 1 Printed in U.S.A.

Gastrointestinal Microecology of BALB/c Nude Mice JAMES F. BROWN AND EDWARD BALISH* Departments of Medical Microbiology and Surgery, University of Wisconsin Center for Health Sciences, Madison, Wisconsin 53706 Received for publication 16 January 1978

The aerobic, facultative, and anaerobic microorganisms cultivable from the stomachs, ilea, ceca, and colons of BALB/c athymic (nu/nu) mice (normal and wasting), thymus-implanted normal nude mice, and their heterozygous (nu/+) littermates were investigated. Ninety-one species representing 23 genera of bacteria and yeasts were isolated from the 27 mice. The wasting nude mice showed significantly lower numbers of lactobacilli in their stomach microbiota than did mice from the other three groups. The littermate animals appeared unique among the four groups in having corynebacteria as a major constituent of their stomach and ileal flora. The normal nude mice appeared to have a more diverse anaerobic stomach flora than their heterozygous littermates. These minor differences are discussed with respect to possible immunological, physiological, and environmental factors as their cause. Because the gastrointestinal microfloras of the mice from the four groups were not radically divergent from each other, it was concluded that loss of T-cell function does not dramatically alter the makeup of the cultivable gastrointestinal microflora in these mice. It has long been recognized that unprotected, congenitally athymic (nude) mice often succumb to bacterial infections. Indeed, the wasting syndrome observed in neonatally thymectomized and nude mice is not seen when these mice are born and raised in a germfree environment (25, 28). The lack of thymic function in nude mice could cause alterations in their microbial flora that enhance their susceptibility to bacterial infections. Recently, Tamura et al. (36) have shown that a probable etiological agent of wasting syndrome in nude mice is a mouse hepatitis virus. It seems probable that viral hepatitis is a factor predisposing unprotected nude mice to bacterial infections. The present study was undertaken to define, compare, and contrast the aerobic and anaerobic bacterial gastrointestinal microflora of nonwasting nude mice, wasting nude mice, thymus-implanted nude mice, and their heterozygous littermates. We also wanted to determine whether the gastrointestinal microflora is significantly affected by the absence of T-cell function.

females, 5 13 weeks of age), 10 were thymus-implanted nude mice (8 males, 2 females, 9 19 weeks of age), and 6 were wasting nude (nu/nu) mice (1 male, 5 females, 9 13 weeks of age). Animals were fed an autoclaved Ralston Purina 5010c diet (Ralston Purina Co., St. Louis, Mo.), watered with acidified, chlorinated water (23), and randomly housed as well as randomly chosen for the bacterial enumeration studies. All animals were taken from a conventional breeding colony derived from specific pathogen free stock. Reconstitution of nude mice. Mice were reconstituted with thymus glands from 1- to 3-day-old BALB/c donors. The thymus tissue was surgically implanted either in the axillary area or just under the renal capsule. Tests to assess the restoration of T-cell function in the implanted mice (e.g., the presence of an antibody response to sheep erythrocytes or rabbit serum) were carried out as described previously (30). Bacterial flora enumeration was done a minimum of 3 weeks after thymus implantation. Thus, the thymicimplant animals were used as an effective experimental control for possible non-thymus-related effects on the gut flora of nude mice. Wasting. Athymic animals were considered to be wasting when they appeared to either lose weight rapidly, become less mobile, exhibit diarrhea, or become distinctively hunchbacked. Sacrifice of animals and isolation of microorganisms. Mice were weighed, etherized, sexed, and placed in an anaerobic glove box (Coy Manufacturing, Ann Arbor, Mich.) via a rapid entry port. Tissues were chosen as follows: stomach (keratinized portion); ileum (a portion 6 to 8 cm proximal to the cecum); cecum (a central portion); and colon (a portion 1 to 2 cm distal to the cecum). Each tissue sample, with its

MATERIALS AND METHODS

Animals. Congenitally athymic (nude) mice and their phenotypically normal littermates were produced by mating homozygous (nu/nu) males to heterozygous (nu/+) females. All of these mice were backcrossed into the BALB/c strain. Of the 27 mice cultured, 6 were nonwasting nude (nu/nu) mice (4 males, 2 females, 3 13 weeks of age), 5 were heterozygous (+/nu) littermates (2 males, 3

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intraluminal contents, was collected via aseptic technique and placed in 10 ml of prereduced transport medium (2). The tissues were ground to a homogeneous suspension in the transport medium with a highspeed Waring blender (Scientific Products Co., Evanston, Ill.). The homogenate was diluted serially in 10fold dilutions (with transport medium as diluent) to a final dilution of 1:108. A volume of 0.1 ml of each dilution was plated on A II agar (1). After removal of the dilution tubes from the glove box, 0.1 ml of each dilution was plated onto sheep blood agar (Grand Island Biological Co., Madison, Wis.), brain heart infusion agar (Difco Laboratories), eosin-methylene blue agar (Difco), Sabouraud dextrose agar (Difco), and phenylethyl alcohol or Columbia CNA agar (Baltimore Biological Laboratory [BBL]). The A II agar plates were incubated in the anaerobic glove box by using a 37°C Plexiglas incubator (4). The aerobic plates were incubated at 37°C in air. The Sabouraud agar plates were incubated at 25°C. All plates were checked for growth at 24 and 48 h, and colony counts were done on appropriate dilutions at 24 h for aerobic plates and at 48 h for anaerobic plates. Sabouraud agar plates were also checked for growth at 7 and 14 days. Tissue dry weight was determined by placing a 3to 5-ml measured portion of undiluted tissue homogenate on an aluminum drying dish and drying overnight in an oven at 70°C. Counts were expressed as the number of viable microorganisms per gram (dry weight) of tissue and contents. The counts were log10 transformed before the statistical analyses. Direct microscopic counts on the undiluted homogenate done at various times during the experiment showed an 86 to 100% recovery in culture of the total microscopic number. The criterion for picking colonies from both the aerobic and anaerobic plates was to pick as many different colonial morphological types as could be discerned on a dilution plate containing at least 30 but no more than 200 colonies. The counts expressing the number of organisms of a particular species per gram (dry weight) of tissue and contents were arrived at by counting all of those colonies that were morphologically indistinguishable from the colony of that type picked for identification as to species. Identification-anaerobes. Colonies picked from A II agar dilution plates were streaked back onto A II agar to insure purity. All bacteria isolated in this manner were then streaked to A II agar plates and sheep blood agar plates. The A II agar plates were incubated under aerobic conditions at 37°C, and the sheep blood agar plates were incubated under 10% CO2 at 37°C. All strains producing growth either in CO2 on sheep blood agar or in air on A II agar were identified by the procedures outlined below for facultative and aerobic isolates. All strict anaerobes were identified in the following manner: each isolate was inoculated into PYG broth (for gas chromatographic analysis of volatile and nonvolatile fatty acids and Gram staining) and litmus milk medium and subjected to a battery of tests selected from the Minitek system (BBL) (35). Gas chromatography was carried out on each anaerobic isolate by using extraction procedures, equipment, and recommended operating conditions

