Independent research by Canadian investigators using state-of-the-art technology confirms that alpha-toxin, a secreted protein from Clostridium perfringens, plays a key role in the development of necrotic enteritis (NE) in broilers and that other proteins may also be involved in the pathogenesis of this complex disease.

Immunization with alpha-toxin provided almost total protection of broiler chickens against NE, while other secreted proteins produced by virulent C. perfringens yielded various degrees of immunity, Dr. John F. Prescott, of the University of Guelph, Ontario, said at a meeting of the Ontario Association of Poultry Practitioners held in 2008 in Guelph.

Necrotic enteritis has become an economically important disease for the broiler industry. Although the cause is known to be C. perfringens, exactly how this bacterium causes NE has been uncertain, Prescott explained.

The major culprit has been assumed to be alpha-toxin. Earlier this year, however, Australian researchers published an article about a novel C. perfringens toxin they identified, called NetB. In widely publicized findings, they indicated that NetB was the main cause of NE and, on the basis of their carefully conducted research, discounted the role of alpha-toxin.

Canada study

Studies by Prescott and his colleagues, however, show that immunization with alpha-toxin provided the best protection against a severe C. perfringens challenge with a virulent strain that contains the NetB gene.

In their studies, which Prescott reviewed at the Ontario meeting, several proteins secreted by C. perfringens were evaluated for their ability to protect broilers against the virulent strain of the organism. The proteins were alpha-toxin, glyceraldehyde- 3-phosphate dehydrogenase, pyruvate: ferredoxin oxidoreductase (PFOR), fructose 1,6-biphosphate aldolase and a fifth one called hypothetical protein (HP).

Broilers were immunized two to three times with one of the proteins, then one week after their last immunization, they were challenged with the virulent strain, which was administered in feed at 4 weeks of age.

The severity of the challenges differed; a mild challenge, for instance, involved feeding the virulent strain to birds three times daily for 3 days, and the most severe challenge involved feeding the virulent strain daily to birds continuously for 5 days. The severity of each challenge was confirmed by NE lesion scores in non-immunized but challenged control birds.

All the proteins significantly protected broilers against the relatively mild challenge. For the more severe challenge, alpha-toxin, PFOR and HP provided significant protection, Prescott said.

Alpha-toxin provided best protection

The greatest protection against severe challenge, however, occurred in birds that were primed twice with alpha-toxoid — a toxin that is altered so it is no longer toxic but still initiates immunity — and then boosted with active, purified toxin, Prescott and colleagues found in their study, published in 2007 in the September issue of Clinical and Vaccine Immunology.

In addition, serum and intestinal washings from protected birds had high antigenspecific antibody titers for all proteins used in their study, the researchers found.

This was the first report demonstrating the immunizing ability of C. perfringens secreted proteins in protecting broilers against NE, Prescott and associates wrote.

They concluded that there are certain secreted proteins, in addition to alpha-toxin, involved in immunity to NE in broiler chickens. NetB

NetB may be marker for NE strains

Prescott and associates also used polymerase chain reaction to test the virulent challenge strain and found it was positive for the NetB gene.

“The fact...that immunization with alpha-toxin strongly protected birds against experimental NE caused by a NetB-containing isolate suggests that alpha-toxin actually is critical to the development of NE, and perhaps that NetB may only initiate infection,” he said.

“I know that the Australian workers think that the success of antibody against alpha-toxin in protecting so well against NE is because it may interfere with the secretion of all proteins by this organism, including, for example, the secretion of NetB. It will be hard to prove this, and actually may not matter if alpha-toxin immunization works so well,” he added.

In addition, unpublished observations from Ontario show that genetically unrelated isolates from sick birds in flocks with NE “were systematically NetB-positive, whereas isolates from healthy birds at slaughter were usually negative” for NetB, he said.

“Almost but not quite all [C. perfringens] isolates from birds with NE or from flocks experiencing NE have NetB, so it’s a good marker for a strain of C. perfringens that causes necrotic enteritis,” Prescott said.

Asked by Intestinal Health why the search continues for other secreted proteins when it has already been shown that alpha-toxin can protect broilers from NE, Prescott said, “It will help us understand NE better, though I agree that alpha-toxin should be the main focus. On the basis of the findings of protection of birds following immunization, alpha-toxin apparently has a central role in NE,” but there may be an advantage to using more than one protein.

Proteins differ, he added, in their structure, in their activity, including toxicity, and in their targets.

Favors vaccine

Methods for controlling NE might include probiotics to provide bacterial competition for C. perfringens or killing C. perfringens with novel antibiotics, but Prescott favors immunization.

“I think a vaccine probably has the most promise because it should be the most reliable. I like the idea of an oral vaccine because it could also be used to deliver other antigens and products,” said Prescott, who has been experimenting with an orally administered, attenuated salmonella vaccine vector with C. perfringens antigens.

Even though there is still much to be learned about NE, he predicts rapid advancement in the quest to conquer the disease, thanks to large-scale genome sequencing and other technologies. Due to these advances, “scientists working on NE around the world have made more strides in the last 3 to 4 years than in the previous 25 years,” he said.

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