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C. difficile is the primary cause for nosocomial infective diarrhoea. For a successful infection, C. difficile must navigate between resident gut bacteria and the harsh host environment. The perturbation of the intestinal microbiota by broad-spectrum antibiotics alters the composition and the geography of the gut microbiota, deterring colonization resistance, and enabling C. difficile to colonize. This review will discuss how C. difficile interacts with and exploits the microbiota and the host epithelium to infect and persist. We provide an overview of C. difficile virulence factors and their interactions with the gut to aid adhesion, cause epithelial damage and mediate persistence. Finally, we document the host responses to C. difficile , describing the immune cells and host pathways that are associated and triggered during C. difficile infection.

SummaryThe anaerobic pathogen Clostridioides difficile, a primary cause of antibiotic-associated diarrhoea, faces a variety of stresses in the environment and in the mammalian gut. To cope with environmental stresses, it uses the alternative sigma factor B (σB) to modulate gene transcription, which is regulated by an anti-sigma factor, RsbW. To understand the role of RsbW in C. difficile physiology, a rsbW mutant (ΔrsbW) where σB is ‘always on’, was generated. ΔrsbW did not have deleterious fitness defects but tolerated acidic environments and detoxified reactive oxygen and nitrogen species better. ΔrsbW was defective in spore and biofilm formation, adhered better to human gut epithelia and was less virulent in a Galleria mellonella infection model. A transcriptomic analysis to understand this unique phenotype showed a change in expression of some σB-controlled genes along with several non-σB controlled genes. Interestingly, sinRR’ locus that encodes a pleiotropic regulator, was highly upregulated in ΔrsbW indicating a potential indirect role for σB or RsbW in control of sinRR’. Furthermore, the unexpected lower intracellular levels of σB observed suggest post translational control mechanisms. Our study thus provides new insight into the regulatory role of RsbW and the complexity of regulatory networks in C. difficile.ImportancePathogens, like C. difficile, face a range of stresses in the environment and within the host. Alternate transcriptional factors such as sigma factor B (σB) enables the bacterium to respond quickly to different types of stresses and are conserved across bacteria. Anti-sigma factors like RsbW control the activation of genes via these pathways. Such transcriptional control systems provide pathogens like C. difficile a route to tolerance and detoxification of harmful compounds. In this study we investigate the role of RsbW in C. difficile physiology. We demonstrate distinctive phenotypes for a rsbW mutant in growth, persistence and virulence. Our data suggest new σB regulatory circuits in C. difficile. Understanding bacterial responses to external stress is key to designing better strategies to combat this highly resilient bacterial pathogen.

A lot of phenolic compounds are widespread in industrial effluents and they are considerable environmental pollutants. Being a compound commercially available, the effect of a bearing-wastewater phenolic compound 3,4-dimethylphenol (3,4-DMP) on Ca2+ homeostasis and its related physiology has not been explored in cultured human kidney cell models. The aim of this study was to explore the effect of 3,4-DMP on [Ca2+]i and viability in HK-2 human proximal renal tubular epithelial cells. In terms of Ca2+ signaling, 3,4-DMP (5–100 μM) induced [Ca2+]i rises only in HK-2 cells and Ca2+ removal reduced the signal by 40%. In Ca2+-containing medium, 3,4-DMP-induced Ca2+ entry was inhibited by 20% by a modulator of store-operated Ca2+ channels (2-APB), and by a PKC activator (PMA) and inhibitor (GF109203X). Moreover, 3,4-DMP-induced Mn2+ influx suggesting of Ca2+ entry. In Ca2+-free medium, inhibition of PLC with U73122 abolished 3,4-DMP-induced [Ca2+]i rises. Furthermore, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished 3,4-DMP-evoked [Ca2+]i rises. Conversely, treatment with 3,4-DMP abolished thapsigargin-evoked [Ca2+]i rises. Regarding to cell viability, 3,4-DMP (60–140 μM) killed cells in a concentration-dependent fashion in HK-2 cells. Chelation of cytosolic Ca2+ with BAPTA-AM partially reversed cytotoxicity of 3,4-DMP. Collectively, our data suggest that in HK-2 cells, 3,4-DMP-induced [Ca2+]i rises by evoking Ca2+ entry via PKC-sensitive store-operated Ca2+ entry and PLC-dependent Ca2+ release from the endoplasmic reticulum. 3,4-DMP also caused cytotoxicity that was linked to preceding [Ca2+]i rises. Our findings provide new insight into the cytotoxic effects of 3,4-DMP and the possible mechanisms underlying these effects.

