[Comparative metabolomics analysis of metabolic pathways in the high-yielding mutant strain of avilamycin].

Avilamycin (AVI) is an oligosaccharide antibiotic that has strong inhibitory effect on Gram-positive bacteria. It is widely used in livestock and poultry farming. However, the use of traditional breeding techniques and immature fermentation process have become the key factors limiting its commercialization. In this study, we used comparative metabolomics techniques to examine the difference in intracellular metabolism between a high-yield AVI mutant strain modified by ribosome engineering technology and the parental strain. GC-MS analysis was conducted on mycelia samples taken on days 4, 6, and 8 of fermentation, resulting in the detection of a total of 112 compounds. After comparison with the NIST library, 29 intracellular metabolites were accurately identified. Two-dimensional principal component analysis (PCA) revealed significant differences in metabolites between the mutant strain and the parental strain at different time points. Orthogonal partial least squares-discriminant analysis (OPLS-DA) identified 11 intracellular metabolites that were closely related to AVI biosynthesis. KEGG metabolic pathway enrichment analysis showed that avilamycin synthesis was closely related to carbohydrate metabolism and amino acid metabolism. Six key differential metabolites were selected: L-valine, L-serine, L-alanine, D-galactose, D-cellobiose, and D-glucose. Upregulation of these metabolites in the mutant strain enhanced its metabolic flow for AVI synthesis. After 8 days of fermentation, the mutant strain produced 76.86% more AVI than the parental strain. The findings of this study serve as reference for the future rational optimization of avilamycin fermentation.

Dietary lysozyme and avilamycin modulate gut health, immunity, and growth rate in broilers.

BACKGROUND: Attempts to use dietary lysozyme (LYZ) as an alternative to antibiotics in broilers have been successful, but further research is needed for effective use. Here, we compared the differences between LYZ and avilamycin (AVI) feed additives for growth performance, gut health and immunity of broilers. One-day old, one hundred and twenty broiler chicks (Ross 308) were randomly allocated into three groups consisting forty birds in each group. Standard diet without supplementation was applied as the control group (I), while the chicks of the other groups were supplemented with 100 mg of AVI per kg diet (AVI, group II), and 90 mg LYZ per kg diet (LYZ, group III) for five consecutive weeks. RESULTS: Body weight, feed conversion ratio, body weight gain, and European production efficiency factor were markedly (p < 0.05) increased in both AVI and LYZ groups in relation to CON group, but the feed intake and protein efficiency ratio were not affected. Both AVI and LYZ significantly (p < 0.001) upregulated the mRNA expression of ileal interleukin-18 (IL-18), interferon-gamma (IFN-γ), and interleukin-10 (IL-10), interleukin-2 (IL-2), and glutathione peroxidase (GSH-PX) genes compared to CON group. However, IL-2, IL-10, IL-18, and GSH-PX genes were markedly (p < 0.01) upregulated in LYZ compared to the AVI group. LYZ treated group had a significant increase (p < 0.05) in the serological haemagglutination inhibition titers of H5N1 vaccination and a significant decrease (p < 0.0001) in coliform counts compared to control and AVI groups, but all growth parameters were nearly similar between AVI and LYZ groups. The VH and VH/CD were markedly higher in LYZ than AVI and control groups. CONCLUSION: Exogenous dietary lysozyme supplementation by a dose of 90 mg/kg broilers' diet induced better effects on intestinal integrity, fecal bacterial counts, immune response, and growth performance which were comparable to avilamycin. Therefore, dietary lysozyme could safely replace avilamycin in the broiler chickens' diet. However, further experimental studies regarding the use of lysozyme in commercial broilers, both in vitro and in vivo, targeting more communities of intestinal microbiome and explaining more details about its beneficial effects need to be conducted.

Optimal Regimens and Clinical Breakpoint of Avilamycin Against Clostridium perfringens in Swine Based on PK-PD Study.

