Effects Of P-arsanilic Acid Nanoparticles On Growth Performance, Antibacterial Activities And Approach To Its Mechanism In Weaning Piglets

The purpose of this study was to evaluate the antibacterial effects of p-arsanilic acid nanoparticles(p-ASAP) against E. coli, S. choleraesuis, S. typhimurium, and S. aureus in vitro, and the effects of p-ASAP on the growth performance, arsenic retention and excretion in weaning piglets.The size of p-ASAP prepared based on the ionic gelation of p-arsanilic acid with ethanol were determined by atomic force microscopy(AFM). The antibacterial activity of p-ASAP was evaluated by calculation of minimum inhibitory concentration (MIC) and minimum bactericidal concentration(MBC) determined by a broth dilution method recommended by the NCCLS(2000). The CSH(Cell Surface Hydrophobicity) of E. coli was determined by the method of MATH (Microbial Adhesion To Hydrocarbon).The antibacterial process of p-ASAP against 5. choleraesuis was elucidated by AFM observation at intervals. p-ASAP were added to bacterial cultures grown to the late exponential phase(a final concentration of 6μg/mL). Samples were removed onto the surface of a piece of mica plate after 30, 60, 90, 120, and 180 minutes for AFM observation.In animal trial, A total of 216 weanling pigs (Duroc×Landrace×Yorkshire) at an average initial body weight of 7.0±0.4kg were used to investigate the effects of p-ASAP on the growth performance, retention and excretion of arsenic. The pigs were allocated to six dietary treatments in a randomized complete block design (three replicates of 12 pigs per replicate) for 35 days. The dietary treatments were: (1) basal diet, (2) basal diet + 90mg/kg p-arsanilic acid(p-ASA), (3) basal diet + 10 mg/kg p-ASAP, (4) basal diet + 30 mg/kg p-ASAP,(5) basal diet + 60 mg/kg p-ASAP, or (6) basal diet +90 mg/kg p-ASAP. Diets were formulated to meet or exceed requirements suggested by the NRC (1998) for 10-20 kg pigs. All diets were fed as mash, and no other antibiotic was included. The content of arsenic in basal diets was 0.25 mg/kg. All pigs were given ad libitum access to feed and water. Average daily gain (ADG), average daily feed intake (ADFI) and gain/feed ratio were measured.At the end of feeding trial, eight pigs each from treatment 1,2 and having the best growth performance group among treatment 3,4,5 and 6 were slaughtered under general anaesthesia and then immediately eviscerated. The samples were collected for laboratory analysis.Results showed that p-ASAP could inhibit the growth of various bacteria tested and exhibited higher antibacterial activity than p-arsanilic acid, the MIC and MBC values were less than 2.0μg/mL and 8.0μg/mL, respectively. AFM suggested that the exposure of S. choleraesuis to the p-ASAP led to the disruption of cell membranes and the leakage of cytoplasm. The mechanism for the antibacterial activity of p-ASAP may involve the enhanced their affinity to S. choleraesuis and the antibacterial activity of As(V) or As(III) reduced from As(V) by bacterial enzymes.The CSH result showed that the CSH of E.coli were decreased significantly with the supplementation of p-ASAP, the CSH was decreased by 38.82%(p<0.05) with supplementation of 90mg/L p-ASA, and by 47.74%(p<0.05),59.66%(p<0.05), 74.38% (p<0.01) and 79.86%(p<0.07) with the supplementation of 10,30,60, 90mg/L p-ASAP.The results of animal feeding trial showed that supplementation with p-arsanilic acid or various levels of p-ASAP all improved (p<0.05) growth performance as compared with control. With the supplementation of 10,30,60, 90mg/kg p-ASAP, the AFI were increased by 12.0% (p<0.05),13.98% (p<0.05),9.80% (p<0.05) and 12.99% (p<0.05);the ADG were increased by 28.99(p<0.05), 29.23(p<0.05), 21.13(p<0.05) and 23.58%(p<0.05); The feed conversion rate were increased by 16.66%(p<0.05), 15.15% (p<0.05),12.12%(p<0.05) and 10.6%(p<0.05), the diarrhea frequency were decreased by 52. 