Absence Of Protozoa Research Articles

Effects of Rumen Protozoa of Brahman Heifers and Nitrate on Fermentation and In vitro Methane Production.

Two experiments were conducted assessing the effects of presence or absence of rumen protozoa and dietary nitrate addition on rumen fermentation characteristics and in vitro methane production in Brahman heifers. The first experiment assessed changes in rumen fermentation pattern and in vitro methane production post-refaunation and the second experiment investigated whether addition of nitrate to the incubation would give rise to methane mitigation additional to that contributed by defaunation. Ten Brahman heifers were progressively adapted to a diet containing 4.5% coconut oil distillate for 18 d and then all heifers were defaunated using sodium 1-(2-sulfonatooxyethoxy) dodecane (Empicol). After 15 d, the heifers were given a second dose of Empicol. Fifteen days after the second dosing, all heifers were allocated to defaunated or refaunated groups by stratified randomisation, and the experiment commenced (d 0). On d 0, an oral dose of rumen fluid collected from unrelated faunated cattle was used to inoculate 5 heifers and form a refaunated group so that the effects of re-establishment of protozoa on fermentation characteristics could be investigated. Samples of rumen fluid collected from each animal using oesophageal intubation before feeding on d 0, 7, 14, and 21 were incubated for in vitro methane production. On d 35, 2% nitrate (as NaNO3) was included in in vitro incubations to test for additivity of nitrate and absence of protozoa effects on fermentation and methane production. It was concluded that increasing protozoal numbers were associated with increased methane production in refaunated heifers 7, 14, and 21 d after refaunation. Methane production rate was significantly higher from refaunated heifers than from defaunated heifers 35 d after refaunation. Concentration and proportions of major volatile fatty acids, however, were not affected by protozoal treatments. There is scope for further reducing methane output through combining defaunation and dietary nitrate as the addition of nitrate in the defaunated heifers resulted in 86% reduction in methane production in vitro.

Effect of diet and absence of protozoa on the rumen microbial community and on the representativeness of bacterial fractions used in the determination of microbial protein synthesis1

Accurate estimates of microbial synthesis in the rumen are vital to optimize ruminant nutrition. Liquid- (LAB) and solid-associated bacterial fractions (SAB) harvested from the rumen are generally considered as microbial references when microbial yield is calculated; however, factors that determine their composition are not completely understood. The aim of this study was to evaluate the effect of diet and absence or presence of rumen protozoa on the rumen microbial community. It was hypothesized that these treatments could modify the composition and representativeness of LAB and SAB. Twenty twin lambs (Ovis aries) were used; one-half of the twins were kept protozoa-free, and each respective twin sibling was faunated. At 6 mo of age, 5 animals from each group were randomly allocated to the experimental diets consisting of either alfalfa hay as the sole diet, or 50:50 mixed with ground barley grain. After 15 d of adaptation to the diet, animals were euthanized, rumen and abomasum contents were sampled, and LAB and SAB isolated. The presence of protozoa buffered the effect of diet on the rumen bacterial population. Faunated animals fed alfalfa hay had a greater abundance of F. succinogenes, anaerobic fungi and methanogens, as well as an enhanced rumen bacterial diversity. Cellulolytic bacteria were more abundant in SAB, whereas the abomasal abundance of most of the microorganisms studied was closer to those values observed in LAB. Rumen and abomasal samples showed similar bacterial DNA concentrations, but the fungal and protozoal DNA concentration in the abomasum was only 69% and 13% of that observed in the rumen, respectively, suggesting fungal and protozoal sequestration in the rumen or possible preferential degradation of fungal and protozoal DNA in the abomasum, or both. In conclusion, absence of protozoa and type of diet extensively modified the chemical composition of LAB and SAB as a consequence of changes in the microbial composition of these fractions.

