Gnotobiotic Silage

Selected strains of lactic acid bacteria isolated from grass silage were found to flourish when inoculated into irradiation-sterilized forage under gnotobiotic conditions. The acid content and pH of these silages resembled naturally fermented silage. Inoculation of gnotobiotic silage with Clostridium sporogenes and C. tyrobutyricum failed to cause any noticeable deterioration of silage quality.     

Gnotobiotic techniques

A brief review is given of the development of equipment used for rearing germfree animals in various laboratories and of our exparience with designing germfree isolators and with the technology of rearing germfree piglets is presented. The philosophy of isolation as applied to the animal as a biological unit, the elimination of contamination and the theoretical prerequisites of isolation were considered by Reyniers (1943, 1959) and Trexler (1960).     

悉生蚕的研究与应用

悉生蚕是指蚕体内外栖息的其他生物体是已知的蚕,不仅培育方法较哺乳动物简单,且彻底克服蚕的季节性和地域性饲育限制,为蚕在实验动物科学中的广泛应用打下基础。随着现代生物技术的发展,悉生蚕作为生物反应发生器或转基因工程动物的应用已逐步进入实用化研究阶段。     

Experimental Dental Caries on Gnotobiotic Inbred Mice

The purpose of this study in mono-infected gnotobiotic BALB/cA and C3H/HeN mice was to evaluate the cariogenicity of Enterococcus faecalis. The caries incidence and mean caries score in the BALB/cA mice were significantly higher than those in the C3H/HeN. In both of the mouse strains, the mean number of E. faecalis isolated from the cecum content was almost the same, however, the mean number of E. faecalis from the maxilla of BALB/cA was significantly higher than that of C3H/HeN. These results indicate that C3H/HeN has some factors that prevent E. faecalis from attaching to the tooth surfaces.     

Mycophenolic Acid in Silage

We examined 233 silage samples and found that molds were present in 206 samples with counts between 1 × 10³ and 8.9 × 10⁷ (mean, 4.7 × 10⁶) CFU/g. Mycophenolic acid, a metabolite of Penicillium roqueforti, was detected by liquid chromatography-mass spectrometry in 74 (32%) of these samples at levels ranging from 20 to 35,000 (mean, 1,400) μg/kg. This compound has well-known immunosuppressive properties, so feeding with contaminated silage may promote the development of infectious diseases in livestock.     

Silage and animal health

The process of preserving crops by fermentation in silos is under the control of the farmer to a much lesser degree compared to the level of control by the manufacturer over the production of other fermented foods, such as cheese and yoghurt. Additives designed to direct the extent and pattern of the fermentation are relatively unpopular in most countries, and their use is not guaranteed to remove the risk of undesirable components in silage. Hazards to animal health associated with silage fall into three categories: (1) undesirable micro-organisms e.g. Listeria, enterobacteria, clostridia and moulds; (2) undesirable chemicals, e.g. mycotoxins, and (3) excess acidity and other metabolic disorders. In some regions of Europe, the production of silage is discouraged or prohibited because of the risk of undesirable microbes. The princIpal risk in these areas is that of the secondary fermentation of cheese made from milk contaminated by bacterial spores, rather than a direct hazard of contaminated silage to animal health. With the possible exception of high dry matter silage conserved in large bales, respiratory hazards to animals from moulds and their spores generally are less from silage than hay. Mycotoxins and phytoestrogens may survive the ensiling period and constitute risks to animal health. Relatively little is known about the epidemiology of diseases that may be linked to undesirable chemicals and excess acidity in silage. Therefore, research is needed to define epidemiologically and mechanistically the risks to animal health and to the human food chain from silages contaminated with pathogenic bacteria and mycotoxins, and to understand more completely the relationships between the physical and chemical compositions of silage and metabolic disorders in animals.     

Fat Absorption from the Small Intestine of Gnotobiotic Chicks

The small intestine of 3-week-old gnotobiotic chicks was divided into five equal segments for analysis. Since it has been found that most fat absorption occurs in the third and fourth segments, they were selected for study. The major fatty acids in the diet and the feces of chicks were palmitic, stearic, oleic, and linoleic acids. Consequently, these fatty acids were selected for quantitative analyses in the absorption studies. Germ-free chicks were contaminated at 7 days of age with bacteria isolated from the third and fourth intestinal segments, except for the Escherichia coli contaminant, which had been previously isolated from a chicken with air sac disease. There were two different degrees of response by chicks to the bacterial contaminants in this study. For this reason, the chicks were placed retrospectively into two categories according to their response to environmental conditions. The first category (A) contained germ-free chicks and chicks monocontaminated with E. coli, Staphylococcus, or Lactobacillus. The second category (B) was composed of conventional chicks and chicks monocontaminated with Streptococcus faecalis or Clostridium welchii, dicontaminated with S. faecalis and C. welchii, and accidentally polycontaminated with coliforms and anaerobic gram-positive bacilli. Chicks in category A demonstrated increased absorption of palmitic and stearic acids and total fat as compared to category B chicks. Conversely, category B chicks showed slightly increased absorption of oleic and linoleic acids. Thin-layer chromatographic analyses demonstrated that cholesterol esters were affected similarly to fatty acids. The site of absorption of fatty acids, cholesterol esters, and total fat was unchanged, regardless of the environment.     

