PSII-15 Dietary Supplementation of Bacillus Subtilis Modified Intestinal Microbiome of Weaned Pigs Differently to Antibiotics

2021 ◽  
Vol 99 (Supplement_1) ◽  
pp. 161-162
Author(s):  
Cynthia Jinno ◽  
Yijie He ◽  
Yanhong Liu
Keyword(s):  
Bacillus Subtilis ◽  
Hypervariable Region ◽  
Alpha Diversity ◽  
Basal Diet ◽  
Positive Control ◽  
Negative Control ◽  
Intestinal Microbiome ◽  
Weaned Pigs ◽  
E Coli ◽  
Downstream Analysis

Abstract The objective of this study was to compare the dietary effects of Bacillus subtilis and antibiotics in intestinal microbiota of pigs experimentally infected with F18 E. coli. Forty-eight weaned pigs (6.17 ± 0.36 kg BW) were individually housed and randomly allotted in one of four treatments with 12 replicates per treatment: negative control (NC), positive control (PC), antibiotics, and B. subtilis (probiotics). Pigs in NC and PC were fed with basal diet without or with E. coli, respectively. Pigs with antibiotics and probiotics were challenged with E. coli and supplemented with 50 mg/kg of carbadox or 500 mg/kg of B. subtilis, respectively. After 7 days habituation period, pigs were inoculated with F18 E. coli at 1010 CFU/3 mL dose for three consecutive days. All pigs were euthanized to collect feces and digesta from jejunum, ileum, and colon on d 21 post-infection to perform 16S rRNA sequencing at the V4 hypervariable region. Downstream analysis was performed using QIIME2 (2019.4) and R. Kruskal-Wallis test followed by Conover test was used to analyze data in R. Colon digesta and feces have greater (P < 0.05) alpha diversity than ileal and jejunal digesta. No difference was observed among treatments at different intestinal sites. Bray-Curtis PCoA plots displayed pronounced clusters of all treatment groups throughout all sites. Bacteroidetes and Proteobacteria were more (P < 0.05) abundant but Firmicutes were less (P < 0.05) abundant in ileal digesta of pigs fed with antibiotics than pigs in probiotics. Firmicutes were more (P < 0.05) abundant in colon and feces of NC than of antibiotics. Bifidobacterium was least (P < 0.05) abundant throughout all sites and Prevotella 1 was most (P < 0.05) abundant in colon of pigs fed with antibiotics compared with other treatments. In conclusion, both B. subtilis and carbadox supplementation modified gut microbiota of weaned pigs challenged with F18 E. coli. However, the impacts are different and need further investigation.

scholarly journals Efficacy of organic acids as an alternative to antibiotic growth promoters in weaned pigs

2018 ◽  
Vol 17 (06) ◽  
pp. 8-14
Author(s):  
Tung M. Che
Keyword(s):  
Organic Acids ◽  
Antibiotic Treatment ◽  
Average Daily Gain ◽  
Basal Diet ◽  
Positive Control ◽  
Negative Control ◽  
Growth Promoters ◽  
Weaned Pigs ◽  
E Coli ◽  
Diarrhea Incidence

The objective of the experiment was to compare effects of dietary supplementation of organic acids (ProHacid Advance, PRO) and antibiotics on growth performance, diarrhea incidence, frequency of antibiotic treatment, and E. coli shedding in weaned pigs. A total of 224 crossbred weaned pigs [(Yorkshire x Landrace) x Duroc; 29 days old)] were allotted to 1 of 4 treatments with 7 replicate pens of 8 pigs each according to sex, litter origin and weight in an RCBD. The 4 dietary treatments included (1) basal diet + 50 mg/kg neomycin and 10 mg/kg enramycin (positive control, PC), (2) basal diet without antibiotics (negative control, NC), (3) As 2 + 0.25% PRO, and (4) As 2 + 0.5% PRO. Over a 4-week study, the results showed that there were no differences in the average daily gain and average daily feed intake of pigs among the 4 treatments (P = 0.328). However, pigs fed the PC diet (1.642) and 0.25% PRO-supplemented diet (1.641) had a lower feed to gain ratio (P < 0.05) than those fed the NC diet (1.808). The diarrhea incidence of pigs fed the 0.25% PRO-supplemented diet (7.36%) was lower (P < 0.01) than that of pigs fed the PC diet (11.61%), NC diet (16.70%), and 0.5% PRO-supplemented diet (10.08%). The frequency of antibiotic treatment of pigs consuming the 0.25% PRO-supplemented diet (4.67%) was lower (P < 0.01) than that of pigs consuming the PC diet (7.33%), NC diet (9.53%) and 0.5% PRO-supplemented diet (7.65%). No differences were found in the number of fecal E. coli among the 4 treatments. In brief, 0.25% PRO added to a nursery pig diet would be considered a potential alternative to the use of antibiotics in feed, but further research on this aspect is needed