Upgrading the efficiency of an external nitrification BNR system – The modified Dephanox process

Two types of external nitrification biological nutrient removal (BNR) plants were successively operated for municipal wastewater treatment. The first plant configuration was based on the conventional Dephanox design. Basic operational parameters were chosen as variables in order to assess their impact on the plant efficiency. The experimental results indicated that minimization of the aerobic mass fraction to values as low as 11% was practically feasible, resulting to high nutrient removal percentages, of 81% and 83%, respectively for nitrogen and phosphorus. However, the activated sludge characteristics with pin flocs formation and the absence of protozoa, led to increased total suspended solids concentration in the plant effluent (42 mg TSS/L in average). By increasing the aerated mass fraction to 16–17%, the sludge quality characteristics were improved and the pin flocs phenomenon was moderated. The increase in the aerated mass fraction was achieved “indirectly”, because of the lower biomass concentration in the anaerobic reactor, by establishing an additional mixed liquor recirculation flow in the conventional Dephanox design. This new configuration, referred as the modified Dephanox process, allowed for maintaining high anoxic P uptake extend with simultaneous enhancement of the plant denitrification potential. Fluorescence in situ hybridization (FISH) demonstrated that the well-known Phosphorus Accumulating Organism (PAO), Candidatus Accumulibacter phosphatis, was present in both the conventional and modified Dephanox process. The new configuration was proven to be more efficient regarding nutrient removal, being an interesting modification of the typical Dephanox design.

Study of the effect of presence or absence of protozoa on rumen fermentation and microbial protein contribution to the chyme1

The aim of this study was to investigate the effect of presence or absence of protozoa on rumen fermentation and efficiency of microbial protein synthesis under different diets. Of 20 twin paired lambs, 1 lamb of each pair was isolated from the ewe within 24 h after birth and reared in a protozoa-free environment (n = 10), whereas their respective twin-siblings remained with the ewe (faunated, n = 10). When lambs reached 6 mo of age, 5 animals of each group were randomly allocated to 1 of 2 experimental diets consisting of either alfalfa hay as the sole diet, or 50:50 mixed with ground barley grain according to a 2 × 2 factorial arrangement of treatments. After 15 d of adaptation to the diet, the animals were euthanized and total rumen and abomasal contents were sampled to estimate rumen microbial synthesis using C(31) alkane as flow marker. Different ((15)N and purine bases) and a novel (recombinant DNA sequences) microbial markers, combined with several microbial reference extracts (rumen protozoa, liquid and solid associated bacteria) were evaluated. Absence of rumen protozoa modified the rumen fermentation pattern and decreased total tract OM and NDF digestibility in 2.0 and 5.1 percentage points, respectively. The effect of defaunation on microbial N flow was weak, however, and was dependent on the microbial marker and microbial reference extract considered. Faunated lambs fed with mixed diet showed the greatest rumen protozoal concentration and the least efficient microbial protein synthesis (29% less than the other treatments), whereas protozoa-free lambs fed with mixed diet presented the smallest ammonia concentration and 34% greater efficiency of N utilization than the other treatments. Although (15)N gave the most precise estimates of microbial synthesis, the use of recombinant DNA sequences represents an alternative that allows separate quantification of the bacteria and protozoa contributions. This marker showed that presence of protozoa decrease the bacterial-N flow through the abomasum by 33%, whereas the protozoa-N contribution to the microbial N flow increased from 1.9 to 14.1% when barley grain was added to the alfalfa hay. Absolute data related to intestinal flow must be treated with caution because the limitations of the sampling and maker system employed.

Long-term defaunation increases the abundance of cellulolytic ruminococci and methanogens but does not affect the bacterial and methanogen diversity in the rumen of sheep1

Protozoa are commensal eukaryotes in the rumen of herbivores. Protozoa are large producers of hydrogen, which is utilized by methanogenic archaea to produce methane, a greenhouse gas. The removal of protozoa from the rumen (defaunation) decreases methanogenesis, but also negatively affects fiber digestion, which is the main function of the rumen. The aim of this study was to examine the effect of long-term defaunation on the structure of the microbiota and particularly methanogenic archaea and fibrolytic bacteria to better understand the microbial mechanisms responsible for the decrease in methanogenesis and fibrolysis. The trial was conducted in 5 adult sheep subjected successively to long-term defaunation (2 yr), refaunation (12 wk), and short-term defaunation (10 wk). Methanogens were enumerated by quantitative PCR targeting the rrs (16S ribosomal RNA subunit) and mcrA (methyl coenzyme-M reductase) genes. The rrs gene was used to quantify the 3 major culturable rumen cellulolytic bacterial species (i.e., Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) and total bacteria. Bacterial and methanogen diversity was also examined by PCR-DGGE (PCR-denaturing gradient gel electrophoresis) analysis targeting the rrs and mcrA genes, respectively. Total rumen bacterial density estimated as rrs copies per gram of DM of rumen content increased in response to long- and short-term defaunation (+1 log, P < 0.001), but without noticeable shifts in diversity. Defaunation increased the rrs copies per gram of DM of rumen content of R. albus and R. flavefaciens (+2 log, P < 0 0.001), but did not affect that of F. succinogenes. Despite a 20% reduction in methane emission in the 2 defaunated periods, the mcrA and rrs copies of methanogens per gram of DM of rumen content increased (+1 log, P < 0.001) in the absence of protozoa, whereas the diversity of the dominant methanogenic community was not modified. This study shows no major difference between long- and short-term defaunation in abundance and diversity of bacteria and archaea. It also provides evidence that monitoring the abundance and diversity of methanogens is not sufficient to comprehend the microbial mechanisms leading to a reduction in methane emissions by ruminants. This study also reports for the first time in sheep a selective effect of defaunation on the abundance of cellulolytic bacterial species.