Background: \ud The SARS-CoV-2 delta (B.1.617.2) variant was first detected in England in March, 2021. It has since rapidly become the predominant lineage, owing to high transmissibility. It is suspected that the delta variant is associated with more severe disease than the previously dominant alpha (B.1.1.7) variant. We aimed to characterise the severity of the delta variant compared with the alpha variant by determining the relative risk of hospital attendance outcomes.\ud \ud Methods: \ud This cohort study was done among all patients with COVID-19 in England between March 29 and May 23, 2021, who were identified as being infected with either the alpha or delta SARS-CoV-2 variant through whole-genome sequencing. Individual-level data on these patients were linked to routine health-care datasets on vaccination, emergency care attendance, hospital admission, and mortality (data from Public Health England's Second Generation Surveillance System and COVID-19-associated deaths dataset; the National Immunisation Management System; and NHS Digital Secondary Uses Services and Emergency Care Data Set). The risk for hospital admission and emergency care attendance were compared between patients with sequencing-confirmed delta and alpha variants for the whole cohort and by vaccination status subgroups. Stratified Cox regression was used to adjust for age, sex, ethnicity, deprivation, recent international travel, area of residence, calendar week, and vaccination status.\ud \ud Findings: \ud Individual-level data on 43 338 COVID-19-positive patients (8682 with the delta variant, 34 656 with the alpha variant; median age 31 years [IQR 17–43]) were included in our analysis. 196 (2·3%) patients with the delta variant versus 764 (2·2%) patients with the alpha variant were admitted to hospital within 14 days after the specimen was taken (adjusted hazard ratio [HR] 2·26 [95% CI 1·32–3·89]). 498 (5·7%) patients with the delta variant versus 1448 (4·2%) patients with the alpha variant were admitted to hospital or attended emergency care within 14 days (adjusted HR 1·45 [1·08–1·95]). Most patients were unvaccinated (32 078 [74·0%] across both groups). The HRs for vaccinated patients with the delta variant versus the alpha variant (adjusted HR for hospital admission 1·94 [95% CI 0·47–8·05] and for hospital admission or emergency care attendance 1·58 [0·69–3·61]) were similar to the HRs for unvaccinated patients (2·32 [1·29–4·16] and 1·43 [1·04–1·97]; p=0·82 for both) but the precision for the vaccinated subgroup was low.\ud \ud Interpretation: \ud This large national study found a higher hospital admission or emergency care attendance risk for patients with COVID-19 infected with the delta variant compared with the alpha variant. Results suggest that outbreaks of the delta variant in unvaccinated populations might lead to a greater burden on health-care services than the alpha variant.\ud \ud Funding: \ud Medical Research Council; UK Research and Innovation; Department of Health and Social Care; and National Institute for Health Research.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections were rising during early summer 2021 in many countries as a result of the Delta variant. We assessed reverse transcription polymerase chain reaction swab positivity in the Real-time Assessment of Community Transmission–1 (REACT-1) study in England. During June and July 2021, we observed sustained exponential growth with an average doubling time of 25 days, driven by complete replacement of the Alpha variant by Delta and by high prevalence at younger, less-vaccinated ages. Prevalence among unvaccinated people [1.21% (95% credible interval 1.03%, 1.41%)] was three times that among double-vaccinated people [0.40% (95% credible interval 0.34%, 0.48%)]. However, after adjusting for age and other variables, vaccine effectiveness for double-vaccinated people was estimated at between ~50% and ~60% during this period in England. Increased social mixing in the presence of Delta had the potential to generate sustained growth in infections, even at high levels of vaccination.

A lot of phenolic compounds are widespread in industrial effluents and they are considerable environmental pollutants. Being a compound commercially available, the effect of a bearing-wastewater phenolic compound 3,4-dimethylphenol (3,4-DMP) on Ca

Interactions of commensal bacteria within the gut microbiota and with invading pathogens are critical in determining the outcome of an infection. While murine studies have been valuable, we lack in vitro models to monitor community responses to pathogens at a single-species level. We have developed a multispecies community of nine representative gut species cultured together as a mixed biofilm and tracked numbers of individual species over time using a quantitative PCR (qPCR)-based approach. Introduction of the major nosocomial gut pathogen, Clostridioides difficile, to this community resulted in increased adhesion of commensals and inhibition of C. difficile multiplication. Interestingly, we observed an increase in individual Bacteroides species accompanying the inhibition of C. difficile. Furthermore, Bacteroides dorei reduced C. difficile growth within biofilms, suggesting a role for Bacteroides spp. in prevention of C. difficile colonization. We report here an in vitro tool with excellent applications for investigating bacterial interactions within a complex community. IMPORTANCE Studying interactions between bacterial species that reside in the human gut is crucial for gaining a better insight into how they provide protection from pathogen colonization. In vitro models of multispecies bacterial communities wherein behaviors of single species can be accurately tracked are key to such studies. Here, we have developed a synthetic, trackable, gut microbiota community which reduces growth of the human gut pathogen Clostridioides difficile. We report that Bacteroides spp. within this community respond by multiplying in the presence of this pathogen, resulting in reduction of C. difficile growth. Defined in vitro communities that can be tailored to include different species are well suited to functional genomic approaches and are valuable tools for understanding interbacterial interactions.

The Galleria mellonella is an effective model for probing Clostridioides difficile interactions with phages. Despite valuable insights from this model, the larvae are not easily amenable to assessing detailed clinical responses to either bacteria or phages. Here, larval survival, colonisation and toxin levels were compared to expression profiles of 17 G. mellonella stress genes to monitor Clostridiodes difficile infection (CDI), and recuperation during phage therapy. The larvae were infected with a ribotype 014/020 isolate and treated with an optimised phage cocktail. Larvae treated prophylactically with phages and the phage-control larval group were protected, showing the highest survival, and low C. difficile colonisation and toxin rates, compared to co-infection, remedial and bacterial-control larval groups. Expression of growth (9) and reproduction (2) genes were enhanced within prophylaxis and phage-control larval groups compared to the co-infection, remedial and bacterial control groups. In contrast, expression of infection (2), humoral (1) and cellular (3) immunity genes declined in the prophylactic and phage-control groups but increased in the co-infection, remedial and bacterial control larvae. The molecular markers augment the survival, colonisation and toxin data and allow detailed monitoring of CDI and recovery. This data support the use of stress marker genes as tools to analyse clinical symptoms in this model.

