J. P. Koopman

The attachment of filamentous segmented micro-organisms to the distal ileum wall of the mouse: a scanning and transmission electron microscopy study.

Scanning electron micrographs are presented of the ileal epithelium of mice aged 5, 15, 20 and 25 days. During this period the villous pattern develops to full maturity. By the twentieth day of life a segmented filamentous micro-organism colonizes the ileal epithelium and is firmly attached via a small segment. During the first days of colonization the segmented filamentous micro-organisms themselves are subcolonized by small rod-shaped bacteria, presumably lactobacilli. At the age of 25 days this subcolonization was no longer observed.

'Normalization' of germfree mice with anaerobically cultured caecal flora of 'normal' mice:

Germfree (GF) mice were inoculated with a cultured flora from 10-1, 10-3, 10-5, and 10-7 dilutions of caecal contents from a normal mouse. GF mice associated with a flora of a normal mouse served as controls. The following intestinal parameters were determined: Colonization resistance (CR), Relative caecal weight (RCW), villus:crypt ratio (jejunum and ileum), IgA-producing cells (jejunum and ileum), ß-aspartyl glycine (faeces), volatile and non-volatile fatty acids (caecum) and bile acids (faeces). Only the 10-1 culture was able to induce similar changes in the GF mice to a normal flora. The GF + 10-5 and GF + 10-7 groups deviated markedly from the controls while the GF + 10-3 group showed in general intermediate values between GF + SPF and GF + 10-1 on the one hand and GF + 10-5 and GF + 10-7 on the other hand. ß-aspartyl glycine was present only in the GF + 10-7 group. Scanning electron microscopy (SEM) of ileal contents revealed segmented filamentous organisms in the ileum of controls and the GF + 10-1 group. The faecal flora consisted mainly of fusiform organisms. In the faeces of the 10-5 and 10-7 groups increasing amounts of non-bacterial matter were found, while in the faeces of the other groups virtually only bacteria were seen.

THE NORMALIZATION OF GERM-FREE RABBITS WITH HOST-SPECIFIC CAECAL-MICROFLORA

Hysterectomy-derived germ-free rabbits were given colonization-resistant caecal flora (CRF) from mice, or microflora obtained from the caecum of an antibiotic-decontaminated conventional rabbit and compared with rabbits conventionally raised with the doe. Bodyweight and the following intestinal parameters were determined for the 3 groups: colonization resistance to E. coli, relative caecal weight, villus:crypt ratio (ileum), β-aspartylglycine (faeces), volatile fatty acids (caecum), and bile acids (faeces). Germ-free rabbits given mouse CRF-flora showed values quite different from control animals (or most parameters, indicating unsuitability of mouse CRF flora to normalize rabbits. In germ-free rabbits given modified (antibiotic-treated) rabbit flora, values for most parameters were intermediate between those found for the other 2 groups. This species-specific caecal flora should be improved to provide full normalization of germ-free rabbits.

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

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 β-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.

Some aspects of the gastrointestinal microflora of germfree mice associated with cultured microfloras.

Attempts are described to normalize germfree mice by association with 3, 21 and 71 different intestinal bacterial cultures isolated from mice with an SPF flora. Germfree mice associated naturally with an SPF flora served as controls. Vital bacterial counts were determined by aerobic and anaerobic culture. Stomach and small intestine contained fewer bacteria per gram than caecum and large intestine. Aerobic vital counts from caecum and large intestine were higher in the experimental groups than in control mice. The aerobic and anaerobic flora in stomach and small intestine comprised mainly Gram-positive nonfusiform shaped rods. In the caecum and colon Gram-positive cocci predominated in the aerobic culture while in the anaerobic culture fusiform-shaped rods were prominent. Scanning electron microscopy of oesophagus, ileum, caecum and faeces demonstrated colonization of the oesophageal epithelium only after association with 71 bacterial strains; the filamentous bacteria present in the ileum of SPF mice were not found in the experimental groups and caecum and faeces contained mainly fusiform-shaped bacteria. Nonbacterial matter decreased in the caecum and faeces with increase in the complexity of the flora.

The use of a rat-derived microflora for providing colonization resistance in SPF rats

To obtain a suitable species-specific microflora for a new rat SPF-unit, germ-free WAG/Rij rats were associated with a flora derived originally from selectively decontaminated Cpb:WU (Wistar) rats. Caecal and ileal contents of these rats had been cultured anaerobically (37°C) for 7 days and harvested. This cultured flora was given to germ-free Cpb : SE (Swiss) mice, which were kept in an isolator system and acted as a source of the flora to associate germ-free Wag/Rij rats. In these associated rats, several parameters indicative of the quality of the intestinal micro flora were investigated and compared to those in rats with a mouse derived anaerobic microflora. Parameters included relative caecal weight, colonization resistance and the concentration of faecal bile acids. The cultured rat-derived microflora normalized the observed intestinal parameters better than the mouse derived microflora, and provided better colonization resistance. We conclude that culturing of intestinal contents of selectively decontaminated animals can be a useful way to obtain a species-specific donor-micro flora which can be used to start new SPF units.

Colonization resistance of the digestive tract and gastro-intestinal transit time in SPF mice

The gastro-intestinal colonization resistance to Escherichia coli was assessed in individual CBA/Rij, C3H/StZ and Swiss/Cpb:SE mice. Gastro-intestinal transit time was determined by feeding small steel balls and x-ray examination of sequentially collected faeces. No correlation was found between transit times measured on 3 subsequent days, nor between them and colonization resistance.

Tail lesions in C3H/He mice

C3H/He mice obtained from different suppliers developed tail lesions shortly after arrival. Histologically no inclusion bodies could be shown. A serological survey of diseased mice was negative for those viruses which may cause skin lesions. The disease could not be transmitted to healthy mice and no virus could be cultured from the skin lesions. It is concluded that the syndrome was induced by stress, in this case transport from the United Kingdom to the Netherlands.

The 'normalization' of germ-free guineapigs with host-specific caecal microflora.

Hysterectomy-derived germ-free guineapigs were given colonization-resistant caecal flora from mice (mCRF) or microflora obtained from the caecum of an antibiotic-decontaminated conventional guineapig (gpCRF) and compared with guinea pigs raised conventionally with the sow. Body weight and the following intestinal parameters were determined for the groups: colonization resistance (CR) to Escherichia coli, relative caecal weight (RCW), β-aspartylglycine (faeces), volatile fatty adds (caecum) and bile acids (faeces). mCRF guineapigs showed values quite different from control animals for CR and RCW, indicating the unsuitability of mouse CRF for normalizing guineapigs. In gpCRF guineapigs CR and RCW values were comparable with controls, indicating the suitability of the guineapig flora for normalizing guineapigs. mCRF guineapigs housed with gpCRF guinea pigs, showed an improvement in CR and RCW, yielding values found in control animals.