Effects of Phage Cocktail, Probiotics, and Their Combination on Growth Performance and Gut Microbiota of Broiler Chickens.

Phages, which are often used therapeutically, have begun to receive interest as alternatives to antibiotic growth promoters (AGPs) for enhancing chicken growth. Another option that has been extensively studied as a growth promoter in chickens is probiotics. To the best of our knowledge, there is currently no study available on the use of phages and probiotics in combination as potential feed additives for broiler chickens. Therefore, this study demonstrated the effects of a phage cocktail, probiotics, and their combination on the growth performance and gut microbiota of broiler chickens. A total of 288 one-day-old male Cobb 500 broilers were randomly allotted to one of six treatments in a completely randomised design. The treatments were (i) C (basal diet (BD) only), (ii) 1ϕ (BD + 0.1% phage cocktail), (iii) 2ϕ (BD + 0.2% phage cocktail), (iv) P (BD + 0.1% probiotic), (v) 1ϕP (BD + 0.1% phage cocktail + 0.1% probiotic), and (vi) 2ϕP (BD + 0.2% phage cocktail + 0.1% probiotic). The 1ϕP treatment had significantly (p < 0.05) better BW (35 days), BWG (22-35 days, 1-35 days), and FCR (1-21 days, 22-35 days, 1-35 days) compared to C. Unique gut microbiota diversity was also found between the ϕP (1ϕP and 2ϕP) and non-ϕP groups (C, 1ϕ, 2ϕ, and P) in ilea, particularly in the 35-day-old chickens. Microorganisms associated with short-chain fatty acid (SCFA) producers were significantly (p < 0.05) more present in the ϕP group than in the non-ϕP group. The predicted genes related to carbohydrate and amino acid metabolism were significantly upregulated in ϕP groups compared to non-ϕP groups. These genes were involved in the digestion and absorption of nutrients, as well as the production of energy. Our findings showed that the 1ϕP treatment could be a potential alternative to AGPs for poultry, as growth performance was enhanced, and gut microbiota was positively modulated.

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.

A 12-residue epitope displayed on phage T7 reacts strongly with antibodies against foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a major threat to the livestock industry worldwide. Despite constant surveillance and effective vaccination, the perpetual mutations of the foot-and-mouth disease virus (FMDV) pose a huge challenge to FMD diagnosis. The immunodominant region of the FMDV VP1 protein (residues 131-170) displayed on phage T7 has been used to detect anti-FMDV in bovine sera. In the present study, the functional epitope was further delineated using amino acid sequence alignment, homology modelling and phage display. Two highly conserved regions (VP1145-152 and VP1159-170) were identified among different FMDV serotypes. The coding regions of these two epitopes were fused separately to the T7 genome and displayed on the phage particles. Interestingly, chimeric phage displaying the VP1159-170 epitope demonstrated a higher antigenicity than that displaying the VP1131-170 epitope. By contrast, phage T7 displaying the VP1145-152 epitope did not react significantly with the anti-FMDV antibodies in vaccinated bovine sera. This study has successfully identified a smaller functional epitope, VP1159-170, located at the C-terminal end of the structural VP1 protein. The phage T7 displaying this shorter epitope is a promising diagnostic reagent to detect anti-FMDV antibodies in vaccinated animals.

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.

Construction of a novel inducible expression vector for Lactococcus lactis M4 and Lactobacillus plantarum Pa21.

A vector that drives the expression of the reporter gusA gene in both Lactobacillus plantarum and Lactococcus lactis was constructed in this study. This vector contained a newly characterized heat shock promoter (Phsp), amplified from an Enterococcus faecium plasmid, pAR6. Functionality and characterization of this promoter was initially performed by cloning Phsp into pNZ8008, a commercial lactococcal plasmid used for screening of putative promoters which utilizes gusA as a reporter. It was observed that Phsp was induced under heat, salinity and alkaline stresses or a combination of all three stresses. The newly characterized Phsp promoter was then used to construct a novel Lactobacillus vector, pAR1801 and its ability to express the gusA under stress-induced conditions was reproducible in both Lb. plantarum Pa21 and L. lactis M4 hosts.

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.

Display of the VP1 epitope of foot-and-mouth disease virus on bacteriophage T7 and its application in diagnosis.

Foot-and-mouth disease (FMD) is a highly contagious epidemic disease threatening the cattle industry since the sixteenth century. In recent years, the development of diagnostic assays for FMD has benefited considerably from the advances of recombinant DNA technology. In this study, the immunodominant region of the capsid protein VP1 of the foot-and-mouth disease virus (FMDV) was fused to the T7 bacteriophage and expressed on the surface of the bacteriophage capsid protein. The recombinant protein of about 42 kDa was detected by the anti-T7 tag monoclonal antibody in Western blot analysis. Phage ELISA showed that both the vaccinated and positive infected bovine sera reacted significantly with the recombinant T7 particle. This study demonstrated the potential of the T7 phage displaying the VP1 epitope as a diagnostic reagent.

Purification of long helical capsid of newcastle disease virus from Escherichia coli using anion exchange chromatography.

NP(Δc375) is a truncated version of the nucleocapsid protein of Newcastle disease virus (NDV) which self-assembles into a long helical structure. A packed bed anion exchange chromatography (PB-AEC), SepFastTM Supor Q pre-packed column, was used to purify NP(Δc375) from clarified feedstock. This PB-AEC column adsorbed 76.2% of NP(Δc375) from the clarified feedstock. About 67.5% of the adsorbed NP(Δc375) was successfully eluted from the column by applying 50 mM Tris-HCl elution buffer supplemented with 0.5 M NaCl at pH 7. Thus, a recovery yield of 51.4% with a purity of 76.7% which corresponds to a purification factor of 6.5 was achieved in this PB-AEC operation. Electron microscopic analysis revealed that the helical structure of the NP(Δc375) purified by SepFast(TM) Supor Q pre-packed column was as long as 490 nm and 22-24 nm in diameter. The antigenicity of the purified NP(Δc375) was confirmed by enzyme-linked immunosorbent assay.