Ciliates rapidly enhance the frequency of conjugation between Escherichia coli strains through bacterial accumulation in vesicles

The mechanism underlying bacterial conjugation through protozoa was investigated. Kanamycin-resistant Escherichia coli SM10λ + carrying pRT733 with Tn phoA was used as donor bacteria and introduced by conjugation into ciprofloxacin-resistant E. coli clinical isolate recipient bacteria. Equal amounts of donor and recipient bacteria were mixed together in the presence or absence of protozoa (ciliates, free-living amoebae, myxamoebae) in Page’s amoeba saline for 24 h. Transconjugants were selected with Luria broth agar containing kanamycin and ciprofloxacin. The frequency of conjugation was estimated as the number of transconjugants for each recipient. Conjugation frequency in the presence of ciliates was estimated to be approximately 10 −6, but in the absence of ciliates, or in the presence of other protozoa, it was approximately 10 −8. Conjugation also occurred in culture of ciliates at least 2 h after incubation. Successful conjugation was confirmed by the polymerase chain reaction. Addition of cycloheximide or latrunculin B resulted in suppression of conjugation. Heat killing the ciliates or bacteria had no effect on conjugation frequency. Co-localization of green fluorescent protein-expressing E. coli and PKH-67-vital-stained E. coli was observed in the same ciliate vesicles, suggesting that both donor and recipient bacteria had accumulated in the same vesicle. In this study, the conjugation frequency of bacteria was found to be significantly higher in vesicles purified from ciliates than those in culture suspension. We conclude that ciliates rapidly enhance the conjugation of E. coli strains through bacterial accumulation in vesicles.

Effect of Nylon Bag and Protozoa on In Vitro Corn Starch Disappearance

An in vitro experiment was carried out to study whether the presence of protozoa in nylon bags can explain the underestimation of the in situ degradation of slowly degradable starch. Corn of a high (flint) and a low (dent) vitreousness variety was ground over a 3-mm screen, weighed in nylon bags with a pore size of 37μ m, and washed in cold water. Samples of washed cornstarch were incubated in 40-mL tubes with faunated and defaunated ruminal fluid. An additional amount of washed corn, in nylon bags, was inserted in each incubation tube. Incubations were carried out for 0, 2, 4, 6, 12, and 24h, and starch residue in tube and nylon bag was determined. In general, starch disappearance from the nylon bag was less than from the tube, and was less with faunated than defaunated rumen fluid, but corn variety did not affect starch disappearance. When no protozoa were present, the disappearance of starch from the bags was higher after 6 and 12h incubation compared with presence of protozoa. However, in the tubes, there was no difference in starch disappearance due to presence or absence of protozoa. Estimated lag time was higher in presence (4.6h) then absence (3.6h) of protozoa. It was concluded that the effect of presence or absence of protozoa on starch disappearance differs within or outside nylon bags. The reduced disappearance rate of starch inside the nylon bags in the presence of protozoa helps to explain the underestimation of starch degradation based on the in sacco procedure when compared with in vivo data upon incubation of slowly degradable starch sources.

