Burk A. Dehority

Phylogeny of the Rumen Ciliates Entodinium, Epidinium and Polyplastron (Litostomatea: Entodiniomorphida) Inferred from Small Subunit Ribosomal RNA Sequences

ABSTRACT. Three complete 18S ribosomal RNA gene sequences from the rumen ciliates, Entodinium coudatum (1,639 bp), Epidinium caudarum (1,638 bp), and Polyplastron multivesiculatum (1,640 bp) were determined and confrimed in the opposite direction. Trees produced using maximum parsimony and distance‐matrix methods (lest squares and neighbour‐joining). with strong bootstrap support, depict the rumen ciliates as a monophyletic group, Entodinium caudatum is the earliest branching rumen ciliate. However, Entodiniwn simplex does not pair with En. caudatum, but rather with Polyplastron multivesiculatum. Signature sequences for these rumen ciliates reveal that the published SSrRNA gene sequence from En. simplex is in fact a Polyoplastron species. The free‐living haptorian ciliates, Loxophyllum, Homalozoon and Spathidium (Subclass Hoptoria), are monophyletic and are the sister group to the rumen cilates. The litostomes (class Litostomatea), consisting of the haptorians and the rumen ciliates, are also a monophyletic group.

Effect of external resistance on bacterial diversity and metabolism in cellulose-fed microbial fuel cells.

External resistance affects the performance of microbial fuel cells (MFCs) by controlling the flow of electrons from the anode to the cathode. The purpose of this study was to determine the effect of external resistance on bacterial diversity and metabolism in MFCs. Four external resistances (20, 249, 480, and 1000 Ω) were tested by operating parallel MFCs independently at constant circuit loads for 10 weeks. A maximum power density of 66 mW m(-2) was achieved by the 20 Ω MFCs, while the MFCs with 249, 480, and 1000 Ω external resistances produced 57.5, 27, and 47 mW m(-2), respectively. Denaturing gradient gel electrophoresis analysis of partial 16S rRNA genes showed clear differences between the planktonic and anode-attached populations at various external resistances. Concentrations of short chain fatty acids were higher in MFCs with larger circuit loads, suggesting that fermentative metabolism dominated over anaerobic respiration using the anode as the final electron acceptor.

Protozoa of the digestive tract of herbivorous mammals

Occurrence of protozoa in the digestive tract of herbivores is dependent upon the occurrence of an environmentally compatible section of the tract and a retention time for gut contents in that section which exceeds the protozoan generation time. In general, herbivores can be classified as pre-gastric (foregut) or post-gastric (hindgut) fermentors. Fermentation in the foregut has evolved through enlargement of the stomach in some way to slow down ingesta passage rate and provide physical separation of the ingesta from the acid-secreting regions, as well as an adequate production of buffered saliva. Hindgut fermentations occur in the caecum-proximal colon area. Most of the protozoa in the digestive tract of herbivorous mammals belong to the class Kinetofragminophorea in the orders Prostomatida, Trichostomatida and Entodiniomorphida. They are anaerobic, live in conjunction with a large bacterial population and can ferment the structural polysaccharides of plants. End products of protozoal fermentation are similar to those of the bacterial population, i.e. volatile fatty acids, lactic acid, carbon dioxide and hydrogen. Additional products of the fermentation are vitamins and microbial protein which are subsequently utilized by foregut fermentors, whereas these products are essentially lost in hindgut fermentors unless they practice coprophagy. Only limited information is available on digestive and metabolic pathways of the gut protozoa, primarily because of our inability to grow them in axenic culture. Specific faunas appear to be associated with site of fermentation, animal species and diet. Diet in turn can affect pH and contents turnover time, both of which are very important in the establishment and growth of a protozoan population in the digestive tract.RésuméLa présence de protozoaires dans le tractus intestinal des herbivores est fonction de la présence d’une section environnementale compatible du tractus et d’un temps de rétention du contenu intestinal dans cette section excédant la durée de génération du protozoaire. De manière générale les herbivores peuvent être classés en fermenteurs pré-gastriques (intestin antérieur) ou en fermenteurs post-gastriques (intestin postérieur). La fermentation dans l’intestin antéroeur a évolué à travers un agrandissement de l’estomac, pour en quelque sorte ralentir le débit du bol alimentaire et permettre une séparation physique du bol alimentaire et des régions à sécrétion acide, ainsi qu’ à travers une production adéquate de salive tamponnée. Les fermentations dans l’intestin postérieur se produisent dans la région du colon caeco-proximal. La plupart des protozaires du tractus intestinal des mammifères herbivores appartiennent à la classe des Ciliés, ordres des Gymnostomatida, des Trichostomatida et des Entodiniomorphida. Ils sont anaerobiques, vivent en conjonction avec une large population bactérienne et peuvent fermenter les polysaccharides structuraux des plantes. Les produits finaux de la fermentation protozoaire sont similaires à ceux de la population bactérienne, c’est-à-dire acides gras volatiles, acide lactique, dioxide de carbone et hydrogène. Les produits additionnels de la fermentation sont des vitamines et des proteines microbiennes qui sont subséquemment utilisées par les fermenteurs “à intestin antérieur” alors que ces produits sont essentiellements perdus chez les fermenteurs “à intestin postérieur” sauf si ils pratiquent la coprophagie. Seulement des informations limitées sont disponibles sur les mécanismes digestifs et métaboliques des protozaires intestinaux, principalement à cause de notre incapacité à les cultiver en culture axénique. Des faunes spécifiques paraissent être associées au site de fermentation, à l’espèce animale et au régime alimentaire. La régime alimentaire peut à son tour affecté le pH et la durée de rétention du contenu, ces 2 facteurs étant très importants dans l’établissement et la croissance d’une population protozoaire dans le tractus intestinal.

