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 held recently in Brisbane, Australia.

Alpha-toxin is a toxic protein secreted by the bacterium Clostridium perfringens. It is also a component of Netvax, a Clostridium perfringens type A toxoid vaccine. Netvax, which is conditionally licensed in the US, 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.

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 alpha-toxin 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).

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.”

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