'Normalization' of germfree mice after direct and indirect contact with mice having a 'normal' intestinal microflora

Transcription

1 286 Laboratory Animals (1986) 20, 'Normalization' of germfree mice after direct and indirect contact with mice having a 'normal' intestinal microflora J. P. KOOPMAN 1, H. M. KENNIS 1, A. LANKHORST 2, G. W. WELLING3, M. P. C. HECTORS 4 & F. NAGENGAST 4 ICentral Animal Laboratory and 4 Department of Gastroenterology, Catholic University, P. O. Box 9101, 6500 H B Nijmegen; 2Department of Laboratory A nimal Science, Faculty of Veterinary Medicine, State University of Utrecht; 3Department of Medical Microbiology, State University of Groningen, The Netherlands Summary Germfree mice were associated via direct and indirect contact with a 'normal' microflora by placing 'normal' mice in an isolator with germfree mice. Relative caecal weights, the ratio of secondary to primary bile acids, the presence of filamentous segmented bacteria in the small intestine and faecal ~-aspartylglycine were normal 5 days after direct contact and 15 days after indirect contact. Enterobacteriaceae were demonstrated by the third day after direct contact and the fourth day after indirect contact. Volatile and non-volatile fatty acids in the caecal contents were variable and appeared to be unrelated to the 'normalization' process of germfree mice after association with a microflora. Keywords: Mice; Germfree life; Enterobacteriaceae The exact mechanism of the 'normalization' process when germfree animals are associated with a microflora is not known. Germfree mice or rats may be associated with a microflora orally or rectally (Syed, Abrams & Freter, 1970; Freter & Abrams, 1972; Koopman et al., 1983); another method is to place a 'normal' animal in a cage or isolator with germfree animals (Freter & Abrams, 1972). Newborn animals are colonized with micro-organisms in a certain succession and in mice the flora is stabilized 5 weeks after birth (Savage, 1977). A comparable succession probably occurs in adult germfree mice which are associated with a microflora. The characteristics of a 'normal' microflora, acquired as the result of associating germfree animals with 'normal' animals, may be studied via the host's responses. In this study we investigated the timescale of the 'normalization' process after placing 'normal' mice in an isolator in direct or indirect contact with germfree mice. Materials and methods Germfree Cpb:SE (Swiss) mice of both sexes, aged Received 20 August 1985; in revised form 8 February Accepted 26 March weeks, were placed in two cages in a germfree plastic isolator. The mice were supplied with autoc1aved SRM food (Hope Farms B.V., Woerden, The Netherlands). Two mice with a specifiedpathogen-free (SPF) flora (Koopman & Janssen, 1974) were placed in one of the cages and served as controls. One animal died as a result of fighting. Two littermates served as controls at the beginning of the experiment. Thereafter the 'normalization' process was studied by investigating animals at 1, 2, 3,4,5,8, 10, 12, 15,22 and 29 days after placing the 'normal' mice in the isolator. The following parameters were studied: (1) relative caecal weight; (2) the number of faecal Enterobacteriaceae; (3) the presence of filamentous segmented bacteria in the small intestine; (4) faecal bile acids; (5) faecal ~-aspartylglycine; (6) volatile and non-volatile fatty acids. The parameters were determined as follows. (1) Relative caecal weight The caeca were removed and weighed with their contents and the wet mass was expressed as a percentage of total body mass. (2) Faecal Enterobacteriaceae The concentration was measured using a plastic tray with 144 cups (12 x 12). each of 1 ml capacity and containing 0 5 ml of nutrient broth (Difco: Detroit, MI, USA). A faecal pellet was then suspended in the first row and tenfold serial dilutions were made with 0 05 ml microdiluter loops (Cooke Engineering Co., Alexandria, VA, USA). After incubation overnight at 37 C the contents of the cups were subcultured on Levine EMB agar plates (Oxoid: Basingstoke, Hants, UK) and examined after incubation for 24 h. (3) Presence of filamentous segmented bacteria in the small intestine The small intestine was divided into nine equal parts. From each of the five distal parts (ileum) a 1 cm piece of intestine was opened and rubbed on an object glass of 5 cm 2 After Gram staining 20 fields

