Bernard S. Wostmann

EFFECTS OF MICROBIAL FLORA ON CARDIAC OUTPUT AND OTHER ELEMENTS OF BLOOD CIRCULATION.

Summary Cardiac output in young adult germfree rats was found to be approximately 30% lower than in conventional controls. Concomitantly, signs of mild hemoconcentration were observed in the germfree group, which may be linked to changed water balance in these animals. The reduced work performance of the heart indicated in the germfree group is associated with the absence of a microbial flora.

Efficiency of digestion in germ-free and conventional rabbits.

1. Germ-free (GF) and conventional (CV) rabbits wearing collars to prevent coprophagy were fed an autoclaved diet with added cellulose. Their faecal excretion was analysed to determine nutrient digestibility. 2. Clearly distinguishable hard faeces were excreted by the GF rabbit only if the diet contained at least 15% cellulose. Unlike CV rabbits, the GF rabbits did not consume their soft faeces even when permitted to do so. Soft faeces made up a larger proportion of the total output of GF than of CV rabbits. Food intake and total dry-matter excretion per kg body-weight were similar in both groups. 3. Although digestibility of dry matter was similar in the two groups, in the GF rabbits there was a higher digestibility of crude fat and true protein and a lower digestibility of crude fibre and nitrogen-free extract. GF rabbits excreted a higher percentage of ingested calcium and phosphorus in the urine than did CV rabbits. 4. The results suggest that intestinal microbes, even without the enhancing effect of coprophagy, aid in the digestion of carbohydrate by rabbits. The greater faecal excretion of crude fat and true protein by CV rabbits could result from poorer digestion and absorption, but could also represent nutrients synthesized by microbes from simpler materials. The reingestion of faecal crude fat and true protein might therefore improve the quality of the total nutrient intake. The results suggest ways of assuring an adequate dietary intake by GF rabbits in the absence of contributions from an intestinal microflora.

Cecal Enlargement, Cardiac Output, and O2 Consumption in Germfree Rats

Summary Germ free rats have cardiac output and O2 consumption values respectively 30% and 24% lower than found in comparable conventional rats. Surgical removal of the enlarged cecum typical for these animals resulted in cardiac output values similar to those obtained with conventional rats, while O2 consumption increased significantly to levels only slightly lower than seen in the conventional control group. No significant difference in body temperature was found between comparable groups of germfree and conventional rats.

Oxidation-Reduction Potentials in Gecal Contents of Germfree and Conventional Rats.∗

Summary Oxidation-reduction potentials in cecal contents of germfree rats were 250 to 300 mV more positive than in the contents of conventional animals. Treatment of germ-free rats with cecal contents from conventional rats showed that 4 hours later these animals demonstrated oxidation-reduction values comparable to those found in conventional rats. The more positive oxidation-reduction potential thus appears directly related to the absence of an intestinal micro-flora.

RECENT STUDIES ON THE SERUM PROTEINS OF GERMFREE ANIMALS

The germfree animal has been reared in the absence of any demonstrable microbial form. Thus it seems logical to expect that those serum protein fractions that normally harbor antimicrobial antibodies will show a certain deficiency or possibly be absent in the germfree animal. In 1958 I reported’ that the absence of a microbial flora caused a deficit in alpha2 , beta, and gamma globulin in the serum of the rat. In the chicken only a lower gamma fraction was found, while the then available germfree guinea pigs showed no gamma globulin whatsoever. These studies have now been extended to include other electrophoretic techniques, while more data are available on guinea pigs, rabbits and, also, ruminants.

SERUM PROTEIN FRACTIONS AND ANTIBODY STUDIES IN GNOTOBIOTIC ANIMALS REARED GERMFREE OR MONOCONTAMINATED

The germfree animal has been described as differing in various aspects from animals reared under conventional animal room conditions.’ These differences have for the most part been ascribed to the respective absence or presence of an associated microbial flora. The role played by specific members of the normal flora in establishing the “physiologic state” of conventional life is obscured by the complex nature of the normal flora. This paper describes some observations made on the serum protein fractions and antibacterial agglutinins found in animals maintained under less complex conditions; that is, either entirely germfree or in association with only single selected strains of intestinal microorganisms (monocontaminated). In another paper,

Electrophoretic studies on serum proteins of young germfree, conventional and antibiotic treated conventional chickens.