Clostridium perfringens causes significant morbidity and mortality in swine worldwide. Avilamycin showed no cross resistance and good activity for treatment of C. perfringens. The aim of this study was to formulate optimal regimens of avilamycin treatment for C. perfringens infection based on the clinical breakpoint (CBP). The wild-type cutoff value (COWT) was defined as 0.25 µg/ml, which was developed based on the minimum inhibitory concentration (MIC) distributions of 120 C. perfringens isolates and calculated using ECOFFinder. Pharmacokinetics-pharmacodynamics (PK-PD) of avilamycin in ileal content were analyzed based on the high-performance liquid chromatography method and WinNonlin software to set up the target of PK/PD index (AUC0-24h/MIC)ex based on sigmoid Emax modeling. The PK parameters of AUC0-24h, Cmax, and Tmax in the intestinal tract were 428.62 ± 14.23 h µg/mL, 146.30 ± 13.41 µg/ml,, and 4 h, respectively. The target of (AUC0-24h/MIC)ex for bactericidal activity in intestinal content was 36.15 h. The PK-PD cutoff value (COPD) was defined as 8 µg/ml and calculated by Monte Carlo simulation. The dose regimen designed from the PK-PD study was 5.2 mg/kg mixed feeding and administrated for the treatment of C. perfringens infection. Five respective strains with different MICs were selected as the infection pathogens, and the clinical cutoff value was defined as 0.125 µg/ml based on the relationship between MIC and the possibility of cure (POC) following nonlinear regression analysis, CART, and "Window" approach. The CBP was set to be 0.25 µg/ml and selected by the integrated decision tree recommended by the Clinical Laboratory of Standard Institute. The formulation of the optimal regimens and CBP is good for clinical treatment and to control drug resistance.

Development of a highly sensitive monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for the detection of avilamycin in feed.

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed using a highly sensitive and specific monoclonal antibody against avilamycin. The immunising antigen synthesised through the carbonyldiimidazole (CDI) method was used to prepare antibodies. The established ic-ELISA, after optimisation of conditions, possessed a half maximum inhibitory concentration (IC50) value of 7.44 ng mL-1 and the detection limit of 0.21 ng mL-1 in the standard curve. The spiked experiments indicated that the limits of detection were 1.86 µg kg-1 and 2.31 µg kg-1 in swine feed and chicken feed, respectively. In addition, the average recoveries ranged from 74.7 to 105.4% with the coefficient of variation less than 11%. The good correlation (R2 = 0.9818) between the result of ic-ELISA and HPLC demonstrated the reliability of the developed ic-ELISA, which showed that the ic-ELISA developed here achieves the aim of strengthening the monitoring of avilamycin residues and the inspection of import and export related products.

Avilamycin production enhancement by mutagenesis and fermentation optimization in Streptomyces viridochromogenes.

Avilamycin, an excellent growth-promoting feed additive, produced by Streptomyces viridochromogenes, was widely used to promote the growth of poultry by inhibiting Gram-positive bacteria. In this work, the methods of combinational mutagenesis of UV (Ultraviolet) and ARTP (Atmospheric and room temperature plasma), and rational screening by high concentrations of CaCl2 were utilized to promote the production of avilamycin. The avilamycin high-yielding mutant strains of Z-6 (29.31 mg/L), A-9 (36.84 mg/L) and F-23 (45.73 mg/L) were screened out, with yields of avilamycin improved by 57.92%, 98.49% and 146.39%, respectively, compared with the wild strain (WT). The performance comparison showed that Z-6, A-9 and F-23 mutant strains had stronger abilities of substrate consumption, cell growth and antibiotic synthesis than WT. Furthermore, the composition of fermentation medium, inoculation parameters, supplementation strategies of oxygen vectors, glucose and precursors (L-valine, D-xylose and sodium acetate) had been optimized and the avilamycin yield of the mutant strain F-23 was significantly enhanced by 41.87% by fermentation optimization. In summary, the strategy of increasing the production of avilamycin in S. viridochromogenes in this work might provide an alternative method to enhance the synthesis of secondary metabolites in other Streptomyces.

Research Note: Snapshot of the transcriptome via RNA sequencing in the ileum of broiler chickens fed subtherapeutic concentrations of avilamycin.