The Galleria mellonella is an effective model for probing Clostridioides difficile interactions with phages. Despite valuable insights from this model, the larvae are not easily amenable to assessing detailed clinical responses to either bacteria or phages. Here, larval survival, colonisation and toxin levels were compared to expression profiles of 17 G. mellonella stress genes to monitor Clostridiodes difficile infection (CDI), and recuperation during phage therapy. The larvae were infected with a ribotype 014/020 isolate and treated with an optimised phage cocktail. Larvae treated prophylactically with phages and the phage-control larval group were protected, showing the highest survival, and low C. difficile colonisation and toxin rates, compared to co-infection, remedial and bacterial-control larval groups. Expression of growth (9) and reproduction (2) genes were enhanced within prophylaxis and phage-control larval groups compared to the co-infection, remedial and bacterial control groups. In contrast, expression of infection (2), humoral (1) and cellular (3) immunity genes declined in the prophylactic and phage-control groups but increased in the co-infection, remedial and bacterial control larvae. The molecular markers augment the survival, colonisation and toxin data and allow detailed monitoring of CDI and recovery. This data support the use of stress marker genes as tools to analyse clinical symptoms in this model.

The innate lymphoid cells (ILCs) are a recently discovered type of innate immune cell. The functions of these cells resemble different T-cell subtypes. These cells play important roles in local injury, inflammation, pathogen infection, or tumours. However, there have been few studies focusing on the role of ILCs in nasal diseases.We reviewed the literature about the roles of ILCs in nasal inflammation, tissue remodeling, and cancer.The ILCs represent a newly identified family of innate immune cells. These cells play important roles in inflammation, immune responses, tissue remodeling, and cancer immunity. The ILCs, especially ILC2s, play important roles in CRSwNP and AR. ILC2s may be involved in the pathogenesis of eosinophilic inflammation in non-allergic nasal diseases, such as non-allergic CRSwNP and non-allergic rhinitis. ILCs also play pro-tumor or anti-tumor roles in cancer immunity for head and neck cancer.LC2s may be a useful therapeutic target for CRSwNP and AR. ILCs may also represent new therapeutic targets to activate anti-tumor immunity in head and neck cancer.

A 14-week long feeding trial was carried out to evaluate the efficiency, safety and effects on egg quality of three experimental diets using 0%, 10% and 18% chopped full-fat dried black soldier fly larvae (DBSFL) to partially or fully replace soybean meal in free range layer diets. The feed was provided in mash form and DBSFL was mechanically chopped (3 – 5 mm) before mixing in the diet. For each of the three diets, 30 pullets were housed in 2 mobile poultry trailers. The weight gain, feed intake, egg production, egg weight, feed conversion ratio, health and welfare parameters, blood biochemistry and hematology, excreta microbiology, digestive tract mass, egg shell weight and thickness, interior egg quality (albumen weight and height, yolk weight and color, and Haugh Units), yolk metal and fatty acid compositions, and sensory evaluation were examined. Data were analyzed by Least Squares ANOVA with repeated measures using JMP®. Results of the study indicated that control hens had significantly better egg production and feed conversion ratio than 18% DBSFL hens probably because of the higher fat content, presence of chitin and bulkiness of the 18% feed. Control hens had better weight gain, and heavier shell and albumen weights than DBSFL eggs. 18% DBSFL hens had lower plasma albumin, total protein and triglycerides, but heavier duodenum weights than the control hens. Despite these apparent short-comings, the 18% hens were still gaining weight during the laying period, maintaining above 90% hen-day egg production, and their eggs were still in the “Large” egg category. There were no obvious differences between the three treatment groups in yolk metal content or sensory characteristics. In conclusion, if the digestibility of DBSFL can be improved, it may fully replace soybean meal in laying hen diets.

Hypoxia-inducible factor (HIF) is considered an important transcription factor due to its roles in glycolysis, angiogenesis, cell differentiation, apoptosis, and other cellular pathways. It takes the role in various physiological and pathological states, such as solid tumors, vascular injury, and atherosclerotic lesion progression. In recent studies, HIF is found as a master regulator of body inflammation and immunity, not only in hypoxia but also in normoxia. Nasal inflammation has a close relationship with anoxia. But the role of HIF in nasal inflammation is still unclear.We searched the Pubmed using the key words: "Hypoxia-inducible factor" and "nasal" or "Hypoxia-inducible factor", and reviewed the related articles.HIF is composed of HIF-α and HIF-β subunits. HIF-a is an adjusting relational subunit, which is divided into three subtypes: HIF-1a, HIF-2a, and HIF-3a. HIF-1a is the key component and best understood. HIF-1a can be activated under hypoxic conditions or by various cytokines and growth factors. HIF-1α accumulation is critical for sustaining human allergic effector cell survival and function. The level of HIF-1a is increased in the patients with allergic rhinitis and become a new therapeutic target. HIF-1a also plays an important role in the pathogenesis of CRS and polyp formation. Some research found that the expression of HIF-1α was increased in CRS with polyps.HIF-1a takes an important role in allergic rhinitis and chronic sinusitis. It will be a key therapeutic target of these diseases in the future.

