The effect of Saccharomyces cerevisiae and Aspergillus oryzae on the digestion of the cell wall fraction of a mixed diet in defaunated and refaunated sheep rumen.

The objective of this study was to determine the effect of two probiotics, Saccharomyces cerevisiae (SC) and Aspergillus oryzae (AO), without their culture medium, on the digestion of plant cell wall components in sheep that had been successively defaunated and refaunated. Six sheep fitted with large rumen cannulae were used to study 1) defaunated sheep with no probiotic, 2) defaunated sheep with SC or AO, 3) refaunated sheep with no probiotic, 4) refaunated sheep with SC or AO. The apparent digestibility of the plant cell walls was not altered (P > 0.05) by the probiotics in defaunated sheep but was increased (P < 0.05) with SC (+16%) in refaunated sheep. Simultaneously, SC stimulated the growth of the protozoal population in the rumen. As noted in most previous experiments, the positive effect of the presence of protozoa on plant cell wall digestion (P < 0.001) was confirmed here. The effect (P > 0.05) of SC or AO on in situ ADF digestion was either not significant or negative in defaunated rumens, whereas it became positive in refaunated rumen after a residence time of 12 h. The improvement of in situ ADF digestion due to the presence of protozoa was significant (P < 0.05 for NDF; P < 0.001 for ADF). However, we could not determine whether this was a direct effect of protozoa or an indirect effect operating via bacteria. The limits of the nylon bag technique for evaluating the microbial activity are discussed in relation to the ability of protozoa to enter and grow in the bags, and the pH regulation inside the bags. The pH values below 5.5 noted inside the bags can significantly alter the bacterial and protozoal populations and limit the validity of the technique. SC increased (P < 0.05) the specific activity of CMCase and xylanase of the solid-adherent bacteria (SAB) isolated from the rumen digesta of refaunated sheep. Stimulation of both the bacterial activity and protozoa numbers could explain the positive effect of SC on cell wall digestion in the rumen since the modelling of marker excretion in faeces showed that the ruminal mean retention time of hay measured from the model of Danhoa et al. was not modified (P > 0.05) by either refaunation or the presence of probiotics. AO increased (P < 0.05) the total retention time of the solid particles in the whole digestive tract and increased the ruminal liquid volume in the refaunated animals but it had no effect on the protozoa population or on the polysaccharidase activity of the SAB, which could explain the absence of effect of AO on the total digestibility of plant cell walls. Both probiotics decreased the liquid turnover in defaunated rumens (P < 0.05) but neither had any effect (P > 0.05) on this parameter after refaunation. The improvement of plant cell wall degradation in the whole digestive tract is probably due to a stimulation of digestion at the rumen level as indicated by the higher activity of the SAB in rumen digesta and the growth of protozoa.

Effects of an asymmetric triazine derivative, HOE 092 V, on Glugea anomala, Moniez, 1887 (Microsporidia) parasitic in the three-spined stickleback Gasterosteus aculeatus.

An asymmetric triazine derivative, HOE 092 V,2-[3,5-alpha-dichloro-4-(4-methyl-sulfonylphenoxy)-phenyl]- 1-methyl-hexahydro-1,2,4-triazine-3,5-dion, was tested in vivo against Glugea anomala parasitizing the connective tissue of sticklebacks (Gasterosteus aculeatus). Naturally infected sticklebacks were incubated in 10-1 plastic aquaria in water containing 0, 2.5, 5, and 10 micrograms HOE 092 V/ml for 2, 3, and 4 h at 22 degrees C. As seen at the ultrastructural level, the drug caused severe damage to all developmental stages of G. anomala except the mature spores. Starting with a dose of 2.5 micrograms/ml, the drug caused significant damage on uni- and multinucleate meronts, sporogonial plasmodia, and sporoblasts. The damage mainly consisted in a decrease in the number of ribosomes, an enlargement of the smooth endoplasmic reticulum and a vacuolization of the cytoplasma. When treatment was done with 5 micrograms for 2 h, multinucleate meronts and sporogonial plasmodia were no longer detectable, and the sporoblasts and the prespore stages except the mature spores had shrunk. After incubation of the infected fish with 10 micrograms HOE 092 V/ml and 4 h exposure, uninucleate meronts were no longer detectable by means of transmission electron microscopy. In the sporoblast mother cells, vacuolization of the cytoplasma and lysis of the nuclei occurred. However, mature spores were not affected. It seems likely that HOE 092 V can be successfully applied in medicinal baths against Microsporidia in fish. The infected fish should be incubated in separate, aerated containers.

