Donald E. Corrier

Effect of Dietary Lactose on Cecal pH, Bacteriostatic Volatile Fatty Acids, and Salmonella typhimurium Colonization of Broiler Chicks

One-day-old broiler chicks were inoculated with volatile fatty acid producing cecal flora from adult chickens. The chicks were divided into four groups and provided 1) no lactose, 2) 2.5% lactose in water, 3) 5% lactose in feed, or 4) 10% lactose in feed, until 10 days of age. All groups were challenged at 3 days of age with 10(6) or 10(8) S. typhimurium. At 10 days, the number of Salmonella in the ceca of the chicks challenged with 10(6) Salmonella was significantly decreased (P less than 0.01) in the groups provided lactose as compared with the controls. A significant decrease (P less than 0.01) in Salmonella numbers occurred in the chicks challenged with 10(8) Salmonella and provided 10% lactose. Providing 2.5% lactose or 5% lactose failed to inhibit Salmonella growth in chicks challenged with 10(8) Salmonella. The pH of the ceca of the groups provided lactose decreased significantly (P less than 0.05) and was accompanied by significant increases (P less than 0.01) in the concentrations of bacteriostatic acetic and propionic acids. Results showed that providing dietary lactose to broiler chicks and inoculation with normal cecal flora decreased cecal pH, increased the concentrations of bacteriostatic volatile fatty acids, and inhibited Salmonella colonization.

In vivo activation of heterophil function in chickens following injection with Salmonella enteritidis-immune lymphokines.

We have previously shown that increased resistance to Salmonella enteritidis organ infectivity in day‐old chicks was conferred by the immunoprophylactic administration of S. enteritidis‐immunc lymphokines (ILK). This resistance was associated with a significant increase in the number of circulating heterophils 4 h after ILK injection. The objective of the present study was to evaluate heterophil function following the administration of ILK in day‐old chicks. Significant increases (P < 0.001) in adherence, chemotaxis, and phagocytosis of S. enteritidis were found with heterophils isolated from ILK‐injected chickens compared to the heterophils isolated from birds injected with either pyrogen‐free saline or lymphokines from non‐immune T cells. After phagocytosis, the heterophils from the ILK‐injected chickens were also able to kill significantly greater numbers of S. enteritidis more rapidly than did the heterophils from the saline‐injected control birds (within 30 min, control cells killed 21.89% of the bacteria whereas ILK‐treated cells killed 88.22%). We also found that the heterophils from the ILK‐injected birds were more efficient killers of S. typhimurium, S. gallinarum, and E. coli These results strongly suggest that the protection against S. enteritidis organ invasion induced by the prophylactic treatment of day‐old chicks with ILK involves activated heterophils which migrate rapidly to the inflammatory stimulus where they phagocytize and kill the bacteria. J. Leukoc. Biol. 57: 56–62; 1995.

Effect of Carbohydrates on Salmonella typhimurium Colonization in Broiler Chickens

The effect of carbohydrates in the drinking water of broiler chickens on Salmonella typhimurium colonization was evaluated. Results indicate that mannose and lactose (2.5%) significantly (P less than 0.05) reduced intestinal colonization of S. typhimurium by at least one-half, as compared with dextrose, maltose, and sucrose. Lactose and mannose also significantly reduced (P less than 0.01) the mean log10 number of S. typhimurium in the cecal contents. Although mannose was the most effective sugar at blocking colonization, lactose may be more practical because it is effective and costs much less than mannose. Provision of carbohydrates in the drinking water had no significant effect on weight gain.

