Effects of supplementing freeze-dried Mitsuokella jalaludinii phytase on the growth performance and gut microbial diversity of broiler chickens.

Inclusion of phytase in animal feedstuff is a common practice to enhance nutrients availability. However, little is known about the effects of phytase supplementation on the microbial ecology of the gastrointestinal tract. In this study, freeze-dried Mitsuokella jalaludinii phytase (MJ) was evaluated in a feeding trial with broilers fed a low available phosphorus (aP) diet. A total of 180 male broiler chicks (day-old Cobb) were assigned into three dietary treatments: Control fed with 0.4% (w/w) of available phosphorus (aP); Group T1 fed low aP [0.2% (w/w)] supplemented with MJ; and T2 fed low aP and deactivated MJ. The source of readily available P, dicalcium phosphate (DCP), was removed from low aP diet, whereby additional limestone was provided to replace the amount of Ca normally found in DCP. For each treatment, 4 replicate pens were used, where each pen consisted of 15 animals. The animals' energy intake and caecal bacterial community were monitored weekly for up to 3 weeks. The apparent metabolizable energy (AME) and apparent digestibility of dry matter (ADDM) of broilers fed with different diets were determined. In addition, the caecal microbial diversities of broilers were assessed using high-throughput next-generation sequencing targeting the V3-V4 region of bacterial 16S rRNA. The results showed that broilers fed with T1 diet have better feed conversion ratio (FCR) when compared to the Control (p < .05) and T2 diets (p < .05), demonstrating the efficiency of MJ as a supplement to low aP diet. Nevertheless, MJ did not significantly affect the microbial population and diversity in broilers' caeca, which mainly consists of members from Bacteroidetes, Firmicutes, and Proteobacteria. Regardless, significant variations in the caecal bacterial composition were observed over time, probably due to succession as the broilers aged. This is the first reported study on the effect of MJ on the microbial diversity of broiler's caeca.

Probiotics: From Isolation to Application.

Probiotics have become highly recognized as supplements for humans and animals because of their beneficial effects on health and well-being. The present review aims to provide an overview of different steps through which microbial strains become applicable probiotics in food and/or feed industries. Isolation of potential probiotic strains is the first step. Lactic acid bacteria are the most frequently used microorganisms as probiotics, which can be isolated from human, animal, plant, and environment. The next steps are identification of the isolates and characterization of them based on the main selection criteria for any potential probiotic microorganism, including resistance to gastric acidity and bile salt, adherence to mucus and/or intestinal epithelial cells and cell lines, and antimicrobial and antagonism activity against potentially pathogenic microbes. There are additional probiotic properties that may be considered for selection of probiotic strains with specific effects, such as cholesterol reduction ability, antioxidant activity, or cytotoxic effect against cancer cells. However, a potential probiotic does not need to fulfill all such selection criteria. As the last step, safety status of probiotics for humans is verified by taxonomy clarification, in vitro and in vivo tests, human trials, and genome sequencing.

Performance, biochemical and haematological responses, and relative organ weights of laying hens fed diets supplemented with prebiotic, probiotic and synbiotic.