145

outlined elsewhere (17). Anaerobic isolates were identified by using the information in Bergey's Manual (5) and the VPI Anaerobe Laboratory Manual (17). Bacteroides fragilis subspecies were identified as such but are listed in the tables as species of the genus Bacteroides, following the recommendations of Cato and Johnson (6). and facultative Identification-aerobes aerobes. All aerobic and facultatively aerobic microorganisms were identified by using information and procedures available in the Manual of Clinical Microbiology (20), Bergey's Manual of Determinative Bacteriology (5), and Diagnostic Microbiology (3). Statistical analyses. A one-way analysis of variance procedure (11) was used to test for significant differences between the four groups of animals with respect to numbers of Lactobacillus sp. found in each of the specific gut areas. Contrasts between the animal treatment groups were made by using Fisher's leastsignificant-difference rule (21). A chi-square test (34) was used to look for significant differences in frequency of culture (number of animals positive per total number of animals sampled) of various genera (and groups) of bacteria when comparing mouse groups. The nonparametric Mann-Whitney test (11) was used to assess significant differences in species diversity between animal treatment groups within gut tract areas and to determine significant differences in species diversity between various gut tract areas irrespective of animal treatment group.

RESULTS Overall, there were 91 identifiable species of bacteria cultured from the 27 mice (Table 1). Several unidentified isolates (Table 2) were also cultured; these isolates could not be identified as to species by the bacteriological techniques and methods of identification outlined above. Table 1 lists the 23 genera and the number of species isolated from the 27 mice. The greatest species diversity occurred in the lactobacilli (19 species) and bacteroides (18 species). For purposes of statistical analysis and discussion, the quantitative data on the microbial flora were combined into the following 13 groups: streptococci, gram-negative facultative rods, nonfermenting gram-negative aerobic rods, aerobic gram-positive rods, yeasts, lactobacilli, staphylococci, bacteroides, bifidobacteria, clostridia, eubacteria, fusobacteria, and anaerobic gram-positive cocci. Table 3 lists the gut area sampled, the various genera cultured from the four mouse groups, the number of species within each genus cultured, the log1o range of the species representing each genus, and the frequency of occurrence of each genus in the animals of each mouse group. Table 3 shows that the most frequently occurring genera in the stomachs of the four mouse

treatment groups were Lactobacillus (A), Strep-

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TABLE 1. Genera isolated from the gastrointestinal tracts of the nude, wasting nude, thymus-implanted nude, and heterozygous littermate mice Genus'

No. of species cultured

19 Lactobacillus 18 Bacteroides Clostridium ......... ............... 9 8 ......... Bifidobacterium ........... Eubacterium 5.................5 4 Fusobacterium Streptococcus ........................ 4 4 Corynebacterium 2 ................. Staphylococcus ... 2 ........... Peptococcus ............ ............ 2 Peptostreptococcus ...... 2 Bacillus 1................... Candida

1 Torulopsis 1.................... Escherichia ............ ... ........ 1 1 Proteus ............................. Klebsiella ....... _................. 1 1 ..... Enterobacter ............... Pasteurella 1................1 Moraxella 1...............1 1 ......... Flavobacterium ............ 1 A cinetobacter 1 Pseudomonas a In the genera Corynebacterium, Bacillus, Torulopsis, and Streptococcus, the microorganisms were sometimes grouped rather than identified as to species in every case (i.e., Corynebacterium sp. [a] and Corynebacterium sp. [y], Bacillus sp., Torulopsis sp., and group D streptococci).

tococcus (G), and Bacteroides (B). The littermate group of mice, however, had a high occurrence of Corynebacterium spp. (V). Four of five littermate mice were culture positive for one or more species of the genus Corynebacterium. In contrast, the thymus-implant group had a significantly lower frequency of occurrence (1/10) of corynebacteria than the littermate mice (X2 = 4.54, P < 0.05). Microorganisms of the genus Corynebacterium were not cultured from the stomachs of the wasting nude mice, but they were cultured from the stomachs of two of six normal nude mice. A chi-square test comparing the frequency of occurrence of Corynebacterium spp. in normal nude versus littermate mice was not significant (X2 = 0.89). Aside from the differences noted above, the four mouse treatment groups had basically the same genera in their stomach flora, with minor (statistically nonsignificant) differences occurring with several miscellaneous genera (Table 3). The most diverse flora occurred in the stomachs of normal nude mice, where 13 genera and 20 species were represented. In comparison the littermate mice had six genera and 10 species in their stomachs (Table 3). The genera comprising the ileal flora of the

APPL. ENVIRON. MICROBIOL.

four mouse groups were much the same as the genera found in the stomachs (Table 3). Again, however, Corynebacterium spp. were seemingly more frequent in the littermate animals (5/5). The chi-square test comparing the frequency of occurrence of Corynebacterium spp. in the ilea of the littermate versus normal nude mouse group, however, was not significant (X2 = 2.75), but was significant (X2 = 7.81, P < 0.01) when the littermates were compared to the thymic implant group. Members of the genus Corynebacterium were not cultured from the ilea of any of six wasting nude mice. The most frequently occurring genera isolated from the ceca of all mouse groups were Lactobacillus (A), Bacteroides (B), and Streptococcus (G). There were no statistically significant differences (by the chi-square test) when intermouse-group comparisons were made on the frequencies of occurrences of the various genera isolated from the cecal contents and tissues. The most frequently occurring genera isolated from the colons of all the mice, irrespective of the mouse group, were Lactobacillus (A), Bacteroides (B), and Streptococcus (G) There were no statistically significant differences (by the chi-square test) when inter-mouse-group comparisons were made on the frequencies of occurrences of any of the genera isolated from the colonic tissue and contents. Tables 4 through 8 show the various species of aerobic, anaerobic, and facultative bacteria cultured from the gastrointestinal tracts of the four groups of mice. The quantitative data in Tables 4 through 8 are arranged to show the species of bacteria that were most numerous in the specimens. The highest logio values observed and the number of animals positive for that species are indicated in parentheses. For the purposes of presentation, all species of lactobacilli are included with the aerobic and facultative microorganisms, even though two were isolated and identified as strict anaerobes (i.e., Lactobacillus catenaforme and L. minutus). Table 4 clearly shows that the most numerous aerobic and facultative bacteria in the stomachs of all groups of animals were the lactobacilli, corynebacteria, and Streptococcus spp. Group D streptococci (most often identified as Streptococcus faecalis) were found consistently in the stomachs of animals from all mouse groups within the 5- to 6-log range. Much less prevalent in the stomachs were the facultatively anaerobic, fermenting gram-negative rods. Figure 1 shows the logio counts of Lactobacillus spp. found in the stomachs of the four animal groups and demonstrates that the wasting nude mice had significantly lower numbers of Lactobacillus spp. in their stomachs than any one of the other