The effects of the nonionic surfactant Tween 80 and a mixture of fibrolytic enzymes on total tract digestion, in situ disappearance (ISD) and ruminal fermentation characteristics of orchardgrass hay and barley grain were investigated in a 4x4 Latin square experiment with 4 non-lactating Holstein cows and 4 diets in 4 periods. Cows were offered a total mixed ration of 50% rolled barley grain and 50% orchardgrass hay treated with either 1) water (control), 2) 0.2% (vol/wt) Tween 80, 3) 0.2% (vol/wt) hydrolytic enzyme, or 4) 0.2% hydrolytic enzyme plus 0.2% Tween 80. Total tract digestibility coefficients of DM, nitrogen, NDF and ADF were not affected (p>0.05) by dietary treatment. Compared to the control, the rate of ISD of DM from orchardgrass hay was faster (p 0.05) by treatments. Cows that consumed diets treated with enzyme plus Tween 80 had higher (p

A direct somatic embryogenesis and secondary embryogenesis protocol was developed for seven cereal species, thus providing a new vista for in vitro plant genetic transformation or propagation. This paper describes a novel process that has been successfully developed for efficient regeneration of a wide range of cereal species and genotypes. This tissue culture and regeneration system does not require formation of callus tissues and takes approximately 2 months to complete, shorter than any of the currently available systems requiring 3-4 months. Rapid induction of direct somatic embryogenesis in barley (Hordeum vulgare), common wheat (Triticum aestivum), durum wheat (T. durum) and derived amphiploids, wild wheat (T. monococcum and T. urartu), rye (Secale cereale) and oats (Avena sativa) was induced from excised immature scutellum on DSEM medium. Newly developed globular embryos were cultured on SEM medium for a second cycle of embryogenesis followed by germination (GEM medium) and regeneration of embryos into normally growing green and fertile plants. In vitro techniques to induce direct somatic embryogenesis, secondary embryogenesis and plant regeneration from these cereals require a specific sequence of defined media and controlled environments. The sequence and the timing of the media used, as well as their hormonal composition and balance are critical aspects of this process. The organic and mineral compositions of these media are not new but are important for supporting and sustaining rapid growth of the tissues.

Effect of a surfactant Tween 80 on the bacterial growth in the rumen was examined on the in vitro pure cultures of Streptococcus bovis, Selenomonas ruminantium, Butyrivibrio fibrisolvens, Prevotella ruminicola, Megasphaera elsidenni, Fibrobacta succinogenes, Ruminanococcus albus and Ruminococcus flavefaciens. Dry matter degradability (DMD), concentrations and compositions of volatile fatty acids (VFA), and the most probable number (MPN) of cellulolytic bacteria and total number of bacteria in the presence of Tween 80 were also examined on the in vitro rumen mixed culture either with barley grain or orchardgrass hay. The growth of S. bovis, S. ruminantium, B. fibrisolvens, P. ruminicola , M. elsidenni and F. succinogenes were significantly higher (p

The study evaluates the enzymatic dry matter (DM) degradability and water holding capacity of leaf and stem fractions of orchardgrass (Dactylis glomerata L.) at different growth stages with or without the presence of surfactant Tween 80. While Tween 80 significantly (p

A series of experiments was carried out to determine the possibility for the non-ionic surfactant (NIS) as a feed additive for ruminant animals. The effect of the NIS on c the enzyme distribution in the rumen fluids of Hereford bulls, 2 the growth of pure culture of rumen bacteria and e rumen anaerobic fungi, f the ruminal fermentation characteristics of Korean native cattle (Hanwoo), and g the performances of Holstein dairy cows were investigated. When NIS was added to rumen fluid at the level of 0.05 and 0.1% (v/v), the total and specific activities of cell-free enzymes were significantly (p

The effects of monensin, Tween 80 and exogenous fibrolytic enzymes on ruminal fermentation and animal performance were studied in vitro and in vivo. In Expt 1, the effects of the surfactant Tween 80 (0.2% wt/wt, DM basis) on ruminal fermentation of alfalfa, corn and orchardgrass silages were investigated using in vitro gas production techniques. Tween 80 did not affect (p>0.05) cumulative gas production at 24 h, but it reduced (p factorial arrangement of treatments. The backgrounding and finishing diets contained barley grain and barley silage in ratios of 57.8:42.2 and 93.5:6.5 (DM basis), respectively. Added alone, none of the additives affected DM intake (p>0.1) in the backgrounding or in the finishing period, but interactive effects were observed in the finishing period (p=0.02) and overall (p=0.04). In the finishing period, T without M tended to reduce DM intake (p=0.11), but T with M increased (p=0.05) DM intake. Monensin increased average daily gain (ADG) during backgrounding (p=0.07) and finishing (p=0.01), and this ionophore also improved overall feed efficiency (p=0.02). Warm carcass weight was increased (p0.10) ADG during finishing, or overall feed efficiency. Whereas T enhanced the positive effects of M on ADG during backgrounding (p=0.04) and overall (p=0.05), it had no impact (p>0.1) on the effects of E. Interactions between M and T suggest that the surfactant may have potential for enhancing the positive effects of monensin on beef production, but this requires further research.