BACKGROUND: The increasing trend of ban on the use of antibiotic growth promoters (AGPs) across the globe in the poultry industry has led to a growing need for alternatives to AGPs. Prebiotic, probiotic and their combination as a synbiotic have been considered as potential alternatives. This study aimed to investigate the effects of a prebiotic (isomaltooligosaccharide, IMO), a probiotic (PrimaLac®), and their combination (synbiotic) on hen performance, biochemical and haematological responses, and relative organ weights from 20 to 52 weeks of age. RESULTS: Supplementation of 1% IMO (PRE), 0.1% PrimaLac® (PRO) and 1% IMO + 0.1% PrimaLac® (SYN) improved (P < 0.05) feed intake and egg production at 20-36 weeks of age; body weight gain, feed conversion ratio and egg mass at 20-36 and 20-52 weeks of age; and egg weight at 20-36, 37-52 and 20-52 weeks of age. Compared to control-fed hens at 20-36 weeks of age, PRO- and SYN-fed hens produced less (P < 0.05) small size eggs while SYN-fed hens produced more large size eggs. From 37 to 52 weeks of age, PRE-, PRO- or SYN-fed hens produced less (P < 0.05) medium size eggs, and more large and extra-large size eggs. PRE, PRO or SYN supplementation decreased (P < 0.05) the serum total cholesterol at 36 weeks of age, and serum low-density lipoprotein (LDL) cholesterol, alanine aminotransferase (ALT) and alkaline phosphatase (ALP) at 36 and 52 weeks of age. At 36 and 52 weeks of age, supplementation of PRE, PRO or SYN increased (P < 0.05) lymphocyte percentage and decreased (P < 0.05) heterophil percentage, leading to a lower heterophil to lymphocyte (H/L) ratio. No significant differences were observed in the relative weights of the heart, liver, ovary, pancreas and spleen of all dietary treatment groups. CONCLUSIONS: Supplementation of PRE, PRO or SYN improved performance, serum total cholesterol, LDL cholesterol, ALT, ALP and H/L ratio of hens from 20 to 52 weeks of age. These results demonstrated the use of PRE, PRO and SYN as alternative feed additives to AGPs for improving the health and productivity of hens, while PRO is the best for commercial layer production to yield maximum profit.

Effects of a Lactobacillus salivarius mixture on performance, intestinal health and serum lipids of broiler chickens.

The ban or severe restriction on the use of antibiotics in poultry feeds to promote growth has led to considerable interest to find alternative approaches. Probiotics have been considered as such alternatives. In the present study, the effects of a Lactobacillus mixture composed from three previously isolated Lactobacillus salivarius strains (CI1, CI2 and CI3) from chicken intestines on performance, intestinal health status and serum lipids of broiler chickens has been evaluated. Supplementation of the mixture at a concentration of 0.5 or 1 g kg-1 of diet to broilers for 42 days improved body weight, body weight gain and FCR, reduced total cholesterol, LDL-cholesterol and triglycerides, increased populations of beneficial bacteria such as lactobacilli and bifidobacteria, decreased harmful bacteria such as E. coli and total aerobes, reduced harmful cecal bacterial enzymes such as ß-glucosidase and ß-glucuronidase, and improved intestinal histomorphology of broilers. Because of its remarkable efficacy on broiler chickens, the L. salivarius mixture could be considered as a good potential probiotic for chickens, and its benefits should be further evaluated on a commercial scale.

Safety Assessment of Two New Lactobacillus Strains as Probiotic for Human Using a Rat Model.

Two previously isolated Lactobacillus strains (L. fermentum HM3 from human milk and L. buchneri FD2 from fermented dates), intended as probiotic for human, were assessed for their safety using acute and subacute oral toxicity tests in rats. In addition, their effects on cecal microflora and harmful bacterial enzymes (ß-glucuronidase and ß-glucosidase) of the tested animals were also determined. The results showed that L. buchneri FD2, L. fermentum HM3, or a mixture of them were safe up to a level of 1010 CFU/kg BW/day in a 14-day or 28-day treatment period. Both strains were well tolerated and there were no observed adverse effects on growth, feed consumption, cellular blood components and vital organs of the treated animals. The Lactobacillus strains were also able to reduce harmful intestinal bacterial enzymes, and decrease pathogenic bacterial populations while increasing beneficial bacterial populations. These results suggest that the two Lactobacillus strains are safe and could be potential probiotic for human.

Modulatory effects of condensed tannin fractions of different molecular weights from a Leucaena leucocephala hybrid on the bovine rumen bacterial community in vitro.