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TABLE 3. Frequency of occurrence, log10 range, and species diversity of genera isolated from BALB/c thymus-implant, wasting nude, heterozygous littermate, and normal nude mice Area

Stomach

Mouse treatment group Thymus implant

Wasting nude

Littermate

Normal nude

Ileum

Thymus implant

A G B C U L, M, Rb V A G D B, E, U V A B, G, M W G A B H, J, M, P, V C, N, 0, T, W

5.0-8.0 6.0-7.5 4.3-10.5 4.5-4.7 4.0-6 ob 8.0 4.5-7.5 4.3-7.5 5.5-11.8

1, 1, 1 1 3 4 2 1, 2, 1, 1, 1 1, 1, 1, 1, 1

A G

5 3 1 1, 1, 1, 1, 2 3, 1, 1, 1 6 2 2 3, 1 1, 1, 1 3 2 5 1 1, 1, 1

D, M, S, T, W B, K, L, V A G D, I B, J, M G

A M

B, H, W A G p J, M, V C, N, 0, T

three mouse groups. Pseudomonas aeruginosa was cultured from the stomachs of 4 of 10 thymus-implant animals and 1 out 6 wasting nude animals and was not cultured from 6 normal nude and 5 littermate mice. The inconsistent results observed with P. aeruginosa may be due to animal-to-animal variation or the inherent sampling error. The frequencies of occurrences of P. aeruginosa between the two positive groups of mice (4/10 versus 1/6), however, were not significantly different by a chi-square test. It is interesting to note, however, that the two mouse groups that contained animals which were undergoing, or had undergone, excessive stress, harbored P.

5.3-9.5

2 4 2 1

4

V

Normal nude

7

sampled 9/10

1 6 2 2 1, 1, 1 2

E

Littermate

No. of animals positive/total no.

1, 1, 1b

C

Wasting nude

Log1o range

of speces

Genera

5 2 1 2, 1,2 1, 1, 1, 1

3.7-7.3 6.3-8.9 6.9-9.4 4.3-8.6 6.3 4.6-7.3 6.0-9.0 4.6-7.7 4.3-7.5 3.0-7.9 5.3-7.6 5.3-6.8 4.3-6.9 3.6-6.9 3.9-6.8 4.6-7.0 3.9-5.3 4.5-7.9 4.0-6.8 3.9-7.6 4.3-7.6 4.8-7.7 4.5-7.8 3.6-5.3 2.5-7.8 3.8-7.0 3.9-6.0 3.8-5.3 4.3-8.3 3.5-6.0

6/10 5/10 5/10 4/10 2/10 1/10 5/6 5/6 5/6 5/6

4/5 3/5 2/5 1/5 5/6

4/6 3/6 2/6 1/6 7/10 6/10 3/10 2/10 1/10 5/6 3/6 3/6 2/6 1/6

5/5 5/5 3/5 2/5 1/5 5/6 5/6 3/6 2/6 1/6

aeruginosa. The relatively unstressed normal (nonwasting) nude mice and the unstressed littermate mice showed no evidence of P. aeruginosa.

Although anaerobes were cultured from the stomachs of all the mice (Table 4), the overall diversity of anaerobic species there was not as great as the cecal or colonic anaerobic diversity (Fig. 2). The bifidobacteria reached as high as 11 logs in the stomach of one wasting nude animal. Two of six wasting animals were culture positive for stomach bifidobacteria in contrast to 0/21 animals from the other three mouse groups. There were no statistically significant differences (by

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149

TABLE 3-Continued Area of 8am-

pie Cecum

Mouse treatment group

Generaa

No. of species

cultured

Log1o range

Thymus implant

A G B, D, E, F, U L, M, T C B G A C D E, F, M G A B E F, H, M, N J, W A B G D M C, F, 0, P, V H, J, N, S

5 3 7, 2, 2, 2, 1 1, 1, 2

7.3-9.2 5.0-7.3 4.9-9.0 3.0-6.0 7.5 6.9-9.3 6.2-7.0 6.0-9.0 8.8-8.9 5.0-6.0 6.7-8.9 6.2-8.5 4.7-9.3 7.0-9.5 9.0-9.5 3.8-8.8 8.2-8.4 7.0-9.0 4.8-9.0 4.5-7.6 7.9-8.7 4.3-7.3 3.9-8.8 4.0-7.9

Wasting nude

Littermate

Normal nude

Colon

Thymus implant

A G B U

1

7 3 3 2 3 2,2, 1 3 7 4 2 1, 2, 1, 1 1, 1 3

6 2 2 1

2, 1, 1, 1, 1 1, 1, 1, 1

No. of animals

positive/total no. sampled

8/10 6/10 4/10

2/10 1/10 5/6 5/6 4/6 4/6 3/6 2/6

5/5 4/5 4/5 3/5 2/5 1/5 5/6 4/6 4/6 3/6 3/6 2/6 1/6

6 2 4 1 1, 2, 1, 1, 2 2 5 5 1 3, 1, 1, 1 3 3 3

6.0-8.8 8/8 5.9-7.0 8/8 7.7-9.0 4/8 4.0-4.7 4/8 D, F, 1, S, T 3.0-9.5 2/8 G Wasting nude 5.5-8.6 5/5 A 4.5-8.4 3/5 B 3.5-7.7 3/5 M 4.5-6.3 2/5 C, D, E, I 5.7-8.9 1/5 Litternate A 4.4-8.3 3/3 B 5.5-8.6 3/3 G 4.9-7.9 3/3 M 1 4.3-5.5 2/3 C, E, F, J, W 1, 1, 1, 1, 1 5.7-9.0 1/3 A Normal nude 4 6.6-8.0 4/4 B 4 6.5-8.7 4/4 G 1 4.9-7.0 3/4 1 p 3.5-5.0 3/4 F, H, M, N, 0, T, U 2.5-7.0 1/4 a Genus equivalents of the letter codes used in the table: A, Lactobacillus; B, Bacteroides; C, Clostridium; D, Bifidobacterium; E, Eubacterium; F, Fusobacterium; G, Streptococcus; H, Staphylococcus; I, Peptococcus; J, Peptostreptococcus; K, Candida; L, Torulopsis; M, Escherichia; N, Proteus; 0, Klebsiella; P, Enterobacter; Q, Pasteurella; R, Moraxella; S, Flavobacterium; T, Acinetobacter; U, Pseudomonas; V, Corynebacterium; and W, Bacillus. b In cases where more than one genus is listed on a line, the column labeled "no. of species cultured" gives the number of species of the listed genera, respectively, as they are listed, left to right. The log10 range in these cases is a composite for the genera listed, and the frequency of occurrence is the same for all genera listed on that line.

the one-way analysis of variance) among the four animal groups when comparing the logio counts of a particular group or genus of stomach bacteria.