Gnotobiotic studies to determine the colonization resistance of the intestines

The literature data and the results of the author studies on determination of intestine colonization resistance are presented. The mechanisms of the colonization resistance defined by the macroorganism factors and representatives of indigenic microflora are discussed. The results of the experiments with animal gnotobiotes aimed at elucidating new aspects of the colonization resistance mechanism: antagonistic interrelations between pathogenic and nonpathogenic bacteria and the role of transitory microflora, factors lowering the colonization resistance are presented. The up-to-date methods for testing the colonization resistance and the ways for its increasing are indicated.     

Establishment of Ureolytic Staphylococci in the Rumen of Gnotobiotic Lambs

Six strains of ureolytic staphylococci isolated from rumen digesta and the rumen wall of conventionally reared sheep were inoculated per os into two germ-free lambs. High numbers of staphylococci established in rumen fluid, and urease activity and normal NH₃ and urea concentrations were maintained for approximately 4 weeks until slaughter. Staphylococci were found also to be associated with the rumen wall, conferring urease activity on this tissue.     

Pneumonia Due to Mycoplasma in Gnotobiotic Mice III. Lesions in the Lungs of Gnotobiotic Mice After Multiple Intranasal Inoculations of Broth Cultures of Mycoplasma pneumoniae

Lung lesions characterized by extensive peribronchial and perivascular round cell infiltration and intrabronchial plugging with polymorphonuclear leukocytes were produced in 5 of 44 Ha/ICR gnotobiotic mice sacrificed 3 to 10 days after three intranasal inoculations of broth cultures of Mycoplasma pneumoniae. After 10 days, no significant lesions were seen, and the proportion of lungs positive for M. pneumoniae dropped off sharply. M. pneumoniae persisted for longer periods (up to 24 days) in the trachea, nasopharynx, and anterior nares. These findings would seem to represent a self-limited respiratory infection due to M. pneumoniae in gnotobiotic mice. Ring forms within granular alveolar pneumocytes were seen by electron microscopy in the lungs of triply inoculated gnotobiotic controls receiving sterile horse-serum broth as well as in the lungs of mice receiving broth cultures of M. pneumoniae. Ring forms must now be considered to be part of the nonspecific cellular reactions of pneumocytes to foreign substances in the lungs of mice rather than intracytoplasmic developmental forms of mycoplasma as previously proposed.     

Increased Enterocyte Production in Gnotobiotic Rats Mono-Associated with Lactobacillus rhamnosus GG

There is increasing scientific and commercial interest in using beneficial microorganisms (i.e., probiotics) to enhance intestinal health. Of the numerous microbial strains examined, Lactobacillus rhamnosus GG has been most extensively studied. Daily intake of L. rhamnosus GG shortens the course of rotavirus infection by mechanisms that have not been fully elucidated. Comparative studies with germfree and conventional rats have shown that the microbial status of an animal influences the intestinal cell kinetics and morphology. The present study was undertaken to study whether establishment of L. rhamnosus GG as a mono-associate in germfree rats influences intestinal cell kinetics and morphology. L. rhamnosus GG was easily established in germfree rats. After 3 days of mono-association, the rate of mitoses in the upper part of the small intestine (jejunum 1) increased as much as 14 and 22% compared to the rates in germfree and conventional counterparts, respectively. The most striking alteration in morphology was an increase in the number of cells in the villi. We hypothesis that the compartmentalized effects of L. rhamnosus GG may represent a reparative event for the mucosa.     

Sterility of Salmonid Roe and Practicality of Hatching Gnotobiotic Salmonid Fish

The ventral surface of spawning salmonid fish was opened aseptically and the roe were removed aseptically. Roe obtained by using this technique were demonstrated to be sterile. Aseptic fertilization and incubation of eggs obtained in this manner resulted in the hatching of gnotobiotic salmonid fish.     

Life Cycle and Mating Behavior of Belonolaimus longicaudatus in Gnotobiotic Culture

The life cycle of Belonolaimus longicaudatus was observed in vitro on excised roots of Zea mays. Roots were cultured on Gamborg''s B5 medium in petri dishes with 1.5% agar adjusted to pH 5.8 and incubated at 28 °C in darkness. Second-stage juveniles (J2) fed on the roots and started the second molt (M2) to the third-stage juveniles 2 days after inoculation (DAI). The third molt (M3) to the fourth-stage juveniles occurred 7 DAI, followed by the fourth molt (M4) to males 13 DAI or to females 14 DAI. Nematode gender differences were observed by the end of the fourth molt. The first male appeared 15 DAI and the first female 17 DAI, after which mating occurred. Males were attracted to females, and mating was observed. Mating was required for reproduction. Fertilized females began to lay eggs 19 DAI and continued egg laying without the further presence of males during a 90-day observation. All of the eggs hatched. Unfertilized females rarely laid eggs, and none of the eggs were able to hatch. Feeding took place between each molt and before egg deposition occurred. The first-stage juveniles molted in the eggs 4 days after deposition, and J2 hatched from eggs 5 days after egg deposition. The life cycle from J2 to J2 was completed in 24 days.