Effect of ruminal protozoa on fecal microbiota in cattle using terminal restriction fragment length polymorphism

ABSTRACTThe effect of the presence of ruminal protozoa on the composition of fecal microbiota in cattle was investigated. Six castrated Holstein cattle (mean bodyweight 137 kg) were divided into two groups: three faunated and three unfaunated. The fecal bacterial composition of the faunated and unfaunated cattle was compared using a culture method and by terminal restriction fragment length polymorphism (T‐RFLP) analysis. Approximately 0.4 to 2.3% of the bacterial cells detected by microscopy formed colonies on medium 10. The major terminal restriction fragments were detected in the T‐RFLP profiles generated by Hha I and Msp I digestion in both the faunated and unfaunated cattle. In particular, the Bacteroides group, the Clostridium coccoides group and the Clostridium leptum subgroup might be the known bacterial groups that protozoa influence by Msp I digestion. From the dendrogram analysis by T‐RFLP patterns, the faunated and unfaunated cattle were divided into two clusters, I and II, respectively. These results suggest that absence of protozoa in the rumen changes the composition of fecal bacteria.

Changes in methane emission and rumen fermentation parameters induced by refaunation in sheep

Elimination of protozoa or defaunation is known to reduce methane emission by ruminants. However, the long-term efficacy of this practice is not well documented. In this work methane and other end products of fermentation were monitored in five adult, rumen-cannulated wethers that had been kept defaunated for two years (Def1) and successively refaunated (Fau, 12 weeks) and defaunated (Def2) during a 6-month-long experiment. Methane was measured for periods of 4 days at about 4-weekly intervals using the SF6 tracer technique, and rumen content samples were taken regularly throughout the experiment. Refaunation was performed by single administration of a mixed protozoal inoculum (103 cells). A noticeable protozoal population was observed at 12 days after inoculation, which was followed by an exponential increase that peaked at 12 ± 3 × 105 cells/mL rumen fluid at 4 weeks and decreased thereafter to 8 ± 4 × 105 cells/mL at the end of the 12-week Fau period. Production of methane, which was 33 ± 8 L/animal.day in Def1, increased (P &lt; 0.05) up to 42 ± 5 L at the end of the Fau period and dropped back at the end of Def2 to 35 ± 7 L (Def1 v. Def2, P &gt; 0.05). Even though protozoal numbers were comparable to conventional animals 20 days after inoculation, the increase in methane production was observed later, at 8 weeks, suggesting that protozoa are not the only microbial factor favouring methanogenesis and that the ecosystem needs a relatively long adaptation time following protozoal introduction to optimise this function. The presence of protozoa did not affect total volatile fatty acids and acetate concentration in the rumen but increased butyrate, while decreasing propionate concentration (Fau v. Def1, Def2, P &lt; 0.05). These results show that the decrease in methane emissions induced by defaunation in sheep was stable for a period of up to 2 years. In the absence of protozoa, methane decreased by ~20% in both short- and long-term defaunated animals.

Persistence of defaunation effects on digestion and methane production in ewes

The effect of defaunation on feed digestion and on methane production of ewes was determined 10 and 25 weeks after defaunation. When fed a lucerne diet (800 g/day), the absence of protozoa did not change the apparent digestibility of dry matter, excretion of macro-minerals, or methane production 10 or 25 weeks post-treatment. Defaunation did, however, increase microbial protein flow by 22% (estimated from allantoin excretion) and the molar proportions of acetate and butyrate in the rumen, while decreasing excretion of copper and manganese. The fermentation data contrasts with previous studies that found defaunation reduced methanogenesis and rumen acetate proportions, but supports the hypothesis that a reduction in rumen acetate percentage is required to achieve reduced methanogenesis through defaunation.

Impact of protozoan grazing on nitrification and the ammonia- and nitrite-oxidizing bacterial communities in activated sludge

In activated sludge, protozoa feed on free-swimming bacteria and suspended particles, inducing flocculation and increasing the turnover rate of nutrients. In this study, the effect of protozoan grazing on nitrification rates under various conditions in municipal activated sludge batch reactors was examined, as was the spatial distribution of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) within the activated sludge. The reactors were monitored for ammonia, nitrite, nitrate, and total nitrogen concentrations, and bacterial numbers in the presence and absence of cycloheximide (a protozoan inhibitor), allylthiourea (an inhibitor of ammonia oxidation), and EDTA (a deflocculating agent). The accumulations of nitrate, nitrite, and ammonia were lower in batches without than with protozoa grazing. Inhibition of ammonia oxidation also decreased the amount of nitrite and nitrate accumulation. Inhibiting protozoan grazing along with ammonia oxidation further decreased the amounts of nitrite and nitrate accumulated. Induction of deflocculation led to high nitrate accumulation, indicating high levels of nitrification; this effect was lessened in the absence of protozoan grazing. Using fluorescent in situ hybridization and confocal laser scanning microscopy, AOB and NOB were found clustered within the floc, and inhibiting the protozoa, inhibiting ammonia oxidation, or inducing flocculation did not appear to lower the number of AOB and NOB present or affect their position within the floc. These results suggest that the AOB and NOB are present but less active in the absence of protozoa.