scholarly journals 140 Effects of Bacillus subtilis probiotics on growth performance, diarrhea, and fecal β-hemolytic coliforms of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli

2019 ◽  
Vol 97 (Supplement_2) ◽  
pp. 79-80
Author(s):  
Yijie He ◽  
Kwangwook Kim ◽  
Cynthia Jinno ◽  
Lauren Kovanda ◽  
Seijoo Yang ◽  
...  
Keyword(s):  
Body Weight ◽  
Bacillus Subtilis ◽  
Growth Performance ◽  
Feed Efficiency ◽  
Control Diet ◽  
Positive Control ◽  
Negative Control ◽  
Enterotoxigenic Escherichia Coli ◽  
Weaned Pigs ◽  
E Coli

Abstract The objective of this experiment was to investigate the effects of Bacillus subtilis on growth performance, diarrhea and fecal β-hemolytic coliforms of weaned pigs experimentally infected with a strain of E. coli (F18, express genes of LT, STb, and SLT 2 toxins). Weaned pigs (n = 48, 6.17 ± 0.36 kg BW) were individually housed in disease containment rooms and randomly allotted to one of four dietary treatments: negative control (NC, control diet without E. coli challenge), positive control (PC, control diet with E. coli challenge), and supplementation of 50 mg/kg of carbadox or 500 mg/kg of Bacillus subtilis probiotics. The experiment lasted 28 d with 7 d before and 21 d after the first E. coli inoculation. The F18 E. coli were given to pigs at 1010 CFU/3 mL dose for three consecutive d. Diarrhea score was daily recorded for each pig to calculate frequency of diarrhea. Fecal samples were collected on d 0, 3, 7, 14, and 21 PI to analyze β-hemolytic coliforms. Data were analyzed using the Mixed Procedure of SAS. Pigs supplemented with carbadox had greater (P < 0.05) body weight on d 7, 14, and 21 PI than pigs in the PC and probiotics group. Supplementation of probiotics enhanced pig body weight on d 21 PI, compared with the PC. E. coli challenge reduced (P < 0.05) ADG and feed efficiency from d 0 to 21 PI, while supplementation of antibiotics or probiotics enhanced ADG and feed efficiency from d 0 to 21 PI. Pigs in carbadox and probiotics groups had reduced (P < 0.05) frequency of diarrhea throughout the experiment and fecal β-hemolytic coliforms on d 7 PI than pigs in the PC. In conclusion, supplementation of Bacillus subtilis could enhance disease resistance and promote growth performance of weaned pigs under disease challenge condition.

scholarly journals PSVIII-7 Effects of Bacillus subtilis on colon digesta metabolomic profiles in weanling pigs experimentally infected with a pathogenic E. coli

2020 ◽  
Vol 98 (Supplement_3) ◽  
pp. 203-203
Author(s):  
Kwangwook Kim ◽  
Yanhong Liu
Keyword(s):  
Bacillus Subtilis ◽  
Positive Control ◽  
Negative Control ◽  
High Dose ◽  
Post Inoculation ◽  
Weaned Pigs ◽  
E Coli ◽  
Gut Barrier Function ◽  
Pathogenic Escherichia Coli ◽  
Lysine Degradation