2 'Normalization' of germfree mice 287 were examined with a light microscope at a magnification of 1000x at two different loci (ten fields per locus). The filamentous segmented bacteria were easily recognized. The number of positive fields was counted and a score was given: no fields positive, 0; 1-6 fields positive, 1; 7-13 fields positive, 2; fields positive, 3. A mean score per segment was calculated. (4) Faecal bile acids Faecal bile acids were determined as described by Koopman et al. (1981). (5) ~-Aspartylglycine Faecal supernatants (50 I-Ll)were subjected to high voltage paper electrophoresis on Whatman 3 MM chromatography paper at ph 3 5 for min at 3000 V, following the techniques described by Welling and Groen (1978) and Welling (1982). (6) Volatile and non-volatile fatty acids Volatile and non-volatile fatty acids were measured as described earlier (Koopman et al., 1981). Results The relative caecal weights of germfree mice associated with a 'normal' mouse flora via direct and indirect routes are plotted in Fig. 1. The relative caecal weights did not reach 'normal' values until 5 and 15 days respectively. Faecal Enterobacteriaceae could be demonstrated by the third day after direct contact and the fourth day after indirect contact c.Q> ~ 5 iii u ro 4 u.~ 3....!l! D: Days after placing normal mice in an isolator with germfree mice Fig. I. Relative caecal weights of germfree mice after direct and indirect contact with 'normal' mice:., direct contact (mean values of two mice per day); 0, indirect contact (mean values of two mice per day); f.., controls (n = 3); shaded zone, range of relative caecal weights of control animals: Initially the concentrations were 1O~cells per gram of faeces in both groups, decreasing to 10 6 cells/g, the same value as that found in the control animals. Segmented filamentous bacteria were found on the fourth and the tenth day, respective Iy, after direct and indirect contact (Fig. 2) and thereafter colonized the small intestine in large numbers. Volatile and non-volatile fatty acids in caecal contents are presented in Table 1 but were variable in controls and appeared to be unrelated to the 'normalization' process of germfree mice after association with a microflora. The faecal concentration of bile acids is presented in Table 2. The ratio of secondary to primary bile acids increased from relatively low to relatively high after contact with a microflora. In the direct contact group a normal value was reached after 5 days, while in the other group this took 12 days. The total amount of bile acids rose in both groups. In the direct contact group a peak was seen on day 8 with a subsequent decline. ~- Aspartylglycine concentrations in the faeces are presented in Fig. 3, although only one mouse is represented by each point. Initially high values followed by a marked decrease were found in both groups. After 5 and 15 days respectively ~- aspartylglycine could no longer be demonstrated. Discussion 'Normalization' of germfree mice occurred at 5 and 15 days after direct and indirect contact with 'normal' mice as monitored by the relative caecal weight, the ratio of secondary to primary bile acids, the presence of segmented filamentous bacteria in the ileum and faecal13-aspartylglycine. Volatile and non-volatile fatty acids were not suitable as 'normalization' indicators. Earlier we had found that the concentration of volatile fatty acids can vary widely in normal animals and we concluded that volatile and non-volatile fatty acid concentrations are of limited value in assessing the degree of 'normalization' of a microflora (Koopman et al., 1984b). From this study it appeared that direct contact between germfree and 'normal' mice leads rapidly to 'normalization'. In earlier association studies at least 1 month was used as a stabilization period (Koopman et at., 1984b, 1984c). This was based on the establishment of successive components of the flora in newborn animals. Savage (1977) found a so-called climax community in mice 5 weeks after birth. In our study we used young adult animals and it was probable that a climax community was reached earlier due to the absence of the suckling period. When mice were maintained in only indirect contact with 'normal' mice in an isolator, then

3 288 Koopman, Kennis, Lankhorst, Welling, Hectors & Nagengast 3 day]. day4 day5 day8 I,~LLLdlJ6J I :;: DIRECT CONTACT :!:L'''L~-_--da-~-,2--~--d-a-Y-, Small intestine segments Fig. 2. Time of appearance of segmented filamentous bacteria in thc ileum after direct and indirect contact with a 'normal' mouse: " based on the presence in 20 fields (magnification, 1000x); 0, negative; I, 1-6 fields positive; 2, 7-13 fields positive; 3, fields positive. Table 1. Volatile and non-volatile fatty acids in caecal contents* Days Volatile fully acids (flttlo/lg) Non-volatile fatly acids (!,.Iff/ol/g) after initial Ox Sacc Lactate COli tact Clt C 3 i-c. C. i-cs Cs Co Total D L D+L Direct contact I III to to II 29 n.t.t n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. Indirect contact I I W ] ] Controls 'Based on pooled samples from two mice per group. tcz, acetic acid; C 3, propionic acid; i-c 4, isobutyric acid; C 4, n-butyric acid; i-cs, isovaleric acid; Cs, II-valerie acid; C 6, II-caproic acid; Ox, oxalic acid; Succ, succinyl acid. :j:n.t., not tested.

