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Clostridium perfringens α-Toxin and NetB Toxin Antibodies and Their PossibleRole in Protection Against Necrotic Enteritis and Gangrenous Dermatitis inBroiler ChickensAuthor(s): K. W. Lee, H. S. Lillehoj, M. S. Park, S. I. Jang, G. D. Ritter, Y. H. Hong, W. Jeong, H. Y.Jeoung, D. J. An and E. P. LillehojSource: Avian Diseases, 56(1):230-233. 2012.Published By: American Association of Avian PathologistsDOI: http://dx.doi.org/10.1637/9847-070711-ResNote.1URL: http://www.bioone.org/doi/full/10.1637/9847-070711-ResNote.1

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Research Note—

Clostridium perfringens a-Toxin and NetB Toxin Antibodies and Their Possible Rolein Protection Against Necrotic Enteritis and Gangrenous Dermatitis in

Broiler Chickens

K. W. Lee,A H. S. Lillehoj,AF M. S. Park,A S. I. Jang,A G. D. Ritter,B Y. H. Hong,C W. Jeong,D H. Y. Jeoung,D D. J. An,D andE. P. LillehojE

AAnimal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, USDA, Beltsville, MD 20705BMountaire Farms, Millsboro, DE 19966

CDepartment of Animal Science and Technology, Chung-Ang University, Anseong 456-756, South KoreaDNational Veterinary Research and Quarantine Service, Anyang City, Kyunggido, 430-824, South Korea

EDepartment of Pediatrics, University of Maryland School of Medicine, Baltimore, MD 21201

Received 7 July 2011; Accepted and published ahead of print 16 September 2011

SUMMARY. Necrotic enteritis (NE) and gangrenous dermatitis (GD) are important infectious diseases of poultry. AlthoughNE and GD share a common pathogen, Clostridium perfringens, they differ in other important aspects such as clinical signs,pathologic symptoms, and age of onset. The primary virulence factors of C. perfringens are its four major toxins (a, b, e, i) and thenewly described NE B-like (NetB) toxin. While neutralizing antibodies against some C. perfringens toxins are associated withprotection against infection in mammals, the serologic responses of NE- and GD-afflicted birds to these toxins have not beenevaluated. Therefore, we measured serum antibody levels to C. perfringens a-toxin and NetB toxin in commercial birds from fieldoutbreaks of NE and GD using recombinant toxin-based enzyme-linked immunosorbent assay (ELISA). Initially, we used thisELISA system to detect antibody titers against C. perfringens a-toxin and NetB toxin that were increased in birds experimentally co-infected with Eimeria maxima and C. perfringens compared with uninfected controls. Next, we applied this ELISA to field serumsamples from flock-mated birds with or without clinical signs of NE or GD. The results showed that the levels of antibodies againstboth toxins were significantly higher in apparently healthy chickens compared to birds with clinical signs of NE or GD, suggestingthat these antitoxin antibodies may play a role in protection against NE and GD.

RESUMEN. Nota de Investigacion—Anticuerpos contra las toxinas a y NetB de Clostridium perfringens y su posible papel en laproteccion contra la enteritis necrotica y dermatitis gangrenosa en pollos de engorde.

La enteritis necrotica (NE) y la dermatitis gangrenosa (GD) son enfermedades infecciosas importantes de la avicultura comercial.Aunque la enteritis necrotica y la dermatitis gangrenosa comparten un patogeno comun, Clostridium perfringens, estas dosenfermedades difieren en otros aspectos importantes tales como los signos clınicos, cambios patologicos, y la edad de inicio. Losprincipales factores de virulencia de C. perfringens son sus cuatro principales toxinas (a, b, e, i) ademas de la toxina recientementedescrita que es similar a la toxina B de enteritis necrotica (NetB). Mientras que los anticuerpos neutralizantes contra algunas toxinasde C. perfringens se asocian con la proteccion contra la infeccion en los mamıferos, las respuestas serologicas contra estas toxinas enlas aves afectadas por enteritis necrotica y dermatitis gangrenosa no han sido evaluadas. Por lo tanto, se midieron los niveles sericosde anticuerpos contra las toxinas a y la NetB de C. perfringens en aves comerciales de brotes de campo de la enteritis necrotica y ladermatitis gangrenosa utilizando un ensayo de inmunoabsorcion con enzimas ligadas (ELISA) utilizando una toxina recombinante.Inicialmente, se utilizo este sistema ELISA para determinar los tıtulos de anticuerpos contra las toxinas a y NetB de C. perfringensque se incrementaron en aves experimentalmente infectadas de manera simultanea con Eimeria maxima y C. perfringens encomparacion con los controles no infectados. A continuacion, se aplico esta prueba de ELISA en el estudio de muestras de suero deparvadas de aves con o sin signos clınicos de enteritis necrotica y de dermatitis gangrenosa. Los resultados mostraron que los nivelesde anticuerpos contra las toxinas fueron significativamente mayores en pollos aparentemente sanos, en comparacion con las aves consignos clınicos de enteritis necrotica y dermatitis gangrenosa, lo que sugiere que estos anticuerpos antitoxina puede jugar un papelen la proteccion contra la enteritis necrotica y la dermatitis gangrenosa.