Abstract Our previous studies have shown that supplementation of Bacillus subtilis enhanced growth rate, improved gut barrier function, and modified colon microbiome of weaned pigs infected with pathogenic Escherichia coli (E. coli). The objective of this experiment was to investigate the effects of Bacillus subtilis on colon digesta metabolomic profiles of weaned pigs experimentally infected with F18 E. coli. Forty-eight pigs (6.73 ± 0.77 kg BW) were individually housed in disease containment rooms and randomly allotted to one of the four treatments (12 pigs/treatment). Four treatments included negative control (NC), positive control (PC), low-dose (1.28 × 109 CFU Bacillus subtilis/kg feed), and high-dose (2.56 × 109 CFU Bacillus subtilis/kg feed). The experiment lasted 18 d [7 d before and 11 d after first inoculation (d 0)]. The F18 E. coli inoculum was orally provided to all pigs with the dose of 1010 cfu/3 mL for 3 consecutive days, except NC. Twenty-four pigs (6 pigs/treatment) were euthanized on d 5 post-inoculation (PI) and the remained pigs were euthanized on d 11 PI to collect colon digesta for the analysis of metabolomic profiles by gas chromatography time of flight-mass spectrometer (GCTOF-MS). All processed data were statistically analyzed and evaluated by online MetaboAnalyst tool. No significant differences were observed in the metabolites between NC and PC on d 5 and 11 PI. Compared with PC, low- and high-dose Bacillus subtilis reduced (Fold change &gt; 1.5; FDR &lt; 0.20) four metabolites (proline, 2-hydroxyglutaric acid, lysine, and glutamic acid) and two metabolites (ribose, and D-xylulose) in colon digesta on d 5 PI, respectively. These metabolites were related to aminoacyl-tRNA-biosynthesis, arginine and proline metabolism, and lysine degradation. In conclusion, supplementation of Bacillus subtilis modified the levels of microbial metabolites associated with amino acid metabolism in colon digesta of pigs.

scholarly journals Metabolomic Profile of Weaned Pigs Challenged with E. coli and Supplemented with Carbadox or Bacillus subtilis

Metabolites ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 81
Author(s):  
Yijie He ◽  
Yanhong Liu ◽  
Peng Ji
Keyword(s):  
Bacillus Subtilis ◽  
Basal Diet ◽  
Positive Control ◽  
Negative Control ◽  
Enterotoxigenic Escherichia Coli ◽  
Post Inoculation ◽  
Metabolomic Profile ◽  
Weaned Pigs ◽  
Ileal Mucosa ◽  
Flight Mass Spectrometry

This study explored the metabolomic profiles in ileal mucosa and colon digesta in response to enterotoxigenic Escherichia coli F18 (ETEC) infection and dietary use of probiotics and low-dose antibiotics. Weaned pigs (n = 48, 6.17 ± 0.36 kg body weight) were randomly allotted to one of four treatments. Pigs in the negative control (NC) were fed a basal diet without ETEC challenge, whereas pigs in the positive control (PC), antibiotic, and probiotic groups were fed the basal diet, basal diet supplemented with 50 mg/kg of carbadox, or 500 mg/kg of Bacillus subtilis, respectively, and orally challenged with ETEC F18. All pigs were euthanized at day 21 post-inoculation to collect ileal mucosa and colon digesta for untargeted metabolomic profiling using gas chromatography coupled with time-of-flight mass spectrometry. Multivariate analysis highlighted a more distinct metabolomic profile of ileal mucosa metabolites in NC compared to the ETEC-challenged groups. The relative abundance of 19 metabolites from the ileal mucosa including polyamine, nucleotide, monosaccharides, fatty acids, and organic acids was significantly different between the NC and PC groups (q < 0.1). In colon digesta, differential metabolites including 2-monoolein, lactic acid, and maltose were reduced in the carbadox group compared with the probiotics group. In conclusion, several differential metabolites and metabolic pathways were identified in ileal mucosa, which may suggest an ongoing intestinal mucosal repair in the ileum of ETEC-challenged pigs on day 21 post-inoculation.

