Immunization of broiler chickens against Clostridium perfringens-induced necrotic enteritis

Abstract

Necrotic enteritis (NE) in broiler chickens is caused by Clostridium perfringens. Currently, no vaccine against NE is available and immunity to NE is not well characterized. Our previous studies showed that immunity to NE followed oral infection by virulent rather than avirulent C. perfringens strains and identified immunogenic secreted proteins apparently uniquely produced by virulent C. perfringens isolates. These proteins were alpha-toxin, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase (PFOR), fructose 1,6-biphosphate aldolase, and a hypothetical protein (HP). The current study investigated the role of each of these proteins in conferring protection to broiler chickens against oral infection challenges of different severities with virulent C. perfringens. The genes encoding these proteins were cloned and purified as histidine-tagged recombinant proteins from Escherichia coli and were used to immunize broiler chickens intramuscularly. Serum and intestinal antibody responses were assessed by enzyme-linked immunosorbent assay. All proteins significantly protected broiler chickens against a relatively mild challenge. In addition, immunization with alpha-toxin, HP, and PFOR also offered significant protection against a more severe challenge. When the birds were primed with alpha-toxoid and boosted with active toxin, birds immunized with alpha-toxin were provided with the greatest protection against a severe challenge. The serum and intestinal washings from protected birds had high antigen-specific antibody titers. Thus, we conclude that there are certain secreted proteins, in addition to alpha-toxin, that are involved in immunity to NE in broiler chickens.

FIG. 1.

Recombinant Clostridium perfringens histidine-tagged proteins purified from Escherichia coli cells. (A) Coomassie-stained purified proteins; (B) reactivities of purified proteins to immune serum from chickens immune to NE. Lanes 1, alpha-toxin (45 kDa); lanes 2, GPD (40 kDa); lanes 3, FBA (35 kDa); lanes 4, tPFOR (67 kDa); lanes 5, HP (90 to 100 kDa); lanes M, molecular mass standards.

FIG. 2.

Summary of mean lesion scores for birds from all immunized groups across different experiments, together with those for the concurrent unimmunized controls. VC, vehicle-only controls, A-tox, alpha-toxin; Sup, culture supernatant of C. perfringens; G+H, combination of GPD and HP; Exp, experiment; +, the birds in this group were challenged for 3 days and necropsied on day 6; ++, the birds in this group were given a severe challenge, like the one used in experiment 3; *, the immunized group had significantly fewer chickens with lesions than the unimmunized vehicle-only control group (Fisher's exact test, P ≤ 0.05).

FIG. 3.

Serum IgY ELISA titers of broiler chickens immunized intramuscularly with Clostridium perfringens purified proteins. Serum was collected at three time points: day 0, for determination of the preimmunization titer; day 10 (midexperiment); and day 20, for determination of the prechallenge titer. Exp, experiment. In experiment 4A, the birds were challenged for 3 days and necropsied on day 6. In experiment 4B, the birds were given a severe challenge, like the one used in experiment 3. *, significant titer values compared to the preimmunization titers (P ≤ 0.01).

FIG. 4.

Intestinal IgY and IgA ELISA titers of broiler chickens immunized intramuscularly with Clostridium perfringens purified proteins. The samples analyzed were from pooled intestines collected from at least 10 chickens in each group. Exp, experiment. In experiment 4A, the birds were challenged for 3 days and necropsied on day 6. In experiment 4B, the birds were given a severe challenge, like the one used in experiment 3.