A feeding trial was conducted to compare intercropped barley/annual ryegrass silage (B/RS) and barley silage (BS) on average daily gain (ADG), dry matter intake (DMI) and carcass characteristics of beef steers in a randomized complete block design experiment. Both B/RS and BS were prepared in plastic tube silos, ensiled for 168 days and then used in a feeding trial. Steers ( n =120) were housed in individual pens and fed a backgrounding diet containing 350 g steam-rolled barley grain (SBG) and 600 g kg −1 B/RS or 600 g kg −1 BS and 50 g kg −1 supplement until they weighed 445 kg, after which they were adapted to a finishing diet consisting of 860 g SBG and 100 g kg −1 B/RS or 100 g kg −1 BS and 40 g kg −1 supplement. Steer weight and back-fat depth (determined by ultra-sonography) were measured every 3 weeks. Steers were slaughtered when back-fat depth was greater than 7 mm and carcass data were obtained. Content of ADF in B/RS was lower ( P P P

The potential development of potato (Solanum tuberosum) as a low-cost eukaryotic system for the production of a commercially valuable enzyme feed supplement was examined. AFibrobacter succinogenes 1,3-1,4-β-glucanase [1,3-1,4-β-D-glucan 4-glucanohydro-lase] gene under the control of the constitutive cauliflower mosaic virus 35S promoter was transferred into the potato cultivar, Desiree. The presence of the β-glucanase cDNA in the plant genome of independent transgenic potato lines was confirmed by PCR and Southern analysis. Northern analysis identified the presence of the β-glucanase mRNA in the leaf tissue of transgenic plants. Furthermore, western analysis showedF. succinogenes β-glucanase accumulations of 0.1% and 0.05% of total soluble protein in the leaves and tubers, respectively. Specific activities of the enzyme in leaves (1693 units mg-1 β-glucanase) and tubers (2978 units mg-1 β-glucanase) were comparable to that previously reported for the enzyme produced in bacteria. Lyophilization of leaves had no effect on the specific activity of the β-glucanase, and only marginally influenced the specific activity of the enzyme expressed in tubers. Relative to the control line (cv. Desiree), tuber yields were significantly reduced by 28%-72% in all lines expressing theF. succinogenes β-glucanase, and microscopy showed that expression of the β-glucanase caused changes in cell wall structure. Results of this study demonstrate that a 1,3-1,4-β-glucanase can be expressed in potato tissues, and that potato plants have the potential to be used for the commercial production of heterologous enzymes.

The distribution and activities of hydrolytic enzymes (cellulolytic, hemicellulolytic, pectinolytic and others) in the rumen compartments of Hereford bulls fed 100% alfalfa hay based diets were evaluated. The alfalfa proportion in the diet was gradually increased for two weeks. Whole rumen contents were processed into four fractions: Rumen contents including both the liquid and solid fractions were homogenized and centrifuged, and the supernatant was assayed for enzymes located in whole rumen contents (WRE); rumen contents were centrifuged and the supernatant was assayed for enzymes located in rumen fluids (RFE); feed particles in rumen contents were separated manually, washed with buffer, resuspended in an equal volume of buffer, homogenized and centrifuged and supernatant was assayed for enzymes associated with feed particles (FAE); and rumen microbial cell fraction was separated by centrifugation, suspended in an equal volume of buffer, sonicated and centrifuged, and the supernatant was assayed for enzymes bound with microbial cells (CBE). It was found that polysaccharide-degrading proteins such as β-1,4-D-endoglucanase, β-1,4-D-exoglucanase, xylanase and pectinase enzymes were located mainly with the cell bound (CBE) fraction. However, β-D-glucosidase, β-D-fucosidase, acetylesterase, and α-L-arabinofuranosidase were located in the rumen fluids (RFE) fraction. Protease activity distributions were 37.7, 22.1 and 40.2%, and amylase activity distributions were 51.6, 18.2 and 30.2% for the RFE, FAE and CBE fractions, respectively. These results indicated that protease is located mainly in rumen fluid and with microbial cells, whereas amylase was located mainly in the rumen fluid. (Asian-Aust. J. Anim. Sci. 2002. Vol 15, No. 12 : 1725-1731)

The effects of protozoa on the degradation of plant cell walls (CW) during different growth stages of the fungus Anaeromyces mucronatus have been investigated. Since fungi show a marked lag in their in vitro cultures and many protozoa rapidly die during a prolonged incubation time, the effects of protozoa may vary according to the growth phase of the fungi. Therefore, the approach adopted was (i) to inoculate CW with fungus monoculture, (ii) to inoculate CW with fungus-protozoa coculture, or (iii) to sequentially inoculate fungal cultures that had been grown in CW for 24 (initial stage of growth), 48, and 72 h (late stage of growth) with mixed protozoa. When a fungus was associated with protozoa, a growth phase dependent effect was observed. Ruminal protozoa adversely affected the growth and activity when introduced in the initial growth stage of A. mucronatus, but a synergetic interaction was detected when added to late growth stage cultures. Although there is no immediate explanation for these results, the data suggested that protozoa can engulf the fungal zoospores, which are in ruminal fluids and (or) attached to small feed particles, but cannot engulf the fungal thallus that is tightly attached to feed particles by a rhizoidal system. Our data indicated that the protozoa did not influence cellulolysis by the fungi in exponential and (or) stationary phase, but they had a marked inhibitory effect on fungi that were in lag phase. Inhibition during lag phase could result from the protozoal predation of fungal zoospores that had failed to attach to substrates.Key words: rumen fungi, rumen protozoa, cellulose digestion, cellulase activity, interactions.