scholarly journals PSIX-5 Dietary supplementation of Bacillus subtilis modified fecal microbiome of weaned pigs in comparison to antibiotics

2020 ◽  
Vol 98 (Supplement_3) ◽  
pp. 183-183
Author(s):  
Cynthia Jinno ◽  
Yijie He ◽  
Xunde Li ◽  
Yanhong Liu
Keyword(s):  
Bacillus Subtilis ◽  
Relative Abundance ◽  
Hypervariable Region ◽  
Negative Control ◽  
Rrna Gene ◽  
Post Inoculation ◽  
Weaned Pigs ◽  
Fecal Samples ◽  
Fecal Microbiome ◽  
E Coli

Abstract The objective of this study was to investigate the effects of supplementing Bacillus subtilis on fecal microbiota of pigs experimentally infected with F-18 Escherichia coli (E. coli), in comparison to carbadox. Forty-eight weaned pigs (6.17 ± 0.36 kg BW) were individually housed and randomly allotted to one of four treatment (n =12): negative control (NC), positive control (PC), antibiotics (50 mg/kg of carbadox), and direct fed microbials (DFM, 500 mf/kg of Bacillus subtilis). The experiment lasted 28 days with 7 days before and 21 days after first E. coli inoculation (d 0). Pigs in the NC, PC, and DFM groups were orally inoculated with F18 E. coli for 3 consecutive days with 1010 CFU/3 mL/dose. Fecal samples were collected on d -7 and 0 before E. coli inoculation, and d 7 and 21 post inoculation (PI). DNA were extracted from all fecal samples to perform 16S rRNA gene sequencing at the V4 hypervariable region. All data were analyzed with QIIME2 (2019.4) and R. Chao1 index was greatest (P &lt; 0.05) in feces collected on d 0 before E. coli inoculation and lowest (P &lt; 0.05) on d -7 feces. Pigs supplemented with DFM had lower (P &lt; 0.05) Chao1 index than pigs fed with antibiotics on d 21 PI. Bray-Curtis PCoA displayed separate clusters among days but overlaps among treatments. Bacteroidetes and Proteobacteria were most (P &lt; 0.05) abundant on d -7 and lowest (P &lt; 0.05) on d 21 PI. However, Actinobacteria and Firmicutes were most (P &lt; 0.05) abundant on d 21 PI. Pigs in the NC and DFM groups had greater (P &lt; 0.05) relative abundance of Firmicutes than pigs fed with antibiotics on d 0 and 7. Supplementation of antibiotics reduced (P &lt; 0.05) the relative abundance of Lactobacillaceae compared with other treatments on d 0 PI. In conclusion, both animal age and dietary treatments influenced the fecal microbiome of weaned pigs.

scholarly journals Growth performance and gut health of Escherichia coli-challenged weaned pigs fed diets supplemented with a Bacillus subtilis direct-fed microbial

2020 ◽  
Vol 4 (3) ◽  
Author(s):  
Sangwoo Park ◽  
Jung Wook Lee ◽  
Kevin Jerez Bogota ◽  
David Francis ◽  
Jolie Caroline González-Vega ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Growth Performance ◽  
Feed Efficiency ◽  
Basal Diet ◽  
Feed Additives ◽  
Growth Promoter ◽  
Gut Health ◽  
Weaned Pigs ◽  
E Coli