BACKGROUND: Condensed tannin (CT) fractions of different molecular weights (MWs) may affect rumen microbial metabolism by altering bacterial diversity. In this study the effects of unfractionated CTs (F0) and five CT fractions (F1-F5) of different MWs (F1, 1265.8 Da; F2, 1028.6 Da; F3, 652.2 Da; F4, 562.2 Da; F5, 469.6 Da) from Leucaena leucocephala hybrid-Rendang (LLR) on the structure and diversity of the rumen bacterial community were investigated in vitro. RESULTS: Real-time polymerase chain reaction assay showed that the total bacterial population was not significantly (P > 0.05) different among the dietary treatments. Inclusion of higher-MW CT fractions F1 and F2 significantly (P < 0.05) increased the Fibrobacter succinogenes population compared with F0 and CT fractions F3-F5. Although inclusion of F0 and CT fractions (F1-F5) significantly (P < 0.05) decreased the Ruminococcus flavefaciens population, there was no effect on the Ruminococcus albus population when compared with the control (without CTs). High-throughput sequencing of the V3 region of 16S rRNA showed that the relative abundance of genera Prevotella and unclassified Clostridiales was significantly (P < 0.05) decreased, corresponding with increasing MW of CT fractions, whereas cellulolytic bacteria of the genus Fibrobacter were significantly (P < 0.05) increased. Inclusion of higher-MW CT fractions F1 and/or F2 decreased the relative abundance of minor genera such as Ruminococcus, Streptococcus, Clostridium XIVa and Anaeroplasma but increased the relative abundance of Acinetobacter, Treponema, Selenomonas, Succiniclasticum and unclassified Spirochaetales compared with the control and lower-MW CT fractions. CONCLUSION: This study indicates that CT fractions of different MWs may play an important role in altering the structure and diversity of the rumen bacterial community in vitro, and the impact was more pronounced for CT fractions with higher MW. © 2016 Society of Chemical Industry.

Characterization of Conjugated Linoleic Acid-Producing Lactic Acid Bacteria as Potential Probiotic for Chicken

Aims: Dietary intake of conjugated linoleic acid (CLA) by human is insufficient to exhibit properties of anti-cancer, antiinflammatory, anti-atherosclerosis, anti-obesity and enhancing immune system. Thus, enrichment of CLA in chicken by bacteria is a suggestion to solve the problem. It would be an advantage to have bacteria capable of producing CLA and has probiotic potential in chicken. Thus, probiotic properties of CLA-producing bacteria were accessed in this study. Methodology and results: In this study, 47 lactic acid bacteria (LAB) isolated from gastrointestinal tract of chickens were screened for conjugated linoleic acid (CLA) production. Lactobacillus salivarius strain P2, Enterococcus faecium strain P1 and Lactobacillus agilis strain P3 were shown to produce 21.97, 23.35 and 31.08 µg/mL of CLA in MRS broth containing free linoleic acid (0.5 mg/mL) and 2% (w/v) Tween 80, respectively. Lactobacillus salivarius strain P2, E. faecium strain P1 and L. agilis strain P3 were found to be able to tolerate 0.3% oxgall (Difco, France) and pH 2.5. Lactobacillus agilis strain P3 and L. salivarius strain P2 showed better acid tolerance compared to E. faecium strain P1. Besides that, L. agilis strain P3 and L. salivarius strain P2 were resistant to two out of eight types of antibiotics tested, able to produce 220.04 mM lactic acid and 200.17 mM of lactic acid, respectively. Enterococcus faecium strain P1 was resistant to five out of eight types of antibiotic tested, produced 90.39 mM lactic acid and showed hemolytic activity. Only L. agilis strain P3 can produce acetic acid at a concentration of 2.71 mM. Conclusion, significance and impact of study: These results showed that the CLA-producing L. salivarius strain P2 and L. agilis strain P3 could be potential probiotic bacteria for chickens, which may eventually lead to production of chicken with better meat quality.

Chemical Compositions of Egg Yolks and Egg Quality of Laying Hens Fed Prebiotic, Probiotic, and Synbiotic Diets.