The effect of absence of protozoa on methane emissions by lambs

Livestock are one of the largest single sources of methane emission, equivalent to 15-20 % of total anthropogenic methane. Selective suppression of the rumen protozoa has been suggested to be promising approach to reduce methane release (Moss et al., 2000) as up to 25 % of the methanogens in the rumen are associated with protozoa (Newbold et al., 1995). However, contradictory results have been reported between in vitro and in vivo data and short and long term defaunation experiments (Ranilla et al., 2003). This study was carried out to investigate the effect of the absence of protozoa in the rumen on enteric methane production by lambs.

Archaeal community structure diversity in the rumen of faunated and defaunated sheep

Elimination of protozoa from the rumen is usually accompanied by a decrease in methane production. However, the possible changes brought about by defaunation upon the structure of the methanogenic population are not known. Archaeal communities of rumen samples from 2 faunated (F), 2 recently defaunated (3 months, D−), and 2 long-term defaunated (+ 1 year, D+) sheep were profiled by PCR-denaturing gradient gel electrophoresis (DGGE) using 16S rDNA primers. Dendrogram analysis of the whole densitometric curves separated F and D+ in different clusters. Samples from D− animals did not show high similarity and were not separated into an independent cluster although they tended to be placed at an intermediate distance between F and D+. Principal components analysis (PCA) also confirmed these results. Bands explaining most of the differences in PCA could be correlated ( P < 0.05) to the presence/absence of protozoa. As measured by the Shannon index (H) based on DGGE operational taxonomic units, D+ populations had a lower diversity than F or D− populations ( P < 0.05). Shifts in the archaeal community structure of the rumen in the presence or absence of protozoa were identified. Population changes that follow defaunation seem to develop slowly as evidenced by the differences between recent and long-term defaunated animals.

Planktonic Replication Is Essential for Biofilm Formation byLegionella pneumophilain a Complex Medium under Static and Dynamic Flow Conditions

Legionella pneumophila persists for a long time in aquatic habitats, where the bacteria associate with biofilms and replicate within protozoan predators. While L. pneumophila serves as a paradigm for intracellular growth within protozoa, it is less clear whether the bacteria form or replicate within biofilms in the absence of protozoa. In this study, we analyzed surface adherence of and biofilm formation by L. pneumophila in a rich medium that supported axenic replication. Biofilm formation by the virulent L. pneumophila strain JR32 and by clinical and environmental isolates was analyzed by confocal microscopy and crystal violet staining. Strain JR32 formed biofilms on glass surfaces and upright polystyrene wells, as well as on pins of "inverse" microtiter plates, indicating that biofilm formation was not simply due to sedimentation of the bacteria. Biofilm formation by an L. pneumophila fliA mutant lacking the alternative sigma factor sigma(28) was reduced, which demonstrated that bacterial factors are required. Accumulation of biomass coincided with an increase in the optical density at 600 nm and ceased when the bacteria reached the stationary growth phase. L. pneumophila neither grew nor formed biofilms in the inverse system if the medium was exchanged twice a day. However, after addition of Acanthamoeba castellanii, the bacteria proliferated and adhered to surfaces. Sessile (surface-attached) and planktonic (free-swimming) L. pneumophila expressed beta-galactosidase activity to similar extents, and therefore, the observed lack of proliferation of surface-attached bacteria was not due to impaired protein synthesis or metabolic activity. Cocultivation of green fluorescent protein (GFP)- and DsRed-labeled L. pneumophila led to randomly interspersed cells on the substratum and in aggregates, and no sizeable patches of clonally growing bacteria were observed. Our findings indicate that biofilm formation by L. pneumophila in a rich medium is due to growth of planktonic bacteria rather than to growth of sessile bacteria. In agreement with this conclusion, GFP-labeled L. pneumophila initially adhered in a continuous-flow chamber system but detached over time; the detachment correlated with the flow rate, and there was no accumulation of biomass. Under these conditions, L. pneumophila persisted in biofilms formed by Empedobacter breve or Microbacterium sp. but not in biofilms formed by Klebsiella pneumoniae or other environmental bacteria, suggesting that specific interactions between the bacteria modulate adherence.