Biological Control of Salmonella typhimurium in Young Chickens

The effect of dietary lactose and anaerobic cultures of cecal microflora of mature chickens on the colonization of young broiler chickens by Salmonella typhimurium was evaluated. Newly hatched chicks were given either no treatment (controls), anaerobic cecal cultures, lactose (2.5%) in the drinking water, or both anaerobic cultures and lactose. Chicks were challenged per os at 3 days of age with either 10(6) or 10(8) S. typhimurium resistant to nalidixic acid and novobiocin. On day 10, the cecal contents of the chicks were examined for S. typhimurium, pH, short-chained volatile fatty acids (VFAs), undissociated VFAs, and lactic acid. Chicks given either lactose alone or cecal anaerobes alone had significantly (P less than 0.05) fewer S. typhimurium recovered from their ceca than the controls. Chicks given the combination of dietary lactose and cecal anaerobes had significantly fewer S. typhimurium recovered from their ceca than the chicks given dietary lactose or cecal anaerobes alone. Chicks given lactose had significant (P less than 0.05) increases in the lactic acid concentration of their cecal contents. Increased lactic acid concentrations were directly correlated to decreased cecal pH values and caused a reduction in the total concentration of VFAs but a significant (P less than 0.05) increase in the undissociated form of some VFAs.

Short-Chain Volatile Fatty Acids Modulate the Expression of the hilA and invF Genes of Salmonella Typhimurium

The ability of Salmonella typhimurium to invade the intestinal mucosal cells is an important step in pathogenesis. This invasion process requires genes encoded on the Salmonella pathogenicity island 1 (SPI1). Two transcriptional activators, HilA and InvF, encoded in SPII regulate the expression of invasion genes in response to environmental stimuli such as osmolarity, oxygen tension, and pH. During its pathogenic life cycle, Salmonella typhimurium is also exposed to short-chain fatty acids (SCFA), especially acetate, propionate, and butyrate, in the intestinal lumen, as well as the SCFA used as food preservatives. The effects of SCFA on the expression of hilA and invF-lacZY transcriptional fusions were examined to determine the potential role of SCFA in the pathogenesis of Salmonella typhimurium. Growth rates were reduced by increasing SCFA concentrations at pH 6 but not at pH 7. At pH 7, hilA and invF expression was induced by acetate but not by propionate or butyrate, while at pH 6, all SCFA induced hilA and invF expression at 1 h. In general, hilA and invF expression levels when compared to respective control responses were higher at 1 h than at 4 and 8 h in the presence of most SCFA concentrations at pH 6. However, expression levels at 4 and 8 h were either similar or higher than the 1-h responses for the hilA-lacZY fusion strain in the presence of acetate while exposure to 20 mM propionate yielded similar levels of expression at 1, 4, and 8 h. The pH-dependent manner of induction suggests that entry of SCFA into the cell was necessary for induction. We speculate that SCFA may serve as an environmental signal that triggers the expression of invasion genes in the gastrointestinal tract.

Effect of dietary lactose on salmonella colonization of market-age broiler chickens.

The effect of providing lactose in feed and inoculation with volatile fatty acid-producing anaerobic cultures (AC) of cecal flora on Salmonella typhimurium colonization was evaluated in broilers. One-day-old chicks were divided into four groups and provided 1) no lactose, no AC; 2) AC, no lactose; 3) AC and lactose on days 1-10; or 4) AC and lactose on days 1-40. All groups were challenged per os with 10(6) Salmonella on day 3 and with 10(8) Salmonella on day 33. Salmonella growth in the cecal contents was significantly decreased (P less than 0.01) on day 10 in the chicks provided lactose from day 1-10. However, after the removal of lactose from the diet, the chicks were susceptible to Salmonella colonization. The number of Salmonella in the ceca was significantly reduced (P less than 0.05) in the chicks provided lactose throughout the 40-day growing period. Dietary lactose decreased the pH of the cecal contents and was accompanied by marked increases in the concentrations of undissociated bacteriostatic volatile fatty acids in the cecal contents.