Effects of microbial synergism on fibre digestion in the rumen

The primary sources of energy found in forages are the structural polysaccharides, cellulose, hemicellulose and pectin. These three components generally account for about 400-600 g/kg forage dry matter (Lagowski et al. 1958; Waite & Garrod, 1959; Chesson & Forsberg, 1988). The inability of most animals to digest these structural polysaccharides has resulted in some of them adopting a microbial population which can. In essence the animal provides an environmentally suitable area for growth of these micro-organisrfls, which in turn digcst the forage structural carbohydrates and thereby supply energy to the host (Hungate, 1972; Dehority, 1986). Since most of these digestive tract microorganisms have complex nutritional requirements and can only utilize one or two of the major polysaccharides, synergism between the various organisms can be important for the efficient use of forages by the ruminant animal. Microbial synergism is defined as increased growth or productivity resulting from the combination of two or more micro-organisms, which exceeds the additive effects of their separate activities. In general, this occurs through crossfecding of hydrolysis products. utilization of end-products or production of an essential nutrient. One of the best examples of crossfeeding of hydrolysis products in the rumen is probably the utilization of cellodextrins by non-cellulolytic rumen bacteria (Scheifinger & Wolin, 1973: Bryant & Wolin, 1975; Russell, 1985). End-product utilization is best exemplified by the rumen methanogens, which use hydrogen and carbon dioxide to generate energy through production of methane (Stumm et al. 1982: Russell & Wallace, 1988; Wolin & Miller, 1988). Conversion of succinate, a normal end-product of many rumen bacteria, to propionate would be another example of this type of synergism (Scheifinger & Wolin, 1973; Russell & Wallace, 1988; Wolin & Miller, 1988). Nutritional interdependence. production of a nutrient by one species which is essential for a second species, generally involves the vitamins, amino acids and branched-chain fatty acids (Miura et al. 1980; Wallace, 1985; Wolin & Miller, 1988).

The effects of feeding monensin and yeast culture, alone or in combination, on the concentration and generic composition of rumen protozoa in steers fed on low-quality pasture supplemented with increasing levels of concentrate

Sixteen zebu-cross steers, kept under grazing conditions, were supplemented with increasing levels of concentrate (0.4, 0.7 and 1.0% of body weight) over three periods of 41, 41 and 42 days, respectively. The animals were divided into four groups, a control plus groups supplemented with either monensin, yeast culture or a combination of monensin and yeast culture. Samples of rumen contents were taken on Day 0 and at the end of each period to determine the concentration and generic composition of rumen protozoa. After 124 days on treatment, protozoal concentrations increased in the controls (p<0.01), yeast culture treatment (p<0.03) and monensin treatment (p<0.06). However, protozoal concentrations did not change in the steers fed the combination of monensin+yeast culture. No marked differences were observed in the generic composition of protozoa among the four treatments. Entodinium was the predominant genus in all groups: control (87.7%); monensin (78.1%); yeast culture (69.6%) and the combination of both, (89.3%). The lower percentage of Entodinium in the monensin and yeast culture treatments was primarily replaced by an increase in the percentage of Dasytricha. The rest of the ciliate population, which belonged to ≈9 genera, was observed in similar proportions for all treatment groups.

Antibiosis between ruminal bacteria and ruminal fungi.

ABSTRACT Cellulose digestion, bacterial numbers, and fungal numbers were monitored over time in vitro by using a purified cellulose medium with and without antibiotics (penicillin and streptomycin). All fermentations were inoculated with a 1:10 dilution of whole rumen contents (WRC). Without antibiotics, cellulose digestion was higher (P < 0.01) at 24, 30, 48, and 72 h; fungi had almost disappeared by 24 h, while bacterial concentrations increased over 100-fold in 24 h and then decreased gradually up to 72 h. In those fermentations with added antibiotics, fungal concentrations increased 4-fold by 30 h and up to 42-fold at 72 h; bacterial concentrations were markedly reduced by 24 h and remained low through 72 h. Similar results were obtained with ground alfalfa as a substrate. In further studies, the in vitro fermentation of purified cellulose without antibiotics was stopped after 18 to 20 h, and the microbial population was killed by autoclaving. Antibiotics were added to half of the tubes, and all tubes were reinoculated with WRC. After 72 h, extensive cellulose digestion had occurred in those tubes without antibiotics, as compared to very low cellulose digestion with added antibiotics. The extent of this inhibition was found to increase in proportion to the length of the initial fermentation period, suggesting the production of a heat-stable inhibitory factor or factors. The inhibitory activity was present in rumen fluid, could be extracted from lyophilized rumen fluid (LRF) with water, and was stable in response to proteolytic enzymes. In addition, the water-extracted residue of LRF was found to contain growth factor activity for rumen fungi in vitro.