A 16-wk feeding experiment was conducted to investigate the effects of a prebiotic, isomaltooligosaccharide (IMO), a probiotic, PrimaLac®, and their combination as a synbiotic on the chemical compositions of egg yolks and the egg quality of laying hens. One hundred and sixty 16-wk-old Hisex Brown pullets were randomly assigned to 4 dietary treatments: (i) basal diet (control), (ii) basal diet + 1% IMO (PRE), (iii) basal diet + 0.1% PrimaLac® (PRO), and (iv) basal diet + 1% IMO + 0.1% PrimaLac® (SYN). PRE, PRO, or SYN supplementation not only significantly (P < 0.05) decreased the egg yolk cholesterol (24- and 28-wk-old) and total saturated fatty acids (SFA; 28-, 32-, and 36-wk-old), but also significantly (P < 0.05) increased total unsaturated fatty acids (UFA; 28-, 32-, and 36-wk-old), total omega 6 and polyunsaturated fatty acids (PUFA), including linoleic and alpha-linolenic acid levels in the eggs (28-wk-old). However, the total lipids, carotenoids, and tocopherols in the egg yolks were similar among all dietary treatments in the 24-, 28-, 32-, and 36-wk-old hens. Egg quality (Haugh unit, relative weights of the albumen and yolk, specific gravity, shell thickness, and yolk color) was not affected by PRE, PRO, or SYN supplementation. The results indicate that supplementations with IMO and PrimaLac® alone or in combination as a synbiotic might be useful for improving the cholesterol content and modifying the fatty acid compositions of egg yolk without affecting the quality of eggs from laying hens between 24 and 36 wk of age.

Deciphering chicken gut microbial dynamics based on high-throughput 16S rRNA metagenomics analyses.

BACKGROUND: Chicken gut microbiota has paramount roles in host performance, health and immunity. Understanding the topological difference in gut microbial community composition is crucial to provide knowledge on the functions of each members of microbiota to the physiological maintenance of the host. The gut microbiota profiling of the chicken was commonly performed previously using culture-dependent and early culture-independent methods which had limited coverage and accuracy. Advances in technology based on next-generation sequencing (NGS), offers unparalleled coverage and depth in determining microbial gut dynamics. Thus, the aim of this study was to investigate the ileal and caecal microbiota development as chicken aged, which is important for future effective gut modulation. MATERIAL AND METHODS: Ileal and caecal contents of broiler chicken were extracted from 7, 14, 21 and 42-day old chicken. Genomic DNA was then extracted and amplified based on V3 hyper-variable region of 16S rRNA. Bioinformatics, ecological and statistical analyses such as Principal Coordinate Analysis (PCoA) was performed in mothur software and plotted using PRIMER 6. Additional analyses for predicted metagenomes were performed through PICRUSt and STAMP software package based on Greengenes databases. RESULTS: A distinctive difference in bacterial communities was observed between ilea and caeca as the chicken aged (P < 0.001). The microbial communities in the caeca were more diverse in comparison to the ilea communities. The potentially pathogenic bacteria such as Clostridium were elevated as the chicken aged and the population of beneficial microbe such as Lactobacillus was low at all intervals. On the other hand, based on predicted metagenomes analysed, clear distinction in functions and roles of gut microbiota such as gene pathways related to nutrient absorption (e.g. sugar and amino acid metabolism), and bacterial proliferation and colonization (e.g. bacterial motility proteins, two-component system and bacterial secretion system) were observed between ilea and caeca, respectively (P < 0.05). CONCLUSIONS: The caeca microbial communities were more diverse in comparison to ilea. The main functional differences between the two sites were found to be related to nutrient absorption and bacterial colonization. Based on the composition of the microbial community, future gut modulation with beneficial bacteria such as probiotics may benefit the host.

Effects of condensed tannin fractions of different molecular weights from a Leucaena leucocephala hybrid on in vitro methane production and rumen fermentation.

