Campylobacter is one of the leading causes of food-borne gastroenteritis in humans worldwide(2–4), which accounts for approximately 96 million cases of human illness per year on a global scale(15). Poultry is the most common species associated with human Campylobacter illness, and most chicken flocks became Campylobacter-positive when reached slaughter age, making it an important reservoir for human infection. After Campylobacter infect humans, the clinical symptoms could include mild abdominal pain, headaches, fever, vomiting and sever watery and bloody diarrhea, and the infection can sometimes lead to serious sequelae such as Guillain-Barré syndrome, Miller Fisher syndrome and reactive arthritis(15), although most cases were self-limiting. Still, a number of patients required medication and hospitalization treatment, which brings great health and economic burden to the public(3, 4, 15). Therefore, the control of Campylobacter colonization in chickens at the farm level should be meaningful work.
Malic acid can be industrially produced at present and has the advantages of no pollution/residue, lack of toxicity and easy of application(16). In previous studies, it was found that malic acid could cause a 6 log reduction of Campylobacter in laboratory broth and a 4 log reduction in chicken juice after 24 h of exposure at 4 °C(13). The contamination of Campylobacter on chicken legs was also observed to decrease 1.18 log after treatment with malic acid solution at 4 °C for 8 days(14). These results foreshadow the promising application of malic acid in the poultry industries, but the antimicrobial effect of malic acid towards chicken feeding still stayed unclear. Malic acid is commonly recognized as a mild acid and has wide application in the food industry(16), which makes it possible to be applied in animal feeding. Our pre-experiment found that the effect of malic acid against the growth of Campylobacter in vitro was obvious (Supplementary Figure S1). Besides, an in vivo study showed that malic acid had a more stable effect to control the Campylobacter contamination when compared to other acids (Supplementary Figure S2). The minimum inhibitory concentration of malic acid against Campylobacter was also found to be lower than for other acids(17), suggesting that a more effective and economical potency may exist.
Organic acids exploit their antimicrobial activity in the undissociated form, which is closely related to the pH of the medium(18). Previous studies have indicated that the well bactericidal effect of organic acids on Campylobacter strains shows up at a pH of 4.0(19), and thus we decided to adjust the pH to 4.0 using malic acid in this study. There is no evidence of the vertical transmission of Campylobacter in chicken flocks, while the cross contamination from the environment seems to be an important infection source(4, 20). Malic acid could be supplemented into the feed or drinking water of the broilers. The dry conditions of the feed is lethal to Campylobacter, which is thus recognized as not being a potential source for contamination(21). Meanwhile, water is an important vehicle for spreading Campylobacter that is prominent in chicken flocks(4, 21, 22). Thus, malic acid was added into the drinking water in our study.
Although most studies reported the effectiveness of using organic acid to control the contamination of pathogenic bacteria during animal rearing(17, 22–24), some results showed its limited or variable effect which could not be ignored(25–27). The bacteriostatic or bactericidal effect of the organic acid may depend on the manner of use (concentration, vehicle, and duration) and is also related to the status of the host. In our study, we found that supplementation of malic acid into the drinking water for the chicken flocks at slaughter age was effective in two chicken lines, the Campylobacter load in the cloaca was found to be decreased by 1.05–1.55 log cfu/g, while in the caeca was decreased by 1.56 log cfu/g. However, in a long period daily rearing for three weeks, the effect of malic acid was not consistent, with significant decontamination only found in the first week of application. Our present study suggests that application of acidified drinking water to the broilers at slaughter age could effective limited the Campylobacter infection load. Limitation of its use to the last week before slaughter also has other advantages, such as cost savings, thus making it more economical. In addition, Campylobacter infection of the broilers at slaughter age are epidemiologically more relevant to human infection, thus making the decontamination more meaningful. Nevertheless, the reason why a long period of application of acidified drinking water has a limited decontamination effect on Campylobacter still requires further investigation.
Malic acid is a flavoring agent, and it is also an intermediate in metabolic cycles, including the tricarboxylic acid (TCA) cycle and the glyoxylic acid cycle (GAC). Feeding diets with malic acid was found to increase the weight gain and feed consumption in Japanese quails(11). For dairy cows, the feed efficiency was improved, the milk yield was increased and the fat content in the milk was subtly changed(12). The growth and feed utilization were found to be improved in tilapia, but an excess supply would compromise the beneficial effect(10). Despite that inconsistent findings were reported(28), these results showed the potential benefits of feeding malic acid to animals when applied with suitable nutritional and managerial measures. In our study, the broiler body weight as well as intestinal indices, including length, weight, pH, and microflora were not influenced by the malic acid supplementation, indicating that the chicken performance was not affected at least. The bacteriostatic effect was found on Campylobacter, while not on the normal intestinal microflora, which may be attributed to the acid-intolerance characteristics of the pathogens when compared to the gut microflora (especially for the probiotics)(29). The propagation of the pathogens was inhibited, which will in turn promote the growth of microflora as it reduces microbial competition for nutrients(22, 30).
A previous study showed that feeding malic acid to cows could influence the composition of milk(12), and thus its potential effect on chicken meat was analyzed. Our results showed that the protein content of the chicken meat was not influenced by the malic acid treatment, suggesting that the nutrition was maintained. Compared to the control group, the moisture was increased by 5.12% and 5.92% in thigh and breast meat, respectively, while the fat content was decreased by 1.60% in thigh meat. This contributes to the tenderness, juiciness and taste sensory of the meat and promotes the acceptability and preference of the customers(31, 32). The changes in the chicken meat composition may be attributed to the influence of malic acid on the sense of chicken taste, and could affect the feed intake, nutrient digestion and conversion. Organic acids could exert beneficial effects on disease resistance, which was observed in tilapia(33, 34). In this study, the malic acid-supplemented drinking water was provided to one flock, which suffers from respiratory disease, and after three weeks of treatment with acidified water, the mortality was found to be decreased from 52–32%, which is also an additional benefit for chicken rearing.
This study showed that the malic acid-supplemented drinking water could benefit chicken production while also improving food safety. Our study only applied malic acid, and many improvements could be considered in the subsequent research, such as the use of malic acid in combination with prebiotics or bacteriophages(35) as well as with nutritional, managerial and biosecurity measures(30). Additionally, the bactericidal effect of malic acid was observed as not only restricted to Campylobacter but also on other food-borne pathogens(9), which foreshadows its promising application improving the safety of poultry production.