Abstract This study was conducted to investigate the effects of a direct-fed microbial (DFM) product (Bacillus subtilis strain DSM 32540) in weaned pigs challenged with K88 strain of Escherichia coli on growth performance and indicators of gut health. A total of 21 weaned pigs [initial body weight (BW) = 8.19 kg] were housed individually in pens and fed three diets (seven replicates per diet) for 21 d in a completely randomized design. The three diets were a corn-soybean meal-based basal diet without feed additives, a basal diet with 0.25% antibiotics (neo-Oxy 10-10; neomycin + oxytetracycline), or a basal diet with 0.05% DFM. All pigs were orally challenged with a subclinical dose (6.7 × 108 CFU/mL) of K88 strain of E. coli on day 3 of the study (3 d after weaning). Feed intake and BW data were collected on days 0, 3, 7, 14, and 21. Fecal scores were recorded daily. On day 21, pigs were sacrificed to determine various indicators of gut health. Supplementation of the basal diet with antibiotics or DFM did not affect the overall (days 0–21) growth performance of pigs. However, antibiotics or DFM supplementation increased (P = 0.010) gain:feed (G:F) of pigs during the post-E. coli challenge period (days 3–21) by 23% and 24%, respectively. The G:F for the DFM-supplemented diet did not differ from that for the antibiotics-supplemented diet. The frequency of diarrhea for pigs fed a diet with antibiotics or DFM tended to be lower (P = 0.071) than that of pigs fed the basal diet. The jejunal villous height (VH) and the VH to crypt depth ratio (VH:CD) were increased (P &lt; 0.001) by 33% and 35%, respectively, due to the inclusion of antibiotics in the basal diet and by 43% and 41%, respectively due to the inclusion of DFM in the basal diet. The VH and VH:CD for the DFM-supplemented diet were greater (P &lt; 0.05) than those for the antibiotics-supplemented diet. Ileal VH was increased (P &lt; 0.05) by 46% due to the inclusion of DFM in the basal diet. The empty weight of small intestine, cecum, or colon relative to live BW was unaffected by dietary antibiotics or DFM supplementation. In conclusion, the addition of DFM to the basal diet improved the feed efficiency of E. coli-challenged weaned pigs to a value similar to that of the antibiotics-supplemented diet and increased jejunal VH and VH:CD ratio to values greater than those for the antibiotics-supplemented diet. Thus, under E. coli challenge, the test DFM product may replace the use of antibiotics as a growth promoter in diets for weaned pigs to improve feed efficiency and gut integrity.

scholarly journals PEMBERIAN RANSUM KOMBINASI HERBAL DENGAN MINERAL ZINK TERHADAP PERFORMANS AYAM BROILER YANG DIINFEKSI Escherichia coli

ZOOTEC ◽  
2013 ◽  
Vol 33 (1) ◽  
pp. 35
Author(s):  
M. N. Regar ◽  
R. Mutia ◽  
S. D. Widhyari ◽  
Y. H. S. Kowel
Keyword(s):  
Escherichia Coli ◽  
Sweet Potato ◽  
Basal Diet ◽  
Positive Control ◽  
Negative Control ◽  
E Coli ◽  
Garlic Powder ◽  
Ipomea Batatas ◽  
Sweet Potato Leaves ◽  
Better Than

ABSTRAKPenelitian ini dilakukan untuk menguji pengaruh pemberian kombinasi herbal dengan mineral zink dalam ransum broiler yang diinfeksi Escherichia coli (E. coli). Dua ratus ekor d.o.c (day old chick) dibagi secara acak ke dalam lima perlakuan, masing-masing perlakuan diulang empat kali sehingga terdapat 20 unit percobaan, setiap unit percobaan terdiri dari 10 ekor d.o.c. Anak ayam percobaan dipelihara selama 35 hari. Ransum perlakuan terdiri dari R1 = Pakan basal/ ayamsehat (kontrolnegatif); R2 = Pakan basal/ ayamdiinfeksiE.coli (kontrolpositif); R3= Pakan basal + serbukkunyit 1.5% + ZnO 180 ppm/ ayamdiinfeksiE.coli; R4 = Pakan basal + serbukbawangputih 2.5% + ZnO 180 ppm/ ayamdiinfeksiE.coli; R5 = Pakan basal + antibiotik/ ayamdiinfeksiE.coli.Penelitian menggunakan rancangan acak lengkap. Peubah yang diamati konsumsi ransum, pertambahan bobot badan, dan konversi ransum. Hasil dari penelitian ini menunjukkan bahwa pemberian kombinasi kunyit 1.5% dengan ZnO 180 ppm dan kombinasi bawang putih 2.5% dengan zink 180 ppm dalam ransum mampu memperlihatkan performa yang lebih baik.Kata Kunci:Daun ubi jalar, Pertambahan berat badan, Konsumsi ransum, Konversi ransum, Ayambroiler.ABSTRACTTHE EFFECT OF SUPPLEMENTA-TION SWEET POTATO LEAVES MEAL (Ipomea batatas) ON BROILER PERFORMANCE.This experiment was conducted to study the combination of herbal with zink in poultry diet on the performance of Escherichia coli – challenged broiler. Two hundred d.o.c unsexed were devided into five treatments and four replications, with ten chicks in each replicates. The treatments were R1 (basal diet as a negative control/ healhty chickens), R2 (basal diet as a positive control/ Escherichia coli challenged), R3 (basal diet +1.5% turmeric powder + ZnO 180 ppm/ Escherichia coli challenged), R4 (basal diet + 2.5% garlic powder + ZnO 180 ppm/ Escherichia coli challenged), and R5 (basal diet + antibiotic/ Escherichia coli challenged). Data were collected during 35 days, diet and water were offered ad libitum. The results of this research indicated that chickens fed basal diet + 1.5% turmeric powder + ZnO 180 ppm/ challenged Escherichia coli and chickens fed basal diet + 2.5% garlic powder +ZnO 180 ppm/ challenged Escherichia coli showed performances better than control .Keywords: Sweet potato leaves, Body weight, Consumtion ration, Convertion ration, Broiler