Table 5 lists the bacteria cultured from the ilea of the four groups of mice. The composition of the ileal flora changes relative to the stomach flora within each animal group. The corynebac-

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TABLE 4. Microorganisms cultured form the stomachs of BALB/c nude mice, heterozygous littermates, wasting nudes, and thymus-implanted nudes Mouse group

Normal nude (6 mice, 3-13 weeks old)

Aerobic/facultative organism

Lactobacillus delbrueckii (9.0)a (1) b

L. fermentum (8.0) (2) Corynebacterium sp. (y)c (7.5) (2) Streptococcus mitis (7.3) (4) L. lactis (7.0) (2) Staphylococcus aureus (6.8) (1) Streptococcus faecalis (6.7) (3) Group D streptococci (6.0) (1) L. acidophilus (6.0) (1) Proteus mirabilis (5.5) (1) Escherichia coli (5.0) (3) Enterobacter (4.9) (2) Klebsiella pneumoniae (4.5) (1) Acinetobacter sp. (4.0) (1) Bacillus sp. (3.0) (1) Littermate (5 mice, 5-13 weeks old)

L. lactis (9.4) (1)

Thymus implanted (5 mice, 5-13 weeks old)

L. minutus (9.5) (2) L. brevis (8.2) (2) L. helveticus (8.2) (3) L. plantarum (8.2) (1) S. mitis (8.0) (3) L. lactis (7.7) (3) L. fermentum (7.4) (4) L. acidophilus (7.4) (6) Corynebacterium sp. (y) (7.0) (1) E. coli (6.0) (3) S. faecalis (5.9) (3) Moraxella sp. (5.5) (2) A. calcoaceticus subsp. lwoffi

Corynebacterium sp. (a) (8.9) (4) S. mitis (8.3) (2) L. fermentum (8.2) (2) L. acidophilus (8.0) (2) Lactobacillus sp. 1 (7.2) (1) Corynebacterium sp. (a) (7.0) (2) E. coli (6.3) (2) Bacillus sp. (6.3) (1) S. faecalis (6.2) (1)

Anaerobic organism

Clostridium sp. Ml (7.9) (1) Bacteroides distasonis (7.7) (1) Eubacterium sp. 1 (?)d (7.0) (1) B. ruminicola subsp. ruminicola (6.3) (2) Eubacterium aerofaciens (6.0) (1) Peptostreptococcus productus

(5.3) (1)

P. intermedius (5.0) (2)

B. succinogenes (8.3) (2) Eubacterium lentum (8.0) (1)

Clostridium glycolicum (10.5) (1) Clostridium sp. Ml (8.0) (1) B. furcosus (7.0) (1) B. distasonis (7.0) (1) B. coagulans (7.0) (1) B. capillosus (6.5) (1)

(5.0) (2) Pseudomonas aeruginosa (4.7) (4)

Torulopsis sp. (4.0) (2) L. acidophilus (7.5) (1) Bifidobacterium infantis (11.8) S. mitis (7.5) (3) (1) L. plantarum (6.7) (4) B. distasonis (7.3) (1) Lactobacillus sp. 1 (6.5) (1) B. longum subsp. longum (5.5) (1) L. casei subsp. casei (6.5) (2) Eubacterium sp. 1 (5.3) (1) L. fernentum (5.3) (2) L. catenaforme (5.0) (1) S. faecalis (4.3) (3) P. mirabilis (3.5) (1) P. aeruginosa (3.7) (1) a Log1o counts per gram (dry weight) of tissue and contents (highest value recorded in any of the animals of that group). b Number of animals in that group which were positive for the given isolate. c y, Gamma- (non-) hemolytic isolates; a, alpha- (green) hemolysis. d?, Isolate which was not definitely identified, but which is listed as such, by best available evidence.

Wasting nudes (6 mice, 9-13 weeks old)

GASTROINTESTINAL MICROECOLOGY OF NUDE MICE

VOL. 36, 1978

151

-9

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III

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FIG. 1. Log10 counts of Lactobacillus spp. found in the stomachs of normal (I), thymus-implanted (II), and wasting (III) nude mice and their heterozygous littermates (IV). The bar graphs show the mean ± the standard error of each group. Group 3 had significantly lower counts (P < 0.05) by a one-way analysis of variance than the other three groups. n, Counts of each of the individual species of Lactobacillus found within a particular animal group.

teria appeared to be more prevalent in the ilea than in the stomachs, and there was no longer a statistically significant difference between any of the mouse groups in the number of lactobacilli found there. Escherichia coli reached high levels (6 to 7 logs) in the ilea of some animals from all four groups. Other coliforms were conspicuously absent in the ilea of animals from the four mouse groups studied. Non-lactose-fermenting, gram-negative facultative rods were rarely isolated from any of the gut areas cultured. Anaerobes reached 10 to 11 logs in the stomachs of the wasting nudes and thymus implants (Table 4), but only 6 to 7 logs in the ilea from the four groups of animals. Bacteroides spp. and the clostridria were the most common strict anaerobes cultured from the ilea (Table 5). The littermates did not appear to have as many strict anaerobic species in their ilea as the three groups of nude mice. A one-way analysis of variance test revealed no significant differences in the ileal counts between any two of the animal

groups when they were tested separately for each of the 13 major bacterial groups or genera. Table 6 shows the aerobic and facultative bacteria cultured from the ceca. Lactobacilli and streptococci were the most numerous facultative bacteria isolated from the ceca of the four mouse groups (Table 6). E. coli was the major species of Enterobacteriaceae cultured from the ceca of the mice (Table 6). The diversity of facultative, gram-negative fermenting rods increased in the ceca as reflected by the occurrence of detectable levels of Proteus mirabilis, Enterobacter aerogenes, and Klebsiella pneumoniae. Acinetobacter spp. were isolated only from the ceca of thymic-im-

planted animals. Staphylococci occurred sporadically throughout all gut areas of all the animals cultured and were never found at greater than 107/g (dry weight) of tissue and contents in any gut area cultured. Staphylococcus aureus occurred in the ceca of 3 of the 27 animals cultured (Table 6).