Key words: necrotic enteritis, gangrenous dermatitis, Clostridium perfringens, broiler chicken

Abbreviations: ELISA 5 enzyme-linked immunosorbent assay; GD 5 gangrenous dermatitis; NE 5 necrotic enteritis;OD450 5 optical density at 450 nm; PBS 5 phosphate-buffered saline; PBS-T 5 phosphate-buffered saline containing 0.05%Tween 20

Necrotic enteritis (NE) is an acute or chronic enterotoxemia inchickens, turkeys, and ducks caused by Clostridium perfringens, agram-positive, anaerobic, spore-forming bacterium, and character-ized by a fibronecrotic enteritis of the small intestine (1,14,17).Gangrenous dermatitis (GD) is caused by C. perfringens and, to alesser extent Clostridium septicum, resulting in red to black patches of

moist, gangrenous skin over the breast, abdomen, wing tips, orthighs with feather loss and sloughing of the epidermis. In additionto their unique clinical signs and symptoms, NE generally occurs at17–18 days of age whereas GD develops at 4–7 wk (10,16).Combined with C. perfringens, co-infection with immunosuppressiveviruses, reuse of litter, high stocking density, and high viscosity dietsare other common risks factors that have been proposed tocontribute to both diseases (5,7,8).FCorresponding author. E-mail: Hyun.Lillehoj@ARS.USDA.GOV

AVIAN DISEASES 56:230–233, 2012

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An increasing incidence of NE in Europe subsequent to thediscontinued use of in-feed antibiotic growth promoters has beenreported (15). Use of antibiotic ionophores (e.g., salinomycin),however, is still permitted to control avian coccidiosis resulting frominfection by Eimeria protozoa. Some coccidiostats also possess anti-bacterial activity against clostridia and can prevent NE. Ironically,however, GD is not uncommon in commercial poultry feedingprograms containing ionophore coccidiostats (13). In addition,Ritter (12) first reported that NE-afflicted flocks were protectedfrom later GD outbreaks and speculated that early exposure to C.perfringens may induce an immune response to clostridial toxins thatprevented later infections. Unlike mammals (20), however, thepresence of antibodies against C. perfringens toxins has not beencorrelated with protection against bacterial infection in chickens. Inthis communication, we provide evidence that healthy chickensraised on farms with NE- or GD-afflicted chickens have higher titersof antitoxin antibodies compared with their diseased cohorts.

For this purpose, we developed an enzyme-linked immunosorbentassay (ELISA) using the recently developed C. perfringens recombi-nant toxin proteins a-toxin and NE B-like (NetB) toxin (6). TheELISA system was validated using serum samples from NE birds thatwere induced by dual challenge with Eimeria maxima and C.perfringens. Next, we applied the ELISA to field NE- or GD-afflictedchickens to monitor their antibody status.

MATERIALS AND METHODS

Serum samples. Peripheral blood samples were collected from chickenson commercial farms with clinically confirmed outbreaks of NE (NE-afflicted farm) or GD (GD-afflicted farm) (n 5 12 each). Clinical signs ofNE in 14-day-old birds included ballooning of the intestine with ulcerativeto necrotic lesions in the jejunum. Clinical signs of GD in 35-day-old birdsincluded discoloration of the skin over the breast, abdomen, and wings asa result of congestion, necrosis, and emphysema (gas accumulation),discoloration of muscle with serosanguineous fluid, and fibronecroticenteritis. As controls, blood samples were obtained from 12 apparentlyhealthy birds raised on the NE-afflicted farm and from eight healthychickens from the GD-afflicted farm. Sera were isolated by centrifugationand stored at 220 C.