The effect of Saccharomyces cerevisiae and Aspergillus oryzae on fermentations in the rumen of faunated and defaunated sheep; protozoal and probiotic interactions.

We measured the effect of the direct addition to the rumen of Saccharomyces cerevisiae (SC 50 mg/day) and Aspergillus oryzae (AO 3 g/day) on the fermentation processes in fistulated sheep. The measurements were carried out on animals whose rumens were first defaunated and then refaunated. The animals received a ration composed of hay (600 g/day), barley (600 g/day) and soybean meal (150 g/day), fed twice daily in two equal meals. The number of fungi and total, viable or cellulolytic bacteria were lower after the inoculation of protozoa in defaunated rumens. The probiotics stimulated the development of total bacteria but reduced the population of cellulolytic bacteria. The addition of the probiotics and the presence of protozoa each incurred a decrease in the redox potential values. The association of both treatments had an additive effect on this parameter. The two probiotics and the protozoa stabilized the rumen pH after the meal, maintaining it above the value of 6 for a longer period of time. The positive effects on pH were accumulated in the refaunated animals receiving probiotics. The ammonia nitrogen concentration was considerably increased by the presence of the protozoa; the probiotics increased the ammonia concentration only in the refaunated sheep. The methane and hydrogen proportions in the fermentation gases were invariably higher in the refaunated animals. The probiotics had no clear effect either on the gas composition or the concentration and the composition of the mixture of volatile fatty acids; only the concentration of isovalerate was significantly increased by probiotics and only in refaunated animals. The protozoa did, however, considerably increase the concentrations of acetate, butyrate and isoacids and decreased the concentration of caproate.

Effect of ruminal inoculation of Isotricha alone or a mixed B-type fauna in a defaunated rumen on the digestion of a hay-maize diet (70:30) in sheep.

Two adult sheep (75 kg live weight) fitted with rumen cannulas were defaunated by the emptying method during the first period of the experiment. They were inoculated with the genus Isotricha alone during the second period, and with a mixed ciliate population (Entodinium, Eudiplodinium, Epidinium) during the third. They were fed a diet of grass hay (840 g) and pelleted maize grains (360 g) in 8 meals per day, every 3 h. Defaunation was successful and no accidental contamination occurred during the experiment. The protozoa had no significant effect on the volume of rumen digesta, nor on the turnover of the particulate phase. The addition of Isotricha and of the mixed fauna increased the ADF digestibility of the diet but, in the same animals, lowered the in sacco degradation of wheat straw. The ruminal pool sizes of dry matter (DM), organic matter (OM), nitrogen (N), neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid lignin detergent (ADL) remained unchanged after protozoa inoculations. The concentration of total volatile fatty acids (VFA) was not altered by faunation with Isotricha or a mixed fauna. The molar proportion of acetate increased at the expense of all the other VFAs (mainly propionate with the mixed fauna). Correspondingly, the proportion of methane in the rumen gases increased and that of CO2 decreased in inoculated animals. The ammonia concentration was highest in animals with a mixed fauna and lowest in those inoculated with Isotricha alone. This trend is explained in terms of the specific effect of the different genera of protozoa on nitrogen metabolism.

Digestion of a dehydrated lucerne/barley diet (70:30) in defaunated, Isotricha-monoinoculated and mixed-fauna-inoculated rumen in sheep.