Antibiotics have played a critical role in sustaining and improving livestock production in the past decades, but the emergence of antimicrobial resistance has led several countries to ban or limit their use. Since then, in-feed alternatives have gained a lot of attention but the development of efficacious alternatives implies a better understanding of the mode of action of antibiotic growth promoters (AGP) when administered at subtherapeutic concentrations. In the present study, 120 broiler chickens per group (8 pens/group) were fed for 35 d with either basal feed (control group) or feed supplemented with avilamycin (AGP group; 10 g/1,000 kg of feed). At the end of the trial, the ileum from the small intestine of 5 birds per group was sampled, and RNA were isolated for profiling their transcriptome via RNA sequencing (RNA-Seq). As expected, the growth of chickens in the AGP group was significantly higher than in the control group. Overall, 66 differentially expressed genes (false discovery rate ≤ 0.05 and fold change ≥ 2 or ≤ -2) were found in the ileum of chickens fed avilamycin in comparison with the control group. The functional analysis showed reduced activity of genes related to signaling by interleukins, with IL-22, SOCS3, and certain antimicrobial peptides found multiple times in these pathways in the AGP group at day 35. In addition, higher activity was predicted in a module of genes related to lipid metabolism and transport in the avilamycin group. The use of RNA-Seq allowed a snapshot of the whole transcriptome at day 35 and aimed at delivering additional data on the host-centric hypothesis regarding the mode of action of AGP (i.e. immunomodulation, reduction of the immunological stress).

Determination of total avilamycin residues as dichloroisoeverninic acid in porcine muscle, fat, and liver by LC-MS/MS.

A sensitive and reliable method for determining the total avilamycin residues was developed using LC-MS/MS. Avilamycin (consisting of avilamycin A and 15 other minor factors) and its metabolites in porcine muscle, fat, and liver were analysed as the marker residue dichloroisoeverninic acid (DIA), in accordance with the maximum residue limit (MRL) established by international organisations such as Codex Alimentarius Commission and other regulatory bodies. The analytes were extracted from samples with acetone, hydrolysed to DIA, partitioned into ethyl acetate, and cleaned up prior to the LC-MS/MS analysis. The method was validated at Codex MRL and 0.01 mg/kg. The results show excellent recoveries ranging from 100 to 108%, with the relative standard deviations <6%. Matrix effects were negligible for all types of samples, indicating effective sample clean-up. The absence of interfering peaks close to the retention time in blank samples demonstrates high selectivity. Overall, this method is reliable and suitable for regulatory-purpose analysis.

Performance, gut morphology and microbiology effects of a Bacillus probiotic, avilamycin and their combination in mixed grain broiler diets.

1. This study aimed to determine the effect of avilamycin (AGP) and a multi-strain Bacillus probiotic (DFM) on the performance, gut histology and microbiology of broilers fed on a mixed grain diet. 2. A total of 800 chicks were allocated to four treatments: a control diet, control+AGP, control+DFM, or control+AGP+DFM. Bodyweight, feed intake and FCR were measured at d 0, 21 and 42. Samples were taken at d42 to determine villus height (VH), crypt depth (CD) and ratio (VH:CD). Mucosal E. coli and Lactobacilli counts were measured at d42. 3. At d42, DFM and AGP+DFM significantly increased weight over the control, with AGP returning an intermediate value. FCR followed a similar pattern. DFM and AGP+DFM significantly increased VH and CD in all gut sections compared to the control. 4. DFM and AGP+DFM reduced E. coli counts compared to control, with AGP reducing caecal counts only, while Lactobacilli counts were increased. 5. Divergent histology and microbiology between treatments highlight the different modes of action of AGP and DFM for improving broiler growth and feed efficiency.

Use of Bacillus Subtilis PB6 as a potential antibiotic growth promoter replacement in improving performance of broiler birds.