Experiments were conducted to study the effect of administration of anaerobic fungal cultures or their enzymes on ruminal fermentation, microbial populations and enzyme activities in the rumen, and general nutrient digestion in sheep. Twelve mature sheep receiving 560 g of orchard grass hay and 240 g of concentrate per day were randomly assigned to three different treatments with four sheep per treatment. Control (FM) animals received 200 ml of the defined medium for fungal growth through rumen cannulae at 8.00 h each day (as a control treatment without added fungal enzymes and viable cells). The second group of animals (FE) were given 200 ml of supernatant from fungal culture incubated with an anaerobic ruminal fungus for 7 days (as a treatment for fungal enzymes in cultures without viable fungus). The third group (FC) was administered with an anaerobic fungal culture incubated for 7 days (as a direct-fed microbials (DFM) treatment with fungal enzymes and viable cells). Animals were exposed to each treatment for 15 days before rumen collection and digestion trials began. Direct administration of cultures of a polycentric fungal strain, Orpinomyces strain KNGF-2 isolated from a Korean native goat, to the rumen of sheep (FC) generally increased nutrient digestibility and nitrogen (N) retention resulting from an increase in numbers of bacteria and fungi in the rumen and by altering the patterns of volatile fatty acids (VFA) production. However, administration of fungal enzyme (FE) failed to improve nutrient digestibility and N retention, and ruminal fermentation parameters. Results indicate that these proteins may be rapidly degraded by ruminal microbes or that these enzymes could reduce the growth or activity of ruminal microbes. These are the first report of the effect of DFM originating from an anaerobic fungal culture. Such a development is particularly significant because this study demonstrates that the introduction of a microorganism with superior fibrolytic activity into the rumen can improve nutrient utilization in ruminants.

Condensed tannins (CT) are polyphenolic secondary plant products that are widespread in the plant kingdom. By definition, CT bind to protein and are regarded as "antinutritional" compounds which reduce protein digestibility. Variations in CT chemistry alter protein binding capacities among polymers from different plant species and developmental stages. Condensed tannins interact with proteins in feed, saliva and microbial cells, with microbial exoenzymes, and with endogenous proteins or other feed components, which alters digestive processes as compared with diets free of CT. Tannin levels exceeding 40 to 50 g kg−1 DM in forages may reduce protein and DM digestibility of the forages by ruminants. At low to moderate levels, CT increase the quantity of dietary protein, especially essential amino acids, flowing to the small intestine. Unlike alfalfa, legumes that contain CT do not cause bloat. Dietary CT may provide a means to beneficially manipulate protein digestion and/or prevent pasture bloat in ruminants. Research efforts are also currently being directed to genetically modify alfalfa to de-repress its CT biosynthetic pathway, or to move genes encoding steps of this pathway into alfalfa from other legumes. The effects of CT in forage legumes on digestion in ruminants and the potential use of tannin-rich forages for preventing bloat in grazing systems are discussed. Key words: Tannins, bloat, by-pass protein, cattle, grazing, alfalfa

The studies that led to the development of a bloat-reduced alfalfa began in 1970. Initial work focused on the causes of bloat, the characteristics of bloat-safe and bloat-causing legumes and the development of screening methodologies. The characteristics that were examined as causal factors of bloat in legume species included soluble proteins, saponins, absence of condensed tannins, and rapid breakdown of cells in the rumen. The factor or combination of factors responsible for causing bloat in animals grazing legumes have not been clearly identified; however, of the above, rapid cell breakdown was considered to be the most promising line of research to pursue in selecting a bloat-safe legume. A 4-h nylon-bag rumen digestion technique was developed, which was used to assess the initial rate of digestion (IRD) of fresh leaf material in large numbers of alfalfa genotypes. Using this technique on a population of four alfalfa cultivars, four cycles of recurrent selection for low initial rate of digestion (LIRD) were carried out over an 11-yr period. The resulting selected population, LIRD-4, had a vegetative stage initial rate of digestion that was approximately 85% of that of unselected alfalfa. To determine whether this reduction in IRD would reduce or eliminate bloat, grazing experiments comparing LIRD-4 and unselected cv. Beaver were conducted at three locations over 3 yr. Over all grazings, LIRD-4 caused significantly fewer incidences of bloat than Beaver; thus it is a bloat-reduced, rather than a bloat-safe cultivar. LIRD-4 was released in 1997 and named AC Grazeland Br. Key words: Bloat, cattle, alfalfa, low initial rate of digestion, grazing, bloat-reduced alfalfa

and Cheng, K.-J. 2000. Bloat in cattle grazing alfalfa cultivars selected for a low initial rate of digestion: A review. Can. J. Plant. Sci. 80: 493‐502. The occurrence of frothy bloat limits the practice of alfalfa grazing in spite of the availability of strains bred specifically for pasture. Bloat is a chronic condition, endemic to cattle. Prophylactics and management techniques are ava ilable to reduce its incidence but they are expensive, difficult to administer, conflict with traditional grazing management regi mens and do not eliminate bloat in all circumstances. A program to breed and evaluate a bloat-reduced strain of alfalfa was initiate d in 1980 to overcome some of these limitations. A review of the results of grazing and feeding trials using alfalfas with low initi al rates of digestion (LIRD) shows that this new strain reduces the incidence and severity of frothy bloat on pasture. Their effec tiveness in controlling bloat was related to feeding or grazing management practices, the maturity of the plants and the season of use. Graziers may reduce the risk of occasional livestock losses from bloat by using LIRD cultivars, like AC Grazeland, or managing species/cultivar mixtures in ways that reduce the initial rate of digestion. Other bloat preventive strategies, including coseeding with bloat-free legumes and using bloat-controlling prophylactics in combination with a LIRD alfalfa, are being investigated.

