Evaluation of Coated Butyrate to Reduce Campylobacter Infection in Broilers01 March 2015
This experiment from Nutriad shows that supplementation of a diet with coated butyrate resulted in a statistically significant decrease in Campylobacter counts in broiler caeca from day 29 onwards.
During the last years, Campylobacter (Figure 1) has gained considerable interest as an important cause of human food-born gastroenteritis.
There is increasing evidence that the consumption of Campylobacter jejuni-contaminated poultry and poultry products is the main contributor to human campylobacteriosis cases. Campylobacter may reside within the intestinal tract of broilers as a commensal, resulting in rapid horizontal transmission of the human pathogen in a flock, as well as in carcass contamination during slaughter.
Reducing the number of intestinal Campylobacter via the inclusion of pathogen-controlling feed additives in the broiler diet, could therefore provide a valuable strategy to produce a safer poultry end product. Indeed, studies predict that the reduction of intestinal Campylobacter in broilers by 2-log10, would result in a 30-fold reduction of human campylobacteriosis cases (Rosenquist et al., 2003).
One of the feed additives that deserves special attention in this context, is butyrate.
Sodium butyrate is the salt of a short-chain fatty acid that is well-studied for its beneficial effects in several regions of the gastro-intestinal tract (GIT) of animals and humans. For example, butyrate can stimulate the production of digestive enzymes and the development of intestinal villi, while it reduces inflammatory responses and inhibits the growth of colon cancer cells (Guilloteau et al., 2012).
The discovery of these biological effects has triggered a series of studies focusing on the potential of butyrate in human medicine, and on its function as performance enhancer in several livestock animals. Interestingly, butyrate also has the potential to have an effect on gut microbiota composition: it stimulates the proliferation of lactic acid bacteria in the upper part of the small intestine, while in the lower parts of the GIT, it induces secretion of antibacterial peptides and, most importantly, can interfere with virulence mechanisms of Salmonella (Gantois et al., 2006). These effects have been documented to result in a significant reduction of colonisation and shedding of this pathogen in poultry and pigs (Van Immerseel et al., 2005).
However, for butyrate to be able to exert its effects on microbial control, it has to be delivered to the distal regions of the GIT, where Salmonella resides. This is not a trivial task, as butyrate is a small molecule that is rapidly taken up by cells in the stomach and the proximal part of the small intestine. Therefore, in recent years, a number of feed additive companies have invested in the development of a protective fat-based matrix. Many of these coatings only mask the pungent smell of butyrate, but some of them have the additional property that they can protect the embedded butyrate from gastric absorption, and allow the gradual release of butyrate throughout the intestines.
Given its gut health promoting effects and its documented success in controlling Salmonella, a preliminary Campylobacter study was set up at the University of Bologna (Italy) with a product that assures post-gastric precision delivery of butyrate. This product was evaluated for its efficacy to reduce the number of C. jejuni in the caeca of infected broilers.
A total of 72 commercial day-old broilers were randomly divided into three treatment groups of 24 birds each, and reared in three separate isolator units of 1.2 square metres each. Birds were fed a basal diet containing corn and soybean ranging between 31.50 to 13 per cent and 22.66 to 16.5 per cent, respectively.
The first treatment group only received the basal diet, without supplementation of feed additives; the diet of the second group was supplemented with 0.3 per cent coated butyrate, while the third group received a supplement of 0.5 per cent coated butyrate.
Ten cloacal swabs were randomly collected from individual birds belonging to each group and tested for the presence of Campylobacter on days 10 and 18. The swabs were added to medium to prepare a cellular suspension, which was incubated and plated on mCCDA under microaerobic conditions. The analysis of Campylobacter was also performed for feed and drinking water on days 10 and 18 after hatching.
Cloacal swab analysis was negative at day 10, while feed and drinking water were Campylobacter-negative on days 10 and 18.
An experimental infection with C. jejuni was planned at day 20. However, cloacal swab analysis was found to be positive for all three groups on day 18. Using selective media, microscopic analysis and a multiplex PCR-protocol, the Campylobacter strains isolated from the birds were all identified as C. jejuni. As the estimated Campylobacter counts in all groups were comparable to the bacterial load that would have been used for infection (more than 5-log Colony Forming Units per swab), it was decided to continue the trial without additional inoculation at day 20.
C. jejuni colonisation was monitored by euthanising the chicks by cervical dislocation on days 20, 29 and 39.
Each time, six or nine caeca were aseptically removed for microbiological analysis, and 10g caecal content per bird was serially diluted. Dilutions were plated on mCCDA medium. After incubation at 42°C for 48 hours under microaerobic conditions, colonies were inspected microscopically and enumerated. The number of CFUs per gram of caecum content were transformed into log10 values and analysed using the ANOVA test. The mean values for each group were compared using the Scheffe test. Differences at P values less than 0.01 were considered statistically significant.
Results and Discussion
The mean count of Campylobacter in the caeca of broilers fed with the basal diet without supplementation of coated butyrate did not statistical significantly change over the period of analysis (see Figure 2).
The mean caecal Campylobacter counts of the groups fed 0.3 per cent or 0.5 per cent of butyrate, however, showed a statistically significant reduction over time compared to the control group. On day 29, the 0.3 per cent butyrate group displayed a mean count of 3.77-log CFU per gram, while the number of Campylobacter in the 0.5 per cent group was found to be below the detection level of 3-log CFU per g. No Campylobacter counts above this detection level were found in the caecal contents of both the 0.3 per cent and the 0.5 per cent group on day 39. All samples with values under the detection limit were also Campylobacter-negative as determined by PCR-analysis.
These mean count values were reflected in the number of caeca with detectable levels of Campylobacter at day 20 (6 out of 6, for all treatment groups), day 29 (9/9, 3/9 and 0/9 for the control group, the 0.3 per cent group and the 0.5 per cent group, respectively) and day 39 (9/9 for the control group and 0/9 for both butyrate groups).
The effect of the coated butyrate used in this study on caecal Campylobacter counts in broilers might be mediated through various biological mechanisms. Butyrate might kill Campylobacter by enhancing the formation of antimicrobial butyric acid in the intestine or by modulating the activity of bacterial virulence genes, as seen in Salmonella. Alternatively, butyrate might exert its effect through empowering the intestinal barrier, thereby reducing Campylobacter invasion and/or translocation over the intestinal epithelium.
Further research will be required to elucidate butyrate's mode of action, as well as to establish the optimal dosage and timing at which this particular coated butyrate should be applied to reduce Campylobacter count during broiler production.
In this trial, the supplementation of the coated butyrate under investigation resulted in a statistically significant decrease of Campylobacter counts in broiler caeca from day 29 onwards.
A reduction of minimum two log-units of intestinal Campylobacter in commercial broilers, as seen in this study, is estimated to result in a 30-fold decrease of the most commonly reported human food-borne disease worldwide (Rosenquist et al., 2003).
As retailers and governmental organisations worldwide are continuously increasing pressure on poultry production companies to take measures to increase food safety, the use of this precision-delivery coated butyrate seems to be a promising strategy to reduce Campylobacter at the farm level.