BACKGROUND: Molecular weights (MWs) and their chemical structures are the primary factors determining the influence of condensed tannins (CTs) on animal nutrition and methane (CH4 ) production in ruminants. In this study the MWs of five CT fractions from Leucaena leucocephala hybrid-Rendang (LLR) were determined and the CT fractions were investigated for their effects on CH4 production and rumen fermentation. RESULTS: The number-average molecular weight (Mn ) of fraction F1 (1265.8 Da), which was eluted first, was the highest, followed by those of fractions F2 (1028.6 Da), F3 (652.2 Da), F4 (562.2 Da) and F5 (469.6 Da). The total gas (mL g(-1) dry matter (DM)) and CH4 production decreased significantly (P < 0.05) with increasing MWs of the CT fractions, but there were no significant (P > 0.05) differences between the CT fractions and control on DM degradation. However, the in vitro N disappearance decreased significantly (P < 0.05) with the inclusion of CT fraction F1 (highest MW) compared with the control and other fractions (F2-F5). The inclusion of CT fraction F1 also significantly decreased (P < 0.05) total volatile fatty acid and acetic acid concentrations compared with the control. The acetic/propionic acid ratio was significantly decreased (P < 0.05) by fraction F1 but not by the control and other fractions (F2-F5). CONCLUSION: The CT fractions of different MWs from LLR could affect rumen fermentation and CH4 production, and the impact was more pronounced for the CT fraction with a higher MW.

Probiotic potential of Lactobacillus strains with antimicrobial activity against some human pathogenic strains.

The objective of this study was to isolate, identify, and characterize some lactic acid bacterial strains from human milk, infant feces, and fermented grapes and dates, as potential probiotics with antimicrobial activity against some human pathogenic strains. One hundred and forty bacterial strains were isolated and, after initial identification and a preliminary screening for acid and bile tolerance, nine of the best isolates were selected and further identified using 16 S rRNA gene sequences. The nine selected isolates were then characterized in vitro for their probiotic characteristics and their antimicrobial activities against some human pathogens. Results showed that all nine isolates belonged to the genus Lactobacillus. They were able to tolerate pH 3 for 3 h, 0.3% bile salts for 4 h, and 1.9 mg/mL pancreatic enzymes for 3 h. They exhibited good ability to attach to intestinal epithelial cells and were not resistant to the tested antibiotics. They also showed good antimicrobial activities against the tested pathogenic strains of humans, and most of them exhibited stronger antimicrobial activity than the reference strain L. casei Shirota. Thus, the nine Lactobacillus strains could be considered as potential antimicrobial probiotic strains against human pathogens and should be further studied for their human health benefits.

Polymerization degrees, molecular weights and protein-binding affinities of condensed tannin fractions from a Leucaena leucocephala hybrid.

Condensed tannins (CTs) form insoluble complexes with proteins and are able to protect them from degradation, which could lead to rumen bypass proteins. Depending on their degrees of polymerization (DP) and molecular weights, CT fractions vary in their capability to bind proteins. In this study, purified condensed tannins (CTs) from a Leucaena leucocephala hybrid were fractionated into five different molecular weight fractions. The structures of the CT fractions were investigated using 13C-NMR. The DP of the CT fractions were determined using a modified vanillin assay and their molecular weights were determined using Q-TOF LC-MS. The protein-binding affinities of the respective CT fractions were determined using a protein precipitation assay. The DP of the five CT fractions (fractions F1-F5) measured by the vanillin assay in acetic acid ranged from 4.86 to 1.56. The 13C-NMR results showed that the CT fractions possessed monomer unit structural heterogeneity. The number-average molecular weights (Mn) of the different fractions were 1265.8, 1028.6, 652.2, 562.2, and 469.6 for fractions F1, F2, F3, F4, and F5, respectively. The b values representing the CT quantities needed to bind half of the maximum precipitable bovine serum albumin increased with decreasing molecular weight--from fraction F1 to fraction F5 with values of 0.216, 0.295, 0.359, 0.425, and 0.460, respectively. This indicated that higher molecular weight fractions of CTs from L. leucocephala have higher protein-binding affinities than those with lower molecular weights.