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Co

Area of Gut Cultured (St=Stomach, Il=Ileum, Ce=Cecum, Co=Colon) FIG. 2. Diversity of anaerobes cultured from four areas of the gastrointestinal tract of BALB/c background mice. St and II were each significantly different from the Ce or Co, by the diversity scores in Fig. 2, via a Mann- Whitney ranked-scores test (P < 0.05). An St and Il contrast and Ce and Co contrast were not significant by the same test. Each point represents the datum from one animal.

The anaerobic species isolated from the ceca of the 27 animals are shown in Table 7. The colonic and cecal diversity of anaerobes (as measured by the number of different anaerobic species found per mouse per gut area) is significantly greater (P < 0.05, Mann-Whitney rankedscore test [33]) than either the stomach or ileal diversity (Fig. 2). Although there appeared to be fewer anaerobic species in the ceca of littermate mice, no significant differences were observed among the four mouse groups with respect to the diversity of anaerobes in any gut area (Mann-Whitney test). The bacteria responsible for the increased diversity of the anaerobes found in the colons and ceca of all the mice were primarily Bacteroides spp., although Peptostreptococcus spp., Eubacterium spp., Fusobacterium spp., and Clostridium spp. also contributed to the diversity observed. The population levels of anaerobes in the ceca of the animals reached 9 to 10 logs (Table 7). Quantitative differences in the cecal flora among the four mouse groups are not apparent statistically by the one-way analysis of variance. Table 8 shows the results of aerobic and anaerobic cultures of the colons of 20 mice. Lactobacilli and streptococci were the most numerous facultative bacteria isolated. The anaerobic colon flora is roughly similar to the anaerobic

cecal flora, both qualitatively and quantitatively. There appeared to be more species of bacteria in the cecal than in the colonic flora of all of the mice. There were, however, no significant quantitative differences (one-way analysis of variance) between the four groups of mice with respect to the major genera or groups of bacteria found in the colons. As noted previously, there was a difference between the colon (or cecum) and the stomach (or ileum) in the diversity of anaerobic species isolated (Fig. 2). There was, however, no statistical difference between any two gut areas in the diversity of the aerobic and facultative species cultured. Also, in all 27 animals cultured, the frequencies of occurrences of all 13 major groups or genera of bacteria changed only slightly from stomach to ileum to cecum to colon. For instance, in comparing the flora from the stomachs of wasting nude mice (Table 3) with their cecal flora (Tables 7 and 8), it is apparent that: (i) the frequencies of occurrences of the 13 major groups of bacteria are about the same; (ii) with the aerobic and facultative microorganisms, there is a quantitative shift from prevalence of Lactobacillus spp. in the stomachs to prevalence of streptococci and coliforms in the ceca; (iii) although there is an increase in diversity of species of anaerobic flora in the ceca as compared with the stomachs, the 13 major bacterial

TABLE 5. Aerobic, facultative, and anaerobic microorganisms cultured from the ilea of BALBIc nude mice, heterozygous littermates, wasting nudes, and thymus-implanted nudes Mouse group

Normal nude (6 mice, 3-13 weeks old)

Littermate (5 mice, 5-13 weeks old)

Aerobic/facultative organism

Corynebacterium sp. (y) a (8.3) b (2) Lactobacillus fermentum (7.0) (2) Corynebacterium sp. (a) (6.9) (1) Lactobacillus sp. 1 (6.9) (1) L. casei subsp. casei (6.5) (1) L. lactis (6.5) (3) Escherichia coli (6.3) (2) Streptococcus mitis (6.0) (2) L. plantarum (6.0) (2) S. faecalis (5.9) (3) Proteus mirabilis (5.8) (1) Enterobacter aerogenes (5.3) (1) Acinetobacter sp. (4.0) (1) Klebsiella pneumoniae (3.5) (1) Lactobacillus sp. 4 (7.8) (1) Corynebacterium sp. (y) (7.7) (2) S. mitis (7.6) (2) L. fermentum (7.5) (3) L. lactis (6.7) (1) L. acidophilus (6.2) (1) S. faecalis (5.6) (4) S. salivarius (5.5) (1) L. cellobiosus (4.5) (1) Corynebacterium sp. (a) (4.5) (1) E. coli (3.6) (2) Staphylococcus epidermidis (3.6) c

Anaerobic organism

Peptostreptococcus intermedius (6.0) (1) Clostridium sp. Ml (6.0) (1)

Eubacterium sp. 1 (7.6) (1)

(1)

Bacillus sp. (2.5) (1)

Thymus implanted (10 mice, 8-19 weeks old)

L. lactis (7.6) (2) L. plantarum (7.5) (3) L. casei subsp. rhamnosus (7.3) (2) Bacillus sp. (6.9) (2) L. acidophilus (6.8) (2) S. mitis (6.8) (4) Corynebacterium sp. (y) (6.5) (1) S. faecalis (6.3) (2) E. coli (6.0) (1) Corynebacterium xerosis (5.6) (2) S. salivarius (5.3) (1) L. delbrueckii (5.3) (2) C. pseudodiphtheriticum (5.3) (1) Pasteurella multocida (4.9) (1) Torulopsis sp. (4.0) (2) Flavobacterium sp. (3.9) (2) Candida parasilosis (3.9) (1) Acinetobacter calcoaceticus (3.6) (2)

Clostridium sp. Ml (8.8) (1) Bacteroides capillosus (7.0) (1) B. furcosus (6.8) (1)

Bifidobacterium longum subsp. longum (6.6) (1) B. pneumosintes (6.0) (1) P. intermedius (4.8) (1)

B. fragilis, other (7.0) (1) Streptococcus faecalis (7.9) (2) P. intermedius (6.3) (1) Escherichia coli (7.6) (1) B. infantis, other (5.0) (1) L. plantarum (7.0) (3) Eubacterium sp. 2 (5.3) (1) Streptococcus mitis (6.9) (4) B. longum subsp. longum (4.9) (1) L. salivarius (6.8) (1) B. adolescentis (4.0) (1) L. leischmannii (5.9) (1) P. constellatus (3.9) (1) L. lactis (5.6) (2) Eubacterium rectale (3.9) (1) L. acidophilus (5.5) (1) L. fermentum (4.6) (1) y, Gamma- (non-) hemolytic isolates; a, alpha- (green) hemolysis. ' Logio counts per gram (dry weight) of tissue and contents. 'Number of animals in that group which were positive for the given isolate. d Either one or both subspecies are included here (i.e., A. calcoaceticus subsp. anitratum or A. calcoaceticus subsp. lwoffi).