the response of the germfree rat to a purified antigen, bovine serum albumin, is described. Earlier work on the comparison of serum proteins of germfree and conventional rats has characterized the germfree rat as having lower but nevertheless detectable levels of alphaz , beta, and gamma-globulin fraction~.~J A similar comparison in chickens showed the principal difference to be a decreased gamma globulin level under germfree conditions? The characterization of other germfree-reared species from this standpoint, for example, mouse, guinea pig, rabbit, sheep, and goat have been reported and reviewed by Wostmann?J In general, all germfree-reared animal species studied to date have shown varying degrees of hypogammaglobulinemia, depending on the species, age and, possibly, dietary factors. Germfree rat and chicken serum have been found to be devoid of many, but not necessarily all, antibacterial agglutinins demonstrable in the serum of conventional animals.4s6 The agglutinins that have been demonstrated in germfree animals are believed to be formed in response to dead bacteria and probably other antigenic substances indigenous to the autoclaved diet. These dietary antigens may thus be responsible, a t least in part, for the levels of the probable antibody-carrying serum protein fractions seen in germfree animals. Exposure of young adult germfree rats to the normal intestinal flora of conventional rats resulted in a gradual increase in various globulin fractions.6 The increase occurred first in the alphaz fraction at one week post-contamination followed by an increase in beta globulin by the second week. Gamma globulin stayed at the low germfree level and only started to rise after a two to

Diffusion chambers for inducing immunity to Trichinella spiralis in mice.

Summary The effect of feeding procaine penicillin G (50 mg/kg diet) on the electrophoretic pattern of serum of young conventional chickens was studied and compared with data obtained from germfree animals. While the effect of the antibiotic on morphological characteristics of conventional chicken made itself felt after one month, up to the age of 2 1/2 months no differences were seen in the electrophoretic pattern. Both groups showed an equal increase in gamma globulin over the level found in serum of the germfree animal. At 4 months, however, the antibiotic-treated birds showed lower gamma globulin values than the untreated group, though the level was still substantially higher than found in germfree serum.

Ferritin-conjugated antibody studies on the larvae of Trichinella spiralis.

Abstract Mice exposed for 7 days to Trichinella spiralis larvae in diffusion chambers, were found to be immune to challenge infections. Immunity was reflected by a significant reduction of muscle larvae and adults. Immunity persisted for at least 6 months after removal of the diffusion chambers. Diffusion chambers allow the investigator to control length of host exposure to the parasite. Furthermore, for experimental purposes, immunization is carried out without the pathological effects that occur when live infections are used to stimulate resistance. Finally, the diffusion-chamber technique permits the host to come into direct contact with the living parasites, thereby exposing it to a great variety of antigenic stimuli.

SERUM PROTEINS IN GERMFREE VERTEBRATES

The antibody binding sites of the larva of Trichinella spiralis were investigated using the ferritin-conjugated antibody technique. It has been shown from this study that ferritin-conjugated hyperimmune gamma globulin produced during the course of infection, attached specifically to the outer surface of the cuticle. Nonimmune ferritin-conjugated gamma globulin did not show specific attachment to any portion of the larvae. Ferritin particles were not observed at any site along the gut tract of larvae exposed to either immune or nonimmune gamma globulin-ferritin conjugates. The results demonstrate the antigenic nature of the cuticle of T. spiralis larvae during viable infection. Fluorescent antibody techniques have been employed for detection and localization of antigenic sites in several parasitic helminths, with conflicting results. Jackson (1959) failed to demonstrate attachment of fluorescein-tagged antibody on the cuticle of Trichinella spiralis. With the same system, Sadun (1963) described selective positive fluorescence of larval cuticle, but suggested that this reaction may have been due to leakage of antigenic materials from the internal portion of the larva onto the cuticle. Sulzer (1965), using whole formalinized or cuticular preparations of larvae treated with pepsin, showed that the cuticle was an effective antigen suitable for use in the indirect immunofluorescence test. The ferritin-conjugated antibody technique has been useful in revealing the antibody binding sites of infectious agents. Morgan et al. (1961) used ferritin-conjugated antibody for visualizing stages in the replication cycle of Received for publication 20 December 1966. * This research was supported, in part, by USPHS grant 5 TI GM 1248-03, NSF grant G.B. 3752, and by the Cancer Society of St. Joseph County, Inc., South Bend, Indiana. t This work is in partial fulfillment for the degree of Doctorate of Philosophy in the Department of Microbiology, University of Notre Dame, Notre Dame, Indiana. influenza virus in cell cultures. Shands (1965) applied this technique to investigate the site of somatic antigens on the cell wall of gramnegative bacteria. Matsubayashi et al. (1966) used ferritin antibody conjugates to locate parasite-derived antigens in the limiting membrane of the vacuole surrounding Toxoplasma organisms in peritoneal exudate cells from