Effects of dietary prebiotics, probiotic and synbiotics on performance, caecal bacterial populations and caecal fermentation concentrations of broiler chickens.

BACKGROUND: In view of a worldwide attempt to restrict or ban the use of antibiotics as growth promoters in animal production, probiotics, prebiotics and combinations of both, as synbiotics, have been suggested as potential alternatives. In this study, the effects of a prebiotic (isomalto-oligosaccharides, IMO), a multi-strain probiotic (consisting of 11 Lactobacillus strains), and a combination of these dietary additives as a synbiotic on the performance, caecal bacterial populations and concentrations of caecal volatile fatty acids and non-volatile fatty acids of broiler chickens were evaluated. RESULTS: Supplementation of 1g kg⁻¹ probiotic (PRO); 5 g kg⁻¹ prebiotic IMO (PRE05); 10 g kg⁻¹ prebiotic IMO (PRE10); synbiotic consisting of 1g kg⁻¹ probiotic + 5 g kg⁻¹ prebiotic IMO (SYN05); or synbiotic consisting of 1 g kg⁻¹ probiotic + 10 g kg⁻¹ prebiotic IMO (SYN10) significantly (P < 0.05) improved weight gain of broiler chickens at 22-42 and 1-42 days of age, and feed conversion rate from 1 to 21, 22-42 and 1-42 days of age. The supplementation of probiotic (PRO), prebiotics (PRE05 and PRE10) or synbiotics (SYN05 and SYN10) also significantly (P < 0.05) increased the caecal populations of lactobacilli and bifidobacteria, and decreased the caecal Escherichia coli at 21 days of age, and increased the caecal VFA at 21 and 42 days of age. In all parameters studied, synbiotics did not show a two-fold synergistic effect, when compared to those of probiotic or prebiotic alone. CONCLUSION: The results of the study indicated that prebiotic IMO (5 g kg⁻¹ or 10 g kg⁻¹), probiotic and their combinations as synbiotics were effective in improving the performance of broiler chickens and in increasing the caecal beneficial bacteria and fatty acids.

Evaluation of a lytic bacteriophage, Φ st1, for biocontrol of Salmonella enterica serovar Typhimurium in chickens.

In this study, a Salmonella Typhimurium lytic bacteriophage, Φ st1, which was isolated from chicken faecal material, was evaluated as a candidate for biocontrol of Salmonella in chickens. The morphology of Φ st1 showed strong resemblance to members of the Siphoviridae family. Φ st1 was observed to be a DNA phage with an estimated genome size of 121 kbp. It was found to be able to infect S. Typhimurium and S. Hadar, with a stronger lytic activity against the former. Subsequent characterisation of Φ st1 against S. Typhimurium showed that Φ st1 has a latent period of 40 min with an average burst size of 22 particles per infective centre. Approximately 86.1% of the phage adsorbed to the host cells within the initial 5 min of infection. At the optimum multiplicity of infection (MOI) (0.1), the highest reduction rate of S. Typhimurium (6.6 log10 CFU/ml) and increment in phage titre (3.8 log10 PFU/ml) was observed. Φ st1 produced adsorption rates of 88.4-92.2% at pH7-9 and demonstrated the highest bacteria reduction (6.6 log10 CFU/ml) at pH9. Φ st1 also showed an insignificant different (P>0.05) reduction rate of host cells at 37 °C (6.4 log10 CFU/ml) and 42 °C (6.0 log10 CFU/ml). The in vivo study using Φ st1 showed that intracloacal inoculation of ~10¹² PFU/ml of the phage in the chickens challenged with ~10¹° CFU/ml of S. Typhimurium was able to reduce (P<0.05) the S. Typhimurium more rapidly than the untreated group. The Salmonella count reduced to 2.9 log10 CFU/ml within 6h of post-challenge and S. Typhimurium was not detected at and after 24h of post-challenge. Reduction of Salmonella count in visceral organs was also observed at 6h post-challenge. Approximately 1.6 log10 FU/ml Φ st1 was found to persist in the caecal wall of the chicks at 72 h of post-challenge. The present study indicated that Φ st1 may serve as a potential biocontrol agent to reduce the Salmonella count in caecal content of chickens.