The intestinal gut health is one of the primary determinants of broiler growth and performance. Among the various enteric diseases, necrotic enteritis (NE) is an enterotoxemic disease caused by Clostridium perfringens, which can result in severe economic losses in poultry farming. Antibiotics like bacitracin methylene disalicylate (BMD) and avilamycin (AVL) are commonly used antibiotic growth promoters (AGP) in poultry feed to control necrotic enteritis in birds. Bacillus subtilis PB6 was reported to prevent necrotic enteritis and improve performance in birds. This paper investigated the influence of Bacillus subtilis PB6 in improving the performance of broiler birds in comparison with BMD and avilamycin. A 35 day trial was conducted with 240 day-old commercial broiler chicks (VenCobb 400), which were divided into four treatment groups, where each treatment group was composed of 6 replicates each containing 10 birds, for a total of 60 birds per treatment. The treatment groups included a negative control (no AGP), Bacillus subtilis PB6, BMD, and avilamycin. The parameters analyzed included body weight, feed conversion ratio (FCR), mortality, villus histomorphometry, and European efficiency factor (EEF). Bacillus subtilis PB6 significantly (P < 0.05) improved body weight and FCR (8 points) compared to the control. The group supplemented with B. subtilis PB6 or BMD had higher (P < 0.05) body weight compared to all other treatment groups. The supplementation of B. subtilis PB6 significantly improved the villus height (P < 0.05) compared to control and other AGP groups. The EEF was found to be the highest in the B. subtilis PB6 supplemented group at 35th day as compared to other treatment groups. The combined data from this study indicate that supplementation of B. subtilis PB6 improves overall performance of broilers compared to BMD and avilamycin, and can be used as potential AGP replacement in poultry farming.

Preparation and Quality Evaluation of Avilamycin Self-emulsifying Microemulsion / 中国药学杂志

OBJECTIVE: To optimize the formulation of avilamycin self-microemulsifying drug delivery system (SMEDDS) using central composite design-response surface method and evaluate its quality. METHODS: The compositions of avilamycin SMEDDS were screened by solubility experiment and self-emulsifying grading test. The formulation was optimized using Design Expert Software, taking particle size and Zeta potential as dependent variables and the usage amounts of oil, surfactant and cosurfactant as independent variables. RESULTS: The optimized formulation was quickly and conveniently obtained as follows: 36.67% propylene glycollaurate, 42.83% cremophor RH40 and 20.50% diethylene glycol monoethyl ether.The average diameter of the preparation was (28.34±0.06)nm, the Zeta potential was (-1.98±0.24)mV and PDI was (0.15±0.005). CONCLUSION: The central composite design and response surface method is useful for the formula optimization of avilamycin self-microemulsifying drug delivery system.The prediction accuracy of the established mode1 is good.

Prebiotic and antimicrobials on performance, carcass characteristics, and antibody production in broilers / Prebiótico e antimicrobianos sobre o desempenho, características de carcaça e produção de anticorpos em frangos de corte

ABSTRACT: To evaluate the effect of supplementation with mannan oligosaccharides, avilamycin and halquinol, alone or in combination, on the performance, carcass characteristics and antibody production in broilers (1-49 days old), male broiler chicks (n=1440; Cobb 500; one day old) were housed and distributed into a completely randomized design into six treatments (eight replicates; 30 animals per pen). To produce the experimental diets, three types of performance enhancer additives were used. Halquinol (HAL), avilamycin (AVI) and mannan oligosaccharides (MOS) were included (alone or in combination) in the basal diet (instead of corn starch). Effects of diet were observed on results of animal performance in the period 1-21 and 1-42 days old. Broilers fed with a diet without growth promoter showed lower weight gain in relation to those fed with diets with antimicrobials, MOS or a combination of them. In the period 1-49 days old, feed conversion increased in broilers fed with rations without promoter. At the end of the experimental period no influence of diets was observed on the carcass yield and cuts, and titles of specific antibodies to avian infectious bronchitis. The use of MOS and/or antimicrobials (AVI or HAL), alone or in combination, improves feed conversion of broilers reared until 49 days of age.