Ten Jersey steers (484 ± 55 kg) were fed a basal diet of 70% rolled barley grain and 30% alfalfa silage [dry matter (DM) basis] with or without additives, in a replicated 5 × 5 Latin square experiment. Treatments (additives per kilogram dietary DM) were: C, no additives (control); T, Tween 80 (2 g kg−1); SH, salinomycin (13 mg kg−1); TSM, Tween 80 (2 g kg−1) plus salinomycin (6.5 mg kg−1); and TSL, Tween 80 (2 g kg−1) plus salinomycin (3.25 mg kg−1). Ruminal pH, concentrations of ammonia, total free amino acids, reducing sugars and total volatile fatty acids (VFA), and fluid phase viscosity were unaffected (P > 0.05) by treatment. Ruminal carboxymethylcellulase, xylanase and amylase activities, numbers of protozoa, and outflow rates of the liquid and solid phases of ruminal contents did not differ (P > 0.05) among treatments. Ruminal lactic acid bacteria populations tended (P 0.05) by T, TSL and TSM. Partitioning of ruminal digesta and microbial protein flow were similar (P > 0.05) among treatments. Compared with C, the ruminal rate of degradation (in situ) of alfalfa DM tended to be higher (P 0.05) by treatment, as were blood glucose and urea levels. Tween 80 and salinomycin did not affect ruminal fermentation or nutrient digestibility in steers fed a barley grain/alfalfa silage diet. Key words: Tween 80, salinomycin, ruminal fermentation, digestibility, steer

Two cellulase cDNAs, celB29 and celB2, were isolated from a cDNA library derived from mRNA extracted from the anaerobic fungus, Orpinomyces joyonii strain SG4. The nucleotide sequences of celB2 and celB29 and the primary structures of the proteins encoded by these cDNAs were determined. The larger celB29 cDNA was 1966 bp long and encoded a 477 amino acid polypeptide with a molecular weight of 54 kDa. Analysis of the 1451 bp celB2 cDNA revealed an 1164 bp open reading frame coding for a 44 kDa protein consisting of 388 amino acids. Both deduced proteins had a high sequence similarity in central regions containing putative catalytic domains. Primary structure analysis revealed that CelB29 contained a Thr/Pro-rich sequence that separated the N-terminal catalytic domain from a C-terminal reiterated region of unknown function. Homology analysis showed that both enzymes belong to glycosyl hydrolase family 5 and were most closely related to endoglucanases from the anaerobic fungi Neocallimastic patriciarum, Neocallimastix frontalis and Orpinomyces sp. The classification of CelB29 and CelB2 as endoglucanases was supported by enzyme assays. The cloned enzymes had high activities towards barley b-glucan, lichenan and carboxymethylcellulose (CMC ), but not Avicel, laminarin, pachyman, xylan and pullulan. In addition, CelB29 and CelB2 showed activity against pnitrophenyl-b-d-cellobioside (pNP-G 2 )t op-nitrophenyl-b-d-cellopentaoside (pNP-G 5 ) but not p-nitrophenyl-b-d-glucopyranoside (pNP-G 1 ) with preferential activity against p-nitrophenyl-b-d-cellotrioside (pNP-G 3 ). Based on these results, we proposed that CelB29 and CelB2 are endoglucanases with broad substrate specificities for short- and long-chain b-1,4-glucans. © 2000 Elsevier Science B.V. All rights reserved.

The effects of Tween 80 on the stability and substrate binding capacity of two commercially available enzyme preparations were studied in vitro. After 1 wk of incubation, endoglucanase activity was higher (P −1) Tween 80 than without this surfactant. Tween 80 increased (P −1) by the lambs, as compared to untreated diet. With the forage diet, neutral detergent fibre digestibility was higher with Tween 80 alone than with enzymes + surfactant. In the feedlot experiment, treating the forage diet with surfactant reduced (P −1) of lambs fed the concentrate diet was lower (P

Microbial enzymes extracted from mixed ruminal microorganisms were incubated for 2 h with casein and Tween 60 or Tween 80 at 10 concentrations ranging from 0 to 2.0% (vol/vol) to determine the effects of these nonionic surfactants on protease activation and thiol reactivity (unmasking of thiol groups). Rate and extent of protein adsorption to cellulosic substrate (barley straw) was measured in the presence of 0, 0.05, 0.10, 0.25, and 0.50% (vol/vol) Tween 80. Degradation of cellulose by a rumen bacterial fraction was measured over 48 h of incubation with and without Tween 60 or Tween 80 at 0.25% (vol/vol). Maximum accelerations of protease activity achievable with Tween 60 and Tween 80 (calculated from a Michaelis-Menten kinetics model) were 99.2 and 166.8%, respectively. Concentrations of Tween 60 and Tween 80 at which half the maximal velocities were attained were 0.28 and 0.20% (vol/vol), respectively. Tween 80 increased (P0.05) the rate and extent of adsorption of microbial protein to barley straw, and the effect was related to concentration of Tween 80 up to 0.10% (vol/vol). Initial rates of cellulose degradation with no surfactant, 0.25% Tween 60, or 0.25% Tween 80 were 0.60, 0.87, and 1.04 micrograms/ml per h, respectively. These nonionic surfactants were effective for enhancing rumen microbial protease and cellulase activities. Thus, further study is warranted to determine their potential for improving ruminant feeding.