scholarly journals Ameliorative Effect of Bacillus subtilis on Growth Performance and Intestinal Architecture in Broiler Infected with Salmonella

Animals ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 190 ◽  
Author(s):  
Alaeldein M. Abudabos ◽  
Muttahar H. Ali ◽  
Mohammed A. Nassan ◽  
Ahmad A. Saleh
Keyword(s):  
Bacillus Subtilis ◽  
Basal Diet ◽  
Positive Control ◽  
Negative Control ◽  
Broiler Chicks ◽  
Feed Conversion ◽  
Negative Effects ◽  
Villus Height ◽  
Bacillus Subtilis Atcc ◽  
Ameliorative Effect

A total of 600 day-old broiler chicks (Ross 308) confirmed for the absence of Salmonella were randomly allocated to five treatments each with 10 replicates: negative control (basal diet only); positive control (basal diet) + infected with Salmonella; T1, Salmonella infected + avilamycin; T2, Salmonella infected + Bacillus subtilis (ATCC PTA-6737; 2 × 107 CFU/g) and T3, Salmonella infected + B. subtilis (DSM 172999; 1.2 × 106 CFU/g). The results revealed that feed intake (FI) and body weight (BW) were significantly (p < 0.01) lower in T1 compared to T2. The feed conversion ratio (FCR) was significantly (p < 0.01) lower in T2 and T3 compared to other treatments. Similarly, the performance efficiency factor (PEF) was also significantly (p < 0.01) higher in T2 and T3 compared to positive control. Villus height was significantly (p < 0.01) higher in T2 compared to all other treatments. However, villus width and surface area were significantly (p < 0.01) higher in T1. In conclusion, dietary supplementation with B. subtilis improved growth and intestinal health by reversing the negative effects of Salmonellosis.

scholarly journals Effect of dietary organic acids supplementation on growth performance, nutrient digestibility, and gut morphology in weaned pigs

2021 ◽  
Author(s):  
Jinyoung Lee ◽  
Jong Woong Kim ◽  
Heidi N. Hall ◽  
Charles M. Nyachoti
Keyword(s):  
Growth Performance ◽  
Phase 1 ◽  
Basal Diet ◽  
Positive Control ◽  
Negative Control ◽  
Nutrient Digestibility ◽  
Feed Ratio ◽  
Weaned Pigs ◽  
Gut Morphology ◽  
Two Phases