RESUMO: Para avaliar o efeito da suplementação com mananoligossacarídeo, avilamicina e halquinol, isoladamente ou combinados, sobre o desempenho, características de carcaça e produção de anticorpos em frangos de corte (1-49 dias de idade), pintos machos de corte (n=1440; linhagem Cobb 500; idade: um dia) foram alojados e distribuídos em delineamento experimental inteiramente casualizado com seis tratamentos (oito repetições; 30 animais por unidade experimental). Para compor as rações experimentais, três tipos de aditivos melhoradores de desempenho foram usados. Halquinol (HAL), avilamicina (AVI) e mananoligossacarídeo (MOS) foram incluídos (isoladamente ou em associação) na ração basal (em substituição ao amido de milho). Foi observado efeito das rações sobre os resultados de desempenho dos animais no período 1-21 e 1-42 dias de idade. As aves alimentadas com ração sem melhorador de desempenho apresentaram menor ganho de peso em relação àquelas que consumiram rações com antimicrobianos, MOS ou uma combinação deles. No período 1-49 dias de idade, a conversão alimentar aumentou nas aves alimentadas sem melhorador de desempenho. No final do período experimental, não foi observado efeito das rações sobre o rendimento de carcaça e cortes e títulos de anticorpos específicos contra bronquite infecciosa aviária. A utilização de mananoligossacarídeo e/ou antimicrobianos (avilamicina e halquinol), isoladamente ou combinados, melhora a conversão alimentar de frangos de corte criados até 49 dias de idade.

Efficacy of avilamycin for the prevention of necrotic enteritis caused by a pathogenic strain of Clostridium perfringens in broiler chickens.

The efficacy of avilamycin for the prevention of necrotic enteritis (NE) was investigated in a 35-day floor pen study of 2200 broiler cockerels using a Clostridium perfringens (Cp) feed inoculum challenge model. Treatments consisted of (1) nonmedicated, nonchallenged; (2) nonmedicated, challenged; (3) avilamycin at 15 ppm, challenged; (4) avilamycin at 30 ppm, challenged. Avilamycin was administered in the feed from day 7 to day 30 of the study. Challenge inoculum was administered on day 14 and delivered approximately 10(9) CFU Cp/bird. NE mortality rates from day 14-35 were significantly (P < 0.0001) lower in birds treated with avilamycin at 15 and 30 ppm when compared to nonmedicated, challenged birds. Treatment with avilamycin also resulted in a significant reduction in ileal Cp count on day 21 (P < 0.0001) and NE lesion scores on day 17 (P < 0.006) when compared to nonmedicated, challenged birds. The performance of birds treated with avilamycin was also improved when compared to nonmedicated, challenged birds. Cockerels that received either 15 or 30 ppm avilamycin had a significantly (P < 0.0001) increased body weight on day 35 and average daily gain from days 0-35 than nonmedicated, challenged birds. Furthermore, birds treated with avilamycin had an improved feed conversion rate from days 0-35 compared to both nonmedicated, nonchallenged birds and nonmedicated, challenged birds. This study confirms that avilamycin is effective at controlling mortality related to NE in growing broiler chickens.

Effect of different doses of coated butyric acid on growth performance and energy utilization in broilers.

We recently applied four dietary treatments in experiments I and II to determine the effect of protected calcium butyrate (BP) on growth performance and nutrient digestibility in broiler chickens. A group of one-day-old male Ross 308 broiler chicks (total 960, 480 per trial) were used in the study. In experiment I, the basal diets were fed with protected BP inclusion (0.2, 0.3, or 0.4 g/kg of finished feed) (BP) or without (C). In experiment II, 4 different diets were tested: 1) basal diet with no supplementation (C), 2) basal diet supplemented with protected BP (0.3 g/kg) (BP), 3) basal diet supplemented with avilamycin (6 mg/kg, active substance) a common antibiotic growth promoter (AGP) (Av), and 4) basal diet supplemented with the combination of both avilaymicin and BP. In experiment I, considering the entire study period, the use of BP improved feed conversion ratio (P<0.05) irrespective of the dose. Apparent total tract crude fat digestibility and apparent metabolizable energy corrected for nitrogen (AMEN) were improved after BP supplementation (P<0.05). In experiment II, A or AB diets improved (P<0.05) body weight gain compared to the control treatment. The diets Av, BP, and AvB improved (P<0.05) feed conversion ratio compared to the control treatment. Birds from the treatment diet were characterized by having the thickest mucosa (P<0.05). On days 14, 35, and 42, the use of AB diets improved AMENcontent compared to the control treatment (P<0.05). The apparent ileal digestibility of amino acid data showed that Av or AvB treated birds were characterized by higher Asp, Glu, Cys, Gly, and Ala ileal digestibility than the control animals (P<0.05). The use of Av, BP, or AvB increased ileal digestibility of Thr, Ser, and Pro (P<0.05). There is an indication that BP, alone or in combination with avilamycin, improve the digestion and absorptive processes and consequently birds performance results.