Rumen ciliate protozoa of the blue duiker (Cephalophus monticola), with observations on morphological variation lines within the species Entodinium dubardi.

ABSTRACT. Protozoal concentrations were determined in rumen and cecal contents of 20 blue duikers (Cephalophus monticola). Ten animals of each sex were fed either a high concentrate or high roughage diet. Rumen protozoa were present in 19 of the 20 animals and concentrations ranged from 4.5 to 33.7 × 106 per g of rumen contents. At the higher concentrations, protozoal cells equaled between 30–40% of the total rumen contents volume. No protozoa were found in cecal contents. Weight of rumen contents was higher in females than in males (P < 0.01), and rumen protozoa concentrations were higher in males (P < 0.05) and in those animals fed the high concentrate diet (P < 0.05). All the protozoa were identified as belonging to a single species, Entodinium dubardi. However, an average of about 30% of the E. dubardi cells varied from the typical morphology of this species. These cells appeared to be on variation lines leading toward 7–10 other non‐caudate species of Entodinium. The present data were used to evaluate and discuss the concept of variation lines within E. dubardi.

Effect of pH on viability of Entodinium caudatum, Entodinium exiguum, Epidinium caudatum, and Ophryoscolex purkynjei in vitro

Abstract. Cultures of Entodinium caudatum, Entodinium exiguum, Epidinium caudatum, and Ophryoscolex purkynjei were grown and transferred in poorly buffered media prepared using different concentrations of sodium bicarbonate and a nitrogen gas phase. By transferring every 12 or 24 h, culture pH was gradually decreased until the protozoa disappeared. The cultures were transferred by placing half of the culture into an equal volume of fresh medium, resulting in pH fluctuations similar to those in the rumen, resulting from fermentation, eating, and saliva production. All four species appeared to maintain their concentrations around pH 5.8, but numbers decreased as pH values fell below 5.6. The four species were similar in that they all survived above pH 5.3. These results differ from previous reports in which Entodinium species appeared to be more tolerant to low pH than all other species of rumen ciliates. No adaptation to low pH was observed in Epidinium caudatum cultures after recovery from pH 5.4 medium containing only one or two viable cells.

Influence of Diet on the Rumen Protozoal Fauna of Indigenous African Wild Ruminants

Abstract A study was carried out to determine if the protozoal fauna of indigenous African wild ruminants was different from that found in their domestic counterparts and if the animals diet influenced the number and types of protozoa. Samples of rumen contents were collected in 1997 and 2001 from various indigenous African wild ruminants in Kenya. All three ruminant feeding types were sampled: browsers or concentrate selectors (giraffe and Guenthers dik-dik); intermediate or adaptable mixed feeders (impala, Thomsons gazelle, Grants gazelle and eland); grass or roughage eaters (hartebeest and wildebeest). Total concentration of ciliate protozoa and percentage generic distribution were determined. In general, protozoal concentrations were higher in concentrate selectors, followed by the intermediate or opportunistic mixed feeders and lowest in the grass and roughage eaters. Both Thomsons and Grants gazelle were protozoa-free in the 2001 samples. Entodinium percentages were considerably higher in concentrate selectors and intermediate mixed feeders, compared to roughage eaters. Two genera of protozoa previously found in only a few African ruminants, Epiplastron and Opisthotrichum, were observed in several additional animal species and represent new host records. A difference was noted in the protozoal species composition of the indigenous wild ruminants from that previously observed in African domestic ruminants.

A new family of entodiniomorph protozoa from the marsupial forestomach, with descriptions of a new genus and five new species.

ABSTRACT. A unique group of entodiniomorph protozoa was found in forestomach contents from quokka (Setonix brachyurus), western grey kangaroo (Macropus fuliginosus), red kangaroo (Macropus rufus) and euro (Macropus robustus erubescens). A new genus, Macropodinium n.g., containing five new species, is described. Three species are described from forestomach contents of the quokka: Macropodinium baldense n. sp., Macropodinium moiri n. sp. and Macropodinium setonixum n. sp. A single species, Macropodinium ennuensis n. sp., is described from the red kangaroo and euro. The last species, Macropodinium yalanbense n. sp., is described in forestomach contents from the western grey kangaroo. At least three distinct features in the new genus are incompatible with any of the described families in the order Entodiniomorphida. On this basis, the new family Macropodiniidae has been created.