NE experimental model. The NE disease model was successfullyreproduced by dual infection with C. perfringens and E. maxima (11) usingpreviously described methods (18). Both C. perfringens and E. maximachallenge strains were isolated from field NE- or coccidiosis-afflicted

chickens. In brief, 30 1-day-old broiler chicks (Ross strain) wereprovided with a nonmedicated, low-protein (17% protein) basal dietand switched to commercial, nonmedicated, high-protein (24%protein) diet at 18 days of age. At 14 days, chickens were uninfected(n 5 15/group) or orally infected (n 5 15/group) with 1.0 3 104

sporulated oocysts of E. maxima. At 4 days postinfection, the E.maxima-infected chickens were orally infected with 1.0 3 109 of apathogenic C. perfringens field isolate (11). In general, gut lesionscharacteristic of NE developed at 2 days following C. perfringensinfection. At 0, 2, 7, and 14 days following C. perfringens infection,blood samples were obtained from uninfected or E. maxima–C.perfringens co-infected birds (n 5 3) and serum was stored at 220 C.Two birds in the NE group were dead at 2 days following C. perfringensinfection and were eliminated from this study.

C. perfringens recombinant a-toxin and NetB toxin ELISA.Clostridium perfringens a-toxin and NetB toxin genes were expressed inEscherichia coli and their corresponding recombinant proteins werepurified as described (6). Recombinant bacteria were cultured overnightwith shaking at 37 C and induced for 5 hr at 37 C with 1.0 mMisopropyl b-D-thiogalactopyranoside (Amresco, Solon, OH). The cellswere harvested by centrifugation at 15,000 3 g for 10 min at 4 C,resuspended in phosphate-buffered saline (PBS), and disrupted bysonication on ice (Misonix, Farmingdale, NY). The sonicate wasrecentrifuged and the pellet was solubilized in 8 M urea. Both thesupernatant and solubilized pellet were applied to an Ni-NTA-agarosecolumn (Qiagen, Valencia, CA), the column was washed with PBS, andrecombinant proteins were eluted with PBS containing 0.25 Mimidazole, pH 9.2. Flat-bottom, 96-well microtiter plates (CorningCostar, Corning, NY,) were coated overnight at 4 C with 100 mg/well ofa- or NetB toxins in 0.1 M carbonate buffer, pH 9.6. The plates werewashed twice with PBS containing 0.05% Tween 20 (PBS-T), blockedfor 1 hr at 22 C with 100 ml/well of PBS containing 1.0% bovine serumalbumin, washed with PBS-T, and incubated for 2 hr at 22 C with100 ml/well of appropriately diluted serum samples. The plates werewashed with PBS-T, incubated for 2 hr at 22 C with horseradishperoxidase-conjugated goat anti-chicken IgY secondary antibody(1:1000; Sigma, St. Louis, MO), washed in PBS-T, and incubated for10 min with 0.01% tetramethylbenzidine substrate (Sigma) in 0.05 Mphosphate-citrate buffer, pH 5.0. Bound antibodies were detected bymeasuring optical density at 450 nm (OD450) using a microplate reader(Bio-Rad, Richmond, CA). All values were corrected for backgroundreactivity in the absence of recombinant toxins.

Statistical analysis. Data were expressed as the mean 6 SD values.Comparisons of the mean values were performed with the Student’st-test and differences between groups were considered statisticallysignificant at P , 0.05.

Fig. 1. Antibody levels against C. perfringens a-toxin and NetB toxin in experimental NE in chickens. Broiler chickens were uninfected (control)or co-infected with E. maxima and C. perfringens (NE). Serum samples (n 5 3) were taken at 0, 2, 7, and 14 days following C. perfringens infectionand analyzed by ELISA with purified recombinant a-toxin and NetB toxin. Data points not sharing common letters are significantly different(P , 0.05).

C. perfringens toxin-specific antibodies in NE and GD birds 231

RESULTS AND DISCUSSION

Clostridium perfringens a-toxin and NetB toxin are well-characterized virulence factors that have been implicated in thepathogenesis of NE and GD in chickens (3,7,8). Initially, wemeasured antibody titers against both toxins using an experimentalmodel of NE by oral infection of chickens with live Eimeriaparasites, feeding a high-protein diet, and subsequent oral infectionwith virulent C. perfringens at 5 days following Eimeria infection(11,18). This co-infection model system consistently reproduces theintestinal lesions that are characteristically seen in NE that isacquired in the field. Antibody titers against C. perfringens a-toxinand NetB toxin were increased in birds experimentally co-infectedwith E. maxima and C. perfringens compared with uninfectedcontrols, thus validating the antitoxin antibody responses in birdsinfected with the bacterial pathogen (Fig. 1).