Two adult Texel sheep fitted with permanent rumen fistulae were defaunated for 12 weeks in a preliminary experiment. They were then reinoculated with Isotricha sp for 11 weeks, and finally with a mixed fauna (Entodinium, Epidinium, Eudiplodinium and Isotricha) for 10 weeks. They were fed a diet composed of dehydrated lucerne (700 g/d), pelleted barley grain (300 g/d), grass hay (100 g/d) and wheat straw (50 g/d) in one daily meal. Isotricha and the ciliates of the mixed fauna developed rapidly, reaching a maximum concentration 9-17 d after inoculation. Their concentration then fell for 2-3 d and finally stabilized at values close to 10(4)/ml for Isotricha, Eudiplodinium and Epidinium and 2.5 x 10(5)/ml for Entodinium. The estimated ciliate biomass in the rumen was 1.8 g l-1 for a volume of 10.2 ml l-1 in the Isotricha-monoinoculated sheep and 4.7 g l-1 for a volume of 25.0 ml l-1 in mixed-fauna-inoculated sheep. The concentration of Isotricha was unaffected by the inoculation of mixed fauna during the third part of the experiment. No difference in total rumen bacteria counts was observed between defaunated, monoinoculated and completely refaunated animals. The total adenylic nucleotide concentration in filtered rumen juice was 4 times higher in faunated sheep just before feeding and 4-9 times lower after feed intake as compared to defaunated animals; the energy charge was always higher in faunated animals. These results are discussed in relation to the digestive activity and biomass of the protozoa. The in situ degradation of lucerne stems was the highest in the mixed-faunated sheep over a retention time interval of 6-18 h. The values obtained in Isotricha-monoinoculated sheep were intermediate between the defaunated and the mixed-faunated states. The digestibility in the whole digestive tract of dietary dry matter, organic matter, nitrogen and neutral detergent fibre was unaffected by the addition of either Isotricha or the mixed fauna in defaunated rumens. The total volatile fatty acid concentration was higher (p < 0.05) in the defaunated sheep between 6 and 18 h after feeding. The molar proportion of butyrate was significantly (p < 0.05) increased by the presence of mixed fauna in the rumen while that of propionate and valerate was lower. A non-significant decrease in acetate was also observed. The concentration of NH3-N in the rumen was significantly increased (p < 0.05) by the presence of the mixed fauna in the rumen but was unaffected or slightly reduced by Isotricha alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Establishment of ciliate protozoa in the rumen of conventional and conventionalized lambs: influence of diet and management conditions.

The establishment of ciliate protozoa in the rumen was studied in conventional lambs reared under different conditions of management. The role of the microflora in the kinetics of this establishment was also investigated in conventionalized lambs. In lambs reared under farm conditions ciliate protozoa appeared in the following order: Entodinium (15-20 days), Polyplastron, Eudiplodinium, and Epidinium (20-25 days), and Isotricha (50 days). Entodinium was the most abundant (10(5)-10(6) ciliates mL-1). During the 3rd month, ciliates disappeared spontaneously in about 60% of the lambs during a period that varied from 1 to 4 weeks. In lambs fed only cow's milk Entodinium spp. and Polyplastron multivesiculatum became established at low levels. The results obtained with the conventionalized lambs demonstrate that the establishment of the ciliates in the rumen requires that the bacterial flora be well established beforehand.

Besnoitia sp. from goats in Kenya.

The eyelids of goats in Kenya contained several, conspicuous white cysts which were up to 1.5 mm in size. By histological and electron microscopical studies it was confirmed that these cysts belong to the genus Besnoitia.

Three new types of globidium of sheep: an in vivo and in vitro investigation.

The abomasum and duodenum of sheep contained three types of globidium. The size and fine structure of these globidium differed from those described in previous investigations. The highly motile parasites failed to develop in cell lines of goat, sheep and dog origin.

Two types of globidium-cysts of goats.

In the abomasum of naturally infected goats two types of globidium were found. An electron microscopic study revealed that the parasites were different from those described from sheep in former publications.

[Effect of rumen ciliates on the digestive utilization of various carbohydrate-rich diets and on the end-products formed in the rumen. II. Utilization of inulin, saccharose and lactose]. / Influence des ciliés du rumen sur l'utilisation digestive de différents régimes riches en glucides solubles et sur les produits terminaux formés dans le rumen. II. -- Régimes contenant de l'inuline, du saccharose et du lactose.