Grazing of a common species of soil protozoa ( Acanthamoeba castellanii) affects rhizosphere bacterial community composition and root architecture of rice ( Oryza sativa L.)

We performed a controlled experiment with rice seedlings ( Oryza sativa L.) growing in Petri dishes on homogeneous nutrient agar containing a simple rhizosphere food web consisting of a diverse bacterial community and a common soil protozoa, Acanthamoeba castellanii, as bacterial grazer. Presence of amoebae increased bacterial activity and significantly changed the community composition and spatial distribution of bacteria in the rhizosphere. In particular, Betaproteobacteria did benefit from protozoan grazing. We hypothesize that the changes in bacterial community composition affected the root architecture of rice plants. These effects on root architecture affect a fundamental aspect of plant productivity. Root systems in presence of protozoa were characterized by high numbers of elongated (L-type) laterals, those laterals that are a prerequisite for the construction of branched root systems. This was in sharp contrast to root system development in absence of protozoa, where high numbers of lateral root primordia and short (S-type) laterals occurred which did not grow out of the rhizosphere region of the axile root. As a consequence of nutrient release from grazed bacteria and changes in root architecture, the nitrogen content of rice shoots increased by 45% in presence of protozoa. Our study illustrates that interactions over three trophic levels, i.e. between plants, bacteria and protozoa significantly modify root architecture and nutrient uptake by plants.

Effects of protozoa on Methane production in rumen and hindgut of calves around time of weaning

Effects of the presence or absence of ciliate protozoa on methanogenesis in the rumen and hindgut were investigated in young calves during a 7-week period. Ten Holstein calves, aged 7 days, were divided in two groups (n = 5) and fed an increasing amount of a commercial milk replacer and small amounts of a calves starter. One group was inoculated with ciliate fauna on two occasions, week 5 and 6, while the second remained ciliate-free. The absence of protozoa in the rumen decreased rumen empty weight ( − 23%, P < 0.01), and rumen pool size of N ( − 36%, P < 0.01) and crude fat ( − 37%, P < 0.05). Rumen bacteria of non-faunated calves contained a higher proportion of total amino acid-N per 16 g N ( + 3%, P < 0.01) and D-alanine-N per 16 g N ( + 13%, P < 0.05) compared to faunated calves. Further results contain a reference for a higher bacterial mass in the ciliate-free rumen with an increased number of bacteria adherent to rumen mucosa. The CH4 production in the rumen increased exponentially with the increase in protozoa population size (R2 = 0.68). In presence of 46 · 104 protozoa per ml rumen fluid, the in vitro CH4 production of rumen fluid per mol total VFA was about 34% higher in faunated than in non-faunated calves (P < 0.001). Hydrogen (2H) recovery of rumen fermentation was positively correlated (R2 = 0.55) to the CH4 production rate. Methanogens were attached on rumen mucosa. Methanogenesis, induced by rumen mucosa attached bacteria, was stimulated by ruminal protozoa. In the absence of protozoa in the rumen, the acetate - propionate ratio and butyrate proportion of VFA were reduced. In vivo in the absence of protozoa not only the whole animal CH4 production ( − 30%, P < 0.05) but also the digestibility of carbohydrates ( − 4%, P < 0.05) was reduced. Thereby no difference was observed in the intake of ME per kg DM between the groups. In conclusion, the methanogenesis in the rumen, but not in hindgut, is associated with the development of the ruminal protozoa population. The level of methanogenesis (mol/mol VFA) in the hindgut amounts to 20% of the ruminal methanogenesis.

Role of biofilms in the survival of Legionella pneumophila in a model potable-water system.

