Why are researchers investigating the role of alpha-toxin in the development of necrotic enteritis? Two scientists share insights from studies that could impact the management of NE, a serious and costly disease.

Strong evidence that alpha-toxin plays a role in the development of necrotic enteritis (NE) has been demonstrated in studies utilizing an alpha-toxin test kit and immunohistochemistry, Dr. Joan Schrader said at the World’s Poultry Congress.

Alpha-toxin is a toxic protein secreted by the bacterium Clostridium perfringens. It is also a component of C. perfringens type A toxoid, a conditionally licensed US vaccine that is administered to breeders for control of NE in progeny chicks. The vaccine was developed by Intervet/ Schering-Plough Animal Health, said Schrader, a scientist with the company.

The recent availability of a commercial diagnostic test-strip kit designed to detect C. perfringens and alpha-toxin in feces provided a new way to evaluate the role of alpha-toxin in the development of NE, she said. Schrader also conducted immunohistochemistry to physically demonstrate alpha-toxin at the lesion site.

The test utilizes monoclonal antibodies to both C. perfringens type A and alpha-toxin bound to a paper strip. When the strip is exposed to these antigens in solubilized chicken feces, one line develops color in the presence of C. perfringens type A, and a second line develops color in the presence of alpha-toxin.

Study details and results

For the study, 52 commercial, day-old broiler chicks were placed in floor pens at the company’s R&D facility in Elkhorn, Nebraska. Thirty-five test chicks were housed in one hut, and the remaining chicks were housed in another hut and were used as controls.

Chicks were fed a non-medicated starter ration for the first 5 days and were then switched to a high-protein diet for the remainder of the study. When the test chicks were 19, 20 and 21 days of age, a C. perfringens type A challenge was performed by oral gavage.

At 23 days of age, fecal material was collected from the caudal rectum/cloaca of each chicken and tested according to the kit instructions. Three strips were tested for each sample, Schrader said.

Chickens were also scored for NE lesions, which were used to determine the true prevalence of NE, and the ability of the test strips for detecting C. perfringens and alpha-toxin was determined, she said.

The overall prevalence of positive test, according to lesion score, for C. perfringens in birds was 33% for score 0 (6/18), 18% for score 1 (14/78), 19% for score 2 (9/48), 61% for score 3 (11/18) and 88% for score 4 (16/18). The incidence of positive test strips for C. perfringens was not different between birds positive or negative for NE (Table 1), Schrader said.

Table 1. Incidence of positive test strips for C. perfringens was not different between birds positive or negative for NE.

CP = Clostridium perfringens
Note: Alpha-toxin was only detected in chickens positive for NE

Alpha-toxin was not detected by the test strips until lesion scores reached 3 or 4: The test kit was able to detect alpha-toxin in 37% (7/18) of tests among chickens with lesion scores of 3 and in 71% (13/18) of tests among chickens with lesion scores of 4, she said.

The study showed a good correlation between lesion score and the detection of alpha-toxin, with higher lesion scores resulting in greater detection of alphatoxin with the test kit, Schrader said.

In addition, the finding that high lesion scores correlated with positive test-strip results for C. perfringens and alpha-toxin at the site of NE lesions “supports the hypothesis that the severity of the gross lesions is directly proportional to the number of C. perfringens present and amount of alpha-toxin produced,” she said.

Immunohistochemistry results

Schrader then performed immunohistochemistry on NE lesions, a technique that has been widely used to detect the presence of disease agents in tissues. To perform the test, a very thin-sliced tissue sample is fixed to a slide. An “anti-antibody” that has fluorescent or pigmented material is added to the slide and binds to the antibody in question if that antibody is present. In this case, “There was clearly a positive binding of antibodies,” Schrader explained (see Figure 1).

Figure 1. An “anti-antibody” with fluorescent or pigmented material binds to the antibody in question if that antibody is present.

The results of the study, Schrader concluded, “strongly demonstrate the involvement of alpha-toxin in the disease of necrotic enteritis.”

Although the test kit was useful for the purposes of her study, she said, it would not be particularly helpful in the field for producers trying to detect subtle, subclinical NE that can go unnoticed but eat away at performance. It would pick up birds with overt, clinical NE and high lesion scores, and “by then you’d already know the birds are sick.”

US study suggests alpha-toxin plays role in cause of NE

Vaccination of broilers with recombinant alpha-toxin protected broilers against an experimental challenge with Clostridium perfringens, suggesting that alpha-toxin plays a role in the pathogenesis of necrotic enteritis (NE).