Three diets rich in inulin, saccharose and lactose, respectively, were given to 10 rumen-fistulated sheep. Two animals were defaunated, two were inoculated with either Polyplastron multivesibulatum or Entodinium sp., and two others were inoculated with both. The latter two were bred in conventional conditions. All animals ingested the same amounts of carbohydrates in the three diets (21-22 g/kg P0.75/day). Dietary nitrogen content was similar (table 1). The ciliate population was improved with the inulin diet (fig. 1; table 2). With a mixed population, the Entodinium improved with the inulin diet (fig 1; table 2). With a mixed population, the Entodinium sp. genus was always predominant. Holotrich protozoa (mainly Isotricha) in the rumen of the conventional sheep represented 15 to 30% of the total ciliate biomass, indicating that they were able to metabolize these soluble sugars. We also observed that P. multivesiculatum can ferment cellulose and all the soluble carbohydrates proposed in these diets. However, Entodinium sp. development occurred mainly in the presence of the sugard produced during carbohydrate hydrolysis by other ciliates or bacteria. The highest organic matter digestibility, noted in faunated animals (table 3) was confirmed by the VFA concentration in the rumen (table 4). This could be explained either by an activation of bacterial metabolism due to predation or by the direct effect of ciliates on fermentations, or both. Modifications in the VFA composition varied with ciliate inoculation, showing that ciliate metabolism may vary with the nature of the energy in the diet or that the observed results depended on various opposite effects in which the intensity of each component was influenced by the diet. In general, the acetic acid molar proportion increased and propionic acid decreased when there was a considerable Entodinium sp. population. The effect on butyric acid was low with these diets. Higher ammonia and lactic acid concentrations were observed in the rumen of faunated than defaunated sheep, irrespective of the ciliate inoculum.

A descriptive study of rumen digestion in meroxenic lambs according to the nature and complexity of the microflora.

We studied in meroxenic lambs, i.e. in lambs with a simplified digestive microflora, the effect of the microflora on the quantities of solid feed intake and on the main digestive parameters in the rumen. Axenic lambs were inoculated with a more or less complex flora, obtained by diluting (10(-8), 10(-7), 10(-8) a pool of rumen fluid taken either from young conventional lambs before weaning from adult sheep (Pool A) or from meroxenic lambs (Pool B). A few of these lambs then were inoculated with a genus of protozoa (Entodinium sp. or Polyplastron multivesiculatum). The results show that the main digestive parameters depended on the nature of the inocula which the lambs had received. Food consumption and volatile fatty acid concentration of the rumen fluid, low in lambs inoculated with the 10(-8) dilution, were higher in lambs inoculated with a more complex microflora (10(-6) and 10(-7) dilutions). The VFA concentration measured in these lambs however was approximately two times lower than that observed in conventional animals at the same age and fed the same feed. Food intake and the development of the fermentation pattern were favoured by an early inoculation of the animals. The complexity of the microflora appears to have influenced the composition of the VFA mixture. The latter was found to consist mainly of acetic acid in lambs inoculated with the 10(-8) dilution. In lambs which received the 10(-6) dilution, the composition of the VFA mixture was similar to that observed in conventional lambs. In all animals, except in lambs 10(-8), the ammonia nitrogen concentration of the rumen fluid was found to be higher during the first month after birth (between 100 and 200 mg/l). A subsequent decrease in ammonia nitrogen concentration was observed at two and a half months of age (20 to 40 mg/l). The establishment of protozoa ciliates in the rumen of these lambs was followed by an increase in butyric acid and ammonia nitrogen concentration.

Ecological factors determining establishment of cellulolytic bacteria and protozoa in the rumens of meroxenic lambs.

Ecological factors that control the establishment of cellulolytic bacteria and ciliate protozoa in the lamb rumen were studied in meroxenic animals. Axenic lambs received dilutions of rumen liquor from either conventional lambs and sheep (pool A) or meroxenic lambs (pool B). The total number of bacteria established in the rumen was between 10(9) and 5 x 10(10) g-1. In lambs inoculated with dilutions (10(-6), 10(-7), 10(-8)) of pool A, cellulolytic bacteria did not become established. However, subsequent inoculation with Bacteroides succinogenes, resulted in colonization in lambs that had received 10(-6) and 10(-7) dilutions of pool A. However, B. succinogenes became established in only one of three lambs that received the 10(-8) dilution. Similar results were obtained for the protozoan Entodinium sp. With pool B, lambs were inoculated earlier and cellulolytic bacteria were established directly from the 10(-6) and 10(-7) inocula. Polyplastron multivesiculatum establishment occurred readily when inoculated into the lambs which had received the 10(-6) dilution of pool B. Results obtained in this study suggest that establishment of cellulolytic bacteria and protozoa requires an abundant and complex flora and is favoured by early animal inoculation.

