| Campylobacter is one of the most common food-borne intestinal pathogens in the world. In order to reduce the risk of people exposing to the pathogen, it is a traditional measure to control Campylobacter in the primary production stage of poultry by supplement antibacterial agent in feed or drinking water. Since the potential safety hazard such as drug resistance and residue caused by the abusing of antibiotic, natural additives have been widely used in feed of animals which act as substitute for antibiotics. In this study, organic acids used-in-feed were selected by in vitro experiments and the effect of them on Campylobacter were explored. The acids selected in vitro were supplied in drinking water of broilers in order to evaluate the effect of acids on Campylobacter loading and intestinal microflora of broilers. Assessments of the application of acids were made by the detection of intestine, production processing and distribution of acid tolerance genes.1. Selection and analysis of the effects of organic acids on CampylobacterInoculation concentration of Campylobacter and the concentration of organic acids for evaluation were selected.7 kinds of organic acids, propionic acid, citric acid, lactic acid, and so on chosen from<GB 2760-2011 using standards of standard food additives for national food safety> were co-cultured with Campylobacter in vitro, in order to select the most effective bactericide for Campylobacter. The inhibition efficiency, the effects of organic acids on structure and metabolism of Campylobacter were explored as well.In vitro assay, the inoculation concentration of Campylobacter was chosen at 5×108 CFU/mL, the concentration of organic acids for evaluation was chosen at 0.1%(w/v). The result of selection of organic acids showed that, propionic acid, tartaric acid and malic acid were most effective to inhibit Campylobacter. The comparison results of inhibition efficiency of organic acids suggested that 0.1% propionic acid,0.1% tartaric acid and 0.15% malic acid could reduce the concentration to undetectable levels within the monitoring time, respectively. The inhibitory effect of organic acids on Campylobacter from strongest to weakest was propionic acid, tartaric acid, malic acid. In the view of scanning electron microscope, cell walls of Campylobacter co-cultured with 0.1% propionic acid for 20 h showed shrinking distinctly, even with some holes on the surface. While in the groups of tartaric acid and malic acid, the cell walls of most bacteria obviously shrinking and round-shaped appeared on poles, some bacteria broke up in view. A large number of leaking of the contents detected by UV spectrophotometer showed bacterial cells of Campylobacter damaged by treatment of 0.1% propionic acid and 0.15% malic acid when co-cultured for 20 h, respectively. Concentrations of total soluble sugar in supernatant of propionic acid and malic acid treatment groups showed significantly higher than control, indicating that glycometabolism of Campylobacter may be affected in these two groups. The results of phosphorus metabolism showed that after 30 h phosphorus concentration in culture supernatant of 0.15% malic acid treatment group was significantly reduced, indicating that malic acid could affect phosphorus metabolism of Campylobacter.2. The effect of organic acids on Campylobacter loading in poultry on-farmThe chicken drinking water (pH 4.0) containing 0.253% propionic acid,0.059% tartaric acid and 0.075% malic acid were prepared and applied, respectively. Cloaca swabs were collected on the day of supplement of acids and 1,2,3 weeks post administered for monitoring positive rates and dynamics of Campylobacter loading in cloaca. The total number of colonies in the gut was detected at the same time, for the purpose of the comparison of potency of organic acids in aspects of antibiotic effect and intestinal microflora balance. The detection of Campylobacter loading in cloaca showed that malic acid could significantly reduce Campylobacter loading in cloaca by 0.41-0.48 log (P<0.05), the inhibition rate up to 61.10%?66.88% when supplied for 1 week, but the impact of other two acids were instable. No significant difference was found in the second week in Campylobacter loading between treatment and control groups (P>0.05). In the third week, Campylobacter loading of all treatment groups were significantly higher than control, except the AA broilers in propionic acid group. The detection result of total colonies in the gut of broilers suggested that the number of all treatment groups were significantly higher than control in the first week of supplement (P<0.05). No significant difference was found in the 2-3 weeks after supplement between propionic acid, malic acid treatment groups and control (P>0.05). Malic acid was most effective to reduce Campylobacter loading with minimum effect on the total number in the gut of broilers when applying in drinking water for 1 week.The market-aged broilers drinking water containing 0.15%,0.225%,0.4%,0.8% and 1.2% (w/v) malic acid were prepared and applied, respectively. Cloaca swabs and intestines were collected on the day of supplement of acids and 2,5 days post administered for monitoring positive rates and dynamics of Campylobacter loading in cloaca and intestine. The results of the detection of cloaca swabs of slaughter-day-old broilers showed Campylobacter loading in cloaca in 0.15% malic acid group was significantly reduced by 1.55 log (P<0.05), the inhibition rate up to 97.18% on the fifth day of supplement.1.2% malic acid could made Campylobacter loading in cloaca of AA broilers markably reduce by 0.75 log (P<0.05), no more than that of 0.15% group, whose antibacterial rate was 82.22%. No significant difference was found in other treatment groups (P>0.05). The detection of Campylobacter loading in cecum indicated that Campylobacter loading in cecum in 0.4% and 0.8% malic acid groups were respectively reduced by 1.56 log and 1.35 log, inhibition rates were 97.25% and 95.53%, significantly lower than that of control (P<0.05). The detection of Campylobacter loading in jejunum suggested that 0.8% malic acid could markedly decrease the positive rate of Campylobacter in jejunum by 80% on the fifth day after supplement (P<0.05). The detection of total number of colonies in the gut showed that 0.15% malic acid had no significant effect on gastrointestinal microflora.3. The effect of organic acids on gut, production performance of poultry and acid tolerance genes in Campylobacter isolatesIn this study, intestine and production performance affected by organic acids were detected. In addition, detection of the dynamic distributions of 10 acid tolerance genes in Campylobacter isolates were also carried out by PCR. The commercialization performance of propionic acid, tartaric acid and malic acid were assessed in aspects of practical use and changes of the distributions of acid tolerance genes in Campylobacter isolates.The detection result of total weight of intestine showed that organic acids made no significant effect on it no matter supplied in drinking water of broilers daily or on slaughter age. The detection result of length of intestine of broilers showed that propionic acid could significantly increase the length of cecum by 12.89%(P<0.05), while malic acid and tartaric acid made no significant difference on that by daily supplement (P>0.05). Malic acid made no significant difference on the length of intestine on slaughter-age broilers (P>0.05). The result of pH value of intestine of broilers demonstrated that the pH value of Ma chickens' jejunum in tartaric group was decreased by 2.44%(P<0.05), pH value of cecum of AA broilers was decreased by 5.47 in malic acid group (P<0.05). Production performance of broilers showed that organic acids could increase the weight and average daily gain (ADG) of broilers and decrease cull-and-death rate when supplied for 5 days, without significant difference (P>0.05). Malic acid made no significant effect on weight or ADG of broilers (P>0.05). The result of conventional components of muscle of broilers showed that no significantly adverse impact on meat quality by the daily using of propionic acid or malic acid (P<0.05). While the supplement of tartaric acid daily may markedly affect the contents of crude fat and ash (P<0.05).0.15% malic acid could increase the content of moisture in both breast and leg and decrease the content of crude fat in the leg of broilers on slaughter age significantly. The results above suggested that the use of 0.15% malic acid could markedly improve the special flavor and taste of chicken.The results of the distribution of acid stress related genes in 161 Campylobacter isolates showed that no changes in distributions of genes associated with acid resistance in Campylobacter isolates were induced by long-term using of organic acids. |
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