In a first experiment, ground alfalfa hay and rolled barley grain were incubated in buffered ruminal fluid with and without Yucca schidigera extract (YE, 0 or 10 mg ml−1). Gas and total VFA production from barley grain were increased (P

Differential agar media for the detection of microbial phytase activity use the disappearance of precipitated calcium or sodium phytate as an indication of enzyme activity. When this technique was applied to the study of ruminal bacteria, it became apparent that the method was unable to differentiate between phytase activity and acid production. Strong positive reactions (zones of clearing around microbial colonies) observed for acid producing, anaerobic bacteria, such as Streptococcus bovis, were not corroborated by subsequent quantitative assays. Experimentation revealed that acidic solutions generated false positive results on the selected differential medium. Empirical studies undertaken to find a solution to this limitation determined the false positive results could be eliminated through a two step counterstaining treatment (cobalt chloride and ammonium molybdate/ammonium vanadate) which reprecipitates acid solubilized phytate. This report discusses the application of the developed two step counterstaining treatment for the screening of phytase producing ruminal bacteria as well as its use in phytase zymogram assays.

The effects of exogenous fibrolytic enzymes, α-bromoethanesulfonate (BES) and monensin on fermentation and digestibility of grass hay were examined using two rumen simulation technique (RUSITEC) devices, each equipped with eight 820-mL fermenters with a liquid dilution rate of 0.73 d−1. Grass hay (10 g d−1) was fed either untreated or pre-treated with 0.2 g each of cellulase and xylanase, and in combination with no chemical or daily dosing of 20.5 µmol monensin, 20.5 µmol BES or 41 µmol BES. Pretreatment of hay with the fibrolytic enzymes increased (P

In vitro studies demonstrated that cell-free ruminal fluid inhibits the saponin-based hemolytic activity of yucca extract, and that the inhibitory factor isolatable with the protein fraction of ruminal fluid is not heat labile. Hemolysis assay is not reliable for assessing saponin activity in ruminant nutrition applications. Key words: Yucca extract, ruminal fluid, hemolysis, saponins

Yearling cattle fed pasture-only for the entire grazing season were compared with cattle receiving pasture plus steam-rolled barley (SRB; 4 kg head−1 d−1) or pasture plus SRB (4 kg head−1 d−1) treated with Tween 80 at a rate of 4 kg t−1, for 74 d prior to slaughter. Heifers were more likely (P = 0.015) to grade A or higher than steers. Feeding supplemental grain on pasture increased pasture gains (P 0.05) improve carcass quality, chemical analysis, shear force, carcass value or gross return per head. Treatment of SRB with Tween-80 did not influence (P > 0.05) cattle performance or carcass quality. From the results reported in this study, it is possible to finish cattle on pasture to Canada A grade standards. Key words: Forage-finishing, carcass quality, beef, pasture, grain

Barley silage was sprayed with water or with a 2:1 combination of commercial cellulase and xylanase preparations, or the enzymes were introduced directly into the rumen, in a digestibility study (replicated incomplete 3 × 3 Latin square) using 10 sheep. Apparent digestibilities of dry matter (DM) and neutral detergent fibre (NDF) were lower (P 0.05) intake of DM, organic matter or digestible organic matter, or digestibilities of DM or NDF, ruminal pH, xylanase activity, endoglucanase activity or ruminal cellulolytic bacterial populations. Treating the silage portion of an 82.5% barley silage backgrounding diet with the enzyme mix at 0, 1.25, 3.5 or 5.0 L t−1 DM tended to linearly increase (P = 0.08) final weights of steers (n = 24). Average daily gain tended to be (P = 0.06) and feed intake and feed efficiency were (P = 0.04 and P = 0.03, respectively) quadratically related to these enzyme concentrations from days 0 to 56, but not overall (days 0 to 120). In contrast, treatment of both portions (forage and concentrate) of a 70% barley-ryegrass silage finishing diet at 3.5 L t−1 DM increased (P 0.1) by enzyme supplementation. In this study, treating the total mixed ration improved feedlot cattle performance more than treating the silage component alone. Key words: Enzymes, beef cattle, growth, barley silage

Canola meal that contains a high level of protein (40% crude protein) was used as compost material for the isolation of feather-degrading bacteria. After 7 and 14 days, bacteria were isolated from compost amended and unamended with soil. Eighty bacterial isolates from canola meal compost were then grown on milk-agar and isolates that produced proteolytic enzymes were identified by the formation of clear haloes around the colonies. A feather medium was chosen for a secondary selection of feather-degrading isolates. Of the eight isolates that hydrolyzed milk protein, five isolates hydrolyzed feathers. Their keratinolytic activities were subsequently confirmed by an assay using azo-keratin as substrate. Seven of the eight bacteria that hydrolyzed milk protein were Bacillus spp, and all five isolates that hydrolyzed feathers were strains of Bacillus licheniformis. Protease inhibition studies indicated that serine proteases are the predominant proteolytic enzymes produced by these feather-degrading isolates.