Legionellae can infect and multiply intracellularly in both human phagocytic cells and protozoa. Growth of legionellae in the absence of protozoa has been documented only on complex laboratory media. The hypothesis upon which this study was based was that biofilm matrices, known to provide a habitat and a gradient of nutrients, might allow the survival and multiplication of legionellae outside a host cell. This study determined whether Legionella pneumophila can colonize and grow in biofilms with and without an association with Hartmannella vermiformis. The laboratory model used a rotating disc reactor at a retention time of 6.7 h to grow biofilms on stainless steel coupons. The biofilm was composed of Pseudomonas aeruginosa, Klebsiella pneumoniae and a Flavobacterium sp. The levels of L. pneumophila cells present in the biofilm were monitored for 15 d, with and without the presence of H. vermiformis, and it was found that, although unable to replicate in the absence of H. vermiformis, L. pneumophila was able to persist.

Protozoan feeding on natural and cultured bacteria deposited on inert polymeric and mineral membrane filters

Early stages of development of marine bacterial biofilms were represented by cultured or freshly‐collected natural bacteria radiolabelled and deposited on 0.2 μm pore size polycarbonate (Nuclepore) or aluminium oxide (Anopore) filters. A proportion of the bacteria gradually resuspended themselves, but few were lost from the mineral filters; larger quantities of bacteria were lost from the surfaces of filters (especially the mineral ones) in the presence of un‐specialised protozoa than in the absence of protozoa. The extent of grazing on deposited bacteria and the assimilation of bacterial biomass was measured separately for the flagellates Paraphysomonas imperforata, Pteridomonas danica and Cafeteria menbergensis and the ciliate Uronema marinum. Deposited bacteria were grazed similarly by all four protozoa to the extent of about 25% from polymeric filters and 7% from mineral filters. The proportion of the consumed biomass of cultured Vibrio natriegens that was assimilated by protozoa ranged between 10 and 20%, while between 25% (Cafeteria) and 65% (Pteridomonas) of the consumed natural bacterial biomass was assimilated.

Reducing rumen methane emissions through elimination of rumen protozoa

Methanogens living on and within rumen ciliate protozoa may be responsible for up to 37% of the rumen methane emissions. In the absence of protozoa, rumen methane emissions are reduced by an average of 13% but this varies with diet. Decreased methane emissions from the protozoa-free rumen may be a consequence of: (1) reduced ruminal dry matter digestion; (2) a decreased methanogen population; (3) an altered pattern of volatile fatty acid production and hydrogen availability; or (4) increased partial pressure of oxygen in the rumen. The decline in methanogenesis associated with removal of protozoa is greatest on high concentrate diets and this is in keeping with protozoa being relatively more important sources of hydrogen on starch diets, because many starch-fermenting bacteria do not produce H2. Because protozoa also decrease the supply of protein available to the host animal, their elimination offers benefits in both decreasing greenhouse gas emissions and potentially increasing livestock production. Strategies for eliminating protozoa are reviewed. None of the available techniques is considered practical for commercial application and this should be addressed.

Influence of Rumen Ciliate Protozoa and Protein Level on Rumen Fermentation, Enzyme Activities and Blood Metabolites in Crossbred Cattle Calves

Pal, D.T., Kamra, D.N., Pathak, N.N. and Bisht G.S. 1998. Influence of rumen ciliate protozoa and protein level on rumen fermentation, enzyme activities and blood metabolites in crossbred cattle calves. J. Appl. Anim. Res., 13: 153–159. The influence of ciliate protozoa in the rumen on fermentation and enzyme activities in rumen fluid and blood metabolites of crossbred (Bos taurus X Bos indicue) cattle calves was studied in animals fed at two levels of protein. Sixteen crossbred male calves, separated from their dams at 24h to 48h of birth, were divided into two groups—faunated and defaunated and given a diet of normal protein or lower protein (75% of the requirement). The calves were reared in isolation stalls to avoid contact with faunated calves. On 3 months of age ruminal contents from all calves were collected at 4h post feeding and analysed The pH, total volatile fatty acids (TVFA), carboxymethylcellulase (CMC), xylanase and protease activities were similar in both faunated and fauna-free calves. Ammonia-nitrogen, TCA-soluble nitrogen, the urease activity, plasma protein and urea levels were significantly lower in ciliate-free animals than those in faunated animals, whereas, plasma glucose level was significantly higher in defaunated animals. Blood haemoglobin level was not affected either by the presence or absence of protozoa or by the protein level in the diet. The results of the present experiment indicate that the rumen ciliate protozoa affect the nitrogen metabolism of the rumen microbes considerably.