Wasting nude (6 mice, 9-13 weeks old)

a

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TABLE 6. Aerobic and facultative microorganisms cultured from the ceca of BALB/c nude mice, heterozygous littermates, wasting nude mice, and thymus-implanted nudes Mouse group

Aerobic/facultative organism

Normal nude (6 mice, 3-13 weeks old)

Lactobacillus plantarum (9.0) a (1) b L. salivarius subsp. salivarius (8.5) (1) Streptococcus mitis (7.6) (3) Escherichia coli (7.3) (3) S. faecalis (7.3) (2) L. lactis (7.0) (2) Enterobacter aerogenes (7.0) (2) Proteus mirabilis (6.3) (1) Staphylococcus aureus (6.0) (1) S. epidermidis (4.3) (1) Klebsiellapneumoniae (4.0) (2)

Littermate (5 mice, 5-13 weeks old)

Lactobacillus sp. 2 (9.3) (1) Lactobacillus sp. (?)' (9.0) (1) S. mitis (8.5) (4) Bacillus sp. (8.4) (1) Group D streptococci (8.0) (1) Corynebacterium sp. (y) d (7.9) (2) L. acidophilus (7.8) (2) Corynebacterium sp. (a) (7.5) (1) L. salivarius subsp. salivarius (7.0) (1) S. faecalis (6.2) (3) S. aureus (6.0) (1) Lactobacillus sp. 2 (?) (6.0) (1) E. coli (5.9) (2) L. helveticum (5.2) (1) S. epidermidis (5.0) (1) L. fermentum (4.7) (1)

Thymus implanted (10 mice, 8-19 weeks old)

L. minutus (9.2) (4) L. acidophilus (8.7) (3) L. luct's (8.4) (4) L. salivarius subsp. salivarius (8.0) (2) L. fermentum (7.5) (4) S. faecalis (7.3) (6) L. plantarum (7.3) (4) Corynebacterium sp. (y) (7.0) (1) S. mitis (6.5) (2) S. epidermidis (6.0) (1) E. coli (6.0) (2) Pseudomonas aeruginosa (5.8) (4) S. salivarius (5.0) (1) Corynebacterium pseudodiphtheriticum (5.0) (2) Torulopsis sp. (4.0) (2) Acinetobacter calcoaceticus subsp. lwoffi (3.0) (2) A. calcoaceticus subsp. anitratum (3.0) (2)

Wasting nude (6 mice, 9-13 weeks old)

L. plantarum (9.0) (2) S. salivarius (7.0) (2) E. coli (6.7) (2) L. fermentum (6.5) (1) S. mitis (6.4) (4) S. faecalis (6.2) (5) S. epidermidis (6.0) (1) L. lactis (6.0) (1) P. mirabilis (5.5) (1) E. aerogenes (5.0) (1) a Logio counts per gram (dry weight) of tissue and contents. 'Number of animals in that group which were positive for the given isolate. c ?, Isolate which was not definitely identified, but which is listed as such by best available evidence. d y, Gamma- (non-) hemolytic isolates; a, alpha- (green) hemolysis.

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TABLE 7. Anaerobic microorganisms isolated from the ceca of BALB/c nude mice, heterozygous, littermates, wasting nudes, and thymus-implanted nudes Mouse group

Anaerobic organism

Normal nude (6 mice, 3-13 weeks old)

Bacteroides nodosus (9.0) a (1) b Bacteroides sp. (?)c (9.0) (1) B. distasonis (9.0) (1) Clostridium paraputrificum (8.8) (1) Bifidobacterium breve (8.7) (2) Fusobacterium sp. 1 (8.5) (2) Clostridium sp. Ml (8.3) (1) B. infantis (8.0) (1) Fusobacterium sp. 2 (8.0) (1) Paptostreptococcus productus (7.9) (1) B. pneumosintes (7.9) (1) B. ruminicola subsp. brevis (7.5) (1) B. ruminicola subsp. ruminicola (4.8) (1)

Litternate (5 mice, 5-13 weeks old)

B. vulgatus (9.5) (2) Eubacterium sp. 1 (9.5) (1) Eubacterium sp. 2 (9.0) (2) Fusobacterium sp. 1 (8.9) (2) B. oralis (8.8) (1) Fusobacterium sp. 2 (8.8) (2) B. distasonis (8.7) (1) P. intermedius (8.2) (1) B. succinogenes (7.0) (1)

Thymus implanted (10 mice, 8-19 weeks old)

Eubacterium sp. 1 (9.0) (1) B. vulgatus (8.9) (1) B. distasonis (8.0) (1) B. ochraceus (8.0) (1) B. clostridiiformis subsp. clostridiiformis (8.0) (1) Bifidobacterium adolescentis (8.0) (1) Bifidobacterium sp. (?) (8.0) (1) P. productus (7.9) (1) Fusobacterium symbiosum (7.9) (1) Fusobacterium sp. 1 (7.8) (1) Eubacterium sp. (?) (7.5) (1) Clostridium difficile (7.5) (1) B. oralis (7.5) (1) B. pneumosintes (7.0) (1) B. furcosus(7.0) (1)

Wasting nude (6 mice, 9-13 weeks old)

B. ovatus (9.3) (2) B. distasonis (9.3) (2) C. sphenoides (8.9) (2) Fusobacterium mortiferum (8.9) (1) F. aquatile (8.8) (1) C. ramosom (8.8) (1) Eubacterium sp. (?) (8.6) (1) B. ruminicola subsp. ruminicola (8.5) (2) Bacteroides sp. (?) (8.1) (3) B. clostridiiformis subsp. clostridiiformis (7.0) (3) B. ruminicola (6.0) (2) B. breve (6.0) (1) B. longum subsp. longum (5.0) (1)

a Logio counts per gram (dry weight) of tissue and contents. b Number of animals in that group which were positive for the given isolate. c?, Isolate which was not definitely identified, but which is listed as such, by best available evidence.

represented roughly equally in both stomachs and ceca. Thus, in general, the major genera (or groups) are represented in equal fregroups are

quency in all areas of the gut tracts sampled (Table 3). The diversity (the number of species) of these individual genera expands as one pro-

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TABLE 8. Aerobic, facultative, and anaerobic microorganisms cultured from the colon of BALB/c nude mice, their heterozygous littermates, wasting nudes, and thymus-implanted nude mice Anaerobic organism Aerobic/facultative organism Mouse group Bacteroides sp. (?)M (8.7) (2) Normal nude Lactobacillus casei subsp. rhamnosus (8.0)a (2)b Bacteroides distasonis (8.6) (1) (4 mice, 3-13 weeks old) L. lactis (7.6) (2) B. corrodens (7.0) (2) L. plantarum (7.5) (1) Fusobacterium sp. (?) (7.0) (1) Streptococcus mitis (7.0) (3) L. casei subsp. casei (6.6) (1) Proteus mirabilis (6.6) (1) Enterobacter aerogenes (5.0) (3) Acinetobacter calcoaceticus subsp. lwoffii (4.0) (1) Klebsiella pneumoniae (4.0) (1) Pseudomonas aeruginosa (3.9)