To gain further characterization of the serologic responses againsta-toxin and NetB toxin, the levels of serum antibodies weremeasured in birds from commercial production farms with clinicalsymptoms of NE or GD and in disease-free chickens raised underthe same field conditions. NE- or GD-afflicted chickens hadsignificantly lower antibody levels against both toxins compared withthe clinically healthy controls (Tables 1, 2). To the best of ourknowledge, this is the first report to quantify toxin antibody levels inNE- or GD-affected chickens naturally infected with C. perfringens.Given that C. perfringens is a spore-forming bacterium, it is likelythat all of the chickens examined, both healthy and diseased, wereexposed to the bacteria from used, contaminated litter. Indeed, ourprior study demonstrated that broiler chickens exposed to used litterfrom a GD endemic farm had detectable C. perfringens-reactiveantibodies that gradually and persistently increased between 14 and43 days postexposure, whereas bacterial antibodies were absent inbirds raised on fresh, uncontaminated litter (K. W. Lee, unpubl.obs.). The higher levels of toxin-reactive antibodies in apparentlyhealthy chickens compared to chickens with symptoms of NE or GDsupports the hypothesis that these antibodies protect the exposedbirds from developing overt disease. It is unclear, however, whatfactors may influence the production of high-level, antitoxinantibodies in healthy chickens or may contribute to lower antibodytiters in the diseased animals.

The presence of higher antitoxin antibody titers in C. perfringens-exposed, healthy chickens compared with diseased birds is consistentwith the epidemiologic study of Heier and colleagues (2). Theseinvestigators observed that the levels of maternal antibodies againstC. perfringens a-toxin were inversely associated with mortality incommercial broiler flocks. Lovland et al. (9) provided evidence thatbroilers hatched from breeder hens previously immunized with a C.perfringens toxoid vaccine displayed signs of protective immunityagainst experimental NE compared with chickens hatched fromunvaccinated hens. As expected, immunization of breeder hensinduced high levels of serum IgG antibodies against C. perfringens a-toxin. Recently, recombinant subunit vaccines incorporating C.perfringens-derived antigenic proteins were used to induce protectiveimmunity against experimental challenge infection (4,19). The

immunized birds produced antigen-specific IgY antibodies in serumand antigen-specific IgY and IgA antibodies in the intestine. Inconclusion, these results and those of the current study highlight theimportance C. perfringens toxin antibodies in protection againstbacterial infection and raise the possibility of increasing theresistance of chickens to NE or GD by measures that enhanceantitoxin antibody levels.

REFERENCES

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2. Heier, B. T., A. Lovland, K. B. Soleim, M. Kaldhusdal, and J. Jarp. Afield study of naturally occurring specific antibodies against Clostridiumperfringens alpha toxin in Norwegian broiler flocks. Avian Pathol. 45:724–732. 2001.

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Table 2. Antibody levels against C. perfringens toxins in healthy andGD-afflicted chickens.

Toxin

Healthy (n 5 8) GD (n 5 12)

P valueMean SD Mean SD

a-Toxin 1.038A 0.053 0.910 0.052 0.001NetB 0.906 0.061 0.698 0.056 0.0001

AELISA OD450 values corrected for background reactivity.

Table 1. Antibody levels against C. perfringens toxins in healthy andNE-afflicted chickens.

Toxin

Healthy (n 5 12) NE (n 5 12)

P valueMean SD Mean SD

a-Toxin 0.748A 0.114 0.636 0.100 0.024NetB 0.779 0.124 0.596 0.084 0.002

AELISA OD450 values corrected for background reactivity.

232 K. W. Lee et al.

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ACKNOWLEDGMENTS

This project was supported, in part, by a trust agreement establishedbetween ARS-USDA and Animal Plant and Fisheries Quarantine andInspection Agency, Republic of Korea, and the ARS in-house project(1265-32000-086-00D) and the Next-Generation BioGreen 21 Program(No. PJ008084), Rural Development Administration, Republic of Korea.We thank Marjorie Nichols and Stacy Torreyson for technical assistance.

C. perfringens toxin-specific antibodies in NE and GD birds 233