Protection Against Necrotic Enteritis in Broiler Chickens by Regulated Delayed Lysis Salmonella Vaccines.

Necrotic enteritis (NE), caused by Gram-positive Clostridium perfringens type A strains, has gained more attention in the broiler industry due to governmental restrictions affecting the use of growth-promoting antibiotics in feed. To date, there is only one commercial NE vaccine available, based on the C. perfringens alpha toxin. However, recent work has suggested that the NetB toxin, not alpha toxin, is the most critical virulence factor for causing NE. These findings notwithstanding, it is clear from prior research that immune responses against both toxins can provide some protection against NE. In this study, we delivered a carboxyl-terminal fragment of alpha toxin and a GST-NetB fusion protein using a novel attenuated Salmonella vaccine strain designed to lyse after 6-10 rounds of replication in the chicken host. We immunized birds with vaccine strains producing each protein individually, a mixture of the two strains, or with a single vaccine strain that produced both proteins. Immunization with strains producing either of the single proteins was not protective, but immunization with a mixture of the two or with a single strain producing both proteins resulted in protective immunity. The vaccine strain synthesizing both PlcC and GST-NetB was able to elicit strong production of intestinal IgA, IgY, and IgM antibodies and significantly protect broilers against C. perfringens challenge against both mild and severe challenges. Although not part of our experimental plan, the broiler chicks we obtained for these studies were apparently contaminated during transit from the hatchery with group D Salmonella. Despite this drawback, the vaccines worked well, indicating applicability to real-world conditions.

A low gastric pH mouse model to evaluate live attenuated bacterial vaccines.

The low pH of the stomach serves as a barrier to ingested microbes and must be overcome or bypassed when delivering live bacteria for vaccine or probiotic applications. Typically, the impact of stomach acidity on bacterial survival is evaluated in vitro, as there are no small animal models to evaluate these effects in vivo. To better understand the effect of this low pH barrier to live attenuated Salmonella vaccines, which are often very sensitive to low pH, we investigated the value of the histamine mouse model for this application. A low pH gastric compartment was transiently induced in mice by the injection of histamine. This resulted in a gastric compartment of approximately pH 1.5 that was capable of distinguishing between acid-sensitive and acid-resistant microbes. Survival of enteric microbes during gastric transit in this model directly correlated with their in vitro acid resistance. Because many Salmonella enterica serotype Typhi vaccine strains are sensitive to acid, we have been investigating systems to enhance the acid resistance of these bacteria. Using the histamine mouse model, we demonstrate that the in vivo survival of S. Typhi vaccine strains increased approximately 10-fold when they carried a sugar-inducible arginine decarboxylase system. We conclude that this model will be a useful for evaluating live bacterial preparations prior to clinical trials.

Low-pH rescue of acid-sensitive Salmonella enterica Serovar Typhi Strains by a Rhamnose-regulated arginine decarboxylase system.

For Salmonella, transient exposure to gastric pH prepares invading bacteria for the stresses of host-cell interactions. To resist the effects of low pH, wild-type Salmonella enterica uses the acid tolerance response and the arginine decarboxylase acid resistance system. However, arginine decarboxylase is typically repressed under routine culture conditions, and for many live attenuated Salmonella vaccine strains, the acid tolerance response is unable to provide the necessary protection. The objective of this study was to enhance survival of Salmonella enterica serovar Typhi vaccine strains at pHs 3.0 and 2.5 to compensate for the defects in the acid tolerance response imposed by mutations in rpoS, phoPQ, and fur. We placed the arginine decarboxylase system (adiA and adiC) under the control of the ParaBAD or PrhaBAD promoter to provide inducible acid resistance when cells are grown under routine culture conditions. The rhamnose-regulated promoter PrhaBAD was less sensitive to the presence of its cognate sugar than the arabinose-regulated promoter ParaBAD and provided tighter control over adiA expression. Increased survival at low pH was only observed when adiA and adiC were coregulated by rhamnose and depended on the presence of rhamnose in the culture medium and arginine in the challenge medium. Rhamnose-regulated acid resistance significantly improved the survival of ΔaroD and ΔphoPQ mutants at pHs 3 and 2.5 but only modestly improved the survival of a fur mutant. The construction of the rhamnose-regulated arginine decarboxylase system allowed us to render S. Typhi acid resistant (to pH 2.5) on demand, with survival levels approximately equivalent to that of the native arginine decarboxylase system.

Safety and protective efficacy of live attenuated Salmonella Gallinarum mutants in Rhode Island Red chickens.

Salmonella enterica serovar Gallinarum is the causative agent of fowl typhoid, an important systemic disease of poultry with economic consequences in developing nations. A live attenuated orally applied S. Gallinarum vaccine could provide a low cost method for controlling this disease. We constructed S. Gallinarum strains in which the expression of the crp, rfc and rfaH genes, important for virulence of Salmonella Typhimurium in mice, were under the control of an arabinose-regulated promoter. We evaluated the virulence of these strains compared to wild-type S. Gallinarum and to mutants carrying deletions in these genes. We found that rfc mutants were fully virulent, indicating that, unlike the S. Typhimurium mouse model, the rfc gene is dispensable in S. Gallinarum for virulence in birds. In the case of rfaH, the deletion mutant was attenuated and protective, while the strain with arabinose-regulated rfaH expression retained full virulence. The strain exhibiting arabinose-regulated crp expression was attenuated. Its virulence was not affected by the inclusion of 0.2% arabinose in the drinking water. Birds immunized with this strain were protected against a lethal S. Gallinarum challenge and against colonization with the human pathogen Salmonella Enteritidis. This work shows that an arabinose-regulated crp strain provides a basis for further development of a fowl typhoid vaccine.