This study was conducted to investigate the effects of dietary supplementation with different organic acid (<b>OA</b>) mixtures on growth performance, nutrient digestibility, and gut morphology in weaned pigs. A total of 56 weaned pigs (7.93 ± 1.04 kg body weight) were assigned to four dietary treatments with seven replicates per treatment for a 35-d study conducted over two phases; phase 1 (d 1 to 14) and phase 2 (d 14 to 35). Diets consisted of 1) a corn-soybean meal-basal diet without any additive (negative control, <b>NC</b>); 2) NC + formic and propionic acids (<b>TRT1</b>); 3) NC + butyric, formic, and propionic acids (<b>TRT2</b>); and 4) NC + antibiotic (positive control, <b>PC</b>). During the overall period, the gain to feed ratio of pigs fed the PC and TRT1 diets tended to be higher (<i>P</i> = 0.059) than that of the NC diet. However, OA supplementation had no effects on nutrient digestibility. Jejunal villus height to crypt depth ratio was higher (<i>P</i> < 0.05) in pigs fed the TRT1 diet than those fed the NC diet. In conclusion, dietary OA supplementation tended to increase overall feed efficiency and improved gut morphology in weaned pigs.

scholarly journals POS0384 A NOVEL MECHANISM LINKING MUCOSAL BACTERIA WITH AUTOANTIBODY RESPONSES IN RA: ACETYLATED BACTERIAL LYSATE AS A MODEL ANTIGEN

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 422.1-422
Author(s):  
M. Volkov ◽  
A. S. B. Kampstra ◽  
K. van Schie ◽  
J. Kwekkeboom ◽  
T. Huizinga ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Activation ◽  
Positive Control ◽  
Negative Control ◽  
B Cell Activation ◽  
Human Fibrinogen ◽  
Post Translational Modifications ◽  
E Coli ◽  
Bacterial Proteins ◽  
Citrullinated Proteins

Background:Rheumatoid arthritis (RA) is characterized by autoantibodies against post-translationally modified proteins (AMPA) such as citrullinated, carbamylated and acetylated proteins. Importantly, these antibodies are highly multireactive, as they often recognize more than one of these post-translational modifications. Despite extensive research, the antigens inducing the breach of tolerance remain unknown, although microbial antigens are often suspected. Various bacteria are known to be capable of acetylation, therefore, it is intriguing to know what mechanisms can underlie the breach of tolerance towards acetylated proteins and development of anti-acetylated protein antibodies (AAPA).Objectives:To investigate whether acetylated proteins of bacterial origin (1) are recognized by human derived AMPA and AMPA expressing B cells; and (2) can induce AMPA development when used to immunize mice.Methods:Acetylated E. coli proteins were acquired with two separate methods (Figure 1A): by culturing E. coli in a condition promoting auto-acetylation (intrinsically acetylated bacterial proteins, IABP), or by directly acetylating lysate-derived proteins via a chemical reaction (extrinsically acetylated BP, EABP). Acetylated ovalbumin (AcOVA) served as positive control for AAPA induction in mice, non-acetylated BP (NABP) and phosphate buffer saline (PBS) served as negative control. Mice were immunized with these proteins and the resulting antibody response was studied by ELISA. Furthermore, EABP/IABP/NABP were investigated for recognition by human-derived AAPA with ELISA and AAPA-expressing B cells with spleen tyrosine kinase (Syk) phosphorylation assay; acetylated human fibrinogen and native fibrinogen served as positive and negative control.Results:Repetitive immunization of mice with EABP resulted in an AMPA response recognizing acetylated, carbamylated and citrullinated proteins. AMPA titers in these mice exceeded the titers in the positive control mice immunized with AcOVA and were substantially higher than in the NABP-immunized mice (Figure 1B). Human-derived monoclonal AAPA recognized EABP and IABP (not shown). B cell activation (measured by Syk phosphorylation) assay indicated that AAPA expressing B cells recognized EABP and (to a lesser extent) IABP, but not NABP (Figure 1C).Conclusion:Acetylated bacterial proteins are potent antigens that can induce cross-reactive AMPA responses in mice and they are recognized by human AAPA. This suggests that acetylated bacterial proteins could possibly be involved in the breach of tolerance in RA.Acknowledgements:We thank Dr. Can Araman and Prof. Chunaram Choudhary for their advice regarding optimization of bacterial auto-acetylation.Disclosure of Interests:None declared

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