Philippe Gouet

Establishment of the Microflora and Anaerobic Fungi in the Rumen of Lambs

SUMMARY: The establishment of different bacterial populations and fungi in the rumen was investigated in lambs reared under different conditions of diet and management. The rumen was rapidly colonized by an abundant microflora after birth. By day 2 strictly anaerobic bacteria predominated (109c.f.u. ml−1); their population increased slightly during the first week of life and again when the animals began to ingest solid feed (3 weeks). The composition of the microflora in the 2-10-d-old lambs was quite different from that of adult sheep. The aerobic and facultatively anaerobic bacterial count was 10-100-fold lower than the strictly anaerobic count during the first week, and decreased steadily afterwards. In flock-reared lambs, cellulolytic and methanogenic bacteria appeared very early after birth (3-4 d). At the end of the first week the population of these bacteria reached a level close to that generally observed in a mature rumen. The cellulolytic bacteria were also able to survive in the rumen of lambs fed cows milk exclusively. Anaerobic fungi appeared later (8-10 d). They were present in all lambs studied until 3 weeks of age, and then disappeared in most of them when a solid diet was given.

Quantitative Determination of H2-Utilizing Acetogenic and Sulfate-Reducing Bacteria and Methanogenic Archaea from Digestive Tract of Different Mammals

Abstract. Total number of bacteria, cellulolytic bacteria, and H2-utilizing microbial populations (methanogenic archaea, acetogenic and sulfate-reducing bacteria) were enumerated in fresh rumen samples from sheep, cattle, buffaloes, deer, llamas, and caecal samples from horses. Methanogens and sulfate reducers were found in all samples, whereas acetogens were not detected in some samples of each animal. Archaea methanogens were the largest H2-utilizing populations in all animals, and a correlation was observed between the numbers of methanogens and those of cellulolytic microorganisms. Higher counts of acetogens were found in horses and llamas (1 × 104 and 4 × 104 cells ml−1 respectively).

Effects of glycerol on the growth, adhesion, and cellulolytic activity of rumen cellulolytic bacteria and anaerobic fungi.

The effect of glycerol on the growth, adhesion, and cellulolytic activity of two rumen cellulolytic bacterial species,Ruminococcus flavefaciens andFibrobacter succinogenes subsp.succinogenes, and of an anaerobic fungal species,Neocallimastix frontalis, was studied. At low concentrations (0.1–1%), glycerol had no effect on the growth, adhesion, and cellulolytic activity of the two bacterial species. However, at a concentration of 5%, it greatly inhibited their growth and cellulolytic activity. Glycerol did not affect the adhesion of bacteria to cellulose. The growth and cellulolytic activity ofN. frontalis were inhibited by glycerol, increasingly so at higher concentrations. At a concentration of 5%, glycerol totally inhibited the cellulolytic activity of the fungus. Thus, glycerol can be added to animal feed at low concentrations.

Effects of live Saccharomyces cerevisiae cells on zoospore germination, growth, and cellulolytic activity of the rumen anaerobic fungus, Neocallimastix frontalis MCH3.

The effects of a live yeast strain of Saccharomyces cerevisiae have been investigated on zoospore germination, metabolism, and cellulolytic activity of the anaerobic rumen fungus Neocallimastix frontalis MCH3. The addition of yeast cells to a vitamin-deficient medium stimulated the germination of fungal zoospores, increased cellulose degradation and hydrogen, formate, lactate, and acetate production. Responses depended on the concentration of yeast cells added and on their viability. Yeast supplementation provided vitamins such as thiamine, which is essential for fungal growth and activity. These results demonstrate that yeasts could enhance plant cell wall colonization by N. frontalis. With certain diets, yeasts could therefore be a good tool to optimize the microbial degradation of lignocellulosic materials, but more research is needed to understand their mechanisms of action, so that they can be used with maximum efficiency as feed supplements.