Desempenho e características bioquímicas de leitões submetidos a dietas com aditivos probióticos, prebióticos, simbióticos e antibióticos / Performance and biochemical parameters of piglets and the use of alternative additives as antibiotic replacement

Avaliaram-se o desempenho e as características bioquímicas de leitões desmamados, utilizando-se aditivos alternativos em substituição aos antibióticos. Foram utilizados 480 leitões machos, distribuídos em delineamento inteiramente ao acaso, composto por cinco tratamentos. A ração foi à base de milho, farelo de soja e um núcleo comercial para leitões. Os tratamentos consistiram em T1: ração sem aditivos, T2: dieta basal com antibiótico, T3: dieta basal com adição de prebiótico, T4: dieta basal com adição de probiótico e T5: dieta basal com adição de simbiótico. Foram avaliados peso corporal, ganho de peso, consumo de ração e conversão alimentar. Em 50 amostras de sangue de leitões, sendo 10 amostras de cada tratamento, avaliaram-se as concentrações séricas de proteínas totais, de albumina e globulina, a relação albumina:globulina e a fosfatase alcalina, creatinina, alanina aminotransferase, aspartato aminotransferase, gama- glutamiltransferase, colesterol, ureia, cálcio e fósforo e a relação cálcio:fósforo. A substituição de antibióticos por aditivos alternativos não influenciou o desempenho nem as características bioquímicas dos leitões.

The experiment was conducted to evaluate the performance and biochemical parameters in weaned piglets using alternative supplements to replace antibiotics. A total of 480 male pigs were used, randomly distributed in delineation with five treatments. The feed was based on corn, soybean meal and a commercial core for piglets. The treatments were T1: diet without supplements or basal diet, T2: basal diet with antibiotics, T3: basal diet with added probiotic, T4: basal diet with added probiotic and T5: basal diet with addition of symbiotic. In the experiment we evaluated performance characteristics, body weight, weight gain, feed intake and feed conversion. In the second part of the experiment, we evaluated the biochemical parameters of piglets used in the first experiment, fifty blood samples, ten samples of each treatment, were taken, and after separation of the clot, the serum was frozen at -20°C. Subsequently we analyzed the serum levels of total protein, albumin, globulin, albumin/globulin, ALP, creatinine, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, cholesterol, urea, calcium, phosphorus and calcium/phosphorus ratio. No significant differences in biochemical parameters or in the performance of piglets submitted to different diets were found.

Study on Breeding of High-yield Avilamycin-producing Strains by Nitrogen Ion Implantation / 微生物学通报

To obtain high-yield avilamycin-producing strains,low energy N~+ ion implantation technology and screening of streptomycin-re- sistant mutants are used in the study on breeding mutation.The results show that,“saddle”region,which range is from 3?10~(15) to 5?10~(15) ions/cm~2,has got better induced mutation action.It also means that the strain's resistant mutation and yield mutation closely correlate to each other,and the method of streptomycin resistant screening is feasible.We have isolated a high-yield strain SVT-45 which the productivi- ty is 195% higher than the original strain's in the rotation-flask experiments.These results showed that the ion implantation was an effective method for microbe mutagensis.