(1)

Staphylococcus epidermidis (3.5)

(1) Escherichia coli (2.5) (1) Littermate (3 mice, 5-13 weeks old)

Lactobacillus sp. (?) (8.3) (1) L. acidophilus (8.0) (2) S. mitis (8.0) (2) Bacillus sp. (6.5) (1) E. coli (5.5) (2) S. salivarius (4.9) (1) S. faecalis (4.5) (1) L. fernentum (4.4) (1)

Eubacterium sp. (?) (9.0) (2) B. capillosus (8.6) (1) Clostridium sp. (8.6) (1) B. vulgatus (7.0) (1) Fusobacterium sp. (5.7) (1) B. succinogenes (5.5) (2)

Thymus implanted (8 mice, 13-15 weeks old)

L. acidophilus (8.8) (4) L. catenaforme (8.6) (2) L. fermentum (7.5) (4) L. leichmannii (7.5) (3) L. plantarum (7.3) (2) S. mitis (7.0) (4) L. lactis (6.0) (2) S. faecalis (6.0) (4) P. aeruginosa (4.7) (4) Flavobacterium sp. (4.3) (2) Acinetobacter calcoaceticus subsp. Iwoffii (3.5) (2) A. calcoaceticus subsp. anitratum (3.0) (2)

Peptococcus prevotii (9.5) (1) B. ruminicola subsp. ruminicola (9.0) (1) Fusobacterium bullosum (8.3) (1) B. coagulans (8.0) (1) Fusobacterium sp. (?) 1 (8.0) (1) Bacteroides sp. (?) (7.8) (1) Bifidobacterium bifidus (7.3) (1) B. vulgatus (7.0) (1)

Wasting nude (5 mice, 9-13 weeks old)

S. faecalis (8.6) (3) L. fermentum (8.4) (1) L. plantarum (8.3) (2) L. casei subsp. casei (8.0) (1) S. mitis (6.0) (3) E. coli (5.0) (2) L. buchneri (4.5) (1) L. lactis (4.3) (1)

Bacteroides sp. (?) (7.7) (2) B. ruminicola subsp. ruminicola (7.6) (1) B. pseudolongum (7.0) (1) Peptostreptococcus internedius (6.9) (1) B. hypermegas (6.8) (1) Clostridium sardiensis (6.7) (1) C. biejerneckii (6.5) (1) B. clostridiiformis subsp. clostridiiformis (6.0) (1) Eubacterium sp. (?) (6.0) (1) C. difficile (5.7) (1)

a Log1o counts per gram (dry weight) of tissue and contents. Number of animals in that group which were positive for the given isolate. Isolate which was not definitely identified, but which is listed as is, by best available evidence.

b

C?,

ceeds from stomach to cecum, perhaps reaching Despite rigorous attempts to isolate yeasts peak diversity in the cecum (Fig. 2, Tables 4 to from the animals, Torulopsis spp. and Candida 8). parapsilosis were isolated only sporadically and

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seen only in the stomachs, ilea, and ceca of the thymus-implant mice at very low dilutions. DISCUSSION The bacterial flora data presented here and that collected from several sources (26, 31) are generally in agreement with respect to the major groups and genera of microorganisms cultivable from the mouse gut. The major bacterial species cultured from the stomachs of nu/+ littermate mice were the lactobacilli. This observation agrees with the data presented by Tannock and Savage (37) for ARSHa/ICR mice. The ileal, cecal, and colonic data reported here for the littermate mice agree with the published data on the flora of several different strains of mice, even though minor strain-to-strain differences in the mouse gut flora have been reported (32). Reported levels of anaerobes, lactobacilli, streptococci, and coliforms in the fecal contents of various strains of mice (10, 13-15, 18, 19, 26, 27, 33, 37) agree reasonably well with values for colonic tissue and contents reported here for the heterozygous littermate mice. Considering the variety of techniques employed to culture and identify the various microorganisms, there appears to be a good deal of agreement on the major genera cultivable from the various areas of the murine gastrointestinal tract. There are still many other bacteria in the gastrointestinal tract of these mice that we were able to see by scanning electron microscopy (9), but unable to grow and identify (e.g., segmented filamentous microbes, spirals) with the media that we used in this study. Mitsuoka (26) and Savage (32) reviewed data on several different mammals and found that the rat and mouse fecal flora were very similar and consisted of high levels (8 to 10 logs) of Bacteroides spp., Bifidobacterium spp., Peptostreptococcus sp., Lactobacillus spp., and Corynebacterium spp., with lesser numbers (3 to 6 logs) of Streptococcus spp., Staphylococcus spp., Bacillus spp., Enterobacteriaceae, Veillonellae, and Clostridium spp. Mitsuoka also reported the presence of Spirillaceae in feces. Our data on the colonic flora of heterozygous (nu/+) littermate mice agree closely with those reported by Mitsuoka (26) and Savage (32) with some important differences: we found Clostridium spp. at higher numbers and we did not culture Bifidobacterium spp. or Spirillaceae from the colonic tissue and contents of the heterozygous littermate mice. Because Mitsuoka reported data on fecal flora, it may be that our data differ from his because of our technique of collecting tissue and contents from the animals immediately after exsanguination. The immunological status of nude mice has

157

been investigated in sufficient detail to allow several theoretical statements about their ability to respond to their normal flora. An outstanding feature of nude mice is, of course, the absence of functional T-cells. This immunodeficiency naturally predisposes nude mice to an altered response to infectious agents which require a cellmediated component to effect immunity or to alter the course of infection (12). The response of nude mice to Listeria infections, BCG infections, mycoplasma infections, and even parasitic infections in some cases (8) is one of relatively high initial resistance and then inability to clear the infection normally. The initial resistance factor in nude mice has been tentatively identified as the presence of activated macrophages (7, 24, 29), presumably turned on by components of the normal gastrointestinal flora, because germfree nude mice do not have activated macrophages (29). Our study, however, showed that coliforms (a most likely source of macrophage activation via endotoxin) were a minor (but consistent) component of the nude mouse gut flora. The presence of activated macrophages undoubtedly plays a role in intracellular opportunistic infections in nude mice. The effect, if any, of macrophages on bacteria at the flora-host interface of the gut mucosa is unknown. Although nude mice apparently have an arsenal of activated macrophages (29), the net effect is not to prevent (but perhaps to delay) the onset of bacterial infections. Because all of the functions of secretory antibodies have not been fully elucidated, a possible effect of secretory immunoglobuHin A (IgA) on the normal microbial flora must be considered. The expression of the systemic IgA response is T-cell dependent in mice (22), as evidenced by the depressed levels of IgA in the sera of nude mice. The local secretory IgA response in the gut of nude mice is depressed severely (18), indicating considerable T-dependency of that parameter. If secretory IgA plays a role in the qualitative or quantitative regulation of the gastrointestinal flora, then athymic mice should have a severely limited capacity to regulate their gastrointestinal tract flora. Our quantitative data on lactobacilli in the stomachs of wasting versus heterozygote nude mice reinforce the observations of Tannock and Savage (37) that stressed mice have lower counts of Lactobacillus spp. in their stomachs. Our wasting nude mice were naturally stressed, being unusually susceptible to changes in temperature and other environmental influences such as ease of access to water and food in their cages. Although the study by Tannock and Savage involved food and water deprivation, the stressful conditions resulting when athymic mice under-