Degradation and fermentation of cellulose by the rumen anaerobic fungi in axenic cultures or in association with cellulolytic bacteria

Three rumen anaerobic fungi—Neocallinastix frontalis MCH3,Piromyces (Piromonas) communis FL, andCaecomyces (Sphaeromonas) communis FG10—were cultured on cellulose filter paper alone or in association with one of two rumen cellulolytic bacteria,Ruminococcus flavefaciens 007 andFibrobacter succinogenes S85. Cocultures ofN. frontalis orP. communis andR. flavefaciens were markedly less effective than the fungal monocultures in degrading cellulose but more effective than the bacterial monocultures.R. flavefaciens had an antagonistic effect against both of the fungal species. In contrast, no interaction was observed between the two fungal species andF. succinogenes. Cellulose was more effectively degraded by the cocultureC. communis-R. flavefaciens than by the corresponding fungal and bacterial monocultures. The effectiveness of degradation of the cocultureC. communis-F. succinogenes was comparable to that of the bacterial strains but greater than that of the fungi; no interaction was observed between these two microorganisms.

Inhibition of the cellulolytic activity of Neocallimastix frontalis by Ruminococcus flavefaciens

A study was made of the antagonistic effect of Ruminococcus flavefaciens on the cellulolytic activity of Neocallimastix frontalis. An extracellular factor inhibiting the cellulolytic activity of the fungus was detected in the bacterial supernatant. The antagonistic factor, which precipitated with ammonium sulphate at 40% saturation, was temperature-sensitive and was destroyed at temperatures above 60 degrees C. After separation by anion-exchange chromatography, sequential precipitation, dialysis and SDS-PAGE, two protein species of 100 and 24 kDa were identified as being involved in this antagonistic effect. It is known whether the proteins are two subunits of a single protein or represent two different proteins. The inhibitory factor, which is not a bacterial cellulase, did not affect fungal growth, but it inhibited the activity of the fungal cellulases.

Competition Between Ruminal Cellulolytic Bacteria for Adhesion to Cellulose

Abstract. Competition for adhesion to cellulose among the three main ruminal cellulolytic bacterial species was studied using differential radiolabeling (14C/3H) of cells. When added simultaneously to cellulose, Ruminococcus flavefaciens FD1 and Fibrobacter succinogenes S85 showed some competition; however, both species were surpassed competitively by Ruminococcus albus 20. When R. flavefaciens FD1 and F. succinogenes S85 were already adherent, R. albus 20 adhesion occurred without inhibition but involved R. flavefaciens FD1 detachment.

Degradation of wheat straw and maize stem by a monocentric and a polycentric rumen fungi, alone or in association with rumen cellulolytic bacteria

Abstract Two rumen anaerobic fungi, Neocallimastix frontalis MCH3 and Orpinomyces (Neocallimastix) joyonii TP 90-9 were cultured on wheat straw or maize stem alone or in association with one of two rumen cellulolytic bacteria, Ruminococcus flavefaciens 007 and Fibrobacter succinogenes S85. Observations by scanning electron microscopy showed that the behaviour of the different microorganisms was qualitatively similar. The phloem and inner parenchyma of the wheat straw and maize stem were completely degraded after 48 h in both the monocultures and cocultures. Quantitatively, the two fungal species degraded comparable amounts of wheat straw and maize stem as the two cellulolytic bacteria. In the coculture of N. frontalis or O. joyonii with F. succinogenes , wheat straw or maize stem were digested to the same extent as in the fungal monocultures. The associations of N. frontalis with R. flavefaciens on the two substrates and of O. joyonii with R. flavefaciens on wheat straw were less efficient than the fungal monocultures. R. flavefaciens exerted an inhibitory effect on the cellulolytic activity of N. frontalis and O. joyonii while F. succinogenes did not interact with the two fungal species.

Light and electronic microscopic studies of the changes in the intestinal mucosa of gnotobiotic calves infected with a wild rotavirus

Summary Experimental infection of four gnotobiotic calves by a rotavirus isolated from the faeces of diarrheic calves caused non-fatal diarrhoea without dehydration. Lesions of the alimentary mucosa appear well before the onset of diarrhoea and are characterized by a release of mucus in the anterior part of the intestine. By the time diarrhoea begin, different types of rotavirus particules are present in the enterocytes of the mucosa, and mucus contents of distal intestine are affected. At this stage of the disease, the virus causes epithelial cells in the small intestine to desquamate and changes the secretion of mucus in the small as well as the large intestine. The very intense virus multiplication is completed within 18 h. Intracellular rotavirus disappear very quickly after the onset of diarrhoea, and cannot be detected even during the acute phase of the disease. Regeneration of the mucosa takes place slowly, and the cellular and structural lesions are still visible more than 4 days after the diarrhoea stopped.