[Attempt to obtain and maintain rumen entodiniomorph ciliates in axenic cultures]. / Essai d'obtention et de survie de ciliés entodiniomorphes du rumen en cultures axéniques.

A method to obtain and maintain rumen ciliates in either axenic cultures or cultures with a reduced flora is described; it consists of two or three incubating periods with different antibiotic associations (separated by washing) and renewing of nutrient medium. On these conditions, Polyplastron multivesiculatum is obtained in an axenic state and can survive for 5 days. Entodinium sp. can survive for 10 to 14 days, but with a reduced bacterial population composed only of strict aerobic of facultative anaerobic species.

[Effect of rumen ciliates on the digestion of different carbohydrates in sheep. I.--Utilization of cell wall carbohydrates (cellulose and hemicellulose) and of starch]. / Influence des ciliés du rumen sur la digestion de différents glucides chez le mouton. I.--Utilisation des glucides pariétaux (cellulose et hémicelluloses) et de l'amidon.

Two diets rich in cell-wall carbohydrates or starch were given to 10 rumen-fistulated sheep; two sheep were defaunated and the others were inoculated with Polyplastron multivesiculatum (P) or Entodinium sp. (E), or both (P + E), or with conventional fauna. Ciliate biomass was greater when the animals were fed a high starch diet than when the diet was rich in cell-wall carbohydrates (table 2). With both diets, the Entodinium genus in the mixed fauna sampling predominated. We showed that Polyplastron was directly involved in cell-wall carbohydrate breakdown, while Entodinium capacity to digest cellulose remained low. We noted that with a diet rich in cellulose and hemicellulose, bacterial cellulolytic activity was improved by the presence of ciliates in the rumen but was decreased with the "starch" diet (table 3). The greater VFA concentration observed in the faunated animals expressed ciliate effect on the fermentations as well as activation of bacterial metabolism. With a high starch diet, the Entodinium sp. ciliates may have a buffering effect on the pH values in the rumen by limiting bacterial fermentation after food intake and by prolonging starch digestion during the day (table 4). The composition of the VFA mixture was modified by ciliate inoculation. The molar proportion of butyric acid always increased, while that of acetic and propionic acids evolved differently according to the diets and the ciliates (table 4). The higher ammonia concentration in the rumen liquor observed in faunated animals (table 4) could be explained either by the breakdown of both feed and bacterial proteins ingested by ciliates or by a lower ammonia nitrogen incorporation by fewer bacteria. Statistical analyses were used to explain the specific effect of P and E and also the interactions between them and between each of them and the diets.

Fine structure of trophozoites and cysts of the pathogenic diplomonad Spironucleus muris.

Using light and electron microscopy, we studied the ultrastructure of Spironucleus muris (syn. Hexamita muris) from spontaneously and experimentally infected normal and athymic mice. Kinetosomes and cytoskeletal fibers arranged in two-fold rotational symmetry are similar to the species Spironucleus elegans from amphibians. Taxonomic proposals and a possible evolutionary scheme for diplomonad genera accepted at the last International Congress of Parasitology are given. The genus Hexamita should be divided into two new genera: Hexamita (usually free-living) and Spironucleus (exclusively parasitic). We consider previous descriptions of hexamitiasis in rodents as dealing with spironucleosis. We distinguish a fresh and an old cyst of the parasite on the basis of structure, light refraction, location in the host, and infectivity. The transition from a fresh to an old cyst parallels the dessication of feces. The trophozoites can damage the microvilli and penetrate into the epithelium. We explain the differences in findings concerning intestinal lesions and forms of the disease (from acute to chronic to latent) by differences in the virulence of different parasite strains and by differences in inherent host resistance.