23%(p<0.01), 65.39%(p<0.07),58.81%(p<0.07) and 64.21%(p<0.07) compared to control group respectively, when compared to 90mg/kg p-ASA, the diarrhea frequency were decreased by 38.68%(p<0.05), 55.57% (p<0.05), 47.13%(p<0.05) and 54.05%(p<0.05); but there was no significant difference on other indicators between p-arsanilic acid and p-ASAP treated pigs.The result of intestinal content bacteria culture showed that p-ASAP had strong antibacterial effects on gut flora of weanling pigs. Compared with control group, the number of Bifidobacterium, Lactobacillus, E.coli and Salmonella in jejunum were decreased by 21.23%(p<0.05),21.18%(p<0.0J),41.63%(p<0.01) and 41.01%(p<0.07); by 17.91%(p<0.05), 20.11%(p<0.05), 38.54%(p<0.07) and 33.12%(p<0.07) in ileum; 13.75%(p<0.05), 14.82%(p<0.05), 33.85%(p<0.07) and 30.18%(p<0.07) in colon respectively with supplementation of 10mg/kgp-ASAP.Compared to 90mg/kg p-ASA group, the number of E.coli and Salmonella in jejunum were decreased by 16.96%(p<0.05) and 14.69%(p<0.05); by 19.96%(p<0.05) and 12.39%(p<0.05) in ileum; 14.81%(p<0.05) and 1.59%(p<0.05) in colon respectively with supplementation of 10mg/kgp-ASAP.The results of intestine morphology observation indicated that supplementation of 10mg/kg p-ASAP could improve the morphology of intestine in weanling piglets. Compared to control group, the villus length were increased by 41.06%(p<0.07) and 18.02%(p<0.07), crept depth decreased by 22.12%(p<0.05) and 13.82%(p<0.05)) in jejunum and ileum respectively, Compared to 90mg/kg p-ASA group, the villus length were increased by 20.0%(p<0.05), in jejunum and ileum respectively.The enzymes analysis indicated that supplementation of 10mg/kg p-ASAP increased the activity of lactase, amylase, lipase and total proteinase of duodenum contents by 27.15%(p<0.05),49.35%(p<0.03),38.94%(p<0.0.5) and 38.39%(p<0.0.5); the activity maltase, saccharase and lactase of mucosa by 55.74%(p<0.07),47.16% (p<0.01) and 63.88%(p<0.07) respectively; Compared to 90mg/kg p-ASA group, activity of maltase and saccharase increased by 17.48% (p<0.05),29.69%(p<0.05).The analysis of intestinal mucosa Nuclear factor-κB indicated that with the supplementation of 10mg/kg p-ASAP, the activity of NF-κB was decreased by 68.53%(p<0.01); Compared to 90mg/kg p-ASA group, the activity of NF-κB was decreased by 33.33%(p<0.05).The results of serum parameters analysis indicated that supplementation of 10mg/kgp-ASAP decreased the content of TNF-αand IL-6 by 78.29%(p<0.01) and 61.42%(p<0.07) compared to control group respectively.The arsenic analysis results indicated that both p-ASA and p-ASAP supplemented in feed were mainly excreted from feces with a little absorbed. Ingested arsenic can accumulate in all tissues in each treated pigs, the largest residual concentration of arsenic was in the livers, the next largest in the kidneys, followed by skin, hair, fattiness, bone, skeletal muscles and finally blood. Tissues of the control contained less than 0.5μg/g arsenic. Supplementation of 10mg/kg p-ASAP reduced (P<0.05) the arsenic residues in tissues compared with 90mg/kg p-arsanilic acid.The results suggest that p-ASAP has strong antibacterial activity, low level (10mg/kg) of p-ASAP has the equal antibacterial activity and growth promotion to 90mg/kg p-ASA in weanling piglets. It can inhibit the growth and reproduction of E.coli and Salmonella in intestine, decrease the activity of NF-κB of intestinal mucosa, improve the morphology and functions of digestive tract, decreased the diarrhea frequency and promote the growth of weanling piglets. It imply that p-ASAP can be applied as organic-arsenic feed additive instead of p-arsanilic acid for its higher biological activity in pig production.