In the study, broiler chicks were vaccinated subcutaneously with recombinant alpha-toxin at 5 and 15 days of age, then 10 days later were challenged with C. perfringens, the cause of necrotic enteritis. The challenge was administered twice daily for 4 consecutive days by mixing C. perfringens cultures with feed.

Non-vaccinated birds challenged with C. perfringens developed NE at the rate of 87.8%, but only 54.9% of vaccinated birds developed lesions. In addition, non-vaccinated birds had lesion scores averaging 2.37, compared to 1.35 in vaccinated birds, write K. K. Cooper and colleagues at the University of Arizona, Tucson, in the June 2008 issue of Veterinary Microbiology.

Vaccination also produced an antibody response — post-vaccination anti-alpha-toxin titers in vaccinated birds were more than 5-fold greater than in non-vaccinated birds. After challenge, vaccinated birds had average IgG (IgY) titers >15-fold higher compared to those of non-vaccinated birds, the investigators say.

NE in poultry has re-emerged as a concern for poultry producers due in part to the ban on in-feed antimicrobial growth promoters, but the results of this study, say the investigators, suggest that alpha-toxin may serve as an effective immunogen and, as such, may play a role in pathogenesis of necrotic enteritis.

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 nonimmunized 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.

NetB may be marker

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 alphatoxin 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.

NE is a complicated disease

Finding ways to prevent or control necrotic enteritis ( NE) in broilers is challenging because Clostridium perfringens, the bacterium that causes the disease, has chameleon-like qualities, and other factors, such as management, may be involved.

At the World’s Poultry Conference this summer in Brisbane, Dr. John Prescott, of the University of Guelph, called C. perfringens “an absolute thug.”

The bacterium is “exquisitely adapted as an environmental anaerobe to grow very rapidly in injured or dead animal tissue. Consider that Escherichia coli doubles every 20 minutes. In contrast, C. perfringens is the fastest growing organism known and, under optimal conditions, doubles every 8 to 10 minutes,” he said.

“It is superbly designed to take advantage of injured tissue,” he said. It secretes multiple toxins and enzymes that maximize the destruction of tissues.

Dr. Joan Schrader, a scientist with Intervet/Schering-Plough Animal Health who has researched NE and helped develop the company’s Clostridium perfringens type A toxoid for broilers, agrees.

“It’s as though virulent C. perfringens has an arsenal of toxins it can produce, and depending on the environment the bacterium is in, it will use the toxins that are most advantageous for the circumstances. It’s very much a multifactorial disease,” she says.

Schrader echoes Prescott’s opinion, saying that while “alphatoxin is a key player, other secreted proteins from C. perfringens may be involved in development of this complicated disease.”

In addition, secreted proteins may be only part of the story.

In his OAPP talk, Prescott pointed to published evidence that dietary components might adversely affect intestinal motility or damage intestinal mucosa, which in turn affect C. perfringens toxin production or the growth of C. perfringens. Coccidial infection can be a contributing factor too, he said.

“The interaction of [C. perfringens] with other intestinal microflora, including non-NE isolates, and the effect of other microflora on intestinal innate immunity” may be important, he said.

There’s no question, he and Schrader say, that NE is a complex infection.

Alpha-toxin gene linked to necrotic enteritis in India

A study conducted on broilers from India confirmed that Clostridium perfringens type A was the cause of necrotic enteritis (NE) and that alpha-toxin may play a significant role in development of the disease, say Arunava Das of the Bannari Amman Institute of Technology, and associates.

After six broilers died at 2 to 3 weeks of age on a poultry farm in Meghalaya, India, investigators performed scanning electron microscopy (SEM) and evaluated intestinal contents and liver samples.

SEM revealed massive necrosis and complete destruction of the intestinal villi within the intestinal mucosa. Bacterial isolation confirmed that C. perfringens was the cause. Polymerase chain reaction (PCR) testing of 10 clinical isolates showed they all harbored the alpha-toxin gene of C. perfringens; four were positive for the beta2 toxin gene; and none were positive for the beta, epsilon, iota or enterotoxin genes.

All isolates derived from NE belonged to C. perfringens type A and there was 97.6% to 100% homology among the C. perfringens isolates, they write in a recent issue of the International Journal of Poultry Science (7 (6): 601-609, 2008).

The study confirms that C. perfringens type A is the most predominant one associated with necrotic enteritis in broiler chickens in this region of India and that the alpha-toxin gene might play a significant role in the pathogenesis of the disease in broiler chickens, the investigators conclude.

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