Anaerobic cellulolytic rumen fungal populations in goats fed with and without Leucaena leucocephala hybrid, as determined by real-time PCR.

The effect of Leucaena leucocephala hybrid-Bahru (LLB), which contains a high concentration of condensed tannins, on cellulolytic rumen fungal population in goats was investigated using real-time PCR. The fungal population in goats fed LLB was inhibited during the first 10 days of feeding, but after 15 days of feeding, there was a tremendous increase of fungal population (157.0 µg/ml), which was about fourfold more than that in control goats (39.7 µg/ml). However, after this period, the fungal population decreased continuously, and at 30 days of feeding, the fungal population (50.6 µg/ml) was not significantly different from that in control goats (55.4 µg/ml).

The complete genome sequence of EC1-UPM, a novel N4-like bacteriophage that infects Escherichia coli O78:K80.

BACKGROUND: Bacteriophage EC1-UPM is an N4-like bacteriophage which specifically infects Escherichia coli O78:K80, an avian pathogenic strain that causes colibacillosis in poultry. The complete genome sequence of bacteriophage EC1-UPM was analysed and compared with other closely related N4-like phage groups to assess their genetic similarities and differences. RESULTS: Bacteriophage EC1-UPM displays a very similar codon usage profile with its host and does not contain any tRNA gene. Comparative genomics analysis reveals close resemblance of bacteriophage EC1-UPM to three N4-like bacteriophages namely vB_EcoP_G7C, IME11 and KBNP21 with a total of 44 protein coding genes shared at 70% identity threshold. The genomic region coding for the tail fiber protein was found to be unique in bacteriophage EC1-UPM. Further annotation of the tail fiber protein using HHpred, a highly sensitive homology detection tool, reveals the presence of protein structure homologous to various polysaccharide processing proteins in its C-terminus. Leveraging on the availability of multiple N4-like bacteriophage genome sequences, the core genes of N4-like bacteriophages were identified and used to perform a multilocus phylogenetic analysis which enabled the construction of a phylogenetic tree with higher confidence than phylogenetic trees based on single genes. CONCLUSION: We report for the first time the complete genome sequence of a N4-like bacteriophage which is lytic against avian pathogenic Escherichia coli O78:K80. A novel 928 amino acid residues tail fiber protein was identified in EC1-UPM which may be useful to further the understanding of phage-host specificity. Multilocus phylogenetic analysis using core genes of sequenced N4-like phages showed that the evolutionary relationship correlated well with the pattern of host specificity.

Lovastatin in Aspergillus terreus: fermented rice straw extracts interferes with methane production and gene expression in Methanobrevibacter smithii.

Lovastatin, a natural byproduct of some fungi, is able to inhibit HMG-CoA (3-hydroxy-3 methyl glutaryl CoA) reductase. This is a key enzyme involved in isoprenoid synthesis and essential for cell membrane formation in methanogenic Archaea. In this paper, experiments were designed to test the hypothesis that lovastatin secreted by Aspergillus terreus in fermented rice straw extracts (FRSE) can inhibit growth and CH4 production in Methanobrevibacter smithii (a test methanogen). By HPLC analysis, 75% of the total lovastatin in FRSE was in the active hydroxyacid form, and in vitro studies confirmed that this had a stronger effect in reducing both growth and CH4 production in M. smithii compared to commercial lovastatin. Transmission electron micrographs revealed distorted morphological divisions of lovastatin- and FRSE-treated M. smithii cells, supporting its role in blocking normal cell membrane synthesis. Real-time PCR confirmed that both commercial lovastatin and FRSE increased (P < 0.01) the expression of HMG-CoA reductase gene (hmg). In addition, expressions of other gene transcripts in M. smithii. with a key involvement in methanogenesis were also affected. Experimental confirmation that CH4 production is inhibited by lovastatin in A. terreus-fermented rice straw paves the way for its evaluation as a feed additive for mitigating CH4 production in ruminants.