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go "wasting" are probably very similar. In humans, emotional stress has been implicated as a factor contributing to alterations in the normal gut flora (16). In general, our data seem to indicate that there is very little qualitative or quantitative difference between the normal microbial flora in the ilea, ceca, and colons of athymic and thymusintact mice. The question of qualitative differences is perhaps the easiest to answer because gross shifts in genera or groups of bacteria would be easily discerned. The question of quantitative differences is much more difficult to answer because our experimental design and the resulting analyses were not tailored to suit this purpose fully. Therefore, in answering the question of whether the lack of a thymus significantly affects the normal microbial flora of the mouse, our data suggest that the best answer is that there is probably very little, if any, primary influence on the ileal, cecal, or colonic flora. Secondary influence would be taken here to mean a shift in flora secondary to some effect of the athymic condition, e.g., wasting due to viral hepatitis (36). Primary would then, of course, refer to the direct influence of T-cells (or lack of influence of T-cells) on the normal microbial flora at the host-microbe interface (including the lack of T-B-cell interactions in the athymic mice, for instance). The stomach flora of nude mice appeared to be different from the stomach flora of the heterozygous littermates. The nude mice had a more diverse microbial flora in their stomachs. The diversity was manifested in the greater numbers of both the genera and species present. There appeared to be a lack of some control mechanism in the nude mouse stomach that allowed for a more diverse microbial flora. Although this effect need not be immunological (e.g., it could be due to a deficiency of stomach acidity in the nudes), it could be an indicator that nude mice lack some flora control mechanism that is operative in the stomach of heterozygous mice. Whether or not the changes noted in the stomach flora (lactobacilli) of the wasting animals are due to primary or secondary influences of the athymic condition is not known for certain, because the experiment as designed does not control for these variables. It is our interpretation, however, that the depression in counts of lactobacilli seen in the stomachs of the wasting animals is the result of secondary influences; e.g., the athymic condition allowed wasting to occur (via bacterial, parasitic, or viral infections) which caused the shift in flora (via stress). Evidence for this hypothesis is drawn mainly from the study by Tannock and Savage (37) on

APPL. ENVIRON. MICROBIOL.

stressed mice and Holdeman et al. on humans (16). Our data also indicated a significantly higher frequency of occurrence of Corynebacterium spp. in the stomachs and ilea of the littermate mice as compared with the other mouse groups. Perhaps the fact that the littermate animals were the only animals in the study which were born with thymic function may explain this result. The nornal gastrointestinal flora of mice is a stable, self-regulating ecosystem which has evolved in intimate association with its host. This inherent stability and regulatory capacity of the normal flora as a whole is probably responsible for its relative resistance to host immunological mechanisms. LITERATURE CITED 1. Aranki, A., and R. Freter. 1972. Use of anaerobic glove boxes for the cultivation of strictly anaerobic bacteria. Am. J. Clin. Nutr. 25:1329-1334. 2. Aranki, A., S. A. Syed, E. B. Kenney, and R. Freter. 1969. Isolation of anaerobic bacteria from human gingiva and mouse cecum by means of a simplified glove box procedure. Appl. Microbiol. 17:568-576. 3. Bailey, W. R., and E. G. Scott. 1974. Diagnostic microbiology, 4th ed. C. V. Mosby Co., St. Louis. 4. Balish, E. B., J. Brown, and T. Wilkins. 1977. Transparent plastic incubator for the anaerobic glove box. Appl. Environ. Microbiol. 33:525-527. 5. Buchanan, R. E., and N. E. Gibbons (ed.). 1974. Bergey's manual of determinative bacteriology, 8th ed. The Williams & Wilkins Co., Baltimore. 6. Cato, E. P., and J. L. Johnson. 1976. Reinstatement of species rank for Bacteroides fragilis, B. ovatus, B. distasonis, B. thetaiotamicron, and B. vulgatus: designation of neotype strains for Bacteroides fragilis (Veilon and Zuber) Castellani and Chalmers and Bacteroides thetaiotamicron (Distaso) Castellani and Chalmers. Int. J. Syst. Bacteriol. 26:230-237. 7. Cheers, C., and R. Wailer. 1975. Activated macrophages in congenitally athymic "nude" mice and in lethally irradiated mice. J. Immunol. 115:884-887. 8. Clark, I. A., and A. C. Allison. 1974. Babesia microti and Plasmodium berghei Yoelii infections in nude mice. Nature (London) 252:328-329. 9. Davis, C. P., D. Clever, E. Balish, and C. E. Yale. 1977. Bacterial localization in the gastrointestinal tracts of beagle dogs. Appl. Environ. Microbiology 34:194-206. 10. Dubos, R., R. W. Schaedler, R. Costello, and P. Hoet. 1965. Indigenous, normal, and autochthonous flora of the gastrointestinal tract. J. Exp. Med. 122:67-75. 11. Dunn, 0. J., and V. A. Clark. 1974. Applied statistics: analysis of variance and regression, p. 112-115. John Wiley & Sons, New York. 12. Emmerling, P., H. Finger, and J. Bockemuhl. 1975. Listeria monocytogenes infection in nude mice. Infect. Immun. 12:437-439. 13. Gillmore, J. A., and F. B. Gordon. 1974. Effect of exposure to hypertoxic, hyobaric, and hyperbaric environments on concentrations of selected aerobic and anaerobic fecal flora of mice. Appl. Microbiol. 29:358-367. 14. Hagen, C. A., P. W. Barbera, W. H. Blair, A. M. Shefner, and S. M. Poiley. 1968. Similarity of intestinal microflora of BDF, conventional mice from different sources and of different ages. Lab. Anim. Care 18:550-556.

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