Effect of Mixed Cecal Microflora Maintained in Continuous Culture and of Dietary Lactose on Salmonella typhimurium Colonization in Broiler Chicks

Mixed cecal microflora obtained from a mature chicken were maintained in vitro in continuous-flow (CF) culture. The effect of the CF culture and dietary lactose on Salmonella typhimurium cecal colonization in broiler chicks was evaluated. When averaged across four replicates, chicks treated with the culture alone (1.75 log10 decrease) or with 5% dietary lactose alone (2.98 log10 decrease) were protected against S. typhimurium. Optimum protection against S. typhimurium was observed when birds were treated with the culture in combination with dietary lactose (4.27 log10 decrease). Dietary lactose resulted in reduced cecal pH. A large increase in cecal propionic acid was observed in the birds given the CF culture. A significant correlation (P < 0.001) was observed between the cecal concentration of undissociated propionic acid and protection against S. typhimurium colonization (r = -0.78). The results indicated that indigenous cecal flora that protect against Salmonella colonization can be maintained without loss of efficacy in CF culture.

Intracloacal Salmonella typhimurium infection of broiler chickens: reduction of colonization with anaerobic organisms and dietary lactose.

The combined effect of treatments with dietary lactose plus anaerobic organisms on cecal colonization of broiler chicks by Salmonella typhimurium was evaluated. Chickens treated with a combination of anaerobic organisms and 7% dietary lactose were resistant to cecal colonization by S. typhimurium. The number of recoverable S. typhimurium cells per gram of cecal contents taken on days 10 and 15 after infection was significantly reduced. Treatment with anaerobes without the addition of lactose did not effectively control cecal colonization. Intracloacal inoculations with bacterial concentrations that varied by 10,000-fold resulted in roughly similar levels of colonization. The treatments resulted in reduced cecal pH and elevated levels of undissociated volatile fatty acids. Statistically significant correlations (P less than 0.01) were observed between the S. typhimurium concentrations in cecal material and the concentrations of undissociated fatty acids (r = -0.79, and between the bacterial counts and pH (r = 0.72).

Provision of lactose to molting hens enhances resistance to Salmonella enteritidis colonization.

Older leghorn hens, more than 50 weeks of age, were divided into three groups designated 1, unmolted controls; 2, molted; or 3, molted treated with lactose. Forced molt was induced by 14 days of feed removal. Lactose was provided to the hens in group 3 as 2.5% (wt/vol) of the daily drinking water. Each hen in all groups was challenged orally with 10(5) Salmonella enteritidis (SE) cells on day 7 of feed removal. The study was repeated in three replicated trials. The concentrations of acetic, propionic, and total volatile fatty acids (VFA) in the cecal contents of the molted hens in groups 2 and 3 decreased significantly (P < 0.05) on days 6 and 14 of molt compared with the unmolted controls. Forced molt had no apparent effect on pH or on the oxidation-reduction potential of the ceca. Compared to the unmolted controls, SE cecal and spleen and liver colonization was significantly increased (P < 0.05) in the molted hens in group 2. Compared to the molted hens in group 2, SE cecal and spleen and liver colonization was significantly decreased (P < 0.05) in two of three trials in the hens in group 3 provided with lactose. The results suggested that the increased susceptibility of molting hens to SE colonization may be associated with decreased fermentation and production of VFA by cecal bacteria or by a depletion of the number of VFA-producing bacteria present in the ceca. The results further suggest that providing lactose in the drinking water during molting may significantly enhance resistance to SE colonization.

Effect of Anaerobic Cecal Microflora and Dietary Lactose on Colonization Resistance of Layer Chicks to Invasive Salmonella enteritidis

The effect of oral inoculation with anaerobic cultures of cecal microflora and providing lactose in the feed on colonization resistance to invasive Salmonella enteritidis was evaluated in newly hatched leghorn chicks. Salmonella colonization of the ceca, tissue invasion and organ colonization, horizontal transmission, and seroconversion were significantly decreased (P less than 0.01) in chicks inoculated with cecal flora. The addition of lactose to the feed, in the absence of cecal microflora, failed to provide protection. Dietary lactose enhanced colonization resistance in chicks that were inoculated with anaerobic cultures of cecal flora. The results indicated that establishment of normal cecal flora in layer chicks together with the addition of lactose to the diet markedly increases resistance to cecal colonization and organ invasion, and decreases horizontal transmission of S. enteritidis.