Lovastatin-enriched rice straw enhances biomass quality and suppresses ruminal methanogenesis.

The primary objective of this study was to test the hypothesis that solid state fermentation (SSF) of agro-biomass (using rice straw as model); besides, breaking down its lignocellulose content to improve its nutritive values also produces lovastatin which could be used to suppress methanogenesis in the rumen ecosystem. Fermented rice straw (FRS) containing lovastatin after fermentation with Aspergillus terreus was used as substrate for growth study of rumen microorganisms using in vitro gas production method. In the first experiment, the extract from the FRS (FRSE) which contained lovastatin was evaluated for its efficacy for reduction in methane (CH4) production, microbial population, and activity in the rumen fluid. FRSE reduced total gas and CH4 productions (P < 0.01). It also reduced (P < 0.01) total methanogens population and increased the cellulolytic bacteria including Ruminococcus albus, Fibrobacter succinogenes (P < 0.01), and Ruminococcus flavefaciens (P < 0.05). Similarly, FRS reduced total gas and CH4 productions, methanogens population, but increased in vitro dry mater digestibility compared to the non-fermented rice straw. Lovastatin in the FRSE and the FRS significantly increased the expression of HMG-CoA reductase gene that produces HMG-CoA reductase, a key enzyme for cell membrane production in methanogenic Archaea.

Effect of condensed tannins on bovine rumen protist diversity based on 18S rRNA gene sequences.

Molecular diversity of protists from bovine rumen fluid incubated with condensed tannins of Leucaena leucocephala hybrid-Rendang at 20 mg/500 mg dry matter (treatment) or without condensed tannins (control) was investigated using 18S rRNA gene library. Clones from the control library were distributed within nine genera, but clones from the condensed tannin treatment clone library were related to only six genera. Diversity estimators such as abundance-based coverage estimation and Chao1 showed significant differences between the two libraries, although no differences were found based on Shannon-Weaver index and Libshuff.

Lovastatin production by Aspergillus terreus using agro-biomass as substrate in solid state fermentation.

Ability of two strains of Aspergillus terreus (ATCC 74135 and ATCC 20542) for production of lovastatin in solid state fermentation (SSF) using rice straw (RS) and oil palm frond (OPF) was investigated. Results showed that RS is a better substrate for production of lovastatin in SSF. Maximum production of lovastatin has been obtained using A. terreus ATCC 74135 and RS as substrate without additional nitrogen source (157.07 mg/kg dry matter (DM)). Although additional nitrogen source has no benefit effect on enhancing the lovastatin production using RS substrate, it improved the lovastatin production using OPF with maximum production of 70.17 and 63.76 mg/kg DM for A. terreus ATCC 20542 and A. terreus ATCC 74135, respectively (soybean meal as nitrogen source). Incubation temperature, moisture content, and particle size had shown significant effect on lovastatin production (P < 0.01) and inoculums size and pH had no significant effect on lovastatin production (P > 0.05). Results also have shown that pH 6, 25°C incubation temperature, 1.4 to 2 mm particle size, 50% initial moisture content, and 8 days fermentation time are the best conditions for lovastatin production in SSF. Maximum production of lovastatin using optimized condition was 175.85 and 260.85 mg/kg DM for A. terreus ATCC 20542 and ATCC 74135, respectively, using RS as substrate.