4 'Normalization' of germfree mice 289 Table 2. Faecal concentrations of bile acids* Primary Secondary Ratio of Unidentified Total bile acidst bile acids* secondary bile acids bile acids Days after (fwloj/g (f!mol/g to primary (f!mol/g (f!flioi/g initial contact dry weight) dry weight) bile acids dry weight) dry weight) Direct contact ] ] 93 Indirect contact ] ] Controls ] 'Based on pooled samples from two mice per group. tcholic acid, u-muricholic acid, ~-muricholic acid. *Deoxycholic acid, w-muricholic acid, lithocholic acid, hyodeoxycholic acid. Controls: first line, pooled sample of two normal mice from the beginning of the experiment; second line, sample from a 'normal' mouse placed in the germfree isolator at the end of the experiment. '0 c >20.Q 20 -;UllJ =.~ c u llj» 15 0» u~ -'- I1lI1l llj"'- >.;; 111 Qj a: 5 \T-:---r-T":::::-' 'T Days after placing normal mice in an isolator with germfree mice Fig. 3. Relative concentrations of ~-aspartylglycine in the faeces of germfree mice after direct and indirect contact with 'normal' mice:., direct contact (one mouse per sample); 0, indirect contact (one mouse per sample). 'normalization' occurred later than after direct contact but was achieved by about 15 days. This means that the micro-organisms with ecological importance can be transported by air or fomites. Most intestinal micro-organisms are strictly anaerobic (Koopman, Janssen & van Druten, 1977) and therefore during transport the micro-organisms were apparently protected against inactivation by atmospheric oxygen. It is probable that organic matter such as faeces and dander provided this protection. This is in agreement with the reported protection of anaerobic bacteria by blood or serum (Carlsson, Fr61ander & Sundquist, 1977; Koopman & Kennis, 1982). It may be concluded that 'normalization' is possible without intimate contact between the animals. It would be of interest to know the function(s) of the filamentous segmented organisms in the intestinal ecology. In an earlier study we found that relative caecal weight and bile acids were similar in animals with and without this organism (Koopman et at., 1984a).

5 290 Koopman, Kennis, Lankhorst, Welling, Hectors & Nagengast References Carlsson, J., Fr6lander, F. & Sundquist, G. (1977). Oxygen tolerance of anaerobic bacteria isolated from necrotic dental pulps. Acta Odol1lologica Scandinavica 35, Freter, R. & Abrams, G. D. (1972). Function of various intestinal bacteria in converting germfree mice to the normal state. Infection and Immunity 6, Koopman, J. P. & Janssen, F. G. J. (1974). The suitability of an intestinal flora with colonization resistance factor for SPF mice, rats and gerbils. Zeilschrift fur Versuchstierkunde 16, Koopman, J. P. & Kennis, H. M. (1982). Influence of diluting fluids and exposure to the air on the viability of the anaerobic bacterial flora of the mouse cecum. Zeilschrift fiir Versuchstierkunde 24, Koopman, J. P., Janssen, F, G. J. & van Druten, J. A. M. (1977). Isolation of the cecal micro flora of mice and comparison between the gastro-intestinal and the fecal microfloras. Zeilsclzrifl fur Versuchslierkunde 19, Koopman, J. P., Welling, G. W., Huybregts, A. W. M., Mullink, J. W. M. A. & Prins, R. A. (1981). Association of germ-free mice with intestinal microfloras. Zeilschrifl fur Versuchstierkunde 23, Koopman, J. P., Kennis, H. M., Stadhouders, A. M. & de Boer, H. (1983). Some aspects of the gastrointestinal microflora of germfree, mice associated with cultured microfloras. Laboratory Animals 17, Koopman, J. P., Kennis, H. M., Hectors, M. P. c., Lankhorst, A., Stadhouders, A. M. & de Boer, H. (1984a). Reciprocal 'normalization' of intestinal parameters by indigenous intestinal microflora of the rat and mouse. Zeitschrift fur VersuchSlierkUllde 26, Koopman, J. P., Kennis, H. M., Mullink, J. W. M. A., Prins, R. A., Stadhouders, A. M., de Boer, H. & Hectors, M. P. C. (1984b). 'Normalization' of germfree mice with anaerobically cultured caecal flora of 'normal' mice. Laboratory Animals 18, Koopman, J. P., Kcnnis, H. M., Mullink, J. W. M. A., Prins, R. A., Stadhouders, A. M., de Boer, H., Hectors, M. P. C. & van der Logt, J. T. M. (1984c). Association of germfree rats with different microfloras. Zeilsohrifl fur Versuchstierkunde 26, Savage, D. C. (1977). Microbial ecology of the gastrointestinal tract. Annual Review of Microbiology 31, Syed, S. A., Abrams, G. D. & Freter, R. (1970). Efficiency of various intestinal bacteria in assuming normal functions of enteric flora after association with germ-free mice. Infection and Immunity 2, Welling, G. W. (1982). Comparison of methods for determination of ~-aspartylglycine in faecal supernatants of leukaemic patients treated with antimicrobial agents. Journal of Chromatography 232, Welling, G. W. & Groen, G. (1978). ~-Aspartylglycine, a substance unique to caecal contents of germ-free and antibiotic treated mice. Biochemical Journal 175,